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Maturano Y.P.,Institute Biotecnologia | Nally M.C.,Institute Biotecnologia | Toro M.E.,Institute Biotecnologia | de Figueroa L.I.C.,PROIMI | And 3 more authors.
World Journal of Microbiology and Biotechnology | Year: 2012

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae. Samples were periodically extracted during vinification, seeded on Petri dishes and incubated at 25 and 37 °C; the latter temperature was assayed for possible inactivation of killer toxin production. Colonies developed under the described conditions were randomly transferred to killer phenotype detection medium. Significant differences in the killer/sensitive ratio were observed between both incubation temperatures in all microvinifications. These results suggest that 37 °C seems a better option to determine the biomass of sensitive yeasts, in order to avoid underestimation of sensitive cells in the presence of killer yeasts during fermentations. Incubation at a toxin-inhibiting temperature clearly showed the real ratio of killer to sensitive cells in fermentation systems. © 2012 Springer Science+Business Media B.V.


Maturano Y.P.,Institute Biotecnologia | Rodriguez Assaf L.A.,Institute Biotecnologia | Toro M.E.,Institute Biotecnologia | Nally M.C.,Institute Biotecnologia | And 5 more authors.
International Journal of Food Microbiology | Year: 2012

Saccharomyces and non- Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non- Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non- Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non- Saccharomyces species survived during 15-18. days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity β-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non- Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for β-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments. © 2012 Elsevier B.V.


Nally M.C.,National University of San Juan | Pesce V.M.,National University of San Juan | Maturano Y.P.,National University of San Juan | Toro M.E.,National University of San Juan | And 3 more authors.
Postharvest Biology and Technology | Year: 2013

Sour rot is an important disease of grapes caused by an etiologic complex of microorganisms in which filamentous fungi play a key role. Yeasts are used for biocontrol of pathogenic filamentous fungi on fruits. The major objective of this study was to assess in vivo on detached berries the effect of viticultural yeasts on phytopathogenic fungi involved in grape sour rot. Yeasts that were found to be effective in vivo against the fungi were assayed for their possible pathogenicity in humans: growth at 42. °C, pseudohyphal formation, adhesion, and phospholipase and protease activity. A total of 234 yeasts belonging to 14 genera were assayed against the following pathogens: Aspergillus caelatus, Aspergillus carbonarius, Aspergillus terreus, Aspergillus versicolor, Fusarium oxysporum, Penicillium comune, Rhizopus stolonifer and Ulocladium sp. Forty-three (16 Saccharomyces and 27 non- Saccharomyces) showed antagonistic properties against some of the fungi assayed in grapes at 25. °C. Yeast isolates determined as biocontrol agents under in vivo conditions were isolated from fermenting musts (35), viticultural soils (6) and grape berries (2). Twenty biocontrol agents did not show phenotypical characteristics associated with pathogenicity in humans. © 2013 Elsevier B.V.


Maturano Y.P.,Institute Biotecnologia Unsj | Assof M.,Instituto Nacional de Tecnologia Agropecuaria | Fabani M.P.,Institute Biotecnologia Unsj | Nally M.C.,Institute Biotecnologia Unsj | And 5 more authors.
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2015

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4–5 of the fermentation process, and highest populations were observed in MSD2 (10 % S. cerevisiae/90 % D. vanrijiae) and MSC1 (1 % S. cerevisiae/99 % C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation. © 2015, Springer International Publishing Switzerland.


Nally M.C.,National University of San Juan | Pesce V.M.,National University of San Juan | Maturano Y.P.,National University of San Juan | Munoz C.J.,National University of Cuyo | And 4 more authors.
Postharvest Biology and Technology | Year: 2012

Botrytis cinerea, the causal agent of gray mold, is an important disease of grapes. Yeasts are members of the epiphytic microbial community on surfaces of fruits and vegetables and because some yeasts inhibit fungi they are used as biocontrol agents. The major objective of the present work was to isolate yeasts from grapes, vineyard soil, and grape must and select them for their ability to prevent gray mold onset after harvest. Yeasts that were found effective against the fungus were also assayed for their possible pathogenicity in humans. Two antagonism experiments were performed to study the effect of yeasts on B. cinerea, an in vitro study with Czapeck Yeast Extract Agar and an in vivo study with grape berries at 2°C and 25°C; both experiments were conducted at different yeast concentrations (105, 106 and 107cfu/mL). Antagonists were subsequently assayed for their ability to colonize and grow in fruit wounds. The biocontrol yeasts were also examined for their possible pathogenicity in humans: phospholipase and proteolytic activity, growth at 37°C and 42°C, pseudohyphal formation and invasive growth. A total of 225 yeasts belonging to 41 species were isolated from must and grape berries and 65 of them, representing 15 species, exhibited in vitro inhibition of B. cinerea at 25°C. These 65 biocontrol yeasts were subsequently assayed in vivo and 16 of them (15 Saccharomyces cerevisiae and 1 Schizosaccharomyces pombe) showed antagonistic properties against B. cinerea at 25°C. Only one isolate (S. cerevisiae BSc68) was able to inhibit mycelial growth of B. cinerea on grape berries at both 2°C and 25°C. The biomass of this strain in grape wounds increased 221.5-fold at 25°C after 3d and 325.5-fold at 2°C after 10d of incubation. An increase in the concentration of certain yeasts significantly enhanced their antagonistic activity. All yeast isolates determined as biocontrol agents under in vivo conditions were isolated from fermenting musts. Twelve biocontrol agents (S. cerevisiae) revealed one or more phenotypical characteristics associated with pathogenicity in humans but none of them showed all characteristics together. The fact that there exist few reports on S. cerevisiae and none on Sch. pombe as biocontrol agents against B. cinerea makes our results even more relevant. © 2011 Elsevier B.V.


PubMed | Instituto Nacional de Tecnologia Agropecuaria, PROIMI and Institute Biotecnologia Unsj
Type: Journal Article | Journal: Antonie van Leeuwenhoek | Year: 2015

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as -glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. -Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of -glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.


Niccio J.N.,Federal University of Grande Dourados | Ucha M.A.,Federal University of Grande Dourados | Faccenda O.,State University of Mato Grosso do Sul | Guimares J.A.,EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária | Marinho C.F.,PROIMI
Florida Entomologist | Year: 2011

The frugivorous larvae of Tephritidae and Lonchaeidae are key pests of fruit trees and vegetable crops in Brazil and in many other South American countries. Their most important natural enemies are parasitoids of the families Braconidae and Figitidae (Hymenoptera). The aim of this study was to evaluate the incidence of parasitoids in larvae of fruit flies (Tephritidae) and frugivorous Lonchaeidae that infest several species of native and exotic fruit trees in the South Pantanal Region, Mato Grosso do Sul, Brazil. Ninety-two species of fruits from 36 families and 22 orders were sampled. From 11 species of host fruits, we obtained 11,197 larvae of Tephritoidea, and in some samples there occurred Braconidae, Figitidae or Pteromalidae parasitoids. The Braconidae totaled 99.45%, represented by 3 species: Doryctobracon areolatus (Szpligeti) 92.45%, Utetes anastrephae (Viereck) 6.17%, and Opius bellus (Gahan) with 0.82%. The Figitidae were represented by Lopheucoila anastrephae (Rohwer) (0.28%), and Pteromalidae by Spalangia endius (Walker) (0.28%). Lopheucoila anastrephae emerged from puparia of Neosilba spp. (Lonchaeidae) infesting pods of Inga laurina (Swartz) Willdenow. Doryctobracon areolatus was associated with 2 species of Anastrepha:A. rheediae Stone in Rheedia brasilensis Planchon & Triana and A. zenildae Zucchi in Sorocea sprucei saxicola (Hassler) C. C. Berg. In Ximenia americana L. 14% of the larvae of Anastrepha spp. were parasitized and D. areolatus reached more than 96% of total parasitism in this host fruit. The braconids were specific to Tephritidae, and the Figitidae species collected in this work were associated only with larvae of Neosilba spp. (Lonchaeidae). Copyright © 2011 BioOne All rights reserved.


Vazquez S.,University of Buenos Aires | Vazquez S.,CONICET | Nogales B.,University of the Balearic Islands | Ruberto L.,University of Buenos Aires | And 6 more authors.
International Biodeterioration and Biodegradation | Year: 2013

In Antarctica, the environmental conditions and the restrictions imposed by the Antarctic Treaty prevent inoculation with foreign bacteria. Therefore, our aim was to investigate native bacterial consortia which might serve to design bacterial formulas suitable for soil bioremediation processes at cold temperatures. Two bacterial consortia, M10 and J13, were isolated from diesel contaminated Antarctic soils. Their ability to use hydrocarbons was evaluated in vitro and by the detection of three catabolic genes (alkB, nahAc, xylE). Both consortia showed similar 16S rRNA gene profiles, suggesting the presence of the same phylotypes. Total 16S rDNA was cloned from M10 grown on diesel. Sixty clones were screened, grouped by restriction profiles of PCR-amplified inserts and sequenced. T-RFLP (Terminal-Restriction-Fragment-Length-Polymorphism) of clones showed that all phylotypes from the entire consortia were recovered. A culture-dependent approach was used to isolate M10 components able to utilise aliphatic and aromatic hydrocarbons. Pseudomonas, Stenotrophomonas, Pedobacter and Brevundimonas genera were detected. The combination of dependent and independent culture methods allowed elucidating the taxonomic composition of these native bacterial consortia. Further work will assess whether combining the isolates obtained as a defined mixed culture can enhance bioremediation of contaminated soils. © 2012.


Nieto-Penalver C.G.,PROIMI | Nieto-Penalver C.G.,National University of Tucuman | Bertini E.V.,PROIMI | De Figueroa L.I.C.,PROIMI | De Figueroa L.I.C.,National University of Tucuman
Archives of Microbiology | Year: 2012

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14- HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium. © Springer-Verlag 2012.


PubMed | National University of San Juan and PROIMI
Type: | Journal: International journal of food microbiology | Year: 2015

The aim of this study was to determine the putative modes of action of 59 viticultural yeasts (31 Saccharomyces and 28 non-Saccharomyces) that inhibited fungi isolated from sour and grey rot in grapes. Inhibition of fungal mycelial growth by metabolites, enzyme activities (laminarinases, chitinases), antifungal volatiles, competition for nutrients (siderophores, Niche Overlap Index (NOI)), inhibition of fungal spore germination and decreased germinal tube length and induction of resistance were assayed. Biofungicide yeasts were classified into antifungal patterns, according to their mechanisms of action. Thirty isolates presented at least two of the mechanisms assayed. We propose that inhibition of fungal mycelial growth by metabolites, laminarinases, competition for nutrients, inhibition of fungal spore germination and decreased germinal tube length, and antifungal volatiles by Saccharomyces and non-Saccharomyces viticultural yeasts is used as putative biocontrol mechanisms against phytopathogenic fungi. Twenty-four different antifungal patterns were identified. Siderophore production (N)and a combination of siderophore production and NOI>0.92 (M)were the most frequent antifungal patterns observed in the biofungicide yeasts assayed. Elucidation of these mechanisms could be useful for optimization of an inoculum formulation, resulting in a more consistent control of grey and sour rot with Saccharomyces and non-Saccharomyces biocontrol yeasts.

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