Dissemination and survival of commercial wine yeast in the vineyard: a large-scale, three years study

The use of commercial wine yeast strains as starters has been extensively generalised over the past two decades. In this study, a large-scale sampling plan was devised over a period of three years in six different vineyards to evaluate the dynamics and survival of industrial yeast strains in the vineyard. A total of 198 grape samples were collected at various distances from the wineries, before and after harvest, and yeast strains isolated after spontaneous fermentation were subsequently identified by molecular methods. Among 3780 yeast strains identified, 296 isolates had a genetic profile identical to that of commercial yeast strains. For a large majority (94%), these strains were recovered at very close proximity to the winery (10-200m). Commercial strains were mostly found in the post harvest samples, reflecting immediate dissemination. Analysis of population variations from year to year indicated that permanent implantation of commercial strains in the vineyard did not occur, but instead that these strains were subject to natural fluctuations of periodical appearance/disappearance like autochthonous strains. Our data show that dissemination of commercial yeast in the vineyard is restricted to short distances and limited periods of times and is largely favoured by the presence of water runoff.ENOSAFE (Nº 762, Programa AGRO, medida 8.1) and the grant nº 657 C2 from the cooperation agreement between the Portuguese Institute for International Scientific and Technological Cooperation (ICCTI) and the French Embassy in Lisbon and the Marie Curie Fellowship of the European Community programme of Quality of Life under Contract QLK4-CT-2001-51873

Survey of molecular methods for the typing of wine yeast strains

A survey of the genetic polymorphisms produced by distinct methods was performed in 23 commercial winery yeast strains. The microsatellite typing, using 6 different loci, an optimized interdelta sequence analysis and RFLP of mitochondrial DNA generated by the enzyme Hinf I had the same discriminatory power: among the 23 commercial yeast strains, 21 distinct patterns were obtained. Karyotype analysis originated 22 patterns, thereby allowing the discrimination of one of the three strains that were not distinguished by the other methods. Due to the equivalence of the results obtained in this survey, any of the methods can be applied at the industrial scale.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/BIO/38106/2001 (Eixo 2, Medida 2.3, QCAIII - FEDER).Instituto de Cooperação Científica e Tecnológica Internacional (ICCTI) - grant nº 657 C2.Comunidade Europeia (UE) - contract number QLK4-CT-2001-51873

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, all revealed to belong to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.ENOSAFE - (Nº 762, programa AGRO, 657 C2

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects – the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer’s yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculent Saccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI - BD11306/97

Association between Grape Yeast Communities and the Vineyard Ecosystems

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Acores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viti-cultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

Genetic Diversity and Population Structure of Saccharomyces cerevisiae Strains Isolated from Different Grape Varieties and Winemaking Regions

We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8–43 strains per fermentation) was associated with high percentage (60–100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0–40%) corresponded to a rather low strain diversity (1–10 strains per fermentation)

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains

Characterization of fusion products from protoplasts of yeasts and their segregants by electrophoretic karyotyping and RAPD

In order to characterize fusion products from yeast protoplasts and their segregants, with important features to the wine making industry, electrophoretic karyotyping and RAPD (Random Amplified Polymorphic DNA) were utilized. Electrophoretic karyotyping was performed by the CHEF ("contour-clamped homogeneous electric field electrophoresis") method, which allowed the detection of chromosomal band complementation in fusion products and the presence of patterns of both parental and intermediary strains in segregants. By utilizing two primers, an amplification pattern of DNA fragments was obtained. While fusion products (diploid) showed a pattern of complementary bands, segregants showed bands of either parental strains or even intermediary bands<br>Com o objetivo de caracterizar os produtos de fusão de protoplastos de leveduras com características de importância para a indústria vinícola e seus segregantes, foram empregadas as técnicas de separação de bandas cromossômicas por eletroforese e de RAPD (amplificação ao acaso de DNA polimórfico). O cariótipo eletroforético foi realizado pelo método CHEF ("contour-clamped homogeneous eletric field eletrophoresis"), constatando-se a complementação de bandas cromossômicas no produto de fusão e padrões de ambos os parentais e padrões intermediários nos segregantes. A análise do padrão de amplificação dos fragmentos de DNA com dois primers evidenciou um padrão de bandas complementares nos produtos de fusão (diplóide) e padrão de bandas de um e de outro parental ou mesmo bandas intermediárias nos segregantes

Coordination des soins ville-hôpital en oncologie : une étude qualitative auprès de médecins généralistes et d'oncologues pour déterminer des pistes d'amélioration

CONTEXTE : le cancer est une pathologie fréquente avec 382 000 nouveaux cas en 2018. La prise en charge ambulatoire des cancers se développe notamment avec l’utilisation croissante des thérapies orales, nécessitant un relai dans le suivi. Ces changements entrainent une nécessaire coopération entre le médecin traitant et l’oncologue afin d’assurer le suivi du patient pendant la phase de traitement et au-delà avec la prise en charge de l’après-cancer.OBJECTIF : ce travail a pour objectif d’évaluer les difficultés rencontrées et d’envisager les possibilités et propositions d’amélioration pour optimiser la prise en charge des patients en cancérologie. L’objectif secondaire est d’étudier dans une moindre mesure la coopération entre tous les intervenants médicaux et paramédicaux pendant la phase de traitement.MATÉRIELS ET MÉTHODES : pour cette étude, il a été réalisé une étude qualitative auprès de treize médecins (9 généralistes et 4 oncologues) à l’aide d’entretiens semi-dirigés. La grille d’entretien était composée de 7 items.RÉSULTATS : l’analyse des entretiens a permis de décrire les motifs de recours aux soins ambulatoires des patients et les difficultés en résultant lors de la prise en charge. Il est ressorti une nécessité de meilleure communication bilatérale entre oncologues et médecins généralistes, la nécessité d’identifier le parcours de soins du patient, son référent et demieux appréhender les effets secondaires des traitements. Enfin, il a été montré le rôle central du médecin traitant dans ses différents champs de compétence pour la prise en charge des patients atteints de cancer.DISCUSSION : cette étude s’inscrit dans un projet impliquant différents professionnels libéraux pour améliorer leur coopération. Elle permet d’ouvrir des perspectives pour optimiser la prise en charge ambulatoire des patients en cancérologie. En ce sens, nous émettons des suggestions qui répondent de façon pratique aux difficultés rencontrées