Antarctic Soil Yeasts with Fermentative Capacity and Potential for the Wine Industry

Citation: Navarro, L.; Gil i Cortiella, M.; Gutiérrez-Moraga, A.; Calisto, N.; Ubeda, C.; Corsini, G. Antarctic Soil Yeasts with Fermentative Capacity and Potential for the Wine Industry. Foods 2023,12, 4496. https:// doi.org/10.3390/foods12244496 Academic Editors: Vania Patrone and François Bourdichon Received: 10 November 2023 Revised: 1 December 2023 Accepted: 4 December 2023 Published: 16 December 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Antarctic Soil Yeasts with Fermentative Capacity and Potential for the Wine Industry Laura Navarro 1, Mariona Gil i Cortiella 2, Ana Gutiérrez-Moraga 1,3, Nancy Calisto 1,4 , Cristina Ubeda 5,* and Gino Corsini 1,* 1Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago 8900000, Chile; [email protected] (L.N.); ana.gutierr[email protected] (A.G.-M.); nancy[email protected] (N.C.) 2Instituto de Ciencias Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Santiago 8900000, Chile; [email protected] 3Departamento de Producción Agropecuaria, Facultad de Ciencias Agropecuarias y Medioambiente, Universidad de La Frontera, Temuco 4811230, Chile 4Centro de Investigación y Monitoreo Ambiental Antártico (CIMAA), Departamento de Ingeniería Química, Universidad de Magallanes, Avenida Bulnes 01855, Punta Arenas 6210427, Chile 5Departamento de Nutrición, Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, C/P. García González No 2, 41012 Sevilla, Spain *Correspondence: [email protected] (C.U.); [email protected] (G.C.) Abstract: Low fermentation temperatures are usually employed to obtain high-quality wines. This is especially interesting for white wine production since it prevents the loss of volatile compounds and a browning appearance; however, available fermentative yeasts do not usually tolerate low temperatures. Therefore, an interesting place to find new yeasts with cryotolerance is the Antarctic continent. From soil samples collected in Antarctica, 125 yeasts were isolated, of which 25 exhibited fermentative activity at 10 ◦ C. After a fingerprinting assay, we classified the candidates into nine isotypes and sequenced internal transcribed spacer regions for their identification. These yeasts were identified as part of the Mrakia genus. Sugar and alcohol tolerance tests showed that some of these Antarctic soil yeasts were able to grow up to 9% alcohol, and 25% sugar was reached; however, they exhibited longer latency periods compared to the control Saccharomyces cerevisiae. The optimal growing temperature for the isolated Antarctic yeasts was between 10 ◦ C and 15 ◦ C. A comprehensive analysis of the results obtained showed that the isolates 10M3-1, 4M3-6, and 4B1-35 could be good candidates for fermentation purposes due to their alcohol, sugar tolerance, and growth features. Our results prove that it is possible to isolate fermentative yeasts from Antarctic soil with promising characteristics for their potential use in the wine production industry. Keywords: Antarctic yeast; fermentative yeast; psychrophilic yeast; wine; Mrakia 1. Introduction Wine has been produced since ancient times, and today it continues to expand to many different places all over the world. Due to this, the wine industry has become a highly competitive market, with several wineries increasing their experimentation to produce wines with distinguishable features to diversify and create new products. Multiple factors modulate the characteristics of a wine, such as weather, soil, vineyard practices, winemaking practices/conditions, microorganisms involved in the process, etc. [ 1 – 3 ]. Among these factors, the yeast employed to carry out the fermentation is one of the elements that has a significant impact on the final product, especially in terms of the aromatic characteristics of the wine [ 4 ]. Therefore, wineries are currently focusing on the search for novel fermentative yeast strains or the utilization of non-traditional options to obtain wines with greater typicality [ 5 ]. In the case of the food industry, the most widely used yeasts are of the Foods 2023,12, 4496. https://doi.org/10.3390/foods12244496 https://www.mdpi.com/journal/foods Foods 2023,12, 4496 2 of 14 Saccharomyces type. They are employed to carry out fermentation processes, for example, to produce alcoholic beverages such as wine and beer. These yeasts can endure the specific conditions of this process, i.e., high osmolarity, high ethanol content, and low pH [ 6 , 7 ]. On the other hand, non-Saccharomyces yeast strains are less used in alcoholic beverage production, mainly due to their challenges in surviving high concentrations of alcohol produced during fermentation [ 8 ]. However, the interest in this kind of yeast in the industry has grown, as it has been observed that they have a positive effect on the organoleptic characteristics, increasing aromatic complexity and enhancing wine quality [ 9 – 11 ] when used along with Saccharomyces strains in co-inoculation experiments. Some authors have successfully tested some non-Saccharomyces yeast strains from the gender Metschnikowia, Kluyveromyces, or Candida to produce wines with lower alcohol levels [ 3 , 12 , 13 ]. In addition to the yeast strain employed for alcoholic fermentation, there are other critical conditions during wine production that directly influence the organoleptic properties of this beverage. The temperature reached during the alcoholic fermentation strongly affects the organoleptic characteristics of the final product, especially in the case of white wines [ 6 , 14 – 17 ]. This is partially due to low temperatures producing wines with a higher aromatic potential due to a lower loss of aromas by evaporation. It has been observed that lower temperatures during fermentation affect white, rosé, and red wines in a similar way, resulting in wines with more fruity and floral profiles. This effect is primarily due to the increased production of esters, making the wines more aromatic and complex [ 14 , 16 , 18 , 19 ]. Moreover, conducting alcoholic fermentation at lower temperatures also prevents the appearance of undesirable browning. This oxidative process involves sugars, lipids, amino acids, and phenols [ 20 , 21 ], leading to a decrease in the sensory quality of wines (loss of color, flavor, and aroma) and an increase in astringency [ 22 ]. Despite the advantages of a low fermentation temperature, conducting alcoholic fermentation under these conditions also entails some drawbacks. Yeast may stop growing or die due to the low temperatures, ceasing to be the dominant culture, which creates an opportunity for other microorganisms, such as fungi or bacteria, to take advantage and colonize the environment, leading to wine spoilage. Metabolic enzymes present in yeasts work in a concrete range of temperatures. The typically employed yeast in the wine industry, Saccharomyces cerevisiae, has an optimal growth temperature of around 32 ◦ C; therefore, the fermentation temperature is always above 15 ◦ C [ 23 ]. As a result, the fermentation temperature during wine production is a compromise between the quality of the wine and the viability of the microorganism. Accordingly, the selection of yeast strains with the ability to ferment grape must, at low temperatures, leads to less-oxidized wines with a high concentration of volatile compounds. Hence, the isolation of a new fermentative yeast from low-temperature climates could be a proper approach [ 24 ]. The Antarctic media have a wide microbial diversity, enabling the isolation of adequate candidates. Most microorganisms studied in cold environments are bacteria [ 25 – 27 ], but it has been suggested that yeasts could be better adapted to low temperatures than bacteria [ 28 ]. Yeast has been isolated from the Arctic, Alpine glaciers, Antarctic ecosystems, and Argentinean Patagonia [ 17 , 24 , 29 – 33 ]. Various cold adaptation features have been described in Antarctic yeast. These include a shift from a respiratory metabolism at 23 ◦ C to a fermentative metabolism when grown in cold conditions (0 ◦ C), achieved through the downregulation of citrate cycle genes and the ETC genes of Rhodotorula frigidialcoholis [ 34 ]. The metabolic enzymes of the yeast strains isolated from Antarctic media are expected to work at low temperatures. Some of these cold-active enzymes are well-known, being an attractive research topic in many fields [ 35 ]. They tend to share similar characteristics, such as improved flexibility and thermal compensation of the yeast [36]. In the search for fermentative yeast, Ballester-Tomás et al. (2017) [ 37 ] implemented a study of a strain of Candida sake isolated from the sub-Antarctic region. They compared some volatile compounds in the wines fermented with Saccharomyces cerevisiae strain QA23 to the compounds in wines produced with C. sake. They observed that wines fermented Foods 2023,12, 4496 3 of 14 with C. sake had a lower ethanol and glycerol content, whereas the wines fermented with this sub-Antarctic strain presented larger amounts of higher alcohols. While it is well-known that fermenting grape juice at low temperatures significantly enhances the final quality of white wine, and extensive research has been conducted on Antarctic yeasts, little is known about the potential of psychrophilic Antarctic yeasts as fermenting agents. Therefore, the objectives of the present study were to isolate and identify yeasts from different Antarctic locations, for potential use in the wine industry, with the ability to ferment at various temperatures and resistance against different alcohol concentrations and osmotic pressures. 2. Materials and Methods 2.1. Yeast Isolation Six soil samples collected from Fildes Bay (62 ◦ 11 ′ S, 58 ◦ 57 ′ W) and three soil samples from Collins Glacier (62 ◦ 9743 ′ S; 58 ◦ 27,978 ′ W) were processed for yeast isolation. The soil was suspended in sterile water, and inoculums were seeded into YM agar plates (3% yeast extract (Becton Dickinson and Company, Pont de Claix, France), 3% malt extract (Becton Dickinson and Company, Pont de Claix, France), 5% peptone (Merck, Darmstadts, Germany), 2% agar (Becton Dickinson and Company, Pont de Claix, France), supplemented with 1% of glucose (Sigma-Aldrich Co., St. Louis, MO, USA) and 100 µ g/mL of kanamycin (Caisson Labs, Smithfield, UT, USA) and ampicillin. Plates were cultured at 4, 10, and 18 ◦ C. Non-filamentous colonies were selected and transferred into fresh YM agar plates (1% glucose). Isolates were analyzed macroscopically and microscopically for subsequent confirmation of their yeast nature. 2.2. Macroscopic and Microscopic Analyses The colony growth in YM agar plates with 1% glucose (Sigma-Aldrich Co., St. Louis, MO, USA) of different microorganisms isolated was analyzed and registered using an optical microscope Motic BA310 with a 40 × objective and an additional 4.5 × magnification for imaging capture [38]. 2.3. Fermentative Capacity Test To determine the fermentative capacity, we used a modified YM medium containing 3% yeast extract (Becton Dickinson and Company, Pont de Claix, France), 3% malt extract (Becton Dickinson and Company, Pont de Claix, France), 5% peptone (Merck, Darmstadts, Germany), and 2% agar (Becton Dickinson and Company, Pont de Claix, France). The medium was supplemented with lactose (Sigma-Aldrich Co., St. Louis, MO, USA) and sucrose (Sigma-Aldrich Co., St. Louis, MO, USA) at a concentration of 10 g/L, 1 g/L of glucose, and 25 mg/L of phenol red—a pH variation indicator (Sigma-Aldrich Co., St. Louis, MO, USA). In assay tubes, the media were inoculated with the respective Antarctic yeast strains, and these were cultured at 10 ◦ C for 14 days. As a control yeast for the fermentative test, Saccharomyces cerevisiae EC-1118 (Lallemand-South America, Montreal, QC, Canada) was employed. 2.4. Sugar and Alcohol Tolerance Test The sugar tolerance capacity of the yeasts was analyzed with a multi-plate culture test with the YM medium, supplemented with different concentrations of glucose and fructose (Sigma-Aldrich Co., St. Louis, MO, USA) in a 1:1 ratio. These sugar concentration ranges started with 5% w/v, increasing by 5% until reaching a monosaccharide concentration of 25% w/v. Additionally, tolerance to alcohol was determined using cultures with ethanol (Sigma-Aldrich Co., St. Louis, MO, USA) at 0, 3, 6, and 9% v/v. Tolerance was determined by measuring the optical density of the samples at 600 nm every 48 h for 8 days. As a control yeast for the sugar and tolerance tests, Saccharomyces cerevisiae EC-1118 (Lallemand-South America) was employed. Foods 2023,12, 4496 4 of 14 2.5. DNA Isolation To isolate the genomic DNA, the following protocol was performed. The samples were obtained from a culture plate and solved in 200 µ L of sterile water. Then, 400 µ L of lyticase enzyme (Sigma-Aldrich Co., St. Louis, MO, USA) at a concentration of 1000 U/mL was added. The samples were incubated at 30 ◦ C for 2 h. Then, 10 µ L of proteinase K (Promega, Madison, WI, USA) at a concentration of 20 mg/mL was added and the samples were incubated for 2 h at 50 ◦ C. A heat shock protocol was performed twice: 30 min at − 20 ◦ C and then 10 min at 80 ◦ C. The samples were then vortexed at the maximum speed for 5 min. Next, the Zymo Research Quick-DNA Fungal and Bacterial miniprep kit Cat.No.: D6005 (Zymo Reasearch, Irvine, CA, USA) was used following the instructions of the manufacturer. 2.6. Molecular Characterization of Antarctic Fermentative Yeasts The arbitrary primers used for fingerprinting corresponded to (GTG)5 and (GACA)4. The reaction mix for the PCR contained 50 µ L: 25 µ L GoTaq Green Master Mix from Promega, provided by Fermelo Biotech (Región Metropolitana, Chile); 1 uL of each primer at 1 nM; 2 µ L of genomic DNA; and water to reach a total reaction volume of 50 µ L. The PCR reaction started with an initial denaturation cycle at 95 ◦ C for 3 min, followed by 45 cycles at 95 ◦ C for 1 min for denaturation, 50 ◦ C for 1 min for annealing, and 72 ◦ C for 90 s for extension, with a final extension at 72 ◦C for 10 min. For the identification of the samples, the amplification and sequencing of the internal transcribed spacer (ITS) were carried out. The primers used in the amplification of the ITS region were ITS1 (5 ′ -TCCGTAGGTGAACCTGCGG-3 ′ ) and ITS4 (5 ′ -TCCTCCGCTTATTGATATGC- 3). To amplify the ITS region, the reaction mix for the PCR contained 50 µ L: 20 µ L GoTaq Green Master Mix from Promega, provided by Fermelo Biotech (Chile); 1 µ L of each primer at 1 nM; 2 µ L of DNA; and 19 µ L of water to reach a total reaction volume of 50 µ L. The PCR reaction started with an initial denaturation cycle at 94 ◦ C for 5 min, followed by 45 cycles at 94 ◦ C for 1 min, 50 ◦ C for 1 min for annealing, and an extension at 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 5 min. The PCR products were purified for sequencing purposes using the E.Z.N.A. Gel Extraction Kit from Omega Biotech, provided by Grupo SIBI (Santiago, Chile). The results from the amplification of the ITS region were analyzed using the Basic Local Alignment Search Tool (BLAST), optimized for highly similar sequences. 2.7. Optimal Growth Temperature The growth of each selected yeast was analyzed in both solid and liquid media. In the liquid media, growth was determined using optical density. An initial culture was used to inoculate the samples of the YM medium with 100 µ L in a total volume of 8 mL; each culture was performed in triplicate. The cultures were then incubated at 4, 10, 15, 18, and 21 ◦ C. The optical density was measured at 600 nm every 48 h. Growth in the solid medium was also analyzed. For this purpose, YM plates were prepared, and a 5 µ L spot of each Antarctic yeast was placed in duplicate. The plates were cultured at 4, 10, 15, 18, and 21 ◦ C. The plates were analyzed for colony growth every 48 h for 2 weeks. As a control yeast for optimal growth temperature determination, Saccharomyces cerevisiae EC-1118 (Lallemand-South America) was employed. 3. Results 3.1. Yeast Isolation From all the soil samples, a total of 296 microorganisms were isolated. Following the macro and microscopic analyses, it was determined that 125 of the samples were indeed yeasts, as indicated by the observed cell size in microscopy (Figure 1). All the yeast was isolated from cultures at 4 and 10 ◦C; at 18 ◦C, no yeast was isolated. Foods 2023,12, 4496 5 of 14 Foods 2023, 12, x FOR PEER REVIEW 5 of 16 yeasts, as indicated by the observed cell size in microscopy (Figure 1). All the yeast was isolated from cultures at 4 and 10 °C; at 18 °C, no yeast was isolated. Figure 1. Process of isolation of the yeasts from soil sampling areas in Antarctica, Collins Glacier, and Fildes Bay, and the results of the isolation process, including images showing an example of the isolated yeasts from each location. 3.2. Fermentative Capacity Test The 125 yeasts isolated were analyzed to determine their fermentative capacity. This was performed at 10 °C with a pH variation indicator. This indicator turns from red at a neutral pH to yellow at an acidic pH. In fermentation processes, the reduction of the pH value is characteristic. From the 125 yeast samples, 25 (20%) were capable of fermenting optimally at 10 °C. Out of these 125 yeasts, 12.8% of the fermentative isolates (16 isolates) were obtained from the soil sample from Fildes Bay, and 7.2% (9 isolates) were from the Collins Glacier (Figure 2). This indicates that we recovered a greater number of fermentative yeasts in the soil of the Collins Glacier (9/33, 27.2%) compared to the soil of Fildes Bay (16/92, 17.4%). Figure 2. Percentage of fermentative yeasts of each soil sample from Collins Glacier and Fildes Bay. 3.3. Molecular Characterization of Antarctic Fermentative Yeasts As a first step in the molecular characterization of the Antarctic fermentative yeasts, we conducted a fingerprinting analysis using the primers (GACA)4 and (GTG)5. This methodology was used to determine which of the 25 strains were identical and, therefore, Figure 1. Process of isolation of the yeasts from soil sampling areas in Antarctica, Collins Glacier, and Fildes Bay, and the results of the isolation process, including images showing an example of the isolated yeasts from each location. 3.2. Fermentative Capacity Test The 125 yeasts isolated were analyzed to determine their fermentative capacity. This was performed at 10 ◦ C with a pH variation indicator. This indicator turns from red at a neutral pH to yellow at an acidic pH. In fermentation processes, the reduction of the pH value is characteristic. From the 125 yeast samples, 25 (20%) were capable of fermenting optimally at 10 ◦ C. Out of these 125 yeasts, 12.8% of the fermentative isolates (16 isolates) were obtained from the soil sample from Fildes Bay, and 7.2% (9 isolates) were from the Collins Glacier (Figure 2). This indicates that we recovered a greater number of fermentative yeasts in the soil of the Collins Glacier (9/33, 27.2%) compared to the soil of Fildes Bay (16/92, 17.4%). Foods 2023, 12, x FOR PEER REVIEW 5 of 16 yeasts, as indicated by the observed cell size in microscopy (Figure 1). All the yeast was isolated from cultures at 4 and 10 °C; at 18 °C, no yeast was isolated. Figure 1. Process of isolation of the yeasts from soil sampling areas in Antarctica, Collins Glacier, and Fildes Bay, and the results of the isolation process, including images showing an example of the isolated yeasts from each location. 3.2. Fermentative Capacity Test The 125 yeasts isolated were analyzed to determine their fermentative capacity. This was performed at 10 °C with a pH variation indicator. This indicator turns from red at a neutral pH to yellow at an acidic pH. In fermentation processes, the reduction of the pH value is characteristic. From the 125 yeast samples, 25 (20%) were capable of fermenting optimally at 10 °C. Out of these 125 yeasts, 12.8% of the fermentative isolates (16 isolates) were obtained from the soil sample from Fildes Bay, and 7.2% (9 isolates) were from the Collins Glacier (Figure 2). This indicates that we recovered a greater number of fermentative yeasts in the soil of the Collins Glacier (9/33, 27.2%) compared to the soil of Fildes Bay (16/92, 17.4%). Figure 2. Percentage of fermentative yeasts of each soil sample from Collins Glacier and Fildes Bay. 3.3. Molecular Characterization of Antarctic Fermentative Yeasts As a first step in the molecular characterization of the Antarctic fermentative yeasts, we conducted a fingerprinting analysis using the primers (GACA)4 and (GTG)5. This methodology was used to determine which of the 25 strains were identical and, therefore, Figure 2. Percentage of fermentative yeasts of each soil sample from Collins Glacier and Fildes Bay. 3.3. Molecular Characterization of Antarctic Fermentative Yeasts As a first step in the molecular characterization of the Antarctic fermentative yeasts, we conducted a fingerprinting analysis using the primers (GACA)4 and (GTG)5. This methodology was used to determine which of the 25 strains were identical and, therefore, rule them out. This allowed us to select only nine strains from the 25 isolates, using repetitive region DNA profile analyses by PCR fingerprinting (Figure 3). Foods 2023,12, 4496 6 of 14 Figure 3. Fingerprinting assay to discard overrepresentation of the isolates. (A) Yeasts 10M3-1 (1), 10M3-4 (2), 10M3-9 (3), 10M3-13 (4), 10M3-19 (5), 10M3-20 (6), 10M3-21 (7), 10M3-22 (8) and 4M3-6 (9); (B) Yeasts 4B1-19 (1), 4B1-20 (2), 4B1-21 (3), 4B1-22 (4), 4B1-23 (5), 4B1-24 (6), 4B1-25 (7), 4B1-26 (8) and 4B1-35 (9); and (C) Yeasts 4C2-38 (1), 4C2-39 (2), 10C2-3 (3), 10C2-4 (4), 10B2-15 (5), 10B2-13 (6) and S. cerevisiae EC1118 (7) Selected yeasts are highlighted by a rectangle in each respective lane. We identified nine isotypes (Table 1), each represented by a specific strain: 4B1-35, 4B1-23, 4B1-19, 4C2-38, 10C2-3, 10B2-15, 10M3-1, 10M3-19, and 4M3-6. The results are summarized in Table 1. Also, these yeasts were analyzed by optic microscopy, using an amplification of 400 × and an additional amplification of 4.5 × , resulting in a total magnification of 1800×(Figure 4). Table 1. Classification of isolated yeasts using the ITS region sequencing. Isotype Yeasts Selected Yeast I1 4B1-26, 4B1-25, 4B1-24, 4B1-22, 4B1-21, and 4B1-20 4B1-35 I2 4B1-23 4B1-23 I3 4B1-19 4B1-19 I4 4C2-38, 4C2-39, and 4C2-4 4C2-38 I5 10B2-13 and 10C2-3 10C2-3 I6 10B2-15 10B2-15 I7 10M3-1, 10M3-4, 10M3-9, 10M3-12, 10M3-21, and 10M3-22 10M3-1 I8 10M3-19 10M3-19 I9 4M3-6 and 10M3-20 4M3-6 Foods 2023, 12, x FOR PEER REVIEW 7 of 16 Figure 4. Optic microscopy of the 9 isotypes using a total amplification of 1800×. Graphical scale 10 μm. In addition, these nine representative yeast strains of different isotypes were analyzed by amplification and sequencing of the 18S rRNA ITS region using primers IT1 and ITS4 (Table 2). The results were obtained using BLAST and showed that these Antarctic yeast strains belong to the Mrakia genus, although some samples resulted in more than a single hit in the database with the maximum identity percentage and query cover. Table 2. Identification of the Antarctic isolates using bioinformatics. Yeast (Isotype) Identification Isotype ITS Length (bp)/Gen- Bank Sequence Length (bp) Identity (%)/Covered (%) GenBank Accession Number 4B1-35 (I1) Mrakia blollopis CPCC 300333 550/639 100/100 MZ683240.1 Mrakia psychrophilia T5Mp 550/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 550/601 100/100 EU224267.1 Mrakia psychrophilia AB16-13 550/610 99.81/100 LC125322.1 Mrakia psychrophilia BF-OTU180 550/813 99.81/100 AM901861.1 4B1-23 (I2) Mrakia robertii YH18 504/ 635 100/100 MT048630.1 Mrakia robertii YH01 504/631 100/100 MT048621.1 Mrakia robertii GR2-2-4-9 504/1207 100/100 LC514985.1 Mrakia robertii A2(b) 504/ 1210 100/100 MH481688.1 Mrakia robertii AI 504/1212 100/100 MH481684.1 4B1-19 (I3) Mrakia robertii YH18 513/ 635 100/100 MT048630.1 Mrakia robertii YH01 513/631 100/100 MT048621.1 Mrakia robertii GR2-2-4-9 513/1207 100/100 LC514985.1 Mrakia robertii A2(b) 513/1210 100/100 MH481688.1 Mrakia robertii AI 513/1212 100/100 MH481684.1 4C2-38 (I4) Mrakia blollopis CPCC 300333 491/639 100/100 MZ683240.1 Mrakia sp. I12F-02259 491/619 100/100 JX852329.1 Mrakia psychrophilia T5Mp 491/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 491/601 100/100 EU224267.1 Mrakia psychrophilia AB16-13 491/610 99.80/100 LC125322.1 10C2-3 (I5) Mrakia blollopis CPCC 300333 513/639 100/100 MZ683240.1 Mrakia psychrophilia AB16-13 513/610 99.81/100 LC125322.1 Mrakia blollopis MKOTU31 513/606 99.60/100 KP714624.1 Figure 4. Optic microscopy of the 9 isotypes using a total amplification of 1800 × . Graphical scale 10 µ m. Foods 2023,12, 4496 7 of 14 In addition, these nine representative yeast strains of different isotypes were analyzed by amplification and sequencing of the 18S rRNA ITS region using primers IT1 and ITS4 (Table 2). The results were obtained using BLAST and showed that these Antarctic yeast strains belong to the Mrakia genus, although some samples resulted in more than a single hit in the database with the maximum identity percentage and query cover. Table 2. Identification of the Antarctic isolates using bioinformatics. Yeast (Isotype) Identification Isotype ITS Length (bp)/GenBank Sequence Length (bp) Identity (%)/Covered (%) GenBank Accession Number 4B1-35 (I1) Mrakia blollopis CPCC 300333 550/639 100/100 MZ683240.1 Mrakia psychrophilia T5Mp 550/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 550/601 100/100 EU224267.1 Mrakia psychrophilia AB16-13 550/610 99.81/100 LC125322.1 Mrakia psychrophilia BF-OTU180 550/813 99.81/100 AM901861.1 4B1-23 (I2) Mrakia robertii YH18 504/ 635 100/100 MT048630.1 Mrakia robertii YH01 504/631 100/100 MT048621.1 Mrakia robertii GR2-2-4-9 504/1207 100/100 LC514985.1 Mrakia robertii A2(b) 504/ 1210 100/100 MH481688.1 Mrakia robertii AI 504/1212 100/100 MH481684.1 4B1-19 (I3) Mrakia robertii YH18 513/ 635 100/100 MT048630.1 Mrakia robertii YH01 513/631 100/100 MT048621.1 Mrakia robertii GR2-2-4-9 513/1207 100/100 LC514985.1 Mrakia robertii A2(b) 513/1210 100/100 MH481688.1 Mrakia robertii AI 513/1212 100/100 MH481684.1 4C2-38 (I4) Mrakia blollopis CPCC 300333 491/639 100/100 MZ683240.1 Mrakia sp. I12F-02259 491/619 100/100 JX852329.1 Mrakia psychrophilia T5Mp 491/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 491/601 100/100 EU224267.1 Mrakia psychrophilia AB16-13 491/610 99.80/100 LC125322.1 10C2-3 (I5) Mrakia blollopis CPCC 300333 513/639 100/100 MZ683240.1 Mrakia psychrophilia AB16-13 513/610 99.81/100 LC125322.1 Mrakia blollopis MKOTU31 513/606 99.60/100 KP714624.1 Mrakia psychrophilia AB16-12 513/606 99.61/100 LC125311.1 Mrakia psychrophilia AS 2.1971 513/601 100/98 EU224267.1 10B2-15 (I6) Mrakia robertii YH18 554/ 635 100/100 MT048630.1 Mrakia robertii YH01 554/631 100/100 MT048621.1 Mrakia robertii GR2-2-4-9 554/1207 100/100 LC514985.1 Mrakia robertii A2(b) 554/ 1210 100/100 MH481688.1 Mrakia sp. isolate J-39 554/1212 100/100 KY782313.1 10M3-1 (I7) Mrakia blollopis CPCC 300333 528/639 100/100 MZ683240.1 Mrakia psychrophilia BF-OTU180 528/813 99.82/100 AM901861.1 Mrakia psychrophilia AB16-13 528/610 99.82/100 LC125322.1 Mrakia psychrophilia AB16-2 528/610 99.64/100 LC125311.1 Mrakia psychrophilia T5Mp 528/595 100/98 JQ857018.1 10M3-19 (I8) Mrakia blollopis CPCC 300333 502/639 100/100 MZ683240.1 Mrakia psychrophilia T5Mp 502/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 502/601 100/100 EU224267.1 Mrakia psychrophilia AB16-13 502/610 99.80/100 LC125322.1 Mrakia psychrophilia BF-OTU180 502/813 99.80/100 AM901861.1 4M3-6 (I9) Mrakia blollopis CPCC 300333 492/639 100/100 MZ683240.1 Mrakia sp. I12F-02259 492/619 100/100 JX852329.1 Mrakia psychrophilia T5Mp 492/595 100/100 JQ857018.1 Mrakia psychrophilia AS 2.1971 492/601 100/100 EU224267.1 Mrakia blollopis MKOTU31 492/606 99.50/100 KP714624.1 3.4. Sugar and Alcohol Tolerance of Antarctic Fermentative Yeasts The Antarctic fermentative yeasts were subjected to an assay to assess sugar and alcohol tolerance. The nine different isolated yeasts withstood the experimental growing conditions, displaying a longer latency period and slower growth or proliferation compared to the control, Saccharomyces cerevisiae EC-1118. However, as the data show, there was an increase in the optical density (OD) over time in every condition. Therefore, there was observable growth, and the isolated Antarctic yeasts exhibited tolerance to conditions of high sugar content and high alcohol concentration (Figures 5and 6). Foods 2023,12, 4496 8 of 14 Foods 2023, 12, x FOR PEER REVIEW 9 of 16 Figure 5. Sugar tolerance. Yeast growth represented by the optical density under a wide range of sugar concentrations in the culture medium, ranging from 5% w/v to 25% w/v. (A) Saccharomyces cerevisiae EC-1118, (B) I1, (C) I2, (D) I3, (E) I4, (F) I5, (G) I6, (H) I7, (I) I8, and (J) I9. Figure 5. Sugar tolerance. Yeast growth represented by the optical density under a wide range of sugar concentrations in the culture medium, ranging from 5% w/vto 25% w/v. (A)Saccharomyces cerevisiae EC-1118, (B) I1, (C) I2, (D) I3, (E) I4, (F) I5, (G) I6, (H) I7, (I) I8, and (J) I9. Foods 2023,12, 4496 9 of 14 Foods 2023, 12, x FOR PEER REVIEW 10 of 16 Figure 6. Ethanol tolerance. Yeast growth is represented by the optical density under a wide range of alcohol concentrations in the culture medium, ranging from 0% to 9% v/v. (A) Saccharomyces cerevisiae EC-1118, (B) I1, (C) I2, (D) I3, (E) I4, (F) I5, (G) I6, (H) I7, (I) I8, and (J) I9. As can be observed in Figure 5, the typical yeast employed in wine production, S. cerevisiae, grew quickly in all tested sugar concentrations. In general, the isolated strains showed a shorter latency period at the lowest sugar concentration tested in the medium. Strains 10M3-1, 4M3-6, 4B1-35, 4C2-38, 10B2-15, and 10C-23 showed a similar growth pat- Figure 6. Ethanol tolerance. Yeast growth is represented by the optical density under a wide range of alcohol concentrations in the culture medium, ranging from 0% to 9% v/v. (A)Saccharomyces cerevisiae EC-1118, (B) I1, (C) I2, (D) I3, (E) I4, (F) I5, (G) I6, (H) I7, (I) I8, and (J) I9.