Non-transgenic strains of the yeast saccharomyces cerevisiae with lower alcohol production efficiency

Abstract

The present invention relates to a group of strains of yeast of the species S. cerevisiae that have been genetically improved in a non-transgenic manner, by means of an intraspecific genetic improvement programme, in order to achieve lower efficiency in converting sugar into alcohol. The invention further relates to the use thereof to produce beverages.

Description

[0001] Non-transgenic Saccharomyces cerevisiae yeast strains with lower efficiency in alcohol production.

[0002] Description of what is known in the field

[0003] The yeast Saccharomyces cerevisiae, also known as Saccharomyces cerevisiae or simply "yeast," plays a central role in the fermentation of grape must and is considered an essential element in the wine production process. Globally, there are more than one hundred yeast strains available commercially for winemaking. These strains, mostly collected from viticulture-related environments, are used as starter cultures for wine fermentation. Wine strains, considered fundamental oenological industrial inputs, play a crucial role in the production process. Currently, the wine strain industry generates an impressive economic volume, mobilizing approximately USD 1.358 billion annually, according to data from Global Yeast Market Grow CAGR 9-2020 (https: / / www.businesswire.com).

[0004] Consistent with this, new yeast strains are constantly being sought to improve different aspects of wine fermentation, for which new strategies are needed to explore and utilize the wide genetic diversity of S. cerevisiae within the framework of the legislation of some of the main wine importing countries (China, the United Kingdom, Brazil, and Japan) (ODEPA, 2019). One way to overcome the constant need for new wine strains with superior fermentation capacities is to prospect for strains in wild environments (e.g., native forests, rivers, or flowers) and utilize their genetic diversity to generate new industrial strains with exceptional properties through genetic improvement.

[0005] One of the main problems currently facing the wine industry is high alcohol levels in wine, a result of high sugar concentrations in the grapes at harvest (musts with levels of 25 or more degrees Brix (°Bx)), a recurring issue in recent years. This situation has been correlated with global climate changes that cause a mismatch between the appropriate harvest time according to oenological parameters and the sugar concentration in the grapes. If the climate is warmer than ideal, the vines have a faster phenological development, resulting in earlier sugar ripening and a loss of acidity through respiration while the flavors develop. The result is unbalanced or "flaccid" wines (high alcohol content with little retained acidity for freshness) (Jones et al., 2005). Over the last 30 years, the average alcohol concentration in wines has increased by an average of 2 °GL.(Tilloy et al., 2014).

[0006] This condition has made the alcohol content of wines a relevant issue for the wine industry today, forcing the sector to implement strategies that allow for reducing alcohol content without harming the attributes that make wine highly competitive. High alcohol content in wines affects competitiveness in several ways: commercially, wines with high alcohol levels increase the tax burden; sensorially, it masks flavors and odors, and, to a lesser extent, diminishes the perception of sweetness and acidity; productively, it inhibits the winemaking process, causing slowdowns and fermentation stops (Escudero et al., 2007, Buescher et al., 2001); and socially, it partly contributes to lower wine consumption; for example, per capita wine consumption in Chile is falling by an average of 2% per year (Wine Intelligence, Vinitrac®).

[0007] Currently, strategies seeking to reduce alcohol content in wines include viticultural, pre-fermentation and winemaking practices, and post-fermentation and processing technologies, including the physical removal of alcohol after fermentation (Gutiérrez-Gamboa et al., 2021; Schmidtke et al., 2012; Zhu et al., 2021; Varela et al., 2015). For example, at the pre-fermentation level, the dilution of the must with others of lower sugar content, the addition of enzymes that degrade sugars, the addition of a fraction of water, crop management and early fruit harvesting to the detriment of oenological maturity are used. On the other hand, post-fermentation actions involve directly intervening in the wine through methods that reduce the alcohol content. Reverse osmosis processes are widely used, allowing the separation of, among other things, the alcoholic fraction of the wine, and then reconstructing a product with a lower alcohol concentration.However, these strategies are inefficient, affect the wine's organoleptic properties, face legal restrictions, and / or significantly increase production costs.

[0008] In this context, efforts have been made in recent years to generate microbiological solutions, such as the use of mixed cultures and yeasts that are less efficient in alcohol production (Varela and Varela, 2019), which avoid adding pre- or post-fermentation steps that lead to diluting the wine or altering it through physical processes. Regarding commercial yeasts, there are only two examples designed to reduce alcohol levels in wine. The first one (Maurivin AWRI Obsession) corresponds to a different species of yeast (Metschnikowia pulcherrimd), which in any case must be co-cultivated with a commercial strain of S. cerevisiae (Contreras et al., 2014). The second corresponds to a strain of S. cerevisiae obtained through adaptive evolution (Lallemand lONYSwf™), which however presents a reduction in alcohol in only one variety of wine (Shiraz), leaving a large market without a solution (Tilloy et al., 2014).Finally, no wine yeast obtained through intraspecific hybridization (that is, crossing strains of the same species) is marketed worldwide that has the particularity of lower alcohol production.

[0009] Other viable microbiological alternatives to reduce the alcohol content in wines have basically involved the generation of S. cerevisiae strains with lower ethanol production yields generated through genetic engineering, the use of unconventional Saccharomyces yeasts to reduce ethanol concentration, or the use of non-Saccharomyces yeasts that naturally exhibit lower ethanol production rates (Varela and Varela, 2019). However, these strategies have faced problems such as increased fermentation times, complexity, uncertainty, and public and commercial rejection of genetically modified organisms (GMOs), among others.

[0010] As mentioned above, most commercial yeast strains worldwide have been the result of collection and selection studies of naturally occurring yeast strains. This is different in the case of the present patent application, where a genetic improvement program was developed. Therefore, the yeast strains generated do not previously exist in nature, and the probability of them being generated naturally is zero. Carrying out a genetic improvement program involves the formation of hybrids through a sequential and rational design of crosses between individuals, guided by the individual phenotypic values ​​they present for the trait under improvement and associated traits. This differentiates this strategy from both genetic improvement using random methods and that carried out through genetic engineering.

[0011] An exhaustive search for other similar patents revealed those contained in documents CN113564061, ES2330709, ES2356011, WO9831784, WO2022112214, ZA919818 and WO201511411. Although these patents claim yeast strains capable of producing wines with a lower alcohol content, none of these strains were obtained through a genetic improvement program, so the present patent application is clearly different from them.

[0012] For its part, the use of the genetic diversity present in the species to generate new industrial strains through a genetic improvement program has been successfully carried out in obtaining yeast strains efficient in the consumption of nitrogen sources for use in fermentation of nitrogen-deficient musts, as described in patent CL 65,379. However, in this patent the genetic improvement program was directed to solve a technical problem different from the present application, where strains were sought for use in musts with low nitrogen content, clearly differentiating itself from the present application as well.

[0013] It should be noted that to date, there is no genetic improvement program focused on obtaining yeast strains with lower efficiency in converting sugar into alcohol, while maintaining their fermentation conditions in terms of residual sugar, for use in the production of alcoholic beverages. This patent application is the first of its kind.

[0014] In conclusion, and in light of the background presented, the present patent application corresponds to totally non-transgenic S. cerevisiae yeast strains, obtained through a genetic improvement program (which implies the non-use of genetic engineering), genetically improved to have a lower efficiency in the conversion of sugar into alcohol while maintaining their fermentation conditions in terms of residual sugar, for use in the production of alcoholic beverages, distancing itself from other publications and patents that have only been based on the isolation and characterization technique.

[0015] Thus, the present invention fully meets the criteria of novelty, inventive step, and industrial applicability, all of which are required for a patent application to be granted. In this regard, it is worth noting that this set of yeast strains was developed without the use of genetic engineering, so they are not considered genetically modified organisms (GMOs) by law. Therefore, they can be directly transferred to industry and used in all possible markets, particularly those where GMOs are largely prohibited (such as Chile and Europe).

[0016] Finally, this patent does not fall under any of the patentability exclusion criteria established in global laws. First, since the yeast strains belonging to the claimed population were developed through a genetic improvement program, they are not part of the biological material present in nature. Furthermore, without the work carried out, the probability of any of the claimed strains generating spontaneously in nature is zero, mainly due to the geographic isolation between the parents used and the fact that two rounds of selective crosses were carried out, defined by the criteria applied.And second, because this genetic improvement program corresponds to a modified microbiological process (considering the micromanipulation technology used and the planning of defined and controlled crosses), different from the essentially biological procedures used, for example, for the production of plants and animals.

[0017] Description of the figures:

[0018] Figure 1. Radial chart for the descriptive attributes of A) aroma and B) flavor of the white wines produced.

[0019] Figure 2. Radial chart for the descriptive attributes of A) aroma and B) flavor of the red wines produced.

[0020] Detailed description of the invention

[0021] This invention corresponds to a set of yeast strains genetically improved in a non-transgenic manner for lower efficiency in the conversion of sugar into alcohol through a genetic improvement program, of the species S. cerevisiae, as well as its use for the production of alcoholic beverages.

[0022] These strains were developed through a genetic improvement program (see Example 1), which sought to obtain yeast strains with lower ethanol production yields while maintaining their fermentation conditions in terms of residual sugar. This genetic improvement program was carried out in two stages. In the first stage, the improvement was directed towards reducing ethanol production yields, using as a base population a first filial generation (population "F1") previously generated through random crosses from a large group of strains isolated in various winemaking environments in Chile and Argentina (population "FO") (Kessi-Pérez et al., 2020). Through directed crosses, a second filial generation (population "F2") composed of different families and hybrids was generated from this "F1" population through directed crosses (see Example 1).1), which were identified using highly informative molecular markers (see Example 1.2). It was found that the trait being improved has a high heritability (0.62) (see Example 1.3), resulting in the F2 population producing strains with lower ethanol production yields (see Example 2.1); specifically, individuals in the F2 population had an average yield of 0.452 [g ethanol / g sugar], while individuals in the F1 population had an average yield of 0.506 [g ethanol / g sugar], representing a decrease of 10.7%.

[0023] In a second stage, improvement was directed towards reducing the residual glucose content (see Example 1.1), this time starting from the F2 generation to generate a third filial generation (population "F3"), whose individuals were molecularly identified using the same markers (see Example 1.2). It was found that the trait under improvement has a high heritability (0.94) (see Example 1.3), obtaining strains in population F3 with lower ethanol production yields (see Example 2.1); specifically, individuals in population F3 presented an average residual glucose of 4.30 [g], while individuals in population F2 presented an average residual glucose of 14.61 [g], representing a decrease of 70.6%.

[0024] Finally, among the strains belonging to the F3 population, those with the lowest ethanol production yields and lowest residual sugar values ​​were identified. Those that were also resistant to the levels of sulfites regularly used in the wine industry (see Example 2.2) were identified, as this is an important characteristic for ensuring their industrial applicability. Residual sugar is the amount of sugar remaining in the wine once the winemaking process is complete. The concentration is generally expressed as grams of residual sugar per liter of wine (g / L).From this analysis, strains C2-1B4, C7-1B7, C7-2B2, C7-2C2, C7-3A10 and C 10-215 were selected, which showed especially low values ​​of ethanol production yield (between 0.369 and 0.462 [g ethanol / g sugar]), maintaining their fermentation yield in terms of residual glucose (0.00 and 2.47 [g]) and their ability to grow in high concentrations of sulfites, when evaluated in synthetic grape must at laboratory scale (see Example 2).

[0025] Wine was produced on a pilot scale using these strains under winery conditions, using natural Sauvignon Blanc (white wine) and Carmenere (red wine) grape musts (see Example 3). All strains were successfully implanted (see Example 3.2), and it was found that one of the improved strains (C7-1B7) had a lower than expected alcohol content in Sauvignon Blanc must, while all improved strains had a lower than expected alcohol content in Carmenere must, with strain C2-1B4 being particularly notable (see Example 3.3). Finally, an independent panel of nine expert judges performed a sensory analysis of the wines produced with the improved strains in terms of affective and descriptive qualities (aroma and flavor). No statistically significant differences were found between the wines produced with the improved strains.Both the white and red wines produced by the improved strains obtained good scores in terms of affect, while at the descriptive level, both the white and red wines produced by the improved strains stood out for their overall aroma, while in terms of flavor, both were characterized by their low bitterness. Taken together, these results indicate that these strains are capable of producing wines that can appeal to a segment of consumers and are therefore potentially marketable.

[0026] In conclusion, the industrial application of these improved yeast strains is equivalent to that of any other commercial yeast strain used in the production of alcoholic beverages, requiring no technical adaptation of the fermentation process. Furthermore, it is worth noting that these yeast strains were developed without the use of genetic engineering, so they are not considered GMOs by law. Therefore, they can be directly transferred to industry and used in all possible markets, particularly those where GMOs are largely prohibited (such as Chile and Europe).

[0027] It is also important to consider that the process for obtaining the yeast strains of the invention do not constitute essentially biological procedures and are very far from understanding natural phenomena, since the selection of the strains for crossing could not have occurred through a natural phenomenon (mainly due to the geographical isolation existing between the strains used) and also that the crossing process involved a hierarchical mating method, which involves laboratory techniques such as induction of sporulation of individuals, directed mating of spores and evaluation of the character of hybrids through the use of molecular markers.

[0028] Application example

[0029] The following examples illustrate the invention, but should not be considered as limiting its scope.

[0030] Example 1: Genetic improvement program modified for application in microorganisms

[0031] Example 1.1: Design of the genetic improvement program

[0032] To carry out the genetic improvement program, a first filial generation (population "Fl") composed of 195 hybrid individuals previously generated and characterized (Kessi-Pérez et al., 2020) was used as a base population. In the first round of improvement, 19.5% of the strains from the Fl population that showed lower ethanol production performance were selected to be used as parents to produce a second filial generation (population "F2"), composed of 162 hybrid individuals, which was generated through a hierarchical mating strategy in which defined and controlled crosses were carried out. To achieve this, each parental strain selected from the Fl population was induced to sporulate in SP medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose, 2% agar) to obtain aseas.From these plates, individual spores were removed using a micromanipulator and placed in order on plates with YPD medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar), in order to generate defined and controlled crosses. The colonies obtained from these "spore matings" were transferred to a new plate with YPD medium and evaluated to verify their hybrid character (different from both parents) using molecular markers, identifying the presence of different molecular markers present in each of the parents.

[0033] Subsequently, a second round of improvement was carried out, in which 10.5% of the strains of the F2 population that showed a lower amount of residual glucose were selected to be used as parents to produce a third filial generation (population "F3"), composed of 132 hybrid individuals, which was generated in the same way as the F2 population was constructed through a hierarchical mating strategy in which defined and controlled crosses were carried out.

[0034] Example 1.2: Identification of hybrid strains in F2 and F3 populations using molecular markers

[0035] For the molecular confirmation and identification of hybrids from the F2 and F3 populations, 19 microsatellite molecular markers were used, 12 of which had been previously used (Kessi-Pérez et al., 2020) and 7 more that were added during the present investigation (Table 1). Using between 1 and 4 of these for each cross made, 162 of the 632 potential hybrids evaluated in the F2 population (25.6%) and 132 of the 344 potential hybrids evaluated in the F3 population (38.4%) were confirmed as hybrids. Each of these confirmed hybrids is characterized by these molecular markers, information that is kept confidential.

[0036] Table 1. Molecular markers used. In addition, strains C2-1B4, C7-1B7, C7-2B2, C7-2C2, C7-3A10 and C10-2I5 have been characterized at the level of their mitochondrial DNA (using the mtDNA-RFLP technique) and their electrophoretic karyotype (using the PFGE technique), while strains C2-1B4 and C7-1B7 also have their ITS region sequenced. All this information at the molecular level is also kept confidential.

[0037] Example 1.3: Estimating the heritability of traits to be improved

[0038] The 162 individuals belonging to the F2 population and the 132 individuals belonging to the F3 population were evaluated in synthetic must at the laboratory level, carrying out triplicate microfermentations as previously described (Kessi-Pérez et al., 2020) and then evaluating ethanol production and sugar consumption by HPLC. Using the data obtained, it was found that the heritability for ethanol production performance was 0.62 while the heritability for residual sugar content was 0.94 (1.00 being the maximum possible value). These high values ​​validate the strategy used, as they demonstrate that the traits under study are highly heritable and, therefore, can be improved through a genetic improvement program such as the one carried out.

[0039] Example 2: Evaluation of genetically improved strains at laboratory level

[0040] Example 2.1: Fermentations in synthetic must at laboratory level

[0041] All strains belonging to populations F2 and F3 were evaluated in synthetic must with high sugar content (250 g / L total, 125 g / L glucose and 125 g / L fructose, corresponding to approximately 24.8 °Bx) at laboratory level, performing triplicate microfermentations as previously described (Kessi-Pérez et al., 2020) and evaluating five parameters of oenological importance by HPLC: residual glucose, residual fructose, acetic acid production, glycerol production and ethanol production. In addition to them, the commercial strain Lalvin EC1118™ (Lallemand Inc., Canada) was used as a control, which corresponds to an industrial strain widely used worldwide for wine production.With regard to the first trait under improvement (ethanol production yield), it was found that there was a marked decrease (10.7%) in the average value of the F2 population compared to the F1 population (when selection for the trait was applied), while the F3 population showed a slight increase (3.5%) compared to the F2 population (when no selection for the trait was applied) (Table 2). With regard to the second trait under improvement (residual glucose content), it was found that there was already a marked decrease (25.9%) in the average value of the F2 population compared to the F1 population (when no selection for the trait was applied), while the F3 population showed an even more marked decrease (70.6%) compared to the F2 population (when selection for the trait was applied) (Table 2).

[0042] In addition to the above, the existence of individuals in the F3 population that correctly completed alcoholic fermentation (in terms of residual glucose content) was confirmed, having a low performance in ethanol production (Table 3).

[0043] Table 2. Average population values ​​for traits under improvement.

[0044] Table 3. Selected strains from the F3 population.

[0045] Example 2.1: Sulfite resistance evaluation

[0046] The 17 strains belonging to the F3 population previously selected based on their ability to correctly complete alcoholic fermentation (in terms of residual glucose content) with a low yield in the conversion of sugar into ethanol were grown in synthetic must in microculture to evaluate their resistance to different concentrations of sulfite (0, 20, 40, 80 and 100 ppm), which corresponds to an antimicrobial substance used by the wine industry to prevent the growth of contaminating microorganisms. From this group of strains, 6 of them were selected (C2-1B4, C7-1B7, C7-2B2, C7-2C2, C7-3A10 and C10-2I5), which presented the highest levels of resistance to this substance (retaining at least 50% of their growth capacity at 40 ppm of sulfite).

[0047] In this sense, the results obtained from the evaluation of genetically improved populations in synthetic must at laboratory level clearly demonstrate the existence of strains belonging to the F3 population with a lower yield in ethanol production, without losing fermentation capacities (in terms of residual sugar content) and capable of resisting sulfite concentrations used in the wine production process, as a result of the genetic improvement program carried out.

[0048] Example 3: Evaluation of genetically improved strains on a pilot scale under winery conditions

[0049] Example 3.1: Winemaking conditions

[0050] The 6 previously selected strains (C2-1B4, C7-1B7, C7-2B2, C7-2C2, C7-3A10 and C10-215) were used for pilot-scale wine production under winery conditions, using natural Sauvignon Blanc (white wine) and Carmenere (red wine) grape musts. In all cases, each fermentation tank was inoculated with IxlO. 6 [cells / mL] and were supplemented with both ammonium phosphate and other necessary nutrients to ensure proper fermentation performance. Fermentations with Sauvignon Blanc must were carried out at 14 °C, while those with Carmenere must were carried out at 18 °C and also included a malolactic fermentation step using the commercial Oenococcus oeni strain Lalvin VP41 ™ (Lallemand Inc., Canada). Finally, all produced wines were physically and chemically stabilized, bottled, and stored at 4 °C until final evaluation.

[0051] Example 3.2: Microbiological analysis of the wines produced

[0052] The analysis of the implantation of the different strains was carried out by isolating 11 yeast colonies once each fermentation was completed and identifying them by RFLP-mtDNA, finding that none of the improved strains had implantation problems in either of the two musts used, and could be implanted successfully (Table 4), which indicates that these strains are able to grow without problems in both types of must, a necessary condition to be able to carry out the fermentation process.

[0053] Table 4. Implementation of improved strains.

[0054] Example 3.3: Chemical analysis of the wines produced

[0055] Both the initial musts and the produced wines were evaluated in the manner routinely performed by the wine industry, that is, by determining the initial sugar concentration using Brix degrees and commissioning the final chemical analysis to a certified independent company (in this case, Vinotec Chile SA). The Sauvignon Blanc must had an initial sugar concentration of 22.8 °Bx, with an estimated probable alcohol content of 13.3 °GL, while the Carmenere must had an initial sugar concentration of 24.4 °Bx, with an estimated probable alcohol content of 14.4 °GL. The results obtained for the produced wines are shown in Table 5; from them, it can be concluded that all the improved strains were able to complete fermentation (low concentration of reducing matter).Furthermore, it can be observed that one of the improved strains (C7-1B7) had a lower alcohol content than expected in Sauvignon Blanc must, while all the improved strains had a lower alcohol content than expected in Carmenere must, with strain C2-1B4 standing out.

[0056] Table 5. Chemical analysis of the wines produced.

[0057] Example 3.4: Sensory analysis of the wines produced

[0058] An independent panel of nine expert judges (winemakers who conduct wine tastings periodically) performed a sensory analysis of the wines produced with the improved strains. Two types of sensory tests were performed: an affective (or preference) test and a descriptive one. The affective test, which is recommended for evaluating more than two samples at a time, measures satisfaction through ratings using a 5-point hedonic scale (1 being "I dislike" and 5 "I like it a lot"), while the descriptive test measured various aroma attributes (Mineral, Aroma, Overall, Spice, Floral, Strawberry, Ripe Fruit, Vegetable, and Reduced) and flavor (Bitterness, Acidity, Alcohol, and Sweetness, as well as Structure and Persistence in the case of red wines) (Noble, 1987), using a perceptual intensity scale from 0 ("Absence") to 5 ("Extreme").The results show that there are no statistically significant differences at the sensory level, neither at the affective level (Table 6) nor at the descriptive level (Table 7 and Figures 1-2), between the wines produced by the improved strains. Both the white and red wines produced by the improved strains obtained good scores in affective terms, being evaluated on average between 3 (“I am indifferent”) to 4 (“I like”) points, except for the white wine produced by the C7-1B7 strain which was evaluated between 4 (“I like”) and 5 (“I like it a lot”) points (Table 6).Furthermore, both white and red wines produced by the improved strains stood out for their overall aroma, with more vegetal tones in the whites and ripe fruit in the reds. In terms of flavor, both were characterized by their low bitterness, with the white wines standing out for their acidity and the red wines for their balance in terms of alcohol, sweetness, structure, and persistence (Table 7 and Figures 1-2). Taken together, these results indicate that these strains are capable of producing wines that can appeal to a segment of consumers and are therefore potentially marketable.

[0059] Table 6. Results by strain of the affective test of the wines produced.

[0060] All values ​​correspond to the mean ± standard deviation. Statistical analysis for each must consisted of independent ANOVA tests; in both cases, P values ​​for the F ratio were greater than 0.05, so no statistically significant differences were observed between strains.

[0061] Table 7. Results by attribute of the descriptive test of the wines produced.

[0062] ND: Not determined. All values ​​correspond to the average of the improved strains ± their standard deviation.

[0063] LITERATURE

[0064] Buescher, WA, Siler, CE, Morris, JR, Threlfall, RT, Main, GL, Cone, GC (2001). High alcohol wine production from grape juice concentrates. Am J Enol Vitic, 52:345- 351.

[0065] Contreras, A., Hidalgo, C., Henschke, P.A., Chambers, P.J., Curtin, C., Varela, C. (2014). Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine. Appl Environ Microbiol, 80:1670-8.

[0066] Escudero, A., Campo, E., Fariña, L., Cacho, J., Ferreira V. (2007). Analytical characterization of the aroma of five premium red wines. Insights into the role of odor families and the concept of fruitiness of wines. J Agrie Food Chem, 55:4501-10.

[0067] Gutierrez-Gamboa, G., Zheng, W., Marfnez de Tida, F. (2021). Strategies in vineyard establishment to face global warming in viticulture: a mini review. J Sci Food Agrie, 101:1261-1269.

[0068] Jones, G., White, M., Cooper, O., Storchmann, K. (2005). Climate change and global quality. Climatic Change, 73:319-343.

[0069] Kessi-Pérez, E.I., Molinet, J., García, V., Aguilera, O., Cepeda, F., López, M.E., ... Martínez, C. (2020). Generation of a non-transgenic genetically improved yeast strain for wine production from nitrogen-deficient musts. Microorganisms, 8:1194.

[0070] Noble, A., Arnold, R., Masuda, B., Pecore, S., Smith, J., Stem, P. (1984). Progress Towards a Standardized System of Wine Aroma Terminology. Am J Enol Vitic, 35:107- 109.

[0071] ODEPA. (2019). Boletín del vino: producción, precios y comercio exterior. Avance a diciembre 2018.

[0072] Schmidtke, L. M., Blackman, J.W., Agboola, S.O. (2012). Production technologies for reduced alcoholic wines. J Food Sci, 717:R25-41.

[0073] Tilloy, V., Ortiz-Julien, A., Dequin, S. (2014). Reduction of ethanol yield and improvement of glycerol formation by adaptive evolution of the wine yeast Saccharomyces cerevisiae under hyperosmotic conditions. Appl Environ Microbiol. 80:2623-32.

[0074] Varela, C., Dry, P.R., Kutyna, D.R., Francis, I.L., Henschke, P.A., Curtin, C.D., Chambers, P.J. (2015). Strategies for reducing alcohol concentration in wine Aust J Grape Wine Res, 21:670-679.

[0075] Varela, J, Varela, C. (2019). Microbiological strategies to produce beer and wine with reduced ethanol concentration. Curr Op Biotech, 56:88-96.

[0076] Zhu, X., Torija, M., Mas, A., Beltran, G., Navarro, Y. (2021). Effect of a multistarter yeast inoculum on ethanol reduction and population dynamics in wine fermentation. Foods, 10: 10.3390 / foods 10030623.

Claims

CLAIMS 1. Saccharomyces cerevisiae yeast strains called C2-1B4 and C7-1B7, CHARACTERIZED by being non-transgenic and genetically improved in their fermentation conditions, deposited in CChRGM under access codes RGM 3513 and 3514, respectively.

2. S. cerevisiae yeast strains according to claim 1, CHARACTERIZED in that the genetic improvement comprises a lower efficiency in the conversion of sugar into alcohol while maintaining its fermentation conditions in terms of residual sugar.

3. S. cerevisiae yeast strains according to Claim 1, CHARACTERIZED in that the fermentative condition is in alcoholic beverages.

4. Use of the S. cerevisiae yeast strains according to Claim 1, CHARACTERIZED in that it is used for the production of alcoholic beverages obtained by alcoholic fermentation.

5. Use of the S. cerevisiae yeast strains according to Claim 3, CHARACTERIZED in that they are used in the production of alcoholic beverages from musts with high sugar content.

6. Use of the S. cerevisiae yeast strains according to Claim 3, CHARACTERIZED in that the alcoholic beverage is wine.

7. Use of the S. cerevisiae yeast strains according to Claim 6, CHARACTERIZED in that the grape is of the Sauvignon blanc and Carmenere varieties.

Images