Facultad de Ciencias Agrarias

La biodiversidad como indicador de sustentabilidad en humedales "mallines" de la provincia de Neuquén. Fernández, C.; Aramayo, V.; Elosegui, F.; Smith, P.; Gandullo, R. Cátedra de Botánica Agrícola Sistemática, Facultad de Ciencias Agrarias, Universidad Nacional del Comahue. Ruta 151 Km 12,5. CC 85. Cinco Saltos,

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.

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.

Aims: To investigate whether the presence of Pichia guilliermondii impacts on the production of volatile phenols from mixed wine fermentations with Dekkera bruxellensis and Saccharomyces cerevisiae. Methods and Results: Four inoculation strategies were performed in small-scale fermentations involving P. guilliermondii, D. bruxellensis and S. cerevisiae using Syrah grape juice supplemented with 100 mg l )1 of p-coumaric acid. High pressure liquid chromatography was used for the quantification or volatile phenols. Significant high levels of 4-ethylphenol and 4-ethylguaicol (720 and 545 lg l )1 , respectively), as well as the highest levels of 4-vinylphenol (>4500 lg l )1 ), were observed when P. guilliermondii species was inoculated from the beginning of the fermentation. Conclusions: The metabolic interaction occurring between the high vinylphenol producer species P. guilliermondii and D. bruxellensis exhibiting a high vinylphenol reductase activity resulted in an increased production of volatile phenols in wine. Significance and Impact of the Study: Pichia guilliermondii must be considered a very important spoilage yeast in the wine industry capable of producing large amounts of volatile phenols.

Aims: Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic acids, precursors of these phenolic compounds has been described in D. bruxellensis, no such pathway has been described in P. guilliermondii. Methods and Results: A molecular and physiological characterization of 14 D. bruxellensis and 15 P. guilliermondii phenol-producing strains was carried out. Both p-coumarate decarboxylase (CD) and vinyl reductase (VR) activities, responsible for the production of volatile phenols, were quantified and the production of 4-vinylphenol and 4-ethylphenol were measured. All D. bruxellensis and some P. guilliermondii strains showed the two enzymatic activities, whilst 11 of the 15 strains of this latter species showed only CD activity and did not produce 4-EP in the assay conditions. Furthermore, PCR products obtained with degenerated primers showed a low homology with the sequence of the gene for a phenyl acrylic acid decarboxylase activity described in Saccharomyces cerevisiae. Conclusions: D. bruxellensis and P. guilliermondii may share a similar metabolic pathway for the degradation of cinnamic acids. Significance and Impact of the Study: This is the first work that analyses the CD and VR activities in P. guilliermondii, and the results suggest that within this species, there are differences in the metabolization of cinnamic acids.

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.

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin — but not other cell wall polysaccharides — prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

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