Bioactive volatiles of brewer's yeasts: Antifungal action of compounds produced during wort fermentation on Aspergillus sp.

Previous investigations proved the potential of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 as suitable biocontrolling agents against Aspergillus sp. through the production of soluble and volatile bioactive antifungal compounds. The present study delves into those finding by means of the identification of the volatile compounds produced by brewer's strains that demonstrated fungistatic and fungicidal effects against Aspergillus flavus and A. parasiticus when cultured in brewer's wort agar plates. Traditional brewer's yeasts such as S. cerevisiae MBELGA62 and Saccharomyces pastorianus SAFS235 synthetize volatiles that fully inhibited mycelial development for up to 9 days at 30 °C. The non-conventional brewer's strains P. kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122 increased the lag phase by >100% and significantly reduced the fungal growth rate by 27.5-43.0% and 15.4-31.4%, respectively. In this context, 2-phenylethanol, 2-phenylethyl acetate and benzyl alcohol were identified as the main antifungal agents involved in Aspergillus sp.'s inhibition.

Comparative genomics of infective Saccharomyces cerevisiae strains reveals their food origin.

Fungal infections are less studied than viral or bacterial infections and often more difficult to treat. Saccharomyces cerevisiae is usually identified as an innocuous human-friendly yeast; however, this yeast can be responsible for infections mainly in immunosuppressed individuals. S. cerevisiae is a relevant organism widely used in the food industry. Therefore, the study of food yeasts as the source of clinical infection is becoming a pivotal question for food safety. In this study, we demonstrate that S. cerevisiae strains cause infections to spread mostly from food environments. Phylogenetic analysis, genome structure analysis, and phenotypic characterization showed that the key sources of the infective strains are food products, such as bread and probiotic supplements. We observed that the adaptation to host infection can drive important phenotypic and genomic changes in these strains that could be good markers to determine the source of infection. These conclusions add pivotal evidence to reinforce the need for surveillance of food-related S. cerevisiae strains as potential opportunistic pathogens.

Identification and assessment of non-conventional yeasts in mixed fermentations for brewing bioflavored beer.

The increasing demand for more flavored and complex beers encourages the investigation of novel and non-conventional yeasts with the ability to provide a combination of bioflavoring and low ethanol yields. The present study identified 22 yeasts isolated from different brewing sources, including the fermentation by-products known as yeast sludges, and characterized a selection of strains to find the more suitable for the aforementioned aims. HPLC and GC-FID analysis of its brewing products were performed. The most promising results were obtained with the non-conventional yeasts Pichia kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122. The former, isolated from a Belgian wheat beer sludge, was capable of growing in wort (17.0°Bx., 20 °C) with very low ethanol yields (1.19 % v/v). Besides, upon mixed fermentations with Saccharomyces cerevisiae, was suitable to produce volatile compounds such as ethyl acetate, 2-phenyl ethanol and isoamyl alcohol, with characteristic fruity notes. M. guilliermondii MUS122, isolated from a golden ale beer sludge, partially attenuated the wort with low production of ethanol and biomass. In addition, provided some fruity and floral nuances to the aroma profile of mixed fermentations with brewer's yeast. The results suggest that these strains favor the development of more fruity-flowery aroma profiles in beers. Furthermore, they are suitable for use in mixed fermentations with Saccharomyces brewer's strains, although the ethanol level did not decrease significantly.

Bioactive Compounds, Nutritional Quality and Antioxidant Capacity of the Red-Fleshed Kirkwood Navel and Ruby Valencia Oranges.

Kirkwood Navel and Ruby Valencia are two spontaneous bud-mutations of the ordinary Washington Navel and Valencia late oranges characterized by the red coloration of their flesh. The purpose of this study was to analyze the physiological features, internal fruit quality, contents of relevant bioactive compounds and antioxidant capacity in the pulps of the red-fleshed fruits compared with the ordinary oranges during late development and maturation. In general, the content of sugars, organic acids, vitamin C, tocopherols, total phenolics and flavonoids, the hydrophilic antioxidant capacity and their changes during maturation were similar in the red-fleshed oranges and in the corresponding blond oranges. However, the mature Ruby fruits contained lower concentrations of sugars, malic and succinic acid and higher levels of citric acid than the ordinary Valencia. The major difference between the pulps of the Kirkwood and Ruby oranges and those of the ordinary oranges was the higher lipophilic antioxidant capacity and SOAC (singlet oxygen absorption capacity) of the former. Together, the high and unique content and composition of carotenoids in Kirkwood and Ruby may contribute to an enhanced antioxidant capacity without any detrimental effects on other fruit-quality attributes, making these varieties good sources of phytochemicals for the fresh-fruit and juice-processing citrus industries.

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties.

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: ß-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions.

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.

Genome structure reveals the diversity of mating mechanisms in Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrids, and the genomic instability that promotes phenotypic diversity.

Interspecific hybridization has played an important role in the evolution of eukaryotic organisms by favouring genetic interchange between divergent lineages to generate new phenotypic diversity involved in the adaptation to new environments. This way, hybridization between Saccharomyces species, involving the fusion between their metabolic capabilities, is a recurrent adaptive strategy in industrial environments. In the present study, whole-genome sequences of natural hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii were obtained to unveil the mechanisms involved in the origin and evolution of hybrids, as well as the ecological and geographic contexts in which spontaneous hybridization and hybrid persistence take place. Although Saccharomyces species can mate using different mechanisms, we concluded that rare-mating is the most commonly used, but other mechanisms were also observed in specific hybrids. The preponderance of rare-mating was confirmed by performing artificial hybridization experiments. The mechanism used to mate determines the genomic structure of the hybrid and its final evolutionary outcome. The evolution and adaptability of the hybrids are triggered by genomic instability, resulting in a wide diversity of genomic rearrangements. Some of these rearrangements could be adaptive under the stressful conditions of the industrial environment.

Improving the Cryotolerance of Wine Yeast by Interspecific Hybridization in the Genus Saccharomyces.

Fermentations carried out at low temperatures (10-15°C) enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. Notwithstanding, as Saccharomyces cerevisiae is the main species responsible for alcoholic fermentation, other species of the genus Saccharomyces, such as cryophilic species Saccharomyces eubayanus, Saccharomyces kudriavzevii and Saccharomyces uvarum, are better adapted to low-temperature fermentations during winemaking. In this work, a Saccharomyces cerevisiae × S. uvarum hybrid was constructed to improve the enological features of a wine S. cerevisiae strain at low temperature. Fermentations of white grape musts were performed, and the phenotypic differences between parental and hybrid strains under different temperature conditions were examined. This work demonstrates that hybridization constitutes an effective approach to obtain yeast strains with desirable physiological features, like low-temperature fermentation capacity, which genetically depend on the expression of numerous genes (polygenic character). As this interspecific hybridization approach is not considered a GMO, the genetically improved strains can be quickly transferred to the wine industry.

Mitochondrial introgression suggests extensive ancestral hybridization events among Saccharomyces species.

Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several worldwide distributed wild Saccharomyces species and their hybrids isolated from different sources and geographic origins. We demonstrated the existence of several recombination points in mitochondrial region COX2-ORF1, likely mediated by either the activity of the protein encoded by the ORF1 (F-SceIII) gene, a free-standing homing endonuclease, or mostly facilitated by A+T tandem repeats and regions of integration of GC clusters. These introgressions were shown to occur among strains of the same species and among strains of different species, which suggests a complex model of Saccharomyces evolution that involves several ancestral hybridization events in wild environments.

Saccharomyces uvarum is responsible for the traditional fermentation of apple chicha in Patagonia.

Apple chicha is a fresh low alcoholic beverage elaborated by aboriginal communities of Andean Patagonia (Argentina and Chile). In the present work, we identified the yeast microbiota associated with this fermentation, and characterized genetically those belonging to the genus Saccharomyces. Both Saccharomyces cerevisiae and S. uvarum were found in the analyzed fermentations. Phylogenetic and population structure analyses based on genes sequence analysis were carried out for both S. cerevisiae and S. uvarum strains obtained in this study and a set of additional strains from diverse origins. The results demonstrate that S. cerevisiae strains from apple chicha belong to the big group of wine/European strains of this species, while S. uvarum strains were included in the Holartic population of this species. Additionally, some S. uvarum strains from chichas evidenced as an admixture of both pure Holartic and pure South American populations. Our results suggest that Holartic strains could have been introduced in South America together with the domestication of apple trees by Mapuche communities. This Holartic population suffered admixis with the naturally present South American population of this species, originating strains bearing genetic features from the two populations, detectable in both chichas and natural habitats.

Increased mannoprotein content in wines produced by Saccharomyces kudriavzevii×Saccharomyces cerevisiae hybrids.

Several wine quality aspects are influenced by yeast mannoproteins on account of aroma compounds retention, lactic-acid bacterial growth stimulation, protection against protein haze and astringency reduction. Thus selecting a yeast strain that produces high levels of mannoproteins is important for the winemaking industry. In this work, we observed increased levels of mannoproteins in S. cerevisiae×S. kudriavzevii hybrids, compared to the S. cerevisiae strain, in wine fermentations. Furthermore, the expression of a key gene related to mannoproteins biosynthesis, PMT1, increased in the S. cerevisiae×S. kudriavzevii hybrid. We showed that artificially constructed S. cerevisiae×S. kudriavzevii hybrids also increased the levels of mannoproteins. This work demonstrates that either natural or artificial S. cerevisiae×S. kudriavzevii hybrids present mannoprotein overproducing capacity under winemaking conditions, a desirable physiological feature for this industry. These results suggest that genome interaction in hybrids generates a physiological environment that enhances the release of mannoproteins.

Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity.

Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae×S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters).

Stabilization process in Saccharomyces intra and interspecific hybrids in fermentative conditions

We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30-50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively (AU)

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Saccharomyces eubayanus and Saccharomyces uvarum associated with the fermentation of Araucaria araucana seeds in Patagonia.

Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species in A. araucana seeds and bark samples, led us to postulate a potential role for them as the original yeasts responsible for the elaboration of Mudai before the introduction of commercial S. cerevisiae cultures. The molecular and genetic characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus and S. pastorianus), allowed their ecology and evolution us to be examined.

On the complexity of the Saccharomyces bayanus taxon: hybridization and potential hybrid speciation.

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99-100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus. Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains.

Stabilization process in Saccharomyces intra and interspecific hybrids in fermentative conditions.

We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30-50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively.

Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking.

Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains. In this work we evaluated the usefulness of three different hybridization methods: spore to spore mating, rare-mating and protoplast fusion for the generation of intra- and inter-specific stable hybrids, being the first report about the comparison of different methods to obtain artificial hybrids to be used in fermentations. Spore to spore mating is an easy but time-consuming method; hybrids generated with this technique could lack some of the industrially relevant traits present in the parental strains because of the segregation occurred during meiosis and spore generation prior to hybridization. Hybrids obtained by protoplast fusion get the complete information of both parents but they are currently considered as genetically modified organisms (GMOs). Finally, hybrids obtained by rare-mating are easily obtained by the optimized methodology described in this work, they originally contain a complete set of chromosomes of both parents and they are not considered as GMOs. Hybrids obtained by means of the three methodological approaches showed a high genetic variability; however, a loss of genetic material was detected in most of them. Based on these results, it became evident that a last crucial aspect to be considered in every hybridization program is the genetic stabilization of recently generated hybrids that guarantee its invariability during future industrial utilization. In this work, a wine yeast genetic stabilization process was developed and vegetatively stable hybrids were obtained.

Exclusion of Saccharomyces kudriavzevii from a wine model system mediated by Saccharomyces cerevisiae.

This study investigated the competition and potential hybrid generation between the species Saccharomyces cerevisiae and S. kudriavzevii in a wine-model environment. Our main goal was to understand why S. kudriavzevii has not been found in wine fermentations whilst their hybrids are present. Auxotrophic mutants (Ura(-) and Lys(-)) were used to favour the selection of hybrids and to specifically differentiate the two species in mixed fermentations carried out at different temperatures (17 °C, 24 °C and 31 °C). Both yeasts showed a reduction in their maximum specific growth rates in mixed fermentations, indicating a clear antagonistic effect between the two microorganisms. Temperature played an important role in this competition. In this way, S. kudriavzevii was less affected at 17 °C, but S. cerevisiae was clearly the best competitor at 31 °C, preventing the growth of S. kudriavzevii. Population levels of S. kudriavzevii always significantly decreased in the presence of S. cerevisiae. Ethanol was measured throughout the fermentations and in all cases S. kudriavzevii growth was arrested when ethanol levels were < 5 g/l, indicating that this compound did not influence the competitive exclusion of S. kudriavzevii. Killer factors were also discarded due to the K(-) R(-) phenotype of both strains. Finally, no prototrophic interspecific hybrids were isolated in small-scale fermentations at any temperature assayed. Our results show that the lack of competitiveness exhibited by S. kudriavzevii, especially at high temperatures, explains the absence of this species in wine fermentations, suggesting that natural S. cerevisiae × S. kudriavzevii hybrids most likely originated in wild environments rather than in industrial fermentations.