Synthesis and regulation of flavor compounds derived from brewing yeast: Esters

Las levaduras, durante el proceso de elaboración de cerveza, producen más de 500 compuestos químicos; estos pueden impactar tanto negativa como positivamente en las características organolépticas de la cerveza. En los últimos años, y en particular gracias al avance de la biología molecular y la genómica, se han logrado progresos notables en el conocimiento de las bases moleculares y celulares de la síntesis y regulación de muchos de estos compuestos que inciden en lo que se denomina flavor (aroma y sabor) de la cerveza. Este artículo está enfocado en los ésteres responsables del aroma y el sabor floral y frutado de la cerveza. La formación de estos ésteres depende de diversas enzimas y de factores como la concentración de nutrientes presente en el mosto, la cantidad de oxígeno y dióxido de carbono disuelto, la temperatura de fermentación y, principalmente, la genética de la levadura utilizada. En esta revisión se brinda información de cómo se originan los ésteres y cómo los diferentes parámetros fermentativos impactan en las concentraciones finales de estos compuestos y en la calidad del producto terminado.During brewing process yeast produce more than 500 chemical compounds that can negatively and positively impact beer at the organoleptic level. In recent years, and particularly thanks to the advancement of molecular biology and genomics, there has been considerable progress in our understanding about the molecular and cellular basis of the synthesis and regulation of many of these flavor compounds. This article focuses on esters, responsible for the floral and fruity beer flavor. Its formation depends on various enzymes and factors such as the concentration of wort nutrients, the amount of dissolved oxygen and carbon dioxide, fermentation temperature and mainly the genetics of the yeast used. We provide information about how the esters originate and how is the impact of different fermentative parameters on the final concentrations of these compounds and the quality of the end product.Fil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales.; Argentin

Non-conventional yeasts as tools for innovation and differentiation in brewing

En la elaboración de cerveza las levaduras cumplen un rol fundamental. Además de ser responsables de llevar a cabo la fermentación, generando principalmente etanol y dióxido de carbono, también son capaces de metabolizar y producir numerosos compuestos orgánicos que tienen un impacto determinante en el aroma y el sabor final de la cerveza. Las especies Saccharomyces cerevisiae y Saccharomyces pastorianus son utilizadas tradicionalmente para la producción de cervezas ale y lager, respectivamente. No obstante, el continuo crecimiento en el mercado de la cerveza artesanal y el aumento del interés y las exigencias de los consumidores han orientado los esfuerzos hacia la producción de cervezas diferenciales e innovadoras. En este punto, las levaduras no convencionales han cobrado gran protagonismo como herramientas para el desarrollo de nuevos productos. En el presente trabajo se describe y desarrolla la potencial aplicación en el sector cervecero de diferentes especies de levaduras no convencionales pertenecientes a los géneros Brettanomyces, Torulaspora, Lachancea, Wickerhamomyces, Pichia y Mrakia, entre otras, así como también levaduras del género Saccharomyces distintas a las levaduras cerveceras tradicionales. Se detallan las condiciones de fermentación de este tipo de levaduras y su capacidad de asimilar y metabolizar diferentes componentes del mosto y de aportar características particulares al producto final. Este trabajo provee el estado del arte sobre levaduras no convencionales, lo que resulta de gran relevancia para evaluar su aplicación en la producción de cervezas artesanales con características sensoriales diferenciales, cervezas bajas en calorías, cervezas sin alcohol y cervezas funcionales.Yeasts play a crucial role in brewing. During fermentation, besides ethanol and carbon dioxide, yeasts produce a considerable number of organic compounds, which are essential for beer flavor. In particular, Saccharomyces cerevisiae and Saccharomyces pastorianus are traditionally used in the production of ale and lager beers, respectively. Nowadays, the continuous growth of the craft beer market motivates the production of differential and innovative beers; leading specialists and brewers focus on non-conventional yeasts as tools for new product development. In this work, we describe the potential application of non-conventional yeast species such as those of the genera Brettanomyces, Torulaspora, Lachancea, Wickerhamomyces, Pichia and Mrakia in the craft brewing industry, as well as non-traditional brewing yeasts of the Saccharomyces genus. Furthermore, the fermentation conditions of these non-conventional yeasts are discussed, along with their abilities to assimilate and metabolize diverse wort components providing differential characteristics to the final product. In summary, we present a comprehensive review of the state-of-the-art of non-conventional yeasts, which is highly relevant for their application in the production of novel craft beers including flavored beers, non-alcoholic beers, low-calorie beers and functional beers.Fil: Burini, Julieta Amalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Eizaguirre, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; Argentin

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31–43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).Centro de Investigación y Desarrollo en Fermentaciones Industriale

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31–43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).Centro de Investigación y Desarrollo en Fermentaciones Industriale

Análisis metagenómico de la biodegradación de hidrocarburos aromáticos policíclicos en sedimentos marinos subantárticos

Los hidrocarburos aromáticos policíclicos (HAPs), algunos de ellos tóxicos y mutagénicos, son compuestos hidrofóbicos altamente persistentes en el ambiente. Estudios previos realizados en la región Costera Patagónica, particularmente en un sitio crónicamente contaminado con hidrocarburos en Bahía Ushuaia, demostraron un alto potencial para la degradación microbiana de estos compuestos. Sin embargo, estos estudios sólo se orientaron al análisis de fragmentos de genes codificantes para enzimas oxigenasas de anillos aromáticos involucradas en el primer paso de la degración de HAPs. Con el objetivo de conocer la relevancia ecológica de los microorganismos con capacidad de degradar HAPs en este ambiente y revelar los mecanismos que utilizan para ello, en el presente trabajo se emplearon diferentes técnicas independientes del cultivo de microorganismos. La construcción y análisis de una biblioteca metagenómica en fósmidos permitió identificar fragmentos de ADN pertenecientes a microorganismos del phylum Proteobacteria, dos de los cuales podrían provenir de miembros estables de la comunidad microbiana altamente especializados en la degradación de compuestos aromáticos. Asimismo, se detectaron genes completos codificantes para enzimas de la vía alta de degradación de HAPs y de la vía baja de degradación por gentisato y catecol, sugiriendo que las poblaciones degradadoras de los sedimentos intermareales de Bahía Ushuaia utilizan diferentes rutas degradativas para el metabolismo de estos compuestos. Entre estos genes, aquellos codificantes para enzimas oxigenasas de clase A y extradiol dioxigenasas representan potenciales marcadores funcionales para el estudio de la abundancia y dinámica de los microorganismos degradadores de hidrocarburos aromáticos.Facultad de Ciencias Exacta

Selection of Saccharomyces eubayanus strains from Patagonia (Argentina) with brewing potential and performance in the craft beer industry

Saccharomyces eubayanus is a genetically diverse yeast isolated for the first time in Patagonia (Argentina), which has received special attention in research and brewing applications for being identified as one of the parents of the lager-brewing yeast. In the search for different and innovative products, the beer market has focused part of its efforts on innovation through the application of non-conventional and/or wild yeasts, as is the case of S. eubayanus. This study aimed to characterize the brewing potential of S. eubayanus and select the most promising strains in terms of beer production. Fifty S. eubayanus strains from different Patagonian locations were analyzed in small-scale fermentations. Based on its genetic characteristics and fermentative behavior a candidate from each of the five geographically structured subpopulations of S. eubayanus was selected for lab-scale fermentations. Fermentation performance, sugar consumption and production of aroma compounds were evaluated, and one strain was selected to assess for the first time the behavior of this wild yeast at a craft brewing scale. In these conditions, a decrease in fermentation capacity was observed, showing that S. eubayanus requires different handling compared to traditional brewing yeasts and set the basis for further fundamental and applied research at larger productive scales. This work supports and encourages the use of native non-conventional yeast in brewing for the production of innovative beers with greater product differentiation, giving rise to the possibility of granting a regional character to the products.Fil: Burini, Julieta Amalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Eizaguirre, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; Argentin

Substrate specificities of aromatic ring-hydroxylating oxygenases of an uncultured gammaproteobacterium from chronically-polluted subantarctic sediments

Aromatic ring-hydroxylating oxygenases (RHOs) are multicomponent enzymes that catalyze the vicinal hydroxylation of aromatic rings to produce cis-dihydrodiols, a key step in the aerobic biodegradation of aromatic compounds. In this work, we describe the characterization of three RHOs of an uncultured gammaproteobacterium from chronically polluted Subantarctic intertidal sediments. Sequences encoding the α and β subunits of these RHOs, classified as class A type III, and one set of the corresponding electron transfer partners, were identified in a 34 Kb fragment from a metagenomic fosmid library. Structural modeling and docking analyses suggested that the active sites of these enzymes accommodated different set of substrates. The three enzymes, including the electron transfer components, were expressed in Escherichia coli and purified. The enzyme with the largest predicted catalytic pocket and wider diameter channels presented remarkably relaxed substrate specificity, including 2?4 ring PAHs (phenanthrene, pyrene, fluoranthene and naphthalene). The other two RHOs were stricter in their substrate specificity, and hydroxylated biphenyl and naphthalene more efficiently. These results suggest the evolution of compatible RHO enzymes within a single catabolic gene cluster in this microorganism. This work increases our understanding of the PAH-degrading capabilities of uncultured bacteria from cold coastal environments.Fil: Musumeci, Matias Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Lozada, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Ferreira, Flavia Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Entre Ríos. Universidad Nacional de Entre Ríos. Centro de Investigaciones y Transferencia de Entre Ríos; ArgentinaFil: Dionisi, Hebe Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentin

Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments

We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes.Fil: Guibert, Lilian Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Borglin, Sharon. Lawrence Berkeley National Laboratory; Estados UnidosFil: Jansson, Janet K.. Pacific Northwest National Laboratory; Estados UnidosFil: Dionisi, Hebe Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; ArgentinaFil: Lozada, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentin

Aromatic hydrocarbon degradation genes from chronically polluted subantarctic marine sediments

With the goal of identifying functional biomarkers associated with polycyclic aromatic hydrocarbon (PAH) biodegradation in chronically-polluted Subantarctic marine sediments, we amplified from environmental metagenomic DNA, cloned and sequenced gene fragments encoding dioxygenase enzymes associated with the biodegradation of these compounds. In this chapter, we describe and classify 22 different groups of dioxygenase genes identified in this environment, most of them presenting low to intermediate sequence similarities with previously described sequences (33 to 68% identity at the amino acid level). In addition, we show that two of these novel genes were present at high abundances in the sediments, suggesting that populations carrying these genes could have an important ecological role in the microbial community.Fil: Dionisi, Hebe Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; ArgentinaFil: Lozada, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; ArgentinaFil: Marcos, Magalí Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; ArgentinaFil: Di Marzio, Walter Dario. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentin

Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67 % amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16 S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes. © 2012 Springer Science+Business Media, LLC.Fil: Guibert, Lilian Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Loviso, Claudia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Marcos, Magalí Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Commendatore, Marta Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Dionisi, Hebe Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Lozada, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentin