Eur Food Res Technol DOI 10.1007/s00217-012-1874-9
ORIGINAL PAPER
Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.) Gustavo Cordero-Bueso • Braulio Esteve-Zarzoso Juan Mariano Cabellos • Mar Gil-Dı´az • Teresa Arroyo
•
Received: 22 September 2012 / Revised: 5 November 2012 / Accepted: 10 November 2012 Ó Springer-Verlag Berlin Heidelberg 2012
Abstract Non-Saccharomyces yeast species assume an important role in wine flavor. Notwithstanding, the chemical basis for the flavor characteristics of wines from some grape varieties is not yet defined. The value of this work lies in the use of Malvar white grape, an autochthonous variety from Madrid (Spain) winegrowing region to conduct spontaneous fermentations. This is the first time that a comparative characterization of a wide range of non-Saccharomyces species and a comprehensive analysis of these yeast-derived volatiles has been carried out in this grape variety. b-glucosidase and pectinase (polygalacturonase) extracellular activities were tested on agar plates as primary selection criteria among the 504 non-Saccharomyces isolated from Malvar spontaneous fermentations during four consecutive harvests. Analysis of the wines obtained after fermentation using the selected yeast strains indicates that non-Saccharomyces yeasts isolated along the fermentative process seem that could have a positive impact, showing a high variability in the volatile compounds contributing to the organoleptic characteristics of Malvar wines. Torulaspora delbrueckii CLI 918 was defined as the yeast strain with potential interest for its contribution to the aromatic wine profile with flowery and fruity aromas and could be used in mixed starter cultures with
G. Cordero-Bueso (&) J. M. Cabellos M. Gil-Dı´az T. Arroyo Departamento de Agroalimentacio´n, Instituto Madrilen˜o de Investigacio´n Y Desarrollo Rural Agrario y Alimentario, Autovı´a A2 km 38.2, Alcala´ de Henares, 28800 Madrid, Spain e-mail:
[email protected] B. Esteve-Zarzoso Biotecnologia Enolo`gica, Departament de Bioquimica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i Virgili, Marcelli Domingo s/n, 43007 Tarragona, Spain
Saccharomyces cerevisiae. However, Hanseniaspora guilliermondii increased the volatile acidity and ethyl acetate, but this species along with the genus Pichia and Candida seem to provide a high quantity of extracellular enzymes which may be beneficial in wine making. Keywords Yeast identification Volatile compounds b-Glucosidase Polygalacturonase Malvar wine
Introduction The Appellation of Origin ‘‘D.O. Vinos de Madrid’’ is relatively new, created in 1990. The most cultivated varieties are Aire´n and Malvar (white), and Garnacha and Tempranillo (red) (all of them Vitis vinifera L.), with a total extension of 11.758 ha. Malvar is an autochthonous variety for this Appellation of Origin, whereas Aire´n, Garnacha and Tempranillo have major extensions all over the Iberian Peninsula [1–4]. Part of the economic development of this area is based on wine production. Thus, winemakers are constantly searching for new techniques to modulate wine style. Exploiting indigenous yeasts strains associated with the grape berries is emerging as a new commercial option to avoid the competitive market. It is well known that non-Saccharomyces yeasts possess low fermentation capacity and cannot raise must fermentation alone, due to their sensitivity to ethanol and their less competitiveness in media with high sugar concentration [5–7]. Subsequently, ethanol-tolerant species such as those assigned to the genera Saccharomyces could take over fermentation. Several studies showed that non-Saccharomyces species achieved a larger population in the early stages of fermentation before they died off [8–11]. Alternatively, the ability of some non-Saccharomyces strains to grow at 14 %
123
Eur Food Res Technol
(v/v) ethanol is noteworthy [12]. Species such as Hanseniaspora guilliermondii, Torulaspora delbrueckii, Schizosaccharomyces pombe, Zygosaccharomyces bailii, Candida zemplinina, Candida stellata, Lachancea thermotolerans (formerly Kluyveromyces thermotolerans) and Candida apicola were able to survive at the middle or even until the end of fermentation [4, 12–17]. Furthermore, using cultureindependent techniques such as DGGE, real-time PCR or fluorescence ‘‘in situ’’ hybridization (FISH), non-Saccharomyces yeast communities have been detected during all fermentation process [18–20]. Nevertheless, it well established that spontaneous fermentations are not the result of the action of a single species [21]. Oftentimes, non-Saccharomyces yeasts have been associated with high volatile acidity, ethyl acetate production and off-flavors compounds [17, 22]. Fleet [9] reviewed numerous studies which have been emphasized the potential positive role that they play in the organoleptic characteristics of wine. Yeasts with low fermentative capacity affect the characteristics of the wine due to the production of secondary metabolites which can favor or harm the final product’s quality [7, 12, 16]. Fatty acids produced by non-Saccharomyces could interfere negatively and inhibit to S. cerevisiae [23]. Furthermore, many fermentative metabolites such as the esters hexyl acetate, isoamyl acetate and 2-phenylethyl acetate make the greatest contribution to the characteristic fruity odors of wine fermentation bouquet [24, 25]. Moreover, several authors have even reported that some yeasts produce extracellular enzymes [26–32]. Thus, some of the chemical compounds cited above could be the result of the extracellular enzymes secreted to the media by non-Saccharomyces yeasts present during the early stages of must fermentations [33]. Hence, they require specific research and understanding to prevent any unwanted consequences they might cause or to exploit their beneficial contributions to the biotechnological process [9]. Nowadays, the inclusion of non-Saccharomyces wine yeast species as part of mixed starter cultures together the genus Saccharomyces strains has been suggested as a way of taking advantage of spontaneous fermentations without running the risks of stuck fermentations or wine spoilage [9, 25, 34, 35]. Notwithstanding, the majority of these mixed starter cultures used in must fermentations have been developed selecting only some of the most abundant yeast species isolated at the beginning of the fermentation process such as C. zemplinina, Hanseniaspora uvarum yeast species [20], Lachancea thermotolerans and T. delbrueckii [36] among other works. Hence, new challenges to enhance the appeal and value of wine elaborated by traditional technology are being achieved by selecting and using novel autochthonous non-Saccharomyces and Saccharomyces strains. Thus, nowadays is possible to find some commercial
123
non-Saccharomyces yeasts. Greater understanding of yeast biochemistry and physiology is enabling the selection and development of yeast strains that have defined specific influences on process efficiency and wine quality [9]. Species characterization was carried out in Malvar grape juice to reveal new data relevant to actual wines. Furthermore, despite that Malvar grapevine variety is commonly used in commercial wineries in the Appellation of Origin ‘‘D.O. Vinos de Madrid,’’ it has been poorly studied [1, 37]. Thus, in the present study, we characterized the enological relevance of 12 selected non-Saccharomyces wine yeast species isolated from spontaneous fermentations of white wines from grape berries from Malvar variety, with the final aim of selecting those strains and species with a biotechnological interest to propose their use in wine mixed starter cultures to improve the organoleptic properties of the regional Malvar wines. Non-Saccharomyces strains were selected according to their enzymatic profile. These strains were characterized on natural must to obtain a better knowledge of their effect in the white wine grape variety Malvar.
Materials and methods Fermentation procedure and yeast isolation Spontaneous fermentations were conducted during 2006, 2007, 2008 and 2009 harvests at the IMIDRA’s experimental cellar located in the Madrid winegrowing region, Spain (40°80 1.586400 N,3°220 26.975400 W, and 743 m altitude). Grapes were collected from Malvar (Vitis vinifera L.) grapevine white variety to elaborate the wines, which were obtained in accordance with the winery standard vinification practices. Approximately 500 L of grape juice was placed in duplicate stainless steel vats every vintage (coded as F1-06, F2-06, F1-07, F2-07, F1-08, F2-08, F1-09 and F2-09). Musts were racked, homogenized and dislodged statically (at 4 °C) adding pectolitic enzymes (Enozym Altair, Agrovin, Spain) (0.01 g L-1) and 50 mg L-1 of sulfur dioxide (SO2). Musts obtained each year showed 1,087, 1,089, 1,095 and 1,092 g L-1 of density, the pH values were 3.27, 3.48, 3.56 and 3.34, and the titratable acidity (expressed as g L-1 of tartaric acid) was 6.80, 5.11, 7.33 and 7.85, respectively. Spontaneous fermentations were conducted at controlled temperature of 20 °C, monitoring daily the evolution of the density. Wine sampling and yeast isolation during spontaneous fermentations Samples were taken from every vat during the vinification process at different densities; D1 = initial density,
Eur Food Res Technol
D2 = 1,085–1,070 g L-1, D3 = 1,060–1,050 g L-1, -1 D4 = 1,030–1,025 g L , D5 = 1,010–1,000 g L-1 and D6 = 990 g L-1. Thus, forty-eight 500 mL sterile plastic flasks were filled with the must/wine from the center of the vessels, kept under refrigeration and transported to the laboratory. Samples, after dilutions, were spread onto YPD plates (yeast extract 1 % (w/v), meat peptone 1 % (w/v), glucose 2 % (w/v) and agar 2 % w/v). YPD media was supplied with 0.5 g L-1 chloramphenicol (Sigma) and 0.15 mg L-1 biphenyl (Sigma) in order to avoid molds and bacterial growth. The plates were incubated for 48 h at 28 °C. Yeast colonies were counted, and 30 colonies were randomly selected from each fermentation sample for their identification. DNA extraction and identification of yeast colonies Yeast identification was carried out by means of PCR– RFLP of the 5.8S-ITS ribosomal region as previously described by Cordero-Bueso et al. [2]. Restriction patterns of the ribosomal region were compared to those previously described by Guillamo´n et al. [38] and Esteve-Zarzoso et al. [39]. Furthermore, these identifications were also corroborated using the partial 26S-rRNA gene sequences (D1/D2 domains) of two representatives of each restriction pattern, using the primers NL1 and NL4 [40]. Purification and sequencing of PCR products were carried out by Macrogen Inc. facilities (Seoul, South Korea) using an ABI3730 XL automatic DNA sequencer. The BLAST search (Basic Local Alignment Search Tool, http://www. ebi.ac.uk/blastall/nucleotide.html) was used to compare the sequences obtained with databases from the European Molecular Biology Laboratory (EMBL). We considered identification to be correct when gene sequences showed identities of 98 % or higher. Extracellular enzymatic activities b-Glucosidase enzymatic activity was carried out by replica plating of the isolated yeasts onto selective medium as described by Strauss et al. [28]. The pH media was adjusted to 5.0 before autoclaving (121 °C, 20 min). Two milliliters of a filter-sterilized (1 % w/v) ammonium ferric citrate was added to 100 mL of media. The inoculated plates were incubated at 30 8C for 5 days. Hydrolysis of arbutin by yeasts was identified as a dark brown color in the media. One mg mL-1 of a pure enzyme extracted from almonds (5.2 U mg-1 E.C.3.2.1.21, Sigma-Aldrich, Germany) was used as positive control. In order to establish a rapid semiquantitative method to measure the b-glucosidase activity, a colored plate scale from 0 to 7 (cream until dark brown) was elaborated.
Isolates were also screened for polygalacturonase production by the method proposed by Strauss et al. [28]. Colonies were rinsed off the plates with Milli-Q water before staining the plates with 0.1 % (w/v) Ruthenium Red (E. Merck, Darmstadt, Germany). A purple halo around the strain identified pectinase activity, according to the intensity of this, a color scale was elaborated: no color (0), pink (1), pink/purple (2) and purple (3). Enological characterization of the selected nonSaccharomyces yeast strains Triplicate fermentors with 400 mL of the same filter-sterilized must from Malvar grape variety collected at 2010 harvest (density 1,094 g L-1, pH 3.30, titratable acidity 6.86 g L-1) were inoculated with a final concentration of 106 cells mL-1 of pure selected yeast culture (12 nonSaccharomyces and 1 Saccharomyces cerevisiae used as control pattern), from a pre-culture grown for 48 h in the same must. Furthermore, must was supplied with a commercial complex fermentation activator (Actipasa, Agrovin, Spain) containing ammonium sulfate and thiamin to replace the nutrients removed during filtration. Fermentors and fermentation conditions were the same as those previously described by Cordero-Bueso et al. [2]. When sugar content was lower than 2 g L-1 or after 30 days of fermentation, analysis of principal enological parameters and volatile compounds analysis were conducted. Enological parameters of the fermentation assays Alcoholic degree measurement from the different wine fermentations was obtained using near-infrared reflectance method proposed by Linstromberg and Baumgarten [41]. The amount of fermentable sugars was monitored by following the official method of the International Organization of Vine and Wine [42]. The fermentative capacity was calculated as the difference between the initial and final sugar content. The rate of sugar consumption was obtained from the linear regression equation of the log part of the curve depicting the amount of reducing sugars as a function of time. Fermentation velocity (VF) was measured checking daily the sugar percentage lost during the fermentation. Also, V50 amount of sugar daily transformed by the yeasts when 50 % of the sugar content had been used up was evaluated. pH, free and total sulfur dioxide, titratable acidity and volatile acidity were measured following the Commission Regulation [43] analysis methods. Glycerol and 2,3-butanodiol compounds were determined using the Feuilles verts 588 (FV) method following the official method of the International Organization of Vine and Wine [42].
123
Eur Food Res Technol
Characterization of the volatile fraction of the fermentations Six major volatile compounds were determined by gas chromatography coupled to flame ionization detector (GC–FID) and 17 minor volatiles by gas chromatography coupled to mass spectrometry (GC–MS) following the procedure of Gil et al. [1].
where only a reduced number of colonies were found. However, it was frequently found at the later middle stages and the only one isolated at the end of the fermentation process (D = 990 g L-1) (see Table 1). Interestingly, the S. cerevisiae yeast strain more frequently isolated (implantation rate up to 80 %) in the different fermentations (data not shown) and used as control in this work belongs to the most common genotype isolated and previously described as autochthonous of the ‘‘D.O. Vinos de Madrid’’ [2].
Statistical analysis Enzymatic activities One-way ANOVA and principal component analysis (PCA) were carried out with 20.0 SPSS (Inc. Chicago, USA) for Windows statistical package (significance level P = 0.05).
Results Microbiological analysis Over the four vintages, a total of 1,440 isolates were obtained. Molecular identification using ITS-5.8S amplification and restriction analysis showed 13 different yeast species (504 isolates were identified as non-Saccharomyces and 936 as Saccharomyces cerevisiae). The D1/D2 region of the 26S-rDNA of two yeast strains of the species identified was also sequenced in order to confirm that the species were well identified. These species identified using both molecular methods were C. stellata, H. guilliermondii, K. fluxuum (formerly P. fluxuum), L. fermentati (formerly Z. fermentati), L. thermotolerans (formerly K. thermotolerans), M. guilliermondii (formerly P. guilliermondii), P. kudriavzevii (formerly I. orientalis), P. membranifaciens, S. cerevisiae, P. carsonii (formerly D. carsonii), S. pombe, T. delbrueckii and W. anomalus (formerly P. anomala). Table 1 shows the viable counts onto non-selective YPD agar and the number of colonies belonging to the different species isolated during the different fermentation processes over the 4 years of study. In general terms, the higher yeast diversity was found during the first days of fermentation showing similar results over the 4 years. The yeast species M. guilliermondii was exclusively found in must fermentations of the vintage of 2006, while the species K. fluxuum, P. kudriavzevii and P. carsonii were isolated during the harvests of 2006 and 2007, and in some cases, they were only isolated from one of the duplicate vats sampled. We can consider these species as punctual presence, however, the other species isolated were all found in both tanks sampled. L. thermotolerans, L. fermentati, W. anomalus and T. delbrueckii were the most frequent non-Saccharomyces species. These yeast species were also isolated along each fermentation process. In all cases, the predominant species was S. cerevisiae. Its presence was little common at the first stages of the fermentation,
123
A preliminary experiment was carried out over all isolates identified as non-Saccharomyces by testing their capacity for producing extracellular b-glucosidases and pectinases (polygalacturonases). Those which showed a high b-glucosidase and pectinase activities were selected to perform inoculated fermentation assays. Table 2 shows the results of the production of both extracellular hydrolytic enzymes (b-glucosidase and pectinase) by the different yeast strains which showed higher production of extracellular enzymes of interest in enology. The amount and range of extracellular enzymes produced varied within the different isolates of the same species. Only those strains (one for species) which showed strong b-glucosidase and/or pectinase activities were selected for the subsequent individual fermentations assays. The yeast strains CLI 679 (classified as P. membranifaciens), CLI 1219 (classified as L. thermotolerans), CLI 1218 (classified as W. anomalus) and CLI 921 (classified as H. guilliermondii) were the strains with higher b-glucosidase activity in the assay conditions. Pectinases belong to the group of carbohydrases which catalyze the breakdown of pectic substances. Our results clearly indicated that the isolates belonging to Candida and Pichia (with the exception of P. membranifaciens) genus had lead important quantities of pectinases (polygalacturonases) (Table 2). The strains CLI 920 (C. stellata) and CLI 1216 (P. kudriavzevii) showed the highest values. Significant pectinase activity was also found in W. anomalus (CLI 1218) and H. guilliermondii (CLI 921). Table 2 is also shown the accession number of the D1/D2 region sequenced and deposited in GenBank of the selected nonSaccharomyces yeast strains. Characterization of the principal enological parameters using the non-Saccharomyces yeasts A reference strain of Saccharomyces cerevisiae isolated from the same fermentations of Malvar was included in the characterization studies to relate these data to similar studies and to obtain a better understanding of the results. Table 3 shows the means and standard deviations of the principal enological parameters related to the triplicate
Eur Food Res Technol Table 1 Viable counts and distribution of the 13 yeast species isolated and identified from the duplicate vats (F1 and F2) in the harvests of 2006, 2007, 2008 and 2009 during the spontaneous fermentation (D1–D6) of Malvar musts Vintage
2006
Sample
CFU mL-1 YPD agar
C. ste.
H. gui.
Dl
4.61 9 106
7
1
D2
3.86 9 107
K. flu.
L. fer.
L. the.
M. gui.
P. kud.
P. mem.
P. car.
S. cer.
S. pom.
W. ano.
1
2
5
7
1
4
T. del
F1-06 16
2
13
8
1 2
3 1
3
D3
2.24 9 l0
6
D4
1.12 9 107
1
D5
1.23 9 106
30
D6
1.01 9 105
30
9
27
3 3 2
F2-06
2007
Dl
3.81 9 106
3
D2
3.46 9 107
2
D3
22
1
1
3
11
5
2.97 9 107
6
2
D4
2.45 9 106
1
D5
1.96 9 105
30
D6 F1-07
1.31 9 105
30
Dl
3.38 9 106
D2
5.96 9 106
D3
3
1
3 1
2
3
4
2
18
2
2
26
15 3
17
1.14 9 107
6
4
D4
5.50 9 107
3
D5 D6
1
3
2 7 2
6 2
16
2
26
1
1.75 9 105
29
1
1.91 9 104
30
F2-07
2008
Dl
9.98 9 105
6
D2
4.36 9 107
1
7
19 3
16
3
6
2
3 9
1
D3
6.87 9 10
13
1
D4
7.51 9 106
2
26
2
D5
1.75 9 105
1
29
D6
8.74 9 105
F1-08 Dl
3.51 9 106
6
12
2
7
D2
6.66 9 107
2
8
16
4
D3
7.97 9 107
9
20
D4
8.91 9 106
1
28
D5
5.76 9 104
30
D6
5.70 9 104
30
2
3
1
1
30 3
1 1
F2-08 Dl
2.99 9 106
D2
8.11 9 106
D3
2.02 9 107
2
D4
7.51 9 106
5
D5
1.75 9 105
30
D6
8.74 9 104
30
5
15
1
9 6
1
4 17
1
4 3
1
19
2
22
3
123
Eur Food Res Technol Table 1 continued Vintage
2009
Sample
CFU mL-1 YPD agar
C. ste.
H. gui.
K. flu.
L. fer.
L. the.
M. gui.
P. kud.
P. mem.
P. car.
S. cer.
Dl
3.89 9 106
2
2
D2
5.43 9 107
2
D3 D4
7.12 9 107 8.91 9 106
D5
1.62 9 105
30
D6
5.90 9 104
30
S. pom.
W. ano.
T. del
1
F1-09 12 3
4
4
5
3
22
1
3
2
23 30
1
F2-09 Dl
3.51 9 106
2
1
D2
6.66 9 107
1
2
D3
7
7.97 9 10
D4
8.91 9 106
29
D5
9.86 9 104
30
D6
5.79 9 104
Total
17 1 2
7
2
2
6
3
1
11
1
5
24
1
1 1
30 37
17
6
59
240
3
4
25
5
936
18
50
40
C. ste.: Candida stellata, H. gui.: Hanseniaspora guilliermondii, K. flu.: Kregervanrija fluxuum, L. fer.: Lachancea fermentati, L. the.: Lachancea thermotolerans, M. gui.: Meyerozyma guilliermondii, P. kud.: Pichia kudriavzevii, P. mem.: Pichia membranifaciens, P. car.: Priceomyces carsonii, S. cer.: Saccharomyces cerevisiae, S. pom.: Schizosaccharomyces pombe, T. del.: Torulaspora delbrueckii, W. ano.: Wickerhamomyces anomalus
Table 2 Selected strains according to b-glucosidase (scale 0–7) and pectinase (scale 0–3) activities
GenBank ID of the sequences are also included
Species
Strain
Enzymatic activities b-Glucosidase
Pectinase
C. stellata
CLI 920
JQ707776
2
3
H. guilliermondii
CLI 921
JQ707775
4
2
K. fluxuum
CLI 622
JQ804984
2
1
L. fermentati
CLI 1220
JQ804981
1
0
L. thermotolerans
CLI 1219
JQ707778
6
0
M. guilliermondii
CLI 1217
JQ707780
1
0
P. kudriavzevii
CLI 1216
JQ707777
3
3
P. membranifaciens
CLI 679
JQ707779
7
1
P. carsonii
CLI 1221
JQ707781
0
1
S. pombe
CLI 1085
JQ804983
1
0
T. delbrueckii W. anomalus
CLI 918 CLI1218
JQ707782 JQ804982
0 5
1 3
small-scale fermentations carried out with the selected strains. Only T. delbrueckii was able to consume the reducing sugars present in musts, showing after 30 days of fermentation similar quantities of these compounds than S. cerevisiae. In addition, S. pombe reached similar levels leaving on must 3.8 g L-1 of the reducing sugars. Regarding the other yeast species, they were not able to consume all the reducing sugars present on must, showing values ranging from 30.7 g L-1 for L. fermentati to 175 g L-1 for P. carsonii. This behavior was also observed
123
GenBank ID
for the alcoholic degree, seven of the species studied achieved at the end of fermentation around the 3 % of ethanol. With respect to the volatile acidity, only H. guilliermondii reached a significantly value of 0.9 g L-1. Volatile compounds analysis After fermentations, 23 volatile compounds were analyzed (Table 4). The statistical analysis data obtained for S. cerevisiae were different of those obtained for the
Eur Food Res Technol Table 3 Data (mean ± S.D) of the triplicate fermentations of the sterilized Malvar must inoculated with the different yeast strains isolated after fermentation Parameters
S. cer.
Alcoholic degree*
12.50 ± 0.08e
Fermentative capacity
C. ste. 3.5 ± 0.4b
d
12.00 ± 0.07
3.0 ± 0.9
e
VF
2.4 ± 0.6 a
pH
3.6 ± 0.9
a
8.0 ± 1.0
15 ± 2.7ab à
Volatile acidity
Titratable acidity }
Reducing sugar
4.90 ± 0.07
Glycerol
2,3-butanodiol Parameters
Fermentative capacity
Glycerol
2,3-butanodiol
b
144.0 ± 20.1b
243.0 ± 61.9bc
195.0 ± 11.2ab
P. kud. b
P. mem. b
P. car. b
S. pom. a
3.4 ± 0.1
3.0 ± 1.2
2.6 ± 0.2
0.0 ± 0.0
3.7 ± 0.6bc
3.2 ± 1.1b
2.9 ± 0.2ab
0.8 ± 0.2a
a
a
2.4 ± 0.6
1.3 ± 0.4
a
1.2 ± 0.4
a
3.9 ± 0.7
3.7 ± 2.2
abc
abc
3.9 ± 0.1
a
22.0 ± 1.8ef
21.0 ± 1.0def
24.0 ± 4.7f
16.1 ± 2.4abc
0.1 ± 0.0
a
3.6 ± 0.9
ab
3.5 ± 0.5 b
3.9 ± 0.3 b
b
0.1 ± 0.0
a
3.7 ± 1.1
a
157.6 ± 56.2
175.0 ± 22.2
b
b
196.0 ± 34.2
190.4 ± 10.3
4.6 ± 0.3 a
138.0 ± 26.2
9.0 ± 1.7
7.0 ± 1.2ab
17.0 ± 1.2bcd
18.0 ± 1.0bcde
18.0 ± 3.9bcde
325.0 ± 67.7
a
0.2 ± 0.1b
ab
3.5 ± 0.2a
3.9 ± 1.0
a
3.8 ± 1.4
132.2 ± 18.2b
1.3 ± 0.8
b
b
4.9 ± 1.1 cd
b
0.2 ± 0.0
3.9 ± 0.3
b
4.0 ± 0.7
3.6 ± 1.1a
bc
3.7 ± 0.8
ab
141.5 ± 23.2
ab
1.5 ± 0.2a
a
5.5 ± 1.3
0.2 ± 0.0
b
3.8 ± 1.2
1.5 ± 0.1a
d
b
b
de
15.0 ± 3.2
10.0 ± 1.2
140.6 ± 43.2
ab
3.4 ± 0.6bc
d
c
6.0 ± 1.4
0.1 ± 0.0
12.6 ± 2.2d
c
3.9 ± 0.2
8.0 ± 0.4
a
12.4 ± 0.7d
a
8.0 ± 2.0
a
3.1 ± 0.2b
4.8 ± 1.2
8.0 ± 1.8
a
11.9 ± 2.2
c
a
W. ano.
11.8 ± 0.2
10.8 ± 3.3
–
a
T. del. de
–
a
2.5 ± 1.2
4.0 ± 2.0
30.7 ± 12.2
b
210.4 ± 54.5ab
0.1 ± 0.0
Titratable acidity }
142.5 ± 57.2
b
522.4 ± 101.2f
a
§
Reducing sugar
117.8 ± 71.2
b
189.7 ± 33.2ab
abc
}
139.8 ± 34.9
a
373.5 ± 67.5de
4.5 ± 0.2
Volatile acidity
a
3.9 ± 0.6b
a
à
b
3.8 ± 1.2
3.2 ± 0.8
Free SO 2 Total SO 2
4.0 ± 0.9ab
3.7 ± 0.4
a
4.3 ± 0.9
b
pH
a
3.9 ± 0.1
b
0.1 ± 0.0a
0.3 ± 0.0
ab
4.5 ± 0.5
5.3 ± 1.2
VF
0.2 ± 0.0
3.8 ± 0.1
b
22.0 ± 2.2ef
c
3.6 ± 1.3
b
V50
12.0 ± 3.4a
b
7.1 ± 1.6
M. gui. *
17.0 ± 3.2bcd
ab
3.8 ± 1.1
8.0 ± 0.8abc
8.0 ± 0.4
0.9 ± 0.2
ab
3.6 ± 0.3a
abc
9.0 ± 0.2
d
0.2 ± .0.0
b
1.5 ± 0.3
}
18.0 ± 2.0bcde
1.5 ± 0.1a
a
3.6 ± 0.3
bc
9.0 ± 1.0
b
0.14 ± 0.01 §
Alcoholic degree
20.0 ± 2.2cdef
a
3.7 ± 1.2
bc
1.6 ± 0.3a
b
2.2 ± 0.7
a
3.7 ± 0.3
abc
6.0 ± 0.3
2.2 ± 1.4
a
3.7 ± 0.8bc
b
5.3 ± 2.1
a
0.2 ± 0.0
a
3.81 ± 0.01
Free SO 2 Total SO 2
2.5 ± 0.3
a
3.3 ± 0.4b
d
11.7 ± 1.4
a
1.3 ± 0.2
ab
6.7 ± 0.3
3.5 ± 0.9
a
L. the.
10.5 ± 1.5d
bc
5.6 ± 3.2
ab
L. fer.
3.3 ± 0.2b
c
3.7 ± 1.2
18.00 ± 0.04
K. flu.
5.3 ± 0.9c
bc
e
V50
H. gui.
3.2 ± 1.7b
4.3 ± 0.1 ab
196.0 ± 88.8
ef
446.0 ± 81.2
218.0 ± 13.8ab
Fermentation by Saccharomyces cerevisiae was considered as control. Different letters (a–f) indicate significant differences at significance level (p = 0.05) * % v/v;
mg L-1; à: g L-1; acetic acid; §: g L-1; Tartaric acid; }:g L-1
V50 = fermentation velocity consumption of 50 % of the sugar content; VF = fermentation in velocity (% of daily sugar consumption) S. cer.: Saccharomyces cerevisiae, C. ste.: Candida stellata, H. gui.: Hanseniaspora guilliermondii, K. flu.: Kregervanrija fluxuum, L. fer.: Lachancea fermentati, L. the.: Lachancea thermotolerans, M. gui.: Meyerozyma guilliermondii, P. kud.: Pichia kudriavzevii, P. mem.: Pichia membranifaciens, P. car.: Priceomyces carsonii, S. pom.: Schizosaccharomyces pombe, T. del.: Torulaspora delbrueckii, W. ano.: Wickerhamomyces anomalus
fermentations carried out with the different selected nonSaccharomyces. In all cases, acetaldehyde levels achieved were higher than the threshold proposed by Duarte et al. [44]. Among these values, the highest value corresponds to the must fermented by T. delbrueckii (136.6 mg L-1), while in the other fermentations, the values showed are in accordance with the sugars consumption, the highest values corresponds to the higher sugar consumption, with the exception of the fermentations by L. fermentati which showed 56.7 mg L-1 of this compound. Regarding acetoin, the concentrations found were lower than the odor threshold (Table 4) with the exception of the must fermentations by H. guilliermondii. Although ethyl acetate concentrations showed higher values than the threshold proposed in the majority of the
fermentations, only in fermentations by H. guilliermondii has been detected as a significant value, twenty times more than the control (S. cerevisiae). Higher major alcohols Higher major alcohols are produced mainly during the first two stages of the alcoholic fermentation. Isobutanol and 1-propanol were present in all fermentations analyzed. Hence, they had no odor significance for them, with the only exception of T. delbrueckii, which isobutanol concentration was 63.5 mg L-1. Isoamylic alcohols were the higher major alcohols with major influence onto the fermentations. As in the case of acetaldehyde, the values
123
123 50.75 ± 8.8 6.9 ± 2.1ab
Acetaldehyde
Acetoin
0.60 ± 0.05 1.01 ± 0.25c
Ethyl hexanoate
Ethyl octanoate
a
cd
Butyric acid 1.98 ± 0.10 4.97 ± 0.23 6.11 ± 0.20 3.70 ± 0.77d
Hexanoic acid
Octanoic acid Decanoic acid P MCFA
9.4 ± 0.3
47.2 ± 2.3i
Ethyl acetate
1-Propanol
28.4 ± 0.2d
47.5 ± 3.4
25.1 ± 11.1bcd
43.3 ± 27.3d
Acetoin d
nd
19.8 ± 0.9b
Acetaldehyde a
P. mem.
P. kud.
1.2 ± 0.1a
12.9 ± 0.2
nd a
27.5 ± 2.1c
P. car.
0.88 ± 0.15ab
a
bc
bc
a
a
0.42 ± 0.03 0.12 ± 0.01a
0.34 ± 0.00
0.8 ± 0.13
0.31 ± 0.10
0.03 ± 0.00
0.01 ± 0.00
Compound (mg L-1)
14.78 ± 1.20d
b
i
e
Isovaleric acid P SCFA
0.08 ± 0.06
0.49 ± 0.20bcd
1.90 ± 0.04f a
0.03 ± 0.01
Diethyl succinate
a
0.22 ± 0.08
ab
nd
d
Ethyl lactate
0.03 ± 0.00
a
0.09 ± 0.02ab
0.01 ± 0.00
0.09 ± 0.01
b
c
2.01 ± 0.37
ab
c
0.7 ± 0.00
0.03 ± 0.01
Ethyl decanoate P Fatty acid esters
0.08 ± 0.06
0.33 ± 0.01
Ethyl butyrate
d
e
8.38 ± 0.20
0.56 ± 0.03
a
a
Phenyl ethyl acetate P Higher alcohol acetates
0.01 ± 0.00a
0.14 ± 0.01b
Hexyl acetate
a
0.02 ± 0.01 0.01 ± 0.00a
a
0.16 ± 0.00 7.66 ± 0.18d
2.7 ± 1.7
2.5 ± 0.3
bc
cd
85.3 ± 10.3
17.4 ± 0.0
30.1 ± 0.2
e
Isobutyl acetate Isoamyl acetate
c
11.59 ± 0.27
a
e
10.79 ± 0.29
a
0.81 ± 0.01bc
1-Hexanol
2-Phenylethanol P Higher minor alcohols
0.2 ± 0.0a
247.65 ± 0.66
e
175.23 ± 1.74
Isoamylic alcohols P Higher major alcohols
k
37.39 ± 0.58
37.8 ± 2.2
Isobutanol
g
g
35.02 ± 1.83
53.9 ± 0.1
e
1-Propanol
f
b
7.4 ± 2.1ab
18.7 ± 1.1
C. ste.
39.5 ± 1.8
e
Ethyl acetate
c
S. cer.
Compound (mg L-1) a
g
c
ab
ab
ab
a
a
ab
18.4 ± 0.0b
10.7 ± 0.1
a
31.8 ± 12.9
39.9 ± 0.1d
S. pom.
2.44 ± 0.37bc
a
f
ab
0.50 ± 0.00 0.71 ± 0.03b
1.23 ± 0.14
0.52 ± 0.25
0.08 ± 0.00
ab
0.44 ± 0.08abc
0.01 ± 0.00
nd
0.35 ± 0.13
0.08 ± 0.00
b
0.18 ± 0.02b
0.05 ± 0.01
0.04 ± 0.01
0.31 ± 0.10
0.23 ± 0.11
nd
s 0.08 ± 0.01b
5.5 ± 3.5
a
5.2 ± 0.1
c
0.3 ± 0.0a
85 ± 5.4
bc
34.5 ± 0.0
26.1 ± 0.1
24.4 ± 0.1
c
874.4 ± 1.1
f
189.2 ± 23.2e
8.4 ± 0.4
H. gui.
cd
c
a
a
b
a
a
ab
31.6 ± 0.0e
22.3 ± 0.1b
11.7 ± 2.2abc
136.6 ± 0.4f
T. del.
0.74 ± 0.15ab
a
bc
ab
0.38 ± 0.09 0.08 ± 0.00a
0.28 ± 0.01
0.54 ± 0.13
0.18 ± 0.08
abc
0.36 ± 0.12ab
0.01 ± 0.00
s
0.19 ± 0.06
0.04 ± 0.01
ab
0.05 ± 0.02a
0.02 ± 0.00
0.08 ± 0.01
0.11 ± 0.00
0.02 ± 0.00
a
0.03 ± 0.01a
0.03 ± 0.01 0.03 ± 0.01a
2.6 ± 1.3
a
2.2 ± 0.0
a
0.4 ± 0.0ab
bcd
de
i
d
b
91.5 ± 15.3
15.7 ± 3.3
29.4 ± 1.3
46.4 ± 3.1
48.2 ± 9.2
s
20.0 ± 1.0
K. flu. e
i
g
b
d
a a
b
b
bc
b
a
b
43.1 ± 0.0
h
21.6 ± 0.0b
nd
19.0 ± 0.3b
W. ano.
1.82 ± 0.30abc
a
e
a
0.36 ± 0.12 0.52 ± 0.18ab
0.94 ± 0.03
0.41 ± 0.09
0.14 ± 0.00
ab
0.27 ± 0.03ab
0.14 ± 0.06
0.04 ± 0.00
0.5 ± 0.17
0.13 ± 0.01
b
0.14 ± 0.01ab
0.13 ± 0.02
0.10 ± 0.03
0.46 ± 0.14
0.33 ± 0.01
a
0.04 ± 0.01a
0.02 ± 0.00 0.07 ± 0.01b
5.2 ± 3.1
4.8 ± 1.2
c
0.4 ± 0.0ab
122.5 ± 23.7
66.2 ± 1.0
37.1 ± 0.0
19.2 ± 0.0
50.2 ± 0.1
de
8.2 ± 0.9ab
56.7 ± 0.3
L. fer. b
a
b
b
de
c
c
ab
b
ab
a
b
ab
Alcohol, ripe fruit
Fruit, solvent
Flowery, wet
Pleasant, fruity
ODE
0.53 ± 0.05ab
a
ab
0.20 ± 0.07 0.12 ± 0.00a
0.21 ± 0.06
d
1.4 ± 0.15
0.59 ± 0.1
e
0.81 ± 0.32e
0.08 ± 0.02
nd
0.26 ± 0.09
0.06 ± 0.01
b
0.08 ± 0.03ab
0.04 ± 0.01
0.08 ± 0.02
0.21 ± 0.05
0.07 ± 0.01
nd
0.12 ± 0.03 0.03 ± 0.02ab
8.1 ± 4.1
7.0 ± 0.0
d
1.1 ± 0.0c
70.6 ± 3.3
19.7 ± 0.0
25.8 ± 0.1
25.1 ± 0.2
25.3 ± 0.0
b
9.8 ± 2.6ab
16.4 ± 0.1
L. the
306.0*
12.26*
150.0*
0.0025
OTH (mg L-1)
0.59 ± 0.08ab
0.25 ± 0.10a 0.23 ± 0.04a
0.11 ± 0.03a
0.95 ± 0.35c
0.23 ± 0.04bc
0.72 ± 0.13de
0.07 ± 0.01b
0.02 ± 0.00a
0.26 ± 0.10ab
0.03 ± 0.00a
0.11 ± 0.04ab
0.02 ± 0.00a
0.10 ± 0.10b
0.09 ± 0.01a
0.05 ± 0.01a
0.02 ± 0.01a
nd 0.02 ± 0.00a
4.1 ± 2.5a
3.8 ± 0.0b
0.3 ± 0.0a
73.8 ± 11.9bc
11.3 ± 0.7b
28.1 ± 2.4d
34.4 ± 0.0f
50.2 ± 0.5de
4.0 ± 0.7a
14.1 ± 0.2ab
M. gui.
Table 4 Data (mean ± S.D) of volatile composition related to the single fermentations by non-Saccharomyces yeast strains and the autochthonous S. cerevisiae used as control performed using Malvar must
Eur Food Res Technol
a
a
a
0.07 ± 0.01ab 0.03 ± 0.00a 0.22 ± 0.08ab
0.06 ± 0.01ab
0.02 ± 0.00a
0.17 ± 0.06ab
Ethyl octanoate
Ethyl decanoate P Fatty acid esters
a
abcd
a
0.06 ± 0.00a
a
0.27 ± 0.01
0.19 ± 0.03a
Octanoic acid
Decanoic acid P MCFA 0.93 ± 0.31
ab
e
12.5 ± 6
b
0.75 ± 0.17
ab
0.13 ± 0.03a
0.18 ± 0.03
a
0.44 ± 0.05
c
1.51 ± 0.15d
0.86 ± 0.27f
0.65 ± 0.17
cde
0.09 ± 0.00
nd
0.15 ± 0.05ab
0.04 ± 0.00a
0.07 ± 0.01ab
0.01 ± 0.00a 0.03 ± 0.00a
0.11 ± 0.01
a
0.04 ± 0.00a
0.01 ± 0.00a
0.04 ± 0.01
ab
0.02 ± 0.00a
a
10.5 ± 0.1
2.0 ± 0.0
d
18.2 ± 4.2a
7.7 ± 0.0
a
9.3 ± 0.1a
P. car.
3.23 ± 0.81
c
0.44 ± 0.13ab
0.80 ± 0.02
a
1.99 ± 0.07
g
0.7 ± 0.23abc
0.19 ± 0.01abc
0.51 ± 0.22
bcd
0.03 ± 0.00
a
0.10 ± 0.01
b
0.5 ± 0.17b
0.16 ± 0.09c
0.18 ± 0.07b
0.06 ± 0.00b 0.10 ± 0.00ab
0.31 ± 0.08
ab
0.21 ± 0.09a
0.03 ± 0.01a
0.02 ± 0.00
a
0.05 ± 0.00b
b
g
56.8 ± 23.9
45.3 ± 0.3
11.5 ± 0.9
e
83.6 ± 16.9bc
47.4 ± 6.2
h
17.8 ± 0.2b
S. pom.
j
h
a
f
15.07 ± 4.22
d
2.88 ± 0.43c
9.88 ± 5.43
c
2.31 ± 0.10
h
0.76 ± 0.23bc
0.22 ± 0.10abc
0.54 ± 0.21
bcde
0.08 ± 0.00
b
0.10 ± 0.01
b
1.3 ± 0.52c
0.19 ± 0.08c
0.11 ± 0.05ab
0.17 ± 0.10c 0.83 ± 0.23d
2.04 ± 0.61
d
0.53 ± 0.13b
0.12 ± 0.01b
1.37 ± 0.11
c
0.02 ± 0.01a
12.9 ± 7.8
12.0 ± 0.6
0.9 ± 0.0
c
241.2 ± 59.1e
146.1 ± 0.3
63.5 ± 0.4
T. del.
a
a
1.11 ± 0.20ab
0.21 ± 0.04a
0.59 ± 0.14a
0.31 ± 0.00bc
0.58 ± 0.23ab
0.13 ± 0.03ab
0.45 ± 0.11
abc
0.02 ± 0.00
nd
0.13 ± 0.05a
0.01 ± 0.00a
0.06 ± 0.01ab
0.04 ± 0.00a 0.02 ± 0.00a
0.19 ± 0.04ab
0.03 ± 0.01a
0.01 ± 0.00a
0.04 ± 0.00a
0.11 ± 0.03b
2.3 ± 1.3
2.1 ± 0.8
a
0.2 ± 0.0a
104.3 ± 7.5cd
28.4 ± 0.0f
32.8 ± 0.2f
W. ano.
Fatty
Rancid, harsh
Cheese
Blue cheese
Cheese
Apple, fruity
Lactic
Pleasant, soap
Sweet, soap
Acid fruit Green apple
Pleasant, flowery
Fruity, green, pear
Banana
Sweet fruit
Roses
Green grass
Bitter, harsh
Fusel, alcohol
ODE
1.00*
0.500*
0.420*
0.033*
0.173*
0.20
0.157
0.200*
0.005*
0.020* 0.014
0.250*
0.020à
0.030*
1.60*
14.00*
8.00*
30.00*
40.00*
OTH (mg L-1)
S
traces, * thresholds from Gil et al. [1],
thresholds from Duarte et al. [44],
à
threshold from Falcue et al. [64]
S. cer.: Saccharomyces cerevisiae, C. ste.: Candida stellata, H. gui.: Hanseniaspora guilliermondii, K. flu.: Kregervanrija fluxuum, L. fer.: Lachancea fermentati, L. the.: Lachancea thermotolerans, M. gui.: Meyerozyma guilliermondii, P. kud.: Pichia kudriavzevii, P. mem.: Pichia membranifaciens, P. car.: Priceomyces carsonii, S. pom.: Schizosaccharomyces pombe, T. del.: Torulaspora delbrueckii, W. ano.: Wickerhamomyces anomalus
SCFA small chain fatty acids, MCFA medium chain fatty acids
nd non-detected,
Odor descriptors (ODE) and odor thresholds (OTH) described in the literature are included. Different letters (a–g) indicate significant differences at significance level (p = 0.05)
0.7 ± 0.04
a
0.20 ± 0.11
0.67 ± 0.22
d
ab
0.24 ± 0.01
0.38 ± 0.03a
0.84 ± 0.04bc
Hexanoic acid
0.17 ± 0.02abc
0.39 ± 0.09d
0.21 ± 0.07
0.45 ± 0.11
Isovaleric acid P SCFA
0.08 ± 0.02
0.25 ± 0.12
c
0.36 ± 0.01
b
0.02 ± 0.00
c
a
Butyric acid
Diethyl succinate
Ethyl lactate
0.09 ± 0.01b 0.03 ± 0.00a
0.07 ± 0.02b 0.02 ± 0.00a
Ethyl butyrate Ethyl hexanoate
0.02 ± 00
0.11 ± 0.01
a
0.01 ± 0.00a
0.01 ± 0.00a
Phenyl ethyl acetate P Higher alcohol acetates a
s
0.04 ± 0.00a
Hexyl acetate
0.01 ± 0.01
nd
2.4 ± 1.3
2.1 ± 0.6
0.3 ± 0.0
0.02 ± 0.00
a
0.04 ± 0.01a
2.3 ± 1.1
a
1.9 ± 0.1
a
0.4 ± 0.1
a
77.8 ± 8.7bc
Isoamyl acetate
Isobutyl acetate
2-Phenylethanol P Higher minor alcohols
1-Hexanol
ab
102 ± 12.3bcd
16.2 ± 0.0
e
22.6 ± 0.1
32.2 ± 0.3f
Isobutanol
Isoamylic alcohols P Higher major alcohols
33.2 ± 0.0f c
P. mem.
P. kud.
Compound (mg L-1)
Table 4 continued
Eur Food Res Technol
123
Eur Food Res Technol
achieved of this compound are closely related to the sugar consumption obtained in the fermentations by H. guilliermondii, L. fermentati, T. delbrueckii and S. pombe. The total amount of higher alcohols ranged from 7.7 to 247.65 mg L-1, this variation principally is due to the isoamyl alcohol produced, which represent in some cases close to 60 % of the total higher alcohols. Higher minor alcohols This group of volatiles showed minor concentrations than the higher major alcohols. The 1-hexanol was detected in all cases in lower concentrations than the threshold value, except in the case of fermentations by S. pombe which showed the higher value (Table 3). The 2-phenyl ethanol which provides a typical floral chemical composition to the wines was detected in higher concentration than the threshold only in the fermentations by S. pombe (Table 4). Regarding the total amount of these compounds is possible to differentiate two groups, one which includes fermentations by S. pombe and the other one includes all other fermentations, with a statistical significance value of P \ 0.005. Acetate esters Higher alcohol acetates were found to vary between the ranges of 0–0.12, 0.01–1.37, 0–0.12 and 0.01–0.53 mg L-1, respectively, for isobutyl acetate, isoamyl acetate, hexyl acetate and phenyl ethyl acetate. Isoamyl acetate and phenyl ethyl acetate, which have banana and fruity flavors, showed odor significance for the fermentations by T. delbrueckii and L. fermentati (Table 4). Ethyl esters of fatty acids These compounds appear mainly at the first phases of the alcoholic fermentation (Table 4). Important variations in the concentrations found, but in the majority of the cases they showed higher values than the threshold value proposed (Table 4). The total amount produced of these compounds can group the fermentations in five different clusters with significant differences (Table 4), in which the lower value was found in fermentations by W. anomalus (0.13 mg L-1) and the higher one was detected in the fermentation by S. cerevisiae (2.01 mg L-1). Ethyl lactate and diethyl succinate The real contribution to the ‘‘D.O. Vinos de Madrid’’ white wines of these compounds has been previously described as insignificant [1, 37]. Only fermentations by P. membranifaciens exceeded the OTH for both compounds (Table 4).
123
Small and medium chain fatty acids (SCFA and MCFA) Fatty acids are formed basically during the first two phases of the alcoholic fermentations. The small chain fatty acids (SCFA) variation between the fermentations by the nonSaccharomyces yeasts ranged from 0.38 mg L-1 of the fermentations by P. membranifaciens to 1.51 mg L-1 of the fermentations by S. pombe. Regarding the medium chain fatty acids (MCFA), not all compounds in the different fermentations reached the threshold value proposed by Duarte et al. [44]. Thus, only the fermentation conducted by T. delbrueckii showed higher values than the threshold in the three compounds analyzed. Principal component analysis (PCA) The PCA score plots for the enological parameters (Table 3) and the volatile compounds (Table 4) produced by the different yeasts strains and the variance explained by each principal component (PC1 and PC2) which is in brackets are shown in Fig. 1. The main components for PC1 were isovaleric acid (0.970), V50 (0.966), isoamylic (0.962), ethyl butyrate (0.942), hexanol (0.916) and ethyl lactate (0.894). PC1 differentiates Saccharomyces from the non-Saccharomyces yeasts species. PC2 is mostly formed by the phenyl ethanol (0.737), isobutyl (0.726) and isoamyl (0.725). A clearly separate disposition of the principal nonSaccharomyces species according to their capability of transformation the sugar content into ethanol (Fig. 1).
Discussion The value of this work lies in a better knowledge of Malvar grape musts which were used to conduct the yeast isolations and afterward to use it as substrate to study the effect of different wine yeast species isolated during wine fermentation. Furthermore, the selection criteria based on the extracellular enzymatic activities used for the non-Saccharomyces yeasts, ensures the selection of the biotechnological potential yeast strains of interest in enology. Numerous extracellular enzymatic assays were also conducted (manuscript in preparation) despite in this paper is only showed the yeast strains b-glucosidase and pectinase activities which resulted to be the highest. Moreover, countless references reports the beneficial or detrimental influence of several yeast species studied such as K. fluxuum, M. guilliermondii, P. kudriavzevii or P. carsonii on the volatile composition of musts from grape berries. The initial concentration of yeast population at early fermentation phases can range between 104 and 106 CFU m L-1. Non-Saccharomyces species can proliferate to the final population of 106–109 CFU m L-1 in
Eur Food Res Technol Fig. 1 PCA score plots for yeast strains studied in the plan made of the first two principal components (PC1 against PC2). Cste: Candida stellata, Hgui: Hanseniaspora guilliermondii, Kflu.: Kregervanrija fluxuum, Lfer: Lachancea fermentati, Lthe: Lachancea thermotolerans, Mgui: Meyerozyma guilliermondii, Pkud: Pichia kudriavzevii, Pmem: Pichia membranifaciens, Pcar: Priceomyces carsonii, Scer: Saccharomyces cerevisiae, Spom: Schizosaccharomyces pombe, Tdel: Torulaspora delbrueckii, Wano: Wickerhamomyces anomalus
fermenting musts [45]. It is accepted that the content of wild non-Saccharomyces yeasts begins to decrease rapidly after 2 days of their intensive activity in contrast to Saccharomyces species, which takes over the fermentation process [9, 45]. In this study, the viable cell count obtained from the spontaneous must fermentations ranged from 104 to 108 CFU m L-1. Regarding the number of isolates, data showed higher values for non-Saccharomyces compared to the Saccharomyces population isolated during the early and middle phases of the Malvar spontaneous fermentations. Only a few isolates were identified as S. cerevisiae during the first stages of spontaneous fermentations. This is in agreement with the opinion that S. cerevisiae is rarely isolated from grapes and their presence in spontaneous fermenting grapes is in direct association with those socalled residential [46]. However, this quantity of isolates dropped sharply as fermentation process went by and other non-tolerant species die off owing to their low tolerance to the ethanol produced, S. cerevisiae became the predominant species at the end of the spontaneous fermentation. As stated in results, interestingly, in our study, the predominant S. cerevisiae resulted on an autochthonous yeast strain. Recently, Cordero-Bueso et al. [47] pointed out that a commercial yeast strain did not displace autochthonous Saccharomyces and no implantation was produced in the fermentation, as the presence of indigenous yeast strains was not subsequently affected. The presumable effect of these yeast species on the wine flavor was performed by means of a comparative
characterization of yeast-derived volatiles. Moreover, to give better context to the volatile composition of the wines produced by the non-Saccharomyces yeasts studied, the autochthonous S. cerevisiae yeast strain isolated from the same Malvar wines has been included, since the volatile composition of wines obtained from Malvar grape variety is little well known in the scientific literature [1, 37]. Several culture media to isolate non-Saccharomyces yeasts from wine fermentations have been previously described [48], notwithstanding, in this work, we used a general culture medium (YPD) because we are only interested in those yeasts species capable to survive during the early stages of the wine fermentation. In spite of this, 12 non-Saccharomyces yeast species were isolated during the early and middle phases of the wine fermentations. The fact of isolating them by using this culture medium supports our interest in the selection of yeast species able to resist the wine fermentation conditions. Identification was done by ITS-5.8S-ITS restriction analysis and confirmed by sequencing of D1/D2 region. Regarding the presence of the species C. stellata in the earlier stages of fermentation was corroborated using both methods of molecular identification, digestion of ITS-5.8S fragment amplified by PCR and sequencing the D1/D2 region of 26S-rDNA. This is not in agreement with the results obtained by Andorra` et al. [18, 19], in which the authors were able to detect Candida zemplinina during all fermentation process using independent culture techniques. But Csoma and Sipiczki [49] pointed out the possibility of misidentification of C. stellata
123
Eur Food Res Technol
and C. zemplinina. They also described the first one as the most osmotolerant during the fermentation process and the second one as the most resistant to ethanol. Thus, this supports that our species isolated during the first stages of the fermentation was C. stellata and not C. zemplinina. All species considered in this study were characterized because of their isolation at early and middle stages of Malvar wine fermentations until S. cerevisiae dominance. Several authors [21, 26, 28, 30, 32] reported that extracellular enzymes produced by non-Saccharomyces yeasts during the initial phase of wine fermentation can influence in the final organoleptic composition of the wines and might be harnessed to catalyze desired biotransformation during wine fermentations. The action of grape enzymes and Saccharomyces enzymes is insufficient to carry on this transformation completely [28, 32]. From an enological point of view, two of the most important enzymatic activities involved in wine making are pectinases which increase juice extraction from grapes, improve wine clarification and facilitate wine filtration, extraction of phenolic and aromatic compounds [32], and glucosidases which hydrolyze non-volatile glucosidase precursors as well [50]. The addition of exogenous enzymes to solve wine filtration and clarification problems (proteases, pectinases and glucanases) or to increase aroma (glucosidases) is a frequent practice in wineries. These enzymes are normally produced by bacteria or filamentous fungi; although commercial preparations of such enzymes are available, they are complex undefined mixtures of enzymes, and sometimes are not active on wine conditions (low pH, high sugar concentration or high ethanol content, etc.). For these reasons, an alternative to the commercial enzymes could be the use of specific enzymes contained in yeasts forming part of the wine ecosystem. Our results showed that the strains P. membranifaciens (CLI 679), CLI 1219 (classified as L. thermotolerans), W. anomalus (CLI 1218) and H. guilliermondii (CLI 921) were the species with a higher b-glucosidase activity in the assay conditions. These results are in agreement with those found by Strauss et al. [28], but in our case, we used the yeast species isolated during wine fermentation, this fact ensures the production of these compounds during winemaking processes. Regarding pectinase production, our results show that yeasts of the species C. stellata, P. kudriavzevii and W. anomalus were good pectinase producers (Table 2). Thus, we considered that pectinase activity from yeast strains remaining during the early phases of the wine fermentations; it may also provide advantages to the final quality of the Malvar wines. Inoculation of the selected yeast strains in Malvar must allowed to differentiate two groups of yeast species, those which were able to transform sugars to ethanol, reaching alcoholic degrees higher than 10 % (v/v), or those that
123
were not able to do it and only produce low alcoholic levels, around 3 % (v/v). Regarding the global enological data obtained, as we expected, only H. guilliermondii reached a significantly value of volatile acidity; in this aspect, this species could be considered not adequate to the elaboration of wine. However, glycerol production by S. cerevisiae exceeded the threshold taste level of sweetness (5.2 g L-1) proposed by Noble and Bursick [51]. Although C. stellata is a common producer of glycerol, in our case, this yeast species was not able to ferment on natural must. So, this was the reason that the levels achieved of glycerol are lower than those exhibited by S. cerevisiae, contrary to the previously described in the literature [52–54]. Despite this finding, it may be due to the presence of C. zemplinina and not C. stellata. The higher amounts of 2,3-butanodiol were also found in the species H. guilliermondii and T. delbrueckii, this compound is a precursor of acetoin or diacetyl which has a subtle butter aroma. Thus, high diacetyl levels would contribute offflavors. Nonetheless, the values above indicated were low taking into account the usual range described for wines [55]. The volatile compounds analyzed in the fermentations by the autochthonous Saccharomyces cerevisiae yeast strain are in accordance with those previously described in Malvar wines [1, 37]. Taking into account the different non-Saccharomyces yeast strains, the volatile compounds analyzed ranged from the positive aspects (increasing flowery and fruity aromas) to the negative one (increasing harsh aromas). However, sometimes the analysis of solute concentration in relation to odor threshold does not always imply a ‘‘positive’’ or beneficial impact on wine organoleptic properties; excessive concentrations usually contribute spoilage odors, and the presence of some compounds produces masking or other sensory affects. Excessively high concentrations of, for example, acetaldehyde produced by T. delbrueckii and ethyl acetate produced by H. guilliermondii would certainly contribute off-flavors. In our study, acetaldehyde has been found in high levels in relation to other carbonyl compounds, but its mean content was lower than in other non-oxidized white wines previously studied (30–100 mg L-1) [56, 57]. Regarding the esters of acetate showed in this study are lower than those exhibited by Andorra` et al. [20]. This difference, besides of the protocol of extraction and analysis, may be also due to the grape variety used, both fermentations protocols were conducted under aeration conditions. This aeration procedure has been described as one of the main factors that affect the production of these compounds ranging until ten times more than fermentations conducted under non-aerated conditions [34, 58]. To avoid the previous discussions about the real interaction between Saccharomyces yeast strains and the nonSaccharomyces yeast species [59–62], we decided to study
Eur Food Res Technol
the real contribution of the non-Saccharomyces wine yeast strains individually in order to assay in the future the possible sequential inoculation of these non-Saccharomyces yeasts with selected or commercial S. cerevisiae yeast strains. As we expected, it is well known in the literature that only a few of the non-Saccharomyces yeast strains analyzed showed the possibility of dryness in the grape must. Future applications of these yeasts and using a selected strain of S. cerevisiae in Malvar wines may be improve the aromatic composition of the Malvar wines. In addition to the use of inoculations of non-Saccharomyces such as C. zemplinina and S. cerevisiae [63] which demonstrated variation on the chemical composition of the final wines depending of the type of inoculation (co-inoculation or sequential inoculation). As stated in results, the PCA of the yeast strains behavior divided the yeast strains in two groups was well differentiated, a group formed by S. cerevisiae, T. delbrueckii and L. fermentati which are all capable of consuming the residual sugars in the grape musts and a second one without this capacity. Although there is a clear difference between both groups of yeast strains, when fermentative yeast strains were removed from the PCA, the PCA score plots for other nonSaccharomyces yeast strains were not related to a single parameter (data not shown), these results indicates that there is not a clear relation between certain metabolites produced and the non-Saccharomyces yeast species used. Identification and characterization of non-Saccharomyces yeast strains isolated from Malvar wines belonging to the Madrid winegrowing region have never been characterized before. Extracellular enzymatic activities of interest in enology have been used as selection criteria on the basis of the non-Saccharomyces yeast strains. Yeast-derived volatile fermentation products have been shown to constitute a significant difference within the non-Saccharomyces yeast strains used in this study. Different concentrations of the analyzed compounds have been found between the different fermentations. Characterization of these yeasts strains indicates that autochthonous non-Saccharomyces yeasts could have a positive impact on the organoleptic characteristics of these wines, showing a high variability in the volatile compounds that contribute to the wine from Malvar grape berries aroma. T. delbrueckii (CLI 918) was defined as the yeast strain with potential interest for its contribution to the aromatic wine profile with flowery and fruity aromas and could be used in mixed starter cultures with S. cerevisiae. However, H. guilliermondii CLI 1217 increased the volatile acidity and ethyl acetate, but this species along with the genus Pichia and Candida seem to provide a high quantity of important enzymes which may be beneficial in wine making. Nevertheless, biotechnological profiles based on single strains of a given species should be confirmed by the study of additional strains.
Acknowledgments We are grateful to Margarita Garcı´a Garcı´a for her support at the laboratory and Matthew Tyndale-Tozer for revising the manuscript. This work was financed by the Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA-RM 2006-00012-00-00), and by the Instituto Madrilen˜o de Investigacio´n y Desarrollo Rural Agrario y Alimentario (IMIDRA—FP—IA-07-Lev).
References 1. Gil M, Cabellos JM, Arroyo T, Prodanov M (2006) Characterization of the volatile fraction of young wines from the Denomination of Origin ‘‘Vinos de Madrid’’ (Spain). Anal Chim Acta 563:145–153 2. Cordero-Bueso G, Arroyo T, Serrano A, Tello J, Aporta I, Ve´lez MD, Valero E (2011) Influence of the farming system and vine variety on yeast communities associated with grape-berries. Int J Food Microbiol 45:132–139 3. Cordero-Bueso G, Arroyo T, Serrano A, Valero E (2011) Influence of different floor management strategies of the vineyard on the natural yeast population associated with grape berries. Int J Food Microbiol 148:23–29 4. Tello J, Cordero-Bueso G, Aporta I, Cabellos JM, Arroyo T (2012) Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts. J Appl Microbiol 112:316–328 5. Torija MJ, Roze`s N, Poblet M, Guillamo´n JM, Mas A (2001) Yeast population dynamics in spontaneous fermentations: comparison between two different wine-producing areas over a period of three years. Antonie Leeuwenhoek 79:345–352 6. Romancino DP, Di Maio S, Muriella R, Oliva D (2008) Analysis on non-Saccharomyces yeast populations isolated from grape musts from Sicily (Italy). J Appl Microbiol 105:2248–2254 7. Oco´n E, Gutie´rrez AR, Garijo P, Lo´pez R, Santamarı´a P (2010) Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time. Food Microbiol 27:1023–1027 8. Gonza´lez SS, Barrio E, Querol A (2007) Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Islands, Spain). J Appl Microbiol 102:1018–1025 9. Fleet GH (2008) Wine yeast for the future. FEMS Yeast Res 8:979–995 10. Lopandic K, Tiefenbrunner W, Gangl H, Mandl K, Berger S, Leitner G, Abd-Ellah A, Querol A, Gardner RC, Sterflinger K, Prillinger H (2008) Molecular profiling of yeasts isolated during spontaneous fermentations of Austrian wines. FEMS Yeast Res 8:1063–1075 11. Francesca N, Chiurazzi M, Romano R, Aponte M, Settani L, Moschetti G (2010) Indigenous yeast communities in the environment of ‘‘Rovello bianco’’ grape variety and their use in commercial white wine fermentation. World J Microbiol Biotechnol 26:337–351 12. Tofalo R, Chaves-Lo´pez C, Di Fabio F, Schirone M, Felis GE, Torriani S, Paparella A, Suzzi G (2009) Molecular identification and osmotolerant profile of wine yeast that ferment a high sugar grape must. Int J Food Microbiol 130:179–187 13. Kapsopoulou K, Mourtzini A, Anthoulas M, Nerantzis E (2007) Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World J Microbiol Biotechnol 23:735–739 14. Bely M, Stoeckle P, Masneuf-Pomare`de I, Dubourdieu D (2008) Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae on high-sugar fermentation. Int J Food Microbiol 122:312–320 15. Barrajo´n N, Are´valo-Villena M, Rodrı´guez-Arago´n LJ, Briones A (2009) Ecological study of wine yeast in inoculated vats from La Mancha Region. Food Control 20:778–783
123
Eur Food Res Technol 16. Renault P, Miot-Sertier C, Marullo P, Hena´ndez-Orte P, Lagarigue L, Lonvaud-Funel A, Bely M (2009) Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: potential applications in the wine industry. Int J Food Microbiol 134:201–210 17. Moreira N, Pina C, Mendes F, Couto JA, Hogg T, Vasconcelos I (2011) Volatile compounds contribution of Hanseniaspora guilliermondii and Hanseniaspora uvarum during red wine vinifications. Food Control 20:778–783 18. Andorra` I, Landi S, Mas A, Guillamo´n JM, Esteve-Zarzoso B (2008) Effect of oenological practices on microbial populations using culture independent techniques. Food Microbiol 25:849–856 19. Andorra` I, Landi S, Mas A, Esteve-Zarzoso B, Guillamo´n JM (2010) Effect of fermentation temperature on microbial population evolution using culture independent and dependent techniques. Food Res Int 43:773–779 20. Andorra` I, Berradre M, Roze`s N, Mas A, Guillamo´n JM, EsteveZarzoso B (2010) Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations. Eur Food Res Technol 23:215–224 21. Esteve-Zarzoso B, Manzanares P, Ramo´n D, Querol A (1998) The role of non-Saccharomyces yeasts in industrial winemaking. Int Microbiol 1:143–148 22. Swiegers JH, Pretorius IS (2005) Yeast modulation of wine flavor. Adv Appl Microbiol 57:131–175 23. Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS (2005) Yeast and bacteria modulation of wine aroma and flavour. Aus J Grape Wine Res 11:139–173 24. Herna´ndez-Orte P, Cersosimo M, Loscos N, Cacho J, Garcı´aMoruno E, Ferreira V (2008) The development of varietal aroma from non-floral grapes by yeast of different genera. Food Chem 107:1064–1077 25. Viana F, Gil JV, Genove´s S, Valle´s S, Manzanares P (2008) Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol 25:778–785 26. Charoenchai C, Fleet GH, Henschke PA, Todd BEN (1997) Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Aus J Grape Wine Res 3:2–8 ´ beda JF, Briones AI (2000) Typing of non-Sac27. Ferna´ndez M, U charomyces with enzymatic activities of interest in wine-making. Int J Food Microbiol 59:29–36 28. Strauss MLA, Jolly NP, Lambrechts MG, van Rensburg P (2001) Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J Appl Microbiol 91:182– 190 29. Cordero-Otero RR, Iranzo JFU, Briones-Perez AI, Potgieter N, Villena MA, Pretorius IS, van Rensburg P (2003) Characterization of the beta glucosidase activity produced by oenological strains of non-Saccharomyces yeasts. J Food Sci 68:2564–2569 30. Palmieri R, Spagna G (2007) Glucosidase in cellular and acellular form for winemaking application. Int J Food Microbiol 138:19–25 31. De Benedictis M, Bleve G, Grieco F, Tristezza M, Tufariello M, Grieco F (2011) An optimized procedure for the enological selection of non-Saccharomyces starter cultures. Antonie Leeuwenhoek 99:189–200 32. Manzanares P, Valle´s S, Viana F (2011) Non-Saccharomyces yeasts in the winemaking process. In: Carrascosa AV, Mun˜oz R, Gonza´lez R (eds) Molecular wine microbiology, 1st edn. Academic Press Elsevier, London, pp 85–110 33. Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M (2011) Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol 28:873–882
123
34. Rojas V, Gil JV, Pinaga F, Manzanares P (2003) Acetate ester formation in wine by mixed cultures in laboratory fermentations. Int J Food Microbiol 86:181–188 35. Ciani M, Beco L, Comitini F (2006) Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations. Int J Food Microbiol 108:239–245 36. Ciani M, Comitini F, Mannazzu I, Dominizio P (2010) Controlled mixed culture fermentation: a new perspective on the use of nonSaccharomyces yeast in winemaking. FEMS Yeast Res 10:123–133 37. Santos JP, Arroyo T, Aleixandre M, Lozano J, Sayago I, Garcı´a M, Ferna´ndez MJ, Are´s L, Gutie´rrez J, Cabellos JM, Gil M, Horrillo MC (2004) A comparative study of sensor array and GCMS: application to Madrid wines characterization. Sens Actuat B 102:299–307 38. Guillamo´n JM, Sabate´ J, Barrio E, Cano J, Querol A (1998) Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region. Arch Microbiol 169:387–392 39. Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A (1999) Identification of yeast by RFLP analysis of the 5.8 rRNA and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 49:329–337 40. Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Leeuwenhoek 73:331–371 41. Linstromberg WW, Baumgarten HE (1987) Organic chemistry, a brief course. DC Heath, Lexington, Toronto 42. Office International de la Vigne et du Vin (1990) Recopilacio´n de los Me´todos Internacionales de Ana´lisis de los Vinos OIV Parı´s, France 43. Commission Regulation (EEC) (1990) No 2676/90 of 17 September 1990 determining community methods for the analysis of wines 44. Duarte FW, Dias RD, Oliveira JM, Vilanova M, Teixeira JA, Almeida e Silva JB, Schwan RF (2010) Raspberry (Rubus idaeus L) wine: yeast selection, sensory evaluation and instrumental analysis of volatile and other compounds. Food Res Int 43:2303–2314 45. Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of wine grape berries. Int J Food Microbiol 153:243–259 46. Mortimer R, Polsinelli M (1999) On the origins of wine yeast. Res Microbiol 150:199–204 47. Cordero-Bueso G, Arroyo T, Serrano A, Valero E (2011) Remanence and survival of commercial yeast in different ecological niches of the vineyard. FEMS Microbiol Ecol 77:429–437 48. Angelo J, Siebert KJ (1987) A new medium for the detection of wild strains in brewing culture yeast. J Am Soc Brew Chem 45:135–140 49. Csoma H, Sipiczki M (2008) Taxonomic reclassification of Candida stellata strains reveals frequent occurrence of Candida zemplinina in wine fermentation. FEMS Yeast Res 8:328–336 50. Rodrı´guez ME, Lopes C, Valles S, Giraudo MR, Caballero A (2007) Selection and preliminary characterization of b-glucosidases producer Patagonian wild yeast. Enzyme Microb Technol 41:812–820 51. Noble AC, Bursick GF (1984) The contribution of glycerol to perceived viscosity and sweetness in white wine. Am J Enol Viticult 39:110–112 52. Ciani M, Ferraro L (1996) Enhanced glycerol content in wines made with immobilized Candida stellata cells. Appl Environ Microbiol 62:128–132 53. Ciani M, Comitini F (2006) Influence of temperature and oxygen concentration on the behavior of Candida stellata in mixed fermentation with Saccharomyces cerevisiae. World J Microbiol Biotechnol 22:619–623
Eur Food Res Technol 54. Garcı´a V, Va´squez H, Fonseca F, Manzanares P, Viana F, Martı´nez C, Ganga MA (2010) Effects of using mixed wine yeast cultures in the production of Chardonnay wine. Rev Arg Microbiol 42:226–229 55. Ribe´reau-Gayon P, Dubourdieu D, Done`che B, Lonvaud A (2006) The microbiology of wine and vinifications, Handbook of enology, vol 1 (pp. 1–51) John Wiley and Sons, West Sussex 56. Escudero A, Asensio E, Cacho J, Ferreira V (2002) Sensory and chemical changes of young white wines stored under oxygen. An assessment of the role played by aldehydes and some other important odorants. Food Chem 77:325–331 57. Go´mez-Mı´nguez MJ, Cacho JF, Ferreira V, Vicario IM, Heredia FJ (2007) Volatile components of Zalema white wines. Food Chem 100:1464–1473 58. Valero E, Moyano L, Milla´n MC, Medina M, Ortega JM (2002) Higher alcohols and esters production by Saccharomyces cerevisiae. Influence of the initial oxygenation of the grape must. Food Chem 78:57–61 59. Nissen P, Nielsen D, Arneborg N (2003) Saccharomyces cerevisiae cells at high concentrations cause early growth arrest of
60.
61.
62.
63.
64.
non-Saccharomyces yeasts in mixed cultures by a cell–cell contact-mediated mechanism. Yeast 20:331–341 Nissen P, Arneborg N (2003) Characterization of early deaths of non-Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae. Arch Microbiol 180:257–263 Arneborg N, Siegumfeldt H, Andersen G, Nissen P, Daria V, Rodrigo P, Glu¨ckstad J (2005) Interactive optical trapping shows that confinement is a determinant of growth in a mixed yeast. FEMS Microbiol Lett 245:155–159 Pe´rez-Nevado F, Albergaria H, Hogg T, Girio F (2006) Cellular death of two non-Saccharomyces wine-related yeasts during mixed fermentation with Saccharomyces cerevisiae. Int J Food Microbiol 108:336–345 Rantsiou K, Dolci P, Giacosa S, Torchio F, Tofalo R, Torriani S, Suzzi G, Rolle L, Cocolin L (2012) Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations. Appl Environ Microbiol 78:1987–1994 Falque´ E, Ferna´ndez E, Dubourdieu D (2001) Differentiation of white wines by their aromatic index. Talanta 54:271–281
123
