Footprint of Nonconventional Yeasts and Their Contribution in Alcoholic Fermentations

FOOTPRINT OF NONCONVENTIONAL YEASTS AND THEIR CONTRIBUTION IN ALCOHOLIC FERMENTATIONS

14

Maurizio Ciani, Laura Canonico, Lucia Oro, Francesca Comitini Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy

14.1 Introduction Fermentation is an ancient method for preserving foods that is strictly correlated to complex multispecies microbial communities. However, over thousands of years, to obtain specific desired products humans mostly optimized the technological process with poor attention to microbial aspect. Indeed, for a long time it was thought that the technological process was the principal aspect that mainly defines the organoleptic characteristics of the final product (Khan et  al., 2013). In the last few years, more emphasis is given to the microbial component, intended as responsible for the specificity of the final product (Bourdichon et al., 2012). Indeed, researchers are now agreeing that peculiar physiological characteristics of the single species and the overall metabolic interactions of the microbial communities is key to control the safety, flavor, texture, and aroma of fermented foods and beverages. Among the fermented beverages, wine is the result of the complex interactions among yeast, bacteria, and other fungi that origin in vineyards and continues with the fermentation process in winery. Different yeast species are predominant on the surface of grape skins and in the winery environment (Albergaria and Arneborg, 2016), and Saccharomyces cerevisiae is recognized as the main responsible for this process (Pretorius, 2000). In recent years, yeasts with fermentative aptitude, belonging to non-Saccharomyces species are being studied in

Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00014-X © 2020 Elsevier Inc. All rights reserved.

435

436  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

applied research, evidencing the important role in wine fermentation under certain controlled conditions (Ciani et al., 2010, 2016). Indeed, the microbial interactions between selected strains of S. cerevisiae and non-Saccharomyces yeasts may give positive effects to alcoholic fermentation (Nissen et al., 2003; Pérez-Nevado et al., 2006; Albergaria et al., 2010; Branco et al., 2014; Kemsawasd et al., 2015; Wang et al., 2015). Hanseniaspora uvarum, Hanseniaspora guilliermondii, Starmerella bacillaris (formerly Candida zemplinina and Candida stellata), Metschnikowia pulcherrima, Lachancea thermotolerans (previously classified as Kluyveromyces thermotolerans), and Torulaspora delbrueckii are the main fermentative yeast species naturally associate to vineyard environment (Bauer and Pretorius, 2000). On the other hand, Saccharomyces species, main responsible for wine fermentation, are rarely isolated from the microbial community naturally associated with fruits (Goddard et al., 2010). Moreover, focusing attention on the microbial aspect of “terroir,” recent works highlight that peculiar flavors and aromas in wine may be in part due to regionally structured microbial diversity (Tofalo et al., 2014; Bokulich et al., 2016; Morrison-Whittle and Goddard, 2017). Studies based on the relationship between fermentation variability and managed or unmanaged ecosystems represent a new and interesting target of current applied research. The microbial conversion of fermentable sugars into alcohol during fermentation is also the key process that also characterizes the beer production. Although there is a great diversity of beers, they can be classified into two major types, ale and lager, according to the yeast used and fermentation conditions (Petruzzi et al., 2016). As well as in oenological field, also in brewing is well known that the choice of yeast has a great impact on the quality of the final product. Indeed the inoculated starter strain produces metabolites such as acetate, ethyl esters, and higher alcohols that give characteristic flavor to beer (Pires et al., 2014). Recently, on increased number of studies focused on the use of non-Saccharomyces strains for the production of different beers as low-alcohol beers, functional, and distinctive beers (Basso et al., 2016; Varela, 2016). Although this current research topic has just begun to be explored, there is growing evidence of the enormous potential for the application of non-Saccharomyces yeast in the brewing industry. Indeed, several non-Saccharomyces yeast species are found during spontaneous fermentations of American coolship ales and Belgium lambic beer, such as Meyerozyma guilliermondii, Debaryomyces spp., Pichia spp., Wickerhamomyces anomalus, Brettanomyces spp., Candida krusei, Cryptococcus keutzingii, and Rhodotorula mucilaginosa (Bokulich et  al., 2012; Spitaels et  al., 2014). More recently, an hunting campaign was conducted in various natural habitat with

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   437

the aim to isolate yeast strains with peculiar features, in particular yeast able to produce sour beer without the use of lactic acid bacteria (Osburn et al., 2018). However, little has been studied on the fitness of non-Saccharomyces yeasts of single or in mixed starter cultures for the production of beer.

14.2  Spontaneous Fermentations: Failures or Success? In most societies, fermented beverages and foods have an important role for their economic and cultural significance and the development of fermentation technologies is deeply established in their history. Yeast is the main protagonist for several industrial fermentation processes, including the fermented beverages. Historically, these fermentative processes developed from unknown, uncontrolled, and spontaneous reactions due to a complex mixture of microbes present in the diverse natural niches. Yeast species play an important role in the winemaking processes: transforming sugar to ethanol, producing specific secondary metabolites, and finally, contributing to wine flavor characteristics. Spontaneous wine fermentation is an ecologically complex process, and it is well established that the yeast population change as the fermentation proceeds (Fleet, 2008; Wang and Liu, 2013). The main yeast involved in this transformation process belonging to the Saccharomyces genus. However, other wine yeasts can also be isolated during the process, and can influence on analytical and aromatic wine composition (Andorrà et al., 2010; Wang et al., 2015). Indeed, grape must is a nonsterile nutrient rich substrate that allows growth and fermentation activity of various yeasts. As a consequence, spontaneous fermentation is carried out through a sequential occupation of the substrate by different yeast species. Indeed, a series of microbiological analyses of the yeast flora associated with natural fermentation of grape juice, revealed that in most enological areas, a sequential fermentation takes place: initially, apiculate yeasts (Hanseniaspora/Kloeckera) are the most abundant, although after 3–4 days, they are replaced by S. cerevisiae (Martini, 1993; Pretorius, 2000). In addition, during the various stages of fermentation, it is possible to isolate other yeast genera, such as Candida, Pichia, Zygosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, and Metschnikowia (Heard and Fleet, 1985; Pardo et  al., 1989). Because of such spontaneous processes may be inconsistent, inefficient and/or lead to the formation of off-flavors, the applied research and industrial production utilize specific starter cultures, often domesticated strain of S. cerevisiae (Verstrepen et al., 2006).

438  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

The spontaneous fermentation in brewing process occurs in some specific beer style. An example of spontaneous fermentation in beer production is a lambic beer where the wort, after the boiling, is left in the air and it come inoculated naturally by yeasts and bacteria. In beer process there is a succession of four different microbial phases related to evolution of sequential microbial colonization that lasts for 1–3 years. The Enterobacteriaceae phase that starts after 3–7 days of fermentation is characterized by different bacteria, Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Hafnia alvei and different yeasts H. uvarum and Saccharomyces uvarum. The second phase is dominated by elliptical yeast belong to S. cerevisiae, S. bayanus/pastorianus, and S. uvarum species. The acidification phase is characterized by Pediococcus spp. and occasionally Lactobacillus spp., while Brettanomyces spp. becomes prevalent after 4–8 months of fermentation. The final phase is the maturation in which the wort is gradually attenuated and lactic acid bacteria decrease. However, in lambic beer and gueuze beer (a blend of old and new lambic), Dekkera/Brettanomyces yeast is the main responsible in the phase of acidification and maturation. These yeasts produce peculiar flavor compounds like ethyl phenol, ethyl guaiacol, isovaleric acid, acetic acid, ethyl acetate responsible of a typical aroma of these beers. Another example of beer brewed with a spontaneous fermentation is American coolship ale, a sour beer produced in the United States, with the same practices of lambic beer. In this type of beer, the fermentation begins with the contribution of Enterobacteriaceae and oxidative yeasts, followed by Saccharomyces spp. and Lactobacillales. Only after 1 year of fermentation Dekkera/Brettanomyces dominates the process with the contribution of other yeasts species such as Candida spp., Pichia spp., and lactic acid bacteria (Bokulich et al., 2012).

14.3  Saccharomyces cerevisiae the Old Hero of Fermentation Winemaking, brewing, and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the principal biotransformation process and Saccharomyces cerevisiae is definitely the main microorganism involved. Throughout the early decades of the 20th century the place for S. cerevisiae in fundamental research was affirmed, and there were several good reasons for this. The close relationship with this yeast in food and beverage production over millennia is due to its safety, as confirmed by its “Generally Recognized as Safe” (GRAS) designation by the US Food and Drug Administration. In addition, it is inexpensive, easy to grow and can be stored for long periods in suspended animation. Perhaps the most

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   439

i­ mportant aspect is that it has accessible genetics that can be followed through sexual and asexual cycles (Barnett, 2007; Chambers and Pretorius, 2010). S. cerevisiae is the main microorganism responsible for the fermentation of wine. However, results of the studies on the origin of S. cerevisiae were controversial and long debated, although now it was widely proven a direct association with artificial, manmade environments such as wineries and fermentation plants that exclude a natural origin for S. cerevisiae. Indeed, fermentative species of Saccharomyces (e.g., S. cerevisiae) occur at extremely low populations on grapes and are rarely isolated from intact berries and vineyard soils (Martini et al., 1996; Pretorius, 2000). In contrast, S. cerevisiae is abundant on grape juice and must-coated surfaces of winery equipment, forming an important component of a so-called “residential” or “winery” yeast flora. Extensive ecological investigations using molecular approaches have been carried out to explore yeast genetic diversity. Different molecular tools have been used for that purpose also to understand the S. cerevisiae population dynamics. Recently, also the impact of human activity on yeast diversity has been assessed at gene and genome level, evidencing several events of domestication (Fay and Benavides, 2005). In this regard, S. cerevisiae isolates from worldwide area showed low differentiation if compared to Saccharomyces paradoxus populations that revealed well delineated variation along geographic boundaries (Liti et  al., 2009) Microsatellite analysis has been used to differentiate the population genetic structure of wine and vineyard S. cerevisiae strains (Legras et al., 2007; Schuller and Casal, 2007). Recently FrancoDuarte et  al. (2014) used polymorphic microsatellites to genetically characterize a wide group of S. cerevisiae strains, from different geographical area relating the data with important enological traits. In winemaking and brewing, the use of commercial S. cerevisiae strains is becoming a common practice, due to certain advantages to the process performances and product quality. To guarantee the desirable features of the yeast strains selected some criteria are mandatory, such the ethanol tolerance, the exhaustion of sugar, potential and high fermentation activity, the growth at high sugar concentrations, the low production of hydrogen sulfide, and low volatile acidity, the resistance to killer toxin and good enzymatic profile. All these characteristics should go together with adequate flavor wines. However, the use of commercial S. cerevisiae strains may reduce the biodiversity of yeast strains that perform spontaneous fermentation, and consequently, to reduce the resulting product complexity (Frezier and Dubourdieu, 1992). Recently, many studies focused on the selection of local autochthonous strains to be used as starters contributing to the sensory characteristics of final product (Le Jeune et al., 2006). However, in the last years, wineries are facing new challenges due to current market

440  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

demands and climate change effects on the wine quality. New yeast starters formed by nonconventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii) can contribute to solve some of these challenges (Pérez-Torrado et  al., 2017). They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae exhibits a high competing capability compared to non-Saccharomyces. The main strategy of S. cerevisiae to displace other microbial species present in grape juice is the vigorous fermentative capacity in both the presence (Crabtree effect) and absence of oxygen. In this manner S. cerevisiae consumes sugar resources faster, and the ethanol produced becomes toxic for their competitors.

14.4  The New Concept of Nonconventional Fermentation: Non-Saccharomyces Yeasts 14.4.1 Wine Most of the non-Saccharomyces species coming from wine-related environments have limited fermentation potential, such as low fermentation power and rates, as well as low SO2 resistance (Ciani and Maccarelli, 1998; Ferreira et al., 2001; Jolly et al., 2006). Moreover, the production of acetic acid, ethyl acetate, acetaldehyde, and acetoin at high concentrations generally prevents the use of these strains as starter cultures both in wine and beer industry. However, over the last decade, many studies have been revaluating the involvement of non-Saccharomyces yeasts during alcoholic fermentation and their role on the metabolic impact and aroma complexity of the final product (Jolly et al., 2006; Domizio et al., 2007; Varela and Borneman, 2017). In particular, the reevaluation of the role of non-Saccharomyces yeasts in winemaking and in brewing is related to the use of controlled mixed fermentations using S. cerevisiae/non-Saccharomyces yeast species in simultaneous or sequential inoculation. Indeed, it was demonstrated that mixed fermentations using controlled inoculations of S. cerevisiae starter cultures and non-Saccharomyces yeasts represent a feasible way toward improving the complexity of products enhancing particular and specific characteristics. Fermentations carried out using mixed and controlled inoculums can improve the quality of the final product, and can assure both a more standard fermentation process and an enhancement of the analytical composition of wine, by taking advantage of several metabolic pathways of non-Saccharomyces yeast strains. However, from the applicative point of view, non-Saccharomyces yeasts give their significant contribution during the early stages of fermentation. With the increase in alcohol concentration, indigenous or

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   441

commercial strains of S. cerevisiae take over and complete the fermentation process. During spontaneous grape juice fermentation, there is a sequential succession of yeasts. Initially, species of Hanseniaspora (Kloeckera anamorph counterpart), Starmerella, Issatchenkia, Pichia, Zygosaccharomyces, Torulaspora, Lachancea, Schizosaccharomyces, Candida, Metschnikowia, Debaryomyces, and Cryptococcus are found in fresh must. Of these, the most common yeast species, present at highest numbers is H. uvarum/guilliermondii and consequently several studies in the last decade have been developed aiming to evaluate the effects of apiculate strains on the quality of the final fermentation product (Comi et al., 2001). In grape must, H. uvarum and H. guilliermondii were found at high cell densities, up to 106–108 cells/ mL, during the first 4–6 days of fermentation, until the ethanol level of approximately 4%–7% v/v was produced (Zott et al., 2008). Apiculate yeasts have become an object of increasing interest, as their proliferation in competition with S. cerevisiae can impact on the sensory quality of wine, because of higher esters production (Viana et  al., 2009). Because of their high diffusion on wine grapes, and at the beginning of wine fermentations, the influence of H. guilliermondii on wine aromas was studied (Moreira et al., 2005). Rojas et al. (2003) showed that a selected strain of H. guilliermondii promotes the esterification of a variety of alcohols such as ethanol, isoamyl alcohols, geraniol, and 2-phenylethanol. Several authors suggested that the presence of apiculate yeasts during fermentation positively contributed to a more complex aroma of the wine because of high production of aromatic compounds (Romano and Suzzi, 1996; Ciani and Maccarelli, 1998). These oenological properties were found to be strain dependent and several H. uvarum strains of oenological origin were reported to have beta-d-glucosidase (Palmeri and Spagna, 2007; Rodríguez et al., 2007) and beta-d-xylosidase activity. Both these glycosidases are important for enzymatic release of aromatic compounds in winemaking. Among non-Saccharomyces yeasts, T. delbrueckii (anamorph Candida colliculosa) is drawing considerable attention in wine industry. T. delbrueckii is a typical representative of the natural flora on the grape surface and, just like S. cerevisiae, can be found in most wine producing regions (Jolly et  al., 2006). T. delbrueckii, formerly known as Saccharomyces rosei or Saccharomyces delbrueckii is an Ascomycetous yeast that has been reclassified many times which resulted in the description of numerous taxa that are now known as synonyms (Kurtzman et  al., 2011). Currently, there are six accepted species assigned to the Torulaspora genus, T. delbrueckii, Torulaspora globosa, Torulaspora franciscae, Torulaspora microellipsoides, Torulaspora maleeae, and Torulaspora pretoriensis (Kurtzman et  al., 2011). However, strains of T. delbrueckii show variation in their ­ability

442  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

to f­erment and assimilate carbon compounds, thus contributing to the uncertain description of this species (Limtong et al., 2008). T. delbrueckii has previously been suggested for vinification of musts low in sugar and acid, and has been used for the production of red and rosé wines in Italy (Castelli, 1955) and for Sauvignon Blanc in South Africa (Jolly et al., 2006). However, T. delbrueckii is also a common food and beverage spoilage organism. Stantford and James (2003) noted that T. delbrueckii is a frequent contaminant in soft drink manufacturing plants, where it may result in product spoilage even if this species is relatively sensitive to preservatives. L. thermotolerans showed a peculiar ability to produce lactic acid, glycerol, and 2-phenyl ethanol during fermentation of grape musts (Kapsopoulou et  al., 2007; Comitini et  al., 2011; Gobbi et  al., 2013). This ability is of increasing interest due to a progressive reduction of the total acidity of wines caused by global climate change and variations in viticulture and oenology practices. W. anomalus is widespread in nature, occurring in soil, plant material and as an opportunistic pathogen of humans and animals. Presumably, the primary habitat of this species is plants. Sláviková et  al. (2009) reported W. anomalus to be one of the most frequent yeasts isolated from leaves and needles of 10 different tree species growing in the Small Carpathian mountain range of Slovakia. Although W. anomalus is noted for its ability to grow under stressful environmental conditions, such as extremes of pH, low water activity and anaerobic conditions, few biotechnological applications have been reported (Passoth et  al., 2006). W. anomalus has been tested extensively for biocontrol of spoilage yeasts and mold growth that develops during postharvest storage of fruits or during fermentation (Fredlund et al., 2002). Comitini et al. (2004) for the first time studied the effect of a killer toxins produced by W. anomalus and active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces. The fungicidal effect exerted by W. anomalus killer toxin against Dekkera bruxellensis was stable for at least 10 days in wine, suggesting a potential application as antimicrobial agents during wine aging and storage. S. bacillaris (synonym Candida zemplinina) (Duarte et  al., 2012) exhibits strong fructophilic character and shows the ability to produce low quantities of ethanol and acetic acid and high amounts of glycerol. C. zemplinina was described by Sipiczki (2004) as highly osmotolerant and cryotolerant species isolated from botrytized grape musts in the Tokaj wine region of Hungary. Several ecology studies have reported the presence of this species during spontaneous must fermentations in different countries (Rantsiou et al., 2012; Bokulich et al., 2013, 2014; Milanović et  al., 2013), suggesting the frequent involvement of this species in the fermentation process. Indeed, S. bacillaris (formerly C. stellata) is a common member of the first few days of the ­spontaneous

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   443

fermentation of grape must remaining active much longer than most other non-Saccharomyces yeasts. Its presence during fermentation is thought to contribute to a more complex and better aroma of wine because of the higher production of specific aroma compounds (Soden et al., 2000) and glycerol (Ciani et al., 2000) and positive interactions with S. cerevisiae in the production and degradation of metabolites (Ciani and Ferraro, 1998). Since its growth rate is significantly lower than that of S. cerevisiae and its cells are sensitive to ethanol, S. bacillaris is usually overgrown by more ethanol-tolerant Saccharomyces strains. Another yeast species typically present on the surface of grape berries and that receiving attention as a biocontrol agent, is M. pulcherrima (Janisiewicz et al., 2001). Among the various fruits, the grape berry surface represents an optimal and nutrient-rich habitat for M. pulcherrima, which is considered common wine yeast on overripe grape berries, used to produce ice wine and botrytized grapes. Moreover, M. pulcherrima is generally present during the first stages of grape juice fermentation (Prakitchaiwattana et al., 2004; Oro et al., 2014). The evaluation of oenological aptitude showed low fermentation power and fermentation rate but exhibited some positive features such as polysaccharides and glycerol production and glycosidase activity (Comitini et  al., 2011).This non-Saccharomyces yeast is not normally associated with volatile acidity production, but can form relatively high concentrations of esters (Bisson and Kunkee, 1991). These esters, as well as other metabolites could have a positive benefit wine derived from neutral cultivar characteristics (Jolly et al., 2017). Many ecological studies have been carried out on the biological control of M. pulcherrima against different postharvest fungal pathogens on ­apple, such as Botrytis cinerea, Penicillium, Monilia, and Alternaria spp. (El-Ghaouth et al., 1998; Saravanakumar et al., 2008). This yeast species apparently has a broad-spectrum killer activity affecting blue mold (Penicillium sp.) and Botrytis sp. Sipiczki (2006) demonstrated that the pulcherrimin of Metschnikowia species inhibits the growth of Botrytis cinerea in grapes as well as a number of other molds, yeasts, and bacteria that occur in wine fermentations. He provided evidence that the inhibition is due to sequestration of iron away from the target species. The yeast is thought to excrete a soluble precursor, which then combines with ferric ions to form the characteristic insoluble maroon pigment. Zygosaccharomyces spp. is one of the most damaging foodborne spoilage yeast. Tools for controlling its growth are limited due to its resistance to preservatives and osmotic stress. Zygosaccharomyces contamination may result in spoilage of grape juice concentrates or in re-fermentation and CO2 production in sweet wines. Zygosaccharomyces bailii is usually found in food and beverage

444  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

i­ ndustries using processed raw materials such as fruit juices, glucose syrups, and concentrated juice (Sancho et  al., 2000). For its osmophilic characteristic and high resistance to high ethanol concentration, Zygosaccharomyces yeasts was found in “mother sediment” of Vinsanto wine where it was not possible to isolate yeasts belonging to the genus Saccharomyces (Domizio et al., 2007). Indeed, Vinsanto is made with dried grapes with very high sugar content with aging period of 2 years or more (Domizio et  al., 2011), The influence of Zygosaccharomyces yeasts in the analytical and aromatic profile of the Vinsanto wine is not completely elucidated. In this regard, a screening for the enological aptitude of some potential spoilage non-Saccharomyces wine yeast showed that Z. bailii and Z. florentina (reclassified as Zygotorulaspora florentina) exhibited positive features as increased amount of polysaccharides in wine (Domizio et al., 2011). Another non-Saccharomyces yeast usually found in high sugar habitats is Schizosaccharomyces pombe. Schizosaccharomyces yeasts are characterized by malo-alcoholic fermentation and they are capable to completely metabolize the malic acid present in grape must and wine. It has been recognized to improve some of sensory parameters of the wine, especially those related to wine color stability due to the correlation between the amount of pyruvic acid released into the medium and the formation of vitisin A (a pyranoanthocyanin), natural polyphenol, found in grapes. Other studies carried out with the aim of deacidifying the grape must or wine through malic acid degradation successfully used mixed fermentations of S. pombe and S. cerevisiae. Pichia kluyveri is widely distributed in nature and is commonly isolated from rotted fruit and the fleshy parts of plants (Phaff et  al., 1987). Although this yeast is common in natural fermentations of agricultural products such as the coffee bean. Pichia kudriavzevii and its anamorph Candida krusei are widely distributed in nature often occurring in soil, on fruits and in various natural fermentations. P. kudriavzevii was found in spontaneous wine fermentation and was proposed in mixed fermentation to reduce ethanol content in wine (Wang and Liu, 2013; Contreras et al., 2015a, b).

14.4.2 Beer In the brewing industry, the choice of raw materials is crucial to achieve a product with distinctive characteristics. This aspect has pushed the brewers to experiment on the ingredients such as water, malt, hop, and yeast (Bernstein, 2010; Brungard, 2014; So, 2014; Osburn et al., 2016). In the last few years, brewer’s payed attention to yeasts selected not only for their good fermentation efficiency

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   445

but also for the characteristic of aroma and flavors given to the final product. Recent genetic investigations have also been focused on methods to enhance the fermentation efficiency and aromatic profile of selected S. cerevisiae (Saerens et al., 2010; Steensels et al., 2012; Steensels and Verstrepen, 2014). Several studies proposed the isolation of new starter yeasts from natural matrices (Marongiu et al., 2015; Mascia et  al., 2015), and the selection of wine yeast strains (Canonico et al., 2014). In the last few years, there was a worldwide growth of microbrewery that has reinforced and encouraged the selection and use of different yeast genera, with pronounced impacts on aroma and flavor. To achieve this, non-Saccharomyces yeasts could represent a large source of biodiversity with consequent wide-ranging of the fermentation products able to generate new beer styles. Within non-­Saccharomyces yeasts, different genera and species have been proposed. T. delbrueckii is a yeast species widely studied in winemaking for its ability to produce fruitiness and positive aromatic flavor and, for this reason, it was also evaluated for beer production, in both pure and in mixed cultures with different S. cerevisiae starter strains (Canonico et  al., 2016a, b, 2017). Beers obtained with T. delbrueckii pure cultures were characterized by a distinctive analytical, aromatic profile and a low alcohol content (2.66%; v/v) (Canonico et  al., 2016a, b). Generally, T. delbrueckii affected the aromatic compounds with the production of some fruity esters. Investigating on the possible use in beer production of T. delbrueckii, Michel et al. (2016) found that a strain was able to produce amyl alcohols as well as fruity and floral aroma. Furthermore, two strains were found to be suitable for producing low-alcohol beer owing to their inability to ferment maltose and maltotriose but still produced good flavor. Another non-Saccharomyces suitable for beer production is L. thermotolerans. The capability to ferment maltose is variable within this species. Domizio et al. (2016) found a strain of L. thermotolerans not only able to ferment brewer’s wort but also to increase the production of glycerol, lactic acid and decrease the pH value in comparison with S. cerevisiae starter strain. In this way, they propose the use of L. thermotolerans in pure culture to producing sour beers without the use of lactic acid bacteria. In this regard, Osburn et al. (2018) investigated on various non-Saccharomyces species isolated from natural environments for their ability to produce lactic acid and ethanol. These authors found that Lachancea fermentati, Schizosaccharomyces japonicus, and Hanseniaspora vineae, exhibited a sour character (Osburn et al., 2018). In particular, a strain of H. vineae exhibited high levels of attenuation after only 2 weeks and the final product were slightly sour but clean and highly drinkable, with notes of apple cider. Also W. anomalus was capable of heterolactic fermentation of sugar

446  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

into lactic acid, ethanol, and CO2. Larger-scale brewing with four strains demonstrated that these yeasts are highly attenuative, flocculate, yield appreciable levels of lactic acid, and produce pleasant aromatic and flavor compounds (Osburn et al., 2018). Saccharomycodes ludwigii is a species that does not utilize maltose or maltotriose in beer wort but ferments only glucose, fructose, and sucrose. For these reasons the final beer is low alcohol or alcohol free (De Francesco et al., 2015; Petruzzi et al., 2016). Another non-Saccharomyces yeasts proposed to produce low alcohol beers is Zygosaccharomyces rouxii (Sohrabvandi et al., 2010; Mohammadi et al., 2011; Mortazavian et al., 2014) and Pichia (Saerens and Swiegers, 2014). These investigations showed that some non-Saccharomyces yeasts could be profitable used to ferment wort leading into beers with different flavor and ethanol content. However, it is necessary taking in account two features: (i) the inter and intraspecific variability of non-Saccharomyces yeasts; (ii) the most of the studies are carried out at laboratory scale. For these reasons, further investigations are necessary to understand the behavior and the use of non-Saccharomyces yeasts in brewing process. The main distinctive features of nonconventional yeasts in beer are illustrated in Table 14.1.

Table 14.1  Distinctive Features of Nonconventional Yeasts in Beer Species

Impact on Main Characters and Aroma Profile

Brettanomyces spp.

Overattenuation of wort Increased acidity Increased on overall aromatic component Low alcohol beer Lactic acid production Reduced pH value Lactic acid production Reduced pH value Lactic acid production Reduced pH value Increased glycerol content Low alcohol or alcohol free beers Low alcohol or alcohol free beers Beer flavor enhancement Low alcohol or alcohol free beers

Torulaspora delbrueckii Hanseniaspora vinae Schizosaccharomycs japonicus Lachancea thermotolerans/fermentati

Saccharomycodes ludwigii Zygosaccharomyces rouxii Pichia kluyveri

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   447

14.5  The Attractive Biotechnological Tool of Mixed Fermentations In the past, the contribution of non-Saccharomyces yeasts in winemaking has always been considered negative for their lower enological attitude (capacity to produce alcohol to lead the fermentation process and to produce undesirable compounds) compared to S. cerevisiae. In recent decades, however, there has been a reevaluation of the role of non-Saccharomyces yeast and several investigations have been carried out to better understand the impact of non-Saccharomyces strains on the chemistry and sensory properties of wine (Ciani and Maccarelli, 1998; Pretorius, 2000; Swiegers and Pretorius, 2005). In this regard, numerous studies have found a wide intraspecific variability of oenological characters, the possession of peculiar and positive oenological characters and, above all, a different behavior in coculture due to interactions with S. cerevisiae. All these aspects have highlighted a significant role of these nonconventional yeasts in determining the analytical and sensory profile and the aromatic complexity of wine. Table 14.2 illustrates the impact of the main nonconventional species on the aromatic profile and main characters of wines. The controlled multistarter fermentation with S. cerevisiae is the most profitable modality to use of these selected nonconventional wine yeasts. Several objectives can be pursued with the use of controlled mixed cultures with nonconventional yeasts: (i) enhancement of flavor and aroma complexity; (ii) distinctive features; (iii) ethanol reduction; and (iv) control of spoilage microflora.

14.5.1  Enhancement of Flavor and Aroma Complexity Several investigations focused the attention on the enhancement of flavor and aroma complexity of wine using nonconventional yeasts in mixed fermentation. In this regard, T. delbrueckii, low frequently isolated on the surface of the grape, was one of the most studied species to increase flavor and aroma complexity in alcoholic beverages. Indeed, T. delbrueckii shows a positive effect on the taste and aroma of alcoholic beverages exhibiting a low production of acetaldehyde, acetoin, acetate, and ethyl acetate. In winemaking, several investigations agree that T. delbrueckii impact on aromatic composition and sensory attributes of wines in both simultaneous and sequential fermentation. Indeed, these investigations found an increase acetate ester, (Cordero-Bueso et  al., 2013), thiols (3-sulfanylhexan-1-ol and 3-sulfanylhexyl acetate) (Zott et  al., 2011; Renault et al., 2016), terpenes (α terpineol and linalool) (Cus and Jenko, 2013), 2 phenyl-ethanol (Comitini et al., 2011). Moreover, results

448  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Table 14.2  Distinctive Features of Nonconventional Mixed Fermentations in Wine Species

Impact on Aroma Profile

Impact on Main Characters

Torulaspora delbrueckii

Increased on overall aromatic component, terpens, thiols, tropical fruits, floreal notes

Hanseniaspora uvarum/vinae Hanseniaspora vinae Hanseniaspora guilliermondii Starmerella bacillaris/bombicola (Candida stellata) Metschnikowia pulcherrima

Increased of volatile compounds (acetals, terpens) Phenyl ethyl acetate fruity, floral notes Increased of esters from various alcohols

Volatile acidity reduction, increased polysaccharides, enhancement of foam persistence Glycosidases activity

Lachancea thermotolerans Zygotorulaspora florentina Zygosaccharomyces bailii Schizosaccharomycs pombe/japonicus Wickerhmomyces anomalus Debaryomyces vanrijiae Kazachstania gamospora Zygosaccharomyces kombuchanensis Pichia kluyveri Pichia kudriavzevii

– –

Increased overall aroma complexity

Enhancement glycerol, reduction of volatile acidity

Increased of “fruity” characters (peach and pear) Increased “fruity” and “spicy” characters

Increased polysaccharides glycerol production and glycosidase activity Lactic acid production, increased total acidity Volatile acidity reduction – Malic acid degradation, deacidification activity increased mannoproteins Lactic acid production – –

Increased of “fruity” and “floral” notes Increased of ethyl esters Increased of polyphenols content Increased of isoamyl acetate Increased in esters and fatty acids Increased of “flavor persistence,” “flavor intensity” Increased of “flavor persistence,” “flavor intensity” Increased volatile thiols (exotic fruits) –

– – Deacidification activity

of sensory evaluations of final wines revealed an impacts on sensory attributes such as increased “aroma intensity,” complexity, persistence, and “fruity” aroma, depending on grape variety (Azzolini et al., 2015). Even in the brewing industry, T. delbrueckii was recently proposed to enhance and differentiate the aroma profile of final beer. Indeed, the success of craft beers induces brewers to look for new alternatives to impact on aroma and flavor and generate differentiated products (Basso et  al., 2016). In this contest, mixed fermentation using T. delbrueckii and S. cerevisiae fully converted the fermentable sugars

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   449

exhibiting distinctive analytical and aromatic profiles producing desirable fruity attributes (Canonico et al., 2016a, b, 2017; Michel et al., 2016). Also H. uvarum or H. vineae was proposed in mixed fermentation to enhance the aromatic profile of wines. Indeed, these species determined and increased in terpenes and acetate esters production with an increase in positive aromatic characters such as “floral” and “fruity” (Hu et al., 2016; Tristezza et al., 2016). Moreover, mixed fermentation trials in the presence of H. uvarum and S. cerevisiae starter cultures increased isoamyl acetate content, while the use of Hanseniaspora osmophila increased 2-phenylethyl acetate production (Moreira et  al., 2010; Medina et  al., 2013). Also sequential fermentation of Macabeo must, carried out with Hanseniaspora vineae and S. cerevisiae, showed an enhancement of phenyl ethyl acetate and more fruity and flowery notes in comparison with wines fermented with S. cerevisiae. Thus, this compound is associated with fruity, floral, and honey aromas (Lleixà et al., 2016). Another nonconventional yeast with interesting fermentation behavior is M. pulcherrima, species, generally recovered during the initial stages of alcoholic fermentation. M. pulcherrima is a high producer of β-glucosidase (Rodríguez et  al., 2007), and its presence in mixed cultures can provide significant enhancements in the wine of higher alcohols, esters, and terpenoids. Its aromatic profile in mixed fermentation was characterized by “citrus/grape fruits” a smoky and flowery attributes in Riesling and Macabeo grape varieties, respectively (Benito et al., 2015; González-Barreiro et al., 2015). According to a report by Kurita (2008), mixed inoculations using W. anomalus (formerly Pichia anomala) resulted in positive enhancement of isoamyl acetate. Cañas et al. (2014) also studied the effect of mixed fermentations with W. anomalus. The obtained results showed that wines elaborated by sequential inoculation presented higher levels of acetates and ethyl esters, compounds that supply a fruity note, higher levels of linear alcohols, which are responsible for herbaceous notes and lower concentrations of organic acids, that contribute to increase the aromatic quality, in comparison with wines produced by a S. cerevisiae pure culture. Debaryomyces vanrijiae also determined an increase in esters and fatty acids (Maturano et al., 2015). Dashko et  al. (2015) using Kazachstania gamospora and Zygosaccharomyces kombuchaensis in sequential fermentations of Ribolla gialla grape juice, found an increase in “flavor persistence,” “flavor intensity,” and several fruity attributes. Z. bailii in simultaneous fermentation positively influence the aroma profile in chardonnay wine enhancing the ethyl esters production (Garavaglia et al., 2015) while Zygotorulaspora florentina increased fruity and floral notes in Sangiovese grape juice (Lencioni et al., 2016). Finally, Pichia kluyveri was proposed in both wine and beer fermentation to enhance the aroma profile of the final

450  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

product (Benito et al., 2015; Saerens et al., 2017). In particular, in wine mixed fermentation with S. cerevisiae, P. kluyveri increased varietal thiols concentrations in Sauvignon Blanc and overall impression and peach-apricot characters (Benito et al., 2015).

14.5.2  Distinctive Features The use of nonconventional yeasts in controlled mixed fermentation has been proposed and applied also to take advantage of some their specific fermentative features. For example S. pombe and/or Schizosaccharomyces japonicus has been proposed for a long time as biological deacidification agent (Magyar and Panyik, 1989; Ciani, 1995) and could be profitable used since they have characteristics that are beneficial for winemaking. In addition, more recently works showed that these yeasts species in mixed fermentation determined and increased in the production of pigments and large amounts of polysaccharides (Domizio et al., 2017; Escott et al., 2018). The ability of grape juice/wine deacidification was also found in Pichia kudriavzevii, another nonconventional wine yeast isolated from Patagonian (Moreno et  al., 2014). On the other hand, the characteristic to produce organic acids during the fermentation may be a desired feature in some winemaking environments and process conditions. In this regard, L. thermotolerans in simultaneous and sequential fermentation exhibited the ability to produce lactic acid determining an increase in total acidity of wine, desired feature in grape juices deficient in acidity generally coming from wines of warm climates (Kapsopoulou et al., 2007; Gobbi et al., 2013). The production of lactic acid during alcohol fermentation by L. thermotolerans was also exploited in beer production. Indeed, this yeast species was proposed as starter culture to produce sour beer (Domizio et al., 2016). In this regard, with the aim to produce sour beer without lactic acid bacteria, Osburn et  al. (2018) found that other nonconventional species, such as H. vineae, W. anomalus, S. japonicus, and Lachancea fermentati, are able to produce lactic acid during fermentation. Another positive trait desired and pursued by nonconventional yeast involvement is the reduction of volatile acidity. A low volatile acidity (mainly acetic acid) is one of the fundamental characters to select strain for the oenological use. Indeed, volatile acidity plays a significant role in wine aroma since excessive concentrations of acetic acid are highly detrimental to wine quality. The amount of volatile acidity produced by S. cerevisiae is usually low (up to 0.50 g/L), but may be higher during fermentation of high-sugar media. Indeed, S. cerevisiae produce acetic acid as response to osmotic due to the upregulation of genes encoding for aldehyde dehydrogenases (Blomberg and Adler, 1992). Some non-Saccharomyces species do not respond in the same way to osmotic stress and for these reasons

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   451

researchers have been proposed to reduce the volatile acidity in wines in particular in those with initial sugar content. T. delbrueckii and C. stellata (now reclassified as Starmerella bombicola) exhibited a very low production of volatile acidity (Ciani and Maccarelli, 1998). In this regard, T. delbrueckii, showed in mixed fermentation with S. cerevisiae a consistent reduction of volatile acidity in high sugar fermentation (Bely et al., 2008). Similarly, C. stellata in mixed and sequential fermentation with S. cerevisiae showed a reduction of volatile acidity (Ciani and Ferraro, 1998). A reduction of acetic acid production was obtained in sweet wine fermentations using C. zemplinina (now reclassified as Torulopsis bacillaris) in simultaneous and sequential fermentation with S. cerevisiae (Rantsiou et al., 2012). Glycerol is a desired fermentation by product and is quantitatively the major end product other than ethanol and carbon dioxide. The amount of glycerol formed during fermentation by the yeast species S. cerevisiae is in the range of 7%–10% compared to that of ethanol. Among nonconventional wine yeasts, the high glycerol producer species were used in mixed fermentation to enhance the glycerol content in wines. In this regard, immobilized cells of C. stellata showed an increase of glycerol content of about 100% in mixed fermentation (Ciani and Ferraro, 1998). In addition, S. bacillaris (formerly C. stellata) yeast in mixed fermentation with S. cerevisiae starter culture has been widely investigated and several studies demonstrated an increase in glycerol content in mixed wines, related with the mouth feel and complexity of wine flavor. An increase of glycerol content in wine was also found in mixed fermentation with M. pulcherrima (Comitini et al., 2011). Polysaccharides production is another relevant and important feature that could be improved with the use of nonconventional yeasts in winemaking. Indeed, S. cerevisiae releases low amounts of polysaccharides, generally ranging from 50 to 150 mg/L (Giovani et al., 2010). Several studies have shown that nonconventional wine yeasts are generally characterized by the capacity to release a high quantity of polysaccharides (Comitini et  al., 2011; Domizio et  al., 2011; Gobbi et  al., 2013). The possibility to increase the content of mannoproteins naturally by the use of these yeasts could represent a valuable possibility to enhance the overall quality of wines. In this regard, M. pulcherrima, Saccharomycodes ludwigii, L. thermotolerans, S. pombe, S. japonicus showed high polysaccharides production and could be profitable used in mixed fermentation (Domizio et al., 2017).

14.5.3  Ethanol Reduction The increase in alcohol levels in wine is one of the main challenges affecting the winemaking industry nowadays. Indeed. Over the last few decades, there has been a progressive increase in the ethanol c­ ontent

452  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. Consequently, there is a rising interest in ethanol reduction in wine. In this context, microbiological approach for decreasing ethanol concentrations appears a promising way since it takes advantage of the differences in energy metabolism among the wine yeast species. In particular, there is a growing interest to investigate on nonconventional wine yeast. Indeed, they show a wide variability in ethanol yield that could be a potential tool for the reduction of alcohol content in wine. Recent works investigated on interspecies and/or intraspecies variability in ethanol yield among nonconventional wine yeasts (Magyar and Tóth, 2011; Contreras et al., 2014; Gobbi et al., 2013; Contreras et al., 2015a, b). Low ethanol yield was found in some strains of C. zemplinina (Magyar and Tóth, 2011) and in strains belong to Hanseniaspora, and Zygosaccharomyces genera (Gobbi et al., 2013). Ethanol yield like other fermentation features is a species-related trait but, similarly to other fermentation parameters, a pronounced intraspecies variability was also evident (Ciani and Maccarelli, 1998; Comitini et  al., 2011; Domizio et  al., 2011). A low ethanol yield was also found in strains of M. pulcherrima, Schizosaccharomyces malidevorans, and C. stellata) (Contreras et al., 2014). The regulatory respiro-fermentative metabolism in yeasts might be used as strategy to reduce the ethanol concentration in wine. In addition to a low ethanol yield, among non-Saccharomyces wine yeasts some strains/species showed and sugar consumption by respiration (Crabtree negative). Both these approaches have indicated the promising use of nonconventional wine yeast to limit ethanol production. Since most nonconventional yeast are incapable of completing alcoholic fermentation S. cerevisiae wine strain should be added in simultaneous or sequentially. In this regard several works recently investigated on the combination of selected nonconventional yeasts such as M. pulcherrima, T. bacillars, T. bombicola, Z. rouxii, T. delbrueckii, and P. kudriavzevii. These yeast species are able to divert the carbon flux toward multiple metabolites rather than ethanol, with the high fermentative ability of S. cerevisiae strains (Englezos et al., 2015; Canonico et al., 2016a, b; Varela, 2016). The different respiro-fermentative regulatory mechanisms of some nonconventional yeasts compared to S. cerevisiae was evaluated to reduce the ethanol content through partial and controlled aeration of the grape juice in simultaneous and sequential fermentation (Contreras et al., 2015a, b; Quirós et al., 2014; Rodrigues et al., 2016). Results in terms of ethanol reduction are promising and ranging from 0.3% to 2.2% v/v depending on the strain and the fermentation conditions. However, in simultaneous fermentation aeration condition showed consistent increase of volatile acidity since S. cerevisiae in this condition has the tendency to produce large amount of acetic acid

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   453

(Morales et al., 2015). On the other hand, these nonconventional yeast species produce very little volatile acidity even under oxygenated conditions (Quirós et al., 2014; Rodrigues et al., 2016). For these reasons, sequential fermentation using before the nonconventional yeast with moderate aeration condition followed by the inoculum of S. cerevisiae in strict anaerobiosis condition could be a suitable strategy to avoid increase in acetic acid content and obtain at the same time a reduction in ethanol content in wine. In a recent work carried out at pilot scale level using T. delbrueckii or M. pulcherrima in sequential fermentation and in aerated conditions a consistent reduction of ethanol content in the final wines was obtained. However, sensory and aroma analysis revealed that the quality of mixed fermentations was affected by the high levels of some yeast amino acid related by-products and further investigations and set up of fermentation conditions needed (Tronchoni et al., 2017).

14.5.4  Antimicrobial Activity Another possible applicative feature of non-Saccharomyces yeasts in winemaking is regard to the control of undesired microorganisms. During the various stages of fermentation, a punctual and timely control of potential spoilage microorganisms is needed. Indeed, in winemaking and brewing processes, a wide number of yeasts can participate during the various production phases determining, sometimes, undesired organoleptic features of the final product. Moreover, nowadays there is an increasing interest in the use of natural antimicrobial agents in foods and beverages to control spoilage microflora, thus reducing the chemical additives. In this context, killer yeasts and their secreted toxins appear to represent an interesting solution as antimicrobial agents, for the partial or complete substitution of the use of synthetic agents. Indeed, one of the topical subjects in winemaking is the reduction in the use of SO2 and its partial or complete substitution with natural antimicrobials, which would be more compatible with the requests of consumers for safe and unspoiled food products. Killer toxins are proteins or glycoproteins naturally produced by yeasts that kill sensitive cells and some of them were purified, characterized. The mode of action of most of the killer toxins were well studied even if the modalities to kill the sensitive cells in some of the newly discovered killer toxins are still unknown (Liu et al., 2015). Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental applicative aspects, particularly in winemaking. In relation to the ecological aspect, during the years, killer strains were isolated from various oenological sources, including grape berries, grape musts, and wines. Afterwards several studies have been carried out evaluate the possible application in winemaking

454  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

(Santos et  al., 2011; Comitini et  al., 2011). At present, the control of wild spoilage yeast at the pre-fermentative stage is generally achieved by the addition of SO2 to freshly pressed must. At this stage, apiculate yeasts and in particular H. uvarum are widely present that needed to be controlled. About this, Tetrapisispora phaffii represents an interesting application killer phenomenon since its killer toxin is able to control the proliferation of apiculate yeasts during the prefermentation phase (Comitini and Ciani, 2010). During fermentation and mainly in the aging stages, another undesirable yeast is B. bruxellensis that is responsible for undesired odors in wine and considered the current major concern for winemakers, since an effective method to control their growth has not yet been developed. To reduce the Brettanomyces proliferation high doses of sulfur dioxide were commonly employed but the efficiency of this chemical compound is subject to wine composition and physicochemical characteristics. Recently, Mehlomakulu et  al. (2017) focused on the identification and characterization of killer toxins from Candida pyralidae that show a potential antimicrobial effect against B. bruxellensis in wine. They were active and stable with winemaking conditions and the activity of these killer toxins was not affected by the ethanol and sugar concentrations typically found in grape juice and wine. Also Belda et  al. (2017) studied and characterized two killer toxins from Pichia membranifaciens (PMKT1 and PMKT2), which is able to inhibit B. bruxellensis while S. cerevisiae was fully resistant. Also Kwkt and Pikt, two killer toxins produced by Kluyveromyces wickerhamii and W. anomalus, respectively, showed an antimicrobial activity against Brettanomyces/Dekkera wine-spoilage yeast (Oro et al., 2016). Villalba et  al. (2016) addressed their study on the identification and partial characterization of a new killer toxin from T. delbrueckii with potential biocontrol activity of B. bruxellensis and also other spoilage non-­Saccharomyces yeasts such as Pichia guilliermondii, Pichia manshurica, and P. membranifaciens. Finally, another interesting modality in the control of undesirable microflora during fermentation was revealed by Nissen et  al. (2003). These authors showed that early death of L. thermotolerans and T. delbrueckii in mixed-culture fermentations with S. cerevisiae was not induced by ethanol or any other toxic compound but rather by a cell-to-cell contact mediated mechanism. Subsequent studies (Renault et al., 2013) supported the previous assumption that death of T. delbrueckii is mediated by a cell-to-cell contact mechanism. On the other hand, Albergaria and Arneborg (2016) well described how S. cerevisiae establishes antagonistic interactions against several wine-related microbial species (both yeasts and bacteria), mediated by the secretion of antimicrobial peptides that play an important role in its dominance within high-sugar ecosystems.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   455

14.6  Conclusion: The Power of Yeast Wine and beer, as some of the oldest biotechnological fermentations, has been consumed by humans for thousands of years. Despite the discovery that these fermented beverages tasted good, it must have taken many years for humans to understand the mechanism behind the fermentation process, mainly regarding the microbiological aspect. Only at the end of 19th century, Louis Pasteur considered one of the founders of microbiology, demonstrated the growth of yeast and its capacity to cause fermentation in a synthetic medium; this observation invalidated the concept that yeast or ferments originated from the action of oxygen on the nitrogenous elements of the fermentable liquid. From that time until now, the applied research, supported by innovative and advanced techniques, has reached significant advances, useful to manage the fermentation process. Nowadays, it is clearly established that alcoholic fermentation is the main biotransformation and S. cerevisiae is the primary microorganism involved, although a wide variety of microbial species may participate contributing to the properties of final products. Certainly, the fermented industry has changed dramatically in the past 50 years. The size of production capacity and consequent productivity has increased several fold. As a consequence, small and traditional facilities, as many microbreweries and small organic winery underwent a drastic reduction. Therefore, the fermented beverages industry is now dominated by big producers, moved only from the economic and profitable purpose. For this reason, the attention was focused on the standardization of the technological process that was managed in order to increase the production. In winemaking field, natural fermentation that is clearly a hazardous and uncontrolled process was replaced with the practice of pure fermentation. The use of selected starter cultures of S. cerevisiae can thus play an important role in the control of microbial process. Inoculated cultures of S. cerevisiae are expected to suppress either indigenous S. cerevisiae and non-Saccharomyces species and to dominate the fermentation. Moreover, the use of antiseptic agents, such as SO2, to which most of the non-Saccharomyces yeasts are scarcely resistant, should guarantee the dominance of the inoculated strains. With the commercial availability of active dry cultures of S. cerevisiae, the inoculation of grape must has become an attractive and convenient practice. Although the extensive use of starter cultures was an important advance in wine biotechnology, the generalized use of selected cultures represents a simplification of microbial fermentation communities promoting a standardization of the analytical and sensory properties of final wines.

456  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Recently in brewing there was a worldwide growth of microbrewery that have introduced into the market niche beer products with peculiar and distinctive characteristics. This trend reinforced and encouraged the selection and the use of different yeast genera, with pronounced impacts on aroma and flavor. In this, context, the involvement of non-Saccharomyces yeasts in mixed fermentation with S. cerevisiae starter cultures could be a practical way to improve the complexity and to enhance the particular characteristic of beer and wine (Fig. 14.1). However, in this regard the interactions among the different starter cultures performed during fermentation and the modalities of inoculation need to be further investigated. Although the current interest on non-Saccharomyces yeasts was triggered by their positive production of aroma and sensory-active compounds, and also their peculiar ethanol yield, one of the most invariant traits of S. cerevisiae, was exploited. The possibility, at the industrial level, of using non-Saccharomyces yeasts for reducing alcohol levels will require an improved understanding of the metabolism of these alternative yeast species, as well as the interactions with different yeast starters during the fermentation of grape must. The investigations on the yeast interactions could be of interest in the

Biotechnological progress Pure fermentaon

Selected Saccharomyces cerevisiae manage industrial wine and beer

Standardized product

Mixed fermentaon

Non-convenonal yeasts for nonconvenonal wine and beer

Disncve features increased aroma

Fig. 14.1  The use of nonconventional yeasts in biotechnological process to obtain distinctive features.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   457

development of new bioactive products or methods to prevent/control microbial contamination. The killer toxins or other antimicrobial compounds produce by non-Saccharomyces yeasts could be exploited to control spoilage yeasts. The interaction of different yeast strains is a very complex applicative topic; however, if S. cerevisiae remains the main manager of alcoholic fermentation, non-Saccharomyces species may have a complementary role on aroma, ethanol production, and microbial control in wine.

References Albergaria, H., Arneborg, N., 2016. Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions. Appl. Microbiol. Biotechnol. 100, 2035–2046. Albergaria, H., Francisco, D., Gori, K., Arneborg, N., Gírio, F., 2010. Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-­Saccharomyces wine-related strains. Appl. Microbiol. Biotechnol. 86, 965–972. Andorrà, I., Landi, S., Mas, A., Guillamón, J.M., Esteve-Zarzoso, B., 2010. Effect of oenological practices on microbial populations using culture-independent techniques. Food Microbiol. 25, 849–856. Azzolini, M., Tosi, E., Lorenzini, M., Finato, F., Zapparoli, G., 2015. Contribution to the aroma of white wines by controlled Torulaspora delbrueckii cultures in association with Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 31, 277–293. Barnett, J.A., 2007. A history of research on yeasts 10: foundations of yeast genetics1. Yeast 24, 799–845. Basso, R.F., Alcarde, A.R., Portugal, C.B., 2016. Could non-Saccharomyces yeasts contribute on innovative brewing fermentations? Food Res. Int. 86, 112–120. Bauer, F.F., Pretorius, I.S., 2000. Yeast stress response and fermentation efficiency: how to survive the making of wine—a review. S. Afr. J. Enol. Vitic. 21, 27–51. Belda, I., Ruiz, J., Alonso, A., Marquina, D., Santos, A., 2017. The biology of Pichia membranifaciens killer toxins. Toxins 9, 112. Bely, M., Stoeckle, P., Masneuf-Pomarède, I., Dubourdieu, D., 2008. Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation. Int. J. Food Microbiol. 122, 312–320. Benito, A., Calderon, F., Palomero, F., Benito, S., 2015. Combine use of selected Schizosaccharomyces pombe and Lachancea thermotolerans yeast strains as an alternative to the traditional malolactic fermentation in red wine production. Molecules 20, 9510–9523. Bernstein, J.M., 2010. Imbibe Magazine, Portland, OR. vol. 2010. https://www.bjcp.org/. Bisson, L.F., Kunkee, R., 1991. Microbial interactions during wine production. In: Zeikus, J.G., Johnson, E.A. (Eds.), Mixed Cultures in Biotechnology. McGraw-Hill, New York, pp. 37–68. Blomberg, A., Adler, L., 1992. Physiology of osmotolerance in fungi. Adv. Microb. Physiol. 33, 145–212. Bokulich, N.A., Bamforth, C.W., Mills, D.A., 2012. Brewhouse-resident microbiota are responsible for multi-stage fermentation of American coolship ale. PLoS ONE 7, 35507. Bokulich, N.A., Ohta, M., Richardson, P.M., Mills, D.A., 2013. Monitoring seasonal changes in winery-resident microbiota. PLoS ONE 8, e66437. Bokulich, N.A., Thorngate, J.H., Richardson, P.M., Mills, D.A., 2014. Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proc. Natl. Acad. Sci. 111, 23–29.

458  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Bokulich, N.A., Collins, T.S., Masarweh, C., Allen, G., Heymann, H., Ebeler, S.E., Mills, D.A., 2016. Associations among wine grape microbiome, metabolome, and fermentation behavior suggest microbial contribution to regional wine characteristics. MBio 7. 00631-16. Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J.C., Gerds, M.L., Hammes, W.P., 2012. Food fermentations: microorganisms with technological beneficial use. Int. J. Food Microbiol. 154, 87–97. Branco, P., Francisco, D., Chambon, C., Hébraud, M., Arneborg, N., Almeida, M.G., Albergaria, H., 2014. Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions. Appl. Microbiol. Biotechnol. 98, 843–853. Brungard, M., 2014. Calcium and magnesium in brewing water. New Brew. 31, 80–88. Cañas, P.M.I., García-Romero, E., Manso, J.M.H., Fernández-González, M., 2014. Influence of sequential inoculation of Wickerhamomyces anomalus and Saccharomyces cerevisiae in the quality of red wines. Eur. Food Res. Technol. 239, 279–286. Canonico, L., Comitini, F., Ciani, M., 2014. Dominance and influence of selected Saccharomyces cerevisiae strains on the analytical profile of craft beer refermentation. J. Inst. Brew. 120, 262–267. Canonico, L., Agarbati, A., Comitini, F., Ciani, M., 2016a. Torulaspora delbrueckii in the brewing process: a new approach to enhance bioflavour and to reduce ethanol content. Food Microbiol. 56, 45–51. Canonico, L., Comitini, F., Oro, L., Ciani, M., 2016b. Sequential fermentation with selected immobilized non-Saccharomyces yeast for reduction of ethanol content in wine. Front. Microbial. 7, 278. Canonico, L., Comitini, F., Ciani, M., 2017. Torulaspora delbrueckii contribution in mixed brewing fermentations with different Saccharomyces cerevisiae strains. Int. J. Food Microbiol. 259, 7–13. Castelli, T., 1955. Yeasts of wine fermentations from various regions of Italy. Am. J. Enol. Vitic. 6, 18–20. Chambers, P.J., Pretorius, I.S., 2010. Fermenting knowledge: the history of winemaking, science and yeast research. EMBO Rep. 11, 914–920. Ciani, M., 1995. Continuous deacidification of wine by immobilized Schizosaccharomyces pombe cells: evaluation of malic acid degradation rate and analytical profiles. J. Appl. Microbiol. 79, 631–634. Ciani, M., Ferraro, L., 1998. Combined use of immobilized Candida stellata cells and Saccharomyces cerevisiae to improve the quality of wines. J. Appl. Microbiol. 85, 247–254. Ciani, M., Maccarelli, F., 1998. Oenological properties of non-Saccharomyces yeasts associated with winemaking. World J. Microbiol. Biol. 14, 199–203. Ciani, M., Ferraro, L., Fatichenti, F., 2000. Influence of glycerol production on the aerobic and anaerobic growth of the wine yeast Candisa stellata. Enzyme Microb. Technol. 27, 698–703. Ciani, M., Comitini, F., Mannazu, I., Domizio, P., 2010. Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking. FEMS Yeast Res. 10, 123–133. Ciani, M., Capece, A., Comitini, F., Canonico, L., Siesto, G., Romano, P., 2016. Yeast interactions in inoculated wine fermentation. Front. Microbial. 7, 555. Comi, G., Romano, P., Cocolin, L., Fiore, C., 2001. Characterization of Kloeckera apiculata strains from the Friuli region in Northern Italy. World J. Microbiol. Biotechnol. 17, 391–394. Comitini, F., Ciani, M., 2010. The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking. Lett. Appl. Microbiol. 50, 50–56.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   459

Comitini, F., De Ingeniis, J., Pepe, L., Mannazzu, I., Ciani, M., 2004. Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/ Brettanomyces spoilage yeasts. FEMS Microbiol. Lett. 238, 235–240. Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., 2011. Selected non-Saccharomyces wine yeasts in controlled multistarter f­ermentations with Saccharomyces cerevisiae. Food Microbiol. 28, 873–888. https://doi. org/10.1016/j.fm.2010.12.001. Contreras, A., Hidalgo, C., Henschke, P.A., Chambers, P.J., Curtin, C., Varela, C., 2014. Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine. Appl. Environ. Microbiol. 80, 1670–1678. Contreras, A., Curtin, C., Varela, C., 2015a. Yeast population dynamics reveal a potential ‘collaboration’ between Metscknikowia pulcherrima and Saccharomyces uvarum for the production of reduced alcohol wines during Shiraz fermentation. Appl. Microbiol. Biotechnol. 99, 1885–1895. Contreras, A., Hidalgo, C., Schmidt, S., Henschke, P.A., Curtin, C., Varela, C., 2015b. The application of non-Saccharomyces yeast in fermentations with limited aerationa sastrategy for the production of wine with reduced alcohol content. Int. J. Food Microbiol. 205, 7–15. Cordero-Bueso, G., Esteve-Zarzoso, B., Cabellos, J.M., Gil-Díaz, M., Arroyo, T., 2013. Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.). Eur. Food Res. Technol. 236, 193–207. Cus, F., Jenko, M., 2013. The influence of yeast strains on the composition and sensory quality of Gewürztraminer wine. Food Technol. Biotechnol. 51, 547. Dashko, S., Zhou, N., Tinta, T., Sivilotti, P., Lemut, M.S., Trost, K., Piskur, J., 2015. Use of non-conventional yeast improves the wine aroma profile of Ribolla gialla. J. Ind. Microbiol. Biotechnol. 42, 997–1010. De Francesco, G., Turchetti, B., Sileoni, V., Marconi, O., Perretti, G., 2015. Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low-alcohol beer. J. Inst. Brewing 121, 113–121. Domizio, P., Lencioni, L., Ciani, M., Di Blasi, S., Pontremolesi, C.D., Sabatelli, M.P., 2007. Spontaneous and inoculated yeast populations dynamics and their effect on organoleptic characters of Vinsanto wine under different process conditions. Int. J. Food Microbiol. 115, 281–289. Domizio, P., Romani, C., Lencioni, L., Comitini, F., Gobbi, M., Mannazzu, I., Ciani, M., 2011. Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation. Int. J. Food Microbiol. 147, 170–180. Domizio, P., House, J.F., Joseph, C.M.L., Bisson, L.F., Bamforth, C.W., 2016. Lachancea thermotolerans as an alternative yeast for the production of beer. J. Inst. Brewing 122, 599–604. Domizio, P., Liu, Y., Bisson, L.F., Barile, D., 2017. Cell wall polysaccharides released during the alcoholic fermentation by Schizosaccharomyces pombe and Schizosaccharomyces japonicus: quantification and characterization. Food Microbiol. 61, 136–149. Duarte, F.L., Pimentel, N.H., Teixeira, A., Fonseca, A., 2012. Saccharomyces bacillaris is not a synonym of Candida stellata: reinstatement as Starmerella bacillaris comb. nov. Antonie Van Leeuwenhoek 102, 653–658. El-Ghaouth, A., Wilson, C.L., Wisniewski, M., 1998. Ultrastructural and cytochemical aspects of the biological control of Botrytis cinerea by Candida saitoana in apple fruit. Phytopathology 88, 282–291. Englezos, V., Rantsiou, K., Torchio, F., Rolle, L., Gerbi, V., Cocolin, L., 2015. Exploitation of the non-Saccharomyces yeast Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: physiological and molecular characterizations. Int. J. Food Microbiol. 199, 33–40.

460  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Escott, C., Del Fresno, J.M., Loira, I., Morata, A., Tesfaye, W., del González, M.C., SuárezLepe, J.A., 2018. Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts. Food Chem. 239, 975–983. Fay, J.C., Benavides, J.A., 2005. Evidence for domesticated and wild populations of Saccharomyces cerevisiae. PLoS Genet. 1, e5. Ferreira, F.L., Bota, D.P., Bross, A., Mélot, C., Vincent, J.L., 2001. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA 286, 1754–1758. Fleet, G.H., 2008. Wine yeasts for the future. FEMS Yeast Res. 8, 979–995. Franco-Duarte, R., Mendes, I., Umek, L., Drumonde-Neves, J., Zupan, B., Schuller, D., 2014. Computational models reveal genotype–phenotype associations in Saccharomyces cerevisiae. Yeast 31, 265–277. Fredlund, E., Druvefors, U., Boysen, M.E., Lingsten, K.J., Schnürer, J., 2002. Physiological characteristics of the biocontrol yeast Pichia anomala J121. FEMS Yeast Res. 2, 395–402. Frezier, V., Dubourdieu, D., 1992. Ecology of yeast strain Saccharomyces cerevisiae during spontaneous fermentation in a Bordeaux winery. Am. J. Enol. Vitic. 43, 375–380. Garavaglia, J., de Souza Schneider, R.D.C., Mendes, S.D.C., Welke, J.E., Zini, C.A., Caramão, E.B., Valente, P., 2015. Evaluation of Zygosaccharomyces bailii BCV 08 as a co-starter in wine fermentation for the improvement of ethyl esters production. Microbiol. Res. 173, 59–65. Giovani, G., Canuti, V., Rosi, I., 2010. Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides. Int. J. Food Microbiol. 137, 303–307. Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., Ciani, M., 2013. Lachamcea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiol. 33, 271–281. Goddard, M.R., Anfang, N., Tang, R., Gardner, R.C., Jun, C., 2010. A distinct population of Saccharomyces cerevisiae in New Zealand: evidence for local dispersal by insects and human-aided global dispersal in oak barrels. Environ. Microbiol. 12, 63–73. González-Barreiro, C., Rial-Otero, R., Cancho-Grande, B., Simal-Gándara, J., 2015. Wine aroma compounds in grapes: a critical review. Crit. Rev. Food Sci. Nutr. 55, 202–218. Heard, G.M., Fleet, G.H., 1985. Growth of natural yeast flora during the fermentation of inoculated wines. Appl. Environ. Microbiol. 50, 727–728. Hu, K., Qin, Y., Tao, Y.S., Zhu, X.L., Peng, C.T., Ullah, N., 2016. Potential of glycosidase from non-Saccharomyces isolates for enhancement of wine aroma. J. Food Sci. 81, M935–M943. Janisiewicz, W.J., Tworkoski, T.J., Kurtzman, C.P., 2001. Biocontrol potential of Metchnikowia pulcherrima strains against blue mold of apple. Phytopathology 91, 1098–1108. Jolly, N.P., Augustyn, O.P.H., Pretorius, I.S., 2006. The role and use of non-­Saccharomyces yeasts in wine production. S. Afr. J. Enol. Vitic. 27, 15–39. Jolly, N.P., Augustyn, O.P.R., Pretorius, I.S., 2017. The effect of non-Saccharomyces yeasts on fermentation and wine quality. S. Afr J. Enol. Vitic. 24, 55–62. Kapsopoulou, K., Mourtzini, A., Anthoulas, M., Nerantzis, E., 2007. Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 23, 735–739. Kemsawasd, V., Viana, T., Ardö, Y., Arneborg, N., 2015. Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation. Appl. Microbiol. Biotehnol. 99, 10191–10207. Khan, R.S., Grigor, J., Winger, R., Win, A., 2013. Functional food product development opportunities and challenges for food manufacturers. Trends Food Sci. Technol. 30, 27–37. Kurita, O., 2008. Increase of acetate ester-hydrolysing esterase activity in mixed cultures of Saccharomyces cerevisiae and Pichia anomala. J. Appl. Microbiol. 104, 1051–1058.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   461

Kurtzman, C.P., Fell, W.F., Boekhout, T., Robert, V., 2011. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman, C.P., Fell, J.W., Boekhout, T. (Eds.), The Yeasts: A Taxonomic Study. 5th ed... Elsevier Science Publishers, Amsterdam, pp. 2011,87–110. Le Jeune, C., Erny, C., Demuyter, C., Lollier, M., 2006. Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation. Food Microbiol. 23, 709–716. Legras, J.L., Merdinoglu, D., Cornuet, J., Karst, F., 2007. Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history. Mol. Ecol. 16, 2091–2102. Lencioni, L., Romani, C., Gobbi, M., Comitini, F., Ciani, M., Domizio, P., 2016. Controlled mixed fermentation at winery scale using Zygotorulaspora florentina and Saccharomyces cerevisiae. Int. J. Food Microbiol. 234, 36–44. Limtong, S., Youngmanitchai, W., Kawasaki, H., Seki, T., 2008. Candida phangngensis sp. nov., an anamorphic yeast species in the Yarrowia clade, isolated from water in mangrove forests in Phang-Nga Province, Thailand. Int. J. Syst. Evol. Microbiol. 58, 515–519. Liti, G., Carter, D.M., Moses, A.M., Warringer, J., Parts, L., James, S.A., Tsai, I.J., 2009. Population genomics of domestic and wild yeasts. Nature 458, 337–341. Liu, G.L., Chi, Z., Wang, G.Y., Wang, Z.P., Li, Y., Chi, Z.M., 2015. Yeast killer toxins, molecular mechanisms of their action and their applications. Crit. Rev. Biotechnol. 35, 222–234. Lleixà, J., Martín, V., Portillo, M.D.C., Carrau, F., Beltran, G., Mas, A., 2016. Comparison of fermentation and wines produced by inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae. Front. Microbiol. 7, 338. Magyar, I., Panyik, I., 1989. Biological deacidification of wine with Schizosaccharomyces pombe entrapped in Ca-alginate gel. Am. J. Enol. Vitic. 40, 233–240. Magyar, I., Tóth, T., 2011. Comparative evaluation of some oenological properties in wine strains of Candida stellata, Candida zemplinina, Saccharomyces uvarum and Saccharomyces cerevisiae. Food Microbiol. 28, 94–100. Marongiu, A., Zara, G., Legras, J.L., Del Caro, A., Mascia, I., Fadda, C., Budroni, M., 2015. Novel starters for old processes: use of Saccharomyces cerevisiae strains isolated from artisanal sourdough for craft beer production at a brewery scale. J. Ind. Microbiol. Biotechnol. 42, 85–92. Martini, A., 1993. Origin and domestication of the wine yeast Saccharomyces cerevisiae. J. Wine Res. 4, 165–176. Martini, A., Ciani, M., Scorzetti, G., 1996. Direct enumeration and isolation of wine yeasts from grape surfaces. Am. J. Enol. Vitic. 47, 435–440. Mascia, I., Fadda, C., Dostálek, P., Karabín, M., Zara, G., Budroni, M., Del Caro, A., 2015. Is it possible to create an innovative craft durum wheat beer with sourdough yeasts? A case study. J. Inst. Brewing 121, 283–286. Maturano, Y.P., Mestre, M.V., Esteve-Zarzoso, B., Nally, M.C., Lerena, M.C., Toro, M.E., Combina, M., 2015. Yeast population dynamics during prefermentative cold soak of Cabernet Sauvignon and Malbec wines. Int. J. Food Microbiol. 199, 23–32. Medina, K., Boido, E., Fariña, L., Gioia, O., Gomez, M.E., Barquet, M., Gaggero, C., Dellacassa, E., Carrau, F., 2013. Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae. Food Chem. 141, 2513–2521. Mehlomakulu, N.N., Prior, K.J., Setati, M.E., Divol, B., 2017. Candida pyralidae killer toxin disrupts the cell wall of Brettanomyces bruxellensis in red grape juice. J. Appl. Microbiol. 122, 747–758. Michel, M., Kopecká, J., Meier-Dörnberg, T., Zarnkow, M., Jacob, F., Hutzler, M., 2016. Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model. Yeast 33, 129–144. Milanović, V., Comitini, F., Ciani, M., 2013. Grape berry yeast communities: influence of fungicide treatments. Int. J. Food Microbiol. 161, 240–246.

462  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Mohammadi, A., Razavi, S.H., Mousavi, S.M., Rezaei, K., 2011. A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces cerevisiae, Saccharomyces ludwigii and Saccharomyces rouxii on brewer’s spent grain. Braz. J. Microbiol. 42, 605–615. Morales, P., Rojas, V., Quirós, M., Gonzalez, R., 2015. The impact of oxygen on the final alcohol content of winef ermented by a mixed starter culture. Appl. Microbiol. Biotechnol. 99, 3993–4003. Moreira, N., Mendes, F., Hogg, T., Vasconcelos, I., 2005. Alcohols, esters and heavy sulphur compounds production by pure and mixed cultures of apiculate wine yeasts. Int. J. Food Microbiol. 103, 285–294. Moreira, N., de Pinho, P.G., Santos, C., Vasconcelos, I., 2010. Volatile sulphur compounds composition of monovarietal white wines. Food Chem. 123, 1198–1203. Moreno, P.I., Vilanova, I., Villa-Martínez, R., Garreaud, R.D., Rojas, M., De Pol-Holz, R., 2014. Southern annular mode-like changes in southwestern Patagonia at centennial timescales over the last three millennia. Nature Conserv. 5, 4375. Morrison-Whittle, P., Goddard, M.R., 2017. From vineyard to winery: a source map of microbial diversity driving wine fermentation. Environ. Microbiol. https://doi. org/10.1111/1462-2920.13960. Mortazavian, A., Razavi, S., Mousavi, S., Malganji, S., Sohrabvandi, S., 2014. The effect of Saccharomyces strain and fermentation conditions on quality parameters of non-­ alcoholic beer. J. Paramed. Sci. 5, 21–26. Nissen, P., Nielsin, D., Arneborg, N., 2003. Viable Saccharomyces cerevisiae cells at high concentrations cause early growth arrest of non-Saccharomyces yeasts in mixed cultures by a cell-cell contact-mediated mechanism. Yeast 20, 331–341. Oro, L., Ciani, M., Comitini, F., 2014. Antimicrobial activity of Metscknikowia pulcherrima on wine yeasts. J. Appl. Microbiol. 116, 1209–1217. Oro, L., Ciani, M., Bizzaro, D., Comitini, F., 2016. Evaluation of damage induced by Kwkt and Pikt zymocins against Brettanomyces/Dekkera spoilage yeast, as compared to sulphur dioxide. J. Appl. Microbiol. 121, 207–214. Osburn, K., Ahmad, N.N., Bochman, M.L., 2016. Bio-prospecting, selection, and analysis of wild yeasts for ethanol fermentation. Zymurgy 39, 81–88. Osburn, K., Amaral, J., Metcalf, S.R., Nickens, D.M., Rogers, C.M., Sausen, C., Caputo, R., Miller, J., Hongde, L., Tennessen, J.M., Bochman, M.L., 2018. Primary souring: a novel bacteria-free method for sour beer production. Food Microbiol. 70, 76–84. Palmeri, R., Spagna, G., 2007. β-Glucosidase in cellular and acellular form for winemaking application. Enzyme Microb. Technol. 40, 382–389. Pardo, I., García, M.J., Zúñiga, M., Uruburu, F., 1989. Dynamics of microbial populations during fermentation of wines from the Utiel-Requena region of Spain. Appl. Environ. Microbiol. 55, 539–541. Passoth, V., Fredlund, E., Druvefors, U.Ä., Schnürer, J., 2006. Biotechnology, physiology and genetics of the yeast Pichia anomala. FEMS Yeast Res. 6, 3–13. Pérez-Nevado, F., Albergaria, H., Hogg, T., Girio, F., 2006. Cellular death of two non-­ Saccharomyces wine-related yeasts during mixed fermentations with Saccharomyces cerevisiae. Int. J. Food Microbiol. 108, 336–345. https://doi.org/10.1016/j.ijfoodmicro. 2005.12.012. Pérez-Torrado, R., Barrio, E., Querol, A., 2017. Alternative yeasts for winemaking: saccharomyces non-cerevisiae and its hybrids. Crit. Rev. Food Sci. Nutr. 58 (11), 1780–1790. Petruzzi, L., Carbo, M., Sinigaglia, M., Bevilacqua, A., 2016. Brewers’ yeast in controlled and uncontrolled fermentation, with a focus on novel, non-conventional and superior strains. Food Rev. Int. 32, 341–363. Phaff, H.J., Starmer, W.T., Tredick-Kline, J., Aberdeen, V., 1987. Pichia barkeri, a new yeast species occurring in necrotic tissue of Opuntia stricta. Int. J. Syst. Evol. Microbiol. 37, 386–390.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   463

Pires, E.J., Teixeira, J.A., Brányik, T., Vincente, A.A., 2014. Yeast: the soul of beer’s aroma and a review of flavour-active esters and higher alcohols produced by the brewing yeast. Appl. Microbiol. Biotechnol. 98, 1937–1949. Prakitchaiwattana, C.J., Fleet, G.H., Heard, G.M., 2004. Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast Res. 4, 865–877. Pretorius, I.S., 2000. Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast, 675–729. Quirós, M., Rojas, V., Gonzalez, R., Morales, P., 2014. Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration. Int. J. Food Microbiol. 181, 85–91. Rantsiou, K., Dolci, P., Giacosa, S., Torchio, F., Tofalo, R., Torriani, S., Suzzi, G., Rolle, L., Cocolin, L., 2012. Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations. Appl. Environ. Microbiol. 78 (6), 1987–1994. Renault, P.E., Albertin, W., Bely, M., 2013. An innovative tool reveals interaction mechanisms among yeast populations under oenological conditions. Appl. Microbiol. Biotechnol. 97, 4105–4119. Renault, P., Coulon, J., Moine, V., Thibon, C., Bely, M., 2016. Enhanced 3-sulfanylhexan1-ol production in sequential mixed fermentation with Torulaspora delbrueckii/­ Saccharomyces cerevisiae reveals a situation of synergistic interaction between two industrial strains. Front. Microbiol. 7. Rodrigues, A.J., Raimbourg, T., Gonzalez, R., Morales, P., 2016. Environmental factors influencing the efficacy of different yeast strains for alcohol level reduction in wine by respiration. LWT—Food Sci. Technol. 65, 1038–1043. Rodríguez, M.E., Lopes, C.A., Valles, S., Giraudo, M.R., Caballero, A., 2007. Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts. Enzyme Microb. Technol. 41, 812–820. Rojas, V., Gil, J.V., Piñaga, F., Manzanares, P., 2003. Acetate ester formation in wine by mixed cultures in laboratory fermentations. Int. J. Food Microbiol. 86, 181–188. Romano, P., Suzzi, G., 1996. Origin and production of acetoin during wine yeast fermentation. Appl. Environ. Mircobiol. 62, 309. Saerens, S., Swiegers, J.H., 2014. Enhancement of beer flavor by a combination of Pichia yeast and different hop varieties, U.S. Patent 20140234480 A1. Saerens, S.M.G., Duong, C.T., Nevoigt, E., 2010. Genetic improvement of brewer’s yeast: current state, perspectives and limits. Appl. Microbiol. Biotechnol. 86, 1195–1212. Saerens, S. and Swiegers, J.H., Chr Hansen A.S., 2017.Production of low-alcohol or ­alcohol-free beer with Pichia kluyveri yeast strains. U.S. Patent 9,580675. Sancho, T., Giménez-Jurado, G., Malfeito-Ferreira, M., Loureiro, V., 2000. Zymological indicators: a new concept applied to the detection of potential spoilage yeast species associated with fruit pulps and concentrates. Food Microbiol. 17, 613–624. Santos, A., Navascués, E., Bravo, E., Marquina, D., 2011. Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. Int. J. Food Microbiol. 145, 147–154. Saravanakumar, D., Ciavorella, A., Spadaro, D., Garibaldi, A., Gullino, M.L., 2008. M. pulcherrima strain MACH1 outcompetes Botrytis cinerea, Alternaria alternata and Penicillium expansum in apples through iron depletion. Postharv. Biol. Technol. 49, 121–128. Schuller, D., Casal, M., 2007. The genetic structure of fermentative vineyard-associated Saccharomyces cerevisiae populations revealed by microsatellite analysis. Antonie Van Leeuwenhoek 91, 137–150. Sipiczki, M., 2004. Species identification and comparative molecular and physiological analysis of Candida zemplinina and Candida stellata. J. Basic Microbiol. 44, 471–479.

464  Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS

Sipiczki, M., 2006. Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion. Appl. Environ. Microbiol. 72, 6716–6724. Sláviková, E., Vadkertiová, R., Vránová, D., 2009. Yeasts colonizing the leaves of fruit trees. Ann. Microbiol. 59, 419–424. So, A., 2014. Developing new barely varieties: a work in progress. New Brew. 31, 60–68. Soden, A., Francis, I.L., Oakey, H., Henschke, P.A., 2000. Effects of co-fermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of Chardonnay wine. Aust. J. Grape Wine Res. 6, 21–30. Sohrabvandi, S., Hadi Razavi, S., Mousavi, S., Mortazavian, A., 2010. Characteristics of different brewer’s yeast strains used for non-alcoholic beverage fermentation in media containing different fermentable sugars. Int. J. Biotechnol. 8, 178–185. Spitaels, F., Wieme, A.D., Janssens, M., Aerts, M., Daniel, H., Van Landschoot, A., De Vuyst, L., Vandamme, P., 2014. The microbial diversity of traditional spontaneously fermented lambic beer. PLoS ONE 9, e95384. Stantford, M., James, S.A., 2003. Non-alcoholic beverages and yeasts. In: Boekhout, T., Robert, V. (Eds.), Yeasts in Food: Beneficial and Detrimental Aspects. Woodhead publishing Ltd, Cambridge, pp. 309–345. Steensels, J., Verstrepen, K., 2014. Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations. Annu. Rev. Microbiol. 68, 61–80. Steensels, J., Snoeka, T., Meersmana, E., Nicolino, M.P., Aslankoohi, E., Christiaens, J.F., Gemayel, R., Meert, W., Newa, A.M., Pougacha, K., Saels, V., van der Zande, E., Voordeckers, K., Verstrepen, K.J., 2012. Selecting and generating superior yeasts for the brewing industry. Cerevisia 37, 63–67. Swiegers, J.H., Pretorius, I.S., 2005. Yeast modulation of wine flavor. Adv. Appl. Microbiol. 57, 131–175. Tofalo, R., Perpetuini, G., Fasoli, G., Schirone, M., Corsetti, A., Suzzi, G., 2014. Biodiversity study of wine yeasts belonging to the “terroir” of Montepulciano d’Abruzzo “Colline Teramane” revealed Saccharomyces cerevisiae strains exhibiting atypical and unique 5.8 S-ITS restriction patterns. Food Microbiol. 39, 7–12. Tristezza, M., Tufariello, M., Capozzi, V., Spano, G., Mita, G., Grieco, F., 2016. The oenological potential of Hanseniaspora uvarum in simultaneous and sequential co-­ fermentation with Saccharomyces cerevisiae for industrial wine production. Front. Microbiol. 7, . Tronchoni, J., Curiel, J.A., Sáenz-Navajas, M.P., Morales, P., de-la-Fuente-Blanco, A., Fernández-Zurbano, P., Gonzalez, R., 2017. Aroma profiling of an aerated fermentation of natural grape must with selected yeast strains at pilot scale. Food Microbiol. 70, 214–223. Varela, C., 2016. The impact of non-Saccharomyces yeasts in the production of alcoholic beverages. Appl. Microbiol. Biotechnol. 100, 9861–9874. Varela, C., Borneman, A.R., 2017. Yeasts found in vineyards and wineries. Yeast 34, 111–128. Verstrepen, K.J., Chambers, P.J., Pretorius, I.S., 2006. The development of superior yeast strains for the food and beverage industries: challenges, opportunities and potential benefits. In: Yeasts in Food and Beverages. Springer, Berlin Heidelberg, pp. 399–444. Viana, F., Gil, J.V., Vallés, S., Manzanares, P., 2009. Increasing the levels of 2-phenylethyl acetate in wine through the use of a mixed culture of Hanseniaspora osmophila and Saccharomyces cerevisiae. Int. J. Food Microbiol. 135, 68–74. Villalba, M.L., Sáez, J.S., del Monaco, S., Lopes, C.A., Sangorrín, M.P., 2016. TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts. Int. J. Food Microbiol. 217, 94–100. Wang, C., Liu, Y., 2013. Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang. China. Food Microbiol. 33, 172–177.

Chapter 14  FOOTPRINT OF NONCONVENTIONAL YEASTS   465

Wang, C., Mas, A., Esteve-Zarzoso, B., 2015. Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation. Int. J. Food Microbiol. 206, 67–74. Zott, K., Miot-Sertier, C., Claisse, O., Lonvaud-Funel, A., Masneuf-Pomarede, I., 2008. Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking Int. J. Food Microbiol. 125, 197–203. Zott, K., Thibon, C., Bely, M., Lonvaud-Funel, A., Dubourdieu, D., Masneuf-Pomarede, I., 2011. The grape must non-Saccharomyces microbial community: impact on volatile thiol release. Int. J. Food Microbiol. 151, 210–215.

Further Reading Benito, S., Palomero, F., Morata, A., Calderón, F., Palmero, D., Suárez-Lepe, J.A., 2014. Selection of appropriate Schizosaccharomyces strains for winemaking. Food Microbiol. 42, 218–224.