Nonconventional yeasts and hybrids for low temperature handcrafted sparkling ciders elaboration in Patagonia.

Yeasts play a crucial role in transforming apple must into cider. While Saccharomyces cerevisiae (Sc) has been traditionally associated to cider fermentations worldwide, cryotolerant species such as Saccharomyces uvarum (Su) as well as natural S. cerevisiae × S. uvarum (Sc×Su) hybrids have also been detected in ciders fermented at low temperatures. This study aimed to evaluate the ability of two Patagonian cryotolerant yeast strains (Su and Se) and their interspecific hybrids with a Sc to conduct handcrafted apple must fermentations and a second fermentation process (champenoise method). The main chemical parameters and sensory quality of the resulting sparkling beverages was also analysed. Firstly, Sc×Se and Sc×Su hybrids were evaluated in their fermentative features at laboratory scale. Hybrids were compared with their respective parental species evidencing significant differences in the physicochemical and aromatic composition of the obtained base ciders. Both Su parental strain and the hybrid Sc×Se were selected for performing pilot scale fermentations (250 L) using natural (non-sterilized) apple juice at two different temperatures: 20 °C and 13 °C. Sc parental strain was also evaluated for comparative purposes. All base ciders obtained were then subjected to a second fermentation. A high implantation capacity of both Su and the hybrid was evidenced at the lowest evaluated temperature, while commercial Sc strain was not detected at the final fermentation stage, independently from the temperature. All sparkling ciders exhibited distinct physicochemical profiles. Ciders inoculated with commercial Sc (but effectively fermented with local Sc strains) allowed the development of malolactic fermentation (MLF) in processes carried out at both temperatures. Contrarily, no MLF was observed in ciders inoculated with either Su or the hybrid. Sparkling ciders fermented with Su displayed the highest concentrations of 2-phenylethanol and 2-phenylethyl acetate, regardless of the fermentation temperature. Conversely, ciders fermented with the hybrid at 20 °C exhibited the highest concentrations of ethyl octanoate and ethyl decanoate, contributing to floral and fruity notes in the beverage. Sensory analysis conducted with untrained individuals revealed a preference for sparkling ciders produced with the hybrid strain at both 20 °C and 13 °C. The cider fermented at 20 °C exhibited floral notes, sweetness, and a full body, while ciders fermented at 13 °C displayed moderate acidity and a well-balanced profile. Conversely, a trained panel described the cider fermented at 20 °C with Su as a fruity and acidic beverage, whereas the ciders fermented at 13 °C exhibited intense bitterness and acidity. This study highlights the potential of cryotolerant Saccharomyces species and hybrids in the development of new starter cultures for producing artisanal sparkling ciders with distinctive properties.

Convergent adaptation of Saccharomyces uvarum to sulfite, an antimicrobial preservative widely used in human-driven fermentations.

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VIIXVI and XIXVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.

Evaluation of cryotolerant yeasts for the elaboration of a fermented pear beverage in Patagonia: Physicochemical and sensory attributes.

The production of pome fruits as pears and apples, as well as their derived industries, is of great economic importance in North Patagonia. The elaboration of fermented beverages as cider or perry has evidenced a substantial diversification during the last years, with the evaluation of different fruit varieties, yeast starters and technological changes. In this work, two cryotolerant yeasts belonging to the species Saccharomyces uvarum were evaluated at laboratory and pilot scale in sterile and no-sterile pear must. One of the strains was originally isolated from apple chicha (strain NPCC1314) and the other from apple cider (strain NPCC1420) in Patagonia. Both physicochemical and sensory features of the fermented products were evaluated. Both strains were able to successfully complete the fermentations, although strain NPCC1420 showed the better kinetic properties including a faster sugar consumption than the strain NPCC1314. Both strains showed excellent implantation capacity, but the fermented products showed different chemical profiles. The perry fermented with the strain NPCC1314 was characterized by better sensory attributes as assessed by trained panelists and a greater acceptance for untrained public than the same fermented with the strain NPCC1420. The two strains were able to consume sorbitol, both in pear must and in agar-plates supplemented with sorbitol as the sole carbon source. This ability is described for the first time in S. uvarum, at least for the two strains evaluated in this work.

Purification and characterization of Saccharomyces eubayanus killer toxin: Biocontrol effectiveness against wine spoilage yeasts.

Microbiological contamination by spoilage yeasts species are frequent during winemaking, and biological control using antagonistic yeasts is considered a more beneficial alternative to conventional synthetic antimicrobials. Saccharomyces eubayanus killer toxin (SeKT) was produced and purified in a synthetic optimized medium. Purification procedure allowed the identification of SeKT as protein with an apparent molecular mass of 70 kDa and activity at physicochemical conditions suitable for winemaking process. Purified SeKT reduced the levels of volatile phenols produced by the spoilage yeasts Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii and Pichia manshurica in wine-like medium. The putative mode of action of SeKT on sensitive yeast strains comprises cell wall disruption through ß-glucanase and chitinase activities as well as necrotic and apoptotic death in a toxin dose dependent manner. Thus, SeKT appears to be a promising biocontrol agent against spoilage yeasts during wine aging and storing.

Inheritance of winemaking stress factors tolerance in Saccharomyces uvarum/S. eubayanus × S. cerevisiae artificial hybrids.

Stress has been defined as any environmental factor that impairs the growth of a living organism. High concentrations of ethanol, sugars and SO2 as well as temperature variations occurring during winemaking processes are some recognized stress factors that yeasts must overcome in order to avoid stuck or sluggish fermentations. At least two of these factors -sugar and ethanol concentrations- are strongly influenced by the global warming, which become them a worry for the future years in the winemaking industry. One of the most interesting strategies to face this complex situation is the generation of hybrids possessing, in a single yeast strain, a broader range of stress factors tolerance than their parents. In the present study, we evaluated four artificial hybrids generated with S. cerevisiae, S. uvarum and S. eubayanus using a non-GMO-generating method, in their tolerance to a set of winemaking stress factors. Their capacity to overcome specific artificial winemaking situations associated with global warming was also analyzed. All four hybrids were able to grow in a wider temperature range (8-37 °C) than their parents. Hybrids showed intermediate tolerance to higher ethanol, sugar and sulphite concentrations than their parents. Additionally, the hybrids showed an excellent fermentative behaviour in musts containing high fructose concentrations at low temperature as well as under a condition mimicking a stuck fermentation.

Saccharomyces uvarum isolated from patagonian ciders shows excellent fermentative performance for low temperature cidermaking.

Saccharomyces uvarum has been recovered from natural habitats and traditionally fermented beverages (apple chicha) in Patagonia. However, this species has never been obtained from industrially relevant beverages like wine or cider in the same region. In this work, different strains belonging to the cryotolerant species S. uvarum were recovered from spontaneous cider fermentations carried out at low temperature in Red Delicious apple must. The strain S. uvarum NPCC1420 obtained from this cider and selected for its physiological and technological features, evidenced a better adaptation to the cidermaking process than a previously selected strain obtained from a less industrialized product called apple chicha. Some differences, like a higher ethanol and sulphite tolerance, seemed to be associated with differential domestication pressures suffered by each different strain. Moreover, the most important fermentative features of the strain NPCC1420 were a higher competition capacity than the strain NPCC1314 in non-sterile apple must, as well as significantly higher amounts of glycerol, 2-phenylethanol and 2-phenylethyl acetate than the strain isolated from apple chicha.

Interspecific hybridisation among diverse Saccharomyces species: A combined biotechnological solution for low-temperature and nitrogen-limited wine fermentations.

Lack of the prezygotic barrier in the Saccharomyces genus facilitates the construction of artificial interspecific hybrids among different Saccharomyces species. Hybrids that maintain the interesting features of parental strains have been applied in industry for many beneficial purposes. Two of the most important problems faced by wine makers is nitrogen deficiency in grape must and low-temperature fermentation. In our study, hybrids were constructed by using selected low nitrogen-demanding cryotolerant S. eubayanus, S. uvarum strains and S. cerevisiae. The fermentation capacity of the hybrid strains was tested under four conditions by combining two temperatures, 12 °C and 28 °C, and two nitrogen concentrations, 60 mg/L and 300 mg/L. The hybrid strains obtained combined characters of both parental strains and conferred better fermentation rates under low-temperature or low-nitrogen conditions. The hybrid strains also produced larger amounts of acetate esters and higher alcohols, which increase aroma intensity and complexity in wine. Nitrogen sources were more rapidly consumed by the hybrid strains, which allows greater competition ability under nitrogen-deficiency conditions. Therefore, the interspecific hybridisation between low nitrogen-demanding cryotolerant strains and S. cerevisiae is a potential solution for low-temperature or low-nitrogen fermentations.

Fermentative behaviour and competition capacity of cryotolerant Saccharomyces species in different nitrogen conditions.

The selection of yeasts with low nitrogen requirement is a current need in winemaking. In this work, we analysed nitrogen requirements of strains belonging to the cryotolerant species S. uvarum, S. eubayanus and S. kudriavzevii, in order to evaluate their potential for conducting the fermentation of low nitrogen content grape musts. Our result demonstrated that S. eubayanus is the species less influenced by the increasing nitrogen concentrations in both growth and fermentation conditions. Strains showing the best behaviours, S. eubayanus NPCC 1285 and S. uvarum NPCC 1317, were selected to be tested in mixed cultures with S. cerevisiae T73 at different temperatures (12 °C, 20 °C and 28 °C) in synthetic grape must with different nitrogen concentrations (60, 140 and 300 mg/L YAN). The cryotolerant strains dominated the fermentations carried out at 12 °C while S. cerevisiae prevailed at 28 °C independently from the nitrogen concentration. At intermediate temperature, 20 °C, S. eubayanus mono and mixed cultures showed the best fermentative behaviour especially with low and intermediate nitrogen concentration. In summary, cryotolerant Saccharomyces species, particularly S. eubayanus, could be interesting tools to avoid fermentations stucks caused by low nitrogen content in grape musts.

Saccharomyces cerevisiae × Saccharomyces uvarum hybrids generated under different conditions share similar winemaking features.

Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are post-zygotic. Depending on several factors, including the involved strains, the hybridization mechanism and stabilization conditions, hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13 and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilization based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results demonstrated that, irrespective of the S. uvarum parent and temperature used for hybrid generation and stabilization, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterized by low ethanol production, high glycerol synthesis and the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame. Copyright © 2017 John Wiley & Sons, Ltd.

Physiological characterization of Saccharomyces uvarum and Saccharomyces eubayanus from Patagonia and their potential for cidermaking.

A diversity of yeast strains belonging to the cryotolerant fermentative species S. uvarum and S. eubayanus have been recovered from natural habitats and traditional fermentations in North Patagonia. The aim of this work was to evaluate the most relevant physiological features in a set of Patagonian strains belonging to S. uvarum and S. eubayanus, in order to analyze their potentiality to be used as starter cultures for cidermaking elaborated at low temperature. We evidenced that S. uvarum strains isolated from natural habitats (Araucaria araucana bark) showed similar physiological features to S. eubayanus strains obtained from the same habitat, and different from S. uvarum strains from fermentative environments (apple chichas). We also confirm the capacity of S. uvarum to produce high glycerol levels, low acetic acid and elevated production of the higher alcohol 2-phenylethanol and 2-phenylethyl acetate and demonstrated similar properties in S. eubayanus. Finally, we evidenced for the first time the antagonistic activity of S. eubayanus and selected three strains (two S. uvarum and one S. eubayanus) bearing the best combination of features to be used as a starter culture in cidermaking.

TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts.

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin ­ but not other cell wall polysaccharides ­ prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

Diversity and killer behaviour of indigenous yeasts isolated from the fermentation vat surfaces in four Patagonian wineries.

The diversity and killer behaviour of the yeast biota associated with surfaces of four Patagonian wineries were analyzed in the present study. These wineries were different in their technological and ecological features. Following liquid enrichment of samples from fermentation vat surfaces yeast isolates were identified by pheno- and genotyping and characterized using killer sensitivity patterns. Out of 92 isolated yeasts, 25% were Saccharomyces cerevisiae; 18% were Kloeckera apiculata and 11% were Pichia anomala; other six species representing a low percentage were also found. A particular biota composed mainly by S. cerevisiae (57%) and P. anomala (37%) was found in the winery located far from the other three wineries. As a whole, the wineries using spontaneous fermentation showed a major percentage of S. cerevisiae and a minor percentage of K. apiculata. The present study showed a pronounced heterogeneity in killer behaviour: killer, 35%, neutral, 25% and sensitive, 40%. In particular, S. cerevisiae isolates showed a higher sensitivity to killer reference yeasts than non-Saccharomyces isolates. On the other hand, most of the non-Saccharomyces yeasts isolated from fermentation vats were resistant to Saccharomyces toxins.