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.

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.

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers.

The burgeoning interest in archaic, traditional, and novel beer styles has coincided with a growing appreciation of the role of yeasts in determining beer character as well as a better understanding of the ecology and biogeography of yeasts. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts for production of beers with novel flavour profiles and other desirable properties. Yeasts isolated from spontaneously fermented beers as well as from other food systems (wine, bread, and kombucha) have shown promise for brewing application, and there is evidence that such cross-system transfers have occurred naturally in the past. We review here the available literature pertaining to the use of nonconventional yeasts in brewing, with a focus on the origins of these yeasts, including methods of isolation. Practical aspects of utilizing nondomesticated yeasts are discussed, and modern methods to facilitate discovery of yeasts with brewing potential are highlighted.

Phylogeography of the wild Lager-brewing ancestor (Saccharomyces eubayanus) in Patagonia.

Saccharomyces eubayanus is the close relative of the Lager-brewing yeast and was firstly found in North Patagonia associated with Nothofagus trees. In recent years additional strains were found in North America, Asia and New Zealand, and genomic analyses showed the existence of two main populations of this yeast, both of them present in Patagonia. Here, we performed the most comprehensive study of S. eubayanus in Patagonia natural environments (400 samples) and confirmed that this region has the highest isolation success rate for this species described worldwide (more than 10-fold). The genetic characterization of 200 isolates (COX2, DCR1, intFR) revealed five geographically structured subpopulations. We hypothesized that marine ingressions and glaciations, which shaped the Patagonian landscape, contributed on population differentiation. The first large screening of fermentation performance of 60 wild S. eubayanus strains indicated which subpopulations would be more suitable for beer production.

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.

Saccharomyces eubayanus and Saccharomyces arboricola reside in North Island native New Zealand forests.

Saccharomyces is one of the best-studied microbial genera, but our understanding of the global distributions and evolutionary histories of its members is relatively poor. Recent studies have altered our view of Saccharomyces' origin, but a lack of sampling from the vast majority of the world precludes a holistic perspective. We evaluate alternate Gondwanan and Far East Asian hypotheses concerning the origin of these yeasts. Being part of Gondwana, and only colonized by humans in the last ∼1000 years, New Zealand represents a unique environment for testing these ideas. Genotyping and ribosomal sequencing of samples from North Island native forest parks identified a widespread population of Saccharomyces. Whole genome sequencing identified the presence of S. arboricola and S. eubayanus in New Zealand, which is the first report of S. arboricola outside Far East Asia, and also expands S. eubayanus' known distribution to include the Oceanic region. Phylogenomic approaches place the S. arboricola population as significantly diverged from the only other sequenced Chinese isolate but indicate that S. eubayanus might be a recent migrant from South America. These data tend to support the Far East Asian origin of the Saccharomyces, but the history of this group is still far from clear.

Identification and characterization of non-saccharomyces spoilage yeasts isolated from Brazilian wines.

The industry of fine wines and also locally consumed table wines is emerging in Brazil with an increasing volume and economic impact. Enologists in this region currently lack information about the prevalence and characteristics of spoilage yeasts, which may contaminate and potentially undervalue Brazilian wines. Herein, we analyzed 50 local red wines including 27 fine wines (V. vinifera) and 23 table wines (V. labrusca). Presumptive spoilage yeasts were isolated on differential medium, and classified by RFLP-PCR and sequencing of the ITS1-5.8S-ITS2 and D1/D2 26S rDNA loci. The prevalence of spoilage yeasts in fine wines (11 %) was comparable to that reported in European and US wines, and significantly lower than that observed for local table wines (70 %). The majority of isolates belonged to Brettanomyces bruxelliensis, followed by Pichia guillermondii, and more rarely Candida wickerhamii and Trigonopsis cantarelli. The Brettanomyces isolates varied greatly in off-flavor production, displayed ethanol tolerance (>10 % by volume), tolerated sulfite (≥0.68 mg/l mSO2), and 39 % of them grew on ethanol as sole carbon source. We discuss the causes and consequences of spoilage yeasts in relation to the Brazilian wine industry.

Rapid identification of Saccharomyces eubayanus and its hybrids.

The Saccharomyces species Saccharomyces eubayanus was recently discovered in Patagonia. Genomic, genetic and phylogenetic data all suggest that it is one of the two parents of the hybrid yeast S. pastorianus (S. cerevisiae being the other). Saccharomyces eubayanus genomes can also be found in strains of the hybrid species S. bayanus. Here, we describe a novel pair of PCR primers targeted against the S. eubayanus FSY1 gene that will specifically detect S. eubayanus and hybrids containing this allele of the gene. The primer pair can be used to identify the species using a rapid, inexpensive colony PCR method suitable for a preliminary identification of wild isolates.

Reconstruction of the evolutionary history of Saccharomyces cerevisiae x S. kudriavzevii hybrids based on multilocus sequence analysis.

In recent years, interspecific hybridization and introgression are increasingly recognized as significant events in the evolution of Saccharomyces yeasts. These mechanisms have probably been involved in the origin of novel yeast genotypes and phenotypes, which in due course were to colonize and predominate in the new fermentative environments created by human manipulation. The particular conditions in which hybrids arose are still unknown, as well as the number of possible hybridization events that generated the whole set of natural hybrids described in the literature during recent years. In this study, we could infer at least six different hybridization events that originated a set of 26 S. cerevisiae x S. kudriavzevii hybrids isolated from both fermentative and non-fermentative environments. Different wine S. cerevisiae strains and European S. kudriavzevii strains were probably involved in the hybridization events according to gene sequence information, as well as from previous data on their genome composition and ploidy. Finally, we postulate that these hybrids may have originated after the introduction of vine growing and winemaking practices by the Romans to the present Northern vine-growing limits and spread during the expansion of improved viticulture and enology practices that occurred during the Late Middle Ages.

Kefir grains as a starter for whey fermentation at different temperatures: chemical and microbiological characterisation.

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

Efecto de diámetro de esfera y densidad celular en la producción de etanol con levadura inmovilizada en alginato / Effect of pellet diameter and cell density on ethanol production with alginate immobilized yeast

Las células inmovilizadas tienen aplicación potencial en la producción de biocombustibles posibilitando la reutilización de biomasa, el empleo de diversas configuraciones de reactores y sistemas de cultivo, el manejo de altas densidades celulares alcanzando altas productividades volumétricas, y la simplificación de operaciones de procesamiento de salida. El objetivo del presente estudio fue evaluar la influencia del diámetro de las perlas y la densidad celular en la producción de etanol con Saccharomyces uvarum inmovilizada en alginato al 2% (p/v). Para ello se evaluaron tres diámetros de perlas de 2, 2,5 y 3 mm. Las células inmovilizadas fueron cultivadas en medio con 12% (p/v) de glucosa en biorreactores de columna sin agitación a 28 ºC, y se operaron cuatro lotes consecutivos de 48 horas cada uno. En cada lote se cuantificó el consumo de glucosa y se determinó la cantidad de etanol producido. Los rendimientos máximos de etanol para las esferas de 2, 2,5 y 3 mm de diámetro fueron 81, 83 y 97% del rendimiento teórico. La máxima productividad volumétrica de etanol fue 1,2 g/L-1/h-1 con un consumo de glucosa de 99,8% al término del lote, correspondiente a las columnas con perlas de 3 mm y con una producción de 0,017 g de etanol por esfera. La producción de etanol acumulada en cada sistema fue 178, 189 y 200 g/L-1 para 2, 2,5 y 3 mm respectivamente, encontrándose una relación directa con el diámetro de perla e inversa respecto a la densidad celular. Los rendimientos de etanol obtenidos son superiores a los reportados para la misma especie.

Immobilized cells have a potential use in biofuel production. They also allow re-using biomass, using diverse reactor configurations and culture systems, handling high cell densities to obtain high volumetric productivities and to simplify the downstream processing. The purpose of this work was to evaluate the influence of bead diameter and cell density on ethanol production using immobilized Saccharomyces uvarum in 2% (w/v) alginate. For that, three bead diameters (2, 2.5 and 3 mm) were evaluated. Immobilized cells were cultured on a 12% (w/v) glucose medium in column bioreactors without agitation at 28 °C for four 48 h–repeated batches. For each batch, both glucose consumption and ethanol produced were measured. Maximum yields for 2, 2.5 and 3 mm bead diameters were 81, 83 and 97% of theoretical yield. Maximum volumetric productivity of ethanol was 1.2 g/L-1/h-1 with 99.8% glucose consumption at the end of the batch, corresponding to the 3 mm bead diameter and the ethanol production per bead was 0.017 g. Accumulated ethanol production for each system was 178, 189 and 200 g/L-1 for 2, 2.5 y 3 mm bead diameter, respectively, being this directly related to bead diameter and inversely related to cell density. Ethanol yields were higher than those reported for the same species.

Preservation of probiotic strains isolated from kefir by spray drying.

AIMS: This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage. METHODS AND RESULTS: Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk (D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica. The three micro-organisms studied varied considerably in their ability to survive to spray drying processes. Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased. CONCLUSIONS: Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures. SIGNIFICANCE AND IMPACT OF STUDY: It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

Probióticos são definidos como microrganismos (bactérias e leveduras) que exercem um efeito benéfico na saúde do hospedeiro quando ingeridos em quantidades adequadas. A seleção desses agentes bioterapêuticos normalmente é feita por testes in vitro simulando o ambiente gastrointestinal que determina a capacidade de sobrevivência no trato digestivo. Neste trabalho, a possibilidade de extrapolação dos dados obtidos nos testes in vitro para as condições in vivo foi estudada utilizando cinco linhagens de Saccharomyces cerevisiae isoladas da floresta Atlântica brasileira. O conteúdo de trealose e a sobrevivência após a exposição a diversos estresses fisiológicos geralmente encontrados no trato gastrointestinal de humanos foram determinados para as cinco linhagens e os resultados comparados com a Saccharomyces boulardii, um probiótico conhecido. Esses resultados foram completados com a capacidade de colonização do trato gastrointestinal de camundongos gnotobióticos pelas leveduras. Pelos resultados obtidos, concluimos que os testes in vitro não são confirmados pelos ensaios in vivo, indicando que essa extrapolação não pode sempre ser feita.

Study of flocculent yeast performance in tower reactors for bioethanol production in a continuous fermentation process with no cell recycling.

The purpose of this study was to assess the retention ability of 12 different Saccharomyces sp. yeast strains with flocculent characteristics when inoculated in a continuous ethanol fermentation process. The system was comprised of two reactors connected in series with no cell recycling. The feeding substrate used was a synthetic medium containing glucose. The parameters assessed were total reducing sugars of the feeding substrate, total reducing sugars and ethanol at the outlet of the first and second reactors and quantification and classification of yeast population in the two reactors. The system reached yield levels of 83.53% of theoretical yield with a maximum total reducing sugars conversion of 92.68%. The conversion in this system was lower than expected. The dominant yeast in the process in both reactors, contrary to expectation, was the Saccharomyces CP6 strain which was unable to form pellets in spite of its flocculate growth.

Diversity of Saccharomyces strains on grapes and winery surfaces: analysis of their contribution to fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years.

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

Chemical and microbiological characterisation of kefir grains.

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.

Levaduras aisladas de mostos de uva en la zona de Oltrepo Pavese, Italia, y su sensibilidad in vitro frente a compuestos de cobre y zinc / Yeast isolated from musts of grapes in Oltrepó Pavese, Italy, and their in vitro sensibility to zinc and copper compounds

Se investigó durante el periodo 1989-90 en la región de Oltrepó Pavese (Italia), la micota de los mostos provenientes de uvas negras y blancas. De un total de 28 especies levaduriformes aisladas, incluidas en 10 géneros, Saccharomyces, fué el género dominante con las especies S.italicus y S.cerevisiae. Tambien se detectaron frecuentemente levaduras anascosporógenas, tales como Candida valida, Torulopsis (C) holmii y Kloeckera apiculata. Se efectuaron estudios de sensibiliadad in vitro frente a compuestos de CuSO4, ya sea en cepas de S. cerevisiae aisladas de los mostos, como de Alternaria, Botrytis, Cladosporium y Sporobolomyces, aisladas del filoplano de la uva. Las concentraciones de CuSO4 a 0.007M y a 0,062M, fueron fúngicidas para S.cerevisiae y Sporobolomyces roseus respectivamente