1. Introduction
Hanseniaspora is an
Ascomycete characterized to own bipolar budding, which confers its typical apiculate form under microscopic observation [
1]. Traditionally, this genus is included within the denominated no-
Saccharomyces yeasts, frequently isolated during the first stages of fermented beverage production, as in the case of wine or cider. They can also be found at the surface of the raw material (grapes or apples) as well as in the industries and the machinery of harvesting and processing of these fruits [
2].
Nowadays, there are available diverse methodologies to correctly identify the different species from
Hanseniaspora. Traditionally, the identification of yeast species has been based on the accomplishment of different tests to discriminate among them centered in the analysis of morphologic, physiological and biochemical traits [
3]. Different miniaturized systems useful for yeast identification are commercially available, generally based on several tests of assimilation or fermentation of substrates that, although are not conclusive, can be used to complement other studies. The progress in the molecular techniques has allowed the development of other methods of yeast identification, like 5.8S rDNA RFLPs, to typify different
Hanseniaspora strains; or the amplification and sequencing of D1/D2 region of the 26S rDNA gene that allows comparing the results with the existing data bases [
4].
The non-
Saccharomyces yeasts predominate during the first 3–4 days of alcoholic fermentation, until the concentration of ethanol and its ability to capture sugars favors the implantation of
Saccharomyces cerevisiae [
5].
Hanseniaspora participates in this process, and also in the production of some other compounds, such as alcohols, esters or organic acids that can modify the organoleptic characteristics of the wine [
6]. The production of these compounds depends on the conditions of fermentation, the species and even on the implied yeast strains [
5]. Exocellular enzymes of related yeast species in different ecosystems have been previously reviewed [
7]. Winemaking has been the process more deeply studied regarding the influence of
Hanseniaspora (
H. guilliermondii,
H. uvarum,
H. osmophila and
H. vineae), and other non-
Saccharomyces species. The modification of the characteristics of the wine is attributed to the capacity of certain non-
Saccharomyces yeasts to produce and secrete hydrolytic enzymes able to transform grape compounds [
8]. These compounds are present in varying amounts as non-volatile flavor glycosylated precursors [
9], mainly disaccharides 6-
O-α-
l-arabinofuranosyl-β-
d-glucopyranoside, 6-
O-α-
l-rhamnopyranosyl-β-
d-glucopyranoside, 2-
O-β-
d-xylosyl-
d-glucopyranoside and 6-
O-β-
l-apiofuranosil-β-
d-glucopyranoside [
10]. The action of enzymes produced by wine yeasts
i.e., β-glucosidase or β-xylosidases can contribute to liberate flavor from these compounds [
10]. Several research groups have already made trial fermentations to study the compounds generated by
Hanseniaspora [
11,
12,
13]. Yeast proteases contribute to the reduction of turbidity in wine and other fermented beverages [
2]. The use of these and other enzymes from non-
Saccharomyces yeasts has been previously reported [
2,
8].
In this study, we screened several Hanseniaspora strains, regarding their ability to produce enzymes (mainly β-glucosidases, β-xylosidases, proteases) with potential for commercial application either in winemaking or in other biotechnological processes. We also assessed the effect of adding those Hanseniaspora strains to fermented grape musts regarding the increase of some volatile compounds such as terpenes that might have a positive impact on the final flavor of wines.
3. Results
3.1. Molecular Analysis
Amplification of D1/D2 rDNA domain of the studied strains was sequenced and compared, obtaining a sequence homology with the D1/D2 fragments deposited in database >98%. The size of the amplicons ranged between 372 and 855 bp. The analysis of the restrictions of each one of these fragments, carried out with the above described enzymes showed great inter- and intraspecific variability, which allowed us to propose different groups to include the
Hanseniaspora strains (
Table 1).
Most of the formed groups displayed an amplificon size between 750 and 800 bp. The greater intraspecific variability occurs within species H. uvarum, with 5 different groups (IV). The size of amplicons of both groups of H. vineae (III) was very different from the one from the rest of the species. H. guilliermondii shows three groups (I–III). H. valbyensis, H. osmophila and H. occidentalis, displaying a characteristic restriction profile for each species.
3.2. Physiological Characterization
Characterization of physiological traits of the 26 strains
Hanseniaspora yeasts was carried out using miniaturized systems API 20C AUX (
Table 2) and RapID Yeast Extra (
Table 3).
Briefly, the API 20C AUX strips assay revealed that genus
Hanseniaspora was not able to assimilate
l-arabinose, inositol, lactose nor maltose.
H. osmophila strains were not able to assimilate any of the other substrates included in the strips, under the assayed conditions while
H. vineae strains were only able to assimilate cellobiose. All
H. guilliermondii strains were able to assimilate this saccharide and also 2-keto-glutarate and sucrose. Upon this global profile, there are strain specific differences for the other substrates, attributed to individual strain characteristics. According to RapID Yeast Plus strips (
Table 2), none of
Hanseniaspora species used in this study had lipase, α-galactosidase, β
-galactosidase, phosphatase, phosphatidylcoliesterase nor urease. Moreover,
N-acetyl-glucosamine was not assimilated. As has been reported above some other minor differences were observed, probably due to strain rather than intraspecific differences. The use of these yeast identification tests, although in some cases is species-specific, has been used for a preliminary characterization of assimilation of carbon compounds.
Table 1.
Restriction profiles of Hanseniaspora strains amplified with primers ITS1 and ITS4.
Table 1.
Restriction profiles of Hanseniaspora strains amplified with primers ITS1 and ITS4.
Group | Species and Laboratory Code | N° CECT | Amplified rDNA Size (bp) | Size of Restriction Fragments (bp) |
---|
HaeIII | HinfI | CfoI |
---|
| H. uvarum | | | | | |
I | HU1 | 1444 | 748 | 748, 500, 413, 156 | 320 | 295 |
II | HU2-HU4-HU7-HU8 | 10387, 10509, 11106, 11107 | 786 | 335, 234, 136 | 372 | 335, 303 |
III | HU3-HU9 | 10389, 11156 | 748 | 748 | 345, 201, 177 | 321, 132 |
IV | HU5 | 10603 | 855 | 307, 233, 176 | 345 | 337, 307 |
V | HU6 | 11105 | 372 | 372, 273, 120 | 206, 190 | 136, 129 |
| H. vineae | | | | | |
I | HV1-HV3 | 11326, 11338 | 577 | 577 | 293 | 520 |
II | HV2 | 11330 | 673 | 288, 194, 104 | 319 | 300, 270, 96 |
| H. guilliermondii | | | | | |
I | HG1-HG3 | 11102, 11104 | 489 | 489 | 265 | 480 |
II | HG2 | 11103 | 363 | 363, 269 | 194, 183 | 200 |
III | HG4-HG5 | 11027, 11029 | 755 | 755 | 356, 212, 190 | 336, 147 |
| H. valbyensis | | | | | |
| HVA1-HVA2-HVA3 | 1445, 10122, 11339 | 763 | 763 | 239, 211, 165, 123 | 646, 123 |
| H. osmophila | | | | | |
| HO1-HO2-HO3 | 1119,11206, 11207 | 769 | 434, 170, 153 | 361 | 279, 184, 157 |
| H. occidentalis | | | | | |
| HOC1-HOC3-HOC4 | 11341, 11472, 11329 | 795 | 673, 128 | 276, 239, 134 | 340, 125 |
In a second stage, qualitative analyses were carried out. The most relevant enzymatic assays were tested in individual plates, obtaining the results shown in
Table 4.
β-Glucosidase activity was detected in all Hanseniaspora strains, with strong dark halos in HVA3, HO2, HOC3 and HOC4 strains. Another interesting activity is the protease, since it was detected in the majority of the strains. Protease we detected in H. guilliermondii HG2. Lipase and esterase activities were not detected in any strain, agreeing with the data previously collected in the RapID Yeast Extra strips. Lipase activity was only detected in some H. vineae and H. uvarum strains, whereas the esterase activity was only detected in some H. guilliermondii and H. occidentalis strains. Polygalacturonase and pectinase activities were detected, although with low intensity, in a limited number of strains; a moderated activity in 6 of the 26 analyzed strains was observed.
Quantitative analysis of the glycosidase enzymatic activities is shown in
Figure 1. No remarkable levels were detected in the α-arabinoside and α-rhamnosidase activities in any of the 26 strains. The HU7, HU8, HV1, HV3, HO2 and HOC1 strains showed high levels of β-glucosidase and β-xylosidase activity, while other strains as HU4, HO1 and HVA3 exhibited high levels of β-glucosidase.
Table 2.
Substrate assimilation of Hanseniaspora strains incubated in the API 20C AUX system.
Table 2.
Substrate assimilation of Hanseniaspora strains incubated in the API 20C AUX system.
Strain | Substrate |
---|
Glucose | Glycerol | 2-Ketoglutarate | Arabinose | Xylose | Adonitol | Xylitol | Galactose | Inositol | Sorbitol | Methyl-α-d-glucopyranoside | N-acethyl-glucosamine | Cellobiose | Lactose | Maltose | Sucrose | Trehalose | Melezitose | Raffinose |
---|
HU1 | + | + | + | - | - | - | - | + | - | - | - | + | + | - | - | + | - | - | - |
HU2 | + | + | - | - | - | - | + | - | - | + | + | - | - | - | - | + | - | + | + |
HU3 | + | - | + | - | - | - | - | - | - | - | - | - | + | - | - | + | + | - | + |
HU4 | + | + | - | - | + | + | + | + | - | + | + | + | + | - | - | + | + | + | - |
HU5 | + | + | - | - | + | - | - | + | - | - | - | - | + | - | - | + | - | + | + |
HU6 | + | + | + | - | - | + | + | + | - | - | - | - | + | - | - | + | + | + | + |
HU7 | + | + | + | - | - | - | - | - | - | - | - | - | + | - | - | + | - | - | + |
HU8 | + | + | + | - | - | + | - | - | - | - | - | - | + | - | - | + | - | - | + |
HU9 | + | - | + | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - |
HV1 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | + |
HV2 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - |
HV3 | + | + | - | - | - | - | - | - | - | + | + | - | + | - | - | + | - | + | + |
HG1 | + | - | + | - | - | - | - | - | - | - | - | - | + | - | - | + | + | + | + |
HG2 | + | + | + | - | - | + | + | + | - | + | + | + | + | - | - | + | - | + | - |
HG3 | + | + | + | - | - | + | + | - | - | + | + | + | + | - | - | + | - | + | + |
HG4 | + | - | + | - | - | - | - | - | - | - | - | - | + | - | - | + | - | - | - |
HG5 | + | - | + | - | - | - | - | - | - | - | - | - | + | - | - | + | + | - | + |
HVA1 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | + | + |
HVA2 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - |
HVA3 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - |
HO1 | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
HO2 | + | - | - | - | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - |
HO3 | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
HOC1 | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
HOC3 | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
HOC4 | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Table 3.
Results of Hanseniaspora strains incubated in the RapID Yeast Plus system.
Table 3.
Results of Hanseniaspora strains incubated in the RapID Yeast Plus system.
Strain | Substrate |
---|
Glucose | Maltose | Sucrose | Trehalose | Raffinose | Fatty acid ester | N-Acetyl-glucosamine | α-Glucoside | β-Glucoside | β-Galactoside | α-Galactoside | β-Fucoside | Phosphate | Phosphatidylcholine | Urea | Proline-β-naphthylamide | Histidine β-naphthylamide | Leucyl-glycine β-naphthylamide |
---|
HU1 | + | + | − | − | − | − | − | − | − | − | − | − | − | − | − | + | + | − |
HU2 | + | − | + | − | + | − | − | + | − | − | − | − | − | − | − | + | + | + |
HU3 | + | + | + | + | + | − | − | − | + | − | − | − | − | − | − | − | + | − |
HU4 | + | − | − | − | − | − | − | + | + | − | − | − | − | − | − | − | + | − |
HU5 | + | − | + | − | + | − | − | − | − | − | − | − | − | − | − | − | + | − |
HU6 | + | + | − | − | + | − | − | + | − | − | − | − | − | − | − | + | + | + |
HU7 | + | − | − | − | + | − | − | − | + | − | − | − | − | − | − | − | − | − |
HU8 | + | − | − | − | + | − | − | − | − | − | − | − | − | − | − | − | − | − |
HU9 | + | − | − | − | − | − | − | − | + | − | − | − | − | − | − | − | + | − |
HV1 | + | + | + | − | + | − | − | + | + | − | − | + | − | − | − | + | + | + |
HV2 | + | − | − | − | − | − | − | + | + | − | − | + | − | − | − | − | + | + |
HV3 | + | + | + | − | + | − | − | + | + | − | − | + | − | − | − | − | + | + |
HG1 | + | + | + | + | + | − | − | + | + | − | − | + | − | − | − | − | + | + |
HG2 | + | − | − | − | − | − | − | + | + | − | − | − | − | − | − | + | + | + |
HG3 | + | + | + | + | + | − | − | + | + | − | − | + | − | − | − | − | + | + |
HG4 | + | + | + | + | − | − | − | + | + | − | − | + | − | − | − | − | + | − |
HG5 | + | + | + | + | + | − | − | − | + | − | − | + | − | − | − | − | − | − |
HVA1 | + | − | − | − | − | − | − | + | + | − | − | + | − | − | − | − | − | − |
HVA2 | + | − | − | − | − | − | − | + | + | − | − | + | − | − | − | − | − | − |
HVA3 | + | + | + | + | − | − | − | − | + | − | − | + | − | − | − | − | − | − |
HO1 | + | − | − | − | − | − | − | + | + | − | − | − | − | − | − | − | + | + |
HO2 | + | + | − | − | − | − | − | + | + | − | − | − | − | − | − | − | + | + |
HO3 | + | + | + | + | − | − | − | + | + | − | − | − | − | − | − | − | + | + |
HOC1 | + | − | + | − | − | − | − | + | + | − | − | + | − | − | − | − | + | + |
HOC3 | + | − | + | − | − | − | − | + | + | − | − | + | − | − | − | − | + | + |
HOC4 | + | + | + | + | + | − | − | − | + | − | − | + | − | − | − | − | + | + |
Table 4.
Qualitative detection of enzymes in Hanseniaspora strains.
Table 4.
Qualitative detection of enzymes in Hanseniaspora strains.
| Protease | Esterase | Lipase | β-Glucosidase | Polygalacturonase | Pectinase |
---|
HU1 | − | + | − | + | − | + |
HU2 | − | + | + | ++ | − | − |
HU3 | + | − | − | + | − | − |
HU4 | + | + | + | ++ | − | + |
HU5 | − | + | + | + | − | − |
HU6 | − | − | − | ++ | + | − |
HU7 | + | + | + | ++ | + | + |
HU8 | + | + | + | ++ | − | − |
HU9 | + | − | − | ++ | − | − |
HV1 | + | + | + | ++ | + | − |
HV2 | − | − | − | ++ | − | − |
HV3 | + | + | + | ++ | + | + |
HG1 | − | + | − | + | − | − |
HG2 | ++ | + | − | ++ | − | − |
HG3 | + | + | − | ++ | + | + |
HG4 | + | − | − | ++ | − | − |
HG5 | + | − | − | ++ | + | − |
HVA1 | + | − | − | + | − | − |
HVA2 | − | − | − | ++ | − | − |
HVA3 | − | − | − | +++ | − | − |
HO1 | + | − | − | + | − | − |
HO2 | − | − | − | +++ | − | − |
HO3 | + | − | − | + | − | − |
HOC1 | + | − | − | + | − | − |
HOC3 | + | + | − | +++ | − | + |
HOC4 | − | − | − | +++ | − | − |
Figure 1.
Quantitative enzymatic activities in Hanseniaspora strains. From left to right, for each strain: α-rhamnosidase, α-arabinosidase, β-xylosidase and β-glucosidase. Units: nkat (nmol pNP released min−1 106 yeast−1).
Figure 1.
Quantitative enzymatic activities in Hanseniaspora strains. From left to right, for each strain: α-rhamnosidase, α-arabinosidase, β-xylosidase and β-glucosidase. Units: nkat (nmol pNP released min−1 106 yeast−1).
The tests of the lipase and esterase activities showed low levels in all the strains, which allowed us to determine that Hanseniaspora genus is not a good producer of this enzyme.
Once all isolates were identified, they were inoculated on YPD casein agar plates to detect the production of an exocellular protease; only five
Hanseniaspora isolates showed an interesting level of this enzymatic activity and were selected for quantitative determination (
Table 5).
Table 5.
Protease activity (nkat) in selected Hanseniaspora isolates.
Table 5.
Protease activity (nkat) in selected Hanseniaspora isolates.
Hanseniaspora Isolate | Activity (nkat) |
---|
HG1 | 20.7 ± 2.2 |
HG3 | 37.9 ± 1.4 |
HVA1 | 35.8 ± 1.1 |
HOC3 | 40.5 ± 1.8 |
HOC4 | 31.7 ± 2.1 |
In four of the strains (HG3, HVA1, HOC3 and HOC4), protease levels were greater than 30 nkat.
3.3. Effect of Sugars and Ethanol on Glycosidase Activities
Hanseniaspora strains HU7, HU8, HV1 and HV3 were selected on the basis of their higher levels of activity in β-glucosidase and β-xylosidase quantitative analysis. β-Glucosidase (
Figure 2a) and β-xylosidase (
Figure 2c) maintained 80% of their activities up to concentrations of 10% (
v/
v) ethanol. In the presence of different amounts of glucose, β-glucosidase (
Figure 2b) and β-xylosidase (
Figure 2d) reached a stable residual activity at concentrations close to 100 mM. However, strain HU7 exhibited a 60% of residual β-xylosidase activity even in the presence of 500 mM glucose (a concentration higher than the typically found in wine).
Figure 2.
Influence of ethanol (a,c); and glucose (b,d) on the β-glucosidase (a,b); and β-xylosidase (c,d) activities in (♦) HU7, (□) HU8, (▲) HV1 and (○) HV3 strains.
Figure 2.
Influence of ethanol (a,c); and glucose (b,d) on the β-glucosidase (a,b); and β-xylosidase (c,d) activities in (♦) HU7, (□) HU8, (▲) HV1 and (○) HV3 strains.
3.4. Determination of Volatile Compounds Liberated from Wine
Muscat juice was used for vinification with a commercial
S. cerevisiae strain. Subsequently, the four non-
Saccharomyces isolates were individually inoculated (in triplicate assays) and volatile compounds were determined (
Table 6).
Table 6.
Terpene and other volatile compounds in Muscat wine.
Table 6.
Terpene and other volatile compounds in Muscat wine.
Compound | Control b | Non-Saccharomyces Yeast Inoculated a |
---|
H. uvarum HU8 | H. uvarum HU7 | H. vineae HV1 | H. vineae HV3 |
---|
TERPENES | | | | | |
cis-5-Vinyltetrahydro-1,1,5-trimethyl-2-furanmethanol | 29.7 (1.2) | 30.3 (3.1) | 33.6 (3.3) | 33.9 (3.3) | 35.9 (3.3) |
trans-5-Vinyltetrahydro-1,1,5-trimethyl-2-furanmethanol | nd | nd | nd | 3.8 * (1.3) | 6.3 * (3.3) |
Linalool | 20.0 (0.9) | 30.3 * (3.9) | 36.3 * (3.3) | 38.8 * (3.6) | 33.6 * (3.9) |
Ho-trienol | 24.0 (3.2) | 51.3 * (5.3) | 35.1 * (3.3) | 38.0 (3.3) | 30.0 (3.6) |
cis-6-Vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol | nd | 36.1 * (3.6) | nd | 3.9 (3.6) | nd |
trans-6-Vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol | nd | 36.3 * (1.3) | nd | 3.9 (0.9) | nd |
Terpineol | 53.3 (3.4) | 66.3 * (3.6) | 65.1 * (1.3) | 51.1 (5.6) | 36.3 (3.3) |
Nerol | 24.6 (2.8) | 35.8 (1.1) | 33.3 (3.1) | 35.1 (3.5) | 36.1 (1.3) |
Geraniol | 59.8 (5.0) | 61.3 (3.6) | 56.9 (1.6) | 59.1 (3.3) | 58.3 (3.3) |
2,6-Dimethyl-3,7-octadien-2,6-diol | 43.2 (4.7) | 86.9 * (3.1) | 80.3 * (3.1) | 68.3 * (3.3) | 69.0 * (3.8) |
2,6-Dimethyl-7-octene-2,6-diol | nd | 58.8 * (3.1) | 53.0 * (3.3) | 35.6 * (3.6) | 39.6 * (3.3) |
2,6-Dimethyl-2,7-octadien-1,6-diol | 12.0 (0.6) | 13.3 (0.9) | 6.8 (3.6) | 10.3 (3.6) | 5.8 (1.5) |
OTHER VOLATILE COMPOUNDS | | | | | |
4-Vinylphenol | 63.2 (1.2) | 89.6 * (3.3) | 65.6 * (5.8) | 56.9 (3.5) | 59.3 (3.1) |
2-Methoxy-4-vinylphenol | 89.0 (6.1) | 103.0 * (5.3) | 105.3 * (6.5) | 88.6 (3.9) | 83.6 (3.3) |
2-Phenylethanol | 1890.2 (43.4) | 3056.5 * (39.8) | 3636.8 * (36.8) | 3308.8 * (36.6) | 3333.3 * (33.6) |
2-Phenylethyl acetate | 28.0 (4.1) | 56.3 * (6.3) | 33.3 (1.3) | 33.6 (3.3) | 16.3 (3.5) |
We were not able to detect significant increase in the level of nerol and geraniol (sweet rose) after the addition of non-Saccharomyces strains. The concentration of cis-5-vinyltetrahydro-1,1,5-trimethyl-2-furanmethanol was not increased despite the addition of any of the four non-Saccharomyces yeasts, while the other oxides (trans-5-vinyltetrahydro-1,1,5-trimethyl-2-furanmethanol, cis-6-vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol and trans-6-vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol) were not detected in controls and in inoculations. Trans-5-vinyltetrahydro-1,1,5-trimethyl-2-furanmethanol (faint burning odor) was detected in the HV1 and HV3 inoculation tests while cis-6-vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol and trans-6-vinyltetrahydro-2,2,6-trimethyl-2H-pyran-3-ol (floral, fresh) only were detected in HU7 and HU8 ones. Moreover, 2,6-dimethyl-2,7-octadien-1,6-diol levels were not affected.
Linalool (floral, with a touch of spiciness) and derivative compounds and aromatic alcohols were detected at the same level of control wine, after the addition of any of the four isolates. Terpineol (odor similar to lilac) increased only after the addition of
H. uvarum isolates, but not when
H. vineae strains were added. The same results were recorded for 4-vinylphenol (barnyard, medicinal and mousy) and 2-methoxy-4-vinylphenol (wine-like and curry). Linalool, 2-phenyl ethanol and 4-vinylphenol compounds are associated with fruity and medicinal characteristics, respectively [
22].
The analysis of the other compounds revealed an increase in concentration when yeasts were added, therefore assessing the effect of glycosidases. Wines treated with
Hanseniaspora strains produced more 2-phenyl ethanol as previously described [
23,
24].
H. uvarum HU8 provided improvements in 2-phenylethyl acetate production as previously reported for other
Hanseniaspora strains [
25,
26,
27].
4. Discussion
The genus
Hanseniaspora is one of the most abundant among the non-
Saccharomyces yeasts present on the surface of intact grapes [
22], and it is also considered to be one of the main producers of glycolytic [
16] and other enzymatic activities [
28]. Some authors consider substrate assimilation miniaturized systems not to be appropriate tools for the identification of non-
Saccharomyces yeasts isolated from natural ecosystems, although they can complement RFLP analysis [
3]. The results of our work show that in some of the studied species (
H. valbyensis,
H. osmophila and
H. occidentalis), the amplification of the ITS1–ITS2 region generates fragments of similar size, corresponding with the physiological profiles. In the other three species, the results of the two methods are not comparable. This may be because the substrate assimilation miniaturized identification methods are not fully precise for the identification process of the six species of yeasts analyzed [
29]. The miniaturized systems help to characterize the strains, but as they are strain dependant, they vary among isolates included in the same species (by molecular typing).
Previous studies pointed to the existence of a unique restriction profile for all strains of the genus
Hanseniaspora, after the digestion of a standard amplicon of approximately 750–775 bp [
3,
29,
30]. Our results show some differences, as
H. osmophila,
H. occidentalis, and
H. valbyensis showed a unique restriction profile for all of the assayed strains. However, in the other three species, several restriction profiles within a single species can be observed. Among the
H. guilliermondii strains, only the group HG4–HG5 showed a similar profile to that previously reported [
30]. The restriction profiles of the
H. vineae strains were different from those described by other authors [
3,
29]. In
H. uvarum we find greater diversity, obtaining a larger number of profiles. Only the group formed by HU3 and HU9 strains showed a profile similar to that described in other publications. Moreover, amplicons from HU6 and HG2 strains were different in size from those of other strains (372 bp). Some of the strains included in this study were further used for amplification of the region D1/D2 [
4] and subsequent sequencing, yielding coincident homologies in some of the strains. According to these data, we conclude that there is a dispute in the identification of this genus of yeasts, and further studies are needed in order to clarify this situation.
The search for enzymatic activities in non-
Saccharomyces yeast is a crucial point due to the contribution of these enzymes to the improvement of the aroma of wines. Interesting studies have been reported that demonstrate the role of glycosidases on microbial hydrolysis of glycosides during the winemaking process [
32]. Some of the aromatic precursors found in wine, such as some terpenes, are in the glycosylated form, which is not volatile and cannot contribute to the aroma of wine. The
Hanseniaspora strains analyzed in this study have no relevant α-rhamnosidase or α-arabinosidase activities, but β-glucosidase and β-xylosidase were frequently detected; strains with increased activity (HU7, HU8, HV3, HO2 and HOC1) could be selected for further study, similar to those described above [
33,
34].
Previous studies suggest the importance of the proteolytic activity of yeasts in relation to the reduction of turbidity in wine and other fermented beverages [
2]. Our work confirms that some strains are capable of producing
Hanseniaspora proteolytic enzymes. These strains belong to the species
H. guilliermondii,
H. occidentalis and
H. valbyensis. This activity has already been described by other authors in some strains of
Kloeckera apiculata (
H. uvarum) [
2], although increased activity has also been reported in other yeasts, such as
Candida pulcherrima and
Pichia anomala [
28]. Yeast proteases may liberate amino acids and peptides from grape protein during fermentation, which can benefit the growth of microorganisms during or after alcoholic fermentation. Another aspect is that yeast cells may release nitrogen-containing metabolites into the media. The composition of amino acids, peptides and proteins in wine is based on grape-related compounds transferred and transformed during the winemaking process and breakdown products through protease activity from yeasts and compounds released by yeasts [
35]. Results obtained in our laboratory in previous work allow us to conclude that protease activity in
Pichia and
Wickerhamomyces isolates was very low [
18], in accordance with results obtained by other authors [
2,
28]. These authors suggested that
Hanseniaspora isolates could be a more interesting group of yeasts to obtain this enzymatic activity, but some contradictory data have been obtained. Many of these studies have been conducted with
H. uvarum (
K. apiculata) isolates and, on the basis of the results obtained in our work, the exocellular protease of this species has a very low activity. On the other hand, assays performed by these authors have used acidic buffers and we have shown that protease from
Hanseniaspora yeasts is pH dependent, showing maximum activity at pH 6.0.
The discovery of pectinase in yeast is an asset to winemakers, because of its importance in winemaking. However, studies related to the search for pectinase activities are often contradictory [
2,
28]. Our results point to a low production of pectinase and polygalacturonase by
Hanseniaspora strains, as already described [
8,
36]. Remarkable lipase and esterase activities were not found in these yeasts and, in fact, some studies support the absence of lipolytic enzymes in the genus
Hanseniaspora [
28].
Both glycosidases and proteases have direct applications in the wine industry, but in wine some conditions are present that may affect their activity. The high concentration of sugars in the must as well as the ethanol concentration in the wine may inhibit glycosidase enzymes [
37]. In this work, we have studied the influence of both factors on the stimulation of the enzymatic conditions in must and wine. Our results are in agreement with Strauss
et al. [
2] who detected a significant negative influence of glucose on β-glucosidase and β-xylosidase activities. This negative influence was greater than that exerted on ethanol enzymes, suggesting that they may be less effective in wine.
Monoterpenes, benzene derivatives, aliphatic components and norisoprenoids are habitually involved in Muscat grape wine and juice. These compounds have been identified in the glycosidically bound form: consequently, their release could enhance wine aroma. Volatile compounds in Muscat wine were analyzed by GC/MS. Muscat wine (13.2%
v/
v initial alcohol) exhibited only a moderated overall terpene increase (1.1 to 1.3-fold) when inoculated with these yeasts. These results are conditioned by the effect of ethanol on glycolytic enzymes. Basically, the use of these strains offered an increase in the levels of ho-trienol, 2-phenylethanol and 2,6-dimethyl-3,7-octadien-2,6-diol in wine. The sum of ho-trienol, linalool and terpineol seems to play a central role in the aromatic definition of the wines of Alvarinho and Loureiro cultivars [
38]. 2-Phenylethanol also contributes by adding floral and fruity notes to these wines, and its presence is connected to the metabolic activity of the non-
Saccharomyces yeasts [
39]. Our results are similar to the interpretations of Fernandez-González
et al. [
40], who have shown the ability of several wine yeasts to hydrolyze norisoprenoids, benzenoids glycosides and terpenoids; among wine yeasts
H. uvarum was able to hydrolyse both glycoconjugated forms of furanic and pyranic oxides of linalool. Our results open the opportunity to the use of these strains for improving the aromatic characteristics of wines, in regard to the liberation of terpenes. The production of wines with the addition of non-
Saccharomyces strains has been habitually related to high concentrations in vinyl-phenols (4-vinyl-phenol, 4-vinyl-guayacol) reaching concentrations of up to 1 mg/L [
41,
42]. The concentration of 4-vinyl-phenol in the studied wines was under 90 μg/L, which enables the use of our selected strains in winemaking.