Identificación y caracterización de levaduras vínicas, un mundo por descubrir

La elaboración de vinos finos consta de dos fases fundamentales, la fermentación y la maduración biológica. Durante la fase de fermentación, Sacharomyces cerevisiae es el principal responsable de la transformación del azúcar en alcohol. En la fase de maduración, son también levaduras de la especie Sacharomyces cerevisiae, pero un subgrupo muy especial con capacidad de formar un biofilm en la superficie del vino que, soportando elevadas concentraciones de alcohol, confieren al vino fino sus característicos aromas y sabores. Estas levaduras se conocen como levaduras de flor. Tras identificar y cuantificar mediante técnicas moleculares las poblaciones de levaduras presentes durante la elaboración de vinos fino en cinco bodegas de la D.O. Montilla Moriles hemos caracterizado su papel en la maduración del vino y seleccionado aquellas con las características más destacadas para la elaboración de estos caldosUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Localización y caracterización de los genes implicados en la síntesis de la bacteriocina Lcn972 en aislados silvestres de Lactococcus Lactis

Motivación: Las bacteriocinas son proteínas sintetizadas por bacterias con actividad antimicrobiana frente a especies relacionadas con la cepa productora. La bacteriocina Lactococina 972 (Lcn972) inhibe la formación del septum en células en división. Su modo de acción se basa en la inhibición de la biosíntesis de péptidoglicano uniéndose de manera específica al lípido II (Martínez et al., 2000).El espectro de actividad es bastante específico, afectando sólo a Lactococcus en división.Los genes que codifican para la síntesis de Lcn 972 así como para la resistencia a la misma se encuentran en un plásmido, pBL1, de 10.9 kD. Lcn 972 es producida por Lactococcus Lactis IPLA 972 (Martínez et al., 1995) aunque recientemente, se han aislados nuevos productores (Alegría et al., 2010). El objetivo de este trabajo es la identificación y caracterización de los genes implicados en la síntesis de Lcn 972 en los nuevos productores aislados.Métodos: Para localizar el gen estructural, se llevó a cabo un análisis del DNA plasmídico de los nuevos productores por hibridación DNA-DNA con el gen estructural de Lcn 972 y, tras el aislamiento de los plásmidos, se secuenciaron. La actividad de la bacteriocina de los diferentes productores fue examinada por el test de difusión en agar y su producción fue cuantificada mediante el método ELISA.Resultados: En 4 de las 5 nuevas cepas productoras, los genes para la producción e inmunidad de Lcn 972 se localizaron en  un plásmido diferente, de mayor tamaño que pBL1. Tras la secuenciación, se comprobó que tanto el gen estructural como la región promotora permanecen idénticas en todas las cepas productoras. La diferencia entre ambos plásmidos radica en la existencia de una secuencia de insercción en el de mayor tamaño. En una de las cinco nuevas cepas productoras, dichos genes se localizaron en pBL1. No se encontraron diferencias significativas en la producción ni en la actividad de la bacteriocina entre ambos plásmidos.Conclusiones: Existen nuevos productores de Lcn 972. En uno de ellos, los genes para la producción e inmunidad de Lcn 972 se encuentran en pBL1. En otras cepas aisladas productoras de Lcn 972, los genes responsables se encuentran en un plásmido de mayor tamaño a pBL1. No hay diferencias contundentes de producción y actividad entre los diferentes plásmido

Isolation, identification and characterization of natural yeasts for brewing

In recent years, there has been a worldwide increasing interest in craft beers and in microbreweries. Producers and consumers continuously require new beers with new aroma, flavors, alcohol or sugar content, etc. that can be in part conferred by using other yeast instead of the ones supplied by the yeast companies [1]. The objective of this study is to carry out an analysis on the Saccharomyces biodiversity in Andalusia, studying them at the genetic as well as at the phenotypic level. Thus yeasts with different properties to those currently used in the brewing industry could be found. With this, we would be able to create a catalogue of autochthonous yeasts with different features for microbreweries, so they could chose the one that suits their procedures or their beer best, taking into account the fermentation conditions and the characteristics that they can contribute to the final product [2]. For wild type yeast isolation, we have collected wine must from several wineries of Andalusia as well as fruits. Natural microbial population in these samples were incubated in grape or malt must at 16 or 22 ºC, taking samples at different times along the fermentation. Later, isolated colonies for different samples were grown and Saccharomyces strains were selected and analysed by PCR of microsatellite regions to distinguish different strains.  At this time ninety strains of S. cerevisiae have been identified with different genetic patterns. Now we will study them phenotypically in order to analyze their growth capacity in different conditions and sugar sources. After this, beer will be produced with selected strains to evaluate their effect on the organoleptic characteristics. At this point of the project, we can conclude that the results seem promising and that there are numerous yeasts present in nature in Andalusia that we can isolate and characterize to make them available to microbreweries. This means that they could use this autochthonous yeasts to make their craft beers without the need to buy commercial yeasts from abroad, thus providing more authenticity to its product

New strategies to find chromatin silencers in the pathogenic fungus Ustilago maydis

Ustilago maydis is a smut fungus that infects maize, causing tumors, stunted growth and consequently reduced yields leadingto economic losses [2]. A key aspect of the pathogenic development of U.maydis is the action of effectors, which are secretedvirulence factors with principal roles in plant defense suppression and host's metabolism alterations. Many genes encodingeffector proteins are grouped in silenced clusters in the genome highly induced during infection. It has been shown thatintroduction of resistance marker genes with high expression promoters in these clusters de-repress the surrounding region ofthe insertion point [3]. Consequently, it is suggested that these clusters are subjected to chromatin silencing. However, U. maydislacks the canonical factors involved in chromatin silencing. The main purpose of this project is to find regulators that control thesilencing state of these regions. To achieve this goal we are going to perform a screening in a U.maydis strain harboring anantibiotic resistant marker gene inside a silenced cluster. In order to do this strain, we decided to introduce in one of theseclusters an antibiotic marker that will be controlled by an endogenous promoter followed of a different resistance marker genewith a high expressed promoter that will disturb the silencing of the region of insertion. Once we obtained this strain, we restoredsilencing by removing the high expressed gene, which is flanked by two direct repeat sequences, expressing the flippaserecombinase [1]. We are currently performing the first steps of the mutagenesis assay using the recently generated strain

Effect of bioestimulant on the antioxidant system of Solanum Lycopersicum.

During vegetal growth, Oxidative stress is one of the main causes that affect plant development. Plant subjected to biotic or abiotic stress (drought, heat, UV light, salinity…) generate Reactive Oxygen Species (ROS) inside chloroplasts. ROS cause lipid peroxidation, DNA damage, protein denaturation, carbohydrate oxidation, pigment degradation and deterioration off enzymatic activity, even leading to plant death. To counteract the harmful effects of ROS species, plants have developed an antioxidant system that is based on scavenging free radicals generated by different stresses and eliminating them. This reduces cell and plant damage. Plant bioestimulant is any substance or microorganism applied to plants in order to improve nutritional efficiency, tolerance to abiotic stress and qualitative attributes of the crop, regardless of its nutrient content [3]. They are beneficial by triggering the production of plants defence metabolites. The active principles present in the bioestimulant extracts have great potential for the antioxidants effects. Among the main active ingredients polyphenols, polyalcohols and hydroxybenzoic acids have been described [2].The objective of this work is to analyse the effects of active principles present in four different Biostimulants produced by Biopharma Research on the tomato metabolism. To do it, Solanum lycopersicum cv. Micro-Tom seeds were germinated in long day conditions directly in pots. Plants were grown 21 days until they reach vegetative development stage. Four different Biostimulants were applied by irrigation once a week for two consecutive weeks. One week after the last application, physical, biochemical and molecular parameters during plant growth will be studied to determine the most effective biostimulant. The physical parameters analysed have been: 1) dry and fresh weight of the aerial and subterranean part of the plant; 2) agronomic and nutritional characteristics of tomato; 3) chlorophyll quantification 4) accumulation of hydrogen peroxide in the tissue and 5) total protein. The results showed that biostimulant “BT2” turned out to be one of the most interesting since at the end of the cycle the plant continued to produce flowers and after its application the tomatoes ripened earlier. Tomato plants are highly susceptible to water deficit notably during the growth phase, but also at flowering and during the fruit growth[1]. In the first assay we focused on the analysis of the physical parameters. Now, in the second assay we will induce water stress to the plant and we will apply biostimulant and then we will study hormones production and gene expression of the tomato antioxidant system. We have observed that in depend on the composition of the bioestimulant, they will differently affect plant development. It has been observed that the plants treated with biostimulants reduce H2O2 accumulation in comparate the control plant

Proceso de elaboracion de vino en Bodegas Jose y Miguel Martin. Fermentación por inversion térmica

In order to get wines of excellence, rigorous quality control are required during every step of the production. Continuous analysis for parameters such as grape maturation, alcohol content, density, pH, total acidity or volatile acidity are required to ensure wine quality. Moreover, some of these analysis are also required to reach the characteristics requested by the client and to fulfil current legislation (1).Jose and Miguel Martin S.L. is a wine company located in Bollullos Par Del Condado, Huelva. It is dedicated to the production and aging of wines and their derivatives, as well as to the manufacture of oak barrels. To ensure the quality of their wines, this company have a full equipped laboratory where we have been analysing the evolution of grape, must and wine. Initially, a grape maturation control was carried out to determine the optimum stage for grape harvesting. Once in the wine cellar, sugar must density as well as SO2 content was measured every day for many samples. Later, during fermentation, sugar density and yeast population (2), was analysed for every fermenter and for finished wines alcohol content, total and volatile acidity and heat and tartaric stability was also controlled. With these data, wine corrections were done to get the final desired characteristics for the wines (1, 3). Furthermore, a study to determine the effect of fermentation temperature on the organoleptic characteristics of wine was performed. A drop in temperature along fermentation, known as thermal inversion, was applied. With this, concentration for varietal thiol compounds (Ac3MH), as well as for other fermentative esters should been modified (4, 5). Thus, temperature-controlled fermenters were used to study the differences in the resulting wines produced with Zalema grape must. To this, one of the fermenter was kept at a constant temperature of 18 degrees during the whole fermentation, while in the other the temperature was lowered to 14 degrees, 4 days after the yeast fermentation started, remaining constant until the end of this process. Full analysis of chemical parameters was performed to the initial must before fermentation, to the wine when fermentation was completed, and later after wine clarification. Stability tests, both for protein and tartaric acid, was also assayed. Once the wine were stabilized and clarified, they have been stored and a final tasting will be carried out, to compare the organoleptic characteristics of both wines. The wine produced by thermal inversion should present an intermediate aromatic profile, with noticeable concentrations of varietal thiols (Ac3MH) and fermentative esters, while the wine produced by fermentation at 18ºC should present a predominance of varietal thiols (4, 5

Online_Appendix – Supplemental material for Diagnostic and Interventional Nephrology in Spain: A snapshot of current situation

<p>Supplemental material, Online_Appendix for Diagnostic and Interventional Nephrology in Spain: A snapshot of current situation by R Haridian Sosa Barrios, Jose Ibeas, Ramon Roca Tey, Manuel Ceballos Guerrero, Angels Betriu Bars, Ignacio Cornago Delgado, Manuel Lanuza Luengo, Vicente Paraíso Cuevas, Pedro L Quirós Ganga and Maite E Rivera Gorrín in The Journal of Vascular Access</p