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[EN] Winemaking is a step in which several and complex microbiological and physicochemical conversions occur. The alcoholic fermentation is mainly conducted by Saccharomyces yeast species, although some non-Saccharomyces yeast could contribute to the final quality of wines with their presence during the elaboration. Some of them can produce wine spoilage developing during vinification even staying during ageing. The Brettanomyces genus is one of the most dangerous microorganisms for wine quality. The anamorphic species Brettanomyces bruxellensis has the ability of developing in wines, surviving into the natural wood pores of barrels during the ageing. It is considered a potentially spoiler microorganism when generate undesirable phenolic volatile compounds. The most employed tool for ensuring the wine microbial stability is the sulphur dioxide addition. This traditional task is usually aimed to prevent wine oxidation and to ensure the inactivation of most of the wine-borne spoilage microorganisms. Actually, the sulphur dioxide is added to must, after alcoholic fermentation, after malolactic fermentation and during the ageing. Specifically, the ageing of wine in wood barrels is the most complex stage because the solid sulphur dioxide has to be burnt inside the barrel, generating an inadequate and non-safe atmosphere for winery workers. Although very useful, the sulphur dioxide can cause allergic reactions so the maximal legal dose to be added is going to be probably reduced in a near future. Current consumers demand quality, free-additive wines, so alternative and new technologies should be investigated, in order to both reduce the sulphur dioxide addition and avoid the wine spoilage by this yeast. This has made oenological industry search for alternative non-thermal technologies as pulsed electric field (PEF) and high hydrostatic pressure (HHP). Two different PEF treatments were tested in a continuous-flow system for inactivating B. bruxellensis yeast in a red wine., [ES] La vinificación es una etapa de complejos cambios microbiológicos y fisicoquímicos. La fermentación alcohólica es dirigida principalmente por la especie Saccharomyces cerevisiae, aunque hay otras especies no-Saccharomyces también presentes que pueden influir en la calidad de los vinos. Entre las levaduras de la vinificación, puede existir alguna con una influencia negativa en el perfil organoléptico de los vinos. La levadura Brettanomyces bruxellensis es capaz de sobrevivir en el interior de los poros de la madera de la barrica en el envejecimiento y en las posteriores etapas de almacenamiento en botella. Además, actúa en detrimento de la calidad del vino cuando sintetiza fenoles volátiles indeseables para el perfil aromático del vino. La forma tradicional de asegurar la estabilidad microbiológica del vino ha sido el empleo del dióxido de azufre. El sulfitado del mosto o vino es una práctica enológica dirigida a asegurar la inactivación de microorganismos perjudiciales para el vino así como prevenir la oxidación del mismo. El sulfitado se lleva a cabo en mosto, tras fermentación alcohólica y maloláctica y durante el envejecimiento en barrica y en botella. Cuando se realiza mediante la quema de una forma sólida de dióxido de azufre dentro de la barrica, se genera una atmósfera insegura para la salud de los empleados. Aunque sigue siendo muy importante en enología, el dióxido de azufre puede provocar alergias tras su consumo, por lo en un futuro cercano las dos is máximas legales pueden sufrir una reducción importante. Los consumidores actuales demandan vinos de calidad, naturales y libres de aditivos. Así, se deben investigar tecnologías alternativas para reducir el empleo de conservantes y evitar el deterioro de la calidad del vino por algunos microrganismos. Ésta nueva situación ha hecho que la industria enológica busque nuevas técnicas de conservación no basadas en la aplicación de calor, como por ejemplo los pulsos eléctricos de alto voltaje (PEAV) y las alt, [FR] Vinification est une étape complexe de microbiologiques et physico-chimiques des changements. La fermentation alcoolique est entraînée principalement par l'espèce Saccharomyces cerevisiae, bien que d'autres espèces non-Saccharomyces aussi présents qui peuvent influer sur la qualité des vins. Parmi la levure de vin, il peut y avoir certaines avec une influence négative sur le profil organoleptique des vins. La Levure Brettanomyces bruxellensis est capable de survivre dans les pores du bois du canon au cours du vieillissement et dans les étapes ultérieures de la bouteille de stockage. Il agit également préjudiciable à la qualité du vin lorsque synthétise phénols volatils indésirables pour le profil de saveur du vin. La manière traditionnelle pour assurer la stabilité microbiologique du vin a été l'utilisation de dioxyde de soufre. Le sulfitage du moût ou du vin est une pratique œnologique visant à assurer l'inactivation des micro-organismes nocifs pour le vin et éviter l'oxydation. Le sulfitage est effectué dans le moût, après la fermentation alcoolique et malolactique au cours du vieillissement et dans le fût et en bouteille. Quand elle est réalisée en brûlant une forme solide du dioxyde de soufre à l'intérieur du canon, une atmosphère dangereuse pour la santé des employés est générée. Bien qu'il soit encore très important en œnologie, le dioxyde de soufre peut provoquer des allergies après la consommation, dans un proche avenir, les doses maximales légales peuvent subir une réduction significative. Les consommat eurs d'aujourd'hui exigent des vins de qualité, naturels et sans additifs. Donc, devrait étudier les technologies alternatives pour réduire l'utilisation de conservateurs et d'éviter la détérioration de la qualité du vin par certains micro-organismes. Cett e situation nouvelle a fait le look de l'industrie du vin pour les nouvelles techniques de conservation ne sont pas basées sur l'application de la chaleur, tels que des champs électriques pulsés (CEP)

© 2015. High Hydrostatic Pressure (HHP) technology was tested on two strains of Brettanomyces bruxellensis isolated from Rioja red wines for analyzing the microbial inactivation reached in different oenological conditions. With this purpose, these strains were inoculated in synthetic wine with different pH and ethanol content. The inactivations reached with 100MPa, 200MPa and 300MPa applied from one to seven minutes were tested just after the treatment and after a week.Current consumers demand quality, free-additive wines. This has made oenological industry search alternative non-thermal technologies. The present results supported that high pH and high ethanol wines shortly treated at 100MPa made B.bruxellensis undetectable. Moreover, low pH and low ethanol wines content should be at least treated at 200MPa, and wines with intermediate ethanol content should be carefully analyzed in terms of strain composition before applying HHP. Finally, treatments at 300MPa managed a complete inactivation regardless of oenological conditions.

El vino es el resultado de multitud de interacciones complejas entre una gran diversidad de microorganismos. El proceso de transformación de la uva en vino consta habitualmente de dos etapas fermentativas: la fermentación alcohólica, llevada a cabo por levaduras y la fermentación maloláctica, en la que las bacterias lácticas son los microorganismos principales. En este estudio se profundizó en el conocimiento de las comunidades de bacterias lácticas de vinos de Tempranillo de la Denominación de Origen Calificada Rioja (D.O.Ca. Rioja) a lo largo del proceso de elaboración. Para ello, se emplearon tanto técnicas de Biología molecular cultivo-dependientes como cultivo-independientes, resultando así complementaria la información obtenida. Se detectaron un gran número de especies de bacterias lácticas y de cepas de Oenococcus oeni, observándose en esta región un ecosistema diverso y maduro, donde tanto las especies como las cepas se fueron sucediendo como resultado de la adaptación a las condiciones de cada año, de cada bodega y de cada etapa de la vinificación. El estudio se completó con la comparación de las poblaciones dominantes de O. oeni de dos regiones vitivinícolas españolas (D.O.Ca. Rioja y D.O. Castilla-La Mancha). Dichas poblaciones se perfilaron como una única población panmíctica en la que algunas cepas de O. oeni parecían estar desarrollando una evolución clonal pero sin llegar a diferenciarse subpoblaciones alopátricas. La variabilidad de la microbiota observada implica que el control microbiológico sea indispensable para el desarrollo adecuado de las fermentaciones y para garantizar la calidad del vino. Para llevar a cabo este control microbiológico se han empleado tradicionalmente técnicas como el filtrado, la clarificación, así como la adición de sustancias químicas como el sulfuroso. Precisamente con la idea de reducir los aditivos habitualmente empleados en enología, en este trabajo de tesis doctoral también se estudiaron diferentes tecnologías emerge

[EN] Phenolic compounds are known to have a great importance on grape and wine quality and health properties. Viticultura! practices have a strong influence on grape phenolic composition and thus on its quality. Recently, there has been an increasing interest in foliar fertilization, which entails a fast and efficient assimilation of the products. The purpose of the present work was to study the influence of foliar applications of phenylalanine and urea to vines, at two different doses, on grape detailed phenolic composition. Previous studies have investigated the foliar application of urea in order to enhance grape nitrogen content and aroma composition while, on another note, phenylalanine is the precursor of most phenolic compounds. The experiment was conducted during 2013 in a cv. Tempranilla vineyard in La Rioja (north region of Spain). The foliar treatments were carried out at veraison and one week later at doses of 0.9 and 1.5 kg N/ha. The results showed that, in general, grapes from the treatments with phenylalanine and urea presented higher phenolic content than control grapes. Contrary to our expectations, despite being a precursor in phenolic biosynthesis, phenylalanine applications did not show a stronger influence on these compounds than urea treatments. In particular, it was noticeable that the application of the lowest dose of urea induced high synthesis of several phenolic compounds, especially anthocyanins and flavonols. This fact could be related to the time of application, since anthocyanins and flavonols are mainly formed after veraison. In conclusion, foliar application of phenylalanine and urea to vineyard could be a useful and interesting management tool for improving grape quality., [ES] Los compuestos fenólicos tienen una gran importancia en la calidad de la uva y del vino y en sus propiedades saludables. Las prácticas vitícolas son un factor determinante en la composición fenólica de la uva y por tanto en su calidad. En los últimos años, el empleo de la fertilización foliar ha despertado un interés creciente, debido a que posibilita una rápida y eficiente asimilación de los compuestos aplicados en el viñedo. El objetivo de este trabajo fue evaluar el efecto de la aplicación foliar, en la viña, de fenilalanina y urea (a dos dosis diferentes), en la composición fenólica de la uva. La urea es un compuesto nitrogenado que se ha aplicado en algunos estudios para mejorar la composición nitrogenada y aromática de la uva, mientras que la fenilalanina es el aminoácido precursor de la mayoría de compuestos fenólicos. El trabajo se llevó a cabo en el año 2013 en un viñedo de la variedad Tempranilla en La Rioja (España). Los tratamientos foliares se realizaron en envero y una semana después, aplicándose fenilalanina (0,9 y 1,5 kg N/ha) y urea (0,9 y 1,5 kg N/ha). Los resultados mostraron que, en general, las uvas procedentes de los tratamientos foliares con fenilalanina y urea tenían un contenido fenólico mayor que la uva control. Contrariamente a nuestras expectativas, la aplicación foliar de fenilalanina, a pesar de ser un precursor en la síntesis de los compuestos fenólicos, no mostró mayor influencia en la formación de dichos compuestos que la observada en el tratamiento con urea. En concreto, la aplicación de la menor dosis de urea indujo mayor síntesis de numerosos compuestos fenólicos, principalmente antocianas y flavonoles. Este hecho podría estar relacionado con el momento del tratamiento foliar, puesto que ambos grupos de compuestos fenólicos se acumulan mayoritariamente después del envero. En conclusión, la aplicación foliar de fenilalanina y urea podría ser una herramienta útil e interesante para mejorar la calidad de la uva., [IT] E ben noto che i composti fenolici svolgano un ruolo importante sulla qua lita dell'uva e del vino e sulle proprieta salutistiche. Le pratiche viticole esercitano una notevole influenza sulla composizione fenolica dell'uva e quindi sulla sua qualita. Recentemente, un crescente interesse e rivolto alla fertilizzazione fogliare, che favorisce una rapida ed efficiente assimilazione dei prodotti. Lo scopo del presente lavoro e quello di studiare !'influenza delle applicazioni fogliari, a due differenti dosi, di fenilalanina e urea alla vite sulla composizione fenolica dell'uva. Studi precedenti hanno studiato l'applicazione fogliare dell'urea allo scopo di incrementare il contenuto di sostanze azotate dell'uva e di composti aromatici, mentre la fenilalanina e il precursore della maggior parte dei composti fenolici. L'esperimento e stato condotto nel 2013 sulla cv. Tempranilla in un vigneto nella regione La Rioja (zona settentrionale della Spagna). 1 trattamenti fogliari so no stati condotti durante l'invaiatura e una settimana dopo, utilizzando dosi di 0,9 e 1,5 kg N/ha. 1 risultati mostrano che, in genere, le uve ottenute dalle vigne trattate con fenilalanina e urea mostravano contenuti pi u elevati in composti fenolici rispetto alle uve di controllo. Contrariamente alle nostre attese, nonostante la fenilalanina sia un precursore nella biosintesi dei composti fenolici, le applicazioni fogliari di fenilalanina, nonostante questa sostanza si a un precursore dei composti fenolici, non han no mostrato un'influenza maggiore sulla formazione di questi composti rispetto ai trattamenti con urea. In particolare, e stato notato che l'applicazione della dose pi u bassa di urea ha indotto la sintesi pi u elevati di diversi composti fenolici, specialmente antocianine e flavonoli. Questo risultato potrebbe essere correlato al momento del trattamento poiché antocianine e flavonoli si forma no principalmente dopo l'invaiatura. In conclusione, l'applicazione fogliare di fenilalani

[EN] Ethephon is known to be a growth regulator that, when applied to the plant, it is metabolized into ethylene. In this study, it has been evaluated the use of a commercial plant growth regulator (Fruitel180, 18% w/v ethephon) with the aim to balance both grape sugar and phenolic ripeness. The tri al was peformed on Vitis vinífera L. Tempranilla red wine grapevine grown in La Rioja (north Spain). A completely randomized experimental design was set up consisting in four replicates of 40 vines per treatment. The following treatments were compared: treatment 1 (control or nonapplied vines), treatment 2 (application of 1.25 L/ha Fruitel180), and treatment 3 (application of 2.5 L/ha Fruitel180). Treatments were foliar applied at veraison (20%). Grape ripening was controlled by berry sampling once a week until harvest. During ripening, grapes from vines treated with ethephon always showed lower sugar content than those from control. Moreover, grapes from the treatments had higher anthocyanin and total polyphenol content than control grapes. However, there was not a marked difference between the two doses applied. Grapes were vinified in 100L tanks obtaining for each treatment four wines (one wine per field replicate). lt was observed that fermentation time was shorter in wines from the ethephon treatments, in particular in the case of treatment 2. This result could be explained bythe lower sugar content in grapes from the treatments in comparison with control grapes, besides the different grape composition. Wine physicochemical parameters after malolactic fermentation showed significant differences regarding alcoholic strength and volatile acidity, which were lower in wines made from grapes treated with Fruitel than in control wines, although there were not differences between the two doses. Moreover, wines from the ethephon treatments showed a certain tendency towards greater color intensity values with respect to control wines, although not statistically different. As, [ES] El etefón es un regulador del crecimiento, que aplicado sobre la planta, se descompone en el interior de los tejidos vegetales liberando etileno. En este trabajo se ha evaluado la utilización en campo de un producto fitorregulador del crecimiento vegetal en uva (Fruitel 180) con un contenido en etefón del 18% p/v que puede resultar beneficioso para uniformizar la maduración tecnológica y fenólica de la uva. El ensayo se realizó en una parcela de uva de la variedad Tempranilla de D.O.Ca. Rioja (España) con un diseño experimental de bloques al azar con cuatro repeticiones por tratamiento y 40 cepas por repetición. Se realizaron los siguientes tratamientos: Tratamiento 1 (Testigo, sin aplicación de Fruitel 180), tratamiento 2 (aplicación de 1.25 L/ha de Fruitel180) y tratamiento 3 (aplicación de 2.5 L/ha de Fruitel180). Los tratamientos se aplicaron mediante pulverización foliar cuando el porcentaje de envero se situó en torno al 20%. Se controló la evolución de la maduración de la uva una vez por semana hasta la vendimia, observando en todos los muestreos que las uvas procedentes de las cepas tratadas presentaron valores más bajos en el contenido en azúcares. Asimismo, tanto el contenido total de antocianas como de polifenoles, que aumentaron durante la maduración, fue significativamente mayor en las uvas tratadas, aunque no se encontraron diferencias claras entre las dosis del tratamiento. Se llevaron a cabo vinificaciones por cuadriplicado en depósitos de 100 litros, observando que tanto la fermentación alcohólica como la maloláctica fueron ligeramente más rápidas en los vinos tratados con Fruitel, especialmente con la dosis doble. Este resultado pudo ser consecuencia del menor contenido en azúcares de la uva tratada en el momento del encubado, así como de su distinta composición. Los resultados de los parámetros analíticos de los vinos después de la fermentación maloláctica mostraron diferencias significativas en el grado alcohólico y en la acidez volátil, que, [FR] L'éthéphon est connu pour etre un régulateur de croissance qui, lorsqu'il est appliqué a la plante, il est métabolisé en éthylime. Dans cette étude, il a été évalué l'utilisation d'un régulateur de croissance commercial (Fruitel 180, 18% p/v éthéphon) avec l'objectif de rapprocher l'accumulation des sucres et des poliphenoles dans le raisin. L'étude a été mené a un vignoble cv Tempranilla situé a La Rioja (nord de I'Espagne). Un plan expérimental completement aléatoire a été mis en place avec quatre répétitions par traitement et 40 vignes par répétition. Les suivantes traitements ont été comparés : traitement 1 (controle, aucune demande Fruitel), traitement 2 (application de 1,25 L 1 ha Fruitel180) et traitement 3 (application de 2,5 L / ha Fruitel 180). Les traitements ont été appliqués par pulvérisation foliaire lorsque le pourcentage de veraison était d'environ 20%. L'évolution de la maturation des raisins a été suivie une fois par semaine jusqu'a la récolte. Pendant la maturation, les raisins provenant de vignes traitées avec l'étephon ont toujours montré des teneurs en sucre inférieurs a ceux de controle. En revanche, les raisins traités ont montré teneurs plus élevées d'anthocyanes et de polyphenols que les raisins controles. Cependant, aucune différence n'a été constatée entre les deux doses utilisées. Les raisins ont été vinifiés en cuves de 100 litres (quatre vins pour chaque traitement). 11 a été noté que la durée de la fermentation était plus courte dans les vins provenant des traitements avec l'éthéphon, en particulier dans le cas de traitement 2. Ce résultat pourrait etre conséquence d'une teneur en sucre inférieur dans les raisins des traitements en comparaison avec les raisins controle. Les résultats des parametres physico-chimiques des vins apres fermentation malolactique ont montré des différences significatives dans le teneur en alcool et l'acidité volatile, qui étaient plus faibles dans les vins issus de raisins traités avec Fruitel que dans

Las condiciones climatológicas en las que tiene lugar una vendimia influyen en el estado de las uvas, en el desarrollo de la vinificación, en los microorganismos presentes y, en consecuencia, en las características de los vinos finalmente obtenidos. Una de las principales problemáticas que pueden ocurrir en los vinos tintos es su alteración por levaduras de la especie Brettanomyces bruxellensis. En este trabajo se analizaron 100 vinos tintos jóvenes de una campaña climatológicamente conflictiva (2013), y se compararon con 100 vinos de una campaña normal (2012). Se encontraron diferencias significativas en algunos parámetros analíticos y también en la incidencia y desarrollo de levaduras alterantes del género Brettanomyces, que fue mayor en la campaña de 2013. Algunos vinos en los que Brettanomyces se encontraba en niveles superiores a 102 ufc/ml, no desarrollaron carácter >Brett> al someterlos a un test odorimétrico. Esto indicaría que la contaminación de un vino con esta levadura no es el único factor determinante de la alteración. La existencia en los vinos de sustancias susceptibles de ser transformadas en compuestos responsables del carácter >Brett>, y/o la presencia de clones de Brettanomyces capaces de sintetizarlos, podrían ser importantes. En dicha prueba odorimétrica, los vinos que desarrollaron la alteración presentaron valores superiores de pH, Ac. volátil y Azúcares, así como menor Grado alcohólico que los vinos que no la desarrollaron. Puesto que la contaminación por Brettanomyces no es fácilmente evitable, especialmente en campañas conflictivas, el control del carácter >Brett> debe realizarse mediante medidas que prevengan la multiplicación de dichas levaduras. Pero también se debería seguir investigando sobre la importancia que compuestos minoritarios y clones de Brettanomyces presentes en un vino tienen en el desarrollo de la alteración.

Los compuestos fenólicos tienen gran importancia en la calidad del vino. La aplicación de elicitores sobre los racimos es una de las prácticas vitícolas más prometedoras en cuanto a la mejora de la composición fenólica. Sin embargo, esta técnica precisa de un deshojado previo, con el consecuente consumo de tiempo v dinero. En el presente trabajo se estudió el efecto de la aplicación foliar de metil jasmonato en la composición fenólica de uvas v vinos de la variedad Tempranilla. El tratamiento dio lugar a un incremento del contenido de antocianas v estilbenos, tanto en la uva como en el vino, además de aumentar el contenido de flavonoles en el vino. Asimismo, se observó una mejora de los parámetros cromáticos del vino. Los resultados obtenidos sugieren que esta herramienta podría ser una práctica vitícola sencilla v de fácil aplicación para mejorar la calidad de la uva v del vino.

© 2015 Elsevier Ltd. Four different treatments of pulsed electric field (PEF) technology were tested in a continuous-flow system for inactivating 25 species of yeast, lactic acid bacteria and acetic acid bacteria associated with wines. Overall, the reached inactivation varied from 0.64 to 4.94 log units. Curiously, it was determined that the application of higher specific energy did not provide significant declination of yeast population, although significantly different inactivations were determined for bacteria. The inactivation of bacteria highly depended on each species and results corroborated different sensitivity of two Oenococcus oeni strains what could be related with their genetic characteristics.

The importance of phenolic compounds for grape and wine quality has drawn attention to studying different practices with the aim of increasing their content. Cluster application of elicitors is a viticultural practice that has shown promising results in recent years. However, cluster application requires a previous defoliation, which is time-consuming and expensive. In the present study, methyl jasmonate was foliar applied to Tempranillo grapevines in order to study its effect on grape and wine phenolic composition. Methyl jasmonate foliar application increased anthocyanin and stilbene content in both grape and wine, besides enhancing wine flavonol content. This treatment induced the synthesis of 3-O-glucosides of petunidin and peonidin and trans-p-coumaroyl derivatives of cyanidin and peonidin. For stilbenes, trans-piceid content was considerably increased in both grape and wine. The results obtained suggest that methyl jasmonate foliar application could be a simple and accessible practice to enhance grape and wine quality.

© 2015 Elsevier Ltd. All rights reserved. The aim of this work was to study and compare the effect of pulsed electric field (PEF) treatment on the content of individual anthocyanins, phenolic acids, flavanols, and flavonols of three grape varieties from two vintages. For this purpose, the treatment showing to induce the highest release of phenolic compounds out of four different PEF applications assayed during the 2011 vintage was applied in 2012. The same continuous system was used over three grape varieties: Graciano, Tempranillo, and Grenache. Additionally, the influence of PEF on the physicochemical composition of the grapes was also studied. The effect of PEF treatment in the amount of individual phenolic compounds released and in the values of the physicochemical parameters analysed was qualitatively similar yet quantitatively different between the two years of study. Considering that the initial values of the physicochemical parameters were dependent on the vintage, it was observed that the lower the initial values the higher their increase after PEF treatment. Hence, the lower the initial values the higher the benefit obtained by PEF treatment. Furthermore, the influence of PEF treatment depended on the variety treated. For instance, Tempranillo was the variety showing the most significant differences in the content of individual anthocyanins upon PEF treatment, while Grenache variety showed major differences in the content of gallic acid, catechin, and epicatechin. Graciano variety showed almost no differences in the amount of individual phenolic compounds released after PEF application. For the three varieties, colour intensity, TPI, and total anthocyanins were increased by PEF application during the two assayed vintages, as well as antioxidant activity in 2012. Industrial relevance Pulsed electric field treatment is a suitable technology for the extraction of phenolic compounds. However, its suitability depends on the grape variety treated, and its quanti

© 2014 Elsevier Ltd. In this work the influence of the foliar application of proline, phenylalanine, urea and two commercial nitrogen fertilizers, with and without amino acids, on grape aroma composition was studied. The treatments were applied in a Tempranillo vineyard, at a dose of total N of 900g/ha, at veraison and a week later. Enological parameters and volatile compound content were determined in grapes. The results showed that terpenoids synthesis decreased with all treatments carried out in the vineyard, and there were not significant differences either in the amount of C13 norisoprenoids or in esters between treated and control samples. The treatment with phenylalanine increased the presence of benzenoid compounds and decreased C6 compounds. Therefore, this treatment could be considered the best from the point of view of grape aroma composition. The treatments with the commercial nitrogen fertilizers did not affect the total compound content present in the samples.

© 2015 Elsevier Ltd. The Oenococcus oeni populations of Tempranillo wines from Castilla La Mancha and La Rioja winemaking regions were analysed from one to three years and up to ten wineries. The objective was to evaluate the genetic variability and the O.oeni population structure. For this purpose a MLST scheme based on four loci (. gyrB, purK, pgm and recP genes) and PFGE with SfiI restriction enzyme were developed for later combination. The results showed an O.oeni population completely adapted to winemaking regions. Apurifying selection influenced the genes evolution, especially recP that along with purK were the most interesting loci to analyse the genetic variability of the isolates. In this way linkage disequilibrium and intergenic and intragenic recombination were determined between isolates. PFGE typing with UPGMA data were not coincident with the phylograms assessed for MLST by Maximum likelihood and combination of both techniques differentiated all the isolates as strains. Those results led the research to conclude that O.oeni population from CM and LR was a panmictic population with a slight clonal evolution, so subpopulations could not be described. A broader study including more winemaking regions with different grape varieties and distinct ways of elaborating would be interesting to complete the knowledge about O.oeni populations.

© 2015 Elsevier Ltd. All rights reserved. Grapevines may require the input of nitrogen to grow and to guarantee an appropriate grape composition. Recently there has been a growing interest in foliar fertilization, which entails a fast and efficient assimilation of the products. The aim of this work was to study the influence of foliar applications of phenylalanine and urea, at two different doses, on grape anthocyanins, flavonols, flavan-3-ols, phenolic acids, and stilbenes. All treatments were applied at veraison and one week later at doses of 0.9 and 1.5 kg N/ha. The results showed that the synthesis of phenolic compounds was favoured by foliar applications of phenylalanine and urea. The application of the lowest dose of urea was the most effective treatment, increasing the content of several anthocyanins and flavonols. Moreover, none of the foliar treatments worsened the grape phenolic composition. In conclusion, foliar application of phenylalanine and especially urea, could be an interesting management tool for improving grape quality and their health-promoting properties.

Brettanomyces bruxellensis is a major cause of wine spoilage due to the production of ethyl phenols, and it has become a major worldwide oenological concern in recent years. The most critical factor in volatile phenol production is the presence of microorganisms responsible for biosynthesis. In this work, carried out during three consecutive harvests, grapes and the first step in grape processing (stemming–crushing) have been evaluated as the origin of wine contamination by these spoilage yeasts. Results showed that there was nil or minimal presence of Brettanomyces yeasts in grapes and on the stemmer, in levels that the method was not able to detect. This shows that the main contamination of wines by this microorganism occurs in later stages of the vinification process and/or during storage. The contamination of many wines with Brettanomyces, either via the fruit or from the winery environment, during the early stages of vinification and before the start of aging, was confirmed by analyzing 100 recently made red wines, in which this yeast was detected in a high percentage of wines (27 %). However, the level of Brettanomyces yeasts found in the samples was low, with values which would not be sufficient to cause organoleptic defects. Consequently, this study confirms that many wines are still tainted by Brettanomyces when the winemaking phase comes to an end. It was also found that wines with problems during fermentation had a higher level of Brettanomyces. This is why it is essential to ensure strict controls during fermentation processes and conservation to prevent wine becoming spoiled or tainted.

[EN] Must nitrogen composition plays a key role in the alcoholic fermentation, affecting the fermentation kinetics and the formation of secondary metabolites, especially alcohols and esters. Thus, grapevine might require the input of fertilizers, in particular nitrogen compounds to grow and to guarantee an appropriate grape composition that ensures the correct development of the alcoholic fermentation and the quality of the final product. New fertilization methods have arisen due to the pollution problems caused by traditional soil fertilization. In the last few years, there has been a growing interest in foliar fertilization, which entails a fast and efficient assimilation of the products, contributing then to sustainable eco-friend ly agriculture. As far as we know, there are not previous studies about foliar application of amino acids in the vineyard. Nevertheless, urea foliar application has been reported to modify grape composition. On the other hand, proline is one of the most important amino acids in grapes. Phenylalanine is not present in such high quantities, but it is the precursor of some interesting compounds such as 2-phenylethanol and resveratrol. Moreover, new nitrogen fertilizers have recently appeared on the market, alt hough their effects on grape composition are still unknown. The aim of this work was to study the influence of different nitrogen foliar applications on grape amino acid composition. The nitrogen sources applied to Tempranillo grapevine were proline, pheny lalanine, urea, and two commercial nitrogen fertilizers, without and with amino acids in their formulations. All treatments were applied at veraison and one week later at a dose of 0.9 kg N/ha. Treatments were performed in triplicate and were arranged in a complete randomized block design, with three vines for each replication. Oenological parameters and amino acids were determined in musts. Oenological parameters were analysed according to ECC methods while amino acids were analys, [ES] La composición nitrogenada de la uva juega un papel importante en la fermentación alcohólica, ya que afecta a su cinética y a la formación de compuestos secundarios, especialmente alcoholes y ésteres. Por tanto, el viñedo puede requerir el uso de fertilizantes, principalmente compuestos nitrogenados, que aseguren un correcto desarrollo de la fermentación y la calidad del producto final. En los últimos años han surgido nuevos métodos de fertilización debido a los problemas ambientales causados por el abonado tradicional. En este sentido, hay un interés creciente en la fertilización foliar, que asegura una rápida y eficiente asimilación de los productos, contribuyendo así a una agricultura sostenible y respetuosa con el medio ambiente. No se han encontrado estudios previos sobre la aplicación foliar de aminoácidos en el viñedo. Sin embargo, se ha observado que la aplicación foliar de urea modifica la composición nitrogenada de la uva. Por otra parte, la prolina es uno de los aminoácidos más importantes en la uva. La fenilalanina, aunque su concentración en la uva no sea alta, es precursora de algunos compuestos de interés como 2-feniletanol y resveratrol. Además, en el mercado han aparecido abonos comerciales con aminoácidos, cuyo efecto en la composición de la uva no se ha testado todavía. El objetivo de este trabajo fue estudiar la influencia de la aplicación de diversas fuentes nitrogenadas en la composición aminoacídica de la uva. Las fuentes nitrogenadas aplicadas en un viñedo de Tempranillo fueron prolina, fenilalanina, urea y dos abonos comerciales, uno sin aminoácidos y otro con aminoácidos en su formulación. Todos los tratamientos se efectuaron en el envero y una semana después, aplicándose una dosis de 0.9 kg N/ha. Los tratamientos se realiz aron por triplicado bajo un diseño de bloques al azar, consistente de tres plantas por réplica. Los parámetros enológicos se analizaron según los métodos oficiales mientras que los aminoácidos se analizaron por HPLC, [IT] [IT] La composizione azotata del mosto svolge un ruolo fondamentale nella ferm entazione alcolica, poichè influenza la cinetica fermentativa e la produzione di composti secondari, in particolare di alcoli ed esteri. In conseguenza di ciò, potrebbe essere necessario aggiungere fertilizzanti alla vite, in particolare composti azotati, al fine di facilitare la crescita della pianta ed assicurare una composizione del mosto che garantisca il corretto andamento del processo fermentativo e la qualità del prodotto. A causa dei problemi di inquinamento causati dai sistemi tradizionali di fertilizzazione del suolo, si stanno diffondendo nuovi metodi di fertilizzazione. Negli ultimi anni, vi è un crescente interesse verso la concimazione fogliare, che comporta una rapida ed efficiente assimilazione dei prodotti, contribuendo anche ad un’agricoltu ra sostenibile ed “eco-friendly”. In base alle nostre conoscenze, fino ad ora non sono stati condotti studi sull’applicazione fogliare degli amminoacidi in vigna. Inoltre, è stato riportato che l’applicazione fogliare di urea modifica la composizione dell’uva. D’altro canto, la prolina rappresenta uno degli amminoacidi principali delle uve. La fenilalanina sebbene non sia presente in quantità così elevate, è il precursore di alcuni composti interessanti, come 2-feniletanolo e resveratrolo. Inoltre, recentemente sul mercato sono comparsi nuovi fertilizzanti azotati, benchè l’effetto di questi composti sulla composizione dell’uva sia del tutto sconosciuto. Lo scopo di questo lavoro è stato quello di studiare l’effetto dell’applicazione fogliare di diverse font i azotate sul contenuto in amminoacidi dell’uva. Le fonti azotate utilizzate per la varietà Tempranillo sono state prolina, fenilalanina, urea e due fertilizzanti azotati commerciali, con e senza amminoacidi nelle loro formulazioni. Tutti i trattamenti son o stati applicati nella fase di invaiatura e una settimana più tardi, utilizzando una dose di 0,9 kg N/ha. I trattamenti, Nel mosto sono stati determinati i parametri enologici e gli amminoacidi. I parametri enologici sono stati analizzati seguendo i metodi ECC, mentre gli amminoacidi sono stati analizzati mediante HPLC. Per i parametri enologici, nessuna differenza è stata riscontrata tra i campioni di controllo e quelli provenienti dalle vigne trattate. Per quanto riguarda la composizione amminoacidica, gli amminoacidi principali in tutti i campioni sono stati prolina, arginina, alanina, acido glutammico e serina, mentre quelli presenti in misura minore erano glicina, metionina, isoleucina e lisina. I risultati ottenuti dimostrano che il trattamento con prolina esercita una scarsa influenza sulla composizione azotata del mosto, poiché aumenta solo la concentrazione di treonina. Comunque, le applicazioni fogliari di fenilalanina ed urea incrementano la sintesi della maggior parte degli amminoacidi da parte della pianta, esercitando un effetto simile. Inoltre, lo spruzzo di fertilizzanti commerciali azotati sulle foglie determina anche un aumento nella concentrazione di amminoacidi dell’uva, indipendentemente dalla loro presenza o assenza nelle formulazioni. La spiegazione di ciò potrebbe essere che il fertilizzante commerciale con amminoacidi contiene principalmente prolina (43%), la cui applicazione in questo studio non ha svolto nessun effetto. Sulla base di questi risultati, è possibile concludere che i trattamenti più efficace sono stati quelli con fenilalanina ed urea, seguiti dai fertilizzanti azotati commerciali, mentre l ’applicazione di prolina non ha esercitato alcun effetto. Questi risultati sono di interesse enologico poiché si ha un miglioramento della composizione azotata del mosto, garantendo una migliore cinetica fermentativa e probabilmente migliorando la qualità del vino, poiché gli amminoacidi sono i precursori di diversi composti volatili.

The presence and distribution of genotypes from malolactic starter cultures between the autochthonous microbiota in fermenting Rioja wines have been studied in this paper. The commercial cultures characterization allowed to identify different species and common pulsed field gel electrophoresis and randomly amplified polymorphic DNA genotypes in several brands of Oenococcus oeni starter cultures. Four indistinguishable genotypes were found between the commercial and the autochthonous O. oeni strains. These four genotypes appeared during different vintages in seven of the 10 sampled wineries despite bacterial starter cultures had never been used. Therefore, the detection of commercial LAB genotypes indistinguishable from the autochthonous ones involved their removal from a selection process. These genotypes represented the 17.7% of autochthonous O. oeni isolated in this region during three consecutive years. This fact demonstrated that performing similar studies as previous selection criteria is advisable to avoid large work and the proposal of one commercialized bacteria. © 2014 The Society for Biotechnology, Japan.

The aim of this work was to study the influence of foliar application of different nitrogen sources on grape amino acid content. The nitrogen sources applied to Tempranillo grapevines were proline, phenylalanine, urea, and two commercial nitrogen fertilisers, both without and with amino acids in their formulations. All treatments were applied at veraison and one week later. Proline treatment did not affect the must nitrogen composition. However, phenylalanine and urea foliar application enhanced the plants' synthesis of most of the amino acids, producing similar effects. In addition, the spray of commercial nitrogen fertilisers over leaves also induced a rise in grape amino acid concentrations regardless of the presence or absence of amino acids in their formulation. The most effective treatments were phenylalanine and urea followed by nitrogen fertilisers. This finding is of oenological interest for improved must nitrogen composition, ensuring better fermentation kinetics and most likely enhancing wine quality. © 2014 Elsevier B.V. All rights reserved.

Oenococcus oeni is usually the main lactic acid bacteria (LAB) responsible for conducting malolactic fermentation (MLF) in wines. Pulsed Field Gel Electrophoresis (PFGE) is one of the most common methods used to identify different genotypes among the wine LAB populations. Although PFGE is a powerful typing tool, it is time-consuming and its results are not easily exchangeable between laboratories so typing methods such as Multilocus Sequence Typing (MLST) have been developed. In this study, thirty O.oeni isolates from Rioja Tempranillo wines were characterized performing SfiI and ApaI PFGE and MLST with eight housekeeping genes. Using the latter technique, six new alleles have been described for five genes. PFGE was slightly more efficient than MLST because of the number of genotypes and of the index of diversity (ID) that each technique discriminated. This has been the first time that PFGE and MLST results have been combined to shape a unique dendrogram. Thus, the combination of results from both typing methods allowed the discrimination of twenty-two PFGE-ST genotypes showing the highest ID of these research (0.947). According to these results, the future application of the combination of PFGE and MLST results could be successful for reliable O.oeni strain typification. © 2013 Elsevier Ltd.

The purpose of this paper was to study the effect of pulsed electric fields (PEF) on the stilbene content of three grape varieties. For this purpose, four different PEF treatments were applied using a continuous system over three varieties, Graciano, Tempranillo and Grenache, destemmed and crushed. In addition, the influence of PEF on their physicochemical composition was studied. PEF treatments did not affect the pH or total acidity of Graciano, however, musts from Tempranillo and Grenache had higher pH values and lower total acidity. In the three varieties, all treatments resulted in an increase of potassium content, deeper colour intensity and total polyphenol index and lower tonality, more pronounced in the treatments with higher time and energy. The stilbene content of the must significantly increased with respect to the control. This increase depended on the variety and the treatment applied. Tempranillo increased up 200% the total stilbene concentration, Grenache 60% and Graciano 50%. For the three varieties, the treatment with the highest time and energy was the most effective on the total stilbene extraction. These results indicate that PEF could be a suitable technology for obtaining musts with deeper colour and higher phenolic content, including resveratrol and piceid.

[ES] La enología moderna exige elaborar vinos de calidad, que además sean beneficiosos para la salud. El sector enológico está obligado, para ser competitivo, a desarrollar y adoptar nuevos procesos que permitan satisfacer dichas exi- gencias manteniendo la calidad sensorial y reduciendo aquellos compuestos que puedan ser perjudiciales para la salud. En esta publicación se presentan algunos de los trabajos de investigación que se están desarrollando en la actualidad en el CIDA y otros que se han llevado a cabo por el grupo firmante en los últimos años, con el objetivo de optimizar los procesos fermentativos y mejorar la estabilidad microbiológica y la calidad sanitaria y sensorial de los vinos. Se han estudiado diversas técnicas como la aplicación de pulsos eléctricos de alto voltaje, el uso de lisozima y plata coloidal o la inoculación con bacterias lácticas, útiles para reducir los niveles de anhídrido sulfuroso y de aminas biógenas en los vinos., [EN] The modern winemaking requires making quality wines, which are also beneficial to health. The wine industry is bound to be competitive, to develop new processes to meet these requirements while maintaining the sensory quality and reducing compounds that may be harmful to health. This publication presents some of the research being developed at present in CIDA and others that have been carried out by the signer group in recent years with the aim of optimizing fermentation processes and improving microbiological stability and sensory quality of wines. Several techniques have been studied such as the application of pulsed electric field technology, the use of lysozyme and colloidal silver or inoculation with lactic acid bacteria, which may be useful in reducing levels of sulfur dioxide and biogenic amines in wines.

Indigenous lactic acid bacteria (LAB) communities have been analyzed for three years (2006, 2007 and 2008) during alcoholic (AF) and malolactic (MLF) fermentations of Tempranillo wines in ten wineries of La Rioja. The results showed that analytical composition of wines and physical–chemical conditions of elaboration influenced the LAB populations, the MLF duration and the percentage of each isolated species and strains. The highest diversity of LAB species was observed during AF in all the wineries. Oenococcus oeni was present in all studied stages of the fermentation process, being the predominant species at final AF stage. The study of 925 isolates of O. oeni by Pulsed Field Gel Electrophoresis (PFGE) allowed the detection of a total of 112 distinct genotypes. Most fermentation stages of both AF and MLF showed mixed O. oeni strain populations, so that there were different genotypes able to share their ecological niche or tank in spontaneous MLF. The frequency of participation of each genotype varied either from year to year or from winery to winery. Otherwise, seven genotypes were detected in the three studied years and in at least three out of the ten studied wineries, being four of them also present in the three studied subzones of this region. These results suggest the existence of an endemic microbiota in this region, the adaptation of indigenous O. oeni strains to the winery conditions every year and the interest of selecting predominant genotypes in order to preserve the biodiversity and peculiarity of these wines.

This study presents a novel microwave technology application which investigates the ability of a new equipment prototype, based on high frequency microwaves, to reduce microbial populations in oak wine barrels. American and French oak staves from highly contaminated barrels were treated. It was demonstrated that microwave treatment was able to significantly decrease the main wine associated microorganism populations from the first 8 mm of oak surfaces. However, the reduction in viable counts depended on the studied microorganism. Thus, counts were reduced by 36%–38% for total yeast, from 35% to 67% for Brettanomyces and around 91%–100% for lactic acid bacteria and acetic acid bacteria, using a very short treatment time (3 min). Nowadays, there is not a convenient method for decontaminating barrels and microwave technology may be a more efficient treatment than currently available methods. Findings from this research suggested that microwave would be beneficial for wine industry and environment by increasing barrel functionality, reducing frequency of replacement, improving microbiological control of oak wood, as well as minimizing of preservative use.

The aim of this work was to study the influence of pulsed electric field (PEF) treatments on the volatile composition of three grape varieties. In order to do this, grapes were crushed and destemmed, and four PEF treatments were applied. The process was carried out continuously using a semi-industrial equipment. Volatile compounds were analyzed by SPME-GC¿MS. The results showed that the effect of PEF treatment was different depending on variety. However, the aromatic profile of the studied varieties was not affected in any case. PEF enhanced the aromatic composition of Grenache, increasing the quantity of monoterpenoids. Moreover, a positive effect was observed in the amount of ß-ionone, total esters and benzenoid compounds, and little difference was observed between treatments for C6 compounds. However, the volatile composition of Tempranillo and Graciano was not favored by PEF. In conclusion, from the point of view of the volatile composition and taking into account the treatments used in this study, it can be concluded that PEF is an appropriate technology to improve the aromatic quality of Grenache variety. Industrial relevance: Pulsed electric field is a non-thermal technology that enhances the volatile content in Grenache. Consequently, this technology could be used in the food industry to improve the aromatic quality of Grenache must and therefore the overall quality of Grenache wine, as this variety is one of the most widely grown in the world.

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 101 colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.

Diversity of lactic acid bacteria (LAB) species has been analyzed for three consecutive years (2006, 2007, and 2008) during alcoholic and malolactic fermentations of Tempranillo wine in a winery at La Rioja. The results showed differences in malolactic fermentation duration, and in both diversity of LAB species and diversity of Oenococcus oeni genotypes. O. oeni was shown to be the predominant species (73% of total isolates). Monitoring the different strains of O. oeni using pulsed-field gel electrophoresis of chromosomal DNA digested with SfiI and ApaI allowed detection of a total of 37 distinct genotypes, most of them comprised at least two isolates. Six appeared in more than one vintage, one of them being present in the three studied years. Moreover, four genotypes were indistinct of the strains isolated from the air of this same winery in 2007 vintage. The frequency of participation of each genotype varied from year to year, thus dominant genotypes at one year were minority or not present at another year. This suggests that distinct indigenous O. oeni strains are better adapted to the different winery conditions every year. Predominant genotypes that appeared in more than one vintage and lead to quality wines with low histamine contents could be considered as interesting for selecting of new malolactic starter cultures. © 2011 Springer Science+Business Media, LLC.

The aim of this work was to study the effect of pH on biogenic amine formation during the elaboration and conservation (7 months) of Tempranillo wines. Grapes at two pHs (3.4 and 3.7) were vinified to achieve this purpose. After alcoholic fermentation the wines were either inoculated with a commercial malolactic starter or not inoculated. Identification and clonal distribution of lactic acid bacteria and amino acid concentration were determined in an attempt to explain the biogenic amine generation. The results showed that the pH of the must influenced the clonal distribution of the Oenococcus oeni strains which conducted the malolactic fermentation and also the concentration of amino acids in the wines after alcoholic fermentation. These aspects could account for the higher biogenic amine formation in wines with the lowest pH during malolactic fermentation. In these wines, inoculation with a malolactic starter was favourable since it produced a lower biogenic amine concentration after malolactic fermentation. Furthermore, the pH also influenced the concentration of amino acids after malolactic fermentation and the lactic acid bacteria distribution during the conservation of the wines. These aspects allow explaining the greater formation of histamine, tyramine and putrescine in the wines with the lowest pH vinified via lactic acid bacteria inoculation after seven months of conservation. © 2011 Elsevier Ltd.

Ecology of the lactic acid bacteria (LAB) during alcoholic fermentation (AF) and spontaneous malolactic fermentation (MLF) of Tempranillo wines from four wineries of La Rioja has been studied analyzing the influence of the winemaking method, processing conditions, and geographical origin. Five different LAB species were isolated during AF, while, during MLF, only Oenococcus oeni was detected. Although the clonal diversity of O. oeni strains was moderate, mixed populations were observed, becoming at least one strain with distinct PFGE profile the main responsible for MLF. Neither the winemaking method nor the cellar situation was correlated with the LAB diversity. However, processing conditions influenced the total number of isolates and the percentage of each isolated species and strains. The winemaking method could cause that genotypes found in semicarbonic maceration did not appear in other wineries. Four genotypes of O. oeni were isolated in more than one of the rest wineries. These four together with other dominant strains might be included in a future selection process. © 2012 Lucía González-Arenzana et al.

Malolactic fermentation (MLF) of Tempranillo Rioja wines (Spain) inoculated with two lactic acid bacteria (LAB) strains were studied and compared with spontaneous MLF. Inoculation with selected Oenococcus oeni lyophila shortened MLF duration up to 19days and lead to wines with more fresh and fruity characters, especially when implantation was 100%. We demonstrated modifications in the concentration of volatile and nitrogenous compounds and a good correlation between analytical and sensory attributes was also noted. In addition, the low initial amino acid concentration and the consumption of these compounds by the inoculated yeast strain during alcoholic fermentation resulted in wines with very low biogenic amines levels (under 3.75mgL-1) after MLF and 3month storage period in all cases. The results showed the significance of choose the most suitable starter to elaborate quality wines and suggest the control of amino acid content in must and wine to prevent the formation of biogenic amines. © 2011 The Authors. International Journal of Food Science and Technology © 2011 Institute of Food Science and Technology.

Lysozyme and the reduction of metabisulfite addition to red wine were evaluated during a winemaking process and after malolactic fermentation (MLF). Treatment, with lysozyme, of the must from Tempranillo grapes and at the end of alcoholic fermentation (AF) caused the 100% implantation of the inoculated bacterial strain and shortened the duration of MLF by 7 days. At the end of the MLF, wines treated with lysozyme showed lower volatile acidity, color intensity, and biogenic amine content. The differences in color intensity disappeared during wine stabilization. The lysozyme addition after MLF led to lower histamine concentrations in wines. These phenomena occurred irrespective of the lactic acid bacteria (LAB) proliferation control and of the Oenococcus oeni dominant strain identified at this period. The results of this study show the significance of preventive use of lysozyme in vinification of red wine to maintain low histamine levels and ensure a successful implantation of inoculated Oenococcus oeni starters.