Comparing these results with those obtained in the same vineyard in previous years, some qualitative and quantitative differences can be observed [
15]. In samples from untreated vineyards, yeast population increased during the ripening process, being at its maximum in the final stage. At the beginning of this process, the predominant specie is
Metschnikowia pulcherrima. However, while the ripening process progresses, the population of this specie decreases, being replaced by
Aureobasidium pullulans,
Cryptococcus uzbekistanensis and
Rhodotorula mucilaginosa, although the numbers of these yeasts are lower than
M. pulcherrima population at the beginning of the process.
R. mucilagionsa is a pigmented single-cell yeast that belongs to Basidiomycota division. Its different color, red-orange colonies, are the result of pigments that they produce in which the function is to block certain wavelengths that may cause a harmful effect [
21]. On the other hand,
C. uzbekistanensis is not a capsulated round yeast and was first identified by Maksimova and Chernov [
25]. These species are also replaced, with
M. pulcherrima being the only one that presents during all of the ripening process. Thereby, in the middle of the process, the species
Cryptococcus adeliensis and
Cryptococcus sp. CF-285748 appear. Although
Cryptococcus genus is mentioned as one of the typical one pertaining to non-
Saccharomyces yeasts and, therefore, present in the surface of grapes [
26,
27], these two species are not typical ones, but both have been isolated in other studies [
28,
29,
30]. There are not many references regarding the isolation and characterization of
Cryptococcus sp. CF-285748; however,
C. adeliensis is described by Scorzetti et al. [
31] as a species from the
Cryptococcus genus. This yeast is not present during fermentation but can use a wide variety of carbon sources and produces starch during its growth. Furthermore, the low presence of this species during the ripening process can be due to the fact that during the incubation of the replicas the incubation temperature was 28
C, being 25
C its optimal growth temperature; the growth of this yeast is weak at 30
C [
31]. Finally, in the last phase of ripening, a new species appears,
Quambalaria cyanescens, of which the population number is well above the rest of isolated microorganisms. This microorganism has been found in a wide range of ecological niches, being symbiont in species of
Corymbia and
Eucalyptus [
32]. Hence, although this last species is well described [
32], there are not studies regarding its presence in the must nor in the grape surface. In fact, one of the objectives of this study was to confirm previous results obtained in our laboratory. On the other hand, the fact that this yeast only appears at the end of the ripening process may be due to the application of antifungal treatments during most parts of the period in which the samples were collected, except in the last week, during which there was no treatment applied. This hypothesis seems to fit with certain studies about antifungal treatments that produced a decrease in the yeast population [
20,
33,
34]. Some of the microorganisms described in this work are not the common type of yeast founded on grape surface. This fact may be caused by several factors. First, there is a limited number of works studying the yeast-like microbiota present during the ripening of grapes and its dynamics during this process [
35]. On the other hand, the origin of the grapes should be considered, which could explain one part of the dissimilarity in terms of diversity of non-
Saccharomyces yeasts isolated in the different studies. Regarding the population dynamics,
A. pullulans is one of the main yeasts isolated in unripe grapes, although its population decreases along the ripening process, being undetectable when grapes are harvested [
36]. This result agrees with our data, as
A. pullulans appears at the beginning of the ripening process, but it has not been isolated in the later phases of the process. It is also reported the presence of
R. mucilaginosa and
Cryptococcus sp. during the midpoint of the ripening process, although the abundance of
R. mucilaginosa in our study is smaller than the one observed in other studies [
36].
If only glycosides with the most flavorant aglycons were considered, the most abundant in grape juice are apiosylglycosides (more than 50%), followed by rutinosides (6% to 13%), and lastly, glucosides (4 to 9%). All glycosides are not existing in all cultivars and their amounts also differ according to the original grape. The glycoside flavor potential remains rather constant during fermentation and in drinks as well. This fact opened a new field of rigorous investigation on the chemistry of glycoconjugated flavor compounds to exploit this significant flavor source [
37]. Terpene glycosides can be enzymatically hydrolyzed to enrich wine flavor by release of free aromatic complexes from natural glycoside precursors. This procedure is carried out with several enzymes, which act consecutively according to two phases: firstly,
-L-rhamnosidase,
-L-arabinosidase or
-D-apiosidase make the cleavage of the terminal sugar and rhamnose, arabinose or apiose and the corresponding
-D-glucosides are released; then, the deliverance of monoterpenol takes place after action of a
-D-glucosidase [
37]. The sensorial features of the wines produced with Muscat grapes are connected to the level of terpene alcohols, so an improvement of such a level, as a result of hydrolytic processes conducted by non-
Saccharomyces yeasts is expected. Isolates from
Hanseniaspora uvarum and
H. vineae have been proved to be candidates to be used in vinification procedures to improve wine olfactive properties [
38]. Optimal conditions to induce
-glucosidase activity have also been determined [
38]. On the other hand, a total of 17
Pichia and
Wickerhamomyces isolates belonging to the species
P. fermentans,
P. membranifaciens and
W. anomalus have been tested for exocellular
-glucosidase production. W. anomalus and
P. membranifaciens were obtained from enological ecosystems in Utiel-Requena Spanish region and characterized by physiological and molecular techniques (PCR-RFLP and sequencing). They were proved to be the most interesting species to be used as a source of enzymes because they show tolerance to high levels of ethanol and glucose, making them of great interest to be used in vinification procedures [
39]. Interest in the health benefits of red wine has augmented over the last several years due to the occurrence of resveratrol, which can be found mainly as the glucoside form. Yeasts-endowed
-glucosidase activity improves free-resveratrol concentration in wine. After screening 308 non-
Saccharomyces yeast strains for
-glucosidase, Gaensly et al. [
40] found 14 yeasts, which increased the resveratrol concentration up to 102% without any noteworthy difference, and nine of these yeast strains also created high ethanol contents. Four autochthonous
H. uvarum -glucosidase-producer strains displayed adequate oenological characteristics and hydrolyzed resveratrol-glucosides during the alcoholic fermentation of
V. labrusca grape must. Extracellular proteolytic activity of yeasts may increase the nitrogen sources for the grown of microorganisms during alcoholic fermentation [
41]. Initial low content in nitrogen sources may lead to stop fermentations. On the other hand, yeasts can produce esters, higher alcohols and volatile fatty acids, compounds contributing to the fermentation bouquet of beverages, as primary metabolites from sugar and amino acid metabolism [
42]. Proteolytic activity of some
H. guilliermondii strains have been studied as a biotechnological solution to reduce turbidity due to proteins in fermented beverages [
43]. Our findings confirm that some
Hanseniaspora strains can synthesize proteolytic enzymes [
44], but protease activity in
Pichia and
Wickerhamomyces isolates was too low [
39], according to results obtained by other authors [
43]. Usually described non-
Saccharomyces yeasts are an interesting source of enzymes to be used in wine-making. Isolation of non-usual yeasts in the surface of unripened grapes opens a new door to obtain new enzymes with new potential to be used in biotechnological processes. Further studies should be developed to characterize these “new” enzymes and determine their potential use in enology.