OENO One Biological 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 1 Fondazione Edmund Mach, Centro di Trasferimento Tecnologico. Via Mach 1. 38010 San Michele all’Adige, Trento (Italy). 2 Università di Trento, Dipartimento C3A. Via Mach 1. 38010 San Michele all’Adige, Trento (Italy). Gallo Adelaide 1 Guzzon Raffaele * 1 Paolini Mauro 1 Malacarne Mario 1 Nardin Tiziana 1 Ongaro Marco 2 Roman1. Tomas * raffaele.guzzon@fmach.it Abstract

In this work, Lachancea thermotolerans was exploited as a biological acidifier of Vino Santo di Gambellara, a traditional Italian wine made from long-dried grapes. The drying of grapes before winemaking is a traditional technique widely applied in Italy in the production of sweet wines, but in recent years, global warming reduces dramatically the acidity of grapes, causing microbial instability during drying and winemaking. The ability of L. thermotolerans to convert sugars in lactic acid was already applied in the acidification of red wines, but the peculiar features of dried grape (e.g., osmotic stress, lack of nutrients, presence of mould-derived toxic compounds during drying) impose specific tests. L. thermotolerans was employed in sequential fermentation in combination with a strain of Saccharomyces cerevisiae. Considering the lack of information about the nutritional requirement of the non-Saccharomyces yeast, three protocols of nitrogen supplementation (mineral, organic and organic at high dosage) were tested. Alcoholic fermentation experiments were followed by plate counts onto differential media to discriminate between the two yeast species and by chemical analysis. Moreover, a GC-MS-MS approach carried out a complete characterisation of the volatile profile of wines. Results evidenced a long permanence of L. thermotolerans during alcoholic fermentation, which remained over the 7 log units until the 14th day of fermentation. The nitrogen supplementation protocol influenced cell growth and fermentative activity. Inorganic nitrogen supplementations allowed the accomplishment of alcoholic fermentation and the maintenance of pH below 3.35, with respect to the control wine (made only by S. cerevisiae), which was over pH 3.50. L. thermotolerans also influenced the wine's volatile aroma profile. Statistical differences were found in the main families of the yeast-derived aroma: acetate, esters, lactates, fatty acids and C6 compounds.

Keywords: L. thermotolerans, straw wine, Vino Santo, yeast assimilable nitrogen (YAN), lactic acid, mixed fermentation, wine acidification.

 Introduction

The progressive southernisation of the climate is a fact in many European wine regions. The responses to this phenomenon have been various, the evolution of agronomic practices, the use of different cultivars of Vitis vinifera or winemaking strategies aimed at minimising the impact of climate change on wine composition (Ollat et al., 2016). However, there are wines more sensitive to sudden climatic changes because of having an ancient tradition, refractory to changes due to the artisanal productive process or their peculiar organoleptic profile.

The production of Vino Santo, sweet wines obtained from post-harvest dried grapes and subject to a prolonged oxidative ageing in little casks called “Caratelli”, is traditional in some Italian regions: Tuscany (Domizio and Lencioni, 2011), Emilia Romagna (Laureati et al., 2020), Veneto (Torriani et al., 2011) and Trento province (Guzzon et al., 2014). Although peculiarities related to the different regional traditions, there are some common traits in the production and in the organoleptic profile of the different Vino Santo. Grapes of traditional Italian white varieties, the most employed are Trebbiano, Garganega, Malvasia di Candia or Nosiola, are chosen among sound bunches and dried in the attic of the winery, in a ventilated room called “Vinsantaia” (Laureati et al., 2020). To favour the water evaporation and avoid the growth of grey mould (Botrytis cinerea), grapes are conserved in small, perforated boxes, racks or hanging from vertical nets for 3–4 months, reaching a sugars concentration of 35–40 % g/g (Domizio and Lencioni, 2011). Traditionally spontaneous alcoholic fermentation takes place after grape pressing by a consortium of Saccharomyces and non-Saccharomyces yeasts (Stefanini et al., 2016) that colonises the Caratelli. Alcoholic fermentation stops naturally for the simultaneous presence of several limiting factors, such as volatile acidity, osmotic stress, high ethanol content and lack of nutrients (Guaragnella & Bettiga, 2021), and the Vino Santo ages some years (4–10 years) in barrels under oxidative conditions because the “Caratelli” are not fully filled. This sweet wine typically has an alcohol degree of up to 14 % (v/v) and a residual sugar content between 50 and 100 g/L (Laureati et al., 2020).

The organoleptic equilibrium and the durability of Vino Santo are based on maintaining an adequate acidity that regulates microbial evolution during grape drying and through the winemaking process. The acidity of Vino Santo cannot be represented only by acetic acid, accumulated by yeast due to osmotic stress (Erasmus et al., 2004; Bely et al., 2003) because the wine would be unbalanced. To obtain a high-quality Vino Santo, it is necessary to maintain a composite acidic profile based on the contribution of tartaric, lactic and succinic acids. The control of acetic acid accumulation can be done in many ways. Apart from careful hygiene and the use of osmotolerant yeast strains, the nitrogen supplementation of grapes must play a key role. Bely et al. (2008) demonstrated that the production of volatile acidity is inversely correlated with cell concentration during fermentation. High nitrogen availability in the first stage of winemaking is essential because it stimulates cell growth, improves the redox-equilibrating process, and consequently reduces volatile acidity formation.

Despite Vino Santo's capability to age for a very long time (Domizio and Lencioni, 2011), there are numerous microorganisms able to spoil Vino Santo. Moulds, already during grape dying (Guzzon et al., 2018; Lorenzini et al., 2013), acetic or lactic bacteria and some kinds of non-Saccharomyces yeasts can create organoleptic faults, interfere with fermentation and accumulate toxic molecules. The artisanal production of the Vino Santo, as well as a peculiar organoleptic profile, discourage relevant changes in the production process. Therefore, it is necessary to resolve the main critical issues of this wine-producing process in a discreet and sustainable way; in this sense, a relevant contribution can be given using non-Saccharomyces yeasts (Mateo and Maicas, 2016).

Lachancea thermotolerans is a yeast widespread in the oenological environment, surviving up to 13 % (v/v) of ethanol (Morata et al., 2018). As with other non-Saccharomyces yeasts, it is common to combine this yeast with a co-starter, generally a Saccharomyces cerevisiae strain. The timing and ratio of S. cerevisiae inoculum regulate the activity of L. thermotolerans, thanks to different phenomena of antagonism: cell–cell contact mechanisms, secretion of antimicrobial peptides and mere competition for nutritional sources (Nissen and Arneborg, 2003; Kemsawasd et al., 2015). There are two features of L. thermotolerans that arouse interest in the production of straw wines. The main is the production of L(+)-lactic acid, already during alcoholic fermentation (Banilas et al., 2016; Hranilovic et al., 2018b; Morata et al., 2018). With oxygen deficiency, typical of alcoholic fermentation, L. thermotolerans activates genes involved in the pentose phosphate pathway and the citric acid cycle. However, in opposition to what occurs in S. cerevisiae, the hyperactivation of genes encoding for enzymes of the lactate dehydrogenase class (LDH) was observed which allows the production of lactic acid from glucose and fructose. It can be assumed that the high expression of these genes is linked to the lack of production of alcohol dehydrogenase enzymes (only two genes involved are expressed, compared to seven of the other yeasts); therefore, the production of lactic acid is an alternative way for the reduction of acetaldehyde to ethanol to maintain intracellular redox balance, in the absence of oxygen (Gatto et al., 2020; Vicente et al., 2021). The highest accumulation of lactic acid so far reported reached 16 g/L, the maximum observed among non-genetically modified yeasts (Banilas et al., 2016). The acidification induced by L. thermotolerans positively affects the microbiological stability of the wine and the organoleptic balance, avoiding alterations or exogenous acidifications. Another characteristic of grapes subjected to withering is the excessive sugar content, which leads to high concentrations of ethanol and the massive production of fermentation bioproducts, such as acetic acid, which is a symptom of the osmotic stress of common winemaking yeasts. Several studies (Comitini et al., 2011; Gobbi et al., 2013; Morata et al., 2019; Sgouros et al., 2020) have reported significantly lower ethanol contents in co-fermentations with L. thermotolerans and S. cerevisiae with reductions ranging from 0.2 % to 0.9 % (v/v). Other interesting aspects are the increase in the concentration of glycerol, the decrease in the acetic acid content and a peculiar modulation of the volatile compounds in the wine.

In this work, we evaluated for the first time the use of this yeast in the production of Vino Santo di Gambellara, a traditional sweet wine produced in the Veneto region. The evolution of sequential fermentation made by combining L. thermotolerans and S. cerevisiae was followed by microbiological and chemical determinations, also evaluating the aromatic profile of obtained wines by a comprehensive GC-MS-MS approach. Among the different technological variables, particular attention was paid to the nutrition protocol furnished to the yeasts, considering the need to increase the knowledge regarding the nutritional requirements of non-Saccharomyces yeasts employed in oenology.

 Materials and methods 1. Winemaking protocol and nitrogen supplementation schema

According to the traditional winemaking of Vino Santo, grapes of cv. Garganega were hand-harvested in small boxes of about 25 kg, then further transferred on grids in a ventilated attic to allow a natural drying of bunches. During the period of grape drying, the temperature remained in the interval between 5 and 15 °C, and the relative humidity of the atmosphere was 80±30 %. After drying, grapes were transferred to the experimental winery of the Edmund Mach Foundation (Italy) and further crushed using a vertical pneumatic press (Alfa 40, Polisnelli Enologia, Italy). Grape must was statically decanted at 10 °C for 48 h, adding pectolytic enzyme (10 mg L-1, Rapidase Clear Extreme, Oenobrands, France) and potassium metabisulfite salt (30 mg L-1, Lafood, Italy). After decanting, the grape must was subdivided into aliquots to perform the experimental plan detailed in Table 1. For alcoholic fermentation starters, two Active Dry Yeast strains were employed at an inoculum dose of 250 mg L-1: S. cerevisiae var. bayanus (PDM, Oenobrands) and L. thermotolerans (LaktiaTM, Lallemand Inc, Canada). Active dry yeast was inoculated in grape must after rehydration according to the OIV standards (OIV, 2023a). Nitrogen supplementation of grape must was performed by two different products, a source of organic nitrogen (Natuferm Bright, Oenobrands) and diammonium phosphate salt (Oenobrands). Table 1 lists the amount of nitrogen furnished at each trial and the timing of supplementation; each trial was performed in triplicate. Alcoholic fermentation occurred in stainless steel containers of 10 L of volume at a temperature of 21±2 °C. After fermentation, wines were decanted and stored at 4±2 °C in stainless steel kegs with minimal ullage filled by argon until analysis.

Table 1. Experimental plan performed in the work to evaluate the potential of L. thermotolerans in the acidification of Vino Santo during alcoholic fermentation, in function of different nitrogen supplementation. Each trial was performed in 3 replicates in a volume of about 80 L. Organic nitrogen: Natuferm Bright, Oenobrands; DAP: diammonium phosphate salt.
titre du tableau
Experimental trial S. cerevisiae L. thermotolerans Nitrogen supplementation (Each dose 300 mg L-1)
Day 1 Day 5 Day 7 Day 10 Day14
SC Inoculum at Day 0 NO Organic N Organic N - - DAP
LTNM Inoculum at Day 5 Inoculum Day 0 DAP DAP - - DAP
LTNO Inoculum at Day 5 Inoculum Day 0 Organic N Organic N - - DAP
LTNO+ Inoculum at Day 5 Inoculum Day 0 Organic N ×3 Organic N ×3 Organic N Organic N DAP

2. Microbiological analysis and monitoring of alcoholic fermentation

Microbiological analyses were performed following the OIV methods (OIV, 2023b). Total yeasts were enumerated onto Wallerstein Laboratory agar medium (WL Agar, Oxoid, UK), while non-Saccharomyces yeasts, including L. thermotolerans, were differentiated using Agar Lysine Medium (Oxoid). Both samples were incubated aerobically at 25±2 °C for 3 days. The amount of live and dead cells in active dry yeast samples was determined by flow cytometry analysis using a Cube6 apparatus (Sysmex, Germany) coupled with a live & dead staining of samples, according to Guzzon and Larcher (2015). Flow cytometry analyses revealed that the S. cerevisiae var. bayanus employed in this work has a concentration of live cells of 9.8±1.1 × 109 cell/g, while the strain of L. thermotolerans showed a concentration of live cells of 2.5±2.1 × 1010 cell/g. Dead cells in both cases resulted below the 109 cell/g. Considering the ratio of dilution due to the inoculum of yeast in wine, the nominal concentration of S. cerevisiae at the beginning of fermentation is about 2.3 × 106 cell/mL and 6.3 × 106 cell/mL for L. thermotolerans.

The evolution of alcoholic fermentation was monitored by measuring the weight loss due to CO2 evolution and the °Brix with an optical refractometer (ATC, Polsinelli Enologia, Italy). pH and titratable acidity were measured by a pH meter and an automatic titrator (Crison Instruments, Spain). The fermentation rate (V) was calculated as the first derivative of the weight loss expressed as g L-1 day-1. At the end of alcoholic fermentation, reducing sugars, ethanol and organic acids were determined by enzymatic assay (Miura One, Exacta-Optech, Italy).

3. GC-MS/MS analysis of wines

The analysis of volatile compounds in wine was performed according to the method reported by Paolini et al. (2018). A total of 50 mL of wine was diluted to 100 mL with H2O milliQ after adding 100 μL of internal standard (n-heptanol), and volatile compounds were extracted by solid-phase extraction (SPE) using ENV+ cartridges. The GC-MS/MS Analysis was performed on an Agilent (California, USA) Intuvo 9000 coupled with an Agilent 7000 Series Triple Quadrupole mass spectrometer working in electron impact (EI) mode at 70 eV. Chromatographic separation was achieved by injecting 2 µL in split mode (1:5) into a DB-Wax Ultra Inert capillary column (20 m, 0.18 mm di × 0.18 µm film thickness) and using helium (He) as carrier gas (0.8 mL min-1). The oven temperature was programmed starting at 40 °C for 2 minutes, increased to 55 °C at 10 °C min-1, then increased to 165 °C at 20 °C min-1, and finally increased to 240 °C at 40 °C min-1, maintaining this temperature for 5 minutes. The mass spectrum was acquired in MRM (multiple reaction monitoring) mode, setting the instrument in a dynamic system. The injector, transfer line and source temperatures were 260 °C, 250 °C and 230 °C, respectively.

 4. Amino acid analysis by HPLC

“The quantification of amino acids was performed with the method reported by Gallo et al. (2023) by creating o-phthalaldehyde (OPA) adducts. The measurements were carried out using an HPLC 1260 Infinity system from Agilent Technologies (Santa Clara, USA), which had a fluorescence detector (Ex = 336 nm, Em = 445 nm). Separation was accomplished using a Chromolith Performance RP-18e column (100 × 4.6 mm; Merck, Darmstadt, Germany) with a Guard Cartridge Chromolith RP-18e (10 × 4.6 mm; Merck, Darmstadt, Germany) at a temperature of 40 °C. The mobile phase consisted of sodium acetate 0.05 M (pH 6.9; eluent A) and methanol (eluent B). The flow rate was set at 2 mL/min. The analytical gradient used was as follows: 100 % A for 1 minute, 80 % A and 20 % B over 10 minutes, 60 % A and 40 % B over 5 minutes, and 100 % B over 4 minutes, held for 5 minutes. This was followed by a gradient of 90 % A, 10 % B in 0.5 minutes, held for 2 minutes, and 100 % A for 3 minutes. Prior to separation, a 10 μL sample was automatically derivatised with a mixture of OPA (4.5 g/L; Sigma-Aldrich, St. Louis, USA) in sodium tetraborate 0.1 M, adjusted to pH 10.5, methanol (10 %; Sigma-Aldrich, St. Louis, USA), and 2-mercaptoethanol (2 %; Sigma-Aldrich, St. Louis, USA). Agilent OpenLab CDS 3.1 software was used for data acquisition and processing.”

 5. Statistical treatment of data

The wine data were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s HSD test post-hoc (HSD) in R (R Foundation for Statistical Computing, Austria) version 1.4.1103 in RStudio (RStudio, Massachusetts, USA).

 Results 1. Grapes drying and grape must feature.

Table 2 reports the evolution of the main parameters of grapes during withering from November to April. In addition to the acidity (as g L-1 of tartaric acid) and sugars concentration (as °Brix), the weight of a berry sample (n = 30) was measured, considering it a good index of the evolution of water loss from bunches. During drying, the sugar concentration inside berries increased by about 42 % in the function of grape drying time and relative humidity, while the temperature of the environment, in the interval of the test, appeared less relevant in driving the evaporation process. The rate of sugar concentration and weight loss followed a nonlinear trend, with two peaks, the first one observed between December and January and the second one before the crushing, from March and April. (Table 2). The total acidity of grapes showed a peculiar trend, with a decrease in the first month and a stabilisation in the final part of the drying process. At the end of withering, grapes were crushed, and grape must was characterised for the main oenological quality control parameters. The total soluble solids level in the grape must was 39.0° Brix, with a pH of 3.35 and titratable acidity equal to 6.01 g L-1 (expressed as tartaric acid), yeast-assailable nitrogen was below 40 mg/L.

Table 2. Evolution of main characters of grapes cv. Garganega during natural dying and relative humidity of the environment (Data obtained by analysing a sample of 30 berries randomly sampled).
titre du tableau
Sampling data Sugars content Total acidity Weight of 30 berries Mean berry weight Relative humidity
Day/month °Brix g L-1 of tartaric acid g %
28/11 20.5 6.5 46.1 1.5 87.0
19/12 21.0 6.1 49.0 1.6 83.5
7/01 24.0 6.8 36.8 1.2 80.0
27/01 23.5 7.1 32.1 1.4 76.5
18/02 25.0 7.6 33.8 1.1 67.5
06/03 26.2 8.0 35.5 1.2 63.0
20/03 27.1 7.8 35.0 1.2 57.5
09/04 34.0 8.0 31.2 0.9 48.0

2. Evolution of alcoholic fermentation and microbial populations

Considering the importance of nitrogen supplementation in the experimental plan, the characterisation of the two nitrogen preparations employed was performed. For diammonium phosphate salt, we estimated that the addiction of 300 mg/L corresponds to about 60 mg N/L; regarding organic supplementation (Natuferm Bright), Table S1 shows the amino acid composition of preparation. Considering the observed abundance of each amino acid and the dose of addiction, it is possible to estimate that each dose of Natuferm Bright furnishes at wine 9.2 mg/N/kg. Nevertheless, it is known that organic formulates deriving from yeast autolysates contain further compounds influencing the nitrogen metabolism of yeasts (e.g., di- and tri-peptides, oligopeptides, vitamins) and a YAN comparison would be underestimated in terms of nutrient supplementation.

Figure 1. Evolution of alcoholic fermentation (A) and V (B) of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n=3).

Figure 1. Evolution of alcoholic fermentation (A) and V (B) of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n=3).

Diammonium phosphate salt in Figure 1A shows the trend of alcoholic fermentations as weight loss (g L-1day-1). In the SC experiment, the lag phase lasted 4 days; after that, the maximum fermentation rate (Vmax) of 4.30±0.22 g L-1day-1 was reached 13 days before S. cerevisiae inoculum. Experiments conducted by sequential inoculum of L. thermotolerans and S. cerevisiae (LTNM, LTNO and LTNO+, Figure 1) showed a reduction of the lag phase. The promptest activation of alcoholic fermentation was observed in the experiment LTNM (lag phase duration 3.2 days), while LTNO and LTNO+ required 3.8 days to start sugar consumption. The readiness in the activation of alcoholic fermentation did not correspond to a higher sugar degradation rate which, independently from the nutritional protocol applied, remained lower than that observed in the SC trial for both experiments conducted with the sequential inoculation of the two yeasts. The LTNM trial reached a maximum fermentation rate of 3.16±0.18 g L-1day-1 after 8 days, the LTNO experiment showed a Vmax of 3.06±11 g L-1day-1 after 9.8 days, and LTNO+ Vmax was 3.77±0.26 g L-1day-1, measured at the 9th day of fermentation. The inoculation of S. cerevisiae performed 5 days after the beginning of the test did not alter the behaviour of the LTNM and LTNO experiments, which maintained a relevant delay with respect to the test performed only by S. cerevisiae in terms of sugar degradation rate (SC, Figure 1). In the LTNO+ experiment, the evolution of alcoholic fermentation resulted comparable to that of the SC test, especially in the last days of winemaking. Another difference observed among the four experiments concerns the amount of CO2 produced, which corresponds to the level of sugar consumption. SC and LTNO+ experiments produced 98.30 and 95.92 g L-1 of CO2, respectively, after 55 days of fermentation, while the LTNM and LTNO CO2 production was in the range between 77.87 and 79.90 g L-1 at the same time (Figure 1A).

In wine, the yeast population was monitored in the first two weeks of alcoholic fermentation, obtaining an estimation of the populations of L. thermotolerans (when present) and S. cerevisiae by the difference between plate count onto a non-selective (WL agar) and selective (Lysine agar) medium. (Figure 2). The different types of inoculum and nutritional protocols influenced the development of the yeasts. In the SC experiment, the population of S. cerevisiae reached the maximum density (6.0±0.4 × 107 CFU mL-1) on the 7th day of fermentation; the population of non-Saccharomyces yeast was in the order of 4 log units and disappeared (Detection limit 50 CFU/mL) after 24 hours of fermentation. In the LTNM experiment (Figure 2), the population of L. thermotolerans was over the 7 log units for the entire duration of observations, while S. cerevisiae increased after the 6 log units only after the inoculum was performed on day 5. This trend is amplified in the experiments conducted by organic nitrogen supplementation, with a spread among non-Saccharomyces and Saccharomyces yeast in the order of 5 log units after 24 hours and 1 log units after 7 days of alcoholic fermentation. However, the maximum density of S. cerevisiae was reached in the LTNO+ experiments on the 14th day of fermentation (1.1±0.2 × 107 CFU mL-1).

Figure 2. Estimation of populations of S. cerevisiae and L. thermotolerans during alcoholic fermentation of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n=3), different letters in the different trials (SC, LTNM, LTNO and LTNO+) in the same day of sampling and yeast group indicate data that are significantly different. ANOVA and Tukey’s HSD test α = 5 % ANOVA and Tukey tests (p = 0.05).

Figure 2. Estimation of populations of S. cerevisiae and L. thermotolerans during alcoholic fermentation of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n=3), different letters in the different trials (SC, LTNM, LTNO and LTNO+) in the same day of sampling and yeast group indicate data that are significantly different. ANOVA and Tukey’s HSD test α = 5 % ANOVA and Tukey tests (p = 0.05).

3. Grape must acidification and chemical features of wines

The evolution of pH was monitored daily during the first 25 days after grape crushing, corresponding to the hypothesised period of activity of L. thermotolerans. As reported in Figure 3, the experiments performed only with S. cerevisiae (SC) started from a pH of 3.36±0.02 and increased up to 3.51±0.01 after 25 days, while in the experiments performed by sequential inoculum of L. thermotolerans and S. cerevisiae, the wine pH remained below 3.50. More in detail, the LTNM experiment showed after 25 days of fermentation, a pH value of 3.30±0.02, less than that of the grape must. In the LTNO and LTNO+ experiments, the acidification was less efficient, reaching a pH value of 3.43±0.01 and 3.46±0.02, respectively. In both cases, it is interesting to observe that the acidification trend is not linear; in the first days of alcoholic fermentation, a sudden decrease in pH was observed, probably due to the lactic acid production due to L. thermotolerans, followed by an increase attributable at the precipitation of tartaric acid salts during alcoholic fermentation. This hypothesis is supported by the acidic profile of the resulting wines, which revealed an accumulation of lactic acid only in the test performed in the presence of LT (Table 3). Table 3 lists the chemical features of the wines. The addition of L. thermotolerans significantly (p ≤ 0.05) reduced the sugar consumption and ethanol accumulation, considering that wines were sampled at the same time; also, the volatile acidity was lowered in the trials performed by sequential inoculum of the two yeasts. Titratable acidity increased due to the accumulation of lactic acid, whose content in wines made by non-Saccharomyces yeast was statistically different (p ≤ 0.05) with respect to SC trials.

Figure 3. Evolution of pH during alcoholic fermentation of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n = 3).

Figure 3. Evolution of pH during alcoholic fermentation of grape must obtained from dried grapes of Garganega suitable for production of Vino Santo di Gambellara. Mean data ± SD (n = 3).

Table 3. Main chemical parameters of Vino Santo obtained by different oenological protocols that consider the use of L. thermotolerans as natural acidifiers (Mean ± SD, n=3). Different superscript letters indicate data statistically different (ANOVA and Tukey’s HSD test α = 5 %).
titre du tableau
SC LTNM LTNO LTNO+
EtOH (% vol) 15.85±0.15a 12.54±0.39b 13.15±1.08b 14.18±0.99ab
pH 3.51±0.02a 3.30±0.01b 3.43±0.04a 3.46±0.05a
Titratable acidity (g L-1 of tartaric acid) 7.00±0.00c 8.87±0.15a 8.07±0.25b 7.83±0.32b
Volatile acidity (g L-1 of acetic acid) 1.80±0.03a 1.65±0.01b 1.68±0.09ab 1.75±0.05ab
Reducing sugars (g L-1) 150.23±3.65b 195.53±5.73a 184.03±21.65ab 165.93±20.35ab
Lactic acid (g L-1) 0.01±0.00c 2.51±0.24a 1.70±0.20b 1.57±0.15b
Malic acid (g L-1) 0.61±0.01a 0.54±0.03b 0.56±0.02ab 0.53±0.04b

A total of thirty-three free volatile compounds were identified and quantified in wines (Table 3). These were represented mainly by yeast-derived metabolites. Acetates, Ethyl Esters, Alcohols and Fatty acids underwent statistically significant differences among treatments. The profile of volatile compounds was significantly altered by the presence of L. thermotolerans in all main aroma groups. The family of acetates resulted in significantly differences both among yeast and nutrition treatments. The lowest concentration of acetates (sum of isobutyl acetate, isopentyl acetate, 2-phenylethyl acetate, ethyl phenyl acetate) was detected in SC wines (0.225 mg L-1). On the other hand, LTNO wines were in all the cases characterised by higher values of acetates. On the other hand, calculating the sum of ethyl esters of medium-chain fatty acids (ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate), emerged higher concentrations of these compounds in SC wines compared to LT treatments. The concentrations of the ester precursors were mainly differentiated between yeast species rather than nutrient treatments. However, when considering ethyl lactate, the situation is overturned; in fact, LTNM, LTNO and LTNO+ were about 10 to 30 times higher than the control. Regarding ethyl esters of short-chain fatty acids, ethyl butyrate in LTNO was higher than in the controls. Medium-chain fatty acid concentrations resulted in a significant difference in yeast strain management, following the trend found in their derivative ethyl esters, as seen previously: higher values in SC wines. Likewise, the same tendency was detected in the quantification of short-chain fatty acids. Another strain-derived difference was noticeable in C6 compounds, such as 1-hexanol, higher in SC wines (0.218 mg L-1). Regarding alcohols, 2-phenylethanol was found to be higher in LT-fermented wines than in the controls; among the LT fermentations, LTNM significantly decreased compared to LTNO. The family of terpenes were not present in relevant amounts in all wines, according to the features of native grapes.

Discussion

The main objective of this work is to improve the acidic profile of Vino Santo, without relevant changes in the productive process or in the features of obtained wines to ensure, at the same time, wine quality, microbiological control and recognisability of this ancient oenological production. Previous experiences in the biological acidification of different wines, such as Merlot (Hranilovic et al., 2021), Emir (Balikci et al., 2016) and, more generally, wines made from warm regions (Morata et al., 2019), suggested that L. thermotolerans could be a promising solution in the fermentation of grape must made from dried bunches. At the same time, the peculiar features of Vino Santo require an in-depth study of the interaction among different yeast species, S. cerevisiae and L. thermotolerans, and the nutritional requirement of the last one in terms of nitrogen sources.

In our experiments, the early inoculum of L. thermotolerans ensured the prompt activation of alcoholic fermentation, while the pure culture of S. cerevisiae was slower (SC, Figure 1A). This behaviour is not surprising because the sensitivity of S. cerevisiae to the osmotic stress typical of high-sugar grape must made from dried grapes is already known. On the contrary, L. thermotolerans, as reviewed by Vicente et al. (2022), can better adapt to high osmotic pressure environments (García et al., 2021), such as over-ripe grapes; then, S. cerevisiae ensures a prompt activation of alcoholic fermentation useful to protect grape must from the early proliferation of spoilage microorganisms (Morata et al., 2021). However, in the continuation of the fermentation, the poor fermentative capacity of the non-Saccharomyces yeast emerges, resulting in a low fermentation rate, both in terms of sugar consumption and in the Vmax value, which resulted in a mean of 23 % lower than that of the SC trial. This difference is more relevant if we consider the addition of S. cerevisiae on the 5th day, resulting that the Vmax measured after 10 days is due to the contribution of both yeasts. The lowering of fermentative activity observed in the LT tests confirms the presence of negative interactions between the two species of yeast utilised in this work, as already observed by some authors (Ciani et al., 2006; Hranilovic et al., 2018a; Hranilovic et al., 2021). A different hypothesis regarding the nature of these interactions was advanced (Renault et al., 2013; Kemsawasd et al., 2015; Ciani et al., 2016). In this work, we observe the role of nitrogen supplementation in reducing these detrimental effects, suggesting the key role of the competition among yeasts for nutritional sources. The non-mixed nitrogen supplementation protocols employed in this study (only inorganic or organic nitrogen) did not ensure the optimal evolution of sugar consumption, according to the observation of Roca-Mesa et al. (2020). On the contrary, in the thesis LTNO+, the high organic nitrogen supplementation reduces the gap with the SC trial regarding fermentation behaviour and Vmax (Figure 1).

The microbiological analysis performed on the active dry yeast samples and the wines during alcoholic fermentation (Figure 2) ensures additional information about the interaction between yeasts during mixed fermentation. The L. thermotolerans strain showed a slightly higher concentration in terms of viable cells per gram compared to the S. cerevisiae strain, which justified the high cell concentration measured already after 24 hours from the inoculum. In SC trials, the population of S. cerevisiae after 24 hours was in the order of 6 log units in accordance with the inoculum ratio, reaching after 5 days 7 log units and remaining constant for the entire duration of sugars degradation. The presence of a high concentration of Saccharomyces counteracted the growth of non-Saccharomyces yeasts made from the raw material. In all the experiments, L. thermotolerans showed good tolerance to the wine limiting factors, remaining up to 7 log units for the entire duration of alcoholic fermentation. S. cerevisiae suffered, however, from the competition of the other yeast. Two days after the inoculum of S. cerevisiae (Day 7, Figure 2), it did not reach 7 log units, remaining significantly less concentrated (p ≤ 0.05) than that observed in the SC test. In synthesis, the presence of L. thermotolerans allowed a faster activation of sugar degradation but, at the same time, created an environment less favourable for the activity of S. cerevisiae; the chemical analysis performed on the resulting wines can aid in explaining how these differences impact the composition of wines and their organoleptic profile.

Regarding the composition of wines (Table 4), the early inoculation with L. thermotolerans ensured the main objective of this work was achieved. Compared to the SC test, wines produced by the two yeasts showed a higher content of lactic acid that resulted in a pH below 3.50. This pH value is generally recognised as critical for wine microbiological stability (Bartowsky, 2009; Rankine, 2006). This result is even more interesting if we consider that the malolactic fermentation did not occur, as demonstrated by the malic acid residue. The sequential fermentation of L. thermotolerans and S. cerevisiae positively impacted the other parameters: the wines had a more moderate alcohol content and a lower volatile acidity, according to that observed by Vilela (2018).

Table 4. Volatile profile of Vino Santo obtained by different oenological protocols that consider the use of L. thermotolerans as natural acidifiers. (Mean ± SD, n = 3). Different superscript letters indicate data statistically different (ANOVA and Tukey’s HSD test α = 5 %).
titre du tableau
SC LTNM LTNO LTNO+
µg/L
Isobutyl acetate 20±1b 30±1a 31±2a 30±1a
Isopentyl acetate 169±16c 229±20ab 253±8a 212±11b
2-phenylethyl acetate 34±4b 39±1ab 44±0a 36±2b
Ethyl phenyl acetate 3±0b 3±0ab 4±0a 3±1ab
Acetate (sum) 225±21c 302±19b 332±8a 281±12ab
Ethyl butyrate 49±49b 56±6ab 65±4a 55±3ab
Ethyl-2-methylbuthyrate 5±0a 5±0a 5±1a 5±0a
Ethyl isovalerate 0±0b 1±0a 1±0a 1±0a
Ethyl pentanoate 0±0a 1±0a 1±0a 0±0a
Ethyl hexanoate 72±5a 37±10b 37±3b 37±4b
Ethyl octanoate 71±17a 22±9b 18±2b 24±1b
Ethyl decanoate 49±8a 21±1b 16±5b 21±3b
Ethyl dodecanoate 5±1a 3±0b 2±0b 3±0b
Ethyl lactate 2239±83d 58043±7215a 33939±4527b 26101±2424c
Esters + lactate (sum) 2494±643c 58192±7190a 34089±4516b 26250±2417b
Butanoic acid 146±6a 122±5b 137±3a 137±6a
Isobutytric acid 268±27a 156±9b 161±9b 131±7b
Valeric acid 8±0a 4±0b 4±1b 5±1b
Isovaleric acid 312±13a 213±12c 245±5b 237±15bc
Hexanoic acid 267±3a 102±19b 99±11b 104±14b
Octanoic acid 143±4a 33±12b 31±6b 38±6b
Nonanoic acid 3±0a 3±0a 1±0c 2±0b
Decanoic acid 98±9a 29±8b 22±3b 26±1b
Fatty acids (sum) 1245±28a 662±43b 702±28b 680±48b
1-hexanol 218±7a 142±10b 134±11b 147±12b
Trans-3-hexen-1-ol 7±0a 8±1a 8±0a 8±0a
Cis-3-hexen-1-ol 2±0a 2±0a 1±0ab 1±0ab
Terpinen-4-ol 7±0a 7±0a 8±0a 7±0a
3-methylthio-1-propanol 92±4b 224±48ab 279±95a 226±54ab
2-phenylethanol 27,897±2364c 33,557±886b 40,070±1717a 35,835±1300ab
Benzaldehyde 142±3a 78±5b 103±15b 145±17a
Benzothiazole 1±0a 1±0a 1±0a 1±0a
Diethyl-succinate 653±32b 782±96ab 890±66a 937±46a
Beta-damascone 97±9a 76±11a 83±19a 77±13a
Guaiacol 2±0a 2±0a 2±0a 2±0a
Benzyl alcohol 43±7a 65±2b 59±9ab 37±4a
Geranic acid 22±2c 29±1b 33±1a 29±1b
Zingerone 9±1a 1±0b 2±0b 2±0b

The volatile profile of wines (Table 4) was influenced both by the yeast specie and the nutrition protocol in LT wines. As previously reported (Vaquero et al., 2021; Hranilovic et al., 2021), L. thermotolerans produced a higher concentration of acetate esters compared to S. cerevisiae. Acetate esters are derived from higher alcohols, which can be formed by yeast catabolism through the Ehrlich pathway from amino acids (Labuschagne and Divol, 2021). Among the LT wines, LTNO showed a greater value of the total acetates than LTNM, perhaps due to the presence of amino acids in the organic nutrient composition (Mendes-Ferreira et al., 2011). However, no statistically significant differences were noticed between LTNO+ and the other LT treatments. The level of ethyl esters of medium chain fatty acids was yeast-dependent, with higher values in SC wines, and related to the availability of their respective precursors, hexanoic, octanoic and decanoic acid (Dennis et al., 2012), which followed the same trend. Those observations agreed with some previous studies (Vaquero et al., 2021; Sgouros et al., 2020; Hranilovic et al., 2021). However, only ethyl hexanoate and ethyl octanoate were above the ODT. Moreover, branched-chain fatty acids (isobutyric acid, valeric acid and isovaleric acid) resulted higher in SC wines, with some differences in concentration also among the nutrient treatments; LTNM always reported lower values, results in contrast with previous works (Whitener et al., 2017; Sgouros et al., 2020). The correspondent ethyl esters were influenced by the yeast strain, in agreement with previous studies (Mallouchos et al., 2003; Sgouros et al., 2020; Hranilovic et al., 2021). SC wines had, in general, either equal or significantly lower concentrations with respect to LT wines. Whereas, as expected, ethyl lactate was the ester predominant in wines fermented by L. thermotolerans, probably due to the high availability of lactic acid as its precursor. Ethyl lactate is formed because of the esterification of D-lactic or L-lactic acid, rarely produced by S. cerevisiae (Ishida et al., 2006). Ethyl lactate gives the wine buttery and fruity notes. In this case, the concentrations, even if important, did not reach the perception threshold (146 mg L-1; Moyano et al., 2010). This result is in accordance with previous studies (Whitener et al., 2017; Vaquero et al., 2021; Sgouros et al., 2020; Hranilovic et al., 2021).

Figure 4. Principal component analysis (PCA) of compositive profile of Vino Santo di Gambellara obtained by different yeast inoculum and nitrogen supplementation protocol. Projection in the plane of first two factors of variables (Compositive data) and cases (Experimental trials).

Figure 4. Principal component analysis (PCA) of compositive profile of Vino Santo di Gambellara obtained by different yeast inoculum and nitrogen supplementation protocol. Projection in the plane of first two factors of variables (Compositive data) and cases (Experimental trials).

Besides the univariate analysis, the chemical dataset was also subjected to multivariate analysis with the principal component analysis (PCA; Figure 4). Wines are clearly separated in the space depending both on yeast and nutrient treatments. The first two principal components accounted for 70.3 % of the total variation in the samples. Along the first principal component, which accounts for 57.6 % of the variance explained, we observe a clear distinction between wines fermented with different yeasts. In fact, wines fermented by L. thermotolerans occupy the left side, in correspondence with higher values of lactic acid, titratable acidity (TA), and some acetates. While the SC samples are in the far-right, this is driven mainly by higher values of fatty acids, 1-hexenol and ethanol, as a confirmation of results seen in Table 3. The major distinction between nutrient treatments is observable along PC2 (12.72 % of the variance explained). LTNM is in the left-highest quadrant, correlated to the highest accumulation of lactic acid, residual sugars, ethyl lactate and titratable acidity. Finally, the two experiments made by organic nutrition of yeasts (LTNO and LTNO+) are placed in the left lower quadrant, corresponding to greater values of some acetates and esters.

In conclusion, L. thermotolerans showed promising potentiality in restoring an adequate acidic profile in wines made from dried grapes, also giving peculiar character due to the synthesis of specific volatile metabolites. These results are in accordance with previous studies on different wines (Whitener et al., 2017, Vaquero et al., 2021; Roca-Mesa et al., 2020; Sgouros et al., 2020; Hranilovic et al., 2021); however, further investigations into the nutrition management of L. thermotolerans and volatile metabolites would be interesting as a confirmation of the present work.

 Acknowledgements

We thank Dr Paola Vagnoli and the staff of Lallemand Italy for their support in this work and for the supply of the commercial strain of L. thermotolerans.

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