Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains.

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the "1002 Yeast Genomes Project" population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

Volatile compounds of traditional sorghum beer (tchapalo) produced in Côte d'Ivoire: comparison between wild yeasts and pure culture of Saccharomyces cerevisiae.

In recent years, there had been growing demand for distinctive high quality beer. Fermentation management has a fundamental role in beer quality and the levels of aroma compounds. Use of non-conventional yeast has been proposed to enhance beer flavor. In this study, the bioflavor of traditional sorghum beer from Côte d'Ivoire was investigated. The flavor profile of two beers fermented with wild yeasts and with pure culture of Saccharomyces cerevisiae respectively were studied. The main flavor components of the beer fermented by pure culture of Saccharomyces cerevisiae were different from those of the beer fermented with wild yeasts. The total level of esters and higher alcohols were (173.51 and 128.85 mg/L) respectively in the beer fermented with wild yeasts. These levels were significantly higher than those in the beer fermented with pure culture of Saccharomyces cerevisiae which were 13.08 and 78.26 mg/L for higher alcohols and esters respectively. On the other hand, the beer fermented with pure culture of Saccharomyces cerevisiae had an acid content higher than beer fermented with wild yeasts, i.e. 9.3 mg/L and 7.53 mg/L respectively.

Pathogenic budding yeasts isolated outside of clinical settings.

Budding yeasts are distributed across a wide range of habitats, including as human commensals. However, under some conditions, these commensals can cause superficial, invasive, and even lethal infections. Despite their importance to human health, little is known about the ecology of these opportunistic pathogens, aside from their associations with mammals and clinical environments. During a survey of approximately 1000 non-clinical samples across the United States of America, we isolated 54 strains of budding yeast species considered opportunistic pathogens, including Candida albicans and Candida (Nakaseomyces) glabrata. We found that, as a group, pathogenic yeasts were positively associated with fruits and soil environments, whereas the species Pichia kudriavzevii (syn. Candida krusei syn. Issatchenkia orientalis) had a significant association with plants. Of the four species that cause 95% of candidiasis, we found a positive association with soil. These results suggest that pathogenic yeast ecology is more complex and diverse than is currently appreciated and raises the possibility that these additional environments could be a point of contact for human infections.

Using wild yeasts to modulate the aroma profile of low-alcoholic meads.

In recent years, ample research has focused on applying wild (especially non-Saccharomyces) yeasts in producing alcoholic beverages. Common characteristics of wild yeast strains include simultaneous high production of fruity and floral aroma compounds and low ethanol production. In this study, mead starter cultures were selected based on preliminary screening of wild yeast strains from a Brazilian culture collection (n = 63) for their ability to produce aroma-active compounds. The selected strains included one strain of Saccharomyces cerevisiae and three non-Saccharomyces strains (Pichia jadinii, Torulaspora delbrueckii, and Kluyveromyces lactis). These strains were used to ferment honey must prepared with Aroeira honey, adjusted to 24°Brix, which took 36 days to complete. Single culture fermentations and co-fermentations with S. cerevisiae and non-Saccharomyces strains were carried out. The quality of the produced beverages was evaluated by sugar consumption and production of alcohols and organic acids, analyzed with high-performance liquid chromatography. The volatile organic compound composition was analyzed with gas chromatography-mass spectrometry. Meads with various ethanol amounts (4.7-11.0% v/v) and residual sugar contents (70.81-160.25 g l-1) were produced. In addition, in both single-strain fermentation and co-fermentation with S. cerevisiae, meads produced with either Torulaspora delbrueckii or Kluyveromyces lactis had a roughly three-fold higher content of honey-aroma compound phenethyl acetate and a higher hedonic impression score than meads produced with only S. cerevisiae. These results demonstrated non-Saccharomyces yeasts' ability to increase aroma complexity and improve the sensory quality of low-alcoholic meads.

Effects of feedstock and co-culture of Lactobacillus fermentum and wild Saccharomyces cerevisiae strain during fuel ethanol fermentation by the industrial yeast strain PE-2.

Even though contamination by bacteria and wild yeasts are frequently observed during fuel ethanol fermentation, our knowledge regarding the effects of both contaminants together is very limited, especially considering that the must composition can vary from exclusively sugarcane juice to a mixture of molasses and juice, affecting the microbial development. Here we studied the effects of the feedstock (sugarcane juice and molasses) and the co-culture of Lactobacillus fermentum and a wild Saccharomyces cerevisiae strain (rough colony and pseudohyphae) in single and multiple-batch fermentation trials with an industrial strain of S. cerevisiae (PE-2) as starter yeast. The results indicate that in multiple-cycle batch system, the feedstock had a minor impact on the fermentation than in single-cycle batch system, however the rough yeast contamination was more harmful than the bacterial contamination in multiple-cycle batch fermentation. The inoculation of both contaminants did not potentiate the detrimental effect in any substrate. The residual sugar concentration in the fermented broth had a higher concentration of fructose than glucose for all fermentations, but in the presence of the rough yeast, the discrepancy between fructose and glucose concentrations were markedly higher, especially in molasses. The biggest problem associated with incomplete fermentation seemed to be the lower consumption rate of sugar and the reduced fructose preference of the rough yeast rather than the lower invertase activity. Lower ethanol production, acetate production and higher residual sugar concentration are characteristics strongly associated with the rough yeast strain and they were not potentiated with the inoculation of L. fermentum.

Estudo de linhagens de leveduras Saccharomyces cerevisiae oriundas da biodiversidade ambiental na fermentação alcoólica / Study of yeast strains Saccharomyces cerevisiae from biodiversity environmental in alcoholic fermentation

Linhagens de leveduras Saccharomyces cerevisiae, nativas e oriundas do processo industrial de produção de etanol foram isoladas e comparadas com a levedura padrão CAT-1, selecionada e amplamente utilizada na indústria de etanol, em sistema descontínuo de fermentação, com reciclo celular. As linhagens foram reativadas em meio sólido de manutenção em placas de Petri e em seguida foram colocadas em crescimento em meio de caldo de cana-deaçúcar clarificado e diluído à 4º Brix. Após a obtenção da massa celular, na concentração de 1,0 x 108 cél./mL, as linhagens foram inoculadas em meio de caldo de cana-de-açúcar a 20º Brix e assim procedeu-se por seis ciclos fermentativos consecutivos. Ao final de cada ciclo procedeu-se as determinações do teor de etanol, pH, produção de biomassa, viabilidade e concentração de células, açúcares redutores totais residuais no vinho e acidez. Com os resultados obtidos foram calculados a eficiência fermentativa, viabilidade celular e brotamento. Os dados foram analisados estatisticamente utilizando-se a análise de variância (ANOVA) e as médias comparadas pelo teste de Tukey a 5% de probabilidade. Os resultados mostram que a linhagem utilizada em processo industrial, a CAT-1, se destacou diante das duas linhagens isoladas e selecionadas (18 e 19) em relação à eficiência fermentativa, mostrando um bom desempenho no processo de fermentação para a produção de etanol. Por outro lado observou-se que as linhagens 18 e 19 apresentaram desempenho fermentativo semelhante e, de modo geral, características adequadas à produção de etanol.

Native strains of Saccharomyces cerevisiae from the industrial ethanol process were isolated and have been tested with standard CAT-1 strain, which was selected, and widely used in ethanol production in southest of Brazil in batch fermentation system with cells recycles (Melle-Boinot). The strains were reactivated in synthetic culture media and then inoculated into clarified sugar cane juice at 4° Brix for growth until it reaches a cell concentration 1.0 x 108/mL. After this, the cells were inoculated in a sugar cane juice at concentration 20°Brix by six fermentative cycles. After each cycle were carried out the following analyses: ethanol concentration, pH, biomass, viability and budding cells. The data were analyzed statistically using variance analyses (ANOVA) and averages compared by Tukey test at 5% probability. The results show the best performance for that CAT-1 in fermentative efficiency than strains 18 and 19, mainly by the ethanol concentration. The 18 and 19 strains showed similar results of performance with characteristics for the ethanol production.