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Seminario Tcnico
Colaboran:
ndice
Ponencias 01. Relacin entre el contenido nitrogenado 4
en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
Prof. Gemma Beltran Universidad Rovira i Virgili Prof. Jose Manuel GuillaMn Universidad Rovira i Virgili Instituto de Agroqumica y Tecnologa de Alimentos IATA-CSIC
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Formacin de compuestos voltiles azufrados por levaduras vinicas. Una revolucin en el sector enlogico: Levaduras de vinificacin que no producen Sulfuro de hidrgeno (H2S) Nitrogen management is critical for wine Managing Director, flavour and style Unravelling the genetic blueprint of wine yeast When the heat is on, yeast fermentation runs out of puff Taking control of alcohol A la recerca del codi digital dels llevats del vi Effect of Micro-oxygenation on Color and Anthocyanin-Related Compounds of Wines with Different Phenolic Contents Efectos de la microoxigenacin en vino tinto
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isak S. Pretorius The Australian Wine Research Institute, PO Box 197, Glen Osmond, Adelaide, SA 5064, Australia
02. Los nuevos retos en microbiologa del vino. Levaduras no productoras de SH2
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01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
Prof. Gemma BeltRan Universidad Rovira i Virgili Prof. Jose Manuel GUillaMn Universidad Rovira i Virgili instituto de agroqumica y tecnologa de alimentos iata-CSiC
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3. Efecto de la concentracin y tipo de nitrgeno sobre la cintica fermentativa 4. Relacin entre el contenido en nitrgeno y la sntesis de compuestos aromticos Bibliografa
Seminario tcnico
investigacin:
Coautor de 50 artculos cientficos en revistas internacionales de las reas de Tecnologa de Alimentos, Biotecnologa y Microbiologa. Coautor de 20 artculos en revistas de divulgacin del sector agroalimentario y vitivincola. 80 comunicaciones a Congresos nacionales e internacionales, 24 de ellas como ponente invitado o comunicacin oral. Autor de 5 captulos de libros especializados y 37 captulos en libros de congresos. Autor de 3 patentes de levaduras vnicas en explotacin. Director de 3 tesis doctorales (2 de ellas con formato de doctorado europeo)
Posicin actual:
Profesora ayudante doctor del Departamento de Bioqumica y Biotecnologa de la Universidad Rovira i Virgili, Facultad de Enologa, desde diciembre del 2007. Miembro del grupo de investigacin de Biotecnologa Enolgica de la Facultad de Enologa de la URV, dentro de la lnea de estudio de las levaduras vnicas.
lneas de investigacin
Estudio del metabolismo de las levaduras en condiciones de fermentacin vnica. Efecto de las bajas temperaturas de fermentacin y del metabolismo del nitrgeno. Estudio de las poblaciones de levaduras y bacterias acticas durante los procesos de elaboracin de vino. Utilizacin de marcadores moleculares para la caracterizacin e identificacin de los microorganismos del vino.
A lo largo de su carrera investigadora Gemma Beltran ha colaborado en proyectos sobre el estudio las levaduras y sus requerimientos nutricionales, analizando los factores que pueden condicionar a una mejora en la fermentacin alcohlica, tanto los que se derivan del mosto (niveles de nitrgeno disponible) como de varias prcticas enolgicas (fermentaciones a bajas temperaturas, rehidratacin). Sus investigaciones predoctorales se centraron principalmente en el estudio la represin catablica por nitrgeno a lo largo de la fermentacin (Beltran et. al. 2004, 2005), as cmo en el efecto de las bajas temperaturas de fermentacin sobre los cambios en la expresin gnica global y el metabolismo de la levadura vnica (Beltran et al. 2006, 2007, 2008). Adems, en su estancia post-doctoral realizada en el grupo de Biologa Molecular de levaduras del profesor Stefan Hohmann (Suecia), estuvo involucrada en estudios de sealizacin de nutrientes, en concreto estudiando el metabolismo de la tiamina y la ruta de la represin catablica por glucosa, como parte de un proyecto europeo de investigacin. Gemma Beltran es autora o co-autora de un total de 15 publicaciones internacionales y 9 publicaciones nacionales, ha participado en un total de 12 proyectos investigacin o contratos de transferencia, asistido a 22 congresos cientficos, dirigido tesis de master y est actualmente dirigiendo una tesis doctoral. Actualmente es profesora de Bioqumica y Microbiologia Enolgica en la Facultad de Enologa, y desde su reincorporacin al grupo de Biotecnologa Enolgica de la URV en diciembre de 2007 ha estado colaborado en proyectos de investigacin y transferencia con el sector vitivincola, centrndose nuevamente en el estudio de los requerimientos nitrogenados de las levaduras vnicas de primera y segunda fermentacin.
Ponencias
31 de octubre de 1975
Captulo
Resumen:
La cantidad y calidad del componente nitrogenado de los mostos no determina nicamente un buen crecimiento de la levadura y una buena capacidad fermentativa, sino que influye directamente sobre la sntesis de un buen nmero de compuestos voltiles o aromas. En un trabajo de nuestro grupo (Beltran et al., 2005) comprobamos que las adiciones de nitrgeno asimilable en forma de aminocidos, y despus de la fase exponencial de crecimiento, aumentaban los niveles de alcoholes superiores y de algunos steres. En un estudio similar, Vilanova y cols. (2007) observaron tambin incremento en la sntesis de alcoholes superiores y en steres de acetato cuando los mostos eran suplementados con concentraciones de amonio no superiores a los 300 mg/l. Sin embargo, la concentracin y fuente nitrogenada no solo tiene influencia en la sntesis de compuestos con impacto positivo en las calidades organolpticas de los vinos, sino que tambin varios trabajos han sealado una relacin directa entre el nitrgeno asimilable y la sntesis de sulfhdrico. Otros factores nutricionales y ambientales relacionados con la sntesis de este compuesto han sido: la concentracin de sulfatos y sulfitos (en forma de SO2) y la deficiencia en vitaminas (en concreto el cido pantotnico y la tiamina).
muy frecuentes las situaciones industriales en las que el nitrgeno se convierte en un factor limitante para el desarrollo de las levaduras y para la finalizacin de la fermentacin alcohlica. Son varios los autores que han tratado de establecer una concentracin mnima donde se pone en peligro la fermentacin alcohlica. Estos valores no coinciden entre los distintos trabajos, fluctuando de los 120-140 g Nitrgeno asimilable/l (Bely et al, 1990) hasta los 200 o 267 (Mendes-Ferreira et al, 2004), es decir ms del doble. Dichas diferencias pueden ser debidas a varios factores, pero entre los ms importantes podemos destacar dos: la cepa de levadura, perfectamente conocido por los productores de levaduras que incluso las catalogan como con elevado o bajo requerimiento nitrogenado o nutritivo y la forma qumica en que el nitrgeno est disponible en el mosto. Evidentemente otros factores como la forma de vinificacin, variedad de uva, etc. tambin son relevantes, pero su efecto se puede considerar como incluido en la forma qumica y cantidad de nitrgeno en el medio. El nitrgeno en el mosto puede estar presente en dos formas claramente diferenciadas: la inorgnica, bsicamente como amonio, y la orgnica, formada por aminocidos, pptidos y protenas. No todas estas formas son igualmente disponibles, ya que, por ejemplo, los pptidos y las protenas no se suelen considerar como autnticas fuentes de nitrgeno, y los aminocidos son muy variables como fuentes nitrogenadas, ya que mientras algunos son consumidos vidamente (glutamina, por ejemplo), otros no lo son en absoluto en condiciones anaerobias como la prolina. Curiosamente la prolina, junto con la arginina, son los dos aminocidos mayoritarios en mostos. La levadura necesita la presencia de oxgeno para llevar a cabo la utilizacin de la prolina, adems este proceso de metabolizacin de la prolina se lleva a cabo principalmente en la mitocondria, que se encuentra muy poco funcional en condiciones fermentativas. Sin embargo, no se ha estudiado si en condiciones de fermentaciones ms aireadas, tales como durante la maceracin en tintos, puede haber un consumo mayor de este aminocido. El nitrgeno en forma de amonio suele ser altamente disponible para las levaduras, por lo que es una forma qumica que se suele utilizar de forma abundante en la industria enolgica. La presencia de nitrgeno en cualquiera de estas formas qumicas es fuertemente variable, dependiendo de diversos factores, entre ellos la variedad, grado de maduracin de la uva, caractersticas edafo-climticas en las que se desarrolla sta y diversos aspectos tecnolgicos (tipo de vinificacin, prensado, etc). En resumen, debemos considerar como nitrgeno asimilable (NA) por las levaduras en las condiciones de vinificacin al amonio como fuente inorgnica (representa hasta el 40% del NA), y todos los aminocidos excepto la prolina. Sin embargo, la levadura vnica Saccharomyces cerevisiae no consume este ni-
1. introduccin:
La transformacin de la uva en vino es un proceso biotecnolgico donde los microorganismos presentes, fundamentalmente las levaduras, utilizan los nutrientes del mosto para su crecimiento, produciendo toda una gama de metabolitos que convierten un lquido empalagoso en una solucin hidroalcohlica de sabor y aroma ms agradable. La principal reaccin bioqumica durante la fermentacin alcohlica es la conversin de los azcares en etanol. Sin embargo, no es la nica. Un pequeo porcentaje de los azcares van destinados a la sntesis de metabolitos secundarios (glicerol, succnico, lctico, actico, alcoholes superiores, steres, etc), de menor concentracin que el etanol pero que son los determinantes de la calidad del vino. Tambin hay una pequea parte de los azcares del mosto que va destinada a la sntesis de componentes celulares, es decir, a la sntesis de biomasa. En esta sntesis de biomasa no solo intervienen los azcares sino que es imprescindible el componente nitrogenado. Aunque el componente nitrogenado es cuantitativamente el segundo nutriente ms abundante en el mosto, presenta una gran desproporcin con el componente carbonatado (azcares). Mientras que la suma de glucosa y fructosa se encuentra del orden de 200 a 300 g/litro, concentraciones de mil veces menores (200-300 mg/litro) suelen ser valores muy habituales para el nitrgeno asimilable. Por tanto, son
Seminario tcnico
2. efecto de la concentracin y tipo de nitrgeno sobre el crecimiento de las levaduras y su cintica fermentativa.
Varela y cols. (2004) propusieron que la velocidad de fermentacin depende por un lado del estado metablico de las clulas y por otro del nmero de clulas alcanzados durante la fermentacin alcohlica. Dicho de otro modo, de la viabilidad (nmero de clulas fermentando) y la vitalidad de cada una de estas clulas. Ambos componentes tienen una dependencia importante del nitrgeno disponible en el mosto. En este punto nos vamos a centrar en como afecta el nitrgeno a la divisin celular o produccin de biomasa y en el siguiente apartado abordaremos como afecta a la vitalidad o actividad celular. En un trabajo reciente de nuestro grupo hemos comprobado como el nitrgeno es el substrato limitante del crecimiento celular hasta alcanzar valores cercanos a los 200 mg/l, aunque esto depende de la fuente de nitrgeno utilizada. Para ello hacamos crecer las levaduras en un mosto sinttico (similar al natural) donde iba cambiando nicamente la concentracin de N y el tipo de fuente de N. Como puede verse en la figura 2, el nmero de clulas o biomasa (medido por la densidad ptica (O.D.) a 595 nm) iba aumentando conforme aumentaba la concentracin de nitrgeno, en este caso en forma de arginina como nica fuente de nitrgeno. Esto fue as hasta niveles de 180 mg de
Figura 1. Representacin grfica del mecanismo de Represin Catablica por nitrgeno (nCR) segn Cooper (2002)
Ponencias
01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
Figura 2. evolucin de la OD de la cepa PDM en mosto sinttico con arginina como nica fuente de nitrgeno y con concentraciones crecientes entre 20 y 140 mg n/l.
N/l (en la grfica solo se representa hasta 140 mg de N/l), por encima de esta cantidad ya no determinamos diferencias en la biomasa obtenida. Como se comenta anteriormente, no solo tiene importancia la cantidad sino la calidad o tipo de nitrgeno. En un experimento similar determinamos la biomasa producida para la misma concentracin de diferentes fuentes de nitrgeno. Como puede verse en la figura 3, la glutamina permite un mayor crecimiento celular que la arginina, y resulta sorprendente que la menor cantidad de biomasa producida se obtiene con la utilizacin de amonio, tericamente una de las fuentes de nitrgeno ms interesante para la levadura. Varela y cols (2004) determinaron el efecto que tena la cantidad de clulas en un mosto deficiente en N
(50 mg/l) sobre la velocidad y tiempo de fermentacin. Para ello aadan dos y 5 veces ms de la dosis recomendable de levadura seca activa a los mostos. El resultado fue que en estas fermentaciones, con mayor concentracin de clulas, se alcanzaban velocidades de fermentacin similares a las obtenidas en un mosto rico en nitrgeno (300 mg/l). De esta manera mostraban una relacin directa entre mayor concentracin de biomasa y mayor velocidad fermentativa en mostos pobres en nitrgeno. Desde un punto de vista aplicado para la industria enolgica, esto sugiere dos posibles soluciones frente a un mosto pobre en nitrgeno. La primera y ms habitual solucin consiste en la suplementacin de los mostos con nitrgeno, fundamentalmente en forma de sales de amonio. Como posible alternativa, sera la adicin de una mayor cantidad de levadura seca activa o la adicin de biomasa de otros
Figura 3. Comparativa de crecimiento en las tres fuentes de nitrgeno para una cantidad idntica de 140 mg n/l.
Seminario tcnico
Figura 5. efecto de la adicin de una mezcla de amonio y aminocidos (240 mg de n/l) sobre la velocidad fermentativa, medida como reduccin de la densidad del mosto. la densidad a que se realizaba la adicin viene reflejada en el pie de la figura.
Figura 6. Consumo de n total, amonio y aminocidos (expresados como mg de n/l) en las distintas fermentaciones.
Ponencias
01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
que la mayor parte de la fermentacin alcohlica se produce en la fase estacionaria o de no proliferacin celular. A pesar de que durante este periodo no hay multiplicacin celular, las levaduras necesitan nitrgeno para mantener su metabolismo activo. El medio de fermentacin es un ambiente cambiante y hostil que requiere una readaptacin continua para las nuevas condiciones. Esta readaptacin al medio supone un recambio proteico continuo en la maquinaria enzimtica celular. Para este recambio de protenas celulares, es necesaria la disponibilidad de nitrgeno en el medio de fermentacin. Taillandier y col. (2007) establecieron la cantidad de nitrgeno consumido por la levadura por gramo de azcar consumido (mg N/g de glucosa o fructosa). Como siempre hay que contar con las diferentes necesidades de las distintas cepas, esta cantidad tena un rango de 0,61 a 0,91 mg de N por gramo de azcar. Es decir, una relacin mg N/g azcar prcticamente asegura cubiertas las necesidades nitrogenadas y es una relacin muy sencilla para el enlogo poder calcular las diferentes necesidades de sus mostos. Una relacin similar establecieron Bisson y Butzke (2000), pero en este caso, entre necesidad de N asimilable frente a grado alcohlico probable (GAP) (figura 7). Figura 7. Relacin entre grado alcohlico probable (GaP) y cantidad necesaria de n asimilable (mg de n/l) para alcanzar dicha cantidad (Bisson y Butzke, 2000).
cols (2004) observaron una activacin del enzima fosfofructoquinasa, enzima clave de la glucolisis, con la adicin de amonio.
Sin embargo, el aumento de la cintica fermentativa no debe ser solo entendido por la disponibilidad de nitrgeno para facilitar el recambio proteico celular, sino que se ha demostrado que algunas fuentes de nitrgeno, en concreto el amonio, pueden actuar como activadores de la actividad de enzimas glucolticos claves. En concreto, se ha visto un incremento importante del transporte de azcares por activacin de las permeasas implicadas (genes HXT) (Bely et al., 1990). Taillandier y cols (2007) tambin informaron de una mayor actividad fructoflica (mayor consumo de la fructosa frente a la glucosa) de la levadura en presencia de mayor concentracin de nitrgeno, probablemente por un cambio en la afinidad de los transportadores de hexosas. Igualmente, Berthels y
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Seminario tcnico
Figura 8. Relacin entre la acumulacin de algunos compuestos voltiles y la concentracin inicial de nitrgeno en vinos producidos a partir de mosto sinttico con dos cepas Saccharomyces cerevisiae (cepa M522 () y cepa KU1 ()). (Carrau y col., 2008)
Ponencias
11
01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
tabla 1. Relacin entre el momento de la adicin de nitrgeno y la produccin de metabolitos secundarios por la levadura. las fermentaciones se realizaron con mosto sinttico limitante en nitrgeno (60 mg/l Yan), al que se aadi 240 mg/l de nitrgeno (mezcla amonio y aminocidos) en distintos momentos de fermentacin (densidad 1080 g/l o inicio, 1060, 1040, 1020, 1000 g/l y no suplementado). (Beltran y col. 2005)
Figura 9. Relacin entre la concentracin de metabolitos secundarios (normalizados con el valor de mayor produccin en cada caso) y el momento de la suplementacin de nitrgeno. las fermentaciones se realizaron con mosto sinttico limitante en nitrgeno (60 mg/l Yan), al que se aadi 240 mg/l de nitrgeno (mezcla amonio y aminocidos) en distintos momentos de fermentacin (densidad 1080 g/l o inicio, 1060, 1040, 1020, 1000 g/l y no suplementado).
Figura 10. Ruta de sntesis de alcoholes superiores a partir de aminocidos (via de ehrlich) o a partir de los azcares (via anablica)
COOH H C NH2
COOH H C O
H C O
NADHNAD+
H H C OH
R R R R NH3 CO2 Aminocido Aldehido Alcoholsuperior cetocido Desaminacin Descarboxilacin Reduccin ViadeEhrlich:viacatablicadeaas
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Seminario tcnico
sulfato. En carencia de nitrgeno, disminuye el pool de nitrgeno intracelular y por tanto la sntesis de los precursores nitrogenados O-AS y O-AH, con lo que el sulfito producido no podr utilizarse para la sntesis de aminocidos y se excretar en forma de cido sulfhdrico (SH2). As pues, el ratio de formacin del H2S parece estar regulado por los requerimientos celulares de aminocidos azufrados, y por el mantenimiento de los pools de nitrgeno intracelular. Jiranek y col. (1995) observaron que en fermentaciones vnicas existe una relacin directa entre la limitacin de nitrgeno y la sntesis de sulfhdrico. Algunos de sus resultados se muestran el la Figura 12 y Tabla 2. En la Figura 12 observamos que la produccin de sulfhdrico por parte de la levadura aumenta en el momento en que el nitrgeno del medio se consume (en presencia de sulfito). Este efecto es mucho ms pronunciado en fase exponencial de crecimiento, cuando la levadura tiene ms requerimientos nitrogenados, que en fase estacionaria, donde disminuyen las necesidades nitrogenadas, y tambin la produccin de sulfhdrico. Estos autores relacionaron tambin este aumento en la produccin con la actividad sulfito reductasa, la cual es mayor en fase exponencial de crecimiento. Por otro lado, si un medio limitante en nitrgeno se suplementa con amonio o con la mayora de aminocidos, la produccin de sulfhdrico disminuye. Pero este no es el caso de los aminocidos azufrados (principalmente Cistena, de manera individual o en combinacin con Metionina), la adicin de los cuales da lugar a un aumento incluso mayor en la produccin de H2S por parte de las levaduras (Tabla 2). Esto es debido a la liberacin del sulfhdrico como subproducto
Ponencias
13
01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
Figura 12. Relacin entre la produccin de H2S y el momento en que se produce la limitacin de nitrgeno durante la fermentacin. las fermentaciones se realizaron con la cepa aWRi77 en mosto sinttico con distintas concentraciones de nitrgeno inicial: 3.3 (), 16.6 () o 24.9 () mM. las flechas indican el momento en que se aadi sulfito (132 M) a los medios, 1 hora antes del consumo total de nitrgeno. (Jiranek y col. 1995)
tabla 2. Produccin de H2S por parte de la levadura aWRi77, 6 horas despus de haber suplementado un medio limitante en nitrgeno con aminocidos o amonio de manera individual. la suplementacin se realiz una hora antes de que el nitrgeno inicial fuera consumido. los datos estn normalizados al valor obtenido por el cultivo suplementado con amonio (Jiranek y col. 1995)
de la degradacin / transaminacin de estos aminocidos para ser utilizados como fuente de nitrgeno. En un trabajo reciente, Mendes-Ferreira y col. (2009) analizaron en varias cepas la relacin entre la concentracin de nitrgeno en el medio y la produccin de varios compuestos aromticos, entre ellos el cido sulfhdrico. Los resultados obtenidos variaban entre las distintas cepas, la Figura 13 muestra los resultados de la cepa UCD522, considerada alta productora de sulfhdrico. La sntesis de H2S se produce en el momento en que se consume el nitrgeno del medio, pero curiosamente el medio con una cantidad ptima de nitrgeno (267 mg/l), el cual se consume a las 48 horas de fermentacin, presenta valores mayores que el medio muy limitante en nitrgeno (66 mg/l). El exceso de nitrgeno disminuye la produccin de
sulfhdrico, pero este exceso puede provocar por otro lado la inestabilidad del vino y la produccin de otros compuestos indeseados. Pero el cido sulfhdrico no es el nico compuesto azufrado que podemos encontrar en los vinos, a partir de este compuesto se pueden formar mercaptanos o tioles, algunos de los cuales aportan olores desagradables a los vinos, los temidos caracteres de reduccin. H2S es un compuesto altamente reactivo, y se Figura 13. Perfil de fermentacin y de liberacin de cido sulfhdrico de la cepa UCD522 en medio sinttico y con distintas concentraciones de nitrgeno inicial: 66 mg/l (a), 267 mg/l (b) y 402 mg/l (c). (Mendes-Ferreira y col. 2009)
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Seminario tcnico
Bibliografa
alberts, e., C. larsson, G. liden, C. niklasson, l. Gustafsson (1996) Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl Environ Microbiol 62: 31873195 alberts, e., G. lidn, C. larsson, l. Gustafsson (1998) Anaerobic redox balance and nitrogen metabolism in Saccharomyces cerevisiae. Recent Res. Dev. Microbiol. 2:253-279. Bell, S.J., P.a. Henschke (2005) Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 11:242-295. Beltran, G., B. esteve-Zarzoso, n. rozes, a. Mas, y J. M. Guillamon. influence of the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. J agric. Food Chem, 53: 996-1002, 2005. Bely,M., Sablayrolles,J.M., Barre,P. (1990). Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions. J.Ferment.Bioeng. 70, 246-252. Bely, M., a. rinaldi, D. Dubourdieu (2003) Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. J. Biosci Bioeng. 96:507-512 Bisson,l.F., Butzke,C.e. (2000). Diagnosis and rectifications of stuck and sluggish fermentations. Am.J.Enol. Vitic. 51, 168-177. Carrau, F.M., K. Medina, l. Farina, e. Biodo, P.a. Henschke, e. Dellacassa (2008) Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains. FEMS Yeast Res. 8:11961207. Cooper,t.G. (2002). Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS Microbiol.Rev. 26, 223-238. Hernndez-Orte, P., J.F. Cacho, V. Ferreira (2002) Relationship between varietal aminoacid profile of grapes and wine aromatic composition. Experimetns with model solutions and chemometric study. J. Agric. Food Chem. 50: 2891-2899. Jiranek V, langridge P y Henschke Pa (1995a) Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen. Appl Environ Microbiol 61: 461467. Mendes-Ferreira,a., Mendes-Faia,a., leao,C. (2004). Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. J.Appl.Microbiol. 97, 540-545.
Ponencias
15
01. Relacin entre el contenido nitrogenado en mosto/uva y la sntesis de aromas: efecto sobre la produccin de sulfhdrico
Mendes-Ferreira, a., C. Barbosa, V. Falc, C. leao, a. Mendes-Faia (2009) The production of hydrogen sulphide and other aroma compounds by wine strains of Saccharomyces cerevisiae in synthetic media with different nitrogen concentrations. J. Ind. Microbiol. Biotechnol. DOI 10.10007/s10295-009-0527-x Oshita, K., M. Kubota, M. uchida, M. Ono (1995) Clarification of the relationship between fusel alcohol formation and amino acid assimilation by brewing fusel alcohol formation and amino acid assimilation by brewing yeast using 13C-labeled amino acid. Proceedings of the European Brewing Convention. Bruxelles, 387-394. Swiegers J. H. y i. S. Pretorius (2007) Modulation of volatile sulfur compounds by wine yeast Appl Microbiol Biotechnol 74:954960 thaillandier, P., ramon-Portugal, F., Fuster, a. y Strehaiano P. (2007) Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiology 24: 95-100.
thibon, C., P. Marullo, O. Claisse, C. Cullin, D. Dubourdieu, t. tominaga (2008) Nitrogen catabolic repression controls the release of volatile thiols by Saccharomyces cerevisiae wine fermentation. FEMS Yeast Res. 1-11. Varela C, Pizarro F, agosin e (2004) Biomass content governs fermentation rate in nitrogen-deficient wine musts. Appl Env Microbiol 70:33923400. Vermeulen, C., l. Gijs, S. Collin (2005) Sensorial contribution and formation pathways of thiols in foods: a review. Food Rev. Int. 21:69-137. Vilanova, M., M. ugliano, C. Varela, t. Siebert, i.S. Pretorius, P.a. Henschke (2007) Assimilable nitrogen utilisation and production of volatile and on-volatile compounds in chemically defined medium by Saccharomyces cerevisiae wine yeasts. Appl. Microbiol. Biotechnol. 77:145-157.
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Seminario tcnico
Ponencias
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02. los nuevos retos en microbiologa del vino. levaduras no productoras de SH2
isak S. Pretorius the australian Wine Research institute, PO Box 197, Glen Osmond, adelaide, Sa 5064, australia
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3. Optimizacin de la fermentacin 4. Optimizacin de la calidad del vino: evitar la produccin de sabores y aromas indeseables 5. Optimizacin de la calidad del vino: mejora del aroma deseable, el sabor y el color 6. Aumento de las opciones de los vinicultores mediante el desarrollo de cepas de levaduras vincolas mejoradas y novedosas 7. Conclusiones y previsiones futuras 8. Agradecimientos 9. Lecturas complementarias
Seminario tcnico
Resumen
Un mundo sin levaduras, esos humildes hongos unicelulares que nos ayudan a confeccionar alimentos y bebidas, tambin nos negara la capacidad de hacer avanzar las fronteras de la ciencia y de la tecnologa. La mayor parte de todo el abanico de funciones desempeadas por las levaduras es realizada por una sola especie, la Saccharomyces cerevisiae. De todas sus funciones, esta levadura es bien conocida por su capacidad para impulsar la transformacin del mosto en vino. Si bien la Saccharomyces cerevisiae es conocida como la levadura vinica, no se pueden considerar todas sus cepas iguales, dadas sus diferencias en el desarrollo de la fermentacin y atributos organolpticos. En la enologa moderna, donde es esencial disponer de fermentaciones rpidas y fiables para producir vinos estables en el tiempo y con estilo predeterminado y una calidad predecible, se prefiere la inoculacin en el mosto de cepas seleccionadas de Saccharomyces cerevisiae. Actualmente seguimos profundizando en el conocimiento de las interacciones asociadas con el vino que se producen entre nutrientes, precursores de sabores derivados de la uva, condiciones de la fermentacin y cepas especficas de levaduras. El presente documento aborda cmo los enologos pueden gestionar correctamente la fermentacin a travs de la dinmica de las levaduras en los mostos con el fin de optimizar su rendimiento y evitar el desarrollo de sabores y aromas indeseables, y sacar partido de cepas especializadas de levaduras de nuevo desarrollo y de las estrategias de inoculacin para mejorar la generacin de sabores.
1. introduccin
Imagnese, si puede, un mundo sin levaduras. Imagnese que se encuentra entre las muchas personas que asumen la comida y la bebida que hay en sus mesas sin preguntarse cmo ha llegado hasta all, tan sana y rica. Imagnese un mundo sin nada para subir la masa al cocer el pan, sin nada con lo que hacer cerveza, vino o bebidas de alta graduacin. Imagnese tambin un mundo en el que nuestra capacidad para hacer avanzar las fronteras de la ciencia y de la tecnologa en varios frentes de manera sana y relativamente barata estuviera muy limitada. Y por ltimo imagnese un mundo con muy pocas alternativas con las que convertir los residuos agrcolas ricos en azcar en bioetanol para el uso como fuentes de energa renovables. Si puede imaginarse todo eso, estar viendo un mundo en el que nuestro avance tecnolgico y nuestro patrn de utilizacin de energa, nuestra gastronoma, jovialidad y buen estilo de vida se veran afectados considerablemente. Lo que necesitamos y explotamos parar evitar tal mundo inimaginable es un humilde hongo unicelular que durante milenios nos ha ayudado a confeccionar
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02. Los nuevos retos en microbiologa del vino. Levaduras no productoras de SH2
alimentos y bebidas, y que en los ltimos tiempos ha sido moldeado por seleccin artificial para realizar una ms vasta, si cabe, variedad de funciones primero como agente de fermentacin vital en panificadoras, fbricas de cerveza, bodegas de vino y destileras y, en los tiempos ms recientes, como herramienta cientfica en los laboratorios de investigacin y como minifbricas para la produccin de productos biotecnolgicos de bajo volumen y gran valor tales como enzimas, productos qumicos, protenas teraputicas y otros productos farmacuticos importantes comercialmente. Lo que es an ms increble es que la mayora de esta gama de diversas funciones es realizada por una nica especie de levadura, la Saccharomyces cerevisiae. No obstante, de todas estas funciones y productos, esta extraordinaria levadura es probablemente mejor conocida por su capacidad para transformar la dulce, azucarada y poco sabrosa uva en el producto distintivo y rico en sabor que conocemos como vino. Los trminos levadura y fermentacin provienen etimolgicamente de palabras que se refieren al efecto de hervir o burbujear producido cuando el azcar se convierte bioqumicamente en alcohol etlico y dixido de carbono, pero la fermentacin producida por las levaduras es mucho ms que eso. De hecho, es la responsable de la mayora de los cambios asociados con la biotransformacin del mosto en vino el aroma, el sabor, la sensacin en el paladar, el color y la complejidad qumica son producidos por la levadura conforme diversifica y ampla su mundo con los productos de su metabolismo. Tradicionalmente el vino se elaboraba con las de levaduras ambientales que realizaban una fermentacin espontnea; la inoculacin deliberada de cultivos de levaduras puras es una tcnica relativamente reciente. En la fermentacin espontnea, existe una sucesin de levaduras indgenas provcedentes del viedo y de la bodega, pero las etapas finales estn dominadas de manera invariable por cepas de Saccharomyces cerevisiae tolerantes al alcohol. Hace mucho tiempo esta importante especie conocida universalmente como la levadura del vino, evolucion su capacidad para fabricar, acumular, tolerar y, bajo ciertas condiciones de crecimiento, incluso consumir alcohol, mientras que simultneamente produce metabolitos que mejoran el sabor y el aroma, tan importantes en nuestra apreciacin sensorial del vino. No obstante no todos los miembros de la tribu Saccharomyces cerevisiae pueden considerarse iguales, dado que existen diferencias en su robustez, rendimiento de la fermentacin y en los atributos sensoriales que introducen en el vino que las hacen nicas. En la vinicultura moderna, donde es esencial disponer de fermentaciones rpidas y fiables para producir homogneamente vino segn unas especificaciones definibles de sabor, unos estilos predeterminados y
una calidad predecible, se prefiere la inoculacin de cepas seleccionadas de Saccharomyces cerevisiae en el mosto. Las funciones principales de estas cepas seleccionadas son establecer un dominio numrico y metablico en la fase temprana de la fermentacin del vino y catalizar la conversin completa y eficiente de los componentes de la uva a alcohol, dixido de carbono y metabolitos que mejoran su sabor y aroma sin desarrollar sabores y aromas indeseables. La eleccin de la cepa de levadura a inocular en el caldo y la gestin eficaz de las interacciones entre la levadura, el mosto y las condiciones de fermentacin son factores importantes que determinan la duracin de la fermentacin y la composicin qumica y las propiedades organolpticas de un vino. En los apartados siguientes se aborda brevemente cmo los enlogos pueden controlar la dinmica de la levadura en los caldos para optimizar el desempeo de la fermentacin y por tanto evitar el desarrollo de muchos sabores y aromas indeseables y cmo pueden aprovecharse de cepas especializadas de reciente desarrollado para gestionar correctamente la fermentacin del vino.
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Seminario tcnico
3. Optimizacin de la fermentacin
Cuando se prepara el mosto a partir de uvas sanas y maduras, con una poblacin baja de microorganismo indgenas y un buen equilibro de nutrientes, si a este mosto se inocula un cultivo de levaduras vigoroso, la fermentacin empieza y finaliza rpidamente, dejando una pequea cantidad de azcar residual. Bajo estas condiciones ideales, las fermentaciones subptimas son relativamente infrecuentes. No obstante, cuando se producen, es necesario recuperar recursos considerables y pueden conducir a la prdida de la calidad del vino por un deterioro oxidativo, microbiolgico o autoltico de las levaduras. Se pueden producir fermentaciones subptimas en cualquier tipo de vino o producto en fermentacin, y stas pueden ocurrir incluso cuando se utilizan las cepas ms robustas fisiolgicamente hablando. Si bien las levaduras son enormemente adaptativas al ambiente del mosto, las condiciones fisicoqumicas y nutritivas no siempre son favorables para una actividad fisiolgica vigorosa y, a menos que la levadura se pueda adaptar a condiciones cambiantes durante la fermentacin, una reduccin del estado fisiolgico puede conducir a una fermentacin incompleta. Habitualmente se observan cuatro tipos de fermentaciones subptimas: (i) inicio con retraso; (ii) fermentacin continuamente lenta; (iii) fermentacin ralentizada; y (iv) fermentacin incompleta o interrumpida. Las fermentaciones que comienzan despus de un tiempo, pero que a menudo finalizan por ellas mismas de manera normal, son habitualmente el resultado de la utilizacin de un cultivo activador con una calidad deficiente. Las fermentaciones que constantemente son lentas, que pueden llegar a su finalizacin, habitualmente son el resultado de una cantidad reducida de nitrgeno asimilable. Las fermentaciones ralentizadas e interrumpidas, sin embargo, parecen ser causadas por varios factores relacionados con las interacciones entre la fisiologa de las levaduras, el estado de los nutrientes del mosto, la presencia de inhibidores y las condiciones de fermentacin. Concentraciones elevadas de azcar en el mosto, lo cual viene determinado principalmente por la ma-
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02. Los nuevos retos en microbiologa del vino. Levaduras no productoras de SH2
durez de la uva en su vendimia, es probablemente la causa principal de fermentaciones ralentizadas e interrumpidas, y esto se debe a su vez primordialmente a la relacin entre la concentracin inicial de azcar y la produccin final de etanol; cuanto ms azcar hay en la uva, ms alcohol habr en el vino. Este problema se magnifica por la tendencia a utilizar uvas muy maduras o demasiado maduras en las bodegas. Por tanto, cada vez es ms importante elegir cepas de levaduras con alta tolerancia al etanol. El riesgo de que aparezcan problemas de fermentacin aumenta cuando el mosto tiene un contenido subptimo de nutrientes, especialmente de nitrgeno asimilable y vitaminas. Es importante saber que estas ltimas se pueden perder durante la vendimia y el procesamiento del mosto. Un elevado ndice de azcar con respecto a otros nutrientes puede provocar una reduccin de la cantidad de biomasa y una bajada de la velocidad de fermentacin, as como el inicio precoz de la inactivacin del transporte de azcar en las levaduras. La clarificacin del mosto es un factor importante en la fermentacin de vinos blancos debido al profundo efecto que la disminucin de los slidos de las uvas tiene sobre la definicin de la variedad de la uva, sobre el sabor y aroma del vino y sobre la aparicin de aromas fenlicos indeseables. No obstante, el exceso de clarificacin elimina lpidos importantes necesarios para que las levaduras puedan tolerar el etanol; en la prctica se adquiere una solucin de compromiso determinada por la experimentacin para establecer el nivel de slidos de la uva que favorecen una tasa satisfactoria de fermentacin manteniendo la calidad del vino en un nivel aceptable. La incapacidad de finalizar la fermentacin de mostos demasiado clarificados, con una elevada cantidad de azcar y una fermentacin anaerbica, pueden evitarse parcialmente introduciendo un paso de aireacin breve durante la fermentacin. La aireacin, unida a la adicin de nitrgeno asimilable despus de que haya finalizado la fase de crecimiento de las levaduras, revigoriza considerablemente la fermentacin. Este paso de aireacin no tiene un impacto detectable sobre el sabor del vino e incluso es beneficioso ya que minimiza el riesgo del desarrollo de sabores y aromas indeseables que podran originarse a partir de una fermentacin incompleta. No obstante, cuando la fermentacin se frena debido a una cantidad elevada de azcar, el procedimiento de rescate ms exitoso depende de la adaptacin secuencial de un cultivo fresco de levaduras a los inhibidores del vino en fermentacin interrumpida. Este procedimiento, que comienza preparando un cultivo activador de una levadura altamente tolerante al etanol, implica multiplicar sucesivamente por dos el volumen de cultivo, por adicin al vino en fermentacin interrumpida, a la vez que se mantienen la aireacin y
el aporte de nutrientes. Despus de dos a cuatro ciclos de adaptacin al vino en fermentacin interrumpida, el cultivo de rescate altamente adaptado puede finalizar la fermentacin en presencia de concentraciones de etanol y de cidos grasos voltiles normalmente inhibidoras. Los procedimientos que mantienen la levadura en suspensin hasta que vuelve a comenzar la produccin de CO2 mejoran ms la tasa y la probabilidad de que se complete la fermentacin.
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Seminario tcnico
5. Optimizacin de la calidad del vino: mejora del aroma deseable, el sabor y el color
De lo anterior se desprende claramente que existen muchas herramientas para minimizar el riesgo de desarrollar sabores y aromas indeseables durante la fermentacin, pero debemos considerar qu estrategias podemos emplear para que las fermentaciones proporcionen caractersticas positivas al vino. La rehidratacin de la levadura con nutrientes de levaduras inactivas puede estimular la produccin de aromas afrutados. De manera similar, niveles modera-
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02. Los nuevos retos en microbiologa del vino. Levaduras no productoras de SH2
gunas variedades no aromticas como la Chardonnay, la Semillon y la Sauvignon Blanc. Entre los ejemplos de compuestos derivados de la fruta con sabor y aroma potenciales se encuentran los alcoholes monoterpenos florales linalool y geraniol, as como los norisoprenoides tales como la -damascenona. En variedades no aromticas, estos sabores y aromas se desarrollan durante la fermentacin, y la acidez del vino, la cual permite una hidrlisis qumica de los precursores no voltiles de aromas lenta, se ha considerando durante mucho tiempo una ruta importante de la formacin de estos compuestos. No obstante, trabajos recientes demuestran la existencia de al menos otras cuatros rutas (en las que estn implicadas las levaduras). De estas, la hidrlisis de glucoconjugados por glucsidos extracelulares de las levaduras parece ser la ms importante cuantitativamente durante la fermentacin. El color es otro atributo sensorial del vino muy importante y, en el vino tino, ste est ampliamente determinado por los antocianinos y pigmentos derivados de la antocianina. Existen numerosos factores viticulturales y viniculturales implicados en la formacin y en la estabilizacin del color del vino tinto, no obstante el papel de la levadura solamente se ha estudiado recientemente. La eleccin de la cepa de levadura no slo afecta a la profundidad del color, sino que tambin a su matiz; los tonos rojo-morados. Existe un estudio en curso para comprender el mecanismo bioqumico que afecta al desarrollo del color del vino. Los conocimientos extrados de este estudi permitirn desarrollar estrategias genticas para personalizar cepas de levadura con el fin de ofrecer a los consumidores vinos de un aspecto excepcional.
cida como tecnologa de modificacin gentica). A este respecto, el desarrollo de cepas de levaduras de vino es una fuente importante de una nueva diversidad gentica para mejorar las opciones disponibles de los vinicultores. Por ejemplo, la produccin de hbridos intra- e inter-especficos del gnero Saccharomyces permite la generacin de cepas novedosas de levaduras con las propiedades de fermentacin robusta de la levadura vincola comercial y las diversas propiedades sensoriales ofrecidas por otras especies. El sector est actualmente evaluando desde un punto de vista vincola los hbridos interespecficos de Saccharomyces kudriavzevii y Saccharomyces cariocanus con Saccharomyces cerevisiae. Se espera que estos hbridos aporten aromas multicapa diferenciadores en ciertos estilos de vinos. Otro ejemplo de aplicacin satisfactoria de estrategias sin modificacin gentica en nuestro programa de desarrollo de cepas es la produccin y la comercializacin de cepas novedosas de Saccharomyces cerevisiae con la capacidad de fermentar robustamente y de producir simultneamente niveles ptimos de tioles afrutados deseables y cantidades mnimas de H2S. En primer lugar, la investigacin de los beneficios de las estrategias de co-inoculacin ha contribuido al desarrollo de dos productos comercializados mixtos (una mezcla de cepas de un proporcin especfica) que mejoran los aromas afrutados provenientes de tioles. En segundo lugar, se han utilizado estrategias sin modificacin gentica para desarrollar cepas de vino que fermentan excepcionalmente bien sin producir cantidades detectables de H2S. Estas cepas comerciales que producen menos H2S obtuvieron resultados excelentes a escala industrial. Con el fin de empezar a investigar las bases genticas de las diferencias entre cepas de la levadura vnica, el Australian Wine Research Institute (Instituto de Investigacin Enolgica de Australia) obtuvo el genoma de una cepa de levadura vincola y lo compar con el de la cepa de Saccharomyces cerevisiae de laboratorio. La cepa de vino result ser muy diferente a su homloga de laboratorio. La secuenciacin del genoma de otras cepas industriales proporcionar una visin ms profunda de las variaciones presentes en las cepas vincolas y debera resaltar variaciones comunes y especficas de cepas que puedan asociarse con caractersticas industriales importantes. As como las investigaciones fundamentales sobre levaduras pasaron de estrategias clsicas reduccionista a estudios que implican todo el genoma, estos ltimos pasarn tambin al siguiente nivel de investigacin biolgica conocido como la biologa de sistemas. La denominada revolucin -mica promete, con la ayuda de modelos matemticos, la consecucin de una comprensin total de las clulas de la levadura vnica en toda su complejidad. Esta metodologa permitir el
6. aumento de las opciones de los vinicultores mediante el desarrollo de cepas de levaduras vincolas mejoradas y novedosas
Actualmente es indudable que las levaduras desempean un papel esencial a la hora de dotar al vino de sus propiedades sensoriales. Durante siglos de vinicultura se han aislado y utilizado, accidental o deliberadamente, cepas de Saccharomyces cerevisiae por su capacidad para convertir eficazmente el mosto de uva en vino a pesar de su exposicin a un esfuerzo osmtico, de nutrientes y etlico. Estas levaduras han desarrollado una serie de caractersticas especficas de cada cepa, como la produccin de diversos sabores y aromas y diferentes perfiles de utilizacin de nutrientes, lo cual es reflejo de su historia y ahora puede emplearse para adecuar cepas especficas a las condiciones y estilos concretos de cada vino. Adems de esta diversidad de opciones disponibles en la actualidad, se han mejorado muchas cepas de levadura vincola utilizando mtodos genticos clsicos (mutagnesis, hibridacin, evolucin adaptativa), y en los ltimos tiempos mediante ADN recombinate (tambin cono-
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Seminario tcnico
8. agradecimientos
El Australian Wine Research Institute Ltd (AWRI, Instituto de Investigacin Enolgica de Australia), un miembro del Wine Innovation Cluster (Grupo de Innovacin del Vino) de Adelaida, recibe el apoyo de viticultores y vinicultores australianos a travs de su agencia de inversiones, la Grape and Wine Research and Development Corporation (Corporacin para la Investigacin y el Desarrollo de la Uva y el Vino), la cual lo financia segn una metodologa matching funds con el Gobierno australiano. Me gustara agradecer la contribucin investigadora indicada en este documento a todos los miembros actuales y pasados del departamento de biociencia del AWRI: Caroline Abrahamse, Eveline Bartowsky, Jenny Bellon, Etjen Bizaj, Paul Chambers, Anthony Borneman, Antonio Cordente, Peter Costello, Chris Curtin, Angus Forgan, Paul Henschke, Wanphen Jitjaroen, Robyn Kievit, Ellie King, Dariusz Kutyna, Jane McCarthy, Jean Macintyre, Simon Schmidt, Tina Tran, Hentie Swiegers, Maurizio Ugliano, Cristian Varela y Gal Winter.
9. lecturas complementarias
1. Alexandre, H., P.J. Costello, F. Remize, J. Guzzo, & M. Guilloux-Benatier. 2004. Saccharomyces cerevisiaeOenococcus oeni interactions in wine: current knowledge and perspectives. International Journal of Food Microbiology 93:141154. Bartowsky, E.J. & I.S. Pretorius. 2009. Microbial formation and modification of flavour and off-flavour compounds in wine. In: H. Knig, G. Unden & J. Frlich (eds.), Biology of Microorganisms on grapes, in must and wine. Springer, Heidelberg, Alemania. Captulo 11, pp. 209-231. 8. 3. Bataillon, M., A. Rico, J.-M. Sablayrolles, J. -M. Salmon & P. Barre. 1996. Early thiamin assimilation by yeasts under enological conditions: impact of alcoholic fermentation kinetics. Journal of Fermentation and Bioengineering 82:145150. Bauer, F.F. & I.S. Pretorius. 2000. Yeast stress response and fermentation efficiency: how to survive the making of wine A review. South African Journal of Enology and Viticulture 21:27-51. Bell, S.J. & P.A. Henschke. 2005. Implications of nitrogen management for grapes and wine. Australian Journal of Grape and Wine Research 11:242-295. 9. 6. Bellon, J., A. Heinrich & P. Chambers. 2006. Yeast research increases choice for winemakers and consumers. Australian & New Zealand Grapegrower & Winemaker (514):102-103. Bellon, J., L. Rose, B. Currie, J. Ottawa, S. Bell, H. Mclean, C. Rayment, C. Treacher & P. Henschke. 2008 Summary from the winemaking with nonconventional yeasts workshops, 13th AWITC. Australian & New Zealand Grapegrower & Winemaker (528):72-77. Berthels, N.J., R.R. Cordero Otero, F.F. Bauer, I.S. Pretorius and J.M. Thevelein. 2008. Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different Saccharomyces cerevisiae wine yeast strains. Applied Microbiology and Biotechnology 77:1083-1091. Bisson, L.F. & C.E. Butzke. 2000. Diagnosis and rectification of stuck and sluggish fermentations. American Journal of Enology and Viticulture 51:168-177.
7. 2.
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10. Bisson, L.F. 1999. Stuck and sluggish fermentations. American Journal of Enology and Viticulture 50:107-119.
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11. Bohlscheid, J.C., J.K. Fellman, X.D. Wand, D. Ansen & C.G. Edwards. 2007. The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentation. Journal of Applied Microbiology 102:390400. 12. Borneman, A.R., P.J. Chambers & I.S. Pretorius. 2007. Yeast Systems Biology: modelling the winemakers art. Trends in Biotechnology 25:349355. 13. Borneman, A.R, P.J. Chambers & I.S. Pretorius. 2009. Systems biology as a platform for wine yeast strain development. In: H. Knig, G. Unden & J. Frlich (eds.), Biology of Microorganisms on grapes, in must and wine. Springer, Heidelberg, Alemania. Captulo 22, pp. 395-414. 14. Borneman, A.R., A.H. Forgan, P.J. Chambers & I.S. Pretorius. 2008. Unravelling the genetic blueprint of wine yeast. Australian and New Zealand Wine Industry Journal 23:21-23. 15. Borneman, A.R., A. Forgan, P.J. Chambers & I.S. Pretorius. 2008. Comparative genome analysis of a Saccharomyces cerevisiae wine strain. FEMS Yeast Research 8:1185-1195. 16. Borneman, A.R, P.J. Chambers & I.S. Pretorius. 2009. The way forward: modelling the winemakers art using yeast systems biology. In: P. Romano & G.H. Fleet (eds.), Yeast in Wine Fermentation. Springer, Heidelberg, Alemania (en prensa). 17. Boulton, R.B., V.L. Singleton, L.F. Bisson & R.E. Kunkee. 1998. Principles and Practices of Winemaking. USA: Aspen Publishers, Inc. 18. Chambers, P.J., Bellon, J.R., Schmidt, S.A., Varela, C. & Pretorius, I.S. 2008. Non-genetic engineering approaches to isolating and generating novel yeasts for industrial applications. In: G. Kunze & T. Satyanarayana (eds.), Diversity and Potential Biotechnological Applications of Yeasts. Elsevier (en prensa). 19. Cordente, A.G., A. Heinrich, I.S. Pretorius & J.H. Swiegers. 2009. Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production. FEMS Yeast Research (en prensa). 20. Curtin, C.D., J.R. Bellon, A.D. Coulter, G.D. Cowey, E.M.C. Robinson, M.A. de Barros Lopes, P. W. Godden, P.A. Henschke & I.S. Pretorius. 2005. The six tribes of Brett in Australia: Distribution of genetically divergent Dekkera bruxellensis strains across Australian winemaking regions. Australian and New Zealand Wine Industry Journal 20:2835.267:159-166.
21. De Barros Lopes, M., J.R. Bellon, N.J. Shirley & P.F. Ganter. 2002. Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species. FEMS Yeast Research 1:323-331. 22. Edwards, C. & J.C. Bohlscheid. 2007. Impact of pantothenic acid addition on H2S production by Saccharomyces cerevisiae under fermentative conditions. Enzyme & Microbial Technology 41:14. 23. Eglinton, J.M. & P.A. Henschke. 1993. Can the addition of vitamins during fermentation be justified? Australian Grapegrower and Winemaker (352):4749,5152. 24. Eglinton, J.M. & P.A. Henschke. 1999. Restarting incomplete fermentations: the effect of high concentrations of acetic acid. Australian Journal of Grape and Wine Research. 5:7178. 25. Fleet, G.H. 2003. Yeast interactions and wine flavour. International Journal of Food Microbiology 86:11-22. 26. Gockowiak, H. & P.A. Henschke. 1992. Nitrogen composition of grape juice and implications for fermentation: results of a survey made in N-E Victoria. Australian & New Zealand Grapegrower& Winemaker (340):131, 133138. 27. Henschke, P.A. 1997. Stuck fermentation: causes, prevention and cure. Allen, M.; Leske, P.; Baldwin, G., eds. Advances in juice clarification and yeast inoculation: proceedings of a seminar; 15 August 1996; Melbourne, Vic. Adelaide S.A: Australian Society of Viticulture and Oenology; 1997: 3038, 41. 28. Henschke, P.A. & V. Jiranek. 1991. Hydrogen sulfide formation during fermentation: effect of nitrogen composition in model grape must. Rantz, J.M., ed. Proceedings of the international symposium on nitrogen in grapes and wine; 18-19 June 1991; Seattle, Washington, USA. Davis, CA: American Society for Enology and Viticulture; pp. 172-184. 29. Houtman, A.C. & C.S. Du Plessis. 1981. The effect of juice clarity and several conditions promoting yeast growth on fermentation rate, the production of aroma components and wine quality. South African Journal of Enology and Viticulture 2:7181. 30. Howell, K.S., J.H. Swiegers, G.M. Elsey, T.E. Siebert, E.J. Bartowsky, G.H. Fleet, I.S. Pretorius, & M.A. de Barros Lopes. 2004. Variation in 4-mercapto-4methyl-pentan-2-one release by Saccharomyces cerevisiae commercial wine strains under diffe-
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02. Los nuevos retos en microbiologa del vino. Levaduras no productoras de SH2
lian & New Zealand. Grapegrower Winemaker 54. Torrea, D., & P.A. Henschke. 2004. Ammonium su(528):6871. pplementation of grape juice-effect on the aroma profile of a Chardonnay wine. Technical Review 52. Swiegers, J.H, S.M.G. Saerens & I.S. Pretorius I.S. 150:5963. 2008. The development of yeast strains as tools to adjust the flavour of fermented beverages to 55. Ugliano, M., P.A. Henschke & I.S. Pretorius. 2007. market specifications. In: D.H. Frenkel & F. BelanNitrogen management is critical for wine flavour ger (eds.), Biotechnology in Flavour Production. and style. Australian and New Zealand Wine InBlackwell Publishing, Oxford, Reino Unido. Capdustry Journal 22:24-30. tulo 1, pp.1-55. 56. Vilanova, M., M. Ugliano, C. Varela, T. Siebert, I.S. Pretorius & P.A. Henschke. 2007. Assimilable ni53. Swiegers J.H., R.L. Willmott, T.E. Siebert, K. Lattey, trogen utilisation and production of volatile and B.R. Bramley, I.L. Francis, E.S. King & I.S. Pretorius. non-volatile compounds in chemically defined 2009. The influence of yeast on the aroma of Saumedium by Saccharomyces cerevisiae wine yeasts. vignon Blanc wine. Food Microbiology 26:204Applied Microbiology and Biotechnology 77:145211. 157.
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Seminario tcnico
Los radicales formados durante este proceso intermedio pueden ser directamente reducidos por los fenoles y son mejores oxidantes que el oxgeno (Waterhouse y Laurie, 2006). Los principales productos secundarios de la reduccin del oxgeno en el vino sern quinonas y aldehdos, fundamentalmente el acetaldehdo. Las quinonas pueden participar en reacciones de polimerizacin que llevarn a la formacin de grandes polmeros, normal-
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la tcnica de la micro-oxigenacin
Estas observaciones pueden ser la base cientfica que explica los resultados de la micro-oxigenacin, tcnica que naci en la dcada de los 90. Est basada en el aporte controlado de pequeas cantidades de oxgeno al vino mediante un microdifusor poroso, de forma continua y lenta, siendo la velocidad de aporte de oxigeno inferior a la velocidad de consumo, evitando la acumulacin de ste en el vino. Los efectos positivos de la micro-oxigenacin sobre los vinos tintos se pueden resumir en: Estabilizacin del color Reduccin de olores a reduccin y sabores herbceos Suavizacin del vino por disminucin de astringencia Estos efectos se deben a la formacin de esos productos finales de la oxidacin en los vinos. As, las quinonas formadas pueden fijar compuestos azufrados y contribuir a la disminucin de los olores a reduccin en los vinos. Adems, las quinonas formadas pueden comenzar un proceso de polimerizacin de compuestos fenlicos, lo que puede modificar el sabor y la astringencia de los vinos. De igual forma, otro de los productos que aparecen, el acetaldehdo, participa en las reacciones de estabilizacin de color de los vinos tintos a travs de la formacin de compuestos fenlicos unidos por puente de etilo (tanino-tanino y antociano-tanino fundamentalmente) y favoreciendo la formacin de piranoantocianos, compuestos ms estables que los antocianos nativos de los vinos.
mente de color pardo, responsables del pardeamiento. Pero tambin, las quinonas pueden reaccionar y formar aductos con otros compuestos del vino, como los tioles, contribuyendo al control del problema de reduccin en los vinos (Waterhouse y Laurie, 2006; Vidal y Aagaard, 2008). Los aldehdos, en el caso del vino tinto sobre todo, pueden participar en reacciones que sern de inters pues favorecen la estabilizacin del color de los vinos (Timberlake y Bridle, 1976). Ya se haba observado desde hace tiempo una diferencia importante entre los resultados en el vino de una oxidacin lenta frente a una oxidacin rpida, que sugiere la existencia de unas reacciones adicionales, que ocurren de forma lenta y que aumentan el conjunto de sustratos oxidables en el vino (Singleton, 1986). As, lentamente, dos semiquinonas radicales se pueden unir covalentemente. Este proceso se llama polimerizacin regenerativa y lleva a la generacin de una hidroquinona reoxidable. Tiene un potencial de reduccin menor que sus constituyentes originales e incrementar la capacidad de tomar oxgeno del vino. Es decir, la polimerizacin regenerativa lenta lleva a unidades no oxidables (quinonas) a incorporarse en hidroquinonas reoxidables lo que incrementar los sustratos oxidables del vino y proteger a los sustratos originales. Si el oxigeno se aade rpidamente, esta reaccin no ocurre, se agotan tambin rpidamente los fenoles para reaccionar, con formacin de muchas quinonas y, por tanto, el pardeamiento de los vinos.
a) Control de la reduccin
Uno de los motivos por los que a este defecto se le llama olor a reduccin es por que normalmente aparece
Compuestos azufrados de bajo Pm Sulfuro de hidrgeno (H2S) Metanotiol (MeSH) Etanotiol (EtSH) Sulfuro de dimetilo (DMS) Sulfuro de dietilo (DES) Disulfuro de carbono (CS2) Disulfuro de dimetilo (DMDS) Disulfuro de dietilo (DEDS) Metil tioacetato (MeSAc) Etil tioacetato (EtSAc) Metionol
Descripcin del olor Huevos podridos Goma quemada, putrefaccin Cebolla, goma, terroso Col cocida, esparragos Ajo, goma Dulce, goma, azufrado Vegetal, col, cebolla (a alta concentracin) Olor desagradable, cebolla Azufrado, huevo, queso Azufrado, cebolla, ajo Col cocida
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Seminario tcnico
O2
H3C-S-S-CH3 12 ppb
Como se observa, los compuestos azufrados no desaparecen sino cambian a otras formas con umbrales de percepcin ms altos. Pero esta reaccin es reversible y en ambiente reductor se puede volver a formar tioles. Pero no hay que olvidar que otra reaccin es posible en presencia de oxgeno y es la reaccin entre las quinonas formadas por oxidacin de fenoles con los grupos SH de los compuestos azufrados (Waterhouse y Laurie, 2006).
As, estudios realizados micro-oxigenando vinos tintos han mostrado que los vinos con micro-oxigenacin tenan menos niveles de metanotiol y etanotiol que los vinos testigo, disminuyendo su concentracin por debajo de su umbral aromtico y adems no se detect acumulacin de disulfuros (McCord, 2003).
Es muy importante mantener el suficiente aporte de oxigeno para producir continuamente quinonas que atrapen las nuevas molculas azufradas que se pueden ir formando durante el envejecimiento del vino, tanto por hidrlisis de tiosteres o por condiciones anaerobias.
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Figura. Comparacin de la concentracin de etanotiol, metanotiol y dimetilsulfuro en vinos control, micro-oxigenados, micro-oxigenados y con aplicacin de duelas de roble y microoxigenados y con aplicacin de segmentos de roble (adaptado de McCord, 2003).
que son mas estables que los antocianos originales, dando lugar a una mayor estabilidad e intensidad del color de los vinos micro-oxigenados (Timberlake y Bridle, 1976; Francia-Aricha et al., 1997; Salas et al., 2004). La correcta aplicacin de la tcnica para conseguir los mayores beneficios va a depender del periodo de aplicacin y el tipo de vino a micro-oxigenar. Fundamentalmente, las mayores ventajas las vamos a encontrar cuando el vino est equilibrado, la proporcin de
antocianos-taninos sea correcta y haya una cantidad importante de antocianos libres. Si no hay suficientes antocianos libres en el vino para reaccionar se puede acumular acetaldehdo y verse favorecida la polimerizacin de taninos, hasta que las molculas formadas sean tan grandes que precipiten. Los diversos ensayos realizados han puesto de manifiesto que la etapa comprendida entre fermentacin alcohlica y fermentacin malolctica es la que mejores resultados suele dar por una alta presencia de antocianos libres, cido pirvico y ausencia de SO2. Esto se observa en los trabajos de Cano-Lpez et al. (2006), donde se ha puesto de manifiesto que se observa un incremento en la intensidad de color fundamentalmente en el primer periodo de microoxigenacin (entre la fermentacin alcohlica y el inicio de la fermentacin malolctica) en los vinos microoxigenados. Al finalizar la fermentacin malolctica se observ en todos ellos un descenso, probablemente consecuencia de la variacin del pH y la precipitacin de materia coloidal que arrastra materia colorante. Aunque el aporte de oxigeno durante el segundo periodo (se comenz cuando la fermentacin alcohlica ya esta finalizada) fue menor, se observ de nuevo un aumento de intensidad de color en los vinos microoxigenados, diferencindose todos los vinos en color, y observndose tambin el efecto de la dosis de oxgeno aplicada. El incremento de color se adscribi a una mayor concentracin de piranoantocianos y com-
Figura. evolucin del grado medio de polimerizacin de los taninos en vinos micro-oxigenados y sus correspondientes testigos (W1: vino de bajo contenido polifenlico, W2: vino de contenido polifenlico medio, W3: vino de alto contenido polifenlico). adaptado de Cano-lpez et al. 2008
Concentracin de las principales familias de antocianos y compuestos derivados en vinos de Monastrell micro-oxigenados (Cano-lpez et al., 2006). Compuestos identificados t0 Control Antocianos monomricos (mg L-1) Aductos directos (g L-1) Suma de Piranoantocianos Compuestos unidos por etilo (g L-1) Pico polimrico (mg L-1) 277.94.2c 190087a 14872374b 500846a 19.70.2a Control 1850.9b 212151b 9974225a 550314a 19.20.7a tf Dosis 1 1787.4b 194584a 1452499b 588318b 21.62ab Dosis 2 15916.1a 1858110a 1409116b 6262145c 23.41.1b
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Seminario tcnico
puestos unidos por puente de etilo en los vinos microoxigenados (Cano-Lpez et al., 2006). Tambin los efectos de la micro-oxigenacin sobre los taninos del vino han sido estudiados (Cano-Lpez et al., 2008). Se han observado diferentes comportamientos con respecto a la evolucin del grado medio de polimerizacin. Este parmetro siempre se incrementa en el primer periodo (fin de fermentacin alcohlica-inicio de fermentacin malolctica) para
Bibliografa
Cano-lpez, M.; Pardo-Mnguez, F.; lpez-roca, J.M.; Gmez-Plaza, e. (2006). Effect of microoxygenation on anthocyanin and derived pigment content and chromatic characteristics of red wines. American Journal of Enology and Viticulture 57, 325-331. Cano-lpez, M.; Pardo-Mnguez, F.; Schmauch, G.; Saucier, C.; teissedre, C.; lpez-roca, J.M.; GmezPlaza, e. (2008). Effect of Micro-oxygenation on Color and Anthocyanin-Related Compounds of Wines with Different Phenolic Contents. Journal of Agricultural and Food Chemistry, 56, 5932-5941 Francia-aricha, e.; Guerra, M.t.; rivas-Gonzalo, J.; Santos-Buelga, C. (1997). New anthocyanin pigments formed after condensation with flavanols. Journal of Agricultural and Food Chemistry, 45, 2262-2266. rauhut. D (2002). Volatile sulfur compounds. Impact on reduced sulfur flavor defects and atypical aging in wine. 31st Annual New York Wine Industry Conference. Salas, e.; atanasova, V.; Poncet-legrand, e.; Meudec, e.; Mazauriz, J.; Cheynier, V. (2004). Demostration of occurrence of flavanol-anthocyanin adducts in wine and model solutions. Analytica Chimica Acta, 213, 367-370. Singleton, V. (1987). Oxygen with phenols and related reactions in musts, wines and model systems. Observations and practical implications. American Journal of Enology and Viticulture, 38, 69-77. timberlake, C.; Bridle, P. (1976). Interaction between anthocyanins, phenolic compounds and acetaldehyde and their significance in red wines. American Journal of Enology and Viticulture, 27, 97-105. Vidal, S., aagaard, O. (2008). Oxygen management during vinification and storage of Shiraz wine. Wine Industry Journal, 23, www. Winebiz.com.au Waterhousse, a.; laurie, F. (2006). Oxidation of wine phenolics. A critical evaluation and hypothesis. American Journal of Enology and Viticulture, 57, 306-312. Zoecklein, B. (2006). Sulfur compounds: impact on aroma, flavor and texture and practical management. Enology notes, 113 Zoecklein, B. (2007). Factors impacting sulfur-like off odors in wine and winery options. 8th Annual Enology and Viticulture British Columbia Wine Grape Council Conference.
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04. los compuestos voltiles no son todos perjudiciales! los tioles varietales y el sulfuro de dimetilo
Rmi GUeRin-SCHneiDeR institut Franais de la Vigne et du Vin entaV-itV France
Seminario tcnico
Rmi Guerin-Schneider
Ingeniero Agrnomo y Enlogo, Schneider, completa su formacin con una Tesis Doctoral, en el ao 2001, sobre el potencial aromtico de los vinos de Muscadet. Ingeniero de Investigacin y Desarrollo el Instituto francs de la Via y del Vino desde 2001, est encargado del Proyecto tiles y mtodos de caracterizacin de la calidad de uvas y vinos Particularmente especializado en el anlisis de compuestos aromticos de vinos y de sus precursores, Remi Schneider, es autor de numerosas publicaciones cientficas centradas en la puesta apunto de metodologas analticas sensibles y fiables, particularmente empleando dilucin isotpica, as como mtodos de estimacin del potencial aromtico de uvas procedentes de viedos de alta produccin. Desde el ao 2006, coordina un programa de investigacin (UMT Qualinnov ) agrupando equipos de el INRA y del IFV.
Publicaciones principales
R. SCHNEIDER, A. RAZUNGLES, C. AUGIER, R. BAUMES, 2001. Monoterpenic and Norisoprenoidic Glycoconjugates of Vitis vinifera L. cv. Melon B. as Precursors of Odorants in Muscadet Wines. J. Chromatogr., 936, 145-147. R. SCHNEIDER, A. RAZUNGLES, F. CHARRIER, R. BAUMES, 2002. Effet du site, de la maturit, et de lclairement des grappes sur la composition aromatique des baies de Vitis vinifera L. cv. Melon B. dans le vignoble du Muscadet. Bulletin de lOIV,855-856, 269-283. A. BELANCIC-MAJCENOVIC, R. SCHNEIDER, J.P. LEPOUTRE, V. LEMPEREUR, R. BAUMES, 2002. Synthesis and Stable Isotope Dilution Assay of Ethanethiol and Diethyl Sulfide in Wine using Solid Phase Microextraction. Effect of Aging on their Levels in Wine. J. Agric. Food Chem., 50, 6653-6658. R. SCHNEIDER, Y. KOTSERIDIS, J.L. RAY, C. AUGIER, R. BAUMES, 2003. Quantitative Determination of Sulfur-Containing Wine Odorants at sub-ppb Levels. Part II : Development and Application of a Stable Isotope Dilution Assay. J. Agric. Food Chem., 51, 3243-3248. R. SCHNEIDER, F. CHARRIER, M. MOUTOUNET, R. BAUMES, 2004. Rapid Analysis of Grape Aroma Glycoconjugates using Fourrier-Transform Infrared Spectrometry and Chemometric Techniques. Analytica Chimica Acta, 513, 91-96. C. PROUTEAU, R. SCHNEIDER, Y. LUCCHESE, F. NEPVEU, R. RENARD, C. VACA-GARCIA, 2004. Improving Headspace Solid phase Microextraction of 3-Isobutyl-2-methoxypyrazine by Experimental Design with Regard to Stable Isotope Dilution Gas Chromatography- Mass Spectrometry Analysis of Wine. Analytica Chimica Acta, 513, 223-227. DAZ-MAROTO M.C., SCHNEIDER R., BAUMES R., 2005. Formation pathways of ethyl esters of branched short-chain fatty acids during wine aging. J. Agric. Food Chem., 53, 3503-3509. DAGAN L., SCHNEIDER R., LEPOUTRE J.P., BAUMES R., 2006. Stability of sotolon in acidic and basic aqueous solutions. Application to the synthesis of a deuterated analogue for its quantitative determination in wine. Analytica Chimica Acta, 563, 365-374. SCHNEIDER R., CHARRIER F.,RAZUNGLES A., BAUMES R., 2006. Evidence for an alternative biogenetic pathway leading to 3-mercaptohexanol and 4-mercapto-4-methylpentan-2-one in wines. Analytica Chimica Acta, 563, 58-64. M. SUBILEAU, R. SCHNEIDER, JM. SALMON, E. DEGRYSE, 2008 Nitrogen catabolite repression modulates the production of aromatic thiols characteristicof SauvignonBlanc at the level of precursor transport, FEMS Yeast Res, 8, 771780
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04. Los compuestos voltiles no son todos perjudiciales! Los tioles varietales y el sulfuro de dimetilo
Los compuestos azufrados voltiles estn presentes en gran nmero en los vinos. Se acostumbra a separarlos en dos clases distintas, los compuestos sulfurosos ligeros, cuyo punto de ebullicin es inferior a 90C, y los compuestos sulfurosos pesados que presentan un punto de ebullicin ms elevado. Esta clasificacin, prctica por su simplicidad, aporta sin embargo escasa informacin acerca de la naturaleza de los compuestos y de su origen. Si nos adentramos con ms detalle en el tema, encontramos tres grandes familias de compuestos en trminos de estructura qumica: los tioles, los mono-o polisulfuros y los tiol-ster. De manera general, las funciones qumicas azufradas confieren a estos compuestos olores desagradables, intensos, que son percibidos negativamente. No obstante, cuando su peso molecular aumenta, el efecto negativo del grupo funcional sulfuroso se atena y las percepciones olfativas de los compuestos son ms agradables. As pues, la mayor parte de los compuestos sulfurosos ligeros, principalmente en productos en curso de fermentacin son perjudiciales para la calidad del aroma, mientras que los compuestos sulfurados ms pesados, pueden contribuir de manera favorable a mejorar el perfil aromtico de los vinos. Es concretamente el caso de los tioles varietales (Tominaga et al. 1998) y del sulfuro de dimetilo (Sgurel, 2005).
ritariamente presente en la piel (Murat et al., 2001b; Peyrot des Gachons et al., 2002a). As pues, la maceracin pelicular afecta principalmente al P3MH, cuyas cantidades recuperadas en el mosto son ms importantes en relacin a una vinificacin clsica (Murat et al., 2001b; Peyrot des Gachons et al., 2002a). Cabe sealar igualmente la presencia en los mostos de Sauvignon Blanc de un conjugado del 3 mercaptohexanol o glutathion que ser el precursor biogentico del conjugado a la cisteina correspondiente, por hidrlisis de los dos aminocidos asociados a la cisteina del glutation (Peyrot des Gachons et al., 2001). Ms recientemente (Fedrizzi et al, 2009) un conjugado de la 4MMP al glutathion, ha sido formalmente identificado en un mosto de Sauvignon. An que poco abundantes y poco numerosos, estos precursores cisteinicos contribuyen sin embargo en gran manera al aroma del vino. Son en efecto el origen de cuatro tioles extremadamente olorosos, ausentes en la uva, pero responsables en el vino de notas olfativas reconocibles cuando sus contenidos son los suficientes el 3-sulfanilhexan-1-ol (3MH), el acetato de 3-sulfanylhexyle (ac3MH), el 4-metil-4-sulfanilpentan-2-one (4MMP), presentando umbrales de percepcin olfativa muy bajos, respectivamente de 60 ng/L, 4,2 ng/L y 0,8 ng/L en solucin hidroalcohlica (Tominaga et al., 2000). Estos presentan respectivamente olores de pomelo, de corteza de ctricos y de boj, que participan de manera importante en el afrutado de numerosos vinos blancos y rosados. Si la 4MMP parece especfica de un reducido nmero de variedades (Sauvignon, Bacchus, Scheurebe), el 3-sulfanyl-hexanol y su acetato han sido observados en un gran nmero de variedades. Estos participan no solamente en el aroma de los vinos de numerosas variedades blancas como pueden ser la Gewrztraminer, Scheurebe, Colombard, Petit Manseng, Melon Blanc sino igualmente en tintas, como en la Garnacha, Merlot, Cabernet franc, Cabernet Sauvignon, Syrah, Mourvdre, Cinsault (Darriet et al., 1993; Darriet et al., 1995; Gth, 1997a; Tominaga et al., 2000; Tominaga et al., 1996; Gth, 1997a; Bouchilloux et al., 1998; Kotseridis et Baumes, 2000; Lopez et al., 2003; Murat et al., 2003; Schneider et al., 2003; Fretz et al., 2005, Ferreira et al., 2002, Schneider et Masson, 2009). Estudios recientes (Ferreira et al 2002; Masson et Schneider, 2009), han demostrado que el mercaptohexanol era por otra parte un aroma clave en los vinos rosados de Garnacha, y ms concretamente en los Rosados de Provence (Masson et Schneider, 2009). Esta ubicuidad del 3MH es en parte debida a una segunda va de formacin de este tiol durante la fermentacin, no proveniente de S-conjugados a la cisteina de la uva, sino al aadido de fuentes sulfurosas al (E)-2-hexenal en un mosto en fermentacin (Schneider et al., 2006). Esta va de formacin ha sido por otra
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Seminario tcnico
2. el sulfuro de dimetilo
El sulfuro de dimetilo (DMS) es un compuesto sulfuroso ligero evidenciado por Du Plessis et Loubster (1974) en el vino, en el que sus umbrales de percepcin olfativa eran del orden de 25 g/l. Sus contenidos en los vinos jvenes eran ms bien inferiores a este umbral, pero podan alcanzar los 900 g/l en vinos ms evolucionados (Dagan 2006 y referencia mencionada). El DMS es uno de los constituyentes importantes del aroma de trufas, una nota olfativa a menudo citada para el bouquet de reduccin de los grandes vinos
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04. Los compuestos voltiles no son todos perjudiciales! Los tioles varietales y el sulfuro de dimetilo
Los anlisis de uvas efectuados a da de hoy han revelado un potencial en DMS extremadamente heterogneo, y a veces muy elevado en algunas muestras de uva de la variedad Petit y Gros Manseng en estado de sobremaduracin, de hasta 4,5 mg/L (Dagan, 2006), mucho ms elevado que los contenidos encontrados en la Garnacha y el Syrah en maduracin tecnolgica (Sgurel et al., 2004). Para las muestras de estas cuatro variedades, las nicas estudiadas a da de hoy, el PDMS es dependiente de la variedad, del terreno y de la aada, y sus contenidos aumentan de manera muy importante en estado de sobremaduracin en el caso de la variedad Manseng, los nicos estudios realizados a da de hoy para este parmetro. Sin embargo, en algunas de las muestras de estas cuatro variedades, el PDMS de las uvas, quizs mucho ms elevado que en los vinos correspondientes, la simple degradacin qumica no puede explicar estas prdidas a veces considerables de transmisin de PDMS. Las causas de estas prdidas son a da de hoy desconocidas, pero existen varias hiptesis que podran explicarlas. La hiptesis de la degradacin de la SMM por las levaduras es muy plausible, ya que ha sido descubierta recientemente una nueva permeasa, capaz de transportar de manera especfica la SMM, que permitira a la Saccharomyces cerevisiae utilizar la SMM como fuente de azufre (Rouillon et al., 1999), pero la evolucin de la SMM en la levadura no ha sido todava estudiada. Sea cual sea, el DMS formado sera casi enteramente eliminado por arrastre por el gas carbnico durante la fermentacin
Conclusin
Los compuestos sulfurados juegan un papel importante en el aroma de los vinos. Si los compuestos sulfurados ligeros, excepto el DMS, son sistemticamente vectores de defectos organolpticos cuando superan sus umbrales de deteccin olfativa, los compuestos sulfurados ms pesados pueden conferir notas olfativas muy interesantes y refinadas. Es especialmente el caso de los tioles varietales, caracterizados por sus notas de ctricos y de boj de los Sauvignon, que de una manera general aportan frescor y afrutado a los vinos. El caso del DMS se muestra mucho ms complejo si cabe, por una parte su efecto realzante en bajas concentraciones, pero que en elevadas concentraciones puede ser percibido negativamente en algunos tipos de vinos blancos. As pues, disponemos a da de hoy de numerosos elementos tecnolgicos para controlar y dominar los contenidos de estos compuestos en los vinos, no hay que olvidar que el aroma de un vino es un conjunto complejo, y que al exacerbar uno u otro de sus componentes, nos arriesgamos a desequilibrar el perfil aromtico y ofrecer simplemente una caricatura.
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Seminario Tcnico
Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
Beltran, G.; Esteve-Zarzoso, B.; Rozs, N.; Mas, A. And Guillamn J.M. Unitat dEnologia del Centre de Referncia de Tecnologia dAliments, Department Bioqumica i Biotecnologia, Facultat dEnologia de Tarragona, Universitat Rovira i Virgili, Ramn y Cajal, 70, 43005 Tarragona, Spain, and Bodegas Miguel Torres, Miquel Torres i Carb 6, 08720 Vilafranca del Peneds, Barcelona, Spain Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. In the present study, we have supplemented nitrogen-deficient fermentations with a mixture of ammonium and amino acids at various stages throughout the alcoholic fermentation. The timing of the nitrogen additions influenced the biomass yield, the fermentation performance, the patterns of ammonium and amino acid consumption, and the production of secondary metabolites. These nitrogen additions induced a nitrogen-repressed situation in the cells, and this situation determined which nitrogen sources were selected. Glutamine and tryptophan were the main amino acids consumed in all the fermentations. Ammonium is the preferred nitrogen source for biomass production but was hardly consumed when it was added in the final stages of the fermentation. The higher ammonium consumption in some fermentations correlated with a greater synthesis of glycerol, acetate, and acetaldehyde but with a lower synthesis of higher alcohols. KEYWORDS: Saccharomyces cerevisiae; Alcoholic Fermentation; Amino acids; Ammonium; GAP1; MEP2
INTRODUCTION
The nitrogen composition of grape musts affects the growth and metabolism of yeast, the fermentation rate, and the completion of fermentation (1). Nitrogen deficiencies are one of the main causes of stuck or sluggish fermentations. One way of avoiding these problems is to add nutritional supplements, usually inorganic forms of nitrogen such as ammonium salts, to grape must prior to fermentation (2-4). These additions are generally made empirically in wine cellars, and the initial nitrogen concentration in the must or the nitrogen requirements of the usual yeast strain used in the cellar are not determined. Yeasts respond metabolically to differences in nitrogen availability, so this lack of control of nitrogen leads to differences in wine composition. Nitrogen affects yeast cells in two ways: it increases biomass production and stimulates the rate of sugar utilization. Nitrogen additions during the period of cell growth have resulted in maximum cell populations. Later additions during the stationary phase have had no effect on the cell population but have increased the specific fermentation rate, thus reducing the length of the fermentation (2, 5, 6). Nitrogen supplementation affects the pattern of nitrogen uptake. Ammonium is a preferred yeast nitrogen source, and when plentiful, it represses the expression of catabolic pathways by degrading other nitrogenous compounds (7, 8). This mechanism, called nitrogen catabolite repression (NCR), has recently been studied during wine fermentations (9). It inhibits the uptake of arginine and alanine and stimulates the consumption of branched-chain and aromatic amino acids. Changes in the nitrogen uptake patterns influence the production of aroma and spoilage compounds (particularly hydrogen sulfide) and the amount of urea, the major precursor of the carcinogen ethyl carbamate (10-13). The volatiles identified in wines are usually dominated by fermentation products. Organic acids, higher alcohols, and esters are the main group of flavor compounds coming from yeast metabolism (12). Higher alcohols can be produced either by the catabolic conversion of the branched-chain amino acids (via Ehrlich) or by the anabolic formation of these amino acids de novo from a sugar substrate (14). An excess of higher alcohols (above 400 mg L-1) can be regarded as a negative influence on the quality of wine, but at the concentrations generally found in wines (below 300 mg L-1), they usually contribute to the desirable complexity of wine. Furthermore, these alcohols, together with the acids in wine, are substrates for ester formation. Most esters, with the exception of ethyl acetate, impart a pleasant smell of fruits and flower notes in the wine (15).
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Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
As mentioned, in winemaking, most of the nitrogen additions are made empirically and do not take into account the different nitrogen needs of the cell during wine fermentation, the proper timing of these additions, or the nitrogen source added. In this study, we supplemented nitrogen-deficient fermentations with a mixture of ammonium and amino acids at different stages of the alcoholic fermentation. We then studied the effect of these additions on the fermentation kinetics, the consumption of organic and inorganic nitrogen throughout the fermentation, and the influence of this consumption on the organoleptic profile of the wines. We also monitored the effect of the nitrogen supplementations on the NCR system and the effect of the nitrogen-repressed situation on nitrogen uptake.
In the latter stages of the fermentation, the sugar consumption was assayed by enzymatic kits (Roche Applied Science, Germany). Fermentation was considered to be complete when the residual sugars were below 2 g L-1. Cell growth was determined by absorbance at 600 nm. Absorbance values were corrected for the initial absorbance reading obtained for juice. Cells were harvested at different points during the fermentation so that mRNA could be analyzed. Flasks were magnetically stirred to resuspend settled biomass, transferred to centrifuge tubes, and centrifuged at 5000 rpm for 5 min at room temperature to prevent temperature shock. Cell pellets were transferred to 1.5 mL Eppendorf tubes and frozen immediately in liquid nitrogen. They were kept at -80 C until they were analyzed. The supernatant of these samples was stored at -20 C for extracellular metabolites and nitrogen content analysis. Nitrogen Content Analysis. YAN was analyzed by the formol index method (18), and the ammonium content was quantified using an enzymatic method (Roche Applied Science, Germany). The individual amino and imino acids were analyzed by OPA and FMOC derivatizations, respectively, using the Agilent 1100 Series HPLC equipped with a low-pressure gradient quaternary pump, a thermostated autosampler, a DAD ultraviolet detector, and a fluorescence detector (Agilent Technologies, Germany). The sample (2 L) was injected into a 4.6 mm 250 mm 5 m Hypersil ODS column (Agilent Technologies, Germany). The gradient solvent system was: solvent A (16 mM sodium acetate and 0.022% triethylamine, adjusted to pH 7.2 with 1-2% acetic acid, and 0.6% tetrahydrofuran) from 100% at t ) 0 to 0% at t ) 18 min, and solvent B (20% of 66 mM sodium acetate, adjusted to pH 7.2 with 1-2% acetic acid, 40% acetonitrile and 40% methanol) from 0% at t ) 0 to 100% at t ) 18 min. The analysis temperature was 40 C, and the flow rate was 1.5 mL min-1. Several dilutions of each sample were analyzed and averaged using the analysis software. The concentration of each amino acid was calculated using external and internal standards and expressed as mg L-1. The software used was Agilent ChemStation Plus (Agilent Technologies, Germany). Ethanol, Glycerol, and Organic Acid Analysis. Ethanol, glycerol, and organic acids were analyzed in all the samples at the end of the fermentation process. Analytical HPLC was carried out on a Hewlett- Packard HP 1050 connected to a Hewlett-Packard Integrator 3395 equipped with an HP 1047 RI detector (Agilent Technologies, Wilmington, DE) (19). The wine sample (450 L) was mixed with 50 L of formic acid (internal standard), and 25 L was injected into a 300 mm 7.8 mm AMINEX HPX-87H column (BioRad, Hercules, CA). The solvent used was sulfuric acid 2.5 mM at 0.5 mL min-1. The analysis temperature was 60 C. The con-
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The PE5700 cycler provided cycle-by-cycle measurement of the fluorescence emission from each PCR reaction. Analysis resulted in the assignation of a threshold cycle (Ct) value to each PCR reaction. The Ct value is the cycle number at which an increase in reporter fluorescence above a baseline signal can first be detected. The threshold was positioned to intersect the exponential part of the amplification curve of positive reactions, as recommended by Applied Biosystems. The Ct value is inversely proportional to the log of the amount of template in the PCR reaction; the lower the
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All PCR reactions were mixed in 96-well optical plates (Applied Biosystems) and cycled in a PE Applied Biosystems 5700 thermal cycler under the following conditions: 50C for 2 min, 95C for 10 min, and 40 cycles at 95C for 15 s and at 60 C for 60 s.
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Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
Ct value, the higher the concentration of template in the PCR reaction. Assuming a 100% effective PCR amplification, a difference of one Ct value corresponds to a 21 ) 2-fold difference in the amount of template. All samples were analyzed in duplicate, and the expression values were averaged by the analysis software (Applied Biosystems). The coefficient of variation in all samples analyzed was less than 10%.
and amino acid nitrogen. Unlike their effect on the O. D. values, the nitrogen additions clearly stimulated the fermentation regardless of when they were made. In the nitrogen-deficient fermentation, yeast consumed the total YAN after the first day (data not shown). However, nitrogen was not completely consumed in the control fermentation. The nitrogen additions were all carried out when the initial YAN had already been depleted, and the later the nitrogen was added, the lower the amount of YAN was consumed (Table 2). The ammonium consumed was 54% of the total YAN consumed in the control fermentation (Table 2), but this proportion decreased when nitrogen was added later in the fermentation. Ammonium was proportionally preferred as the nitrogen source when the additions were made in the first half of the fermentations (N1060 and N1040). In later additions (N1020 and N1000), the small amount of nitrogen consumed was mostly from amino acids. The consumption of amino acids was monitored at different points during the fermentations. The yeasts pattern of amino acid utilization changes with the time of YAN supplementation (Table 2). The amino Figure 1. Effect of nitrogen additions on O. D. measures ( = 600 nm) throughout synthetic grape must fermentations. The arrows indicate the time of addition.
RESULTS
Effect of Nitrogen Addition on Fermentation Kinetics and Nitrogen Consumption. Five fermentations started with a nitrogen content of 60 mg L-1, which is low enough for a fermentation to be sluggish but high enough for it to finish. Four of these nitrogen-deficient fermentations were supplemented at different points with 240 mg L-1 of YAN; the first one at a density of 1060 g L-1, and the second, third, and fourth at 1040, 1020, and 1000 g L-1, respectively. The remaining fermentation was not supplemented, but subjected to nitrogen deficiency throughout the process. As a fermentation control, we used the same medium with a nondeficient amount of nitrogen (300 mgN L-1) (9). Figure 1 shows the effect of nitrogen additions on O. D. measures throughout the fermentations studied. The nitrogendeficient fermentations had lower O. D. values than the control fermentation. When nitrogen was added in the first half of the fermentations (density of 1060 and 1040), these effects were almost overcome, and the O. D. values were similar to those of the control fermentation. Additions at densities of 1020 and 1000, however, had minimal effects on O. D. measures. Table 1 summarizes the evolution of the fermentation and nitrogen consumption, measured as ammonium
Table 1. Determination of Yeast Assimilable Nitrogen (YAN) in the Fermentation Media, Represented by the Amino Acid Fraction (YAN aas) and bythe Ammonium fraction (YAN NH4+)
control fermentation density () 1080 1060 1040 1020 1000 990 (endb) time (h) 0 30 56 96 168 312 YAN NH4+ (mg N L- 1) 120 70 52 36 35 41 N addition at =1020 density () 1080 1060 1040 1020 1000 990 (endb)
a
N addition at F ) 1060 YAN aas (mg N L- 1) 168 98 95 98 98 102 time (h) 0 36 62 96 168 312 YAN NH4+ (mg N L- 1) 25 0.5 (90a) 50 45 42 50 YAN aas (mg N L- 1) 41 4 (145a) 105 104 108 110 time (h) 0 36 78 120 192 336
N addition at = 1040 YAN NH42+ (mg N L- 1) 25 0.1 0.1 (92a) 68 65 66 no N addition time (h) 0 36 78 150 264 504 YAN NH4+ (mg N L- 1) 26 0 0 0 1 1 YAN aas (mg N L- 1) 40 5 2 1 1 1 YAN aas (mg N L- 1) 40 4 4 (136a) 104 110 114
N addition at = 1000 time (h) 0 36 78 150 264 456 YAN NH4+ (mg N L- 1) 26 0 0 0 0 (99a) 100 YAN aas (mg N L- 1) 40 7 4 4 2 (147a) 130
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NH4+ and aas YAN content just after the nitrogen addition. b End of fermentation.
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full N media content 47.4 10.3 9.9 3.8 4.7 3.8 2.8 5.1 7.5 1.8 1.9 47.9 1.4 11.8 3.1 12.1 4.2 179.5 120.0
control consumption total 25.5 6.6 9.4 3.7 4.2 3.2 2.4 3.0 2.8 1.8 1.1 0.6 0.4 0.5 0.2 65.5 79.2
N1060 consumption total 34.9 6.8 3.6 1.5 2.6 1.8 1.2 1.6 2.9 1.1 0.7 5.9 0.2 2.3 1.2 68.4 64.9 post-add 22.1 4.2 1.4 1.5 1.1 0.6 0.4 0.4 0.8 0.5 34.5 39.5
N1040 consumption total 24.5 7.7 2.6 1.2 1.8 1.4 1.3 1.1 1.7 1.0 1.0 9.0 0.3 2.0 1.7 58.2 51.4 post-add 11.8 4.1 0.7 1.2 0.8 0.7 0.5 0.1 0.6 0.9 0.9 22.2 26.0
N1020 consumption total 29.0 8.1 2.0 0.8 1.4 0.8 1.2 1.0 1.7 0.6 1.0 8.8 0.3 2.7 2.7 62.1 29.5 post-add 16.2 4.5 0.2 0.8 0.4 0.2 0.4 0.3 0.9 0.7 0.2 24.6 4.1
N1000 consumption total 23.1 7.8 1.9 0.4 1.2 0.7 0.9 1.1 1.9 0.5 0.1 8.2 0.3 2.5 0.3 3.1 53.9 25.4 post-add 10.3 4.1 0.1 0.4 0.2 0.1 0.1 0.1 0.2 15.7
no N consumption total 12.8 3.6 1.8 1.0 0.6 0.8 1.0 1.8 0.3 0.2 8.2 0.3 2.5 0.4 3.2 0.1 38.6 25.4
In the fermentations with nitrogen additions, post-add represents the nitrogen taken up after this addition.
acids can be grouped in different sets according to the preference of the cell in the different conditions. The amino acids that are most consumed are glutamine and tryptophan. Together they represented 32% of the total assimilable amino acids of the synthetic grape must (Table 2), and regardless of the fermentation conditions, their consumption accounted for 50% to 65% of the total amino acids consumed. On the other hand, the consumption of arginine, glutamate, glycine, alanine, and aspartate, which were approximately 44% of the total assimilable amino acids in the medium (Table 2), was together hardly 2% of the total amino acids consumed in the control fermentation. The consumption of these amino acids was much higher in the fermentations supplemented with nitrogen. However, yeast cells only consumed these amino acids before the nitrogen addition: that is, when the fermentations were nitrogen-deficient. Last, there is one other set of amino acids, consisting of threonine, histidine, leucine, isoleucine, phenylalanine, valine, and methionine, which was consumed proportionally more in the control fermentation than in the supplemented fermentations. GAP1 and MEP2 Gene Expression. The expression of the nitrogen transporters GAP1 and MEP2 was analyzed and quantified relative to the expression of the housekeeping actine gene. Time zero was the expression of yeast before inoculation (and after rehydration). Both genes were repressed in the first hours after inoculation in the must-like medium (Figure 2). In the nitrogen-deficient fermentation, these genes started to be activated/de-repressed after 30 h, when nitrogen was almost depleted. The expression of both genes increased continuously during the first days of fermentation and peaked after 4 and 6 days for GAP1
Figure 2. Gene expression of ammonium permease (MEP2) and general amino acid permease (GAP1) at time zero (before inoculation) and at different points during the control fermentation and the nitrogendeficient fermentation (without nitrogen addition). The data were quantified by calculating the ratio between the concentration of the studied genes normalized with the concentration of the housekeeping ACT gene, and expressed as a percentage (the quantity ratio 1 was set as 100%). YAN and ammonia consumption throughout the fermentations are also indicated.
and MEP2, respectively. The expression of the genes decreased in the last days of fermentation, after several days without a nitrogen source. On the other hand, the presence of residual nitrogen in the control fermentation repressed these genes throughout.
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Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
Figure 3. Relative gene expression of GAP1 and MEP2 at different points in the first 24 h after the nitrogen addition. Time zero represents the point just before this addition. The data were calculated as in Figure 2.
Alcohols and Acids (g L 1) 98.7 97.2 98.0 98.0 6.56 6.57 6.32 6.11 1.17 1.22 0.98 0.80 0.33 0.28 0.26 0.25 0.41 0.41 0.38 0.41 0.13 0.21 0.27 0.26 0.04 0.05 0.04 0.03 Higher Alcohols (mg L 1) 37 33 28 20 11 13 16 16 50 48 81 97 11 21 42 46 109 115 167 179 Fatty Acids (mg L 1) 0.39 0.40 0.43 0.63 0.73 0.67 0.63 0.37 0.30 0.12 0.09 0.09 2.06 1.58 1.40 2.30 1.95 1.84 0.39 0.37 0.21 0.16 0.14 0.09 6.70 5.63 5.02 0.39 0.72 0.44 0.10 1.53 2.34 0.19 0.16 5.87
Figure 3 shows the gene expression of GAP1 and MEP2 in the first 24 h after the nitrogen addition. They were both repressed in all the fermentations. However, the later the addition took place in the fermentation process, the longer it took for the genes to be repressed. When nitrogen was added at the end of the fermentation (density 1000), the effect was negligible because of the low expression at this point. Analytical Profile. We analyzed the residual sugars, ethanol, glycerol, and acids in the wines obtained from the different fermentations and such flavor compounds as higher alcohols, volatile fatty acids, and esters, which arose from yeast metabolism (Table 3). The later the nitrogen addition was, the lower the concentration of glycerol, acetic acid, and acetaldehyde was. The higher alcohol content was lower when excess nitrogen was available at the beginning of the fermentation (control fermentation and N1060). These different concentrations were accounted for by the increase in isoamyl alcohol and 2-phenylethanol. The concentration of these compounds increased considerably in the fermentations with nitrogen additions in the later phases (or no addition), and were approximately 2 and 5 times higher than in the control fermentation. The increase in isoamyl alcohol did not lead to a corresponding clear increase in its ester (isoamyl acetate) in these fermentations, and the phenyl-2-ethanol acetate ester only increased slightly. In fact, the differences in the concentration of the total acetate esters between the fermentations were due to the concentration of ethyl acetate, which was more than 95% of the total acetate esters.
Acetate Esters (mg L 1) ethyl acetate 35 35 32 25 19 28 isobutyl acetate 0.023 0.024 0.012 0.016 0.011 0.011 isoamyl acetate 0.49 0.46 0.39 0.69 0.39 0.29 hexyl acetate 0.006 0.005 phenyl-2-ethanol acetate 0.21 0.37 0.41 0.47 0.41 0.29 35.73 35.86 32.81 26.18 19.81 28.59 ethyl butyrate ethyl isobutyrate ethyl hexanoate ethyl octanoate ethyl decanoate Fatty Acid Esters (mg L 1) 0.224 0.220 0.163 0.006 0.005 0.006 0.089 0.071 0.23 0.022 0.022 0.060 0.002 0.004 0.019 0.343 0.322 0.478 0.232 0.005 0.31 0.081 0.024 0.652 0.164 0.004 0.23 0.059 0.019 0.446 0.124 0.007 0.085 0.020 0.004 0.240
Values are the average of two determinations and the coefficient of variation in all the compounds analyzed was less than 10% with the exception of decanoic acid (18%), dodecanoic acid (38%), ethyl octanoate (16%), and ethyl decanoate (29%).
Its concentration was higher in the control fermentation and the N1060 and 1040 fermentations, which correlated with a higher acetate concentration. The differences in the concentration of fatty acids and their esters were smaller in the final products of the fermentations.
DISCUSSION
The addition of nitrogen to grape musts, especially in the form of ammoniacal nitrogen, is a common winemaking practice that prevents nitrogen-related fermentation problems. Several studies, in which grape musts were supplemented with diammonium phosphate, have proved that nitrogen supplements can optimize fermentation performance (2-4). In the present study, we supplemented a nitrogen-deficient synthetic must with a mixture of ammonium and amino acids at different stages of the alcoholic fermentation. Then we studied the effect of these additions on
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Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
Higher alcohols were also affected by the changes in nitrogen utilization. These compounds can be produced either by the catabolic conversion of the branched-chain amino acids (via Ehrlich) or by the anabolic formation of these amino acids de novo from a sugar substrate (14, 15). Our results show that the anabolic route is of greater importance because the increase in isoamyl alcohol and 2-phenyl ethanol was inversely proportional to the consumption of leucine and phenylalanine, respectively. Furthermore, the closer the nitrogen concentration is to the growth-limiting level, the higher the yield of fusel alcohols is. There is also an inverse correlation between ammonium consumption and the production of fusel alcohols (12). A greater concentration of higher alcohols did not seem to determine an increase in esters. In contrast, the acetate concentration seemed to determine a greater concentration of acetate esters, especially ethyl acetate. In conclusion, our study shows the quantity and quality of the nitrogen demands of the wine strain QA23. Although further studies should be carried out with other wine strains, our data show that cell growth and fermentation have different preferred nitrogen sources. Nitrogen additions always improved fermentation performance but had a minimal effect on biomass production when added in the second half of the fermentation. These nitrogen additions subjected the cells to NCR and changed the profile of nitrogen consumption. The differences in the pattern of nitrogen consumption were related to different aroma compound compositions in the wines. In our opinion, this study is a starting point for further investigation into using an ammonium/amino acid mixture as nitrogen supplementation in the wine industry and the effect that these additions have on yeast physiology, fermentation performance, and wine quality.
LITERATURE CITED
(1) Bisson, L. F. Influence of nitrogen on yeast and fermentation of grapes. In Proceedings of the International Symposium on Nitrogen in Grapes and Wine; Ranz, J. M., Ed.; American Society of Enology and Viticology: Davis, CA, 1991. (2) Bely, M.; Sablayrolles, J. M.; Barre, P. Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions. J. Ferment. Bioeng. 1990, 70, 246- 252. (3) Jiranek, V.; Langridge, P.; Henschke, P. A. Yeast nitrogen demand: selection criterion for wine yeasts for fermenting low nitrogen musts. In Proceedings of the International Symposium on Nitrogen in Grapes and Wine; Ranz, J. M., Ed.; American Society of Enology and Viticology: Davis, CA, 1991. (4) Monteiro, F. F.; Bisson, L. F. Nitrogen supplementation of grape juice. I. Effect on amino acid utilization during fermentation. Am. J. Enol. Vitic. 1992, 43, 1-10. (5) Bely, M.; Sablayrolles, J. M.; Barre, P. Description of alcoholic fermentation kinetics: its variability and significance. Am. J. Enol. Vitic. 1990, 41, 319-324. (6) Mendes-Ferreira, A.; Mendes-Faia, A.; Leao, C. Growth and fermentation patterns of Saccharomyces cereVisiae under different ammonium concentrations and its implications in winemaking industry. J. Appl. Microbiol. 2004, 97, 540545. (7) Cooper, T. G. Nitrogen metabolism in Saccharomyces cereVisiae. In The molecular Biology of the Yeast Saccharomyces; Stratherm, J. N., Jones, E. W., Broach, J. R., Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1982. (8) ter Schure, E. G.; van Riel, N. A.; Verrips, C. T. The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cereVisiae. FEMS Microbiol. ReV. 2000, 24, 67- 83. (9) Beltran, G.; Novo, M.; Rozes, N.; Mas, A.; Guillamon, J. M. Nitrogen catabolite repression in Saccharomyces cereVisiae during wine fermentations. FEMS Yeast Res. 2004, 4, 625-632. (10) Jiranek, V.; Langridge, P.; Henschke, P. A. Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cereVisiae strains by assimilable nitrogen. Appl. EnViron. Microbiol. 1995, 61, 461467.
ACKNOWLEDGMENT
This work was supported by grant AGL20000205-P4-03 from the Comisin Interministerial de Ciencia y Tecnologa, Spain. The authors wish to thank the language service of the Rovira i Virgili University for revising the manuscript.
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Influence of the Timing of Nitrogen Additions during Synthetic Grape Must Fermentations on Fermentation Kinetics and Nitrogen Consumption
(32) Pierce, J. S. The Margaret Jones Memorial Lecture: Amino acids in malting and brewing. J. Inst. Brew. 1982, 88, 228-233. (33) Forsberg, H.; Ljungdahl, P. O. Sensors of extracellular nutrients in Saccharomyces cereVisiae. Curr. Genet. 2001, 40, 91-109. (34) Regenberg, B.; During-Olsen, L.; Kielland-Brandt, M. C.; Holmberg, S. Substrate specificity and gene expression of the amino acid permeases in Saccharomyces cereVisiae. Curr. Genet. 1999, 36, 317-328. (35) Marini, A. M.; Soussi-Boudekou, S.; Vissers, S.; Andre, B. A family of ammonium transporters in Saccharomyces cereVisiae. Mol. Cell Biol. 1997, 17, 4282-4293. (36) Bakker, B. M.; Overkamp, K. M.; van Maris, A. J.; Kotter, P.; Luttik, M. A.; van Dijken, J. P.; Pronk, J. T. Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cereVisiae. FEMS Microbiol. ReV. 2001, 25, 15-37.
(37) Radler, F.; Schutz, H. Glycerol production of various strains of Saccharomyces. Am. J. Enol. Vitic. 1982, 33, 36-40. (38) Watanabe, M.; Uehara, M.; Shinohara, T. Effect of cell number on the formation of glycerol and some volatile components by yeast. J. Brew. Soc. Jpn. 1982, 77, 346. (39) Albers, E.; Larsson, C.; Liden, G.; Niklasson, C.; Gustafsson, L. Influence of the nitrogen source on Saccharomyces cereVisiae anaerobic growth and product formation. Appl. EnViron. Microbiol. 1996, 62, 3187-3195. (40) Michnick, S.; Roustan, J. L.; Remize, F.; Barre, P.; Dequin, S. Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cereVisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase. Yeast 1997, 13, 783-793. Received for review August 2, 2004. Revised manuscript received November 12, 2004. Accepted November 17, 2004.
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1- Introduccin
El azufre es un elemento abundante en la naturaleza y presente en muchos compuestos. Puede presentarse en forma oxidada (sulfato) o reducida (sulfuro). El azufre es uno de los elementos ms importantes necesarios para la vida, particularmente como componente de los aminocidos cistena y metionina, y tambin es un componente de cofactores esenciales. Los microorganismos pueden metabolizar los compuestos de azufre a travs de dos rutas. En la ruta de reduccin asimilativa del azufre se toma sulfato y se utiliza para la biosntesis de compuestos orgnicos como la cistena y la metionina. En la ruta de reduccin disimilativa del sulfato, la molcula de sulfato se reduce como parte de una ruta respiratoria a sulfito o sulfuro. Ninguno de estos metabolitos se vuelve a metabolizar y la mayora se excreta (Kappler y Dahl 2001). La ruta anterior es realizada por bacterias reductoras de sulfato que estn ampliamente distribuidas en ambientes anaerbicos como el suelo, los sedimentos, agua de mar y agua dulce y en la boca e intestino de muchos animales (Purdy et al. 2002). Los microorganismos tambin son capaces de descomponer los aminocidos con azufre cistena y metionina para formar sulfuros y posteriormente otros compuestos voltiles de azufre y tioles (Dainty et al. 1989; Russell et al. 1995; Bonnarme et al. 2000; Seefeldt y Weimer 2000; Morales et al. 2005). La levadura de vino Saccharomyces cerevisiae es responsable de la produccin de varios compuestos voltiles de azufre que afectan a la calidad sensorial del vino. Estos importantes compuestos voltiles de azufre son: (1) sulfuro de hidrgeno (aroma a huevos podridos); (2) metanotiol (metilmercaptano; aroma a col cocida); (3) dimetilsulfuro, dimetildisulfuro y dimetiltrisulfuro (aromas a col, coliflor y ajo); (4) metiltioesteres (tioacetato de S-metilo, tiopropanato de S-metilo y tiobutanato de S-metilo; aromas a coliflor cocida, queso y cebolleta); y (5) los tioles voltiles afrutados del vino (aromas a maracuy, pomelo, grosella espinosa, guayaba y aromas a boje; Swiegers y Pretorius 2005; Swiegers et al. 2006). Durante la fermentacin del vino, la reduccin asimilativa de sulfato por la levadura (para biosintetizar cistena y metionina) puede producir un exceso de iones HS-, lo que genera la formacin H2S en el vino (Jiranek et al. 1995; Spiropoulos et al. 2000; Mendes- Ferreira et al. 2002; Swiegers et al. 2005a). Esto es probablemente uno de los problemas ms comunes en una bodega y, si no se trata, el vino resultante estar contaminado provocando una prdida de calidad y la posibilidad de que sea rechazado por los consumidores (Vos y Gray 1979; Henschke y Jiranek 1991; Rauhut 1993). Los vinos fermentados finalizados a menudo se tratan con sulfato de cobre con el fin de que reaccione con los compuestos de azufre para formar complejos estables y, por tanto, eliminar los efectos negativos del H2S y de los mercaptanos. No obstante no es deseable utilizar sulfato de cobre en el vino. La concentracin de H S producido durante Applied Microbiol Biotechnol, Enero de 2007 (on line)
Resumen
Los compuestos de azufre en el vino constituyen una espada de doble filo. Por una parte, ciertos compuestos voltiles que contienen azufre como el sulfuro de hidrgeno ofrecen un aroma similar a huevos podridos, por lo que pueden afectar negativamente a la calidad sensorial del vino pero, por otra parte, algunos compuestos de azufre como el 3-mercaptohexanol, que proporciona notas afrutadas, puede tener un impacto positivo sobre el sabor y el aroma del vino. Adems, estos compuestos se pueden volver ms o menos atractivos o repulsivos dependiendo de sus concentraciones absolutas y relativas. Por consiguiente es un desafo interesante que los vinicultores modulen las concentraciones de tales compuestos determinantes de la calidad del vino segn las preferencias de los consumidores. La levadura del vino, Saccharomyces cerevisiae, desempea un papel clave en la produccin de compuestos voltiles de azufre. A travs de la ruta de la secuencia de reduccin del sulfato se forma HS-, el cual puede conducir a la formacin de sulfuro de hidrgeno y diversos mercaptanos. Por tanto, limitar la formacin del in HS es un objetivo importante en la ingeniera metablica de esta levadura. La levadura del vino tambin es responsable de la transformacin de precursores de azufre no voltiles presentes en la uva, en tioles voltiles con propiedades aromatizantes. En particular, 4-mercapto-4-metilpentan-2-ona, 3-mercaptohexanol y acetato de 3-mercaptohexilo son los tioles voltiles ms importantes que aaden aromas afrutados al vino. El presente documento revisa brevemente el proceso metablico implicado en la produccin de importantes compuestos voltiles de azufre y las principales estrategias a la hora de conseguir desarrollar cepas de levaduras como herramienta para ajustar el aroma del vino a las especificaciones del mercado. Palabras clave: Aroma . Sabor . Bebidas fermentadas . Tioles . Vino . Levadura
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la fermentacin del vino depende de varios factores como la presencia de compuestos de azufre, la cepa de la levadura, las condiciones de fermentacin y los nutrientes del zumo de uva (Henschke y Jiranek 1991; Rauhut 1993; Spiropoulos et al. 2000). No obstante, algunas cepas parecen producir H2S intrnsecamente sin que les afecten las condiciones ambientales, lo que posiblemente indica un defecto metablico (Jiranek et al. 1995; Spiropoulos et al. 2000; MendesFerreira et al. 2002). Por otra parte, las levaduras de vino pueden producir compuestos voltiles de azufre beneficiosos para la calidad del vino. Algunos de estos son el furfuriltiol, un compuesto identificado en el aroma del Pinot Manseng blanco, tintos de Burdeos, y en las duelas tostadas (Tominaga et al. 2000). El furfuriltiol es un aromatizante extremadamente potente (umbral de percepcin de 0,4 ng/l) y tambin se ha identificado en el caf tostado, la carne, el pan de trigo y en las palomitas de maz. Se cree que la levadura forma el furfuriltiol mediante la transformacin del furfural liberado durante la fermentacin a partir de las duelas de roble tostadas (Tominaga et al. 2000). Otros tioles voltiles que mejoran el aroma producidos por la levadura a partir de los precursores de la uva son el 4-mercapto-4-metilpentano- 2-ona (4MMP), el 3-mercaptohexan-1-ol (3MH) y el acetato de 3-mercaptohexilo (3MHA). Los tioles voltiles son extremadamente potentes y tienen umbrales de percepcin bajos: 0,8 ng/l (4MMP), 60 ng/l (3MH) y 4 ng/l (3MHA). En los Sauvignon Blanc, estos compuestos son de particular importancia para diversos caracteres dado que ofrecen aromas a boje (4MMP), maracuy, pomelo, grosella espinosa y guayaba (3MH y 3MHA; Tominaga et al. 1995, 1998a,b; Dubourdieu et al. 2006). No obstante, tambin se han identificado 4MMP, 3MH y 3MHA en distintas concentraciones en vinos elaborados a partir de uvas Colombard, Riesling, Semillon, Merlot y Cabernet Sauvignon, por lo que tambin podran afectar al aroma y la calidad de tales vinos (Tominaga et al. 1995, 1998a,b; Murat et al. 2001b). El desafo de la enologa moderna es limitar (o eliminar) la produccin de H2S y mercaptanos indeseables y, al mismo tiempo, mejorar la produccin de tioles voltiles beneficiosos. El uso de sulfato de cobre introduce un interesante dilema para el vinicultor, ya que es utilizado para tratar vinos contaminados con H2S y mercaptanos, pero al mismo tiempo reduce la concentracin de tioles voltiles deseables ya que el in Cu2+ no discrimina entre los dos tipos de compuestos de azufre. Por tanto, la mejor solucin yace en el desarrollo de levaduras que produzcan durante la fermentacin concentraciones absolutas y relativas de tioles voltiles que mejoren el aroma sin la formacin de H2S ni mercaptanos (Pretorius 2000; Pretorius y Bauer 2002). En el siguiente apartado de este documento se detallan los elementos metablicos de la
levadura del vino que resultan en la produccin de compuestos voltiles de azufre. Adems, se discuten los ltimos avances en la modificacin del metabolismo del azufre de la levadura del vino para mejorar el sabor y el aroma del vino.
2.- Mecanismo de formacin de azufre voltil 2.1 Formacin de sulfuro de hidrgeno a travs de la ruta de secuencia de reduccin de sulfato.
La levadura del vino puede formar metablicamente H2S a partir de compuestos inorgnicos de azufre como los sulfatos y sulfitos, as como de compuestos orgnicos como la cistena y el glutatin (Henschke y Jiranek 1993; Rauhut 1993; Hallinan et al. 1999; Spiropoulos et al. 2000). En general, la uva debe ser deficiente en compuestos orgnicos de azufre, ya que stos pueden activar la sntesis de tales compuestos de azufre a partir de fuentes inorgnicas habitualmente abundantes en el mosto (Henschke y Jiranek 1993; Park et al. 2000; Moreira et al. 2002). En la S. cerevisiae, H2S es el producto de la ruta de la secuencia de reduccin de sulfato (SRS) (Fig. 1; Yamagata 1989; Rauhut 1993). En la ruta SRS, el H2S procede del in HS-, un intermedio metablico de la reduccin del sulfato o sulfito necesario para la sntesis de compuestos orgnicos de azufre. Si durante la fermentacin estas reacciones se producen en presencia de un Fig. 1 Un diagrama que representa la ruta SRS y la biosntesis de aminocidos en S. cerevisiae. Adenosil 5-fosfosulfato (APS); 3-fosfoadenosil 5-fosfosulfato (PAPS); O-acetilserina (O-AS); O-acetlilhomoserina (O-AH)
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sidera procedente de la descomposicin de la metionina (Rauhut 1993). La concentracin de disulfuro de dimetilo encontrada en algunos vinos est muy por encima del umbral sensorial de 25 g/l (vino blanco) y 60 g/l (vino tinto; Rauhut 1993). En bajas concentraciones, se considera que el disulfuro de dimetilo es un compuesto beneficioso que contribuye al aroma asociado al tiempo de maduracin en botella (Rauhut 1993; Ribreau-Gayon et al. 2000).
ltimo estudio no se observ que la sobreexpresin de estos genes produjera un aumento de la liberacin de 4MMP. No obstante, recientemente, nuestro grupo sobreexpres un gen heterolocigoso que codifica una enzima carbono-tio-lasa en una levadura comercial ampliamente utilizada (VIN13) y demostr como conclusin que, en vinos en fermentacin modelos, la cepa VIN13 modificada liberaba diez veces ms 4MMP y 3MH de sus precursores sintetizados qumicamente que la cepa control VIN13. Adems, el vino Sauvignon Blanc producido por la levadura modificada tuvo un muy potente aroma no detectado en el vino control. Por tanto, es posible modificar levaduras para utilizar ms precursores de tioles derivados de la uva y por tanto mejorar el aroma del vino (Swiegers et al. sin publicar). Se ha demostrado anteriormente que la cantidad liberada de 4MMP en el vino depende de qu cepa de levadura se utilice para llevar a cabo la fermentacin (Dubourdieu et al. 2006). Por tanto, la gentica y la fisiologa de la cepa determinan su capacidad para liberar tioles voltiles. Algunos estudios del primer trabajo indicaron que las cepas comercialmente disponibles de S. cerevisiae VL3 y EG8 liberan ms tioles que las cepas VL1 y 522d. Adems, las cepas de Saccharomyces bayanus liberan ms 4MMP que las cepas de S.cerevisiae VL3 y EG8. Los vinos confeccionados con cepas hbridas de S.bayanus/S. cerevisiae muestran un contenido superior de tioles voltiles (Murat et al. 2001b). Estos hallazgos se han confirmado al observar que diferentes cepas comerciales tienen capacidades diferentes de liberacin de 4MMP a partir del precursor Cis-4MMP en vinos modelo en fermentacin (Howell et al. 2004). Howell et al. (2004) han identificado cepas comerciales de levaduras que liberan an ms tioles que VL3. En un estudio de seguimiento, el Sauvignon Blanc producido por siete cepas diferentes de levadura del vino (VL3 entre ellas) mostr perfiles nicos de tioles voltiles, siendo la cepa VIN7 la que produjo mayor concentracin de 4MMP y 3MHA, mientras que VIN13 produjo mayores concentraciones de 3MH (Swiegers et al. 2006). Se ha demostrado que durante la fermentacin, el 3MHA se forma a partir del 3MH por la accin de la alcohol acetiltransferasa que forma steres y que es codificada por el gen ATF1 (Swiegers et al. 2005b). La sobreexpresin del gen ATF1 en la cepa de levadura VIN13 produjo un aumento significativo de la cantidad de 3MHA producido. Por otra parte, la sobreexpresin del gen IAH1, el cual codifica un enzima que descompone steres, produjo una reduccin en la concentracin de 3MHA. Tambin se investig la capacidad de diferentes levaduras comerciales para convertir 3MH en 3MHA durante la fermentacin. Se observaron grandes variaciones en la concentracin de 3MHA y, en la mayora de los casos, no correspondan con la capacidad de liberacin de 4MMP de las levaduras (Swiegers et al. 2005b). Por tanto, queda puesto de manifiesto que la seleccin de la cepa de levadura es de mxima importancia en la modulacin
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3. Conclusin
El objetivo final de todo vinicultor es conseguir un vino con unas caractersticas ptimas de calidad, precio y apariencia al consumidor (Pretorius 2006). Para lograr este objetivo, es necesario un control exhaustivo del mtodo de produccin, de las condiciones de fermentacin, de la eleccin de la cepa de levadura y de la produccin de componentes que afectan favorablemente a la calidad organolptica del vino. Los compuestos voltiles de azufre son aromatizantes y saborizantes potentes que pueden tener un efecto
Fig. 2 El vino es una mezcla muy compleja de compuestos derivados de la uva, de los microorganismo y, en algunos casos, del roble, que definen en gran medida su apariencia, aroma, sabor y sensaciones en el paladar (Swiegers et al. 2005). Los compuestos derivados de la uva distinguen al vino por su variedad y lo dotan de su estructura bsica. La fermentacin de los azcares por parte de las levaduras no slo produce etanol y dixido de carbono, sino que tambin una gama de metabolitos menores pero importantes organolpticamente que proporcionan el carcter del vino. Estos metabolitos voltiles que son steres, alcoholes superiores, carbonilos, cidos grasos voltiles y compuestos de azufre, son derivados del metabolismo de los azcares y de los aminocidos. Los tioles voltiles, particularmente 4MMP, 3MH y 3MHA, contribuyen de manera importante al aroma de los vinos, particularmente en variedades como la Sauvignon Blanc. Durante la fermentacin del vino, la S. cerevisiae favorece la divisin de precursores no voltiles con cistena (Cis-4MMP y Cis-3MH) existente el zumo de uva para liberar 4MMP y 3MH. La ausencia de tioles voltiles en la uva indica la importancia de la fermentacin de la levadura para su formacin (Swiegers et al. 2006)
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considerable sobre la calidad del vino. Las levaduras de vino son las principales generadoras de compuestos voltiles de azufre, los cuales son producidos a partir de fuentes de azufre y precursores derivados de las uvas (y en algunos casos por productos aadidos por el vinicultor, e.g., SO2). A da de hoy se han conseguido avances significativos para dilucidar las rutas metablicas responsables de la formacin de compuestos voltiles de azufre. Este conocimiento se est utilizando actualmente para desarrollar cepas de levaduras de vino que: (1) produzcan nada o mucho menos sulfuro de hidrgeno y mercaptanos asociados y (2) tengan una mayor capacidad de producir compuestos voltiles de azufre deseables que aporten aromas afrutados al vino. En algunos casos, se ha aplicado ingeniera gentica con el objetivo de evaluar la viabilidad del diseo de tales cepas para lograr los resultados deseados. Si bien los consumidores actuales son an muy resistentes a consumir bebidas producidas por microorganismos modificados genticamente, la reciente comercializacin de una levadura genticamente modificada en la industria vitivincola norteamericana podra indicar la aceptacin gradual de esta tecnologa (Husnik et al.2006). No obstante, aunque en el futuro cercano no se utilice para producir vino co-
mercial ninguna de estas levaduras genticamente modificadas con metabolismos del azufre alterados, sirven como modelos y prototipos tiles para aportar ms informacin que podra aplicarse para desarrollar cepas no modificadas genticamente utilizando tcnicas ms convencionales (por ejemplo, mutagnesis, hibridacin y evolucin adaptativa). Por tanto, existe un gran consenso sobre la posibilidad de confeccionar a medida levaduras de vino sin acudir a ingeniera gentica. Sin embargo, para personalizar levaduras no modificadas genticamente con el fin de que los vinicultores ajusten las concentraciones de compuestos que determinan la calidad del vino, el conocimiento fundamental requerido continuar generndose ampliamente a partir de informacin obtenida mediante cepas prototipo modificadas genticamente. Agradecimientos: la investigacin en el Australian Wine Research Institute (Instituto de Investigacin Enolgica de Australia) es posible gracias al apoyo de viticultores y vinicultores australianos a travs de su organismo inversor, la Grape y Wine Research Development Corporation, la cual financia segn una metodologa matching funds junto al Gobierno australiano.
Referencias
Amerine MA, Berg HV, Kunkee RE, Ough CS, Singleton VL, Webb AD (1980) The technology of winemaking, 4th edn. AVI Technical Books, Westport, CT Bonnarme P, Psoni L, Spinnler HE (2000) Diversity of L-methionine catabolism pathways in cheese-ripening bacteria. Appl Environ Microbiol 66:55145517 Dainty RH, Edwards RA, Hibbard CM, Marnewick JJ (1989) Volatile compounds associated with microbial growth on normal and high pH beef stored at chill temperatures. J Appl Bacteriol 66:281289 Darriet P, Tominaga T, Lavigne V, Boidron J, Dubourdieu D (1995) Identification of a powerful aromatic compound of Vitis vinifera L. var. Sauvignon wines: 4-mercapto-4-methylpentan-2-one. Flavour Fragr J 10:385392 Donalies UEB, Stahl U (2002) Increasing sulfite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1. Yeast 19:475484 Dubourdieu D, Tominaga T, Masneuf I, Peyrot des Gachons C, Murat ML (2006) The role of yeasts in grape flavor development during fermentation: the example of Sauvignon Blanc. Am J Enol Vitic 57:8188 Giudici P, Kunkee RE (1994) The effect of nitrogen deficiency and sulfur-containing amino acids on the reduction of sulfate to hydrogen sulfide by wine yeasts. Am J Enol Vitic 45:107112 Hallinan CP, Saul DJ, Jiranek V (1999) Differential utilization of sulfur compounds for H2S liberation by nitrogen-starved wine yeast. Austral J Grape Wine Res 5:8290 Henschke PA, Jiranek V (1991) Hydrogen sulfide formation during fermentation: effect of nitrogen composition in model grape must. Proceedings of the international symposium on nitrogen in grapes and wine, Seattle, USA. American Society for Enology and Viticulture, Davis, CA, pp 172184 Henschke PA, Jiranek V (1993) Yeastgrowth during fermentation. In: Fleet GH (ed) Wine microbiology and biotechnology. Harwood Academic, Chur, Switzerland, pp 2754 Howell KS, Swiegers JH, Elsey GM, Siebert TE, Bartowsky EJ, Fleet GH, Pretorius IS, de Barros Lopes MA (2004) Variation in 4-mercapto-4-methyl-pentan2-one release by Saccharomyces cerevisiae commercial wine strains. FEMS Microbiol Lett 240:125129
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Captulo
Spiropoulos A, Tanaka J, Flerianos I, Bisson LF (2000) Characterization of hydrogen sulfide formation in commercial and natural wine isolates of Saccharomyces. Am J Enol Vitic 51:233248 Swiegers JH, Pretorius IS (2005) Yeast modulation of wine flavour. Adv Appl Microbiol 57:131175 Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS (2005a) Yeast and bacterial modulation of wine aroma and flavour. Austral J Grape Wine Res 11:139173 Swiegers JH, Willmott R, Hill-Ling A, Capone DL, Pardon KH, Elsey GM, Howell KS, de Barros Lopes MA, Sefton MA, Lilly M, Pretorius IS (2005b) Modulation of volatile thiol and ester aromas in wine by modified wine yeast. Proceedings of the Weurman flavour research symposium, Roskilde, Denmark, 21 24 June 2005, Developments in Food Science, Elsevier, Amsterdam, The Netherlands Swiegers JH, Francis IL, Herderich MJ, Pretorius IS (2006) Meeting consumer expectations through management in vineyard and winery: the choice of yeast for fermentation offers great potential to adjust the aroma of Sauvignon Blanc wine. Austral NZ Wine Ind J 21:3442 Sutherland CM, Henschke PA, Langridge P, de Barros Lopes M (2003) Subunit and cofactor binding of Saccharomyces cerevisiae sulfite reductasetowards developing wine yeast with lowered ability to produce hydrogen sulfide. Austral J Grape Wine Res 9:186193
Thornton RJ, Bunker A (1989) Characterisation of wine yeasts for genetically modifiable properties. J Inst Brew 95:181184 Tominaga T, Masneuf I, Dubourdieu D (1995) A Scysteine conjugate, precursor of aroma of white sauvignon. J Int Sci Vigne Vin 29:227232 Tominaga T, Peyrot des Gachons C, Dubourdieu D (1998a) A new type of flavour precursors in Vitis vinifera L. cv. Sauvignon Blanc: S-cysteine conjugates. J Agric Food Chem 46:52155219 Tominaga T, Furrer A, Henry R, Dubourdieu D (1998b) Identification of new volatile thiols in the aroma of Vitis vinifera L. var. Sauvignon Blanc wines. Flavour Fragr J 13:159162 Tominaga T, Blanchard L, Darriet P, Dubourdieu D (2000). A powerful aromatic volatile thiol, 2-furanmethanethiol, exhibiting roast coffee aroma in wines made from several Vitis vinifera grape varieties. J Agric Food Chem 48:17991802 Vermeulen C, Gijs L, Collin S (2005) Sensorial contribution and formation pathways of thiols in foods: a review. Food Rev Int 21:69137 Vos PJA, Gray RS (1979) The origin and control of hydrogen sulphide during fermentation of grape must. Am J Enol Vitic 30:187197 Yamagata S (1989) Roles of O-acetyl-L-homoserine sulfhydrylases in micro-organisms. Biochimie 71:11251143
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Una revolucin en el sector enlogico: Levaduras de vinificacin que no producen Sulfuro de hidrgeno (H2S)
Cordente, T.; Swiegers, H.; (Australian Wine Research Institute AWRI), Heinrich, A.; (Mauri Yeast Australia) El artculo ntegramente reproducido a continuacin se publico por primera vez en la revista The Australian and New Zealand Grapegrover ans Winemaker Magazine en noviembre de 2007. Publicado con el permiso de la citada revista . Las cepas de levadura descritas en este artculo como su correspondiente proceso de desarrollo estn pendientes de patente.
manteniendo a la vez una excelente capacidad de fermentacin? El Instituto de Investigacin Vitivincola de Australia (AWRI) ha desarrollado, en colaboracin con la Sociedad Mauri Yeast Australia, nuevas cepas de levadura que producen cantidades indetectables de H2S. Estas levaduras son Advantage, Platinum y Distinction.
Introduccin
La produccin excesiva de Sulfuro de Hidrgeno (H2S) que tiene lugar en el transcurso del proceso de fermentacin del mosto constituye un problema bastante comn en lo que a vinificacin se refiere (Monk 1986; Henschke and Jiranek 1991). El principal motivo de ello radica en la baja concentracin de elementos nitrogenados en el medio. Con el objeto de paliar este problema, los enologos pueden, en el transcurso del proceso de fermentacin, aumentar el grado de nitrgeno en el mosto, aadindole fosfato diamnico (DAP) o utilizando sulfato de cobre despus de la fermentacin con el fin de suprimir el H2S del vino. Pero a pesar del aporte en DAP, la levadura sigue produciendo H2S en algunos medios y todo ello sin hablar del hecho de que la mayora de los enlogos preferira no tener que recurrir al uso del sulfato de cobre. Los vinos que contienen H2S desprenden un olor desagradable (a huevo podrido), pero adems, debido a su olor, el H2S, no solo reduce las cualidades organolpticas del vino sino tambin oculta las notas aromticas favorables de ste. Cabra imaginar que en el futuro podamos llevar a cabo una fermentacin con levaduras de vinificacin que no produzcan H2S detectable sea cual sea el contenido en nitrgeno asimilable del medio y sin ningn aporte en DAP? Por otro lado, Estas cepas de levadura seran capaces de realzar aromas favorecedores
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Una revolucin en el sector enlogico: Levaduras de vinificacin que no producen Sulfuro de hidrgeno
Estos resultados demuestran que estas tres cepas Advantage, Platinum y Distinction, fermentan mostos empobrecidos en nitrgeno asimilable sin producir cantidades de H2S detectables por la nariz humana. Asimismo, estas tres cepas de levadura presentan cinticas de fermentacin comparables con las de la cepa de levadura PDM, conocida por su robustez (Tabla 1, Figura 2).
cantidad de H2S que sobrepasaba el lmite de la percepcin sensorial. Las catas llevadas a cabo al finalizar el anlisis demostraron que los vinos presentaban caractersticas sensoriales positivas. Adems, las cepas revelaron unas cinticas de fermentacin comparables a la obtenida con la cepa Maurivin PDM .
En resumen
La produccin de H2S es perjudicial para la produccin de vinos de calidad. Felizmente, hoy en da, existen cepas de levaduras llamadas Advantage, Platinum Distinction.. Estas cepas se adaptan especialmente a los mostos que, empobrecidos en nitrgeno, evitan la produccin de H2S. Por lo general se trata de cepas polivalentes que permiten la fermentacin de una amplia gama de variedades a la vez que una produccin de vinos de calidad con caractersticas sensoriales considerablemente mejoradas. Adems, al no tener que recurrir al DAP (o emplearlo en cantidades inferiores) se facilita as el procesos de vinificacin.
Agradecimientos
Los autores dan las gracias al Dr. Nick Yap, al Dr. Dan Johnson y al Profesor Sakkie Pretorius por su valiosa colaboracin a lo largo de todo este proyecto as como por la crtica constructiva que hicieron de este artculo. El Dr. Toni Cordente se ha beneficiado del apoyo econmico del grupo AB Mauri. El Dr.Hentie Swiegers es investigador en el Instituto de Investigacin Vitivincola de Autralia, subvencionado por la asociacin de los vinificadores y viticultores Australianos a travs de su organismo de inversin: la sociedad de Desarrollo de la Investigacin sobre Uva y Vino as como por los fondos del gobierno de Australia.
Referencias
Monk, P.R. Formation, utilization and excretion of hydrogen sulfide by wine yeast. (1986) Wine Industry Journal, Noviembre 10-16. Henschke PA, Jiranek V (1991) Hydrogen sulfide formation during fermentation: effect of nitrogen composition in model grape must. Proceedings of the international symposium on nitrogen in grapes and wine, Seattle, USA. American Society for Enology and Viticulture, Davis, CA, Pg.172184
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Figura 1. Cintas reactivas cuyo color negro indica la presencia de pequeas cantidades de H2S
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Nitrogen management is critical for wine Managing Director, flavour and style
Ugliano, M.; Henschke, P.A.; Herderich, M.J.; Pretorius, I.S. The Australian Wine Research Institute, PO Box 197, Glen Osmond (Adelaide), South Australia 5064, Australia Winemaking begins in the vineyard is a mantra that has widespread support amongst winemakers. It conveys the concept of vineyard or, in French jargon, terroir as an intrinsic property of grape, and consequently the corresponding wine. There is no doubt that many great wines are associated with great vineyards. So, where do yeast fi t in? The perception that fermentation yeast faithfully transform grape must into wine has been changing in the detail over the past decades. This is a result of science uncovering the many roles that yeast perform, and the wider selection of strains available that promote these various attributes. For example, whereas most strains produce a relatively similar, generic, fermentation bouquet only some strains possess a strong ability to hydrolyse cysteine conjugates responsible for Sauvignon Blanc character, meaning that only selected strains can enhance varietal expression (Swiegers et al. 2006). Winemakers today have many options through fermentation management to enhance the varietal characteristics of their wine, or to express further regional attributes. Furthermore, yeast strongly respond to their environment. It is well known that temperature aff ects the rate of fermentation, that grape solids enhance survival and that high osmotic stress, as imposed by a Botrytis-aff ected must, leads not only to increased glycerol production but also to higher volatile acidity. The latter example highlights the remarkable ability of yeast to adapt to stressful (i.e. high sugar) environments. However, there is an accompanying metabolic adaptation which can have positive or negative fl avour implications. At the time of inoculation, yeast are subjected to a range of stresses to which the cell must adapt in order to exploit its new environment. Some of the known stresses are osmotic pressure, oxidative conditions,
sulphite toxicity and temperature shock (Bauer and Pretorius 2000). Nutrients, whether present in sub- or super-optimal concentration, can also induce stress and metabolic responses. The primary response is aimed at protecting the cell from committing to reproduction when key nutrients are lacking or dealing with potential toxicity when the concentration is outside the normal range. The metabolic response often involves a cascade of biochemical reactions, some of which can lead to altered metabolism of nutrients such that the yeast will secrete end-products in diff erent amounts (Albers et al. 1998). Some of the end products that have sensory properties can lead to changes in the fl avour profi le of the wine. H2S formation is an all-too-well known example relating to nitrogen depletion stress. Clearly, the vineyard environment and intervention by the viticulturist shape the development of the vine and especially the composition of the grape. Because the viticulturist attempts to balance a long list of priorities in order to produce fruit to specifi cation, most attention will focus on those factors that cannot be modifi ed once the fruit has been harvested. Therefore, yeast nutrients, especially nitrogen, might not be optimised for fermentation, largely in the belief that nutrients can be easily corrected in the winery. Given that we estimate that up to 500t of diammonium phosphate (DAP) could be used each year to produce Australian wine, is this winemaking input being used eff ectively? Our current state of knowledge on the implications of controlling vineyard nitrogen as opposed to fermentation nitrogen on fermentation performance and wine composition has been recently reviewed by Bell and Henschke (2005). In this article, we will focus on the role that fermentation nitrogen has in modulating metabolism and some of the changes that this can have on wine fl avour. We will fi rst summarise current best practice for managing fermentation nitrogen and then describe the main fl avour changes that are aff ected by nitrogen. Finally, we will consider the fl avour implications of nitrogen for white and red wine fermentations.
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based on studies with Chardonnay and Shiraz; however, it should stimulate winemakers to experiment with these and other varieties.
gen measurements yields YAN (Figure 1). This procedure is used by NATA-accredited laboratories such as AWRIs Analytical Service laboratory (http://www.awri. com.au/analytical_ service/analyses/yeast_assimilable_ nitrogen/), which can provide a timely service during vintage. The so-called Formol Titration is a simpler, rapid method for measuring YAN (Shively and Henick-Kling 2001; Gump et al. 2002; Filipe-Ribereiro and MendesFaia 2005), although the use of formaldehyde, a toxic volatile reagent, requires a well-trained analyst and suitable laboratory. YAN determination with midinfrared (MIR) spectrometry, which is most rapid, has recently been developed by the AWRI (Dambergs et al. 2005). YAN measurements, ideally, should be performed directly on juice or must samples at the point of inoculation to avoid over-estimation due to processing losses which inevitably occur between vineyard and the fermentor. Furthermore, juice samples taken from grape musts can under-estimate total berry YAN due to an important proportion of amino acid contained in the grape skin. Refer to the review by Bell and Henschke (2005) for a detailed discussion of these points. Nevertheless, an early warning for low YAN can be achieved by sampling in the vineyard one to two weeks prior to harvest, such as during maturity sampling.
Figure 1. Summation of free amino nitrogen and ammonia nitrogen levels provides a useful estimate of the yeast assimilable nitrogen level.
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Glycerol Concentration Malic acid Succinic acid Acetic acid SO2 100 200 YAN 300 400
Figure 2. Effect of yeast assimilable nitrogen on production or utilisation of major metabolic products of sugar fermentation and sulphur assimilation.
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Ethanol is the major product of sugar fermentation. However, while DAP addition increases yeast growth and the rate of fermentation, it has little to no practical eff ect on fi nal ethanol yield. Theoretically, DAP-grown yeast are forced to synthesise amino acids for cell growth when compared with amino acid grown yeast. This decreases the proportion of sugar available for ethanol production (Albers et al. 1996), but in our experiments this has a minor aff ect on ethanol yield. Glycerol and acetic acid, which are important to wine composition and fl avour, respond relatively strongly to juice YAN concentration (Albers et al. 1998; Torrea et al. 2005; Vilanova et al. 2007). Figure 2 summarises general trends observed in synthetic juice and white wine fermentations. Both glycerol and acetic acid production depends strongly on the yeast strain used. For example, when using yeast Vitilevure M05 DAP addition increases glycerol production whereas the reverse is the case for AWRI 796 (Vilanova et al. 2007). Both yeast, however, produce the lowest concentrations of acetic acid at moderate YAN concentrations (range of 200-250mg/L) while higher concentrations are produced at both lower and higher concentrations of YAN. Malic acid consumption does, however, increase with increasing DAP concentration, irrespective of yeast strain. On the contrary and depending on the strain, succinic acid concentration can increase with increasing DAP addition (Coulter et al. 2004). In general, YAN can aff ect TA and the balance of organic acids which can aff ect fl avour (Sowalsky and Noble 1998). Sulphur dioxide production during fermentation can also be stimulated by initial YAN concentration, but the response seems to be yeast strain dependent. Experimental work in synthetic media and wort suggests that low SO2 is produced in low YAN media but increases when initial YAN availability is higher (Duan et al. 2004; Osborne and Edwards 2006). SO2 production contrasts with H2S production, which is generally lowered by increasing YAN. Increased risk of MLF inhibition has also been associated with high YAN addition but this inhibition has not been conclusively correlated with SO2 production (Osborne and Edwards 2006). Nevertheless, until better information is available, consideration should be given to limiting high YAN conditions when malolactic fermentation (MLF) is required.
tation-derived volatiles in the aroma character of wine (Smyth et al. 2005). Several studies have indicated that both the total available nitrogen and the balance of amino acids and ammonia can signifi cantly aff ect the production of different groups of fermentationderived volatile compounds. From a practical point of view, the problem of juice nitrogen composition is primarily linked to the frequent occurrence of juices with suboptimal concentrations of nitrogen, and higher risk of slow or stuck fermentation. As this problem is frequently corrected in the winery through the addition of DAP, several studies have investigated the implications of this common winery practice on the volatile composition of wine (Ayrapaa 1971, Rapp and Versini 1991; Carrau 2003; Torrea and Henschke 2004; Hernandez-Orte et al. 2005, 2006; Vilanova et al. 2007). Due to the variety of yeast strains and fermentation conditions employed, it is somewhat diffi cult to extrapolate from the literature defi nitive conclusions concerning the eff ect of DAP addition on wine aroma. Nevertheless, some general trends relating to DAP supplementation and wine volatile composition are summarised in Figure 3. Higher alcohols, which are directly related to amino acid metabolism in the cell, exhibit a characteristic behaviour. Therefore, when total nitrogen is increased by adding ammonium to a medium containing very low levels of YAN, the production of higher alcohols is initially increased, but then tends to decrease after a peak between 200-300mg/L YAN. This activity depends on various factors, including yeast strain and fermentation conditions.Higher alcohols are characterised by fusel-like odours, and are generally thought to contribute to the complexity of wine fermentation bouquet. However, when present in very high concentrations they can have a negative impact on wine aroma, mainly because they mask fruity characters. Several authors have reported that ammonium supplementation can improve wine sensory quality
Higher alcohols
Ethyl acetate Acetate esters Fatty acids ethyl esters Branched chain esters 100 200 300 YAN 400 500
Figure 3. Relationship between initial YAN concentration and fi nal concentration of volatile compounds after fermentation.
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characterised by fruit-like odours. Nevertheless, relatively small variations in the concentration of ethyl fatty acid esters, such as those introduced by variations in YAN content, are more likely to aff ect the aroma of wine than if proportionally similar variations would occur for higher alcohols. This is due to the fact that some of the possible sensory modifi cations associated with changes in the concentration of specifi c aroma compounds depend, among other factors, on the ability of that aroma compound to generate an olfactory stimulus at a given concentration. This complex relationship is often simplifi ed by means of the concept of odour threshold, defi ned as the minimum concentration at which a given compound can be detected by the sense of smell (Guth 1997). This is referred to as the odour activity value (OAV). Some of the fermentation-derived volatile compounds, such as esters, that are generally associated with the fruity character of wine, are extremely powerful odourants (i.e. have a very low odour threshold) and can, therefore, impart specifi c sensory attributes even when present in low concentrations. On the contrary, compounds like higher alcohols possess a much higher odour threshold and, therefore, are likely to generate variations in the aroma profi le of a wine only when their concentration varies to a very large extent. In Figure 4, a theoretical relationship between the OAV of selected volatile compounds, belonging to the chemical classes of Figure 3, and DAP supplementation is illustrated. It appears clear then that the range of variations potentially introduced by DAP in the concentration of acetates and fatty acid ethyl esters (isoamyl acetate and ethyl octanoate are used as reference compounds for these two classes) can have a dramatic impact on the volatile character of wine, whereas variations in compounds such as higher alcohols (isoamyl alcohol), although quantitatively extremely large, are likely to have a limited impact. Although it has to be stressed that OAVs only give a projection of the potential of a given compound to contribute to the overall aroma of a wine, the trends shown in Figure 4 provide a good indication of which one of the compositional changes associated with DAP supplementation is likely to have a greater impact on wine aroma.
IMPLICATIONS OF NITROGEN FOR WINE FERMENTATIONS 3.1 Implications of nitrogen for white wine fermentations
Interestingly, the results obtained in various winemaking trials conducted at the AWRI with sub-optimal YAN juices have indicated that, under typical winemaking conditions, DAP supplementation is an extremely powerful tool for modulating the production of esters which, based on the previous discussion, are probably
Figure 4. Theoretical relationship between initial YAN concentration and Odour Activity Values of selected yeast-derived aroma compounds.
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the most sensorially-interesting group of compounds generated during fermentation. Figures 5 and 6 show the variations in volatile compounds and the sensory profi le of Chardonnay wines made at diff erent DAP concentrations. In good agreement with the trends shown in Figure 3, DAP had a positive eff ect on ester production, while it lowered the formation of higher alcohols. However, the wines obtained with moderate nitrogen supplementation of the juice were preferred by panellists compared with those obtained without or with high DAP addition. This preference might be due to a combination of higher acetates, ethyl fatty acid ester concentrations and moderate levels of ethyl acetate, the latter being associated with unwanted, solvent-like characteristics when present at very high concentrations. DAP addition to low YAN juices also suppresses the production of H2S and mercaptans by many wine yeasts, which although not quantifi ed in this study, no doubt contributed to the preference of the moderate YAN wines. The impact of DAP addition on the production of fruity thiols, such as 4MMP, 3MH and 3MHA, still needs to be determined. These results highlight the complexity of predicting wine aroma from compositional data. They also underline the importance of measuring YAN and adding the appropriate amount of DAP, if necessary, before or during fermentation in order to reduce the potentially negative eff ects that inadequate or excessive DAP supplementation can have on wine aroma. Particularly, the risk of excessive formation of ethyl acetate has to be considered as this ester is relatively stable during wine ageing, compared with other acetate and ethyl fatty acid esters, which tend to decrease signifi cantly after several months of bottle storage.
12,000
160mg/L YAN
320mg/L YAN
480mg/L YAN
10,000
Concentration g/L
8000
Cheesy
Musk
6000
Acetic
Floral
4000
Nail lacquer Stewed fruit
2000
Stale beer Tropical Honey Bruised apple Citrus
Figure 5. Volatile compounds of wines obtained from a low YAN (160mg/L) Chardonnay juice supplemented with two increasing concentrations of DAP, to a fi nal YAN of 320mg/L and 480mg/L, respectively. Fermentations were carried out at 18C using S. cerevisiae AWRI 796.
Figure 6. Sensory characteristics of wines obtained from a low YAN (160mg/L) Chardonnay juice (green line) supplemented with two increasing concentrations of DAP, to a fi nal YAN of 320mg/L (red line) and 480mg/L (blue line), respectively. Fermentations were carried out at 18C using S. cerevisiae AWRI 796.
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CONCLUSION
This work shows that the concentration of yeast assimilable nitrogen is not only important for ensuring that adequate yeast growth and fermentation kinetics are achieved, but also can aff ect the production of the major metabolites arising from sugar fermentation. Whereas ethanol concentration is little aff ected, that of glycerol and various carboxylic acids can be markedly modulated. These changes are likely to aff ect wine fl avour. Most importantly, however, is the fi nding that YAN can strongly infl uence production of some of the volatile metabolites, especially the acetate and ethyl esters, which are known to be positive to wine aroma when in balance. The impact of higher alcohols, which can be negative when present in high concentration, can also be modulated by YAN. These various yeast metabolites were also found to vary in
ACKNOWLEDGEMENTS
We thank our many colleagues for supporting this project, especially Tracey Siebert and Dimitra Capone for help with analysis of fermentation volatiles; Mariola Kwiatkowski and Meagan Mercurio for analysis of phenolic compounds; Kate Lattey, Belinda Bramley and Dr Leigh Francis for carrying out the sensory analyses; Industry Development and Support and Analytical Service team members for help with chemical analyses; and Dr Paul Chambers, Dr Cristian Varela and Biosciences team members for many helpful discussions. Professor Francisco Carrau, of Uruguay, and visiting scientists Drs Diego Torrea and Mar Vilanova, from Spain, have made important contributions to this project. We are especially grateful to Mike Farmilo, of Boars Rock winery, for supplying a large number of juice samples and donating must for the Shiraz fermentation trials, and Russell Johnstone and Inca Pearce, of Orlando Wines, and Louisa Rose and Simon Dillon of The Yalumba Wine Company, for supplying white wine juices. Current work on nitrogen management also involves collaboration with Dr Sally-Jean Bell and Marcel Essling. Rae Blair is thanked for her editorial assistance. This project is supported by Australias grapegrowers and winemakers through their investment agency, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. The AWRI is a member of the Wine Innovation Cluster.
10,000 9000 8000 Concentration g/L 7000 6000 5000 4000 3000 2000 1000 0 Fatty acids ethyl esters Acetates Ethyl acetate Higher alcohols/100 100mg/L YAN 250mg/L YAN 400mg/L YAN
Figure 7. Volatile compounds of wines obtained from low YAN (100mg/L YAN) Shiraz grapes supplemented with two increasing concentrations of DAP, to a fi nal YAN of 250mg/L and 400mg/L respectively. Fermentations were carried out at 22C using S. cerevisiae AWRI 796, with cap plunging three times per day.
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REFERENCES
1 Albers, E.; Larsson, C.; Liden, G.; Niklasson, C. & Gustafsson, L. (1996) Infl uence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl. Env. Microbiol. 62: 3187-3195. 2 Albers, E.; Lidn, G.; Larsson, C.; Gustafsson, L. (1998) Anaerobic redox balance and nitrogen metabolism in Saccharomyces cerevisiae. Recent Res. Develop. Microbiol. 2: 253-279; 1998. 3 yrp, T. (1971) Biosynthetic formation of higher alcohols by yeast. Dependence on the nitrogenous nutrient level of the medium. J. Inst. Brew. 77: 266-276. 4 Bauer, F.F.; Pretorius, I.S. (2000). Yeast stress response and fermentation effi ciency: how to survive the making of wine A review. S. Afr. J. Enol. Vitic. 21: 27-51. 5 Bell, S.-J.; Henschke, P.A. (2005) Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 11: 242-295. 6 Blateyron, L.; Ortiz-Julien, A; Sablayrolles, J.M. (2003) Stuck fermentations: oxygen and nitrogen requirements importance of optimising their addition. Aust. N.Z. Grapegrower Winemaker 478: 73-79. 7 Butzke, C. (1998). Survey of Yeast Assimilable Nitrogen status in musts from California, Oregon, and Washington. Am. J. Enol. Vitic. 49: 220-224. 8 Carrau, F.M. (2003) Characterization of yeast in relation to the ability to utilize nitrogen Studies of aroma compounds. PhD thesis, Universidad de la Republica, Uruguay. 9 Coulter, A.D.; Godden, P.W.; Pretorius, I.S. (2004) Succinic acid-how is it formed, what is its eff ect on titratable acidity, and what factors infl uence its concentration in wine? Aust. N.Z. Wine Ind. J. 19: 16-20, 22-25. 10 Dambergs, R.G.; Kambouris, B.; Cynkar, W.; Janik, L.J.; Cozzolino, D.; Henschke, P.A.; Gishen, M. (2005) A comparison of near infrared and midinfrared spectroscopy for the analysis of yeast-assimilable nitrogen in grape juice. Blair, R.J.; Williams, P.J.; Pretorius, I.S. (eds.) Proceedings of the twelfth Australian wine industry technical conference; 2429 July 2004; Melbourne, Vic. Australian Wine Industry Technical Conference Inc., Adelaide SA: 334.
11 Diaz-Maroto, M.C.; Schneider, R.; Baumes, R. (2005) Formation pathways of ethyl esters of branched short-chain fatty acids during wine aging. J. Agric Food Chem., 53, 3503-3509. 12 Duan, W.; Roddick, F.A.; Higgins, V.J.; Rogers, P.J. (2004) A parallel analysis of H2S and SO2 formation by brewing yeast in response to sulphur-containing amino acids and ammonium ions. J. Am. Soc. Brew. Chem. 62: 35-41. 13 Dukes, B.C.; Butzke, C.E. (1998) Rapid determination of primary amino acids in grape juice using an o-phthaldialdehyde/N-acetyl-L-cysteine spectrophotometric assay. Am. J. Enol. Vitic. 49: 125-134. 14 Eglinton, J.; Siebert, T.; Kwaitkowski, M.; Francis, L.; Henschke P. (2005) Compositional and sensory implications of practical strategies for ensuring complete fermentation with non-conventional yeast. Blair, R.J.; Williams, P.J.; Pretorius, I.S.(eds.) Proceedings of the twelfth Australian wine industry technical conference; 24-29 July 2004; Melbourne, Vic. Australian Wine Industry Technical Conference Inc., Adelaide SA: 288. 15 Filipe-Ribeiro, L.; Mendes-Faia, A. (2007) Validation and comparison of analytical methods used to evaluate the nitrogen status of grape juice. Food Chem. 100: 1272-1277. 16 Gockowiak, H.; Henschke, P.A. (1992) Nitrogen composition of grape juice and implications for fermentation: results of a survey made in N-E Victoria. Aust Grapegrower Winemaker 340: 131, 133-138. 17 Gump, B.H.; Zoecklein, B.W.; Fugelsang, K.C.; Whiton, R.S. (2002) Comparison of analytical methods for prediction of prefermentation nutritional status of grape juice. Am. J. Enol. Vitic. 53: 325-329. 18 Guth, H. (1997) Quantitation and sensory studies of character impact odorants of diff erent white wines varieties. J. Agric. Food Chem., 45: 3027-3032. 19 Guth, H.; Sies, A. (2002) Flavour of wines: Towards an understanding by reconstitution experiments and an analysis of ethanols eff ect on odour activity of key compounds. Blair, R.J.; Williams, P.J.; Hj, P.B. (eds.). Proceedings of the eleventh Australian Wine Industry Technical Conference, 7-11 October 2001, Adelaide SA. Australian Wine Industry Technical Conference Inc., Adelaide SA 128-139. 20 Henschke, P.A. (1996) Hydrogen sulphide production by yeast during fermentation. Proceedings Eleventh international oenological symposium, Sopron, Hungary (International Association for Winery Technology and Management: Breisach, Germany) pp. 83-102.
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liver gold medal wines when other S. cerevisiae strains dont even leave the starting blocks? Research at the Australian Wine Research Institute (AWRI) is beginning to unravel the mysteries of the variation across the S. cerevisiae species, and early results on what they tell us about wine yeast are tantalising. The variation in performance that we see across strains of S. cerevisiae is inheritable; this means that it is genetically determined. The starting point for characterising this variation, therefore, should focus on yeast genetics. Fortunately, S. cerevisiae was the first organism of its type to have its genetic make-up (its genome) sequenced, and this was done over ten years ago on a strain, known as S288c, chosen for its laboratory friendly characteristics (for more information on what genes and genomes are, see the breakout box). Scientists love this yeast because it is very easy to work with, but it would not win any medals in the winemaking arena; in fact, it is probably not even up to amateur status. Nevertheless, if you want to find out what makes wine yeast so different from other S. cerevisiae strains, you have to have something to compare it with, and S288c is a good starting point. Another strain of S. cerevisiae, YJM789, recently had its genome sequenced. The genome of this yeast, an opportunistic pathogen isolated from the lungs of an AIDS patient, turned out to be quite different to that S288c. Thus we had two different versions of S. cerevisiae to compare a wine yeast against, and this is what we found. It turns out that our wine yeast is a little more different to the two previously sequenced strains than they are to each other (Figure 2). About 0.6% of the letters of the wine yeast sequence are different to what is found in the laboratory strain. This might seem like a small difference, but if you consider that genetic differences between humans and chimpanzees amount to only about 1-2%, it is really quite large. Perhaps of greater interest, however, is that there are extra DNA sequences in the wine yeast; enough to carry at least 27 genes that are not present in the two yeasts it was compared against. In fact, some of the sequences in this extra DNA do not resemble anything found in other species of Saccharomyces; they appear to be more like genes found in very distant fungal relatives. We do not yet know how they got into the wine yeast genome, but we are curious to find out whether or not they play a part in distinguishing wine yeast from other S. cerevisiae, particularly in the winemaking stakes. Some of the wine yeast-specific genes encode proteins that are probably associated with the cell wall,
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a feature of yeast that is undoubtedly important for resilience in inhospitable environments. We are curious to find out whether these genes impact on robustness of wine yeast, a feature that is crucial for completing fermentations. Do these genes, for example, make the yeast more or less vulnerable to becoming stuck or sluggish in a ferment? We have also identified genes that probably encode proteins associated with amino acid uptake (a neutral amino acid transporter) and metabolism (an aspartate transaminase). Because amino acid metabolism is associated with flavour development, it is tempting to suggest that these genes will impact on sensory attributes in wine, but, of course, this will have to be tested. Then there are lots of genes that we cannot guess the function(s) of yet, and these might turn out to be the most exciting of all; time and experimental work will tell. Interestingly, we also found some sizeable rearrangements in the genome that we are also curious about,
but cannot even guess what their significance will be. What does the future hold now that we have this rich source of information on a wine yeast? We will, of course, ascertain as far as possible, which of the unique features of a wine yeast genome are important in a winemaking context. However, we also plan to build on data gathered from this project by sequencing and comparing the genomes of several other wine yeast strains that are known to have different winemaking properties This will enable us to work out what is common to all wine yeasts (i.e. what constitutes the core requirements of a wine yeast) and what differences between them drive production of wines with differing qualities (e.g. different propensities to deliver fruity flavours and aromas). Once we understand what makes a wine yeast tick, and the significance of variation among wine yeasts, we will be much better placed to develop strains of
1(a)
1(b)
Figure 1. No two humans have the same genetic make-up. An elite athlete, for example, is born with a genome that has gold medal stamped all over it (Figure 1(a)). Similarly, not all wine yeasts are the same; different wine yeasts have different genetic make-up (Figure 1(b)). This is why some wine yeast strains are more robust that others and different strains impart different sensory properties to wines. Understanding what, in the genome of a wine yeast, determines its robustness and its ability to produce desirable (and undesirable) sensory attributes will enable the development of improved strains that will assist winemakers to craft wines for an increasingly demanding and constantly changing market.
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ACKNOWLEDGEMENTS
The AWRI, a member of the Wine Innovation Cluster in Adelaide, is supported by Australias grapegrowers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. Systems biology research at the AWRI is performed using resources provided as part of the National Collaborative Research Infrastructure Strategy, an initiative of the Australian Government, in addition to funds from the South Australian State Government. We gratefully acknowledge the contribution of the Australian Genome Research Facility, a member of Bioplatforms Australia, where the actual sequencing of the wine yeast genome was carried out. We also thank Sharon Mascall and Rae Blair for editorial assistance, and JeffEglinton for the preparation of the illustrations. The detailed results of this work are published in the peer-reviewed journal FEMS Yeast Research.
Figure 2. The genome sequences of three strains of Saccharomyces cerevisiae has revealed how similar they are to each other. The numbers in circles on the arrows separating the different strains represent the number of letters in the genome that differ between the strains. The 60,399 differences between a wine yeast and a laboratory yeast is equivalent to about 0.6% of the genome. this microorganism that can complete the marathon of fermentation without becoming stuck or sluggish en route, while producing gold medal wines; and all of this should be possible without the need of performance enhancing additives. Just like our Olympians at the Australian Institute of Sport, the wine sector has gold-medal aspirations. Backed by sound science and robust research it should be gold all of the way for Australian winemakers.
FURTHER READING
Borneman, A.R, Forgan; A., Chambers, P.J. and Pretorius, I.S. (2008) Comparative genome analysis of a Saccharomyces cerevisiae wine strain. FEMS Yeast Research 8:1185-1195. Borneman, A.R.; Chambers, P.J. and Pretorius, I.S. (2007) Yeast Systems Biology: modelling the winemakers art. Trends in Biotechnology 25:349-355. Goffeau A.; Barrell B.G.; Bussey H.; Davis R.W.; Dujon B.; Feldmann H.; Galibert F.; Hoheisel J.D.; Jacq C.; Johnston M,; Louis E.J.; Mewes H.W.; Murakami Y.; Philippsen P.; Tettelin H. and Oliver S.G. (1996) Life with 6000 genes. Science 274: 546, 563-567.
enes are recipes for making proteins. For example, your cells carry a gene/recipe for making the protein insulin, which is a hormone that regulates blood sugar level. You also have genes/recipes that instruct your cells how to make proteins that control how tall you can grow, the colour of your eyes, the general shape of your body, etc. It is these recipes of life that dictate whether you will have athletic potential or not, and, because you inherited them from your parents you end up looking like them. If genes are recipes then genomes are recipe books. The human genome carries all of the recipes required for making proteins to build a human body from conception to adulthood, and repair and defend that body during its life. All of our physiology and anatomy is shaped by a collection of 20,000-25,000 genes that comprise the human genome. And, unless you have an identical twin, your recipe book diff ers a little from everyone elses. The language of the genes is very diff erent the languages we use to communicate with each other. It is based on an alphabet of only four letters (A, T, G and C) and its lexicon is limited to three letter words, which means there are only 64 words in the genetic dictionary. However this is more than enough to string together sets of instructions for building all of the proteins (enzymes, hormones, muscles, antibodies, cartilage etc.) we require for life. The paper on which the words that make up the recipes of life is written is known as DNA, and when we read an entire recipe book of an organism, decoding what is recorded in its DNA, we say we are sequencing its genome. What we end up with in this process is a long sequence of millions of A, T, G, and Cs, with no spaces or obvious punctuation marks, that we have to decipher. Thankfully, sophisticated computational aids can do most of this for us.
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problem and research has identified a multitude of potential causes, any combination of which can be involved. Our inability to measure or assess many of these factors in real time further exacerbates the problem. As a result, it can be difficult to work out why it happens. The definition is straightforward. At some point during fermentation, the process slows or stops too soon, preventing the remaining sugar in the wine from being converted to alcohol and carbon dioxide (Figure 1). Yeast activity or a lack of it is usually the culprit. The yeast might be feeling quite stressed due to its environment, or there might be something wrong with the yeast itself, preventing it from doing its job. Wine yeast works best when the temperature is not too hot, or not too cold and there are plenty of nutritious side-dishes to go with the main carbohydraterich course. Like any selfrespecting organism, it enjoys sanitary conditions and relative freedom from the irritating excesses of unwanted dinner guests (Figure 2) that can spoil the best parties. Coming up for air turns out to be surprisingly important for fermenting yeast a sniffof oxygen will repay the winemaker many fold with renewed enthusiasm to get back to the job at hand. Inebriating agents render it incompetent. Too much ethanol, for example, can stop yeast from growing by starving it of nutrients and increasing the toxicity of other compounds. The advice from the AWRI is that stuck fermentation is usually avoidable if winemakers keep the basics in balance: moderation of sugar levels, adequate supply of key nutrients including nitrogen and oxygen, effective sulfur dioxide, pH control, avoiding over-clarification, vigilant barrel management and clean conditions from harvester to fermentor. But this year, the heat created a new set of problems.
20 Alcohol Biomass 16
% Ethanol
8 4 4
Figure 1. Optimal and sub-optimal fermentation profi les. Four types of sub-optimal fermentation are commonly observed: delayed on-set; continuously slow ferments; sluggish ferments; and incomplete or stuck ferments.
Inside the wineries, in the fermentors, dehydrated and shrivelled fruit was hard to process, causing blockages in heat exchangers. The first week of the heatwave was bad enough with rapidly rising sugar levels, as has been seen in previous hot vintages. But, the second week stressed the already drought stressed vines beyond their limit producing heavily dehydrated fruit, perhaps as the parched vines deprived sunburnt berries of moisture. Outside, in the trucks queued up with their precious loads, the microbes took hold. Micro-organisms had a field day in the mountains of warm berries. Delays, lack of refrigeration and the un-
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During Marchs heatwave, winemakers reported fruit arriving at the crusher with temperatures in the mid to high 30s. On one particular day, fruit arrived at one winery where temperatures topped 40C. Some cooling systems could not cope. The scramble to harvest and deliver the rapidly ripening fruit made matters worse.
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relenting heat offered an opportunity for growth. The combination of heat stress, dehydration and mechanical harvesting had left more fruit damaged than usual. Enemy microbes had the perfect breeding ground. Left to grow unchecked, some bugs can wreak havoc. The grape apiculate yeast Hanseniaspora uvarum can develop quickly, for example, robbing grape must of important nutrients. One of its targets is thiamine, for which it has an insatiable appetite. Thiamine is essential for the efficient production of ethanol by Saccharomyces yeast. There is no easy procedure to detect vitamin deficiencies until the ferment becomes sluggish and responds to supplements. When excessive growth of wild yeast (Figure 2) is observed between the harvester and fermentor it is important to add thiamine and other vitamins to the starter culture. For musts from vineyards with a history of fermentation problems, the addition of complex inactivated yeast nutrients can also be beneficial. Hanseniaspora uvarum can also produce off-flavours including the vinegary taste of acetic acid. Research has shown that hot conditions can dramatically increase the production of acetic acid and, this year, winemakers have reported high levels of volatile acidity (VA) to the AWRI. Lactic acid bacteria also thrive in warm conditions, producing acetic acid as well as other unwanted compounds from the grape sugars and acids. Growth of certain lactic acid bacterial strains is enough to leave the pluckiest wine yeast running for cover. Spoilage in addition to stuck fermentation is often the result.
Accumulation of acetic acid beyond a gram per litre starts to affect yeast growth and becomes more toxic as the alcohol level builds. When this years stuck ferments were tested at the AWRI, sensory analysis revealed that a number of them were affected by a mousy off-flavour. They had high concentrations of acetic acid, low concentrations of malic acid many ferments underwent malolactic fermentation and they contained various unwelcome micro-organisms. The results after analysing one red wine with stuck fermentation were typical of those tested (Table 1, see page 27). Lactic acid bacteria are also known to produce mousy off- flavour in grape bins when given a long journey under warm conditions without sufficient protective sulfites. The winemakers usual weapons against enemy microbes are sulfites, and clarification treatments for whites. But a review of research into stuck fermentation published by the AWRI shows that in extreme conditions, adding sulfur dioxide to grape bins might not be enough. Sulfur dioxide has a tendency to hook up with sugar, sapping its effectiveness. The high levels of sugar caused by this years heatwave would have had such an effect. The higher levels of spoilage noted this year signals that the levels of sulfites normally used to control wild yeast and bacteria was often inadequate under these exceptional conditions. As a result, winemakers needed to increase their use of sulfites and clean their harvester bins regularly to keep the bugs at bay. Without these kinds of control measures, the development of large populations of indigenous microorganisms could have depleted nutrients. This would have made it very hard for the wine yeast to complete the fermentation, which with the higher sugar levels would have been an even harder task.
Debaryomyces
Schizosaccharomyces Kluyveromyces
Pichia
Table 1. Results of analysis of a typical high VA 2008 red ferment. Analysis Alcohol Acetic acid Glucose + fructose Malic acid pH Microbiological Result 12.0% v/v 1.94g/L 47.0g/L <0.05g/L 3.81 Yeast: Saccharomyces sp., non-Saccharomyces sp. Bacteria: Lactobacillus sp. Acetobacter sp. Sensory Volatile, mousy off-flavour
Cryptococcus Metschnikowia
Dekkera
Zygosaccharomyces
Saccharomyces
Figure 2. Grapes harbour a variety of yeasts and bacteria, and depending on the addition of sulfi tes, grape temperature and time taken for transport to the winery, the hygienic status of grape processing machinery, pre-maceration, pressing and clarifi cation procedures, a variable proportion of grape yeasts survive in the juice or must. Together with microorganisms associated with processing equipment, these yeasts can proliferate in juice or must, depending on physicochemical and nutrient conditions.
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tions are high, this threshold increases. More research is needed to determine a threshold for red wine ferments. Recent research at the AWRI is showing that YAN can affect wine style, especially so in white wines. Low YAN ferments tend to give more complex flavour profiles, sometimes a bit dirtier, whereas moderate YAN levels give cleaner, fruitier styles in Chardonnay. But this trend is yeast strain and variety dependent. Some yeast strains actually make more H2S at moderate YAN levels compared with low YAN levels; so it is important to know the characteristics of your favourite yeast. Until we have better information, it appears that moderate diamonnium phosphate (DAP) addition may actually reduce the formation of the characteristic tropical fruit thiols in Sauvignon Blanc and related varieties. Using complex inactivated yeast nutrients in this latter case appears to be a better option. Research has also shown that agrochemicals might also have played a role in the increased incidence of stuck fermentations. While residues of copper from fungicides, are unlikely to cause problems on their own, they might frustrate fermentation when other factors come into play. Late application of agrochemicals to fruit that was harvested weeks ahead of schedule, together with the dry conditions, could have contributed to slightly higher residue levels. One favourite suspect often held responsible for stuck fermentation by winemakers is not proven guilty, however, by AWRI research. Every year, the AWRI handles enquiries about glucose-to-fructose ratios, as winemakers suspect that fructose is somehow to blame when fermentation grinds to a halt. Research has shown, and indeed has been known for nearly a century, for example, that the Saccharomyces yeast tends to consume glucose more quickly than fructose and that the last few grams of sugar feeding the final stages of fermentation will be fructose. Under normal circumstances, when there are no other factors affecting fermentation, the yeast will use the fructose. But as far as the AWRI is aware, there is no conclusive evidence that higher amounts of fructose will cause stuck fermentation. Until proven otherwise, it appears that the high fructose:glucose ratio is a symptom rather than cause of stuck fermentation. Sluggish and stuck ferments from the 2008 vintage are characterised by higher residual sugar concentrations than usual. Under these tougher conditions more robust rescue procedures are needed. By all reports, the AWRIs acclimatisation procedure is working well and has benefited from several improvements, such as fining musts with yeast hulls and racking offyeast lees before reinoculation, and rehydrating the initial yeast preparation with complex yeast nutrients. The important principal behind this procedure is that the rescue culture is sequentially acclimatised to the high
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Optimal 50 400 250 80 Time (hr) 150 120 100 160 200
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Figure 3. The risk of fermentation problems is increased when grape must is sub-optimal in nutrient content, especially for assimilable nitrogen, and vitamins, the latter of which can be lost during grape harvest and must processing. A high ratio of sugar to other nutrients can lead to low biomass and fermentation rate, and the early onset of sugar transport inactivation in yeast. The general rule of thumb is that musts with YAN levels of <150mg/L are more likely to result in slow fermentations, and musts with <50mg/L are more likely to result in stuck ferments (see also Figure 1).
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Region A
Region A
Region B
Figure 4(a). The average YAN concentration in juices from two regions in South Australia for the 2007 vintage and the 2008 vintage. For region A, n = 855 in 2007 and 1272 in 2008; for region B, n = 414 in 2007 and 788 in 2008. Figure 4(b). Standard deviation in average YAN concentration from wine regions A and B was greater in 2008 than in 2007. concentrations of inhibitors in the stuck wine, especially ethanol. It is, however, critically important that the yeast not be starved of sugar or nitrogen during the stepwise acclimatisation procedure. The culture should also be briefly aerated at each step. Fresh yeast lees from successfully completed ferments can also be useful for reinvigorating or restarting ferments when a relatively small amount of residual sugar remains. A small addition of DAP and limited aeration, once the yeast is actively fermenting, promotes good activity. This procedure provides a more convenient rescue option when yeast lees are available. Looking ahead, the AWRI is also alerting winemakers to the longer term effects of the 2008 heatwave. If red wines contain higher than usual amounts of residual sugar, and other nutrients added as fermentation stimulants, for example, the Brettanomyces/Dekkera yeast, can strike later in the maturation cycle or even after bottling of unfiltered wines. The higher alcohol concentrations of 2008 wines might slow the growth of this spoiler meaning that greater vigilance should be maintained further on down the process chain. Malolactic fermentation is also adversely affected by high alcohol levels such that Brettanomyces/Dekkera yeast can have a greater opportunity while the wine is unprotected by sulfites. The more alcohol tolerant lactobacilli, which are capable of forming biogenic amines, will also thrive if the pH is not maintained below 3.5, which is more favourable to Oenococcus. The impact of March 2008 might continue if not managed with greater care due to the perturbed nature of this years wine chemistry.
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ACKNOWLEDGEMENTS
The Australian Wine Research Institute, a member of the Wine Innovation Cluster in Adelaide, is supported by Australias grapegrowers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. We thank Sharon Mascall and Rae Blair for editorial assistance, and JeffEglinton for the preparation of illustrations.
Henschke, P.A. (1997) Stuck fermentation: causes, prevention and cure. Allen, M.; Leske, P.; Baldwin, G., eds. Advances in juice clarification and yeast inoculation: proceedings of a seminar; 15 August 1996; Melbourne, Victoria. Adelaide, South Australia: Australian Society of Viticulture and Oenology; 1997: 30-38, 41. Sablayrolles, J.M.; Dubois, C.; Manginot, C.; Roustan, J.L. and Barre, P. (1996) Effectiveness of combined ammoniacal nitrogen and oxygen additions for completion of sluggish and stuck fermentations. Journal of Fermentation and Bioengineering 82:377-381. Schmidt, S.A.; Tran, T.; Chambers, P.J.; Herderich, M.J. and Pretorius, I.S. (2006) Developing indicators of wine yeast performance: an overview of the impact of ethanol stress. Australian and New Zealand Wine Industry Journal 21:24-30. Ugliano, M.; Henschke, P.A.; Herderich, M.J. and Pretorius, I.S. (2007) Nitrogen management is critical for wine flavour and style. Australian and New Zealand Wine Industry Journal 22:24-30. These articles can be obtained by contacting the AWRIs John Fornachon Memorial Library. Email library@awri.com.au, telephone (08) 8303 6600. Further information is also available on the AWRIs website www.awri.com.au A summary of factors affecting sub-optimal fermentation is presented by Dr Paul Henschke in a webcast at www.awri.com.au. This site is accessible to all Australian grape and wine levy payers.
FURTHER READING
Bell, S.J. and Henschke, P.A. (2005) Implications of nitrogen management for grapes and wine. Australian Journal of Grape and Wine Research 11:242-295. Bisson, L.F. and Butzke, C.E. (2000) Diagnosis and rectification of stuck and sluggish fermentations. American Journal of Enology and Viticulture 51:168177. Bisson, L.F. (1999) Stuck and sluggish fermentations. American Journal of Enology and Viticulture 50:107119. Eglinton, J.M. and Henschke, P.A. (1999) Restarting incomplete fermentations: the effect of high concentrations of acetic acid. Australian Journal of Grape and Wine Research. 5:71-78.
Summary
The 2008 vintage in southern Australia experienced an increase in High sugar levels led to high amounts of ethanol, which can be toxic cases of stuck fermentation. to yeast, leading to higher residual sugar levels in wine. Higher The March heatwave was a key factor. Yeast does not thrive in ex- nutrients can stimulate Brettanomyces/Dekkera yeast during matreme heat or cold, or indeed when subjected to rapid changes in turation. temperature. Low YAN levels after persistent drought also had an impact. Heat stress, transport delays and refrigeration problems gave Simple strategies such as keeping bins and equipment clean, mounwanted microbes a head start, further compromising yeast ac- nitoring pH and using the right yeast and nutrients, can give winetivity. makers the upper hand.
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flavour development in relation to sugar ripeness, this approach can undermine the full-bodied character and ripe fruit flavours for which some Australian wines are known. Removing sugar from grape must before fermentation is another way to lower ethanol but is relatively expensive to carry out. A third strategy used successfully at a number of wineries around the world is to remove alcohol from wine after fermentation. Even this has its drawbacks: it adds to production costs; might impact wine flavour under certain conditions, and not all international markets might accept Australian wine that has undergone this procedure. Another strategy is to target wine yeast. The hunt is on for strains of wine yeast that convert less of the sugar they consume into ethanol (see, for example, Figure 1). Commercially available Saccharomyces cerevisiae strains have been categorised by some yeast manufacturers according to the concentrations of ethanol they produce. But how different are these yeasts? Do strains really differ in terms of the ethanol they produce? To find out, scientists at the AWRI checked how much ethanol is produced by six commercial wine yeasts described by manufacturers as low- or high-ethanol strains. We also investigated two of our own novel wine yeasts produced at the AWRI to see how efficient they were at fermentation and how much ethanol they produced. The two strains came from early stage experimental work at the AWRI aimed at generating yeast that produce reduced amounts of ethanol.
A
Ethanol C0 2
Ethanol
Figure 1. Sugar metabolism of wine yeast. A: Consumption of sugars leads mainly to the production of ethanol and carbon dioxide and, to a lesser extent, glycerol. B: Modifying yeast metabolism so more glycerol is synthesised reduces the amount of ethanol produced.
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acid as well as alcohol in the synthetic wine (CDGJ) and Chardonnay using advanced analysis techniques. The concentrations of ethanol produced by the six commercial wine strains are shown in Figure 2. In synthetic wine, ethanol concentrations varied between 11.3% v/v and 11.6% v/v with a maximum difference of 0.3% v/v. In Chardonnay, ethanol content varied between 12.4% v/v and 12.9% v/v, with a maximum difference of 0.5% v/v. The differences look small but are statistically significant. They are also supported by a previous research study that tested 113 strains of wine yeast grown in synthetic grape juice. Although there are no published data on final ethanol concentrations in red varieties, our research shows it is very likely that 0.5% v/v is the maximum difference possible by choosing a currently available, commercial wine yeast strain. Commercial strains do not give rise to large differences in ethanol concentrations. As a result, we are trying to generate novel strains of wine yeast that metabolise sugar in such a way that less ethanol is produced while maintaining high wine quality. Using a non-genetically modified (non-GM) approach, known as adaptive evolution, we are working to create the right conditions to persuade yeast to produce less ethanol. Even though the science is not straightforward, early results have been promising. So far, two prototype yeast strains have been produced AWRI 1689 and AWRI 1690. Both generate less ethanol than their parent strain N96 (similar to stra-
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Figure 2. Ethanol content of wine produced from chemically defined grape juice (CDGJ) and Chardonnay juice, fermented with different commercial wine yeast strains. We tested the eight different yeasts using chemically defined grape juice (CDGJ), containing specified levels of sugar and yeast assimilable nitrogen, and Chardonnay juice. Fermentation took place in controlled laboratory conditions and, once complete, samples were collected for analysis. The researchers measured residual sugar, ethanol, glycerol, malic acid and acetic
Table 1. Product concentrations in wine made from Chardonnay juice using N96 and wine yeast variants obtained by adaptive evolution. Strain N96 AWRI 1689 AWRI 1690 Ethanol [% v/v] 12.9 0.1 12.7 0.1 12.6 0.1 Glycerol [g/L] 5.8 0.1 7.0 0.0 7.1 0.0 Acetic acid [g/L] 0.1 0.0 0.1 0.0 0.0 0.0 Fermentation efficiency* [g sugar per 1% ethanol] 16.1 0.1 16.4 0.1 16.5 0.1
* Initial and residual sugar concentrations were used to calculate fermentation efficiency. Table 2. Product concentrations in wine made from Chardonnay juice using a range of different commercial wine yeast strains. Strain AWRI 796 Maurivin B AWRI R2 PDM UCD 522 N96 Ethanol [% v/v] 12.4 0.1 12.7 0.1 12.7 0.1 12.8 0.1 12.8 0.0 12.9 0.1 Glycerol [g/L] 7.7 0.2 6.4 0.0 6.4 0.0 5.9 0.1 6.3 0.1 5.8 0.1 Acetic acid [g/L] 0.1 0.0 0.5 0.0 0.1 0.0 0.2 0.0 0.1 0.0 0.1 0.0 Fermentation efficiency* [g sugar per 1% ethanol] 16.8 0.2 16.5 0.1 16.4 0.1 16.3 0.1 16.2 0.0 16.1 0.1
* initial and residual sugar concentrations were used to calculate fermentation efficiency.
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FURTHER READING
De Barros Lopes, M.; Eglinton, J.M.; Henschke, P.A.; Hj, P.B. and Pretorius, I.S. (2003) The connection between yeast and alcohol production in wine: Managing the double edged sword of bottled sunshine. Australian and New Zealand Wine Industry Journal 18:27-31. Godden, P.W. and Gishen, M. (2005) Trends in the composition of Australian wine 1984-2004. In: Blair, R.J.; Francis, M.E. and Pretorius, I.S. (ed) Advances in wine science commemorating 50 years of The Australian Wine Research Institute, The Australian Wine Research Institute, pp115-139. Guth, H. and Seis, A. (2002) Flavour of wines: towards an understanding by reconstitution experiments and an analysis of ethanols effect on odour activity of key compounds. Blair, R.J.; Williams, P.J. and Hj, P.B., (eds). In: Proceedings of the eleventh Australian Wine Industry Technical Conference; 7-10 October 2001. Adelaide, SA Australian, Australian Wine Industry Technical Conference Inc.; pp128-139. Jenson, I. (1997) Differences in alcohol production by various yeast strains: myth or fact? Allen, M.; Leske, P. and Baldwin, G. (eds.) Advances in juice clarification and yeast inoculation: Proceedings of a seminar; 15 August 1996; Melbourne, Vic. Adelaide, SA: Australian Society of Viticulture and Oenology; pp24-25. Palacios, A.; Raginel, F. and Ortiz-Julien, A. (2007) Can the selection of Saccharomyces cerevisiae yeast lead to variations in the final alcohol degree of wines? Australian and New Zealand Grapegrower and Winemaker 527, 71-75. Piskur, J.; Rozpedowska, E.; Polakova, S.; Merico, A. and Compagno, C. (2006) How did Saccharomyces evolve to become a good brewer? Trends in Genetics 22, 183-186.
ACKNOWLEDGEMENTS
The Australian Wine Research Institute, a member of the Wine Innovation Cluster in Adelaide, is supported by Australias grapegrowers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. The authors wish to thank Rae Blair and Sharon Mascall for editorial assistance.
Summary
Hot weather and mature fruit make high alcohol levels in wine more likely. The 2008 vintage is set to hit a sugar high, with high levels of ethanol. Winemakers want to keep alcohol under control for cost, taste and health reasons. Scientists at the AWRI have pioneered a new, GM-free approach to persuade yeast to produce less ethanol during fermentation. Studies have shown that the maximum variation in ethanol levels from current, commercial yeast strains is 0.5% v/v. Innovation at the AWRI is driving the evolution of new, low ethanol yeast in the right direction.
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sistible vi a partir del most del ram. La majoria dels llevats del vi sn de la mateixa espcie, Saccharomyces cerevisiae, per no tots els membres daquest grup sn capaos de produir vi i, entre aquelles soques que ho sn, hi ha una considerable variaci de com deficients sn i com fidedignament fan la seva feina i com poden produir vins de qualitat. Aix ens porta a la segent pregunta: Qu marca a un llevat del vi? Qu existeix en el funcionament intern daquest atleta delit, que li permet crixer en un ambient tan hostil i aconseguir vins amb medalles dor mentre altres soques de S. cerevisiae no passen de la lnia de sortida? Estudis portats a terme a lAustralian Wine Research Institute (AWRI) comencen a desvetllar misteris de les variacions entre soques de S. cerevisiae, essent els primers resultats obtinguts realment prometedors. La variaci en el rendiment que observem entre soques de S. cerevisiae s un atribut heretable; aix significa que est genticament determinat. Per tant, el punt de partida per caracteritzar aquesta variaci shauria denfocar prioritriament en la gentica dels llevats. Sortosament, S. cerevisiae va ser el primer organisme del seu tipus en tenir el seu genoma seqenciat. Aix va assolirse fa uns deu anys en una soca coneguda com S288c, escollida per la seva idonetat a les condicions del laboratori (per una informaci ms detallada sobre qu sn els gens i els genomes, consulteu requadre). Als cientfics els encanta aquest llevat ja que s molt fcil treballar amb ell, per daltra banda, no seria una soca capa de guanyar cap medalla en el camp de la producci de vi, de fet, s possible que ni tan sols arribi a nivells dels que en direm amateurs . No obstant, per discernir qu s el que fa un llevat del vi tan diferent daltres soques de S. cerevisiae s necessari disposar dalguna cosa amb qui comparar-la, i S288c s un bon punt de partida. El genoma duna altra soca de S. cerevisiae, YJM789, sha seqenciat recentment. El genoma daquest llevat, un patogen oportunista allat dels pulmons dun pacient amb SIDA, va resultar fora diferent al dS288c. Aix, ara disposem de dues versions diferents de S. cerevisiae amb les que poder comparar el llevat del vi, i aix va ser el que vam trobar.
Algunes de les seqncies de DNA addicional trobades en el llevat del vi, no sassemblen a res trobat en daltres espcies de Saccharomyces
La variaci individual
Aquestes diferncies en el rendiment dels individus duna mateixa espcie no sn tan sols exclusives de lsser hum. De fet, podem apreciar- les contnuament a la natura. Prenem, per exemple, lhumil llevat que els enlegs utilitzen per produir el complex i irre-
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% de les lletres gentiques de la seqncia del llevat del vi sn diferents de les de la soca de laboratori. Aix pot semblar una molt petita diferncia, per si considerem que la distncia gentica entre dues espcies com els humans i els ximpanzs s nicament dun 1-2 %, el 0,6 % seria una xifra fora considerable per a dues soques duna mateixa espcie. Daltra banda, potser el que sigui ms interessant daquests resultats, s que shan trobat seqncies addicionals al llevat del vi, que sn suficients per afegir com a mnim 27 gens que no estan presents en les soques de Saccharomyces amb que va ser comparada. De fet, algunes de les seqncies en aquest DNA addicional no sassemblen a res trobat en daltres espcies de Saccharomyces. De fet, aquestes seqncies sn ms similars a gens trobats en fongs fora llunyans filogenticament. Encara no sabem com van arribar al genoma del llevat del vi, per tenim una certa curiositat per avaluar si aquestes seqncies estan involucrades en la diferenciaci entre llevats del vi i altres S. cerevisiae, especialment en all referent a les caracterstiques necessries per produir vi. Alguns dels gens especfics en el llevat del vi codifiquen, probablement, protenes associades amb la paret cellular, un tret del llevat que s, sense cap mena de dubte, important per a la seva resistncia en ambients inhspits. Ens agradaria descobrir si aquests gens tenen alguna mena dimpacte en la robustesa del
En el futur, serem capaos desbrinar quins daquests trets nics del genoma dun llevat del vi sn determinants en lmbit de la producci vincola
llevat del vi, una caracterstica que s de gran importncia per completar la fermentaci. Ens preguntem, per exemple, si aquests gens fan al llevat del vi, ms o menys vulnerable a fermentacions lentes o a parades fermentatives. Tamb hem identificat gens que probablement codifiquen protenes associades amb la captura daminocids (un transportador daminocids neutre) i el seu metabolisme (una aspartat transaminasa). Pel fet que el metabolisme daminocids est directament relacionat amb el desenvolupament de compostos associats a sabor i aroma, s temptador suggerir que aquests gens podrien tenir un impacte en els atributs sensorials del vi, encara que aix shauria dinvestigar i confirmar. A ms a ms, existeixen multitud gens dels quals encara no podem inferir les seves funcions. Aquests podrien resultar ser els ms interessants de tots; el temps i el treball experimental ens ho diran.
De manera molt interessant, tamb hem trobat alguns reordenaments cromosmics en el genoma daquest llevat i sobre els quals tamb estem intrigats, encara que de moment no podem fer-nos una idea de quina ser la seva veritable importncia i abast.
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Els membres de lequip investigador australi. Desquerra a dreta: Paul Chambers, Angus Forgan, Sakkie Pretorius i Anthony Borneman. vat del vi? Per suposat, serem capaos de determinar quins daquests trets nics del genoma dun llevat del vi sn determinants en lmbit de la producci de vi. Tot i aix, tamb ens plantegem acumular ms dades a partir daquest projecte mitjanant la seqenciaci i comparaci dels genomes daltres llevats del vi, aquelles que posseeixen diferents propietats per la seva capacitat per produir vi. Aix ens permetr determinar qu s com a tots els llevats del vi (per exemple, quins sn els requeriments fonamentals dun llevat del vi) i quines diferncies entre elles condueixen a la producci de vins amb diferents qualitats (per exemple, la predisposici a produir uns determinats aromes i matisos frutals). Un cop puguem entendre que s all que determina el dest dun llevat del vi, i el significat profund daquestes variacions entre soques de llevats del vi, estarem en una millor posici per desenvolupar variants daquest microorganisme que puguin completar la marat de la fermentaci sense alentir-la o evitant quedar parades durant el seu curs, produint al mateix temps vins mereixedors duna medalla dor; i tot aix seria possible sense afegir additius que incrementin el rendiment. De manera similar als nostres esportistes olmpics, el sector del vi tamb ha de tenir aspiracions a medalles dor. Amb el suport duna cincia de qualitat i una recerca robusta, haurien de ser tot ors per als productors de vi. Research and Development Corporation, a ms duna subvenci equivalent del Govern australi. La recerca en lrea de la biologia de sistemes al AWRI es realitza amb recursos provinents en part del National Collaborative Research Infrastructure Strategy, una iniciativa del Govern australi, amb fons addicionals del Govern de lEstat de South Australia. Agram tamb la contribuci de la Australian Genome Research Facility, membre de Bioplatforms Australia, on es va portar a terme la seqenciaci del genoma del llevat del vi. Tamb volem agrair a Sharon Mascall i Rae Blair per lajut en la redacci, i a Jeff Eglinton per la preparaci de les illustracions. Els resultats cientfics detallats daquest treball han estat publicats a la revista FEMS Yeast Research.
Bibliografia
Borneman, A.R.; Forgan, A.H.; Chambers, P.J.; Pretorius, I.S.: Comparative genome analysis of a Saccharomyces cerevisiae wine strain, FEMS Yeast Research 2008; 8: 1185-1195. Borneman, A.R.; Forgan, A.H.; Chambers, P.J.; Pretorius, I.S.: Unravelling the genetic blueprint of wine yeast, Australian and New Zealand Wine Industry Journal 2008; 23: 21-23. Borneman, A.R.; Forgan, A.H.; Chambers, P.J.; Pretorius, I.S.: Cracking the genetic code of wine yeast, Wine Business Monthly 2008, octubre: 41-43. Borneman, A.R.; Chambers, P.J.; Pretorius, I.S.: Yeast Systems Biology: modelling the winemakers art, Trends in Biotechnology 2007; 25: 349-355. Goffeau, A.; Barrell, B.G.; Bussey, H.; Davis, R.W.; Dujon, B.; Feldmann, H.; Galibert, F.; Hoheisel, J.D.; Jacq, C.; Johnston, M.; Louis, E.J.; Mewes, H.W.; Murakami, Y.; Philippsen, P.; Tettelin, H.; Oliver, S.G.: Life with 6000 genes,
Agraments
Els membres de lequip del AWRI responsables de discernir el mapa gentic del llevat del vi sn els doctors Anthony Borneman, Angus Forgan, Paul Chambers i Sakkie Pretorius. LAustralian Wine Research Institute, un dels membres del Wine Innovation Cluster a Adelaida, disposa del suport econmic dels productors de ram i de vi a travs del seu organisme dinversi, el Grape and Wine
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Effect of Microoxygenation on Color and AnthocyaninRelated Compounds of Wines with Different Phenolic Contents
Cano-Lpez, M.; Pardo-Mnguez, F.; Schmauch, G.; Saucier, C.; Teissedre, P.L.; Lpez-Roca, J.M.; and Gmez-Plaza, E. Departamento de Tecnologa de Alimentos, Nutricin y Bromatologa, Facultad de Veterinaria, Universidad de Murcia, Spain, Bodegas San Isidro, Jumilla, Murcia, Spain, and Facult dOenologie, Universit Victor Segalen, Bordeaux 2, France Several factors may affect the results obtained when micro-oxygenation is applied to red wines, the most important being the moment of application, the doses of oxygen, and the wine phenolic characteristics. In this study, three red wines, made from Vitis vinifera var. Monastrell (2005 vintage) and with different phenolic characteristics, were micro-oxygenated to determine as to how this technique affected the formation of new pigments in the wines and their chromatic characteristics. The results indicated that the different wines were differently affected by micro-oxygenation. In general, the micro-oxygenated wines had a higher percentage of new anthocyanin-derived pigments, being that this formation is more favored in the wines with the highest total phenol content. These compounds, in turn, significantly increased the wine color intensity. The wine with the lowest phenolic content was less influenced by micro-oxygenation, and the observed evolution in the degree of polymerization of tannins suggested that it might have suffered overoxygenation. KEYWORDS: Wine; color; anthocyanins; anthocyanin-derived compounds; micro-oxygenation.
while their interactions with other compounds largely determine the color changes observed in maturing wines. The wine phenolic content depends on the grape characteristics and on the winemaking process. Factors such as phenolic maturity of the grapes, length of maceration, frequency of pumping over, etc. determine the final wine phenolic content (13). The importance of oxygen in the evolution of wine color has been studied for many years, both as regards its role in polyphenol oxidation (47) and as a promoter of the formation on new anthocyanin-derived compounds (5, 811). Indeed, one way of manipulating the phenolic structure of a wine is to use the micro-oxygenation (MO) technique, which relies on the formation of microbubbles through the injection of gaseous oxygen into the wine using a microdiffuser (12). These bubbles rise through the wine, dissolving as they travel to the surface. Empirical results have indicated that MO benefits include the stabilization of wine color, the softening of tannins, and the lessening of vegetative aromas (13, 14). Reactions involving wine polyphenols are the key to these processes, which include changes in proanthocyanin chain length and the consequent effect on wine astringency and the linking of anthocyanins and tannins to form more stably colored forms. Dissolved oxygen leads to the formation of acetaldehyde that, in turn, reacts with flavanols and anthocyanins to induce the formation of a very reactive carbocation that quickly reacts with another flavanol molecule or with anthocyanin, producing ethyl bridge-linked compounds (15, 16). They are unstable (17) and undergo reactions that lead to the formation of new compounds such as flavanyl pyranoanthocyanins. These end products are more stable and colored than the original compounds (11). Moreover, incorporation of anthocyanin into tannin structures also may lead to a decrease in astringency (18). Since its commercial adoption, MO has become common practice and is now used worldwide, although most of the available information is from empirical results. Only a few scientific publications can be found that are related to the effects of MO (8, 1923). Several factors may affect the final results obtained by MO, the most important being the moment of application, doses of oxygen, and wine characteristics. Wines have marked differences in their respective abilities to consume oxygen. As a general rule, this facility is directly related to their relative concentration of polyphenols since phenolic compounds are the main consumers of oxygen (60%) together with ethanol (20%) and SO2 (12%) (22). In our first studies, we tested the use of different doses of oxygen and the effect of the moment of application on wine chromatic characteristics (23, 24). Here, we report the results of a commercial scale MO experiment where the influence of applying oxygen to three wines made from Monastrell grapes of J. Agric. Food Chem. 2008, 56, 59325941
INTRODUCTION
Phenolic compounds are responsible for many of the organoleptic characteristics of wines. Among them, anthocyanins are responsible for the color of red wine,
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Effect of Micro-oxygenation on Color and Anthocyanin-Related Compounds of Wines with Different Phenolic Contents
Table 1. Evolution of Some Physicochemical Parameters during MO of Monastrell Winesa t0 W1 pH titratable acidityb volatile acidityc free SO2 (mg/L) total SO2 (mg/L) acetaldehyde (mg/L) 3.72 5.87 0.39 25 39 22.2 W2 pH titratable acidityb volatile acidityc free SO2 (mg/L) total SO2 (mg/L) acetaldehyde (mg/L) 3.66 6.31 0.33 25 41 29.1 W3 pH titratable acidityb volatile acidityc free SO2 (mg/L) total SO2 (mg/L) acetaldehyde (mg/L) 3.63 6.45 0.40 27 48 32.3 t1 t2 t3 W1 W1 W1 W1 W1 W1 (C) (MO) (C) (MO) (C) (MO) 3.78 5.76 0.40 26 32 13.5 3.76 5.35* 0.41 22* 28* 12.8 3.85 5.38 0.43 37 50 15.1 3.83 5.17* 0.47 37 49 16.6 3.83 5.06 0.42 22 48 21.3 3.79* 4.90* 0.34 14* 46 32.6*
W2 W2 W2 W2 W2 W2 (C) (MO) (C) (MO) (C) (MO) 3.71 5.83 0.33 31 35 23.0 3.67 5.96 0.34 22* 33 24.7 4.00 5.60 0.29 38 61 32.2 4.00 5.73* 0.32 33 61 23.4* 3.83 5.08 0.37 16 51 41.4 3.80 4.90 0.33 11* 35* 45.2
When MLF was complete for all wines, SO2 was added to leave the wines with a free SO2 content close to 30 mg/L, and MO was resumed (January 19, 2006). At this moment, the oxygen supply was reduced to avoid accumulation of acetaldehyde (3 mL/L/month). After 2 months, the oxygen supply was reduced again (1.5 mL/L/month) before it was completely stopped. The wine was analyzed immediately before the MO system began (t0), at the beginning of MLF (t1), when MO began again after MLF (t2), and 15 days after the MO system was stopped (t3) since wines take 8-10 days to absorb the oxygen, depending on temperature and phenolic composition of the wines (26). The MO system (Agrovin S.A., Alcazar de San Juan, Spain) was comprised of an oxygen cylinder (food grade) and a dosing chamber that allowed the doses of oxygen flowing through nine different microdiffusers to be programmed. General Determinations. pH and titratable acidity were measured using an automatic titrator (Metrohm, Herisau, Switzerland). Volatile acidity was determined according to the OIV Official Methods (27). Acetaldehyde and malic acid concentration were determined using enzymatic test kits from Roche Boehringer, Mannheim, Germany. The SO2 concentration (free and total) was measured iodometrically by the Ripper procedure (28) modified to detect the end point potentiometrically with an automatic titrator (Metrohm, Herisau, Switzerland). Identification and Quantification of Anthocyanins. Wines were analyzed by direct injection of the samples previously filtered through a 45 m nylon filter (Teknokroma, Barcelona, Spain). The HPLC analyses were performed on a Waters 2690 liquid chromatograph (Waters, Milford, MA), equipped with aWaters 996 diode array detector and a 250 mm 4 mm i.d., 5 m particle size Lichrocart RP-18 column (Merck, Darmstadt, Germany), using as solvents 4.5% aqueous formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.8 mL/ min. Elution was performed with a gradient starting with 10% B to reach 14.5% B at 30 min, 15.2% B at 45 min, 18% B at 60 min, 25% B at 100 min, and 25-100% B in 30 min. Chromatograms were recorded at 520 nm. Compounds were identified by comparing their UV spectra recorded with the diode array detector and those reported in the literature (11, 29). In addition, an HPLC-MS analysis was conducted to confirm each peak identity. An LC-MSD-Trap VL-01036 liquid chromatograph-ion trap mass detector (Agilent Technologies, Waldbronn, Germany) equipped with an electrospray ionization (ESI) system was used. Elution was performed with the HPLC analysis conditions detailed previously. The heated capillary and voltage were maintained at 350 C and 4 kV, respectively. Mass scans (MS) were measured from m/z 300-1100. Mass spectrometry data were acquired in the positive ionization mode. Anthocyanins and anthocyanin-
W3 W3 W3 W3 W3 W3 (C) (MO) (C) (MO) (C) (MO) 3.66 5.70 0.37 32 44 25.6 3.66 5.56 0.38 25* 35 23.2 4.01 5.66 0.33 26 64 28.7 4.02 5.27* 0.37 29 64 28.4 3.77 5.09 0.37 16 61 22.3 3.78 4.89* 0.39* 11* 38* 35.0*
a An asterisk indicates significant differences between control and microoxygenated wines for each time considered. b Expressed as g/L of tartaric acid. c Expressed as g/L of acetic acid.
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Figure 1. Development of monomeric anthocyanins and ethyl-linked compounds in W1, W2, and W3 wines (SD). Each point corresponds to t0, t1, t2, and t3.
an intermediate situation, the maceration time being intermediate between W1 and W3. As seen in Table 1, there was little effect of MO on pH, and small differences were observed in titratable acidity after MLF (t2) and at the end of the experiment (t3). With regard to concerns about potential microbial spoilage, the volatile acidity did not increase during the studied period. When the MO application was finished, a higher concentration of acetaldehyde was detected in the W1 and W3 MO wines, as compared to their control counterparts, and similar quantities of acetaldehyde were found in W2 (C) and W2 (MO). Excessive oxidation may result in increased levels of acetaldehyde, a compound that at sensory threshold levels adversely affects wine flavor and aroma. Sensory detection limits for red wines are typically in the range of 40-100 ppm (37, 38). Our results showed that, even in wines containing the highest content of acetaldehyde, its sensory detection limit barely was reached. MO promoted a slightly lower content of free in the wines, but differences were small. Total was also lower in W2 and W3 MO wines. Boulet Moutounet (12) reported no effect of MO on SO2 SO2 and SO2
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Figure2. Development of carboxypyranoanthocyanins and sum off lavanyl- and vinylpyranoanthocyanins in W1, W2, and W3 wines (SD).Each point corresponds to t0, t1, t2, and t3. content, whereas Prez-Magario et al. (39) reported small decreases due to MO, results that are similar to our findings. The results of Tao et al. (21) showed that SO2 had a moderating effect on the interaction of oxygen with wine polyphenols since it has the ability to reduce oxidized polyphenols and to remove peroxide. The levels of SO2 in MO wine affect the rate of development of wine polyphenol chemistry, including the formation of polymeric pigments and changes in tannin structure, affecting wine astringency. Anthocyanins and Derived Compounds. The predominant compounds detected in HPLC analyses were the monoglucosides of malvidin, petunidin, delphinidin, peonidin, and cyanidin and their respective acetyl and coumaryl derivatives. The combined concentration of all these compounds diminished with time (Figure 1) the decrease being larger in the MO wines, as also found by Atanasova et al. (8), a decrease that might be explained by the various reactions where anthocyanins are involved, including degradation or polymerization reactions. The most significant decrease was detected between t0 and t2, except in W1, whose concentrations barely changed during this period. This was probably due to the fact that W1 wines were MO for 17 days before MLF started, whereas W2 and W3 were MO for 44 days since the starting of MLF was delayed in these wines. Acetaldehyde-mediated condensation between anthocyanin- 3-glucoside and (epi)catechin leads to ethyl bridge-linked compounds. Three possible isomers were elucidated as well as another compound in which the flavanyl moiety is a dimer. Throughout the experiment, the concentration of ethyl-linked compounds was higher in the MO wines (Figure 1). These are compounds with a purple color, less sensitive to bleaching by SO2 and pH than monomeric anthocyanins, and their formation is favored by oxygen (8, 40). A very large increase was observed in W2 (MO) from t0 to t1, after which it fell sharply. These compounds are unstable and have a tendency to increase in size in the presence of available acetaldehyde. The behavior is in accordance with their reactivity; they are rapidly formed, but also, they can be rapidly broken down, releasing ethyl-flavanol units that, in turn, may react again with anthocyanins or dimers, giving more condensed products or even polymers (11). Pyroanthocyanins are compounds formed when a pyran ring is introduced between the C4 and the
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Figure 3. Development of direct anthocyanin-flavanol adducts and polymeric compounds in W1, W2, and W3 wines (SD). Each point corresponds to t0, t1, t2, and t3. hydroxyl group attached to C5 in the anthocyanin molecule. Some compounds result from the addition of anthocyanin and pyruvic acid (carboxypyranoanthocyanins). Several of these compounds were detected in our samples: petunidin 3-glucoside pyruvate, vitisin A (malvidin 3-(glucoside)pyruvate), acetyl vitisin A (malvidin 3-(acetylglucoside) pyruvate), and coumaryl vitisin A (malvidin 3-(coumarylglucoside) pyruvate). Another pyranoanthocyanin resulting from the cycloaddition of acetaldehyde to malvidin 3-glucoside, and referred to as vitisin B, also was found, and its quantification was included with the carboxypyranoanthocyanins. Pyranoanthocyanins are considered to be important compounds concerning the color of red wine since the cycloaddition process seems to strongly increase the stability of the products, and, in this way, vitisin A has been reported as being more stable than malvidin 3-glucoside or ethyl-linked compounds and more resistant to oxidation (41). The sum of carboxypyranoanthocyanins and vitisin B increased from t0 to t1 (Figure 2), with MO wines increasing more (except for W1). At the end of the experiment, carboxypyranoanthocyanins showed lower concentrations in the control wines than in the MO wines, the greatest increases being detected in W2. Other authors found a strong decrease in vitisin A and related compounds during the first year of wine storage, after which the concentration remained relatively constant (4244). Such a decrease, observed mainly in our study between t1 and t2, was ascribed to their incorporation into polymeric compounds (42). The concentration of carboxypyranoanthocyanins in wines is a result of a balance between the formation reactions and their incorporation in the polymeric compounds. The formation of vitisin A and related compounds requires the presence of free monoglucosides and pyruvic acid. Oxygen or reactive oxygen species are also necessary for the reaction to proceed since all cycloaddition pathways require an oxidation step to recover the flavylium moiety within the final structures (4547). Therefore, the MO process seemed to enhance the formation of vitisin-type compounds by providing oxygen. This result is contrary to that of Atasanova et al. (8), who stated that the addition of pyruvic acid to anthocyanins is not influenced by oxygen. W2, with a higher anthocyanin monoglucoside content (as quantified by HPLC), was the wine showing the highest final concentration of carboxypyranoanthocyanins. Another group of anthocyanins, constituted by hydroxyphenyl- pyranoanthocyanins, results from
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Figure 4. Development of color intensity and hue in W1, W2, and W3 wines (SD). Each point corresponds to t0, t1, t2, and t3. the reactions of anthocyanins and vinyl derivatives (48, 49). Malvidin 3-glucoside- 4-vinylphenol, pinotin A (malvidin 3-glucoside-4-vinylcatechol), and malvidin 3-glucoside-4-vinylguaiacol were detected in our samples. The presence of vinyl derivatives in wine was attributed to enzymatic decarboxylation of phenolic acids by yeast enzymes (48). However, Schwarz and Winterhalter (50) demonstrated that pinotin A also could be formed as a result of the direct addition of caffeic acid to malvidin-3-glucoside, without the need for prior decarboxylation of cinnamic acid derivatives by wine yeasts (51). Also, flavanylpyranoanthocyanin was detected in our samples. We found that the concentration of vinyl and flavanylpyranoanthocyanins increased with time (Figure 2), the increases being larger in the MO wines, especially from t2 to t3. We also detected compounds formed by direct reactions between anthocyanins and flavanols (Figure 3). These may result from the addition of flavanols to anthocyanins (47, 52). Little differences were detected in the direct adducts between control and MO wines. A broad peak at the end of the chromatogram was observed (polymeric peak). It absorbed at around 540, indicating that it contained flavylium units. Its concentration increased with time while its max value decreased, perhaps because anthocyaninethyl- flavanol adducts would first have been formed during winemaking and then transformed into flavanyl-pyranoanthocyanins during wine aging, compounds that present lower max values. The contribution of these polymeric pigments to the overall color of aged red wines may be superior to the contribution of either genuine monomeric anthocyanins or pyranoanthocyanins (50). The area of this peak, as shown in Figure 3, is larger, in general, in MO wines, although no significant differences were observed between W3 (C) and W3 (MO) at the end of the experiment. Chromatic Characteristics of Wine. Figure 4 reflects the evolution of color intensity and hue. The wines behaved in a similar way, independently of their phenolic content. An increase from t0 to t1 was observed in all wines and was higher in MO wines, these wines also being darker in color (see Table S2 in the Supporting Information). A decrease during MLF was then detected, probably due to an increase in pH and the degradation of pigmented compounds by lactic bacteria. After MLF, CI increased, although MO wines maintained a statistically higher CI value. The higher CI in MO wines was probably due to the contribution of
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Figure 5. Development of WC, WCP, and CRDSO2 in W1, W2, and W3 wines (SD). Each point corresponds to t0, t1, t2, and t3. the newly formed pigments, for example, ethyllinked pigments, since the absorbance of these molecules at 620 nm is higher than that of genuine anthocyanins (the percentage of blue color in MO wines was higher than in control wines). Pyranoanthocyanins also may have participated in the higher CI as they show both higher absorbance at 420 nm and contribute to the redness of wines. The hue evolved in a very similar way in all the wines, with a tendency to increase, but more so in control wine, demonstrating that no detectable browning occurred in the MO wines. Figure 5 reflects the evolution of WC, WCP, and CRDSO2. A very small decrease in WC was observed in all wines with no differences due to MO. WCP decreased with time, and the decreases were concomitant with the loss of free anthocyanins usually observed during wine evolution, meaning that the formation of anthocyanin-derived pigments did not compensate for free anthocyanin degradation. Figure 6. Development of abs280 and tannins in different wines (SD). Each point corresponds to t0, t1, t2, and t3. A continuous increase in pigments resistant to SO2 bleaching was observed. The formation of pyranoanthocyanins, which are very stable compounds toward
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Figure 7. Development of mean degree of polymerization of tannins in W1, W2, and W3 wines (SD). Each point corresponds to t0, t1, t2, and t3.
Figure 8. Graphical plot representing the distribution of the wine samples in the plane defined by principal components 1 and 2 as regards their chromatic characteristics (MO wines: solid symbols; control wines: open symbols; W1: circles; W2: squares; and W3: triangles). The variance explained by each principal component is shown in parentheses. SO2 and pH due to the substitution of C4 (47), was probably associated with this evolution. Ethyl-linked compounds also should be resistant to sulfite addition and may have contributed to the observed changes, explaining as to why the values were higher in MO wines.
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Having obtained this grouping, a principal components analysis was conducted with the same data and using the same variables to find as to which variables were mainly responsible for the grouping found (Figure 8). The first two principal components explained 83% of the variance. The representation of these two principal components showed that all MO wines had lower values in component 1 than their corresponding control wines mainly due to lower values of L*, H*, and hue and higher values of CI and anthocyanin-derived compounds, among others. The wines with the highest phenolic content showed negative values of PC1, while W2 differed from the other wines especially in the highest values of PC2, due to the high WC and anthocyanin monoglucoside values. W2 (MO) and W3 (C) were again very close. MO favored reactions that led to the greater formation of new pigments, which, in turn, increased CI and CRDSO2 in all the wines, regardless of their phenolic content. W1 wine was less influenced by MO and the observed increases in mDP for W1 (MO) suggested overoxygenation. This wine presented an anthocyanin content that was too low to promote any great level of condensation reactions. W3 was favored by MO, but the most evident results were found in W2 (MO), with its high anthocyanin content that favored the formation of more stable derived pigments and led to a wine close to W3 (C). Supporting Information Available: Figure of differentiated clusters and tables of wine characteristics at the end of alcoholic fermentation and evolution of CIELab and color. This material is available free of charge via the Internet at http://pubs.acs.org.
(5) Singleton, V. A survey of wine aging reactions, especially with oxygen. In Proceedings of the 50th AnniVersary Annual Meeting; Rantz, J. M., Ed.; American Society for Enology and Viticulture: Davis, CA, 2000; pp 323-336. (6) Singleton, V. Oxygen with phenols and related reactions in must, wines, and model systems: Observations and practical implications. Am. J. Enol. Vitic. 1987, 38, 6977. (7) Danilewicz, J. Review of reaction mechanism of oxygen and proposed intermediate reduction products in wine: Central role of iron and copper. Am. J. Enol. Vitic. 2003, 54, 7385. (8) Atanasova, V.; Fulcrand, H.; Cheynier, V.; Moutounet, M. Effect of oxygenation on polyphenol changes occurring in the course of wine-making. Anal. Chim. Acta 2002, 458, 1527. (9) Jordao, A. M.; Ricardo-da-Silva, J. M.; Laureano, O. Effect of oak constituents and oxygen on the evolution of malvidin-3- glucoside and (+)-catechin in model wine. Am. J. Enol. Vitic. 2006, 57, 377381. (10) Salmon, J. M. Interactions between yeast, oxygen, and polyphenols during alcoholic fermentations: Practical implications. Food Sci. Technol. 2006, 39, 959965. (11) Alcalde-Eon, C.; Escribano-Bailon, M. T.; SantosBuelga, C.; Rivas-Gonzalo, C. Changes in the detailed pigment conposition of red wine during maturity and ageing. A comprehensive study. Anal. Chim. Acta 2005, 563, 238254. (12) Boulet, J.; Moutounet, M. Micro-oxigenacin de los vinos. In Enologa: Fundamentos Cientficos y Tecnolgicos; Flanzy, C., Ed.; MundiPrensa Ediciones: Madrid, 2000; pp 638-642. (13) Parish, M.; Wollan, D.; Paul, R. Micro-oxygenation. A review. Aust. Grapegrower Winemaker 2000, 438, 4750. (14) Kamio, I.; Kamio, X.; Roig, G.; Otero, B.; Yerle, S. Microoxygenation dans la Pninsule Ibrique: Repenser lelevage des vins du sud. ReV. Oenol. 2008, 126, 2227. (15) Dallas, C.; Ricardo da Silva, J. M.; Laureano, O. Products formed in model wine solutions involving anthocyanins, procyanidin B2, and acetaldehyde. J. Agric. Food Chem. 1996, 44, 24022407. (16) Es-Safi, N.; Fulcrand, H.; Cheynier, V.; Moutounet, M. Studies on the acetaldehyde-induced condensation of (-)-epicatechin and malvidin-3-glucoside
LITERATURE CITED
(1) Prez-Magario, S.; Gonzlez-San Jos, M. L. Evolution of flavanols, anthocyanins, and their derivatives during the aging of red wines, elaborated from grapes harvested at different stages of ripening. J. Agric. Food Chem. 2004, 52, 11811189. (2) Bautista-Ortn, A. B.; Fernndez-Fernndez, J. I.; Lpez-Roca, J. M.; Gmez-Plaza, E. The effect of grape ripening stage on red wine color. J. Int. Sci. Vigne Vin 2006, 40, 1424. (3) Bautista-Ortn, A. B.; Fernndez-Fernndez, J. I.; Lpez-Roca, J. M.; Gmez-Plaza, E. Wine-making of highly colored wines: Extended pomace contact and run-off of juice prior to fermentation. Food Sci. Technol. Int. 2004, 10, 287295. (4) Ribreau-Gayon, P.; Glories, Y. Phenolics in grapes and wines. In The Sixth Australian Wine Industry Technical Conference; Lee, T. H., Ed.; Australian Industrial Publishers: Adelaide, Australia, 1986; pp 247-256.
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(37) Peynaud, E. The Taste of Wine; Wiley: New York, 1996. (38) Amerine, M.; Roessler, E. Wines, Their Sensory EValuation; W. H. Freeman: New York, 1982. (39) Perez-Magario, S.; Snchez-Iglesias, M.; OrtegaHeras, M.; Gonzlez-Huerta, C.; Gonzlez-Sanjos, M. L. Color stabilization of red wines by microoxygenation treatment before malolactic fermentation. Food Chem. 2007, 101, 881893. (40) Rivas-Gonzalo, J.; Bravo-Haro, S.; Santos-Buelga, C. Detection of compounds formed through the reaction of malvidin-3- monoglucoside and ca-
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(36) Fernandez, K.; Kennedy, J. A.; Agosin, E. Characterization of Vitis Vinifera L. Cv. Carmanere grape and wine proanthocyanidins. J. Agric. Food Chem. 2007, 55, 36753680.
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techin in the presence of acetaldehyde. J. Agric. Food Chem. 1995, 43, 14441449. (41) Morata, A.; Gomez-Cordoves, C.; Colomo, B.; Suarez, J. A. Pyruvic acid and acetaldehyde by different strains of Saccharomyces cereVisiae: Relationship with vitisin A and B formation in red wines. J. Agric. Food Chem. 2003, 51, 74027409. (42) Schwartz, M.; Quast, P.; von Baer, D.; Winterhalter, P. Vitisin A content in Chilean wines from Vitis Vinifera cv. Cabernet Sauvignon: A contribution to the color of aged red wines. J. Agric. Food Chem. 2003, 51, 62616267. (43) Revilla, I.; Gonzlez-San Jos, M. L. Evolution during the storage of red wines treated with pectolytic enzymes: New anthocyanin pigment formation. J. Wine Res. 2001, 12, 183197. (44) Asestorfer, R.; Markides, A.; Iland, P.; Jones, G. Formation of vitisin A during red wine fermentation and maturation. Aust. J. Grape Wine Res. 2003, 9, 4046. (45) Lee, D.; Swinny, E.; Asenstorfer, R.; Jones, G. Factors affecting the formation of red wine pigments. In Red Wine Color. ReVealing the Mysteries. ACS Symposium Series 886; Waterhouse, A., Kennedy, J. A., Eds.; American Chemical Society: Washington, DC, 2004; pp 125-142. (46) Pozo-Bayon, M. A.; Monagas, M.; Polo, M. C.; Gomez-Cordoves, C. Occurrence of pyroanthocyanins in sparkling wines manufactured with red grape varieties. J. Agric. Food Chem. 2004, 52, 13001306. (47) Fulcrand, H.; Atanasova, V.; Salas, E.; Cheynier, V. The fate of anthocyanins in wine. Are they determining factors? In Red Wine Color. ReVealing the Mysteries. ACS Symposium Series 886; Waterhouse, A., Kennedy, J. A., Eds.; American Chemical Society: Washington, DC, 2004; pp 68-88. (48) Monagas, M.; Nuez, V.; Bartolom, B.; GomezCordobes, C. Anthocyanin-derived pigments in Graciano, Tempranillo, and Cabernet Sauvignon wines produced in Spain. Am. J. Enol. Vitic. 2003, 54, 163169.
(49) Schwartz, M.; Winterhalter, P. A novel synthetic route to substituted pyranoanthocyanins with unique color properties. Tetrahedron Lett. 2003, 44, 75837587. (50) Schwartz, M.; Winterhalter, P. Novel aged anthocyanins from Pinotage wines: Isolation, characterization, and pathway of formation. In Red Wine Color. ReVealing the Mysteries. ACS Symposium Series 886; Waterhouse, A., Kennedy, J. A., Eds.; American Chemical Society: Washington, DC, 2004; pp 179- 197. (51) Schwarz, M.; Hofmann, G.; Winterhalter, P. Investigations on anthocyanins in wines from Vitis Vinifera cv. Pinotage: Factors influencing the formation of Pinotin A and its correlation with wine age. J. Agric. Food Chem. 2004, 52, 498504. (52) Salas, E.; Atanasova, V.; Poncet-Legrand, C.; Meudec, E.; Mazauric, J. P.; Cheynier, V. Demostration of the occurence of flavonol-anthocyanin adducts in wine and in model disolutions. Anal. Chim. Acta 2004, 513, 325332. (53) Nikfardjam, M.; Dykes, S. I. Micro-oxygenation research at Lincoln University. Part 3: Polyphenolic analysis of Cabernet Sauvignon wines under the application of micro-oxygenation. Aust. NZ Grapegrower Winemaker 2003, 467, 4144. (54) Vidal, S.; Cartalade, D.; Souquet, J. M.; Fulcrand, H.; Cheynier, V. Changes in proanthocyanidin chain length in winelike model solutions. J. Agric. Food Chem. 2002, 50, 22612266. (55) Cheynier, V.; Remy, S.; Fulcrand, H. Mechanisms of anthocyanin and tannin changes during winemaking and aging. In The ASEV 50th AnniVersary Annual Meeting; Rautz, J., Ed.; ASEV: Davis, CA, 2000; pp 337-344. (56) Du Toit, W.; Marais, J.; Pretorius, I. S.; du Toit, M. Oxygen in must and wine: A review. S. Afr. J. Enol. Vitic. 2006, 57, 7694. (57) Du Toit, W.; Lisjak, K.; Marais, J.; du Toit, M. The effect of micro-oxygenation on the phenolic composition, quality, and aerobic wine-spoilage microorganisms of different South African red wines. S. Afr. J. Enol. Vitic. 2006, 27, 5767. Received for review February 28, 2008. Revised manuscript received May 9, 2008. Accepted May 9, 2008. This work was made possible by financial assistance from the Fundacin Sneca (PB/31/FS/02).
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valores mximos en el vino microoxigenado con dosis media. Estos parmetros muestran que se ha favorecido la formacin de nuevos pigmentos en los vinos. Por otro lado, el contenido de acetaldehdo libre en los vinos microoxigenados es menor que en el testigo, manifestando que el acetaldehdo generado forma parte de nuevos pigmentos, no interfiriendo en las caractersticas organolpticas de los vinos microoxigenados. Al final del tratamiento se realiz un anlisis organolptico de los vinos, que junto a los resultados obtenidos se concluye que la dosis media result ser la mas adecuada al finalizar la microoxigenacin.
INTRODUCCIN
Los compuestos fenlicos en un vino tinto determinan sus caractersticas sensoriales, principalmente el color y la astringencia. La composicin fenlica depende de la composicin de la uva, factores edafoclimticos, procesos de vinificacin y diferentes prcticas enolgicas. El oxigeno juega un papel importante en diversos procesos bioqumicos en los mostos y en los vinos, tanto durante la fermentacin alcohlica como en las reacciones de oxidacin y/o polimerizacin de compuestos polifenlicos que se producen durante el envejecimiento del vino. Por ello, tradicionalmente se han efectuado aportes de oxigeno, bien mediante remontados al inicio de la fermentacin alcohlica; bien mediante trasiegos tras la fermentacin alcohlica y fermentacin malolctica cuyo objetivo es eliminar y/o evitar la formacin de compuestos sulfhdricos y retirar las las gruesas (1). Durante el envejecimiento del vino en barrica de roble se produce la cesin de compuestos fenlicos y aromas propios de la madera al vino, adems de una microoxigenacin natural, al difundirse pequeas cantidades de oxgeno a travs del esquive de la barrica,
RESUMEN
La microoxigenacin est basada en el aporte controlado de pequeas cantidades de oxigeno de forma continua y lenta; siendo la velocidad del aporte de oxigeno inferior a la velocidad de su consumo, evitando la acumulacin de oxigeno. El objetivo de esta tcnica es favorecer la condensacin entre taninos y antocianos y la formacin de nuevos pigmentos; proporcionando as un incremento y estabilizacin del color del vino. Se ha estudiado el efecto de la tcnica aplicando distintas dosis de oxigeno (dosis baja, media y alta), a vinos de Monastrell. Los resultados obtenidos muestran que la aplicacin de oxigeno no afecta a la sanidad del vino. Los vinos microoxigenados presentan mayor intensidad colorante que el testigo. La tonalidad es similar en todos ellos, manifestando la mnima oxidacin de polifenoles producida. Se destaca el incremento de la fraccin de color debido a pigmentos polimricos, el ndice de ionizacin y de PVPP en los vinos microoxigenados, presentando sus
Figura 1.- Estructura del polmero por condensacin directa, A) tanino-antociano y B) compuesto bicclico formado por la reaccin entre el flavano y Mv-3Glu. (Remy et al. 2000). Enlogos Efecto de la microoxigenacin en vinos tintos. Enologos 34,46-50, 2005
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entre las uniones de las duelas y los poros de la madera. Este oxigeno se ha demostrado que influye notablemente en la composicin fenlica del vino, ya que interviene en reacciones de oxidacin y/o polimerizacin, generando nuevos pigmentos que incrementan y estabilizan el color del vino (7,8,9,10,11,12,13,14, 15). La generacin de estos pigmentos sigue los siguientes mecanismos: a) Reacciones de condensacin directa entre antocianos (A) y taninos (T): Los nuevos compuestos se forman mediante procesos de adicin nucleoflica dando lugar a estructuras A+-T y T-A+. Han sido encontradas en vinos envejecidos (10, 16), ya que es una reaccin lenta (17). Ambas estructuras son mas complejas que los antocianos monmeros, de color similar a los antocianos pero resistentes a la decoloracin por SO2, especialmente la estuctura A+-T. La formacin de estos compuestos genera una disminucin de la astringencia de los vinos durante el envejecimiento (Figura 1).
b) Reaccin de condensacin mediante puentes de etilo. En estas reacciones est involurado el acetaldehdo, producto que aparece en el vino producido en pequeas cantidades por las levaduras durante el metabolismo de azcares (18) y por la oxidacin de etanol. El resultado son productos enlazados por puente de etilo, incluyendo taninos (T-etil-T), aductos de taninos-antocianos (T-etil-A) y oligmeros de antocianos (A-etil-A). La reaccin de polimerizacin finaliza cuando en el otro extremo del polmero se encuentra unido un antociano (20, 21). Su formacin es ms rpida que la de los pigmentos polimricos por condensacin directa (17). La presencia de estos pigmentos produce incremento y estabilizacin del color del vino; su color es malva, son resistentes a las variaciones de pH, a la decoloracin del sulfuroso y a las oxidaciones (19, 24), aunque se ha demostrado que su estabilidad en el tiempo es pequea y evolucionan hacia otros tipos de compuestos polimricos (Figura 2).
Figura 2.- Compuestos formados en presencia de acetaldehdo y de otros metabolitos de las levaduras. (Cheynier 2005).
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MATERIALES Y MTODOS
a) Condiciones de microoxigenacin de los vinos Para la experiencia de microoxigenacin se ha utilizado un vino tinto de la variedad Monastrell, elaborado por Bodegas BSI de Jumilla (Murcia), que presentaba las siguientes caractersticas cromticas iniciales: intensidad colorante (IC): 14.7, tono: 0.51, ndice de polifenles totales (IPT): 67.40 y antocianos totales: 443.63 mg/L Tras la fermentacin alcohlica el vino se dispuso en depsitos de 17.500L., uno de ello se utiliza como testigo, en los otros se aplic tres dosis diferentes de oxgeno y en tres periodos distintos de la elaboracin. La adicin de oxigeno se ha realizado durante tres periodos:
Figura 3.- Estructura qumica Vitisina 1) A y 2) B. (Cheynier 2005). c) Nuevos pigmentos de bajo peso molecular. Estos pigmentos se forman mediante la cicloadicn de metabolitos producidos por las levaduras, como el acetaldehdo, cido pirvico o vinilfenoles, con el antociano, generando pigmentos como la Vitisina A y B y los antocianovinilfenoles. Han sido identificados en vinos envejecidos (8,9,12,13,15,22) en botella y en barrica y en vinos microoxigenados (7). Son pigmentos ligeramente anaranjados, ms resistentes a las variaciones de pH, al sulfuroso (9) y a las oxidaciones que los antocianos; estabilizando as el color del vino (12, 23) (Figura 3). Debido a la importancia de la presencia de pequeas cantidades de oxgeno y de acetaldehdo en la formacin de estos pigmentos, naci en la dcada de los 90 la tcnica de la microoxigenacin, basada en el aporte controlado de pequeas cantidades de oxigeno al vino mediante un microdifusor poroso, de forma continua y lenta, siendo la velocidad de aporte de oxigeno inferior a la velocidad de consumo, evitando la acumulacin en vino y generando la mxima cantidad posible de este aldehdo. Determinadas experiencias han observado que tanto la crianza en barrica como la microoxigenacin producen resultados similares en cuanto al incremento de color y disminucin de astringencia, pudiendo con esta tcnica reproducir, e incluso acelerar, parte de las transformaciones que sufren los compuestos fenlicos durante la crianza en barrica (3, 4). En este estudio se pretende conocer el efecto de esta tcnica, aplicando distintas dosis de oxigeno, sobre el color de un vino tinto de la variedad Monastrell.
- Durante dos semanas, entre el final de la fermentacin alcohlica y el inicio de malolctica, con dosis aplicadas de 5, 10 y 15 mL/L/mes. La cantidad de oxigeno aplicada en este periodo, es la mas alta, para favorecer la generacin de acetaldehdo. - Despus de la fermentacin malolctica, durante un periodo de 45 das, las dosis aplicadas fueron 3, 5 y 7 mL/L/mes. - Tras un anlisis organolptico de los vinos, se continu microxigenando 15 das ms, suavizando los vinos microxigenados, aunque en este periodo las dosis en los tres casos se reduce a la mitad, puesto que el contenido de antocianos haba descendido en todos los vinos. La Figura 4 muestra un esquema del sistema de microoxigenacin. Cada salida de oxigeno esta conectada a un microdifusor poroso que se encuentra suspendido en el interior del depsito sin llegar a tocar el fondo, de tal manera que las microburbujas se disuelvan en el vino antes de que lleguen a la superficie. b) Determinaciones fisico-qumicas y espectrofotomtricas El pH, la acidez total (g/L de cido tartrico) y la acidez voltil se determinaron segn los mtodos oficiales. Las medidas de absorbancia se realizan en un espectrofotmetro Helios Alpha (Thermospectronic). Las muestras son centrifugadas y se ajust el pH a 3.6. La intensidad de color (IC) se calcul como la suma de absorbancias a 620 nm, 520 nm y 420 nm; otras variables calculadas son los porcentajes de rojo, amarillo y azul del color del vino (30). El tono como el coeficiente entre la absorbancia a 420 nm y 520 nm (25). El anlisis del ndice de polifenoles totales (IPT), antocianos totales y taninos totales (g/L), y los ndices
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MANMETRO
MICRODIFUSOR
de PVPP, ionizacin y HCl se efectu siguiendo los mtodos descritos por Ribereau-Gayon et al. (29). La fraccin de color debida a los pigmentos polimricos se calcul de acuerdo con los mtodos descritos por Boulton (27).
RESULTADOS Y DISCUSIN
El Grfico 1 muestra la evolucin de pH, acidez total y acidez voltil durante la microoxigenacin. Dichos parmetros son similares en los vinos microoxigenados y el testigo, manifestando que el aporte de oxigeno no altera la sanidad del vino. Se observa un incremento en la IC (Grfico 2a) en el primer periodo de microoxigenacin (Octubre-Diciembre/03) en los vinos microoxigenados. Tras la fermentacin malolctica (Enero/04) se observ en todos ellos un descenso, probablemente consecuencia de la variacin del pH y la precipitacin de materia coloidal que arrastra materia colorante. Aunque el aporte de oxigeno durante el segundo periodo fue menor, se observ de nuevo un aumento de IC en los vinos microoxigenados, diferencindose todos los vinos en color. Este incremento de color en presencia de pequeas cantidades de oxigeno durante los primeros meses de la elaboracin tambin ha sido encontrado por otros autores (3,5,6, ). Por otra parte, la evolucin del tono, representado por el Grfico 2b; es similar en los distintos vinos, indicando que los compuestos fenlicos sufren una mnima oxidacin durante el tratamiento. Mientras que la IC vari para los diferences vinos, el % rojo tras la microoxigenacin (Grfico 3) se mantuvo constante en todos los vinos; sugiriendo que ambos parmetros no estn relacionados directamente, tal y como se ha indicado en otros trabajos (31,32), sino que la IC depende tambin de la absorbancia de los compuestos que aportan tonos amarillos y azules. El
Grfico 1.- Evolucin durante la microoxigenacin de a) pH, b) acidez total y c) acidez voltil de los vinos durante la microoxigenacin.
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Grfico 2.- Evolucin de a) intensidad colorante (I.C.) y b) tono durante la microoxigenacin. Grfico 3 muestra la variacin de la componente amarilla y azul en los vinos tras la microoxigenacin. Se observa un incremento de la componente azul en los vinos microoxigenados, siendo mxima para el vino donde se aplic la dosis media de oxgeno; mientras que la componente amarilla se mantiene constante en todos ellos excepto en el testigo. Estos resultados sealan la presencia de pigmentos polimricos unidos mediante acetaldehdo en los vinos microoxigenados, ya que como propone Gmez-Cordovs et al. (23) existe una correlacin directa con la componente azul y la polimerizacin de polifenles. El contenido de antocianos totales (Grfico 4) desciende por igual en todos los vinos durante la experiencia. El incremento de color y la disminucin de antocianos totales en los vinos microoxigenados son datos aparentemente contradictorios, pero tiene su explicacin cuando se analiza la evolucin de la fraccin de color debido a pigmentos polimricos, como muestra el Grfico 5. El color debido a los pigmentos polimricos incrementa durante la aplicacin de oxigeno, al igual que los resultados recogidos por Atanasova et al. (7). Su evolucin es similar a la variacin del color (Grfico 2a), dicha relacin tambin fue observada por Castellari et al. (6), presentando al finalizar la microoxigenacin su valor mximo en el vino con dosis media. El incremento de color en los vinos microoxigenados puede explicarse tambin mediante el anlisis del ndice de ionizacin y PVPP, como muestra el Grfico 6. El ndice de ionizacin indica el porcentaje de antocianos que presentan color en el vino. Los vinos microoxigenados presentan mayor valor que el testi-
Grfico 3.- Variacin al inicio y al final de la microoxigenacin de los distintos vinos, % rojo, % amarillo y % azul.
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Grfico 4.- Evolucin de antocianos totales (mg/L) de los vinos durante la microoxigenacin.
Grfico 7.- Evolucin de A) IPT y B) taninos totales (g/L) de los vinos durante la microoxigenacin.
Grfico 6.- ndice de ionizacin y de PVPP en el vino inicial y en todos los vino tras la microoxigenacin.
Los Grficos 7a y 7b muestran la evolucin de IPT y taninos totales. Ambos parmetros son similares en todos los vinos; lo que indica que no se produce ninguna precipitacin de taninos durante la aplicacin de esta tcnica. El anlisis del contenido en taninos (procianidinas), representado por el Grfico 7b, muestra que los niveles se mantiene durante la microoxigenacin y es semejante en todos los vinos. No obstante, el ndice de HCl, indicador del porcentaje de la polimerizacin de taninos presentes en vino; es superior en los vinos microoxigenados que en el testigo tras el periodo de microoxigenacin (Grfico 8), al igual que a Cabanillas
et al.(2001), presentando el mximo en el vino microoxigenado con la dosis media. Confirmando que la presencia de oxigeno favorece la polimerizacin de taninos, traducindose en una disminucin de astringencia. Tras la microoxigenacin se realiz el anlisis organolptico de los vinos, concluyendo que: - El vino testigo era ligeramente spero en boca con taninos agresivos. - Los vinos microoxigenados con dosis baja y media en boca eran redondos y con gusto agradable a mitad
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BIBLIOGRAFA
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(20) Es-Safin.E, Fulcrand H., Cheynier V., Mountounet M.(1999). Studies on the Acetaldhyde-Induced concensacion of (-)- epicatequina and Malvini-3 Glucoside in a Model Solution system. J. Agric. Food Chem.,47,2096-2102. (21) Es-Safin.E, Cheynier V., Mountounet M.(2002). Role of Aldehydic Derivates in the Condensation of Phenolic Compounds with emphasis on the sensorial Propieties of fruit-derived foods. J. Agric. Food Chem.,50,5571-5585. (22) Fancia- Aricha E.M., Guerra M.T., Rivas- Gonzalo J.C., Santos-Buelga C. (1997) New anthocyanin Pigments formed after condensation with flavanols J.Agric. Food Chem.,45,2262-2266. (23) Sarni-Manchado P., Fulcrand H., Souquet JM., Cheynier V., Moutounet M.(1996). Stability and Color of unreported wine anthocyanin-derived Pigments. J.Food Science,61,938-941. (24) Escribano-Bailon T., lvarez-Garca M., RivasGonzalo J., Heredia F.J., Santos-Buelga C.(2001). Color and Stabity of Pigments-Derived from Acethaldehido medianted condensation between Mv-3-O-Glu and (+)-catequin. J.Agric. Food Chem.,49,1213-1217. (25) Sudraud P.(1958). Interpretationdes courbes dabsortion des vins rouges. An Technol Agric.,7,203-208. (26) OIV (1990).Rcueil des Mthodes Internationales dAnalyse des vins et mots. Office International de la Vigne et du Vin, Paris, Francia.
(27) Boulton R.B. (2001). The copigmentationof anthocyanins and its role in the color in red wine: a critical review. Am. J. Enol. Vitic. 52, 67-87. (28) Ribreau-Gayon J, Peynaud E., Sudraud P., Ribreau-Gayon P. (1982) Trait denologie. Sciences et Thechniques et du Vin.Tome I. Analise et contrle des Vins.Ed.Dunod. Paris. Francia. (29) Ribreau-Gayon P., Gories Y., Maujean A., Dubourdieu D.(1998). Trait dOenologie.2. Chimie du Vin. Stabilisation et traitaments. Editorial Dunod. (30) Glories Y. (1978). Recherches sur la materie colorant des vins rouges. Thesis de la Universite de Bourdeaux, Universit de Bourdeaux. (31) Revilla I., Gonzlez-SanJos M.L. and Gmez Cordovs (1999). Modificaciones cromticas del vino timto de crianza segn el tipo de varrica en que envejece. Food Sci. Tech. Int., 5(2): 177-181. (32) Prez-Prieto L.J., De la Hera-Orts M.L., Lpez Roca J.M., Fermandez-fernndez J.I. y Gmez Plaza E. (2003). Oak-matured wines: Influence of the caracteristics of the barrel on wine colour and sensory characteristics. J. Sci. Food Agric., 83: 14451450. (33) Gmez- Cordovs C and Gonzlez-SanJos M.L (1995). Interpretation of color Varibles during the aging of red wines: Relathionship with Families of phenolic compounds. J.Agric. Food Chem., 43(3): 557-561. (34) Cheynier V. (2005). Polyphenols in foods are more complex than often thought. Am. J. Clin. Nutr., 81 (suppl): 223S-229S.
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