OENO One2494-1271Use of non-Saccharomyces yeasts in the prise de mousse of Lambrusco. Microbial evolution through alcoholic fermentation and effect on wine volatile profileGuzzonRaffaeleraffaele.guzzon@fmach.it1MauroPaolini1MalacarneMario1RomanThomas1NaselliVincenzo2FrancescaNicola2LarcherR.11Centro di Trasferimento Tecnologico. Fondazione Edmund Mach. Via Mach 1, 38010 San Michele all’ Adige, Italy.2Department of Agricultural and Forestry Science. Food and Agricultural Microbiology Unit. University of Palermo. Viale delle Scienze 4, 90128 Palermo, Italy.652024
Lambrusco is a sparkling wine largely produced in the north of Italy, especially in the Emilia region. The possible application of two non-Saccharomyces yeasts in the secondary bottle fermentation (Champenoise method) was tested in combination with a commercial strain of S. cerevisiae to obtain wines having a distinctive volatile profile. Our results demonstrated that the gradual increase of ethanol content in the pied de cuve ensured the adaptation of Hanseniaspora guilliermondii and Torulaspora delbrueckii at the bottle fermentation, with survival comparable with that of S. cerevisiae, in the order of 6 log units. The simultaneous presence of two yeast species reduced the maximum fermentation rate, without any relevant alteration in the main oenological parameters of resultant wines. GC MS-MS analysis of the volatile profile of wines (46 compounds) highlighted differences in wine made from a pure culture of S. cerevisiae from wines obtained by mixed yeasts. Acetates, esters, and fatty acids are the classes of volatile compounds mostly affected by using different yeasts in the bottle fermentation of Lambrusco wines. This work provided for the first time information about the volatile profile of Lambrusco and suggests an innovative application of non-Saccharomyces yeast in the production of sparkling wines by champenoise methods.
Introduction
Lambrusco wine is produced in Emilia (Italy) by different
V. vinifera cultivars, the most widespread are
Lambruscosalamino, L. grasparossa,
L. Sorbara, and
L. marani. Lambrusco is one of the major wine productions in Italy. In 2021, about 170 million bottles were produced, corresponding to 490 million euros in value (
I numeri del vino, 2021). Today, Lambrusco is a red, sparkling, dry or semi-sweet wine, consumed in the early months after the harvest. To obtain this Lambrusco style the winemaking follows the Charmat method, with a one-step alcoholic fermentation in an autoclave and early isobaric bottling. On the contrary, traditionally Lambrusco wines were made with the Champenoise method (
Favaro, 2017), but in the last decades bottle-fermented Lambrusco almost disappeared because was considered a poor-quality wine. Recently many Lambrusco producers have rediscovered this technique, as it could yield more long-lasting and complex wine, which has found increasing appreciation from consumers. To date, knowledge regarding the Lambrusco wines is scarce, in-depth studies are necessary to improve the quality of Lambrusco made through the Champenoise method.
The Champenoise method is the oldest technique for producing sparkling wines (
Buxaderas and Lopez-Tamames, 2012). Yeast finds during bottle-fermentation harsh conditions for its development. The resistance mechanisms activated by yeast in bottle fermentation are partly like that of primary alcoholic fermentation, due to the occurrence of the same limiting factors (ethanol, low pH, nitrogen starvation), but it showed some peculiarity related to environmental bottle conditions (low temperature, CO
2, overpressure) (
Porras-Agüera et al., 2021;
Penacho et al., 2012). Ethanol is the main factor that activates gene transcription during bottle fermentation, inducing the expression of genes involved in respiratory metabolism, in the response to oxidative stress, autophagy, and peroxisomal function (
Marks et al., 2008;
Rossignol et al., 2003). In addition, low temperature seems to influence the gene transcription profile of yeast during sparkling wine production (
García-Ríos et al., 2017).
An accurate adaptation of yeast is essential to stimulate the mechanisms of stress response (
Guzzon and Larcher, 2015). Equally fundamental is the supplementation of the wine with the nutritional factors that have been depleted during the first alcoholic fermentation (
Costa et al., 2018). If these requirements are ensured, bottle fermentation occurs within a couple of weeks. Subsequently, the ageing of wine begins in contact with the yeast cells which, lysing gradually, release a wide range of chemical fractions: polyphenols, peptides, proteins, amino acids, and lipids (
Gnoinski et al., 2021;
Moreno Arribas et al., 1996). The release of cell fractions during bottle ageing modifies the organoleptic profile of sparkling wine and guarantees its longevity, thanks to the antioxidant activity of some cell compounds such as gallic acid,
trans-resveratrol, catechin, and epicatechin, procyanidins and B complex vitamins (
Martínez-García et al., 2021;
Perez-Magarino et al., 2015;
Stefenon et al., 2010).
For their high resistance to ethanol, yeast strains for bottle fermentation are generally chosen from within the
Saccharomyces genus. However, there is evidence that some non-
Saccharomyces yeasts are resistant to ethanol concentrations over 10 % (
Guzzon et al., 2022;
Catrileo et al., 2020;
Antoce et al., 2011;
Xue et al., 2009). Non-
Saccharomyces yeasts represent a promising resource in oenology (
Di Gianvito et al., 2022;
He et al., 2022). Biocontrol, increasing wine mouthfeel by releasing mannoproteins, and improving wine aromatic profile thanks to overexpressed enzymatic activities, are just some of the applications of these microorganisms that are now available on the market as active dry yeasts (
OIV, 2017). Among the non
-Saccharomyces yeasts, two species deserve particular interest considering the scope of this work:
Torulaspora delbrueckii and
Hanseniaspora uvarum.
T. delbrueckii have been involved in fermentation with
S. cerevisiae in winemaking since the 1990s, when its positive contribution to the aromatic profile of wine (
Moreno et al., 1991) and its ability to maintain biological activity at low temperatures were highlighted (
Charoenchai et al., 1998). The application of
T. delbrueckii in sparkling wine production was suggested by
Canonico et al. (2018). More recently,
Silva-Sousa et al. (2022) compared the activity of 40 strains of
T. delbrueckii, confirming the preference of this yeast for low fermentation temperature and noting ethanol resistance up to 18 %. The use of
Hanseniaspora spp. in winemaking is less widespread, but its presence in the wine environment is ascertained (
Mancic et al., 2022). Its enzymatic pathway is broad and of great interest in terms of impact on the volatile profile of wines. An ethanol resistance of up to 12.5 % suggests the possible use of
Hanseniaspora in sparkling wine production (
Moreira et al., 2011).
In this work,
H. uvarum and
T. delbrueckii have been used in mixed culture with
S. cerevisiae as a starter of bottle fermentation of
L. Sorbara and
L. marani. The activity of the three yeast species was monitored according to international OIV standards (2010), and the wines obtained were characterised by GC MS-MS analysis to highlight the peculiarities due to the contribution of different yeasts.
The preparation of pied de cuve and bottle fermentation was monitored by plate count (WL Agar medium for total yeast and Lysine Agar medium for non-
Saccharomyces yeast, both purchased by Oxoid, UK) and microscopical yeast count, according to the OIV methods (2010). The attribution of species to the non-
Saccharomyces yeast growth onto the plate was confirmed by specie-specific PCR, as proposed by van Breda et al. (2013) and
Santiago-Urbina et al. (2015) on a representative number of colonies grown onto WL/Lysine Agar.
4. Chemical analysis
Basic chemical analysis of wines (Sugars, pH, ethanol, total acidity, acetic and malic acids, yeast assailable nitrogen, and total/free SO
2) was performed by a WineScan SO
2 FT-IR analyser (FOSS, DK). The analysis of volatile compounds was performed according to the method proposed by
Paolini et al. (2018), while volatile compounds were sampled by solid-phase extraction (SPE) using ENV+ cartridges. The GC-MS/MS analysis was performed by an Agilent (CA) Intuvo 9000 apparatus, coupled with an Agilent 7000 Series Triple Quadrupole mass spectrometer working in electron impact mode at 70 eV. The mass spectrum was acquired in Multiple Reaction Monitoring mode, setting the instrument in a dynamic system.
5. Statistics
ANOVA One-way, Partial Least Squares regression (PLS), and Principal Component Analysis (PCA) on the set of data were performed by TIBCO Statistica® 14.1.0 (TIBCO Software, CA).
Results1. Evolution of yeast during pied de cuve preparation and bottle fermentation
Table 2 shows the evolution of the four pied de cuve produced by different yeasts in terms of cell density and sugars/ethanol concentration. The pied de cuve produced by pure
S. cerevisiae reached a cell concentration of 1.5 × 10
8 CFU/mL in 48 hours. In the pied de cuve made by mixed yeast culture, the concentration of each strain was initially lower because the mixing of strains was performed immediately prior to the bottling, to avoid competition between the two species.
H. uvarum and
T. delbrueckii reached a cellular concentration over the 7 log units (Table 2).
T. delbrueckii appeared more able to accumulate ethanol, and the 10.2 % v/v was reached after 48 hours with a cellular concentration of 7.7×10
7 CFU/mL.
H. uvarum reached 8.5 × 10
7 CFU/mL with an ethanol content of 9.5 % v/v. At bottling time, the total yeast concentration was 1.3 ± 0.5 × 10
6 CFU/mL in the test performed by pure
S. cerevisiae, 2.2 ± 0.6 × 10
6 CFU/mL in the tests performed by
S. cerevisiae and
T. delbrueckii, and 2.1 ± 0.2 × 10
6 CFU/mL in the test performed by
S. cerevisiae and
H. uvarum. For both mixed cultures, the ratio between
Saccharomyces and non-
Saccharomyces was adjusted to 1:1.
Pied de cuve
Sampling time
Yeast
Ethanol
Sugars
h
×10
7 CFU/mL
% v/v
g/L
Pure
S. cerevisiae
8
9.8
5.5
93.4
24
12.5
9.8
51.5
48
15.1
10.8
33.4
S. cerevisiae 50 %
8
5.7
5.0
95.2
24
7.9
9.4
71.2
48
7.6
10.6
44.1
H. uvarum
8
5.0
3.8
94.6
24
7.7
8.2
53.5
48
8.5
9.5
30.6
T. delbrueckii
8
6.1
5.2
94.9
24
6.8
9.6
68.6
48
7.7
10.2
46.8
Monitoring yeast viability continued during bottle fermentation. In Figure 1, the concentration of
S. cerevisiae,
T. delbrueckii, and
H. uvarum was represented at 3, 8, and 12 days after wine bottling, as the mean of both tests performed on
L. Sorbara and
L. marani wines (n = 6), because the differences observed between the two varieties were non-statistically significant (One-way ANOVA,
p > 0.05). At the beginning of bottle fermentation in the test by pure
S. cerevisiae, the total concentration of yeast was 4.4 ± 0.1 × 10
6 CFU/mL with a concentration of non-
Saccharomyces below the 4 log units. The concertation of yeast increased on the 8
th day (5.8 ± 0.9 × 10
6 CFU /mL), and after that, a reduction in cell concentration was observed (3.2 ± 0.4 × 10
6 CFU /mL), probably due to the exhaustion of sugars. In the test performed by
S. cerevisiae and
H. uvarum, the total yeast concentration remained at the interval between 2.1 ± 0.7 and 3.4 ± 0.4 × 10
6 CFU/mL and 9.8 ± 2.3 × 10
5 and 1.2 ± 0.4 × 10
6 CFU/mL for
H. uvarum. Worth noting was the concentration of
H. uvarum, over the 6 log units after 12 days of fermentation, in the presence of high ethanol content, in the harsh environment of sparkling wine. As regards fermentation by
S. cerevisiae and
T. delbrueckii, the non-
Saccharomyces yeast started from the lowest concentration (3.9 ± 0.1 × 10
5 CFU/mL) but settled between the 5 and 6 log units for the entire duration of bottle fermentation. At the beginning of the test, the total yeast has a concentration of 2.2 ± 0.1 × 10
6 CFU/mL, reaching the maximum after 12 days of fermentation. The statistical treatment of data revealed significant differences (One-way ANOVA,
p < 0.05) in the concentration of
S. cerevisiae in the mixed fermentation, compared to the tests performed by pure yeast culture on the 3
rd and 8
th days.
2. Dynamics of bottle alcoholic fermentation and chemical features of wines
Figure 2A represents the evolution of alcoholic fermentation, with the data expressed as the mean between tests performed on
L. Sorbara and
L. marani (n = 6, 3 for each wine). In the test performed by pure
S. cerevisiae, the alcoholic fermentation started quickly, and the residual sugar was about 15 g/L (2
nd day) and 10 g/L (5
th day). In the tests performed by mixed yeast culture, the sugar degradation followed a linear trend, with a delay compared to the previous test in the first two points of observation. After that, the fermentation rate increased and the evolution of alcoholic fermentation in the 3 trials proved comparable. In both cases, sugar degradation finished in 20 days. The maximum rate of fermentation (V
max) was reached on the 2
nd day of fermentation (Figure 2B), corresponding to 6.15 ± 0.22 g/L/day for the test performed by pure
S. cerevisiae, while in the tests performed by mixed yeast culture, the V
max was 2.95 ± 0.48 g/L/day
in the case of
H. uvarum+S. cerevisiae and 2.50 ± 0.37 g/L/day in the case of
T. delbrueckii + S. cerevisiae. The statistical analysis revealed significant differences (One-way ANOVA,
p < 0.05) in the number of sugars degraded and in the fermentation rate until the 10
th day of fermentation, comparing pure fermentation and mixed fermentation.
The volatile concentrations are shown in Table 1. Thirty-two aromatic compounds belonging to different chemical classes were identified and quantified. Due to the absence of a large number of aromatic precursors in the Lambrusco grapes, the majority of volatile molecules identified have a fermentative origin, and only seven of them are varietal compounds. Among these were identified the monoterpenes linalool, linalool oxides, and alpha-terpineol. These compounds are directly involved in the floral bouquet, but in the Lambrusco wines their concentration was lower the odour threshold: 0.05 mg/L for linalool, 6 mg/L for linalool oxides, and 0.4 mg/L for alpha-terpineol (
Fariña et al., 2015). Hexanol, cis-3-hexen-1-ol, and trans-3-hexen-1-ol are the C6-molecules formed during the pre-fermentative stage of winemaking. 1-hexanol in alcoholic beverages imparts green and floral notes, with a sensory threshold of about 1 mg/L (
Berger, 2007). As reported in Table 1, only the L. Sorbara wines have a concentration of 1-hexanol that exceeds slightly the threshold limit. Cis-3-hexen-1-ol and trans-3-hexen-1-ol generate unpleasant herbaceous off-odours but they usually occur in low levels and do not contribute to wine aroma. In both the Lambrusco varieties, the concentration of cis-3-hexen-1-ol and trans-3-hexen-1-ol was under the threshold value of 0.4 and 1 mg/L, respectively (
Fariña et al., 2015).
2-phenylethanol and benzyl alcohol are derived from grapes through the metabolism of yeast during the fermentation process. 2-phenylethanol is generally a positive contributor to wine aroma, characterised by a pleasant rose-like aroma.
L. Sorbara shows a high content of 2-phenylethanol above its sensory threshold (10 mg/L) (
Fariña et al., 2015). The organic compounds originated from the alcoholic fermentation and quantified in the wine samples were acids, acetates, and ethyl esters. As shown in Table 1, a total of eight acids were identified. Hexanoic acid, octanoic acid, and decanoic acid showed the highest concentration exceeding their odour threshold (0.4, 0.5, and 1 mg/L, respectively) (
Fariña et al., 2015). At high concentrations (20 mg/L), these compounds have been associated with an unpleasant scent, but low amounts contribute positively to increasing the complexity of the wine aroma. Among the acetates and ethyl esters characterised by fruity descriptors, isopentyl acetate, ethyl hexanoate, ethyl octanoate, diethyl-succinate, and ethyl lactate were the major compounds. Isopentyl acetate and ethyl hexanoate were the only ones that showed a concentration that exceeded their odour threshold (0.03 and 0.014 mg/L, respectively) for all samples, while the odour threshold of ethyl octanoate (0.5 mg/L) was exceeded in only one (
Fariña et al., 2015).
The statistical treatment of data in Table 1 by different approaches facilitates the compression of differences among wines due to the yeast’s activity. ANOVA and Tukey tests found significant differences in volatile compounds between base wines and wines after secondary fermentation. In some cases, we also found differences among the 3 different experimental hypotheses and between wines made by SC with respect to those made by non-
Saccharomyces yeasts. PCA analysis considered the entire set of compounds. Figure 4 represents the distribution of cases on the plan designed by the first two variables that explained 99.4 % of the entire variability in
L. Sorbara samples and 93.9 % in
L. marani. Wines before bottle fermentation proved to be separated from other samples, with negative coordinates from 1
st and 2
nd variables. This result was linked to the lesser content in all classes of volatile compounds, except for esters in
L. marani wines. Lambrusco samples made from bottle fermentation by pure
S. cerevisiae were characterised by a high content of acetates, while wines made from mixed yeast cultures are clearly distinguishable by the higher content of fatty acids and alcohol. The results seem to confirm that the wine volatile profile after bottle fermentation can be modulated by using different yeasts and they will be discussed in the next chapter.
Discussion
The production of sparkling wines using the Champenoise method is complex and expensive, justified by the greater value that these wines have for consumers than wine produced through the Charmat method. This comparison acquires even greater importance in the case of Lambrusco, which is generally positioned on the market in a medium-to-low range of price. The use of non-
Saccharomyces yeasts to obtain wines having peculiar organoleptic features has now been widely tested. Promising applications involved "difficult" wines, where the activity of
S. cerevisiae was partially compromised. In this sense, worthy experiences were conducted on wines made from dried grapes where osmotic stress could alter the response of
S. cerevisiae (
Azzolini et al., 2012). Other interesting studies concerned the enhancement of wines made from autochthonous grape varieties (
Arslan et al., 2018;
Zhang et al., 2018), or the correction of deficiencies in grape musts, exploiting alternative ways of consuming carbohydrates, as in the case of
L. thermotolerans (
Morata et al., 2018). Generally, the activity of non-
Saccharomyces yeasts is limited to the first days of alcoholic fermentation, because it was believed that they did not tolerate high doses of ethanol or, worse still, could alter wines. One of the main points of interest of this work is the use of non-
Saccharomyces yeasts for the entire duration of bottle alcoholic fermentation, based on the assumption that an adequate adaptation of yeasts could activate their resistance to limiting factors, such as ethanol, ensuring their prolonged survival in wine. This hypothesis is based on several reports (
Yoshida et al., 2021;
Ma and Liu, 2010), which demonstrate that resistance mechanisms of yeasts are induced by cell growth in the presence of stress factors at the sub-lethal level. In our experience, the progressive increase of ethanol content in pied de cuve did not counteract yeast growth, which reached 7 log units/mL in the presence of 10 % ethanol.
T. delbrueckii and
H. uvarum proved capable of surviving in wine, during bottle fermentation, for at least 12 days, maintaining a cellular concentration between 5 and 6 log units/mL. The lack of dominance of
S. cerevisiae, which in mixed fermentation reached a concentration lower than that of the test by pure strain, suggests a possible competition among different yeast species, as reviewed by
Di Gianvito et al., 2022.
The evolution of bottle fermentations agreed with the hypothesis of an interaction between different yeasts in mixed cultures, even if all wines regularly concluded the consumption of sugars and no alterations of the main oenological parameters (acidity, acetic acid, pressure, and alcohol) are observed. Mixed fermentations show an initial delay, compared to fermentations performed by pure
S. cerevisiae, and a lower V
max. Further studies are necessary to understand what actions should be taken to optimise yeast performance in mixed fermentations. To our knowledge, this work is the first time that the volatile profiles of wines made from Lambrusco grapes are analysed with advanced mass spectrometry techniques. We have no prior scientific references to use as a comparative basis, and we must, therefore, rely on tasting notes, technical publications (
Favaro et al., 2017), and the product specification of the main Lambrusco DOC (https://lambrusco.net) to draw some confirmations about the aromatic profiles of the base wines.
L. Sorbara is generally recognised as a wine characterised by a delicately floral aroma. The high content of 2-phenylethanol would seem to confirm this description (
Cordente et al., 2021). On the other hand,
L. marani is known not to have a profile distinguishable from other varieties of Lambrusco, so much so that it is often used in blends. The high concentration of medium-chain fatty acids and esters suggests an aromatic profile based on fruity notes (
Cameleyre et al., 2015). Bottle fermentation by ordinary
S. cerevisiae enhances the contribution of acetates in the wine profile (
Martínez-García et al., 2017;
Verzeletti et al., 2016), while the presence of non-
Saccharomyces yeast has resulted in wines characterised by floral and spicy aromas, typical of higher alcohols and fatty acids, if present in small quantities (
Carpena et al., 2020).
In conclusion, bottle fermentation influences the aromatic profile of Lambrusco wines, without depletion of molecules that could be related to the distinctive traits of each grape variety. The wine obtained with a pure culture of
S. cerevisiae appeared to be a transition point, in terms of organoleptic profile, between the base wine and the bottle-fermented Lambrusco obtained by mixed yeast cultures. Further studies will be necessary to establish whether these variations in the aromatic profile are due to an effective contribution of non-
Saccharomyces yeasts or the altered behaviour of
Saccharomyces, due to a competition between different species, which can be hypothesised considering the synthetic parameters of the fermentations. A key role can be played by the nutritional integration of the base wine, calibrated on the characteristics of each species, as well as a refinement of the inoculum ratio of the different yeast species (
Gallo et al., 2023).
A.oAntoceI.cNamolosanuF.mRadoi2011Comparative study regarding the ethanol resistance of some yeast strains isolated from Romanian vineyards.Romanian Biotechnological Letters,165981-5988E.ArslanZ.dÇelikT.Cabaroğlu2018Effects of pure and mixed autochthonous Torulaspora delbrueckii and Saccharomyces cerevisiae on fermentation and volatile compounds of Narince wines.Foods,7147https://doi.org/10.3390/foods7090147M.AzzoliniB.FedrizziE.TosiF.FinatoP.VagnoliC.ScrinziG.Zapparoli2012Effects of Torulaspora delbrueckii and Saccharomyces cerevisiae mixed cultures on fermentation and aroma of Amarone wine.European Food Research and Technology,235303-313https://doi.org/10.1007/s00217-012-1762-3R. GBerger2007Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability.S.BuxaderasE.Lopez-Tamames2012Sparkling wines: features and trends from tradition.Advances in Food and Nutrition Research,661-45https://doi.org/10.1016/B978-0-12-394597-6.00001-XM.CameleyreG.LytraS.TempereJ.cBarbe2015Olfactory impact of higher alcohols on red wine fruity ester aroma expression in model solution.Journal of Agriculture and Food Chemistry,6344L.CanonicoF.ComitiniM.Ciani2018Torulaspora delbrueckii for secondary fermentation in sparkling wine production.Food Microbiology,74100-106https://doi.org/10.1016/j.fm.2018.03.009M.CarpenaM.Fraga-CorralP.OteroR.aNogueiraP.Garcia-OliveiraM.aPrietoJ.Simal-Gandara2020Secondary aroma: influence of wine microorganisms in their aroma profile.Foods,1051https://doi.org/10.3390/foods10010051D.CatrileoA.Acuna-FontecillaGodoy L2020Adaptive laboratory evolution of native Torulaspora delbrueckii YCPUC10 with enhanced ethanol resistance and evaluation in co-inoculated fermentation.Frontiers in Microbiology,11595023https://doi.org/10.3389/fmicb.2020.595023C.CharoenchaiG.hFleetP.aHenschke1998Effects of temperature, pH, and sugar concentration on the growth rates and cell biomass of wine yeasts.American Journal of Enology and Viticulture,49283-288https://doi.org/10.5344/ajev.1998.49.3.283A.gCordenteD.eNandorfyM.SolomonA.SchulkinR.KolouchovaI.lFrancisS.aSchmidt2021Aromatic higher alcohols in wine: implication on aroma and palate attributes during chardonnay aging.Molecules,264979https://doi.org/10.3390/molecules26164979G. P.CostaK. P.NicolliJ.eWelkeManfroiVC.aZini2018Volatile profile of sparkling wines produced with the addition of mannoproteins or lees before second fermentation performed with free and immobilized yeasts.Journal of Brazilian Chemical Society,291866-1875https://doi.org/10.21577/0103-5053.20180062P.Di GianvitoV.EnglezosK.RantsiouL.Cocolin2022Bioprotection strategies in winemaking.International Journal of Food Microbiology,364109532https://doi.org/10.1016/j.ijfoodmicro.2022.109532L.FariñaV.VillarG.AresF.CarrauE.DellacassaE.Boido2015Volatile composition and aroma profile of Uruguayan Tannat wines.Food Research International,69244-255https://doi.org/10.1016/j.foodres.2014.12.029C.Favaro2017Everyone Calls it Lambrusco.A.GalloR.GuzzonM.OngaroM.PaoliniT.NardinM.MalacarneT.RomanR.Larcher2023Biological acidification of “Vino Santo di Gambellara” by mixed fermentation of L.thermotolerans and S. cerevisiae. Role of nitrogen in the evolution of fermentation and aroma profile. OENO One,57205–217https://doi.org/10.20870/oeno-one.2023.57.3.7376E.García-RíosM.MorardL.PartsG.LitiJ. MGuillamón2017The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae, 18, 159.https://doi.org/10.1186/s12864-017-3572-2G.bGnoinskiD.cCloseS.aSchmidtF.lKerslake2021Towards Accelerated Autolysis?Dynamics of phenolics,proteins, amino acids, and lipids in response to novel treatments and during ageing of sparkling wine.R.GuzzonR.Larcher2015The application of flow cytometry in microbiological monitoring during winemaking: two case studies.Annals of Microbiology,651865-1878https://doi.org/10.1007/s13213-014-1025-6R.GuzzonElena FA.Toffanin2022Investigation by high-throughput sequencing methods of microbiota dynamics in spontaneous fermentation of Abruzzo (south Italy) wines.Agronomy-Basel,133104https://doi.org/10.3390/agronomy12123104Y.HeX.yWangP.hLiY.cLvH.lNanL.kWenWang Z2022Research progress of wine aroma components: A critical review.Food Chemistry,402134491https://doi.org/10.1016/j.foodchem.2022.134491I Numeri Del Vino2021http://www.M. and LiuMaZ2010Mechanisms of ethanol tolerance in Saccharomyces cerevisiae.Applied Microbiology and Biotechnology,87829-845https://doi.org/10.1007/s00253-010-2594-3S.MancicS.sStojanovicB.DanilovicN.DjordjevicM.MalicaninM.LazicI.Karabegovic2022Oenological characterization of native Hanseniaspora uvarum strains.Fermentation-Basel,892https://doi.org/10.3390/fermentation8030092V. D.MarksS. J. H.SuiD.ErasmusG. K.Van Der MerweJ.BrummW. W.WassermanJ.BryanH. J. JVan Vuuren2008Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response.FEMS Yeast Research,813552https://doi.org/10.1111/j.1567-1364.2007.00338.xR.Martínez-GarcíaT.García-MartínezA.Puig-PujolJ.cMauricioJ.Moreno2017Changes in sparkling wine aroma during the second fermentation under CO2 pressure in sealed bottle.Food Chemistry,2371030-1040https://doi.org/10.1016/j.foodchem.2017.06.066R.Martínez-GarcíaJ.cMauricioT.García-MartínezR.aPeinadoJ.Moreno2021Towards a better understanding of the evolution of odour-active compounds and the aroma perception of sparkling wines during ageing.Food Chemistry,357129784https://doi.org/10.1016/j.foodchem.2021.129784A.MorataI.LoiraW.TesfayeM.aBañuelosGonzález CJ.aSuarez Lepe2018Lachancea thermotolerans applications in wine technology.Fermentation,453https://doi.org/10.3390/fermentation4030053N.MoreiraC.PinaF.MendesJ.aCoutoT.HoggI.Vasconcelos2011Volatile compounds contribution of Hanseniaspora guilliermondii and Hanseniaspora uvarum during red wine vinifications.Food Control,22662-667https://doi.org/10.1016/j.foodcont.2010.07.025J.jMorenoC.MillanJ.mOrtegaM.Medina1991Analytical differentiation of wine fermentations using pure and mixed yeast cultures.Journal of Industrial Microbiology. 7,181-189. https://doi.org/10.1007/BF01575881V.Moreno ArribasE.PueyoM.cPolo1996Peptides in musts and wines.Changes during the manufacture of cavas (Sparkling wines). Journal of Agriculture and Food Chemistry. 44,3783-3788. https://doi.org/10.1021/jf960307aOiv2009International Oenological Codex.COEI-2-CONBAC Methods of microbiological analysis. Bacteriological control. OIV ed. (Dijon,F).Oiv2010Compendium of international methods of analysis.OIV-MA-AS4-01 Microbiological analysis of wines and musts. Detection,differentiation and counting of microorganismsResolution OIV-Oeno 206/2010Oiv2017International Oenological Codex.F-COEI-1-NOSACC Non-Saccharomyces spp. Selected yeasts. OIV ed. (Dijon,F).M.PaoliniL.TonidandelS.MoserLarcher R2018Development of a fast gas chromatography tandem mass spectrometry method for volatile aromatic compound analysis in oenological products.Journal of Mass Spectrometry,53801-810https://doi.org/10.1002/jms.4259V.PenachoE.ValeroR.Gonzalez2012Transcription profiling of sparkling wine second fermentation.International Journal of Food Microbiology,153176-182https://doi.org/10.1016/j.ijfoodmicro.2011.11.005S.Perez-MagarinoM.Ortega-HerasM.Bueno-HerreraL.Martinez-LapuenteZ.GuadalupeAyestaran B2015Grape variety, aging on lees and aging in bottle after disgorging influence on volatile composition and foamability of sparkling wines.LWT-Food Science and Technology,6147-55https://doi.org/10.1016/j.lwt.2014.11.011J.aPorras-AgüeraJ.Moreno-GarcíaT.García-MartínezJ.MorenoJ.cMauricio2021Impact of CO2 overpressure on yeast mitochondrial associated proteome during the "prise de mousse" of sparkling wine production.International Journal Food Microbiology,348109226https://doi.org/10.1016/j.ijfoodmicro.2021.109226T.RossignolL.DulauA.JulienB.Blondin2003Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation.Yeast,201369–1385https://doi.org/10.1002/yea.1046J.aSantiago-UrbinaArias-Garcia JRuiz-Teran F2015Yeast species associated with spontaneous fermentation of taberna, a traditional palm wine from the southeast of Mexico.Annals of Microbiology,65287-296F.Silva-SousaT.FernandesF.PereiraD.RodriguesRito TC.CamarasaR.Franco-DuarteM.jSousa2022Torulaspora delbrueckii phenotypic and metabolic profiling towards its biotechnological exploitation.Journal of Fungi,8569C.aStefenonM.ColomboC.dBonesiV.MarzarottoR.VanderlindeM.SalvadorJ.aHenriques2010Antioxidant activity of sparkling wines produced by Champenoise and Charmat methods.Food Chemistry,11912-18V.Van BredaN.JollyJ.Van Wyk2013Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains.International Journal of Food Microbiology,16380-88https://doi.org/10.1016/j.ijfoodmicro.2013.02.011A.VerzelettiS.EcheverrigarayA.CardosoR.VanderlindeA.pLongaray Delamare2016Evolution of aromatic compounds during the second fermentation and aging of Brazilian sparkling wine.B.XueE.jLiR.pFuY.lLiu2009Study on stress resistance of wine-related yeasts.Proceedings of the Sixth International Symposium on Viticulture and Enology, 147-153.M.YoshidaS.KatoS.FukudaS.Izawa2021Acquired resistance to severe ethanol stress in Saccharomyces cerevisiae protein quality control.Applied and Environmental Microbiology, 87(6).https://doi.org/10.1128/AEM.02353-20B.qZhangY.LuanC.qDuanG.lYan2018Use of Torulaspora delbrueckii co-fermentation with two Saccharomyces cerevisiae strains with different aromatic characteristic to improve the diversity of red wine aroma profile.Frontiers in Microbiology,9, https://doi.