Isolation, Selection, and Identification Techniques for Non-Saccharomyces Yeasts of Oenological Interest

ISOLATION, SELECTION, AND IDENTIFICATION TECHNIQUES FOR NON-SACCHAROMYCES YEASTS OF OENOLOGICAL INTEREST

15

Loira Iris⁎, Morata Antonio⁎, Bañuelos María Antonia†, Suárez-Lepe José Antonio⁎ ⁎

Dpto. Química y Tecnología de Alimentos, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain, †Dpto. Biotecnología—Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain

Abbreviations B. bruxellensis Brettanomyces bruxellensis C. magnolia Candida magnolia Dekkera bruxellensis (Teleomorph of B. bruxellensis) D. bruxellensis H. guilliermondii Hanseniaspora guilliermondii H. osmophila Hanseniaspora osmophila H. uvarum Hanseniaspora uvarum (Teleomorph of K. apiculata) H. vineae Hanseniaspora vineae I. orientalis Issatchenkia orientalis K. apiculata Kloeckera apiculata L. thermotolerans  Lachancea thermotolerans (formerly Kluyveromyces thermotolerans) M. pulcherrima Metschnikowia pulcherrima P. fermentans Pichia fermentans P. guilliermondii Pichia guilliermondii P. membranaefaciens Pichia membranaefaciens Starmerella bacillaris (synonym Candida zemplinina) S. bacillaris S. bombicola Starmerella bombicola (formerly Candida stellata) S. cerevisiae Saccharomyces cerevisiae S. ludwigii Saccharomycodes ludwigii S. pombe Schizosaccharomyces pombe T. delbrueckii Torulaspora delbrueckii W. anomalus Wickerhamomyces anomalus (formerly Pichia anomala) Z. bailii Zygosaccharomyces bailii Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00015-1 © 2020 Elsevier Inc. All rights reserved.

467

468  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

15.1 Introduction The use of non-Saccharomyces yeasts is increasingly common in oenology since they can contribute to enhance wine quality by modifying certain physicochemical parameters. In the past, many of the yeast species reviewed in this chapter were considered spoilage yeasts in the wine industry (Kalathenos et al., 1995; Loureiro and Malfeito-Ferreira, 2003). But nowadays, thanks to research and scientific advances, the current tendency is toward getting the maximum benefit from their metabolome, making them useful in the field of oenology instead of considering them as microbial contamination. Moreover, the choice of yeast or yeasts to be used in the winemaking process is an additional tool that allows the winemaker to differentiate their wines in the market (Eglinton et  al., 2003). This chapter highlights the potential usefulness of several yeast species, Schizosaccharomyces pombe, Torulaspora delbrueckii, and Lachancea thermotolerans among others, that in the past were not considered for winemaking applications. In this review, different non-Saccharomyces yeast isolation techniques are discussed, the most common selection criteria commented and recent advances in available techniques for its identification reviewed. Hereinafter, isolation of non-Saccharomyces yeasts is going to be considered not only as a technique for obtaining new yeast strains from grapes, must under fermentation, and environments in the cellar, but also as a method of population count in order to differentiate the number of yeast cells corresponding to certain non-Saccharomyces species within the set of yeasts involved in wine fermentation. In this way, we can have an idea of the degree of participation of each species along the fermentation process and, therefore, of its influence on the final composition of the wine. In addition, the selection criteria will be considered in terms of the main uses and potential applications in the field of oenology of the different genera and species of non-Saccharomyces yeasts. That is, when speaking of selection criteria will refer to the harnessing of secondary metabolic characteristics provided by each nonSaccharomyces yeasts for a new concept of wine making adapted to the new situation of climate change, as well as to the current demands of consumers. Finally, the main methods and tools available for the identification of non-Saccharomyces yeasts will be reviewed. We have tried to organize the chapter in these three major sections (isolation, selection, and identification), however, in some cases, isolation and identification may occur simultaneously in some of the techniques discussed, such as is the case of the chromogenic media.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   469

15.2 Non-Saccharomyces Yeasts Isolation Techniques Traditional yeast extract-peptone-dextrose (YEPD) growth medium with agar, sometimes abbreviated as YPD, is the most commonly used for yeast isolation from either grapes, musts, or wines (CorderoBueso et al., 2011; Viana et al., 2011; Lleixà et al., 2016). However, this method is global rather than specific, so that all kinds of yeasts can be grown on it. So that can be used for total yeast counts. It is also very useful to perform a preliminary screening test and, after that, proceed with the selection and identification of the yeast species. Other media frequently used in oenology for total yeast count are WL agar (a differential yeast growth medium made of 50 g/L glucose, 5 g/L tryptone, 4 g/L yeast extract, 0.55 g/L potassium dihydrogen phosphate, 0.425 g/L potassium chloride, 0.125 g/L calcium chloride, 0.125 g/L magnesium sulfate, 0.0025 g/L ferric chloride, 0.0025 g/L manganese sulfate, 0.022 g/L bromocresol green, and 15 g/L agar) (Urso et  al., 2008; Barquet et al., 2012; Gobbi et al., 2013; Medina et al., 2013; Wang et al., 2015) and YM agar (yeast malt agar made of 10 g/L glucose, 5 g/L peptic digest of animal tissue, 3 g/L yeast extract, 3 g/L malt extract, and 20 g/L agar) (de Ponzzes-Gomes et  al., 2014; Pantelides et  al., 2015). Some of these nutrient agar media, as WL, also allow wine yeast identification on the basis of the color and morphology of their colonies (Pallmann et al., 2001). Nowadays, available isolation techniques allow detecting, differentiating between yeasts and even, in some cases, quantitating the non-Saccharomyces yeasts.

15.2.1  Selective and Differential Culture Media A differential isolation technique would allow the quantification of non-Saccharomyces yeasts present in a mixed culture by means of plate culturing. In this way, it would be possible to know the active and viable population of non-Saccharomyces yeasts at a given moment, either at the beginning or at the end of fermentation, and along the same. Selective culture media based in lysine as sole nitrogen source let differentiate between T. delbrueckii and Saccharomyces cerevisiae (Ciani et al., 2006; Loira et al., 2014), Hanseniaspora vineae and S. cerevisiae (Viana et al., 2011; Lleixà et al., 2016), Hanseniaspora uvarum and S. cerevisiae (Ciani et al., 2006), Starmerella bombicola and S. cerevisiae (Di Maio et al., 2011), since no S. cerevisiae strain can utilize lysine to grow (Kurtzman et al., 2011). Nevertheless, on the whole, plate culture media as isolation technique are never 100% reliable, as demonstrated in the study published by Belda et al. (2016) in which growth of S. cerevisiae in lysine agar medium

470  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

was observed. In general, non-Saccharomyces yeasts can use this amino acid for the growth of the colonies. However, in S. pombe is a strain-dependent skill (Kurtzman et  al., 2011). So, this synthetic lysine medium specific for certain non-Saccharomyces yeasts can be used to ensure their detection (Ortiz-Barrera et al., 2015). A lysine agar medium is currently being commercialized by Oxoid Ltd. (England) in dehydrated format with a total amount of 1 g/L of lysine in the formula (Oxoid, 2016). Fig. 15.1 shows a compilation of the results obtained from different culture media used to isolate and/or to characterize some yeast species of oenological interest. It can be seen that all the species tested can grow in YEPD medium with no problem, proving once again the effectiveness of this culture medium for total counts. However, when temperature in the stove is set at 37°C using the same culture medium, only three yeast species could effectively grow, specifically S. cerevisiae, S. pombe, and Wickerhamomyces anomalus. l-lysine and Nitrate agar were tested as culture media with a sole nitrogen source to assess the ability of the different yeast species to assimilate various forms of this specific nutrient. From the results obtained, it could be concluded that all the non-Saccharomyces yeasts species tested except S. pombe, were able to use lysine for the growth of the colonies and, however, only Candida magnolia and W. anomalus showed a good colonies formation in nitrate agar medium. When the chromogenic medium CHROMagar Candida was tested, different colors in the colonies were obtained.

Fig. 15.1  Yeast growth on solid media. Serial tenfold dilutions of saturated cultures were spotted onto different media. YEPD growth medium. For assessing the assimilation of nitrogen compounds: l-lysine agar medium (Oxoid) and Nitrate agar (Yeast Carbon Base, YCB, and 7.8 g/L potassium nitrate). Chromogenic media for rapid yeast identification: BBL-CHROMagar Candida medium. For assessing the assimilation of carbon compounds: Yeast Nitrogen Base (YNB) and 2 g of carbon source in 100 mL of deionized water: Hexoses: d-glucose (Gluc), d-fructose (Fruc), d-galactose (Gal); Disaccharides: lactose (Lac), maltose (Mal), sucrose (Suc). For evaluating lithium tolerance: YNB, 20 g/L of glucose and Lithium Cloride (20 and 100 mM). Growth was recorded after 3–7 days at 28°C (except for growth at 37°C).

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   471

Thus, S. cerevisiae colonies showed a purple color, L. thermotolerans colonies were characterized by a salmon-brown color, C. magnolia colonies showed a distinctive blue color, Starmerella bacillaris had salmon colonies surrounded by a violet halo, W. anomalus colonies were characterized by a soft pink color, Kloeckera apiculata showed brown colonies with a soft violet halo and Rhodotorula sp. colonies had a yellowish color. A selective media can be formed by the addition of certain antibiotic or antiseptic compound to a traditional culture media formulation. Thus, a selective medium based on YEPD containing a final concentration of 50 mg/L SO2 was used (Ciani et  al., 2006) to determine the S. cerevisiae population along mixed and sequential fermentations with non-Saccharomyces yeasts, namely H. uvarum and T. delbrueckii. Cycloheximide is another proved antimycotic compound useful to prepare selective culture media (Moreira et al., 2005; Pérez-Nevado et al., 2006; Di Maio et al., 2011). In 2001, Rodrigues et al. (2001) developed a new medium calledDekkera/Brettanomyces Differential Medium (DBDM) to isolate Brettanomyces yeasts from wine samples. This medium uses ethanol as only carbon source and cycloheximide (actidione) as antimicrobial agent to improve the selectivity toward Brettanomyces yeasts. Its full composition is as follows: 6.7 g/L YNB, 6% v/v ethanol, 10 mg/L cycloheximide, 100 mg/L p-coumaric acid, 22 mg/L bromocresol green, and 20 g/L agar. So far, despite the false positives caused by actidione-resistant yeasts (among them K. apiculata, S. pombe, and Pichia guilliermondii), DBDM is the existing most effective and selective medium for Dekkera/Brettanomyces isolation (Benito et  al., 2012b).

15.2.2  Chromogenic Media Chromogenic media allow yeast identification through detecting the enzymatic interaction with a chromogenic substrate included in the solid culture medium formulation. This methodology can be also used for yeast isolation from a mixed culture, since every yeast species grow with a characteristic color in the colony (Fig. 15.1). So, compounds with different absorbance are released when the chromogens are degraded by specific enzymes. In a relatively recent study, van Breda et al. (2013) confirmed the potential of the CHROMagar Candida medium (CHROMagar, 2016) to differentiate between T. delbrueckii and S. cerevisiae yeast species, as the first one develops colonies with a color ranging from white to yellow, while the second one forms purple colonies. Same violet-purple colonies of S. cerevisiae were previously obtained by Giusiano and Mangiaterra (1998) and Ghelardi et al. (2008). The color of the colonies depends largely on

472  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

the enzymatic activity of the yeast, and, therefore, may depend not only on the genera but also on the species and in some cases also on the strain. Some authors also tested the use of bromocresol green (Rodrigues et al., 2001) and methylene blue (de Siloniz et al., 1999; Pérez-Nevado et al., 2006; Elmacı et al., 2014) as pH or redox indicators components of the culture media for visual identification of the yeast species and its metabolism (e.g., acid-producing strains or killer activity). In an experiment carried out by de Siloniz et  al. (1999), black to violet colonies were formed by Zygosaccharomyces spp., violet colonies by T. delbrueckii, and metallic green by S. cerevisiae, when methylene blue was used as differential agent in the formulation of the agar plates.

15.2.3  Other Parameters Useful for Yeast Isolation in Culture Media Incubation temperature selection is another way to control the growth in agar plates of different species of yeast. H. uvarum cannot grow at 37°C, while Hanseniaspora guilliermondii and S. cerevisiae can (Moreira et  al., 2005). Growth of most non-Saccharomyces yeasts is inhibited at this same temperature (Bañuelos et  al., 2016). Some exceptions are the Pichia and Schizosaccharomyces genera (see Fig. 15.1).

15.3 Non-Saccharomyces Yeasts Selection Criteria: Usefulness of Non-Saccharomyces in Winemaking Processes In this section, different selection criteria are going to be shown from the point of view of the usefulness of non-Saccharomyces yeasts for winemaking processes, fermentation included when possible. Even if the yeast species has not enough fermentative power to complete must fermentation by itself, it can develop another role in the same fermentation stage or in different winemaking processes, such as aging on lees or second fermentation of sparkling wines, helping to modify the chemical composition of the wine (Kulkarni et al., 2015; Liu et al., 2015).

15.3.1  Fermentative Power Together with fermentation kinetics, the fermentative power is an essential parameter to characterize the yeast. Apart from estimating the alcoholic degree which the non-Saccharomyces can reach, the fermentative power will define the type of fermentation in which the

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   473

Table 15.1  Fermentative Power and Ethanol Tolerance of Certain Non-Saccharomyces Yeast Species Yeast Species

Fermentative Power (% v/v)

Ethanol Tolerance (% v/v)

Candida cantarelli Candida pulcherrima Candida stellata Candida zemplinina

2 4 7.5–9.6 4–7.3

5–7 5-7 11 5–7

Kloeckera apiculata

4–6.5

9

Lachancea thermotolerans Metschnikowia pulcherrima Pichia fermentans

4–10.5

5–10

3.5–5

5–7

3–6

5–7

10–20

5–7 15–20

Pichia guilliermondii Saccharomyces cerevisiae Saccharomycodes ludwigii Schizosaccharomyces pombe Starmerella bombicola Torulaspora delbrueckii

Wickerhamomyces anomalus

11–14 10–15

>10

References Fleet (2003), Comitini et al. (2011) Zohre and Erten (2002), Fleet (2003) Gao and Fleet (1988), Magyar and Tóth (2011) Fleet (2003),Comitini et al. (2011), Magyar and Tóth (2011), Rantsiou et al. (2012) Gao and Fleet (1988), Zohre and Erten (2002), Viana et al. (2008) Fleet (2003), Kapsopoulou et al. (2005), Comitini et al. (2011), Gobbi et al. (2013) Fleet (2003), Clemente-Jimenez et al. (2005), Comitini et al. (2011), Morata and Suárez-Lepe (2015) Fleet (2003), Mingorance-Cazorla et al. (2003), Clemente-Jimenez et al. (2005) Fleet (2003) Gao and Fleet (1988), Ciani (1997), Chi and Arneborg (2000), Clemente-Jimenez et al. (2005), Di Maio et al. (2012) Ciani (1997) Fleet (2003), Suárez-Lepe et al. (2012)

4.6

Milanovic et al. (2012)

7.6–9 (Loira et al., 2014) 4.9–9.1 (Comitini et al., 2011)

Loira et al. (2014), Comitini et al. (2011)

5–7

Fleet (2003)

yeast can participate. Thus, low fermentative power yeasts will have to carry out a sequential fermentation or mixed fermentation in order to ensure the total sugar depletion in the wine. Table  15.1 summarizes the fermentative power and the ethanol tolerance of some non-Saccharomyces yeast species of oenological interest.

474  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

15.3.2  Competition Between Yeast Species During Fermentation Different factors can determine the prevalence of some species over others during the winemaking process, such as the lag phase, growth rate, ethanol tolerance, sulfur dioxide tolerance, nutrients requirements, osmotic resistance, killer factor, oxygen limitation, etc. (Holm Hansen et al., 2001; Pina et al., 2004; Zuzuarregui, 2004; De la Torre-González et al., 2016). Also, in controlled fermentations, the winemaker may influence, for example, by choosing the yeast inoculation rate or timing. Lag phase is very important at the beginning of the fermentation process in order to ensure proper establishment of the selected yeast in the fermentation tanks. Those yeast species with long lag phase, such as S. pombe (Mylona et al., 2016), could have problems when used as starter culture because of slow start of fermentation and, therefore, weak competition with other species. A possible solution to that potential problem would be the use of emerging nonthermal technologies, such as high hydrostatic pressure to treat the grape must and obtain a pasteurization effect before yeast inoculation (Bañuelos et al., 2016). When grown in liquid media, using a Bioscreen C MBR (Thermo Fisher Scientific group, Spain) automated apparatus it is possible to observe the differences in the growth curves of the different yeast species (Fig. 15.2). The growth rate and tolerance to antimicrobial compounds, determined from the growth curves in optimal laboratory conditions, can be useful in the selection and characterization of wine yeasts in relation to their association with different strains and species. Among other utilities in oenology, this physiological feature can have an impact on the degree of implementation and competitiveness with other wine yeasts. At this point, it is worth noting that no correlation can be established between growth rate in liquid media and size of the colonies in agar plates. Villalba et al. (2016) discovered a new killer toxin with glucanase and chitinase enzymatic activities able to inhibit the growth of wine spoilage yeasts, such as Brettanomyces bruxellensis, P. guilliermondii, Pichia manshurica, and Pichia membranaefaciens. This toxin is produced by some strains of the species T. delbrueckii. Previously, Mehlomakulu et  al. (2014) have already demonstrated the effectiveness of two killer toxins secreted by Candida pyralidae to control the development of B. bruxellensis in musts and wines. The early deaths of certain non-Saccharomyces yeasts such as L. thermotolerans and T. delbrueckii in mixed cultures with S. cerevisiae can be also explained by a cell-to-cell contact mechanism when S. cerevisiae reaches high cell densities during fermentation, since in the experiments performed by Nissen et al. (2003) no effect due to nutrient depletion or the presence of toxic compounds was observed.

OD600 (log scale)

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   475

1.0

1.0

0.1

0.1

2

6

10

(A) S.cerevisiae M.pulcherrima

14 18 Time (h)

22

K.apiculata S.ludwigii

26

30

2

12

(B) L.thermotolerans W.anomalus

22

32 42 Time (h)

S.bombicola T.delbrueckii

52

62

S.bacillaris S.pombe

Fig. 15.2  Growth curves of wine yeasts in the absence or in the presence of potassium metabisulfite. (A) Growth curves were determined in YPD at pH 3.7 (B) or in the same medium with 100 mg/L of potassium metabisulfite. Growth was monitored in microtiter plates using a Bioscreen C system with automatic recording of OD (600 nm) every 30 min.

15.3.3  Alcoholic Strength Reduction Currently, the International Code of Oenological Practices published by the International Organization of Vine and Wine (OIV) establishes a classification of wine by ethanol content in dealcoholized wines, low alcohol wines, and reduced alcohol wines (OIV-OENO 18/73, n.d.; OIV-ECO 432/2012, n.d.; OIV-ECO 433/2012, n.d.; OIVOENO 394B-2012, n.d.) (Fig. 15.3). Despite their effectiveness and ease of controlling the amount of alcohol to be removed, most physicochemical techniques currently available in the market for wine total or partial dealcoholization (e.g., reverse osmosis, dialysis, freeze concentration, spinning cone columns, etc.) usually require intense practices on wine, which can demean its final quality (high risk of losing some aroma and flavor

Fig. 15.3  Different treatments for alcohol management and types of wines regarding their alcohol content.

476  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

compounds) and a significant investment by the winery for the acquisition of the equipment (de Barros Lopes et al., 2003). Regarding microbiological techniques, some authors studied the possibility of using certain Crabtree-negative non-Saccharomyces yeast strains, among them Pichia stipitis, P. membranaefaciens, W. anomalus, Williopsis saturnus, C. zemplinina, H. uvarum or S. bombicola, in a pre-fermentative aerobic stage to reduce the levels of sugar in the grape must by sugar respiration and thus limit the potential alcohol content (PAC) (Smith, 1995; Erten and Campbell, 2001). However, wines produced with this biotechnology will have a golden or brownish orange color indicative of oxidation. Likewise, the use of genetic engineering with the aim of creating yeasts able to reduce the synthesis of ethanol by redirecting the carbon flux toward the production of other metabolites with interest in wine, such as glycerol, organic acids, and esters (Malherbe et al., 2003; Heux et al., 2006), despite its positive results, is still not legally permitted in many countries, particularly from the old world. Another microbiological alternative to reduce the concentration of ethanol in wine from its origin and, at the same time, improve their sensory quality, would be through the selection of inefficient yeast strains in pure fermentations and the use of non-Saccharomyces yeasts both in sequential or in mixed fermentations coupled with S. cerevisiae. Glycolytic inefficiency understood as the ability of yeasts to naturally direct the sugar consumption to the production of other metabolites with positive sensory impact instead of synthesizing ethanol (Loira et al., 2012). This feature is observed in those yeasts whose performance in the conversion of sugars into ethanol is lower than the average, that is, the ratio between the amount of sugars consumed (g/L) and the alcoholic degree produced (% v/v) exceeds 16.83 (g/L)/(% v/v) (Ribéreau-Gayon et al., 2000). For the evaluation of this performance, it is very important to only consider the yeasts able to deplete all sugars of the must and, therefore, produce a dry wine. According to the definition provided by the OIV, a dry wine is the one that contains a maximum of either 4 g/L of residual sugar or 9 g/L when the total acidity (expressed in grams of tartaric acid per liter) is no >2 g/L less than the sugar content (OIV-OENO 18/73, OIV-ECO 3/2003, and OIV-OENO 415/2011, n.d.). One of the main limitations of this yeast strains selection method for reducing the alcohol content of wine is the low existing variability in the efficiency of sugars conversion into ethanol described in the literature for the commercial strains of S. cerevisiae (Ehsani et al., 2007; Varela et al., 2008; Schmidtke et al., 2012). This can be justified by the fact that the yeast metabolism is naturally adapted and selected to maximize the production of ethanol, since the alcoholic fermentation is its main pathway for obtaining energy under anaerobic conditions (Piškur et al., 2006; Field et al., 2009). Usually, alcoholic strength reductions between 0.5 and 1% v/v can be

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   477

achieved with this biotechnology, such is the case of the experiment developed by Loira et al. (2012), where an average reduction of about one alcoholic degree was obtained in pure fermentations of a red must from Tempranillo variety (PAC exceeding 15% v/v) with the strain TP2A16 (S. cerevisiae isolated from D.O. Toro, Spain, 2008 harvest). According to these findings, this strain can be considered as inefficient yeast as it requires a consumption of between 18 and 18.5 g/L of sugar to produce one degree of alcohol. As for the use of non-Saccharomyces yeasts in sequential fermentations with the purpose to mitigate the sugar richness of the must so that at the moment of subsequent inoculation with S. cerevisiae the potential alcoholic strength in the medium is lower, several studies have demonstrated its potential feasibility and effectiveness (Table  15.2). Most non-Saccharomyces yeasts grow and develop preferably in the early stages of alcoholic fermentation, primarily due to its low e­ thanol tolerance (not all the species, but many of them are also characterized by low fermentative power) and due to the decreasing levels of oxygen in the medium (Holm Hansen et  al., 2001; Pina et  al., 2004). Production of toxic compounds by S. cerevisiae was also proposed as a hypothesis to explain the early death of non-Saccharomyces yeasts in mixed inoculations (Pérez-Nevado et  al., 2006). In addition to ethanol, acetic acid, medium-chain fatty acids, and acetaldehyde may also limit the growth and development of yeasts when their tolerance ranges are exceeded (Bisson, 1999; Ludovico et al., 2001; Fleet, 2003). Among non-Saccharomyces species, Zygosaccharomyces bailii stands out for its high tolerance to ethanol and acetic acid (Santos et  al., 2008). In general, non-Saccharomyces species are characterized by lower fermentative power than S. cerevisiae. However, certain species such as S. pombe have a high fermentative power, in this case ranging from 10 to 12.6% v/v (Suárez-Lepe et  al., 2012), and are able to complete the fermentation by themselves with a total depletion of the sugars in the must. In a recent study carried out by Benito et  al. (2013), mean reductions of about 0.5% v/v were achieved in the average ethanol content by employing a strain of S. pombe in sequential fermentation with S. cerevisiae. In the sequential fermentations developed by Gobbi et  al. (2013), using L. thermotolerans as starter culture and inoculating S. cerevisiae at 48 h after the beginning of fermentation, an average reduction in the alcoholic strength of approximately 1.5% v/v was achieved, with respect to S. cerevisiae control in pure fermentation. However, the concentration of residual sugars in the wine was high, 18.7 ± 1.5 g/L, which theoretically corresponds to 1% v/v of PAC. In another study about mixed fermentations with T. delbrueclkii and indigenous yeasts, doing tests with different sugar concentrations in the must by selecting different dates of harvest, average reductions in the ethanol content of about 2 degrees (2% v/v) were observed with respect

Table 15.2  Summary of Experiments in Which Reductions in the Alcoholic Strength Were Obtained Thanks to the Use of Non-Saccharomyces Yeasts in Sequential (SF) or Mixed (MF) Fermentations With S. cerevisiae NonSaccharomyces Yeast Species

Type of Fermentation (Time of Second Inoculation or NonSacch:Sacch Ratio)

Reduction Achieved in the Alcoholic Strength (% v/v)

Residual Sugars (g/L)

Starmerella bombicola

MF (109:106 cells/mL; ratio 1000:1) SF (3 days)

0.9 (MF) 1.6 (SF)

0.6 (MF) 0.3 (SF)

MF (109:106 cells/mL; ratio 1000:1 and 109:108 cells/mL; ratio 10:1) SF (3 days) SF (3 days)

1.53 and 2.48 (MF) 2.67 (SF)

0

0.6

0

MF (108:106 cells/mL; ratio 100:1)

3

Total sugar consumption

SF (2×108:106 cells/mL; 3 days)

1.6 (S. bombicola) 1.4 (M. pulcherrima)

<2

MF (107:106 cells/mL; 10:1) SF (2 days)

1 (MF) 1.5 (SF)

MF (107:106 cells/mL; 10:1)

0.8

4.1±0.9 g/L (MF) 18.7±1.5 g/L (~1% v/v PAC) (SF) <3

SF (16 days for Chardonnay and 8 days for Shiraz)

0.9 (Chardonnay) 1.6 (Shiraz)

Starmerella bombicola and Metschnikowia pulcherrima Lachancea thermotolerans Metschnikowia pulcherrima

< 1.5

Other Observations

Source

– – – – – –

Ciani and Ferraro (1998)

–

Non-sterilized must of Vitis vinifera Trebbiano Toscano – Initial sugar richness of the must: 190 g/L – C. stellata immobilized in beads – Synthetic grape juice sterilized at 121°C for 20 min – Initial sugar richness of the must: 200 g/L – S. bombicola immobilized in beads – Must of Vitis vinifera Verdicchio – Initial sugar richness of the must: 202 g/L – Non-Saccharomyces immobilized in beads

Ciani and Ferraro (1996) Ferraro et al. (2000)

Milanovic et al. (2012) Canonico et al. (2016)

– –

Must of Vitis vinifera Sangiovese Initial sugar richness of the must: 222 g/L

Gobbi et al. (2013)

– – –

Initial sugar richness of the must: 219 g/L Pasteurized Sauvignon Blanc grape must Musts from Vitis vinifera Chardonnay (filter sterilized) and Shiraz (non-sterilized) Initial sugar richness of the must: 196 g/L (Chardonnay) and 240 g/L (Shiraz) Second inoculation was performed when 50% of the initial sugars were consumed Sterile must of Vitis vinifera Macabeo

Sadoudi et al. (2012) Contreras et al. (2014)

– – Pichia fermentans

SF (3, 4, and 6 days)

0.9–1.6

No residual sugars reported

−

Schizosaccharomyces pombe Torulaspora delbrueckii

SF (2 days)

0.5

<1.5

MF with indigenous yeasts (106 cells/mL; ratio 1:1)

2 (respect to S. cerevisiae) 3 (respect to indigenous yeasts)

<3

− − − −

MF, mixed fermentation; SF, sequential fermentation; non-Sacch, non-Saccharomyces; Sacch, Saccharomyces.

Must of Vitis vinifera pinot grigio Steam-sterilized at 90°C for 15 min Initial sugar richness of the must: 270 g/L C. stellata immobilized in beads Synthetic grape juice sterilized at 121°C for 20 min Initial sugar richness of the must: 217.6 g/L

−

Unpasteurized must from Vitis vinifera Airen Initial sugar richness of the must: 226 g/L Must of Vitis vinifera Pedro Ximenez Unsterilized must (except control in pure fermentation of S. cerevisiae that was sterilized by filtration) Initial sugar richness of the must: 187–193 g/L

ClementeJimenez et al. (2005) Benito et al. (2013) Moreno et al. (1991)

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   479

to control S. cerevisiae in pure fermentation and of about 3 degrees (3% v/v) when compared to a spontaneous fermentation carried out only by the indigenous yeasts. In this case, residual sugars were below 3 g/L (Moreno et al., 1991). By conducting mixed and sequential fermentations with S. bombicola (immobilized in beads) and S. cerevisiae of a must from Vitis vinifera cv pinot grigio with 270 g/L of sugar richness, Ciani and Ferraro (1998) produced dry wines with an average reduction in ethanol content of 0.9% v/v (mixed fermentation) and 1.6% v/v (sequential fermentation), with respect to control S. cerevisiae in pure fermentation. The species Pichia fermentans has also demonstrated its ability to reduce the alcoholic strength between 0.9 and 1.6% v/v when used in sequential fermentation with S. cerevisiae (Clemente-Jimenez et al., 2005). Similarly, Milanovic et al. (2012), in their study on the use of S. bombicola in mixed fermentation with S. cerevisiae, achieved an average reduction in the ethanol content of 3° (3% v/v) with a complete consumption of the sugars in the must. More recently, Contreras et al. (2014), obtained an average reduction in alcoholic strength of 1.6% v/v by using Metschnikowia pulcherrima in sequential fermentation with S. cerevisiae (inoculated on the fourth day) for the production of red wine from a Vitis vinifera cv. Syrah must with 240 g/L of sugar richness (~14% v/v PAC). Immobilized cells of the species S. bombicola, M. pulcherrima, Hanseniaspora osmophila, and H. uvarum have also proved their viability to reduce ethanol content in a range between 1% and 1.6% v/v when performing sequential fermentations together with S. cerevisiae (Canonico et al., 2016). These results corroborate the previous findings of this same research group (Ciani et al., 2014). All the resulting white wines showed good organoleptic quality, except the ones obtained with H. uvarum in which a high content of ethyl acetate (165.68 and 195.79 g/L) was recorded. Worth mentioning that some of the herein reviewed studies were not originally intended to assess a biotechnology for lowering the ethanol content of the wine, it means, the reduction in the ethanol content was not the main target of the experiment. However, in other studies, no significant differences in the ethanol yield were achieved with this type of fermentations (Ciani et  al., 2006; Erten and Tanguler, 2010). Some authors even observed the opposite effect when using non-Saccharomyces yeasts in mixed and/or sequential fermentation with S. cerevisiae, reporting significant increases in the alcoholic strength (0.8–2.5% v/v) (Toro and Vazquez, 2002; Viana et al., 2009).

15.3.4  Aroma Profile Modulation Due to the large amount of interactions at sensory level established between ethanol and other components of wine, it is not possible to generalize the effect of reducing the alcoholic strength on the perception of wine quality. Thus, it has been found that the alcohol content in wine is closely related to perceptions of burning sensation or pungency, astringency, bitterness, fruity aroma, and depth in mouth (Meillon et al., 2009).

480  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Besides allowing a reduction in the ethanol content (>1.5%), certain non-Saccharomyces yeasts have a positive impact on the aromatic profile of the wine finally obtained when performing sequential fermentations (Rossouw and Bauer, 2016). Several authors agree that the sequential culture is much better than the mixed culture when it comes to allow a greater expression of the metabolism of non-­ Saccharomyces yeasts in the early stages of fermentation (Soden et al., 2000; Loira et al., 2015). However, depending on the kind of wine to be produced and the desired organoleptic properties, the selection of either co-inoculation or sequential inoculation with the appropriate non-Saccharomyces yeast and a proper non-Saccharomyces:Saccharomyces ratio of inoculation has to be done (Table 15.3). Significant increases in the concentrations of desirable compounds in red wine making such as ethyl lactate, 2,3-butanediol, 2-phenylethanol, and 2-phenylethyl acetate can be obtained when non-Saccharomyces yeasts participate in the fermentation process (Clemente-Jimenez et al., 2005; Viana et al., 2009; Gobbi et al., 2013). Certain strains of T. delbrueckii in sequential fermentation with S. cerevisiae, can produce significant amounts of 3-ethoxy-1-propanol (Herraiz et al., 1990; Loira et al., 2014, 2015), an aromatic compound with low perception threshold, 0.1 mg/L (Peinado et al., 2004), and a blackcurrant aroma descriptor (Tao and Zhang, 2010). Likewise, slight reductions in the content of higher alcohols are interesting to avoid exceeding the concentration of 350 mg/L above which becomes a defect in nose (Rapp and Mandery, 1986). Ciani and Ferraro (1996) obtained significant reductions in higher ­alcohol content by performing mixed and sequential fermentations with S. cerevisiae and S. bombicola (275.3 and 236.1 mg/L, respectively) compared to the control S. cerevisiae in pure fermentation (375.4 mg/L). Similarly, carrying out mixed fermentations with H. osmophila and S. cerevisiae, Viana et al. (2009), reported a decrease in the higher alcohols from 452.5 mg/L (control) to 306.2 mg/L. In addition, a higher intensity of fruitiness was detected in the sensory evaluation of these wines with respect to control of S. cerevisiae in pure fermentation. Also with the aim of improving the aromatic quality, other selection criterion for non-Saccharomyces yeasts is the high β-glucosidase activity in order to favor the hydrolysis of the nonvolatile aromatic precursors from the grape (Mendes Ferreira et al., 2001). Non-Saccharomyces species usually display greater β-glucosidase activity than Saccharomyces species, which has been defined as intracellular and strain-dependent (Arévalo Villena et al., 2005). K. apiculata and W. anomalus, among others, were found to have a great expression of the β-glucosidase ­enzyme (Charoenchai et al., 1997; Mendes Ferreira, Climaco, & Mendes Faia, 2001). Pérez et al. (2011) confirmed the effectiveness of an esculin glycerol solid medium (EGA) as semiquantitative measurement technique

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   481

Table 15.3  Compounds Associated to the Particular Metabolism of Non-Saccharomyces Yeasts: A Specific Contribution to the Aroma and Flavor of Wine Non-Saccharomyces Yeast Species Candida zemplinina Hanseniaspora guilliermondii

Hanseniaspora osmophila Hanseniaspora uvarum

Hanseniaspora vineae

Lachancea thermotolerans Metschnikowia pulcherrima

Pichia fermentans

Pichia membranefaciens Saccharomycodes ludwigii Schizosaccharomyces pombe Starmerella bombicola

Torulaspora delbrueckii Wickerhamomyces anomalus

Metabolites

Source

Glycerol 2-Phenylethyl acetate Isoamyl acetate Ethyl acetate 2-Phenylethyl acetate Isoamyl acetate Ethyl acetate Acetoin Acetic acid 2-Phenylethyl acetate Benzyl alcohol Benzyl acetate Ethyl lactate Lactic acid Isoamyl acetate Hexanol 2-Phenylethanol Ethyl caprilate 2,3-Butanediol Glycerol Acetaldehyde Ethyl caprilate 2-Phenylethanol Isoamyl acetate Acetoin Ethyl acetate Pyruvic acid Glycerol Succinic acid Acetoin Ethyl acetate 3-Ethoxy-1-propanol Isoamyl acetate Ethyl acetate

Di Maio et al. (2012) Rojas et al. (2001), Moreira et al. (2005), Viana et al. (2008) Viana et al. (2008), Viana et al. (2009) Romano et al. (2003), Ciani et al. (2006), Canonico et al. (2016) Viana et al. (2011), Medina et al. (2013), Lleixà et al. (2016), Martin et al. (2016) Kapsopoulou et al. (2005), Comitini et al. (2011), Gobbi et al. (2013) Clemente-Jimenez et al. (2004), Comitini et al. (2011)

Clemente-Jimenez et al. (2004, 2005)

Viana et al. (2008) Romano et al. (2003) Benito et al. (2014) Ciani and Ferraro (1996), Ferraro et al. (2000), Romano et al. (2003), Canonico et al. (2016)

Herraiz et al. (1990), Loira et al. (2014, 2015) Rojas et al. (2001)

482  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

to quickly identify glucosidase-active strains in a preliminary screening of native yeasts.

15.3.5  Acidity Regulation The use of certain yeasts, such as T. delbrueckii, L. thermotolerans, S. bombicola, and C. zemplinina either in mixed and/or sequential fermentations can reduce volatile acidity in wine making from musts with high sugar richness (Moreno et  al., 1991; Ciani and Ferraro, 1996; Ciani et  al., 2006; Bely et  al., 2008; Vilela-Moura et  al., 2008; Renault et  al., 2009; Rantsiou et  al., 2012; Gobbi et  al., 2013). T. delbrueckii is also characterized by a high fermentative purity and, as a consequence, has a low production of glycerol, acetaldehyde, acetic acid, and ethyl acetate (Ciani and Picciotti, 1995; Renault et al., 2009). Vilela-Moura et al. (2008) reported the use of selected yeast strains to reduce the volatile acidity of acidic wines (1.13–1.44 g/L of acetic acid) by re-fermentation process. Among the strains studied, three S. cerevisiae and one L. thermotolerans showed the highest efficiency in the degradation activity of acetic acid, reaching values similar to those of the control Z. bailii. Because of its particular metabolism of malic acid degradation due to maloalcoholic fermentation, the species S. pombe becomes interesting for biological deacidification of musts and wines, especially in cold regions where the musts usually have high contents of malic acid (Magyar and Panyik, 1989; Ciani, 1995; Benito et al., 2012a, 2013; Suárez-Lepe et  al., 2012). However, some S. pombe strains are characterized by a high acetic acid production, so proper selection of the strain regarding this feature is convenient in order to avoid an excessive level of volatile acidity in wine (Benito et al., 2012a). Similar results in the reduction of malic acid content were achieved by the employment of a mixed culture of Issatchenkia orientalis (another malic acid-degrading yeast) and S. cerevisiae (mean reduction of 0.77 g/L compared to S. cerevisiae pure control) (Kim et al., 2008). Just as Gobbi et  al. (2013) demonstrated in their study, it is also possible to increase the total acidity of the wine by using certain nonSaccharomyces yeasts in mixed and sequential fermentations with S. cerevisiae (mean increase of about 2 g/L). This biological acidification would significantly improve the sensory balance of the wines produced in warm regions. Other authors reported previously this biotechnological application of non-Saccharomyces yeasts, particularly for L. thermotolerans and S. bombicola species (Mora et al., 1990; Ciani and Ferraro, 1996; Kapsopoulou et al., 2007). Ciani and Ferraro (1996) showed increases in the concentration of succinic acid between 1 and 1.3 g/L compared to control S. cerevisiae (0.32 g/L), when sequential and mixed fermentations with S. cerevisiae and S. bombicola

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   483

were carried out. Similarly, Canonico et al. (2016) reported a production of succinic acid more than twice the value obtained in the control (0.52 ± 0.02 g/L) by using S. bombicola in sequential fermentations. Main results for acidity regulation by non-Saccharomyces yeasts are summarized in Table 15.4.

15.3.6  Stable Pigments Formation Non-Saccharomyces yeasts can directly or indirectly contribute to wine color. In the first case by increasing the formation of certain precursors of stable pigments, such as vinyl phenols, acetaldehyde or pyruvic acid, and in the second case by modifying the pH with the organic acids consumption or production, which alters the color parameters of the wine (color intensity and tonality). Thus, mixed or sequential fermentations with non-Saccharomyces may potentiate the synthesis of certain important metabolites for color stability as acetaldehyde and pyruvic acid, both necessary for vitisins synthesis (Ferraro et al., 2000; Clemente-Jimenez et al., 2005; Benito et al., 2012a; Morata et al., 2012; Gobbi et al., 2013). Similarly, the use of non-Saccharomyces yeasts with high hydroxycinnamate decarboxylase (HCDC) activity, such as P. guilliermondii, improves the formation of vinylphenolic pyranoanthocyanins (Benito et  al., 2011). In addition, the ability of certain non-Saccharomyces yeasts, such as S. bombicola, Candida valida, Candida pulcherrima, and K. apiculata (Strauss et al., 2001), to synthesize and release pectolytic enzymes could allow obtaining wines with more color due to the increased extraction of polyphenolic compounds during wine making, while facilitating subsequent clarification and filtration processes.

15.3.7  Contribution to Aging on Lees Process and Sparkling Wines Production: Release of Polysaccharides Some non-Saccharomyces species could be used as selected biomass for the aging on lees process mainly thanks to their high release of polysaccharides during autolysis (Suárez-Lepe and Morata, 2012). Palomero et  al. (2009), observed a polysaccharides release 3 times higher when using S. pombe or Saccharomycodes ludwigii in comparison with the amount obtained from S. cerevisiae after 5 months of aging. The use of this type of yeasts is of particular interest due to the different polysaccharides composition and thickness of their cell walls. More recently, in a study carried out by Kulkarni et al. (2015), some non-Saccharomyces yeasts, namely S. pombe, S. ludwigii, and Dekkera bruxellensis (Teleomorph of B. bruxellensis), showed higher release of polysaccharides than the S. cerevisiae yeast used as control, when

Table 15.4  Acidity Regulation by the Use of Non-Saccharomyces Yeasts in Mixed and/or Sequential Fermentations Volatile Acidity (g Acetic Acid/L)

Total Acidity (g Tartaric Acid/L)

Control

Mixed/ Sequential Fermentation

Mixed/ Sequential Fermentation

285 (indigenous yeasts) 276 (Td+indigenous) 270

9.50 meq/L

3.99 meq/L

Moreno et al. (1991)

0.71±0.01

360

1.00±0.06

0.40±0.05 (MF) 0.32±0.04 (SF) 0.73±0.09

403

0.95–1.54

0.92–1.62 (MF) 0.37–1.04 (SF)

Ciani et al. (2006) Bely et al. (2008) Rantsiou et al. (2012)

217.6

1.45

0.34–0.48 (MF) 0.19 (SF)

Ciani and Ferraro (1996)

270

0.71±0.01

0.51±0.04

MF (5×105 cells/mL; ratio 1:1) SF (1, 2, 3 days)

160

0.40

0.34 (MF) 0.35 (SF—1 day) 0.36 (SF—2 days) 0.38 (SF—3 days)

7.50

Ciani et al. (2006) Kapsopoulou et al. (2007)

MF (107:106 cells/mL; 10:1) SF (2 days) MF (106 cells/mL; ratio 1:1) SF (106 cells/mL; 2 days)

222

0.44±0.05

7.26±0.35

226

0.37±0.01

0.34±0.05 (MF) 0.25±0.02 (SF) 0.37±0.03 (MF) 0.36±0.03 (SF)

NonSaccharomyces Yeast Species

Type of Fermentation (Time of Second Inoculation or NonSacch:Sacch Ratio)

Torulaspora delbrueckii

MF with indigenous yeasts (106 cells/mL; ratio 1:1)

Candida zemplinina

Starmerella bombicola

Lachancea thermotolerans

Schizosaccharomyces pombe

MF (106 cells/mL; ratio 1:1) SF (106 cells/mL; 4 days) MF (5×106:106 cells/mL; ratio 5:1) MF (105–106 cells/mL; ratio 1:1) SF (105–106 cells/mL; 2 days) MF (109:106 cells/mL; ratio 1000:1 and 109:108 cells/mL; ratio 10:1) SF (3 days) SF (106 cells/mL; 4 days)

Sugar Content in the Must (g/L)

MF, mixed fermentation; SF, sequential fermentation; non-Sacch, non-Saccharomyces; Sacch: Saccharomyces.

Control

6.88±0.07

8.10 (MF) 9.44 (SF—1 day) 11.84 (SF—2 days) 12.60 (SF—3 days) 9.20±0.36 (MF) 9.33±0.40 (SF) 3.06±0.17 (MF) 2.95±0.06 (SF)

Source

Gobbi et al. (2013) Benito et al. (2013)

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   485

subjected to ultrasounds treatment before being added to the wine for an aging on lees process. Giovani et al. (2012) studied the polysaccharides released by different non-Saccharomyces yeast species during the fermentation of a synthetic must and its composition. Besides verifying that the amount of polysaccharides released depends on the yeast species and the number of cells formed and their physiological conditions, they reported that most of the non-Saccharomyces tested can release higher concentrations of polysaccharides than S. cerevisiae. Among the species assayed, S. ludwigii, Z. bailii, and B. bruxellensis were the most efficient at releasing polysaccharides from their cell walls, 556, 618, and 576 mg/L, respectively. Similarly, Domizio et  al. (2014) evaluated eight non-Saccharomyces yeast species and revealed that all of them have greater polysaccharide release capability than S. cerevisiae. They also observed a variable composition in the mannoproteins of each non-Saccharomyces yeast species: degree of polymerization ranging from 8 to 15 mannoses, protein size varies between 25 kDa and >250 kDa (abundant in Metschnikowia, Saccharomycodes, and Torulaspora genera), and molecular mass of N-glycans between 1600 and 4000 Da. It is also feasible to use non-Saccharomyces yeasts to carry out the second fermentation in bottle during the winemaking process of sparkling wines in order to take advantage of their higher polysaccharide release rate (Kulkarni et al., 2015). Also, as expected, the polysaccharide content in wine is higher when several yeast species are used during fermentation, since the amount of biomass formed is higher as well and, therefore, also the polysaccharides released (Comitini et al., 2011).

15.3.8  Increasing Glycerol Contents In sequential fermentations with S. pombe and S. cerevisiae, it was possible to increase the concentration of glycerol in 0.5 g/L, with respect to control of S. cerevisiae in pure fermentation (Benito et  al., 2013). Other studies showed similar results in relation to potentiate the synthesis of glycerol, so that a total concentration of up to 15.9 g/L was achieved in the wine when the species S. bombicola was used in sequential fermentation with S. cerevisiae (Ciani and Ferraro, 1996, 1998; Soden et al., 2000; Milanovic et al., 2012). In recent experiments, this same species showed higher production of glycerol in sequential fermentations (7.63 ± 0.04 and 8.43 ± 0.20 g/L) than control S. cerevisiae (5.87 ± 0.19 and 5.68 ± 0.27 g/L) (Canonico et al., 2016). Similarly, by performing sequential and mixed fermentations with S. cerevisiae, the species Candida cantarelli also increased the concentration of glycerol in a 44.3%–58.2% compared to control S. cerevisiae in pure fermentation (Toro and Vazquez, 2002). Through application of different genetic engineering techniques, it is possible to create yeasts with enhanced synthesis of certain

486  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

s­ econdary metabolites during fermentation, such as glycerol or lactic acid (Michnick et al., 1997; de Barros Lopes et al., 2000; Pretorius and Bauer, 2002). de Barros Lopes et  al. (2000) could increase the glycerol concentration until 16.5 g/L by using a yeast overexpressing the gene encoding the enzyme glycerol-3-phosphate dehydrogenase, whereas with the original strain only a mean concentration of 7.9 g/L of this polyol was produced. However, this enhancement in the synthesis of glycerol was accompanied by an increase in acetic acid concentration (1.02 g/L) with respect to control (0.58 g/L) and a reduction of 0.6% v/v in the alcoholic strength. Ehsani et al. (2009) have also engineered a strain of S. cerevisiae with the aim to reduce acetoin synthesis associated with the production of glycerol, and increase at the same time the content of 2,3-butanediol. Furthermore, by using specific selective culture environments, yeast’s adaptive evolution can be directed to obtain strains with increased ability to synthesize glycerol, for example, by repeatedly subjecting to stressful conditions, such as using media with high osmotic concentration (Kutyna et al., 2010).

15.4 Non-Saccharomyces Yeasts Identification Techniques Once the colonies are properly isolated, identification of yeasts can be carried out according to different criteria: morphological, biochemical, immunological, or genetic (Linares and Solís, 2007).

15.4.1  Cultural and Microscopic Characterization Morphology, texture, and color of the colonies formed can be useful parameters to visually differentiate between yeast species grown in the agar plates (see Figs. 15.1 and 15.4). For instance, Medina et al. (2013) reported an intense brilliant green color for the colonies of H. vineae and a matt creamy color for S. cerevisiae colonies, both grown in WL medium. However, in differential media this is only possible when the population of yeasts is similar in order of magnitude. If one of them has 300 times more colonies, the count is not feasible. In addition, when comparing two yeast species with different size of the colonies in a certain growth medium, such as S. pombe and S. cerevisiae, it is possible to independently count the fastest one, because the other yeast will be forming a background of tiny colonies (Fig. 15.4). However, both size and shape of the colonies formed largely depend on the composition of the culture media. The optic microscope observation allows identifying yeasts species by the size and shape of their cells, as well as by the size of their

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   487

corpuscles (e.g., M. pulcherrima has a single lipid globule that fills most of its size), and also by the type of reproduction both sexual (spore formation) and asexual (budding or binary fission) (Fig. 15.5). Table  15.5 summarizes the visual features most commonly used to identify the yeast species in microscope observations.

15.4.2  Biochemical and Physiological Tests Yeasts are mainly characterized by the biochemical and physiological features, especially regarding the pattern of both assimilation and fermentation of sugars, and assimilation of different nitrogen sources (Kurtzman et al., 2011). Their ability to assimilate specific carbon compounds can be analyzed in agar plates (see Fig. 15.1). All yeasts tested except S. bombicola show positive assimilation of d-glucose, d-fructose, sucrose, and maltose, while only M. pulcherrima, C. magnolia, W. anomalus, and Rhodotorula sp. can assimilate d-galactose. None of the assessed yeasts could assimilate lactose as carbon source for growing. Most of these results are in agreement with Kurtzman et al. (2011). Arbutin excision test is a way to evaluate or confirm the βglucosidase activity of the yeast strains. Some species are known for their high enzyme release, as the W. anomalus AS1 strain (Schwentke et al., 2014). These authors reported the use of this kind of yeasts to increase concentrations of sensory and bioactive compounds by splitting glycosylated precursors or to reduce viscosity by hydrolysis of glycan slimes. From the results shown in Fig. 15.6, it can be concluded that, among the species tested, Rhodotorula sp., M. pulcherrima, and S. bombicola have positive β-glucosidase activity. Among the methods for rapid and automated identification based on biochemical and physiological features, galleries API 20C AUX (bioMérieux España, S.A.) may be highlighted, since it is a useful and easy technique to analyze the yeast ability to grow in 19 sources of carbon. Similarly, API ZYM (bioMérieux España, S.A.) semiquantitative test system, even though it is not specifically designed for yeasts, can be used for screening 19 different enzyme activities. Therefore,

Fig. 15.4  Different size and morphology of wine yeast colonies grown on YPD agar. A. Colonies of S. cerevisiae (Sc) and S. pombe (Sp) cultivated at 28°C for 3 days; B. Different colony morphology of wine yeasts isolated from grape must (Cultivated at 28°C for 3 days). Scale = 1 mm.

488  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Fig. 15.5  (A) Asexual reproduction of wine yeasts. (1) S. cerevisiae; (2) S. ludwigii; (3) S. pombe; (4) T delbrueckii; (5) W. anomalus; (6) L. thermotolerans; (7) M. pulcherrima; (8) K. apiculata; (9) C. magnolia; (10) S. bombicola; (11) S. bacillaris; (12) Rhodotorula sp. (B) Sexual reproduction and spores morphologies. (1) S. cerevisiae; (2) S. ludwigii; (3) S. pombe; (4) T delbrueckii; (5) W. anomalus. Scale: 10 μm.

Table 15.5  Specifications of Wine Yeasts: Yeast Cell Size and Shape, Sexual, and Asexual Reproduction and Taxonomic Classification Yeas species

Size (μm)

Shape of the cell

Asexual reproduction

B. bruxellensis

2–7×3.5–28

Ovoid; Ellipsoidal; Ogival; Cylindrical to elongate

Multilateral budding

Anamorphic ascomycetous

C. zemplinina

2–3×3–5

Ellipsoid to elongated

K. apiculata L. thermotolerans

1.5–5×2.5– 11.5 3–6×6–8

Apiculate; Spherical to ovoid; Elongate Spherical to ellipsoidal

Multilateral budding Bipolar budding

Anamorphic ascomycetous Anamorphic ascomycetous Teleomorphic ascomycetous

M. pulcherrima

2.5–6×4–10

S. bombicola

Sexual reproduction

Multilateral budding

Spherical ascospores 1–4 spores per ascus

Globose to ellipsoid

Multilateral budding

1–2×2–4

Ovoid to elongate

S. cerevisiae

3–8×5–10

Globose; Ovoid; Elongate

Multilateral budding Multilateral budding

S. ludwigii

4–7×8–25

S. pombe

3–5×5–24

Lemon-shaped with blunt tips; Sausage-shaped; Curved; Broad-ovoid; Elongated with a swelling in the middle Globose; Ellipsoidal; Cylindrical

Acicular to filiform ascospores 1–2 spores per peduncle Spherical ascospores 1 spore per ascus Globose to short ellipsoidal ascospores 1–4 spores per ascus Spherical and smooth ascopores 4 spores per ascus

T. delbrueckii

2.5–5.6×3–6.6

Spherical to ellipsoidal

W. anomalus

1.9–4.1×2.1– 6.1

Spherical to elongate

Bipolar budding

Fission

Multilateral budding Multilateral budding

Source: Kurtzman, C., Fell, J.W., Boekhout, T. 2011. The Yeasts: A Taxonomic Study, Elsevier.

Globose to ellipsoidal ascospores 2–4 spores per ascus Spherical ascospores 1–4 spores per ascus Hat-shaped ascospores 1–4 spores per ascus

Taxonomic classification

Teleomorphic ascomycetous

Observations Cells occur singly, in pairs, short chains or small clusters. Pseudohyphae are usually abundantly produced Cells occur singly or in pairs Cells occur singly or in pairs Cells occur singly, in pairs or in short clusters. Conjugation can precede ascus formation Asci are sphaeropedunculate. Cells occur singly

Teleomorphic ascomycetous Teleomorphic ascomycetous

Cells occur singly or in pairs

Teleomorphic ascomycetous

Some strains produce only two spores per ascus. Cells occur singly or in pairs and sometimes in groups of three

Teleomorphic ascomycetous

Cells occur singly, in pairs or in small groups

Teleomorphic ascomycetous Teleomorphic ascomycetous

Cells occur singly or occasionally in pairs. Conjugation tubes are often present. Asci are deliquescent. Cells occur singly, in pairs or in small clusters

Cells occur singly or in small clusters

490  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

Fig. 15.6  Arbutin excision test of some non-Saccharomyces wine yeasts using the Arbutin agar medium formula described in Kurtzman et al. (2011).

OF OENOLOGICAL YEASTS

biochemical and physiological characteristics of yeast strains belonging to the same species can be enzymatically characterized using API-ZYM tests (Nikolaou et  al., 2006; Elmacı et  al., 2014). Enzyme activities, such as alkaline phosphatase, esterase, esterase lipase, α-glucosidase, and β-glucosidase can be detected with these tests (Tofalo et al., 2011) based on the comparison of the developed color with the API-ZYM color reaction chart. Fig. 15.7 illustrates the results of using the API-ZYM test with some Saccharomyces and nonSaccharomyces yeast species. From the color intensity of the wells, it can be concluded that M. pulcherrima has the highest β-glucosidase activity followed closely by K. apiculata and S. ludwigii and lastly C. magnolia and W. anomalus. Regarding lithium tolerance, another useful parameter to characterize the yeast strain, three yeasts, namely T. delbrueckii, C. magnolia, and S. bacillaris, showed moderate resistance to this metal when tested at 20 mM (see Fig. 15.1). Rhodotorula sp. was the only one that showed a high tolerance to lithium, even at the concentration of 100 mM. This physiological feature, suitable to differentiate yeasts, based on lithium transport into the cell was previously reported by Asensio et al. (1976) and Garciadeblas et al. (1993).

15.4.3  Molecular Techniques Identification can be performed both at strain and species level using molecular methods. In general, molecular techniques are based on comparing genomes by the study of specific DNA or RNA fragments extracted from the yeast subjected to identification. Molecular identification techniques rely mostly on rRNA gene sequences because of their taxonomic significance (Loureiro and Malfeito-Ferreira, 2003). The ITS regions and the 5.8S rRNA gene exhibit far greater interspecific differences than the 18S and 26S rRNA genes (Esteve-Zarzoso et al., 1999), so it becomes a better tool to discriminate between non-Saccharomyces yeasts. The main advantages of molecular techniques compared to traditional identification methods, that is, plating or physiological tests, are the improvements in speed and precision (Ivey and Phister, 2011). Moreover, molecular analyses are quick, reliable, accurate, and reproducible. Fig. 15.8 illustrates the polymorphic regions in the genetic sequences (ITS1–5.8S rDNA-ITS2) of different wine yeast species. Direct and indirect methodologies can be carried out to obtain the genetic material either from the original sample where the yeasts are found

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   491

Fig. 15.7  Results of the enzyme assay (API Zym profiles) when comparing different wine yeast.

492  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS

Fig. 15.8  (A) Schematic diagram of the fungal rDNA gene cluster. The 18S, 5.8S, and 25–28S rDNA genes are separated by the internal transcribed spacers 1 (ITS1) and 2 (ITS2). Primers for routine sequencing are shown. (B) Multiple alignments of complete 5.8S rDNA sequence and partial sequences of Internal Transcribed Spacer 1 and 2. The shaded sequences are identity regions. GenBank accession numbers: for S. cerevisiae, KT958553.1; for M. pulcherrima, AY301026.1; for L. thermotolerans, CU928180.1; for S. ludwigii, FM199957.1; for T. delbrueckii, HE616749.1; for W. anomalus, FJ797686.1; for K. apiculata, KT029777.1; for S. pombe, EU916982.1; for S. bombicola, KT002361.1; for S. bacillaris, AY372189.1; for Rhodotorula sp., LT548266.1. The ITS sequences were analyzed and aligned using BLAST and CLUSTAL O software.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   493

(in this case grape, must, or wine), or from pure cultures of the yeasts (Urso et al., 2008; Ivey and Phister, 2011). Table 15.6 compares both methods and provides examples of the techniques most commonly used in wine yeast identification. One of the main disadvantages of direct analysis is the inability to differentiate between viable and dead cells, since both may have stable and well-preserved genetic material (DNA or RNA) (Hierro et al., 2006; Ivey and Phister, 2011). In order to speed up and simplify the DNA extraction method for molecular techniques application, several authors developed new rapid and low-cost yeast DNA extraction protocols (Harju et al., 2004; Looke et al., 2011; Blount et al., 2016). Frequently, yeasts are identified using the methodology developed by Esteve-Zarzoso et al. (1999) based on the restriction fragment length polymorphism technique (RFLP) coupled to PCR or gel electrophoresis of ITS-5.8S rDNA region extracted from the isolated colonies (Viana et  al., 2011). Another alternative method commonly used for yeast ­identification is the sequencing of the amplified D1/D2 domain of the 26S rDNA as described by Kurtzman and Robnett (1998). Several authors

Table 15.6  Features and Types of Direct and Indirect Molecular Techniques Direct methods – Detect targeted microorganisms in a mixed population or identify the microbial diversity present in a sample – Genetic material isolated directly from the sample – Faster – Identification at genus and, sometimes, species level – Detect non-culturable microorganisms – Unable to differentiate between viable and dead cells – - Examples of direct molecular techniques: real-time PCR, DGGE, FISH, and Sequencing

Indirect methods – – – – –

Identification of previously cultured microorganisms Genetic material isolated from the pure culture of the microorganism More sensitive Identification at strain level Examples of direct molecular techniques: RFLP and Sequencing

Real-time PCR, real-time polymerase chain reaction; DGGE, denaturing gradient gel electrophoresis; FISH, fluorescence in situ hybridization; RFLP, restriction fragment length polymorphism. Sources: Cocolin, L., Heisey, A., Mills, D.A. 2001. Direct identification of the indigenous yeasts in commercial wine fermentations. Am. J. Enol. Vitic. 52(1), 49–53; Xufre, A., Albergaria, H., Inácio, J., Spencer-Martins, I., Gírio, F. 2006. Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations. Int. J. Food Microbiol. 108(3), 376–384; Ivey, M.L., Phister, T.G. 2011. Detection and identification of microorganisms in wine: a review of molecular techniques. J. Ind. Microbiol. Biotechnol. 38(10), 1619–1634; Kurtzman, C.P. 2015. Identification of food and beverage spoilage yeasts from DNA sequence analyses. Int. J. Food Microbiol. 213, 71–78.

494  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

have confirmed the effectiveness of these methodologies for the identification of non-Saccharomyces yeasts (Ortiz-Barrera et al., 2015; Lleixà et al., 2016). RFLPs of mtDNA allow identifying strains of a same species. Thereby, by using this identification technique, Viana et al. (2011) demonstrated the presence of different native strains of S. cerevisiae in a non-inoculated must. Barquet et  al. (2012) developed a simple and reproducible new tandem repeat PCR methodology based on the combined use of primer sequences for tRNA genes to discriminate strains of non-Saccharomyces yeasts. They could successfully discriminate strains within M. pulcherrima, H. vineae, H. uvarum, Hanseniaspora opuntiae, and C. zemplinina species. Fig. 15.9 shows the results of a gel electrophoresis after PCR amplification of the genetic material obtained from some wine yeast species, also performing a restriction analysis. Recently, Wang et  al. (2015) proposed the use of two culture-­ independent techniques, namely EMA-PCR-DGGE (Ethidium monoazide bromide-Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) and RT-PCR-DGGE (Reverse TranscriptionPolymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis), to follow the viable population of non-Saccharomyces yeasts, particularly H. uvarum and S. bacillaris (synonym Candida zemplinina), along the fermentation process. However, these techniques are not useful to detect viable cells of non-Saccharomyces yeasts when S. cerevisiae predominates in one or two log units or when there are dead cells with relatively stable rRNA that may affect the results.

15.4.3.1  Real-Time PCR or Quantitative PCR (qPCR) Hierro et al. (2006) were the first to develop a RT-qPCR methodology for detecting and quantifying total viable yeast population from rRNA extracted from a sample of wine. They have designed universal yeast primers from the variable D1/D2 domains of the 26S rRNA gene and found them to have good specificity with all the wine yeasts tested, S. bombicola, D. bruxellensis, H. uvarum, S. cerevisiae, S. ludwigii, S. pombe and T. delbrueckii, among others. In addition, no amplification of acetic acid and lactic acid bacteria was observed. The detection limit was set at 103 CFU/mL. Real-time PCR was also previously reported for D. bruxellensis quantification in wine samples (Phister and Mills, 2003). They obtained a good linearity over six orders of magnitude and a low detection limit in wine, approximately 1 CFU per ml. Similarly, Delaherche et  al. (2004) reported a detection limit in wine of 104 CFU/mL for B. bruxellensis (anamorph of D. bruxellensis) quantification. Due to its significance in wine quality, strains of this spoilage yeast isolated from red wines were also identified and genetically characterized by many other molecular techniques such as ISS-PCR (Intron Splice Site-PCR), PCR-DGGE, and REA-PFGE (Restriction Enzyme Analysis-Pulsed Field Gel Electrophoresis) (Oelofse et al., 2009).

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   495

bp 3000

M

M

bp 3000

2000

2000

1500

1500

1000

1000

900

900

800

800

700

700

600

600

500

500

400

400

300

300

(A) 3000

3000

1000

1000

700

700

500

500

400

400

300

300

200

200

100

100

(B) Fig. 15.9  (A) Agarose gel electrophoresis of PCR product obtained from the amplification of the 5.8S-ITS region using the universal primers ITS1-ITS4 from 11 wine yeast species; (B) Agarose gel showing HinfI restriction endonuclease analysis of the PCR product; M, molecular weight marker (100-bp DNA ladder).

Later, qPCR was also tested for the rapid detection and enumeration of Z. bailii from fruit juices (apple, grape, and cranberry-raspberry) and wine even in the presence of nontarget DNA (Rawsthorne and Phister, 2006, 2009). They have also reported the effective use of ethidium monoazide to eliminate the nonviable population of Z. bailii during qPCR performance. In 2010, Zott et al. (2010) designed specific oligonucleotide primers for real-time quantitative PCR, in order to monitor the wine yeast ecosystem before, along, and after the fermentation. They assessed the population dynamics of the following non-Saccharomyces yeasts: I. orientalis, M. pulcherrima, T. delbrueckii, C. zemplinina, and Hanseniaspora spp.

496  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

15.4.3.2  Fish PNA Peptide nucleic acid (PNA) probes have been also successfully tested in oenology for wine-related yeasts detection and quantification through fluorescence in situ hybridization (FISH) (Stender et al., 2001; Barata et al., 2013). Other authors have also implemented this FISH technology, but targeted to the D1/D2 region of the 26S rRNA, to design fluorescent oligonucleotide probes specific for the identification of non-Saccharomyces yeast species involved in the vinification process and record the population dynamics throughout fermentation (Xufre et al., 2006). Thus, among the yeast species of the wine environment, they could identify S. bombicola, H. uvarum, H. guilliermondii, Kluyveromyces marxianus, L. thermotolerans, and T. delbrueckii.

15.4.3.3  DNA Sequencing and Massive Sequencing Analysis DNA sequencing is the best technique currently available for identifying the wine yeasts. It usually requires a pretreatment of the sample with PCR amplification of DNA or RNA. Nowadays, thanks to the development of bioinformatics and automated sequencing equipment, it is possible to make fast, accurate, and reliable analysis of large DNA sequences in a short time, on the order of millions of base pairs per week (Giraffa and Carminati, 2007). Moreover, nowadays, DNA sequencing is becoming an economically affordable technique to identify previously isolated wine yeasts. Massive sequencing analysis is a high-throughput sequencing method with a very fast development during the first decade of the XXI century. Its success is based on the possibility of performing multiple sequencing reactions simultaneously, which greatly speeds up the identification process, obtaining a lot of information in a short period of time. Extracted DNA from mixed samples can be analyzed by massive sequencing, also called next-generation sequencing, and individually identified by using primers for Saccharomyces and non-Saccharomyces yeast species (Lleixà et  al., 2016). Other authors have also applied effectively this identification technique to monitor the yeast and bacteria populations along spontaneous fermentation (Portillo and Mas, 2016) and to follow the evolution of the microbial communities inhabiting the equipment and surfaces in a pilot-scale winery (Bokulich et al., 2013). Table  15.7. summarizes some of the most recent applications of molecular methods for non-Saccharomyces yeasts identification and typing in winemaking industry. In view of this table, it can be concluded that in the last decade, besides DNA sequencing and Real-time PCR, there is a predominance of PCR-RFLP techniques and identification methods based on mtDNA restriction patterns. New possibilities as the use of tandem repeat tRNA PCR method and the application of a denaturing gradient gel electrophoretic separation (DGGE) for non-Saccharomyces screening have also arisen.

Table 15.7  Summary of the Molecular Methods Used in Oenology to Determine the Diversity of Yeasts in Wine Samples or to Monitor Their Evolution Along Fermentation Technique

Type of Genetic Material Used

rtReal-time PCR

D1/D2 domains of the 26S rRNA gene

YEASTF (5′-GAGTCGAGTTGTTTGGGAATGC-3′) YEASTR (5′-TCTCTTTCCAAAGTTCTTTTCATCTT T-3′)

Quantify total viable yeast population in wine samples

Real-time PCR

D1/D2 loop of the 26S ribosomal RNA

ZBF1 (5′-CATGGTGTTTTGCGCC-3′) ZBR1 (5′-CGTCCGCCACGAAGTGGTAGA-3′)

PCR/ rtPCR-DGGE

DNA and 26S rRNA gene

NL1 (5′-GCCATATCAATAAGCGGAGGAAAAG-3′) GC clamp (5′-GCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3′) LS2 (5′-ATTCCCAAACAACTCGACTC-3′)

Detection and quantification of specific yeasts in must and wine Assess the yeast biodiversity and dynamics before and during fermentation

RAPD-PCR PCR fingerprinting REA-PFGE PCR-RFLP

Genomic DNA

M13, M14, Coc, OPA02, and OPA09 Microsatellite primers (GTG)5 and (GAC)5

Sfi I, Apa I and Not I

Genetic characterization of 21 Torulaspora delbrueckii strains

5.8S-ITS region of the rDNA

ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)

HinfI, HaeIII and CfoI

T. delbrueckii, K. apiculata, P. anomala, P.membranifaciens, M. pulcherrima, Z. bailii, and S. cerevisiae among others

Ocón et al. (2010)

PCRmtDNARFLP

ITS1-5.8S-ITS2 rDNA region

Evaluate the presence of different yeasts in the facilities of the wineries (surfaces, equipment and musts) Yeast identification and calculation of percentages and distribution along fermentation

M. pulcherrima, H. guilliermondii, H. osmophila, H. uvarum, H. vineae, S. cerevisiae, T. delbrueckii, and Zygosaccharomyces cidri

Viana et al. (2011)

TrtRNA-PCR

D1/D2 region from 26S rDNA

M. pulcherrima, H. vineae, H. uvarum, Hanseniaspora opuntiae, and C. zemplinina

Barquet et al. (2012)

PCR-RFLP

5.8S-ITS region

Discriminate strains of non-Saccharomyces yeasts at the subspecies level by the combined use of different primers Identification of yeast species able to grow on DBDM medium and not hybridising with the PNA probe

P. guillermondii, Candida parapsilopsis, Rhodotorula mucilaginosa, and Torulaspora globosa

Barata et al. (2013)

Primers

Restriction Enzymes

CfoI, HaeIII, HinfI and DdeI (nonSaccharomyces identification) HinfI (S. cerevisiae identification) TtRNASc (5′-GCTTCTATGGCCAAGTTG-3′) ISSR-MB (5′-CTCACAACAACAACAACA-3′) 5CAG (5′-CAGCAGCAGCAGCAG-3′)

CfoI, HaeIII and HinfI

Application

Yeast Population Identified or Counted (Target Species) S. bombicola, D. bruxellensis, H. uvarum, S. cerevisiae, S. ludwigii, S. pombe and T. delbrueckii, among others Z. bailii

S. cerevisiae, C. zemplinina, H. uvarum, Metschnikowia sp., Debaryomyces hansenii, P. anomala, P. guilliermondii, T. delbrueckii, Z. bailii, and others T. delbrueckii

Source Hierro et al. (2006)

Rawsthorne and Phister (2006) Urso et al. (2008)

Renault et al. (2009)

Continued

Table 15.7  Summary of the Molecular Methods Used in Oenology to Determine the Diversity of Yeasts in Wine Samples or to Monitor Their Evolution Along Fermentation—cont’d Technique PCRSequencing mtDNARFLP

PCR-RFLP Sequencing

Type of Genetic Material Used D1/D2 variable domains of the large subunit of the rRNA gene ITS1-5.8S-ITS2 region of the large-subunit rRNA gene 5.8S rRNA genes D1/D2 domain of the 26S and/or 5.8S rRNA genes

Primers

Application

NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) NL4 (5′-GGTCCGTGTTTCAAGACGG-3′)

HinfI (S. cerevisiae strain identification)

Identification of indigenous Saccharomyces and non-Saccharomyces yeasts from grapes

ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) LR6 (5′-CGCCAGTTCTGCTTACC-3′), NL3A (5′-GAGACCGATAGCGAACAAG-3′) and NL2A (5′-CTTGT TCGCTATCGGTCTC-3′) ITS1 (5′-TCCGTAGGTGAACCTCGCG-3′) ITS4 (5′-TCCTCCGCTTTATTGATATGC-3′)

HinfI, HaeIII and CfoI

Identification of nonSaccharomyces isolated from distillery products and by-products at the species level

HaeIII, HinfI and CfoI

Identification of indigenous yeast species isolated from grape musts by comparison with restriction patterns of sequenced species

PCR-rARNRFLP

ITS1-5.8S rRNA-ITS2 region

PCRSequencing

D1/D2 domain of the gene encoding the subunit 26S rRNA D2 expansion segment region of the nuclear large subunit ribosomal RNA (28S rRNA) gene

NL1 (5′-GCATATCAATAAGCGGAGGAAAAC-3′) NL4 (5′-GGTCCGTGTTTCAAGACGG-3′)

PCR-DGGE

U1/U2 domain of the 28S ribosomal region

Fungi-specific primers U1GC/U2

Massive sequencing analysis

DNA (5–100 ng) Amplification of a 350 bp (on average) 18S rRNA gene fragment

Universal primers FR1 (5′-ANCCATTCAATCGGTANT-3′) FF390 (5′-CGATAACGAACGAGACCT-3′)

PCRSequencing

Restriction Enzymes

Species identification of non-Saccharomyces yeasts Identification of nonSaccharomyces from the grapevine at species level

Detect the predominant yeast species using denaturing electrophoresis Monitoring of the yeast population dynamics

Yeast Population Identified or Counted (Target Species)

Source

S. cerevisiae, Rhodotorula mucilaginosa, Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, W. anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis, and C. zemplinina Pichia galeiformis, Candida lactiscondensi, H. osmophila, T. delbrueckii, L. thermotolerans, and S. ludwigii, among others

de PonzzesGomes et al. (2014)

S. cerevisiae, Aureobasidium pullulans, H. uvarum, T. delbrueckii, W. anomalus, Rhodotorula spp., Rhodosporidium diobovatum, Candida azyma, and L. thermotolerans S. cerevisiae, H.guilliermondii, and H. uvarum

Bagheri et al. (2015)

Aureobasidium pullulans, Cryptococcus magnus, H. uvarum, Candida zeylanoides, Candida sake, Rhodotorula mucilaginosa, and Pseudozyma aphidis H. uvarum, C. zemplinina, H. vineae, and S. cerevisiae

Pantelides et al. (2015)

H. vineae and S. cerevisiae

Lleixà et al. (2016)

Ubeda et al. (2014)

Ortiz-Barrera et al. (2015)

Lleixà et al. (2016)

PCR, polymerase chain reaction; rtReal-time PCR, reverse transcriptase quantitative PCR; rtPCR, reverse transcriptase PCR; DGGE, denaturing gradient gel electrophoresis; ITS, internal transcribed spacer; mtDNA, mitochondrial DNA; RFLP, restriction fragment length polymorphism; RAPD, random amplification of polymorphic DNA; REA-PFGE, restriction endonuclease analysis pulse-field gel electrophoresis.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   499

15.5  Conclusions and Future Prospects Currently, there are a lot of tools available for the isolation, characterization, identification, and selection of wine yeasts. However, some of them have not been tested yet, particularly for the isolation and identification of non-Saccharomyces wine yeasts. One of the main concerns of the current wine industry is the use of native yeasts that guarantees a wine elaboration respectful of the environment and its biodiversity preservation. That is why it is so important to properly isolate and select both Saccharomyces and non-Saccharomyces yeasts from the vineyard and the winery, in order to obtain good quality wines with higher specificity. Recently, molecular methods, more precise and faster than traditional physiological techniques, especially regarding genetic identification of yeast, are beginning to be used to monitor and analyze the microbial diversity of grapes and wines. For example, the design of specific primers to monitor a particular species through qPCR technique and the implementation of massive sequencing to identify the microbial population present in a sample would be interesting to further develop for the identification and quantification of non-Saccharomyces wine yeasts. These two techniques are increasingly affordable and are available to everyone. For winemakers, it is interesting to have a simple and inexpensive method to rapidly perform the non-Saccharomyces yeasts population count and thus can follow more closely the evolution of the microbial community in wine during fermentation and aging, regardless of the type of fermentation conducted (spontaneous, mixed or sequential). Finally, highlight the need to further study the implementation of the various molecular techniques available to detect and quantify non-Saccharomyces yeast at strain and species level.

Acknowledgments This work was funded by the Ministerio de Economía y Competitividad (AGL201340503-R and AGI2013-47706-R).

References Arévalo Villena, M., Úbeda Iranzo, J., Cordero Otero, R., Briones Pérez, A., 2005. Optimization of a rapid method for studying the cellular location of β-glucosidase activity in wine yeasts. J. Appl. Microbiol. 99 (3), 558–564. Asensio, J., Ruiz-Argüeso, T., Rodriguez-Navarro, A., 1976. Sensitivity of yeasts to lithium. Antonie Van Leeuwenhoek 42 (1–2), 1–8. Bagheri, B., Bauer, F., Setati, M., 2015. The diversity and dynamics of indigenous yeast communities in grape must from vineyards employing different agronomic practices and their influence on wine fermentation. S. Afr. J. Enol. Vitic. 36 (2), 243–251. Bañuelos, M.A., Loira, I., Escott, C., Del Fresno, J.M., Morata, A., Sanz, P.D., Otero, L., Suárez-Lepe, J.A., 2016. Grape processing by high hydrostatic pressure: effect on use of non-Saccharomyces. Food Bioprocess Technol. 9, 1769–1778.

500  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Barata, A., Laureano, P., D’Antuono, I., Martorell, P., Stender, H., Malfeito-Ferreira, M., Querol, A., Loureiro, V., 2013. Enumeration and identification of 4-ethylphenol producing yeasts recovered from the wood of wine ageing barriques after different sanitation treatments. J. Food Res. 2 (1), 140. Barquet, M., Martín, V., Medina, K., Pérez, G., Carrau, F., Gaggero, C., 2012. Tandem ­repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies. Appl. Microbiol. Biotechnol. 93 (2), 807–814. Belda, I., Ruiz, J., Alastruey-Izquierdo, A., Navascués, E., Marquina, D., Santos, A., 2016. Unraveling the enzymatic basis of wine “flavorome”: A phylo-functional study of wine related yeast species. Front. Microbiol. 7, 12. 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 (3), 312–320. Benito, S., Morata, A., Palomero, F., Gonzalez, M., Suárez-Lepe, J., 2011. Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies. Food Chem. 124 (1), 15–23. Benito, S., Palomero, F., Morata, A., Calderón, F., Suárez-Lepe, J.A., 2012a. New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines. Int. J. Food Sci. Technol. 47 (10), 2101–2108. Benito, S., Palomero, F., Morata, A., Palmero, D., Suarez-Lepe, J.A., 2012b. Identifying yeasts belonging to the Brettanomyces/Dekkera genera through the use of ­selective-differential media. Afr. J. Microbiol. Res. 6 (34), 6348–6357. Benito, S., Palomero, F., Morata, A., Calderon, F., Palmero, D., Suarez-Lepe, J., 2013. Physiological features of Schizosaccharomyces pombe of interest in making of white wines. Eur. Food Res. Technol. 236 (1), 29–36. Benito, S., Palomero, F., Calderón, F., Palmero, D., Suárez-Lépe, J., 2014. Selection of appropriate Schizosaccharomyces strains for winemaking. Food Microbiol. 42, 218–224. Bisson, L.F., 1999. Stuck and sluggish fermentations. Am. J. Enol. Vitic. 50 (1), 107–119. Blount, B.A., Driessen, M.R., Ellis, T., 2016. GC preps: fast and easy extraction of stable yeast genomic DNA. Sci. Rep. 6, 26863. Bokulich, N.A., Ohta, M., Richardson, P.M., Mills, D.A., 2013. Monitoring seasonal changes in winery-resident microbiota. PLoS One 8 (6), e66437. Canonico, L., Comitini, F., Oro, L., Ciani, M., 2016. Sequential fermentation with selected immobilized non-Saccharomyces yeast for reduction of ethanol content in wine. Front. Microbiol. 7, 1–11. Charoenchai, C., Fleet, G., Henschke, P., Todd, B., 1997. Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Aust. J. Grape Wine Res. 3 (1), 2–8. Chi, Z., Arneborg, N., 2000. Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol. J. Ind. Microbiol. Biotechnol. 24 (1), 75–78. CHROMagar, 2016. Chromagar Candida, Instructions for Use. Available: http://www.chromagar.com/fichiers/1392397721NT_EXT_001_V7_CA.pdf (Accessed 27 June 2016). 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 (6), 631–634. Ciani, M., 1997. Role, enological properties and potential use of non-Saccharomyces wine yeasts. Recent Res. Dev. Microbiol. 1, 317–331. Ciani, M., Ferraro, L., 1996. Enhanced glycerol content in wines made with immobilized Candida stellata cells. Appl. Environ. Microbiol. 62 (1), 128–132. 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 (2), 247–254.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   501

Ciani, M., Picciotti, G., 1995. The growth kinetics and fermentation behaviour of some non-Saccharomyces yeasts associated with wine-making. Biotechnol. Lett. 17 (11), 1247–1250. Ciani, M., Beco, L., Comitini, F., 2006. Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations. Int. J. Food Microbiol. 108 (2), 239–245. Ciani, M., Canonico, L., Oro, L., Comitini, F., 2014. Sequential fermentation using non-Saccharomyces yeasts for the reduction of alcohol content in wine. In: BIO Web of Conferences EDP Sciences. pp. 02015. Clemente-Jimenez, J.M., Mingorance-Cazorla, L., Martı́nez-Rodrı́guez, S., Las HerasVázquez, F.J., Rodrı́guez-Vico, F., 2004. Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must. Food Microbiol. 21 (2), 149–155. Clemente-Jimenez, J., Mingorance-Cazorla, L., Martínez-Rodríguez, S., Las HerasVázquez, F., Rodríguez-Vico, F., 2005. Influence of sequential yeast mixtures on wine fermentation. Int. J. Food Microbiol. 98 (3), 301–308. Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., Ciani, M., 2011. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol. 28 (5), 873–882. 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 (5), 1670–1678. Cordero-Bueso, G., Arroyo, T., Serrano, A., Valero, E., 2011. Remanence and survival of commercial yeast in different ecological niches of the vineyard. FEMS Microbiol. Ecol. 77 (2), 429–437. de Barros Lopes, M., Rehman, A., Gockowiak, H., Heinrich, A., Langridge, P., Henschke, P., 2000. Fermentation properties of a wine yeast over expressing the Saccharomyces cerevisiae glycerol 3 phosphate dehydrogenase gene (GPD2). Aust. J. Grape Wine Res. 6, 208–215. de Barros Lopes, M., Eglinton, J., Henschke, P., Høj, P., Pretorius, I., 2003. The connection between yeast and alcohol reduction in wine: managing the double-edged sword of bottled sunshine. Aust. N. Z. Wine Ind. J. 18 (4), 17–22. De la Torre-González, F.J., Narváez-Zapata, J.A., López-y-López, V.E., Larralde-Corona, C.P., 2016. Ethanol tolerance is decreased by fructose in Saccharomyces and non-Saccharomyces yeasts. LWT- Food Sci. Technol. 67, 1–7. de Ponzzes-Gomes, C.M., de Mélo, D.L., Santana, C.A., Pereira, G.E., Mendonça, M.O., Gomes, F.C., Oliveira, E.S., Barbosa Jr., A.M., Trindade, R.C., Rosa, C.A., 2014. Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco valley. Braz. J. Microbiol. 45 (2), 411–416. de Siloniz, M., Valderrama, M., Payo, E., Peinado, J., 1999. Advances in the development of a methodology to identify common yeast contaminants of high sugar food products. Food Technol. Biotechnol. 37 (4), 277–280. Delaherche, A., Claisse, O., Lonvaud-Funel, A., 2004. Detection and quantification of Brettanomyces bruxellensis and ‘ropy’ Pediococcus damnosus strains in wine by realtime polymerase chain reaction. J. Appl. Microbiol. 97 (5), 910–915. Di Maio, S., Polizzotto, G., Planeta, D., Oliva, D., 2011. A method to discriminate between the Candida stellata and Saccharomyces cerevisiae in mixed fermentation on WLD and lysine agar media. S. Afr. J. Enol. Vitic. 32 (1), 35–41. Di Maio, S., Genna, G., Gandolfo, V., Amore, G., Ciaccio, M., Oliva, D., 2012. Presence of Candida zemplinina in sicilian musts and selection of a strain for wine mixed fermentations. S. Afr. J. Enol. Vitic. 33 (1), 80–87. Domizio, P., Liu, Y., Bisson, L.F., Barile, D., 2014. Use of non-Saccharomyces wine yeasts as novel sources of mannoproteins in wine. Food Microbiol. 43, 5–15. Eglinton, J., Henschke, P., Høj, P., Pretorius, I., 2003. Winemaking properties and potential of Saccharomyces bayanus wine yeast—harnessing the untapped potential of yeast biodiversity. Aust. N. Z. Wine Ind. J. 18 (6), 16–19.

502  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Ehsani, M., Cadière, A., Heux, S., Julien, A., Dequin, S., 2007. Metabolic effect of low ethanol production yeast. Bull. l’OIV 80 (917), 403. Ehsani, M., Fernandez, M.R., Biosca, J.A., Julien, A., Dequin, S., 2009. Engineering of 2,3-butanediol dehydrogenase to reduce acetoin formation by glycerol-­ overproducing, low-alcohol Saccharomyces cerevisiae. Appl. Environ. Microbiol. 75 (10), 3196–3205. Elmacı, S.B., Özçelik, F., Tokatlı, M., Çakır, İ., 2014. Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey. Antonie Van Leeuwenhoek 105 (5), 835–847. Erten, H., Campbell, I., 2001. The production of low-alcohol wines by aerobic yeasts. J. Inst. Brew. 59 (3), 207–215. Erten, H., Tanguler, H., 2010. Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation. Lett. Appl. Microbiol. 50 (5), 474–479. Esteve-Zarzoso, B., Belloch, C., Uruburu, F., Querol, A., 1999. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int. J. Syst. Evol. Microbiol. 49 (1), 329–337. Ferraro, L., Fatichenti, F., Ciani, M., 2000. Pilot scale vinification process using immobilized Candida stellata cells and Saccharomyces cerevisiae. Process Biochem. 35 (10), 1125–1129. Field, Y., Fondufe-Mittendorf, Y., Moore, I.K., Mieczkowski, P., Kaplan, N., Lubling, Y., Lieb, J.D., Widom, J., Segal, E., 2009. Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization. Nat. Genet. 41 (4), 438–445. Fleet, G.H., 2003. Yeast interactions and wine flavour. Int. J. Food Microbiol. 86 (1), 11–22. Gao, C., Fleet, G., 1988. The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata. J. Appl. Bacteriol. 65 (5), 405–409. Garciadeblas, B., Rubio, F., Quintero, F.J., Bañuelos, M.A., Haro, R., Rodríguez-Navarro, A., 1993. Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae. Mol. Gen. Genet. MGG 236 (2–3), 363–368. Ghelardi, E., Pichierri, G., Castagna, B., Barnini, S., Tavanti, A., Campa, M., 2008. Efficacy of chromogenic candida agar for isolation and presumptive identification of pathogenic yeast species. Clin. Microbiol. Infect. 14 (2), 141–147. Giovani, G., Rosi, I., Bertuccioli, M., 2012. Quantification and characterization of cell wall polysaccharides released by non-Saccharomyces yeast strains during alcoholic fermentation. Int. J. Food Microbiol. 160 (2), 113–118. Giraffa, G., Carminati, D., 2007. Molecular techniques in food fermentation: principles and applications. In: Cocolin, L., Ercolini, D. (Eds.), Molecular Techniques in the Microbial Ecology of Fermented Foods. Springer Science & Business Media, pp. 1–29. Giusiano, G., Mangiaterra, M., 1998. Diferenciación e identificación presuntiva rápida de levaduras con el medio CHROM-agar candida. Rev. Argent. Microbiol. 30 (2), 100–103. Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., Ciani, M., 2013. Lachancea 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 (2), 271–281. Harju, S., Fedosyuk, H., Peterson, K.R., 2004. Rapid isolation of yeast genomic DNA: Bust n’grab. BMC Biotechnol. 4 (1), 1. Herraiz, T., Reglero, G., Herraiz, M., Martin-Alvarez, P.J., Cabezudo, M.D., 1990. The influence of the yeast and type of culture on the volatile composition of wines fermented without sulfur dioxide. Am. J. Enol. Vitic. 41 (4), 313–318.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   503

Heux, S., Sablayrolles, J.M., Cachon, R., Dequin, S., 2006. Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions. Appl. Environ. Microbiol. 72 (9), 5822–5828. Hierro, N., Esteve-Zarzoso, B., Gonzalez, A., Mas, A., Guillamon, J.M., 2006. Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine. Appl. Environ. Microbiol. 72 (11), 7148–7155. Holm Hansen, E., Nissen, P., Sommer, P., Nielsen, J., Arneborg, N., 2001. The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae. J. Appl. Microbiol. 91 (3), 541–547. Ivey, M.L., Phister, T.G., 2011. Detection and identification of microorganisms in wine: a review of molecular techniques. J. Ind. Microbiol. Biotechnol. 38 (10), 1619–1634. Kalathenos, P., Sutherland, J.P., Roberts, T.A., 1995. Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine. J. Appl. Bacteriol. 78 (3), 245–250. Kapsopoulou, K., Kapaklis, A., Spyropoulos, H., 2005. Growth and fermentation characteristics of a strain of the wine yeast Kluyveromyces thermotolerans isolated in Greece. World J. Microbiol. Biotechnol. 21 (8–9), 1599–1602. 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 (5), 735–739. Kim, D., Hong, Y., Park, H., 2008. Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine. Biotechnol. Lett. 30 (9), 1633–1638. Kulkarni, P., Loira, I., Morata, A., Tesfaye, W., González, M.C., Suárez-Lepe, J.A., 2015. Use of non-Saccharomyces yeast strains coupled with ultrasound treatment as a novel technique to accelerate ageing on lees of red wines and its repercussion in sensorial parameters. LWT- Food Sci. Technol. 64 (2), 1255–1262. Kurtzman, C.P., Robnett, C.J., 1998. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 73 (4), 331–371. Kurtzman, C., Fell, J.W., Boekhout, T., 2011. The Yeasts: A Taxonomic Study. Elsevier. Kutyna, D.R., Varela, C., Henschke, P.A., Chambers, P.J., Stanley, G.A., 2010. Microbiological approaches to lowering ethanol concentration in wine. Trends Food Sci. Technol. 21 (6), 293–302. Linares, M.J., Solís, F., 2007. Capitulo 11: Identificación de Levaduras. In: Revista Iberoamericana de Micología. Asociación Española de Micología, pp. 1–20. http:// www.guia.reviberoammicol.com/Capitulo11.pdf. Liu, L., Loira, I., Morata, A., Suárez-Lepe, J., González, M., Rauhut, D., 2015. Shortening the ageing on lees process in wines by using ultrasound and microwave treatments both combined with stirring and abrasion techniques. Eur. Food Res. Technol. 242, 559–569. 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. Loira, I., Morata, A., González, C., Suárez-Lepe, J.A., 2012. Selection of glycolytically inefficient yeasts for reducing the alcohol content of wines from hot regions. Food Bioprocess Technol. 5 (7), 2787–2796. Loira, I., Vejarano, R., Bañuelos, M.A., Morata, A., Tesfaye, W., Uthurry, C., Villa, A., Cintora, I., Suárez-Lepe, J.A., 2014. Influence of sequential fermentation with Torulaspora delbrueckii and Saccharomyces cerevisiae on wine quality. LWT Food Sci. Technol. 59 (2), 915–922. Part 1.

504  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Loira, I., Morata, A., Comuzzo, P., Callejo, M.J., González, C., Calderón, F., Suárez-Lepe, J.A., 2015. Use of Schizosaccharomyces pombe and Torulaspora delbrueckii strains in mixed and sequential fermentations to improve red wine sensory quality. Food Res. Int. 76, 325–333. Looke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50 (5), 325–328. Loureiro, V., Malfeito-Ferreira, M., 2003. Spoilage yeasts in the wine industry. Int. J. Food Microbiol. 86 (1–2), 23–50. Ludovico, P., Sousa, M.J., Silva, M.T., Leão, C., Côrte-Real, M., 2001. Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147 (9), 2409–2415. Magyar, I., Panyik, I., 1989. Biological deacidification of wine with Schizosaccharomyces pombe entrapped in ca-alginate gel. Am. J. Enol. Vitic. 40 (4), 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 (1), 94–100. Malherbe, D., Du Toit, M., Otero, R.C., Van Rensburg, P., Pretorius, I., 2003. Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production. Appl. Microbiol. Biotechnol. 61 (5–6), 502–511. Martin, V., Boido, E., Giorello, F., Mas, A., Dellacassa, E., Carrau, F., 2016. Effect of yeast assimilable nitrogen on the synthesis of phenolic aroma compounds by Hanseniaspora vineae strains. Yeast 33 (7), 323–328. Medina, K., Boido, E., Fariña, L., Gioia, O., Gomez, M., 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 (3), 2513–2521. Mehlomakulu, N.N., Setati, M.E., Divol, B., 2014. Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp. Int. J. Food Microbiol. 188, 83–91. Meillon, S., Urbano, C., Schlich, P., 2009. Contribution of the temporal dominance of sensations (TDS) method to the sensory description of subtle differences in partially dealcoholized red wines. Food Qual. Prefer. 20 (7), 490–499. Mendes Ferreira, A., Climaco, M., Mendes Faia, A., 2001. The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components—a preliminary study. J. Appl. Microbiol. 91 (1), 67–71. Michnick, S., Roustan, J., Remize, F., Barre, P., Dequin, S., 1997. Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase. Yeast 13 (9), 783–793. Milanovic, V., Ciani, M., Oro, L., Comitini, F., 2012. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation. Microb. Cell Factories 11 (1), 1. Mingorance-Cazorla, L., Clemente-Jiménez, J., Martínez-Rodríguez, S., Las HerasVázquez, F., Rodríguez-Vico, F., 2003. Contribution of different natural yeasts to the aroma of two alcoholic beverages. World J. Microbiol. Biotechnol. 19 (3), 297–304. Mora, J., Barbas, J., Mulet, A., 1990. Growth of yeast species during the fermentation of musts inoculated with Kluyveromyces thermotolerans and Saccharomyces cerevisiae. Am. J. Enol. Vitic. 41 (2), 156–159. Morata, A., Suárez-Lepe, J.A., 2015. New biotechnologies for wine fermentation and ageing. In: Advances in Food Biotechnology. John Wiley & Sons, pp. 287. Morata, A., Benito, S., Loira, I., Palomero, F., Gonzalez, M., Suarez-Lepe, J., 2012. Formation of pyranoanthocyanins by Schizosaccharomyces pombe during the fermentation of red must. Int. J. Food Microbiol. 159 (1), 47–53.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   505

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 (3), 285–294. Moreno, J.J., Millán, C., Ortega, J.M., Medina, M., 1991. Analytical differentiation of wine fermentations using pure and mixed yeast cultures. J. Ind. Microbiol. 7 (3), 181–189. Mylona, A., Del Fresno, J., Palomero, F., Loira, I., Bañuelos, M., Morata, A., Calderón, F., Benito, S., Suárez-Lepe, J., 2016. Use of Schizosaccharomyces strains for wine fermentation—effect on the wine composition and food safety. Int. J. Food Microbiol. 232, 63–72. Nikolaou, E., Soufleros, E.H., Bouloumpasi, E., Tzanetakis, N., 2006. Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristics and vinification results. Food Microbiol. 23 (2), 205–211. Nissen, P., Nielsen, 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 (4), 331–341. Ocón, E., Gutiérrez, A., Garijo, P., López, R., Santamaría, P., 2010. Presence of non-­ Saccharomyces yeasts in cellar equipment and grape juice during harvest time. Food Microbiol. 27 (8), 1023–1027. Oelofse, A., Lonvaud-Funel, A., Du Toit, M., 2009. Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production. Food Microbiol. 26 (4), 377–385. OIV-ECO 432/2012, n.d. Beverage Obtained by Dealcoholization of Wine. International Code of Oenological Practices. OIV Code Sheet—Issue 2015/01 I.1.6-9. Available: http://www.oiv.int/public/medias/3940/e-code-i-69.pdf (Accessed 9 June 2016). OIV-ECO 433/2012, n.d. Beverage obtained by Partial Dealcoholization of wine. International Code of Oenological Practices. OIV Code Sheet—Issue 2015/01 I.1.610. Available: http://www.oiv.int/public/medias/3941/e-code-i-610.pdf (Accessed 9 June 2016). OIV-OENO 18/73, n.d. Wine Definition. International Code of Oenological Practices. OIV Code Sheet—Issue 2015/01 I.3.1-1. Available: http://www.oiv.int/public/medias/3921/e-code-i-31.pdf (Accessed 9 June 2016). OIV-OENO 18/73, OIV-ECO 3/2003 & OIV-OENO 415/2011, n.d. Complementary Definitions Relating to Sugar Content, International Code of Oenological Practices, OIV Code Sheet—Issue 2015/01 I.1.3-2. Available: http://www.oiv.int/public/medias/3922/e-code-i-32.pdf (Accessed 9 June 2016). OIV-OENO 394B-2012, n.d. Correction of the Alcohol Content in Wines. Available: http://www.oiv.int/public/medias/1432/oiv-oeno-394b-2012-en.pdf (Accessed 9 June 2016). Ortiz-Barrera, E., Miranda-Castilleja, D.E., Arvizu-Medrano, S.M., Pacheco-Aguilar, J.R., Aldrete-Tapia, J.A., Hernández-Iturriaga, M., Martínez-Peniche, R.Á 2015. Enological potential of native non-Saccharomyces yeasts from vineyards established in Queretaro, Mexico. Rev. Chapingo Ser. Hortic., vol. 21, no. 2, pp. 169–183. Oxoid. 2016 Available: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0191&org=107&c=UK&lang=EN (Accessed 22 May 2016). Pallmann, C.L., Brown, J.A., Olineka, T.L., Cocolin, L., Mills, D.A., Bisson, L.F., 2001. Use of WL medium to profile native flora fermentations. Am. J. Enol. Vitic. 52 (3), 198–203. Palomero, F., Morata, A., Benito, S., Calderón, F., Suárez-Lepe, J., 2009. New genera of yeasts for over-lees aging of red wine. Food Chem. 112 (2), 432–441. Pantelides, I.S., Christou, O., Tsolakidou, M., Tsaltas, D., Ioannou, N., 2015. Isolation, identification and in vitro screening of grapevine yeasts for the control of black aspergilli on grapes. Biol. Control 88, 46–53. Peinado, R., Moreno, J., Medina, M., Mauricio, J., 2004. Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures. Biotechnol. Lett. 26 (9), 757–762.

506  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

Pérez, G., Fariña, L., Barquet, M., Boido, E., Gaggero, C., Dellacassa, E., Carrau, F., 2011. A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of esculin glycerol agar (EGA) medium. World J. Microbiol. Biotechnol. 27 (1), 47–55. 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 (3), 336–345. Phister, T.G., Mills, D.A., 2003. Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine. Appl. Environ. Microbiol. 69 (12), 7430–7434. Pina, C., Santos, C., Couto, J.A., Hogg, T., 2004. Ethanol tolerance of five non-­ Saccharomyces wine yeasts in comparison with a strain of Saccharomyces cerevisiae—influence of different culture conditions. Food Microbiol. 21 (4), 439–447. Piškur, J., Rozpędowska, E., Polakova, S., Merico, A., Compagno, C., 2006. How did Saccharomyces evolve to become a good brewer? Trends Genet. 22 (4), 183–186. Portillo, M., Mas, A., 2016. Analysis of microbial diversity and dynamics during wine fermentation of grenache grape variety by high-throughput barcoding sequencing. LWT- Food Sci. Technol. 72, 317–321. Pretorius, I.S., Bauer, F.F., 2002. Meeting the consumer challenge through genetically customized wine-yeast strains. Trends Biotechnol. 20 (10), 426–432. 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. Rapp, A., Mandery, H., 1986. Wine aroma. Experientia 42 (8), 873–884. Rawsthorne, H., Phister, T.G., 2006. A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices. Int. J. Food Microbiol. 112 (1), 1–7. Rawsthorne, H., Phister, T.G., 2009. Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR. Int. J. Food Microbiol. 131 (2), 246–250. Renault, P., Miot-Sertier, C., Marullo, P., Hernández-Orte, P., Lagarrigue, L., LonvaudFunel, A., Bely, M., 2009. Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: potential applications in the wine industry. Int. J. Food Microbiol. 134 (3), 201–210. Ribéreau-Gayon, P., Dubourdieu, D., Donèche, B., Lonvaud, A., 2000. The grape and its maturation. In: Handbook of Enology: The Microbiology of Wine and Vinifications, second ed. vol. 1. Wiley, pp. 261–262. Rodrigues, N., Gonçalves, G., Pereira-da-Silva, S., Malfeito-Ferreira, M., Loureiro, V., 2001. Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces. J. Appl. Microbiol. 90 (4), 588–599. Rojas, V., Gil, J.V., Piñaga, F., Manzanares, P., 2001. Studies on acetate ester production by non-Saccharomyces wine yeasts. Int. J. Food Microbiol. 70 (3), 283–289. Romano, P., Fiore, C., Paraggio, M., Caruso, M., Capece, A., 2003. Function of yeast species and strains in wine flavour. Int. J. Food Microbiol. 86 (1), 169–180. Rossouw, D., Bauer, F.F., 2016. Exploring the phenotypic space of non-Saccharomyces wine yeast biodiversity. Food Microbiol. 55, 32–46. Sadoudi, M., Tourdot-Maréchal, R., Rousseaux, S., Steyer, D., Gallardo-Chacón, J., Ballester, J., Vichi, S., Guérin-Schneider, R., Caixach, J., Alexandre, H., 2012. Yeast– yeast interactions revealed by aromatic profile analysis of sauvignon blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts. Food Microbiol. 32 (2), 243–253. Santos, J., Sousa, M.J., Cardoso, H., Inacio, J., Silva, S., Spencer-Martins, I., Leao, C., 2008. Ethanol tolerance of sugar transport, and the rectification of stuck wine fermentations. Microbiology 154 (2), 422–430.

Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION OF OENOLOGICAL YEASTS   507

Schmidtke, L.M., Blackman, J.W., Agboola, S.O., 2012. Production technologies for reduced alcoholic wines. J. Food Sci. 77 (1), R25–R41. Schwentke, J., Sabel, A., Petri, A., König, H., Claus, H., 2014. The yeast Wickerhamomyces anomalus AS1 secretes a multifunctional exo-β-1,3-glucanase with implications for winemaking. Yeast 31 (9), 349–359. Smith, P., 1995. Biological Processes for the Reduction of Alcohol in Wines. MS dissertation for the Degree of Master of Applied Science at Lincoln University, Christchurch, New Zealand. Soden, A., Francis, I., Oakey, H., Henschke, P., 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 (1), 21–30. Stender, H., Kurtzman, C., Hyldig-Nielsen, J.J., Sorensen, D., Broomer, A., Oliveira, K., Perry-O’Keefe, H., Sage, A., Young, B., Coull, J., 2001. Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes. Appl. Environ. Microbiol. 67 (2), 938–941. Strauss, M., Jolly, N., Lambrechts, M., Van Rensburg, P., 2001. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J. Appl. Microbiol. 91 (1), 182–190. Suárez-Lepe, J., Morata, A., 2012. New trends in yeast selection for winemaking. Trends Food Sci. Technol. 23 (1), 39–50. Suárez-Lepe, J., Palomero, F., Benito, S., Calderón, F., Morata, A., 2012. Oenological versatility of Schizosaccharomyces spp. Eur. Food Res. Technol. 235 (3), 375–383. Tao, Y., Zhang, L., 2010. Intensity prediction of typical aroma characters of cabernet sauvignon wine in Changli county (China). LWT- Food Sci. Technol. 43 (10), 1550–1556. Tofalo, R., Schirone, M., Telera, G.C., Manetta, A.C., Corsetti, A., Suzzi, G., 2011. Influence of organic viticulture on non-Saccharomyces wine yeast populations. Ann. Microbiol. 61 (1), 57–66. Toro, M., Vazquez, F., 2002. Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts. World J. Microbiol. Biotechnol. 18 (4), 351–358. Ubeda, J., Maldonado Gil, M., Chiva, R., Guillamon, J.M., Briones, A., 2014. Biodiversity of non-Saccharomyces yeasts in distilleries of the La Mancha region (Spain). FEMS Yeast Res. 14 (4), 663–673. Urso, R., Rantsiou, K., Dolci, P., Rolle, L., Comi, G., Cocolin, L., 2008. Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods. FEMS Yeast Res. 8 (7), 1053–1062. van Breda, V., Jolly, N., van Wyk, J., 2013. Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains. Int. J. Food Microbiol. 163 (2), 80–88. Varela, C., Kutyna, D., Henschke, P.A., Chambers, P.J., Herderich, M.J., Pretorius, I.S., 2008. Taking control of alcohol. Aust. N. Z. Wine Ind. J. 23, 41–43. Viana, F., Gil, J.V., Genovés, S., Vallés, S., Manzanares, P., 2008. Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol. 25 (6), 778–785. 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 (1), 68–74. Viana, F., Belloch, C., Vallés, S., Manzanares, P., 2011. Monitoring a mixed starter of Hanseniaspora vineae–Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production. Int. J. Food Microbiol. 151 (2), 235–240. Vilela-Moura, A., Schuller, D., Mendes-Faia, A., Côrte-Real, M., 2008. Reduction of volatile acidity of wines by selected yeast strains. Appl. Microbiol. Biotechnol. 80 (5), 881–890.

508  Chapter 15  ISOLATION, SELECTION, AND IDENTIFICATION

OF OENOLOGICAL YEASTS

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., Esteve-Zarzoso, B., Cocolin, L., Mas, A., Rantsiou, K., 2015. Viable and culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during Barbera must fermentation. Food Res. Int. 78, 195–200. Xufre, A., Albergaria, H., Inácio, J., Spencer-Martins, I., Gírio, F., 2006. Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations. Int. J. Food Microbiol. 108 (3), 376–384. Zohre, D., Erten, H., 2002. The influence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation. Process Biochem. 38 (3), 319–324. Zott, K., Claisse, O., Lucas, P., Coulon, J., Lonvaud-Funel, A., Masneuf-Pomarede, I., 2010. Characterization of the yeast ecosystem in grape must and wine using ­real-time PCR. Food Microbiol. 27 (5), 559–567. Zuzuarregui, A., 2004. Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection. Antonie Van Leeuwenhoek 85 (4), 271–280.

Further Reading Cocolin, L., Heisey, A., Mills, D.A., 2001. Direct identification of the indigenous yeasts in commercial wine fermentations. Am. J. Enol. Vitic. 52 (1), 49–53. Kurtzman, C.P., 2015. Identification of food and beverage spoilage yeasts from DNA sequence analyses. Int. J. Food Microbiol. 213, 71–78.