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

Results

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).

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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 (V_max) 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 V_max of 3.06±11 g L^-1day^-1 after 9.8 days, and LTNO+ V_max 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).

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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).

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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 %).

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	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 V_max 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 V_max 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 V_max (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).

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).

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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.

References

• Balikci, E.K., Tanguler, H., Jolly, N.P., & Erten, H. (2016) Influence of Lachancea thermotolerans on cv. Emir wine fermentation. YEAST 33, 312-321. https://doi.org/10.1002/yea.3166
• Banilas, G., Sgouros, G., & Nisiotou, A. (2016). Development of microsatellite markers for Lachancea thermotolerans typing and population structure of wine-associated isolates. Microbiological Research, 193, 1-10. https://doi.org/10.1002/yea.3166
• Bartowsky, E.J. (2009) Bacterial spoilage of wine and approaches to minimize it. Letters in Applied Microbiology. 48, 149-156. https://doi.org/10.1111/j.1472-765X.2008.02505.x
• Bely, M., Rinaldi, A., & Dubourdieu, D. (2003). Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. Journal of Bioscience and Bioengineering, 9, 507-512. https://doi.org/10.1016/S1389-1723(04)70141-3
• Bely, M., Stoeckle, P., Masneuf-Pomarède, I., & Dubourdieu, D. (2008). Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation. International Journal of Food Microbiology. 20, 312-20. https://doi.org/10.1016/j.ijfoodmicro.2007.12.023
• Ciani, M., Beco, L., & Comitini, F. (2006). Fermentation behaviour and metabolic interactions of multi starter wine yeast fermentations. International Journal of Food Microbiology, 108(2), 239-245. https://doi.org/10.1016/j.ijfoodmicro.2005.11.012
• Ciani, M., Capece, A., Comitini, F., Canonico, L., Siesto, G., & Romano, P. (2016). Yeast interactions in inoculated wine fermentation. Frontiers in Microbiology, 7, 1-7. https://doi.org/10.3389/fmicb.2016.00555
• Comitini, F., Gobbi, M., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., & Ciani, M. (2011). Selected non-Saccharomyces wine yeasts in controlled multi starter fermentations with Saccharomyces cerevisiae. Food Microbiology, 28(5), 873–882. https://doi.org/10.1016/j.fm.2010.12.001
• Dennis, E. G., Keyzers, R. A., Kalua, C. M., Maffei, S. M., Nicholson, E. L., & Boss, P. K. (2012). Grape contribution to wine aroma: Production of hexyl acetate, octyl acetate, and benzyl acetate during yeast fermentation is dependent upon precursors in the must. Journal of Agricultural and Food Chemistry, 60(10), 2638-2646. https://doi.org/10.1021/jf2042517
• Domizio, P. & Lencioni, L. (2011). Vino Santo. Advances in Food and Nutrition Research, Vol 63, 41-100. https://doi.org/10.1016/B978-0-12-384927-4.00003-8
• Erasmus, D. J., Cliff, M., & Van Vuuren, H. J. (2004). Impact of yeast strain on the production of acetic acid, glycerol, and the sensory attributes of icewine. American Journal of Enology and Viticulture, 55(4), 371-378. https://doi.org/10.5344/ajev.2004.55.4.371
• Gallo, G., Larcher, R., Cappello, G., Paolini, M., Moser., R., Schneider, R., Carrau., F., Nardin., T., & Roman, T. (2023). Insights into the grape must composition effect on Hanseniaspora vineae performance and metabolic aroma compounds in Chardonnay base wine for sparkling wine production. Journal of Food Composition and Analysis, 105514. In press. https://doi.org/10.1016/j.jfca.2023.105514
• García, M., Crespo, J., Cabellos, J. M., & Arroyo, T. (2021). Growth of non-Saccharomyces native strains under different fermentative stress conditions. Fermentation, 7(3). https://doi.org/10.3390/fermentation7030124
• Gatto, V., Binati, R., L., Lemos Junior, W.J.F., Basile, A., Treu, L., de Almeida, O.G.G., Innocente, G., Campanaro, S., & Torriani, S. (2020). New insights into the variability of lactic acid production in Lachancea thermotolerans at the phenotypic and genomic level. Microbiological Research, 238, 126525. https://doi.org/10.1016/j.micres.2020.126525
• Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., & Ciani, M. (2013). Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiology, 33(2), 271–281. https://doi.org/10.1016/j.fm.2012.10.004
• Guaragnella, N., & Bettiga, M. (2021). Acetic acid stress in budding yeast: From molecular mechanisms to applications. Yeast, 38, 391–400. https://doi.org/10.1002/yea.3651
• Guzzon, R., & Larcher, R. (2015). The application of flow cytometry in microbiological monitoring during winemaking: two case studies. Annals of Microbiology 65, 1865–1878. https://doi.org/10.1007/s13213-014-1025-6
• Guzzon, R., Franciosi, E., & Larcher, R. (2014). A new resource from traditional wines: characterisation of the microbiota of "Vino Santo" grapes as a biocontrol agent against Botrytis cinerea. European Food Research and Technology, 239(1), 117-126. https://doi.org/10.1007/s00217-014-2195-y
• Guzzon, R., Franciosi, E., Moser, S., Carafa, I., & Larcher, R. (2018). Application of ozone during grape drying for the production of straw wine. Effects on the microbiota and compositive profile of grapes. Journal of Applied Microbiology, 125, 513-527. https://doi.org/10.1111/jam.13774
• Hranilovic, A., Albertin, W., Capone, D. L., Gallo, A., Grbin, P. R., Danner, L., Bastian, S. E. P., Masneuf-Pomarede, I., Coulon, J., Bely, M., & Jiranek, V. (2021). Impact of Lachancea thermotolerans on chemical composition and sensory profiles of Merlot wines. Food chemistry 349, 129015. https://doi.org/10.1016/j.foodchem.2021.129015
• Hranilovic, A., Li, S., Boss, P. K., Bindon, K., Ristic, R., Grbin, P. R., Van der Westhuizen, T., & Jiranek, V. (2018a). Chemical and sensory profiling of Shiraz wines co-fermented with commercial non-Saccharomyces inocula. Australian Journal of Grape and Wine Research, 24(2), 166–180. https://doi.org/10.1111/ajgw.12320
• Hranilovic, Ana, Gambetta, J. M., Schmidtke, L., Boss, P. K., Grbin, P. R., Masneuf-Pomarede, I., Bely, M., Albertin, W., & Jiranek, V. (2018b). Oenological traits of Lachancea thermotolerans show signs of domestication and allopatric differentiation. Scientific Reports, 8(1), 1–13. https://doi.org/10.1038/s41598-018-33105-7
• Ishida, N., Suzuki, T., Tokuhiro, K., Nagamori, E., Onishi, T., Saitoh, S., ... & Takahashi, H. (2006). D-Lactic acid production by metabolically engineered Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 101(2), 172-177. https://doi.org/10.1263/jbb.101.172
• Kemsawasd, V., Viana, T., Ardö, Y., & Arneborg, N. (2015). Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation. Applied Microbial and Cell Physiology. https://doi.org/10.1007/s00253-015-6835-3
• Labuschagne, P. W. J., & Divol, B. (2021). Thiamine: a key nutrient for yeasts during wine alcoholic fermentation. Applied Microbiology and Biotechnology, 105, 953-973. https://doi.org/10.1007/s00253-020-11080-2
• Laureati, M., Cattaneo, C., Tateo, F., & Bononi, M. (2020). Identification of the volatile compounds and sensory attributes of long-term aging Vino Santo wine from Malvasia di Candia aromatic grapes. Foods, 9(12), 1736. https://doi.org/10.3390/foods9121736
• Lorenzini, M., Azzolini, M., Tosi, E., & Zapparoli., G. (2013). Postharvest grape infection of Botrytis cinerea and its interactions with other moulds under withering conditions to produce noble-rotten grapes. Journal of Applied Microbiology, 114(3), 762-70. https://doi.org/10.1111/jam.12075
• Mallouchos, A., Komaitis, M., Koutinas, A., & Kanellaki, M. (2003). Wine fermentations by immobilized and free cells at different temperatures. Effect of immobilization and temperature on volatile by-products. Food Chemistry, 80(1), 109-113. https://doi.org/10.1016/S0308-8146(02)00247-9
• Mateo, J., & Maicas, S. (2016). Application of Non-Saccharomyces Yeasts to Wine-Making Process. Fermentation, 2(4), 14. MDPI AG. Retrieved from http://dx.doi.org/10.3390/fermentation2030014
• Mendes-Ferreira, A., Barbosa, C., Lage, P., & Mendes-Faia, A. (2011). The impact of nitrogen on yeast fermentation and wine quality. A review. Ciencia E Tecnica Vitivinicola, 26, 17-32. https://api.semanticscholar.org/CorpusID:59154344
• Morata, A., Bañuelos, M. aA, Vaquero, C., Loira, I., Cuerda, R., Palomero, F., González, C., Suárez-Lepe, J. A., Wang, J., Han, S., & Bi, Y. (2019). Lachancea thermotolerans as a tool to improve pH in red wines from warm regions. European Food Research and Technology, 245(4), 885–894. https://doi.org/10.1007/s00217-019-03229-9
• Morata, A., Loira, I., González, C., & Escott, C. (2021). Non-Saccharomyces as bio tools to control the production of off flavours in wines. Molecules, 26(15). https://doi.org/10.3390/molecules26154571
• Morata, A., Loira, I., Tesfaye, W., Bañuelos, M. A., González, C., & Suárez Lepe, J. A. (2018). Lachancea thermotolerans applications in wine technology. Fermentation, 4(3). https://doi.org/10.3390/fermentation4030053
• Moyano, L., Zea, L., Moreno, J. A., & Medina, M. (2010). Evaluation of the active odorants in Amontillado sherry wines during the aging process. Journal of agricultural and food chemistry, 58(11), 6900-6904. https://doi.org/10.1021/jf100410n
• Nissen, P., & Arneborg, N. (2003). Characterization of early deaths of non-Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae. Archives of Microbiology, 180(4), 257–263. https://doi.org/10.1007/s00203-003-0585-9
• OIV (2023a). COEI-2-CONBAC Bacteriological control. Paragraph 1 “Preliminary rehydration of yeasts”. International Organization of Vine and Wine (OIV), Dijon, F.
• OIV (2023b). OIV-MA-AS4-01 Microbiological analysis of wines and musts. Detection, differentiation and counting of micro-organisms. International Organization of Vine and Wine (OIV), Dijon, F.
• Ollat, N., Touzard, J.M., & van Leeuwen, C. (2016). Climate change impacts and adaptations: new challenges for the wine industry. Journal of Wine Economics, 11(1), 139-149. PMID: 18262301. https://doi.org/10.1017/jwe.2016.3
• Paolini, M., Tonidandel, L., Moser, S., & Larcher, R. (2018). Development of a fast gas chromatography–tandem mass spectrometry method for volatile aromatic compound analysis in oenological products. Journal of Mass Spectrometry, 53, 801–810. https://doi.org/10.1002/jms.4259
• Rankine, B. (2006). Making good wine: a manual of winemaking practice for Australia and New Zealand. Sun Books, South Melbourne (Macmillan Australia).
• Renault, P. E., Albertin, W., & Bely, M. (2013). An innovative tool reveals interaction mechanisms among yeast populations under oenological conditions. Applied Microbiology and Biotechnology, 97(9), 4105–4119. https://doi.org/10.1007/s00253-012-4660-5
• Roca-Mesa, H., Sendra, S., Mas, A., Beltran, G., & Torija, M.-J. (2020). Nitrogen Preferences during Alcoholic Fermentation of Different Non-Saccharomyces Yeasts of Oenological Interest. Microorganisms, 8(2), 157. MDPI AG. http://dx.doi.org/10.3390/microorganisms8020157
• Sgouros, G., Mallouchos, A., Filippousi, M. E., Banilas, G., & Nisiotou, A. (2020). Molecular characterization and enological potential of a high lactic acid producing Lachancea thermotolerans Vinoeyard strain. Foods, 9(5), 1–16. https://doi.org/10.3390/foods9050595
• Stefanini, I., Albanese, D., Cavazza, A., Franciosi, E., De Filippo, C., Donati, C., & Cavalieri, D. (2016). Dynamic changes in microbiota during spontaneous "Vino Santo Trentino' fermentation. Microbial Biotechnology, 9(2), 195-208. https://doi.org/10.1111/1751-7915.12337
• Torriani, S., Lorenzini, M., Salvetti, E., & Fells, G. E. (2011). Zygosaccharomyces gambellarensis sp nov., an ascosporogenous yeast isolated from an Italian 'passito' style wine. International Journal of Systematic and Evolutionary Microbiology. 61, 3084-3088. https://doi.org/10.1099/ijs.0.031146-0
• Vaquero, C., Izquierdo-Cañas, P. M., Mena-Morales, A., Marchante-Cuevas, L., Heras, J. M., & Morata, A. (2021). Use of Lachancea thermotolerans for Biological vs. Chemical Acidification at Pilot-Scale in White Wines from Warm Areas. Fermentation, 7(3), 193. MDPI AG. http://dx.doi.org/10.3390/fermentation7030193
• Vicente, J., Baran, Y., Navascués, E., Santos, A., Calderón, F., Marquina, D., ... & Benito, S. (2022). Biological management of acidity in wine industry: A review. International Journal of Food Microbiology, 375, 109726. https://doi.org/10.1016/j.ijfoodmicro.2022.109726
• Vicente, J., Navascues, E., Calderon, F., Santos, A., Marquina, D., & Benito, S. (2021). An integrative view of the role of Lachancea thermotolerans in wine technology. Foods 10, 278. https://doi.org/10.3390/foods10112878
• Vilela, A. (2018). Lachancea thermotolerans, the non-Saccharomyces yeast that reduces the volatile acidity of wines. Fermentation-Basel, 4, 56. https://doi.org/10.3390/fermentation4030056
• Whitener, M. B., Stanstrup, J., Carlin, S., Divol, B., Du Toit, M., & Vrhovsek, U. (2017). Effect of non‐Saccharomyces yeasts on the volatile chemical profile of Shiraz wine. Australian journal of grape and wine research, 23(2), 179-192. https://doi.org/10.1111/ajgw.12269