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ABSTRACT: Postharvest diseases cause considerable losses of harvested fruits during transportation
and storage. Many yeast species have been reported as good antagonists
against postharvest pear pathogens. In this work, we used a novel selection strategy
that involves the isolation of yeasts from washing fluids, showing biocontrol
activity against a regional Penicillium expansum strain (primary screening),
originally obtained from fruit wounds after long time storage at �1/08C. About
26 isolates representative of the 11 yeast species identified in the 27 selected
washing waters were chosen to be evaluated in a secondary screening against a
regional Botrytis cinerea strain on pear wounds. Among yeasts tested, 38% showed
complete control of P. expansum, but only 15% reduced the decay incidence of
B. cinerea to 60�80% at �1/08C. These results reveal that some of the yeasts found
can be biological alternatives to fungicides in the control of P. expansum and
B. cinerea infections. Based on the data obtained, our strategy seems to be much
more effective than the previously reported methods in obtaining successful
biocontrol agents.
Biocontrol Science and Technology 11/2012; 22(12):1465-1483. · 0.92 Impact Factor
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ABSTRACT: Interspecific hybrids between S. cerevisiae × S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae × S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids.
Comparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae × S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains.
Our data suggest that wine and beer S. cerevisiae × S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions.
BMC Genomics 08/2012; 13:407. · 4.07 Impact Factor
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ABSTRACT: In this paper, a study of the relationship between genetic patterns, obtained by the combination of mtDNA-RFLP and PCR-amplified inter-δ sequence DNA polymorphism analysis, and relevant enological phenotypic data (fermentative power, specific productivity, volatile and total acidity) was carried out on Argentinean Saccharomyces cerevisiae isolates from north Patagonia. The use of a powerful statistical tool, Generalized Procrustes analysis, allowed us to weigh the relationship for each isolate in particular, denoting a good enough degree of agreement between molecular and physiological data for most of the population analysed. The inclusion of a physiological feature, as the killer sensitivity biotype, within identification methods resulted in a higher degree of discrimination among isolates and in better correlation between both characterizations. The combined use of methods based on molecular polymorphisms and killer biotype could be applied so as not to miss any isolate with differential enological properties in selection protocols.
World Journal of Microbiology and Biotechnology 04/2012; 22(8):827-833. · 1.53 Impact Factor
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ABSTRACT: Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed
for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under
laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging
to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage
yeasts.
World Journal of Microbiology and Biotechnology 04/2012; 24(7):945-953. · 1.53 Impact Factor
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ABSTRACT: 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.
International journal of food microbiology 03/2012; 156(2):102-11. · 3.01 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(9):e45527. · 4.09 Impact Factor
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ABSTRACT: To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at -1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.
International journal of food microbiology 06/2011; 147(3):211-6. · 3.01 Impact Factor
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ABSTRACT: In order to detect spoilage yeast species in wines showing off-odors, different yeast isolation protocols were evaluated. Independently of the isolation method, only Saccharomyces cerevisiae and Pichia manshurica were detected. The spoilage capacity of P. manshurica regional isolates was evaluated in red wine and the production of volatile phenols was evidenced. To evaluate the possible source of contamination, yeasts from both grapes and cellar surfaces were obtained. Hanseniaspora uvarum and Zygoascus hellenicus were detected in both sound and damaged grapes from sunny areas. The most frequent species in cellar surfaces was Candida boidinii, Pichia membranifaciens and P. manshurica were detected in filters. The intra-specific genetic characterization of the P. manshurica isolates by mtDNA-RFLP demonstrated that the same strain was detected in both wine and filter. Most P. membranifaciens isolates produced 4-EP (maximum level of 1.895 mg/L) and particularly high levels of 4-EG (maximum level of 10.260 mg/L) were produced by P. manshurica isolates in synthetic wine-like medium. In this work the capacity of P. manshurica and P. membranifaciens species to produce volatile phenols was shown for the first time.
Food Microbiology 05/2011; 28(3):503-9. · 3.28 Impact Factor
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ABSTRACT: The use of selected Saccharomyces and non-Saccharomyces strains as mixed starters in winemaking would have advantages over the traditional spontaneous fermentation, producing wines with predictable and desirable characteristics. The aim of this study was to evaluate the impact of metabolic interactions between Patagonian indigenous Saccharomyces cerevisiae MMf9 and beta-glucosidase producer Candida pulcherrima V(6) strains on alcoholic fermentation behaviour and wine aroma Three inoculation strategies, simultaneous, sequential and final, were assayed at laboratory-scale fermentations using Muscat d'Alexandrie grape juice as substrate. The fermentation and yeast growth kinetics as well as the physicochemical and the sensory quality of wine were evaluated. Results evidenced that the sequential inoculation is the most adequate strategy of strains combination. The kinetic behaviour of sequential fermentation was similar to a successful spontaneous fermentation and its wine showed differential aromatic quality as evidenced through PC analysis using physicochemical and aromatic composition data. This wine presented the highest total concentration of higher alcohol, esters and terpenols and the strongest fruity and floral aroma.
International journal of food microbiology 03/2010; 138(1-2):19-25. · 3.01 Impact Factor
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ABSTRACT: Saccharomyces kudriavzevii, a yeast species described from a pair of strains isolated from decayed leaves in Japan, has recently been isolated from oak barks in Portugal. Some data suggest that these European S. kudriavzevii populations could be closely related to the S. kudriavzevii genetic background present in natural hybrids isolated from wines and beers in different regions of Europe. However, a more exhaustive study of European S. kudriavzevii natural populations is necessary to confirm this observation. In this study, new S. kudriavzevii isolates were recovered from oak trees in different areas in Spain, and identified and characterized according to their molecular and physiological features. The restriction analysis of 35 nuclear genes amplified by PCR demonstrated that all Spanish S. kudriavzevii isolates exhibit three gene alleles different from those present in the Japanese S. kudriavzevii type strain, but identical to the alleles previously reported for natural S. cerevisiaexS. kudriavzevii hybrids. These results clearly support the hypothesis of a European origin for these natural hybrids.
FEMS Yeast Research 02/2010; 10(4):412-21. · 2.40 Impact Factor
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ABSTRACT: Pichia guilliermondii can produce volatile phenols in the initial stages of wine fermentation; however, its response to different wine stress conditions has been poorly studied. In this work, we present an analysis of the response of 23 P. guilliermondii indigenous isolates to physical and chemical wine stress factors and to indigenous wine killer yeasts. Principal coordinates analysis (PCoA), based on data obtained from response patterns, was carried out to evaluate the relationships among the isolates. Major differences among the isolates were detected in media plates containing 8% ethanol and in those containing 280 g/L glucose. PCoA identified 3 clusters of isolates with different stress response patterns, indicating a relationship between the tolerance to these compounds and the origin of the isolates. Pichia guilliermondii isolates were sensitive to the toxins produced by the species Hanseniaspora uvarum, Metschnikowia pulcherrima, Wickerhamomyces anomala (ex Pichia anomala), and Pichia kluyveri, with a maximum level of sensitivity against W. anomala (91% on average). Those isolates obtained from fermenting must proved to be more resistant to killer yeasts than those obtained from grape surfaces. The combined evaluation of the response to physico-chemical and biological factors presented in this work could be a useful standard protocol for the evaluation of the potential spoilage capacity of yeasts in winemaking.
Canadian Journal of Microbiology 07/2009; 55(7):801-9. · 1.36 Impact Factor
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ABSTRACT: The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.
Journal of Industrial Microbiology and Biotechnology 09/2007; 34(8):539-46. · 2.73 Impact Factor
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ABSTRACT: A novel anamorphic yeast species belonging to the genus Candida has been isolated from cellar surfaces in North Patagonia. Morphological and physiological observation and phylogenetic analysis were performed. Pseudomycelium was plentifully produced. No sexual reproduction was observed. From sequence analysis of the 26S rDNA D1/D2 region, Candida bituminiphila and Zygoascus hellenicus were the closest species with 40 and 79 bp substitutions, respectively. C. bituminiphila differed physiologically from the novel species in its ability to assimilate sucrose and erythritol, in not fermenting any sugars, in growing without some vitamin compounds, and in growing at 40 degrees C. All these data support the hypothesis that the new yeast, named Candida patagonica, is a novel species related to C. bituminiphila. The type strain is UNCOMA 159.5 (= CECT 12029 = CBS 10443).
Antonie van Leeuwenhoek 08/2007; 92(1):77-81. · 2.09 Impact Factor
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ABSTRACT: In this work we evaluate the implantation capacity of the selected S. cerevisiae indigenous strain MMf9 and the quality of the produced wines in a traditional (T) and a modern (M) cellar with different ecological and technological characteristics in North Patagonia (Argentina). Red musts were fermented in 10,000 l vats using the indigenous strain MMf9 as well as the respective controls: a fermentation conducted with a foreign starter culture (BC strain) in M cellar and a natural fermentation in T cellar. Since commercial S. cerevisiae starters are always used for winemaking in M cellar and in order to compare the results, natural fermentations and fermentations conducted by the indigenous strain MMf9 were performed at pilot (200 l) scale in this cellar, concomitantly. Thirty indigenous yeasts were isolated at three stages of fermentation: initial, middle and end. The identification of the yeast biota associated to vinifications was carried out using ITS1-5.8S-ITS2 PCR-RFLP. The intra-specific variability of the S. cerevisiae populations was evaluated using mtDNA-RFLP analysis. Wines obtained from all fermentations were evaluated for their chemical and volatile composition and for their sensory characteristics. A higher capacity of implantation of the indigenous MMf9 strain was evidenced in the fermentation carried out in M cellar (80% at end stage) than the one carried out in T cellar (40%). This behaviour could indicate that each cellar differs in the diversity of S. cerevisiae strains associated to wine fermentations. Moreover a higher capacity of implantation of the native starter MMf9 with regard to the foreign (BC) one was also found in M cellar. The selected indigenous strain MMf9 was able to compete with the yeast biota naturally present in the must. Additionally, a higher rate of sugar consumption and a lower fermentation temperature were observed in vinifications conducted by MMf9 strain with regard to control fermentations, producing wines with favourable characteristics. Even when its implantation in T fermentation was lower than that observed in M one, we can conclude that the wine features from MMf9 fermentations were better than those from their respective controls. Therefore, MMf9 selected indigenous strain could be an interesting yeast starter culture in North Patagonian wines.
Journal of Industrial Microbiology and Biotechnology 03/2007; 34(2):139-49. · 2.73 Impact Factor
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ABSTRACT: The aim of this work was to characterize the indigenous wine Saccharomyces cerevisiae diversity within the Argentinean Patagonia. Two cellars with particular enological practices located in different winegrowing areas were selected and 112 indigenous S. cerevisiae isolates were obtained from spontaneous red wine fermentations carried out in them. Thirty-five and 19 patterns were distinguished among the total indigenous isolates using mtDNA-RFLP and killer biotype analysis, respectively. The combination of both typing techniques rendered a higher variability with 42 different patterns, i.e. 42 strains, evidencing a great diversity in S. cerevisiae populations associated with spontaneous red wine fermentations in Northwestern Patagonia. The analysis of the relatedness among strains using Principal Coordinates Analysis from combined data allowed the clustering of the strains into two populations significantly related to their origin fermentations. The combined use of the mtDNA-RFLP analysis together with the killer biotype method proved to be a powerful tool in the fingerprinting of the enological S. cerevisiae strains.
Antonie van Leeuwenhoek 02/2006; 89(1):147-56. · 2.09 Impact Factor
