Food Chemistry: X 21 (2024) 101214 Available online 9 February 2024 2590-1575/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A comparative study of Lachancea thermotolerans fermentative performance under standardized wine production conditions Javier Vicente a, Luka Vladic b,c, Eva Navascués d, Silvia Brezina c, Antonio Santos a, Fernando Calderón d, Wendu Tesfaye d, Domingo Marquina a, Doris Rauhut c, Santiago Benito d,* a Unit of Microbiology, Genetics, Biology Faculty, Physiology and Microbiology Department, Complutense University of Madrid, Ciudad Universitaria, S/N, 28040 Madrid, Spain b Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Gregor-Mendel-Straße 33, 1180 Wien, Austria c Department of Microbiology and Biochemistry, Hochschule Geisenheim University (HGU), Von-Lade-Straße 1, 65366 Geisenheim, Germany d Department of Chemistry and Food Technology, Polytechnic University of Madrid, Ciudad Universitaria, S/N, 28040 Madrid, Spain A R T I C L E I N F O Keywords: Lachancea thermotolerans L-lactic acid Organic acids Aroma compounds Wine Chemical compounds studied in this article: L-Lactic acid (PubChem CID107689) L-Malic acid (PubChem CID222656) Acetic acid (PubChem CID176) Succinic acid (PubChem CID1110) Isoamyl acetate (PubChem CID31276) Ethyl lactate (PubChem CID7344) Phenylethyl Alcohol (PubChem CID6054) A B S T R A C T The study explores diverse strains of Lachancea thermotolerans in single-inoculum wine fermentation conditions using synthetic grape must. It aims to analyze the role of the species without external influences like other microorganisms or natural grape must variability. Commercial strains and selected vineyard isolates, untested together previously, are assessed. The research evaluates volatile and non-volatile chemical compounds in final wine, revealing significant strain-based variations. L. thermotolerans notably produces lactic acid and consumes malic acid, exhibiting moderate ethanol levels. The volatile profile displays strain-specific impacts, affecting higher alcohol and ester concentrations compared to S. cerevisiae. These effects vary based on the specific compounds. Using a uniform synthetic must enables direct strain comparisons, eliminating grape-related, environmental, or timing variables in the experiment, facilitating clearer insights into the behavior of L. thermotolerans in wine fermentation. The study compares for the first time all available commercial strains of L. thermotolerans. 1. Introduction Lachancea thermotolerans is a non-Saccharomyces yeast that possesses the unique ability to significantly produce L-lactic acid under wine making conditions (Jolly et al., 2003, 2014, 2017). This ability allows for efficient acidification of wines, increasing their total acidity and reducing the pH (Blanco et al., 2020; Porter et al., 2019a, 2019b; Vilela, 2018). Such increased acidity is of interest in warm wine regions that suffer from a lack of acidity, among other technical problems. The evolution of climate change can potentially increase the number of re gions facing this problem, further increasing the interest in the use of L. thermotolerans (Benito, 2020). The utilization of L. thermotolerans has emerged as the most reliable biological acidification strategy in winemaking (Vicente et al., 2022). While there are other effective technologies available to enhance wine acidity, the use of L. thermotolerans presents several notable advantages. Firstly, the stability of L-lactic acid surpasses that of other options involving chemical additions, such as the addition of unstable acids like tartaric acid, which precipitates upon combination with potassium ions, or malic and citric acids, which can be metabolized by lactic bacteria, leading to undesirable uncontrolled re-fermentations. Secondly, the implementation of L. thermotolerans does not require initial costly in vestments, unlike other technological alternatives such as inverse osmosis. Furthermore, commercial strains of L. thermotolerans can be readily obtained in any wine region at comparable prices to regular dehydrated S. cerevisiae commercial strains. Despite these advantages, the use of L. thermotolerans, like other non-Saccharomyces yeast species, does present certain concerns when employed on an industrial scale. * Corresponding author. E-mail addresses: javievic@ucm.es (J. Vicente), Luka.Vladic@mail.hs-gm.de (L. Vladic), eva.navascues@upm.es (E. Navascués), Silvia.Brezina@hs-gm.de (S. Brezina), ansantos@ucm.es (A. Santos), fernando.calderon@upm.es (F. Calderón), wendu.tesfaye@upm.es (W. Tesfaye), dommarq@ucm.es (D. Marquina), Doris.Rauhut@hs-gm.de (D. Rauhut), santiago.benito@upm.es (S. Benito). Contents lists available at ScienceDirect Food Chemistry: X journal homepage: www.sciencedirect.com/journal/food-chemistry-x https://doi.org/10.1016/j.fochx.2024.101214 Received 20 November 2023; Received in revised form 26 January 2024; Accepted 6 February 2024 mailto:javievic@ucm.es mailto:Luka.Vladic@mail.hs-gm.de mailto:eva.navascues@upm.es mailto:Silvia.Brezina@hs-gm.de mailto:ansantos@ucm.es mailto:fernando.calderon@upm.es mailto:wendu.tesfaye@upm.es mailto:dommarq@ucm.es mailto:Doris.Rauhut@hs-gm.de mailto:santiago.benito@upm.es www.sciencedirect.com/science/journal/25901575 https://www.sciencedirect.com/journal/food-chemistry-x https://doi.org/10.1016/j.fochx.2024.101214 https://doi.org/10.1016/j.fochx.2024.101214 https://doi.org/10.1016/j.fochx.2024.101214 http://creativecommons.org/licenses/by/4.0/ Food Chemistry: X 21 (2024) 101214 2 These include its moderate fermentative power below 10 % (v/v) and sensitivity to sulfur dioxide (Vicente et al., 2021). Consequently, the available strains of L. thermotolerans should be employed in conjunction with a S. cerevisiae or Schizosaccharomyces pombe strain as these species are reported to be able to ferment over 15 % (v/v) (Benito, 2018), and applied to healthy grapes that do not require substantial additions of sulfur dioxide. The first commercial strain of L. thermotolerans, known as Con certoTM (Hansen), entered the market in 2012. Currently, there are seven strains of L. thermotolerans available in the market (Vejarano & Gil-Calderón, 2021): Concerto (CHR Hansen, Denmark), Laktia (Lalle mand, Canada), Levulia Alcomeno (AEB Group, Italy), EnartisFerm QK (Enartis, Italy), Excellence (X’Fresh Lamothe-Abiet, France), Kluyver omyces thermotolerans (Probiotec, Italy), and Octave (CHR Hansen, Denmark). Furthermore, some manufacturers, such as CHR Hansen, have begun offering two different strains of L. thermotolerans with distinct purposes. This wide range of commercial options facilitates the acquisition of active freeze dried L. thermotolerans by winemakers worldwide, suitable for use in any winery regardless of the wine region. However, no previous study has compared all the commercially avail able strains of L. thermotolerans against each other. There have been several previous studies comparing some of the available commercial strains of L. thermotolerans. Two studies compared the three commercial strains LevuliaTM (AEB), ConcertoTM (Hansen), and LaktiaTM (Lallemand). Another study compared three different commercial strains, namely Excellence X’Fresh (Lamothe-Abiet), Levu liaTM (AEB), and QKAPPA (Enartis) (Vicente et al., 2023a), while one study compared two strains, LaktiaTM (Lallemand) and ConcertoTM (Hansen) (Vaquero et al., 2020). These studies reported varying and sometimes contradictory results regarding the commercial strain that produces the highest concentration of L-lactic acid, with different orders reported in each study. Additionally, different chemical parameters exhibited different production rankings. This phenomenon can be attributed to the varying performance of each strain under different conditions, such as different combinations with S. cerevisiae strains or the use of different grape juices and fermentation conditions. None of these previous studies compared all the commercial strains under the same conditions in pure fermentation without combining them with S. cerevisiae. This study evaluates the fermentative performance of several L. thermotolerans strains in a synthetic grape must (SGM) (Henschke and Jiranek, 1993). The proposed methodology enables the replication of the experiment in any laboratory, thereby facilitating the comparison of new future commercial L. thermotolerans strains or other selected strains under standardized conditions. The study encompasses all commercially available L. thermotolerans strains to date, as well as autochthonous strains isolated from Spanish vineyards and wineries. 1.1. Hypothesis Given the varied and sometimes contradictory results from previous studies comparing different commercial strains of Lachancea thermoto lerans (L. thermotolerans) in terms of L-lactic acid production under winemaking conditions, we hypothesize that there will be significant variability in fermentative performance among the commercially available L. thermotolerans strains and autochthonous strains isolated from Spanish vineyards and wineries. We anticipate observing distinct patterns of L-lactic acid production among these strains in synthetic grape must (SGM), reflecting the diverse genetic makeup and metabolic capabilities of each strain. Furthermore, we predict that the fermenta tion performance of these strains will differ when tested in pure fermentation without the addition of Saccharomyces cerevisiae, high lighting the importance of evaluating L. thermotolerans strains inde pendently to understand their true potential for biological acidification in winemaking. 2. Material and methods 2.1. Microorganisms and fermentations Thirteen strains of Lachancea thermotolerans yeast were utilized in this study: Concerto (CHR Hansen, Denmark), Laktia (Lallemand, Can ada), Levulia Alcomeno (AEB Group, Italy), EnartisFerm QK (Enartis, Italy), Excellence (X’Fresh Lamothe-Abiet, France), and Octave (CHR Hansen, Denmark) as commercially available strains; and NG-108, A11- 612, EM-119, MJ-311, BD-612, L1, and L3 (Complutense University of Madrid, Madrid, Spain) as selected strains. The control strain used was S. cerevisiae AWRI-796 (Maurivin, Australia). All L. thermotolerans strains employed in this study have previously been identified as different strains according to their genotype determined by the micro satellite typing protocol for L. thermotolerans, amplifying six different microsatellites by multiplexed PCR, and resolving them by agarose electrophoresis (Vicente et al., 2023c). Synthetic Grape Must (SGM), prepared according to the original formulation (Henske and Jiranek, 1993), was utilized for all fermenta tions. Briefly, the SGM composition included 200 g/L of equimolar glucose and fructose, 3 g/L of malic acid, and 2.5 g/L of potassium tartrate, with pH adjusted to 3.5. The nitrogen content was adjusted to 140 mg/L from amino acids and 60 mg/L from di-ammonium phos phate. Yeast precultures were incubated in SGM at 25 ◦C and 150 rpm orbital shaking for 24 h. For the fermentations, conducted in triplicate, the final inoculation concentration was 2⋅105 cells/mL in 100 mL bo rosilicate bottles containing 90 mL of SGM. The bottles were then incubated at 25 ◦C. Fermentative kinetics were monitored by measuring weight loss every 24 h, and fermentations were considered complete when the weight loss was less than 0.01 % per day. After fermentation, all wines were centrifuged (7000 rpm for 5 min) and stored at 4 ◦C until further analysis. 2.2. Basic oenological parameters determinations A Y15 Autoanalyzer and its enzymatic kits (Biosystems, Spain) were used to perform determinations of L-malic acid and L-lactic acid. The determination of acetic acid, ethanol, total acidity, glucose + fructose, succinic acid, pH, and glycerol in the resulting wines was carried out using an FTIR autoanalyzer Bacchus 3 (TDI, Spain). 2.3. Volatile compounds The analysis of esters, higher alcohols, and fatty acids was performed using headspace solid phase micro extraction in connection with gas chromatography coupled with mass spectrometry (HS-SPME-GC–MS) by the Department of Microbiology and Biochemistry at Hochschule Gei senheim University (Scansani et al., 2020; Jung et al., 2021). The analytical method is briefly described in the following: 1.7 g of sodium chloride (NaCl; p.a.) were weighted into a 20 mL amber glass headspace vial before 5 mL sample and 10 μL of each in ternal standard solution (1-octanol 600 mg/L (for quantification), cumene 52 mg/L (additionally for control)) were pipetted. Then the vial was tightly closed with a magnetic screw cap. The calibration was performed in model wine (3 %, 6 % or 12 % (v/ v) solutions of ethanol in water (depending on the ethanol content of the samples), 3 g/L tartaric acid, adjusted to pH 3). A multipurpose sampler MPS robotic (Gerstel, Mülheim an der Ruhr, Germany) was applied for HS-SPME injection. SPME extraction was conducted with a 1 cm SPME fiber with 65 μm of polydimethylsiloxane/ divinylbenzene (Supelco) for 20 min (incubation temperature: 40 ◦C, incubation time 10 min). The sample was transferred with a cooled injection system (CIS-4, Gerstel, Mülheim an der Ruhr, Germany) to the gas chromatograph (GC 7890 A, Agilent, Santa Clara, USA): temperature program: 30 ◦C (1 min), 12 ◦C/s to 240 ◦C (4 min); split ratio 1:10. The separation of the volatile com pounds was achieved using a 60 m x 0.25 mm x1 μm gas J. Vicente et al. Food Chemistry: X 21 (2024) 101214 3 chromatographic column (Rxi-5Sil, Restek, Bad Hom-burg, Germany) with the following oven program: 40 ◦C (4 min), with 5 ◦C /min to 210 ◦C and with 20 ◦C/min to 240 ◦C (10.5 min). Helium served as carrier gas (constant flow: 1.2 mL/min). Detection of the volatile substances was conducted with a mass spectrometer MS 5975B (Agilent, Santa Clara, USA) applying EI (70 eV) and scan mode (m/z 35–250). Instru mental control, acquisition of data and quantitative data analysis were carried out using Agilent MassHunter workstation software (Jung et al., 2021). 2.4. Statistical analyses All statistical analyses were conducted using R software version 4.1.2 (R Development Core Team, 2013). Analysis of variance (ANOVA) and Tukey post-hoc tests were utilized to compare the different groups and values. 3. Results and discussion 3.1. Fermentative kinetic and main metabolites in the resulting wines These three parameters—the fermentative kinetics, the residual sugars, and the final ethanol production—should be considered together since the fermentative kinetics were measured as weight loss (CO2 release from the alcoholic fermentation), which is directly proportional to the consumed sugars and the produced ethanol. Despite the fact that the sugar:ethanol ratio in this species is slightly altered due to the absence of a strong Crabtree effect and the conversion of some consumed sugars into lactic acid (Vicente et al., 2021), these parameters are closely related. The fermentative kinetics of all the L. thermotolerans strains were different from that of the S. cerevisiae control, as well as the final residual sugars and ethanol concentration of the resulting wines. All L. thermotolerans strains exhibited a similar fermentative kinetics (Fig. 1), completing the fermentation in 23 days (three days longer than the S. cerevisiae control) and experiencing a weight loss of approximately 2.5 % (compared to the S. cerevisiae control, which had a weight loss of around 3.75 %). Only one strain, Levulia Alcomeno (LEV), exhibited a slower fermentation and a final weight loss of approximately 2.0 %. These results, concerning the fermentative kinetics and the total amounts of released CO2, can be interpreted as an indirect estimation of the fermentative capacity of the strains under investigation. The final ethanol concentration ranged from 6.26 % to 8.59 %, whereas the S. cerevisiae control reached a significantly higher value of 11.26 % (v/v) (Table 1). The commercial strain Levulia exhibited the lowest ethanol concentration, while the autochthonous strain A11-612 showed the highest. Among the commercial strains, Excellence had the highest ethanol concentration at 8.12 % (v/v). The commercial strain Excellence along with the selected strains NG-108, A11-612, and MJ-311, exhibited significantly higher final ethanol concentrations compared to the com mercial strains Levulia and Enartis. Strains EM-119, L1, L3, and the commercial strains Concerto and Laktia did not demonstrate statistically significant differences when compared to all the studied L. thermotolerans strains. Taking into consideration the ethanol production, despite the mod erate Crabtree effect observed in this species, all the L. thermotolerans strains exhibited high levels of glucose and fructose in the final content, ranging from 57.27 to 96.16 g/L (A11-612 and Levulia), indicating that the inoculated strains consumed approximately 52 % to 72 % of the initial sugar concentration. In contrast, the S. cerevisiae control had 17.28 g/L (Table 1) of residual sugars. Previous studies conducted by our research group have demon strated a significant variability in ethanol production among L. thermotolerans strains, ranging from 4.24 % to 10.6 % (v/v) (Vicente et al., 2021). This is consistent with previous findings for the Concerto and Laktia commercial strains, which exhibited ethanol concentrations of 8 % and 6.2 % (v/v) respectively (Vaquero et al., 2020). In accordance with the results of the present study, no statistically significant differ ences were observed, with average ethanol concentrations of 7.47 % and 7.38 % (v/v) respectively. Another metabolite strongly associated with sugar metabolism and alcoholic fermentation is glycerol, which exhibited variation in this study ranging from 2.49 g/L for the Excellence commercial strain to 3.36 g/L for the Octave strain. Our study demonstrated a strain vari ability of 26 %, which is consistent with previous findings ranging from 20 % to 50 % for L. thermotolerans strains (Benito, 2018; Vicente et al., 2021), as observed in S. cerevisiae (Benito, 2018). Several studies have reported no statistically significant differences in glycerol production among different strains. However, it is important to note that these studies employed mixed fermentations with a S. cerevisiae strain, making them incomparable with our results. In internal comparisons of various commercial L. thermotolerans strains (Laktia, Levulia, and Concerto) in sequential fermentation, no statistically significant differences were observed in the final glycerol concentration, despite variations between 6 % and 14 % (Hranilovic et al., 2021, 2022; Snyder et al., 2021). Similar results were obtained in studies comparing other commercial strains (Excellence, Levulia, and EnartisFerm QK) (Vicente et al., 2023a). In such studies, the contribution of L. thermotolerans may go unnoticed due to the masking effect of S. cerevisiae, rendering the differences statisti cally insignificant. Regarding these results, it is evident that the role of L. thermotolerans is influenced by both the natural matrix utilized in the fermentations and the fermentative microbial partner employed. To ensure a proper completion of alcoholic fermentation and prevent high residual sugar levels, it is necessary to combine L. thermotolerans with more fermen tative yeast strains, such as S. cerevisiae or S. pombe. The impact of these yeasts on the fermentative process is somewhat overshadowed by the more robust fermentative yeasts they are combined with (e.g., glycerol production). Nonetheless, the role of L. thermotolerans, which reduces the alcohol concentration due to its alternative metabolism involving Fig. 1. Fermentation kinetics of gravimetrically measured variants by total weight loss during pure fermentation in Synthetic Grape Must (SGM). The Lachancea thermotolerans strains used were CNT (Concerto, CHR Hansen, Denmark), LAKT (Laktia, Lallemand, Canada), LEV (Levulia Alcomeno, AEB Group, Italy), QKK (EnartisFerm QK, Enartis, Italy), EXC (Excellence, X’Fresh Lamothe-Abiet, France), and VINF (Octave, CHR Hansen, Denmark), along with the selected strains NG-108, A11-612, EM-119, MJ-311, BD-612, L1, and L3 (Complutense University of Madrid, Madrid, Spain). The Saccharomyces cer evisiae control strain used was AWRI (AWRI-796, Maurivin, Australia). J. Vicente et al. Food Chemistry: X 21 (2024) 101214 4 lactic acid production, has been extensively investigated when compared to S. cerevisiae (Hranilovic et al., 2021; Snyder et al., 2021; Vicente et al., 2023a; Vicente et al., 2023b). 3.2. The content and pH influence of organic acids One of the main traits of L. thermotolerans is the production of lactic acid that allows the biological management of acidy, together with the decrease in the malic acid content of the resulting wines. The use of natural must in experimental designs is sometimes not adequate, since, if it is sterilized by temperature (i.e., pasteurization or autoclaving) several nutrients could be lost (e.g., amino acids or vitamins) and filtering-sterilization is sometimes difficult due to its particulate content. The indigenous bacterial population naturally present in grape musts, among them, lactic acid, and acetic acid bacteria, may influence the results regarding organic acids content. Here we employed a synthetic media, sterilized through 0.45 μm filters, eliminating any possible bac teria that could interfere with the results. The production of lactic acid exhibits significant variability, with final contents ranging from 0.55 to 5.18 g/L for the L. thermotolerans strains under study (Table 1). Previous studies have reported final concentrations ranging from 0 to 12 g/L, although only a few studies have reported values higher than 6 g/L (Benito, 2018; Vicente et al., 2021). The strain Octave yielded the lowest concentration, while the autochthonous isolated strain A11-612 produced the highest. Among the commercial strains, Excellence and EnartisFerm QK exhibited the highest concentrations, with final average values of 3.22 and 3.13 g/L, respectively. Previous studies have reported varying data on commercial offerings and lactic acid production, including Excellence (2.7 g/L), EnartisFerm QK (0.8 g/L), Levulia (1.0–2.8 g/L), Laktia (1.5–5.8 g/L), and Concerto (0.5–3.4 g/L) (Hranilovic et al., 2021, 2022; Snyder et al., 2021; Vicente et al., 2023a). The differential production of lactic acid directly impacted the final total acidity, which ranged from 4.75 to 9.01 g/L, while the S. cerevisiae control exhibited a final total acidity of 4.47 g/L (Table 1). Some of the studied strains, which produced lower amounts of lactic acid, did not show statistically significant differences compared to the S. cerevisiae control. Previous studies have reported that L. thermotolerans can in crease total acidity in wine conditions, ranging from values close to 0 g/ L up to approximately 5 g/L, depending on the amount of lactic acid produced (Benito, 2018). The impact of lactic acid content on total acidity is closely linked to pH regulation. The L. thermotolerans fer mentations demonstrated lower pH values than the S. cerevisiae controls, with differences ranging from 0.06 to 0.32 pH units. The study utilized a synthetic matrix in which the content of malic acid was known and quantified, and no lactic acid bacteria, which consume malic acid and produce lactic acid, were present. The final concentrations of malic acid in the different L. thermotolerans strains studied varied in the final wines, ranging from 1.39 g/L (A11-612) to 2.5 g/L (Octave), starting from an initial concentration of 3 g/L in the synthetic grape must (SGM). This resulted in reductions ranging from 16 % to 54 % of the total initial concentration. Earlier studies have previously explored this characteristic of L. thermotolerans, presenting data on reductions of approximately 20 %, while a few strains were able to consume over 50 % (Blanco et al., 2020; Hranilovic et al., 2021; Vicente et al., 2021). The parameter of succinic acid has recently become an interesting selection criterion due to its salty taste in concentrations over 100 mg/L, which has the potential to enhance the minerality character of specific wines (Baroň & Fiala, 2012). The use of this yeast species in wine fermentation can significantly impact this organoleptic characteristic, as evidenced by the observed increase in the final concentration of succinic acid. In the case of SGM, a concentration increase was observed, ranging from 0.42 g/L for Concerto to 0.72 g/L for A11-612, whereas the S. cerevisiae control produced 0.30 g/L. Previous studies have examined the role of L. thermotolerans in relation to this parameter in natural Table 1 Final chemical analysis of pure fermentations in Synthetic Grape Must (SGM). The Lachancea thermotolerans strains used were NG-108, A11-612, MJ-311, BD-612, EM- 119, L1, L3 (Complutense University of Madrid, Madrid, Spain), Concerto (CHR Hansen, Denmark), Excellence (X’Fresh Lamothe-Abiet, France), Laktia (Lallemand, Canada), Levulia Alcomeno (AEB Group, Italy), EnartisFerm QK (Enartis, Italy) and Octave (CHR Hansen, Denmark). The Saccharomyces cerevisiae control strain used was AWRI-796 (Maurivin, Australia). Strain Ethanol (%) pH Total Acidity (g/L) Acetic Acid (g/L) Malic Acid (g/L) Lactic Acid (g/L) Succinic Acid (g/L) Glucose + Fructose (g/L) Glycerol (g/ L) NG-108 8,02 ± 1,34c 3,43 ± 0,06bc 5,96 ± 0,85bc 0,1 ± 0,05b 1,95 ± 0,25abc 2,04 ± 1,03b 0,58 ± 0,05ab 67,77 ± 22,01ab 3,02 ± 0,08abcd A11-612 8,59 ± 0,87c 3,24 ± 0,07d 9,01 ± 1,34a 0,14 ± 0,03b 1,39 ± 0,47d 5,18 ± 1,48a 0,72 ± 0,03a 57,27 ± 16,45b 3,07 ± 0,09ab MJ-311 8,27 ± 0,58c 3,42 ± 0,02bc 6,08 ± 0,07bc 0,08 ± 0,04b 2,08 ± 0,03ab 1,99 ± 0,11b 0,6 ± 0,04a 63,49 ± 8,56ab 3,22 ± 0,25abc BD-612 7,62 ± 0,74bc 3,43 ± 0,03bc 5,68 ± 0,47bc 0,04 ± 0,02b 2,05 ± 0,06ab 1,82 ± 0,55b 0,51 ± 0,05a 76,36 ± 13,34ab 2,82 ± 0,5abcd EM-119 7,44 ± 0,65abc 3,43 ± 0,02bc 5,79 ± 0,5bc 0,08 ± 0,04b 2,09 ± 0,11ab 1,7 ± 0,53b 0,58 ± 0,03a 77,49 ± 9,5ab 3,32 ± 0,33a L1 7,46 ± 0,78abc 3,36 ± 0,05 cd 6,84 ± 0,77ab 0,12 ± 0,03b 1,87 ± 0,23bc 2,92 ± 1,02ab 0,59 ± 0,03a 76,14 ± 11,54ab 2,81 ± 0,18 cd L3 7,51 ± 0,59abc 3,34 ± 0,03 cd 6,46 ± 0,6ab 0,12 ± 0,03b 2,01 ± 0,15abc 2,63 ± 0,63b 0,58 ± 0,04a 77,19 ± 10,44ab 2,81 ± 0,23abcd Concerto 7,47 ± 0,61abc 3,49 ± 0,04ab 4,62 ± 0,11c 0,09 ± 0,03b 2,34 ± 0,15ab 0,62 ± 0,17c 0,42 ± 0,04bcd 77,45 ± 11,46ab 3,31 ± 0,31a Excellence 8,12 ± 0,40c 3,31 ± 0,04bcd 6,92 ± 0,77ab 0,21 ± 0,02b 1,96 ± 0,18bcd 3,22 ± 0,83ab 0,56 ± 0,08abc 66,31 ± 6,61ab 2,49 ± 0,24d Laktia 7,38 ± 1,15abc 3,46 ± 0,06ab 5,2 ± 0,49bc 0,18 ± 0,07b 2,4 ± 0,17ab 1,18 ± 0,64bc 0,58 ± 0,06ab 78,45 ± 18,21ab 3,23 ± 0,39abc Levulia Alcomeno 6,26 ± 0,41a 3,40 ± 0,06bc 6,53 ± 0,02bc 0,12 ± 0,05b 2,04 ± 0,11bcd 2,49 ± 0,25b 0,52 ± 0,04 cd 96,16 ± 5,45a 2,65 ± 0,35d EnartisFerm QK 6,45 ± 0,9ab 3,31 ± 0,05 cd 6,97 ± 1,41ab 0,12 ± 0,04b 1,76 ± 0,4cd 3,13 ± 1,37ab 0,65 ± 0,05a 91,3 ± 12,92ab 2,65 ± 0,02bcd Octave 7,64 ± 0,10bc 3,50 ± 0,07ab 4,75 ± 0,25c 0,17 ± 0,04b 2,5 ± 0,12ab 0,55 ± 0,05c 0,57 ± 0,03abc 72,93 ± 1,11ab 3,36 ± 0,39abc AWRI-796 11,26 ± 0,61d 3,56 ± 0,04a 4,47 ± 0,09c 0,55 ± 0,02a 2,48 ± 0,07a 0,03 ± 0,04d 0,3 ± 0,03d 17,28 ± 9,38c 3,68 ± 0,2a Results are mean ± SD of three replicates. Different letters indicate statistical significance between groups. J. Vicente et al. Food Chemistry: X 21 (2024) 101214 5 musts, observing increments ranging from 0.27 to 0.59 g/L (Benito, 2018; Binati et al., 2019; Hranilovic et al., 2021; Vicente et al., 2022). When considering volatile acidity, it is important to take into ac count strains with low acetic acid production under fermentative con ditions, as it is one of the main characteristics that can impact wine quality. The final concentration of acetic acid for the different L. thermotolerans strains studied ranged from 0.04 to 0.21 g/L, without significant statistical differences. In contrast, the S. cerevisiae strain exhibited higher acetic acid production, reaching up to 0.55 g/L (Table 1). These values are conducive to producing high-quality wines, as the concentration remains below the olfactory threshold of 0.8 g/L (Ruiz et al., 2019). Among the commercial strains, all demonstrated acceptable concentrations ranging from 0.09 to 0.21 g/L (Concerto and Excellence, respectively). Previous studies have reported a reduction of approximately 40 % in acetic acid content during mixed fermentations with S. cerevisiae (Vicente et al., 2021). 3.3. The impact of L. Thermotolerans on the volatile profile of wine The production of volatile compounds, including esters, higher al cohols, and fatty acids, may play a crucial role in the interaction be tween species. Therefore, analyzing these compounds under single fermentation conditions, using a neutral medium such as SGM, is crucial to determine the primary volatile compounds produced by this species. Although the interactions between L. thermotolerans and S. cerevisiae in actual fermentative conditions are more complex, we conducted single fermentations of these yeast species in synthetic grape must to evaluate the impact of L. thermotolerans under controlled conditions. It is important to note that the impact of other species, such as S. cerevisiae and non-Saccharomyces yeasts, along with the characteristics of the must variety, can influence yeast performance and the production of various volatile compounds (Zupan et al., 2013; Avbelj et al., 2016). The production of esters by L. thermotolerans is usually lower if compared to the production by S. cerevisiae. The reduction regarding the content in these volatile compounds is from 30 to 60 % for A11-612 and Levulia Alcomeno respectively and are driven by the decrease in the production of acetic acid ethylester (Table 2). A lower concentration of this compound is related with the general lower production of acetic acid that L. thermotolerans presents. This compound is produced both under aerobic and anaerobic conditions, using ethanol and acetate as substrates and is generally related to fruity aromas, such as pineapple or banana (Zang et al, 2020). Since L. thermotolerans shows a lower pro duction of acetic acid than S. cerevisiae, the results agree with this observation. The reported threshold for acetic acid ethyl ester is 12 mg/L (Gómez-Míguez et al., 2007), implying an Odour Activity Value (OAV) of 8.58 units for the S. cerevisiae control at a concentration of 103.56 mg/L. Meanwhile, the highest OAV for L. thermotolerans production is 6.16, and the lowest is 3.41. This discrepancy clearly suggests that the impact of this volatile compound would be 28 % to 60 % lower in pure L. thermotolerans fermentations. Other interesting compounds, despite present in a lower concentra tion, that are usually produced in lower values by L. thermotolerans, if compared to S. cerevisiae, are: butyric acid ethylester (50 %), acetic acid phenylethylester (15 to 50 %), and hexanoic acid ethylester, an ester derived from fatty acids that is produced in around 40 to 70 % less. In all the cases, this decrease in the production of different esters are related to a lower concentration, both regarding the acetic and fatty acids con centration in fermentations carried out by L. thermotolerans. The reduction in the concentration of this compounds influences the aro matic profile of the resulting wines. Regarding the OAVs for the different compounds, only hexanoic acid ethylester and butyric acid ethylester are above its olfactory thresholds in all the cases. Butyric acid ethylester, shows an AOV in the S. cerevisiae fermentation of 11.31 compared to the in L. thermotolerans AOVs that vary between 6.14 and 7.16 for Octave and Concerto respectively. Despite all fermentations showing concen trations of hexanoic acid ethyl ester above the olfactory threshold (14 µg/L) (Gómez-Míguez et al., 2007), the lower production of this com pound by the different L. thermotolerans strains impacts the OAVs in these fermentations, reducing them by up to 62 % compared to the S. cerevisiae control. Other esters are produced at different concentrations by L. thermotolerans in single fermentations, but without statistical differ ences: i-butyric acid ethylester, that is differentially produced depend ing on the strain between an increase in around a 11 % (Levulia Alcomeno) and a reduction in around a 50 % (Excellence); or acetic acid 3-methylbutylester reduced up to a 30 % (Excellence) or increased up to around a 5 % (EM-119). On the contrary, some other esters are usually increased by L. thermotolerans such as propionic acid ethylester, which concentration is doubled or even tripled if compared to S. cerevisiae; or the lactic acid ethylester, being usually increased its concentration about 2.5 times, but with some strains producing up to 10 times more. This increase is related to the increased production of lactic acid, since this product is synthesized directly from the intracellular pool of lactic and acetic acid (Ren et al., 2020). Despite these high differences regarding lactic acid derived esters, the effect on the olfactory profile is not significative due to the high olfactory threshold that this compound has (154,636 µg/L) (Gómez-Míguez et al., 2007). L. thermotolerans is usually linked to an increase in esters production, being wines fermented both by L. thermotolerans and S. cerevisiae usually described as increased in the esters content (Hranilovic et al., 2021; Snyder et al., 2021; Vaquero et al., 2020; Vicente et al., 2021). The production of acetate esters by L. thermotolerans is usually lower compared to that of S. cerevisiae. The reduction in volatile compound content ranges from 30 to 60 % for A11-612 and Levulia Alcomeno, respectively, and is driven by a decrease in the production of acetic acid ethyl ester (Table 2) with a high impact in the OAVs. The lower con centration of this compound is associated with the overall lower pro duction of acetic acid by L. thermotolerans. Acetic acid ethyl ester is produced under both aerobic and anaerobic conditions, using ethanol and acetate as substrates, and is generally associated with fruity aromas, such as pineapple or banana (Zang et al., 2020). The results align with the observation that L. thermotolerans exhibits lower production of acetic acid compared to S. cerevisiae. Other interesting compounds, albeit present in lower concentrations, are typically produced at lower values by L. thermotolerans compared to S. cerevisiae. These include butyric acid ethyl ester (50 % reduction), acetic acid phenylethyl ester (15 to 50 % reduction), and hexanoic acid ethyl ester, an ester derived from fatty acids, which is produced at around 40 to 70 % less. In all cases, the decrease in the production of different esters is associated with lower concentrations of both acetic and fatty acids in fermentations carried out by L. thermotolerans. L. thermotolerans exhibits variations in the production of other esters in single fermentations. For instance, i-butyric acid ethyl ester is differentially produced depending on the strain, with Levulia Alcomeno showing an increase of around 11 % and Excellence showing a reduction of around 50 %. Similarly, acetic acid 3-methylbutyl ester is reduced by up to 30 % in Excellence but increased by around 5 % in EM-119. On the contrary, some other esters are typically increased by L. thermotolerans, such as propionic acid ethyl ester, which is doubled or even tripled compared to S. cerevisiae. Lactic acid ethyl ester is also usually increased by about 2.5 times, with some strains producing up to 10 times more. This increase is related to the elevated production of lactic acid, as it is synthesized directly from the intracellular pool of lactic and acetic acid (Ren et al., 2020). L. thermotolerans is often associated with an increase in ester production, and wines fermented by both L. thermotolerans and S. cerevisiae are typically described as having an increased ester content (Hranilovic et al., 2021; Snyder et al., 2021; Vaquero et al., 2020; Vicente et al., 2021). The statistical analysis revealed that the production of higher alco hols by L. thermotolerans was not significantly different from that of S. cerevisiae. However, an overall increase was observed, ranging from 2 % to 67 %, with strain EM-119 being the greatest producer and NG-108 J. Vicente et al. FoodChemistry:X 21(2024)101214 6 Table 2 Final volatile compound profiles of pure fermentations in Synthetic Grape Must (SGM). The Lachancea thermotolerans strains used were NG-108, A11-612, MJ-311, BD-612, EM-119, L1, L3 (Complutense University of Madrid, Madrid, Spain), Concerto (CHR Hansen, Denmark), Excellence (X’Fresh Lamothe-Abiet, France), Laktia (Lallemand, Canada), Levulia Alcomeno (AEB Group, Italy), EnartisFerm QK (Enartis, Italy) and Octave (CHR Hansen, Denmark). The Saccharomyces cerevisiae control strain used was AWRI-796 (Maurivin, Australia). Olfactory threshold of each compound is indicated (Gómez-Míguez et al., 2007). Strain Acetic acid ethylester [mg/L] i-Butanol [mg/L] Propionic acid ethylester [µg/L] 3-Methyl- butanol [mg/L] 2-Methyl- butanol [mg/L] i-Butyric acid ethylester [µg/L] Butyric acid ethylester [µg/L] Lactic acid ethylester [µg/L] Acetic acid 3-meth ylbutylester [µg/L] Hexanoic acid [mg/L] Hexanoic acid ethylester [µg/L] 2-Phenyl- ethanol [mg/L] Octanoic acid [mg/ L] Acetic acid phenylethylester [µg/L] Odour threshold1 12.264 150.00 – 30.00 40.00 20.00 15.00 154,636 30.00 0.42 14.00 14.00 0.5 250.00 NG-108 47.02 ± 4.38 cd 11.15 ± 1.73b 206.56 ± 14.42abc 41.86 ± 11.29ab 14.07 ± 4.18bc 71.62 ± 3.23a 93.34 ± 5.45b 61.41 ± 40.07b 153.33 ± 8.01a 3.92 ± 0bc 182.15 ± 0.31b 12.62 ± 3.34a 2.02 ± 0b 18.11 ± 0.6bc A11-612 74.21 ± 7.45b 11.97 ± 2.96ab 235.25 ± 27.84abc 66.02 ± 14.9ab 13.59 ± 1.27abc 85.85 ± 4.03a 104.68 ± 4.01b 211.29 ± 107.99a 179.02 ± 20.94a 3.94 ± 0.01b 185.66 ± 0.25b 17.92 ± 1.69a 2.04 ± 0b 18.43 ± 0.71bc MJ-311 47.24 ± 2.23 cd 12.02 ± 2.46ab 234.27 ± 62.46abc 64.65 ± 9.91ab 16.53 ± 2.47abc 114.6 ± 37.43a 99.84 ± 3.54b 50.6 ± 4.05b 165.37 ± 27.23a 3.92 ± 0.01bc 181.36 ± 1.26b 17.13 ± 6.62a 2.02 ± 0.01b 18.54 ± 1.11bc BD-612 58.16 ± 4.78bcd 17.74 ± 1.9ab 221.53 ± 25.92abc 67.95 ± 8.34ab 24.13 ± 6.21a 127.42 ± 29.18a 95.34 ± 1.13b 40.49 ± 9.6b 167.46 ± 8.35a 3.91 ± 0.01bc 180.61 ± 1.66b 13.84 ± 3.94a 2.02 ± 0b 16.89 ± 0.21bc EM-119 60.71 ± 5.01bcd 15.53 ± 1.8ab 272.12 ± 47.2ab 78.35 ± 7.72a 20.02 ± 1.09abc 103.05 ± 17.94a 96.34 ± 0.63b 47.87 ± 18.45b 205.57 ± 33.31a 3.91 ± 0bc 183.07 ± 1.61b 16.59 ± 1.79a 2.02 ± 0.01b 17.37 ± 0.9bc L1 55.79 ± 11.05bcd 11.92 ± 1.29ab 247.62 ± 100.28abc 60.24 ± 6.48ab 16.92 ± 0.63abc 107.12 ± 11.2a 97.12 ± 1.47b 77.63 ± 43.05b 157.2 ± 19.94a 3.91 ± 0.01bc 180.95 ± 0.85b 15 ± 3.14a 2.02 ± 0.01b 15.52 ± 0.1c L3 72.96 ± 4.8b 11.87 ± 2.62ab 257.35 ± 62.8ab 69.51 ± 11.15ab 16.85 ± 2.41abc 91.79 ± 13.47a 95.15 ± 1.81b 69.28 ± 23.04b 160.02 ± 13.89a 3.91 ± 0.01bc 181.35 ± 1.07b 15.11 ± 4.03a 2.02 ± 0.01b 16.38 ± 0.23bc Concerto 46.77 ± 7.54 cd 17.22 ± 3.66ab 278.7 ± 53.46ab 71.7 ± 4.35ab 23.22 ± 3.05ab 127.9 ± 51.52a 107.4 ± 1.7b 21.02 ± 1.23b 182.12 ± 40.2a 3.9 ± 0.01c 181.7 ± 0.65b 15.85 ± 4.52a 2.01 ± 0.01b 17.49 ± 0.98bc Excellence 44.57 ± 8.24 cd 11.19 ± 3.86b 129.12 ± 19.27bc 65.08 ± 22.57ab 17.87 ± 6.35abc 59.65 ± 6.06a 92.74 ± 6.19b 98.86 ± 32.25b 142.56 ± 4.59a 3.92 ± 0.01bc 179.16 ± 1.48b 16.62 ± 9.52a 2.03 ± 0b 17.2 ± 0.98bc Laktia 61.5 ± 9.11bcd 10.32 ± 1.41b 195.74 ± 77.5abc 50.92 ± 5.79ab 11.27 ± 1.13c 108.66 ± 55.88a 94.83 ± 1.57b 40.02 ± 21.29b 182.3 ± 17.01a 3.93 ± 0.02bc 183.48 ± 2.16b 9.8 ± 0.87a 2.05 ± 0.02b 15.97 ± 0.71bc Levulia Alcomeno 41.69 ± 5.18d 17.55 ± 3.53ab 196.3 ± 51.55abc 67.21 ± 8.93ab 20.07 ± 1.79abc 131.95 ± 30.94a 96.67 ± 2.8b 53.88 ± 9.52b 197.01 ± 33.78a 3.93 ± 0bc 183.73 ± 0.18b 14.37 ± 3.69a 2.02 ± 0.01b 17.3 ± 0.65bc EnartisFerm QK 66.68 ± 7.87bc 17.17 ± 3.05ab 313.27 ± 18.16a 74.94 ± 26.66a 17.92 ± 2.83abc 144.98 ± 13.54a 100.46 ± 2.51b 55.7 ± 19.84b 203.36 ± 21.94a 3.91 ± 0bc 181.66 ± 1.98b 15.55 ± 4.1a 2.02 ± 0b 20.48 ± 4.49ab Octave 48.21 ± 7.22 cd 11.95 ± 1.04ab 211.87 ± 41.64abc 64.37 ± 2.73ab 16.04 ± 1.01abc 116.9 ± 21.68a 92.12 ± 2.65b 23.58 ± 0.56b 196.39 ± 13.44a 3.92 ± 0bc 182.5 ± 1.05b 12.21 ± 0.82a 2.02 ± 0b 17.25 ± 0.13bc AWRI-796 103.56 ± 18.4a 19.5 ± 4.23a 100.9 ± 13.37c 36.89 ± 9.41b 15.25 ± 4.87abc 119.33 ± 39.76a 169.71 ± 21.43a 18.07 ± 0.25b 195.71 ± 41.12a 4.05 ± 0.04a 287.91 ± 6.79a 6.47 ± 1.61a 2.19 ± 0.04a 23.32 ± 2.54a Results are mean ± SD of three replicates. Different letters indicate statistical significance between groups. 1 Odour thresholds are in the same units as compounds. J. Vicente et al. Food Chemistry: X 21 (2024) 101214 7 being the least. Detailed analysis of specific compounds showed that 2- phenyl-ethanol was the most important fusel alcohol produced by L. thermotolerans, with increments ranging, depending on the strain, from approximately 1.5 to 2.5 times higher than S. cerevisiae. This compound is generally associated with a floral perception, often described as a rose or honey odor (Liu et al., 2019), and has been identified as one of the key molecules in yeast interactions through volatile compounds (Jagtap et al., 2020; Britton et al., 2023). Other higher alcohols produced in higher concentrations by L. thermotolerans included 3-methyl-butanol (increases ranging from approximately 13 % to 2 times higher) and 2-methyl-butanol (produced at a concentration approximately 60 % higher, although some strains showed slightly lower values). In contrast, the concentration of i-butanol was decreased by 10 % to 50 % compared to S. cerevisiae. The variation in the pro duction of higher alcohols also impacts the olfactory profile of the resulting wines. This effect is particularly notable concerning the con tent of 3-methyl-butanol, with OAVs for this compound ranging from 1.39 to 2.61 in NG-108 and EM-119, respectively, compared to 1.29 OAV units in S. cerevisiae fermentations. The role of this species in the production of other higher alcohols remains unclear. Several strains have shown an increase in these com pounds in some experiments while exhibiting a decrease in others (Hranilovic et al., 2021; Vaquero et al., 2020; Vicente et al., 2023a). The choice of the must, combined with the S. cerevisiae strain in conjunction with this species, may play an important role in regulating the produc tion of these compounds. In this study, L. thermotolerans, fermenting a neutral synthetic must under axenic conditions, appears to increase the content of higher alcohols compared to the S. cerevisiae control, despite its lower fermentative capacity. This finding should be interpreted carefully, as higher alcohols can contribute both desirable and unde sirable aromas to the wine, depending on the specific odor descriptor of each compound and its olfactory threshold. Nevertheless, among several non-Saccharomyces species, L. thermotolerans seems to have the greatest impact on higher alcohols (Castrillo & Blanco, 2023). Regarding fatty acids, L. thermotolerans exhibited a slight, but consistent, decrease of approximately 5 % compared to S. cerevisiae single fermentations. Among them, octanoic acid showed the most sig nificant reduction of approximately 7 %, while hexanoic acid displayed reductions of around 4 %. These compounds are typically associated with cheese or rancid aromas, which are undesirable in certain wines. The impact of L. thermotolerans on fatty acids is still not fully understood. Some studies have reported certain increases (Shekhawat et al., 2017), but most of them agree that L. thermotolerans tends to decrease their concentration when co-inoculated with S. cerevisiae (Hranilovic et al., 2021). The commercial strains employed in this study, previously examined by different researchers, have consistently shown this trend in various matrices (such as Merlot and Tempranillo red wines) and different combinations of species (both with S. cerevisiae and S. pombe) (Hranilovic et al., 2021; Vicente et al., 2023; Vicente et al., 2023). However, it should be noted that under oxygenation conditions, the production of these compounds may increase (Shekhawat et al., 2017). The reduction in the production of fatty acids also has an impact on the olfactory profile. Despite the concentration of these compounds not falling below the olfactory threshold for each compound, significant reductions are observed in the OAVs of both hexanoic and octanoic acids: 9.64 and 4.28, respectively, in S. cerevisiae compared to 9.28 and 4.04, respectively, in L. thermotolerans fermentations. Overall, the results corroborated the hypothesis by demonstrating substantial variability among L. thermotolerans strains in fermentative performance, organic acid production, and volatile compound forma tion, reaffirming the need for individual strain assessment for their distinct roles in winemaking. The results also show that it is possible to select strains that generate higher lactic acid than the nowadays com mercial offer. 4. Conclusion This study presents a comparison of several L. thermotolerans strains using synthetic grape juice, which can be prepared in any laboratory at any time. These results can serve as a baseline for future studies, enabling researchers and yeast manufacturers to compare their future results with newly isolated strains under reproducible conditions. A high strain variability was observed for volatile and non-volatile parameters in L. thermotolerans. This variability, observed not only among com mercial strains but also natural isolates, explains the different perfor mance observed under winemaking conditions. In terms of fermentative performance, a moderate capacity was observed, indicating the need for combination with a strong fermentative yeast such as S. cerevisiae. Certain clear patterns can be linked to the use of L. thermotolerans in relation to organic acids. The high production of lactic acid by some strains is of great interest as it enables pH management through bio logical means. Succinic acid contributes to distinct organoleptic char acteristics that are valuable in certain wines, while the use of L. thermotolerans generally leads to a decrease in volatile acidity during wine fermentation. The impact of this yeast species on volatile com pounds is challenging to summarize when describing individual com pounds. However, a general conclusion can be drawn: under axenic fermentative conditions, L. thermotolerans is characterized by an overall increase in the content of higher alcohols, accompanied by a decrease in the content of esters and fatty acids. Understanding the influence of the environment, as well as abiotic and biotic factors, on yeast metabolism is crucial for regulating fermentative performance and the production of volatile compounds. These traits are influenced by various factors, including must variety, composition, and the naturally occurring mi crobial community present in natural matrices. CRediT authorship contribution statement Javier Vicente: Writing – original draft, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Data curation. Luka Vladic: Formal analysis, Data curation. Eva Navascués: Writing – review & editing, Supervision, Project administration, Methodology, Formal analysis. Silvia Brezina: Formal analysis. Antonio Santos: Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis. Fernando Calderón: Formal analysis. Wendu Tes faye: Writing – review & editing, Formal analysis. Domingo Marquina: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Doris Rauhut: Writing – review & editing, Supervision, Methodology, Formal analysis, Conceptualization. Santiago Benito: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Soft ware, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Declaration of competing interest The authors declare the following financial interests/personal re lationships which may be considered as potential competing interests: Santiago Benito reports financial support was provided by Spanish Ministry of Science and Innovation. If there are other authors, they declare that they have no known competing financial interests or per sonal relationships that could have appeared to influence the work re ported in this paper. Data availability Data will be made available on request. J. Vicente et al. Food Chemistry: X 21 (2024) 101214 8 Acknowledgements A part of the presented research studies was conducted by Luka Vladic during his master’s thesis which is organized in cooperation of four universities and three different European countries and supervised by Prof. Konrad Domig, University of Natural Resources and Life Sci ences (Vienna, Austria), Prof. Santiago Benito, Polytechnic University of Madrid (Spain), Prof. Javier Vicente, Complutense University of Madrid (Spain), and Prof. Doris Rauhut Hochschule Geisenheim University (Germany). Thanks to Erasmus + for funding Luka Vladic’s Mobility for a Traineeship at the Polytechnic University of Madrid, Spain. Funding Funding was provided by the Spanish Ministry of Science and Innovation, and the State Investigation Agency under the framework of Project VinSegCalClim (PID2020-119008RB-I00/AEI/10.13039/ 501100011033) and by the Spanish Center for the Development of In dustrial Technology under the framework of Project IDI-20210391. Javier Vicente conducted this research under a fellowship from Com plutense University of Madrid (CT58/21-CT59/21). Author Contributions S.Be., J.V., D.M., D.R., and J.V. developed the experimental design; J.V., L.V., D.M., A.S., E.N., and S.Be. performed the vinifications; J.V., L. V., and S.Be. performed the formal data analysis and supervised the project; J.V., L.V., and S.Be. wrote the article; D.R., and S.Br., performed gas chromatographic analysis. J.V., L.V., F.C., W.T., and S.Be performed enzymatic and FTIR analysis. All authors discussed the results and contributed to the final manuscript. References Avbelj, M., Zupan, J., & Raspor, P. (2016). Quorum-sensing in yeast and its potential in wine making. Applied Microbiology and Biotechnology, 100, 7841–7852. https://doi. org/10.1007/s00253-016-7758-3 Baroň, M., & Fiala, J. (2012). Chasing after minerality, relationship to yeast nutritional stress and succinic acid production. Czech Journal of Food Sciences, 30(2), 188–193. https://doi.org/10.17221/464/2010-CJFS Benito, S. (2018). The impacts of Lachancea thermotolerans yeast strains on winemaking. Applied Microbiology and Biotechnology, 102(16), 6775–6790. https://doi.org/ 10.1007/s00253-018-9117-z Benito, S. (2020). Combined use of Lachancea thermotolerans and Schizosaccharomyces pombe in winemaking: A review. Microorganisms, 8(5), 655–673. https://doi.org/ 10.3390/microorganisms8050655 Binati, R. L., Innocente, G., Gatto, V., Celebrin, A., Polo, M., Felis, G. E., & Torriani, S. (2019). Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking. Food Research International, 122, 432–442. https://doi.org/10.1016/J.FOODRES.2019.04.043 Blanco, P., Rabuñal, E., Neira, N., & Castrillo, D. (2020). Dynamic of Lachancea thermotolerans Population in Monoculture and Mixed Fermentations: Impact on Wine Characteristics. Beverages, 6(2), 36–56. https://doi.org/10.3390/beverages6020036 Britton, S. J., Rogers, L. J., White, J. S., Neven, H., & Maskell, D. L. (2023). Disparity in pseudohyphal morphogenic switching response to the quorum sensing molecule 2- phenylethanol in commercial brewing strains of Saccharomyces cerevisiae. FEMS Microbes, 4, 1–8. https://doi.org/10.1093/femsmc/xtad002 Castrillo, D., & Blanco, P. (2023). Characterization of indigenous non-Saccharomyces yeast strains with potential use in winemaking. Frontiers in Bioscience, 15(1), 1–16. https://doi.org/10.31083/j.fbe1501001 Gómez-Míguez, M. J., Cacho, J. F., Ferreira, V., Vicario, I. M., & Heredia, F. J. (2007). Volatile components of Zalema white wines. Food Chemistry, 100(4), 1464–1473. https://doi.org/10.1016/j.foodchem.2005.11.045 Henschke, P. A., & Jiranek, V. (1993). Metabolism of nitrogen compounds. In Wine microbiology and biotechnology (pp. 75–86). Amsterdam: Harwood Academic Publishers. 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, 129015–129027. https://doi.org/10.1016/j. foodchem.2021.129015 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. (2022). Impact of Lachancea thermotolerans on chemical composition and sensory profiles of viognier Wines. Journal of Fungus, 8(5), 474–494. https://doi.org/10.3390/ JOF8050474/S1 Jagtap, S., Bedekar, A., & Rao, C. (2020). Quorum sensing in yeast. In Quorum Sensing: Microbial Rules of Life (pp. 235–250). Washington: American Chemical Society. Jolly, N. P., Augustyn, O. P. H., & Pretorius, I. S. (2017). The role and use of non- Saccharomyces yeasts in wine production. South African Journal of Enology and Viticulture, 27(1), 15–40. https://doi.org/10.21548/27-1-1475 Jolly, N. P., Augustyn, O. P. R., & Pretorius, I. S. (2003). The effect of non-Saccharomyces yeasts on fermentation and wine quality. South African Journal of Enology and Viticulture, 24(2), 55–62. https://doi.org/10.21548/24-2-2638 Jolly, N. P., Varela, C., & Pretorius, I. S. (2014). Not your ordinary yeast: Non- Saccharomyces yeasts in wine production uncovered. FEMS Yeast Research, 14(2), 215–237. https://doi.org/10.1111/1567-1364.12111 Jung, R., Kumar, K., Patz, C., Rauhut, D., Tarasov, A., & Schüßler, C. (2021). Influence of transport temperature profiles on wine quality. Food Packaging and Shelf Life, 29, 1–12. https://doi.org/10.1016/j.fpsl.2021.100706 Liu, S., Laaksonen, O., & Yang, B. (2019). Volatile composition of bilberry wines fermented with non-Saccharomyces and Saccharomyces yeasts in pure, sequential and simultaneous inoculations. Food Microbiology, 80, 25–39. https://doi.org/10.1016/j. fm.2018.12.015 Porter, T. J., Divol, B., & Setati, M. E. (2019a). Investigating the biochemical and fermentation attributes of Lachancea species and strains: Deciphering the potential contribution to wine chemical composition. International Journal of Food Microbiology, 290, 273–287. https://doi.org/10.1016/j.ijfoodmicro.2018.10.025 Porter, T. J., Divol, B., & Setati, M. E. (2019b). Lachancea yeast species: Origin, biochemical characteristics, and oenological significance. International Food Research, 119, 378–389. https://doi.org/10.1016/j.foodres.2019.02.003 Ren, J. Y., Liu, G., Chen, Y. F., Jiang, S., Ma, Y. R., Zheng, P., Guo, X., & Xiao, D. G. (2020). Enhanced production of ethyl lactate in Saccharomyces cerevisiae by genetic modification. Journal of Agricultural and Food Chemistry, 68(47), 13863–13870. https://doi.org/10.1021/acs.jafc.0c03967 Ruiz, J., Kiene, F., Belda, I., Fracassetti, D., Marquina, D., Navascués, E., Calderón, F., Benito, A., Rauhut, D., Santos, A., & Benito, S. (2019). Effects on varietal aromas during wine making: A review of the impact of varietal aromas on the flavor of wine. Applied Microbiology and Biotechnology, 103(18), 7425–7450. https://doi.org/ 10.1007/s00253-019-10008-9 Scansani, S., Rauhut, D., Brezina, S., Semmler, H., & Benito, S. (2020). The impact of chitosan on the chemical composition of wines fermented with Schizosaccharomyces pombe and Saccharomyces cerevisiae. Foods, 9(10), 1423–1434. https://doi.org/ 10.3390/foods9101423 Shekhawat, K., Bauer, F. F., & Setati, M. E. (2017). Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 101(6), 2479–2491. https://doi.org/10.1007/s00253-016-8001-y Snyder, E. C., Jiranek, V., & Hranilovic, A. (2021). Impact of Lachancea thermotolerans strain and lactic acid concentration on Oenococcus oeni and malolactic fermentation in wine. OENO One, 55(2), 365–380. https://doi.org/10.20870/oeno- one.2021.55.2.4657 Vaquero, C., Loira, I., Bañuelos, M. A., Heras, J. M., Cuerda, R., & Morata, A. (2020). Industrial performance of several Lachancea thermotolerans strains for pH control in white wines from warm areas. Microorganisms, 8(6), 830–845. https://doi.org/ 10.3390/MICROORGANISMS8060830 Vejarano, R., & Gil-Calderón, A. (2021). Commercially available non-Saccharomyces yeasts for winemaking: Current market, advantages over Saccharomyces, biocompatibility, and safety. Fermentation, 7(3), 171–194. https://doi.org/10.3390/ FERMENTATION7030171 Vicente, J., Navascués, E., Calderón, F., Santos, A., Marquina, D., & Benito, S. (2021). An integrative view of the role of Lachancea thermotolerans in wine technology. Foods, 10(11), 2878–2904. https://doi.org/10.3390/FOODS10112878 Vicente, J., Baran, Y., Navascués, E., Santos, A., Calderón, F., Marquina, D., Rauhut, D., & Benito, S. (2022). Biological management of acidity in wine industry: A review. International Journal of Food Microbiology, 375, 109726–109741. https://doi.org/ 10.1016/J.IJFOODMICRO.2022.109726 Vicente, J., Kelanne, N., Rodrigo-Burgos, L., Navascués, E., Calderón, F., Santos, A., Marquina, D., Yang, B., & Benito, S. (2023). Influence of different Lachancea thermotolerans strains in the wine profile in the era of climate challenge. FEMS Yeast Research, 23, 1–8. https://doi.org/10.1093/FEMSYR/FOAC062 Vicente, J., Kelanne, N., Navascués, E., Calderón, F., Santos, A., Marquina, D., Yang, B., & Benito, S. (2023). Combined use of Schizosaccharomyces pombe and a Lachancea thermotolerans strain with a high malic acid consumption ability for wine production. Fermentation, 9(2), 165–175. https://doi.org/10.3390/FERMENTATION9020165/S1 Vicente, J., Navascués, E., Benito, S., Marquina, D., & Santos, A. (2023). Microsatellite typing of Lachancea thermotolerans for wine fermentation monitoring. International Journal of Food Microbiology, 394, 110186–110192. https://doi.org/10.1016/J. IJFOODMICRO.2023.110186 Vilela, A. (2018). Lachancea thermotolerans, the non-Saccharomyces yeast that reduces the volatile acidity of wines. Fermentation, 4(3), 56–63. https://doi.org/10.3390/ fermentation4030056 Zupan, J., Avbelj, M., Butinar, B., Kosel, J., Šergan, M., & Raspor, P. (2013). Monitoring of quorum-sensing molecules during minifermentation studies in wine yeast. Journal of Agricultural and Food Chemistry, 61(10), 2496–2505. https://doi.org/10.1021/ jf3051363 J. Vicente et al. https://doi.org/10.1007/s00253-016-7758-3 https://doi.org/10.1007/s00253-016-7758-3 https://doi.org/10.17221/464/2010-CJFS https://doi.org/10.1007/s00253-018-9117-z https://doi.org/10.1007/s00253-018-9117-z https://doi.org/10.3390/microorganisms8050655 https://doi.org/10.3390/microorganisms8050655 https://doi.org/10.1016/J.FOODRES.2019.04.043 https://doi.org/10.3390/beverages6020036 https://doi.org/10.1093/femsmc/xtad002 https://doi.org/10.31083/j.fbe1501001 https://doi.org/10.1016/j.foodchem.2005.11.045 http://refhub.elsevier.com/S2590-1575(24)00101-9/h0050 http://refhub.elsevier.com/S2590-1575(24)00101-9/h0050 http://refhub.elsevier.com/S2590-1575(24)00101-9/h0050 https://doi.org/10.1016/j.foodchem.2021.129015 https://doi.org/10.1016/j.foodchem.2021.129015 https://doi.org/10.3390/JOF8050474/S1 https://doi.org/10.3390/JOF8050474/S1 http://refhub.elsevier.com/S2590-1575(24)00101-9/h0065 http://refhub.elsevier.com/S2590-1575(24)00101-9/h0065 https://doi.org/10.21548/27-1-1475 https://doi.org/10.21548/24-2-2638 https://doi.org/10.1111/1567-1364.12111 https://doi.org/10.1016/j.fpsl.2021.100706 https://doi.org/10.1016/j.fm.2018.12.015 https://doi.org/10.1016/j.fm.2018.12.015 https://doi.org/10.1016/j.ijfoodmicro.2018.10.025 https://doi.org/10.1016/j.foodres.2019.02.003 https://doi.org/10.1021/acs.jafc.0c03967 https://doi.org/10.1007/s00253-019-10008-9 https://doi.org/10.1007/s00253-019-10008-9 https://doi.org/10.3390/foods9101423 https://doi.org/10.3390/foods9101423 https://doi.org/10.1007/s00253-016-8001-y https://doi.org/10.20870/oeno-one.2021.55.2.4657 https://doi.org/10.20870/oeno-one.2021.55.2.4657 https://doi.org/10.3390/MICROORGANISMS8060830 https://doi.org/10.3390/MICROORGANISMS8060830 https://doi.org/10.3390/FERMENTATION7030171 https://doi.org/10.3390/FERMENTATION7030171 https://doi.org/10.3390/FOODS10112878 https://doi.org/10.1016/J.IJFOODMICRO.2022.109726 https://doi.org/10.1016/J.IJFOODMICRO.2022.109726 https://doi.org/10.1093/FEMSYR/FOAC062 https://doi.org/10.3390/FERMENTATION9020165/S1 https://doi.org/10.1016/J.IJFOODMICRO.2023.110186 https://doi.org/10.1016/J.IJFOODMICRO.2023.110186 https://doi.org/10.3390/fermentation4030056 https://doi.org/10.3390/fermentation4030056 https://doi.org/10.1021/jf3051363 https://doi.org/10.1021/jf3051363 A comparative study of Lachancea thermotolerans fermentative performance under standardized wine production conditions 1 Introduction 1.1 Hypothesis 2 Material and methods 2.1 Microorganisms and fermentations 2.2 Basic oenological parameters determinations 2.3 Volatile compounds 2.4 Statistical analyses 3 Results and discussion 3.1 Fermentative kinetic and main metabolites in the resulting wines 3.2 The content and pH influence of organic acids 3.3 The impact of L. Thermotolerans on the volatile profile of wine 4 Conclusion CRediT authorship contribution statement Declaration of competing interest Data availability Acknowledgements Funding Author Contributions References