Alcoholic fermentation is a complex ecological and biochemical process where a succession of yeasts of several genera and species are able to convert must sugars into ethanol and carbon dioxide, as well as into important secondary metabolites (Barata et al., 2012; Sun et al., 2014; Albergaria and Arneborg, 2016). Even though Saccharomyces species are present at a low frequency on the surface of healthy grapes, Saccharomyces cerevisiae is considered the primary microorganism in the fermentation process and it is widely used in oenology (Martini et al., 1996; Fleet, 2003). However, during the last decade, non-Saccharomyces yeasts species have been proposed for winemaking as they could contribute to the improvement of wine quality (Ciani et al., 2014; Wang et al., 2015; Masneuf-Pomarede et al., 2016; Puertas et al., 2016). Thus, a new trend has emerged in winemaking using starter cultures composed by non-Saccharomyces yeasts, together with S. cerevisiae or for sequential fermentation with S. cerevisiae.

Molecular methods are showing useful results for detection and faster identification of microorganisms throughout the wine elaboration process (Ivey and Phister, 2011). Classical microbiological methods involving isolation coupled with the enumeration of microbes by plating can lead to misinterpretation of the real number of microorganisms since these methods fail to detect viable but non-culturable (VBNC) organisms (Divol and Lonvaud-Funel, 2005; Quirós et al., 2009; Salma et al., 2013; Wang et al., 2016) and minor populations present are difficult to detect on plates (Cocolin et al., 2013; David et al., 2014). Instead, molecular techniques, generally named culture-independent methods, are used for the identification of microorganism directly in the system through the study of their DNA or RNA without the need for isolation and cultivation, reducing detection time (Andorrà et al., 2008). Real-time quantitative PCR (qPCR) has been widely used in wine for microorganism detection during wine elaboration (Rawsthorne and Phister, 2006; Andorrà et al., 2008, 2010; Tofalo et al., 2012; Wang et al., 2014), providing significant advantages as the low detection level, the speed by which assays are performed, and the ability to quantify yeasts present following alcoholic fermentation.

In a previous work of García et al. (2017), small-scale fermentations were elaborated to study the oenological characterization of five non-Saccharomyces native yeast species under several co-culture conditions in combination with selected strain of S. cerevisiae CLI 889 to improve the organoleptic properties of the regional Malvar wines. There, the best inoculation process was selected depending on the non-Saccharomyces strain inoculated. Preferred sequential inoculations were elaborated with S. cerevisiae CLI 889 in combination with Torulaspora delbrueckii CLI 918 that produced wines with a higher fruity and floral aroma and lower ethanol content; with Candida stellata CLI 920 that increased the aroma complexity and glycerol content; and, with Lachancea thermotolerans 9-6C, produced an increase in acidity and floral and ripe fruit aroma. In the case of Schizosaccharomyces pombe, sequential fermentation was selected according to its fruity aroma score obtained after tasting. However, mixed cultures of S. cerevisiae with Metschnikowia pulcherrima CLI 457 and L. thermotolerans 9-6C was chosen due to a lower volatile acidity observed in final wines. Moreover, an increase of glycerol and ripe fruit aroma in the case of M. pulcherrima was observed, and for L. thermotolerans mixed culture the freshness, citric aroma, and full body were the main aspects to verify at the pilot scale.

Regarding these results, the aim of this work is to study yeast population evolution using real-time PCR during pilot winemaking trials under the best inoculation strategies. Moreover, validation of their positive effect on wine fermentation and wine quality was observed in the previous laboratory scale study (García et al., 2017) using sensory analysis.

In this study, we quantified the evolution of inoculated non-Saccharomyces and Saccharomyces populations during alcoholic fermentation in different combinations between strains of different species in a natural must of a white grape Malvar variety. A rapid culture-independent qPCR method for detection and enumeration of different yeasts was applied in Malvar wine fermentations. Four pairs of primers were designed in this work into the variable D1/D2 domains of the 26S ribosomal DNA gene to the strains S. pombe CLI 1079, C. stellata CLI 920, M. pulcherrima CLI 457, and L. thermotolerans 9-6C; this region has previously been used to develop qPCR methods for several yeasts (Andorrà et al., 2010; Albertin et al., 2014). Two other pair of primers were designed by Zott et al. (2010) to the ITS region of rDNA, and this region is widely used in yeast species identification due to the high degree of interspecies sequence variations (Esteve-Zarzoso et al., 1999; Schoch et al., 2012). These qPCR species-specific primers showed an excellent specificity with all wine yeasts tested and did not amplify other representative wine species. Moreover, standard curves elaborated with the different yeast strains presented high efficiencies, and good detection limits, we enumerated the concentration of 10³ cells mL^-1, and the trials were linear over five orders of magnitude.

The T. delbrueckii CLI 918 strain has been utilized to study the matrix influence in the efficiency of qPCR system. Our results were able to show that the matrix of red wine influences on the PCR amplification or on the DNA extraction and purification, due presumably to its much higher proportion of polyphenols. It is known that wine is a complex matrix that presents various PCR inhibitors (Zoecklein et al., 1999; Phister and Mills, 2003), such as major compounds as polyphenols, tannins, and polysaccharides. The efficiency obtained on qPCR analysis from red wine is lower than from white wine and YPD medium, although these values are similar in all cases and without statistical significance. Some authors have reported problems of amplification with DNA isolated directly from wine (Phister and Mills, 2003; Martorell et al., 2005). The assay performed here helped to check that the wine matrix did not significantly influence in the efficiency of the qPCR analysis. According to our results the construction of standard curves in different matrices do not substantially modify the results, and any matrix can be used to quantify the yeast populations from wine fermentation.

It had long been considered that the non-Saccharomyces yeasts are present at the beginning of alcoholic fermentation, being replaced by S. cerevisiae which has a high capacity to take over the process. In this work, the dynamics of five non-Saccharomyces yeasts in co-inoculation with S. cerevisiae have been analyzed, revealing that these non-Saccharomyces species were present throughout the fermentation process. If they are present during fermentation we expected contribution to the chemical and sensory attributes of the final wines. However, even though these five non-Saccharomyces strains were present during fermentation, S. cerevisiae was the most abundant yeast under any of the co-cultures tested at the end of the fermentations. Different mechanisms have been described to explain the dominance of S. cerevisiae over other competitors during wine fermentation, i.e., cell-to-cell contact (Nissen et al., 2003); competition for nutrients (Taillandier et al., 2014; Kemsawasd et al., 2015b; Lleixà et al., 2016); secretion of toxic compounds (Pérez-Nevado et al., 2006; Branco et al., 2015; Ramírez et al., 2015; Wang et al., 2016), or changes in the medium (Goddard, 2008; Salvadó et al., 2011). These effects caused by S. cerevisiae metabolite production and changes in the medium could provide an explanation for the decrease of M. pulcherrima and C. stellata and the increase and persistence of T. delbrueckii, S. pombe, and L. thermotolerans belong to the fermentation, due to their higher fermentative power (García et al., 2017) in relation to the amount of alcohol produced by the yeast species (Lopes et al., 2006) and, therefore, related to their alcohol tolerance (Ciani et al., 2016). In the case of L. thermotolerans, the enhancement of total acidity produced by this species can also influence in the growth of S. cerevisiae and other yeast species. However, the sensibility to these toxic compounds has been described as species- and strain-specific (Wang et al., 2016).

The multi-starter fermentations, combining both non-Saccharomyces yeasts and S. cerevisiae species able to complete the fermentation, are being studied in depth. All these yeast interaction studies have been increased to explain yeast–yeast interactions and their underlying mechanisms in the increasing use of controlled mixed cultures (Ciani et al., 2010; Ciani and Comitini, 2015). These studies have also been driven by the presence of viable and non-culturable microorganisms in wine samples (Millet and Lonvaud-Funel, 2000; Divol and Lonvaud-Funel, 2005), and may have a false idea about the number of non-Saccharomyces species on microbiological methods based on plating (Serpaggi et al., 2012; Wang et al., 2016). In this study, the counts obtained by qPCR were contrasted with plating in YPD non-selective medium and LYS medium (data not shown), a selective medium for non-Saccharomyces yeasts. Generally, the yeast populations observed in LYS agar were higher than those obtained by qPCR. This greater growth on LYS medium, could be explained by the growth of other non-Saccharomyces yeasts present in the non-sterile Malvar must. This fact is in agreement with the results obtained by Phister and Mills (2003) in a Dekkera bruxellensis study.

Differences on the evolution of Saccharomyces and non-Saccharomyces yeasts have been observed depending on the type of inoculation. In the mixed culture of M. pulcherrima/S. cerevisiae, the M. pulcherrima CLI 457 population started to decrease at 24 h in contrast with the increase of S. cerevisiae counts studied by qPCR. The antagonist effect of M. pulcherrima on several yeasts, including S. cerevisiae, which leads to delays in the fermentation, has been studied (Nguyen and Panon, 1998; Türkel and Ener, 2009). This phenomenon was due to a killer effect linked to pulcherrimin pigment produced by M. pulcherrima strains, Türkel and Ener (2009) found three strains of M. pulcherrima (UMY12, UMY14, and UMY15) that produce the same amount of the pigment pulcherrimin, but their antimicrobial activities showed important variations. Different distinct biotypes within the M. pulcherrima species with respect the pulcherrimin production were identified by Pallmann et al. (2001). However, it has recently been described a difficulty in classifying Metschnikowia fructicola species since this species is not distinguishable from Metschnikowia andauensis and other species of the M. pulcherrima clade because of a possible heterogeneity of rRNA repeats (Cordero-Bueso et al., 2017). For this reason, we keep the original designation for this yeast strain, keeping the same yeast species name described on the published document by Arroyo et al. (2010). However, the variable D1/D2 domain of this strain was sequenced by Macrogen to be identified with 99% of sequence identity as M. pulcherrima and its sequence included in GenBank Database (accession number MF001378). Our results showed that the mixed culture of M. pulcherrima/S. cerevisiae finished with a high level of reducing sugars, in the same way that happened in co-cultures with these strains in laboratory scale fermentations (García et al., 2017), so it could be possible that the M. pulcherrima CLI 457 strain had a negative effect on the fermentative capacity of the S. cerevisiae CLI 889 strain. Instead, sequential fermentation with T. delbrueckii and S. cerevisiae finished with sugar values lower than 4 g L^-1 as at laboratory level (García et al., 2017). Therefore, the fermentative capacity of T. delbrueckii in the first days seems to influence in the low sugar content of final wines, independently of the scale of fermentations, which is in agreement with results obtained by Puertas et al. (2016).

Some authors have reported the competition mechanisms between L. thermotolerans and S. cerevisiae in mixed culture. Hansen et al. (2001) found that oxygen increases the competitiveness between L. thermotolerans CBS 2803 and S. cerevisiae Saint Georges S101 strains in mixed culture. In the same way, Nissen et al. (2004) concluded that S. cerevisiae Saint Georges S101 is able to grow and ferment more efficiently under oxygen-limited conditions present during wine fermentation in comparison with L. thermotolerans CBS 2803 and T. delbrueckii CBS 3085. Although other previous studies (Nissen and Arneborg, 2003; Nissen et al., 2003) showed that the death of L. thermotolerans in mixed culture with S. cerevisiae was induced by a cell-to-cell contact mediated mechanism with the same strains used by Nissen et al. (2004). Finally, Kemsawasd et al. (2015a) concluded that cell-to-cell contact and antimicrobial peptides play a combined role in the death of L. thermotolerans CBS 2803 in mixed fermentation with S. cerevisiae Saint Georges S101 strain. Our strain of L. thermotolerans in mixed culture showed a loss of viability most pronounced, although both populations decreased during fermentation process from day 3.

In sequential cultures, the S. cerevisiae population found in Malvar wine in the first 24 h of fermentation were low, between 10² and 10³ cells mL^-1. It can be seen that native Saccharomyces yeasts of the cellar environmental started to grow on the following days, but when S. cerevisiae CLI 889 was inoculated (day 13), this strain causes a progressive fall in the density until the end of sequential fermentations. It is well-known that S. cerevisiae yeasts are very competitive and normally dominates the fermentation due to its fast growth, efficient glucose competition, good ability to produce ethanol, and a higher tolerance to environmental stresses (Piškur et al., 2006). In this study, the growth of S. cerevisiae CLI 889 after its inoculation may have been affected by environmental factors, such as a low controlled temperature (18°C) during the fermentation process, a different availability of nutrients in the musts, and a wine elaboration without the addition of nutrients. After microsatellites multiplex analysis, the presence of the inoculated S. cerevisiae CLI 889 strain at the end of fermentation together with other S. cerevisiae strains could be confirmed; although in sequential culture, S. cerevisiae CLI 889 was found in lower percentage than in mixed cultures at the end of fermentation.

Nutrient content of the musts can modulate the yeast populations, the time of fermentation and secondary metabolites produced during alcoholic fermentation (Beltran et al., 2005; Andorrà et al., 2012; Kemsawasd et al., 2015b). In grape must, nitrogen is considered the main limiting nutrient for optimized growth and good fermentation performance (Bisson, 1999). We could observe when Malvar must II was used in the elaboration of wines, the fermentation length was increased in the cultures (40 days) compared to the elaborations with must I that finished in 32 days; the higher YAN content of must I (218 mgN L^-1) than must II (100 mgN L^-1) could have influence in the fermentation rate in agreement with other studies (Bely et al., 1990; Monteiro and Bisson, 1992; Beltran et al., 2005). Medina et al. (2012) noticed a negative effect of non-Saccharomyces yeasts on nutrient availability for S. cerevisiae reducing its ability to grow, especially when it was sequentially inoculated. In the tested sequential fermentations, it could be possible that the YAN consumption by non-Saccharomyces would explain the slow growth of S. cerevisiae CLI 889, although S. cerevisiae population was eventually greater at final of fermentation in all cases, since it is well-known that S. cerevisiae strains show a favorable adaptation to the nitrogen-limited wine fermentation environment (Marsit et al., 2015). Additionally, a higher alcohols production (isobutanol, isoamyl alcohol, metionol, and β-phenylethyl alcohol) has been noted in fermentations elaborated with Malvar must II. This is related to the nitrogen concentration, the less nitrogen there is available in the fermentation medium, the more higher alcohols are produced (Beltran et al., 2005; Andorrà et al., 2012). The higher alcohols, along with glycerol, are the end-products of reductive pathway alternatives to the ethanol products. However, we did not detect in all co-cultures a significant decrease in the ethanol content with regard to their controls. Other volatile compounds as acetates, ethyl esters, and 1-propanol have also presented positive correlation with the level of nitrogen in the fermentation process (Rapp and Versini, 1995), this correlation can be observed for most of these compounds when the wines were elaborated with must I.

In terms of glycerol content, we can confirm the use of the tested non-Saccharomyces strains provides an enhancement of glycerol both at laboratory scale and at the pilot scale with the exception of L. thermotolerans 9-6C that did not produce high concentrations with respect to its controls at both scales. It is well-known that several non-Saccharomyces yeasts can considerably increase the glycerol concentrations in wine (Soden et al., 2000; Cominiti et al., 2011; Englezos et al., 2015; Benito et al., 2016b). Glycerol is one of the major compounds produced during wine fermentation, and it is important in yeast metabolism for regulating the redox potential in the cell (Prior et al., 2000). This compound contributes to mouth-feel, sweetness, and complexity in wines (Ciani and Maccarelli, 1998), but its production is usually linked to increased acetic acid production (Prior et al., 2000). In our results, the volatile acidity values measured as grams per liter of acetic acid, were kept low, especially at the pilot scale, with a particular decline in volatile acidity produced by T. delbrueckii CLI 918 in sequential culture.

In respect of the oenological parameters studied, the behavior of the yeast strains and the wine styles were similar regardless of the scale of fermentation tested. However, due to the type of vinification being different, some parameters changed at the pilot scale. Most of the wines can be considered as dry since their sugar content was less than 4 g L^-1 at final of fermentation (Belitz and Grosch, 1999), with the exception of pure culture of S. cerevisiae p-ScI and mixed culture of M. pulcherrima/S. cerevisiae (m-Mp/ScI) (Supplementary Table S1). Generally, volatile acidity values are lower for all co-cultures in this work.

Sequential culture of T. delbrueckii/S. cerevisiae (s-Td/ScI), in comparison with its control (p-ScI), was distinguished for a significant decrease in volatile acidity (0.34 g L^-1) and an increase of glycerol content (Supplementary Table S1). In relation with aromatic compounds, sequential culture of T. delbrueckii presented higher concentration of β-phenylethyl alcohol, and esters, such as ethyl butyrate, ethyl isovalerate, isoamyl acetate, ethyl hexanoate, and 2-phenylethyl acetate (Supplementary Table S2) associated with the fruity and floral character of this wine.

In relation with cultures elaborated with L. thermotolerans 9-6C and S. cerevisiae CLI 889, the effect of L. thermotolerans on oenological and sensorial properties of wines (increase of lactic acid, glycerol, and β-phenylethyl alcohol) depends on the way of inoculation with S. cerevisiae (Kapsopoulou et al., 2007; Gobbi et al., 2013). We observed a higher lactic acid and β-phenylethyl alcohol content in sequential culture due to L. thermotolerans 9-6C growth before S. cerevisiae CLI 889 inoculation. L. thermotolerans seems to be dominant over S. cerevisiae due to the significant enhancement in total acidity and, consequently, a decrease of pH. In contrast, this behavior appears to be softened in mixed culture. This result contrasts with other studies (Gobbi et al., 2013; Benito et al., 2016a) that also observed this pattern of competitiveness in the different inoculation strategies with L. thermotolerans and S. cerevisiae.

Our results showed that C. stellata CLI 920, along with L. thermotolerans 9-6C, are strains that produce lactic acid and, therefore, they increase the total acidity, both at the laboratory scale using sterile Malvar must and at the pilot scale. This production could be related with the higher concentration of ethyl lactate observed in both sequential inoculations since this ester is produced by esterification from acid lactic and ethanol (Inaba et al., 2009; Delgado et al., 2010). Higher concentrations of ethyl lactate after the use of co-cultures with L. thermotolerans and S. cerevisiae have been documented by other authors (Cominiti et al., 2011; Gobbi et al., 2013; Benito et al., 2015, 2016a).

In relation to the S. pombe strain, we tested in this work the S. pombe CLI 1079 yeast strain instead of the CLI 1085 strain used at laboratory scale due to the low growth capacity of this latest strain, that did impossible a successful pied de cuve at the pilot scale. The S. pombe CLI 1079 in sequential culture were able to finish the fermentation with residual sugars less than 4 g L^-1; this strain presented a low consumption of the malic acid at the pilot scale, ending the fermentation with 1.00 g L^-1 of malic acid, a value slightly lower than its control (p-ScII). Additionally, glycerol content was higher than the control. This culture presented an elevated concentration of β-phenylethyl alcohol and the highest values of alcohols. Volatile compounds associated with cheese and butter aromas were higher in sequential culture of S. pombe than the control p-ScII.

We can confirm that the inoculation strategies conducted at the laboratory scale produce a notable improvement in the quality of regional Malvar wines at the pilot scale also. Tasters were able to distinguish the different elaborations with respect the controls and most appreciated wines by tasting panel were those elaborated in sequential cultures with T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures with L. thermotolerans 9-6C in combination with the S. cerevisiae CLI 889 strain. Sequential cultures have produced more different wines with respect to the controls, providing organoleptic properties associated with the non-Saccharomyces strains, but more studies need to be carried out varying the moment of inoculation of S. cerevisiae strain in these cultures to prevent native S. cerevisiae growth on musts, and the reduction of the fermentation time. This work provides the basis for the implementation of new biotechnological strategies for improving Malvar wine quality and it can be tested in commercial wineries.

MG, TA, and BE-Z designed the experiments, analyzed the results, discussion of the results and wrote the manuscript. MG, JC, and JMC performed experiments and analyzed results.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.