Plant extracts applications to the vineyard and their impact

Transcription

UNIVERSIDAD DE CASTILLA-LA MANCHA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
DEPARTAMENTO DE CIENCIA Y TECNOLOGÍA AGROFORESTAL Y GENÉTICA
Plant extracts applications to the vineyard
and their impact on wine aroma
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
At the University of Castilla-La Mancha
In the program of Enology with International Mention
by
Ana María Martínez Gil
Thesis Directors:
M. Rosario Salinas Fernández
Gonzalo L. Alonso Díaz-Marta
Albacete, 2013
UNIVERSIDAD DE CASTILLA-LA MANCHA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
DEPARTAMENTO DE CIENCIA Y TECNOLOGÍA AGROFORESTAL Y GENÉTICA
Aplicación a la vid de extractos vegetales y
su repercusión en el aroma del vino
Memoria presentada por
Ana María Martínez Gil
Para optar al título de
Doctor por la Universidad de Castilla-La Mancha
con Mención Internacional en el programa interuniversitario de Enología
Directores:
M. Rosario Salinas Fernández
Gonzalo L. Alonso Díaz-Marta
Albacete, 2013
Departamento de Ciencia y Tecnología Agroforestal y Genética
D. RICARDO GOMÉZ LADRÓN DE GUEVARA, Director del Departamento de
Ciencia y Tecnología Agroforestal y Genética de la Universidad de Castilla-La
Mancha.
CERTIFICA:
Que la presente memoria de investigación titulada: “Aplicación a la vid de
extractos vegetales y su repercusión en el aroma del vino”, que presenta
Dña. Ana María Martínez Gil para optar al grado de Doctor en Enología, ha
sido realizada bajo la dirección de la Dra. Mª Rosario Salinas Fernández y
del Dr. Gonzalo L. Alonso Díaz-Marta en el Departamento de Ciencia y
Tecnología Agroforestal y Genética de la Universidad de Castilla-La
Mancha. Y para que conste, firma el presente certificado.
Albacete, 24 de enero de 2012
Fdo.: Dr. Ricardo Gómez Ladrón de Guevara
Departamento de Ciencia y Tecnología Agroforestal y Genética
Dña. Mª ROSARIO SALINAS FERNÁNDEZ y D. GONZALO L. ALONSO DÍAZ-MARTA,
Catedráticos de Universidad del Departamento de Ciencia y Tecnología
Agroforestal y Genética de la Universidad de Castilla-La Mancha.
INFORMAN:
Que la presente memoria de investigación titulada: “Aplicación a la vid de
extractos vegetales y su repercusión en el aroma del vino”, que presenta
Dña. Ana María Martínez Gil, para optar al grado de Doctor en Enología,
ha sido realizada bajo nuestra dirección, y a nuestro juicio, cumple todos
los requisitos para proceder a su lectura y defensa pública, por lo que
autorizamos su presentación en el Departamento de Ciencia y Tecnología
Agroforestal y Genética de la Universidad de Castilla-La Mancha. Y para
que así conste, firman el presente certificado.
Albacete, 24 de enero de 2013
Fdo.: Prof. Mª Rosario Salinas Fernández
Prof. Gonzalo L. Alonso Díaz-Marta
“Un experto es aquél que
ha cometido todos los
errores posibles en una
materia muy concreta”
Niels Henrik David Bohr
físico danés (1885-1962)
La realización de este trabajo ha sido posible gracias a la siguiente:
Financiación personal:
 Junta de Comunidades de Castilla-La Mancha, a través de las ayudas
para la formación y contratación de personal investigador (FPI),
cofinanciado por el Fondo Social Europeo (Expediente 422/09). Marzo del
2009 hasta febrero del 2013.
 Junta de Comunidades de Castilla-La Mancha, a través de la ayuda
complementaria “José Castillejo” para personal investigador (Expediente
07/11), cofinanciado por el Fondo Social Europeo, para el desarrollo de una
estancia en el Instituto Nacional de Investigación Agronómica de Francia
(INRA). Junio a agosto del 2011.
 Universidad de Castilla-La Mancha, mediante las ayudas a tesis
concedidas durante los años 2009, 2010 y 2011.
Financiación del trabajo desarrollado:
 Junta de Comunidades de Castilla-La Mancha por la concesión del
proyecto HITO (Haciendo Investigación Tecnológica Orientada) titulado
“Vinos madera hechos desde la viña” (ref.CTR09-0111) a la empresa Dehesa
de Los Llanos (Albacete), cofinanciado por el programa FEDER de la Unión
Europea. Abril a octubre del 2009.
 Ministerio de Ciencia e Innovación por la concesión del proyecto de
investigación titulado “Nueva estrategia para la diferenciación de la calidad
vitivinícola de uvas mediante el empleo de extractos vegetales” (ref. AGL200908950), cofinanciado por el programa FEDER de la Unión Europea. Enero del
2010 hasta junio del 2013.
ÍNDICE / INDEX
Aplicación a la vid de extractos vegetales
y su repercusión en el aroma del vino
ÍNDICE
1. RESUMEN
1
2. JUSTIFICACIÓN
7
3. INTRODUCCIÓN
13
3.1. SITUACIÓN DEL SECTOR VITIVINÍCOLA
3.1.1. Situación del sector en la zona de estudio
3.1.1.1. España
3.1.1.2. Francia
15
18
18
20
3.2. TENDENCIAS DEL CONSUMIDOR Y ADAPTACIÓN DEL SECTOR AL MERCADO
3.3. EL AROMA DE LA UVA Y EL VINO
3.3.1. Técnicas de análisis de aromas de uva y vino
3.3.2. Factores que influyen en el aroma de la uva y del vino
3.3.2.1. Absorción foliar de compuestos
3.3.2.2. Componentes volátiles del ambiente de la vid
3.3.2.3. Aplicaciones foliares a la vid
3.4. EXTRACTOS VEGETALES
3.4.1. Extractos de roble
3.4.2. Extracto de lavandín (hidrolato)
22
23
29
33
34
36
37
39
41
43
4. OBJETIVOS
47
5.PLAN DE TRABAJO
51
6.MATERIALES Y MÉTODOS
59
6.1. MATERIAL VEGETAL
6.1.1. Extractos vegetales
61
61
6.1.1.1. Extractos comerciales a base de roble
6.1.1.2. Extracto de lavandín (hidrolato)
6.1.2. Planta modelo
61
63
65
6.1.2.1. Estudio de fitotoxicidad
6.1.2.2. Preparación de formulaciones y establecimiento de la dosis
6.1.2.3. Preparación de la disolución de referencia
6.1.3. Viñas
66
67
67
68
6.1.3.1. Viñas tratadas en Castilla-La Mancha (España)
6.1.3.2. Viñas tratadas en Languedoc-Roussillon (Francia)
68
69
6.2. TRATAMIENTOS DEL VIÑEDO
6.2.1. Tratamientos con extractos de roble en España
6.2.2. Tratamientos con extractos de roble en Francia
6.2.3. Tratamientos con el extracto de lavandín (hidrolato)
70
71
72
73
i
Aplicación a la vid de extractos vegetales
y su repercusión en el aroma del vino
6.3. VINIFICACIONES
73
6.3.1. Vinificaciones en blanco
73
6.3.2. Vinificación en tinto
76
6.4. MÉTODOS DE ANÁLISIS
78
6.4.1. Parámetros enológicos
78
6.4.2. Análisis de azúcares y ácidos
79
6.4.3. Análisis de aminoácidos y amonio en uvas
80
6.4.4. Extracción de los compuestos volátiles en uvas
81
6.4.5. Análisis de precursores aromáticos glicosídicos en uvas
82
6.4.6. Extracción de los compuestos volátiles de los vinos
84
6.4.7. Análisis de los compuestos volátiles por cromatografía gaseosa y espectrometría
de masas (GC-MS)
85
6.4.7.1 Extracto de roble y uvas y vinos procedentes de su tratamiento
6.4.7.2. Vinos procedentes de las viñas tratadas con el extracto de lavandín
6.4.8. Análisis sensorial de los vinos
6.4.9. Análisis estadístico
85
86
87
88
7.ARTÍCULOS CIENTÍFICOS
89
7.1. ARTÍCULO I
EFFECT OF AN OAK EXTRACT APPLIED TO 'VERDEJO' VINEYARD ON GRAPE COMPOSITION
93
7.2. ARTÍCULO II
EFFECT OF OAK EXTRACT APPLICATION TO VERDEJO GRAPEVINES ON GRAPE AND WINE AROMA
101
7.3. ARTÍCULO III
APPLICATIONS OF AN OAK EXTRACT ON PETIT VERDOT GRAPEVINES. INFLUENCE ON GRAPE AND WINE
VOLATILE COMPOUNDS
117
7.4. ARTÍCULO IV
GLYCOSIDIC AROMA PRECURSORS OF SYRAH AND CHARDONNAY GRAPES AFTER AN OAK EXTRACT
APPLICATION TO THE GRAPEVINES
131
7.5. ARTÍCULO V
LAVANDIN HYDROLAT APPLICATIONS TO PETIT VERDOT VINEYARDS ON THEIR WINES AROMA
COMPOUNDS
145
8. CONCLUSIONES
165
9. BLIBLIOGRAFÍA
171
ii
INDEX
1. ABSTRACT
1
2. JUSTIFICATION
7
3. INTRODUCTION
13
3.1. SITUATION OF THE WINE SECTOR
3.1.1. Situation of the sector in the area of study
3.1.1.1. Spain
3.1.1.2. France
15
18
18
20
3.2. CONSUMER TRENDS AND SECTOR ADAPTATION TO MARKET
3.3. GRAPE AND WINE AROMA
3.3.1. Analytical techniques of aroma in grape and wine
3.3.2. Factors that influence on grape and wine aroma
22
23
29
33
3.3.2.1. Foliar absorption of compounds
3.3.2.2. Volatile compounds of the vineyard enviroment
3.3.2.3. Foliar application to the vineyard
34
36
37
3.4. PLANT EXTRACTS
3.4.1. Oak extract
3.4.2. Lavandin extract (hydrolat)
39
41
43
4. OBJETIVES
47
5. WORK PLAN
51
6.MATERIALS AND METHODS
59
6.1. PLANT MATERIAL
6.1.1. Plant extracts
61
61
6.1.1.1. Aqueous oak extracts
6.1.1.2. Lavandín extract (hydrolat)
61
63
6.1.2. Model plant
65
6.1.2.1. Phytotoxic study
6.1.2.2. Preparation of formulations and dose setting
6.1.2.3. Prepartion of the reference solution
6.1.3. Vineyards
66
67
67
68
6.1.3.1. Treated vineyards in Castilla-La Mancha (Spain)
6.1.3.2. Treated vineyards in Languedoc-Roussillon (France)
6.2. VINEYARD TREATMENTS
6.2.1. Oak extracts treatments in Spain
6.2.2. Oak extracts treatments in France
6.2.3. Lavandin extracts (hydrolat)treatments
i
68
69
70
71
72
73
6.3. VINIFICATIONS
6.3.1. White vinifications
6.3.2. Red vinifications
6.4. METHODS OF ANALYSIS
6.4.1. Oenological parameters
6.4.2. Analysis of sugars and acids
6.4.3. Analysis of amino acids and amonio in grapes
6.4.4. Extration of the volatile compounds in grapes
6.4.5. Analysis of the glycosidic aroma precursors in grapes
6.4.6. Extration of the wine volatil compounds
6.4.7. Analysis of the volatil compounds by gas chromatography and mass
spectrometry (GC-MS)
6.4.7.1 Oak extract and grape and wine from its treatment
6.4.7.2. Wine from treated vineyard with lavandin extract
6.4.8. Sensorial analysis of wine
6.4.9. Stadistical analysis
73
73
76
78
78
79
80
81
82
84
85
85
86
87
88
7.SCIENTIFIC ARTICLES
89
7.1. ARTICLE I
EFFECT OF AN OAK EXTRACT APPLIED TO 'VERDEJO' VINEYARD ON GRAPE COMPOSITION
93
7.2. ARTICLE II
EFFECT OF OAK EXTRACT APPLICATION TO VERDEJO GRAPEVINES ON GRAPE AND WINE AROMA
101
7.3. ARTICLE III
APPLICATIONS OF AN OAK EXTRACT ON PETIT VERDOT GRAPEVINES. INFLUENCE ON GRAPE AND WINE
VOLATILE COMPOUNDS
117
7.4. ARTICLE IV
GLYCOSIDIC AROMA PRECURSORS OF SYRAH AND CHARDONNAY GRAPES AFTER AN OAK EXTRACT
APPLICATION TO THE GRAPEVINES
131
7.5. ARTICLE V
LAVANDIN HYDROLAT APPLICATIONS TO PETIT VERDOT VINEYARDS ON THEIR WINES AROMA
COMPOUNDS
145
8. CONCLUSIONS
165
9. REFERENCES
171
ii
1. RESUMEN
RESUMEN
ABSTRACT
Resumen
Se ha demostrado recientemente que algunas aplicaciones foliares a la vid
y ciertos compuestos volátiles del ambiente en el que se desarrollan las uvas
pueden modificar el aroma del vino. El hecho por el que la vid asimila
compuestos volátiles y los transmite a sus uvas y vinos supone una investigación
innovadora, que puede tener una gran repercusión en el sector vitivinícola que
busca la diferenciación. Existen extractos vegetales procedentes de roble o de
plantas aromáticas (hidrolatos) que poseen compuestos volátiles similares a los
mostrados eficaces, por ello el objetivo principal de este trabajo consistió en
estudiar el efecto que tiene sobre la composición aromática de la uva y de sus
vinos la aplicación a la vid de extractos acuosos de roble y de hidrolato de
lavandín. Para ello, los extractos vegetales fueron formulados y aplicados por
pulverización foliar sobre vides de diferentes variedades blancas y tintas. Las
uvas fueron vendimiadas en el momento óptimo de maduración tecnológica y los
vinos se elaboraron siguiendo los sistemas de vinificación tradicional en blanco y
en tinto. Se analizaron los extractos vegetales, los parámetros enológicos y la
composición aromática de las uvas y de los vinos a lo largo del tiempo, para lo
que se puso a punto un nuevo método de análisis de aromas en uvas por HSSBSE-GC-MS.
Los tratamientos con los extractos de roble no afectaron a la
concentración de compuestos aromáticos libres de la uva, pero sí lo hicieron al
contenido de sus precursores glicosídicos, originarios o no del extracto de roble,
y a la composición volátil de los vinos. Estas observaciones sugieren que la
mayoría de compuestos del extracto de roble son asimilados por las plantas y
almacenados en las uvas en forma de glicósidos, y que después de la
fermentación y a lo largo del tiempo se liberan las agliconas volátiles. Además, a
nivel sensorial se observó que estos vinos mantuvieron sus características típicas
junto con notas a madera que recuerdan a los vinos de crianza.
3
Resumen
Los tratamientos con el hidrolato de lavandín provocaron un incremento
de los principales aromas positivos y la aparición de linoleato de etilo y de
canfor, compuestos que no se encuentran habitualmente en los vinos y que
pueden proceder del lavandín. También se observó que la composición aromática
de los vinos fue más estable a lo largo del tiempo de permanencia en botella.
4
Abstract
It is known that certain foliar applications to the grapevine or volatile
compounds present in the environment, where grapes are grown, may modify the
wine aroma. The fact that the vineyards assimilate volatile compounds and
transmit them to its grapes and respective wines supposes an innovative research,
which might have a huge impact on the wine sector when seeking the
differentiation. There are plant extracts from the oak or aromatic plants
(hydrolats) that have volatile compounds in their composition similar to those
shown effective. Therefore, the principal aim of this work was to study the effect
on the volatile composition of the grapes and their wines by the application of
aqueous oak extracts and lavandin hydrolat to the vineyard. To study it, the plant
extracts were formulated and foliar applied to the vineyards of different white
and red varieties. The grapes were harvested at the optimum technology
maturation moment and processed following traditional vinification systems. The
study analyzed the plant extracts, the oenological parameters and the aroma
composition of grapes and wines throughout time, so that was a method tuned to
determine the aromas of the grapes by HS-SBSE-GC-MS.
The application of oak extracts generally did not affect to the grape free
aroma compounds. Although, they affected to their glycosidic precursors
content, coming or not from oak extract, and the wine volatile composition.
These observations suggest that most of oak extract compounds were assimilated
by the plants and stored as glycosides, which were released to the wine as
volatile aglycons after fermentation and through the time. Sensory analysis
showed that the wines maintained their typical characteristic attributes with
wood notes reminding to the wines stored in barrels.
5
Abstract
The lavandin hydrolat treatments produced an increased on the positive
volatile aroma compounds. The detection of ethyl linoleate and camphor,
compounds not commonly found in wine, corroborated that they may come from
lavandin. It was also observed that the wines aroma composition was more stable
throughout time in the bottle.
6
2. JUSTIFICACIÓN
JUSTIFICACIÓN
JUSTIFICATION
Justificación
El vino es un producto que proporciona un claro ejemplo de mercado
saturado, por ello los enólogos y viticultores buscan un camino más sostenible en
el cual prime la calidad y la diferenciación.
Al ser la calidad de la uva el primer factor que condiciona la
particularidad del vino, en la actualidad se le está dando especial protagonismo a
las prácticas agronómicas, ya que afectan de forma decisiva al aroma y el color
del vino que son las principales cualidades en los que se basa la elección de los
consumidores.
El aroma constituye uno de los factores de calidad más destacables ya que
le proporciona a los vinos un sello identificativo propio. Hay estudios que
demuestran que la composición aromática de las uvas se puede ver afectada por
la presencia de ciertas sustancias de su entorno y por algunas aplicaciones
foliares a la viña, que impactan a nivel sensorial en el vino. Algunos ejemplos
son: la aplicación foliar de ciertos pesticidas que afectan al aroma varietal y
fermentativo de los vinos, el aroma a eucaliptol de vinos procedentes de viñas
cercanas a bosques de eucaliptos, o el olor ahumado de vinos cuyas viñas han
estado expuestas al humo.
En enología uno de los materiales más importante es la madera de roble,
ya que con ella se fabrican las barricas en las que los vinos se someten al proceso
de crianza. En este proceso se genera grandes cantidades de madera que
normalmente se desaprovecha, aunque algunas empresas lo utilizan para
conseguir diferentes tipos de chips y para producir extractos de roble. La madera
de roble posee una composición característica, marcada por compuestos que son
extraídos por el vino durante el periodo de crianza. Estos compuestos comunican
al vino un aroma especial asociado a la madera, cuyos principales responsables
son moléculas de naturaleza química parecida a las sustancias presentes en el
humo, de las que ya se ha demostrado su capacidad para ser asimiladas por las
9
Justificación
uvas de viñas cercanas a zonas que han sufrido incendios forestales, y que se
perciben en el vino.
En Castilla La-Mancha es habitual encontrar viñas en cuyas proximidades
existen plantaciones de cultivos aromáticos, en especial de lavanda y lavandín.
Estas plantas se suelen utilizar para la obtención de sus aceites esenciales en
cuyo proceso se generan importantes cantidades de hidrolatos que son
considerados “subproductos inservibles”. Estos hidrolatos son ricos en
compuestos volátiles, en donde destacan los terpenos, de algunos de los cuales ya
se ha demostrado su capacidad para ser asimilados por las uvas y ser percibidos
en el vino, como es el caso del eucaliptol.
Este trabajo se justifica por el hecho de disponer de madera de roble y de
extractos de lavandín (hidrolatos), con un potencial aromático capaz de impactar
en el aroma de la uva y de sus vinos, y que sin embargo son desaprovechados. Se
impone por tanto la necesidad de conocer si extractos de madera de roble y de
lavandín (hidrolatos) pueden actuar como bioestimulantes del aroma, por ello
pretendemos estudiar el efecto que sobre la composición aromática de la uva y
sus vinos tiene la aplicación a la vid de extractos acuosos obtenidos de roble y de
lavandín. Este trabajo es el inicio de una innovadora línea de investigación que
puede conducir a estrategias de diferenciación en el sector vitivinícola.
10
Justification
Wine is a product that provides a clear example of a saturated market, so
the winemakers and grape growers are seeking a more sustainable path in which
quality and differentiation come first.
The grapes quality is the first factor that determines the wine
characteristics, so at the present special prominence is being given to the
agronomic practices, since they affect decisively to wine aroma and color, which
are the main qualities for consumers’ choice.
Aroma is, without no doubt, one the most remarkable quality factors, as it
provides an identification stamp to wines. There are studies that have
demonstrated that the grapes aroma composition can be influenced by certain
environment substances and by some foliar applications, having an impact on the
sensory characteristics of wine. Some examples are: the foliar application of
certain pesticides which affect to the fermentative and varietal wine aroma, the
vineyards that are grown near eucalyptus forests produce wine with a eucalyptol
aroma or wine with smoky aroma whose vineyards are exposed to smoke.
In Enology one of most important materials is the oak wood, because it is
used to produce the barrels where wines aging process takes place. This process
generates high quantities of wasted wood, although recently some companies use
it to produce different types of chips, and to produce oak extracts. The oak has a
characteristic composition, characterized by compounds that are extracted by the
wine during aging. These compounds give the wine a special aroma associated
with the oak. Many of these are chemical molecules similar to those in smoke,
which have been successfully assimilated by the grapes and perceived in the
wine aroma, when their vineyards are near to wildfires.
In Castilla La-Mancha is common to find vineyards close to aromatic
crop fields, especially lavender and lavandin. These plants produced to obtain
their essential oils, in which process significant amounts of hydrolats are
11
Justification
generated and are considered an "useless byproducts ". These hydrolats are rich
in volatile compounds, especially in terpenes, some of them have already
demonstrated its ability to be absorbed by the grapes and being perceived in the
wine, such as eucalyptol.
This work is justified by the existence of aqueous oak and lavandin
(hydrolat) extracts, which are usually discarded, with a high aroma potential that
may have an impact on the grapes and wines aroma. Consequently, it appears the
necessity to know if really oak and lavandin extracts can act as aroma
biostimulants on grape and their respective wines. This work is the beginning of
an innovative research line that may lead to differentiation strategies for the wine
sector.
12
3. INTRODUCCIÓN
INTRODUCCIÓN
Introducción
3.1. Situación del sector vitivinícola
La superficie vitícola mundial en 2011 ha disminuido en 94 miles de
hectáreas (mha) respecto a 2010, situándose el total mundial en 7,49 millones de
hectáreas (Mha), según datos de la Organización Internacional de la Viña y el
Vino (OIV, 2012). Europa mantiene casi la mitad de la superficie de viñedo del
mundo (47,13% mundial), aunque su superficie plantada se está reduciendo
progresivamente, pasando a 3,53 Mha en el año 2011. La disminución de la
superficie plantada en Europa se debe a factores como la reestructuración del
viñedo y el impacto de la crisis vitícola que, por otra parte, se ha dejado sentir de
forma distinta por zonas y tipos de vino y a la que se ha añadido el programa
europeo de ayuda a los arranques (ICEX, 2012).
China
560mha
Estados Unidos
405mha
Australia
174mha
Brasil
92mha
Chile
202mha
Expansión
Argentina
218mha
Estable
África del Sur
131mha
Nueva Zelanda
37mha
Redución
Francia
807mha
Portugal
240mha
España
1032 mha
Hungría
65mha
Italie
786mha
Bulgaría
72mha
Turquía
500mha
Grecia
111mha
Figura 1. Principales superficies de viñedos en el mundo (OIV, 2012) (mha = miles de hectáreas).
15
Introducción
No obstante, la disminución del viñedo comunitario se ha visto
compensada con el mantenimiento de las áreas de cultivo en el resto del mundo,
con variaciones a la baja en Argentina, Sudáfrica y Turquía, repuntes en China,
Australia y Chile y estabilidad en Estados Unidos y Brasil (Figura 1). China ha
sido el país con mayor expansión, situándose en 2011 como el país con mayor
superficie vitícola fuera de la UE.
Aun así, España, Francia e Italia siguen siendo los países con mayor
extensión de viñedo de la Unión Europea y del mundo. El sector vitivinícola de
estos tres países tiene gran importancia, tanto por el valor económico que genera,
como por la población que ocupa y por el papel que desempeña en la
conservación medioambiental.
A pesar de la disminución de superficie vitícola, la producción mundial
de vino de 2011 (sin contar zumos y mostos) es de 265,8 millones de hectolitros
(Mhl), cifra que representa un aumento de 700 miles de hectolitros (mhl) en
relación a 2010.
La producción en la Unión Europea fue de 156,9 Mhl, similar a la de
2010, representando un 59% del total mundial. A pesar de ser España el país con
más superficie vitícola, el primer país productor de vino es Francia, con 49,6 Mhl
(18,7 % mundial), seguido por Italia, con 41,6 Mhl (15,7 % mundial), y España,
con 38,6 Mhl (14,5 % mundial) (Figura 2).
Fuera de la Unión Europea, el nivel de producción fue de 108,9 Mhl,
siendo EE.UU. el país no europeo con mayor producción de vino (18,7 Mhl),
seguido de Argentina (15,5 Mhl), China (13 Mhl), Australia (11 Mhl) y Chile
(10,6 Mhl) (Figura 2). China está en pleno auge, por lo que se estima que será el
principal productor fuera de la Unión Europea en muy pocos años e incluso a
nivel mundial.
16
Introducción
Francia
49633 mhl
España
38583 mhl
Estados Unidos
18740 mhl
Alemania
9611 mhl
Rusia
6353 mhl
China
13000 mhl
Portugal
5925 mhl
Hungría
2447 mhl
Austria
2814 mhl
República Checa
720 mhl
Brasil
3450 mhl
Bulgaria
1268 mhl
Italia
41580 mhl
Chile
10572 mhl
Rumania
4708 mhl
Grecia
2597 mhl
Australia
11010 mhl
Argentina
15473 mhl
África del Sur
9336 mhl
Aumento
Nueva Zelanda
2350 mhl
Reducción
Figura 2. Distribución mundial de la producción de vino (OIV, 2012) (mhl = miles de hectolitros).
También en 2011 aumentó el consumo mundial de vino, 241,9 Mhl, un
0,7% más que en 2010. Europa es el primer continente en cuanto a
consumidores, no obstante, debido a la coyuntura económica actual, está lejos de
volver al crecimiento de antes de la crisis. Aun así, Francia es el principal
consumidor de vino mundial, con un consumo de 29,94 Mhl (12,3% mundial).
Estados Unidos, con un gran crecimiento en su consumo se sitúa en 2011 como
el segundo consumidor mundial, con 28,5 Mhl. El segundo país consumidor de
vino fuera de la unión europea, ocupando el quinto lugar, es China, con un gran
aumento, en 2011 se sitúa con un volumen de consumo de 17 Mhl, con un
fortísimo incremento de las importaciones y un consumo de casi la totalidad de
su producción (sin apenas exportaciones). El país asiático se ha convertido en un
mercado con un crecimiento espectacular en el sector.
En
2011
las
exportaciones
mundiales
de
vino
representaron
aproximadamente el 42,8% del consumo mundial, siendo Italia, España y
17
Introducción
Francia, los principales países exportadores (ICEX, 2012). Estos tres países
exportaron más de 60,7 Mhl, lo que supone más del 25% del consumo mundial.
3.1.1. Situación del sector en la zona de estudio
Los trabajos del presente estudio se han realizado en viñedos de dos
regiones de los países más importantes en el sector vitivinícola mundial, España
y Francia, ya que entre ambos tienen un 24,5% de la superficie mundial de
viñedo y producen más del 33,2% de la producción mundial de vino. Estos dos
países consumen más del 16,1% y exportan aproximadamente un 35,8%.
3.1.1.1. España
En el año 2011, ocupó la primera posición en el ranking de superficie de
cultivo plantada (1.032 mha), el tercer lugar en cuanto a producción (38,58 Mhl),
el octavo puesto en consumo (10,15 Mhl) y el segundo puesto en volumen de
venta de vino al exterior (22,32 Mhl).
Todas las comunidades autónomas españolas son productoras de vino,
siendo Castilla-La Mancha la región productora más grande de España, seguida
de Extremadura y Cataluña (Figura 3). A pesar de ser estas tres regiones las que
abastecen casi el 80% del mercado nacional, a nivel mundial no son las más
reconocidas.
Los viñedos utilizados en este estudio están localizados en Castilla-La
Mancha. Esta región está situada en el corazón de la Península Ibérica, ocupa la
Submeseta Sur. La gran llanura de la Mancha, hacen de esta la comarca natural
más homogénea y extensa del país. Cuenta con una altitud de 600 a 800 m en la
mayor parte de su territorio y está aislada de las grandes masas de agua por
sistemas montañosos (Sistema Central, Cordillera Ibérica, Sierras Béticas, etc.)
18
Introducción
que confieren a la zona un clima continental, grandes oscilaciones térmicas y
precipitaciones estacionales y escasas.
3,5%
1,7%
3,0%
1,8%
2,2%
Castilla-La Mancha
Extremadura
3,9%
Cataluña
4,7%
La Rioja
6,1%
48,3%
5,2%
Comunidad valenciana
Castilla y León
Galicia
Andalucia
Aragón
8,5%
Murcia
Navarra
11,1%
Otros
Figura 3. Principales comunidades autónomas productoras de vino en España (OeMv 2012).
Ésta es la principal región vitivinícola de España debido a su gran
extensión del cultivo así como a la importancia de sus producciones, siendo
también la zona geográfica con mayor superficie de viñedo del mundo (7,2%)
con 565.000 ha (Winetech, 2012). El mayor factor limitante de la viticultura en
Castilla-La Mancha es el agua, con precipitaciones medias anuales por debajo de
600 mm incluso zonas muy secas donde no se superan los 320 mm. Por ello, para
la mejora de la producción e implantación de nuevas variedades, ha aumentado
el número de hectáreas de plantación con sistema de regadío, llegando a ser más
del 40% del total.
En Castilla-La Mancha además de localizarse la principal producción de
alcohol vínico, también tiene una importante industria de mostos. Esta
comunidad cuenta con el mayor número de bodegas cooperativas de España,
entre el 70% y el 80% de la vinificación de la uva se realiza en ellas.
19
Introducción
Esta comunidad está constituida por cinco provincias: Toledo, Ciudad
Real, Cuenca, Albacete y Guadalajara y posee nueve Denominaciones de Origen
(D.O.): Almansa, Jumilla La Mancha, Manchuela, Méntrida, Mondéjar, Ribera
del Júcar, Uclés y Valdepeñas; ocho Pagos Vitícolas: Casa del Blanco,
Calzadilla, Dehesa del Carrizal, Dominio de Valdepusa, Finca Élez, Florentino,
Guijoso y La Guardia. Además se utiliza vinos de la Tierra de Castilla para la
denominación de los vinos de mesa de la zona que no se encuentran dentro de las
anteriormente citadas. Este trabajo fue realizado con viñedos acogidos a la D.O.
La Mancha en la zona de Albacete.
Castilla-La Mancha durante muchos años ha sido considerada como un
mero abastecedor de vinos a granel o de embotellados de bajo precio. Solamente
en el último lustro está cambiando la imagen, gracias a la implantación de nuevas
estrategias, permanente proyección e innovación, consiguiendo en estos
momentos vinos con calidad notable y con una gran acogida en el mercado
internacional.
3.1.1.2. Francia
En el año 2011 ocupó el segundo puesto en superficie de viñedo plantado
(807 mha), la primera posición en el ranking de producción de vino (49,6 Mhl) y
de consumo (29,94 Mhl) y el tercer puesto en volumen de vino exportado (14,10
Mhl).
En Francia existen doce grandes zonas productoras de vino (Figura 4),
siendo la región de Languedoc-Roussillon la de mayor producción y también
aquella donde se han producido hasta hoy los cambios más interesantes en
materia de viticultura, seguida de la región de Poitou-Charentes y de Bordelais.
La región de Languedoc-Roussillon es donde se localizan las viñas del
estudio que hemos realizado en Francia. Los viñedos de Languedoc-Roussillon
20
Introducción
bordean el Mediterráneo, desde los Pirineos hasta el delta del Ródano. Posee un
clima mediterráneo con veranos calurosos y secos e inviernos suaves y soleados.
Las precipitaciones medias anuales son de 600 mm y la caracteristica principal
de la zona es la gran cantidad de días de viento al año, aproximadamente 300
días anuales. Probablemente es la región vitícola más antigua de Francia y esta
constituida por cinco departamentos: Aude, Gard, Hérault, Lozère y PyrénéesOrientales.
5,1%
Languedoc-Roussillon
0,6%
0,3%
2,5%
Poitou-Charentes
0,2%
Bordelais
6,1%
29,4%
6,1%
Otras zonas Sud-Este
Sud-Oeste
7,9%
Val de Loire
11,1%
Champagne
Bourgogne- Beajolais
12,4%
18,1%
Alsace
Corse
Savoie
Jura
Figura 4. Distribución de la producción de vino en Francia (SeVi, 2011)
El viñedo de Languedoc-Roussillon ocupa un tercio de la superficie
agrícola útil regional, lo que representa cerca de 300 mha de las cuales
aproximadamente 100 mha son dedicadas a las denominación de origen
controlada (AOC). Además de los vinos de AOC, en esta región encontramos
vino de mesa (VDT), vinos del país (VDP) y vinos de calidad producidos en una
región determinada (VQPRD). Los vinos de mesa (generalmente otorgada a los
llamados “de consumo corriente”) que no entran en ninguna otra categoría,
pueden tener la mención vino de mesa francés o la mención mezcla de vinos de
diferentes países de la Comunidad Europea. El vino denominado vino del país
abarca la mayor parte de la producción de esta región, coexistiendo más de 50
21
Introducción
denominaciones. Existen más de 28 vinos de denominación de origen controlado
(AOC) y la mayor parte de sus vinos se producen en cooperativas, unas 300
bodegas.
Esta región produce casi un tercio de la producción total de Francia (14,5
Mhl), sin embargo, no es reconocida por la calidad de sus vinos, por ello el
sector vitivinícola desde hace algunos años viene realizando esfuerzos en
innovación tecnológica y creatividad.
3.2. Tendencias del consumidor y adaptación del sector al
mercado
En los últimos años, los gustos y necesidades de los consumidores de
vino están en continuo cambio. Exigen conocer el origen de la materia prima, los
tratamientos y tecnologías utilizadas, demandan productos de mejor calidad con
una creciente preocupación por el efecto de su consumo sobre la salud, y
solicitan conocer la repercusión del proceso productivo sobre el medio ambiente.
Al mismo tiempo, aunque ciertos sectores de la población tienen sus gustos muy
definidos y siguen decantándose por los productos tradicionales, la tendencia
general de los consumidores va dirigida hacia la demanda de productos nuevos
con características muy específicas.
Simultáneamente, el sector del vino se enfrenta a un mercado saturado,
tanto a nivel nacional como internacional, obligando a todos los integrantes del
mismo a un ejercicio permanente de proyección e innovación para adecuarse a
nuevos desafíos, pues de ello derivará, sin duda, una mejora competitiva.
Consecuentemente, la meta que se persigue en el mundo de la viticultura y de la
enología actual es la elaboración de un producto diferenciado que presente
identidad propia frente a los consumidores, siendo la calidad del vino la que nos
22
Introducción
abre la primera puerta para poder seguir avanzando. Esta estrategia del sector es
ambiciosa y pretende satisfacer el gusto y deleite del consumidor.
Por consiguiente, viticultores y enólogos proponen nuevas formas de
manejo de los viñedos, elaboran con levaduras autóctonas, vinifican mezclas de
uvas buscando el efecto de complementariedad, utilizan nuevas tecnologías
enológicas y manejan una gran diversidad de materiales en la etapa de crianza,
entre otras estrategias. Todas estas formas de proceder tienen gran impacto sobre
la calidad del vino, ya que afectan a los principales factores de elección de los
consumidores como son el aroma, el color y el sabor, los cuales van a manifestar
inequívocamente la buscada diferenciación dando a los vinos un sello
identificativo propio.
El aroma es, posiblemente, una de las características más importantes
ligadas a la calidad y a las preferencias de los consumidores por un determinado
alimento. En el caso del vino, esta característica es aún más importante, ya que
constituye un producto que es fundamentalmente consumido por puro placer
sensorial, y en el que el aroma es su mejor carta de presentación.
3.3. El aroma de la uva y el vino
El aroma de un vino se puede definir como la interacción del sabor y olor
que imparte a cada individuo una experiencia sensorial, debida al impacto de
numerosos grupos de sustancias volátiles en el órgano olfativo (la pituitaria). El
aroma de un vino es una de sus principales características organolépticas, y en
muchas ocasiones constituye la causa de aceptación o rechazo del mismo por
parte del consumidor (Pretorius & Bauer, 2002), de ahí que la composición
aromática de uvas, mostos y vinos haya sido objeto de numerosos estudios.
23
Introducción
El aroma es de una gran complejidad, que se debe en parte a su origen,
pues es el resultado final de una larga secuencia biológica, bioquímica y
tecnológica que se inicia en la cepa y finaliza en la copa, y en parte al elevado
número de compuestos orgánicos volátiles con diferente naturaleza química y
diferentes características organolépticas, y cuyo intervalo de concentración oscila
desde los miligramos/litro (mg/l) a los nanogramos/litro (ng/l). Además, la
personalidad aromática individual de los vinos, no se debe únicamente a unos
pocos compuestos específicos, sino que se debe a diversas combinaciones y
concentraciones de varios compuestos.
En el año 1981, Cordonnier & Bayonove propusieron una clasificación
del aroma del vino según su origen distinguiendo los siguientes tipos:
Aroma varietal: resulta del metabolismo propio de la uva empleada y
depende de la variedad, el suelo, el clima, las prácticas agronómicas y en
general de las características de la zona donde se cultivan las viñas.
Aroma pre-fermentativo: originado desde la vendimia hasta el inicio de la
fermentación (el conjunto de este aroma junto con el aroma varietal se
denomina aroma primario). Este tipo de aroma resulta de los fenómenos
bioquímicos de oxidación e hidrólisis que tiene lugar durante la
extracción del jugo.
Aroma fermentativo o secundario: producido durante la fermentación
alcohólica y maloláctica por el metabolismo de las levaduras y/o
bacterias.
Aroma post-fermentativo o terciario: formado durante el almacenamiento
y envejecimiento de los vinos mediante reacciones químicas y/o
enzimáticas. Las barricas de madera utilizadas en la crianza aportan
también compuestos aromáticos importantes para el aroma de los vinos.
24
Introducción
Existen numerosos factores que pueden afectar a la composición
aromática de la uva y del vino y por consiguiente a la calidad del producto. Entre
otros podemos citar: el estado de maduración y sanitario de la uva, la variedad y
su tipo de cultivo, los tratamientos agronómicos realizados, el tipo de recolección
y transporte a la bodega, las características de los microorganismos empleados en
las fermentaciones, la técnica de vinificación empleada y las condiciones de
envejecimiento del vino (Salinas et al., 1996; Salinas et al., 1998; Bureau et al.,
2000; Bayonove, 2003; Ribéreau-Gayon et al. 2006; Cabrita et al., 2007; Loscos
et al., 2007; Lorenzo et al. 2008; Styger, et al, 2011; Noguerol-Pato et al., 2012).
Los constituyentes volátiles de la etapa prefermentativa se revelan como
consecuencia de los diversos tratamientos que sufre la uva, desde que se decide
realizar la cosecha hasta que se inicia la fermentación. El contacto del mosto de
la uva con las partes sólidas inicia toda una serie de reacciones enzimáticas y
químicas, potenciadas en parte por el oxígeno, dando lugar a compuestos
aromáticos responsables de los olores herbáceos, tales como los alcoholes y
aldehídos de 6 átomos de carbono, a partir de ácidos grasos poliinsaturados,
especialmente de los ácidos linoleico y linolénico (Codornnier & Bayonove,
1981, Ferreira et al., 1995; Oliveira et al., 2006; Cejudo-Bastante et al., 2011).
La formación de estos también puede variar en función del estado de madurez de
la vendimia (Codornnier & Bayonove, 1981; Sánchez-Palomo et al., 2010).
La parte principal, cuantitativamente, del aroma del vino se genera por la
acción de las levaduras durante la fermentación alcohólica, o la acción de las
bacterias lácticas durante la fermentación maloláctica. Los compuestos que
constituyen el aroma fermentativo pertenecen a distintas familias químicas:
alcoholes, ésteres, aldehídos, ácidos, compuestos azufrados, lactonas, fenoles
volátiles etc. La proporción de estos depende de la variedad de uva utilizada, del
tipo de microorganismos, de las condiciones en las que se desarrollen y de las
técnicas y materiales utilizados (Lambrechts & Pretorius, 2000; Lorenzo et al.,
25
Introducción
2008; Díaz-Plaza et al., 2002; Losada et al., 2012). La mezcla de todos los
componentes mayoritarios de la fermentación, a las concentraciones a las que se
encuentran habitualmente en vino, proporcionan el olor típico de bebida
alcohólica que habitualmente se define como “vinoso” y constituye la base
aromática común a todos los vinos. Es un olor ligeramente dulce, picante y
agresivo, alcohólico y frutal. Esta mezcla constituye lo que se denomina un
sistema buffer o tampón aromático. Afortunadamente, el aroma de los vinos es
muy rico en notas aromáticas claramente diferentes al aroma “vinoso”, indicando
que algunas moléculas aromáticas (una a concentración suficiente o un grupo de
moléculas con alguna similitud) son capaces de romper el buffer aromático y
transmitir o inducir la aparición de una nota sensorial diferente (Ferreira, 2007).
Por lo tanto, los aromas fermentativos pueden otorgar tanto características
positivas, como aromas frutales, florales o especiados (ésteres, alcoholes
superiores y fenoles), pero también características negativas, como olor a moho,
grasos, caballo, cuero o huevos podridos (altas concentraciones de bases volátiles
heterocíclicas, ácidos, 4-etilfenol, 4-etilguayacol y compuestos azufrados).
Sin embargo, el aroma de un vino que nos permite tipificarlo es el que
procede de la variedad de la uva, el cual está influido por las condiciones
sanitarias, edafoclimáticas y culturales en las que se ha desarrollado (Bureau et
al., 2000; Koundouras et al., 2006; Zoecklein et al., 2008). También la zona de
cultivo puede darle su impronta y expresarla en el vino, proporcionándole una
característica propia. Por ello, las prácticas vitícolas apuntan principalmente a la
producción de uvas de calidad que reflejen aromas varietales y caracteres típicos
de la zona.
Los compuestos responsables del aroma de la uva constituyen un grupo
muy complejo de sustancias que pueden presentarse en forma libre, es decir,
como moléculas volátiles y por tanto olorosas, o en forma ligada que no son
volátiles y por tanto no huelen, a las que se denomina precursores del aroma. Las
26
Introducción
uvas poseen diferentes grupos de precursores del aroma no volátiles: lípidos
insaturados, ácidos fenólicos, carotenoides, compuestos unidos a la cisteína o al
glutatión y glicoconjugados principalmente.
Los precursores glicosídicos constituyen el grupo más importante
responsable de los atributos varietales de los vinos, especialmente en las
variedades neutras (variedades pobres en aromas libres cuya características
varietales proviene de las diferentes familias de precursores no volátiles), ya que
estas formas son más comunes que los aromas libres (Francis et al., 1996;
Bureau et al., 2000; López et al., 2004; Noguerol-Pato et al., 2012; Salinas et al.,
2012b). Los glicósidos están constituidos por una aglicona volátil unida a una
molécula de glucosa mediante un enlace O-glicosídico, estando siempre ligado
por la parte β-D-glucopiranosa. Los precursores glicosídicos inicialmente fueron
identificados en las uvas por Cordonnier & Bayonove (1974) y se localizan
principalmente en los hollejos (Günata et al., 1985; Wilson et al., 1986; Gómez
et al., 1994; Baumes, 2009), especialmente en forma de disacáridos glicosídicos
(arabinósidos, rutinósidos y apiósidos), aunque, también se pueden encontrar en
forma de monosacáridos como β-D-glucopiranósidos (Williams et al., 1982;
Günata et al., 1988) (Figura 5).
Figura 5: Estructura general de los percusores glicosídicos del aroma (Baumes, 2009).
27
Introducción
La glicosilación es la forma más habitual por la cual las plantas se
protegen de sustancias nocivas o de los efectos adversos del medio. Esto implica
un aumento de la solubilidad del compuesto glicosilado con el fin de facilitar el
transporte celular (Stahl-Biskup et al., 1993; Winterhalter & Skouroumounis,
1997). Podría decirse en el caso de la vid, que los compuestos glicosilados son
transportados hasta la uva como una forma de eliminación.
Esta fracción glicosídica inodora conforma una importante reserva de
aromas del vino que bajo la influencia de diversos factores biológicos,
biotecnológicos y físico-químicos son susceptibles de liberar la aglicona volátil
por hidrólisis enzimática o ácida (Figura 6).
Figura 6: Ruptura del enlace O-glicosídico que mantiene unida la aglicona a la molécula de
glucosa (Salinas & Serrano de la Hoz, 2012a).
Esta aglicona puede pertenecer a diferentes familias químicas,
principalmente terpenos, C13-norisoprenoides, fenoles volátiles, compuestos C6,
entre otros. El proceso de liberación de la aglicona ocurre, en mayor o menor
extensión, durante la etapa de vinificación y a lo largo del envejecimiento y
conservación de los vinos. En general, la liberación de los compuestos volátiles
procedentes de los precursores glicosídicos puede ser realizada por la acción de
enzimas endógenas o exógenas con actividad β-glucosidásica (Günata et al.,
28
Introducción
1990; Cabaroglu et al., 2003; Sánchez-Palomo et al., 2005) por la actividad de
las levaduras (Fernández-González et al., 2003; Delfini et la., 2001; HernándezOrte et al., 2008; Fernández-González & Di Stefano 2004) y de las bacterias
(Ugliano & Moio, 2006; Boido et al., 2002; DÍncecco et al., 2004; Michlmayr et
al., 2012) o por hidrólisis ácida (Williams et al., 1982, Skouroumounis &
Selfton, 2000; López et al., 2004; Salinas et al., 2012b).
3.3.1. Técnicas de análisis de aromas de uva y vino
La cromatografía de gases (GC) es la técnica de elección para el análisis
de los compuestos volátiles, pero sin duda alguna, las técnicas de preparación de
muestra, que incluyen métodos de aislamiento y concentración de compuestos
volátiles, han sido y son imprescindibles para obtener un buen análisis de este
tipo de compuestos, muchos de los cuales se encuentran en concentraciones muy
bajas en mostos y vinos. De hecho, en los últimos años, muchas de estas técnicas
han evolucionado y mejorado para intentar conseguir además de exactitud y
precisión, sensibilidad, rapidez, bajo coste y reducción en la cantidad de
solventes orgánicos empleados.
Los procedimientos de aislamiento de los compuestos volátiles del resto
de la matriz están basados en distintas propiedades físico-químicas de los
analitos como son la volatilidad, la solubilidad en distintas fases orgánicas
inmiscibles con la matriz y la capacidad de ser absorbidos selectivamente sobre
ciertos materiales. Así nos encontramos que hay diferentes técnicas basadas en
estas propiedades:
29
Introducción

La volatilidad de los analitos: la destilación y las técnicas de espacio de
cabeza.

La solubilidad del analito en ciertos disolventes orgánicos: extracción con
el equipo soxhlet, extracción líquido-líquido (LLE), extracción con fluido
supercrítico (SFE), extracción en fase sólida (SPE), extracción asistida
por ultrasonido (EAU), y extracción asistida por microondas (MAE).

La adsorción y la absorción del analito en un determinado material:
extracción en fase sólida (SPE), microextracción en fase sólida (SPME),
extracción con barrita agitadora (SBSE).
Como se ha comentado anteriormente, la preparación de muestra es uno
de los aspectos críticos en el proceso analítico, sobre todo si se pretenden
determinar componentes traza en muestras complejas. La introducción del uso de
adsorbentes y absorbentes comerciales para extraer y purificar los analitos en
disolución supuso un gran paso, ya que la utilización de esta técnica permite
aislar, purificar y preconcentrar con éxito compuestos químicos de manera rápida
y reproducible. Por lo que a continuación nos vamos a centrar en las técnicas
basadas en la adsorción y la absorción del analito:
Extracción en fase sólida (SPE). Esta técnica fue introducida a finales
de los años setenta, con la cual se comenzó a disminuir el uso de disolventes
orgánicos y se evitaron problemas como la separación incompleta de fases. SPE
puede ser aplicado directamente en muestras líquidas para aislar y concentrar los
compuestos de interés. Está basada en la retención selectiva de algunos analitos
en un sólido adsorbente, que actúa como fase estacionaria a través de la cual, tras
un breve acondicionamiento, se hace pasar la muestra. Los analitos se retienen en
la superficie del sólido, seguidamente se realiza una etapa de lavado con la que
se pretende desorber las interferencias que hayan podido quedar retenidas.
Finalmente, los compuestos de interés se eluyen mediante el paso de una
30
Introducción
pequeña cantidad de disolvente que tiene más afinidad por ellos que la fase
estacionaria (Figura 7).
En 1982 Williams et al., comenzaron a usar esta metodología de
extracción para determinar glicósidos en mostos de uva y vino utilizando
cartuchos C18, y en 1985 Günata et al., utilizaron resina XAD-2 con el mismo
fin. A partir de entonces son muchos los trabajos encontrados en el campo de la
enología con esta y otras fases sólidas (Voirin et al., 1992; Bureau et al., 1996;
López et al., 2002; Sánchez Palomo et al., 2006; Campo et al., 2007; Cabrita et
al., 2007; Loscos et al., 2009; García-Carpintero et al., 2012; Lagunas-Allué et
al., 2012).
Acondicionamiento
Elución de
interferencias
Muestra
Elución del analito
de interés
Analito
Interferencias
eluídas
Figura 7: Fases SPE hasta la obtención del extracto a analizar.
Microextracción en fase sólida (SPME). Es una técnica de extracción
desarrollada a principio de los años 90 por Arthur & Pawliszyn (1990). Presenta
numerosas ventajas tales como simplicidad, manipulación prácticamente nula de
la muestra, y no requiere disolventes ni elevadas temperaturas durante la
extracción. Se vale de una fibra de cristal de silicio recubierta con diferentes
materiales poliméricos (PDMS, carbonatos, etc) que puede ser introducida en el
espacio de cabeza del vial (HS-SPME) (Figura 8), o bien directamente en la
31
Introducción
solución de interés (DI-SPME), lo que permite el aislamiento y extracción en una
única etapa de los compuestos odorantes que presenten afinidad por el polímero
de la fibra. Transcurrido el tiempo de adsorción adecuado, la fibra puede
desorberse directamente en el cromatógrafo de gases.
Desde su introducción, esta técnica se ha venido empleando de manera
habitual para el análisis de compuestos del aroma de las uvas y del vino (Mestres
et al., 1999; Rocha et al., 2001; López et al., 2002; Castro et al., 2008; NoguerolPato et al., 2009; Capone et al., 2011; Hjelmeland et al., 2012).
HS-SPME
DI-SPME
Figura 8: Extracción de volátiles mediante SPME: por inmersión (DI-SPME) o por espacio de
cabeza (HS-SPME).
Extracción con barrita agitadora (SBSE). Es una técnica introducida
más recientemente, en el año 1999 por Baltussen et al. Se vale de una pequeña
barra magnética recubierta de un polímero absorbente (polidimetilxilosano,
PDMS) que es comercializada con el nombre de “twister”, la cual puede
colocarse en el espacio de cabeza (HS-SBSE) o directamente dentro de la
muestra líquida (SBSE) (Figura 9). Tras la extracción de los volátiles por
inmersión la barrita debe lavarse con agua destilada para eliminar posibles
compuestos que interfieran, como azúcares o proteínas, y se seca con un pañuelo
de papel. A continuación la barrita es sometida a desorción térmica y los analitos
pasan directamente a la columna cromatográfica.
32
Introducción
Espacio de cabeza
HS-SBSE
Inmersión
SBSE
Figura 9: Extracción de volátiles mediante SBSE: por espacio de cabeza (HS-SBSE) o por
inmersión (SBSE)
Las características de la SBSE son similares a las de la microextracción
en fase sólida o SPME, pero al tener una mayor superficie extractiva la barrita
que la fibra, permite una sensibilidad superior a la SPME, aumentando la
posibilidad de determinar volátiles que aparezcan en concentraciones traza.
Desde su aparición el twister ha sido utilizado con éxito en numerosos campos,
entre ellos en el enológico. Los primeros trabajos en enología se realizaron para
la determinación de plaguicidas en vinos, y a partir de ahí se ha utilizado para
analizar numerosos tipos de compuestos aromáticos (Sandra et al., 2001;
Hayasaka et al., 2003; Díez et al., 2004; Salinas et al., 2004; Alves et al., 2005;
Marín et al., 2005; Zalacain et al., 2007; Maggi et al., 2008; Perestrelo et al.,
2009; Pedroza et al., 2010; Almeida & Nogueira, 2012).
3.3.2. Factores que influyen en el aroma de la uva y del vino
Como se ha comentado anteriormente, en el aroma del vino influyen
numerosos factores a lo largo de todo el proceso de producción, desde la cepa
hasta la copa. Puesto que este trabajo se ha centrado en la aplicación foliar a la
cepa de extractos vegetales y su efecto en el aroma del vino, a continuación nos
referiremos solamente a los factores que en contacto con la parte aérea de la vid
33
Introducción
pueden influir en la composición aromática de la uva y el vino: compuestos
volátiles del ambiente en el que se desarrolla la planta y los tratamientos foliares
aplicados a la misma.
3.3.2.1. Absorción foliar de compuestos
Las hojas son capaces de absorber sustancias exógenas aplicadas vía
foliar, como son los nutrientes, extractos vegetales, biorreguladores, herbicidas y
pesticidas. Esta capacidad le da a la planta la posibilidad de tomar ingredientes
activos aplicados en pulverizaciones, estando limitada por las propiedades físicoquímicas, la masa molecular y la insolubilidad de la sustancias (Faers & Pontzen,
2008). Por ello, moléculas de alto peso molecular como son los taninos, o
sustancias muy insolubles son incapaces de penetrar a través de las hojas (Eichert
& Goldbach, 2008; Fernández & Eichert, 2009).
Los coadyuvantes mejoran la deposición y adhesión de las sustancias en
las hojas. Además, la utilización de estas sustancias en las disoluciones de
aplicación foliar hace que permanezcan en las hojas como una fina película
permitiendo que sea más efectiva la penetración/absorción de las sustancias
activas (Mengel & Kirby, 1987).
La absorción de las disoluciones pulverizadas a las hojas ocurre
directamente a través de la cutícula y de los estomas (Figura 10). La cutícula de
la vid aparentemente es impermeable y repelente al agua por sus propiedades
hidrofóbicas, no obstante, al ser aplicada la disolución por pulverización, ésta se
difunde por los espacios interfibrales de la pared de las células epidermales. La
absorción por los estomas puede ser tan importante como la ruta a través de la
cutícula (Eichert & Goldbach, 2008), estando limitada por el estrés y la noche, ya
que hace que estos estomas se cierren, por lo tanto la aplicación a primeras horas
de la mañana es más apropiada.
34
Introducción
Las sustancias que penetran en la cutícula exterior de las hojas pueden
atravesar las paredes celulares (vía simplástica), especialmente los ingredientes
activos más simples, para ser translocados por el floema, o pueden alcanzar las
paredes celulares pero sin llegar a penetrarlas (vía apoplástica), las cuales se
moverán vía xilema (Figura 10). El movimiento de sustancias desde las hojas
hacia otros órganos de la planta, especialmente a puntos activos de crecimiento,
ocurre principalmente a través del floema.
Figura 10: Vías de absorción de las disoluciones pulverizadas a las hojas.
La absorción foliar está influenciada por numerosos factores: factores
ambientales (temperatura, humedad, luz, viento, etc), factores genéticos, estado
nutricional, edad de la planta, propiedades de la sustancia aplicada, estadío de
desarrollo de la planta, etc.
35
Introducción
3.3.2.2. Componentes volátiles del ambiente de la vid
El efecto que tienen ciertos volátiles presentes en el ambiente donde crece
el viñedo sobre el aroma de sus vinos es un campo aún muy poco estudiado. Sin
embargo, es conocido que muchos enólogos afirman que ciertas plantas que
crecen cerca de las vides influyen en el aroma de los vinos aportándoles notas
distintivas.
A nivel científico, se ha puesto de manifiesto que los vinos procedentes
de viñedos cultivados en zonas cercanas a bosques de eucaliptos manifiestan una
nota aromática característica (Herve et al, 2003). Este fenómeno es debido a que
las uvas poseen mayor concentración de 1,8-cineol (eucaliptol), compuesto
responsable del aroma a eucalipto, cuanto mayor es la proximidad a árboles de
eucalipto, y a que durante la fermentación pasa al vino en cantidades suficientes
para aportarle esta nota aromática (Capone et al., 2011; Capone et al., 2012).
Por otro lado, numerosos estudios han puesto de manifiesto que uvas y
vides cultivadas en las proximidades de zonas que han sufrido incendios,
producen vinos con olor a humo (Kennison et al., 2007, 2008, 2009; Sheppard et
al., 2009). Se observó que los compuestos volátiles presentes en el humo
(guayacol, 4-metilguayacol, siringol, metilsiringol, cresol, etc) eran asimilados
por la planta, almacenados como precursores glicosilados en las uvas (Figura
11) y posteriormente transmitidos al vino (Hayasaka et al., 2010a, 2010b;
Dungey et al., 2011; Singh et al., 2011; Wilkinson et al., 2011; Parker et al.,
2012).
Figura 11: Glicosilación de guayacol tras el contacto de las viñas con humo.
36
Introducción
Además, el efecto del humo sobre la composición aromática de los vinos
depende de la variedad de uva, del momento de aplicación y de la duración de la
exposición (Kennison et al., 2009; Wilkinson et al., 2011).
3.3.2.3. Aplicaciones foliares a la vid
La mayor parte de las aplicaciones foliares a la vid son realizadas para el
control de plagas, aunque también se hacen para nutrir a la planta como
fertilizantes, o mejorar la calidad del producto, denominándose a estas sustancias
bioestimulantes (Pardo-García et al., 2012). Sin embargo, a pesar de las
numerosas aplicaciones foliares habitualmente realizadas, existen muy pocos
estudios sobre su repercusión en el aroma de las uvas y de sus vinos. La mayoría
de ellos son muy recientes, debido por una parte a la preocupación actual por
profundizar en el conocimiento del aroma, y por otra a que ahora se dispone de
un amplio abanico de métodos y de técnicas analíticas sencillas y fiables para el
análisis de los compuestos aromáticos.
Centrándonos en la repercusión en el aroma de los vinos de las
aplicaciones foliares al viñedo de formulaciones comerciales de pesticidas
orgánicos de síntesis, se ha puesto de manifiesto que no sólo afectan a los aromas
fermentativos del vino, sino también al aroma varietal, tanto a la fracción libre
como a la fracción ligada (Aubert et al., 1997a,1997b; Oliva et al., 1999, García
et al, 2004; Oliva et al. 2008; González-Rodríguez et al., 2011; Noguerol-Pato et
al., 2011, González Álvarez et al., 2012a, 2012b). La aplicación de otras
substancias fungicidas, como el cobre, provocan una fuerte disminución de la
tipicidad, debido a su reacción con los tioles varietales (Hatzidimitriou et al.,
1996; Darriet al., 2001; Jackson, 2008). También, el análisis sensorial de vinos
tras la aplicación foliar a las viñas de silicato de potasio y azufre para el control
de oídio, pone de manifiesto diferencias en el aroma (Reynolds et al., 1996).
37
Introducción
Otro ejemplo de aplicaciones foliares a viñas que afectan al aroma de los
vinos son los tratamientos con etanol acuoso durante el envero (Martin et al.,
2008; Zoecklein et al., 2011). Estas disoluciones se aplicaron para evaluar su
impacto en la maduración de las uvas.
Es conocido que la fertilización foliar puede satisfacer con rapidez y
eficacia las necesidades nutricionales, por ello se realizan aplicaciones foliares de
diferentes formas de nitrógeno, en especial de urea, las cuales provocan una
modificación de la composición volátil de los vinos (Lacroux et al., 2008; AncínAzpilicueta et al., 2012), que se puede atribuir a la modificación del perfil
aminoacídico de las uvas (Irti et al., 2005; Lasa et al., 2012). Es sabido que los
aminoácidos de las uvas desempeñan un importante papel como precursores de
compuestos volátiles del vino (Callejón et al., 2010), ya que pueden ser
transformados hasta alcoholes superiores, aldehídos, ésteres y ácidos cetónicos
(Bell & Henschke, 2005; Vilanova et al., 2007). También se ha observado que el
perfil aminoacídico de las uvas puede ser modificado por aplicaciones foliares a
la viña de ciertos fungicidas (Oliva et al., 2011).
Por el contrario, la aplicación foliar a la viña de caolín, arcilla mineral
inerte y reflectante utilizada habitualmente en el control de plagas, muestra un
mínimo efecto sobre los aromas libres y glicosilados de las uvas (Song et al.,
2012) y no altera la composición volátil de los vinos (Ou et al., 2010). Otro
tratamiento que se ha visto que no afecta a las características sensoriales de los
aromas del vino es la aplicación foliar de reguladores del crecimiento, ácido
abscísico, ácido 2-cloroetilfosfónico y ácido indol-3-acético (González et al.,
2012).
La mayoría de las aplicaciones foliares estudiadas afectan al aroma de los
vinos, lo que sugiere que es posible influir en la composición química de la uva
mediante el uso de sustancias aplicadas por vía foliar a la vid, y por tanto que los
38
Introducción
vinos elaborados a partir de ellas tengan un perfil aromático diferenciado. Esta
idea debe ser estudiada ya que profundizaría en el conocimiento del aroma, y
puede suponer la apertura de otras posibilidades aún no explotadas en el sector
vitivinícola.
3.4. Extractos vegetales
En la actualidad se está despertando el interés por la aplicación de
extractos vegetales en agricultura y de forma especial en las viñas. Se están
utilizando como alternativa natural o complementaria a los pesticidas orgánicos
de síntesis. El enfoque agrícola convencional ha tratado de controlar la plagas del
viñedo mediante la aplicación de pesticidas químicos, pero esto ha derivado tanto
en la aparición de resistencia del patógeno (Leroux, 2004; Latorre & Torres,
2012), como en la producción de efectos adversos sobre el medio ambiente y la
salud humana. A ello hay que añadir los efectos tóxicos de sus residuos sobre las
levaduras responsables de la fermentación alcohólica (Calhelha et al., 2006;
Oliva et al., 2007; Čuš & Raspor, 2008), por lo que el uso de pesticidas, en
especial fungicidas, está cada vez más limitado. Además, uno de los productos
habitualmente utilizados en el control fitosanitario del viñedo, el cobre, puede
producir alteraciones del aroma y sabor del vino, causando olores no deseables,
además de acumularse en los suelos y producir fitotoxicidad (Jackson, 2008;
Darriet et al., 2001; Pavlovic, 2011), por lo que es evitado cada vez más por los
viticultores. Por lo tanto, se hace necesaria la búsqueda de alternativas y nuevas
estrategias para el control de plagas, que sean eficaces, de fácil utilización y
económicas. Algunas de estas alternativas en viñas son la aplicación de extractos
de plantas (Jacometti et al., 2010; Harm et al., 2011).
En los últimos años se está dando una gran importancia al uso de
bioestimulantes. Los bioestimulantes son sustancias biológicas que actúan
39
Introducción
potenciando determinadas expresiones metabólicas y/o fisiológicas de las
plantas. Se definen según la EBIC (European Bioestimulant Industry
Consortium) más por lo que hacen que por lo que son (Natale, 2012), ya que la
categoría incluye una gran diversidad de sustancias. La aplicación de estos a las
plantas mejora el desarrollo del cultivo, vigor, rendimiento y la calidad mediante
la estimulación de procesos naturales que benefician el crecimiento y las
respuestas a estrés abiótico. Por ello, en el campo de la viticultura los extractos
vegetales se están utilizando como bioestimulantes, entre otros fines, para
mejorar la calidad de las uvas. Uno de los primeros trabajos realizados fue el de
Carmona et al., (2001), donde se observó un aumento de los polifenoles y del
color en la uva Bobal tras la aplicación a la viña de extractos vegetales. Más
recientemente Parrado et al. (2007) propusieron el uso de extractos de origen
vegetal como bioestimulantes para aumentar el contenido en antocianos de las
uvas y mejorar la calidad del color de los vinos.
A pesar de las múltiples aplicaciones de extractos vegetales sobre la vid,
en la bibliografía exclusivamente se ha encontrado un trabajo (Reynolds et al.,
2005) en el que se estudia su efecto sobre el aroma del vino. Éste únicamente
consta de un análisis sensorial en vinos, lo que pone de manifiesto el gran
desconocimiento del efecto de la aplicación de extractos vegetales en la
composición química del aroma de uvas y vinos.
Existe una amplia gama en el mercado de extractos comerciales de origen
vegetal de uso agrícola, que se usan fundamentalmente con fines fitosanitarios, o
de uso alimentario. No obstante, los extractos procedentes de madera de roble y
los hidrolatos de lavandín fueron los que se eligieron para este trabajo de tesis
doctoral, siendo el primero de ellos usado como aromatizante de zumos y
brandis, mientras que el segundo es un residuo desechado de la industria de los
aceites esenciales. Las razones de su elección se detallan seguidamente.
40
Introducción
3.4.1. Extractos de roble
El roble ha sido utilizado desde hace siglos para la fabricación de barricas
destinadas a la crianza del vino. Durante este proceso, las barricas de roble no
son un mero recipiente sino que contribuyen favorablemente a la evolución
organoléptica, aportando caracteres olfativos y gustativos, marcados por notas de
vainilla, madera y especias que armonizan perfectamente con el afrutado de los
vinos. Además, el roble facilita la combinación de antocianos y taninos, lo que
contribuye a estabilizar el color y suavizar la astringencia del producto.
Los aspectos negativos de la utilización de barricas son el fuerte
desembolso económico necesario para su compra y mantenimiento, y las
pérdidas de producto por evaporación y retención de vino en la madera, por lo
que en los últimos años han surgido alternativas más económicas. Estas se
centran en proporcionar, al igual que la madera, estabilización de la materia
colorante, suavización de la astringencia, y los aromas típicos. Una de las
alternativas a la crianza tradicional más ampliamente utilizada es el uso de chips
o virutas de madera de roble (Zamora, 2003) (Figura 12).
Figura 12: Distintas alternativas al uso de las barricas para dar el carácter “madera” a los vinos.
Los componentes de la pared celular del roble son: celulosa, hemicelulosa
y lignina. Estas macromoléculas polisacáridas (celulosa y hemicelulosa) y
41
Introducción
polifenólicas (ligninas), le aportan a la pared celular características físicoquímicas tales como resistencia a la tracción y a la compresión, rigidez e
impermeabilidad. El resto de componentes constituyen la llamada fracción
extraíble que llegan a representar hasta un 10% de la madera seca, y pueden
presentarse mezclados con los polímeros en la pared celular o como inclusiones
en los lúmenes celulares. Estos son compuestos de difícil clasificación ya que su
naturaleza es muy variada. Los elagitaninos son los más abundantes, pero
también se encuentran otros componentes de estructuras químicas muy
diferentes, polifenoles de bajo peso molecular y compuestos volátiles, algunos de
estos compuestos serán el origen de muchas de las características de interés
organolépticas que se encuentran en los vinos de crianza.
Tabla 1. Principales sustancias volátiles procedentes de la madera de roble.
Nombre
Compuestos
furánicos
β-metil-γoctolactona
Fenoles volátiles
Aldehídos
fenólicos
Fenil cetonas
Furfural
5-Metilfurfural
5-Hidroximetilfurfural
Isómero cis
Isómero trans
Eugenol
Guayacol
Siringol
6-Methoxieugenol
4-Vinilguayacol
4-Etilguayacol
4-Etilfenol
Vanillina
Sirigaldehido
Sinalpadehido
Coniferaldehido
Acetovanillona
Propiovanillona
Vanillato de metilo
Vanillato de etilo
Umbral de
percepción
Descriptor
olfativo en
vinos
Aromas a tostado,
20 mg/l (1)
almendras tostadas
45 mg/l (1)
y caramelo
45 mg/l (1)
20-46 µg/l (2)
Aromas a madera,
coco, vainilla, etc
140-370 µg/l (2)
6 µg/l (3)
Notas ahumadas, a
9,5 µg/l (3)
clavo, especias y
570 µg/l (4)
fenólicos
1,2 mg/l (3)
40 µg/l (3)
Farmacia, cuero y
47 µg/l (5)
animal
230 µg/l (5)
Vainilla
60 µg/l (3)
No participan
apreciablemente en
el aroma
1000 µg/l (3)
Vainilla
3000 µg/l (3)
990 µg/l (4)
Origen
Polisacáridos
Lípidos
Lignina
Lignina
Lignina
(1) Boidron et al., 2006; (2) Brown et al., 2006; (3) Culleré et al., 2004; (4) López et al., 2002; (5)
Chatonnet et al., (1990).
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Introducción
Los compuestos volátiles del roble transmitidos al vino tienen una gran
importancia sensorial, ya que aportan notas aromáticas a “madera”, “coco”,
“especias” “ahumado”, “tostado” contribuyendo a dar complejidad al vino. Los
compuestos responsables de estas notas proceden de la termodegradación de los
polisacáridos (compuestos furánicos), de la termodegradación de la lignina
(fenoles volátiles, aldehídos fenólicos y fenil cetonas) y de la degradación de los
lípidos (whisky lactonas) (Tabla 1). Las concentraciones en el vino son
dependientes de las encontradas en la madera verde, con una fuerte variabilidad
entre árboles individuales, especies y orígenes, así como de las condiciones de la
fabricación de la barrica (secado, tostado, etc).
El extracto de roble no se eligió únicamente porque la madera de roble es
un material habitual de uso enológico y le proporciona características
organolépticas positivas al vino, sino también porque posee en su composición
compuestos comunes con el humo, tales como guayacol, eugenol y siringol.
Como se mencionó anteriormente, se ha demostrado que este tipo de
compuestos, tras la exposición de las viñas al humo, son asimilados por la vid,
glicosilados y liberados en el vino influyendo en el aroma (Kennison et al., 2007,
2008, 2009; Sheppard et al., 2009; Hayasaka et al., 2010a; Hayasaka et al.,
2010b; Dungey et al., 2011; Singh et al., 2011; Wilkinson et al., 2011; Parker et
al., 2012). Esto sugiere que la aplicación por vía foliar a la viña de estos
extractos de roble, probablemente puedan afectar a la composición aromática de
sus uvas y vinos y puedan dar un vino con una aroma diferenciado.
3.4.2. Extracto de lavandín (hidrolato)
España es un país rico en la flora de plantas aromáticas como son
lavanda, lavandín, romero y tomillo. Castilla-La Mancha produce casi el 70% de
lavanda-lavandín del total producido en España (6352 toneladas), seguido de
Castilla y León, Murcia, Valencia, Navarra, Andalucía y Aragón según datos del
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Introducción
Anuario de Estadística Agraria (AEA, 2010). Además, España ha experimentado
un importante aumento de la producción aunque no de superficie de cultivo, esto
ha sido debido al paso de secano a regadío (AEA, 2006-2010). La región de
Castilla-La Mancha, como se mencionó anteriormente, alberga la mayor
superficie de viñedo de Mundo, por lo que es muy común observar campos de
lavanda-lavandín cerca de los viñedos (Figura 13).
Figura 13: Fotografía que muestra la cercanía de campos de lavandín a los viñedos.
El lavandín es una planta herbácea aromática originaria de la zona
mediterránea. El nombre científico de lavandín es el de Lavandula hydrida y es
un híbrido natural entre la lavanda (Lavandula angustifolia P. Miller) y espliego
(Lavandula latifolia L. Medikus), que al ser una especie más rustica y de más
fácil manejo que sus progenitores es más ampliamente cultivada (Meunier,
1992). Es una planta leñosa muy aromática de hasta 40 cm, con hojas alargadas
de color verde azulado y tallos cuadrangulares de los que brotan espigas densas
de flores azuladas, siendo su época de floración el inicio de verano, entre julio y
agosto. Mediante hibridaciones se han obtenido distintas variedades comerciales,
siendo las más extendidas, Super, Abrial y Grosso, por proporcionar mayores
rendimientos de flores y esencias (Meunier, 1992; Férnandez-Pola, 1996).
El lavandín, al igual que la lavanda, ha sido una de las plantas más
utilizadas en los países mediterráneos, debido a que crece en gran abundancia de
forma silvestre y es muy fácil de cultivar. Desde la antigüedad ha sido muy
44
Introducción
utilizado por sus numerosas propiedades, en todas sus formas, flor seca,
infusiones, aceites, siendo esta última la forma más común, con numerosos fines.
Los aceites esenciales son de amplio uso en cosmética y perfumería. En la
extracción de aceites esenciales, durante la destilación de plantas aromáticas
(flores, hojas, tallos y raíces) por arrastre de vapor, se genera una gran cantidad
de agua impregnada de compuestos solubles a la que se le denomina hidrolato.
Los principales componentes volátiles de los hidrolatos son generalmente los
mismos que los presentes en los aceites esenciales, aunque en menor proporción,
siendo en el caso del hidrolato de lavandín los terpenos (Kaloustian et al., 2008;
Paolini et al., 2008; Aazza et al., 2011). Sin embargo, estos hidrolatos son
comúnmente desechados.
Por lo tanto existe una industria que genera gran cantidad de hidrolatos,
los cuales normalmente se desechan, a pesar de que tienen aún una concentración
aprovechable, sobre todo en terpenos. Este tipo de compuestos como ya se ha
mencionado anteriormente en el caso del eucaliptol, son asimilados por la vid y
transmitidos al vino (Herve et al., 2003; Capone et al., 2011; Capone et al.,
2012), lo que nos sugirió que la aplicación a las viñas de un hidrolato de lavandín
podría modificar la composición volátil de sus vinos.
45
4. OBJETIVOS
OBJETIVOS
OBJECTIVES
Objetivos
El objetivo principal de este trabajo fue estudiar el efecto que sobre la
composición aromática de la uva y sus vinos tiene la aplicación a la vid de
extractos acuosos obtenidos de roble y de lavandín.
Para conseguir este objetivo se plantean los siguientes objetivos específicos:
1. Seleccionar y caracterizar los extractos vegetales con capacidad de
trasmitir sus compuestos volátiles a las uvas, y elegir la formulación y
dosificación más adecuada para ser aplicados a cepas de variedades
blancas y tintas por vía foliar.
2. Evaluar el efecto de la aplicación de los extractos vegetales a la vid sobre
los parámetros usados para determinar la aptitud enológica de las uvas.
3. Estudiar el efecto de la aplicación de los extractos de roble a la vid en la
composición aromática de la uva y del vino.
4. Estudiar el efecto de la aplicación del hidrolato de lavandín a la vid en la
composición aromática del vino.
49
Objetives
The main aim of this work was to study how the grape and its wines
aroma composition was behaved when aqueous extracts derived from oak and
lavandin were applied to the vineyard.
To achieve this objective the following specific objectives had been raised:
1. Selecting and characterizing plant extracts capable of transmitting their
volatile compounds to the grapes; and to choose the most appropriate
formulation and dosage to be foliar applied to vineyards of white and red
varieties.
2. Evaluating the effect of plant extracts applications to the vineyard on
grapes parameters used to know the oenological aptitude.
3. Studying the effect of oak extracts applications to the vineyard on the
aroma composition of grapes and their wines.
4. Studying the effect of lavandin hydrolat application to the vineyard on
the wine aroma composition.
50
5.PLAN DE TRABAJO
WORK PLAN
Work Plan
To achieve the objective 1, an extract with an oenological interest was
looked for, resulting promising the oak one. This had compounds in its
composition which are well known assimilated by the vineyards and impact on
the wine. In the same way, the second extract was selected as it is a common
waste byproduct from our area, the lavandin hydrolat, which is known to have
volatile compounds which may be accumulated by the vineyards and pass to the
wine. In both cases, the first task was the selection and characterization of these
extracts. Before their application, it is necessary to formulate and dose them,
which have been included as second task together with its phytotoxicity study.
For this step, a fast growing plant, tomato Micro Tom which is already used in
viticulture, was used for preliminary trials.
The objetive 2 was achieved by the determination of the oenological
parameters, as detailed in Materials and Methods (third task).
To achieve objective 3, three other tasks were proposed as summarized
below. The fourth task consisted of oak extract and reference compounds
applications to the white variety Verdejo vineyards. The determination of
volatile composition on the extract, on the grapes and on the wines (after
alcoholic fermentation (AF) and six months later (6 months)) was carried out.
The fifth task consisted of oak extract and reference compounds applications to
the red variety Petit Verdot vineyards. The determination of volatile composition
of the extract, of the grapes and of the wines (after alcoholic fermentation (AF),
after malolactic fermentation (MLF) and eight months later (8 months)) was
carried out. In order to generalize the conclusions resulting from the above tasks,
and to advance on the knowledge about how oak extract compounds reach the
grapes, an extract was applied on a different geographical area (France) and on
other grape varieties (Syrah and Chardonnay) (sixth task).
53
Work Plan
To achieve the objective 4, a lavandin hydrolat was applied to the red
variety Petit Verdot vineyards and the volatile composition of their wines was
determined (seventh task).
Figures 14, 15 and 16 shown the work plan described previously in terms
on type of extract applied.
54
Work Plan
Aqueous oak extract
(selection and characterization)
Plant fast growing "Tomatoes"
(phytotoxicity study, preparation of formulations and dose setting)
Foliar application of formulations of French
oak extracts to vineyards of Castilla-La
Mancha
Verdejo
Petit Verdot
Foliar application of formulations of
American oak extracts to vineyards of
France
Syrah
Chardonnay
Grapes
Grapes
Grapes
(oenological parameters and
volatile compounds analysis)
(oenological parameters and aroma glycosidic
precursors analysis)
Wine (AF, 6 months)
Wine (AF, MLF, 8 months)
Figure 14. Schematic work plan of oak extracts applications.
55
Work Plan
Oak extract reference compounds (Eugenol and Guaiacol)
(selection of compounds and solution preparation)
Plant fast growing "Tomatoes"”
(phytotoxicity study, preparation of formulations and dose setting)
Foliar application of formulations of
reference solution to vineyards of Castilla-La
Mancha
Verdejo
Petit Verdot
Grapes
Grapes
Wine (AF, 6 months)
Wine (AF, MLF, 8 months)
Figure 15. Schematic work plan of oak extract reference compounds (Eugenol and Guaiacol)
applications.
56
Work Plan
Lavandin plantations in Castilla La Mancha
Distillation to obtain essential oils and
obtaining hydrolat
Lavandin extract (hydrolat)
(volatile composition analysis, design
and establishment of dose
Foliar application of lavandin extract (hydrolat) to
vineyards close to lavandin fields of Castilla-La
Mancha
Petit Verdot
Grapes
(oenological parameters)
Wines (AF, MLF, 6months)
(oenological parameters and volatile compounds analysis)
Figure 16. Schematic work plan of lavandin hydrolat applications.
57
6.MATERIALES Y MÉTODOS
MATERIALES
Y
MÉTODOS
Materiales y Métodos
6.1. Material vegetal
6.1.1. Extractos vegetales
6.1.1.1. Extractos comerciales a base de roble
Para llevar a cabo el estudio se realizó una búsqueda exhaustiva de
extractos comerciales acuosos a base de roble. La empresa Protea France S.A.S
(Gensac la Pallue, Francia) fue la única que encontramos capaz de proporcionar
extractos de roble acuosos. Estos presentaron unas características adecuadas, sin
riesgo de toxicidad al ser utilizados como aditivos alimentarios, pues son
comercializados para bebidas espirituosas y zumos de frutas. La empresa ofreció
diferentes extractos (501, 502, 503, 120 y 103C) obtenidos mediante infusiones
acuosas de “chips” de roble tostado (Figura 17). Estos extractos se prepararon
mediante maceración en agua desmineralizada de “chips” de roble francés
(Quercus sessilis) y de roble americano (Quercus alba) a 100ºC durante 32
horas. La madera procedió de roble secado de forma natural durante al menos 18
meses y sometido a distintos grados de tostado. La concentración de madera por
litro de agua fue de aproximadamente 200 g/l, con un extracto seco de 50 g/l
(datos proporcionado por la empresa).
Figura 17. Fotografía de las muestras de los extractos de roble proporcionados por la empresa
Protea.
61
La composición volátil de los extractos de roble se extrajó según la
técnica SBSE en el caso de los utilizados en España y según una extracción
líquido-líquido (LLE) en el caso del utilizado en Francia. Los volátiles extraidos
se analizaron en ambos casos por cromatografía de gases con detección de
espectrometría de masas (GC-MS). Ambos tipos de análisis se detallarán más
adelante.
Tabla 2. Composición volátil media de los extractos acuosos a base de de roble µg/l.
Compuesto
Intervalo de concentración
cis-Whisky lactona
5,6-2650
trans-Whisky lactona
11,3-750
Furfural
2819-2600
5-Metilfurfural
15,1-350
Eugenol
9,8-90
6-Metoxieugenol
0,97-950
Guayacol
15,2-240
4-Vinilguayacol
1,4-100
4-Etilguayacol
2,7-10
4-Etilfenol
4-10
Siringol
1140-5570
3,4,5-Trimetoxifenol
380
Vainillina
2,3-3810
Siringaldehído
37170
Acetovainillona
0,9-1410
Propiovanillona
1640
Vanillato de metilo
4570
Vanillato de etilo
11980
Ácido homovaníllico
11020
62
El intervalo de concentración de los diferentes compuestos se muestra en
la Tabla 2 que pone de manifiesto la gran variabilidad entre los diferentes
extractos. Además, el pH medio fue de 2,9, el índice de polifenoles totales fue de
437,73 mg/l y el contenido medio de elagitaninos (expresados como ácido
elágico) fue de 790,7 mg/l. Los extractos también fueron sometidos a un análisis
sensorial olfativo por expertos catadores de vinos. La elección de los extractos
para las distintas aplicaciones se baso en que tuvieran una adecuada
concentración en compuestos volátiles y una positiva impresión global por parte
de los catadores.
6.1.1.2. Extracto de lavandín (hidrolato)
El lavandín del que procedió el extracto (Lavandula híbrida, que es un
híbrido natural entre la L. angustifolia y la L. latifolia) fue cultivado en la finca
Dehesa de los Llanos (Albacete) en el año 2010. Las variedades de lavandín
cultivadas en la finca son Grosso, Super, Abrial y Mallieta. El extracto de
lavandín utilizado fue el hidrolato procedente de la destilación para la obtención
del aceite esencial de una mezcla de todas. La recolección del lavandín se realizó
a máquina durante la primera y segunda semana de julio. Se dejaron secar en el
campo durante unos días, recogiéndose la planta casi seca con unos rendimientos
de entre 2.500-3.000 kg de flores por hectárea. La destilación por arrastre de
vapor se realizó el día 17 de julio en la planta de extracción de la misma finca,
empleándose agua a 100ºC a baja presión (0,5 bar) durante una hora y media.
Esta destilación proporcionó por un lado el aceite esencial y por otro el hidrolato.
Para destilar el lavandín se utilizaron 2.000 kg de flor de lavandín seca y 1.200 l
de agua, lo cual dio lugar a unos 75 l de aceite esencial y unos 1.200 l de
hidrolato, con un rendimiento medio 1,67 kg de flor/litro de hidrolato.
63
Tabla 3: Composición volátil del hidrolato de lavandín.
Compuesto
Concentración (mg/l)
Acetato de hexilo
81,48
Butanoato de hexilo
0,02
Hexanoato de etilo
0,34
Heptanoato de etilo
0,93
Octanoato de etilo
50,00
Etanoato de 1-octen-3-ilo
13,02
Piruvato de etilo
3,42
Acetato de isobornilo
6,45
Acetato de lavandulol
88,33
Limoneno
9,98
Linalool
1270,49
Citronelol
9,06
Nerol
19,45
α-Terpineol
221,73
cis-Óxido de linalool
11,63
4-Terpineol
428,30
cis-β-Ocimeno
8,00
α-Bisabolol
1,65
1,8-Cineol (eucaliptol)
27,71
Canfor
119,69
2-Feniletanol
39,33
Alcohol furfurílico
310,58
1-Octen-3-ol
623,47
2(5H)-Furanona
46,58
2-Hidroxi 2-ciclopenten-1-ona
68,00
Octanal
1,74
Nonanal
7,11
Feniletanal
1,65
Furfural
303,33
Benzaldehído
3,33
64
Al ser los hidrolatos el residuo acuoso resultante de la destilación de una
planta para la obtención del aceite esencial, sus principales componentes, además
de agua, son sustancias aromáticas solubles en agua. La mayoría de estos
hidrolatos son normalmente desechados, aunque en algunos casos se utilizan en
cosméticos, como remedios de ingesta oral o en productos domésticos por
ejemplo como aromatizadores en productos de limpieza. La composición volátil
del hidrolato fue analizada mediante SBSE-GC-MS (Tabla 3). La elección se
basó en que era un subproducto normalmente desechado, que en su composición
poseía compuestos, los cuales pueden ser asimilados por la vid y que se obtienen
a partir de plantas cultivadas habitualmente cerca de las viñas de la zona.
6.1.2. Planta modelo
Se usó una planta modelo de crecimiento rápido para abordar los ensayos
de fitotoxicidad, formulación y establecimiento de las dosis de aplicación
(Figura 18). Decidimos usar plantas de tomate (Solanum lycopersicum) de la
variedad Micro Tom (mutación de dwarf y miniature), ya que esta variedad se
suele usar como planta modelo en estudios extrapolables a las uvas (Meissner et
al., 1997; Martí et al., 2006). Su corto ciclo vegetativo (70-90 días desde que se
siembra hasta que madura la fruta) permitió que dichos ensayos se pudieran
realizar con anterioridad al ciclo de desarrollo de la vid, y por tanto, que los
resultados obtenidos se pudieran utilizar como punto de partida para la
aplicación de los extractos al viñedo. Estas plantas fueron obtenidas a partir de
semillas de tomateras proporcionadas por el Departamento de Producción
Vegetal y Tecnología Agraria de la E.T.S.I. Agrónomos de Albacete
(Universidad de Castilla-La Mancha). Esta variedad produce tomates de tamaños
similares a los granos de uva (1-2 cm), y poseen una cutícula y forma de fruto
que permite mantener la misma logística de preparación de muestras y analítica
que para las uvas.
65
Figura 18: Fotografías de la aplicación foliar del extracto de roble en las plantas de tomates
Micro Tom.
6.1.2.1. Estudio de fitotoxicidad
Para el estudio de fitotoxicidad se prepararon disoluciones del extracto en
agua al 1%, 5%, 10%, 20% y 40% y se pulverizaron sobre las plantas en
diferentes estadíos de desarrollo (con 4 hojas, 6 hojas, pleno desarrollo foliar
antes de la floración, en el cuajado y en el envero), hasta cubrirlas en su
totalidad. Las plantas fueron tratadas con las mencionadas disoluciones tres
veces dejando tres días entre aplicación y aplicación. Se observó que ninguna de
las pruebas anteriores produjo síntomas de alteración, por lo que se repitieron las
aplicaciones pero con disoluciones del extracto al 80% y 100% (Figura 19). Las
plantas
fueron
observadas
durante
semanas
para
descartar
cualquier
sintomatología que delatase problemas de fitotoxicidad, problemas nutricionales,
clorosis, acortamiento de tallos, etc. Cuando se dio por concluida esta tarea
ninguna planta tuvo síntomas de alteración respecto al control, por lo que las
disoluciones usadas no mostraron fitotoxicidad como cabía esperar.
Figura 19: Fotografía de algunas plantas tratadas en el estudio de la fitotoxicidad.
66
6.1.2.2. Preparación de formulaciones y establecimiento de la dosis
Únicamente esta tarea se realizó en el caso de los extractos de roble, ya
que para el hidrolato de lavandín se partió de los estudios existentes sobre el uso
de aceites esenciales. Se utilizaron de tres disoluciones del extracto con agua al
25%, 80% y 100% que fueron formulados empleando un coadyuvante para
mejorar su adherencia a las hojas. Se usó el coadyuvante Fluvius (BASF,
España) siguiendo las recomendaciones de la casa. Este producto es un
concentrado soluble formado por una mezcla de copolímeros, que reduce la
tensión superficial, y aumenta la dispersión de la gota mejorando la eficacia de la
formulación, disminuyendo el lavado por lluvias y mejorando su asimilación.
Las diferentes formulaciones se aplicaron a 20 plantas de tomates antes de la
floración y en cuajado. Los parámetros que se controlaron fueron: números de
flores, número de tomates cuajados, número de tomates maduros, peso y tamaño
de los tomates. Los tratamientos que dieron los mejores resultados fueron los del
25% y 100% (Figura 20), por lo que se eligieron estas formulaciones para ser
aplicadas a la vid por pulverización a la parte foliar.
Figura 20: Fructificación de las plantas de tomate Micro Tom tratadas con las distintas
formulaciones.
6.1.2.3. Preparación de la disolución de referencia
Esta tarea se abordó para seguir la evolución individual, desde la uva
hasta el vino, de algunos de los compuestos volátiles presentes en los extractos
67
de roble. Se decidió usar una disolución acuosa de eugenol y guayacol y aplicarla
a las cepas de forma similar al extracto, para que el comportamiento de ambos
compuestos nos sirviera de referencia. Se eligieron eugenol y guayacol por ser
componentes habituales de los extractos de roble, y porque otros autores ya
habían demostrado que ambos compuestos eran asimilados por las uvas
procedentes de viñas expuestas a humo y que se encontraban en mayor
concentración en sus vinos (Kennison et al., 2008).
El punto de partida para la elección de la concentración la marcaron los
artículos de Birti et al. (2009) y Ortiz-Serrano & Gil (2007), en donde se indican
las cantidades medias de ambos compuestos en plantas de tomate. Además, se
consideró que la concentración de la disolución de referencia debía ser de tal
magnitud que tras ser aplicada a las plantas, el contenido de ambos compuestos
en tomate debería ser claramente superior a su nivel natural, con el fin de que el
seguimiento analítico de estos compuestos en tomates no diera lugar a dudas.
Después de varias experiencias y extrapolando a que la superficie foliar media de
una vid es 500 veces superior a la de una planta de tomate, se decidió usar una
concentración de eugenol y guayacol de 6 g/l. Para la preparación de la
formulación se realizaron estudios de fitotoxicidad, formulación y dosificación
en plantas de tomate Micro Tom, de forma similar a los extractos de roble.
6.1.3. Viñas
6.1.3.1. Viñas tratadas en Castilla-La Mancha (España)
Se seleccionó una parcela con Vitis vinifera de la variedad blanca Verdejo
situada en la finca Dehesa de los Llanos (Albacete, Suroeste de España, 38º59'N
de latitud, 1º,51'O) durante la campaña del 2009 y otra parcela de la misma finca
con la variedad tinta Vitis vinifera Petit Verdot durante las campañas de 2009 y
de 2010. Las vides, provistas de un sistema de riego por goteo, estaban
dispuestas en espaldera, con un marco de plantación de 1,4 m entre cepas y 2,8
68
entre filas, estructura en cordón simple y líneas orientadas según la trayectoria
solar en el mes de julio NNE-SSO. Los suelos son de textura franco-arcilloarenosa de baja fertilidad, pH alto (8,4), bajo contenido en materia orgánica (2%)
y alto contenido en caliza activa (6,6% CaCO3). El agua de riego es dura (71,7º),
posee un alto contenido en nitratos (32,4 g/l) y bicarbonatos (250,5 g/l). El clima
es mediterráneo continentalizado con temperaturas extremas en invierno y
verano, temperatura media anual de 13ºC, mínimas de -15ºC (enero) y máximas
de 40ºC (agosto) con una amplitud térmica de 19ºC.
6.1.3.2. Viñas tratadas en Languedoc-Roussillon (Francia)
En la Unidad Experimental del INRA, en Pech Rouge, Gruissan, en el
Sur de Francia (43°10'N de latitud, 3°06'E de longitud) durante la campaña de
2011 se usaron uvas tintas de Vitis vinifera Syrah y uvas blancas Vitis vinifera
Chardonnay. Las vides, provistas de un de sistema de riego por goteo, estaban
dispuestas en espaldera vertical, con marco de plantación de 1 m entre cepas y
2,5 m entre filas. Los suelos de ambas viñas son muy pedregosos y calcáreolimosos-arenosos, de buena estabilidad estructural, cuyas raíces se sitúan poco
profundas debido a la presencia de un estrato salino. Pech Rouge tiene un clima
mediterráneo con influencia marítima, la precipitación media anual es de 600
mm con temperaturas suaves en invierno y verano, la temperatura media anual es
de 19ºC, con mínimas de 2ºC (enero) y máximas de 30ºC (julio), y una amplitud
térmica de 13,5ºC. Este clima se caracteriza por veranos cálidos y secos e
inviernos suaves y húmedos, con la particularidad de ser un área de mucho
viento, con 300 días al año.
69
6.2. Tratamientos del viñedo
Las diferentes aplicaciones al viñedo con los extractos vegetales
(extractos de roble y extracto de lavandín) se hicieron durante el envero, cuando
la flexibilidad del hollejo es más alta y se produce un cambio en el color de los
granos de uva, de forma que las variedades tintas se colorean con pigmentos
rojos y azulados, y el color de las variedades blancas se vuelve más transparente.
El envero, se eligió por ser el principal momento en el que se produce la
asimilación y metabolismo de los azúcares (Conde et al., 2007) y los hollejos
sufren cambios en sus características estructurales y químicas (Nunan et al.,
1998; Mullins et al., 2000). Además, los trabajos de Kennison et al. (2009)
mostraron que el envero es el momento más receptivo en la composición volátil
de los vinos del efecto del humo sobre las viñas expuestas a su influencia.
Cada uno de los preparados con los extractos vegetales fue pulverizado
sobre las hojas a razón de 230-250 ml por planta, volumen necesario para cubrir
la totalidad de la pate foliar de la planta. Los tratamientos se realizaron con un
atomizador, en algunos casos, y con una mochila pulverizadora en otros (Figura
21), entre las 7-8 de la mañana, cuando las temperaturas eran inferiores a 20ºC
para evitar lo máximo posible la pérdida de volátiles por evaporación.
Figura 21: Fotografías de los diferentes modos de aplicación de los extractos: a la izquierda con
atomizador a la derecha con mochila.
70
6.2.1. Tratamientos con extractos de roble en España
Las vides Verdejo y Petit Verdot de la campaña del 2009 fueron tratadas
con los extractos de roble empleando diferentes concentraciones. En primer
lugar, este extracto fue diluído a la cuarta parte con agua y aplicado una vez, el
7º día tras el envero (25%(1)). Esta misma dilución fue aplicada cuatro veces, el
4º, 7º, 10º y 13º día tras el envero (25%(4)). El extracto sin diluir también fue
aplicado una vez, el 7º día tras el envero (100%). Además, se empleó una
disolución de eugenol y guayacol en agua en concentración de 6 g/l que fue
aplicado una vez, el 7º día tras el envero (E+G). Para cada uno de los
tratamientos en la variedad Verdejo se utilizaron filas de 188 plantas, dejando
dos filas sin tratar entre los diferentes ensayos para evitar problemas de deriva.
Para los tratamientos en Petit Verdot se utilizaron 10 plantas, dejando 5 plantas
entre diferentes aplicaciones. Para cada variedad se dejaron el mismo número de
plantas sin tratamiento que fueron usadas como control (188 en Verdejo y 10 en
Petit Verdot). En la Tabla 4 se especifican los diferentes tratamientos.
Tabla4. Número de plantas utilizadas y fechas de los tratamientos en Verdejo y Petit Verdot.
Variedad
Verdejo
Petit
Verdot
Nº plantas
utilizadas
188
10
Comienzo del
envero
25/07
1/08
Control
(C)
St
St
25%(1)
25%(4)
100%
E+G
Vendimia
1/08
29/7
1/08
4/08
7/08
1/08
1/08
28/08
08/08
5/08
8/08
11/08
14/08
08/08
08/08
16/09
St: sin tratamiento
Las uvas fueron recolectadas en su momento óptimo de maduración
tecnológica, estimando para ello la mejor relacción ºBeaumé/Acidez total. El día
71
de la vendimia se escogieron uvas al azar de todos los tratamientos y se
congelaron a -20ºC hasta su posterior análisis.
6.2.2. Tratamientos con extractos de roble en Francia
La parcela usada para cada variedad (Syrah y Chardonnay) fue
fraccionada en 12 mini-parcelas con 27 plantas cada una. Para cada tratamiento y
cada variedad se utilizaron 3 mini-parcelas, distribuidas al azar dentro de cada
parcela. Todos los tratamientos se hicieron por triplicado, por lo que cada uno de
ellos constó de 81 plantas. Las viñas fueron tratadas con el extracto de roble en
tres diferentes momentos tras el comienzo del envero, como se indica en la
Tabla 5. Entre las diferentes aplicaciones se dejó una fila sin tratamiento para
evitar problemas de contaminación. Además, se dejaron 81 plantas de cada
variedad, distribuidas del mismo modo que para los tratamientos, sin tratar para
ser usadas como control. Las uvas fueron recolectadas en su momento óptimo de
maduración tecnológica (mayor relación ºBeaumé/Acidez total). El día de la
vendimia fueron escogidas de todos los tratamientos uvas al azar y se congelaron
a -20ºC hasta su posterior análisis.
Tabla 5. Número de plantas utilizadas y fechas de los tratamientos en Syrah y Chardonnay.
Variedad
Nº plantas
utilizadas
Comienzo
del envero
Control
(C)
T1
T2
T3
Vendimia
Syrah
81
14/07
St
21/07
1/08
8/08
07/09
Chardonnay
81
4/07
St
12/07
21/07
2/08
16/08
T: Tratamiento; St: Sin tratamiento.
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6.2.3. Tratamientos con el extracto de lavandín (hidrolato)
El tratamiento con hidrolato de lavandín unicamente se realizó en vides
de la Dehesa de los Llanos (España), de la variedad Petit Verdot, durante la
cosecha del 2010. Los tratamientos se realizaron a partir del 7º día tras el
comienzo del envero. El envero comenzó el día 6 de agosto, por lo que los
tratamientos se realizaron a partir del día 13 de agosto. Para el tratamiento
llamado hidrolato 1 (H1) se hizo una única aplicación ese día y en el tratamiento
llamado hidrolato 5 (H5) se hicieron cinco aplicaciones los días 13, 20, 27 de
agosto y 3 y 10 de septiembre (Tabla 6). Se utilizaron 6 mini-parcelas de 5
plantas en una misma fila, para cada tratamiento se usaron 2 mini-parcelas
distribuidas al azar utilizando un total de 10 plantas, dejando 1 mini-parcela sin
tratar entre las diferentes aplicaciones para evitar la contaminación. Con la
misma distribución, se dejaron 10 plantas sin tratar para usarlas como control.
Las uvas fueron recolectadas en su momento óptimo de maduración tecnológica.
El día de la vendimia fueron escogidas uvas al azar de todos los tratamientos y se
congelaron a -20ºC hasta su posterior análisis.
Tabla 6. Número de plantas utilizadas y fechas de los tratamientos en Petit Verdot.
Variedad
Nº plantas
utilizadas
Petit Verdot
10
Comienzo
del envero
6/08
Control (C)
St
Hidrolato 1
Hidrolato 5
Vendimia
13/08
13/08
20/08
27/08
3/09
10/09
5/10
St: sin tratamiento.
6.3. Vinificaciones
6.3.1. Vinificaciones en blanco
Las uvas de la variedad Verdejo se recolectaron durante la noche con una
vendimiadora, la temperatura media a la entrada a bodega fue de 19 ± 2ºC. A las
73
uvas se les adicionó 10 g de metabisulfito de potasio por cada 100 kg
inmediatamente después de vendimiarlas. En el momento de entrada de la uva a
la bodega se hizo una selección manual y seguidamente se prensó en una prensa
neumática. El mosto obtenido se introdujo en depósitos de acero inoxidable de
200 l, donde la temperatura se mantuvo a 10°C para facilitar el desfangado.
Durante los siguientes días se controló la temperatura y la turbidez empleando un
turbidímetro (HI 83749, Hanna, USA), hasta que ésta fue adecuada para el
desfangado (valores medios de 300 NTU). Una vez eliminadas las lías, se
adicionó la levadura Saccharomyces cerevisiae de la cepa QA23 previamente
acondicionada según las recomendaciones de la casa comercial (Lallemand,
España), en una dosis de 20 g/hl. La temperatura durante la fermentación se
mantuvo próxima a 13°C y se controló diariamente junto con la densidad.
Durante la fermentación las lías superficiales que iban apareciendo se
iban eliminando. Cuando la densidad se estabilizó en torno a 990 g/l y los
azúcares reductores estaban por debajo de 2,5 g/l se dio por acabada la
fermentación, que tuvo una duración de 17 días en todos los casos.
Tras el final de la fermentación alcohólica se realizó un trasiego con el
que se eliminaron las lías restantes y se corrigió el SO2 libre a 25-35 mg/l. Este
vino permaneció 6 meses en los depósitos. Todos los pasos quedan reflejados en
la Figura 22. Los muestreos para el análisis químico y sensorial se realizaron al
final de la fermentación alcohólica y a los 6 meses de esta. Para el análisis
sensorial las muestras no se congelaron y fueron analizadas en el momento del
muestreo. Las muestras se congelaron a -20ºC para el resto de los análisis.
74
Uva Verdejo
(maduración óptima)
Vendimia nocturna a máquina
Adición de SO2
(10 g/100 kg de uva)
Selección
Prensado
(prensa neumática)
Llenado de los depósitos
(cada tratamiento un depósito de 200 l)
Desfangado estático en frío a 10ºC durante aprox. 24h
(valores medios de 300 NTU)
Eliminación de lías
Siembra de levaduras S. cerevisiae de la cepa QA23 (20 g/hl)
(temperatura controlada 13±1ºC)
Finalización de fermentación alcohólica
(<2,5 g/l azúcares)
Adición de SO2
(SO2 libre corregido a 25-35 mg/l)
Conservación en depósitos “siempre lleno” durante 6 meses
Figura 22. Esquema del proceso de vinificación en blanco.
75
6.3.2. Vinificación en tinto
Las uvas de la variedad Petit Verdot, tanto las procedentes de los
tratamientos con los extractos de roble como con el extracto de lavandín, se
recolectaron a mano, y se les adicionó en las cajas 10 g de metabisulfito de
potasio por cada 100 kg de uva. Las uvas fueron despalilladas y estrujadas
manualmente. El mosto de cada tratamiento fue repartido entre 2 tubos
vinificadores de metacrilato de una capacidad de 5 l cada uno, por lo tanto la
fermentación de cada tratamiento se hizo por duplicado. La vinificación fue
llevada a cabo en un fermentador multitubo (Martínez Solé y Cía, S.A.,
Villarrobledo, España), el cuál reproduce las condiciones de elaboración en
bodega. En el caso de los mostos procedentes de los tratamientos con los
extractos de roble, se hizo una pre-maceración en frío durante 48 horas, para
facilitar la extracción de aromas varietales. Al mosto se adicionó la levadura
Saccharomyces cerevisiae de la cepa QA23 previamente acondicionada según las
recomendaciones de la casa comercial (Lallemand, España), en una dosis de 20
g/hl. La temperatura durante la fermentación alcohólica se mantuvo entre 2224ºC, y se controló diariamente junto con la densidad. La fermentación se dio
por finalizada cuando la concentración de azúcares reductores fue menor de 2,5
g/l, que tuvo una duración de 10 días en el caso de los vinos procedentes de los
tratamientos con los extractos de roble y 14 días en el de los vinos procedentes
de los tratamientos con el extracto de lavandín. Una vez acabada, la parte sólida
se prensó manualmente y se mezcló con el vino flor.
76
Uva Petit Verdot (extracto de roble y extracto de lavandín)
(maduración óptima)
Recolección a mano
Adición de SO2
(10 g/100 kg de uva)
Despalillado/Estrujado manual
Llenado de los tubos de metacrilato
(cada tratamiento dos tubos de 5 l)
Siembra de levaduras S. cerevisiae de la cepa QA23 (20 g/hl)
(temperatura controlada 23±1ºC)
Finalización de fermentación alcohólica
(<2,5 g/l azúcares)
Prensado manual
(mezcla del vino prensa con el flor)
Reposo y eliminación de lías
Siembra de bacterias comerciales Oenococcus oeni Vinoflora CH16 (10 mg/l)
(temperatura controlada 20-25ºC)
Finalización de fermentación maloláctica
(ácido málico ≈0,4 g/l y ácido láctico estable )
Adición de SO2
(SO2 libre corregido a 25-35 mg/l)
Conservación en botellas a 14ºC
(extractos de roble durante 8 meses y extracto de lavandín a durante 6 meses)
Figura 23. Esquema del proceso de vinificación en tinto.
77
El vino se dejo en reposo durante 4 días y se eliminaron las lías. La
fermentación maloláctica se indujo por la siembra de bacterias comerciales
Oenococcus oeni (Vinoflora CH16, Chr Hansen, Buenos Aires), en la dosis
recomendada por el proveedor, 10 mg/l. La fermentación maloláctica se realizó
en el mismo fermentador pero en tubos de 1,5 l a una temperatura controlada de
20-25ºC. Esta fermentación fue monitorizada mediante medidas diarias de la
concentración de los ácidos málico y láctico. Se consideró terminada cuando la
concentración de ácido málico fue aproximadamente 0,4 g/l, y la de ácido láctico
se mantenía estable. Al final de la fermentación maloláctica, la concentración de
SO2 libre fue corregida a 25-35 mg/l. Los vinos procedentes de las viñas tratadas
con los extractos de roble fueron almacenados en botellas a 14ºC durante 8
meses, y los vinos procedentes de los tratamientos con el extracto de lavandín se
almacenaron en botellas a 14ºC durante 6 meses. Todos los pasos quedan
reflejados en la Figura 23.
De cada uno de los vinos se tomaron muestras al final de fermentación
alcohólica, cuando terminó la fermentación maloláctica y después de permanecer
en botella 8 y 6 meses, congelándolas a -20ºC hasta su posterior análisis. El
análisis sensorial se hizo en las muestras recién tomadas, sin congelar.
6.4. Métodos de análisis
6.4.1. Parámetros enológicos
Los parámetros enológicos medidos en uvas y vinos fueron:
Uvas: ºBaumé, alcohol probable, acidez total (g/l de ácido tartárico),
nitrógeno asimilable y pH fueron analizados siguiendo los métodos establecidos
por la UE (D.O.C.E. 1990). En el caso de las uvas procedentes de los
tratamientos realizados en Francia el nitrógeno amínico y el nitrógeno asimilable
fueron medidos según el método de Dubernet et al., (2001). El rendimiento por
78
planta fue obtenido dividiendo la masa total de producción (kg) por el número de
plantas, también fue determinado el peso de 100 bayas, el volumen (ml) y el
calibre de las uvas (calibre digital, Classic Tesa, Suiza), y el % de masa de
vendimia. Todas estas medidas se hicieron en cada muestra por triplicado.
Vinos: grado alcohólico, acidez total (g/l de ácido tartárico), acidez
volátil (g/l de ácido acético), pH, azúcares reductores, antocianos totales e
intensidad colorante fueron analizados siguiendo los métodos establecidos por la
UE (D.O.C.E. 1990). Todas estas medidas se hicieron en cada muestra por
triplicado, además en el caso de los dos trabajos de la variedad Petit Verdot
(extracto de roble y extracto de lavandín) como las fermentaciones se hicieron
por duplicado los resultados son el promedio de seis valores.
6.4.2. Análisis de azúcares y ácidos
El análisis de la glucosa, la fructosa y el acido tartárico en uvas y de los
ácidos málico y láctico en los vinos se realizó en un HPLC (Agilent 1100, Palo
Alto, EE.UU.) provisto de un detector de índice de refracción (Agilent 1200). La
separación cromatográfica se realizó en una columna PL Hi-Plex H (Varian,
Middelburg, Holanda) con un tamaño de partícula de 8 µm (300 x 7.7 mm). La
fase móvil fue ácido sulfúrico 0,004 M con un flujo de 0,4 ml/min. La
temperatura del horno se mantuvo a 75ºC y el detector a 55ºC. Las uvas fueron
estrujadas y el mosto obtenido fue diluido con agua al 50%, pasado por un filtro
de PTFE (0,45 µm de poro, Millipore, Alemania) e inyectado en la columna. Los
vinos únicamente fueron filtrados antes de la inyección. El volumen de muestra
inyectado fue de 10 µl. El tiempo total de análisis fue de 50 minutos. La
identificación se basó en los tiempos de retención y la cuantificación se hizo a
partir de rectas de calibrado (R2 > 0,97) de las disoluciones acuosas de los
patrones comerciales de cada uno de los compuestos (Sigma-Aldrich, Madrid,
España). Las medidas en uvas se hicieron en triplicado, en vinos también se
79
hicieron por triplicado pero como las fermentaciones se hicieron por duplicado
los resultados son el promedio de seis valores.
6.4.3. Análisis de aminoácidos y amonio en uvas
El análisis de los aminoácidos y del amonio de las uvas se hizo
empleando el método de Garde-Cerdán et al., (2009). La derivatización de los
aminoácidos y del amonio se llevó a cabo por reacción de 1,75 ml de tampón
borato 1 M (pH = 9), 750 µl de metanol, 1 ml de la muestra a analizar
(previamente filtrada), 20 µl de estándar interno (ácido 2-aminoadípico, 1 g/l) y
30 µl de derivatizante (etoximetilenmalonato de dietilo, EMMDE). La reacción
de derivatización se realizó en un baño de ultrasonidos durante 30 minutos.
Posteriormente, la muestra se calentó en estufa durante 2 horas a 70-80ºC para la
completa degradación del exceso de EMMDE. El análisis de los aminoácidos y
del amonio, una vez derivatizados, se llevó a cabo en el cromatógrafo líquido de
alta resolución Agilent 1100 (Palo Alto, EE.UU.) ya mencionado, provisto de un
detector de fotodiodos alineados (DAD). La separación cromatográfica se realizó
en una columna ACE HPLC (C18-HL) (Aberdeen, Escocia) con un tamaño de
partícula de 5 µm (250 mm x 4,6 mm), empleando el gradiente que se muestra en
la Tabla 7 (fase A, 25 mM de tampón acetato, pH = 5,8, con 0,4 g de azida de
sodio; fase B, mezcla de acetonitrilo y metanol 80:20 (v/v)). El flujo fue de 0,9
ml/min. Para la detección se emplearon las longitudes de onda, 280, 269 y 300
nm. El volumen de muestra inyectado fue de 50 µl. Las medidas de aminoácidos
y del amonio de las diferentes muestras se hicieron por triplicado.
Tabla 7. Gradiente de fase móvil empleado en el análisis de los aminoácidos y amonio por
HPLC.
Tiempo (min)
0
20
30
30,01
31
31,01
39,51
50
58
63
67
70
75
78
Fase A (%)
90
90
83
91
81
80,5
77
70,6
28
18
0
0
90
90
Fase B (%)
10
10
17
19
19
19,5
23
29,4
72
82
100
100
10
10
80
Los compuestos analizados fueron el amonio y los siguientes 17
aminoácidos: ácido aspártico, ácido glutámico, serina, histidina, glicina, treonina,
arginina, alanina, metionina, valina, cistina, lisina, prolina, tirosina, isoleucina,
leucina y fenilalanina. La identificación se realizó utilizando los tiempos de
retención y los espectros UV-vis de los correspondientes estándares
derivatizados. La cuantificación se hizo a partir de las rectas de calibrado (R2 >
0,98) de los respectivos patrones comerciales (Sigma-Aldrich, Madrid, España)
en HCl 0,1 N, a los que se sometió al mismo proceso de derivatización que a las
muestras.
6.4.4. Extracción de los compuestos volátiles en uvas
Se utilizó la técnica denominada HS-SBSE que se puso a punto en este
trabajo por primera vez. Posteriormente los volátiles se determinaron por GCMS. Se usó un vial específico denominado “twister-headspace” que tiene un
soporte para colocar el twister en el espacio de cabeza de la muestra. Con la
ayuda de una batidora se trituraron las uvas sin romper las pepitas y se
maceraron durante 2 horas a temperatura ambiente en su propio jugo. El método
empleado necesitó ser optimizado previamente en cuanto al tipo de separación
del liquido de las partes sólidas (proceso de colado y centrifugado), en cuanto al
volumen de vial (20 ml y 50 ml), y las temperaturas de extracción (40ºC y 60ºC).
Los mejores resultados se consiguieron con uvas centrifugadas, utilizando
un vial de 50 ml y a una temperatura de 60ºC. El método consistió por tanto en
pesar 250 g de uvas, triturarlas y macerarlas en las condiciones anteriormente
descritas, colar el macerado y centrifurarlo a 3000 rpm durante 30 min.
Posteriormente, se colocó 22 ml de muestra en un vial de 50 ml y se le añadió
0,1 g de NaCl por ml de muestra y 10 µl por cada ml de muestra de γhexalactona (patrón interno preparado en una concentración de 1 µl/ml en etanol
absoluto). En el inserto del espacio de cabeza se colocó un twister de 1 cm
81
(Gerstel, Mülheim y der Ruhr, Alemania), recubierto del absorbente
polidimetilsiloxano y se cerró herméticamente. El vial se introdujo en una estufa
a la temperatura de 60ºC y se agitó durante 1 hora a 500 rpm. Posteriormente el
twister se sacó, se lavó con agua Milli-Q, se secó con un papel de celulosa y se
introdujo en el tubo de desorción térmica para su posterior análisis por GC-MS.
Este método de extracción también fue aplicado para los análisis de los extractos
de roble. Todas las extracciones se hicieron por triplicado. La determinación de
los volátiles extraídos se realizó según se indica posteriormente en el apartado
6.4.7.
6.4.5. Análisis de precursores aromáticos glicosídicos en uvas
Unos 500 g de uva fueron descongelados, triturados con una batidora sin
romper las pepitas, y la masa resultante fue colada antes de ser centrifugada a
7000 rpm a 10ºC durante 20 min. El jugo fue agitado durante 20 minutos con
5g/100 ml en el caso de uvas tintas y con 1g/100 ml en el caso de uvas blancas
de polivinilpolipirrolidona (PVPP, Sigma Aldrich, Francia), y posteriormente
pasada por un filtro de 5 µm de celulosa (Fisherbrand, UK). El jugo se dividió en
tres fracciones de 100 ml, y cada una de ellas fue eluída por un cartucho de SPE
(Strata-X 33u polímero de fase inversa, 500mg/6ml; Phenomenex, EE.UU.)
previamente activado y acondicionado siguiendo las instrucciones del proveedor.
Posteriormente estos cartuchos fueron enjuagados y secados a vacio. La fracción
de precursores glicosídicos se eluyó con 10 ml de acetonitrilo/metanol (5:5, v/v)
y se llevó a sequedad a 45ºC en un rotavapor, y el residuo se diluyó con 1 ml de
tampón fosfato/citrato (dihidrógeno fosfato de sodio 0,2 M/ácido cítrico 0,1 M,
pH 5).
La hidrólisis enzimática de la fracción glicosídica se llevo a cabo con 100
µ/l de disolución de fosfato/citrato con 70 mg/ml de pectinasa AR 2000 (DSM,
Delft, Holanda), en una estufa a 35ºC, durante 16 h (Günata et al. 1985). Las
82
agliconas volátiles liberadas fueron extraídas con 5 ml de un azeótropo de
pentano/diclorometano (2:1, v/v). Después se añadió 200 µl de una solución de
16 mg/l de 4-nonanol (Merck, Germany) en etanol absoluto (patrón interno) y la
disolución resultante se concentró a 40ºC usando una columna Vigreux. Esta
extracción líquido-líquido (LLE) se empleó también en el caso de los extractos
de roble.
La identificación y cuantificación de las agliconas liberadas de la fracción
aromática glicosídica de uvas y de los compuestos volátiles del extracto de roble
utilizado en las experiencias realizadas en Francia, se realizaron en un
cromatógrafo de gases (Agilent 6890, EE.UU.) acoplado a un espectrómetro de
masas de la misma serie, dotado con un cuadripolo. La unidad estaba equipada
con una columna capilar de sílice fundida (30 m longitud, 0,25 mm diámetro
interno y 0,5 µm de espesor, DB-Wax, J & W Scientific, Reino Unido),
conectada al inyector a través de una pre-columna de sílice desactivada (1 m
longitud y 0,53 mm diámetro interno, J & W Scientific, Reino Unido). La
temperatura del inyector se mantuvo a 245ºC y se inyectaron 2 µl de muestra. El
programa cromatográfico se inició a 60ºC durante 3 min hasta 250ºC, con una
rampa 3ºC/min, y se mantuvo esta temperatura durante 10 min. La temperatura
de la línea de transferencia se fijó a 250ºC, la de la fuente se mantuvo a 250ºC y
la del cuadrupolo a 150ºC. El impacto electrónico (EI) se realizó a 70 eV en el
rango de masas 29-350 m/z. La identificación se realizó utilizando la librería
desarrollada por el INRA y la NIST y la cuantificación de cada compuesto se
hizo como equivalentes de 4-nonanol. Los compuestos analizados fueron los
relacionados con el extracto de roble: cis-whisky lactona, trans-whisky lactona,
eugenol, 6-metoxieugenol, guayacol, 4-vinilguayacol, 3,4,5-trimetoxifenol,
siringol, siringaldehído, vainillina, acetovainillona, propiovanillona, vainillol,
vainillato de etilo y ácido homovainíllico, y otros no presentes en el extracto pero
originales de las uvas, 4 compuestos C6, 2 alcoholes, 9 terpenos, 5 fenoles y 4
83
norisoprenoides. Cada medida procedió de tres extracciones diferentes de una
misma muestra de uva.
6.4.6. Extracción de los compuestos volátiles de los vinos
Los compuestos volátiles de los vinos procedentes de los tratamientos con
los extractos de roble se extrajeron según el método descrito por Marín et al.
(2005) y en el caso de los vinos procedentes de los tratamientos con el hidrolato
de lavandín según los método descritos por Zalacain et al. (2007) y Oliva et al.,
(2008). Estos métodos se basan en la técnica SBSE empleando el twister por
inmersión y posteriormente analizados mediante GC-MS. Los compuestos se
extrajeron introduciendo un twister de 1cm en un volumen determinado de
muestra, a la que se le añadió patrón interno. Así, en el caso de los vinos
procedentes de los tratamientos con extracto de roble, el twister se introdujo en
10 ml de vino al que se le añadio 100 µl de γ-hexalactona en etanol absoluto (1
µl/ml). En el caso de los vinos procedentes de los tratamientos con el extracto de
lavandín y del análisis del propio extracto, al cual se le aplicó el mismo método,
el twister se introdujo en 25 ml de muestra (vino o extracto de lavandín), a la que
se le añadió, como patrón interno, 62,5 µl de una disolución procedente de la
mezcla de 1 µl/ml de γ-hexalactona y de 1 µl/ml 3-metil-1-pentanol. Después de
agitar la muestra a 500 rpm a temperatura ambiente durante 60 minutos, el
twister se sacó, se lavó con agua destilada, se secó con un papel de celulosa y se
introdujo en un tubo de desorción térmica para su posterior análisis por GC-MS.
Las extracciones de los vinos procedentes de los tratamientos con los extractos se
realizaron por triplicado en el caso de la variedad Verdejo (n=3), y en el caso de
la variedad Petit Verdot, como además las vinificaciones se hicieron por
duplicado, los resultados para cada compuesto fueron la media de seis (n=6). Las
extracciones de los vinos Petit Verdot procedentes del hidrolato de lavandín se
hicieron por duplicado, y como las fermentaciones se realizaron también por
84
duplicado, los resultados para cada compuesto fueron la media de 4 análisis
(n=4).
6.4.7. Análisis de los compuestos volátiles por cromatografía
gaseosa y espectrometría de masas (GC-MS)
6.4.7.1 Extracto de roble y uvas y vinos procedentes de su tratamiento
La desorción de los compuestos volátiles extraídos por el twister se llevó
a cabo en un equipo de desorción térmica (ATD 400, Perkin Elmer, Estados
Unidos) a 330ºC durante 4 minutos con un flujo de helio de 45 ml/min. Los
compuestos, una vez desorbidos, pasaron a través de una línea de transferencia
mantenida a 330ºC al cromatógrafo de gases (Hewlett-Packard, Palo Alto,
Estados Unidos) que estaba provisto de una columna capilar de sílice fundida
BP21 (SGE, Ringwood, Australia) de 30 m de longitud, 0,25 mm de diámetro, y
0,25 µm de espesor. Las condiciones cromatográficas fueron: temperatura inicial
de 40ºC, que se mantuvo durante 5 minutos, posteriormente se elevó a 150ºC
(5ºC/min) y finalmente se aplicó una rampa de 10ºC/min hasta alcanzar 230ºC,
que se mantuvo durante 15 minutos. El tiempo total de análisis fue de 40
minutos. La detección se hizo por espectrometría de masas por impacto
electrónico a 70 eV, y la detección se hizo por selección de masas iónicas (modo
SIM). Las masas iónicas elegidas para cada uno de los compuestos fueron: ciswhisky lactona 99, trans-whisky lactona 99, furfural 96, 5-metilfurfural 110,
eugenol 164, 6-metoxieugenol 194, guayacol 124 y 109, 4-vinilguayacol 135 y
150, 4-etilguayacol 137 y 152, 4-etilfenol 107 y 122, vainillina 151,
acetovainillona 151 y 166, vainillato de metilo 151 y 182 y siringol 154 y 139.
Estos compuestos fueron elegidos por ser los que forman parte de los extractos
de roble. La temperatura del detector fue de 150ºC. La identificación se realizó
utilizando la librería NIST del cromatógrafo y la realizada por nuestro grupo, así
como por comparación con los espectros de masas de los patrones comerciales
85
(Sigma-Aldrich) y con sus tiempos de retención. Se realizaron 2 calibraciones,
una para la extracción por espacio de cabeza (HS-SBSE), y otra para la
extracción por inmersión (SBSE). Para la cuantificación se utilizaron rectas de
calibrado que se obtuvieron a partir de disoluciones patrón en vino sintético
(12% de etanol (v/v) a pH 3,6 con ácido tartárico) a cinco concentraciones
diferentes obteniéndose valores de R2 > 0.9 tanto para espacio de cabeza como
para inmersión.
6.4.7.2. Vinos procedentes de las viñas tratadas con el extracto de
lavandín
La desorción y posterior análisis cromatográfico se realizaron en un
equipo compuesto por un MultiPurpose Sampler (Gester, Estados Unidos)
acoplado a un cromatógrafo de gases (Agilent 7890A CG-5975 insertado en un
XL MDS Agilent, Little Falls, Estados Unidos). La desorción de los compuestos
volátiles absorbidos en el twister se llevó a cabo a 330ºC durante 4 minutos con
un flujo de helio de 45 ml/min. Los compuestos, una vez desorbidos, pasaron a
una columna capilar de sílice fundida BP21 (SGE, Ringwood, Australia) de 50 m
de longitud, 0,22 mm de diámetro, y 0,25 µm de espesor. Las condiciones
cromatográficas fueron: temperatura inicial de 40ºC, que se mantuvo durante 2
minutos, posteriormente se elevó hasta 150ºC a 10ºC/min y se mantuvo durante 5
minutos y finalmente se aplicó una rampa de 10ºC/min hasta alcanzar 230ºC, que
se mantuvo durante 2 minutos. El tiempo total de análisis fue de 28 minutos. La
detección se hizo por espectrometría de masas por impacto electrónico a 70 eV y
la detección se hizo en SCAN con un rango de masas iónicas comprendido entre
35 a 500 uma, y la temperatura del detector fue de 150ºC. Los compuestos
analizados fueron: 21 ésteres, 5 ácidos, 9 terpenos, 3 fenoles, 11 alcoholes, 2
lactonas y 5 aldehídos. La identificación se realizó utilizando la librería NIST del
cromatógrafo y la realizada por nuestro grupo, así como por comparación con los
espectros de masas de los patrones comerciales (Sigma-Aldrich) y con sus
86
tiempos de retención. Para la cuantificación se utilizaron rectas de calibrado que
se obtuvieron a partir de disoluciones patrón en vino sintético (12% de etanol
(v/v) a pH 3,6 con ácido tartárico) a cinco concentraciones diferentes
obteniéndose valores de R2 > 0,97. Cuando los patrones comerciales no estaban
disponibles se hizo un análisis semi-cuantitativo utilizando la recta de calibrado
del compuesto más similar.
6.4.8. Análisis sensorial de los vinos
Un panel de 8 jueces expertos (3 mujeres y 5 hombres) entre 25 y 50 años
realizó el análisis sensorial de los vinos. Cada uno de los vinos fue catado en
cada uno de los momentos de su evolución, es decir, en Verdejo el análisis se
realizó tras la fermentación alcohólica y tras 6 meses de esta; en la variedad Petit
Verdot procedente de los extractos de roble se hizo tras las fermentaciones
alcohólica y maloláctica y tras 8 meses en botella, y por último, en la variedad
Petit Verdot procedente de los tratamientos con el hidrolato de lavandín se llevo
a cabo después de fermentaciones alcohólica y maloláctica y al cabo de 6 meses
de permanencia en botella. Cada parámetro se puntuó en función de su
intensidad, en una escala de 1 a 7, siendo 1 el valor de menor intensidad y 7 el de
mayor intensidad. La cata consistió en tres fases:
Visual: puntuándose la intensidad de color, los tonos amarillos y verdes
en los vinos de la variedad Verdejo y la intensidad de color, los tonos azules,
rojos y amarillos en los vinos de la variedad Petit Verdot (tanto los procedentes
de los tratamientos con los extractos de roble como con el extracto de lavandín).
Olfativa: puntuándose la intensidad olfativa, las notas aromáticas
fermentativas, varietales, frutales, frutas exóticas, florales, hierba, madera y
especiado en los vinos de la variedad Verdejo y de la variedad Petit Verdot
procedentes de los tratamientos con los extractos de roble. En los vinos Petit
Verdot procedentes del tratamiento con el extracto de lavandín se valoró la
87
intensidad olfativa, las notas aromáticas fermentativas, varietales, frutales, fruta
madura, florales, lavanda y mentoladas.
Gustativa: puntuándose el volumen en boca, la acidez, el amargor, la
astringencia, la persistencia y el equilibrio en todos los vinos.
6.4.9. Análisis estadístico
El tratamiento estadístico de los datos se realizó mediante el programa
SPSS con la versión 17,0 para Windows en los trabajos de la variedad Verdejo, y
con versión 19,0 en los demás trabajos (SPSS, Chicago, Estados Unidos). Se
hicieron análisis de la varianza (ANOVA) para comparar las diferencias entre
medias con el test LSD para una probabilidad del 0,05%. Este análisis de
varianza se le aplicó a los parámetros enológicos de uvas y vinos, a los
compuestos volátiles de uvas y vinos, a las agliconas procedentes de la hidrólisis
ácida de los glicósidos y a los atributos sensoriales con el fin de comparar
diferencias de cada uno de ellos entre las muestras procedentes de los
tratamientos y las muestras control, y también se utilizó para ver diferencias en la
evolución de una misma muestra con el tiempo. También se hicieron análisis
discriminantes para cada variedad con el fin de observar si se podían diferenciar
las uvas y vinos control de sus respectivos tratamientos, así como si las muestras
de las diferentes aplicaciones se separaban de otras aplicaciones. Para llevar a
cabo estos análisis discriminantes en la variedad Verdejo se utilizaron los
resultados de los compuestos volátiles así como los resultados del análisis
sensorial, en los demás trabajo únicamente se utilizaron los resultados de los
compuestos volátiles o de las agliconas procedente de la hidrólisis ácida.
88
7.ARTÍCULOS CIENTÍFICOS
ARTÍCULOS CIENTÍFICOS
SCIENCE PAPERS
Artículos cientifícos
En este apartado se presentan los artículos científicos con los que se ha difundido
el avance del conocimiento adquirido en este trabajo de Tesis Doctoral. Los
cuatro primeros abordan el efecto de la aplicación de los extractos de roble sobre
las viñas de variedades blancas y tintas y el quinto aborda el efecto de la
aplicación del extracto de lavandín (hidrolato) sobre viñas de una variedad tinta.
El artículo I “Effect of an oak extract applied to 'Verdejo' vineyard on
grape composition”, publicado en Acta Horticulturae, estudia la repercusión de
la aplicación de un extracto de roble en viñas Verdejo sobre la composición de la
uva evaluada mediante sus parámetros enológicos y su composición en
aminoácidos y amonio.
El artículo II “Effect of oak extract application to Verdejo grapevines
on grape and wine aroma”, publicado en Journal of Agricultural and Food
Chemistry, aborda los efectos de la aplicación del extracto de roble sobre las
vides de la variedad blanca Verdejo en la composición volátil de sus uvas y de
sus respectivos vinos.
El artículo III “Applications of an oak extract on Petit Verdot
grapevines. Influence on grape and wine volatile compounds”, publicado en
Food Chemistry estudia el efecto de la aplicación de extractos de roble en vides
de la variedad tinta Petit Verdot sobre la composición volátil de las uvas y de sus
vinos.
El artículo IV “Glycosidic Aroma Precursors of Syrah and
Chardonnay Grapes after an Oak Extract Application to the Grapevines”,
publicado en Food Chemistry, se realizó, utilizando las variedades Syrah y
Chardonnay, para corroborar la hipótesis lanzada en los artículos II y III sobre la
asimilación de los compuestos volátiles del extracto de roble y la glicosilación de
estos. Además, para comprobar si otros compuestos no presentes en los
extractos, pero sí habituales en uvas, se veían afectados por la aplicación.
91
Artículos cientifícos
El artículo V “Lavandin Hydrolat Applications to Petit Verdot
Vineyards on their Wines Aroma Compounds”, enviado para su publicación a
una revista SCI (Science Citation Index), estudia el efecto de la aplicación del
extracto de lavandín (hidrolato) sobre las viñas de la variedad tinta Petit Verdot
en el aroma de sus vinos (fermentativos y varietales).
92
7.1. ARTÍCULO I
Proc. XXVIIIth IHC – IS Viti&Climate: Effect of Climate Change on
Production and Quality of Grapevines and Their Products
Eds.: B. Bravdo and H. Medrano
Acta Hort. 931, ISHS 2012
Este primer artículo se centró en estudiar si la aplicación foliar de un
extracto comercial de roble a vides de la variedad blanca Verdejo afectaba a la
composición de la uva. Con este fin, un extracto acuoso de roble francés fue
aplicado en envero mediante pulverización sobre la parte foliar de la vid,
realizándose 3 tratamientos diferentes: 25%(1) (una aplicación de extracto
diluido al 25 %), 25%(4) (cuatro aplicaciones de extracto diluido al 25 %), 100
% (una aplicación del extracto concentrado) y un control (sin tratar). Las uvas
fueron vendimiadas en el momento óptimo de acuerdo con el estudio de la
maduración y se analizaron los siguientes parámetros enológicos: rendimiento,
peso de 100 bayas, ºBaumé, alcohol probable, acidez total, pH, glucosa, fructosa,
ácido tartárico, aminoácidos y amonio.
93
Artículo I
Se observó que el tratamiento 25%(4) afectó a la mayoría de los
parámetros enológicos analizados, y proporcionó el mayor rendimiento por
planta, menor peso de 100 bayas y menores concentraciones de glucosa y
fructosa. Esto pudo ser debido a que este tratamiento, al ser el único que se
aplicó en cuatro ocasiones, indujera más estrés a las plantas, ya que se realizó a
lo largo del envero y durante este periodo el metabolismo de los azúcares es más
activo y se producen los mayores cambios en las características estructurales y
químicas de los hollejos.
Además, todas las aplicaciones del extracto de roble a la vid afectaron a
la concentración de aminoácidos totales de las uvas, siendo el tratamiento
25%(1) el que produjo la menor concentración. Los tratamientos 25%(4) y 100%
disminuyeron el contenido de aminoácidos totales de las uvas en la misma
medida. Sin embargo, la concentración de amonio solamente se vio afectada por
el tratamiento 25%(4). La mayor parte de los aminoácidos estudiados se
encontraron en menor concentración en las uvas de las viñas tratadas que en las
procedentes de las cepas control, a excepción de la tirosina que aumento en las
uvas de todos los tratamientos. Los aminoácidos son una buena fuente de
nitrógeno para los microorganismos, levaduras y bacterias, y se consideran
precursores de algunos de los compuestos aromáticos del vino, en especial los
fermentativos, por lo que influyen en el aroma del vino de una manera decisiva.
Por lo tanto, con este primer trabajo, se pudo observar que la aplicación
foliar de extractos comerciales de roble a viñas de la variedad Verdejo afectan a
la calidad de las uvas, influyendo en sus parámetros enológicos y en su
composición nitrogenada.
94
Acta Horticulturae 931, ISHS 2012
Effect of an Oak Extract Applied to ‘Verdejo’ Vineyard
on Grape Composition
Ana M. Martínez-Gila, Teresa Garde-Cerdána, Laura Martínezb, Gonzalo L. Alonsoa, M. Rosario
Salinasa,*
a
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario,
02071 Albacete, Spain. *E-mail: [email protected]
b
Dehesa de Los Llanos, Carretera de las Peñas de San Pedro, km 5.5, 02006 Albacete, Spain
ABSTRACT
The aim of this work was to study the influence of a commercial oak extract on the
oenological parameters and nitrogen composition of ‘Verdejo’ grapes. The
aqueous extract solution was applied at veraison by spraying over grapevines
according to four different treatments: 25%(1), 25%(4), 100% and control.
Glucose, fructose, tartaric acid, amino acids and ammonium were analysed by
HPLC and the other oenological parameters were analysed according to the
official methods. The results obtained showed that the different treatments did not
affect the titratable acidity and pH of the grapes. The 25%(4) treatment affected
the other oenological parameters (weight of the 100 berries, °Baumé, probable
alcohol) more than 25%(1) and 100% treatments. In addition, grapes from 25%(4)
treatment presented the lowest content in tartaric acid, glucose and fructose;
moreover, all treatments decreased grape nitrogen content, especially the 25%(1).
Consequently, the extract application to the grapevine affect the grape
composition; therefore it will affect the final wine quality, as nitrogen compounds
are precursors of wine aroma compounds.
INTRODUCTION
Research groups have studied the effect on grape and wine composition in
function of different treatments applied to vineyards such as pesticides and other plant
extracts; so, the grapes can be sensitive to the application of oak extracts, this could
influence grape chemistry composition (Oliva et al., 2008; Carmona et al., 2001). There
are available commercial aqueous toasted-oak extracts that can be applied to the plant,
thus modifying the chemistry composition of the grape and wine, as it has been
observed on the aroma composition (Martínez-Gil et al., 2011). Sugars and acids are the
principal components of the grapes, which participate in the alcoholic fermentation, and
so they are directly related with to the oenological capacity, especially glucose, fructose
a
[email protected]
Proc. XXVIIIth IHC – IS Viti&Climate: Effect of Climate Change on
Production and Quality of Grapevines and Their Products
Eds.: B. Bravdo and H. Medrano
Acta Hort. 931, ISHS 2012
339
and tartaric acid. It is well known that the amino acids found in grapes are important as
nutrients for the growth of yeasts, and play a role as precursors of wine aroma
compounds (Bell and Henschke, 2005; Garde-Cerdán et al., 2011). Moreover, the amino
acid composition of the grape is closely related to different vineyard fertilization
strategies (Callejón et al., 2010). Consequently, the aim of this work was to study the
impact of toasted-oak extracts application on grape composition. For this, different
treatments were applied on white Vitis vinifera ‘Verdejo’ grapevines, and the glucose,
fructose, tartaric acid and amino acids content of grapes were studied at harvest.
MATERIAL AND METHODS
Plant Extract
The plant extract chosen for this study was provided by Protea (Gensac la
Pallue, France). This extract is a food additive utilized in spirits and fruit juices, which
guarantees that no toxicity risk exists. The absence of phytotoxicity was validated on
Solanum lycopersicum ‘Micro Tom’ tomato (INRA, Montpellier, France) a cultivar
used as a fast growing plant, it did not show any negative symptomatologic effects;
before treatment of the grapevines.
Grapevine Treatments
White grapes from Vitis vinifera ‘Verdejo’ cultivated in La Mancha (Albacete,
southeast of Spain) during 2009 were used. Different oak extract treatments were
applied to the grapevines during veraison. The vineyards were treated with different
concentrations of the oak extract, a 0.5 ml per litre of adjuvant wetting agent Fluvius
(BASF, Germany) was added to all treatments. In treatments 25%(1) and 25%(4) the
extract was diluted with 75% (v/v) of water. The 25%(1) was applied once at 7 days
postveraison and the 25%(4) was applied four times at 4, 7, 10, and 13 days postveraison. In treatment 100% the extract was applied undiluted once at 7 days postveraison. For each treatment, rows of 188 plants were used, with 2 untreated rows
between the different treatments to avoid contamination. A row of 188 plants was not
treated and served as control. Approximately 230 ml of each formulation was applied
per plant by spraying. The grapes were harvested on 27 August, samples were taken at
random and frozen (-20°C) to analyse later.
Chemical Analysis
°Baumé, probable alcohol, titratable acidity and pH were measured following
the methods established by ECC (1990). The analysis of tartaric acid, glucose and
fructose was done by HPLC (Agilent, Palo Alto, USA) with refractive index detector
(RID). The mobile phase was 0.004 M H2SO4, 0.4 ml/min, 75°C on a PL Hi-Plex H
column (Varian, Middelburg, The Netherlands). 10 l of the samples were directly
injected. The RID was at 55°C and time analysis was 50 min. The analysis of amino
340
acids and ammonium of grape were made using the method described by Garde-Cerdán
et al. (2009).
Statistical Analysis
Data statistical analysis was performed using SPSS Version 17.0. Data were
analyzed statistically using the ANOVA test. Differences between means were
compared using LSD test at 0.05 probability level.
RESULTS AND DISCUSSION
Oenological Parameters
Table 1 shows the yield and oenological parameters of the different grapes. The
treatment that produced the highest fruit yields was the 25%(4), although it had the
lowest weight for 100 berries. Grapes from 25%(4) treatment presented the lowest
°Baumé, and consequently the lowest probable alcohol. Titratable acidity was between
4.2 g/L for 25%(1) and 5.0 g/L for 25%(4), so the lowest °Baumé/titratable acidity ratio
was found in 25%(4) grapes. Hence, the application of oak extract to the vineyard
affected the oenological parameters of the grapes, except the pH, only when it was
repeatedly applied. Probably, the 25%(4) treatment induced plant stress. Since the plant
was sprayed four times with the formulation, this could cause a shield effect, when the
sugar assimilation and the changes in the skin characteristics occur, moreover chemical
and structural characteristics of grape cell walls also change during this period (Conde
et al., 2007).
Table 1. Oenological parameters in grapes after oak extracts application to the grapevine.
Probable
Titratable
Yield
Weight
ºBaumé
Treatments (kg/plant)
ºBaumé
alcohol
acidity
100 berries
/TA
(v/v, %)
(g/l)
Control
3.93
100.7±0.2b 12.4±0.1b 13.0±0.1bc
4.5±0.1a
2.8±0.1b
25%(1)
3.26
112.7±0.8b 12.2±0.2b
12.6±0.1b
4.2±0.2a
2.9±0.1b
25%(4)
4.72
95.4±0.3a 10.8±0.1a
10.8±0.2a
5.0±0.3a
2.2±0.1a
100%
3.90
119.3±0.6b 12.4±0.0b 13.0±0.1bc
4.4±0.2a
2.8±0.2b
pH
3.6±0.1a
3.6±0.0a
3.5±0.2a
3.6±0.0a
TA: Titratable acidity. The oenological parameters are given with their standard derivation (n=3). Different letters
indicate significant differences between the treatments (p<0.05).
Tartaric Acid, Glucose and Fructose
Table 2 shows the tartaric acid, glucose and fructose content in grapes. The
concentration of tartaric acid in the different grapes was not influenced by the
treatments. It ranged between 3.60 and 3.91 g/L for 25%(4) and 25%(1), respectively.
The concentration of total sugars was between 213 and 255 g/L for 25%(4) and 25%(1),
respectively, found inside the usual range. In all samples, the fructose content was 6.5 to
8.5% higher than glucose content and the glucose/fructose ratio was in the range (0.740.97) found by Kliewer (1967) for different grape cultivars. The concentration of these
341
compounds in 25%(4) grapes was the lowest probably due to the plant stress after the
repetitive treatment application.
Table 2. Tartaric acid, glucose, fructose and glucose/fructose ratio in grapes after oak extracts application
to the grapevine
Treatments
Control
25%(1)
25%(4)
100%
Tartaric acid
(g/l)
3.73±0.13a
3.90±0.07a
3.59±0.02a
3.76±0.12a
Glucose
(g/l)
119.58±0.27b
122.04±1.04b
102.43±0.10a
117.64±0.18b
Fructose
(g/l)
127.99±0.27b
133.03±1.04b
110.88±0.10a
127.30±0.18b
Glucose/fructose
0.93
0.91
0.92
0.92
All parameters are given with their standard deviation (n=3). Different letters indicate significant differences between
the treatments (p<0.05).
Amino Acids and Ammonium
Table 3 shows the concentration of amino acids and ammonium in the different
grapes. Extract treatments decreased the total amino acid content in all samples, being
the lowest concentration in 25%(1) grapes. This treatment presented the lowest
concentration of six amino acids (aspartic acid, arginine, lysine, proline, tyrosine, and
phenylalanine) and ammonium. Grapes from 25%(4) and 100% treatments were not
significantly different regarding total amino acid content. The ammonium concentration
in grapes was only affected by the 25%(4) treatment, showing the highest content. This
treatment also presented the highest concentration of arginine, aspartic acid, and
methionine, as well as the lowest concentration of glutamic acid, alanine, valine,
cystine, and phenylalanine. The glycine and threonine were not affected by the different
oak treatments. Therefore, tyrosine concentration, in all cases, increased after extract
application. However, serine, histidine, arginine, valine, isoleucine, leucine, and
phenylalanine content, good nitrogen sources for the yeasts, were found in lower
concentration levels in the grapes treated with extract than in control grapes. The 100%
treatment showed higher concentration of these compounds than the grapes of the other
oak extract treatments, with the exception of arginine. This change in the grape amino
acid content could affect the wine volatile composition (Bell and Henschke, 2005), as
they are precursors of fermentative volatile compounds, i.e., phenylalanine of 2phenylethanol, leucine of 3-methyl-1-butanol, isoleucine of 2-methyl-1-butanol, valine
of isobutanol, tyrosine of tyrosol, and methionine of methionol.
CONCLUSIONS
The oak extract application to grapevine affected the grape quality. The greatest
effect on oenological parameters was found in 25%(4) treatment, and grapes showed the
lowest tartaric acid, glucose and fructose concentrations. All the oak extract treatments
decreased the grape amino acid content, compounds that are wine aroma precursors,
specially the 25%(1) treatment.
342
Table 3. Amino acids and ammonium concentration (mg/l) in grapes after oak extracts application to the
grapevine
Control
25%(1)
25%(4)
100%
Total amino acids
773.7±3.0c
604.4±3.2a
656.4±26.8b
671.9±11.0b
Ammonium
43.7±0.2bc
41.2±0.6ab
49.0±2.0d
45.2±0.8c
Aspartic acid
36.8±0.4c
23.0±0.2a
35.1±1.6c
28.8±0.0b
Glutamic acid
72.6±0.4bc
73.8±1.2c
62.5±2.4a
69.4±0.9b
Serine
60.3±1.0c
47.1±0.7a
48.4±2.2ab
51.1±1.1b
Histidine
24.7±0.3c
19.0±1.3a
19.7±0.9a
22.3±0.2b
Glycine
4.5±0.2b
3.9±0.2a
3.9±0.1a
4.2±0.0ab
Threonine
71.1±3.4b
64.1±0.4a
61.3±3.6a
67.2±2.8ab
Arginine
289.4±0.3d
179.9±0.3a
231.3±8.0c
214.9±2.1b
Alanine
105.3±0.8c
97.9±0.9ab
95.5±4.5a
103.4±2.9bc
Methionine
7.4±0.3a
7.5±0.0a
8.9±0.2b
7.7±0.1a
Valine
25.8±0.0c
20.5±0.3b
19.1±1.0a
23.7±0.1c
Cystine
3.0±0.1b
3.2±0.0c
2.7±0.0a
Lysine
8.3±0.0c
6.4±0.1a
7.9±0.3b
7.4±0.1b
Proline
0.3±0.5ab
1.5±0.3c
0.8±0.1bc
Tyrosine
16.5±0.7a
19.7±0.2b
24.0±0.9c
23.3±0.2c
Isoleucine
11.7±0.1c
9.6±0.3a
9.6±0.5a
11.0±0.1b
Leucine
15.4±0.0c
12.5±0.0a
12.3±0.6a
14.5±0.3b
Phenylalanine
20.9±0.1d
16.3±0.0b
15.3±0.4a
19.2±0.4c
All parameters are given with their standard deviation (n=3). Different letters indicate significant differences between
the treatments (p<0.05).
ACKNOWLEDGEMENTS
Many thanks for the financial support given by the Spanish Ministerio de
Ciencia e Innovación to Project AGL2009-08950 and to contract for T.G.-C. and also to
the Junta de Comunidades de Castilla-La Mancha for the FPI grant for A.M.M.-G. We
wish to thank the Protea (Gensac la Pallue, France) for allowing us to free use its
extracts. Thanks to Etienne Terblanche for proofreading the English manuscript.
Literature Cited
Bell, S.-J. and Henschke, P.A. 2005. Implications of nitrogen nutrition for grapes,
fermentation and wine. Aust. J. Grape Wine Res. 11:242-295.
Callejón, R.M., Troncoso, A.M. and Morales, M.L. 2010. Determination of amino acids
in grape-derived products: a review. Talanta 81:1143-1152.
Carmona, M., Peñaranda, J.A., Carrascal, A., Zalacain, A. and Salinas, M.R. 2001.
Estudio preliminar del aumento de polifenoles y color en uva Bobal empleando
extractos vegetales. Agric. Vergel. 240:703-707.
Conde, C., Silva, P., Fontes, N., Dias, A.C.P., Tavares, R.M., Sausa, M.J., Agasse, A.,
Delrot, S. and Gerós, H. 2007. Biochemical changes throughout grape berry
development and fruit and wine quality. Food 1:1-22.
ECC. 1990. Commission Regulation VO 2676/90 concerning the establishment of
common analytical methods in the sector of wine. Off. J. Eur. Communities L272
(3):1-192.
Garde-Cerdán, T., Lorenzo, C., Lara, J.F., Pardo, F., Ancín-Azpilicueta, C. and Salinas,
M.R. 2009. Study of the evolution of nitrogen compounds during grape ripening.
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Chem. 57:2410-2419.
Garde-Cerdán, T., Martínez-Gil, A.M., Lorenzo, C., Lara, J.F., Pardo, F. and Salinas,
M.R. 2011. Implications of nitrogen compounds during alcoholic fermentation from
some grape varieties at different maturation stages and cultivation systems. Food
Chem. 124:106-116.
Kliewer, W.M. 1967. The glucose-fructose ratio of Vitis vinifera grapes. Am. J. Enol.
Vitic. 18:33-41.
Martínez-Gil, A.M., Garde-Cerdán, T., Martínez, L., Alonso, G.L. and Salinas, M.R.
2011. Effect of oak extract application to Verdejo grapevines on grape and wine aroma.
J. Agric. Food Chem. 59:3253-3263.
Oliva, J., Zalacain, A., Payá, P., Salinas, M.R. and Barba, A. 2008. Effect of the use of
recent commercial fungicides (under good and critical agricultural practices) on the
aroma composition of Monastrell red wines. Anal. Chim. Acta 617:107-118.
344
7.2. ARTÍCULO II
El objetivo de este trabajo fue estudiar el impacto de la aplicación foliar
de extractos acuosos comerciales de roble sobre vides de la variedad Verdejo, en
la composición volátil de sus uvas y vinos. Fue necesario poner a punto el
método de extracción de los volátiles de las uvas para su posterior análisis por
GC-MS. Además, se decidió estudiar este efecto a nivel individual, por lo que se
usó una disolución de referencia constituida por eugenol y guayacol (E+G), ya
que estos compuestos están presentes en los extractos de roble.
Las uvas utilizadas fueron las mismas que en el anterior trabajo (25%(1),
100% y control), a excepción de las procedentes del tratamiento 25%(4), que
fueron descartadas por tener una calidad enológica distinta que podría influir en
el objetivo perseguido en este trabajo. Del mismo modo que con el extracto, se
hizo otro tratamiento aplicando a las vides por pulverización foliar la disolución
de eugenol y guayacol (E+G). Los vinos se elaboraron según el sistema clásico
de vinificación en blanco. Los muestreos se realizaron el día de la vendimia,
después de la fermentación alcohólica y transcurridos 6 meses de esta. Para este
estudio se analizaron los parámetros enológicos de las uvas y de los vinos, los
compuestos volátiles del extracto de roble, de las uvas y de los vinos, y se hizo
101
Artículo II
un análisis sensorial de los vinos. Los volátiles analizados en las muestras fueron
los mismos que se determinaron en el extracto de roble.
El método empleado para la determinación de los volátiles de las uvas se
basó en la técnica de extracción denomina headspace sorptive extraction (HSSBSE) y posterior análisis por cromatografía de gases y espectrofotometría de
masas (GC-MS), según se detalla en el apartado 6.4.4 de M&M de esta memoria.
Las mejores condiciones en la optimización de este método se consiguieron
utilizando uvas centrifugadas, empleando un vial de 50 ml y realizando la
extracción a 500 rpm durante 1 hora a 60ºC.
Los contenidos de volátiles de las uvas procedentes de las vides tratadas
con las diferentes formulaciones del extracto de roble eran similares a las de las
uvas control y no se detectó ninguna de las whisky lactonas ni metoxieugenol.
Sin embargo, el metoxieugenol sí que se encontró en todos los vinos,
aunque las whisky lactonas únicamente fueron detectadas en los vinos
procedentes de las vides sometidas a los distintos tratamientos, lo que sugiere
que su origen fue el extracto de roble. Además, la mayoría de los compuestos
estudiados se encontraron en mayor concentración en los vinos procedentes de
los tratamientos con extracto de roble que en los vinos control. Estos resultados
sugieren que la vid puede asimilar los compuestos volátiles del extracto de roble,
posiblemente en forma de derivados glicosilados con el fin de minimizar sus
efectos tóxicos o bien para aumentar su solubilidad. Durante la elaboración del
vino, y especialmente después de haber transcurrido seis meses desde la
fermentación alcohólica, una importante parte de estos precursores glicosilados
podrían liberar la aglicona, lo que justificaría el aumento en los vinos del
contenido de volátiles del extracto.
El análisis sensorial puso de manifiesto que los vinos procedentes de las
vides tratadas mantenían el aroma típico de los vinos de Verdejo tras finalizar la
102
Artículo II
fermentación alcohólica, seis meses después su color era más verdoso, eran más
astringentes y el aroma presentaba notas de madera que recordaban a los vinos
envejecidos en barricas de roble.
Por otro lado, el tratamiento con la disolución de referencia (E+G)
provocó un aumento de la concentración de eugenol y guayacol en los vinos.
Probablemente estos dos compuestos tras el tratamiento fueron asimilados por la
vid y almacenados en las uvas en forma de precursores, ya que el contenido
volátil de las uvas fue similar a las del control, y posteriormente fueron liberados
durante la fermentación alcohólica y después de seis meses. Este tratamiento
también modificó la concentración volátil de la mayoría de compuestos
estudiados en los vinos, lo que pudo deberse a que la alta concentración de los
compuestos aplicados produjera un estrés en la planta afectando al metabolismo
secundario.
Por lo tanto, con este segundo trabajo se pudo observar que la aplicación
foliar de extractos de roble a viñas de la variedad Verdejo afecta a la
composición volátil y a la percepción sensorial de sus vinos. Esto nos hizo
plantear el siguiente trabajo con el mismo objetivo principal, pero usando una
variedad tinta en la que el sistema de vinificación tradicional implica una
fermentación maloláctica.
103
ARTICLE
pubs.acs.org/JAFC
Effect of Oak Extract Application to Verdejo Grapevines on Grape and
Wine Aroma
Ana M. Martínez-Gil,† Teresa Garde-Cerdan,† Laura Martínez,‡ Gonzalo L. Alonso,† and M.
Rosario Salinas*,†
†
‡
Catedra de Química Agrícola, E.T.S.I. Agronomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain
'Dehesa de los Llanos' Winery, Ctra de las Pe~
nas de San Pedro, km 5.5, aptdo 8, 02080 Albacete, Spain
ABSTRACT: Volatile compounds from a commercial aqueous oak extract application to white Verdejo grapevines at veraison have
been studied. Treated grapes under two types of formulation (25% and 100%) have been analyzed at the optimum maturation time,
and winemaking was then subsequently carried out. The volatile compounds were analyzed by stir bar sorptive extraction-gas
chromatograpy-mass spectrometry. The results suggest that after the grapevine treatments, grapes store the volatiles in the form of
nonvolatile precursors, and some of the volatiles are released during the winemaking process, especially six months after the
alcoholic fermentation. The sensory analysis shows that wines maintain the typical aroma properties of Verdejo wines at the end of
fermentation; but after six months, the wine color is greener and more astringent, and, in terms of aroma, it has wooden notes as if the
wine has been aged in oak barrels.
KEYWORDS: volatiles, oak extracts, grapevines, grapes, wines, Verdejo
’ INTRODUCTION
Varietal aroma of wines is constituted by a complex group of
substances that can occur in both forms: as volatile molecules
(so-called odor-active compounds), or as odorless precursors.
The latter are related to wine aroma potential, as during the
winemaking process and aging, they can be transformed into
odor active-compounds.1,2
The concentration of varietal volatile compounds in wines
varies depending on the grape variety and the “terroir effect”,
which is related to the soil, climate, viticulture, and environment
in which the plant grows. The results of such effects produce
wines with a characteristic and identifiable origin.3 With regard to
the impact of external factors on the aroma, although still a
matter of controversy, the scent of eucalyptus in wines from
vineyards near eucalyptus forests may be due to the absorbtion of
the aroma by the grape plants. However eucalyptol, a compound
that possesses the characteristic odor of eucalyptus, is a terpene
that may originate from chemical transformations of other
terpenes in the grape4 as well as from the combination of certain
wine components.5 On the other hand, there is evidence that
certain fungicide treatments applied to the vineyard can influence
the aroma of wines, especially the varietal component.6
In recent years, some research groups have studied how grapes
from grapevines exposed to smoke from forest fires produced
wines with smoke sensorial notes.7-10 Smoke applications on
grapevines showed that volatile compounds from smoke such as
guaiacol, 4-methylguaiacol, 4-ethylphenol, furfural, and eugenol
were absorbed by the plant and then transmitted to the must and
wine during the winemaking process,7 especially when smoke
application was made seven days after veraison.9 Further, the
sensory characteristics of wines from grapevine exposure to
smoke for 1 h was of the same order as that resulting from wines
in contact with oak, material that contains some compounds that
are also in the smoke.10
r 2011 American Chemical Society
Indeed, oak barrels have been used for a long time to age
wines, especially red wines, as it improves wine characteristics,
especially the aroma. In this sense, the wines in contact with oak
wood extract volatile compounds, which proportionate aromatic
notes of “wood”, “coconut”, “spices”, “toasted”, and “smoke”
associated with compounds such as oak lactones, eugenol,
vanillin, guaiacol, etc.11,12 Some of these compounds are already
present in green wood, and others are formed during the toasting
process of cooperage.13
The aforementioned observations suggest that grapevines may
absorb the aroma compounds present within oak extracts.
Among the constituents of oak are some of the compounds also
in smoke, such as eugenol and guaiacol, which are absorbed by
grape plants and transmitted to their wines. In addition, the
aroma compounds of oak extracts have characteristics of aged
wines and therefore add value to the wines.
Vitis vinifera cv. Verdejo is an important Spanish white cultivar,
the base of Rueda Denomination of Origin wines, which
produces young white wines with fruity attributes (citrus and
tropical characteristics) with hints of green fruit.14 The tropical
fruit character of Verdejo is related to the presence of 3-mercaptohexyl acetate.15 Is it possible to transfer the aromas of oak
extracts to this young wine grape variety? Without a doubt, this
would provide an innovative viticultural strategy in order to
obtain a different type of wine. As a consequence, the aim of this
work was to study the impact of different formulations of a
commercial aqueous oak extract applied to Verdejo grapevines in
relation to the oak volatile composition of grapes and their
respective wines.
Received: October 27, 2010
Accepted: February 8, 2011
Revised:
January 20, 2011
Published: March 11, 2011
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dx.doi.org/10.1021/jf104178c | J. Agric. Food Chem. 2011, 59, 3253–3263
Journal of Agricultural and Food Chemistry
’ MATERIALS AND METHODS
Oak Extract. The aqueous oak extract chosen for this study was
provided by Protea France S.A. (Gensac la Pallue, France). This extract
is a food additive utilized in spirits and fruit juices, which guarantees that
no toxicity risk exists. It was produced by macerating French toasted oak
chips (Quercus sessilis) from natural seasoning for at least 18 months in
demineralized water at 100 °C for 32 h. Before treatment of the
grapevines, the absence of phytotoxicity was confirmed in Solanum
lycopersicum var. Micro Tom tomato (INRA, Montpellier, France), a
cultivar used as a quickly grown plant.
Grapevine Treatments. White grapes from Vitis vinifera variety
Verdejo grown in the La Mancha region (Albacete province, southeast
Spain) during the year 2009 were used. The annual average temperature
was 13 °C, with a minimum of -15 °C (January) and a maximum of
40 °C (August). Grapevines were grown on a trellis system with drip
irrigation system to ensure the plants' water needs, as this region has
300-400 mm of rainfall per year.
Oak extract treatments were applied to the grapevines during
veraison, when the green color of the grape was more transparent and
the flexibility of the skins was high, presenting this aspect in at least half
of the clusters. At veraison, the metabolic activity of the plant is very
important and it has been shown that the volatile composition can be
affected by external phenomena such as smoke.9 Formulations of each of
the treatments were prepared with 0.5 mL of the adjuvant Fluvius
(BASF, Germany) per liter; this is a wetting agent typically used for foliar
herbicide treatment. The extract without dilution (100% treatment) and
diluted with water at 25% (25% treatment) were applied only once on
the seventh day after veraison. In addition, on the same day, a third
treatment consisted of an aqueous solution of eugenol plus guaiacol
(EþG treatment) standard compounds (Sigma-Aldrich, Gillingham,
England) (6 g/L of each compound) was applied. For each of the
treatments, a row of 188 plants was used. A total of 752 plants was
necessary, with two untreated rows between different applications to
avoid contamination. Also, a row of 188 plants was not treated (control).
Around 230 mL of each formulation was applied evenly per plant by
spraying over leaves. The treatments were carried out when the ambient
temperature was below 20 °C, at approximately 7 a.m. Several hours
before harvest, grape sampling was carried out, starting with the first
grapevine of each row, by taking a cluster from every fifth grapevine, for
the entire row (188 plants). Clusters with northern and southern
distribution were alternatively picked, making a total of 11 kg of grapes
for each treatment as well as for the control. Grapes from all clusters were
destemmed and mixed. From this mixture, 300 grapes were randomly
taken to obtain a weight of 100 berries (triplicate analysis). The
remianing grapes were frozen at -20 °C for later volatile composition
analysis.
Winemaking. White Verdejo grapes were harvested on August 27
at the technological ripening moment when the degrees Baume/
titratable acidity ratios were between 2.5 and 3. These grapes were
picked at night with a harvesting machine, the temperature of the grapes
being 19 ( 2 °C. Ten grams of potassium metabisulfite per 100 kg of
grapes was added. For each treatment, all 188 plants were harvested.
Grapes were first destemmed, followed by a pressing process with 55%
yield. The must from each treatment, without skin contact, was put in a
200 L stainless steel tank. One liter of must was removed for oenological
parameters analysis. Saccharomyces cerevisiae strain QA23 was inoculated
at a dose of 20 g/hL according to the recommendation of Lallemand
(Spain). The alcoholic fermentation temperature was maintained
around 13 °C, and the density was measured daily with a densimeter.
The alcoholic fermentation was completed when the reducing sugars
were below 2.5 g/L. At the end of the alcoholic fermentation, the free
SO2 concentration was corrected to 25-35 mg/L. The wines were
stored for six months in the tanks at 17 °C protected from oxygen. For
ARTICLE
each tank, three different wine samples were taken at the end of
the alcoholic fermentation and also six months later and then frozen
at -20 °C until analysis.
Oenological Parameters Analysis. Degrees Baume, reducing
sugars, titratable acidity, volatile acidity, pH, alcohol degree, and yeast
assimilable nitrogen (YAN) from the different samples were measured
following the methods established by ECC.16 Grape yield for the plant
was calculated by dividing the total mass production (kg) by the number
of plants (188).
Extraction of Volatile Compounds from Grapes and Oak
Extract by HS-SBSE. We used as a reference the methods proposed by
Weldegergis and Crough17 and Callej
on et al.,18 which described the
analysis of wines and wine vinegars, respectively, and adapted them to
our samples. Grapes randomly picked from the three different treatments and the control were defrosted, crushed, and macerated for 2 h
and then strained with a colander (must). One aliquot of strained grapes
was centrifuged at 176g for 30 min (centrifuged must). These two
sample types (must and centrifuged must) were used in order to choose
the best extraction conditions for the volatile compounds studied: cisoak lactone, trans-oak lactone, furfural, 5-methylfurfural, eugenol, guaiacol, vanillin, acetovanillone, 6-methoxyeugenol, methyl vanillate, 4-vinylguaiacol, 4-ethylguaiacol, and 4-ethylphenol (Aldrich, Gillingham,
England), characteristic compounds of oak wood, by headspace-stir bar
sorptive extraction (HS-SBSE). The variables studied were vial volume,
50 and 20 mL, and retention temperature, 40 °C and 60 °C. In the 50 mL
vial, 22 mL of sample was added, so the headspace was 28 mL, and in the
20 mL vial, 9 mL of sample was added, so the headspace was 11 mL. In all
cases, 0.1 g of NaCl was added per milliliter of sample. Also, 10 μL of
internal standard γ-hexalactone (Sigma-Aldrich) solution at 1 μL/mL in
absolute ethanol (Merck, Damstard, Germany) was added per milliliter
of sample.19 A polydimethylsiloxane-coated stir bar (twister, 0.5 mm film
thickness, 10 mm length, Gerstel, M€ulheim, and der Ruhr, Germany)
was inserted into the twister-headspace vial and hermetically closed. The
vial was introduced into a heater (Selecta, Barcelona) at the appropriate
temperature and was stirred with a common magnetic stirrer during 1 h
at 500 rpm. Next, the twister was removed, rinsed with distilled water,
dried with a cellulose tissue, and later transferred into a thermal
desorption tube for GC-MS analysis. To check the method, samples
were analyzed in triplicate. Once volatile extraction was optimized, the
method proposed was applied to analyze the oak extract and the
different grape samples. Grapes were separated into three lots, and each
of them was crushed and macerated and the volatile compounds were
extracted (n = 3).
The precision of the method was calculated with the coefficient of
variation, where six extractions were performed on a sample of grapes
(control). Recovery was studied by spiking two concentrations of the
target compounds to the grape matrix. Compounds were then extracted
and quantified according to the extraction method, and their recovery
was calculated. The limit of quantification (LOQ) and limit of detection
(LOD) were estimated as the concentration of the analyte of a standard
that produced a signal-to-noise ratio of 10 and 3 times, respectively.
Extraction of Volatile Compounds from Wines by Immersion SBSE. Volatile wine compounds were extracted according to
Marín et al.19 The twister was introduced into 10 mL of sample to which
100 μL of the same internal standard γ-hexalactone was added. Samples
were stirred at 500 rpm at room temperature for 1 h. The twister was
then removed from the sample, rinsed with distilled water, dried with a
cellulose tissue, and later transferred into a thermal desorption tube for
GC-MS analysis. For each wine, three samples were analyzed (n = 3).
Analysis of Volatile Compounds by GC-MS. In the thermal
desorption tube, the volatile compounds were desorbed from the twister
under the following conditions: oven temperature, 330 °C; desorption
time, 4 min; cold trap temperature, -30 °C; helium inlet flow, 45 mL/
min. The compounds were transferred into a Hewlett-Packard LC 3D
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dx.doi.org/10.1021/jf104178c |J. Agric. Food Chem. 2011, 59, 3253–3263
ARTICLE
Table 1. Oenological Parameters in Grapes after the Different Grapevine Treatments (n = 3)a
treatments
yield (kg/plant)
weight of 100 berries
degrees Baume
YAN (mg N/L)
titratable acidity (g/L)
degrees Baume/TA
pH
control
3.93
100.7 ( 0.2
12.4 ( 0.1
196 ( 3
4.5 ( 0.1
2.8 ( 0.1
3.6 ( 0.1
25%
3.26
112.7 ( 0.8
12.2 ( 0.2
168 ( 4
4.2 ( 0.2
2.9 ( 0.1
3.6 ( 0.0
100%
3.90
119.3 ( 0.6
12.4 ( 0.0
210 ( 7
4.4 ( 0.2
2.8 ( 0.2
3.6 ( 0.0
EþG
3.72
121.4 ( 0.3
12.4 ( 0.1
182 ( 5
5.0 ( 0.3
2.5 ( 0.1
3.6 ( 0.2
a
Control: untreated grapes; 25%: grapevines treated with oak extract diluted to quarter strength; 100%: grapevines treated with oak extract without
dilution; EþG: grapevines treated with eugenol plus guaiacol solution. TA: titratable acidity. YAN: yeast assimilable nitrogen.
Table 2. Relative Area 10-3 of the Grape Volatile Compounds under the Different Conditions Studieda,b
must
vial volume 50 mL
centrifuged must
vial volume 20 mL
vial volume 50 mL
vial volume 20 mL
temp 60 °C
temp 40 °C
temp 60 °C
temp 40 °C
temp 60 °C
temp 40 °C
temp 60 °C
temp 40 °C
furfural
5-methylfurfural
10 ( 1b
5.9 ( 0.8b
10 ( 1b
6.7 ( 0.7bc
5.3 ( 0.5a
1.6 ( 0.2a
5.2 ( 0.5a
2.1 ( 0.3a
21 ( 2d
26 ( 2e
12 ( 1b
8 ( 1 cd
18 ( 2c
9 ( 1d
20 ( 2 cd
9.1 ( 0.9d
eugenol
5.6 ( 0.8bc
6.0 ( 0.8c
2.8 ( 0.4a
1.8 ( 0.2a
10 ( 1e
4.6 ( 0.6b
8 ( 1d
6.3 ( 0.9c
guaiacol
4.4 ( 0.4b
6.6 ( 0.7e
1.8 ( 0.2a
2.6 ( 0.4a
6.6 ( 0.9e
6.2 ( 0.6de
5.5 ( 0.5 cd
4.7 ( 0.6bc
vanillin
3.4 ( 0.4c
3.2 ( 0.3bc
2.8 ( 0.3bc
1.6 ( 0.2a
4.6 ( 0.6d
2.7 ( 0.3b
2.8 ( 0.3bc
2.6 ( 0.3b
acetovanillone
9.3 ( 0.9e
4.1 ( 0.4bc
2.5 ( 0.2a
3.6 ( 0.4ab
11 ( 1f
7.1 ( 0.8d
7.9 ( 0.9d
5.2 ( 0.6c
methyl vanillate
2.5 ( 0.3e
0.89 ( 0.09ab
1.4 ( 0.1c
0.61 ( 0.08a
1.9 ( 0.3d
1.2 ( 0.1bc
0.9 ( 0.2ab
0.75 ( 0.08a
4-vinylguaiacol
2.1 ( 0.2de
2.0 ( 0.2 cd
1.3 ( 0.1b
0.85 ( 0.09a
3.2 ( 0.3 g
2.5 ( 0.3f
2.4 ( 0.2ef
1.7 ( 0.2c
4-ethylguaiacol
4-ethylphenol
2.1 ( 0.3c
1.7 ( 0.2c
4.6 ( 0.5d
2.9 ( 0.3d
0.52 ( 0.05a
1.7 ( 0.2c
0.76 ( 0.08ab
0.85 ( 0.08b
2.1 ( 0.2c
1.0 ( 0.1b
1.1 ( 0.2b
1.0 ( 0.1b
1.0 ( 0.1b
1.0 ( 0.1b
0.82 ( 0.08ab
0.54 ( 0.05a
total
47 ( 2b
47 ( 2b
21.7 ( 0.8a
19.9 ( 0.9a
87 ( 3e
47 ( 2b
57 ( 3d
52 ( 3c
a
All parameters are given with their standard deviation (n = 3). Different letters indicate significant differences (level of significance: p < 0.05) between
columns. b Grapes randomly picked from the four different clusters were used.
mass detector (Palo Alto, CA) with a fused silica capillary column (BP21
stationary phase 30 m length, 0.25 mm i.d., and 0.25 μm film thickness;
SGE, Ringwood, Australia). The chromatographic program was set as
follows: 40 °C (held for 5 min), raised to 150 °C by 5 °C/min, and then
raised to 230 °C by 10 °C/min (held for 5 min). The total analysis time
was 40 min. For mass spectrometry analysis, electron impact mode (EI)
at 70 eV was used and the detection and quantification were carried out
in the selected ion monitoring (SIM) mode. The m/z of ions monitored
in the SIM runs were (italic ions are those used for quantification) as
follows: cis-oak lactone 99, 101, 132, 156; trans-oak lactone 99, 101, 132,
156; furfural 39, 67, 95, 96; 5-methylfurfural 53, 81, 109, 110; eugenol
121, 131, 149, 164; guaiacol 53, 81, 109, 124; vanillin 151, 152, 155, 156;
acetovanillone 108, 123, 151, 166; 6-methoxyeugenol 81, 119, 131, 194;
methyl vanillate 123, 151, 167, 182; 4-vinylguaiacol 77, 107, 135, 150;
4-ethylguaiacol 91, 122, 137, 152; 4-ethylphenol 77, 91, 107, 122. The
detector temperature was 150 °C. Identification was carried out by
comparison with the mass spectrum and retention index of chromatographic standards and data found in the literature. Two calibrations were
performed, one for the headspace extraction, using the optimum
conditions of the method, and one for extraction by immersion. For
all of these compounds, the concentrations of the standards (SigmaAldrich) were between 0.05 and 3500 μg/L in a 12% ethanol (v/v)
solution at pH 3.6, and the quantification was based on five-point
calibration curves (R2 > 0.9 for both extraction methods). Grape results
are given in μg/kg, taking into account the 85% must yield.
Sensory Analysis. A panel of eight expert judges (three females
and five males, with ages between 25 and 50 years old) participated in the
study. At the end of the alcoholic fermentation and six months later,
judges evaluated each wine in triplicate, which were randomly presented.
The sensory analysis was performed by modifying the classic questionnaire of Verdejo wines from Rueda Spanish Origin Apellation, but
adding the wood and spicy attributes. Thus, the analysis was composed
of 17 attributes or descriptors grouped by visual phase (color intensity,
yellow and green tones), olfactory phase (odor intensity, fermentatives,
varietals, fruity, florals, herbaceous, wood, and spicy), and gustatory
phase (mouthfeel, acidity, bitterness, astringency, persistence, and
balance). Panelists rated each attribute on a scale from 1 (absence) to
7 (maximum presence). The sensory analysis of wines from EþG
grapevine treatment was not carried out because, as they are not natural
products, there could be a health risk to the judges.
Statistical Analysis. The statistical elaboration of the data was
performed using SPSS Version 17.0 statistical package for Windows
(SPSS, Chicago, IL). Volatile compound data were processed using
variance analysis (ANOVA). Differences between means were compared using the least significant differences (LSD) test at 0.05 probability
level. Two variance analyses were carried out, one of them related to the
different oak extract treatments, and the other to the EþG solution
treatment. Discriminant analyses of the volatiles composition in the
control wine and the wines obtained from grapes treatment with oak
extract were performed, as well as analyses of their sensory attributes, at
the end of alcoholic fermentation and after six months.
’ RESULTS AND DISCUSSION
The effect of two oak extract applications to Verdejo cultivar
grapevines has been studied. In addition, another grapevine
treatment with a standard solution of eugenol and guaiacol was
followed, as recently studied in the literature.20 These two
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Table 3. Coefficient of Variation (CV (%)), Recovery Indices (R1, R2), Limit of Detection (LOD), and Limit of Quantification
(LOQ) for Each Compound Analyzeda
CV (%)
cis-oak lactone
trans-oak lactone
furfural
C1 (μg/kg)
R1 (%)
b
C2 (μg/kg)
R2 (%)
LOD (μg/kg)
LOQ (μg/kg)
7.1
4.0
72
8.0
89
0.45
1.52
8.0b
15.8
7.0
45.0
78
73
14.0
90.0
80
79
0.85
7.21
2.83
24.03
5-methylfurfural
5.9
0.6
72
1.2
81
0.16
0.53
eugenol
1.4
0.8
84
1.6
104
0.20
0.66
4.1b
0.09
0.2
82
0.4
95
0.03
guaiacol
16.2
0.4
72
0.8
78
0.13
0.41
4-vinylguaiacol
14.3
0.4
77
0.8
92
0.11
0.35
4-ethylguaiacol
0.8
1.2
82
6.0
94
0.35
1.18
4-ethylphenol
vanillin
16.4
16.5
0.8
0.04
75
75
4.0
0.24
89
79
0.23
0.01
0.78
0.03
6-methoxyeugenol
acetovanillone
15.5
0.05
73
0.30
73
0.01
0.04
methyl vanillate
15.9
0.05
76
0.30
81
0.01
0.04
a
C1, C2: two different concentrations of each compound added to the grapes. b Such compounds are not found in grapes, so their CV was calculated
from the samples that were spiked with them.
compounds are present in the oak extract studied, in oak-aged
wines, and in grapes and wines from smoke applications to
grapevines, which opens a new research field on plant responses
to exogenous agents.
Grape Oenological Parameters. The oenological parameters
of grapes from the different treatments (control, 25%, 100%, and
EþG) are shown in Table 1. The control and 100% treatment
produced the highest fruit yields. Also, 100% treatment grapes
showed a high grape weight, but without important differences
with the grapes from the EþG treatment, which showed the
highest values for this parameter. These small differences observed could be due to the plants' natural variation. There were
no differences between the degrees Baume, titratable acidity, and
pH, and therefore neither in the degrees Baume/titratable acidity
ratio. Thus, we conclude that none of the treatments affected
oenological parameters. In all the cases, YAN was higher than 140
mg N/L, which is the concentration needed to complete alcoholic fermentation.21
Selection of HS-SBSE Extraction Conditions. Table 2 shows
the grape volatile composition under the different extraction
conditions. The extraction was higher for centrifuged must, with
the exception of methyl vanillate, 4-ethylphenol, and 4-ethylguaiacol, regardless of temperature and vial volume. The centrifuged must was more limpid than the original must, so the
interchange of the different volatile compounds between the
liquid and the gas phase could be facilitated, improving the
extraction process. Regarding the vial volume, for a sample type
(must and centrifuged must) and temperature given, the extraction of the volatile compounds was higher when a 50 mL vial
volume was used compared to a 20 mL volume, in most of the
cases. Also, Delgado et al.22 found that when vial volume was
increased, extraction was improved. Significant differences have
been found for a 50 mL volume; when the temperature was
increased, the volatiles absorption increased considerably. Theoretically, high temperatures will increase the partial vapor
pressure of analytes in the headspace23 but only up to a limit,
as high temperatures (above 75 °C) will decrease the absorption
of the volatile compounds onto the twister.18 Therefore, the
optimum extraction conditions of the volatile compounds studied from grapes are as follows: crushed, strained, and centrifuged,
Table 4. Volatile Composition of the Aqueous Oak Extracta
concentration (μg/L)
5.6 ( 0.4
cis-oak lactone
trans-oak lactone
11.3 ( 0.9
furfural
2819 ( 200
5-methylfurfural
15.1 ( 0.2
eugenol
6-methoxyeugenol
9.8 ( 0.6
0.97 ( 0.04
guaiacol
15.2 ( 0.5
4-vinylguaiacol
1.4 ( 0.4
4-ethylguaiacol
27 ( 3
4-ethylphenol
vanillin
a
4(1
2.3 ( 0.2
acetovanillone
0.9 ( 0.1
methyl vanillate
1.5 ( 0.5
All parameters are given with their standard deviation (n = 3).
vial volume of 50 mL, with 22 mL of sample, and 60 °C retention
temperature. Table 3 shows the coefficient of variation, recovery
index, and limits of detection (LOD) and quantification (LOQ) for
each compound. The precision of the method was calculated with
the coefficient of variation, and the results fluctuated between 1%
and 16% for the different quantified compounds. The recovery
index was used in order to find out the accuracy of the method. This
index fluctuated between 72% and 104% depending on the different
compounds analyzed. Because of the lack of grape analysis references to these compounds by HS-SBSE, no comparison has been
carried out. However, LOD and LOQ values seem to be adequate
for grape analysis.
Volatile Compounds in Oak Extract. The optimized HSSBSE method was applied to the extraction of volatile compounds from aqueous oak extract; the results are shown in
Table 4. Note that the extract used in this study comes from
toasted wood chips macerated with water by heating at 100 °C
for 32 h (according to the Protea SA procedure). However, up to
now, the literature on wood volatile composition refers to their
extraction by different hydroalcoholic solutions. Lactones are
characteristic compounds of oak wood, which are also generated
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Table 5. Concentration of Volatile Compounds (μg/kg) in Grapes from the Different Grapevine Treatmentsa
control
cis-oak lactone
trans-oak lactone
furfural
25% extract
nd
nd
nd
65.1 ( 11.0a
100% extract
nd
nd
nd
73.3 ( 25.3a
76.1 ( 15.0a
EþG
nd
nd
70.3 ( 0.2a
5-methylfurfural
0.84 ( 0.05ab
1.01 ( 0.32b
0.64 ( 0.07a
0.61 ( 0.11a
eugenol
1.48 ( 0.02b
1.12 ( 0.03ab
0.82 ( 0.22a
1.50 ( 0.21b
6-methoxyeugenol
nd
nd
nd
nd
guaiacol
0.50 ( 0.10ab
0.43 ( 0.06a
0.42 ( 0.09a
0.61 ( 0.02b
4-vinylguaiacol
4-ethylguaiacol
0.70 ( 0.11b
4.78 ( 0.04c
0.69 ( 0.07b
1.20 ( 0.20a
0.64 ( 0.05b
2.21 ( 0.60b
0.37 ( 0.01a
1.45 ( 0.03a
4-ethylphenol
vanillin
2.6 ( 0.5c
2.5 ( 0.3c
1.4 ( 0.1b
0.8 ( 0.1a
0.170 ( 0.051b
0.049 ( 0.001a
0.053 ( 0.002a
0.043 ( 0.008a
acetovanillone
0.23 ( 0.07b
0.13 ( 0.02a
0.12 ( 0.01a
0.10 ( 0.01a
methyl vanillate
0.300 ( 0.051b
0.087 ( 0.008a
0.094 ( 0.001a
0.071 ( 0.010a
a
All parameters are given with their standard deviation (n = 3). nd: not detected. Different letters indicate significant differences (level of significance: p <
0.05) between columns. Control: untreated grapevines; 25%: grapevines treated with oak extract diluted to quarter strength; 100%: grapevines treated
with oak extract without dilution; EþG: grapes treated with eugenol plus guaiacol solution.
during the toasting process.13 These compounds correspond to
the “toasted”, “wood”, or “coconut” aroma characters of the
commercial oak extracts. cis-Oak lactone concentration is higher
than the trans isomer concentration in American oak, but the
concentrations of both lactones are closer to one another in
French oak.24-26 The concentration of trans-oak lactone in the
extract was twice the concentration of cis-oak lactone. This fact
may be attributed to the water maceration process at high
temperature for the toasted wood, which would imply a partial
loss of the cis-oak lactone because it is the most volatile isomer.26
Among the compounds found in the wood that is subjected to
high temperatures, the furanic compounds are the most abundant, emphasizing a larger furfural content,25 which is generated
as a result of the pentose Maillard reaction.26 It is also the most
abundant compound in the extract used, and its content is higher
than that reported in the literature on analysis of the wood used
in wine aging. 5-Methylfurfural comes from the hexose Maillard
reaction, and the concentrations of the extract are within the
range reported in the literature for ethanolic extracts of French
oak.25 The volatile phenols such as eugenol, 6-methoxyeugenol,
guaiacol, vanillin, acetovanillone, and methyl vanillate are formed
by lignin degradation during the toasting process, although
eugenol and vanillin are also present in green wood.27 4-Vinylguaiacol is in the range indicated by the literature mentioned
above, while 4-ethylguaiacol, guaiacol, and eugenol are present in
higher concentrations and 6-methoxyeugenol in lower concentrations. Note that guaiacol content is greater than eugenol
content, a result which coincides with the findings of Guillen
and Manzanos28 in aqueous oak smoke preparations. The
concentration of 4-ethylguaiacol in the extract is almost seven
times higher than that of 4-ethylphenol. Vanillin and derivatives
such as acetovanillone and methyl vanillate were found in very
low concentrations compared to the other compounds.
Volatile Compounds in Grapes. In the literature, we did not
find reports on the volatile composition of Verdejo grapes. This is
a nonaromatic grape variety used for the production of young
wines, which are characterized by a typical floral and fruity
aroma.14 This study focuses on the compounds present in the
oak extract that may have been transmitted to the grapes and
their respective wines by the treatments. Neither oak lactones
nor 6-methoxyeugenol were detected in any of the grape samples
(Table 5). However, other compounds such as eugenol, guaiacol,
4-ethylguaiacol, 4-vinylguaiacol, 4-ethylphenol, vanillin, methyl
vanillate, and acetovanillone were found, which have also been
reported in Verdejo wines,29 suggesting that they may come from
the grapes. Compounds such as furfural and 5-methylfurfural are
generally not the focus of studies in aromas of young wines;
however, the presence of furfural has been reported in Macabeo
young white wines and could therefore come from the grapes.30
The concentrations of furfural, guaiacol, and 4-vinylguaiacol are
similar in the control grapes and in those treated with the extract
(Table 5), but in the latter they contain significantly lower
concentrations of 4-ethylguaicaol and the three vanillin derivatives analyzed compared to the control. The presence of 4-ethylphenol in grapes has not been studied; however, its existence in
grapes as precursors has been suggested.31 When the grapes from
the two oak extract treatments are compared, similar concentrations are observed for all compounds, except for 5-methylfurfural
and 4-ethylphenol, which are in higher concentrations when the
treatment applied was 25%, and for 4-ethylguaiacol, which was
higher in the 100% treatment. Eugenol is a volatile compound
found in Chardonnay grape skins32 and in young white wines,
therefore contributing to the varietal aroma with hints of
aromatic clove spices.30
Grapes treated with the solution of eugenol and guaiacol
(EþG treatment) do not exhibit an increase in the concentrations of these compounds, contrary to what one might expect,
because no significant differences were found compared to the
control (Table 5). Among the other compounds, furfural and
5-methylfurfural are found in concentrations similar to that in the
control but the other compounds tested are in lower concentrations. Flavor compounds in grapes can be present as their free,
odor-active form, or as nonvolatile precursors, mainly glycoconjugates, releasing the aglycone during the winemaking
process.1,2,33 Glycosides of guaiacol were found in both grapes
and wine,10,20,34 and glycosides of eugenol were reported in
grapes.35 Therefore, eugenol and guaiacol, added to the grapevines through the EþG treatment, may have been stored by the
berries as glycosylated precursors, as no increment was observed
because this study focuses only on the volatile compounds or
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Table 6. Oenological Parameters in Wines at the End of the Alcoholic Fermentation and after Six Monthsa
grapevine treatment
pH
titratable acidity (g/L)
volatile acidity (g/L)
alcohol degree (v/v %)
control
3.5 ( 0.0
4.3 ( 0.2
0.3 ( 0.0
13.1 ( 0.2
25%
3.5 ( 0.1
4.2 ( 0.1
0.2 ( 0.0
13.3 ( 0.2
End of Alcoholic Fermentation
100%
3.5 ( 0.1
4.2 ( 0.2
0.2 ( 0.0
13.5 ( 0.1
EþG
3.5 ( 0.0
4.2 ( 0.0
0.2 ( 0.1
13.2 ( 0.3
control
3.6 ( 0.1
4.0 ( 0.1
0.3 ( 0.1
13.8 ( 0.2
25%
3.6 ( 0.0
4.0 ( 0.2
0.3 ( 0.0
13.6 ( 0.3
100%
EþG
3.6 ( 0.0
3.6 ( 0.1
4.0 ( 0.1
3.9 ( 0.1
0.3 ( 0.0
0.3 ( 0.1
14.0 ( 0.1
13.6 ( 0.0
Six Months after Alcoholic Fermentation
a
All parameters are given with their standard deviation (n = 3). Control: untreated grapevines; 25%: grapevines treated with oak
extract diluted to quarter strength; 100%: grapevines treated with oak extract without dilution; EþG: grapes treated with eugenol plus
guaiacol solution.
Table 7. Concentration of Volatile Compounds (μg/L) in Wines from the Different Grapevine Treatmentsa
control wine
wine from 25% treatment
wine from 100% treatment
end of alcoholic
six months after alcoholic
end of alcoholic
end of alcoholic
fermentation
fermentation
fermentation
fermentation
fermentation
fermentation
cis-oak lactone
nd
nd
1.8 ( 0.1a
10.3 ( 0.5c
2.8 ( 0.6a
9 ( 2b
trans-oak lactone
nd
nd
4.8 ( 0.3a
22 ( 3d
8 ( 1b
19 ( 2c
30.1 ( 2.11a
9.22 ( 0.91c
29.1 ( 3.03a
7.91 ( 0.41c
30.1 ( 2.01a
8.80 ( 0.50bc
29.5 ( 0.01a
8.40 ( 1.03c
furfural
5-methylfurfural
eugenol
6-methoxyeugenol
29.1 ( 4.31a
3.40 ( 0.31a
30.0 ( 1.21a
6.6 1 ( 0.32b
4.0 ( 0.3a
11.3 ( 0.5b
14.0 ( 1.1b
31.2 ( 3.4c
14.5 ( 0.9b
28.1 ( 4.1c
2.01 ( 0.20a
0.90 ( 0.04a
1.31 ( 0.11a
17.20 ( 2.01c
1.30 ( 0.13a
8.21 ( 0.41b
guaiacol
2.9 ( 0.2a
3.0 ( 0.1a
8.0 ( 0.6c
4.1 ( 0.2b
8.0 ( 0.6c
4-vinylguaiacol
566 ( 43b
426 ( 17a
533 ( 37b
391 ( 35a
528 ( 21b
446 ( 16a
3.8 ( 0.1b
4-ethylguaiacol
15.2 ( 1.0a
23.0 ( 1.2d
18.1 ( 0.8b
14.8 ( 0.6a
20.3 ( 1.1bc
22.4 ( 2.1 cd
4-ethylphenol
3.0 ( 0.2ab
2.0 ( 0.1a
5.0 ( 0.8bc
4.9 ( 0.7b
3.0 ( 0.2ab
6.1 ( 1.0c
vanillin
acetovanillone
0.50 ( 0.04b
2.0 ( 0.2a
0.44 ( 0.02ab
1.7 ( 0.1a
1.02 ( 0.11c
2.1 ( 0.2a
0.70 ( 0.06b
7.6 ( 0.4c
1.13 ( 0.12d
2.4 ( 0.1a
0.40 ( 0.02a
6.1 ( 0.3b
methyl vanillate
0.26 ( 0.02a
0.36 ( 0.01c
0.26 ( 0.01a
1.10 ( 0.01e
0.29 ( 0.02ab
0.67 ( 0.01d
a
0.05) between columns. Control: untreated grapevines; 25%: grapevines treated with oak extract diluted to quarter strength; 100%: grapevines treated
with oak extract without dilution.
aroma-free forms. If such glycosylation has taken place, the
biosynthesis of other volatile compounds from these grapes
could have been affected and therefore presents significant
differences from that of the control grapes.
Wines Oenological Parameters. Table 6 shows the oenological parameters of wines at the end of the alcoholic fermentation
and after six months. All values were normal for wines from
healthy grapes.36 The treatments did not affect the parameters
analyzed because the values were similar to those of the control
wine. Only the alcohol degree slightly increased at the six month
sampling in comparison with the end of alcoholic fermentation.
Wine Volatile Composition. Neither of the two oak lactones
was found in the control wine, so their origin in other wines can
be attributed to the oak extract treatments (Table 7). At the end
of fermentation, the concentrations of cis-oak lactone were
similar in the wines from the two extract treatments and less
than that of the trans-oak lactone, and the highest concentration
was found in the 25% treatment wine. In these wines, the cis/
trans lactone ratio was between 0.37 (25% treatment) and 0.34
(100% treatment), slightly lower than the ratio of the extract,
however maintaining the pattern of the extract because trans-oak
lactone predominates over the cis-oak lactone.
After six months, both lactone isomer concentrations increased significantly in both types of wines but more importantly
in the grapes from 25% treatment, but without exceeding the
respective perception thresholds (20-23 μg/L, cis isomer; 140
μg/L, trans isomer).37,38 The cis/trans ratio is still more favorable
to the trans-oak lactone in the 25% treatment than in 100%
treatment, as it remains between 0.46 (25% treatment) and 0.44
(100% treatment). The reason for increment oak lactones should
be investigated in future studies; however, these results suggest
that the plant accumulates part of these lactones in the berries as
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Table 8. Concentration of Volatile Compounds (μg/L) in Wines from EþG Grapevine Treatmenta
control wine
wine from EþG treatment
end of alcoholic fermentation six months after alcoholic fermentation end of alcoholic fermentation six months after alcoholic fermentation
cis-oak lactone
nd
nd
3.3 ( 0.2a
17 ( 2b
trans-oak lactone
nd
nd
8 ( 1a
15 ( 3b
furfural
29.1 ( 4.31a
30.0 ( 1.21a
29.0 ( 1.1a
29.4 ( 0.1a
5-methylfurfural
3.40 ( 0.31a
6.6 1 ( 0.32b
9.21 ( 0.01c
8.92 ( 0.22c
4.0 ( 0.3a
11.3 ( 0.5b
42.1 ( 2.0c
158.0 ( 22.3d
6-methoxyeugenol
2.01 ( 0.20a
0.90 ( 0.04a
0.90 ( 0.05a
23.21 ( 1.10b
guaiacol
4-vinylguaiacol
2.9 ( 0.2a
566 ( 43b
3.0 ( 0.1a
426 ( 17a
7.9 ( 0.4b
474 ( 24b
8.0 ( 0.6b
362 ( 1a
4-ethylguaiacol
15.2 ( 1.0a
eugenol
14.8 ( 0.6a
20.1 ( 2.0b
103.2 ( 24.3b
3.0 ( 0.2ab
2.0 ( 0.1a
5.0 ( 0.3b
11.1 ( 2.0c
0.50 ( 0.04b
0.44 ( 0.02ab
0.60 ( 0.03a
acetovanillone
2.0 ( 0.2a
1.7 ( 0.1a
2.3 ( 0.1a
17.3 ( 3.1b
methyl vanillate
0.26 ( 0.02a
0.36 ( 0.01c
0.31 ( 0.02b
1.31 ( 0.30d
4-ethylphenol
vanillin
0.70 ( 0.02b
a
0.05) between columns. Control: untreated grapevines; EþG: grapes treated with eugenol plus guaiacol solution.
nonvolatile precursor forms and later, as a result of the winemaking process and the chemical hydrolysis at the low pH of
wines, are released at the end of the alcoholic fermentation and
mainly after six months. There is no possibility for the formation
of glycoconjugates of the oak lactone ring molecules; although
oak lactone precursors have been described as ring-opened cisand trans-oak lactone glucosides and gallates that can undergo
the lactonization process at wine pH.39
The concentration of furfural and 5-methylfurfural in all samples
(Table 7) were below the values found by other authors in wine aged
in contact with oak24,40 and lower than their olfactory threshold (88
mg/L and 20 mg/L, respectively).41,42 The furanic compounds give
the wine a bitter almond aroma and are considered to enhance the
aroma of the lactones.43 Extract treatments did not affect the furfural
content but increased 5-methylfurfural content, although there were
no differences between the two oak extract formulations used. After
six months, there was a significant increase in 5-methylfurfural in the
control wine while it remained constant in the other wines.
In the control wine a significant increase of eugenol is seen
after six months (Table 7), which shows that it is released from
the soluble precursor forms that must be in the wine. The wines
from the grapevines treated with extracts contain significantly
higher amounts of eugenol than the control wine. After six
months, its concentration increased substantially, exceeding its
olfactory threshold (15 μg/L according to Cutzach et al.44).
These facts suggest the presence in wines of soluble eugenol
precursors from which eugenol is released with age, and that
grapes from oak extract treatments accumulate eugenol as
nonvolatile precursors. In aged wines kept for one year in bottles,
a decrease in eugenol has been described.45 These results suggest
an important difference among wines from grapes treated with
oak extracts and aged wines, given that eugenol from the latter
decreases over time, as its presence in the wine is due to its
extraction from the wood of the barrel in the form of the free
compound, while in the wines from the grapes treated with oak
extract, eugenol is probably mainly in a nonvolatile precursor
form biosynthesized in grapes, which could be released with age.
6-Methoxyeugenol has a spicy aroma and increased significantly after six months due to the effect of the two oak treatments
(Table 7); a greater proportion comes from 25%, exceeding its
olfactory threshold (12 μg/L46).
Lower guaiacol content was found in the control wine than in
the treated grapes, not exceeding its olfactory threshold (9.5 μg/
L, smoke aroma descriptor, according to Ferreira et al.42); in any
of the cases six months after the end of alcoholic fermentation,
the concentrations of guaiacol significantly decreased in the
wines from treated grapes, so its behavior is different from that
observed for eugenol. The major compound in all wines is
4-vinylguaiacol, having concentrations within the range described for white wines30 and exceeding its olfactory threshold
(10 μg/L according to Guth47). The concentration of 4-vinylguaiacol is higher than the ethylphenols concentration in white
wines, contrary to what happens in red wines,48 and decreases
significantly after six months, independently of the grapevine
treatment (Table 5). Ethylphenols content remains constant in
the control wine over time, and in wines made with treated
grapes, their concentrations are slightly higher. The ethylphenols, 4-ethylphenol and 4-ethylguaiacol, may come from the
grapes (Table 5), but they can also be formed by vinylphenol
enzymatic reduction.49 In wines from grapevine oak extract
treatments, the concentrations are higher than in the control
wine (Table 7) which may be due to the contribution of the
extracts, suggesting that there are no soluble precursors, as there
is no release after six months, which is the case especially predominant with eugenol.
After the alcoholic fermentation, the concentrations of vanillin,
acetovanillone, and methyl vanillate, that contributed the spicy aromas
and vanilla, were very low and below those found by other authors in
wines in contact with oak.40,45 After six months, a significant increase
in acetovanillone and methyl vanillate was observed, which was higher
in wines from the 25% treatment, although none of them exceeded
the olfactory threshold (60 μg/L for vanillin, 1000 μg/L for
acetovanillone, and 3000 μg/L for methyl vanillate).50 Therefore,
these two compounds could probably be incorporated into the plant
by the oak extract treatments and stored in the form of nonvolatile
precursors, only to be released in the wines with time.
Eugenol and guaiacol are common compounds in aged wines,
which are extracted from the oak wood into the wine.40,50 These
3259
ARTICLE
Figure 1. Sensory profiles of wines at the end of the alcoholic fermentation and six months after the alcoholic fermentation. *Level of significance: p <
0.05 with control wine.
compounds have also been subject to studies related to the
smoke taint of wines from grapevines exposed to smoke.7,8 This
effect is due to the volatile phenols released from smoked grapes
throughout the winemaking process, making use of a solution of
eugenol and guaiacol as a benchmark to check the evolution of
these compounds in grapes and wines (Table 8). Both eugenol
and guaiacol significantly increase their concentration compared
to its control once the fermentation has finished, with higher
eugenol content than guaiacol. After six months, the content of
eugenol increased to concentrations four times higher, while
guaiacol remained constant. In view of these results and those
observed within the grapes (Table 5), we believe that eugenol
and guaiacol are probably assimilated by grapes mainly in the
form of nonvolatile compounds and that during the winemaking
process both compounds are probably released from their
precursors, eugenol being released in greater proportion than
guaiacol (Table 7). Furthermore, in wines at the end of the
fermentation, a large part of the soluble nonvolatile precursors of
eugenol is maintained and released with time. These results
confirm earlier comments regarding the wines from the grapevines treated with oak extracts. Also, Hayasaka et al.34 showed
that berries and leaves from grapevines exposed to smoke
stored guaiacol from smoke, like β-D-glucopyranoside, and
transfer it to the must where the aglycone is released by
enzymatic and chemical hydrolysis. The application of the
EþG solution to the grapevine has shown increases of other
compounds in relation to the control wine at the end of the
alcoholic fermentation, especially the two lactones, 4-ethylguaiacol and 4-ethylphenol. It is possible that the application of the
EþG solution modifies the biosynthesis of 4-ethylguaiacol and
4-ethylphenol, as they are shikimic derivatives.31 In addition,
4-ethylguaiacol could also come from the enzymatic reduction of
4-vinylguaiacol,49 since its content significantly decreased. However, we cannot provide any explanation for the increase in transand cis-oak lactones (cis/trans ratio of 0.41) so it will be the
subject of future research.
Six months after the end of fermentation, there was a
significant increase in 6-methoxyeugenol, suggesting an increase
in the formation of its putative nonvolatile precursor from
eugenol, as it was not detected in grapes (Table 5). Moreover,
increases in the concentrations of the lactones were also observed, but the cis/trans ratio is higher than 1, which favored the
formation of the cis isomer. The concentrations of ethylphenols
showed a significant increase that could be ascribed to the
significant decrease of vinylphenols. The vanillin derivatives also
increased significantly, especially acetovanillone, indicating that
there were also adjustments in the formation of soluble precursors of these compounds in the berries due to the EþG
treatment. In summary, the results obtained as a consequence of
the EþG treatment for the grapevines are new and should be
confirmed in future studies, given their relevance.
Sensory Analysis. Figure 1 shows the sensory analysis of the
control wines and those made from grapevines treated with oak
extracts. At the end of alcoholic fermentation, the wines showed
all the characteristics of young Verdejo wines, finding significant
differences due to the oak extracts only in the “mouthfeel”
attribute that was greater when the wines came from grapevines
treated with 25% oak extract. This attribute is one of six that have
been evaluated in the gustatory phase and has the highest average
value followed by the “persistent” and “bitterness” attributes,
3260
ARTICLE
Figure 2. Canonical discriminant analysis of volatile compound concentrations and sensory attributes in wines at the end of the alcoholic fermentation
(AF) and after six months.
both showing no significant differences among the three wines. The
predominant attribute of the visual phase was “yellow tone”, and the
lowest attribute was the “colour intensity”. Among the attributes of the
olfactory phase, the highest average scores were for “fruity,” “varietals”,
and “herbaceous”, typical for wines from this grape variety.14 After six
months, wines from the grapevines treated with oak extracts suffer a
major sensory change that is highlighted in the three sensory phases.
Thus, the “yellow tone” is significantly higher in the control wine,
which in turn has the lowest values for the “green tone” attribute.
These results show a significant improvement in color quality of the
wines from treated grapevines, because in young Verdejo wines the
color green is associated with higher quality. In the olfactory phase,
there is a significant increase in the “wood” attribute, especially when
the 25% formulation was used. These results are consistent with those
obtained in the study of the volatiles composition of these wines
(Table 7), because their concentrations of cis- and trans-oak lactones
responsible for the “wood” aromatic note were high, although their
olfactory threshold was not exceeded. However, synergic effects
between these compounds and others such as furfural and 5-methylfurfural have been described, increasing the “lactone” aromatic
perception.43 In the gustatory phase, two attributes, “mouthfeel” and
“astringency”, increased significantly compared to the control wine,
which are slightly higher in wines from the treatment of the grapevines
with the 25% extract formulation. Also, grapes treated with this 25%
formulation had the highest average values for “persistence” and
“balance” attributes. Consequently, treatments with oak extracts on
grapevines produce sensory attributes in the wines that are revealed six
months after the alcoholic fermentation, being characteristic of wines
that have been aged in oak barrels.14
The discriminant analysis applied to wines (control, and 25% and
100% oak extract treatment) in the two sampling times (at the end
of the alcoholic fermentation and after six months of this) (Figure 2)
was carried out by taking into account the volatile compounds
concentration and their sensory attributes. Sample differentiation
was achieved by two canonical functions; the first explained the
97.1% of the total variance and the second explained the 1.9%. The
most important discriminating variables were 4-ethylguaiacol,
6-methoxyeugenol, guaiacol, methyl vanillate, and 4-vinylguaiacol,
followed by the attributes of yellow tone, wood, and astringency.
After the alcoholic fermentation, the wines are quite similar, while
the wines after six months are clearly separated in the graph, thus
showing the full extent of the effect of oak extract treatment. This
statistical analysis corroborates that the wines are differentiated in
relation to their aroma composition only after time and not at the
end of alcoholic fermentation.
In conclusion, the application of aqueous oak extracts to grapevines
of the white Verdejo cultivar affects the aroma composition of grapes
and wines. The results suggest that berries store volatiles, which come
from the oak extract formulations, as nonvolatile precursors, some of
which are released during the winemaking process. This is especially
evident after six months, when the highest release of these volatiles
occurs, significantly impacting the aroma. Sensory analysis shows that
wines from grapevines treated with oak extracts maintain the typical
aroma character of Verdejo wines at the end of fermentation, but after
six months, the color is greener, they are more astringent, and the
aroma of oak wood is highlighted like that of wines aged in barrels.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ34 967 599310. Fax: þ34 967 599238. E-mail:
[email protected].
3261
Funding Sources
Many thanks for the financial support given by the Ministerio de
Ciencia e Innovacion to the Project AGL2009-08950 and to the
contract for T.G.-C and also by the Junta de Comunidades de
Castilla-La Mancha for the FPI scholarship for A.M.M.-G.
’ ACKNOWLEDGMENT
We express our gratitude to Kathy Walsh for proofreading the
manuscript.
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3263
7.3. ARTÍCULO III
Cada variedad posee unas características biológicas y químicas que
influyen en la composición final del vino. La principal diferencia que existe entre
las uvas blancas y tintas es la presencia en estas últimas de antocianos en el
hollejo, además de las técnicas de vinificación empleada para cada una de ellas.
La fermentación en presencia de hollejos, las temperaturas más elevadas de la
fermentación alcohólica, así como la fermentación maloláctica (todo esto
habitual en la vinificación en tinto pero no en blanco), tienen importantes efectos
sobre la composición final del vino, en especial sobre el aroma y el color.
Con este trabajo se ha continuado en la misma línea del anterior, pero
usando la variedad tinta Petit Verdot, con el objetivo de determinar si la
aplicación foliar a la vid de un extracto acuoso de roble influye en la
composición volátil de uvas tintas y en la de sus respectivos vinos que han
sufrido la fermentación maloláctica.
117
Artículo III
Para ello, vides de la mencionada variedad fueron tratadas en envero con
un extracto acuoso de roble francés mediante pulverización sobre la parte foliar,
realizándose 4 tratamientos diferentes: 25%(1) (una aplicación de extracto
diluido al 25 %), 25%(4) (cuatro aplicaciones de extracto diluido al 25 %), 100
% (una aplicación del extracto concentrado), E+G (disolución de referencia
constituida por eugenol y de guayacol) y un control (sin tratar). Los vinos se
elaboraron según el sistema clásico de vinificación en tinto. Los muestreos se
realizaron el día de la vendimia, después de la fermentación alcohólica, después
de la fermentación maloláctica y transcurridos 8 meses de ésta. Para éste estudio
se analizaron los parámetros enológicos de las uvas y de los vinos, y los
compuestos volátiles del extracto de roble, de las uvas y de los vinos. Los
volátiles analizados en las muestras fueron los mismos que se determinan en el
extracto de roble.
Los resultados mostraron que las uvas de los tratamientos únicamente se
diferenciaron de las uvas control por tener mayor concentración de furfural y
guayacol. Al igual que observamos con la variedad Verdejo tras la aplicación de
un extracto similar, las whisky lactonas y el 6-metoxieugenol, no fueron
detectados en las uvas pero sí en los vinos, aunque las lactonas únicamente en los
vinos procedentes de los tratamientos. Por lo tanto los resultados volvieron a
sugerir que el origen de las lactonas en los vinos podría atribuirse al extracto de
roble.
La mayor parte de los compuestos estudiados se encontraron en mayor
concentración en los vinos procedentes de los tratamientos que en los vinos
control. La evolución de estos compuestos dependió del tipo de aplicación y del
compuesto. Con este estudio se confirmaron los resultados observados en
Verdejo, lo que de nuevo sugiere que las uvas acumulan los compuestos volátiles
del extracto, principalmente como precursores no volátiles, y que la mayoría de
ellos se liberan después de la fermentación alcohólica. En todos los muestreos
118
Artículo III
analizados fue posible distinguir el vino control de los vinos procedentes de los
diferentes tratamientos. Únicamente tras la fermentación alcohólica fue posible
diferenciar entre los vinos procedentes de los tratamientos dependiendo del tipo
de aplicación.
Las uvas de los tratamientos E+G mostraron mayor concentración de
algunos compuestos con respecto al control, en especial en eugenol y guayacol,
lo que demuestra un comportamiento diferente según la variedad, ya que Verdejo
no mostró este incremento. Los vinos Petit Verdot tuvieron mayores
concentraciones de eugenol y guayacol que los vinos de Verdejo, lo que puede
deberse tanto a un comportamiento distinto según variedad, ya comentado, como
al diferente proceso de vinificación empleado en cada caso.
Por lo tanto, este artículo muestra que la variedad tinta Petit Verdot
también es receptiva a la aplicación de los extractos de roble, ya que las uvas
pueden acumular los compuestos volátiles de los extractos posiblemente como
glicósidos. Por lo que necesariamente se debe comprobar si la hipótesis
propuesta sobre la asimilación de los compuestos volátiles del extracto de roble
por la vid y su posterior glicosilación es acertada.
119
Food Chemistry 132 (2012) 1836–1845
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Applications of an oak extract on Petit Verdot grapevines. Influence on grape
and wine volatile compounds
Ana M. Martínez-Gil, Teresa Garde-Cerdán, Amaya Zalacain, Ana I. Pardo-García, M. Rosario Salinas ⇑
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain
a r t i c l e
i n f o
Article history:
Received 26 May 2011
Received in revised form 18 October 2011
Accepted 7 December 2011
Available online 16 December 2011
Keywords:
Volatile compounds
Oak extract
Vineyard
Grape
Wine
Petit Verdot
a b s t r a c t
Petit Verdot vineyards were treated at veraison with a commercial aqueous French oak extract in order to
determine if the extract’s volatile components can be transferred to grapes and then to wines. Three different formulations (25% (one application), 25% (four applications) and 100%) were tested, together with
an eugenol and guaiacol standard solution to better follow their behaviour. The volatile compounds of
treated grapes and their wines after alcoholic and malolactic fermentation and after 8 months were analysed by stir bar sorptive extraction and gas chromatography mass spectrometry (SBSE-GC–MS). The
results showed that the grapes stored the volatile compounds mainly as non-volatile precursors, and
some of these were released after winemaking. In the case of wines, it was possible to distinguish the
control versus the ones from vineyard treatments. The different oak extract applications were evident
only after alcoholic fermentation sampling, making it very interesting for young wines.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Wine is a product that provides a clear example of a saturated
market and winemakers are looking for a more sustainable path
in the ‘‘economy of wine’’. One of the most pursued objectives is
to achieve wine quality differentiation, for example by proposing
new ways of vineyard management and winemaking technologies.
To this end, aroma and flavour are arguably a wine’s most important distinguishing marks and key drivers of consumer choice
(Pretorius, 2000; Pretorius & Bauer, 2002). With regard to aroma, recent studies have manifested a growing interest in modulating the
composition of grapes and wines (Diago, Vilanova, Blanco, & Tardáguila, 2010; Martin & Bohlmann, 2004). Moreover, it has been
proven that factors outside the vineyards, as well as various treatments applied to these, are able to modify the aroma composition
of grapes and therefore the wines made from them. A clear example
is the Kennison research (Kennison, Wilkinson, Williams, Smith, &
Gibberd, 2007; Kennison, Gibberd, Pollnitz, & Wilkinson, 2008;
Kennison, Wilkinson, Pollnitz, Williams, & Gibberd, 2009), which
studied how grapevine exposure to smoke fire produced wines with
smoke sensorial notes, or the Martínez-Gil, Garde-Cerdán, Martínez,
Alonso, and Salinas (2011) work, which demonstrated a change in
the aroma profile of white grapes and their wines after applying
an extract to the vineyard.
⇑ Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238.
E-mail address: [email protected] (M.R. Salinas).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.12.016
The grape aroma fraction is constituted by a complex group of
substances that can occur in both free forms: as volatile molecules,
which are odour-active compounds, or as odourless precursors.
The latter, found mainly in grape skins, are related with the wine
aroma potential, as during the winemaking process and aging, they
are released and/or modified by the action of aroma-enhancing
yeasts or by the acidity conditions of the medium and the time
(Howell et al., 2005; Lilly et al., 2006; Swiegers, Pretorius, & Bauer,
2006; Swiegers et al., 2006). The traditional vinification process in
red wines includes maceration with the skins before and during
alcoholic fermentation and subsequent malolactic fermentation.
These two steps have significant effects on the final composition
of wine, both in aroma and colour (Moreno-Arribas & Polo, 2009).
The wine aroma composition may also be influenced by storage
in oak barrels, since barrels provide wines with much appreciated
aromatic notes, such as coconut, vanilla, clove, smoke, and wood.
These are due to the oak lactones, vanillin, eugenol, guaiacol, furanic compounds, etc. (Garde-Cerdán et al., 2010; Pérez-Prieto,
López-Roca, Martínez-Cutillas, Pardo-Mínguez, & Gómez-Plaza,
2003). This practice, usually used with red wines, implies an elevated cost and requires wines to remain in cellars for long periods
of time.
Most red varieties are considered non aromatic, although they
do provide wines with some characteristic aromatic notes. One
example is the Petit Verdot variety, which has been studied in this
work. This variety has commonly been used in wine coupages,
although nowadays it is becoming more popular, especially in
A.M. Martínez-Gil et al. / Food Chemistry 132 (2012) 1836–1845
warm countries, for the production of varietal wines. The latter are
characterised by contributing to the wine aroma composition with
aromatic notes, fresh, ripe fruit, eucalyptus and menthol. Furthermore, Petit Verdot is well suited for oak aging and its wines can
be stored over long periods in barrels.
The aim of this work was to determine if the aroma composition
of a red grape variety, such as Petit Verdot, can be changed by
applying a commercial oak extract to grapevine via foliar. The effect of such treatments on the aroma composition has also been
followed up in the respective wines.
2. Materials and methods
2.1. Oak extract
French toasted aqueous oak extract (103C) supplied by Protea
France S.A.S. (Gensac la Pallue, France) was used for application on
grapevines. No data can be given on the extract preparation,
although this company provides extracts with different toasted
oak material carried out at different maceration times and temperatures. The volatile composition of the oak extract used is defined
by: 0.06 mg/l of cis-oak lactone; 0.03 mg/l of trans-oak lactone;
2.60 mg/l of furfural; 0.35 mg/l of 5-methylfurfural; 0.01 mg/l of
eugenol; 0.95 mg/l of 6-methoxyeugenol; 0.24 mg/l of guaiacol;
0.02 mg/l of 4-vinylguaiacol; 0.01 mg/l of 4-ethylguaiacol; 0.01
mg/l of 4-ethylphenol; 5.57 mg/l of syringol; 3.81 mg/l of vanillin;
1.35 mg/l of acetovanillone; and 1.15 mg/l of methyl vanillate. Since
this extract is a food additive, there is a guarantee that no toxicity
risk exists.
2.2. Grapevine treatments
This study used Petit Verdot, a Vitis vinifera red variety grown in
the La Mancha Region (Albacete Province, southeastern Spain),
from the 2009 harvest. The grapevines were cultivated in trellis
and were fitted with a drip irrigation system to assure adequate
water needs, as this region registers only 300–400 mm of rainfall
per year. The annual average temperature was 13 °C, with a minimum of 15 °C (January) and a maximum of 40 °C (August).
Different oak extract treatments were applied to the grapevines
during veraison. For all treatments, a 0.5 ml per litre of adjuvant
Fluvius (BASF, Germany) was added; since this is a wetting agent
typically used for foliar herbicide treatment. The vineyards were
treated with different concentrations of the extract. First of all, this
extract was diluted with water to four parts. This diluted extract
was applied once on the 7th day post-veraison (25%(1) treatment)
and also four times, on the 4th, 7th, 10th, and 13th days postveraison (25%(4) treatment). Also the undiluted extract was
applied once on the 7th day post-veraison (100% treatment). In
addition, an aqueous solution of eugenol and guaiacol standard
compounds (Sigma–Aldrich, Gillingham, England) (6 g/l of each
compound) was used for a single treatment, 7th day post-veraison
(E + G treatment).
Each treatment was carried out on 10 plants in the same row,
leaving five untreated plants between the different applications
to avoid contamination. 250 ml of each formulation was applied
evenly per plant by spraying over leaves. The treatments were carried out when the environmental temperature was below 20 °C, at
approximately 7 óclock in the morning. Moreover, 10 plants were
not treated (control).
1837
between 2.2 and 2.4. Grape yield per plant was calculated by dividing the total mass production (kg) by the number of plants. Grapes
from the whole clusters were destemmed and mixed. After this,
some berries were randomly separated in order to measure the
weight of 100 berries and their size (caliper digital, Classic Tesa,
Swiss). Also, some grapes were frozen at 20 °C for further analysis.
Vinification was performed in a multitube fermenter (Martínez
Solé y Cía, S.A., Villarrobledo, Spain), which reproduces wine cellar
winemaking conditions. To do this, the remaining grapes were
crushed and half a litre of the must from each treatment was taken
for a grape oenological parameter analysis. Fifty milligram of
potassium metabisulphite per litre was added to the rest of the
vintage mass for each treatment. Then, the must was divided into
two batches of approximately 5 litres each, as the fermentation
was done in duplicate. Skin maceration was performed at 3 °C for
48 h to facilitate the extraction of varietal aromas of the wines
(Flanzy, 2000). After that, a QA23 yeast strain of Saccharomyces
cerevisiae subsp. cerevisiae was inoculated at a dose of 0.2 g/l
according to the recommendation of Lallemand (Spain).
The alcoholic fermentation temperature was maintained at 24 °C
and the density was measured daily. The alcoholic fermentation finished when the reducing sugars were below 2.5 g/l. Free SO2 concentration was corrected to 25–35 mg/l. For each of the fermentations, a
sample was taken and was frozen at 20 °C until analysis.
At the end of the alcoholic fermentation, the wines were
pressed manually and the skins and seeds were removed. Malolactic fermentation was induced using a commercial bacterium strain
of Oenococcus oeni (Lallemand, Spain) in a proportion of 10 mg/l.
The malolactic fermentation was carried out at 25 °C in the same
multitube fermenter as the alcoholic fermentation. The correct
development of malolactic fermentation was monitored by measuring the daily concentrations of malic and lactic acids. The fermentation was considered finished when the concentration of
malic acid was approximately 0.4 g/l. For each wine, a sample
was taken and preserved at 20 °C for subsequent analysis.
At the end of malolactic fermentation, the wines were stored in
bottles at 14 °C for 8 months. After this time a sample was taken
from each of them and was frozen at 20 °C until analysis.
2.4. Oenological parameter analysis
Yeast assimilable nitrogen (YAN), °Baumé, reducing sugars,
probable alcohol, titratable acidity (g/l tartaric acid), volatile acidity (g/l acetic acid), pH, reducing sugars, and alcohol degree from
the different samples were measured in triplicate following the
methods established by ECC (1990).
Malic and lactic acids were analysed in wines using HPLC-RID
(Agilent 1100, Palo Alto, USA) with a column block heater and
refractive index detector (RID) (Agilent 1200). The mobile phase
was 0.004 M H2SO4 flowing at 0.4 ml/min and 75 °C on a PL Hi-Plex
H, 8 lm, 300 7.7 mm column (Varian, Middelburg, The Netherlands). All the samples were filtered (0.45 lm pore filter) and directly injected into the column. Injection volume was 10 ll. The
RID was at 55 °C and the total time of analysis was 30 min. Quantification was based on five-point calibration curves (R2 > 0.97)
using respective standards (Sigma–Aldrich, Madrid, Spain) in
water. The concentration ranges of the calibration were: malic acid
(0.3–10 g/l) and lactic acid (0.02–5 g/l). All the analyses were done
in triplicate; therefore, the result of each wine is the average of six
values (n = 6), since the fermentations were done in duplicate.
2.5. Analysis of volatile composition
2.3. Winemaking
Grapes were harvested on September 16th, at their optimum
maturation moment with the °Baumé/titratable acidity ratio
2.5.1. Volatile compound extraction
Stir bar sorptive extraction (SBSE) was used as the headspace
mode (HS-SBSE) for grape analysis while the immersion mode
1838
was chosen for wine analysis. The extraction of volatile compounds
in grapes by HS-SBSE was carried out according to the Martínez-Gil
et al. (2011) method. Grapes were thawed, crushed and macerated
for 2 h and then centrifugated at 3000 rpm for 30 min. In a 50 ml
vial, 22 ml of sample were added together with 0.1 g of NaCl and
10 ll of internal standard c-hexalactone (Sigma–Aldrich) solution
at 1 ll/ml, in absolute ethanol (Merck, Damstard, Germany) per
ml of sample. A polydimethylsiloxane coated stir bar (twister,
0.5 mm film thickness, 10 mm length, Gerstel, Mülheim and der
Ruhr, Germany) was inserted into the twister-headspace vial and
hermetically closed. The vial was introduced into a heater (Selecta,
Barcelona, Spain) at 60 °C, and was stirred with a common magnetic stirrer for 1 h at 500 rpm.
Wine volatile compounds were extracted by SBSE in immersion
mode according to Marín, Zalacain, De Miguel, Alonso, and Salinas
(2005). For this, the twister was introduced into 10 ml of sample to
which 100 ll of the same internal standard c-hexalactone was
added. Samples were stirred at 500 rpm at room temperature for
1 h.
After both extraction processes, the twister was removed,
rinsed with distilled water and dried with a cellulose tissue, and later transferred to a thermal desorption tube for GC–MS analysis.
2.5.2. GC–MS analysis
The volatile compounds were desorbed from the stir bar following the same methodology as per the Martínez-Gil et al. (2011)
method. The main conditions for analysis were: oven temperature
at 330 °C; desorption time, 4 min; cold trap temperature, 30 °C;
helium inlet flow 45 ml/min. The compounds were transferred into
the Hewlett–Packard LC 3D mass detector (Palo Alto, USA) with a
fused silica capillary column (BP21 stationary phase 30 m length,
0.25 mm i.d., and 0.25 lm film thickness; SGE, Ringwood, Australia). For mass spectrometry analysis, the electron impact mode
(EI) at 70 eV was used and the detection and quantification was
in selected ion monitoring (SIM) mode. The detector temperature
was 150 °C. Identification was carried out by comparison with
the mass spectrum and the retention index of chromatographic
standards and data found in the bibliography (Martínez-Gil et al.,
2011).
All the grape analyses were done in triplicate (n = 3) with three
different extractions. Each wine sample was analysed in triplicate,
so three different extractions were performed, and since each wine
was analysed in duplicate, the results come from an average of six
analyses (n = 6).
2.6. Statistical analysis
Statistical analysis was carried out using SPSS Version 19.0 statistical package for Windows (SPSS, Chicago, USA). The volatile
compound data were processed using variance analysis (ANOVA).
The differences between means were compared using the least significant difference (LSD) test at 0.05 probability level. A discriminant analysis was performed with the oak volatile composition in
the grapes. Another discriminant analysis was done with the concentration of oak volatile compounds in the control wine and in the
wines from grapevines treated with oak extract, at the end of alcoholic and malolactic fermentations and 8 months after the malolactic fermentation.
3. Results and discussion
The starting point of this paper is Martínez-Gil et al. (2011)
where Verdejo white vineyard were treated with an oak extract
and their wines were also evaluated. Due to the satisfactory results
obtained previously, a more complete oak treatment was carried
out this time but with a red variety, such as Petit Verdot. Due to
the variety, Petit Verdot vinification includes as well the malolactic
fermentation, which was avoided with the Verdejo one. Together
with the different oak extract treatments, an application with a
high concentrated standard solution of eugenol and guaiacol was
performed to check easier the behaviour of these compounds in
grapes and their wines after vineyard applications.
3.1. Grape oenological parameters
Table 1 shows the oenological parameters of grapes from all
treatments (control, 25%(1), 25%(4), 100%, and E + G). Significant
differences were observed between the control and the 25%(1)
treatment grape yield, being higher in the control. Among the other
treatments no significant differences were observed for this
parameter. The different treatments did not affect the weight of
100 berries. However, all the grapes from the different treatments
showed a higher berry caliber than the control, with grapes from
the E + G treatment presenting the highest berry size. In all cases,
YAN was higher than or close to 140 mg N/L, which is the concentration needed to complete alcoholic fermentation (Bell & Henschke, 2005). YAN, °Baumé, probable alcohol, and pH of the grapes
from oak extract treatments did not show significant differences
with respect to the control. Titratable acidity and °Baumé/TA were
not affected by any of the treatments applied to the vineyard. Nevertheless, grapes from the E + G treatment presented a higher YAN
and lower values of °Baumé, probable alcohol, and pH than the
control grapes (Table 1). The high concentration of eugenol and
guaiacol in the E + G solution can produce plant stress when sugar
is assimilated and changes are produced in skin characteristics
(Conde et al., 2007). These grapes were the ones with a lower °Baumé and a higher berry caliber. In addition, these grapes presented
the lowest total weight of seeds (data not shown) so the highest
berry size may be due to a major absorption of water that would
logically produce a dilution in the sugar content.
3.2. Petit Verdot grape volatile composition
This study focuses on the volatile compounds that are transferred
to grapes from the different oak extract formulations applied to
vineyards. The compounds which were followed were those ones
presented within the oak extract, such as cis-oak lactone, trans-oak
lactone, furfural, 5-methylfurfural, eugenol, 6-methoxyeugenol,
guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, 4-ethylphenol, syringol,
vanillin, acetovanillone, and methyl vanillate.
Table 2 shows the concentration of the free volatile compounds
in the control grapes and in the grapes from the different grapevine
treatments (25%(1), 25%(4), 100% and E + G). Neither oak lactones
nor 6-methoxyeugenol were detected in any of the samples. However, the treated grapes showed a higher concentration of furfural
than the control and significant differences were only observed between grapes from the 25%(1) and 25%(4) treatments, with concentrations being lower when only one application was done (Table 2).
The concentration of 5-methylfurfural, eugenol, 4-ethylguaiacol
and vanillin did not show significant differences between the
grapes from the oak treatments and control grapes. However,
grapes from the 100% oak extract treatment presented the lowest
concentrations of acetovanillone and methyl vanillate, as well as
the lowest concentration of guaiacol together with the control.
Moreover, the 25%(4) and 100% grapes showed lower concentrations of 4-vinylguaiacol and 4-ethylphenol than the control grapes.
With respect to syringol, we observed that the grapes from the oak
treatments had significantly lower concentrations than the control
grapes.
On the other hand, E + G grapes showed significantly higher
concentrations of eugenol and guaiacol in relation to the control
1839
Table 1
Oenological parameters of grapes on harvest day after the different grapevine treatments.
Treatments
Yield (kg/
plant)
Weight of 100
berries
Caliber of berries
(mm)
YAN (mg
N/L)
°Baumé
Probable alcohol
(v/v,%)
pH
Titratable
acidity (g/l)
°Baumé/TA
Control
25%(1)
25%(4)
100%
E+G
5.64 ± 0.24b
5.07 ± 0.35a
5.26 ± 0.21ab
5.13 ± 0.28ab
5.36 ± 0.32ab
121.26 ± 17.59a
131.28 ± 8.95a
117.99 ± 11.56a
114.91 ± 12.58a
142.52 ± 16.3a
10.68 ± 0.11a
11.37 ± 0.05bc
11.42 ± 0.09c
11.06 ± 0.13b
12.81 ± 0.07d
154 ± 3ab
140 ± 4a
140 ± 5a
168 ± 3bc
182 ± 7c
13.6 ± 0.2b
13.4 ± 0.1b
13.4 ± 0.2b
13.2 ± 0.1b
12.2 ± 0.1a
14.6 ± 0.2b
14.4 ± 0.1b
14.4 ± 0.1b
14.2 ± 0.2b
12.6 ± 0.1a
3.62 ± 0.01bc
3.64 ± 0.04bc
3.66 ± 0.02c
3.51 ± 0.06ab
3.42 ± 0.02a
5.6 ± 0.1a
5.8 ± 0.2a
5.6 ± 0.2a
5.4 ± 0.3a
5.6 ± 0.2a
2.43 ± 0.01a
2.31 ± 0.06a
2.39 ± 0.05a
2.44 ± 0.11a
2.18 ± 0.10a
All parameters are given with their standard deviation (n = 3). Different letters in the same column indicate significant differences (level of significance of p > 0.05) between
treatments. Control: untreated grapes; 25%(1): grapevines treated with oak extract diluted to a quarter applied once; 25%(4) grapevines treated with oak extract diluted to a
quarter applied four times; 100%: grapevines treated with oak extract without dilution applied once; E + G: grapevines treated with eugenol plus guaiacol solution applied
once.
TA, titratable acidity (as g/l tartaric acid); YAN, yeast assimilable nitrogen.
Table 2
Concentration of volatile compounds (lg/kg) in grapes from the different grapevine treatments.
cis-Oak lactone
trans-Oak lactone
Furfural
5-Methylfurfural
Eugenol
6-Methoxyeugenol
Guaiacol
4-Vinylguaiacol
4-Ethylguaiacol
4-Ethylphenol
Syringol
Vanillin
Acetovanillone
Methyl vanillate
Control
25%(1)
25%(4)
100%
E+G
n.d.
n.d.
4.46 ± 0.92a
4.38 ± 0.82ab
1.05 ± 0.16a
n.d.
0.82 ± 0.13a
1.39 ± 0.06b
0.13 ± 0.01a
1.32 ± 0.15c
3.06 ± 0.32b
1.64 ± 0.20a
4.53 ± 0.22b
3.81 ± 0.15b
n.d.
n.d.
8.17 ± 1.77b
4.44 ± 1.18ab
0.73 ± 0.184a
n.d.
4.77 ± 0.10b
1.17 ± 0.22ab
0.11 ± 0.01a
1.12 ± 0.10bc
1.95 ± 0.02a
1.52 ± 0.20a
4.08 ± 0.62ab
3.57 ± 0.52b
n.d.
n.d.
10.82 ± 0.03c
4.13 ± 0.24a
1.14 ± 0.14a
n.d.
5.93 ± 0.083b
1.11 ± 0.16a
0.13 ± 0.01a
0.81 ± 0.09a
2.14 ± 0.26a
1.41 ± 0.09a
4.04 ± 0.11ab
3.13 ± 0.20b
n.d.
n.d.
9.65 ± 0.93bc
5.72 ± 1.00b
1.96 ± 0.14a
n.d.
1.86 ± 0.18a
0.96 ± 0.11a
0.13 ± 0.00a
1.08 ± 0.10b
1.78 ± 0.16a
1.41 ± 0.05a
3.32 ± 0.22a
2.36 ± 0.13a
n.d.
n.d.
9.21 ± 0.83bc
5.73 ± 0.0b
41.51 ± 4.52b
n.d.
45.77 ± 2.34c
2.28 ± 0.05c
0.16 ± 0.02b
1.12 ± 0.12bc
5.53 ± 0.61c
1.73 ± 0.30a
3.89 ± 0.75ab
3.47 ± 0.67b
All parameters are given with their standard deviation (n = 3). n.d.: not detected. Different letters indicate significant differences (level of significance of p > 0.05) between
treatments. Control: untreated grapevines; 25%(1): grapevines treated with oak extract diluted to a quarter applied one time; 25%(4): grapevines treated with oak extract
diluted to a quarter applied four times; 100%: grapevines treated with oak extract without dilution; E + G: grapes treated with eugenol plus guaiacol solution.
grapes, as expected. Also, these grapes had a higher concentration
of furfural, 4-vinylguaiacol, 4-ethylguaiacol, and syringol (Table 2)
without affecting the rest of the compounds.
Hence, the different treatments applied to the vineyard affected
vine metabolism, since changes in the aroma composition of the
grapes were observed, especially when the E + G solution was compared with the different applications of oak extracts.
Discriminant analysis was performed on the oak volatile compounds to determine the possibility of differentiating the grapes
after the different treatments applied to the vineyard. The results
provided two functions, with Function 1 able to discriminate
E + G grapes from the rest of the grapes with a 99.7% variance, with
guaiacol and eugenol as the compounds that contributed most to
differentiation, as expected (Figure not shown). However, it was
not able to separate the control grapes from the grapes treated
with oak extracts.
3.3. Wine oenological parameters
The oenological parameters of Petit Verdot wines from each sampling (at the end of alcoholic and malolactic fermentations, and
8 months after the end of malolactic fermentation) are shown in Table 3. At the end of the alcoholic fermentation, the alcohol degree did
not present significant differences between the wines from oak
extract treatments and the control. This parameter tended to decrease after malolactic fermentation, with the wine from the 100%
treatment showing a lower value compared to the control wine. This
decrement could be due to a possible volatilisation of ethanol and/or
the formation of other compounds, such as ethyl esters. E + G wine
showed the lowest alcohol degree, perhaps since its grapes had
the lowest °Baumé. After alcoholic fermentation, the titratable
acidity was significantly higher in wines from all treatments than
in the control. This acidity decreased after the malolactic fermentation, probably due to the transformation of malic acid into lactic
acid. After 8 months of malolactic fermentation, titratable acidity
was similar in all the wines.
With respect to pH, an increment was observed during malolactic fermentation, as expected. After 8 months, the values of pH
were similar in the control and oak extract wines. Wines from
the E + G treatment had lower pH than the control wine in all
the samplings studied. The volatile acidity increased throughout
wine evolution (Table 3). At the end of alcoholic fermentation,
the values found were in the range of 0.20–0.30 g/l established
for the fermentation from grapes with optimum sanitary conditions (Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2006).
The highest volatile acidity was for the control wine and the lowest
was for E + G wine. After malolactic fermentation, there was an increase of volatile acidity, as expected, probably due to the formation of volatile acids, with normal values around 0.40 g/l
(Ribéreau-Gayon et al., 2006). In general, the volatile acidity of
wines was not affected by the different treatments.
Malolactic fermentation proceeded in the same way in all
wines, since malic and lactic acid concentrations did not show significant differences. In all cases, lactic acid presented a tendency to
decrease after 8 months, probably due to the formation of ethyl
lactate, although a decrement on the alcohol degree was only observed for the 100% treatment (Table 3).
3.4. Wine volatile compounds
Fig. 1 shows the wine volatile composition for the control wine
and the wines from the different oak extract treatments at each
1840
Table 3
Oenological parameters of wines at the end of alcoholic and malolactic fermentations and after 8 months from the end malolactic fermentation.
Treatments
pH
Volatile acidity (g/l)
Titratable acidity (g/l)
End of alcoholic fermentation
Control
13.03 ± 0.07b
25%(1)
13.08 ± 0.03b
25%(4)
13.14 ± 0.22b
100%
12.55 ± 0.46b
E+G
11.31 ± 0.52a
Alcohol degree (%, v/v)
Lactic acid (g/l)
3.64 ± 0.01b
3.67 ± 0.00bc
3.69 ± 0.01c
3.67 ± 0.01bc
3.56 ± 0.01a
0.24 ± 0.03c
0.23 ± 0.01c
0.21 ± 0.02bc
0.17 ± 0.01ab
0.15 ± 0.01a
4.88 ± 0.13a
5.49 ± 0.00bc
5.55 ± 0.08bc
5.44 ± 0.06b
5.66 ± 0.03c
End of malolactic fermentation
Control
12.23 ± 0.01c
25%(1)
12.31 ± 0.16c
25%(4)
12.44 ± 0.09c
100%
12.39 ± 0.18b
E+G
11.00 ± 0.06a
3.95 ± 0.01b
3.76 ± 0.00a
3.86 ± 0.09ab
3.86 ± 0.02ab
3.78 ± 0.00a
0.32 ± 0.01b
0.24 ± 0.01a
0.25 ± 0.03a
0.23 ± 0.04a
0.27 ± 0.01ab
3.74 ± 0.01a
4.84 ± 0.08b
4.43 ± 0.71ab
3.88 ± 0.15a
4.16 ± 0.01ab
1.87 ± 0.03a
1.86 ± 0.02a
1.92 ± 0.01a
1.89 ± 0.10a
1.98 ± 0.01a
0.51 ± 0.01a
0.47 ± 0.02a
0.47 ± 0.01a
0.52 ± 0.02a
0.48 ± 0.03a
8 months after malolactic fermentation
Control
12.49 ± 0.26c
25%(1)
12.39 ± 0.05bc
25%(4)
12.61 ± 0.21c
100%
12.12 ± 0.08b
E+G
11.09 ± 0.08a
3.93 ± 0.01b
3.83 ± 0.08ab
3.93 ± 0.02b
3.91 ± 0.03b
3.78 ± 0.01a
0.40 ± 0.05a
0.40 ± 0.04a
0.55 ± 0.04b
0.47 ± 0.05ab
0.44 ± 0.05ab
4.12 ± 0.19a
4.69 ± 0.41a
4.39 ± 0.28a
4.28 ± 0.21a
4.28 ± 0.02a
1.54 ± 0.02a
1.67 ± 0.06a
1.65 ± 0.14a
1.74 ± 0.04a
1.56 ± 0.10a
0.47 ± 0.01a
0.43 ± 0.02a
0.46 ± 0.01a
0.45 ± 0.01a
0.44 ± 0.04a
–
–
–
–
–
Malic acid (g/l)
2.65 ± 0.01a
2.73 ± 0.08a
2.69 ± 0.06a
2.96 ± 0.07b
2.57 ± 0.04a
All parameters are given with their standard deviation (n = 6). Control: untreated grapes; 25%(1): grapevines treated with oak extract diluted to a quarter; 25%(4) grapevines
treated with oak extract diluted to a quarter applied four times; 100%: grapevines treated with oak extract without dilution; E + G: grapevines treated with eugenol plus
guaiacol solution. At each sampling in the winemaking process, different letters indicate significant differences between the samples (level of significance of p > 0.05).
sampling stage (end of alcoholic and malolactic fermentation and
8 months later). As expected, the oak lactones were not found in
the control wine (Fig. 1a and b). However, both oak lactone isomers
were found in the wines from the oak extract treatments, so the
origin of these compounds could be attributed to the oak extract
applied to the grapevines. These results are similar to the ones
found in the Verdejo wines with similar oak treatments
(Martínez-Gil et al., 2011). The evolution of both isomers depended
on the application to the vineyard, since it was observed that the
wine with the highest concentration of cis and trans-oak lactones
after alcoholic fermentation was 25%(4). However, 8 months after
malolactic fermentation, the 100% wine had the highest concentrations of these two compounds. In addition, the concentration of cis
and trans-oak lactones decreased after 8 months in the 25%(1) and
25%(4) wines, whereas there was an increase of both isomers in the
100% wine. The concentration of both isomers increased with time
in Verdejo wines (Martínez-Gil et al., 2011), where no grape maceration was carried out nor malolactic fermentation. Neither of the
two lactones were found in its free form in grapes, so these results
suggest that the plant accumulates these lactones in berries as
non-volatile precursor forms that are later released, especially during alcoholic fermentation. The concentrations of cis-oak lactone
was higher than the trans-oak lactone in all the wines, as also observed in the extract, and the ratio cis/trans ranged from 1.3 to 2,
according to the results found by other authors for French oak
wood (Díaz-Plaza, Reyero, Pardo, & Salinas, 2002; Waterhouse &
Towey, 1994). This ratio increased during the evolution of the
100% wine and decreased in the 25%(1) and 25%(4) wines, although
after 8 months the three wines presented a similar ratio, which
was around 1.5. The concentration of these two isomers never exceeded the olfactory threshold in red wines in any of the cases (46–
54 lg/l, cis isomer; 370 lg/l, trans isomer) (Brown, Sefton, Taylor, &
Elsey, 2006; Wilkinson, Elsey, Prager, Tanaka, & Sefton, 2004).
However, the presence of these two isomers in the wines suggests
that they can be generated indirectly by the application of oak
aqueous extracts to the vineyards.
In the case of furanic compounds, the concentration of furfural
and 5-methylfurfural was higher in the wines from grapes treated
with oak extract than in the control wine (Fig. 1c and d). This effect
was only observed for 5-methylfurfural in Verdejo treated wines
(Martínez-Gil et al., 2011). In general, both compounds showed a
tendency to decrease with the wine evolution. This might be due
to the fact that these aldehydes are biologically or chemically reduced in wine to give their corresponding alcohols (Garde-Cerdán
& Ancín-Azpilicueta, 2006; Rodríguez-Bencomo, Ortega-Heras,
Pérez-Magariño, & González-Huerta, 2009; Spillman, Pollnitz, Liacopoulos, Pardon, & Selfton, 1998). Furfural and 5-methylfurfural
concentration in all the wines from oak extract treatments and in
all the samples of their evolution, showed an increment of at least
48% when compared to their respective content in the control
wine. Nevertheless, such concentrations were below their olfactory
threshold (20 mg/l for furfural and 45 mg/l for 5-methylfurfural;
Boidron, Chatonnet, & Pons, 1988).
After alcoholic fermentation, the concentration of eugenol increased, with a general tendency to remain constant during the
next sampling (Fig. 1e). Eugenol content in control wines was significantly lower than the wines from vineyards treated with oak
extracts, although its content was similar between the control
grapes and the grapes from the oak extract treatments. Probably,
the grapes accumulated eugenol in the form of non-volatile precursors, depending on the type of treatment, which then passed into
the wine during alcoholic fermentation. However, 8 months after
the end of malolactic fermentation, the content of eugenol was
similar among the wines from the three oak extract treatments
and significantly higher than the content in the control wine. An
increase of at least 50% in the concentration found in the control
wine was observed, with the exception of the 25%(1) wine at the
end of the alcoholic fermentation, which showed an increase of
35%. Similar results were observed for this compound in case of
Verdejo oak treated wine (Martínez-Gil et al., 2011).
At all samplings, the 6-methoxyeugenol content was higher in
the wines from grapes treated with oak extracts than in the control
wine (Fig. 1f). Its behaviour during evolution depended on the type
of treatment carried out on the vineyard; in general, the highest
concentration was quantified in the 100% wine. The concentration
of this compound increased during malolactic fermentation, more
than doubling its initial content in all cases. This suggest that malolactic fermentation favoured the release of this compound from its
precursors, as it has been observed for other compounds since the
lactic acid bacterium O. oeni is able to release terpenes, norisoprenoids, phenol and vanillin derivatives (Hernández-Orte et al.,
2009). After 8 months of malolactic fermentation, the 6-methoxyeugenol concentration continued to increase but more slowly. At
this time, it was observed that the treated wines showed an
1841
(a)
c,β
5
b,αβ
b,β
4
ab,β
3
trans -Oak lactone
3.5
a,α
a,α
3
b,β
a,α
a,α
2.5
C (μg/l)
6
C (μg/l)
(b)
cis -Oak lactone
2
2
1
0
End of alcoholic
fermentation
(c)
End of malolactic
fermentation
8 months after
malolactic
fermentation
End of alcoholic
fermentation
(d)
Furfural
b,β b,β
b,β
b,α
b,αβ
b,α
60
b,α b,α b,α
30
25
20
20
15
10
0
5
0
40
a,α
a,α
End of alcoholic
fermentation
(e)
a,α
End of malolactic
fermentation
b,α
b,β b,α
c,α
b,α
a,α
a,α
b,β
b,α
ab,
b,β
b,β αβ
a,αβ
End of malolactic
fermentation
b,α b,α
b,α
a,α
8 months after
malolactic
fermentation
6-Methoxyeugenol
4
b,α
C (μg/l)
12
a,α
4
bc,γ
b,γ
c,β c,β
3
c,γ
b,β
2
1
c,α
a,α b,α
d,α
a,β
a,γ
0
0
End of alcoholic
fermentation
(g)
End of malolactic
fermentation
60
50
(h)
b,α
b,β
bc,β
c,β
b,α
b,α
End of alcoholic
fermentation
8 months after
malolactic
fermentation
Guaiacol
12
a,α
10
0
C (μg/l)
a,α
8 months after
malolactic
fermentation
16
b,α
a,α
End of malolactic
fermentation
4-Vinylguiaiacol
b,β b,α
30
20
8 months after
malolactic
fermentation
5
c,α
40
a,β
(f)
16
8
b,β
b,β b,β
End of alcoholic
fermentation
8 months after
malolactic
fermentation
Eugenol
End of malolactic
fermentation
5-Methylfurfural
40
35
C (μg/l)
80
C (μg/l)
a,α
a,α
0.5
0
C (μg/l)
a,α
b,α
a,β a,α
a,αβ
1.5
1
C (μg/l)
b,β
ab,β
b,α b,α
ab,α
a,α
b,α b,α
ab,α
a,α
b,α b,α
b,α
a,α
8
25%(1)
4
25%(4)
100%
0
End of alcoholic
fermentation
End of malolactic
fermentation
8 months after
malolactic
fermentation
control
End of alcoholic
fermentation
End of malolactic
fermentation
8 months after
malolactic
fermentation
Fig. 1. Concentration of oak volatile compounds (lg/l) in the wines at the end of alcoholic and malolactic fermentations, and 8 months after malolactic fermentation. Control:
untreated grapevines; 25%(1): grapevines treated once with oak extract diluted to a quarter; 25%(4): grapevines treated four times with oak extract diluted to a quarter;
100%: grapevines treated once with oak extract without dilution. At each sampling of the winemaking process, different letters indicate significant differences between the
samples. For each sample, different Greek letters indicate differences between various winemaking steps (level of significance of p > 0.05). All parameters are given with their
standard deviation (n = 6).
1842
(i)
(j)
4-Ethylguiaiacol
c,α
c,α
b,α
b,α
a,α
a,α
2
c,α
bc,α
c,α c,α
3
6
b,α
a,α
C (μg/l)
C (μg/l)
4
a,α
a,α
a,α
a,αβ a,α
a,α
a,α
a,β a,α a,α
0
0
End of alcoholic
fermentation
End of malolactic
fermentation
(k)
(l)
Syringol
c,β
50
c,α
ab,
b,α
α
a,α
b,β
b,β
60
a,β
a,α
40
a,α
b,α
b,β
b,α
a,α
a,α
0
End of alcoholic End of malolactic
fermentation
fermentation
(m)
8 months after
malolactic
fermentation
End of alcoholic
fermentation
(n)
Acetovanillone
End of malolactic
fermentation
8 months after
malolactic
fermentation
Methyl vanillate
40
35
25
b,α
b,α
20
b,β b,α
b,β
c,α
a,β
b,β
b,β b,α
a,β
a,α
c,α
30
C (μg/l)
30
10
b,α b,α
b,α
b,αβ
b,α b,α
20
0
15
8 months after
malolactic
fermentation
Vanillin
b,γ b,γ
b,α
End of malolactic
fermentation
80
C (μg/l)
100
End of alcoholic
fermentation
8 months after
malolactic
fermentation
150
C (μg/l)
4
a,α a,α
2
1
C (μg/l)
4-Ethylphenol
8
5
20
bc,α
ab,α
a,α
b,α
b,α
ab,α
a,α
b,α
b,α b,α
a,α
100%
0
End of alcoholic
fermentation
End of malolactic
fermentation
End of alcoholic
fermentation
8 months after
malolactic
fermentation
25%(1)
25%(4)
10
5
0
control
End of malolactic
fermentation
8 months after
malolactic
fermentation
Fig. 1 (continued)
increment in their concentration with respect to the control of
more than 135%, even reaching 182% in the 100% treated wine.
6-Methoxyeugenol gives wine a spicy aroma although the concentration in our wines was below its olfactory threshold (1.2 mg/l;
Culleré, Escudero, Cacho, & Ferreira, 2004).
After alcoholic fermentation, the guaiacol concentration in the
wines from the treated grapes was higher than in the control,
but similar among them (Fig. 1g). In general, at the end of malolactic fermentation this compound tended to increase in the wines
from the treated grapes, which can be attributed to the acid medium and the glycoside activity of lactic acid bacteria, whereas its
concentration remained constant in the control. After 8 months,
the concentration in all wines was similar, with guaiacol approximately three times more concentrated in the wines from the
grapes treated with the oak extracts than in the control wine.
The opposite effect was observed for Verdejo wines (Martínez-Gil
et al., 2011). In all the samples, the guaiacol concentration in the
wines exceeded its olfactory threshold (9.5 lg/l, smoke aroma
descriptor, Ferreira, López, & Cacho, 2000).
As with the other compounds, the content of 4-vinylguaiacol, 4ethylguaiacol, and 4-ethylphenol increased during alcoholic fermentation (Fig. 1h–j). After alcoholic fermentation, 4-vinylguaiacol
remained constant, probably since the bacteria O. oeni cannot
decarboxylate the ferulic acid to form 4-vinylguaiacol (Hernández-Orte et al., 2009). The concentration of ethylphenols, 4-ethylguaiacol and 4-ethylphenol, was constant or presented a slight
increase in the case of 25%(1) wine during the period studied.
The sensorial threshold of 4-ethylphenol is 230 lg/l and of 4-ethylguaicol is 47 lg/l (Chatonnet, Boidron, & Pons, 1990), concentrations that the samples studied did not reach. The concentration of
4-ethylguaiacol was higher in wines from grapevine oak treatments than in control wines in all samplings (Fig. 1i), whereas
the concentration of 4-ethylphenol did not present differences between control wine and wines from grapevine oak treatments
1843
Fig. 2. Canonical discriminant analysis of volatile compound concentration in wines (control: untreated grapes; 25%(1): grapevines treated with oak extract diluted to a
quarter applied once; 25%(4) grapevines treated with oak extract diluted to a quarter applied four times; 100%: grapevines treated with oak extract without dilution applied
once) at the end of the alcoholic fermentation (AF), after malolactic fermentation (MLF) and 8 months later (8 months).
Table 4
Concentration of volatile compounds (lg/l) in control and E + G wines.
cis-Oak lactone
trans-Oak lactone
Furfural
5-Methylfurfural
Eugenol
6-Methoxyeugenol
Guaiacol
4-Vinylguaiacol
4-Ethylguaiacol
4-Ethylphenol
Syringol
Vanillin
Acetovanillone
Methyl vanillate
8 Months after malolactic fermentation
Control wine
Wine from E + G treatment
Control wine
Control wine
n.q.
n.q.
31.54 ± 2.60 a,a
17.89 ± 2.32 a,b
6.15 ± 0.77 a,a
0.47 ± 0.06 a,a
17.01 ± 3.09 a,a
8.33 ± 0.68 a,a
1.60 ± 0.19 a,a
3.20 ± 0.32 b,a
48.77 ± 5.94 a,a
31.69 ± 2.86 a,a
9.49 ± 1.17 a,a
16.18 ± 2.42 a,a
4.1 ± 0.52 a,b
1.58 ± 0.19 a,b
42.19 ± 5.13 b,b
23.58 ± 2.37 b,b
364 ± 40 b,a
3.75 ± 0.51 b,a
587 ± 71 b,a
7.12 ± 0.33 a,a
1.73 ± 0.29 a,a
2.43 ± 0.16 a,a
52.92 ± 6.33 a,a
33.68 ± 0.59 a,a
13.38 ± 1.61 b,a
20.58 ± 3.44 a,a
n.q.
n.q.
30.06 ± 1.17 a,a
16.56 ± 2.43 a,ab
7.20 ± 0.43 a,a
1.09 ± 0.09 a,b
13.17 ± 2.51 a,a
8.27 ± 1.06 a,a
1.79 ± 0.28 a,a
3.46 ± 0.49 a,a
54.63 ± 4.95 a,a
26.35 ± 2.64 a,a
14.26 ± 1.14 a,b
19.75 ± 3.08 a,a
4.19 ± 0.47 a,b
1.41 ± 0.2 a,ab
30.18 ± 2.33 a,a
24.50 ± 2.10 b,b
375 ± 37 b,a
9.02 ± 1.02 b,b
610 ± 36 b,a
16.09 ± 1.63 b,b
2.43 ± 0.29 b,b
2.78 ± 0.22 a,b
85.20 ± 8.03 b,b
34.97 ± 2.32 b,a
19.86 ± 3.23 b,b
20.74 ± 2.37 b,a
n.q.
n.q.
27.01 ± 3.70 a,a
11.52 ± 3.59 a,b
6.61 ± 1.06 a,a
1.52 ± 0.18 a,c
13.21 ± 2.07 a,a
8.13 ± 0.82 a,a
1.64 ± 0.28 a,a
3.62 ± 0.54 a,a
77.7 ± 5.16 a,b
30.35 ± 4.36 a,a
14.94 ± 1.21 a,b
16.48 ± 1.10 a,a
3.04 ± 0.15 a,a
1.14 ± 0.12 a,a
26.07 ± 8.07 a,a
15.55 ± 2.07 b,a
398 ± 34 b,a
14.65 ± 0.26 b,c
668 ± 79 b,a
20.96 ± 1.58 b,c
3.47 ± 0.42 b,c
3.09 ± 0.09 a,b
93.87 ± 7.48 a,b
41.92 ± 4.02 a,b
20.20 ± 2.17 a,b
20.68 ± 2.37 a,a
All the parameters are given with their standard derivation (n = 6). The different letters indicate significant differences. At each sampling in the winemaking process, different
letters indicate significant differences between the samples (level of significance of p > 0.05). For each sample, different Greek letters indicate differences between winemaking moments (level of significance of p > 0.05). Control: untreated grapes; E + G: grapevines treated with eugenol plus guaiacol solution.
(Fig. 1j). This could indicate that the oak extract only contributed to
an increase in the concentration of 4-ethylguaiacol, but 4-ethylphenol may only proceed from grapes, since there are no differences between control and treated samples.
Syringol was one of the most dominant volatile phenols in the
wines (Fig. 1k). The content in control grapes was higher than in
oak treated grapes. Nevertheless, the content of this compound
was the highest in the wines from the grapes treated with oak extract, especially in the last two samplings, i.e. the end of the malolactic fermentation and after 8 months. This suggests that grapes
accumulated syringol as non-volatile precursors, e.g. syringol-GG
glycoside, as other authors have observed in berries from grapevines exposed to smoke (Hayasaka, Baldock, Pardon, Jeffery, &
Herderich, 2010). These grapes store syringol, which is then transferred into the wines where the aglycone is released by enzymatic
and/or chemical hydrolysis. This compound was most affected by a
100% treatment, showing the highest concentrations in all the finished wines, with a 76% increment with respect to the control
wine. Hence, the behaviour of this compound depended on the
treatment carried out in the vineyard. Also, none of the wines exceeded their olfactory threshold (570 lg/l, phenolic and medicine
aroma; López, Aznar, Cacho, & Ferreira, 2002).
The levels of vanillin derivatives, vanillin, acetovanillone, and
methyl vanillate, were higher in the wines from treated grapes
than in the control wines, especially at the end of malolactic fermentation and after 8 months (Fig. 1l–n), probably due to the acid
1844
or enzymatic hydrolysis of their precursors as observed by Bureau,
Baumes, and Razungles (2000) and Loscos, Hernández-Orte, Cacho,
and Ferreira (2007). In general, none of the samplings showed significant differences regarding the type of oak extract treatment.
Since the content of these compounds in grapes was similar in
all cases, the results in the wine pointed that these three
compounds could probably be incorporated into the grapes due
to the oak extract treatment, and stored in the form of non-volatile
precursors that are then released in wines. These compounds contributed to the wine aroma with caramel, butterscotch, and vanilla
aromatic notes, although none of the vanillin derivates exceeded
their olfactory threshold (60 lg/l for vanillin, 1000 lg/l for acetovanillone and 3000 lg/l for methyl vanillate; Culleré et al., 2004).
Furthermore, the concentrations of vanillin, acetovanillone, and
methyl vanillate were lower than those found in wines after contact with oak chips (Cejudo-Bastante, Hermosín-Gutiérrez, &
Pérez-Coello, 2011; Rodríguez-Bencomo et al., 2009).
As different behaviour was observed between oak treatments in
all sampling times, a discriminant analysis was carried out in order
to determine clear differences. Fig. 2 shows the results obtained
after performing the discriminant analysis of different wines (control, 25%(1), 25%(4) and 100%) at their different sampling moments
(after alcoholic and malolactic fermentation and after 8 months).
This resulted in function 1, which explained 68.3% of the variance,
and function 2 explaining 23.1% of the variance. The discriminating
variables that contributed more to differentiation with higher
loading were: 6-methoxyeugenol, syringol, cis-oak lactone, guaiacol, 4-ethylguaiacol, eugenol, 5-methylfurfural, and acetovanillone.
It can be seen that function 1 separated the wines from grapevines
submitted to the oak extract treatments from the control wines.
The discriminant was able to differentiate between wines from
the different oak treatments after alcoholic fermentation, whereas
this separation was lower for the wines after malolactic fermentation and 8 months. In relation to the evolution of each wine, there
were differences in the behaviour of the control wines and those
from oak treatments. The discriminant analysis was not able to
separate the control wines after alcoholic and malolactic fermentation, although it did after 8 months. Instead, each wine from the
grapevine treatments showed differences at each time of sampling
(AF, MLF and 8 months), indicating that there was a change in the
oak volatile composition of these wines.
In Table 4, the results are presented for the wines from grapevines treated with the solution of eugenol and guaiacol. These
compounds significantly increased their concentration compared
to the control when fermentation was finished, although this concentration remained constant in the following samplings. Consequently, they were absorbed by the grapevines and accumulated
in grapes, mainly as non-volatile precursors released during alcoholic fermentation, although a small proportion of these compounds were also found in free form in grapes (Table 2). Such
behaviour was also observed when a similar solution was applied
to Verdejo white grapes (Martínez-Gil et al., 2011). However, when
comparing the results of Verdejo wines under the same conditions,
it was observed that the Petit Verdot wines showed higher concentrations of both compounds (eugenol and guaiacol). This was probably due to various factors: the pre-fermentation maceration at
3 °C done to minimise the loss of volatiles, the high fermentative
temperature and the maceration with skins during alcoholic fermentation, as aroma precursors are located mainly in the skins,
and can be enhanced by processes, such as skin contact (MorenoArribas & Polo, 2009). Also, Hayasaka et al. (2010) showed that berries and leaves from grapevines exposed to smoke stored guaiacol
as a b-D-glucopyranoside form, and transferred it to the must
where the aglycone was released by enzymatic and chemical
hydrolysis. The application of E + G solution to the grapevine
showed an increment of other compounds in relation to the control
wine after malolactic fermentation and 8 months later, specially 6methoxyeugenol, syringol, 4-vinylguaiacol, and 4-ethylguaiacol
(Table 4). The 6-methoxyeugenol content increased with time, a
phenomenon also observed in wines treated with oak extract. In
addition, E + G wines also had higher concentrations of 5-methylfurfural, vanillin and acetovanillone than control wines, indicating
that there were also adjustments in the formation of soluble precursors of these compounds in berries due to the E + G treatment.
In general, compounds, such as furfural, 4-ethylphenol, and methyl
vanillate were not affected by this treatment (Table 4). Oak lactones were also found in the E + G wine, and other authors have
found these two isomers in young wines without contact with
wood, suggesting that it may originate from precursors present
in grapes (Bautista-Ortín et al., 2008; Loscos et al., 2007). Furthermore, Verdejo wine treated with E + G solution presented both isomers (Martínez-Gil et al., 2011), suggesting that the application of
this solution might affect the plant metabolism of such
compounds.
4. Conclusions
The application of oak extracts to grapevines of the red Petit
Verdot cultivar affected the aroma composition of grapes and
wines. The results indicated that berries stored volatiles proceeding from oak formulation mainly as non-volatile precursors, and
these compounds were released with the winemaking process.
As soon as the end of the alcoholic fermentation, the different
grapevine oak treatments reveal a clear wine differentiation. Thus,
it is possible to modify the aroma composition of wines by applying different oak treatments to the grapevine, with such effects
being relevant in young wines.
Acknowledgements
We wish to thank the financial support given by the Ministerio
de Ciencia e Innovación to Project AGL2009-08950. Also, we are
grateful for the FPI scholarship from the Junta de Comunidades
de Castilla-La Mancha for A.M.M.-G (EXP 422/09) and to the MICINN for A.I.P.-G (BES-2010-038613). We wish to thank the Dehesa
de Los Llanos estate (winery-Albacete, southeastern Spain) for
allowing us to use its vineyards and Laura-Martínez for her technical assistance. We wish to express our gratitude to Protea France
for supplying for free the oak extracts and Kathy Walsh for proof
reading the English manuscript.
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7.4. ARTÍCULO IV
El origen del presente trabajo fueron los resultados de los anteriores
artículos de la tesis, los cuales demuestran que se produce un cambio en la
composición volátil de los vinos blancos y tintos tras la aplicación foliar de un
extracto de roble a sus vides, a pesar de que la composición volátil de estas uvas
es muy similar a las uvas utilizadas como control.
El objetivo principal fue comprobar si los compuestos volátiles del
extracto de roble aplicados a la vid se encontraban en las uvas en su forma
glicosilada. Además, este estudio a parte de hacer hincapié en las agliconas que
pueden proceder directamente del extracto, por encontrarse en su composición,
también se centró en estudiar las agliconas originarias de la uva, no presentes en
el extracto, que podrían verse afectadas indirectamente tras los tratamientos. Se
sabe que la variedad de uva, el momento, y la dosis de aplicación puede influir
en la composición de la uva. Por ello este trabajo se planteó hacerlo en un lugar
diferente, con condiciones edafoclimáticas distintas a las estudiadas hasta el
momento,
empleando
las
variedades
131
Syrah
y
Chardonnay.
Artículo IV
Para comprobar el efecto del momento de la aplicación, se hicieron 3
tratamientos en cada variedad, que únicamente se diferenciaban en la semana del
envero en que se había llevado a cabo la aplicación (T1, T2 y T3). El trabajo
consistió en la determinación de los compuestos volátiles del extracto de roble,
los parámetros enológicos de las uvas y en cuantificar las agliconas liberadas por
vía enzimática de la fracción glicosilada de las uvas. Los compuestos analizados
fueron los relacionados con el extracto de roble y los compuestos originarios de
la uva agrupados en 5 grupos: 4 compuestos C6, 2 alcoholes, 9 terpenos, 5
fenoles y 4 norisoprenoides.
Se encontraron las agliconas cis y trans whisky lactonas en las uvas de
los tratamientos y no en el control, hecho que puede atribuirse a la glicosilación
de las whisky lactonas volátiles del extracto en las uvas. Este fenómeno sólo es
posible si se abre el anillo lactona de su molécula, dejando el grupo hidroxilo
libre para que tenga lugar la glicosilación, tal como otros autores han descrito.
Las uvas procedentes de los tratamientos tuvieron mayores contenidos de
las agliconas relacionadas con los volátiles del extracto que en las uvas usadas
como control. La proporción de las agliconas en las uvas dependió del tipo de
compuesto, de su concentración en el extracto de roble, de la variedad y del
momento de aplicación del extracto. Así, Chardonnay mostró un mayor
incremento relativo de las agliconas totales respecto a su control que Syrah. La
tasa de glicosilación fue diferente para cada compuesto, requiriéndose, en el caso
de Syrah, una concentración mínima en el extracto para que en las uvas
aumentaran sus respectivos glicósidos. Syrah presentó un incremento más
marcado cuanto más tardío se realizó el tratamiento (T3), en cambio en
Chardonnay cuando se aplicó en la segunda semana del envero (T2).
El estudio mostró también un efecto de los tratamientos en el contenido
de las agliconas originarias de las uvas que no están presentes en el extracto
132
Artículo IV
(Compuestos C6, alcoholes, terpenos, fenoles y norisoprenoides), que dependió
de la variedad. Se observó, en general, una disminución de estos compuestos en
la variedad Syrah y un aumento en la variedad Chardonnay. En ambas
variedades el efecto fue más marcado cuando se realizó el tratamiento en la
segunda semana del envero.
Los resultados de este artículo confirman la hipótesis propuesta en los dos
anteriores artículos sobre la asimilación por la vid de los compuestos de extracto
de roble y su almacenamiento como precursores glicosídicos en las uvas.
133
Food Chemistry 138 (2013) 956–965
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Glycosidic aroma precursors of Syrah and Chardonnay grapes after an oak
extract application to the grapevines
Ana M. Martínez-Gil a, Magaly Angenieux b, Ana I. Pardo-García a, Gonzalo L. Alonso a, Hernán Ojeda b,
M. Rosario Salinas a,⇑
a
b
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Avda, España s/n, 02071 Albacete, Spain
INRA, UE999 Pech Rouge, F-11430 Gruissan, France
a r t i c l e
i n f o
Article history:
Received 5 September 2012
Received in revised form 16 October 2012
Accepted 7 November 2012
Available online 15 November 2012
Keywords:
Chardonnay
Glycosidic aroma
Grape
Precursors oak extract
Syrah
a b s t r a c t
Syrah and Chardonnay grapevines were treated with an oak extract in order to determine the effect on
glycosidic aroma precursors. Grapevines were treated at three different timings of the veraison (treatment 1, 2 and 3). Aglycons were obtained by enzymatic hydrolysis, and these were identified and quantified by means of gas chromatography–mass spectrometry (GC–MS). Results suggest that after the
applications the majority of compounds from the oak extract were assimilated and stored as glycosidic
forms in both cultivars. Also, other compounds not present in the extract were affected, with a different
behaviour observed depending on the timing of application and the variety. In general, C6 compounds,
alcohols, terpenes, phenols and C13-norisoprenoids in Syrah showed a decrease and in Chardonnay an
increase. Thus, this study proved a change in the glycosidic aroma profile in grapes after the oak application, so these treated grapes could produce wines with different aromatic quality.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Glycosidic precursors are a diverse group of odourless compounds initially identified in grapes by Cordonnier and Bayonove
(1974), and reputed to be the most important group responsible
for some of the varietal attributes of wines. This is especially true
for non-floral grapes, since frequently these forms are more
common than free aromas (Bureau, Baumes, & Razungles, 2000;
Francis, Tate, & Williams, 1996; López, Ezpeleta, Sánchez, Cacho,
& Ferreira, 2004; Noguerol-Pato et al., 2012). Grape glycoconjugates are composed of a sugar moiety which always includes
glucose bound to a volatile aglycon by a b-glucosidic bond. The
nature of the aglycon and the glycosidic fraction proportion depends on grape variety, but may be influenced by other factors
such as soil, climatic conditions, viticultural practises and environment where the plant grows (Bureau et al., 2000; Koundouras,
Marinos, Gkoulioti, Kotseridis, & Van Leeuwen, 2006; Zoecklein,
Wolf, Pélanne, Miller, & Birkenmaier, 2008). Some studies also suggest that glycosylation is a storage medium for volatile compounds
which limits the toxicity of some of them since glycosides are
much more water soluble than aglycons and are thus considered
vectors for the transport and accumulation of such compounds in
plants (Stahl-Biskup, Holthuijzen, Stengele, & Schulz, 1993;
Winterhalter & Skouroumounis, 1997). The hydrolysis of these
⇑ Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238.
E-mail address: [email protected] (M. Rosario Salinas).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.11.032
glycoconjugates by acids or enzymes can yield odour-active
aglycons such as monoterpenes, C13 norisoprenoids, phenols,
alcohols and C6 compounds (Günata, Bayonove, Baumes, &
Cordonnier, 1985; Sefton, Francis, & Williams, 1993), being the
source of varietal aromas in wines (D’Incecco et al., 2004; Francis
et al. 1996; Sánchez Palomo, Pérez-Coello, Díaz Maroto, González
Viñas, & Cabezudo, 2006). Enzymatic hydrolysis is the most
adequate technique to generate aglycons without structural
changes, with the enzymatic preparation AR2000 used the most
(Gómez García-Carpintero, Sánchez-Palomo, Gómez-Gallego, &
Gonzálezviñas, 2012; Sefton et al., 1993; Sánchez Palomo et al.
2006).
On the other hand, it has been proven that factors outside the
vineyards, as well as various treatments applied to these, are able
to modify the aroma composition of grapes and their respective
wines. Clear examples are the studies which show that chemical
pesticides not only affect the fermentative aromatic compounds
but also the varietal aroma (Darriet et al. 2001; Oliva, Zalacain,
Payá, Salinas, & Barba, 2008). Moreover, some research studies
have revealed that grape and grapevine exposure to smoke influences the chemical composition and gives the wine smoky sensory
characteristics (Kennison, Wilkinson, Pollnitz, Williams, & Gibberd,
2009; Kennison, Wilkinson, Pollnitz, Williams, & Gibberd, 2011;
Wilkinson et al., 2011). Recently, Martínez-Gil, Garde-Cerdán,
Martínez, Alonso, and Salinas (2011) and Martínez-Gil, GardeCerdán, Zalacain, Pardo-García, and Salinas (2012) have also shown
how the aromatic composition of wines is modified when oak
957
extract applications are carried out on Verdejo and Petit Verdot
grapevines. These studies prove that a change occurs in the free
aroma profile of white and red wines when oak extract is applied
at the onset of veraison, suggesting that grapes can probably store
volatile compounds, mainly as non-volatile precursors. It is
therefore important to complete further studies on the glycosidic
compounds. Also, the studies on smoke application to grapevines
prove the influence of the variety, timing and duration of smoke
exposure on the aromatic composition (Kennison et al., 2009;
Singh et al., 2011). Glycosylation of some volatile phenol
compounds in grapes as a consequence of grapevine exposure to
bushfire smoke has been demostrated (Dungey, Hayasaka, &
Wilkinson, 2011; Hayasaka et al., 2010; Wilkinson et al., 2011).
In consequence, the aim of this study was to determine the impact on the aroma glycosidic precursors of Syrah and Chardonnay
grapes, once an oak extract was applied to grapevines via foliar
at three different times. The study emphasised the aglycons that
could come directly from the extract, as these are found in its composition, and the aglycons that could be modified indirectly after
application, as these are not found in its composition.
2. Materials and methods
2.1. Oak extract
For the different applications to grapevines, an American
toasted aqueous oak extract supplied by Protea France S.A.S.
(Gensac la Pallue, France) was used. It was produced by maceration
American toasted oak chips (Quercus alba) from natural seasoning
for at least 18 months in demineralised water at 100 °C for 32 h.
The volatile composition of the oak extract was analysed and its
composition were defined by: 2.65 mg/l of cis-oak lactone;
0.75 mg/l of trans-oak lactone; 0.09 mg/l of eugenol; 0.23 mg/l of
6-methoxyeugenol; 0.10 mg/l of guaiacol; 0.10 mg/l of 4-vinylguaiacol; 0.38 mg/l of 3,4,5-trimethoxyphenol; 1.14 mg/l of
syringol; 37.17 mg/l of syringaldehyde; 2.95 mg/l of vanillin;
1.41 mg/l of acetovanillone; 1.64 mg/l of propiovanillone;
4.57 mg/l of vanillol; 11.98 mg/l of ethyl vanillate; and 11.02 mg/
l of homovanillic acid, as 4-nonanol equivalents. For analysed these
volatile compounds, they were extracted with 5 ml of azeotrope
pentane-dichloromethane (2/1 v/v). Afterwards, 200 ll of 4-nonanol (Merck, Germany) solution at 16 mg/l in absolute ethanol was
added as internal standard and the resulting solution was concentrated using a Vigreux column at 40 °C. Since this extract is a food
additive, there is a guarantee that no toxicity risk exists.
2.2. Plant materials and treatment
The study was done in a vineyard on the INRA’s Experimental
Unit in Pech Rouge, Gruissan, in the southern of France (43°100 N
latitude, 3°060 E longitude). Grapes from Vitis vinifera L. cv. Syrah
(planted in 1993, clone 174, grafted onto R140) and Chardonnay
(2000, 141, R140) from the 2011 vintage were used. The vines,
on vertical shoot positioning, were separated with 1 m spacing between vines and 2.5 m between rows. Grapevines were grown with
a drip irrigation system to assure water needs. Pech Rouge vineyard has a Mediterranean climate with a strong maritime influence, the mean annual rainfall being about 600 mm. This climate
is characterised by warm, dry and mild summers and wet winters,
with the characteristic of being a very windy area, with 300 days a
year of wind. The vineyards were treated with the commercial extract in three different timings at veraison, according to specifications of Table 1. For all treatments, a 0.5 ml per litre of adjuvant
Fluvius (BASF, Germany) was added to the extract before the
application, since this is a wetting agent typically used for foliar
Table 1
Different timings of treatment to grapevines with commercial oak extract.
Syrah
Chardonnay
Beginning of
veraison
Control
(C)
Treatment
1 (T1)
Treatment
2 (T2)
Treatment
3 (T3)
14th July
4th July
–
–
21th July
12th July
1st August
21th July
8th August
2nd August
herbicide treatments. Around 250 ml of each formulation was applied evenly per plant by spraying over leaves. The treatments
were carried out when the environmental temperature was below
20 °C, and the wind was not too strong. Between the different
applications, a row was left untreated, so between treated rows
was there were five metres of space to avoid contamination. The
grapevines for each variety (Syrah and Chardonnay) were fractionated into 12 plots with 27 plants in each one, distributed with
different orientations at random. All treatments were done in triplicate, so the number of grapevines for each treatment was 81.
After veraison, every five days, maturity was tracked. Syrah grapes
were harvested on September 07th and Chardonnay on August
16th, at their optimum maturation moment with the Baumé/titratable acidity ratio around 3.5 in Syrah and 2.4 in Chardonnay. The
grapes were frozen at 20 °C until analysis.
Reducing sugars, probable alcohol, titratable acidity (g/l tartaric
acid) and pH, from the different samples were measured in triplicate following the methods established by ECC (1990). Amino
and assimilable nitrogen were measured using the Dubernet,
Dubernet, Grasset, and Garcia (2001) method.
2.4. Analysis of glycosidic aroma precursors
2.4.1. Preparation of samples
About 500 g of berries from each treatment were defrosted.
They were then crushed in a blender and the gross product obtained was filtered with a colander before being centrifuged at
7000 rpm at 10 °C for 20 min. The juice was stirred for 20 min in
presence of 5 g/100 ml for red grapes and 1 g/100 ml for white
grapes of resin polyvinylpoly–pyrrolidone (PVPP, Sigma Aldrich,
France), previously hydrated, to eliminate the high levels of phenolic compounds capable of inhibiting the glycosidase activities. The
mixture was filtered again through a 5 lm cellulose filter (Fisherbrand, UK). Three fractions of 100 ml were chosen for extraction of
the glycosylated fraction. The juice was eluted through SPE
cartridges (Strata-X 33u Polymeric Reserved Phase, 500 mg/6 ml;
Phenomenex) previously activated and conditioned following
supplieŕs instructions. After passing the sample, the cartridges
were rinsed and vacuum dried. Then, the fraction was eluted with
10 ml of acetonitrile/methanol (5:5, v/v) and concentrated to dryness at 45 °C in a rotary evaporator, obtaining only the fraction of
glycosilated form The residue was taken up in 1 ml of phosphate/
citrate buffer (hydrogen di-sodium phosphate 0.2 M/citric acid
0.1 M; pH 5) for later hydrolysis.
2.4.2. Enzymatic hydrolysis
This was carried out with 100 ll of a 70 mg/ml solution in the
citrate/phosphate buffer solution of AR 2000 pectinase enzyme
preparation (DSM, Delft, Pays-Bas), in a incubator at 35 °C, for
16 h (Günata et al., 1985). Released aglycons were extracted with
5 ml of azeotrope pentane–dichloromethane (2/1 v/v). Afterwards,
200 ll of 4-nonanol (Merck, Germany) solution at 16 mg/l in
absolute ethanol was added as internal standard and the resulting
solution was concentrated using a Vigreux column at 40 °C.
958
2.4.3. Gas chromatography–mass spectrometry (GC–MS) analysis
The identification and quantification of aglycons released from
the aroma glycosidic fraction of grapes and volatile compounds
of oak extract were performed using a gas chromatograph Agilent
6890, coupled with a mass spectrometer of the same series, with a
quadrupole filter. The unit was equipped with a fused silica capillary column (length 30 m 0.25 mm id., and 0.5 lm phase thickness, DB-Wax, J & W Scientific), connected to the injector via a
deactivated silica pre-column (1 m long and internal diameter
0.53 mm, J & W Scientific). The injector temperature was set from
30 °C to 245 °C at 180 °C/min, and held at 245 °C. Two microlitres
were injected. The oven temperature programme was set from
60 °C (3 min isothermal) to 250 °C at 3 °C/min, then isothermal
for 10 min. The transfer line was set at 250 °C. The source temperature was kept at 250 °C and the quadrupole at 150 °C. EI was recorded at 70 eV in the mass range m/z 29–350. The compounds
were identified by their mass spectra with those of the published
or spectra library and quantified using the 4-nonanol equivalents.
The concentration of each compound was determined by the average of three repetitions, since three different extractions of the
grape were done. Moreover, all the treatments were performed
in triplicate in the vineyard.
2.5. Statistical analysis
This was carried out using the SPSS Version 19.0 statistical
package for Windows (SPSS, Chicago, USA). Oenological parameters and aroma compounds data were processed using the variance
analysis (ANOVA) which takes into account the average of the analytical replicates of the three experiences in field n = 3. Differences
between means were compared using the least significant difference (LSD) test at a 0.05 probability level. A discriminant analysis
was performed on the total aglycon volatiles studied for the two
grape varieties. Other discriminant analyses, one for each variety,
were carried out with the concentration of the glycosidic compounds: (a) those which came directly from the application of
the oak extract, as these are included in its composition and, (b)
those which were modified indirectly by the treatment, as these
are not included in its composition.
The oenological parameters of Syrah and Chardonnay grapes
from the different treatments (Control (C), Treatment 1 (T1), Treatment 2 (T2) and Treatment 3 (T3)) can be observed in Table 2. In
Syrah, only two oenological parameters decreased significantly
with the treatment, i.e. the weight of 200 berries in treatment 3
and pH in treatment 2, which may be due to the natural variation
of the plants. However, Chardonnay did not show any significance
differences. Hence, the oak extract treatments did not affect the
oenological parameters of grapes. Moreover, in all cases, the concentration of sugars and assimilable nitrogen were adequate for
carrying out the complete alcoholic fermentation (Bell & Henschke,
2005).
3.2. Glycosidic aroma precursors of Syrah and Chardonnay grapes
Recently, the application of aqueous oak extracts to grapevines
of white and red varieties have shown how the free aromatic profile is affected, suggesting that grapes store volatiles as nonvolatile
precursors, some of which are released during the winemaking
process (Martínez-Gil et al., 2011, 2012). In this study the aroma
precursor fraction of grapes subjected to oak extract will be assayed in order to distinguish the aglycons that could come directly
from the extract, as these are found in its composition, and the
aglycons that could be modified indirectly after the application,
as these are not included in its composition.
3.2.1. Aglycons modified directly by the application of oak extract, as
these are included in its composition
Table 3 shows the bound volatile compounds, related directly to
the extract, released by enzymatic hydrolysis of different Syrah and
Chardonnay grapes (C, T1, T2 and T3). In Syrah and Chardonnay
control grapes, the two oak lactones (cis and trans-oak lactones)
were not found. As expected, the grapes from the oak extract treatments showed these two isomers, corroborating that these compounds were not found in grapes as free forms, but were
released after vinification (Martínez-Gil et al., 2011, 2012). Thus,
the results obtained in this study ascertained that both oak lactones can be assimilated from the oak extract by plants and stored
as nonvolatile glycosidic precursors. Hayasaka, Wilkinson, Elsey,
Raunkjaer, and Selfton (2007) and Winterhalter (2009) observed
that the formation of lactone precursors is possible when the rings
of these molecules are open. The combination of these two compounds with the sugars depended on the variety, with Syrah being
more susceptible to the assimilation and conjugation than Chardonnay. However, it also depended on the timing of oak extract
application, since the concentration was higher when the application was done in the field on more mature grapes. In general, the
concentration of cis-oak lactone was higher than trans-oak lactone
aglycons, showing a pattern similar to the extract, especially in the
Syrah variety where the ratio cis/trans ranged between 1.6 and 2.2,
while in Chardonnay it was between 1 and 1.9. These lactones provided wines with wood and coconut aromatic notes (Chatonnet,
Table 2
Oenological parameters in grapes on harvest day after the different grapevine treatments.
Weight of 200
berries (g)
Volume of
berries (ml)
Reducing
sugars (g/l)
Probable alcohol
(v/v,%)
Titratable
acidity (g/l)
pH
Amino
nitrogen (mg/l)
Assimilable
nitrogen (mg/l)
Syrah
Control
Treatment 1
Treatment 2
Treatment 3
343.83 ± 12.53b
334.77 ± 7.49ab
346.57 ± 14.81b
317.07 ± 13.81a
1.56 ± 0.04a
1.50 ± 0.05a
1.56 ± 0.07a
1.45 ± 0.07a
218.70 ± 6.70a
224.80 ± 2.71a
222.53 ± 8.76a
217.90 ± 3.50a
13.00 ± 0.40a
13.36 ± 0.16a
13.22 ± 0.52a
13.13 ± 0.51a
3.51 ± 0.10a
3.69 ± 0.09a
3.65 ± 0.04a
3.58 ± 0.13a
3.79 ± 0.04b
3.77 ± 0.06ab
3.68 ± 0.05a
3.73 ± 0.05ab
139.33 ± 16.62a
160.00 ± 32.08a
127.33 ± 6.66a
141.00 ± 18.25a
179.00 ± 17.09a
205.00 ± 40.73a
165.00 ± 9.54a
181.33 ± 23.44a
Chardonnay
Control
Treatment 1
Treatment 2
Treatment 3
278.80 ± 8.49a
295.67 ± 12.28a
291.90 ± 8.72a
280.43 ± 5.05a
1.23 ± 0.04a
1.23 ± 0.07a
1.21 ± 0.01a
1.20 ± 0.07a
209.43 ± 5.79a
206.40 ± 9.24a
211.00 ± 8.13a
208.70 ± 6.97a
12.44 ± 0.34a
12.26 ± 0.55a
12.54 ± 0.48a
12.40 ± 0.41a
4.98 ± 0.28a
5.22 ± 0.24a
4.99 ± 0.15a
4.94 ± 0.11a
3.56 ± 0.09a
3.46 ± 0.06a
3.53 ± 0.04a
3.52 ± 0.02a
189.00 ± 23.52a
181.00 ± 11.14a
182.00 ± 32.45a
169.33 ± 14.57a
257.67 ± 42.83a
260.67 ± 22.90a
255.33 ± 43.82a
222.67 ± 26.76a
All parameters are given with their standard deviation (n = 3). For each variety different letters indicate significant differences between the treatments (level of significance of
p < 0.05). Control: untreated grapes; treatment 1: grapevines treated with oak extract the 21st July for Syrah and 12th July for Chardonnay; Treatment 2: grapevines treated
with oak extract the 1st August for Syrah and 21st July for Chardonnay; Treatment 3: grapevines treated with oak extract the 8th August for Syrah and 2nd August for
Chardonnay. Titratable acidity (as g/l tartaric acid).
All parameters are given with their standard deviation (n = 3). For each variety different letters indicate significant differences between the treatments (level of significance of p < 0.05). Control: untreated grapes; Treatment 1:
grapevines treated with oak extract the 21st July for Syrah and 12th July for Chardonnay; Treatment 2: grapevines treated with oak extract the 1st August for Syrah and 21st July for Chardonnay; Treatment 3: grapevines treated
with oak extract the 8th August for Syrah and 2nd August for Chardonnay.
0.62 ± 0.10b
0.32 ± 0.03b
2.42 ± 0.31b
0.42 ± 0.03b
0.35 ± 0.07c
28.85 ± 4.57a
30.98 ± 5.39ab
0.50 ± 0.06a
7.41 ± 0.50c
8.85 ± 1.37b
16.08 ± 2.40ab
5.10 ± 0.43a
8.55 ± 1.40bc
30.24 ± 2.94b
4.91 ± 0.67b
135.58 ± 8.45b
0.56 ± 0.10b
0.48 ± 0.03c
3.35 ± 0.41c
0.54 ± 0.06c
0.31 ± 0.02bc
29.51 ± 2.13a
41.56 ± 6.47b
0.67 ± 0.06b
9.35 ± 1.15d
8.28 ± 1.47b
20.99 ± 3.90b
7.63 ± 0.94b
10.66 ± 1.89c
46.37 ± 8.55c
4.60 ± 0.59b
184.86 ± 11.97c
Treatment 2
Treatment 1
0.26 ± 0.03a
0.26 ± 0.04a
3.36 ± 0.65c
0.53 ± 0.08c
0.26 ± 0.04b
25.80 ± 4.63a
33.82 ± 5.46ab
0.56 ± 0.11ab
3.60 ± 0.48b
7.02 ± 0.67ab
18.33 ± 2.57b
5.83 ± 1.11a
6.34 ± 1.10ab
20.42 ± 2.85b
3.13 ± 0.60a
129.52 ± 8.36b
N.d
N.d
1.62 ± 0.25a
0.25 ± 0.05a
0.15 ± 0.01a
22.51 ± 4.50a
27.14 ± 4.04a
0.41 ± 0.07a
2.04 ± 0.38a
4.95 ± 0.88a
12.67 ± 1.51a
4.23 ± 0.65a
4.05 ± 0.77a
7.25 ± 4.04a
3.00 ± 0.52a
90.27 ± 6.44a
Control
2.56 ± 0.39c
1.64 ± 0.18b
9.22 ± 0.80a
3.02 ± 0.49a
21.37 ± 0.24a
76.28 ± 12.30a
133.32 ± 3.74a
13.61 ± 0.56a
38.93 ± 2.09c
47.01 ± 0.67d
79.02 ± 5.33b
7.79 ± 1.36a
35.71 ± 2.73c
102.54 ± 7.37c
30.48 ± 1.27c
602.50 ± 16.28c
1.05 ± 0.02a
0.66 ± 0.08a
9.08 ± 1.22a
5.07 ± 0.77b
19.40 ± 2.79a
82.54 ± 13.97ab
137.54 ± 9.04a
17.63 ± 2.21b
25.73 ± 1.96ab
38.80 ± 0.38c
70.31 ± 1.36ab
6.00 ± 1.04a
30.15 ± 1.58b
83.59 ± 2.21b
25.13 ± 1.82b
552.68 ± 17.59b
Treatment 2
Treatment 1
1.70 ± 0.28b
0.76 ± 0.12a
9.87 ± 1.22a
5.05 ± 0.46b
21.92 ± 1.49a
88.57 ± 4.96ab
123.70 ± 12.11a
15.84 ± 1.35ab
28.67 ± 1.70b
32.32 ± 4.26b
63.06 ± 3.52a
6.83 ± 0.85a
31.86 ± 2.94bc
101.41 ± 13.82c
23.05 ± 0.24ab
554.61 ± 20.27b
N.d
N.d
9.19 ± 1.37a
5.58 ± 0.20b
20.81 ± 3.73a
100.07 ± 14.36b
133.01 ± 15.75a
17.37 ± 1.21b
23.45 ± 1.72a
27.59 ± 1.25a
61.09 ± 11.11a
6.44 ± 0.62a
16.29 ± 2.23a
40.58 ± 4.47a
22.59 ± 1.28a
484.06 ± 25.03a
Chardonnay
Control
Treatment 3
Syrah
Compounds
Table 3
Mean concentration (lg/l) of aglycons that can be modified directly by the application of oak extract in Syrah and Chardonnay grapes.
cis-Oak lactone
trans-Oak lactone
Eugenol
6-Methoxyeugenol
Guaiacol
4-Vinylguaiacol
3,4,5-Trimethoxyphenol
Syringol
Syringaldehyde
Vanillin
Acetovanillone
Propiovanillone
Vanillol
Ethyl vanillate
Homovanillic acid
Total
Treatment 3
959
Boidron, & Pons, 1990), with an olfactory threshold in white and
red wines, respectively, of 20–46 lg/l, cis isomer and 140–
370 lg/l, trans isomer (Brown, Sefton, Taylor, & Elsey, 2006). Even
though the olfactory threshold in wine will probably not be surpassed, the presence of these two compounds suggests that an
unnatural compound in grapes can be found after application of
oak extracts to the grapevines.
Eugenol, 6-methoxyeugenol, guaiacol, 4-vinylguaiacol, 3,4,5trimethoxyphenol, and syringol were the phenolic compounds
with the lowest concentration in the oak extract (Table 3). It is
thought Syrah needs a minimum concentration of these compounds in order to affect the content of their glycosides, so grapes
showed no significant differences in the content of eugenol, guaiacol, and 3,4,5-trimethoxyphenol aglycons among samples, and
even a decrease of the concentration of 6-methoxyeugenol, 4vinylguaiacol, and syringol aglycons was observed in treatment
3. The concentration of these six compounds in Chardonnay was
lower than in Syrah grapes. In the Chardonnay variety the concentration of 4-vinylguaiacol was the only one of these compounds
that was not affected by treatment, but eugenol, 6-methoxyeugenol,
guaiacol, 3,4,5-trimethoxyphenol, and syringol were higher in
treated grapes than in control ones. This behaviour depended on
the phenological moment; other authors who studied different
smoke treatments at veraison also found a different effect depending on the application moment and on the compound in question
(Kennison et al., 2009, 2011). In the Chardonnay variety, the
concentration of eugenol and 6-methoxyeugenol compounds increased more than the double in treatment 3 and more than
100% in treatment 1 and 2. Therefore, the treatment had a greater
impact when it was applied closest to beginning of the veraison.
Nevertheless, the concentration of guaiacol was greater when the
application of the oak extract was performed in an advanced state
of maturity, increasing its concentration from 73% to 133%. The
possibility of the biotransformation of this compound into glycosides in the grapes after smoke treatment was demonstrated by
Hayasaka et al. (2010). With respect to the concentration of
3,4,5-trimethoxyphenol and syringol aglycons, significant differences were observed when the application was made 17 days after
the beginning of veraison (T2), increasing its concentration by 50%
when compared to the Chardonnay control grapes. The syringol
accumulated in grapes as non-volatile precursors, e.g. syringolGG glycoside, was also observed in the aforementioned study by
Hayasaka et al. (2010). Eugenol, guaiacol and 4-vinylguaiacol have
in the wine have a low olfactory threshold, 6 lg/l, 9.5 lg/l, and
40 lg/l (Culleré, Escudero, Cacho, & Ferreira, 2004) respectively,
so small changes in the grape compositions due to oak treatment
may have an influence on final wine aroma. The significant increase of eugenol and guaiacol in Chardonnay grapes due to the
treatment could give the wine clove and smoke aromatic notes.
On the other hand, the decrease in the 4-vinylguaiacol concentration in Syrah grapes or zero increase in Chardonnay could be positive as this compound can be transformed into 4-ethylguaiacol by
enzymatic reduction reactions, being harmful to the wine aroma if
it exceeds the perception threshold.
Syringaldehyde and vanillin are the major phenolic aldehydes
that come from the hydrolysis, pyrolysis and oxidation of oak wood
lignin (Baumes, 2009), being the major compounds in the oak extract used. Syringaldehyde, it known by its appreciated aromatic
sweet and dark chocolate notes, while vanillin and it derivatives
by its vanilla, honey and spicy attributes, both compounds with
characteristic notes of oak. Furthermore, these compounds are
found naturally in grapes as part of their varietal aroma (Bureau
et al. 2000; López et al. 2004). Table 3 shows the behaviour, in both
varieties, of syringaldehyde, vanillin and vanillin derivatives such
as acetovanillone, propiovanillone, vanillol, ethyl vanillate and
homovanillic acid. With both varieties, there was no difference in
960
the volume of the treated berries and the volume of their respective control grapes (Table 2). Such increment in the content of berries might be due to their absorption and subsequent union to their
sugar moieties or to the synthesis produced by a change in metabolism after the extract application. It is known that plants may
form glycosylated conjugates from some volatile compounds in order to minimize toxic effects to cells, or to increase their solubility
to facilitate cellular transportation (Winterhalter & Skouroumounis, 1997). In both varieties, the ethyl vanillate was the compound
most affected by all treatments, with a minimum increase of
100% compared to the respective control grapes. The behaviour
of this compound was time-dependent in the different varieties
since Syrah grapes showed the least increase and Chardonnay
the greatest when the application was made after about 17 days
post-veraison. This compound transmits sweet, chocolate and vanillin aromatic notes to the wine, so its increase could be interesting
at the sensory level. The syringaldehyde content in the extract was
higher than ethyl vanillate and homovanillic acid, these latter having similar concentrations between them. Nevertheless the increase of syringaldehyde and homovanillic acid in both varieties
was not as remarkable as that of ethyl vanillate, so the assimilation, storage or synthesis were different depending on the substance studied. This behaviour has been observed in previous
studies, where after applying the same concentration of eugenol
and guaiacol solution to the vineyard, the increase of the substance
in the grapes and wines of each substance was different (MartínezGil et al., 2011, 2012). On the other hand, propiovanillone content
was not affected by the treatments in Syrah and only treatment 2
in Chardonnay showed an increase. This compound had a low concentration in the extract, together with the acetovanillone, and
probably due to this, propiovanillone was the least affected in
grapes. Vanillin showed an increase of about 70% in both varieties,
when the oak extract treatments were done when the phenological
stage was more advanced. Although, the increase of this compound
was not the greatest, due to its low perception threshold in wine
(60 lg/l, Culleré et al., 2004), it could be important in treated
grapes, because it could provide wines with light vanilla notes.
Also, the highest concentration of homovanillic acid and vanillol
were when the application was carried out at a more advanced
stage.
The total aglycon content varied between the control and the
treated grapes depending on the variety and on application time.
In Chardonnay grapes this content was lower than in Syrah, both
in the control and treated grapes. In both varieties, the grapes from
the different treatments showed a significant increase in total aglycons with regard to their respective control grapes. This was more
significant in the Chardonnay variety, with treatment 2 grapes
even showing an increase that more than doubled. This phenomenon indicates that an adequate winemaking process with treated
grapes could obtain a wine with aromas similar to the extract.
The accumulation of each compound in grapes depended on the
target compound as well as on its concentration in the oak extract.
In general, Syrah required a minimum concentration above
1.41 mg/l for each compound within the extract to show a significant concentration increment in the grapes. However, this quantity
was not necessary for Chardonnay grapes, since all compounds increased independently of the treatment used, with the exception of
4-vinylguaiacol.
3.2.2. Aglycons modified indirectly by the application of oak extract, as
these are not included in its composition
Table 4 shows the concentration of the compounds enzymatically released from the glycosylated fraction in Syrah and Chardonnay grapes, whose origin was not the oak extract. Five different
groups of volatile aglycons have been studied: C6 compounds,
alcohols, terpenes, phenols and C13-norisoprenoids.
C6 compounds are derived from the action of lipoxygenase on
the unsaturated fatty acids in grapes, especially linoleic and linolenic acids (Cordonnier & Bayonove, 1981). They are present as free
forms rather than glycosylate, because their formation occurs
mainly in the pre-fermentation stage, being in contact with oxygen
(Ferreira, Hory, & Bard, 1995). Consequently, C6 compounds cannot
be defined as typical aromas of grape varieties but as pre-fermentation aroma compounds of wines (Cordonnier & Bayonove, 1981).
Their olfactive contribution effects on wine flavour change with
their concentrations. At low levels, they contribute positively to
the typical aroma, but at higher concentrations they may be
responsible for herbaceous flavours, so it is important to take a
good pre-fermentative step (Ferreira et al., 1995). The Syrah cultivar presented a higher contribution of these compounds than
Chardonnay (Table 4), being 1-hexanol the most abundant compound in both varieties with more than 50% of the total C6 content.
This compound has a high olfactory threshold (8000 lg/l, green
note, Guth, 1997) so it rarely gives an odour to wines. In Syrah
grapes, no C6 compounds were affected by the treatments
(Table 4). However, in Chardonnay grapes, (E)-2-hexenal concentration increased after the oak extract treatments. This compound
has a low perception threshold (17 lg/l, Noguerol-Pato et al., 2012)
giving wines grass notes. Similar behaviour occurred with the
(E)-2-hexen-1-ol, although in this case it only showed significance
differences in grapes from treatment 2. The Chardonnay grapes
from this treatment therefore presented the highest total concentration of C6 compounds.
The alcohols, benzyl alcohol and 2-phenylethanol, account for
more than 64% of total studied compounds represented in Table 4,
which represents a total minimum of 2451 lg/l in Syrah and
622 lg/l in Chardonnay grapes. Benzyl alcohol was the most abundant compound in all samples. Chardonnay showed an increase of
these two compounds in treated grapes, although only treatment 2
presented significant differences. Syrah grapes from treatment 3
had the highest amount of 2-phenylethanol, so when the application were carried out on more maturated grapes, an increase of
14% was observed. These compounds give positive aroma characteristics to the wine and their formation occurs mainly in the alcoholic fermentation stage. In spite of this, the increase in grapes
could be considered positive, especially, the 2-phenylethanol, with
a rose aroma descriptor, since it has a lower wine odour threshold.
Terpenes are considered to be very important in determining
the flavour and varietal character of Vitis vinifera cultivars, with
an important contribution to the floral and citrus characters of
wines (Ebeler & Thorngate, 2009; Mateo & Jiménez, 2000). These
are present in grapes largely in the skin. The most interesting terpenes (linalool, nerol and geraniol) for organoleptic notes are in relatively small quantities in neutral grapes. Moreover, these three
compounds in free form are used for classification into aromatic
and non aromatic varieties (Bayonove & Cordonnier, 1971; Günata
et al., 1985). The two varieties used in this study (Syrah and Chardonnay) are poor in monoterpenes, both free as glycosylated forms
(Razungles, Gunata, Pinatel, Baumes, & Bayonove, 1993). Razungles
et al. (1993) observed that Syrah contains greater quantities of total glycosylated terpenes than Chardonnay; however these two
varieties have similar amounts of free monoterpenes even slightly
higher in Chardonnay grapes. Also, in our control grapes, the total
of bound terpenes was ten times higher in Syrah than in Chardonnay grapes. Linalool is the only one studied compound studied that
was found in a higher concentration in Chardonnay than in Syrah
grapes (Table 4) by the oak extract effect, but their concentration
in control grapes were similar. In Syrah, all the related linalool
compounds (cis and trans furan-linalool oxide (LOF), linalool, trans
pyran-linalool oxide (LOP) and (Z)-8-hydroxylinalool) showed
higher concentrations in control grapes than in treated grapes.
The oxide forms are less odourous as they have a higher threshold
117.57 ± 19.26a
377.17 ± 36.08a
15.07 ± 1.86b
92.23 ± 3.71a
19.87 ± 2.87b
665.87 ± 85.50b
22.71 ± 2.77b
165.15 ± 25.60b
36.72 ± 6.00b
11.15 ± 1.25c
235.73 ± 26.47c
Phenols
Methyl salicylate
Benzoic acid
Benzaldehyde
Methyl vanillate
Tyrosol
Total
C13-norisoprenoids
3-Hydroxy-b-damascone
3-Oxo-a-ionol
3-Hydroxy-7,8-dihydro-b-ionone
3-Hydroxy-7,8-dihydro-b-ionol
Total
17.86 ± 2.26a
132.17 ± 18.21ab
37.27 ± 4.60b
9.42 ± 0.94b
196.72 ± 18.94b
97.31 ± 12.93a
380.83 ± 28.45a
11.75 ± 1.23a
97.81 ± 4.12a
18.05 ± 1.47ab
605.75 ± 31.58ab
1.68 ± 0.33b
2.14 ± 0.36a
0.73 ± 0.12b
6.74 ± 1.06a
14.31 ± 0.28c
106.90 ± 1.65b
6.88 ± 1.19bc
41.52 ± 0.75b
52.43 ± 3.07bc
233.33 ± 3.95c
1880.79 ± 117.90a
658.69 ± 15.80a
2539.48 ± 118.95ab
21.54 ± 3.77ab
231.67 ± 17.04a
47.47 ± 5.44a
79.43 ± 10.90a
380.11 ± 21.28a
16.12 ± 0.05a
104.41 ± 4.75a
26.07 ± 0.13a
7.78 ± 0.27a
154.38 ± 4.76a
89.39 ± 16.82a
351.98 ± 34.98a
10.09 ± 0.10a
91.00 ± 6.02a
16.31 ± 1.53a
558.77 ± 39.31a
1.49 ± 0.23a
2.01 ± 0.38a
0.51 ± 0.02a
6.52 ± 0.63a
10.04 ± 0.34a
87.58 ± 0.44a
5.80 ± 1.36ab
38.03 ± 1.24a
37.85 ± 4.92a
189.83 ± 5.34a
1818.63 ± 80.25a
632.94 ± 51.83a
2451.57 ± 95.53a
19.05 ± 1.03a
215.74 ± 9.38a
47.43 ± 2.06a
65.68 ± 5.25a
347.90 ± 10.99a
18.65 ± 1.34a
132.06 ± 14.18ab
40.40 ± 3.59b
8.04 ± 0.42ab
199.15 ± 14.69b
97.90 ± 17.56a
355.91 ± 15.69a
11.71 ± 1.60a
123.50 ± 7.01b
18.69 ± 0.64ab
607.24 ± 24.63ab
1.40 ± 0.18a
2.00 ± 0.18a
0.55 ± 0.05a
6.01 ± 0.88a
12.89 ± 0.97b
105.55 ± 1.70b
4.80 ± 0.25a
40.72 ± 2.17ab
43.65 ± 4.53ab
217.57 ± 5.47b
2013.53 ± 98.21a
761.06 ± 9.52b
2774.59 ± 98.67b
25.91 ± 3.28b
238.34 ± 27.76a
46.83 ± 5.80a
73.05 ± 11.43a
384.13 ± 30.75a
7.15 ± 1.21a
56.68 ± 10.72a
8.03 ± 1.18a
4.52 ± 0.73a
76.38 ± 10.88a
1.09 ± 0.19a
105.34 ± 17.06a
0.70 ± 0.07a
20.32 ± 2.67a
N.d
127.45 ± 17.27ab
N.d
N.d
0.93 ± 0.12a
0.57 ± 0.07a
2.34 ± 0.44a
7.64 ± 1.19a
N.d
5.48 ± 0.92a
10.49 ± 1.93a
27.45 ± 2.49a
288.07 ± 47.03a
334.30 ± 53.79a
622.37 ± 71.45a
10.66 ± 1.06a
28.54 ± 4.81a
1.85 ± 0.37a
3.57 ± 0.56a
44.62 ± 4.36a
10.04 ± 1.84b
112.36 ± 22.27b
12.01 ± 1.88b
5.35 ± 0.92ab
139.76 ± 22.44b
1.82 ± 0.33ab
107.30 ± 13.45a
0.93 ± 0.10b
33.56 ± 6.34bc
N.d
143.61 ± 14.87ab
N.d
N.d
1.17 ± 0.21bc
1.10 ± 0.07c
4.08 ± 0.72b
12.11 ± 1.03b
N.d
6.01 ± 0.79ab
20.82 ± 1.50b
45.29 ± 2.12c
334.84 ± 44.22ab
417.05 ± 68.00ab
751.89 ± 8.11ab
16.30 ± 2.13b
29.07 ± 3.02a
2.12 ± 0.25a
4.04 ± 0.23ab
51.53 ± 3.71ab
Treatment 1
13.54 ± 1.04c
120.17 ± 8.82b
14.64 ± 2.42b
6.88 ± 0.41c
155.23 ± 9.21b
2.42 ± 0.91b
112.06 ± 17.58a
1.10 ± 0.05bc
40.85 ± 3.68c
N.d
156.43 ± 17.98b
N.d
N.d
1.36 ± 0.13c
1.09 ± 0.06c
3.75 ± 0.45b
12.01 ± 1.04b
N.d
7.12 ± 0.43b
21.83 ± 1.74b
47.16 ± 2.13c
376.61 ± 17.53b
471.85 ± 24.54b
848.46 ± 30.16b
17.00 ± 1.14b
32.28 ± 3.98a
2.36 ± 0.39a
4.48 ± 0.55b
56.12 ± 4.19b
Treatment 2
11.35 ± 1.61bc
106.08 ± 20.34b
13.12 ± 1.29b
6.37 ± 0.87bc
136.92 ± 20.46b
1.45 ± 0.16ab
87.37 ± 16.55a
1.17 ± 0.14c
30.24 ± 2.94b
N.d
120.23 ± 16.81a
N.d
N.d
1.01 ± 0.12ab
0.83 ± 0.14b
3.29 ± 0.56b
9.57 ± 1.70a
N.d
5.99 ± 0.36ab
18.74 ± 1.67b
39.43 ± 2.48b
317.74 ± 40.83ab
395.37 ± 75.43ab
713.11 ± 85.77ab
16.09 ± 2.98b
29.26 ± 4.08a
2.18 ± 0.28a
4.27 ± 0.26ab
52.50 ± 5.07ab
Treatment 3
All parameters are given with their standard deviation (n = 3). For each variety different letters indicate significant differences between the treatments (level of significance of p < 0.05). Control: untreated grapes; Treatment 1:
grapevines treated with oak extract the 21st July for Syrah and 12th July for Chardonnay; Treatment 2: grapevines treated with oak extract the 1st August for Syrah and 21st July for Chardonnay; Treatment 3: grapevines treated
with oak extract the 8th August for Syrah and 2nd August for Chardonnay.
1.95 ± 0.27b
3.39 ± 0.63b
0.82 ± 0.13b
9.07 ± 1.58b
13.88 ± 0.77bc
108.03 ± 10.55b
7.39 ± 0.63c
45.93 ± 2.02c
61.81 ± 8.17c
252.27 ± 13.64d
2051.09 ± 237.53a
668.67 ± 83.18a
2719.76 ± 251.67ab
Alcohols
Benzyl alcohol
2-Phenylethanol
Total
Terpenes
trans-Furan-linalool oxide (LOF)
cis-Furan-linalool oxide (LOF)
Linalool
trans-Pyran-linalool oxide (LOP)
Nerol
Geraniol
(E)-2,6-Dimethyl-3,7-octadiene-2,6-diol
(Z)-8-Hydroxylinalool
Geranic acid
Total
22.27 ± 3.35ab
221.18 ± 35.66a
49.29 ± 0.69a
74.75 ± 3.15a
367.49 ± 35.96a
Treatment 3
Control
Treatment 2
Chardonnay
Treatment 1
Syrah
Control
C6 compounds
(E)-2-Hexenal
1-Hexanol
(Z)-3-Hexen-1-ol
(E)-2-Hexen-1-ol
Total
Compounds
Table 4
Mean concentration (lg/l) of aglycons that can be modified indirectly by the application of oak extract in Syrah and Chardonnay grapes.
961
962
CONTROL
TREATMENTS
Fig. 1. Canonical discriminant analysis of the aglycons that can be modified indirectly by the application of oak extract in different Syrah grapes (Control: untreated grapes;
Treatment 1: grapevines treated with oak extract the 21st of July; Treatment 2: grapevines treated with oak extract the 01st of August; Treatment 3: grapevines treated with
oak extract the 08th of August).
(Ribéreau-Gayon, Boidron, & Terrier, 1975). In Syrah, geraniol was
the most abundant terpene aglycon followed the geranic acid,
whereas in the Chardonnay variety was the opposite. Control
grapes showed a ratio geraniol/geranic acid of 1.75 and 0.73
respectively for Syrah and Chardonnay. This ratio was increased
to 2.42 by the oak treatments in Syrah, and decreased to 0.51 in
Chardonnay, this could be because geranic acid was more affected
by the all treatments than geraniol. Both compounds have the
same value of the wine perception threshold (40 lg/l, NoguerolPato et al., 2012) but different attributes, while geraniol gives wine
flowery notes such as geranium, geranic acid gives some green and
herbaceous ones. Syrah grapes from treatment 2 showed the
lowest concentration of nerol and geraniol aglycons. Also, the
grapes in this treatment together with treatment 3 showed (E)2,6-dimethyl-3,7-octadiene-2,6-diol and geranic acid concentrations lower than its control. So, in this variety, the oak treatment
decreased the total terpenes composition, with treatment 2 being
the most affected. In contrast, treated Chardonnay grapes showed
a minimum increase of total terpenes of 44% with respect to the
control. The furan-linalool oxides and the (E)-2,6-dimethyl-3,7octadiene-2,6-diol were not found in Chardonnay grapes, which
is in accordance with the Razungles et al. (1993) finding. The
higher concentration of linalool and geraniol was observed for
treatments 1 and 2. Also, an increase of trans LOP, nerol and geranic
acid was also noticed in all the treatments, as well for (Z)-8hydroxylinalool which only had significant differences with
respect to the control for treatment 2. The terpenes in Chardonnay
grapes showed an increase with all treatments, especially when
the application of the oak extract was done when grapes were less
mature. It has been proved that the oak treatments in Chardonnay
improved the aromatic quality due to increased terpene glycoconjugates, which could contribute positive notes to wine, but the
decrease in Syrah could adversely affect their quality.
Phenols were the second most abundant compounds in the glycosylated fraction analysed in Syrah, but in Chardonnay this family
showed concentrations similar to C13 norisoprenoids in some of
the treatments (Table 4). Benzoic acid was the predominant phenol
in both varieties, but its concentration was not affected by the
treatments. Methyl salicylate was only affected in grapes from
treatment 2 of Chardonnay, this compound contributes a wintergreen aroma (40 lg/l, Fan, Xu, Jiang, & Li, 2010), probably this increase in the grapes does not affect to wine aroma. Also, methyl
vanillate content increased in Chardonnay grapes from all treatments and only for treatment 3 in Syrah. However, the perception
threshold of this compound in the wine is high, so the significant
increase observed (33% in Syrah to 50–100% in Chardonnay) could
enhance vanillin flavour. Benzaldehyde, whose flavour reminded
us of bitter almonds, showed two different behaviours, depending
on the variety: a decrease in all samples from Syrah and an
increase in all samples from Chardonnay treatments. Tyrosol decreased in Syrah grapes from treatment 2 but this compound
was not detected in all Chardonnay grapes. Hence, when the treatments with oak extract were carried out about 17 days after veraison, the total volatile phenols in treated grapes of both varieties
presented significant differences with respect to their control.
The tendency of this total was different depending on the variety,
generally decreasing in Syrah and an increasing in Chardonnay.
In grapes, most of the C13-norisoprenoids are present as
glycosidic precursors, the opposite to terpenes, they are found in
similar quantities, in aromatic and neutral grape varieties, and they
are of awarding certain typicity to the wine flavour because they
have very low odour threshold (Sefton et al., 1993; Winterhalter,
Sefton, & Williams, 1990; Wirth, Guo, Baumes, & Günata,
2001). Norisoprenoidic glycoconjugate aglycons (3-hydroxy-bdamascone, 3-oxo-a-ionol, 3-hydroxy-7,8-dihydro-b-ionone and
3-hydroxy-7,8-dihydro-b-ionol) are considered as degradation
products of grape carotenoids (Winterhalter & Skouroumounis,
1997) and during wine ageing they transform into potent
odourants such as b-damascenone (rose, sweet and floral attributes with a very low threshold of 0.005 lg/l, Guth, 1997;
963
CONTROL
TREATMENTS
Fig. 2. Canonical discriminant analysis of the aglycons that can be modified indirectly by the application of oak extract in different Chardonnay grapes (Control: untreated
grapes; Treatment 1: grapevines treated with oak extract the 12th of July; Treatment 2: grapevines treated with oak extract the 21th July; Treatment 3: grapevines treated
with oak extract the 2nd August).
Skouroumounis & Sefton, 2002). Among the C13-norisoprenoid
studies, 3-oxo-a-ionol was found to have the highest amount in
both varieties, as was observed by Bureau et al., (Bureau et al.,
2000) in Syrah variety and by Sefton et al. (1993) in Chardonnay,
followed by the 3-hydroxy-7,8-dihydro-b-ionone. However, 3-hydroxy-7,8-dihydro-b-ionol was the one found in lower concentrations. The content of 3-hydroxy-b-damascone and 3-hydroxy-7,8dihydro-b-ionol was higher in control Syrah grapes than in any
of the treated grapes. Syrah control grapes also showed a higher
3-oxo-a-ionol and dihydro-b-ionone content than grapes from
treatment 2. Thus, the oak extract treatments in Syrah adversely
affect the concentration of C13 compounds, being more remarkable for treatment 2. However, an inverse behaviour was observed
in the treated grapes of Chardonnay because an increase in all C13
compounds was observed. As this family is normally considered a
positive contributor to wine aroma, the oak treatment in this variety could improve the aromatic profile. Chardonnay grapes from
treatment 2 showed the highest concentration of 3-hydroxy-bdamascone and 3-hydroxy-7,8-dihydro-b-ionol; as both of them
are precursors of b-damascenone (Humpf, Winterhalter, & Schreier, 1991), this treatment may provide more floral and exotic fruit
notes. Nevertheless, no significant effect was observed for the total
C13 norisoprenoide aglycons in Chardonnay due to the different
application times.
3.3. Sample discrimination
Taking into account all aglycons studied, a discriminant analysis
was performed on the Syrah and Chardonnay varieties. Results
provided two canonical functions with a 99.9% of variance (figure
not shown). Principally the function 1 separated the Syrah grapes
from the Chardonnay, while function 2 separated the groups of
treated grapes from one another and from their control grapes.
The discriminating variables that contributed more to differentiation were the total phenols and the total terpenes. These results
demonstrated the great difference between the two varieties, so
in order to see the effect of the different treatments more clearly,
other discriminates were done. Two new discriminant analyses,
one for each variety, were performed with the compounds that
can be modified directly by the application of oak extract, that is
to say, compounds which were present in the oak extract. The
two first functions in Syrah explained 99.9% of the variance and
in Chardonnay 95.6%. The discriminant variables held in common
in both discriminants were cis and trans-oak lactones and ethyl
vanillate. This was expected from the previous results, since the
grapes from oak treatments showed the two oak lactones not naturally present in grapes along with ethyl vanillate, which was the
compound that increased most considerably with these applications. Also, there are other variables for Syrah, such as eugenol,
methoxyeugenol, vanillin and propiovanillone, while for Chardonnay there was syringaldehyde. These results also showed that the
application time was differently affected depending on the variety.
When compared with the control, treatment 3 was the most discriminate in Syrah and treatment 2 in Chardonnay.
Finally, Fig. 1 (Syrah) and Fig. 2 (Chardonnay) show the
discriminants performed with the compounds that can be modified indirectly by the application of oak extract, since they are
not in the oak extracts. In both, function 1 in Fig. 1 explained
98.7% of the variance and 99.5% in Fig. 2. The variables that
contributed to the discriminant model were 3-hydroxy-bdamascone, methyl vanillate, (Z)-8-hydroxilinalool, (E)-2,
6-dimethyl-3,7-octadiene-2,6-diol, (E)-2-hexen-1-ol, nerol and
benzyl alcohol (Fig. 1) and geranic acid, 1-hexanol, benzoic acid,
geraniol and benzaldehyde (Fig. 2). Both discriminants (Figs. 1
and 2) showed the possibility of differentiating the control
grapes from the treated grapes. Also, Syrah (Fig. 1) showed a
good discrimination among the grapes from the different treatments, with treatment 1 being the most different. However in
Chardonnay (Fig. 2), this analysis was not able to discriminate
the samples from the different treatments.
964
4. Conclusions
The oak extract applications to Syrah and Chardonnay grapevines affected the grape aroma glycosidic precursors composition.
In general, the results showed that treated grapes of both varieties
had the highest content of the compounds directly related to the
oak extract, which shows that volatile oak extract compounds
can be assimilated by treated grapes and stored as glycosidic precursors. The aglycon content depended on the application timing,
with the highest increase for treatment 3 in Syrah and treatment
2 in Chardonnay. Moreover, these oak treatments affected the
other glycosidic precursors, typical of the varieties and not present
in the extract, with a generally decreased behaviour in Syrah and
increased in Chardonnay. This behaviour depends on the application timing, especially in Syrah, so to perform the treatments at
the appropriate time, it is important to know the responsive phenological stage of each variety. Since the increase of varietal precursor compounds, in general, contributes positive characteristics
and typicity to the wine, the treatment in Chardonnay could enhance their glycosidic aroma profile. Also, depending on the Syrah
wine pursued, the treatment 3 could be interesting, as this showed
the least decrease in varietal aroma precursors but the highest content in compound from the oak.
Acknowledgements
We would like to give thanks for the financial support given by
the Ministerio de Ciencia e Innovación to Project AGL2009-08950,
supported by FEDER funding. Also, we are grateful for the FPI
scholarship and to José Castillejo funding from the Junta de Comunidades de Castilla-La Mancha for A.M.M.-G. (EXP 422/09) and
to the MICINN for A.I.P.-G. (BES-2010-038613). We appreciated
the collaboration with the INRA (Institute National de la Recherche
Agronomique), under an agreement with the european project
WINETech (SOE1/P1/E071). We wish to express our gratitude to
Protea France for donating supplies of oak extracts gifts and Kathy
Walsh for proofreading the English manuscript. The authors wish
to thank Marc Heywang and Jean-Nöel Lacapere (INRA-UE Pech
Rouge) for assistance during the vineyard experimentation.
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7.5. ARTÍCULO V
Al existir extractos vegetales de plantas aromáticas con gran potencial
aromático que sin embargo normalmente son desechados, como es el caso de los
hidrolatos, se planteó el presente trabajo cuyo objetivo principal fue conocer si la
aplicación foliar de un hidrolato de lavandín a viñedos era capaz de afectar a la
composición volátil de sus vinos.
Para ello, se escogieron viñas de la variedad Petit Verdot cercanas a los
campos de lavandín, ya que anteriormente se había observado que eran
receptivas a las aplicaciones externas del extracto de roble, y se realizaron 2
tratamientos: una aplicación (H1), cinco aplicaciones (H5), y un control (sin
tratar). Los muestreos se realizaron el día de la vendimia, después de la
fermentación alcohólica, después de la fermentación maloláctica y transcurridos
6 meses de ésta. Para este estudio se analizaron los parámetros enológicos de
uvas y vinos, los compuestos volátiles del extracto de lavandín y de los vinos.
Los compuestos analizados en estos últimos fueron: 21 ésteres, 5 ácidos, 9
terpenos, 3 fenoles, 11 alcoholes, 2 lactonas, 5 aldehídos y 2 compuestos
desconocidos
145
Artículo V
El estudio del hidrolato mostró que este era rico en compuestos volátiles,
especialmente en terpenos, los cuales son de gran importancia debido a sus
características aromáticas positivas y a sus propiedades biológicas. En éste se
cuantificaron 9 esteres, 11 terpenos, 3 alcoholes, 2 lactonas y 5 aldehídos.
Los vinos procedentes de los viñedos tratados con el hidrolato vieron
favorecida durante su evolución la estabilidad de los principales compuestos
responsables de aroma del vino. Además, estos vinos presentaron un aumento en
compuestos volátiles positivos para el aroma, tales como los terpenos, fenoles,
aldehídos y lactonas, sobre todo después de los seis meses en botella, siendo
mayor la concentración en estos compuestos cuando mayor fue el número de
aplicaciones, tratamiento H5.
Los vinos Petit Verdot procedentes de la zona mostraron una
composición volátil que difiere de la habitual, lo cual puede atribuirse al medio
ambiente, ya que sus viñedos se cultivan cerca de campos de lavandín. Un claro
ejemplo fue que los vinos que procedían de las viñas sin tratar (control)
presentaron un compuesto, canfor, no usual en los vinos pero si en la
composición volátil de las plantas de lavandín. Además se observó que este
compuesto tras los tratamientos con el hidrolato aumentaba su concentración, y
que este aumento era mayor cuanto mayor fue el número de aplicaciones a la
viña, por lo que estos tratamientos podrían potenciar o imitar los caracteres
típicos del terroir, realzando el sello identificativo de la zona.
146
Lavandin Hydrolat Applications to Petit Verdot Vineyards and its impact on their
Wine Aroma Compounds
Ana M. Martínez-Gila; Ana I. Pardo-Garcíaa, Amaya Zalacaina, Gonzalo L. Alonsoa, M. Rosario Salinas a*
a
Cátedra de Química Agrícola. E.T.S.I. Agrónomos. Universidad de Castilla-La Mancha. Campus Universitario. 02071 Albacete. Spain. Tel: +34 967
599310.
ABSTRACT
Petit Verdot vineyards, close to lavandin fields, were treated at veraison with a lavandin hydrolat, subproduct
of essential oils production, in order to determine if such treatment causes changes in wine aroma
composition. Two different foliar hydrolat applications were carried out: H1 where lavandin hydrolat was
applied only once and H5 where it was applied five times. The volatile composition of the wines produced
with such grapes were analysed, after the alcoholic and malolactic fermentations and six months after this,
by stir bar sorptive extraction and gas chromatography mass spectrometry (SBSE-GC-MS). The effect of
lavandin atmosphere on control wines was evident as camphor, unusual wine compound, was detected and
increased with hydrolat treatments. Results also showed that the aroma of wines from treated grapevines was
modified, especially in relation to some positive aroma compounds in H5 wine at six months after malolactic
fermentation. A more stability on some main aroma compounds, such as esters, during evolution was also
observed in wines from treated grapevines than in the control ones. Then, it has been proved how the
application of lavandin hydrolat to Petit Verdot vineyards can be used to modify its aroma profile and
somehow stabilized.
KEYWORDS: lavandin hydrolats, grapevines, aroma compounds, wines, Petit Verdot
1. Introduction
The status of the wine sector nowadays faces
interesting challenges for nationally and
internationally business, not only quality is a
priority but also an identification stamp that
describes itself as a unique product should be
offered to the market.
As the quality of the grapes is the first factor
that conditions the quality of wine, any
innovation on the vine-growing sector will
provide a differentiated final product. For
example, there are many studies on the
agronomic practices having the organic
viticulture as a philosophy. There is also an
increasingly raising concern regarding residues
of fungicides in wine and their effects on human
and environmental health, so there is a new trend
of pest combat with natural compounds.
Castilla-La Mancha produces nearly the 70%
of the lavender-lavandin Spanish production
(6352 tons) (annual of agricultural statistics,
2010, Ministry of Environment and Rural and
Marine, Government of Spain) which is used to
obtaining essential oils. It is well known that
essential oils and their derivates can be used as
antimicrobial, antioxidant, antifungal and insect
repellents, and they are considered as an
alternative to conventional synthetic chemical
pesticides, due to their reduced health risk and
their biodegradability (Kanat & Alma, 2004;
Varona, Kareth, Martín, & Cocero, 2010).
Particular studies were carried out with lavender
EOs which has been proved to enhance the
tolerance of the vines to Mildew (Plasmopara
vitícola) (Harm, Kassemeyer, Seibicke, &
Regneret, 2011) or Botrytis cinerea (Jacometti,
Wratten, & Walter, 2010); however there is not
literature on the agricultural use of lavender
hydrolats and neither of lavandin.
The hydrolats are defined as the distilled
water from the production of essential oils
obtained by steam water distillation from
aromatic plants (flowers, leaves, stems, roots), so
the main components are water-soluble volatile
1
compounds. Hydrolats are normally discarded
because they have no commercial interest as it is
a poor concentration solution in comparison with
their respective EOs, for this reason very few
applications have been reported (Price & Price,
2004; Paolini, Leandri, Desjobert, Barboni, &
Costa, 2008; Aazza, Lyoussi, & Miguel, 2012).
Marín, Alonso, & Salinas, 2007; Tredoux, De
Villiers, Májek, Lynen, Crouch, & Sandra, 2008;
Forde, Cox, Williams, & Boss, 2011; MartínezGil, Garde-Cerdán, Martínez, Alonso, & Salinas,
2011), together with the unique sorbent phase
ratio coating (Baltussen, Sandra, David, &
Cramers, 1999).
Castilla-La Mancha is also the region with the
greatest vineyard surface in the world
(FAOSTAT, 2011), so it is quite common to see
vineyards growing closed to lavender-lavandin
plantations.
All the above consideration has led us to
propose the present study, which aim is to know
if the application a lavandin hydrolat to the
vineyards affects the volatile composition of the
wines from treated grapevines. For this, two
different treatments were carried out to Petit
Verdot grapevines and wine aroma evolution
was followed after the alcoholic and malolactic
fermentation and 6 months after the later.
It is known that some grapevines treatments
or environmental factors can modify the wine
aroma, which supposed an interesting innovative
research. Clear examples are the studies that
show that chemical pesticides not only affect the
fermentative aroma compounds but also in the
wine varietal aroma (Oliva, Zalacain, Payá,
Salinas, & Barba, 2008; Noguerol-Pato,
González-Rodríguez,
González-Barreiro,
Cancho-Grande, & Simal-Gándara, 2011).
Capone, Jeffery, & Sefton, (2012) observed that
wines from grapes that are grow closely of a
eucalyptus forests express an aromatic note to
eucalyptol, while other authors study the impact
of cover crops in vineyard on the aroma
composition wine (Xi, Tao, Zhang, & Li, 2011).
Grapes and grapevines exposed to smoke also
affect the chemical composition and sensory
properties of wine (Kennison, Wilkinson,
Williams, Smith, & Gibberd, 2007; Wilkinson et
al., 2011), or oak compounds are assimilated by
grapes and detected in wines when vineyards are
treated with different oak aqueous extracts
(Martínez-Gil, Garde-Cerdán, Martínez, Alonso,
& Salinas, 2011; Martínez-Gil, Garde-Cerdán,
Zalacain, Pardo-García, & Salinas, 2012;
Martínez-Gil, Angenieux, Pardo-García, Alonso,
Ojeda, & Salinas, 2013).
The analytical technique used to determine
the wine aroma composition is also important
leaning towards rapid and solvent-free extraction
and the non-generation of artifacts while the
isolation of volatile compounds (Noguerol-Pato,
González-Rodríguez,
González-Barreiro,
Cancho-Grande, & Simal-Gándara, 2009;
Noguerol-Pato, González-Rodríguez, GonzálezBarreiro, Cancho-Grande, & Simal-Gándara,
2011; Xi, Tao, Zhang, & Li, 2011). Stir bar
sorptive extraction (SBSE) is a powerful
technique on this extraction basis (Zalacain,
2. Materials and Methods
2.1. Lavandin hydrolats
Lavandin cultivar (Lavandula hybrid, natural
hybrid between L. angustifolia and L. latifolia)
was cultivated in La Mancha Region (Albacete
Province, southeastern Spain). The recollection
mechanized of this plant was carried out on the
first and second week of July in 2010. The
collected plants were allowed to dry in the field
for a few days, collecting the plant almost dry
with yields of between 2500-3000 kg flowers per
hectare. The distillation was carried out on 17
July by steam distillation extraction (water at
100 °C) at low pressure (0.5 bar), and 0.6 liters
of hydrolat were obtained per kilo of plant. This
method gives us the separation between the
essential oil and hydrolat. The lavandin hydrolat
used for the vineyard treatments was obtained
from a mixture of four lavandin varieties
(Grosso, Super, Abrial and Mallieta).
Immediately after having been obtained, the
lavandin hydrolat was stored in tanks at 2-4ºC
until use.
2.2. Grapevine treatments
Vitis vinifera Petit Verdot red variety, grown
closely to lavandin plots, (Albacete Province,
southeastern Spain) were used as starting
materials. Such lavandin plots are the ones used
to produce essential oils generating an important
amount hydrolats as subproduct. The grapevines
were cultivated in trellis through the Smart
Dyson system. The vineyards were fitted with a
drip irrigation system to assure adequate water
2
needs, as this region registers only 300-400 mm
of rainfall per year. The local climate is
continental Mediterranean with extreme
temperatures in winter and summer, annual
average temperature of 13º C, minimum -15º C
(January) and maximum 40 º C (August).
Lavandin hydrolat treatments, season 2010,
were applied to the grapevine leaves during
veraison because at this stage it has been tested
that the volatile composition of grapes can be
affected by similar applications of aqueous oak
extracts (14-16). The 6th of August was the halfveraison, when the color green of the grape
turned to red, and the flexibility of the skins was
high, presenting this aspect at least half of the
clusters. The treatments were commenced 7 days
after the half-veraison. The lavandin hydrolat
was applied once, treatment called Hydrolat 1
(H1), and five times, treatment called Hydrolat 5
(H5). The H1 treatment was applied to the
vineyard only on 13th of August and H5
treatment on 13th, 20th, 27th, of August and 3rd
and 10th of September. To improve the adhesion
of the hydrolat to leaves, 0.5 mL per liter of
adjuvant Fluvius (soluble concentrate composed
of a copolymers mixture: polyether-trisiloxane
modified (19.5% w/w) and polyether siloxanes
(80.5% w/w), BASF, Germany) was added to all
treatments; since this is a wetting agent typically
used for foliar herbicide treatment. The study
used 6 mini-plots of 5 plants in the same row,
each treatment was carried out on 10 plants, so
two of them were randomly distributed by each
treatment, leaving 1 mini-plots untreated
between the different applications to avoid
contamination. The application was performed
with a hand-sprayer, so that the application to
each plant was much focused. To wet the entire
foliar surface per plant 250 mL of each
formulation were necessary, so this volume was
applied evenly by spraying over leaves. The
treatments were carried out when the
environmental temperature was below 20ºC, at
approximately 7 oćlock in the morning.
Moreover, 10 plants were not treated (control
(C)).
2.3. Winemaking
Grapes were harvested on October 5th, at their
optimum maturation moment. Grape yield per
plant was calculated by dividing the total mass
production (kg) by the number of plants and %
of berries was calculated by dividing stemmed
grapes mass (kg) by the total mass production
(kg). 10g of potassium metabisulphite per 100 kg
of grape mass was added. The grapes of the
whole clusters were manually destemmed and
crushed to obtain the must. Then, the must of
each treatment was divided into 2 batches of
approximately 5 liters each one, as the
fermentation was done in duplicate, so 6
vinifications were made. This process was
performed in a multitube fermenter (Martínez
Solé y Cía, S.A., Villarrobledo, Spain) which
reproduces wine cellar winemaking conditions.
The fermentations were carried out with QA23
yeast strain of Saccharomyces cerevisiae subsp.
cerevisiae which was inoculated at a dose of 0.2
g/L according to the recommendation of
Lallemand (Spain). The skins were submerged
throughout
the
alcoholic
fermentations
(maceration step). The alcoholic fermentation
were carried out with an average temperature of
22ºC and daily, the density and temperature were
controlled in the six fermentations. On October
18, the reducing sugars were below 2.5 g/L, so
the alcoholic fermentations were finished. That
day, the wines were pressed manually and the
skins and seeds were removed. For each of the
fermentations, a sample was taken and frozen at
-20ºC until analysis.
Four days after the date of alcoholic
fermentation finalizing, the lees were removed.
Malolactic fermentation was induced using a
commercial bacterium strain of Oenococcus oeni
(Viniflora CH16, Chr Hansen, Buenos Aires) in
a proportion of 10 mg/L on October 22. The
malolactic fermentation was carried out at 20ºC
in the same multitube fermenter as the alcoholic
fermentation. The correct development of
malolactic fermentation was monitored by
measuring the daily concentrations of malic and
lactic acids. Fermentation was considered
finished when the concentration of malic acid
was approximately 0.4 g/L. For each wine, a
sample was taken and preserved at -20ºC for
later analysis.
At the end of malolactic fermentation, on
November 26, free SO2 concentration was
corrected to 25-35 mg/L. The wines were stored
in bottles at 14ºC for six months, after this time a
sample was taken from each of them and frozen
at -20 ºC until analysis.
3
ºBaumé, pH, titratable acidity (g/L tartaric
acid), volatile acidity (g/L acetic acid), alcohol
degree, reducing sugars, anthocyanins and color
index from the different samples were measured
in triplicate following the methods established
by ECC (1990). Malic and lactic acids were
analyzed in wines using HPLC-RID (Agilent
1100, Palo Alto, USA) with a column block
heater and refractive index detector (RID)
(Agilent 1200). The mobile phase was 0.004 M
H2SO4 flowing at 0.4 mL/min and 75ºC on a PL
Hi-Plex H, 8 Pm, 300 x 7.7 mm column (Varian,
Middelburg, The Netherlands). All samples were
filtered (0.45 Pm pore filter) and directly
injected into the column. Injection volume was
10 PL. The RID was at 55ºC and the total time
of analysis was 30 min. Quantification was
based on five-point calibration curves (R2 >
0.97) of respective standards (Sigma-Aldrich,
Madrid, Spain) in water. All the analyses were
done in triplicate, so the result of each wine is
the average of six values (n=6), since
fermentations were done in duplicate.
2.5. Analysis of volatile compounds by gas
chromatography
The wine volatile compounds were extracted
by stir bar sorptive extraction (SBSE) according
to Zalacain, Marín, Alonso, & Salinas, (2007)
and Oliva, Zalacain, Payá, Salinas, & Barba,
(2008) and these were analysed by GC-MS. The
polydimethylsiloxane coated stir bar (0.5 mm
film thickness, 10 mm length, Twister, Gerstel,
Mülheim and der Ruhr, Germany) into 25 mL of
sample, to which 62.5 μL of internal standards hexalactone and 3-methyl-1-pentanol solution at
1 μL/mL, both in absolute ethanol (Merck,
Damstard, Germany) was added. Wine samples
were directly analyzed while hydrolats were
diluted 1:2500 for avoiding saturation effects.
All samples were stirred at 500 rpm at room
temperature for 60 min. The stir bar was then
removed from the sample, rinsed with distilled
water and dried with a cellulose tissue, and later
transferred into a thermal desorption tube for
GC–MS analysis. Thermal desorption was
performed on a TD with a PTV injector
"Programmed-Temperature Vaporisation" CIS-4
Gerstel installed on an Agilent 7890A GC5975C insert XL MDS (Agilent Technologies,
Palo Alto, CA, USA). Volatile compounds were
desorbed from the stir bar at the following
conditions: oven temperature at 330ºC;
desorption time, 4 min; cold trap temperature, 30ºC; helium inlet flow 45 mL/min. The
compounds were transferred into the gas
chromatograph (Agilent 7890A GC-5975C insert
XL MDS Agilent, Little Falls, DE, USA) with a
fused silica capillary column (BP21 stationary
phase, 50 m length, 0.22 mm i.d., and 0.25 m
film thickness; SGE, Ringwood, Australia). The
chromatographic program was set at 40ºC (held
for 2 min), raised to 150ºC at 10ºC/min (held for
5 min) and then raised to 230ºC at 10ºC/min
(held for 2 min). The total time analysis was 28
minutes. For mass spectrometry analysis,
electron impact mode (EI) at 70 eV was used.
The mass range varied from 35 to 500 u and the
detector temperature was 150ºC. The analysis of
volatile compounds in the wines was done in
duplicate, and as the fermentations were done in
duplicate, the results shown for these compounds
were the mean of 4 analyses. Identification was
carried out using the NIST library and by
comparison with the mass spectrum and
retention index of chromatographic standards
designed by us and data found in the
bibliography. When the standards were
available, the quantification was based on fivepoint calibration curves of respective standards
(Aldrich, Steinheim, Germany) (R2 > 0.97) in a
12% ethanol (v/v) solution at pH 3.6; otherwise
semi-quantitative analyses were carried out
using the calibration curves of the most similar
compound.
2.6. Statistical analysis.
Statistical analysis was carried out using
SPSS Version 19.0 statistical package for
Windows (SPSS, Chicago, USA). Volatile
compound data were processed using variance
analysis (ANOVA). Differences between means
were compared using the least significant
difference (LSD) test at 0.05 probability level. A
discriminant analysis was performed with the
total of the volatile compounds in the wines
(esters, fatty acids, terpenoids, phenols, alcohols,
lactones and aldehydes).
Petit Verdot is a red grape variety that produces
wines rich in tannins with an intense color and
aroma, and is a receptive variety to external
applications, such as aqueous oak extract
(Martínez-Gil, Garde-Cerdán, Zalacain, Pardo4
García, & Salinas, 2012). Petit Verdot wines
from the area of this study have a characteristic
aroma likely related to the environment since
their vineyards are grown close to lavandin
fields. In this paper lavandin hydrolat is applied
to these vineyards in order to know its impact on
the wine aroma composition.
Table 1. Volatile composition of the lavandin hydrolat
Lavandin hydrolats (mg/L)
Esters
Hexyl acetate
81.48
Hexyl butanoate
0.02
Ethyl hexanoate
0.34
Ethyl heptanoate
0.93
Ethyl octanoate
50.00
1-octen-3-yl-acetate
13.02
Ethyl piruvate
3.42
Isobornyl acetate
6.45
Lavandulool acetate
88.33
Terpenoids
Limonene
9.98
Linalool
1270.49
Citronellol
9.06
Nerol
19.45
-terpineol
221.73
cis-linalool oxide
11.63
4-terpineol
428.30
cis--ocimene
8.00
-bisabolol
1.65
1,8-cineol
27.71
Camphor
119.69
Alcohols
2-phenylethyl alcohol
39.33
Furanmethanol
310.58
1-octen-3-ol
623.47
total compounds, being the lavandulool acetate,
hexyl acetate, ethyl octanoate and 1-octen-3-yl
acetate the major compounds. The terpenes were
more than double of the total compounds
quantified (56.3%), which is important by the
biological properties that these compounds may
provide to the vineyard, and responsible of the
olfactory properties of the lavandin. Linalool
was the major component identified within this
hydrolat (33.5%), being as well the main
compound in the composition of its flowers
(Salinas, Zalacain, Blázquez, & Alonso, 2007),
EOs (Steltenkamp & Casazza, 1967) and other
hydrolats (Adam, 2006). The terpenes, 4terpineol, -terpineol and camphor were also
abundant and together with linalool supposed the
95.9% of the total terpenes. In accordance with
other authors (Adam, 2006), the quality of the
EOs aroma is determine by the ratio of linalool
and camphor as the highest linalool content the
better its quality, which is observed with the
hydrolat studied (Table 1). The alcohols
represent the 25.8% of the hydrolat aroma
composition, being the most abundant 1-octen-3ol followed by the furanmethanol. Two lactones
were found in the lavandin hydrolat
composition,
2(5H)-furanone
and
2Hydroxycyclopent-2-en-1-one,
which
represented a 3.0%. Aldehydes were 8.4%, being
furfural the most abundant.
Therefore, due to the richness concentration
of volatile compounds present within this
hydrolat, its application to the vineyards may be
positive in order to modify and differentiate the
aroma of the resulting wines.
Lactones
2(5H)-furanone
46.58
2-cyclopenten-1-one, 2-hydroxy
68.00
Aldehydes
Octanal
1.74
Nonanal
7.11
Phenylacetaldehyde
1.65
Furfural
303.33
Benzaldehyde
3.33
3.1. Hydrolat lavandin volatile compounds
The aroma composition of lavandin hydrolat
(mg/L) is shown in Table 1. The volatile
compound families were represented by 9 esters,
11 terpenoids, 3 alcohols, 2 lactones, and 5
aldehydes. Esters represented a 6.46% of the
The oenological parameters of Petit Verdot
grapes from the different treatments (C, H1 and
H5) can be observed in Table 2. The parameters
which are going to define the oenological
aptitude of wines such as ºBaumé, pH and total
acidity did not show significant differences by
the treatments. However, an increase of yield
and berries percentage (%vintage mass) was
observed in vineyards as the number of
treatments increased. ºBaumé/titratable acidity
ratio was measured for harvested at their
optimum maturation moment, showing values
between 1.67 and 1.80.
5
3.3. Wine oenological parameters
were observed between the control wine and
wine from treatments. The malolactic
fermentation was developed correctly and
without significant differences between wines,
as the concentration of lactic acid and malic
acid was similar. An anthocyanins decrease
between 67% and 76% during the evolution of
wine were observed (Table 3); after six months,
the wine that presented the lowest content of
these compounds was from H5 treatment. Color
intensity was not affected by different
treatments, this parameter during the malolactic
fermentation showed a decrement in all the
samples, and this was stable during the bottle
evolution.
At the end of the alcoholic and malolactic
fermentation, alcohol degree presented a lower
value in wine from H5 grapevines (Table 3).
This parameter tended to decrease after
malolactic fermentation, such that after six
months, it did not present significant differences
between the wines from hydrolats treatments
and the control wine. This decrement could be
due to a possible volatilization of ethanol and/or
the formation of other compounds such as ethyl
esters. After alcoholic fermentation, the pH and
the volatile acidity increased while the acidity
decreased, probably due to the transformation of
malic acid into lactic acid, but no differences
Table 2. Oenological parameters in Petit Verdot grapes on harvest day after the different grapevine treatments.
Treatments
Yield
(kg/plant)
%Berries
º Baumé
pH
Titratable acidity
(g/L)a
ºBaumé/TA
Control
3.3a
82.0a
14.6a
3.49a
8.25a
1.77
Hydrolat 1
3.8b
84.6b
14.5a
3.53a
8.66b
1.67
Hydrolat 5
4.5c
89.7c
14.4a
3.44a
8.00a
1.80
%Berries=%vintage mass. As g/L tartaric acid. TA: titratable acidity. Control: grapes from untreated vines; Hydrolat 1: grapes from vine
treated once with lavandin hydrolat; Hydrolat 5: grapes from vine treated five times with lavandin hydrolat.
Table 3. Oenological parameters in wines at the end of alcoholic and malolactic fermentations and after eight months from the end
of malolactic fermentation.
Treatments
Alcohol
degree
(%, v/v)
pH
Volatile
Titratable
Lactic
acidity
acidity
acid
a
b
(g/L)
(g/L)
(g/L)
Malic acid
(g/L)
Anthocyanis
(mg/L)
IC
Control
15.79b
3.96a
0.12a
5.94a
-
3.22a
1072.65b
23.76a
Hydrolat 1
15.60b
3.97a
0.10a
5.99a
-
3.31a
993.73ab
21.99a
Hydrolat 5
15.32a
3.97a
0.11a
5.93a
-
3.31a
914.49a
20.83a
Control
15.28b
4.17a
0.27a
4.37a
1.53a
0.39a
766.90a
19.44a
Hydrolat 1
15.27b
4.20a
0.26a
4.40a
1.57a
0.42a
752.35a
18.55a
Hydrolat 5
14.93a
4.20a
0.24a
4.37a
1.60a
0.43a
685.32a
16.92a
19.50a
Control
15.03a
4.13a
0.29a
4.16a
1.57a
0.35a
630.21b
Hydrolat 1
15.04a
4.15a
0.28a
4.24a
1.62a
0.36a
595.03b
18.54a
Hydrolat 5
14.89a
4.15a
0.30a
4.21a
1.63a
0.42a
520.16a
16.84a
a
As g/L acetic acid. bAs g/L tartaric acid. IC: color index (as sum of absorbances at 620, 520 and 420 nm). All the parameters are given with their
standard derivation (n=3). The different letters indicate significant differences between the samples (level of significance of p<0.05). Control:
wines from untreated vines; Hydrolat 1: wines from vine treated once with lavandin hydrolat; Hydrolat 5: wines from vine treated five times with
lavandin hydrolat.
3.4 Wine volatile compounds
The wine aroma compounds come mainly
from two sources, the grapes and the
microorganisms from the fermentation steps
(yeasts and lactic acid bacteria). They belong to
different chemical families such as esters, acids,
terpenoids, phenols, alcohols, lactones, and
6
aldehydes; and may be influenced by several
factors for example the ones related with the
vineyards, such as the phytosanitary product
(Oliva, Zalacain, Payá, Salinas, & Barba, 2008;
Noguerol-Pato, González-Rodríguez, GonzálezBarreiro, Cancho-Grande, & Simal-Gándara,
2011), cover crops (Xi, Tao, Zhang, & Li,
2011), nearby plants (Capone, Jeffery, &
Sefton, 2012) as well the presence of exogenous
substances such as smoke (Kennison,
Wilkinson, Williams, Smith, & Gibberd, 2007;
Wilkinson et al., 2011) or those provided by oak
extracts treatments (Martínez-Gil, GardeCerdán, Martínez, Alonso, & Salinas, 2011;
Martínez-Gil, Garde-Cerdán, Zalacain, PardoGarcía, & Salinas, 2012; Martínez-Gil,
Angenieux, Pardo-García, Alonso, Ojeda, &
Salinas, 2013).
3.4.1. Esters. This chemical group of
compounds is the main responsible of wine
aroma. These compounds are synthesized
enzymatically by yeasts during alcoholic
fermentation from alcohols and acids, and a
small fraction also comes from the grapes. The
content of esters in Petit Verdot wines
accounted for about 10-30% of the total aroma
fraction (Table 4). Three acetates, ethyl acetate,
sixteen ethyl esters and ethyl linoleate were
found in this chemical group.
3.4.1.1. Acetic esters of higher alcohols: Of
the total esters quantified in Petit Verdot wines,
only 2-14% corresponded to acetates (Table 4),
normally these are found in moderated
quantities, but have intense, rather unusual
positive odors (banana, rose, acid drops, and
apple) and they contribute to the aroma
complexity of wines (Ribéreau-Gayon, Glories,
Maujean, & Dubourdieu, 2006). The wines
showed a decrease of such esters during their
evolution, according with other authors as Du
Plessis (1983) which says that this decrease
follows the same way as normally happens with
the wines fruity character. The decrease of
acetate esters were more accentuated in control
wine, so even though, at the end of the alcoholic
fermentation, it showed the highest acetate
concentration, at the end of malolactic
fermentation these differences did not exist.
Even after six months, hexyl acetate was higher
in wines from treated vines but only in H1 wine
presented significant differences, and 2-phenyl
acetate in wine from H5 (Table 4).
Among acetates studied, the isoamyl one
concentration was higher than its wine odor
threshold (30 μg/L, Ferreira, López, & Cacho,
2000), so could contribute with banana, fruity
and sweet notes. The control wine along time
presented a decrease of total acetates of 78%
while wines from treatments (H1 and H5)
showed only from 38-23%, such decrease could
be due to hydrolysis and esterification seeking
the equilibrium. Other authors suggest that
musts with high antioxidant protection also
showed a lower decrement, especially in terms
of ethyl ester and acetates (Moio, Ugliano,
Genovese, Gambuti, Pessina, & Piombino,
2004; Lambropoulos, & Roussis, 2007). It is
well known that lavandin has compounds with
antioxidant activity, such as chlorogenic acid,
glucosides of hydroxycinnamic acids and
rosmarinic acid, terpenes and other compounds,
which are particularly active in scavenging the
hydroxyl radical (Parejo et al., 2002; TorrasClaveria, Jauregui,
Bastida, Condina, &
Viladomat, 2007; Jacometti, Wratten, & Walter,
2010). So, maybe the most stable acetates
evolution in treated wines respect to the control,
could be related to the treatments.
3.4.1.2. Ethyl acetate: As expected, this was
the most abundant ester in wine together with
ethyl lactate. This compound increased in the
wines after the malolactic fermentation
probably because lactic bacteria possess the
ability to synthesize it (Liu, 2002). At the end of
the alcoholic fermentation, the wines from the
treated grapevines had an ethyl acetate
concentration higher than control wine; contrary
to what it is observed after the malolactic
fermentation and 6 months bottling. Ethyl
acetate in all samples exceeded its olfactory
threshold (7.5 mg/L, Guth, 1997, with fruity
and solvent odor descriptors), but these were
below 80 mg/L, limit for which is emerging as
unpleasant, spite of this, Ribéreau-Gayon,
Peynaud, Ribéreau-Gayon, & Sudraud (1992)
suggest that the wine bouquet is better when
ethyl acetate rate is lower, as it is the case.
7
4355.5 ±169.1 a,
5800.4±319.8 b,
29.45±0.03 a,
Total
Ethyl acetate (*) (mg/L)
7.5±0.9 a,
1776.7±179.3 b,
Ethyl heptanoate (+)
Ethyl octanoate (*)
13.4±0.9 b,
7.7±0.4 a,
10.8±0.5 a,
Ethyl propanoate (+)
6.0±0.4 b,
7670.6±264.3 c,
28.28±3.84 b,
5.1±0.6 a,
6306.1±250.4 b,
nd
Ethyl dihydrocinnamate (+)
Total
Ethyl linoleate (μg/L)
27.28±2.65 b,
5201.0±254.5 a,
5.2±0.2 a,
15.8±0.7 c,
7.6±0.6 a,
10.6±0.2 a,
47.1±3.0 a,
25.9±1.8 a,
188.7±7.5 a,
510.0±20.0 a,
1110.0±45.8 a,
7.8±0.4 a,
557.0±14.9 a,
292.4±9.8 a,
35.4±1.1 a,
45.9±4.1 a,
679.4±24.5 a,
1635.0±247.5 b,
34.55±1.43 b,
4196.2±80.0 a,
3468.0±78.0 a,
143.3±5.8 a,
548.9±16.9 a,
nd
42588.9±3130.4 a,
3.6±0.6 a,
10.9±1.6 b,
8.7±0.2 a,
6.69±0.93 a,
49.2±8.2 a,
29.7±5.9 b,
162.1±36.7 a,
495.0±91.9 a,
970.1±183.9 a,
6.4±0.9 a,
472.7±59.8 a,
353.1±9.7 b,
32.7±5.2 a,
70.8±12.6 a,
992.6±166.2 a,
38925.0±3118.3 a, 67.79±0.65 b,
3545.1±340.6 a,
3005.7±337.4 a,
125.0±21.2 a,
15.04±2.69 b,
39477.6±109.3 a,
4.5±0.1 b,
8.3±0.1 a,
7.5±0.4 a,
9.14±0.64 b,
41.2±7.0 a,
18.2±2.1 a,
118.5±21.1 a,
470.0±42.4 a,
915.0±77.2 a,
6.1±0.6 a,
414.9±27.2 a,
298.8±14.6 a,
41.3±0.4 b,
78.4±10.9 a,
915.8±8.5 a,
36115.0±49.5 a, 52.17±6.04 a, 3669.3±162.5 a,
3100.7±158.6 a,
125.0±7.1 a,
443.6±34.9 a,
12.72±0.69 b,
40736.9±682.4 a,
4.7±0.2 b,
14.4±0.3 c,
7.9±1.4 a,
8.53±0.51 b,
44.0±2.3 a,
30.2±0.9 b,
113.0±5.6 a,
435.0±21.2 a,
860.1±56.6 a,
7.7±0.0 b,
396.2±19.5 a,
313.8±2.1 a,
36.6±3.2 ab,
78.8±0.1 a,
1023.4±29.0 a,
37350.0±678.8 a,
55.57±4.37 a,
3446.3±247.8 a,
2901.6±246.0 a,
135.0±7.1 a,
409.7±29.1 a,
Hydrolat 1
Hydrolat 5
414.4±32.8 a,
Control
360.8±13.6 a,
nd
69353.0±3529.4 ab,
4.0±0.0 a,
12.2±0.4 b,
6.1±1.2 a,
6.61±0.01 a,
39.2±3.6 a,
24.9±2.1 b,
144.6±6.5 c,
455.0±21.2 b,
935.0±49.5 b,
5.4±0.2 a,
507.3±22.4 b,
21.76±2.31 b,
64374.3±2415.2 a,
4.3±0.1 b,
8.5±0.7 a,
7.6±1.2 a,
8.36±0.18 b,
36.0±2.9 a,
13.6±1.0 a,
72.5±7.4 a,
380.0±28.3 a,
800.0±56.6 a,
5.1±0.4 a,
405.3±25.8 a,
297.6±11.6 a,
49.5±3.3 b,
131.0±9.3 a,
156.5±0.7 b,
40.4±0.0 a,
2548.3±118.2 a,
59585.0±2411.2 a,
57.27±0.45 a,
3129.8±95.0 a,
2692.4±87.4 a,
115.0±7.1 a,
322.4±36.6 b,
3230.2±56.5 b,
63425.0±3528.5 a,
72.72±7.84 b,
3247.7±128.5 ab,
2866.1±128.4 ab,
110.0±0.0 a,
271.6±5.1 a,
23.19±0.55 b,
70533.1±1892.5 b,
5.2±0.0 c,
15.9±0.1 c,
12.0±0.6 b,
8.66±0.15 c,
40.3±1.1 a,
26.8±0.3 b,
113.8±4.8 b,
425.1±7.1 b,
890.0±14.1 b,
6.6±0.2 b,
436.0±1.2 a,
305.1±50.4 a,
49.0±0.1 b,
149.2±0.7b,
3736.67±200.1 c,
64290.0±1880.9 a,
50.93±7.84 a,
3359.6±36.2 b,
2917.8±34.9 b,
130.0±0.0 b,
311.7±9.4 ab,
Control
Hydrolat 1
Hydrolat 5
8
All the parameters are given with their standard derivation (n=4). The different letters indicate significant differences. At each sampling in the winemaking process, different letters indicate significant differences between the
samples (level of significance of p<0.05). For each sample, different Greek letters indicate differences between winemaking moments (level of significance of p<0.05). (*) Quantified with pure compound standard. (+) SemiQuantitative compounds. Control: wines from untreated grapes; Hydrolat 1: wines from vine treated once with lavandin hydrolat; Hydrolat 5: wines from vine treated five times with lavandin hydrolat.
11.8±0.8 a,
8.8±1.3 a,
13.8±0.7 b,
Ethyl pyruvate (+)
Ethyl cinnamate (+)
92.3±10.9 b,
31.9±5.5 ab,
41.4±7.0 b,
81.4±14.5 b,
409.8±70.3 b,
793.3±104.1 b,
Isopentyl octanoate (+)
Ethyl dodecanoate (+)
1606.7±187.2 b,
9.9±0.8 b,
766.6±38.6 b,
351.2±2.8 b,
47.8±3.7 b,
85.8±5.7 b,
922.4±53.5 b,
2485.0±120.2 c,
48.40±4.08 c,
Ethyl-9-decenoate (+)
826.7±92.4 b,
425.7±24.3 b,
Ethyl decanoate (*)
Ethyl butanoate
381.8±2.0 c,
38.6±3.3 a,
Ethyl phenylacetate (*)
834.8±57.1 b,
43.1±6.5 a,
Ethyl vanillate (*)
Ethyl hexanoate (*)
653.4±54.0 a,
Diethyl succinate (*)
(*)
1156.7±122.2 a,
Ethyl lactate (*)
Ethyl esters (μg/L)
3646.8±164.9 a,
4773.1±313.1 b,
Isoamyl acetate (*)
133.3±5.8 c,
844.0±63.6 b,
183.3±15.3 b,
575.3±37.1 a,
Hydrolat 1
Hydrolat 5
2-phenyl acetate (*)
Control
Hexyl acetate (*)
Acetates (μg/L)
Volatiles compounds
Table 4. Volatile compounds content in control wines and wines from grapes of treated grapevines with lavandin hydrolat
7030 ±628c,
Total
1.46±0.14 a,
57.59±3.52 a,
Camphor (*)
Total
nd
45,60±4,76 a,
1.87±0.07 a,
nd
45,09±8,51 a,
Vanillin (*)
Total
880 ±90 a,
36,75±4,59 a,
nd
1.69±0.21 a,
35.06±4.59 a,
58.65±1.83 a,
1.77±0.20 a,
0.82±0.02 a,
0.39±0.02 a,
0.87±0.12 a,
7.53±0.77 a,
6.45±0.29 b,
23.61±0.80 a,
5.11±0.50 a,
12.34±1.32 b
4046±183 a,
1470±76 a,
16.58±1.11 a,
340±50 a,
1340±132 a,
111,06±5,57 a,
74.14±2.67 a.
2.10±0.33 a,
34.83±4.87 a,
62.70±2.08 a,
1.14±0.21 a,
0.62±0.05 a,
0.35±0.06 a,
2.87±0.39 a,
15.35±0.59 a,
5.43±0.40 a,
23.48±1.55 a,
7.29±0.92 a,
6.19±0.60 a,
5840±458 b,
1138±147 b,
47.30±10.07 a,
430±71 b,
2520±368 b,
115,75±7,19 a,
71.60±5.88 a.
1.77±0.03 a,
42.38±4.14 a.
76.45±3.10 b,
2.66±0.11 b,
0.67±0.01 a,
0.35±0.02 a,
2.71±0.10 a,
17.92±0.35 b,
7.96±1.03 b,
26.58±2.47 a,
10.68±1.01 b,
6.94±1.13 a,
5025±145 a,
942±48 a,
47.83±6.47 a,
380±0 ab,
2185±49 ab,
1470 ±127 a,
134,97±5,72 b,
91.76±5.66 b.
1.94±0.18 a,
41.27±0.83 a.
65.22±1.12 a,
2.31±0.30 b,
0.60±0.03 a,
0.29±0.03 a,
2.76±0.26 a,
14.79±0.67 a,
6.95±0.25 b,
24.04±0.38 a,
7.60±0.66 a,
5.88±0.06 a,
4913±109 a,
1021±5 ab, 67.17±2.86 b,
343±7 a,
2075±21 a,
1405 ±106 a,
Hydrolat 1
Hydrolat 5
1705 ±219 a,
Control
118,42±6.72 a,
77.94±6.72 a,
2.50±0.05 b,
37.99±0.04 a.
76.54±1.68 a,
1.48±0.09 a,
0.75±0.04 b,
0.38±0.04 a,
4.37±0.40 a,
27.02±0.98 a,
5.85±0.06 a,
20.08±0.64 a,
9.66±1.06 a,
6.96±0.40 b,
5787±85 b,
1179±29 c,
22.46±2.04 b,
385±7 b,
2670±57 b,
1530 ±57 a,
144,29± 11,29 b,
90.02±6.79 a,
2.26±0.04 a,
52.02±9.02 b.
82.08±1.78 b,
2.52±0.47 b,
0.66±0.03 a,
0.36±0.02 a,
4.15±0.27 a,
26.28±0.77 a,
7.29±0.08 b,
23.98±1.33 b,
10.98±0.65 ab,
5.86±0.28 a,
4632±264 a,
574±22 a,
18.42±1.38 a,
305±7 a,
2305±134 a,
1430 ±226 a,
179,45±10,84 c,
123.70±10.45 b.
2.44±0.04 b,
53.31±2.90 b.
91.24±1.76 c,
4.70±0.79 c,
0.63±0.01 a,
0.33±0.03 a,
5.22±0.04 b,
29.39±0.45 b,
7.45±0.12 b,
23.76±0.29 b,
12.82±1.46 b,
6.96±0.21 b,
4972±130 b,
855±8.8 b,
27.56±1.04 c,
295 ±21 a,
2230±127 a,
1565±7 a,
Control
Hydrolat 1
Hydrolat 5
9
All the parameters are given with their standard derivation (n=4). The different letters indicate significant differences. At each sampling in the winemaking process, different letters indicate significant differences between
the samples (level of significance of p<0.05). For each sample, different Greek letters indicate differences between winemaking moments (level of significance of p<0.05). (*) Quantified with pure compound standard. (+)
Semi-Quantitative compounds. Control: wines from untreated grapes; Hydrolat 1: wines from vine treated once with lavandin hydrolat; Hydrolat 5: wines from vine treated five times with lavandin hydrolat.
2.30±0.09 b,
43.22±8.51 a,
Eugenol (*)
43.30±4.76 a,
1.43±0.19 b,
Guaiacol (*)
Volatile Phenols (μg/L)
1.68±0.17 a,
78.65±4.03 b,
1.04±0.17 a,
0.62±0.10 b,
1.27±0.13 b,
17.91±2.58 b,
-ionone (*)
Nerolidol (*)
6.64±0.85 b,
27.07±0.92 b,
1.34±0.22 b,
9.72±1.50 a,
Nerol (*)
0.50±0.05 ab,
5.20±0.57 a,
Citronellol (*)
6.88±0.56 b,
15.15±2.75 b,
-ionone (*)
23.22±2.97 a,
Linalool (*)
-damascenone (*)
9.03±0.08 a,
6.09±0.93 ab,
Limonene (*)
Terpenoids (μg/L)
2517±355 b,
5641±.399 b,
2442±507 b,
Dodecanoic acid (+)
483±35 b,
27.14±4.42 b,
603±55 c,
18.22±2.99 a,
Decanoic acid (*)
2530±262 b,
Nonanoic acid (+)
1620.3±30 a,
1407 ±254 b,
993 ±176 a,
Hydrolat 1
Hydrolat 5
Octanoic acid (*)
Control
Hexanoic acid (*)
Fatty acids(μg/L)
Volatiles compounds
Table 4. (Continued)
993 ±176 a,
7030 ±628c,
Total
Nerolidol (*)
78.65±4.03 b,
57.59±3.52 a,
Total
1.87±0.07 a,
(*)
45,60±4,76 a,
45,09±8,51 a,
Total
880 ±90 a,
36,75±4,59 a,
nd
1.69±0.21 a,
35.06±4.59 a,
58.65±1.83 a,
1.77±0.20 a,
0.82±0.02 a,
0.39±0.02 a,
0.87±0.12 a,
7.53±0.77 a,
6.45±0.29 b,
23.61±0.80 a,
5.11±0.50 a,
12.34±1.32 b
4046±183 a,
1470±76 a,
16.58±1.11 a,
340±50 a,
1340±132 a,
111,06±5,57 a,
74.14±2.67 a.
2.10±0.33 a,
34.83±4.87 a,
62.70±2.08 a,
1.14±0.21 a,
0.62±0.05 a,
0.35±0.06 a,
2.87±0.39 a,
15.35±0.59 a,
5.43±0.40 a,
23.48±1.55 a,
7.29±0.92 a,
6.19±0.60 a,
5840±458 b,
1138±147 b,
47.30±10.07 a,
430±71 b,
2520±368 b,
115,75±7,19 a,
71.60±5.88 a.
1.77±0.03 a,
42.38±4.14 a.
76.45±3.10 b,
2.66±0.11 b,
0.67±0.01 a,
0.35±0.02 a,
2.71±0.10 a,
17.92±0.35 b,
7.96±1.03 b,
26.58±2.47 a,
10.68±1.01 b,
6.94±1.13 a,
5025±145 a,
942±48 a,
47.83±6.47 a,
380±0 ab,
2185±49 ab,
1470 ±127 a,
134,97±5,72 b,
91.76±5.66 b.
1.94±0.18 a,
41.27±0.83 a.
65.22±1.12 a,
2.31±0.30 b,
0.60±0.03 a,
0.29±0.03 a,
2.76±0.26 a,
14.79±0.67 a,
6.95±0.25 b,
24.04±0.38 a,
7.60±0.66 a,
5.88±0.06 a,
4913±109 a,
1021±5 ab, 67.17±2.86 b,
343±7 a,
2075±21 a,
1405 ±106 a,
Hydrolat 1
Hydrolat 5
1705 ±219 a,
Control
118,42±6.72 a,
77.94±6.72 a,
2.50±0.05 b,
37.99±0.04 a.
76.54±1.68 a,
1.48±0.09 a,
0.75±0.04 b,
0.38±0.04 a,
4.37±0.40 a,
27.02±0.98 a,
5.85±0.06 a,
20.08±0.64 a,
9.66±1.06 a,
6.96±0.40 b,
5787±85 b,
1179±29 c,
22.46±2.04 b,
385±7 b,
2670±57 b,
1530 ±57 a,
144,29± 11,29 b,
90.02±6.79 a,
2.26±0.04 a,
52.02±9.02 b.
82.08±1.78 b,
2.52±0.47 b,
0.66±0.03 a,
0.36±0.02 a,
4.15±0.27 a,
26.28±0.77 a,
7.29±0.08 b,
23.98±1.33 b,
10.98±0.65 ab,
5.86±0.28 a,
4632±264 a,
574±22 a,
18.42±1.38 a,
305±7 a,
2305±134 a,
1430 ±226 a,
179,45±10,84 c,
123.70±10.45 b.
2.44±0.04 b,
53.31±2.90 b.
91.24±1.76 c,
4.70±0.79 c,
0.63±0.01 a,
0.33±0.03 a,
5.22±0.04 b,
29.39±0.45 b,
7.45±0.12 b,
23.76±0.29 b,
12.82±1.46 b,
6.96±0.21 b,
4972±130 b,
855±8.8 b,
27.56±1.04 c,
295 ±21 a,
2230±127 a,
1565±7 a,
Control
Hydrolat 1
Hydrolat 5
10
All the parameters are given with their standard derivation (n=4). The different letters indicate significant differences. At each sampling in the winemaking process, different letters indicate significant differences between
the samples (level of significance of p<0.05). For each sample, different Greek letters indicate differences between winemaking moments (level of significance of p<0.05). (*) Quantified with pure compound standard. (+)
Semi-Quantitative compounds. Control: wines from untreated grapes; Hydrolat 1: wines from vine treated once with lavandin hydrolat; Hydrolat 5: wines from vine treated five times with lavandin hydrolat.
nd
2.30±0.09 b,
nd
Vanillin (*)
Eugenol
43.22±8.51 a,
Guaiacol (*)
Volatile Phenols (μg/L)
43.30±4.76 a,
1.68±0.17 a,
1.43±0.19 b,
1.04±0.17 a,
1.46±0.14 a,
-ionone
Camphor (*)
0.62±0.10 b,
1.27±0.13 b,
17.91±2.58 b,
6.64±0.85 b,
27.07±0.92 b,
6.88±0.56 b,
15.15±2.75 b,
(*)
1.34±0.22 b,
9.72±1.50 a,
Nerol (*)
0.50±0.05 ab,
5.20±0.57 a,
Citronellol (*)
-ionone (*)
23.22±2.97 a,
Linalool (*)
-damascenone (*)
9.03±0.08 a,
6.09±0.93 ab,
Limonene (*)
Terpenoids (μg/L)
2517±355 b,
5641±.399 b,
2442±507 b,
Dodecanoic acid (+)
483±35 b,
27.14±4.42 b,
603±55 c,
18.22±2.99 a,
1620.3±30 a,
Nonanoic acid (+)
2530±262 b,
Decanoic acid (*)
1407 ±254 b,
Hydrolat 1
Hydrolat 5
Octanoic acid (*)
Control
Hexanoic acid (*)
Fatty acids(μg/L)
Volatiles compounds
Table 4. (Continued)
3.4.1.3. Ethyl esters: These, together with
acetates, contribute to the typical floral and
fruity aroma of young wines having an
important role in the aroma. Sixteen ethyl esters
were identified in Petit Verdot wines, the most
abundant was ethyl lactate (Table 4). The
behavior of this group of compounds is not
homogeneous as depend of their own acid
hydrolysis and chemical esterification (Ebeler,
2001). After alcoholic fermentation, an increase
respect to the control of at least 40% of ethyl
lactate concentration was observed in wines
from hydrolat treated vines. However, as the
formation of this compound occurs mainly
under malolactic fermentation, showing a
minimum increment of 57.1 mg/L, the
differences between the control and treated
wines were not significant after this stage. The
increase of ethyl lactate during the malolactic
fermentation detracts from wine freshness if it
exceeds its perception threshold (150 mg/L,
Peinado, Moreno, Medina, & Mauricio, 2004),
but this did not occur in any of the wines. After
alcoholic fermentation, diethyl succinate and
ethyl vanillate were higher in wines from H1
treated vine, also, these compounds increased
during wine evolution, but in H1 wines this
increment was the slowest, which caused that
this wine after six months in bottle presented
the lowest concentration of both compounds.
The ethyl vanillate concentrations were below
its odor threshold (990 μg/L, Culleré, Escudero,
Cacho, & Ferreira, 2004, sweet honey and
vanillin notes), nevertheless the increment of
diethyl succinate after six months in bottle made
wines had more than 2500 μg/L, exceeding its
odor threshold (1200 μg/L, Peinado, Moreno,
Medina, & Mauricio, 2004), so this compound
can be considered as contributor to the wine
aroma with light fruity notes. Also, ethyl
phenylacetate showed an increment during the
bottle evolution, being the concentration in
wines from hydrolat treatments a 21% higher
than control wine. This compound gives to the
wines a strong honey-like character when its
concentration is close to 73 μg/L (Tat,
Comuzzo, Battistutta, & Zironi, 2007).
However, most of the ethyl esters decreased
during evolution, such as, ethyl butanoate, ethyl
octanoate, ethyl decanoate, ethyl 9-decenoate,
isopentyl octanoate, ethyl hexanoate, and ethyl
dodecanoate (Table 4). After alcoholic
fermentation, all of them showed higher
concentration in control and H1 wines than H5
ones, probably because the repetitive
application of hydrolat affected the metabolic
pathways of these compounds during berry
maturation, such as amino acids (Martínez-Gil,
Garde-Cerdán, Martínez, Alonso, & Salinas,
2012) or by the yeast inhibition of esters
catabolism. However, these esters content
showed a more accentuated decrement during
control and H1 wines evolution; so, after six
months in bottle the wine from H5 had similar
concentrations of these compounds, except for
ethyl hexanoate and dodecanoate. This
treatment (H5) showed a more stable esters
concentration, which could be due to the
equilibrium or, as mentioned previously in the
ethyl acetates, to the treatments. The H1 wine,
after alcoholic fermentation, showed the highest
concentration of ethyl propanoate and decreased
during the evolution of wine. Such decrement
was lower in treated wines so, after six months
in bottle, these wines showed the highest
concentration, although probably without
affecting directly the aroma, since it have a high
odor threshold (1800 μg/L, Peinado, Moreno,
Medina, & Mauricio, 2004, fruity, banana,
apple aromatic notes). Also, ethyl pyruvate
showed higher concentrations in the H5 wine
after six months in bottle, but probably this
increase did not directly affect to the wine final
aroma for the same reason that occurred with
the ethyl propanoate (5000 μg/L, Zea, Moyano,
Moreno, Cortes, & Medina, 2001, vegetable and
caramel notes). The lowest odor threshold value
for ethyl esters correspond to cinnamic esters
(1.1 μg/L for ethyl cinnamate and 1.6 μg/L for
ethyl dihydrocinnamate, Ferreira, López, &
Cacho, 2000), contributing with fruity, citrus,
honey and cinnamon notes. Both compounds
were treatment dependent, since they showed
concentrations higher in wines from H5
treatment than the control; however, H1 wine
presented a decrement of ethyl cinnamate and
an increment of ethyl dihydrocinnamate. These
two compounds were more stable during
evolution when was performed the H5
treatment. Finally, respect to ethyl esters, ethyl
linoleate was not found in control wine, so it
has been shown separately (Table 4). It is
known that the EOs containing fatty acids
including linoleic acid (Maffei & Peracino,
11
1993), so, probably, the hydrolat linoleic acid
was retained on the skin grapes as the external
covered is of apolar nature. It is possible that
this compound may be release into the wine
during maceration, as in the harvest the grapes
are not washed before the winemaking process,
with their consequence ethanolic esterification.
The presence of the long-chain saturated and
unsaturated fatty acids and their esterified
forms, in the wines is know in other varieties
(Beltran, Novo, Guillamón, Mas & Rozès,
2008), so other possibility could be that the
treatments influence in the synthesis of this
compound. The content of ethyl linoleate in the
resulting wines is similar, as well as its
evolution where a decreased after malolactic
fermentation and increased with time in bottle
was observed.
3.4.2. Fatty acids.
Fatty acids production is governed by the
initial composition of the must and by
fermentation conditions. Six different fatty
acids were found in Petit Verdot wines (Table
4), only three of them (hexanoic, octanoic and
dodecanoic acids with odor thresholds of 420
μg/L, 500 μg/L and 500 μg/L respectively Du
Plessis, 1993; Zea, Moyano, Moreno, Cortes, &
Medina, 2001) may contributed to the aroma of
wine, since its concentration was above their
respective perception threshold, especially
octanoic acid. In general, control wine was the
one with the highest concentration of these three
fatty acids, together with decanoic acid. The
hexanoic acid of each wine had different
behavior during the evolution, so, the
differences between control and wines from
treatments disappeared with time. These
compounds have a negative effect on the overall
wine aroma, as they give cheese, fatty and
rancid notes (Rocha, Rodrigues, Coutinho,
Delgadillo, & Coimbra, 2004). After alcoholic
fermentation, H1 wine showed the highest
concentration of nonanoic acid which increased
in all wines after the malolactic fermentation
and decreased after 6 months, being H5 the one
with the greatest increase. However, the
increment of this acid concentration probably
did not significantly contribute to the final
aroma as were below their perception thresholds
(3000 μg/L for nonanoic acid, Hayasaka,
Baldock, & Pollinitz, 2005). Important
differences have been found between the
different treatments in the total acid content,
since higher concentrations were found in
control wines. Although the presence of fatty
acids is usually related to the occurrence of
negative odors, they are very important for
aroma equilibrium in wines because they resist
the hydrolysis of their corresponding esters
(Bertrand, 1981). Hydrolat treatment affect their
evolution as decrease slightly lower in H1 wine
(above a 1009 μg/L) or even increasing it in H5,
which did not exceed the concentration found in
the control but may affect the balance and avoid
the hydrolysis of aroma esters.
3.4.3. Terpenoids
This group includes terpenes and C13norisoprenoids, which are present in grape skin
especially as glycosides, and they may be
released either chemically or by glycosidase
activities of yeast and bacteria, playing a
significant role in the varietal odour of wines
and contribute substantially to grape bouquet
(Fariña, Boido, Carrau, Versini, & Dellacassa,
2005). The studied wines were found to contain
five different terpenes such as limonene (with
fruity and lemon as odour descriptors), linalool
(flowery, fruity), citronellol (green, lemon,
spicy), nerol (floral, sweet), nerolidol (flowery,
fruity, green, citrus); and three C13norisoprenoids such as -damascenone (fruity,
sweet), -ionone (floral) and -ionone (violet,
balsamic) (Table 4). Although, the majority of
these compounds, except the -damascenone
and -ionone, which had concentrations lower
than its odor threshold, should not be ignored,
as they can enhance some aroma notes by
means of additive or synergistic effects. The
wines from grapevines treated with these
lavandin hydrolats, after alcoholic fermentation,
showed the highest concentration of limonene
and nerol. These compounds could be
proceeding from hydrolat, since it is present on
its composition, with a concentration of 9.8
mg/L and 19.45 mg/L, respectively. Limonene
content
decreased
during
malolactic
fermentation, being more pronounced in wines
from grapevines treated, so after this no
significant differences were observed, even
after six month in bottle the H1 wine showed
the lowest concentration. However, nerol
content on treated wines showed a final
12
increment of at least 25% compared to the
control. Although control and H1 wines were
stable during their evolution, H5 showed the
increment according with time. Linalool
increased during wine evolution in all samples,
probably due the hydrolysis of its glycosidic
precursors from grapes (Fariña, Boido, Carrau,
Versini, & Dellacassa, 2005). In this case, only
H5 wine showed an increment of linalool,
incrementing the concentration respect to the
control wine after six months a 33%. The
citronellol remained constant with time,
showing a slight increase in final wines by the
treatment. The H1 wines had the highest
nerolidol concentration after alcoholic and
malolactic fermentation. This compound
increased during the wine evolution, being the
highest increment in H5 wine. -damascenone
and -ionone are the two compounds that may
directly contribute to wine aroma as their
concentration is higher than their respective
odor threshold (0.05 μg/L and 0.09 μg/L,
respectively, Culleré, Escudero, Cacho, &
Ferreira, 2004). The -damascenone increased
their
concentration
during
malolactic
fermentation and bottle storage in all samples,
probably due to the hydrolysis of their
precursors, such as 3-hydroxy--damascone
(Winterhalter & Skouroumounis, 1997). The H5
treatment probably affect the plant metabolism
during the maturation of the grapes, since, after
six months in bottle, this wine showed the
highest concentration, and this was not found in
the hydrolat. Respect to and -ionone, the H1
wine showed the highest concentration of these
two compounds after alcoholic fermentation.
However, both of them decreased with the time
especially in H1 wines, being the concentration
of -ionone higher in control wine after six
months in bottle whereas no differences were
observed for -ionone.
Camphor is not usually found in the volatile
composition of wines. So, its presence could be
due to that the vines were grown close to the
lavandin area. Other authors have also observed
the influence of different plants near to
vineyards in the wine aroma such as eucalyptus
or cover crops (Xi, Tao, Zhang, & Li, 2011;
Capone, Jeffery, & Sefton, 2012). After
alcoholic
fermentation,
no
significant
differences
were
found
for
camphor
concentration, however after malolactic
fermentation and six month after this, wines
from
treatments
showed
the
highest
concentrations, being the H5 treatment the one
most affected (Table 4). After six months in
bottle was when the greatest differences among
the treated and control wines were observed,
since the H1 wine increased its concentration a
70% and even H5 wine tripled it, so when the
exposure was greater also the concentration was
higher.
The wine from H1 treatment showed the
highest concentration of total terpenoids after
alcoholic fermentation with a later stable
evolution. Control and H5 wines showed a little
increase in terpenoids after the malolactic
fermentation and six months in bottle,
indicating that lactic bacteria and acid
conditions could hydrolyse their glycosylated
fraction. After six months in bottle, wines from
vines treated had the highest concentration of
the total terpenoids contributing positively to
the final aroma of the wine.
3.4.4. Volatile phenols
The main source of these compounds in
young wines is the flavor precursor fraction
present in the grapes, especially on wines that
have made the malolactic fermentation. None of
them (guaiacol, eugenol and vanillin) were
identified in the lavandin hydrolat (Table 1).
However, the application of this hydrolat to the
grapevines had an effect on the concentration of
these volatile compounds in wines (Table 4).
The content of guaiacol although did not
showed significant differences at the end of
alcoholic and malolactic fermentations, but
showed an increment after six months in bottle
of at least of 37% respect to the control. The
guaiacol content was higher than its odor
threshold, so it can contribute to wine with
smoke aroma notes (9.5 μg/L, Ferreira, López,
& Cacho, 2000). Eugenol only showed a
different behavior in wines from H1 treatment,
as the concentration of this was below its
olfactory threshold (6 g/L, Ferreira, López, &
Cacho, 2000, with clove, spicy and sweet
aromatic notes), the small differences found
probably did not influence the wine aroma. The
vanillin was not detected in wines at the end of
the alcoholic fermentation; however after
13
malolactic
fermentation,
a
vanillin
concentration of 70 μg/L in control and H1
wines and 90 μg/L in H5 wine was found,
probably associated to the hydrolysis of their
grapes glycosidic precursors by lactic acid
bacteria. After six months of the malolactic
fermentation, the vanillin content in wines from
treated grapevines were higher than the control,
although only H5 wine showed significant
differences, the perception threshold of this
compound in the wine was exceeded (60 μg/L,
Culleré, Escudero, Cacho, & Ferreira, 2004), so
the significant increase observed (59% in H5
wine) could enhance vanillin flavour on the
wine. Consequently, treatment with lavandin
hydrolat affected to the total volatile phenolic
compounds studied, probably due to the change
produced in the plant secondary metabolism
because hydrolat did not contain these
compounds.
3.4.5. Alcohols
Alcohols synthesis is related to the yeast
metabolism from must amino acids or sugars
content. 1-hexanol and (Z)-3-hexen-1-ol are C6
alcohols formed after the enzymatic oxidation
of linoleic or linolenic acids as precursors
during harvest, transport, crushing and pressing
of grapes. In general, 1-hexanol was affected by
treatment with lavandin extract, as showed
higher concentrations in these wines. The
highest content of (Z)-3-hexen-1-ol was found
in H1 wines, although only significant
differences were found after alcoholic and
malolactic fermentations. These two compounds
provided to wines some green and cut grass
aromatic notes, although concentrations were
lower than their odor threshold (8 mg/L for 1hexanol and 400 μg/L for (Z)-3-hexen-1-ol,
Guth, 1997). Although, 2-phenylethyl alcohol
was highly present within the hydrolat, the H5
wines after alcoholic fermentation had the
lowest concentration and no differences were
found after this step. 2-phenylethyl alcohol
provides to wines with aromatic notes like rose
having an odor threshold of 10000μg/L (Guth,
1997), the Petit Verdot wines contents were
below to this threshold. Isobutanol presented
some differences by the lavandin extract
treatment, but these were lost due to the
increase during the evolution of wine, as the
increment in control wine was more
accentuated, being the concentration after six
months in bottle similar. The concentration of
this compound in all wines was below its
perception threshold (75 mg/L, alcohol, nail,
polis and fermentative notes, Peinado, Moreno,
Medina, & Mauricio, 2004). The 3-methyl-1butanol was the most abundant alcohol,
although the content was lower than its odor
threshold (30 mg/L, alcohol and burnt notes,
Culleré, Escudero, Cacho, & Ferreira, 2004),
and, in general, similar in all wines and
sampling times. Benzyl alcohol increased
during the evolution of wines, being the highest
increment in wines from treated grapevines, so,
after six months in bottle, this was higher in
treated wines than in control wine. No
significant differences were observed for
nonanol, 4-methyl-1-pentanol, 1-octanol and
decanol between the wines from treatments and
control wine, and, in general, their
concentrations remained constant with the time.
Furanmethanol was found in lavandin hydrolat
in an important concentration (Table 1), and it
was also found within the wines, getting after
six months an increment of 53% in H1 wine in
relation to the control, and even the amount of
control was tripled in H5 wine. Even so, this
compound was below its odor threshold (2000
μg/L, with faint burning odor, Culleré,
Escudero, Cacho, & Ferreira, 2004). Total
alcohols accounted for the highest proportion of
volatile compounds in wines (68-87%). The
sum of alcohols only showed a higher content in
wine from H1 after alcoholic fermentation;
however these differences disappeared with
time.
3.4.6. Lactones
Two lactones, 2(5H)-furanone and 2hydroxycyclopent-2-en-1-one, were found in
lavandin hydrolat and in Petit Verdot wines.
Control wine, after alcoholic fermentation, had
the highest concentration of these two lactones,
both decreasing during with time. Their content
in wines from treatments was different, showing
the highest concentration after 6 months
especially in H5 wines. It is known that plants
may form glycosylated conjugates from some
volatile compounds in order to minimize toxic
effects to cells, or to increase their solubility to
facilitate cellular transportation (Winterhalter &
14
Skouroumounis, 1997) Moreover, Hayasaka,
Wilkinson, Elsey, Raunkjaer, & Selfton, (2007)
and Winterhalter (2009) observed that the
formation of glycosylated lactone precursors is
possible when the rings of these molecules are
open. So, it is possible that the treatment with
lavandin hydrolat favored this effect in grapes
and their wines, as lactones were released of
their precursors, especially after six months of
the malolactic fermentation.
3.4.7. Aldehydes
Benzaldehyde (bitter almonds), furfural
(toasty, caramel, sweet), 5-methylfurfural
(warm, spicy), nonanal (green, slight pungent)
and phenylacetaldehyde (floral, sweet, honeylike) were the five volatile aldehydes detected in
wines, which three of them (benzaldehyde,
furfural and phenylacetaldehyde) were also
found in the composition of lavandin hydrolat.
Only phenylacetaldehyde showed a higher
concentration than its odor threshold (1 μg/L,
Culleré, Escudero, Cacho, & Ferreira, 2004), so
the rest of these compounds did not have a great
contribution on wine aroma. Benzaldehyde and
phenylacetaldehyde increased with time in all
samples, but the other aldehydes only increased
with time in H5 wine. After six months of
malolactic fermentation, H5 wine showed
higher concentration of all the studied
aldehydes than the control wine, being the
furfural the most affected because its
concentration was doubled by the treatment.
Probably when treatment was applied
repeatedly with this extract, rich in furfural,
grapes could stored it as non-volatile precursors
and this was released during the wine ageing
process, other authors also show that when
vines were exposure to smoke this compound
increased (Kennison, Wilkinson, Williams,
Smith, & Gibberd, 2007). The same might
occur
with
benzaldehyde
and
phenylacetaldehyde
although
in
lesser
extension.
3.4.8. Other compounds
Figure 1 shows the chromatographic peaks
corresponding to two unknown compounds,
with characteristic m/z data at 186, 127 and
107, in the three sampling studied: a) alcoholic
fermentation, b) malolactic fermentation and c)
6 months after malolactic fermentation. These
compounds showed a huge increase in the
malolactic fermentation, which could be due to
they were secondary products of the bacterium
Oenococcus oeni or they were derived from the
hydrolysis of grapes precursor, anyway there
was a clear influence by the treatment on the
formation of these two compounds, since the
wines from the grapevines treated with lavandin
hydrolats had the highest content of these.
Figure 1. Chromatographic peaks corresponding to two unknown compounds, with characteristic m/z data at 186, 127 and 107, in the three sampling
studied: a) alcoholic fermentation, b) malolactic fermentation and c) 6 months after malolactic fermentation.
15
3.5. Sample discrimination
As a different behaviour was observed
between wines from control and hydrolat
treatments in all sampling times, a discriminant
analysis was carried out according to the
chemical families studied. Figure 2 shows
results obtained after performing the
discriminant analysis of different wines (C, H1
and H5) at their different sampling times (after
alcoholic and malolactic fermentation and after
six months in bottle). The 97% of the variance
is explained by two functions, 73.1% and
23.9%, respectively. The variables that
contributed most to the discriminant in order
were: terpenoids, acids, aldehydes, esters,
alcohols, lactones and phenols. It can be seen
that function 1 separated the wines from
grapevines treated with hydrolat treatments to
the control wines in each sampling, being the
greatest separations at six months in bottle.
Although after alcoholic and malolactic
fermentation, H1 wines were more distanced of
the control, after six months, the discriminant
showed that wine from H5 treatment was the
most different. The function 2 only discriminate
all the wines after alcoholic fermentation to the
rest.
Figure 2. Canonical discriminant analysis of volatile compound in wines (Control: wines from untreated vines; Hydrolat 1: wines from vine treated
once with lavandin hydrolat; Hydrolat 5: wines from vine treated five times with lavandin hydrolat) at the end of the alcoholic fermentation (AF),
after malolactic fermentation (MLF) and six months later (6 months after MLF).
4. Conclusions
Acknowledgements
Petit Verdot control vineyards that are close
to lavandin fields as the ones studied, have an
initial volatile composition that differs from the
usual one. Treated wines volatile composition
was modified as concentration of positive
aroma compounds increased, and also some
main compounds showed a greater stability. In
terms of wine quality, this research suggests
that the treatments produces a wine with a
differentiate aroma profile associate to the zone
where the vineyards grow.
Our thanks for the financial support given by the
Ministerio de Ciencia e Innovación to Project
AGL2009-08950. Also, we are grateful for the FPI
scholarship from the Junta de Comunidades de
Castilla-La Mancha for A.M.M.-G (EXP 422/09) and
to the MICINN for A.I.P.-G (BES-2010-038613). We
wish to thank the Dehesa de Los Llanos estate
(Winery-Albacete, southeastern Spain) for allowing us
to use its vineyards and Laura-Martínez for her
technical assistance. We wish to express our gratitude
to Kathy Walsh for proofreading the English
manuscript.
16
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18
8. CONCLUSIONES
CONCLUSIONES
CONCLUSIONS
Conclusiones
De los estudios realizados en esta Tesis Doctoral se derivan los siguientes logros
y conclusiones:
1. Las aplicaciones foliares a la vid con los extractos vegetales no afectaron
de una forma definida a los parámetros enológicos de uvas y vinos.
2. Las uvas almacenan en forma de glicósidos los volátiles que le llegan de
los extractos de roble cuando se aplican por vía foliar.
3. Parte de los precursores aromáticos glicosídicos cuyo origen es el
extracto de roble se liberan durante la fermentación alcohólica, la
fermentación maloláctica y a lo largo de su evolución, modificando el
aroma del vino.
4. La glicosilación dependió del tipo de compuesto, de la dosis de
aplicación del extracto de roble, de la variedad de uva y del momento de
aplicación.
5. Las aplicación foliar del extracto de roble produjo un cambio en el
contenido de otros compuestos glicosídicos cuyos volátiles no están
presentes en el extracto (Compuestos C6, alcoholes, terpenos, fenoles y
norisoprenoides).
6. Sensorialmente, los vinos procedentes de las vides tratadas con extractos
de roble, mantienen sus características típicas junto con notas a madera
que recuerdan a los vinos de crianza.
167
Conclusiones
7. El canfor, compuesto no habitual en los vinos, se encuentra en los vinos
que han sido elaborados con uvas próximas a plantaciones de lavandín, y
aumenta con la aplicación foliar a las vides de hidrolato de lavandín.
8. La aplicación foliar del hidrolato de lavandín a las vides, afectó al perfil
aromático de sus vinos, favoreciendo la estabilidad de algunos de los
principales compuestos volátiles y aumentando los que tienen un impacto
positivo en el aroma.
168
Conclusions
From the studies carried out in this Doctoral Thesis, the following conclusions
can be made:
1. Foliar plant extracts applications to the vineyards did not affect on a
specific way the grape and wines oenological parameters.
2. The grapes store the volatiles that come from oak extracts as glycosides
when they are foliar applied.
3. A fraction of glycosidic aroma precursors, whose origin is the oak
extract,
are
released
during
alcoholic
fermentation,
malolactic
fermentation and during its evolution, modifying the wine aroma.
4. Glycosylation depends on the type of the compound, on the oak extract
application dose, on the grape variety and on the application moment.
5. The foliar application of oak extract produces a change in other volatile
glycosidic compounds which are not present in the extract (C6
compounds, alcohols, terpenes, phenols and norisoprenoids).
6. At sensory level, the wines from vineyards treated with oak extracts
maintain their typical characteristics with wood notes that reminiscent to
the wines stored in barrels.
169
Conclusions
7. Camphor, an unusual compound in wine, is found in wines which grapes
were grown close to lavandin fields, and increases after lavandin hydrolat
foliar application to the vineyards.
8. Lavandin hydrolat foliar application to the vineyards affected the wine
aroma profile, improving the stability of some main volatile compounds
and increasing the ones which have an aroma positive impact.
170
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Plant extracts applications to the vineyard and their impact

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