Selection Process of a Mixed Inoculum of Non-Saccharomyces Yeasts Isolated in the D.O.Ca. Rioja

The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO2 resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines.


Introduction
Spontaneous fermentations are complex microbial processes performed by the sequential action of indigenous non-Saccharomyces and Saccharomyces yeasts. The high diversity of species involved in the process gives rise to wines with more complex profiles. However, these fermentations are often associated with extended lag phases and long fermentation times, high residual sugar content and organoleptic defects [1]. These disadvantages, together with the lack of predictability of the process, have led winemakers to seek alternatives to spontaneous fermentations. Currently, the most extended practice in winemaking involves the inoculation of the must with selected S. cerevisiae strains in the form of active dry yeasts (ADY). This technique has some advantages, such as the rapid and complete fermentation of the must and great reproducibility of the final product [2]. However, this practice also leads to less complexity and a certain standardization of the wines obtained as a consequence of the reduction of the diversity of the microbial populations involved in the fermentation [3,4].
To avoid homogeneity of the wines inoculated with S. cerevisiae commercial strains, the use of selected non-Saccharomyces yeasts in mixed fermentations with S. cerevisiae has been proposed. This practice offers some of the advantages of spontaneous fermentations such as higher organoleptic complexity, while allowing greater control of the process, This initial list included species that had shown good behavior as inocula in previous studies (Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri and Torulaspora delbrueckii), and species that had not been previously considered but that had been isolated in different D.O.Ca. Rioja enological ecosystems in previous works [40][41][42] (Candida spp. and Criptococcus spp. genera, Debaryomyces hansenii, Sporidiobolus salmonicolor, Williopsis pratensis and Zygosaccharomyces bailii). In addition, during the process, two spontaneously developed mixed inocula in two of the studied cultures were detected and included in the study. Furthermore, a commercial strain of Saccharomyces cerevisiae (Uvaferm VRB, Danstar Ferment AB, Fredericia, Denmark) was used as a fermentative reference or as part of the inoculum in sequential inoculations.

Genetic Characterization of the Strains at Clonal Level
In the species in which several isolates were included, they were identified at a clonal level. Thus total of seventy-one strains were characterized by Random Amplification of Polymorphic DNA ((RAPD)-PCR) using the primers M13, M14, Coc, OPA02 and OPA09, following the methodology described by Renault et al. [23] with some modifications [24].

SO 2 Resistance
Resistance of the non-Saccharomyces strains to different concentrations of SO 2 (20,40,80 and 120 mg/L) was studied in tubes with 5 mL of buffered YPD (1% yeast extract, 2% dextrose, 2% peptone) following the procedure described in González-Arenzana et. al. [43]. The resistance to SO 2 was determined by contrasting the growth of the studied strains in the presence of SO 2 with the growth of the same strains without SO 2 . Measures of growth were conducted by reading the absorbance at 600 nm.

Enzymatic Characterization
The presence of enzymatic activities related to wine aroma (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase and β-glucosidase) and wine color and fining (pectinase, cellulose, xilanase, glucanase, leucine arylamidase, valine arylamidase and cystine arylamidase) was studied in the non-Saccharomyces strains. Enzymatic activities were measured using an API-ZYM test system and different specific media plates as described in Escribano et al. [44]. To carry out the API-ZYM test, yeast cultures were suspended in distilled water until the suspensions reached a turbidity of [5][6] McFarland. 65 µL of the culture suspensions were inoculated in each cupule of the API ZYM test strip.

Pure 100 mL Vinifications
In this assay alcoholic fermentations were performed in sterile, 100 mL Erlenmeyer flasks containing pasteurized (10 min at 70 • C) Viura grape juice (206.45 g/L initial reducing sugar concentration, 0.93 g/L malic acid, 2.36 total acidity expressed as g/L of tartaric acid, 150 mg/L yeast assimilable nitrogen (YAN) and pH 3.56). The fermentation vessels were sealed with Müller valves filled with H 2 SO 4 . Yeasts were precultured in a YPD liquid medium at 25 • C for 48 h before inoculation of the flasks with 10 6 cells/mL. All the fermentations were carried out in duplicate without SO 2 addition at 20 • C and 70 rpm in an orbital incubator. Fermentation activity was measured daily as weight loss from CO 2 release, and fermentations were assessed as stuck when no further weight loss was observed for three consecutive days. Enological parameters and fermentative aromatic compounds were analyzed in final wines and results were compared with those obtained in fermentations performed with a commercial strain of Saccharomyces cerevisiae.

Sequential 2000 mL Vinifications
Vinifications of the following assay were performed in microscale fermenters [45] containing 2000 mL of red Tempranillo must and 500 g of pomace obtained after mechanical treatment of the grapes, in a conditioned chamber at 25 • C. Assays were carried out in duplicate with unsterilized and unsulfited must (14.5% probable degree, 2.26 g/L malic acid, 4.9 total acidity expressed as g/L of tartaric acid, 181 mg/L YAN and pH 3.73). Vinifications were conducted with nine strains belonging to six different species [T. delbrueckii (T18 and T19), M. pulcherrima (M28 and M29), L. thermotolerans (L54 and L57), Z. bailii (Z71), W. pratensis (W87) and C. zeylanoides (C342)] and two mixed inocula of L. thermotolerans and T. delbrueckii in a 70/30 ratio (LT1 and LT2) in sequential inoculations with S. cerevisiae. Non-Saccharomyces yeasts were pre-cultured in YPD liquid medium at 25 • C for 48 h before inoculation of flasks with 10 6 cells/mL. Three days after non-Saccharomyces inoculation, a commercial S. cerevisiae strain was inoculated at a density of 1 × 10 6 cells/mL. Fermentation activity was measured daily as Brix decrease. Wines were pressed 14 days after inoculation, sulfited at 30 mg/L and left to settle for a week. Enological parameters and fermentative aromatic compounds were analyzed in final wines and results were compared with those obtained in fermentations performed only with a commercial strain of Saccharomyces cerevisiae.

Microbial Analysis and Implantation Controls of the Seeded Yeasts
Samples for microbial studies were taken at different times throughout the fermentations. In pure fermentations samples for implantation controls were taken under aseptic conditions on day 7 (tumultuous fermentation) and at the end of fermentation (day 14). In sequential fermentations samples were taken daily under aseptic conditions. Serial dilutions were carried out and the samples were seeded onto plates containing a chloramphenicol glucose agar medium (0.5% yeast extract, 20% glucose, 0.05% chloramphenicol, 17% agar). The plates were incubated at 25 • C for 48 h. Ten colonies from each sample were randomly selected from plates containing between 30 and 300 colony forming units per milliliter (CFU/mL).
The extraction of DNA for the identification of the isolates was conducted from the fresh yeast culture, using the method suggested by López et al. [46]. Domains D1/D2 of the 26S rDNA gene were amplified by PCR using primers and conditions described by Kurtzman and Fell [47]. The PCR products were purified and then sequenced at the facilities of Macrogen Inc. (Amsterdam, The Netherlands) in order to determine which species of yeast each isolate belonged to (according to data retrieved from GenBank: www.ncbi.nlm.nih.gov/blast/blast.org, accessed on 10 February 2017). The identification was considered correct when gene sequences showed identities of at least 98%.

Analysis of Enological Parameters in Wines
Wines were characterized by measuring the alcohol strength, pH, total acidity and volatile acidity according to official European Economic Community EEC methods [48]. Moreover, the malic and lactic acids, glycerol, acetaldehyde and residual sugars were determined by an enzymatic method carried out in a MIURA One enological analyzer (TDI, Barcelona, Spain). Total anthocyanins were measured by decoloring using SO 2 [49].

Analysis of Aromatic Compounds
The analysis of fermentative aromatic compounds in wine was performed by gas chromatography using the method described by Ortega et al. [50] with minor modifications [51]. Extracted samples were injected onto a 6890 series II gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with an automatic injector and a Hewlett-Packard FID detector. Separation was carried out with a DB-Wax capillary column (60 m × 0.32 mm I.D. × 0.5 µm film thickness; J&W Scientific, Folsom, CA, USA). Identification of compounds was carried out by comparison of their retention times with those of pure reference standards using a Hewlett-Packard GCD Series II Gas Chromatograph Electron Ionization Detector.

Statistical Analysis
Analysis of variance (ANOVA) was carried out for all the parameters analyzed for the different assays with the IBM ® SPSS ® Statistics version 23 (company, city, state abbrev if USA, country). Significant differences were established by using the Tukey post hoc test (p ≤ 0.05).

Clonal Characterization of Non-Saccharomyces Strains
In order to evaluate clonal diversity among the most representative non-Saccharomyces yeasts included in the study, genetic characterization was carried out in 71 isolates of five different species (T. delbrueckii, M. pulcherrima, L. thermotolerans, Z. bailii and W. pratensis). RAPD analysis allowed the differentiation of 17 clones among the 21 isolates of T. delbrueckii, 13 clones among the 16 isolates of M. pulcherrima, 15 clones among the 16 isolates of L. thermotolerans and 7 clones among the seven isolates of Z. bailii and the 11 isolates of W. pratensis. Results obtained showed high clonal diversity among the studied species [43].

SO 2 Resistance
Resistance to SO 2 was evaluated after 24 and 48 h in the same isolates in which clonal diversity was assessed. The percentage of isolates in each species able to grow at the same level as the control in different concentrations of SO 2 at 24 h is shown in Figure 1. Strains of T. delbrueckii and Z. bailii showed the best adaptation at 24 h. At that timepoint, 52.4% of T. delbrueckii isolates were able to grow at a similar level as the control in media containing 40 mg/L of SO 2 , and 19.0% were able to grow with a concentration of 80 mg/L. In contrast, isolates of M. pulcherrima showed less tolerance to this compound. 56.3% of the isolates were not able to grow in the medium with a dose of 20 mg/L of SO 2 and the remaining isolates only tolerated that concentration. Isolates of L. thermotolerans also tolerated low levels of SO 2 , but the proportion tolerating at least 20 mg/L was higher than in M. pulcherrima, with 81.3% of the isolates. Results showed better adaptation of the strains at 48 h, but the ability to develop rapidly in presence of SO 2 during vinification is an advantage for the yeast to establish in the medium. For that reason, growth at 24 h was considered as the selection criterion for SO 2 tolerance.

Screening of Enzymatic Activities
Enzymatic activities were studied in non-Saccharomyces strains belonging to ten different genera and species (Candida spp., Criptococcus spp., Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri, Sporidiobolus salmonicolor, Torulaspora delbrueckii, Williopsis pratensis and Zygosaccharomyces bailii). After determination of the enzymatic activities present in the strains, they were classified into different profiles related with wine aroma and color and fining in order to facilitate the selection of the most suitable strains depending on the type of wine or the desired characteristics to be modulated. Profiles related to aroma were named with an "A" and profiles related to color and fining were named with a "C" after the initials of the species or genera.
Enzymatic profiles related to aroma (Table 1) within each species were mainly differentiated by esterase, esterase-lipase and β-glucosidase activities. The β-glucosidase breaks down the terpenes combined with the sugars to release them, thus enhancing the varietal aroma of the wine. Lipases contribute to an increase in the concentrations of free fatty acids and esterases catalyze the synthesis of esters that contribute to the secondary aroma of wines. Esterase and esterase-lipase were the most widespread activities among the studied strains. All the strains of Cryptococcus spp. genera and W. pratensis, S. salmonicolor, P. kluyveri and M. pulcherrima species showed esterase activity. Similarly, esterase-lipase was detected in all the strains of Candida spp., Cryptococcus spp., W. pratensis, S. salmonicolor, P. kluyveri, M. pulcherrima and L. thermotolerans. On the other hand, lipase activity was scarcely detected and only was found in some strains of S. salmonicolor and L. thermotolerans. Table 2 shows enzymatic profiles found in each species/genera based on the enzymes involved in color and clarity of wine. The degradation of structural polysaccharides by enzymes such as pectinase, cellulose, xylanase, and glucanase, can result in greater extraction of substances that enhance color and improve clarification and filterability during winemaking. Regarding these enzymes, the activity most frequently detected among the strains was pectinase. Nevertheless, in many species there were profiles that did not show any of the carbohydrolase activities. The proteolytic enzymes (leucine arylamidase, valine arylamidase, cystine arylamidase) increase the amino acid content, improving the production of aromatic compounds in the wine [55,56] and they are also involved in reducing protein instability in wines [57]. Within this group of aminopeptidases, W. pratensis showed the three activities studied in nine out of ten strains. As happened with carbohydrolases, there were also some strains that did not show any of the aminopeptidase activities. Profile LC6 of L. thermotolerans stood out for being the only one in which all the activities of these groups of enzymes were detected (the four carbohydrolases and the three aminopeptidases). Table 1. Specific enzymatic profiles related to wine aroma.

Species/Profiles N Enzymatic Activities
Esterase

Fermentative Behavior and Aromatic Compound Production of Non-Saccharomyces Strains in Pure 100 mL Vinifications
Taking into account results obtained in the previous steps of the selection process, twenty-four strains belonging to eight different species (three strains of Candida zeylanoides, one strain of Cryptococcus uzbekistanensis, Debaryomyces hansenii and Zygosaccharomyces bailii, five strains of Metschnikowia pulcherrima and Williopsis pratensis and four strains of Lachancea thermotolerans, and Torulaspora delbrueckii) were selected to continue the study. Since Pichia kluyveri and Sporodiobolus salmonicolor strains were not able to grow in liquid medium, they were eliminated from the study.
The establishment of the inoculated yeasts was total in most cases, with the exception of musts inoculated with W. pratensis, two of the strains of C. zeylanoides and the strain of Cr. uzbekistanensis. As these yeasts were not able to develop in the must, they were eliminated from the selection process and data corresponding to them were not included in the results.
Analysis of fermentative features showed that S. cerevisiae was the only yeast capable of consuming all the sugar (Table 3). C. zeylanoides showed the highest levels of acetic acid and acetaldehyde among the yeasts studied, ahead of Z. bailii, which was the next species with the highest values. L. thermotolerans and Z. bailii showed the ability to increase the acidity of the wine due to the production of lactic acid and to the production of malic acid, respectively. The highest levels of glycerol were also found in Z. bailii, followed by those detected in L. thermotolerans and M. pulcherrima. The aromatic composition of wines was determined by analyzing a total of 30 aromatic compounds. Table 4 shows concentrations of the main groups of aromatic compounds, ethyl acetate, acetoin and diacetyl. Results showed a higher concentration of higher alcohols in M. pulcherrima and S. cerevisiae. D. hansenii and C. zeylanoides, both with low fermentation power, produced the lowest concentrations of higher alcohols. The presence of these compounds in concentrations below 350 mg/L contributes to the aromatic complexity of the wine, while levels above 400 mg/L can be negative, since they produce pungent odors [59]. M. pulcherrima showed the highest values of 2-phenylethanol and also reached the highest levels of 2-phenyethyl acetate (data not shown). This rose-scented compound contributes positively to the bouquet of wine [60]. Higher levels of ethyl acetate were observed in M. pulcherrima than in the rest of the species. M. pulcherrima also showed elevated levels of diacetyl and acetoin. Together with M. pulcherrima, C. zeylanoides was the only yeast that exceeded the limit value of 150 mg/L for acetoin, above which it can provide unpleasant buttery aromas [61]. Small differences were also detected in other aromatic compounds among the different species, but especially between S. cerevisiae and non-Saccharomyces yeasts (sum of acetates, esters and acids) [58]. In addition to a comparison between species, differences in analytical parameters of strains within the same species were studied (data not shown) [58]. Species in which different strains were included were T. delbrueckii (four strains), L. thermotolerans (four strains) and M. pulcherrima (five strains). Less significant differences were found among strains within these species than those found among the different species. In T. delbrueckii significant differences between genotypes were detected only in four parameters: malic acid consumed, volatile acidity and concentration of 1-butanol and isovaleric acid. The four clones of L. thermotolerans were differentiated mainly by the acidity parameters (see Table S4 in Supplementary Materials). Lactic acid production, characteristic of L. thermotolerans, varied widely among the clones studied. One of the parameters that showed significant differences between clones in the three species studied was volatile acidity. In addition, in M. pulcherrima and L. thermotolerans, significant differences were observed in ethyl acetate and acetoin, which can also give rise to aromatic defects. These results underline the importance of strain selection for their use in winemaking.

Influence of Sequential Inoculations with Non-Saccharomyces/S. cerevisiae on Wine Aroma and Composition of Anthocyanins and Stilbenes in 2000 mL Vinifications
As it was expected, the previous assay showed that none of the non-Saccharomyces strains included in the study was able to complete fermentation. For that reason, in this stage of the selection process sequential fermentations with a commercial strain of S. cerevisiae were performed in order to ensure complete fermentation of the must.
After analyzing results obtained in the previous steps of the study, nine strains belonging to six different species [T. delbrueckii (T18 and T19), M. pulcherrima (M28 and M29), L. thermotolerans (L54 and L57), Z. bailii (Z71), W. pratensis (W87) and C. zeylanoides (C342)] and two mixed inocula of L. thermotolerans and T. delbrueckii in a 70/30 ratio (LT1 and LT2) were selected to evaluate their influence on wine quality in sequential inoculations with S. cerevisiae. Results were compared with those obtained in wines fermented only with S. cerevisiae. During the development of the study, it was observed that two of the L. thermotolerans strains (L54 and L57) developed in the must together with T. delbrueckii, both in a 30/70 ratio. Due to the interest that this combination of strains could have in winemaking, it was decided to include the mixed inocula in the following assays.
Control of the presence of the inoculated yeasts showed that on day four (24 h after inoculation of S. cerevisiae), Z. bailii, W. pratensis, C. zeylanoides and one strain of M. pulcherrima were not present in the medium. The rest of the yeasts (the other M. pulcherrima strain and the two L. thermotolerans strains) disappeared in the next two days and only the two T. delbrueckii clones remained in the medium until day six. Regarding the L. thermotolerans/T. delbrueckii mixed cultures, it was observed that they maintained percentages similar to initial proportions (30/70) until S. cerevisiae was inoculated ( Figure 2). After that, population of the mixed inocula decreased until they disappeared in seven days. Regarding analytical characteristics of wines after alcoholic fermentation (see Table S5 in Supplementary Materials), in general, wines corresponding to sequential inoculations with non-Saccharomyces yeasts showed an increase in glycerol and acetaldehyde levels, lower volatile acidity and a higher consumption of malic acid compared to wines fermented with S. cerevisiae alone. Table 5 shows aromatic compounds that presented significant differences in wines sequentially fermented with different non-Saccharomyces yeasts and S. cerevisiae. The wines sequentially inoculated with T. delbrueckii and L. thermotolerans exhibited a significant increase in the concentration of higher alcohols compared to those fermented only with S. cerevisiae. Levels of isobutanol were higher in wines made with L. thermotolerans and strains T18 and M28 of T. delbrueckii and M. pulcherrima than in wines fermented only with S. cerevisiae. Wines inoculated with T. delbrueckii showed higher concentrations of 2-phenylethanol, 1-butanol and methionol ((3-methylthio)propionol) than the wines fermented only with S. cerevisiae. In M. pulcherrima samples, high levels of methionol were also found and T. delbrueckii and L. thermotolerans showed concentrations of 1-propanol higher than those detected in S. cerevisiae. No significant differences were found in ester and acid content in the final wines (data not shown). Regarding acetates, wines made with W. pratensis showed the highest levels, but the differences were only significant with respect to strain L54 of L. thermotolerans. The highest levels of diacetyl were detected in T. delbrueckii, except for one of the strains of L. thermotolerans, which showed the highest value of this compound. Diacetyl levels remained below this threshold value in all samples. In wines inoculated with L. thermotolerans the levels of acetoin and ethyl lactate were the highest. Acetoin is a compound with a relatively high detection value (150 mg/L) [61] that was not exceeded in any of the samples.
With regard to total anthocyanins concentration, the wines inoculated only with S. cerevisiae reached a total anthocyanin content of 221 mg/L. The wines obtained by sequential inoculation with W. pratensis, L. thermotolerans, and the mixed inocula LT did not show significant differences in anthocyanin content with respect to S. cerevisiae. On the other hand, in wines inoculated with M. pulcherrima, T. delbrueckii, Z. bailii and C. zeylanoides, a significant increase in anthocyanin content was observed compared to wines fermented with S. cerevisiae alone (Figure 3).  Figure 4 presents the total stilbene content in wines inoculated with the different yeasts included in this part of the study. Vinifications with S. cerevisiae exhibited the lowest level of total stilbenes. In wines inoculated with C. zeylanoides, T. delbrueckii and Z. bailii, the increase in total content of stilbenes was significant with respect to vinifications with S. cerevisiae. In the rest of the genera, significant differences were not observed respect to S. cerevisiae. Table 5. Aromatic compounds (mg/L) with significant differences in wines sequentially fermented with different non-Saccharomyces yeasts and S. cerevisiae at the end of alcoholic fermentation [62].

Clonal Characterization and SO 2 Resistance
A wide clonal diversity was found among the non-Saccharomyces isolates included in this work. As revealed in various studies, non-Saccharomyces yeasts show high intraspecific diversity, which involves the production of different enzymes and compounds during vinification, as well as different enological behavior [28,33,63,64]. This diversity was also reflected in the results obtained in SO 2 resistance, which showed great differences between strains belonging to the same species. Growth respect to a control without SO 2 was evaluated after 24 and 48 h. Nevertheless, growth in the presence of SO 2 at 24 h was taken into account as a selection criterion. Given the great competition between microorganisms that occurs in industrial vinifications, the ability to grow rapidly in the presence of SO 2 represents an advantage for the yeast to establish itself in the medium. Results obtained at this stage confirm the importance of clonal characterization in the selection of yeasts for their use as inoculums in winemaking. For the following selection steps, representative clones were chosen and extremely sulfur sensitive strains were eliminated.

Screening of Enzymatic Activities
Among the enzymatic activities related to aroma, esterase and esterase-lipase were the most widespread activities and were found in strains of all the studied species. These activities are involved in the formation of esters, compounds that provide fruity aromas to wine. Results are in agreement with those obtained in the study carried out by Comitini et al. [7], in which the presence of esterase activity was observed in a large number of strains of different non-Saccharomyces yeast genera. On the other hand, in the present study, lipase activity was scarcely detected and only 33% of the S. salmonicolor strains and 12% of L. thermotolerans showed this activity. These enzymes cause the degradation of lipids, releasing fatty acids. Another activity that allowed the differentiation of the enzymatic profiles was β-glucosidase. This activity was more widespread in strains of Cryptococcus spp. and M. pulcherrima. Fernández et al. [65] indicated that β-glucosidase activity was mainly related to the species M. pulcherrima. Mendes Ferreira et al. [66] showed the potential of this species for the release of terpenes due to its high β-glucosidase activity, which coincides with the results obtained in the present work.
Enzymatic profiles related to aroma shown in Table 1 within each species were mainly differentiated by esterase, esterase-lipase and β-glucosidase activities. For that reason, the strains that would be chosen based on their aromatic potential would be the ones included in the profiles that had all three enzymatic activities: CA5 of Candida spp., CCA1 of Cryptococcus spp., LA5 of L. thermotolerans, MA4 of M. pulcherrima and ZA3 of Z. bailii; and the ones included in the profiles with two of the activities: DA2 of D. hansenii, PKA1 of P. kluyveri, SPA2 of S. salmonicolor, TA5 of T. delbrueckii, WA1 of W. pratensis, and ZA5 of Z. bailii.
Concerning enzymatic activities related to color and fining, pectinase was the activity most detected within the group of carbohydrolases. In their study, Maturano et al. [67] found higher pectinase activity in T. delbrueckii compared to S. cerevisiae. Activities of the group of carbohydrolases favor the degradation of grape structural polysaccharides improving must extraction, clarification, wine filterability, and extraction of substances responsible for color and aroma during maceration [28]. The proteolytic enzymes aminopeptidases are involved in the reduction of proteic instability in wine [57]. Profiles that had more enzymatic activities related with color and limpidity of wine were: CC1 in Candida spp., CCC2 in Cryptococcus spp., DC2 in D. hansenii, LC6 in L. thermotolerans, MC4 and MC6 in M. pulcherrima, PKC1 in P. kluyveri, SPC1 in S. salmonicolor, TC4 and TC6 in T. delbrueckii, WC1 in W. pratensis and ZC1 in Z. bailii. Profile LC6 of L. thermotolerans showed all the carbohydrolase and aminopeptidase activities studied, which means great potential to improve wine characteristics.
All the strains previously indicated passed to the next selection stage due to their potential to improve the aroma, color, clarity and stability of red wines. Once the strains with the potential capacity to improve the characteristics of the wines had been chosen, their fermentative behavior under different conditions was studied.

Fermentative Behavior and Aromatic Compound Production of Non-Saccharomyces Strains in Pure 100 mL Vinifications
As expected, an increase in acidity levels was observed in wines inoculated with L. thermotolerans and Z. bailii. Other authors have described the production of lactic acid by L. thermotolerans [20], giving rise to pH drops and increases in total acidity values. Due to this feature, characteristic of L. thermotolerans, the use of this yeast in vinifications has been proposed as a tool to adjust the acidity of wines [20,68]. Regarding the increase in acidity levels in Z. bailii samples, it was related to the production of malic acid by this yeast. The production of this compound has been described by other authors in Z. rouxii [69]. The highest levels of glycerol were found in Z. bailii, followed by those detected in L. thermotolerans and M. pulcherrima, although differences were not significant respect to S. cerevisiae. This compound, which provides softness, sweetness and complexity to the wine, has been detected in higher concentrations in vinifications with non-Saccharomyces yeasts compared to S. cerevisiae [64]. Other authors have found a high production of glycerol in L. thermotolerans and C. zemplinina [70,71].
Analysis of aromatic composition showed interesting results in M. pulcherrima. This yeast produced high levels of higher alcohols and contributed positively to the aroma, since the concentration remained below 400 mg/L, above which these compounds can cause organoleptic deviations. M. pulcherrima also showed the highest concentrations of 2phenylethanol and 2-phenyl acetate (data not shown). High production of 2-phenylethanol was found in M. pulcherrima in studies carried out by other authors [72][73][74]. Regarding production of esters, Rodríguez et al. [75] observed high concentrations of these compounds in M. pulcherrima. On the other hand, M. pulcherrima showed the highest levels of ethyl acetate, a compound that can give rise to off odors. These results are in agreement with those obtained in previous studies [11]. Besides, in wines inoculated with M. pulcherrima acetoin concentration exceeded the threshold above which unpleasant buttery aromas may appear.
The study of differences within clones of M. pulcherrima, T. delbrueckii and L. thermotolerans revealed variations in some important parameters such as volatile acidity, which can cause aromatic deviations in wine. In L. thermotolerans, clones were differentiated mainly by acidity parameters. Lactic acid production, a trait of great interest in this species, varied widely among the clones studied. Different studies have pointed out the importance of intraspecific heterogeneity in non-Saccharomyces yeasts [32,76,77].

Influence of Sequential Inoculations with Non-Saccharomyces/S. cerevisiae on Wine Aroma and Composition of Anthocyans and Stilbenes in 2000 mL Vinifications
The previous phases in the selection program showed that none of the non-Saccharomyces strains was able to complete fermentation. For that reason, sequential fermentations of the non-Saccharomyces strains with S. cerevisiae were performed in order to validate the behavior of these non-Saccharomyces in the conditions in which they will be used.
The enological parameters studied showed higher glycerol levels in wines sequentially inoculated with M. pulcherrima, T. delbrueckii and the two mixed inocula compared to those inoculated only with S. cerevisiae (data not shown). Previous studies have shown the ability of different non-Saccharomyces yeasts to produce high concentrations of glycerol [64,70,71].
Aromatic compound analysis revealed a differentiation of samples inoculated with T. delbrueckii and L. thermotolerans. Wines sequentially inoculated with these yeasts showed an increase in the concentration of higher alcohols compared to those fermented only with S. cerevisiae. As occurred in the previous stage, the levels remained below the concentration limit that can cause aromatic deviations. In T. delbrueckii and one of the L. thermotolerans strains (L54) the highest levels of diacetyl were detected. This compound can give rise to unpleasant buttery aromas in wine that are detected when the concentration reaches the threshold value of 8 mg/L [61]. Diacetyl levels remained below this threshold value in all samples. On the other hand, acetoin showed the highest values in L. thermotolerans, but they also remained below the threshold value. In wines sequentially inoculated with L. thermotolerans concentrations of ethyl lactate were significantly higher than those found in wines inoculated only with S. cerevisiae and inoculated with other non-Saccharomyces yeasts. In their study, Del Fresno et al. [78] only detected ethyl lactate in fermentations with L. thermotolerans, which is explained by the ability of this yeast to produce lactic acid and the dependence of ethyl lactate formation on the levels of lactic acid present in the medium. As can be observed, T. delbrueckii and L. thermotolerans, the two species that remained the longest in the tanks, showed the most notable differences in the aromatic profile. These results highlight the importance of the implantation ability of the selected inoculum to achieve the modulation of the characteristics of the wine.
Regarding parameters related with wine color, total anthocyan content was significantly higher than that observed for wines inoculated only with S. cerevisiae in M. pulcherrima, T. delbrueckii, Z. bailii and C. zeylanoides. In their work, Chen et al. [79] observed higher concentration of total anthocyanins in sequential fermentations with M. pulcherrima and T. delbrueckii compared to fermentations carried out only with S. cerevisiae. On the other hand, Belda et al. [80] noticed increases in color intensity and total polyphenol index (TPI) in mixed fermentations with M. pulcherrima compared to fermentations only with S. cerevisiae.
Total stilbene content showed higher values in vinifications sequentially inoculated with non-Saccharomyces yeasts respect to those found in vinifications inoculated only with S. cerevisiae, and differences were significant in C. zeylanoides, Z. bailii and T. delbrueckii. Several studies have shown the influence that different yeast strains can have on stilbene levels in wine [81,82]. The presence of enzymatic activities such as β-glucosidase can influence the resveratrol content. Gaensly et al. [83] observed that yeast strains with this enzymatic activity favored the hydrolysis of the transpiceid into trans-resveratrol.

Selection of the Inoculum for Red Winemaking
The review of the characteristics observed during the selection program in M. pulcherrima and the mixed inoculum L. thermotolerans/T. delbrueckii was conducted in order to choose the most suitable option for its use in winemaking. The high implantation and the ability to remain in the tanks for a longer time of the mixed inoculum L. thermotolerans/T. delbrueckii were important features to be taken into account in an inoculum intended for its use in industrial vinifications. On the other hand, the release by the two species of the mixed inoculum of compounds that improve the quality of the wine, such as glycerol and lactic acid, was another important criterion in the selection. The previous results, coupled to the fact that there is no inoculum on the market composed of these two non-Saccharomyces species together, led to the decision to select the mixed inoculum composed of T. delbrueckii and L. thermotolerans (70/30) for its use in winemaking in order to improve the characteristics of red wines.

Conclusions
This study shows the different steps carried out for the selection of a non-Saccharomyces yeast inoculum from the Rioja winegrowing region. Some advantages over fermentations performed only with S. cerevisiae have been proven, which demonstrates the potential of non-Saccharomyces yeasts for use as inocula in winemaking. Differences in important parameters such as SO 2 resistance, enzymatic activities or production of compounds have been observed between strains, highlighting the relevance of clonal screening in selection processes. On the other hand, during the development of the selection program it is important to consider the type of wine yeasts are destined to produce and the specific characteristics that want to be modulated in order to choose the most suitable inoculum. Taking into account all the mentioned points, the mixed inoculum of L. thermotolerans/T. delbrueckii was selected in the final stage for its use in winemaking. Given its characteristics, such as the high implantation capacity, the production of compounds of interest and the modulation of wine acidity, this inoculum is suitable for the production of quality wines.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/fermentation7030148/s1, Table S1: Yeast isolates of each species and place of isolation, Table S2: Percentage (%) of strains growing at the same level as control at 48 h after inoculation, with different dosages of SO 2 , Table S3: Maximum level of SO 2 which allowed a similar growth to control at 24 h and enzymatic activities of the twenty-four strains selected for the vinification trials, Table S4: Average oenological traits of wines (with significant differences) fermented by different Lachancea thermotolerans (Lt) strains and statistical analysis, Table S5: Analytical parameters (mg/L) with significant differences produced by different non-Saccharomyces yeasts at the end of alcoholic fermentation.
Author Contributions: R.E.-V. was in charge of the methodology and the original draft preparation. L.G.-A. and P.G. were also responsible of the methodology. R.L. and P.S. researched and were in charge of resources. A.R.G. was responsible for the project administration, and developed the formal analysis, resources and reviewed and edited the draft. All authors have read and agreed to the published version of the manuscript.