Académique Documents
Professionnel Documents
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M Jess
Ruiz M Jess Ruiz Bejarano
Bejarano
2016 Puerto Real, 2016
Departamento de Qumica Analtica
Facultad de Ciencias
TESIS DOCTORAL
Y para que conste y surtan los efectos oportunos, firmamos la presente en Puerto
Real a 25 de Noviembre de 2015.
HACE CONSTAR:
Y para que conste y surtan los efectos oportunos, firmo la presente en Puerto Real,
a 25 de Noviembre de 2015.
Ruiz Bejarano, M.J., Rodrguez Dodero, M.C., Barroso, C.G. (2010). Optimizing
the process of making sweet wines to minimize the content of Ochratoxin A. Journal of
Agricultural and Food Chemistry 58 (24): 1300613012.
I.I.Objetivos ................................................................................................. 9
II.II.3. Vinificacin.................................................................................. 32
II.II.4. Crianza.......................................................................................... 33
III.I. Muestras.............................................................................................. 99
Bibliografa 281
Justificacin y Objetivos.
Captulo I. Justificacin y Objetivos
CAPTULO I. JUSTIFICACIN Y
OBJETIVOS
Pero la etapa del asoleo natural, que implica un cierto periodo de tiempo,
conlleva junto a importantes cambios en la composicin, importantes riesgos,
entre los que destaca la posible lluvia o el aumento de la humedad durante la
noche, que puede desencadenar una significativa prdida de calidad del
producto, ocasionando incluso infeccin fngica por especies como Botrytis
Cinerea, responsable de la podredumbre gris, o Penicillium y Aspergillus causantes
de la formacin de Ocratoxina A (OTA), metabolito txico, cuyo contenido
mximo en alimentos est regulado en la Unin Europea ((CE) No
1881/2006).
7
I.I.Objetivos
8
Captulo I. Justificacin y Objetivos
uva que se emplean normalmente para los vinos dulces andaluces suelen ser
variedades muy aromticas que dan lugar a vinos con un aroma muy
caracterstico.
I.I.OBJETIVOS
9
I.I.Objetivos
10
Captulo I. Justificacin y Objetivos
Se busca con ello, comprobar la influencia que sobre el producto final tiene el
proceso de secado y pasificado de las uvas estudiado, cmo evolucionan los
vinos a medida que avanza el proceso de envejecimiento, y comparar, tanto
desde el punto de vista analtico como sensorial, los vinos obtenidos.
11
Captulo II.
Introduccin.
Captulo II. Introduccin
15
II.I. Los vinos andaluces
Los vinos andaluces estn regulados por la Ley de proteccin del Origen y la
Calidad de los Vinos de Andaluca (BOJA n 246, 2007), en el cual se
establece, dentro del marco de la normativa de la Unin Europea y del
Estado, una clasificacin de los vinos en diferentes niveles de calidad y
proteccin de Origen con un grado de requisitos creciente, de modo que cada
nivel implica mayores exigencias que el inmediatamente inferior. Atendiendo a
dicha ordenacin, se establecen las siguientes categoras (Tabla 1):
4 Vinos de pagos.
16
Captulo II. Introduccin
17
II.I. Los vinos andaluces
IGP DOP
Vino de la Tierra Denominacin de Origen
Denominacin de Origen Calificada
Vino de Calidad con indicacin geogrfica
Vino de Pago
Vino de Pago Calificado
Tabla 2. Trminos tradicionales para IGP/DOP. (Fuente: MAGRAMA, 2015b)
18
Captulo II. Introduccin
19
II.I. Los vinos andaluces
20
Captulo II. Introduccin
Por otra parte, podr contener uno o varios de los productos siguientes:
mosto de uva concentrado o mezcla de alcohol vnico con mostos de uva
parcialmente fermentada, mosto de uva o mezcla de este producto con vino.
21
II.I. Los vinos andaluces
Los mostos y vinos para la elaboracin de los vinos de licor, as como el mosto
de uva concentrado y el mosto de uva parcialmente fermentado procedente de
uvas pasificadas, utilizados para la elaboracin de vinos de licor con
Denominacin de Origen protegida deben ser originarios de la regin cuyo
nombre lleve el vino de licor con denominacin de origen protegida de que se
trate.
22
Captulo II. Introduccin
Vinos generosos
23
II.I. Los vinos andaluces
Entre los vinos de Andaluca que se acogen a esta normativa, destacan (D.O.
"Jerez-Xrs-Sherry", 2015):
24
Captulo II. Introduccin
Mistelas
25
II.I. Los vinos andaluces
Son vinos dulces elaborados sin ningn aumento artificial del grado alcohlico
natural, siendo el alcohol procedente en su totalidad de la fermentacin y los
azcares residuales tambin naturales. Destacan los vinos elaborados en la
D.O. Mlaga que pueden ser vinos de uva sobremadurada, entre los que se
encuentran los vinos naturalmente dulces, o vinos de uva pasificada.
26
Captulo II. Introduccin
27
II.II. Vinos dulces naturales andaluces. Elaboracin y crianza
28
Captulo II. Introduccin
Aunque las variedades de Moscatel y Pedro Ximnez son las preferentes para
la elaboracin de los vinos dulces naturales de Andaluca, tambin est
permitido el uso de otras variedades en las distintas zonas de produccin. La
variedad Palomino se incluye en la D.O. Jerez-Xrs-Sherry al igual que en la
D.O. Condado de Huelva donde est autorizado adems el uso de Zalema,
Listn y Garrido Fino. Lairen, Doradilla y Rome en la D.O. Mlaga o Layren,
Baladi, Verdejo, Torrontes, Chardonnay, Sauvignon Blanc y Macabeo en
Montilla-Moriles son otras de las variedades permitidas (BOJA n 249, 2011b;
BOJA n 249, 2011a; BOJA n 249, 2011c; BOJA n 155, 2013).
29
II.II. Vinos dulces naturales andaluces. Elaboracin y crianza
30
Captulo II. Introduccin
31
II.II. Vinos dulces naturales andaluces. Elaboracin y crianza
II.II.3. VINIFICACIN
32
Captulo II. Introduccin
II.II.4. CRIANZA
El envejecimiento, con una duracin mnima de dos aos, puede ser esttico o
de aadas o mediante el sistema de criaderas y soleras. ste ltimo es un
sistema dinmico, mediante el cual el vino final es el resultado de una mezcla
compleja de vinos de distintas aadas.
33
II.II. Vinos dulces naturales andaluces. Elaboracin y crianza
34
Captulo II. Introduccin
Entre los diversos agentes qumicos utilizados, la mezcla de K2CO3 con aceite
de oliva o con oleato de etilo a temperatura ambiente son dos de los ms
frecuentes y cuyo uso est ms extendido (Doymaz y Pala, 2002; Serratosa et
al., 2008a).
35
II.III. Secados alternativos al asoleo
En el secado solar, la energa solar se utiliza ya sea como nica fuente de calor
requerido o como sistema de apoyo (Ekechukwu y Norton, 1999). Se clasifican
en tres grupos: los secaderos directos, indirectos y mixtos (Jairaj et al., 2009).
36
Captulo II. Introduccin
37
II.III. Secados alternativos al asoleo
38
Captulo II. Introduccin
39
II.III. Secados alternativos al asoleo
Aunque el secado en cmara permite un mayor control del proceso que los
deshidratadores solares, proporcionando un producto con una ptima calidad
final, conlleva un elevado coste energtico asociado al propio proceso de
deshidratacin y a las prdidas de calor debido a la humedad del aire agotado y
al mal aislamiento trmico de los recintos de secado (Minea, 2013).
40
Captulo II. Introduccin
41
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
42
Captulo II. Introduccin
43
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Por otra parte, genera una mayor predisposicin a la infeccin por bacterias
causantes de enfermedades como el picado actico y facilita el desarrollo de
otros hongos de los gneros Penicillium y Aspergillus, que pueden dan lugar a
la formacin de Ocratoxina A (OTA), metabolito de elevada toxicidad. Sin
embargo, algunos estudios han demostrado que la infeccin por Botrytis Cinerea
es capaz de degradar la Ocratoxina A (Abrunhosa et al., 2002; Valero et al.,
2008b).
44
Captulo II. Introduccin
II.IV.1.2. OCRATOXINA A
45
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
46
Captulo II. Introduccin
47
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
48
Captulo II. Introduccin
La sntesis de los cidos orgnicos tiene lugar en las hojas y en los mismos
racimos a travs de diversas rutas metablicas. Sin embargo durante la
maduracin, especialmente en zonas clidas, se produce un descenso de su
concentracin (Lamikanra et al., 1995; Blouin). Dicha disminucin puede
explicarse por varios factores; en primer lugar, por efecto del fenmeno de
dilucin por el engrosamiento de la baya, ya que desde el envero a la vendimia
la uva dobla prcticamente su volumen.
49
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
50
Captulo II. Introduccin
51
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Poco a poco, las bayas van adquiriendo un tono marrn y un fuerte olor a
vinagre por la formacin de cido actico (Rodrguez-Lpez, 1996). Adems,
se produce la salida del mosto por la fisura que inici la podredumbre,
contaminando las uvas vecinas.
Los daos que ocasiona son importantes. La uva destinada a vino produce
mostos con menor grado alcohlico, mayor acidez voltil total y desviacin de
aromas.
52
Captulo II. Introduccin
53
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
54
Captulo II. Introduccin
55
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
56
Captulo II. Introduccin
57
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
II.IV.2.1.1. Clasificacin
Flavonoides
58
Captulo II. Introduccin
Antocianos
Son los responsables del color rojo azulado del hollejo de las uvas tintas y de la
pulpa de las variedades tintoreras. Mayoritariamente se encuentran como
glucsidos, lo que favorece su estabilidad qumica y su solubilidad en agua
denominndose antocianidinas. La glucosa puede estar, adems, esterificada
con diferentes cidos como actico, p-cumrico y cafico.
Flavan-3-oles
59
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Nombre R1 R2 R3
Catequina H OH H
Galocatequina H OH OH
Epicatequina OH H H
Epigalocatequina OH H OH
Tabla 5. Flavan-3-oles monmeros de la uva.
Flavonoles
Son pigmentos amarillos de los hollejos de las uvas blancas y tintas (Tabla 6).
Las formas glucosiladas son las ms abundantes, encontrndose igualmente
cantidades importantes de derivados glucurnidos (Flanzy, 2003). Los
flavonoles parecen actuar como pantalla solar natural, encontrndose en
mayores concentracines en la piel de uvas expuestas a la luz solar (Price et al.,
1995; Haselgrove et al., 2000).
Nombre R1 R2
Kaempferol H H
Quercitina OH H
Miricetina OH OH
Isoramnetina OCH3 H
60
Captulo II. Introduccin
Flavononoles
Nombre R
Astilbina OH
Engeletina H
No flavonoides
cidos fenlicos
61
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Nombre R R
cido p-cumrico H H
cido cafeico OH H
cido ferlico OCH3 H
cido sinpico OCH3 OCH3
Tabla 8. cidos hidroxicinmicos.
Nombre R R
. p-hidroxibenzoico H H
. protocatquico OH H
cido vainllico OCH3 H
cido glico OH OH
cido sirngico OCH3 OCH3
Tabla 9. cidos hidroxibenzoicos.
Estilbenos
62
Captulo II. Introduccin
Al igual que ocurre con los compuestos voltiles, el perfil polifenlico del vino
vendr determinado por la materia prima de partida y por las posteriores
etapas de vinificacin y envejecimiento.
63
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
64
Captulo II. Introduccin
Entre los antioxidantes del vino y las uvas, algunos actan eliminando las
especies reactivas, como los cidos saliclico y benzoico, que eliminan los
radicales hidroxilos, altamente reactivos, formando compuestos hidroxi
(Zoecklein et al., 2000). Otros, por sus caractersticas estructurales, pueden,
adems, intervenir en reacciones de transferencia de electrones. Es el caso de
los cidos fenlicos, cuya actividad antioxidante se relaciona con el grupo
funcional carboxilo y el nmero y posiciones relativas de los grupos hidroxilo
en la estructura de anillo aromtico; mientras que el cido glico,
trihidroxilado, muestra una elevada actividad antioxidante, la sustitucin con
grupos metoxilo de los grupos hidroxilo de las posiciones 3 y 5, como en el
cido sirngico, reduce la actividad (Balasundram et al., 2006).
65
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
66
Captulo II. Introduccin
67
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Terpenos
68
Captulo II. Introduccin
69
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Norisoprenoides
70
Captulo II. Introduccin
Metoxipirazinas
Precursores aromticos
Por ltimo, algunos compuestos fenlicos, cmo los cidos fenlicos (cafico,
p-cumrico, ferlico) y sus steres, por accin de las levaduras o por estearasas,
pueden dan lugar a fenoles voltiles. Mientras que los vinilfenoles (4-vinilfenol
y 4-vinilguayacol) se forman en su mayora por la accin de S. cerevisiae, siendo
71
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Maceracin pelicular
Esta tcnica debe emplearse en vendimias sanas y con una ptima maduracin
ya que de lo contrario podran transferirse aromas herbceos en el caso de uva
inmadura o aromas terrosos y de hongo en uva con podredumbre (Ribreau-
Gayon et al., 2003a).
72
Captulo II. Introduccin
73
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Otros compuestos que marcan el aroma fermentativo, son los cidos grasos
de cadena corta (C2-C10), sus steres etlicos y acetatos de alcoholes
superiores. La formacin de dichos compuestos est ligada a las condiciones
de fermentacin como temperatura o la presencia de oxgeno.
74
Captulo II. Introduccin
Por otra parte, se produce la cesin por parte de la madera de un gran nmero
de voltiles que vendrn determinados por el tipo de madera empleada,
frecuentemente roble, de su origen y de las condiciones de fabricacin y
utilizacin de las barricas (Garde-Cerdn et al., 2010). En el caso de los vinos
de Jerez, en los que se emplean botas de roble previamente envinadas, la
extraccin de dichas sustancias es menos pronunciada (Flanzy, 2003).
75
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
76
Captulo II. Introduccin
77
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
78
Captulo II. Introduccin
79
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
Entre las variables externas que deben cuidarse para minimizar los efectos
indeseables que puedan restar objetividad a los juicios, se encuentran:
80
Captulo II. Introduccin
81
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
82
Captulo II. Introduccin
Durante el entrenamiento, los jueces son ser calibrados para realizar medidas que
puedan ser reproducidas e interpretadas universalmente. Para ello, se tendrn
en cuenta las siguientes consideraciones generales:
83
II.IV. Parmetros analticos de inters en la elaboracin de vinos dulces naturales andaluces
84
Captulo II. Introduccin
85
II.V. Quimiometra. Tratamiento estadstico de datos
El anlisis univariante de los datos consiste en hacer una revisin del valor de
un slo indicador, por ejemplo, el tipo de envejecimiento al que se ha
sometido un vino. Suele usarse cuando interesa conocer en profundidad qu
significan, por separado, algunos indicadores y es utilizado con fines
descriptivos en las primeras etapas de los estudios realizados.
86
Captulo II. Introduccin
87
II.V. Quimiometra. Tratamiento estadstico de datos
88
Captulo II. Introduccin
89
II.V. Quimiometra. Tratamiento estadstico de datos
90
Captulo II. Introduccin
Por tanto, las aplicaciones del PCA son diversas, pudiendo emplearse para la
representacin de datos multidimensionales, hallar variables latentes as como
encontrar relaciones entre las muestras en funcin de dichas variables.
91
II.V. Quimiometra. Tratamiento estadstico de datos
92
Captulo II. Introduccin
PCA) para asignar grupos a las muestras (Figura 16). Para establecer las reglas
de asignacin de clase (o grupo), las funciones que actan como fronteras
entre grupos se construyen mediante combinacin lineal de las variables
independientes, de tal modo que el conjunto de pesos sea ptimo para una
mxima discriminacin entre los grupos.
El modelo puede construirse usando todas las variables originales, o bien con
solo una seleccin de las mismas tras una serie de etapas secuenciales
(stepwise). Con esta estrategia, es posible construir una funcin usando
nicamente aquellas variables que realmente son tiles para la clasificacin, a la
par que se evala la contribucin individual de cada variable al modelo
discriminante (mayor para mayores F). La estrategia backward (hacia atrs)
toma inicialmente todas las variables y elimina de manera secuencial las que
menos discriminan, mientras la forward (hacia delante) las incluye paso a paso
desde un modelo con 0 variables, en orden decreciente de poder
discriminatorio. En este proceso, pueden eliminarse algunas variables en fases
posteriores, si la informacin que aportan es redundante con la de alguna
combinacin de las variables ya introducidas.
93
II.V. Quimiometra. Tratamiento estadstico de datos
El fin predictivo del LDA exige que la estructura original de los datos cumpla
unos requisitos:
94
Captulo II. Introduccin
95
II.V. Quimiometra. Tratamiento estadstico de datos
96
Captulo III.
Materiales y Mtodos.
Captulo III. Materiales y Mtodos
III.I. MUESTRAS
Los vinos y mostos objeto de estudio fueron vinificados por el propio grupo
de Investigacin en el Centro Andaluz de Investigaciones Vitivincolas (CAIV)
a partir de dos variedades diferentes de uva, Moscatel y Pedro Ximnez
durante tres vendimias: 2008, 2009 y 2010. Las uvas Pedro Ximnez fueron
proporcionadas por una bodega de Jerez, mientras que la variedad Moscatel
proceda de una via situada en Chipiona (Figuras 17 y 18).
99
III.I. Muestras
Precip.
Mes Temperatura (C) Humedad Relativa (%)
(mm)
Max Min Med Max Min Med
V. 2008 Ag. 0,0 38,0 12,3 24,6 85,6 7,8 52,9
Sept. 84,4 34,8 12,7 21,9 88,1 13,1 62,8
V. 2009 Ag. 0,0 37,5 14,2 26,2 84,6 11,7 49,0
Sept. 37,8 35,7 12,3 22,7 86,6 15,3 56,3
V. 2010 Ag. 0,0 41,1 16,8 27,7 96,6 12,7 55,8
Sept. 14,6 38,2 12,3 23,8 96,5 17,4 62,7
Tabla 12. Condiciones climticas en la zona de Jerez. Meses de septiembre y agosto de 2008, 2009
y 2010. Fuente: Estacin Agroclimtica de Jerez, (CAPDER, 2015b).
Para el secado artificial se emple una cmara climtica (Ibercex A.S.L., S.A.,
Espaa) a una temperatura de 40 C y humedad relativa de 10 %. Los racimos
fueron distribuidos uniformemente en bandejas formando una nica capa. En
todos los ensayos se parti de aproximadamente 2000 kg de uva fresca, salvo
en la variedad Pedro Ximnez de la vendimia 2008 donde el volumen de uva
fue menor, por lo que los ensayos se realizaron a pequea escala y el mosto
obtenido no fue posteriormente vinificado. La cmara climtica usada en este
caso fue una de reducidas dimensiones, modelo UV Xenon-Tester (CCI,
Espaa).
100
Captulo III. Materiales y Mtodos
101
III.I. Muestras
Tras 8 das.
Una vez alcanzado el punto ptimo, las pasas fueron despalilladas, molturadas
y prensadas en una prensa vertical. Se realizaron tres ciclos, siendo la mxima
presin en cada uno de ellos de 300 bares. Por ltimo, el pH del mosto fue
ajustado a 3,5 con cido tartrico (Agrovin, Espaa), adicionando, asimismo,
metabisulfito potsico equivalente a una dosis de 120 mg/l de dixido de
azufre antes de proceder a su vinificacin.
102
Captulo III. Materiales y Mtodos
Las condiciones del ensayo 2 (E2) fueron las mismas que en el primero,
adicionando adems nutrientes de levadura (fosfato diamnico, 10 g/hl)
(Actimax Plus+, Agrovin, Espaa).
103
III.I. Muestras
Maceracin
Temperatura
Ensayo Levadura1 Nutrientes enzimas
fermentacin2
pectolticas
E1 S.c. T. A. No No
E2 S.c. T. A. S No
E3 S.c. T. A. No S
E4 S.b. T. B. No No
E5 S.b. T. A. No S
1 S.c.: S. cerevisiae; S.b.: S. bayanus
2T. A.: Temperatura Ambiente; T. B.: Temperatura baja
104
Captulo III. Materiales y Mtodos
Las condiciones de vinificacin seleccionadas fueron las mismas que para los
vinos de la experiencia 4 de la vendimia 2008 (S. bayanus a baja temperatura) ya
que los resultados obtenidos para los vinos de 2008 demostraron que estas
condiciones de vinificacin permitan obtener vinos con mejor perfil sensorial
y bajo contenido en Ocratoxina A y carbamato de etilo. Igualmente, el
envejecimiento se llev a cabo en botas de roble tostado medio de 30 litros
de capacidad durante, al menos, 12 meses.
Tabla 14. Vinos v. 2009 y 2010. (C.: cmara; 1T: asoleo tradicional en el CAIV; 2T: asoleo
tradicional en via).
105
III.II. Control de la seguridad alimentaria
III.II.1.1. REACTIVOS
106
Captulo III. Materiales y Mtodos
(mg/l)
107
III.II. Control de la seguridad alimentaria
III.II.2. OCRATOXINA A
III.II.2.1. REACTIVOS
Para ello, 5 mililitros de la disolucin stock de OTA fue llevada hasta sequedad
bajo corriente de nitrgeno y redisueltos en el mismo volumen de la mezcla de
disolventes tolueno/cido actico glacial (99:1, v/v). A continuacin, se
registra el espectro de absorcin de la disolucin entre 300 y 370 nm en
cubetas de cuarzo de 1 cm de paso de luz. La concentracin, en miligramos
por litros, de OTA vendr dada por:
108
Captulo III. Materiales y Mtodos
A (mg/l)
109
III.II. Control de la seguridad alimentaria
Figura 25. Equipo HPLC-FLD. Figura 26. Rapid Trace SPE Workstation
III.II.2.4.1. Mostos
III.II.2.4.2. Vinos
110
Captulo III. Materiales y Mtodos
111
III.II. Control de la seguridad alimentaria
glucosa) y fueron tratadas de la misma manera que las muestras reales. Cada
una de ellas se analiz por duplicado. Por ltimo, se represent grficamente
las reas de pico de OTA obtenidas en los cromatogramas frente a sus
respectivas concentraciones.
112
Captulo III. Materiales y Mtodos
El equipo estaba compuesto por dos bombas modelo 2150 y un horno modelo
2155 donde se situaba la columna, todos de LKB (Pharmacia, Suecia), un
detector de conductividad Conductomonitor modelo III de Milton Roy
(PMA, USA) y un inyector automtico modelo 717 de Waters (Milford, USA)
(Figura 27).
113
III.II. Control de la seguridad alimentaria
Antes de cada anlisis los mostos y vinos fueron filtrados a travs de filtros de
jeringa de nylon de 0,45 m de dimetro de poro (Millipore, USA).
114
Captulo III. Materiales y Mtodos
115
III.II. Control de la seguridad alimentaria
III.II.4.1. REACTIVOS
116
Captulo III. Materiales y Mtodos
117
III.III. Calidad organolptica
III.III.1.1.1. Reactivos
118
Captulo III. Materiales y Mtodos
El gradiente empleado de 6,5 min fue el siguiente: 0 min. 100% A, 3 min. 90%
A (curva 6), 4 min. 90% A, 6,5 min. 25% A (curva 6). Finalmente la columna
se lav con 100% de B durante 3 minutos y se equilibr con 100% de A
durante otros 3 minutos.
Previo a la inyeccin, todas las muestras fueron filtradas por filtros de nylon
de 0,22 m (Scharlab, Espaa). La identificacin de los compuestos fue
realizada por comparacin de sus espectros UV-Visible y tiempos de retencin
con los correspondientes a las disoluciones estndar de patrones.
119
III.III. Calidad organolptica
Concentracin
Compuestos polifenlicos (nm)
(ppm )
cido glico 280 0,20-20,0
5-Hidroximetilfurfural 280 0,12-120
cido Protocatquico 280 0,10-4,00
2-Furfuraldehdo 280 0,15-15,0
Tirosol 280 0,95-38,0
5-Metilfurfural 280 0,10-2,00
cido cafeico 320 0,12-1,00
Vainillina 320 0,20-20,0
cido p-cumrico 320 0,03-3,00
Siringaldehdo 320 0,20-20,0
120
Captulo III. Materiales y Mtodos
121
III.III. Calidad organolptica
122
Captulo III. Materiales y Mtodos
Reactivos Volumen
123
III.III. Calidad organolptica
Reactivos Volumen
Patrn/ Muestra 0,200 ml
Agua Desionizada 0,800 ml
Nitrato de Sodio 5% 0,060 ml
Mezclar y esperar 5min a temperatura ambiente.
Cloruro de aluminio 10% 0,060 ml
Mezclar y esperar 6 min a temperatura ambiente.
Hidrxido sdico 1M 0,400 ml
Agua Desionizada 0,480 ml
Mezclar y medir la absorbancia a 510 nm.
124
Captulo III. Materiales y Mtodos
Los anlisis se llevaron a cabo siguiendo una variante del mtodo de Huang et
al., (2002) adaptado para un lector de microplacas de fluorescencia FLx800,
(Feliciano et al., 2008) (Figura 30). Estos ensayos miden la capacidad de las
125
III.III. Calidad organolptica
126
Captulo III. Materiales y Mtodos
127
III.III. Calidad organolptica
III.III.2.1. REACTIVOS
128
Captulo III. Materiales y Mtodos
129
III.III. Calidad organolptica
130
Captulo III. Materiales y Mtodos
131
III.III. Calidad organolptica
132
Captulo III. Materiales y Mtodos
Tendr disponibles una serie de tringulos de muestras. Para cada uno, proceda de la siguiente forma:
1. Anote los cdigos de las muestras en la columna Cdigos del tringulo
2. Huela las 3 muestras que se le presentan una vez, o una sola vez ms si es que lo necesita.
3. Indique, rodeando el cdigo con un crculo, cul de ellas es diferente.
4. Especifique el grado de diferencia que encuentra, marcando con una X en la casilla
correspondiente.
5. Describa, si lo desea, en qu consiste la diferencia detectada.
6. Descanse un minuto antes de continuar con el siguiente tringulo.
N de la cabina:
Grado de diferencia
Cdigos del Ausencia Dbil Media Fuerte Muy fuerte
tringulo
Descripcin de la diferencia
Descripcin de la diferencia
Descripcin de la diferencia
Descripcin de la diferencia
Descripcin de la diferencia
MUCHAS GRACIAS
133
III.III. Calidad organolptica
Tendr disponibles una serie de muestras. Para cada una, proceda de la siguiente forma:
1. Destape la copa y huela en su interior.
2. Mueva ligeramente la copa y huela de nuevo.
3. Para cada atributo, seale la marca vertical de la escala de 9 puntos que a su juicio corresponde
con la intensidad del estmulo.
4. Finalmente, punte su impresin general de la muestra en la escala hednica.
5. Descanse un minuto antes de continuar con la siguiente muestra
N DE LA CABINA: CDIGO DE LA
MUESTRA:
Intensidad aromtica | ; | ; | ; | ; |
Afrutado (uva, | ; | ; | ; | ; |
manzana, pera, pltano)
Ctricos | ; | ; | ; | ; |
Frutas maduras | ; | ; | ; | ; |
(pltano, macedonia)
Dulce | ; | ; | ; | ; |
Floral (acacia, | ; | ; | ; | ; |
jazmn, rosa, violeta)
Miel | ; | ; | ; | ; |
Herbceo | ; | ; | ; | ; |
Vinoso | ; | ; | ; | ; |
Lcteo (leche,mantequilla) | ; | ; | ; | ; |
Qumico | ; | ; | ; | ; |
(disolvente, medicinal)
Otros (especificar):
. | ; | ; | ; | ; |
. | ; | ; | ; | ; |
OBSERVACIONES (Opcional)
MUCHAS GRACIAS
134
Captulo III. Materiales y Mtodos
1. Observe, huela y pruebe las N muestras que se le presentan, en el orden de izquierda a derecha.
2. Coloque en la tabla inferior los cdigos de las muestras, por orden creciente de preferencia.
Preferencia Menos Ms
Cdigo
COMENTARIOS:.............................................................................................................
.....................................................................................................................................
.....................................................................................................................................
.....................................................................................................................................
.....................................................................................................................................
...............................................................................................................................
MUCHAS GRACIAS
135
Captulo IV.
Resultados y Discusin.
Captulo IV. Resultados y Discusin
139
IV.I. Parmetros relacionados con la seguridad alimentaria
Tabla 21. Contenidos (media, mg/l desviacin estndar) en cido glucnico en mostos.
Distintos superndices a, b y c indican diferencias en funcin de pasificacin (muestras Ini, C,
1T y 2T). Distintos superndice 1, 2 y 3 indican diferencias en funcin de vendimia (muestras
V08, V09 y V10). *p < 0,05 indica diferencias significativas.
4000
cido glucnico (mg/L)
3000
2000
1000
0
S0 S1 S2 S3 S4 S5
Tiempo pasificacin
Figura 36. Evolucin del contenido en cido glucnico durante el secado de uvas en cmara (C).
S0: Inicial, S1: 2 das, S2: 3 das, S3: 6 das, S4: 7 das, S5: 8 das.
140
Captulo IV. Resultados y Discusin
Los mostos de uva fresca presentaron los menores valores, mientras los
obtenidos de uva pasificada de acuerdo al sistema tradicional (en terraza, 1T, o
en via, 2T) fueron los que alcanzaron los mayores contenidos.
Por otra parte, en estudios a pequea escala del secado de uva en cmara
climtica (Hernndez, 2009), tambin se hallaron menores contenidos de cido
glucnico en mostos procedentes del pasificado artificial frente a aquellos del
141
IV.I. Parmetros relacionados con la seguridad alimentaria
142
Captulo IV. Resultados y Discusin
IV.I.2. OCRATOXINA A
Figura 37. Cromatograma tpico de las muestras estudiadas, mosto y vino. Comparacin con un
blanco.
143
IV.I. Parmetros relacionados con la seguridad alimentaria
Tabla 22. Ocratoxina A en mostos. (C: Cmara climtica; 1T: asoleo tradicional en el CAIV; 2T:
asoleo tradicional en Via.)
144
Captulo IV. Resultados y Discusin
No obstante, gran parte de esta toxina no fue transferida al vino final, cuyo
valores de OTA se encontraron por debajo de los lmites de deteccin y
cuantificacin, en todos los vinos correspondientes a la vendimia 2010.
Este hallazgo est en concordancia con los datos proporcionados por Valero et
al., (2008a), que demostraron que es despus de la vendimia, y particularmente
en el proceso de pasificacin, cuando las especies de Aspergillus se vuelven
predominantes sobre otros hongos presentes en la uva, debido principalmente
a una mayor tolerancia a las altas temperaturas y a la baja actividad del agua
(aw).
145
IV.I. Parmetros relacionados con la seguridad alimentaria
146
Captulo IV. Resultados y Discusin
IV.I.2.2.1. Vinificacin
147
IV.I. Parmetros relacionados con la seguridad alimentaria
IV.I.1.2.2. Envejecimiento
148
Captulo IV. Resultados y Discusin
Experimento 1
4,00
V08. M.C.E1C1
[Ocratoxina A] g/L 3,50
V08. M.C.E1C2
3,00
V08. M.C.E1S
2,50 V08. M.C.E1CH
2,00
1,50
1,00
0,50
0,00
V01 V02 S20d S02 S03 S06 S09 S10 S12
Envejecimiento
Figura 39. Evolucin de vinos Moscatel de la v.2008. Ensayo de vinificacin 1 (E1: S. Cerevisiae, t
ambiente), envejecidas en botas de roble (C), acero inoxidable (S) y con chips (CH).
Adems, se observ que los vinos del mismo ensayo envejecido en diferentes
barricas de roble mostraron una tendencia muy similar en el tiempo mostrando
una dispersin muy baja. En el experimento realizado con virutas de madera
(1_Ch), el contenido de OTA del vino se mantuvo constante, mostrando un
comportamiento similar al envejecimiento en acero inoxidable (Figura 39).
Por tanto, todos los vinos con crianza en madera de roble durante un periodo
de 12 meses mostraron unos contenidos en OTA por debajo de 2,0 g/l,
lmite legal que establece la Unin Europea, por lo que seran aptos para su
consumo.
149
IV.I. Parmetros relacionados con la seguridad alimentaria
Tabla 23. Valores medios (g/l) y desviaciones estndar de la concentracin de OTA en vinos. E1-E5: Experiencias de vinificacin; C: Botas de
roble, S: Depsitos de acero inoxidable, CH: Envejecimiento con chips.
150
Captulo IV. Resultados y Discusin
151
IV.I. Parmetros relacionados con la seguridad alimentaria
152
Captulo IV. Resultados y Discusin
estas muestras, por lo que el menor contenido podra justificarse por los
efectos de combustin ya mencionados.
El cido succnico se hall en las uvas en concentraciones muy por debajo del
rango normal en el vino (500-1500 mg/l), indicando un inicio muy tmido del
proceso fermentativo que se potencia tras la pasificacin, ms en el secado
mediante asoleo que en cmara, probablemente debido a la mayor
disponibilidad de oxgeno en los espacios abiertos. El ao de vendimia result
ser un factor que afect a su presencia en el mosto, no as la variedad.
153
IV.I. Parmetros relacionados con la seguridad alimentaria
Por su parte, el cido actico en las uvas sin secar present concentraciones
bastante menores a las propias para vinos sanos (< 500 mg/l), aunque no hay
que olvidar que se trata de uvas aun sin vinificar. Al analizar los datos para
ambas variedades y vendimias, se observ que el secado de las uvas
increment significativamente estas concentraciones, sin que se pueda concluir
un patrn comn para todas las muestras (variedad x vendimia) en lo que se
refiere al efecto de cada sistema de pasificacin: Moscatel aument en mayor
medida sus niveles de actico tras el secado en cmara, mientras Pedro
Ximnez lo hizo con el secado mediante asoleo tradicional. Y si bien vendimia
contribuy de manera significativa al contenido en actico, al igual que
variedad, la existencia de interaccin estadstica entre estos factores y con el
sistema de pasificacin, impiden establecer patrones de comportamiento. Por
otra parte, la concentracin durante el secado de las uvas no justifica en la
mayora de los ensayos el significativo aumento de su contenido, lo que podra
interpretarse como consecuencia de cierta actividad bacteriana (lctica o
actica).
154
Captulo IV. Resultados y Discusin
Tabla 24. Contenidos (media desviacin estndar) en cidos orgnicos de los mostos (mg/l). Distintos superndices a, b y c indican diferencias en
funcin de la variable pasificacin (muestras Ini, C, 1T y 2T). Distintos superndices 1, 2 y 3 indican diferencias en funcin de la variable ao de
vendimia (muestras V08, V09 y V10). p <0.05 indica diferencia significativa.
PX M pvariedad
p p
V08 V09 V10 V08 V09 V10 V08 V09 V10
vendimia vendimia
. tartrico
Ini 2658181a,1 349211a,2 3849232a,2 0,013 201427a,1 26741a,2 294411a,3 0,000 0,038 0,000 0,031
C 334859b,1 34287a,1 4431260 b,2 0,010 313325b,1 415480b,3 3750145b,2 0,004 0,041 0,006 0,084
1T 347276b 3588145a 0,422 31121b 3706212b 0,058 0,021 0,584
2T 417037 b 7973106c 0,000
p secado 0,011 0,000 0,000 0,000 0,001 0,020
. mlico
Ini 125635a,3 75319a,1 99331a,2 0,001 150216c,2 12833a,1 161334a,3 0,001 0,012 0,000 0,003
C 149237b,3 92716 b,1 118470b,2 0,003 11502a,1 155261b,2 186915b,3 0,001 0,006 0,005 0,005
1T 181644c 144523c 0,009 125125b 193266b 0,005 0,004 0,010
2T 119910 c 220662d 0,002
p secado 0,002 0,000 0,000 0,000 0,025 0,010
. ctrico
Ini 31419a - - 5298 b - - 0,004
C 58128 b - - 7148 c - - 0,023
1T 79621 c - - 4325a - - 0,002
2T
p secado 0,000 0,000
. succnico
Ini 00a,1 423a,2 00a,1 0,000 7543 482a,2 00a,1 0,000 0,001 0,139 1,000
C 4725b,1 1193c,2 556b,1 0,031 7121 1447b,2 627b,1 0,001 0,309 0,043 0,394
1T 1452c 9110c 0,016 9521 979c 0,922 0,077 0,583
2T 1011b 918c 0,238
p secado 0,005 0,000 0,000 0,256 0,003 0,001
155
IV.I. Parmetros relacionados con la seguridad alimentaria
PX M pvariedad
p p
V08 V09 V10 V08 V09 V10 V08 V09 V10
vendimia vendimia
. lctico
Ini 00a,1 8217a,2 00a,1 0,005 1568b,3 805a,2 00a,1 0,000 0,001 0,871 0,849
C 8060a 1860 b 10613b 0,113 00a,1 2491b,3 1835a,2 0,000 0,196 0,000 0,015
1T 2967b 12623b 0,009 00a 2181a 0,000 0,000 0,029
2T 1561b 27036c 0,047
p secado 0,007 0,004 0,001 0,000 0,000 0,000
. frmico
Ini 320a,2 00a,1 00a,1 0,000 813a,3 501a,2 001 0,000 0,001 0,000 0,956
C 8017b,2 1201b,2 314b,1 0,024 882a,3 701b,2 001 0,000 0,870 0,000 0,007
1T 8321b 377b 0,012 1065b 00 0,001 0,028 0,015
2T 1971c 8011c 0,004
p secado 0,050 0,000 0,001 0,012 0,003 0,465
. actico
Ini 1272a,3 00a,1 295a,2 0,000 1026a,2 123a,1 00a,1 0,000 0,035 0,029 0,013
C 10846a,1 3247b,2 53115c,3 0,001 4922c,1 65212b,3 54510c,2 0,001 0,007 0,000 0,410
1T 45014b 37829b 0,088 32611b 1995b 0,004 0,010 0,013
2T 34620b 63334d 0,009
p secado 0,002 0,000 0,000 0,000 0,000 0,000
156
Captulo IV. Resultados y Discusin
157
IV.I. Parmetros relacionados con la seguridad alimentaria
158
Captulo IV. Resultados y Discusin
Tabla 25. Contenidos (media, mg/l desviacin estndar) en cidos orgnicos de los vinos obtenidos bajo las distintas condiciones de fermentacin
ensayadas en la v. 2008. p < 0,05 indica diferencia significativa. Distintos superndices a, b y c indican diferencia en funcin de las condiciones de
vinificacin (muestras E1-E5).
159
IV.I. Parmetros relacionados con la seguridad alimentaria
160
Captulo IV. Resultados y Discusin
161
IV.I. Parmetros relacionados con la seguridad alimentaria
Tabla 26. Contenidos (media, mg/l DE) en cidos orgnicos de los vinos sin envejecer. Distintos superndices a, b y c indican diferencias en funcin
de la pasificacin de los vinos (muestras C, 1T y 2T) (Test de Tukey). p < 0.05 indica diferencia significativa.
PX M pvariedad
V09 V10 pvendimia V09 V10 pvendimia V09 V10
. tartrico C 13608 21533b 0,000 255315 267421 0,041 0,000 0,002
1T 155830a 182220 0,018
2T 204713 214012b 0,035
psecado 0,000 0,000 0,001
. mlico C 10161 8990a 0,000 208024 179415 0,022 0,000 0,000
1T 108022b 189226 0,002
2T 15389 203310c 0,000
psecado 0,000 0,000 0,538
. succnico C 7060 3841b 0,000 6955 5292 0,000 0,139 0,000
1T 50812c 3532 0,006
2T 3921 3292a 0,001
psecado 0,000 0,000 0,000
. lctico C 3993 1502a 0,000 1663 2510 0,001 0,000 0,000
1T 2194b 3063 0,004
2T 2752 3476c 0,007
psecado 0,001 0,000 0,004
. frmico C 240 352a 0,027 840 380 0,000 0,000 0,214
1T 401b 462 0,064
2T 420 640c 0,000
psecado 0,001 0,000 0,034
. actico C 8351 15223b 0,000 8424 13441 0,000 0,206 0,000
1T 105422a 4721 0,001
2T 12781 23019c 0,000
psecado 0,000 0,000 0,000
162
Captulo IV. Resultados y Discusin
163
IV.I. Parmetros relacionados con la seguridad alimentaria
164
Captulo IV. Resultados y Discusin
Tabla 27. Contenidos (media, mg/l desviacin estndar) en cidos orgnicos de los vinos al inicio de la etapa de envejecimiento (S20d S01, segn
las muestras) y tras 12 meses en bota de roble (S12). Diferencias significativas para p < 0,05. En la muestra 1, para cada cido, distintos superndices
indican diferencia en funcin de tipo de envejecimiento (Bota, Acero inoxidable o Chips): superndices a, b y c para S20d/S01 y 1, 2 y 3 para S12.
165
IV.I. Parmetros relacionados con la seguridad alimentaria
. succnico
Bota S20d/S01 4648b 4199 7319 4253 7270 450 5474 243 5454 3867
S12 344123 2905 7613 4740 7793 4800 5615 4852 6761 4702
p 0,000 0,000 0,041 0,001 0,002 0,000 0,118 0,005 0,000 0,003
Acero S20d/S01 4372a 4062 7060 3921 6956 3841 50818 3293 5293 3532
Inox. S12 29342 1743 69114 3831 66511 3884 4972 3304 5182 3562
p 0,000 0,000 0,259 0,015 0,078 0,278 0,466 0,868 0,039 0,289
Chips S20d/S01 4900b
roble S12 21741
p 0,000
ptipo envej pS20d/S01 0,017 0,389 0,057 0,004 0,020 0,013 0,092 0,028 0,040 0,022
pS12 0,001 0,000 0,020 0,000 0,005 0,000 0,003 0,000 0,000 0,000
. lctico
Bota S20d/S01 3134c 22822 46923 3002 15881 189 2652 138 2977 36514
S12 41823 37414 54928 3823 3101 2749 3490 6146 4874 5001
p 0,000 0,001 0,086 0,000 0,116 0,079 0,000 0,010 0,000 0,005
Acero S20d/S01 2145a 1783 3995 2753 1664 1503 2196 3478 2510 3065
Inox. S12 20142 1303 4510 2411 19113 1951 2555 4132 3254 3564
p 0,174 0,008 0,004 0,005 0,125 0,002 0,021 0,007 0,001 0,008
Chips S20d/S01 2425b
roble S12 15131
p 0,004
ptipo envej pS20d/S01 0,000 0,200 0,050 0,012 0,904 0,064 0,009 0,030 0,010 0,029
pS12 0,000 0,000 0,038 0,000 0,006 0,006 0,001 0,000 0,000 0,000
166
Captulo IV. Resultados y Discusin
167
IV.I. Parmetros relacionados con la seguridad alimentaria
168
Captulo IV. Resultados y Discusin
20
Variacin de la seal (%)
10
0
-10 0 2 4 6 8 10 12
-20 Tiempo (horas)
-30
-40
-50
-60
-70
Figura 40. Estabilidad del complejo xantil-CE en disoluciones alcholicas. 20% etanol v/v;
40% etanol v/v; matriz vnica (17% v/v).
169
IV.I. Parmetros relacionados con la seguridad alimentaria
Muestreo E1 E2 E3 E4 E5
S0d 10,22,1 11,03,2 40,81,7 n.d. 13,20,7
S20d 19,56,1 30,38,6 48,61,2 23,91,4 17,21,2
S40d 22,27,1 43,720,2 57,00,3 34,16,6 25,80,3
S2 26,28,1 50,313,6 85,72,9 38,93,6 24,70,9
S4 38,811,5 71,117,0 1033 57,67,0 37,91,2
S7 51,922,2 74,118,7 1092 44,325,8 45,20,7
S8 59,418,1 86,121,3 1231 73,014,1 57,90,3
S12 67,218,8 10019 1462 60,527,7 63,70,3
S18 74,618,7 12110 1821 94,715,7 67,01,1
S28 96,033,8 14716 1946 1069 1252
Tabla 28. Vendimia 2008, variedad Moscatel. Valores medios (g/l) y desviaciones estndar
durante el envejecimiento en madera (E3 y E5) y en barricas y depsitos de acero (E1, E2 y E4).
S0d, S20d y S40d inicial, 20 das y 40 das. S2, S2, S7, S8, S12, S18, S28; 2, 4, 7, 8, 12, 18, 28 meses
respectivamente. n.d.: no detectado.
170
Captulo IV. Resultados y Discusin
100
Concentracin CE (g/l)
80
60
2009 C
40
2009 S
20 2010 C
0 2010 S
-20
0 20 40 2 4 7 8 12 18 28
Das Meses
171
IV.I. Parmetros relacionados con la seguridad alimentaria
Teniendo en cuenta todas las muestras (dos variedades y tres vendimias), los
valores medios de CE oscilaron entre 6,16 g/l para S0d (despus de la
fermentacin alcohlica) y 120 g/l para S28 (despus de 28 meses de
crianza).
172
Captulo IV. Resultados y Discusin
E1 E2 E3 E4 E5
Vinificacin
50,630,2a 80,539,0b 11150c 43,633,9d 47.832,5a
Tabla 29. Valores medios de CE y desviaciones estndar para cada factor considerado. En cada fila, diferentes superndices indican diferencias
significativas a p < 0,01 (Test de Tukey).
173
IV.I. Parmetros relacionados con la seguridad alimentaria
Tabla 30. Anlisis de la varianza para la concentracin de carbamato de etilo de los diferentes
vinos obtenidos.
174
Captulo IV. Resultados y Discusin
175
IV.I. Parmetros relacionados con la seguridad alimentaria
El experimento E4, fermentado con S. bayanus, fue el nico que se llev a cabo
a baja temperatura (10 C) sin uso de nutrientes ni enzimas pectolticas. Ough
et al. (1991) observaron que el empleo de bajas temperaturas durante la
fermentacin alcohlica produca vinos con menor contenido en urea; adems,
varios autores (Daudt et al., 1995; Balcerek y Szopa, 2006; Uthurry et al., 2006)
describieron anteriormente la baja capacidad de S. bayanus para la produccin
de CE. Por otra parte, varios estudios han demostrado que el uso de enzimas
pectolticas en los procesos de maceracin pelicular modifica la composicin
de los mostos, aumentando la concentracin de aminocidos (Kishkovskii et
al., 1974; Aldave, 1992; Bosso y Ponzetto, 1994). Esta conclusin coincide con
el elevado contenido en CE encontrado en el experimento E3, macerado con
enzimas pectolticas y fermentada con S. cerevisiae a temperatura ambiente (
30C). Tanto el experimento E3, fermentado con S. cerevisiae como el
experimento E5, fermentado con S. bayanus, se realizaron bajo las mismas
condiciones de vinificacin. La diferencia encontrada entre el contenido de
carbamato de etilo de estos dos experimentos revela la importante influencia
de la cepa de levadura sobre el contenido en este compuesto.
176
Captulo IV. Resultados y Discusin
Por ltimo, en relacin con el efecto del tipo de envejecimiento, como puede
verse en las Tablas 29 y 30, los valores significativamente ms altos de
carbamato de etilo se obtuvieron para los vinos envejecidos en barricas de
roble. Esta diferencia significativa podra explicarse sobre la base de las
posibles modificaciones microbiolgicas favorecidas por la baja aireacin que
tiene lugar a travs de los poros de la barrica de roble (Blouin y Peynaud,
2005). Esto podra aumentar algunos de los precursores de CE para los vinos
envejecidos en madera (Liu et al., 1994). Dependiendo de la cepa bacteriana,
citrulina y fosfato de carbamilo pueden formarse a partir de arginina y su
posterior reaccin con etanol, durante un perodo concreto de tiempo y bajo
una temperatura dada, conduce a la formacin de CE. Los datos sobre el
contenido de cidos orgnicos (cido mlico, cido lctico y cido actico)
fueron sometidos a un anlisis de varianza considerando "tipo de
envejecimiento" como variable independiente (ver IV.I.3.4. cidos Orgnicos.
Influencia del sistema de envejecimiento). Para todos estos cidos, se encontraron
177
IV.I. Parmetros relacionados con la seguridad alimentaria
178
Captulo IV. Resultados y Discusin
Figura 42. (a); AC vinos Moscatel (M) y Pedro Ximnez (PX), vendimias 2008, 2009 y 2010 y envejecidas en barricas de roble (C) y en depsitos de
acero inoxidable (S). Muestreos; S0d: inicial; S20d, S40d: 20 y 40 das; S2, S4, S7, S8, S12, S18 y S28: 2,4, 7, 8, 12, 18 y 28 meses respectivamente. (b);
Ampliacin del rea indicada.
179
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
180
Captulo IV. Resultados y Discusin
los valores medios para cada vinificacin (E1-E5), as como los valores-p para
cada factor.
Figura 43. Cromatograma, 280 nm. Identificacin de los picos: 1: cido glico, 2: 5-
hydroximetilfurfural, 3: cido protocatquico, 4: a.caftrico 5: furfural, 6: cido cis/trans p-
cutrico, 7: tirosol, 8: compuesto b, 9: compuesto a, 10: compuesto c, 11: metilfurfural, 12:
siringaldehido.
181
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
182
Captulo IV. Resultados y Discusin
183
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 31. Valores medios, mg/l desviaciones estndar para los distintos ensayos de vinificacin. * Diferencias significativas a p < 0,05). Diferentes
letras para la misma fila indican diferencias significativas (Test de Tukey).
184
Captulo IV. Resultados y Discusin
185
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
186
Captulo IV. Resultados y Discusin
Figura 44. ACP por tipo de envejecimiento. 1: Barricas de roble, 2: Depsitos de Acero, 3: Con
chips.
187
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Figura 45. ACP por tiempo de envejecimiento. 1: 20 das, 2: 40 das; 3, 5,9,12,18 y 28 meses.
188
Captulo IV. Resultados y Discusin
189
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
190
Captulo IV. Resultados y Discusin
191
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 32. Valores medios (mg/l) desviaciones estndar para variedad y tipo de secado. Valores p para cada uno de los factores considerados
(* p < 0.05)
Compuestos
Variedad Tipo de secado p
polifenlicos
Cmara Tiempo
P.X. Moscatel Asoleo Variedad Vendimia Secado
climtica de envejec.
cidos hidroxibenzoicos y derivados
cido glico 4,424,46 2,713,53 4,504,39 3,023,93 0,0002* 0,4028 0,0020* 0,0000*
Galato de etilo 4,431,19 2,360,91 4,151,28 3,281,57 0,0000* 0,0061* 0,0000* 0,0000*
cido protocatquico 9,065,52 2,782,11 9,046,11 4,383,16 0,0000* 0,0000* 0,0000* 0,0000*
Vainillina 1,601,55 1,892,14 1,891,85 1,411,59 0,0044* 0,8238 0,0039* 0,0000*
Siringaldehdo 3,523,18 3,373,49 3,863,40 2,923,00 0,6422 0,0070* 0,0347* 0,0000*
cidos hidroxicinmicos y derivados
cido cafeico 0,5700,505 0,3190,504 0,4810,450 0,5070,601 0,0042* 0,1649 0,5812 0,0000*
cido caftrico 5,655,90 0,5190,481 5,254,26 2,366,44 0,0000* 0,7720 0,0022* 0,0780
Cafeato de etilo 0,1860,247 0,0680,140 0,1360,185 0,1680,274 0,0001* 0,8953 0,4184 0,0000*
cido cumrico 0,3100,401 0,1720,250 0,2650,345 0,2700,396 0,0005* 0,4235 0,6613 0,0000*
cido cis/trans p-cutrico 4,973,68 0,3120,177 4,893,72 1,602,85 0,0000* 0,0455* 0,0000* 0,1256
Cumarato de etilo 0,0640,125 0,0420,085 0,0510,088 0,0660,144 0,2811 0,3892 0,1302 0,0000*
Aldehdos furnicos
5-hidroximetilfuraldehdo 6,066,27 6,948,98 6,727,43 5,786,89 0,1348 0,2808 0,9716 0,0000*
2-furaldehdo 6,094,63 5,634,91 6,314,65 5,444,77 0,7874 0,5309 0,3665 0,0000*
192
Captulo IV. Resultados y Discusin
Compuestos
Variedad Tipo de secado p
polifenlicos
Cmara Tiempo
P.X. Moscatel Asoleo Variedad Vendimia Secado
climtica de envejec.
5-metilfuraldehdo 0,7880,462 0,5530,474 0,7430,471 0,6760,486 0,0006* 0,0918 0,7520 0,0000*
Varios
tirosol 11,796,77 4,442,12 11,767,58 6,313,07 0,0000* 0,0003* 0,0000* 0,0001*
a 2,351,25 0,6030,308 2,341,34 1,050,86 0,0000* 0,0000* 0,0000* 0,0000*
b 3,022,54 3,423,69 3,663,19 2,432,39 0,0206 0,0001* 0,0503 0,0000*
c 0,6310,372 0,2990,159 0,6450,416 0,3610,124 0,0000* 0,0010* 0,0000* 0,0000*
d 7,243,07 3,711,79 6,323,17 5,883,22 0,0000* 0,1598 0,1755 0,0000*
193
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
194
Captulo IV. Resultados y Discusin
195
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
196
Captulo IV. Resultados y Discusin
TR MRM
Compuesto Polifenlico PM
(min) [M-H] > MS2
15,7 cido glico 170 169 > 125
19,2 Hidroxitirosol 154 153 >123
19,7 cido Protocatquico 154 153 > 109
20,6 cido Caftrico 312 311 > 179
21,9 cido 2-Furoico 112 111 > 67
23,2 Tirosol 138 137 > 119
23,7 cido p-cutarico 296 295 >163
24,3 cido p-Hidroxibenzoico 138 137 > 93
24,9 cido Gentsico 154 153 > 109
25,2 cido Fertrico 326 325 > 193
25,9 cido Cafeico 180 179 > 135
28,8 cido Dihidroxibenzoico 154 153 > 109
29,3 p-hidroxibenzaldehdo 122 121 > 92
31,9 Galato de etilo 198 197 > 169
34,2 Vainillina 152 151 > 136
197
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
TR MRM
Compuesto Polifenlico PM
(min) [M-H] > MS2
34,2 cido p-coumrico 164 163 > 119
35,5 trans-piceido 390 389 > 227
36,7 cido elgico 302 301 > 284
37,4 Quercetina-3-glucuronido 478 477 > 301
37,8 Quercetina-3-glucsido 464 463 > 301
Dihidroquercetina-3-O-
38,5 450 449 > 303
rhamnosido (astilbina)
40,1 Cafeoiltartrato de etilo 340 339 > 177
41,4 Quercetina-3-rhamnosido 448 447 > 301
41,6 cis-piceido 390 389 > 227
Dihidrokaempferol-3-O-
42,0 434 433 > 269
rhamnosido (engeletina)
44,1 Protocatecato de etilo 182 181 > 153
45,3 cido Saliclico 138 137 > 93
48,8 trans-cafeato de etilo 208 207 > 179
53,2 p-cumarato de etilo 192 191 > 163
198
Captulo IV. Resultados y Discusin
199
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
200
Captulo IV. Resultados y Discusin
Estudios de correlacin
201
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 34. Correlacin de los ndices globales con la actividad antioxidante *p < 0,05
202
Captulo IV. Resultados y Discusin
Con el fin de obtener una posible expresin matemtica que nos indique la
capacidad antioxidante de estos vinos en funcin de los valores de los
indicadores individuales y globales de composicin polifenlica, se procedi al
anlisis por regresin lineal mltiple (RLM). Los datos incluidos en ambos
casos fueron los 29 compuestos polifenlicos recogidos en la Tabla 33, ndice
de polifenoles totales (IPT TPC), ndice de flavonoides totales (IFT TFC),
el rea cromatogrfica total a 280 nm y a 360 nm (A280 y A360) y la suma de
reas de cada uno de los picos obtenidos en el detector electroqumico (AED).
203
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
204
Captulo IV. Resultados y Discusin
205
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
CP2
CP1
206
Captulo IV. Resultados y Discusin
207
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
sta para las muestras de la vendimia 2008 y 2009 sometidas a madera con
respecto a aquellos envejecidos en acero inoxidable, si bien para 2010 estas
diferencias entre ambos sistemas de envejecimiento no fueron significativas.
En el caso del envejecimiento con chips, ste se mostr con valores similares
de AA ORAC a los obtenidos para aquellas muestras envejecidas en bota,
observndose valores menores para los envejecidos en acero inoxidable.
208
Captulo IV. Resultados y Discusin
Para AA HORAC, fue el tipo de fermentacin bajo las condiciones del ensayo
4 (S. Bayanus y baja temperatura) las que mostraron valores ms elevados. En
este caso, tambin se observ mayor AA HORAC en aquellos vinos
procedentes de la variedad Pedro Ximnez.
209
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Ruiz et al., (2010), observaron en uvas de la variedad Pedro Ximnez, que los
mostos de uva secada en cmara climtica exhiban el mismo perfil aromtico
que aquellos secados al sol por el mtodo tradicional.
210
Captulo IV. Resultados y Discusin
211
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 36. Parmetros de identificacin y cuantificacin de los compuestos voltiles en los vinos,
v. 2008.
212
Captulo IV. Resultados y Discusin
vino final. Teniendo en cuenta todos estos factores, con el fin de estudiar
estadsticamente las diferencias en el contenido voltil de todos los vinos
estudiados, los datos fueron sometidos al anlisis de la varianza. Para ello, se
consideraron tres factores independientes: tipo de fermentacin, tiempo de
envejecimiento y tipo de envejecimiento. Como cabra esperar, prcticamente
todos los compuestos voltiles se vieron significativamente afectados (p <
0,01) por estos tres factores (Tabla 37).
steres de etilo
213
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
214
Captulo IV. Resultados y Discusin
cidos
215
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Acetatos
Terpenos y terpenoles
216
Captulo IV. Resultados y Discusin
217
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
218
Captulo IV. Resultados y Discusin
219
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Varios
Entre los compuestos voltiles que han demostrado un claro aumento durante
el envejecimiento de los vinos en madera de roble, algunos autores (Cmara et
al., 2006; Mrquez et al., 2007) hallaron 2-furaldehido y 5-metil-2-furaldehido.
Ambos se forman por la degradacin de los hidratos de carbono durante el
tostado de la barrica y luego se transfieren al vino durante su envejecimiento.
En los vinos sin envejecimiento en madera, pueden proceder de la
deshidratacin de azcares durante el proceso de secado (Franco et al., 2004).
220
Captulo IV. Resultados y Discusin
221
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 37. Medias (reas relativas de pico) y desviaciones estndar de los compuestos voltiles. Anlisis de la varianza. S20d: 20 das de envejecimiento en
madera; S12: 12 meses de envejecimiento en madera.
steres de etilo
222
Captulo IV. Resultados y Discusin
223
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
224
Captulo IV. Resultados y Discusin
225
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
226
Captulo IV. Resultados y Discusin
5-metilfuraldehido
S20d 0,0120,013 0,0110,011 0,0170,010 0,0090,009 0,0230,001
0,0000 a 0,0000 a 0,0000 a
S12 0,0250,032 0,0510,065 0,0350,003 0,0260,028 0,0670,010
227
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
1,1,6-trimetil-1,2-
dihidronafthaleno S20d 0,0040,005 0,0080,001 0,0040,023 0,0020,001 0,0040,001
0,0000a 0,0000a 0,0000a
(TDN)
S12 0,0130,008 0,0520,019 0,0150,006 0,0130,014 0,0460,023
2,6 Di-tert-butil-p S20d 0,1780,039 0,1100,069 0,1270,003 0,1420,037 0,1000,007
cresol (BHT) 0,1128 0,0000a 0,0000a
S12 0,3070,296 0,3350,339 0,0660,050 0,2340,244 0,1190,003
228
Captulo IV. Resultados y Discusin
229
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Por tanto, con el fin de obtener las reglas de clasificacin adecuadas para la
asignacin de categoras a las muestras, se aplic un mtodo de
reconocimiento de modelos supervisado por lo que se llev a cabo un anlisis
lineal discriminante por pasos hacia delante (ALD).
230
Captulo IV. Resultados y Discusin
Como puede apreciarse en la Figura 55, se obtuvo una separacin clara entre
las muestras correspondientes a distintas condiciones de fermentacin. Las
muestras de los Experimentos E3, E4 y E5 estaban claramente diferenciadas,
mientras que los experimentos E1 y E2 fueron en algunos casos
entremezclados. Estos dos ltimos experimentos se llevaron a cabo sin
maceracin pelicular previa, con S. cerevisiae, y fermentados a la misma
temperatura. En el caso del Experimento E2, se emplearon nutrientes (Tabla
13). El 100% de las muestras fueron clasificadas correctamente en el proceso
de validacin por el mtodo de dejar-uno-fuera.
231
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
14 Experiencias
Experiences
1
10 2
3
6 4
Function 2
5
2
-2
-6 5
-10
-21 -16 -11 -6 -1 4 9
Function 1
Figura 55. ALD tipo de fermentacin. Moscatel, v.2008.
232
Captulo IV. Resultados y Discusin
dietlico con signo positivo, y butanoato de etilo y geraniol con signo negativo
fueron los compuestos voltiles ms importantes para la primera funcin. Para
la segunda funcin, stos fueron 3-metilbutanoato de etilo, hexanoato de etilo,
linalol con signo positivo, y el acetato de n-butilo, xido de nerol, epxido
linalol y acetato de feniletilo con signo negativo. El diagrama de dispersin de
las muestras en el plano definido por las dos primeras funciones
discriminantes se muestra en la Figura 57. Como se puede observar, la funcin
1 separa, de forma clara, las muestras con un breve tiempo de crianza (S20d,
S40d y S3, con valores negativos para esta funcin) de aquellos envejecidos
durante un mayor tiempo (S4, S8 y S12). En el proceso de validacin por el
mtodo dejar-uno-fuera, se obtuvo un 98,48% de clasificacin correcta.
7
Botas
Oak de roble
cask
5 Chips
Chips
Depsitos
Stainlees de
steel
3
acero inox.
Function 2
-1
-3
-5
-5 -3 -1 1 3 5 7
Function 1
233
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tiempo
S20d
S40d
S3
S4
S8
S12
234
Captulo IV. Resultados y Discusin
235
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
236
Captulo IV. Resultados y Discusin
steres etlicos
Los steres etlicos de cidos grasos C6, C8 y C10, junto con otros compuestos
voltiles tales como alcoholes superiores, acetatos y ciertos cidos voltiles son
los principales responsables del aroma de fermentacin en vinos (Karagiannis
et al., 2000). En cuanto al factor "tiempo de envejecimiento", ambas variedades
de uva mostraron cambios similares. Como ya se observ para los vinos de
uva Moscatel de la vendimia 2008 envejecidos en barricas de roble, el
contenido de varios steres etlicos (3-metilbutanoato de etilo, pentanodioato
de dietilo y succinato de dietilo) aument significativamente mientras que los
compuestos decanoato de etilo y dodecanoato de etilo disminuyeron a medida
que aumentaba el tiempo de envejecimiento. Cmara et al., (2006) hallaron, en
vinos de Madeira envejecidos en madera de roble durante veinticinco aos,
una disminucin de los steres de etilo de cidos grasos (C6-C16) y de acetatos
as como un elevado incremento de steres etlicos de cidos diprticos como
succinato de dietilo. Chaves et al., (2007) observaron un alto contenido en
acetato de etilo y hexanoato de etilo para vinos dulces Pedro Ximnez
envejecidos en madera. Las reacciones de hidrlisis y esterificacin pueden
afectar a este tipo de compuestos voltiles durante el perodo de
envejecimiento en madera (Cmara et al., 2006; Chaves et al., 2007). Adems, el
alto contenido en etanol que nuestros vinos presentan (17) podran explicar
estos incrementos por reacciones de esterificacin. Sin embargo, hay que tener
237
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
238
Captulo IV. Resultados y Discusin
cidos
Acetatos
239
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Terpenos y terpenoles
240
Captulo IV. Resultados y Discusin
241
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Varios
242
Captulo IV. Resultados y Discusin
243
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Tabla 39. Valores medios (reas cromatogrficas relativas) y desviaciones estndar para cada familia qumica.
steres de etilo 0,4090,946 0,5551,258 0,5781,091 0,4731,018 0,3710,904 0,6281,140 0,4470,961 0,5821,137
cidos 0,0700,081 0,0830,099 0,0750,077 0,0680,085 0,0540,065 0,0860,095 0,0650,072 0,0780,096
Acetatos 0,2640,389 0,3140,373 0,2650,300 0,2820,334 0,2120,193 0,3380,403 0,2990,305 0,2790,350
Terpenos y terpenoles 0,0170,034 0,0180,059 0,0460,077 0,0030,007 0,0130,048 0,0180,044 0,0200,056 0,0090,026
Aldehidos, cetonas y
0,2360,376 0,3300,485 0,3040,453 0,2890,412 0,2780,403 0,3070,443 0,3130,451 0,2650,382
alcoholes
Varios 0,0060,034 0,0320,057 0,0250,044 0,0260,048 0,0260,046 0,0260,048 0,0240,044 0,0280,051
244
Captulo IV. Resultados y Discusin
Tabla 40. Valores medios (reas cromatogrficas relativas) y desviaciones estndar para cada compuesto voltil estudiado.
245
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
246
Captulo IV. Resultados y Discusin
247
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
248
Captulo IV. Resultados y Discusin
249
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
250
Captulo IV. Resultados y Discusin
(X 1000)
8
6
Distance
0
2222222222222222222222222222222222222222 2222222222222222222222222111111
1111
111111111111111
111111111111111111111
251
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
252
Captulo IV. Resultados y Discusin
11
1
1
7
1 1
2 1
2 1 1
2 22 2 111
2 21
Component 2
3 2 2 1 1
2 12 1 111
22 2 2 2 22
1 11
1 1 1 111
11 1 1
2 22 2 11
2 2 2
22 2 2 12 1
2 22 2 11 1 1
-1 2 22 22 2 2 22
2 2
2
1 11
2 22 2
2 2 2
2 2
22 2 22 2
2 2 2 2 22 2 2 2
22 22
2 2 22 2
2 2
-5 2
22
2
2
2
-9
-7 -4 -1 2 5 8 11
Component 1
Figura 59. Anlisis de componentes principales (ACP) de todas las muestras, V.2009 y 2010. 1:
Moscatel; 2: Pedro Ximnez.
Teniendo en cuenta los resultados obtenidos a partir del ACP realizado para
todas las muestras, se decidi llevar a cabo dos nuevos ACPs, pero en este
caso, se estudiaron por separado ambas variedades de uva. Para los vinos
Moscatel, se obtuvieron nueve CPs, que explicaron el 92,8% de la varianza
total, de los que un 53,8% de sta fue explicada por las dos primeras CPs. En
el caso de los vinos de PX, el 87,4% de la varianza total qued explicada con
diez CPs. La Figura 60 muestra la distribucin de los dos vinos varietales sobre
el plano definido por las primeras CPs. Como puede verse, para cada variedad
de uva, los vinos se separaron de acuerdo a tiempo de envejecimiento. Para los
vinos Moscatel, aquellos vinos envejecidos en madera durante 5, 9 y 12 meses
presentaron valores positivos para la CP1 mientras que para los vinos PX, esos
vinos envejecidos durante un mayor perodo de tiempo (5 meses o ms)
mostraron valores negativos para esta CP. Los pesos de cada compuesto
voltil en la CP1 muestran claramente que los compuestos voltiles
relacionados con el envejecimiento en madera (2-furfuraldehdo, benzaldehdo,
5-metilfurfuraldehdo, eugenol y 4-etilfenol) son los principales responsables
253
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
de la CP1, con valores positivos para los vinos Moscatel y valores negativos
para los vinos PX.
9
a
1
6
1 5 12
12
0 9
3 3
Component 2
0 1 3 3
5 9
1 3
9 12
0
55 9
12
-3 0 1
0 1 3
3 12 12
33 9
5 9 9 12 12
5 9
5
-6
-7 -4 -1 2 5 8
Component 1
Vintage 2009
Vendimia 2009
10
b
12
7 12 9
0
9 0 0
1
12 3
4 12 3
12
Component 2
1 0
12
12 55
5 99 9
12 12 9
12
5 3
1 1
5 12 55 3
129 3
99
3 3
9 9 3
9 00
-2 5 3 5
3 1 1 1
5 5 3 1
11 0 0
3 1 1 0
-5 0 0 0
-9 -6 -3 0 3 6 9
Component 1
Vintage 2009
Vendimia 2009
Figura 60. Anlisis de componentes principales para vinos: a) Moscatel; b) Pedro Ximnez. 0, 1,
3, 5, 9 y 12 meses de envejecimiento.
254
Captulo IV. Resultados y Discusin
Para cada variedad de uva, los vinos de la vendimia 2009 mostraron valores
negativos para la CP2, por lo que esta componente parece estar relacionada
con el factor "vendimia". En ambos casos, los principales contribuyentes, con
valores positivos, a esta CP fueron algunos steres de etilo (butanoato de etilo,
hexanoato de etilo, octanoato de etilo, decanoato de etilo y dodecanaoto de
etilo) y cidos (cido octanoico, cido decanoico y cido dodecanoico). La
mayora de estos compuestos voltiles se forman durante la fermentacin
alcohlica y su contenido en el vino depende de la composicin en
aminocidos del mosto (Hernndez-Orte et al., 2002). En el estudio ANOVA
previo, el factor vendimia mostr una influencia significativa en la mayora
de estos compuestos. Como ya se apunt, la gran diferencia entre los valores
de precipitacin de ambas vendimias (MAGRAMA, 2015a) podra explicar el
alto valor de significancia de este factor en el contenido voltil de los vinos
estudiados.
255
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
256
0
5
10
15
20
25
30
35
40
45
50
55
257
Persistencia
Intensidad aromtica
Fresco
Afrutado
Uva
Manzana
Pera
Pltano
Meln
Frutas exticas/tropicales
Pia
Mango
Melocotn
Ciruela
Ctrico
Naranja
Pomelo
Higo
Macedonia
Pltano maduro
Mermelada
Frutas maduras
Orejones
Higo seco
Frutas secas
Frutos secos
Todas frutas maduras
Todas frutas secas
Todas frutas
Dulces
Dulce, melaza
Pasa, uva pasa
Ciruela pasa
Floral
Jazmn
Acacia
Miel
Rosa, pomarosa
Flores silvestres
Flores blancas
Todas flores
Vegetal
Herbceo
Paja cortada y hmeda
Raspn, escobajo
Madera verde
Madera, roble
menta
hierbabuena
Todo vegetal
Qumico
Pegamento, acetato etilo
Disolvente, barniz
Medicinal, aldehdico
Resina
Plstico
Todos qumicos
Tostados
Regaliz
caf
caramelo tostado
chocolate
Todos tostados
Especiado
Vainilla
258
Captulo IV. Resultados y Discusin
En cuanto a las notas positivas, solo las afrutadas y a pasas parecen diferenciar
con alguna significancia estadstica (p < 0,05) los mostos de uvas pasificadas
mediante los distintos sistemas (1T y C). Adicionalmente, los mostos
procedentes de cmara climtica presentaron una menor intensidad del defecto
aromtico, que se defini como olor a humedad. Segn se deduce de los valores
del descriptor impresin general, el mosto PX secado en cmara climtica se
prefiere claramente al de asoleo, sin que se perciba diferencia entre los
correspondientes mostos de Moscatel.
Moscatel PX
259
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Varianza explicada CP1 (40,1%) CP2 (22.6%) CP3 (16,1%) CP4 (7,9%)
260
Captulo IV. Resultados y Discusin
Varianza explicada CP1 (40,1%) CP2 (22.6%) CP3 (16,1%) CP4 (7,9%)
Tabla 42. Pesos de los distintos descriptores sensoriales sobre los componentes principales
(rotacin varimax).
Cuando se proyectaron las muestras sobre los planos de las CPs del nuevo
espacio (Figura 62), se observ que, respecto a la CP1, las muestras de cmara
climtica se situaban a la derecha de aquellas procedentes del asoleo tradicional
en el centro de investigacin (1T). Por su parte, la CP2 situ los mostos de
Moscatel en la mitad superior, atribuyndoles un carcter aromtico ms
complejo y apreciado (Figura 62.(a)). Resulta interesante que solo el mosto PX
de 2008 secado en cmara se ubicase en esta rea. Respecto a las CP3 y CP4
(Figura 62.(b)), permitieron agrupar y diferenciar de manera clara las tres
vendimias en funcin de descriptores aromticos con connotaciones extraas
o directamente defectuosas, de mayor intensidad hacia la derecha y hacia abajo,
respectivamente. De este modo, los mostos de 2010, independientemente de la
variedad o sistema de pasificado, fueron situados sobre el plano CP3-CP4 en
las posiciones ms alejadas de los descriptores negativos (lctico, humedad,
herbceo y qumico), lo cual podra confirmar el efecto principal que ejerce el
ao de vendimia sobre la calidad de los mostos. Adems, es interesante
destacar que los mostos Moscatel de la vendimia 2009 elaborados con uvas
secadas en cmara climticas se localizaron tambin en esta rea.
261
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
2,0
V10.M.C
a) Asoleo en CAIV: 1T
Cmara climtica: C
V10.M.1T
1,5 Asoleo en via: 2T
Uva fresca: Ini
Ctrico
Impresin General
1,0 Floral
Intensidad Aromtica
V09.M.C Miel
V09.M.Ini Afrutado
0,5 V08.PX.C
Fruta madura
V08.M.Ini
HerbceoV08.M.1T
V08.PX.Ini Dulce
PC2
0,0 Hum
icoedad
P
Qum Pasas
V08.M.C
V09.PX.C
V09.PX.Ini Lcteo
V09.PX.2T
-0,5 Vinoso V08.PX.1T
-1,5
-1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0
PC1
1,5
V10.PX.2T
b) 1,0 V10.PX.1T V10.PX.C
V10.M.C V08.PX.1T
V08.M.Ini
V08.M.C V08.PX.C
0,5 V10.M.1T Floral
Dulce Hmedad
V09.M.CImpresin general
Vinoso
Afrutado LcteoV08.M.1T
Ctrico Pasas
Intensidad Aromtica
0,0
Miel
Fruta Madura Herbceo
V08.PX.Ini
-0,5
PC4
V09.M.Ini Qumico
-1,0
V09.PX.Ini
-1,5
V09.PX.C
V09.PX.2T
-2,0 vendimia 2010: V10
vendimia 2009: V09
vendimia 2008: V08
-2,5
-2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0
PC3
Figura 62. ACP de los mostos de uvas pasificadas por ambos sistemas de secado, en cmara
climtica (C) y soleo tradicional (1T, en el centro de investigacin; 2T, en via). Proyecciones de
las muestras sobre (a) CP1-CP2 (b) CP3-CP4.
262
Captulo IV. Resultados y Discusin
Presentan
N de Intensidad
Vinos los vinos
Factor Niveles respuestas de la
comparados diferencia
correctas diferencia
significativa?
Levadura S.cerevisiae E3
11 S 1,7
(TA) S.bayanus E5
No E1
Adicin de N 9 S 2,5
S E2
Maceracin No E1
9 S 1,8
pelicular S E3
T / T.B./S. bayanus E4
7 No -
Levadura T.A./S. cerevisiae E1
Tabla 43. Resultados de las pruebas discriminativas triangulares realizadas sobre los vinos de
2008. Intensidad de la diferencia: 0=Ausencia; 1=Dbil; 2=Media; 3=Fuerte; 4=Muy fuerte.
T.B.: Temperatura baja. T.B.: Temperatura alta.
263
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
Para la comparacin global de los vinos, en una sesin distinta se pidi a los
jueces que los ordenaran de acuerdo a su preferencia. Siendo N=17, =0,05, y
5 el n de muestras, el valor del estadstico F del test de Friedman (anlisis de
la varianza para ordenaciones) a superar es 9,5. Calculado el valor experimental
(9,8), se confirma la existencia de diferencia significativa entre dos o ms de los
vinos. Para determinar cules son significativamente distintos se calcul la
Mnima Diferencia Significativa (MSD) para el riesgo asumido (=0,05). La
secuencia E41 > E11 > E31,2 > E52 > E22 describe el orden de preferencia
decreciente del panel, donde distintos superndices indican vinos con
diferencia significativa en cuanto a la impresin general. De acuerdo a estos
resultados, parece apuntarse la preferencia por los vinos jvenes fermentados a
baja temperatura con levadura especializada (S. bayanus) (E4), si bien la
diferencia con los obtenidos con S. cerevisiae a TA (E1) no resulto significativa.
Los vinos peor calificados fueron aquellos que utilizaron adicin de nitrgeno
(E2), seguidos de los que practicaron maceracin pelicular en presencia de
enzimas pectolticas (E3 y E5).
264
Captulo IV. Resultados y Discusin
Descriptores E1 E2 E3 E4 E5 p
Intensidad
4,10,5 4,20,6 4,40,6 4,10,5 4,20,5 0,97
aromtica
Afrutado 4,20,3 2,70,4 3,80,3 4,10,4 3,80,5 0,11
Ctrico 2,20,72 1,40,51 2,80,32,3 3,10,43 2,70,32,3 0,04
Fruta madura 1,90,4 1,50,6 2,10,4 1,70,5 1,40,4 0,87
Dulce 2,30,6 2,00,6 3,50,6 2,30,7 1,40,5 0,37
Pasas 1,90,5 1,60,5 2,20,4 2,30,4 1,10,3 0,84
Floral 3,60,81,2 3,00,71 3,90,51,2 4,10,62 4,00,82 0,07
Miel 0,90,2 0,80,4 1,50,5 1,20,3 1,10,5 0,54
Herbceo 1,10,4 2,10,6 1,30,6 1,40,5 1,90,7 0,83
Vinoso 2,50,5 2,30,7 2,50,5 2,90,6 2,80,6 0,87
Lcteo 0,20,3 0,50,3 0,50,3 0,50,3 0,50,4 0,99
Humedad 0,60,3 1,10,4 1,10,6 0,90,4 1,50,5 0,86
Qumico 0,80,3 2,10,3 0,60,3 1,00,3 0,80,2 0,43
Impresin
4,40,43 2,50,61 3,10,51,2 4,50,53 3,80,52,3 0,05
general
Tabla 44. ANOVA aplicado a los valores de intensidad (media desviacin estndar) de los
atributos usados para describir el perfil aromtico de los vinos de 2008. p < 0,05 indica diferencia
significativa. Para un descriptor, los vinos con distintos superndices se perciben
significativamente distintos (Test de Tukey).
265
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
los distintos vinos elaborados en cada vendimia bajo las mismas condiciones
de fermentacin, y con cada variedad de uva secada por asoleo (1T y 2T) y en
cmara climtica, se estudiaron mediante ANOVA, con el sistema de secado
como factor de variacin. Previamente, se eliminaron del tratamiento de datos
los descriptores lcteo, madera nueva y herbceo, por presentar muy bajas
puntuaciones (< 1).
Tabla 45. Pesos de los descriptores preseleccionados sobre los cuatro CPs. Entre parntesis, la
varianza explicada por cada CP. Rotacin Varimax.
266
Captulo IV. Resultados y Discusin
relacin de este eje CP3 con la intensidad del olor a roble caracterstico del
contacto con la madera. En cuanto a la interpretacin de la posicin de los
vinos en relacin al eje horizontal CP1, las muestras de cmara climtica de
cada vendimia y variedad, se desplazaron hacia la derecha con el tiempo de
envejecimiento (de C.S0 a C.S12), es decir, aumentaron en intesidad las notas
aromticas que estn relacionadas con el proceso de secado (pasas, fruta
madura, dulce). Sin embargo, los vinos elaborados con uvas asoleadas por el
sistema tradicional, que presentaban valores elevados antes del proceso de
envejecimiento (muestras T.S0) mostraron un comportamiento irregular, con
un ligero aumento o incluso un claro descenso tras 12 meses de
envejecimiento (T.S12).
267
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
3
V08.M
V09.M
V09.PX
2 V10.M
C.S12
V10.PX
C.S12
T.S12
1 RobleT.S12
C.S12
Pasas
Fruta Madura
C.S12 Dulce
C.S0
0 Ctrico T.S12
PC3
P
C.S12 T.S0
C.S0
T.S0
-1 C.S0
C.S0
C.S0
-2 T.S0
PC1
Figura 63. Proyeccin sobre CP1-CP3 de los vinos con (S12) y sin (S0) envejecimiento de las
distintas vendimias y variedades obtenidos con uva secada en cmara climtica (C) y de manera
tradicional (1T y 2T).
Los datos del anlisis sensorial descriptivo de los vinos de 2008 que se
sometieron a envejecimiento en bota de roble, por contacto con chips de roble
y sin presencia de madera, se trataron mediante Anlisis Lineal Discriminante
para seleccionar los descriptores que mejor diferenciasen los sistemas de
envejecimiento. El mtodo usado fue el secuencial hacia adelante, con unos
estadsticos F de entrada y salida de variables en el modelo de 1 y 0
respectivamente. Las variables incluidas en el modelo fueron, de mayor a
menor poder de discriminacin: roble, impresin general, vinoso, pasas, ctrico
(hasta aqu p < 0,05), defecto, miel, afrutado, dulce, frutas maduras. El modelo
permite un 86,9% de clasificacin correcta de las muestras, lo cual confirma
268
Captulo IV. Resultados y Discusin
Tabla 46. Anlisis de componentes principales. Pesos de los CP tras rotacin varimax
normalizada. Entre parntesis, la varianza explicada por cada CP.
269
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
2 Ch Ch
Ch
Ch Ch Ch B
Ch
1 Ch Ch B
S B
B
ChS Ch B PasasB B B
B S Roble B
S S
Ch
0 S Ch B B
SImpresin B
B General
Vinoso
S B
Ch
Ctrico
PC2
-1 S SS S
P
S S
S Ch
-2
S
-3
-4
-3 -2 -1 0 1 2 3
PC1
Figura 64. Proyeccin sobre el plano CP1-CP2 de los vinos Moscatel de 2008 respecto al tipo de
envejecimiento (B, botas de roble; S; depsitos de acero inoxidable, Ch; en contacto con chips).
270
Captulo IV. Resultados y Discusin
Tabla 47. Anlisis de componentes principales. Pesos de los CP tras rotacin varimax
normalizada. Entre parntesis, la varianza explicada por cada CP.
271
IV.II. Parmetros de inters en la calidad organolptica de vinos dulces
4
S01
3 S01 S01
2 S03 S01
S01 S03 S01 S07
S03
S01 S03 S03 S07 S01 S07S07
1 S07S03 S07 S12
Impresin S07 General
MielFruta Madura S12
S01 Roble
S03 Afrutado
Defecto
S07 S12
0 IntensidadCtrico
Aromtica Dulce S12
S03 Floral Pasas S28
PC2
-1 S12
S12 S03
S0
S07
S0
-2
S0 S0 S12
S0
S0 S0
-3
S0 S0
-4
-4 -3 -2 -1 0 1 2 3 4 5
PC1
Figura 65. Proyeccin sobre el plano CP1-CP2 de los vinos de las v. 2008, 2009 y 2010 en
funcin del tiempo de envejecimiento (S0, S01, S03, S07, S12, S18, S28: 0, 1, 3, 7, 12, 18 y 28
meses de envejecimiento en botas de roble, respectivamente).
272
Captulo V.
Conclusiones.
Captulo V. Conclusiones
CAPTULO V. CONCLUSIONES
275
Captulo V. Conclusiones
276
Captulo V. Conclusiones
- Por ltimo, el anlisis sensorial de los mostos y vinos elaborados, reflej una
influencia de la variedad de uva y el ao de vendimia. El secado en cmara
mostr un ligero aumento de las notas afrutado y pasas y menor olor a
humedad. Los vinos elaborados con S. Bayanus a baja temperatura
mostraron mayores notas ctricas y florales en vinos jvenes. El
contacto con madera en las botas mejor la impresin general de los vinos,
mostrando al inicio una intensa maderizacin que fue integrada tras unos
meses de envejecimiento.
277
Bibliografa
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English Summary.
English summary
ENGLISH SUMMARY
III.I. Samples
III.II.2. Ochratoxin A
IV. Conclusions
V. References
305
English summary
In the sunning process, which is one of the most characteristic stages of the
Andalusian sweet wines production, the grapes are dehydrated and
concentrated mainly in sugars by direct exposition to the sun.
307
Justification and Objectives
I.I. OBJECTIVES
The main objective of this thesis is to obtain natural sweet wines from grapes
dried under controlled-conditions with both, composition and organoleptic
properties, similar to those of traditional sweet wines. For that purpose, the
drying will be carried out in a climatic chamber with temperature and humidity
control and a capacity to allow the treatment of enough amounts of grapes for
the subsequent vinification up to 100 liters.
308
English summary
Since, as cited above, the raisining of grapes is one of the most important
stages in the sweet wines production, it becomes necessary to monitor the
parameters related with fungi or microbial infections in the musts obtained
from climate chamber dried grapes and compare them with those from the
traditional sunning.
309
Justification and Objectives
To evaluate the influence of the grapes drying process on the final product and
the evolution of the wines throughout the entire process of aging comparing
the wines obtained from both analytical and sensorial point of view.
310
English summary
In Andalusia, special sweet wines are obtained using dried grapes by direct
exposition to the sun in the process known as asoleo or sunning. They are
elaborated, mainly, in Malaga, Montilla-Moriles, and Jerez in a traditional
method dating many years ago using two grapes varieties, Muscat and Pedro
Ximnez.
After harvesting, Muscat or Pedro Ximnez grapes, in bunches, are spread out
on esparto grass mats to toast in the sun before pressing. The bunches are
turned over on a regular basis and covered at night to avoid the adverse effects
of high air moisture.
When subjected to hours of intense sunshine, the grapes gradually lose water,
resulting in a significant increase in the concentration of sugars, a variation in
the aromatic profile (Franco et al., 2004) and a certain degree of colouring,
among other changes. In this way, concerning phenolic compounds, two
different effects should be described during the raisining: on the one hand, the
increase in their content due to the concentration factor; on the other hand,
oxidative enzymes such as polyphenol oxidases (PPO) catalyse the oxidation
of phenolic compounds in the presence of molecular oxygen, leading to
quinones which subsequently evolves to brown pigments (melanins).
Moreover, the Maillard reactions between free carbonyl and amino groups,
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Backgrounds: Andalusian sweet wines. Winemaking and associated risk
Similarly rains during the sunning process make the grapes more disposed to
infection by bacteria, which cause acetic disease, and facilitates the
development of other molds such as Penicillium and Aspergillus species, which
give rise to the formation of ochratoxin A (OTA) (Suarez and Inigo, 2004), a
highly toxic metabolite.
The growth of these fungi in the grape is subject to the climatic conditions of
the region (Belli et al., 2005; Battilani et al., 2006; Bell et al., 2007). A.
carbonarius, which is highly resistant to solar radiation and high temperatures,
up to a maximum in excess of 40 C, is the major source of OTA in grapes,
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English summary
wines and other enological products (Cabaes et al., 2002; Battilani et al., 2004;
Chiodini et al., 2006; Brera et al., 2008). Furthermore the existence of high
levels of ochratoxin A has been demonstrated in wines originating from grapes
dried in the sun (Valero et al., 2008).
About OTA pathologies and toxic effects (Pavlovi et al., 1979; Fuchs and
Peraica, 2005; Pfohl-Leszkowicz and Manderville, 2007), OTA causes chronic
nephropathies in diverse species of mammals and birds. In the human, it has
been associated with a nephropathy endemic to the Balkans region, although
this is still an unconfirmed hypothesis. OTA also possesses teratogenic,
hepatotoxic, neurotoxic and immunotoxic effects, and is classified by the
International Agency for Research on Cancer as a possible human carcinogen
(class 2B) (IARC, 1993). In the European Union the OTA content is regulated
in foods susceptible to contamination. In the case of wine, other drinks based
on wine and/or musts, the maximum limit is set at 2.0 g/L ((CE) No
1881/2006).
II.II. VINIFICATION
The grapes attain sugar levels above 300 g/L and are crushed and pressed
employing vertical presses. After this, musts with high sugar content are
submitted to partial alcoholic fermentation and fortified with ethanol to ensure
that the wine will contain at least 1518 % alcohol.
In the production of these wines, the fortification may favour the formation of
ethyl carbamate, a known carcinogen that has been found in wines as a by-
product of fermentation.
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Backgrounds: Andalusian sweet wines. Winemaking and associated risk
II.III. AGING
After vinification, the young sweet wine is, generally, submitted to aging in
American oak wood following an oxidative aging. The aging must be
prolonged for a minimum period of two years, being possible to carry out in a
static system or in a traditional dynamic process known as soleras and
criaderas by which wines from different stages of the aging are blended
together in order to conserve the specific characteristics of the wine (D.O.
"Jerez-Xrs-Sherry", 2015).
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English summary
During oxidative aging in wood, wines become gradually darker due to direct
contact with oxygen in the air and are affected by the phenomenon of
concentration produced as a consequence of the transpiration of specific
elements in the wine through the walls of the oak cask (D.O. "Jerez-Xrs-
Sherry", 2015). In addition, the wine acquires aromatic complexity as a result
of important and diverse reactions (Ortega-Heras et al., 2004). New volatile
and nonvolatile compounds are extracted from the wood, modifying the
wines bouquet. As a consequence of all of these processes, the fruity and
fermentative volatile compounds decrease during the process of aging in wood
(Escalona et al., 2002).
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Experimental and results
III.I. SAMPLES
Two grape varieties (Muscat, M and Pedro Ximnez, PX) and three vintages
(2008, 2009 and 2010) were studied. The grapes were subjected to two
different drying procedures: natural sunning (on the terrace of the research
centre, designated 1T, and/or in the vineyard, 2T), and controlled drying in a
climate chamber (C).
In the case of chamber drying, for each grape variety and vintage, about 2000
kg of ripe grape bunches were collected from a local winery in the Jerez-
Xeres-Sherry D.O. (Denomination of Origin) region and dried using a climate
chamber (Ibercex A. S. L., S. A., Spain) at 40 C and relative humidity of 10%.
For the sun drying process, grapes were dried for about 10-15 days, spread out
on esparto grass mats, turned over daily and covered at night. In both
systems, bunches were distributed uniformly forming a single layer and the
drying process was monitored periodically finalizing when the water loss was
about 35% of the original weight and the grapes were at least 20 B.
Dried grapes were de-stemmed, crushed and pressed with a vertical press. The
highest pressure in each of the three cycles was 30 MPa. For those
experiments with skin contact (pellicular maceration), grapes were crushed
without de-stemming and then submitted to maceration. In the next stage, the
pH of the must was adjusted to 3.5 with tartaric acid (Agrovin, Spain). The
concentration of total sulfur dioxide was also set at 120 mg/l by adding
potassium metabisulfite (Agrovin, Spain). Five experimental conditions (in
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English summary
duplicate) of vinification were tested during the vintage 2008 while wines from
2009 and 2010 were elaborated following the experiment E4 conditions:
Experiment E1 the must was fermented with a S. cerevisiae yeast (40 g/hl,
Lalvin D254, Lallemand, Australia) at room temperature (below 30 C).
Experiment E2 the must was fermented as in E1, but with the addition of
yeast nutrients (10 g/hl diammonium phosphate) (Actimax Plus+, Agrovin,
Spain).
When the sugar concentration of the fermenting must was about 90100 g/l,
the fermentation was stopped by adding alcohol up to 1718 % (v/v). Then,
the sweet wines obtained were aged in 30-L medium-toasted oak casks or in
10-L stainless steel vessels. During this aging period, wines were stored in the
same room at approximately 20 C. Sampling commenced at the end of
fermentation and continued periodically for a minimum of 12 months.
Samples were stored at 4 C until they were analysed.
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Experimental and results
Table 1 shows mean values of gluconic acid of the musts obtained during the
initial sampling and after the different drying process. As shown, gluconic acid
contents in musts from raisins were greater than 500 mg/l (limit considered
acceptable for wines from fresh grapes). This threshold was reached after the
first sampling in Muscat 2008. However, for Pedro Ximnez and Muscat
samples from 2009, it happened at the end of the drying step (Figure 1).
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English summary
4000
2000
1000
0
S0 S1 S2 S3 S4 S5
Drying Time
Muscat 2008
Moscatel2008 Moscatel2009
Muscat 2009 PX 2009
PedroXimen2009
Figure 1. Evolution of gluconic acid content during the drying of grapes in climatic chamber (C).
S0: Initial, S1: 2 days, S2: 3 days, S3: 6 days, S4: 7 days, S5: 8 days.
Fresh grape musts showed the lowest values of gluconic acid, while those
obtained from dried grapes according to the traditional system (in terrace, T1,
or vineyard, T2) achieved the highest contents. Moreover, the comparison
between the three vintages showed that the year significantly affected the
presence of gluconic acid in must from both, fresh and raisining grapes,
whether that were dried under controlled conditions or by traditional system.
But it should be noted that, in 2009 and 2010, gluconic acid content in musts
of traditionally dried grapes (T1 and T2) were clearly higher than those
obtained from grapes dried in climatic chamber (C), while from 2008, no
differences were found between them.
Considering the climatic conditions of the three years during the months of
August and September, when the raisining stage takes place, exceptionally high
rainfall values were observed for September in 2008, which could affect the
state of the initial grapes and therefore their evolution during raisining in both
systems: climatic chamber and traditional process.
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Experimental and results
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English summary
III.II.2.1. Musts
The OTA contents in the musts from the two different systems of raisining
tested suggested that a previously existing fungal contamination in the grape
could occur, and this contamination, associated with the conditions of the
drying process itself, led to the growth of the fungus and, consequently, of
ochratoxin A in both systems. This finding is related to the data provided by
Valero et al., (2008) which demonstrated that it is after the harvesting, and
particularly in the process of raisining, when the Aspergillus species becomes
predominant over other fungi present on the grape.
However, the OTA concentration of the must from grapes dried in the
climatic chamber (6.920.01 g/kg) was lower than that obtained in the
natural process (28.80.02 g/kg), the difference being statistically significant
(p < 0.05), so it seems the increased control over the conditions of raisining
resulted in better hygienic conditions for the final must. The must obtained in
the traditional system presented a very high concentration of OTA which was
not suitable for subsequent fermentation.
III.II.2.2. Vinification
During the vinification of the musts from grapes dried in the climatic chamber
a considerable decrease of the OTA content was observed (averages and
standard deviations shown in Table 2). A large part of the solid matter that
was in suspension, and with it part of the toxin present in the grape marc, was
eliminated in the desludging and clarification (Kurtbay et al., 2008), the
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Experimental and results
difference being statistically significant between the two wines before (V01)
and after (V02) these enological practices (p < 0.05). Similar results have been
obtained during the vinification of musts of red grapes (Grazioli et al., 2006).
Similarly, in those wines with prefermentative maceration (experiences 3 and
5), the must was in contact with the solid matter (skins, etc.) for 24 h, and this
facilitated the transfer of the toxin to the must.
III.II.2.3. Aging
During the aging in oak casks (C) a decrease was observed in the OTA content
(Table 2); in all the cases, lower concentrations than in the corresponding
controls (S) were found, the difference being statistically significant (p < 0.05).
The OTA content in these controls hardly varied at all during the assays, while
those of the wines aged in oak casks showed a large decrease at the beginning,
with a slight progressive decrease throughout the aging time in the case of the
wines with the higher contents (experiences 3 and 5). The aging of red wines
in bottle was evaluated by Grazioli et al., (2006), and no short term effect was
found, but there was a decrease of 17% in OTA after 12 months. With regard
to our samples, in four of the seven barrels studied this decrease was greater
than 17% in the first three months, even reducing up to 56.49.3%, as an
average of all the oak cask assays, after 12 months of aging.
In addition, it was observed that wines of the same assay aged in different oak
casks showed a very similar trend over time. In the experiment conducted with
wood shavings (or chips, E1_Ch), the OTA content of the wine remained
constant, showing behavior more comparable to that of the control than the
wine aged in cask.
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English summary
Table 2. Ochratoxin A in wine after vinification (V01 and V02) and aging (S20d and S12).
Therefore, all wines aged in oak for a period of 12 months showed an OTA
content below 2.0 g/l, legal limit established by the European Union, so
these wines would be suitable for consumption.
The acid content of a wine influences on many parameters such as the color
stability, aroma, taste properties and hygienic status (Garcia-Romero et al.,
1993; Lpez-Tamames et al., 1996). In addition, the individualized study of the
different organic acids in musts and wines is indicative of possible
microbiological infections which are, in many occasions, undesirable.
In relation to this, the organic acid content for must and wines from the
different experiments carried out during the three vintages (2008, 2009 and
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Experimental and results
III.II.3.1. Musts
The evolution of organic acids in the grapes during raisining can be described
by two antagonistic effects. On one hand, their concentration increase due to
the evaporation of water in the berries. Moreover, exposed grapes continue
with the respiratory process which takes place during ripening and produce the
combustion of the different acids (Hidalgo-Togores, 2003).
Table 3 shows the organic acids content of the musts at the beginning and
after the different raisining experiences. Overall, the three factors under study;
variety, vintage and type of drying, significantly affected the content of
different organic acids in grapes (p < 0.05), prevailng the concentration effect
during the drying process.
Malic acid levels were higher in dehydrated grapes by sunning than in those
dried in a climatic chamber. However, the differences between both systems
were lower for Muscat variety. It is known that malic acid has a large
susceptibility to high temperatures (Hidalgo-Togores, 2003), so the effects of
combustion may explain the greater variability in the results.
Concerning to citric acid, which only could be determined in vintage 2008 due
to a technical problem; its concentrations were generally within the normal
range. The increased concentrations after the raisining of Pedro Ximnez
grapes were higher than it would be justified by the decrease in the volume of
water, which could be related to a possible growth of Botrytis cinerea. This result
agreed with the contents of gluconic acid described previously for these musts.
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English summary
The presence of succinic, lactic, formic and acetic acids denoted the start of
fermentation activity, which was not surprising particularly in dried grapes, due
to the high sugar content of the musts.
Succinic acid was found in greater concentration in musts from sunning grapes
than in those from grapes dried in a climatic chamber, probably due to the
high availability of oxygen in the open air.
The drying of grapes significantly increased the levels of acetic acid. Although
the factor vintage contributed significantly to the content of acetic acid, like
variety, the existence of statistical interaction between these factors and the
drying system, impeded to establish patterns of behavior. Moreover, the
concentration effect during the drying of the grapes can not justify in most
assays the significant increase in its content, which could be interpreted as a
consequence of some bacterial activity (lactic and acetic bacteria).
Table 4 shows the results obtained for Muscat wines from the vintage 2008.
The contents in tartaric, malic, succinic and lactic acids in wines made under
different fermentation conditions were within normal ranges for healthy wines.
Although, the influence of some of the operational variables studied on the
organic acids contents during the winemaking process was observed (Table 4).
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Experimental and results
higher content in succinic and lower in acetic acid for wines fermented by S.
bayanus. These potential differences may result from the special conditions of
our musts, from dried grapes and consequently with a high concentration of
sugars.
The fermentation temperature affected, in the same way, the content in acids
derived from grapes (malic and citric acid). The remaining acids, which are
originated during the fermentation process, differed depending on the
fermentation temperature of the wines. The addition of ammonium salts as
yeast nutrients during fermentation did not affect the concentrations of acids,
while the maceration with pectolytic enzymes seemed to be related with a
higher level of succinic acid and a decrease in the acetic acid content.
Since the effects of variety and vintage were confirmed as significant in the
study of the musts, results corresponding to unaged wines from each vintage
and variety were independently treated (data not shown). In general, this
influence was kept from musts to wines, judging by the p < 0.05 which
characterized the vintage and variety effects.
In addition, the trends found previously in the must remained, although the
concentrations were not the same: while significant differences in the acid
content were confirmed depending on the drying system of grapes, there was
no clear pattern indicating the direction in which it happened. In view of the
means and calculated p-values, a possible effect of variety, and especially
vintage, might be interacting, so this content could not be ensured a significant
influence of the raisining system.
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English summary
The contents of organic acids changed significantly during the aging in oak
cask. The increases were important in the wines of 2009 and 2010, while the
evolution during 2008 showed a slightly differences depending on the acid and
the different winemaking conditions.
The comparison p-values between the aging systems at the beginning of the
stage (S20d/S01) and after 12 months (S12), showed that time had a
differentiating effect on the contents in organic acids. In this way, the initial
differences were enhanced, even, these appeared where none previously
existed (pS12 << pS20d/S01).
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Experimental and results
Table 3. Organic acids (mean, mg/l standard deviation) for musts..For each column and variety, different letters show significant differences between
drying systems (Ini, C, 1T y 2T) and, for each row, different numbers (1, 2, 3) show a significant difference due to vintage (V08, V09 y V10) at p <
0.01 (Tukeys test).
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Experimental and results
Table 4. Means (mg/l) and standard deviations for organic acids of young wines obtained under different vinification conditions. ANOVA. Different
letters show a significant difference at p < 0.05 (Tukeys test) for each experiment (E1-E5).
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Before and during aging, the wines obtained were periodically sampled, and
ethyl carbamate (EC) content was determined by HPLC with fluorescence
detection. Sampling commenced at the end of fermentation (S0d) and
continued periodically for 28 months (S20d, 20 days; S40d, 40 days; S2, 2
months; S4, 4 months; S7, 7 months; S8, 8 months; S12, 12 months; S18, 18
months and S28, 28 months).
The EC concentration of the wines made from the Muscat grapes (vintage
2008) ranged from not detected (E4, S0d) to 194 g/l (E3, S28) (Table 5). As
could be expected (Kodama et al., 1994), for all experiments, the EC
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Experimental and results
332
English summary
The results obtained from the study of comparison of means using the
Tukeys test (p < 0.01) for all significant factors are shown in Table 6. The EC
concentration of wines increased during aging, with the differences more
important after 4 months of aging. From Table 6, it is possible to conclude
that the differences were significant among wines prepared according to the
several factors.
When considering the grape variety, the wine made from Pedro Ximnez
presented a lower value than that for Muscat (Tables 6). The ratio of proline to
arginine plays a significant role in determining the predisposition of a variety
to produce urea (Bell and Henschke, 2005). In wines made from Chardonnay,
Muscat White and Muscat dAlexandrie, Soufleros et al., (2003) found a high
amino acid concentration, with arginine being the most abundant amino acid.
Data on the amino acid concentration of Pedro Ximnez are scarce: Mauricio
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Experimental and results
et al., (2001) found that Pedro Ximnez musts contained more L-proline than
L-arginine (38.2 and 25.2%, respectively, of the total free amino acids). These
values could explain the lower EC concentration found for this variety in our
study.
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English summary
Figure 2. (a) Dendrogram obtained taking the squared Euclidean distance as metric and Ward
method as amalgamation rule and (b) magnification of the area indicated.
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Experimental and results
As was stated in section III.I. Samples, all musts were obtained from grapes
dried using a climatic chamber (40 C and 10% RH) during vintage 2008 and
the corresponding wines were elaborated following different vinification
conditions. After fermentation, the sampling was carried out periodically for
28 months of aging (S0, 20 days; S1, 40 days; S3, 3 months; S5, 5 months; S9,
9 months; S12, 12 months; S18, 18 months and S28, 28 months).
In the case of the factor fermentation type, the results suggested that the
addition of nutrients, the maceration with pectolytic enzymes and the
employment of different yeasts (S. cerevisiae or S. bayanus) influenced on the
polyphenol content of the final wine.
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English summary
Regarding the type of aging (in oak cask, stainless steel or with chips) a general
trend, with higher levels for those wines aged in casks, was observed. The
accelerated aging with chips showed similar behavior to those wines maturated
in stainless steel tanks.
Finally, the evolution of the wines during the time of aging showed a
significant increase in the polyphenol content. Both hydroxybenzoic and
hydroxycinnamic acids as well as furanic aldehydes with the exception of
caftaric acid and "compound d" increased their concentrations.
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Experimental and results
Table 7. Means (mg/l) and standard deviations for phenolic and furanic compounds of wines obtained under different vinification conditions. Different
letters show a significant difference at p < 0.05 (Tukeys test) for each experiment (E1-E5).
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Experimental and results
Principal component analysis (PCA) was carried out in order to evaluate the
phenolic and furanic compounds which were involved in the differentiation of
the wines studied. As a result, five principal components that explained
87.05% of the total variance were extracted.
Attending to the first two principal components, that explained the 62.28% of
the variability, it was observed that polyphenols with greater weight in the PC1
were gallic acid, ethyl gallate, vanillin, syringaldehyde, caffeic acid, p-coumaric
acid, ethyl caffeate and 2-furaldehyde. Several of these compounds have been
previously related with the aging in wood (Garca Moreno and Barroso, 2002;
Schwarz et al., 2012).
Therefore, the samples corresponding to aging in wood had positive values for
PC1, while those wines subjected to aging in stainless steel or with chips,
presented negative values for this PC (Figure 3).
Figure 3. PCA for type of aging. 1: Oak Cask, 2: Stainless steel tanks, 3: with chips.
The representation of the samples on the plane defined by PC1 and PC2,
showed that the increase of the aging time displaced the samples to positive
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English summary
values of PC1 (data no shown) with those samples more time aged in wood
with higher values from this PC.
In this work, phenolic and furanic compounds of Muscat and Pedro Ximnez
sweet wines from vintages 2009 and 2010 (see III.I. Samples) were
evaluated. The period of aging in oak cask was also monitored at the beginning
and during 12 months (S0, 0 days; S1, 30 days; S7, seven months and S12,
twelve months).
Twelve phenolic compounds and three furanic aldehydes that had been
previously described in oenological products from Jerez-Sherry D.O. (Alonso
et al., 2004; Schwarz et al., 2009; Schwarz et al., 2012) and white wines (Boselli et
al., 2006) were identified. Four unknown polyphenols were also included in the
analysis.
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Experimental and results
content was higher in the Pedro Ximnez grapes. These data were consistent
with the results described by Mrquez et al., (2007).
Although the factor vintage was the least influential of all factors evaluated,
wines from vintage 2009 showed higher concentrations for several of the
compounds studied; ethyl gallate, protocatechuic acid, syringaldehyde,
cis/trans p-coutaric acid, tyrosol and unknown compounds a, b and c were
found in higher amount in wines from vintage 2009.
Lastly, the aging time increased the content of all phenolic and furanic
compounds evaluated, except hydroxycinnamic acid-tartaric acid esters,
caftaric and cis/trans p-coutaric, which showed no significant differences
during their temporary evolution.
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English summary
Table 8. Means (mg/l) and standard deviations for phenolic and furanic compounds according to grape variety and drying type. p-values for each
factor under study. Significant difference at * p < 0.05).
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Experimental and results
344
English summary
When the data matrix was subjected to a principal component analysis, three
major components (PCs) which explained the 83.46% of the total variance
were extracted. As shown in Figure 4, the first principal component, which
explains the 58% of the variance, was related with aging in wood. Those wines
with longer aging time showed higher values for PC1, in which the greatest
weights were for compounds such as gallic acid, 5-methylfuraldehyde, 2-
furaldehyde, p-coumaric and syringaldehyde, that are characteristic of aging
stage (Garca Moreno and Barroso, 2002; Schwarz et al., 2012).
Figure 4. PCA for wines from v.2009 and 2010. (Aging time: 0, 1, 3, 7 y 12 months ).
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Experimental and results
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English summary
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Experimental and results
A PCA was carried out in order to identify possible patterns. Seven significant
Principal Components (PCs) were obtained (eigenvalues>1). Figure 5 shows
the score plot of the first two PCs, which explained 49.76% of the total
variability of the phenolic compounds. As can be seen, PC2 divides the sweet
wines into two groups, sweet wines from cv. Muscat and sweet wines from cv.
Pedro Ximnez, with negative values for this PC for these last ones (Figure 5).
PC2 was related with p-hydroxybenzoic acid and 2-furoic acid (positive sign)
and quercetin-3-O-gluoside, quercetin-3-glucuronide, trans/cis-p-coutaric acid,
gentisic acid, dihydroxybenzoic acid and hydroxytyrosol (negative sign).
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English summary
Figure 6. LDA for aging time. Sweet wines from cv. Muscat.
For aging type, 97.78% of the samples were correctly classified in the check
process (data no shown). The discriminative variables included were: ethyl
protocatechuate, ethyl caffeate, ethyl gallate, trans/cis-p-coumaric acid,
trans/cis-caftaric acid, and dihydroquercetin-3-o-rhamnoside.
Antioxidant activity
In this study, two in vitro assays, ORAC and HORAC have been employed to
analyze the antioxidant activity of Andalusian sweet wines. Possible
correlations between both methods and phenolic content were also studied.
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Experimental and results
No correlation was found between ORAC and HORAC values for the wines
analyzed (r=0.416). This result was according with those obtained by Ou et al.,
(2002). HORAC and ORAC assays measure two different aspects of
antioxidant properties, radical chain breaking and radical prevention
respectively, so that, high HORAC values do not necessarily correspond with
high ORAC values.
As can be seen in Table 10, ORAC was related significantly with all global
indicators evaluated and correlation coefficients greater than 0.6 were
obtained, while HORAC not provided good correlation with any of them,
reaching its highest r-value with AED (0.43). These results agree with those
obtained by Feliciano et al., (2008), in which different tests to measure
antioxidant activity in Muscat wines were evaluated, being ORAC the AA
method that showed highest correlation values with total phenolic and
flavonoid content.
PCA was carried out and eight factors were extracted (Figure 7). The first two
ones represented the 48.5% of variability. PC1 related ORAC to gallic acid,
ethyl gallate, TPC, TFC, A280, A360, and AED while PC2 associated HORAC
with salicylic and 2-furoic acid (in a reverse way). As can be seen, these results
are in agreement with those obtained in the previous correlation study.
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English summary
PC2
PC1
Table 11. ANOVA for AA ORAC and HORAC. F values and p-values for each factor evaluated.
*values are significant at p < 0.05.
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Experimental and results
III.III.2.VOLATILE COMPOUNDS
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In order to study statistically the differences in the volatile content of all the
wines studied, data were submitted to the analysis of variance. For this one,
three independent factors were considered: type of fermentation, time of aging
and type of aging. As can be seen in table 12, practically all the volatile
compounds were significantly affected (p < 0.01) by these three factors.
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Experimental and results
354
English summary
maceration) aged in wood were grouped together with those ones fermented
with the same yeast strain, it could be concluded that, for these wines, the use
of nutrients or pellicular maceration conditioned their later evolution in oak
cask.
Figure 8. Cluster analysis (CA). Dendrogram obtained using squared Euclidean distance and the
Ward method as amalgamation rule.
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Experimental and results
7
Oak cask
5
Chips
3 Stainlees steel
Function 2
-1
-3
-5
-5 -3 -1 1 3 5 7
Function 1
With regard to the criterion time of aging, mainly terpenes, ethyl esters and
acetates were the variables included in the two first discriminant functions
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English summary
The objective of this work was to evaluate the volatile profiles of sweet Sherry
wines obtained from chamber and sun dried grapes after their aging in wood
in order to establish the use of climatic chambers as alternative to the
traditional sun drying. Both ways of drying together with other factors such as
grape variety, vintage and aging time have been considered in this study. Two
grape varieties (Muscat and Pedro Ximnez) and two vintages (2009 and 2010)
were studied. For each grape variety and vintage, musts from grapes dried by
sun and chamber, were separately fermented in duplicate at low temperature
(about 10C) with S. Bayanus (40 g/hl, Uvaferm 43, Lallemand, Australia).
After vinification, the sampling was carried out periodically for one year of
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Experimental and results
aging (S0, 0 days; S1, 30 days; S3, three months; S5, 5 months; S9, nine
months; and S12, twelve months).
For both varieties, volatile compounds found in this study had been previously
identified (Franco et al., 2004; Mrquez et al., 2007; Lpez De Lerma et al.,
2012; Ruiz et al., 2014). Table 13 shows mean values found for the different
chemical families evaluated according to each factor (aging time, grape variety,
vintage and drying type) considering the different values of the remaining
factors. Most of these factors seem to be significant for some of the chemical
families. As could be expected, wines from Muscat grape variety exhibited a
higher content in terpenic compounds (Snchez-Palomo et al. 2005) whereas
aged wines showed higher contents in ethyl esters and aldehydes, ketones and
alcohols (Table 13).
In order to study statistically the differences in the volatile content of all the
wines studied, data were submitted to analysis of variance (ANOVA). For this
one, four independent factors were considered: aging time, grape variety,
vintage, and type of drying. As a result, practically all the volatile compounds
were significant affected (p < 0.01) by one or more factors (data no shown).
Taking into account the results obtained from ANOVA study, in which most
of the volatile compounds were significantly affected by some of the factors
studied, a multivariate study was carried out.
358
English summary
Table 13. Mean values (relative chromatographic area) and standard deviations for each chemical family of volatile compounds.
Ethyl esters 0.4090.946 0.5551.258 0.5781.091 0.4731.018 0.3710.904 0.6281.140 0.4470.961 0.5821.137
Terpenes and
terpenols 0.0170.034 0.0180.059 0.0460.077 0.0030.007 0.0130.048 0.0180.044 0.0200.056 0.0090.026
Aldehydes,
ketones and 0.2360.376 0.3300.485 0.3040.453 0.2890.412 0.2780.403 0.3070.443 0.3130.451 0.2650.382
alcohols
359
Experimental and results
The data matrix was subjected to a cluster analysis of cases. Two main clusters
could be appreciated: one cluster for wines from Muscat grapes and another
one for wines from PX grapes. Later, a clear influence of the factor vintage
could be observed inside of each one (data no shown).
Therefore, considering all volatile compounds studied, it seems that these two
factors, variety and vintage, were more influential in the volatile profile of
wines studied than aging time and drying type. This same differentiation was
observed in a previous study carried out about the characterization of the
volatile fraction of Andalusian sweet wines (Mrquez et al., 2007).
To corroborate the results obtained from cluster analysis (CA) and check
which volatile compounds can allow the differentiation of wines studied, a
principal component analysis (PCA) was performed. 10 PCs (principal
components) which explained the 87.3% of the total variance were extracted.
In the score plot of all wines onto the plane defined by the two first PCs,
wines were differentiated according to grape variety (data no shown) so that, a
new PCA was performed, but in this case, both grape varieties were separately
studied.
For Muscat wines, nine PCs were obtained, which explained the 92.8% of the
total variance, with 53.8% of this one explained by the two first PCs. In the
case of PX wines, the 87.4% of the total variance was explained by ten PCs.
Figure 10 shows the distribution of both varietal wines onto the plane defined
by the first PCs. As can be seen, for each grape variety, wines were separated
according to their aging time.
360
English summary
For both grape varieties, those wines from vintage 2009 showed negative
values for PC2, so this PC seems to be related to the factor vintage. In the
previous ANOVA study, the factor vintage had showed a significant influence
on most of these compounds. The high difference between the precipitation
values of both vintages (MAGRAMA, 2015) could explain the high
significance found for this factor in the volatile content of wines studied.
Taking into account the global volatile profile, the factor drying type was the
least influential on the volatile profiles of sweet Sherry wines studied, with
aging time, grape variety, and vintage showing to be significant on them.
361
Experimental and results
9
a
1
6
1 5 12
12
0 9
3 3
Component 2
0 1 3 3
5 9
1 3
9 12
0
55 9
12
-3 0 1
0 1 3
3 12 12
33 9
5 9 9 12 12
5 9
5
-6
-7 -4 -1 2 5 8
Component 1
Vintage 2009
10
b
12
7 12 9
0
9 0 0
1
12 3
4 12 3
12
Component 2
1 0
12
12 55
5 99 9
12 12 9
12
5 3
1 1
5 12 55 3
129 3
99
3 3
9 9 3
9 00
-2 5 3 5
3 1 1 1
5 5 3 1
11 0 0
3 1 1 0
-5 0 0 0
-9 -6 -3 0 3 6 9
Component 1
Vintage 2009
Figure 10. Principal component analysis (PCA) for: a; Muscat wines; b: Pedro Ximnez wines. 0,
1, 3, 5, 9 and 12: months of aging in wood.
362
English summary
Musts and wines from the three vintages (V08, V09 and V10) were studied.
Must samples were taken at the start and the end of both drying processes; by
sunning (1T and 2T) and controlled drying in a climate chamber (C). In
addition, after vinification, aged wines were periodically monitored for one
year, for wines from vintages 2009 and 2010, and for 28 months in the case of
the v. 2008.
Sensory evaluation of musts and wines, exclusively orthonasal, was carried out
in duplicate by a panel of 14 to 17 assessors, all with a moderate degree of
experience in sensory analysis of sweet wines. Both triangular and ranking
sensory tests, and descriptive profiling, were performed. In addition, to
validate the reproducibility of the judgesopinion, a duplicated discrimination
test was performed and the descriptive profiles of 2 reference samples were
obtained.
Musts obtained by drying grapes in the traditional way were compared to those
dried in a climate chamber, by applying a triangular test (N=14 judges,
=0.05). The result showed that there was a perceptible difference between
musts, and according to the notes of the judges, the intensity of the detected
difference was low-to-medium.
In order to identify the aromatic notes responsible for the difference detected,
an analysis of the variance was applied to the scores obtained in a descriptive
test. The results obtained are shown in Table 14.
363
Experimental and results
Among the positive notes, only fruity and raisin were capable of discriminating
between musts from raisins obtained by the two drying systems (sunning, 1T,
and climatic chamber, C) at a level of statistical significance (p < 0.05). It is
important to note that musts from the climate chamber showed a lower
intensity of the musty off-odour. According to the overall impression, the must
from PX grape dried in the climate chamber was more appreciated than that
from the sunning. However, that difference was not perceived when must
from Muscat grape raisins was evaluated.
364
English summary
process, as it was highly correlated with ripe fruits, sweet and raisin attributes, and
PC2 (22.6% of variance explained) with a higher aromatic complexity
characterized by greater intensity and clear citric and floral notes, all of which
contribute to a better overall impression. On the other hand, both PC3 (16.1% of
variance explained) and PC4 (7.9% of variance explained) were related to
negative aromatic notes (dairy, musty, chemical character).
When all the musts were placed in the new space, it is shown that on PC1
(Figure 11.(a)) musts from grapes raisined in the climate chamber were located
to the right of those from traditional sun drying on the terrace of the research
centre (1T). According to PC2 the Muscat musts (in bold) were located at the
higher values, which means they had a more complex and appreciated
aromatic profile. The sensory profile of the musts was affected by the variety,
as it could be expected. It is interesting that the only PX wine which was
located in this higher value area was the one made with raisins dried in the
climatic chamber in 2008.
Regarding PC3 and PC4, these components could discriminate musts from
different vintages (Figure 11.(b)). Those 2010 musts, independent of variety
and raisining system, were located in the furthest positions to the negative
descriptors (dairy, musty, herbaceous and chemical character). So that, the
major effect that vintage had on the quality of musts was confirmed. It is very
interesting for us that the Muscat must from vintage 2009 elaborated from
climatic chamber dried grapes was also located onto this area.
365
Experimental and results
2,0
V10.M.C
Terrace: 1T
Climate Chamber: C
a) 1,5 V10.M.1T
Vineyard: 2T
Fresh grape: Ini
Citric
Overall impression
1,0 Floral
Aromatic intensity
V09.M.C Honey
Herbaceous
V08.PX.Ini V08.M.1T Sweet
0,0 Musty
P
-1,5
-1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0
PC1
1,5
V10.PX.2T
-0,5
V09.M.IniChemical character
P
-1,0
V09.PX.Ini
-1,5
V09.PX.C
V09.PX.2T
-2,0 2010 vintage: V10
2009 vintage: V09
2008 vintage: V08
-2,5
-2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0
PC3
Figure 11. PCA of the sensory scores of musts from raisins obtained by both drying systems
under study. Projection of the musts onto the new space defined by: a) PC1 and PC2; b) PC3 and
PC4.
366
English summary
E5). The difference was also perceived both when nitrogen was added (E2 vs
E1) and when pellicular maceration with added pectolytic enzymes was used
(E3 vs E1). The intensities of the differences detected were medium-to-strong.
In order to determine which wine was different from another, the least
significant difference was calculated, with =0.05. The ranking of the panel,
from major to minor preference, was E41 > E11 > E31,2 > E52 > E22 .
Different superscript numbers indicate wines that tasters perceived as quite
different from an overall point of view.
Wines were also analyzed by the descriptive test. The results obtained after the
variance analysis of the data (data no shown) suggested that floral and citric
notes were the most sensitive for discriminating among most of these wines.
The preferred wine from the ranking test, that fermented at low temperature
with S. bayanus (E4), scored the highest in citric and floral notes, while that
fermented with added nitrogen (E2) (the lowest rated in the ranking test)
presented very low citric and floral notes, and a clear chemical off-odour. The fruity
and floral scores of wines made under pellicular maceration (E3 and E5) were
both quite high, although they were not the preferred wines.
As has been seen, the sensory profile of the musts was affected by both
vintage and variety. In the case of the wines, they were also affected by the
fermentation stage, so data of all wines fermented under the same conditions
from each vintage and variety were studied separately, in order to identify
those descriptors with the highest discriminating power for drying system.
367
Experimental and results
After removing those descriptors with very low scores, (< 1, that was dairy,
young wood, and herbaceous), the analysis of the variance for the factor drying
system allowed us to select the citric, ripe fruit, sweet, raisin and oak aromatic
notes as discriminators. A PCA was applied to the scores of these descriptors,
and 3 PCs explaining 88.4% of the original variance were extracted. PC1
(55.4% of variance explained) was correlated well with ripe fruit, sweet and raisins
notes, PC2 (20.9%) with citric, while PC3 (12.3% variance explained) was
highly correlated with oak. Figure 12 shows the projection of the wine without
aging (S0) and aged for one year (S12) onto the new plane PC1-PC3. The aged
wines were grouped in the upper part of the graph (S12 wines), and this was
foreseeable since PC3 was highly correlated with the oak note. With respect to
PC1, the samples from climatic chamber for each vintage and variety increased
their scores with the aging time (C.S0 to C.S12), so it means higher intensities
in aromatic notes which were closely related to the drying process (raisin, ripe
fruit and sweet). However, the wines made with sun-dryed grapes, which had a
higher score before the aging process (T.S0 samples), showed an irregular
behaviour, with low increase or even a clear decrease after 12 months of aging
(T.S12 codes).
368
English summary
3
V08.M
V09.M
V09.PX
2 V10.M
C.S12
V10.PX
C.S12
T.S12
1 Oak T.S12
C.S12
Raisin
Ripe fruit
PC3
C.S12 Sweet
C.S0
0 Citric T.S12
P
C.S12 T.S0
C.S0
T.S0
-1 C.S0
C.S0
C.S0
-2 T.S0
PC1
Figure 12. . PCA of the sensory scores for drying system. Projection of the wines with (S12) and
without (S0) aging, onto the new plane defined by PC1 and PC3.
III.III.3.4. Effect of the type and time of aging on the sensory profile of
sweet wines
Sensory data of those 2008 wines aged by contact with oak chips (Ch), in oak
barrel (B) and in stainless steel container without wood (S), were studied by
Linear Discriminant Analysis (LDA) (data no shown). The model obtained
provides a high capacity of discrimination (correct classification for 86.9%).
Those variables with p < 0.05 were graphically represented, by means of their
previously calculated 2 principal components. In the new space, PC1 (39.5%
of variance explained) was well-correlated with both raisin and oak aromatic
notes, and also with the overall impression, while PC2 (22.8% of variance
explained) had a high and negative loading for citric odour.
The wines aged in stainless steel containers (S), had lower scores in PC2; so
they presented higher intensities in the citric aromatic note, a typical
characteristic of the Muscat variety which is preserved in the wines assessed.
369
Experimental and results
Wines aged in oak barrels (B) appeared on the right-hand side on the plane
PC1-PC2 (data no shown); this means that the oak note was perceived more
strongly, as was foreseeable, but the raisin note was also important, and both of
them contribute to a better overall impression. With respect to wines aged in the
presence of oak chips, their locations on the plane gave them, in general, lower
intensities in the descriptors correlated to PC1 and PC2, all of which were
well-appreciated aromatic notes, so this type of aging does not appear to be
the best.
As the best aging system seems to be aging in oak barrels (B), LDA was
applied for all wines aged in this way. The model constructed, which correctly
classifies 75.1% of all the samples, included most of the descriptors (oak,
aromatic intensity, floral, ripe fruit, sweet, citric, raisins, overall impression, honey, off-odour
and fruity), confirming the difficulty in separating the aging time from the other
factors. The PCA of these selected descriptors offered 2 new components; the
first PC (41.2% of variance explained) included high loadings in oak note,
while the second PC (21.5% of variance explained) was well correlated with
ripe fruit and honey, in addition to overall impression. The projection of the
samples onto the new plane PC1-PC2 (Figure 13) showed that the oak note
(PC1) did not always increase with aging time: it reached a maximum
perception after one month, and thereafter the oak intensity slowly decreased.
This could be because the initial maderization was very intense, almost
aggressive, but then it slowly became integrated with the other aromatic notes.
In relation to PC2 (with a high loading in the appreciated honey note, and in
overall impression) the ranking on the axis shows a clear tendency: the scores
increased with the time in contact with the wood of the oak barrel.
370
English summary
4
S01
3 S01 S01
2 S03 S01
S01 S03 S01 S07
S03
S01 S03 S03 S07 S01 S07S07
S07 S03 S07 S12
1 ImpresinS07 General
MielFruta Madura S12
S01 Roble
S03 Afrutado
Defecto
S07 S12
0 IntensidadCtrico
Aromtica Dulce S12
S03 Floral Pasas S28
S12 S12 S18
P
-1 S12
S12 S03
S0
S07
S0
-2
S0 S0 S12
S0
S0 S0
-3
S0 S0
-4
-4 -3 -2 -1 0 1 2 3 4 5
PC1
Figure 13. PCA (PC1-PC2) of sensory data of wines, aged for, at least, one year (S0: Wine before
aging; S01, S03, S07, S12:, S18, S28: 1, 3, 7, 12, 18 and 28 months of aging ).
371
Conclusions
IV. CONCLUSIONS
Taking into account the results obtained from this research, the following
conclusions can be enumerated:
Moreover, the use of the climatic chamber for drying grapes for the
production of sweet wines, combined with the traditional techniques of
vinification (without pellicular maceration nor nutrients), and a final stage
of aging in oak cask had yielded wines with a low content in OTA.
372
English summary
obtained for wines made from Muscat grapes, fermented with S. cerevisiae
and aged in oak cask for up to 28 months (S12, S18 and S28).
- Finally, the sensory analysis of musts and wines showed the influence of the
grape variety and the vintage on this parameter. The artificial drying was
characterized by a slight increase in "fruity" and "raisin" notes and less
musty off-odour. Wines fermented at low temperature with S. bayanus scored
373
Conclusions
the highest in citric and floral notes, and it was the preferred ones. The wood
contact in oak cask improved the overall impression of the wines, showing
a very intense maderization at the beginning of the stage. It took a few
months that the aromatic profiles of wines were well integrated.
374
English summary
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381
Artculos derivados
de la Tesis
13006 J. Agric. Food Chem. 2010, 58, 1300613012
DOI:10.1021/jf103245z
During the drying process of raisins, the grapes are subjected to climatic variations that can result in
heavy infections of some fungal species that produce ochratoxin A (OTA), a powerful toxic metab-
olite, whose maximum permitted content is set by the European Union at 2.0 g/L for grapes, wine
and other drinks derived from the grape. The aim of this paper is to optimize the process of making
sweet wines in order to minimize the content of ochratoxin A. The results reflect a reduction of the
OTA content in grapes dried under controlled conditions in a climatic chamber up to 24% compared
to those sunned in the traditional way. A decrease of the concentrations of OTA is also observed
during the processes of vinification. Those wines with prefermentative maceration reached a higher
OTA content than the wines without maceration, but unexpectedly were not those preferred from a
sensorial point of view. In addition, the process of aging in oak casks has been shown to serve as a
natural method for reducing the OTA content of these wines.
KEYWORDS: Ochratoxin A; food safety; sweet wine; Muscat; climatic chamber; raisining; pellicular
maceration; oak aging; sensory evaluation
fermentation experiences fermentation nutrients pellicular maceration with control with oak
(vessel no.) yeast tempa (diammonium phosphate) pectolytic enzymes (4 C, 24 h) (without aging) with oak casks shavings(chips)
In the human, it has been associated with a nephropathy endemic wines. The chromatographic conditions set were the same in both cases.
to the Balkans region, although this is still an unconfirmed All the analyses were performed in duplicate.
hypothesis. OTA also possesses teratogenic, hepatotoxic, neuro- Sample Preparation. For the preparation of must samples aliquots each
toxic and immunotoxic effects, and is classified by the Interna- of 5 g of the musts were taken. To each was added 75 mg of polyethylene
tional Agency for Research on Cancer as a possible human car- glycol 8000 and 250 mg of sodium bicarbonate. Each preparation was then
cinogen (class 2B). In the European Union the OTA content is mixed using a magnetic agitator for 30 min and centrifuged for 5 min at
4000 rpm. The supernatant obtained was passed through syringe filters of
regulated in foods susceptible to contamination. In the case of
0.45 m (Millipore). The extraction of the wine samples was performed
wine, other drinks based on wine and/or musts, the maximum
using solid phase extraction (SPE) following the method proposed by
limit is set at 2.0 g/L (21, 22). Hernandez et al. (24). Bond Elut C-18 cartridges (500 mg, 3 mL) were
In order to optimize the process of making sweet wines to employed; the solid phase extraction was performed in several stages in a
minimize the content of ochratoxin A, a study of the evolution of Rapid Trace, SPE Workstation system (Zymark). First the cartridge
OTA content throughout the entire process of production of was conditioned with 5 mL of methanol and 5 mL of Milli-Q water, both at
sweet wine, from the raisining of the grapes to vinification and the a flow rate of 2 mL/min. A volume of 10 mL of a 1:2 dilution of the sample
aging of the resulting wine in oak barrels, is presented. An in Milli-Q water was passed through the cartridge at 0.5 mL/min. The
alternative to the traditional sun-drying is proposed. cartridge was then washed with 2 mL of Milli-Q water and 2 mL of
methanol/water (60/40 v/v), and dried under a stream of N2 for 10 min.
MATERIALS AND METHODS Lastly the extract was collected in 2 mL of methanol at a flow rate of
0.5 mL/min.
Samples. The musts analyzed were obtained from grapes of the Muscat
Chromatographic Separation. The HPLC system employed for the
variety, subjected to two different drying procedures: natural sunning on a
analysis was a Waters Alliance 2695, with a 110A C18 Gemini reverse
roof terrace and artificial sunning in a climatic chamber. In the first,
phase column, of 250 mm length 4.6 mm i.d. and particle size of 5 m
bunches were placed on esparto mats exposed to the air, without any type
(Phenomenex). The alkaline mobile phase was composed of acetonitrile/
of protection against environmental conditions. In the artificial sunning,
ammonium chloride-ammonia buffer (205 mM, pH 9.4), in a proportion
bunches were placed in a climatic chamber with controlled temperature of
20:80 (v/v), by isocratic flow at 30 C and at a flow rate of 0.75 mL/min.
40 C and humidity of 10%.
The volume of sample injected was 25 L. A Waters 474 scanning fluores-
In both systems, the loss of weight of the grapes was monitored
periodically and the assays were completed when this loss reached about cence detector was used for the measurement of fluorescence. The wavelengths
30%. Samples were taken at the start and the end of both the traditional of excitation and emission were 333 and 460 nm, respectively. The data
sun-drying and the raisining in the climatic chamber; these samples were acquisition time for each of the samples was set at 14 min; the software
pressed and the must obtained was preserved at -20 C for subsequent employed for the recording and processing of the data was Empower Pro 2002
analysis. (Waters). Figure 1 shows typical chromatograms of the samples studied, a
Five different conditions (two of them by duplicate) were tested for the must and a wine, with a retention time of 10.2 min for OTA.
vinification of the must obtained from grapes dried in the climatic chamber Calibration. For the quantification in musts, a calibration curve was
(experiences 1 to 5, Table 1). The sweet wines obtained in the various obtained from eight different concentrations of a standard solution of
experiments were aged in 30 L oak casks (_OC). In addition, one of them OTA (0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 40.0 g/kg). All the dilutions
was aged by contact with oak shavings (chips, _Ch). were prepared in duplicate, in a matrix of synthetic must (1 g/L of malic
Samplings were performed for each of the assays at the finish of the acid, 6 g/L of tartaric acid and 200 g/L of glucose), and were treated in the
fermentation (V01) and after desludging and clarification (V02 or _C); at same way as the real samples. For the samples of wines, the calibration
the beginning of the aging stage (A01, after 30 days) and after 60 (A02) and curve was obtained in HPLC grade methanol, from known volumes of a
90 (A03) days of aging. working solution of 50 g/L (0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 g/L).
Chemicals. Hydrochloric acid 37%, ammonia 30%, sodium bicarbon- Lastly, the OTA peak areas obtained in the chromatograms were
ate and glucose were obtained from Panreac (Panreac Quimica S.A., represented graphically against their corresponding concentrations. Two
Barcelona, Spain). Polyethylene glycol 8000 and methanol were supplied replicates of all the dilutions were prepared, and these were analyzed in
by Scharlau (Scharlab S.L., Barcelona, Spain). Acetonitrile (HPLC gradient duplicate.
grade), tartaric acid (analytical quality) and acetic acid (analytical quality) Validation of the Analytical Methods. Samples of musts and wines are
were obtained from Merck (Darmstadt, Germany). The OTA standard of great chemical complexity and are difficult to reproduce accurately. In
and malic acid were supplied by Sigma (Sigma-Aldrich, St. Louis, MO, addition, the fluorescence detection is a very sensitive method that could
USA). The ultrapure water employed was purified using a Milli-Q system cause interference from the matrices. As a result, two different studies, one
(Millipore, Bedford, MA, USA). for each type of sample under study, were carried out to ensure the matrix
Analysis of Ochratoxin A. The methods chosen for the determination effects did not affect the quantitation. The method of standard additions to
of ochratoxin A in musts and wines was devised by the Analytical the real samples was used. The slope values of the regression curves
Chemistry of Wine and Agrofood Products Research Group (PAI obtained with standard solutions were then compared with the slope values
AGR122) (23, 24). They are based on an initial extraction followed by obtained by the method of standard additions using the Student t test. In
separation using liquid chromatography (HPLC) and subsequent detec- both studies, for musts and wines, the slopes obtained were similar for a
tion of the toxin by fluorescence. The extraction in musts was performed 95% confidence limit, so it was concluded that matrix effects were absent.
by the addition of sodium bicarbonate and polyethylene glycol. Since wine Regarding the validation of other analytical parameters, it was per-
is a more complex matrix, solid phase extraction (SPE) was used for the formed in a different way for musts and wines. On one hand, two studies of
13008 J. Agric. Food Chem., Vol. 58, No. 24, 2010 Ruz Bejarano et al.
Figure 1. Typical chromatograms of must and wine samples under study, with the one of a blank superimposed, showing the OTA peak at 10.2 min.
the repeatability of wines were carried out, concluding that relative stan- since it minimizes the consumption of samples and the sensorial fatigue of
dard deviations (RSD) both intraday repeatability and interday were low the judges. After presenting the series of wines to the judges, they were
(1.11% and 1.14% respectively). In addition, a series of standard additions asked to assess them from left to right, and then to rank them by preference
(at four levels, in duplicate) from 0.75 to 6 g OTA/L was prepared in from least to most. The test of Friedman was applied for the interpretation
order to obtain the recoveries. These were from 93 to 99%. Finally, the of the results obtained.
limit of detection and limit of quantification were established at 0.22 and Statistical Analysis. One and two factor variance analysis (ANOVA)
0.77 g/L, respectively. On the other hand, samples of grapes and musts and multivariate analysis of data included factor analysis (FA) using
spiked with 2 g/kg of OTA were used for the studies of repeatability per- the statistical computer package Statistica 7.0 (Tulsa, OK, USA) were
formed. The relative standard deviations (RSD %) of the intraday and the performed.
interday studies were both 0.14%. The accuracy of the analysis method
was established by using the method of standard additions. Five levels of
OTA addition, from 0.5 to 5 g/kg, and two samples per level were RESULTS AND DISCUSSION
prepared by evaporation of known volumes of the OTA working solution Musts. The OTA contents in the musts from the two different
under nitrogen stream, followed by dissolution in real samples of grape. systems of raisining tested suggested that a previously existing
The method presented recovery values close to 100% for most of the con- fungal contamination in the grape could occur, and this contam-
centrations analyzed and low values of standard deviations (from 94.7 (
ination, associated with the conditions of the drying process itself,
0.8 to 107.8 ( 0.9). About the limits of detection and quantification, they
were established at 0.13 and 0.43 g/kg of OTA respectively. led to the growth of the fungus and, consequently, of ochratoxin
Sensory Analysis. The sessions were carried out in a standard tasting A in both systems. This finding is related to the data provided by
room (25). All evaluations, exclusively orthonasal, were carried out at Valero et al. (14) which demonstrated that it is after the harvest-
22 C. Twenty milliliters of sample were presented in blue glasses, generally ing, and particularly in the process of raisining, when the Asper-
used for olive oil sensory analysis, and covered with a glass top in order to gillus species becomes predominant over other fungi present on
minimize the possible losses of aroma. The panel judges, all of them the grape, due principally to greater tolerance of high tempera-
laboratory personnel, were submitted to a training period about general tures and low activity of the water (aw).
and specific sensorial aspects. During this stage, the judgment repeatability However, the OTA concentration of the must from grapes
and the panel homogeneity were evaluated. Duplicated discrimination dried in the climatic chamber (6.9 ( g/kg) was lower than that
testing and descriptive profiling were employed to validate the judges re-
obtained in the natural process (28.8 ( g/kg), the difference
producibility, whereas for each descriptor, a two factor ANOVA (judges
samples) allowed study of the homogeneity of the panel. being statistically significant (p < 0.05), so it seems the increased
Triangular test and descriptive profiling were performed. For the first control over the conditions of raisining resulted in better hygienic
case, the triangular test (26), the judges were asked about the quantitative conditions for the final must. The must obtained in the traditional
value of the detected differences. A scale with five points was used: not system presented a very high concentration of OTA which is not
present, poor, regular, strong, and very strong. For the second study, the suitable for subsequent fermentation since it will be difficult to
descriptive profiling (27), as a preliminary stage of generating appropriate achieve a legally acceptable OTA content in the finished wine.
descriptors to define the samples, the judges were presented with a group of With respect to the sensory analysis, the musts obtained from
musts and wines, representative of the complete set, and were asked to the grapes dried by the traditional method of sunning were com-
describe them qualitatively. The final selection included those with a pared by triangular test with those from grapes dried under con-
frequency of appearance greater than 5. A structured nine-point scale was
trolled conditions in the climatic chamber. A significant differ-
employed to evaluate each descriptor (28). The descriptors evaluated were
as follows: aromatic intensity, fruity, citric, ripe fruit, sweet, raisin, floral, ence between them was detected (according to 10 of the 14 judges,
honey, herbaceous, oak, wine character, dairy, wetness, chemical character, p<0.05), with the intensity of difference reported ranging be-
and general impression. A ranking test (29) was carried out to assess the tween weak and moderate with a mean ( standard deviation
judges preferences among the different wines produced. This type of value equal to 1.9 ( 0.8. Then, with the aim of identifying the
discrimination test is indicated when there are several samples to compare, descriptors that best explained the differences detected between
Article J. Agric. Food Chem., Vol. 58, No. 24, 2010 13009
Table 2. Analysis of Variance Applied to the Intensity Values of the Attributes
Used To Describe the Sensory Profiles of the Musts under Studya
descriptors traditional sun drying climatic chamber F p
Experience 1
OC1 3.75 ( 0.03 1.44 ( 0.02 0.64 ( 0.05 0.65 ( 0.05 0.62 ( 0.02
OC2 0.69 ( 0.03 0.58 ( 0.02 0.55 ( 0.02
C 1.12 ( 0.08 1.12 ( 0.10 1.14 ( 0.06
Ch 1.10 ( 0.02 1.08 ( 0.01 1.13 ( 0.01
Experience 2
OCC 3.64 ( 0.01 1.51 ( 0.11 1.11 ( 0.03 0.86 ( 0.02 0.97 ( 0.01
C 1.52 ( 0.01 1.48 ( 0.03 1.60 ( 0.02
Experience 3
OC1 4.09 ( 0.02 0.97 ( 0.01 0.73 ( 0.01 0.58 ( 0.02 0.50 ( 0.02
OC2 0.62 ( 0.02 0.56 ( 0.02 0.52 ( 0.01
C 1.00 ( 0.01 1.01 ( 0.07 1.00 ( 0.02
Experience 5
OC 3.99 ( 0.03 4.44 ( 0.4 3.95 ( 0.01 3.40 ( 0.01
C 4.92 ( 0.2 4.92 ( 0.02 4.98 ( 0.02
a Figure 3. Factor analysis of sensory data of sweet wines made from
V01, wine after fermentation; V02, wine after desludging and clarification; A01,
wine aged for 1 month in oak cask; A02, wine aged for 2 months in oak cask; A03, raisins dried in a climatic chamber. Location of the sensorial attributes onto
wine aged for 3 months in oak cask; C, wine without aging; Ch, wine aged with oak the new space defined by the three major factors: x axis, factor 1 (36.8%);
chips; OC, wine aged in oak cask. y axis, factor 2 (16.1%); and z axis, factor 3 (14.5%).
Table 4. Analysis of Variance Applied to the Intensity Values of the Attributes Used To Describe the Sensory Profiles of the Sweet Wines Produceda
descriptors 1_V02 2_V02 3_V02 4_V02 5_V02 F p
aromatic intensity 4.1 ( 0.5 4.2 ( 0.6 4.4 ( 0.6 4.1 ( 0.5 4.2 ( 0.5 0.114 0.97
fruity 4.2 ( 0.3 2.7 ( 0.4 3.8 ( 0.3 4.1 ( 0.4 3.8 ( 0.5 2.164 0.11
citric 2.2 ( 0.7 1.4 ( 0.5 2.8 ( 0.3 3.1 ( 0.4 2.7 ( 0.3 3.184 0.04
ripe fruit 1.9 ( 0.4 1.5 ( 0.6 2.1 ( 0.4 1.7 ( 0.5 1.4 ( 0.4 0.307 0.87
sweet 2.3 ( 0.6 2.0 ( 0.6 3.5 ( 0.6 2.3 ( 0.7 1.4 ( 0.5 1.312 0.37
raisin 1.9 ( 0.5 1.6 ( 0.5 2.2 ( 0.4 2.3 ( 0.4 1.1 ( 0.3 0.349 0.84
floral 3.6 ( 0.8 3.0 ( 0.7 3.9 ( 0.5 4.1 ( 0.6 4.0 ( 0.8 2.647 0.07
honey 0.9 ( 0.2 0.8 ( 0.4 1.5 ( 0.5 1.2 ( 0.3 1.1 ( 0.5 0.806 0.54
herbaceous 1.1 ( 0.4 2.1 ( 0.6 1.3 ( 0.6 1.4 ( 0.5 1.9 ( 0.7 0.351 0.83
wine character 2.5 ( 0.5 2.3 ( 0.7 2.5 ( 0.5 2.9 ( 0.6 2.8 ( 0.6 0.298 0.87
dairy 0.2 ( 0.3 0.5 ( 0.3 0.5 ( 0.3 0.5 ( 0.3 0.5 ( 0.4 0.033 0.99
wetness 0.6 ( 0.3 1.1 ( 0.4 1.1 ( 0.6 0.9 ( 0.4 1.5 ( 0.5 0.322 0.86
chemical character 0.8 ( 0.3 2.1 ( 0.3 0.6 ( 0.3 1.0 ( 0.3 0.8 ( 0.2 0.908 0.43
general impression 4.4 ( 0.4 2.5 ( 0.6 3.1 ( 0.5 4.5 ( 0.5 3.8 ( 0.5 2.994 0.05
a
Values are given as mean ( standard deviation (n = 17). The differences are significant at p < 0.05. The lowest p (in bold) indicate the most significant descriptors in order to
discriminate the 5 sweet wines produced.
Article J. Agric. Food Chem., Vol. 58, No. 24, 2010 13011
Table 5. Loadings of the Sensory Descriptors from Raisins Dried in a Climatic
Chamber in the New Space Defined by the Three Major Factors 1, 2 and 3a
factor 1 factor 2 factor 3
descriptor (36.8% expl. var.) (16.1% expl. var.) (14.5% expl. var.)
ORIGINAL PAPER
Received: 5 March 2013 / Accepted: 29 June 2013 / Published online: 30 July 2013
Springer-Verlag Berlin Heidelberg 2013
Abstract Several experiments of aging in oak wood and S. cerevisiae and aged in wood or stainless steel. From a
stainless steel of sweet Sherry wines obtained from grapes sensorial point of view, the wines fermented with S. Ce-
cv. Muscat fermented under different enological conditions revisiae and aged in wood (D1C and D2C) were the most
have been carried out. These wines were periodically valued ones.
sampled and their volatile content was analyzed using
SBSEGCMS. A sensory study was also carried out. Data Keywords Sweet Sherry wines
were submitted to different statistical techniques. An Volatile compounds Oak cask Stainless steel
important inuence of the three factors considered, fer- Fermentation conditions
mentation type, aging type, and aging time, have been
observed. In cluster analysis, the grouping obtained dem-
onstrated that the volatile compounds considered possess Introduction
sufcient explanatory power to detect the type of aging
process and differentiate the two yeast strains studied. In The volatile fraction of wine is determined by several
the case of the linear discriminant analysis, high percent- hundreds of chemically different compounds. Alcohols,
ages of correct classication were obtained for the three aldehydes, esters, acids, monoterpenes, and other minor
factors considered. Taking into account the global volatile compounds usually constitute the volatile fraction of this
prole, the factor aging type (oak cask or stainless steel) product. Their presence or absence in a particular wine
has demonstrated the highest inuence on the volatile depends on several factors: environment (climate and soil),
content of the wines, with those wines from Experiments ripeness and grape variety, winemaking conditions, and
D4 and D5 (fermented with S. bayanus) and aged in oak aging. Some of these compounds are already present in the
wood clearly differentiated from those ones fermented with grapes; others are formed during the processes of fermen-
tation and aging [13].
In many cases, the variety of grape employed in making
M. J. Ruiz-Bejarano R. Castro-Mejas (&) a particular wine completely determines the aroma of that
M. C. Rodrguez-Dodero C. Garca-Barroso wine because volatile compounds that are present in the
Analytical Chemistry Department, Faculty of Sciences-CAIV, grape persist throughout the entire process of vinication
University of Cadiz, Agrifood Campus of International
and aging. The extent to which these compounds persist
Excellence, Post Ofce Box 40, 11510 Pol. Ro San Pedro,
Puerto Real, Cadiz, Spain from the grape through to the nished wine will be inu-
e-mail: remedios.castro@uca.es enced by the conditions of its vinication and the aging [4].
M. J. Ruiz-Bejarano However, it should be remarked that the process of
e-mail: mariajesus.ruiz@uca.es vinication itself, and particularly the alcoholic fermenta-
M. C. Rodrguez-Dodero tion, generates the greater part of the aromatic compounds
e-mail: maricarmen.dodero@uca.es present in the wine [5, 6].
C. Garca-Barroso In most wines, all of these compounds are subjected to
e-mail: carmelo.garcia@uca.es the subsequent process of aging in wood. During aging in
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906 Eur Food Res Technol (2013) 237:905922
wood, the wine acquires aromatic complexity as a result of about the volatile prole of Muscat sweet wines and its
important and diverse reactions [7]. Additionally, new relationship, in an individual way, to different vinication
volatile and nonvolatile compounds are extracted from the or aging processes [7, 1618], but not studying both factors
wood, modifying the wines bouquet. As a consequence jointly.
of all of these processes, the fruity and fermentative vol- Several different chemometric tools have been applied
atile compounds decrease during the process of aging in for the analysis of the results.
wood [8].
In Andalusia, special sweet wines are obtained using
dried grapes by direct exposition to the sun. They are Materials and methods
elaborated in Malaga, Montilla-Moriles, and Jerez in a
traditional method dating many years ago using two grapes Samples
varieties, Muscat and Pedro Ximenez. Grapes of these
varieties, in bunches, are spread out on esparto grass mats About 2,000 kg of ripe grape bunches from cv. Muscat
to toast in the sun before pressing. The bunches are turned were collected from a local winery in the Jerez-Xere`s-
over on a regular basis and covered at night to avoid the Sherry D.O. They were dried using a climatic chamber
adverse effects of high air moisture. This traditional pro- (Ibercex A. S. L., S. A., Spain) for 5 days at 40 C and
cess is affected, in many occasions, by the attack of insects relative humidity of 10 %. These bunches were distrib-
and the growth of fungi that can give rise to the formation uted uniformly into the chamber forming a single layer.
of dangerous toxins in the wine [9, 10]. The water loss was daily controlled. At the end of the
During this period, important reactions take place in the drying process, this one was about 35 % with 25
grape. Squadrito et al. [11] and Eberle et al. [12] found Baume.
signicant decreases in terpenols during the sun-drying Grapes were destemmed, crushed, and pressed using a
process of Muscat grapes. vertical press. For those experiments with pellicular mac-
In this way, the grapes attain sugar levels above 300 g/L eration, grapes were crushed without destemming and
and are crushed and pressed employing vertical presses. submitted to maceration for 24 h at 4 C.
After this, musts with high sugar content and, conse- The highest pressure in each cycle was 300 bars. Three
quently, a difcult fermentation are fortied with ethanol cycles were employed.
to ensure that the wine will contain at least 1518 % After that, must pH was adjusted to 3.5 with tartaric acid
alcohol. The young sweet wine is then, generally, sub- (Agrovin, Spain). The concentration of total sulfur dioxide
mitted to aging in American oak wood following a system was also set at 120 mg/L by adding potassium metabisul-
of dynamic oxidative aging [13]. te (Agrovin, Spain).
In these last years, a possible alternative to this tradi- Five different conditions (by duplicate) of vinication
tional sun-drying methodology has appeared. It is being were tested (experimental treatments 15, Table 1).
used for the drying of horticultural products and consists in In Experiment 1, a Saccharomyces cerevisiae yeast
forcing the loss of water by means of forced convection inoculum (40 g/hL, Lalvin D254, Lallemand, Australia)
with hot air in drying chambers [14]. This drying system was employed. The fermentation was carried out at room
avoids the problems mentioned above about fungi and temperature (less than 30 C).
insects that generate a high level of ochratoxin A and The conditions of Experiment D2 were the same as
causes high losses of product during the sun-drying [10]. Experiment D1 with the addition of yeast nutrients
Ruiz et al. [15] found for the grape variety Pedro (diammonium phosphate, 10 g/hL) (Actimax Plus?, Ag-
Ximenez that the musts from chamber-dried grapes exhib- rovin, Spain).
ited the same aroma terms than those obtained from grapes In Experiment D4, must was fermented at low temper-
dried traditionally under the inuence of the sun. These ature (less than 10 C) with S. bayanus (40 g/hL, Uvaferm
authors suggest that the use of chamber-drying can con- 43, Lallemand, Australia).
tribute to improve the drying process of the grapes. In addition, prefermentative cold maceration at 4 C
The objective of this work is to study the evolution, using 3 g/hL of pectolytic enzymes (Enozym Arome, Ag-
under different types of aging, of the main volatile com- rovin, Spain) was tested in Experiments D3 and D5. After
pounds of sweet Sherry wines obtained from grapes cv. 24 h, the grape paste was pressed, using a vertical press
Muscat dried using drying chambers and fermented under under the same conditions mentioned before (3 cycles and
different enological conditions. Therefore, two factors and 300 bars as the highest pressure in each cycle), and fer-
their possible inuence on the volatile prole have been mented. In Experiment D3, the fermentation conditions
considered together: aging and vinication conditions. were the same as Experiment D1 (S. cerevisiae at room
Only a few studies can be found into the bibliography temperature). Must from Experiment D5 was fermented
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Eur Food Res Technol (2013) 237:905922 907
Table 1 Conditions under which the vinication assays and aging experiments were carried out
Vinication Yeast Fermentation Nutrients (diammonium Pellicular maceration Aging
experiments temperaturea phosphate, 10 g/Hl with pectolytic
enzymes (4 C, 24 h) Stainless steel Oak cask Chips
following the conditions of Experiment D4 (S. bayanus at thermal desorption tube and then thermal desorption was
low temperature). carried out.
When the sugar content of the wine was around Previously, taking into account the nature of the sam-
90100 g/L, the fermentation was stopped by adding ples, the possible interference of the high sugar content on
alcohol up to 17. Then, the sweet wines obtained were the SBSE efciency was studied. Consequently, the same
aged in 30-L medium toasted oak casks (samples D1C to model white wine with different amounts of sugar
D5C). In order to compare the evolution of volatile com- (0250 g/L, glucose) was analyzed in triplicate. No inter-
pounds during this period, the same wines were aged in ference was observed (data not shown).
10-L stainless steel (Experiments D1S, D2S, and D4S). In
addition, one of them was aged in contact with 4 g/L of oak Instrumentation
chips for 3 months (Roblemor T, medium toast, Agrovin,
Spain) (sample D1CH). During the aging, all the experi- A commercial TDS-2 thermal desorption unit (Gerstel)
ments were situated in the same room at about 20 C. connected to a programmed temperature vaporization
The sampling was carried out after fermentation and (PTV) injector CIS-4 (Gerstel) by a heated transfer line
periodically for 1 year of aging (S0, 20 days; S1, 40 days; was used for the thermal desorption of the coated stir bars.
S2, 3 months; S3, 4 months; S4, 8 months; and S5, The PTV was installed in an Agilent 6890 GC-5973 MS
12 months). Samples were stored at 4 C until their system (Agilent Technologies, Palo Alto, CA, USA). An
analysis. empty bafed liner was used in the PTV. The thermode-
sorption unit was equipped with a MPS 2L autosampler
Analysis of volatile compounds (Gerstel) capable of handling the program for 98 coated stir
bars. The desorption temperature was programmed from
Chemicals and reagents 40 C to 300 C (held for 10 min) at 60 C/min under a
helium ow (75 ml/min), and the desorbed analytes were
All the aroma standards employed in this work were sup- cryofocused in the PTV system with liquid nitrogen at
plied by Merck (Darmstadt, Germany) and Sigma (Stein- -140 C. Finally, the PTV system was programmed from
heim, Germany). 4-methyl-2-pentanol was used as internal -140 C to 300 C (held for 5 min) at 10 C/s for the
standard. analysis by GCMS. A DB-Wax capillary column (J&W
Scientic, Folsom, CA, USA), 60 m 9 0.25 mm I.D., with
Sample preparation a 0.25 lm coating was used in order to perform the cap-
illary GCMS analysis in the electron impact mode. The
Volatile compounds were analyzed by SBSEGCMS carrier gas was helium at a ow rate of 1.0 ml/min. The GC
according to the method proposed by Alves et al. [19]. oven was programmed as follows: held at 35 C for 10 min
Briey, ve milliliters of ultrapure water, 5 ml of and then ramped at 5 C/min to 100 C. Then, it was raised
wine sample, 30 ll of a solution of 4-methyl-2-pentanol to 210 C at 3 C/min and held for 40 min. The mass
(2.52 g/L in Milli-Q water containing 15 % v/v of ethanol), detector operated in EI? mode at 70 eV in a range of
and a PDMS stir bar (20 mm 9 0.5 mm (length 9 lm 30400 amu.
thickness)) supplied by Gerstel (GmbH, Mulheim a/d Ruhr, Peak identication was carried out using the Wiley 7 N
Germany) were used for the extractions. For each extrac- Edition Library (Wiley Registry of Mass Spectral Data, 7th
tion, the stir bar was stirred for 1 h (800 rpm) at room Edition, 2000) by analogy of mass spectra (with a mini-
temperature (20 C). Then, it was transferred into a glass mum of 90 % of correspondence) and conformed by
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908 Eur Food Res Technol (2013) 237:905922
retention times of standards when they were available. Results and discussion
Additionally, in order to guarantee the identications, the
retention indices were experimentally determined on a Volatile compounds
polar column (DB-Wax) and compared with those found in
the bibliography. Semiquantitative data were obtained by In this research work, a total of 53 individual volatile
measuring the relative quantifying ion peak area in relation compounds have been identied in the different Muscat
to that of the area of 4-methyl-2-pentanol, the internal wines studies (Table 2). In our case, all musts were
standard. obtained from grapes dried using drying chamber (40 C
and 10 % RH). Ruiz et al. [15], in grapes from Pedro
Sensory Analysis Ximenez variety, observed that musts from chamber-dried
grapes exhibited the same aroma terms as those from sun-
The sessions were carried out in a standard tasting room dried grapes.
[20]. All evaluations, exclusively orthonasal, were carried For wines from Muscat grapes, most volatile compounds
out at 22 C. Twenty milliliters of sample was presented in found in this study had been previously identied: from
blue glasses, generally used for olive oil sensory analysis grape pulp and skins [25]; coming from the alcoholic fer-
[21], and covered with a glass top in order to minimize the mentation [26, 27]; and/or appearing after the period of
possible losses of aroma. The panel judges (12 judges), all aging in wood [7, 18, 27].
of them laboratory personnel, were submitted to a training
period about general and specic sensorial aspects. During Analysis of variance (ANOVA)
this stage, the judgment repeatability and the panel
homogeneity were evaluated. Duplicated discrimination Table 3 shows mean values obtained for each vinication
testing and descriptive proling were employed to validate experiment, at the initial (S0, 20 days) and at the nal (S5,
the judges repeatability, whereas for each descriptor, a 12 months) of aging, taking into account, for each case, all
two-factor ANOVA (judges 9 samples) allowed the study different ways of aging. In the case of Experiments D3 and
of the homogeneity of the panel [22]. After this, a D5, only one aging system, aging in oak cask, was studied.
descriptive proling was performed [23]. As a preliminary The process of drying and vinication, together with the
stage of generating appropriate descriptors to dene the later time of aging in wood, determines the prole of
samples, a group of wines, representative of the complete volatile compounds in a nal wine. Taking into account all
set, were presented to the judges. They were asked to of these facts, in order to study statistically the differences
describe them qualitatively. The nal selection included in the volatile content of all the wines studied, data were
those descriptors with a frequency of appearance greater submitted to the analysis of variance. For this one, three
than 5: fruity, citric, sweet, dried fruit/raisin, oral, honey, independent factors were considered: type of fermentation,
herbaceous, young wood/sawmill, oak, wine character, time of aging, and type of aging. As can be seen in Table 3,
dairy, wetness, and chemical character. In order to con- practically all the volatile compounds are signicantly
struct the nal worksheet (Fig. 1), wetness and chemical affected (p \ 0.01) by these three factors.
character were considered jointly in the off-odor descrip-
tor, and aromatic intensity and general impression were Ethyl esters It is well known that several volatile com-
also included. A structured nine-point scale was employed pounds are responsible for the fermentation aroma in wines
to evaluate each descriptor [24]. such as: ethyl ester of C6, C8, and C10 fatty acids, higher
alcohols and their acetates, and certain volatile acids [27].
Statistical analysis In our study, most of ethyl esters showed signicant
increases as the time of aging increased. However, the
Statistical analyses were carried out by using the SPSS effect of the type of aging did not exhibit a clear inuence
version 15.0 for Windows statistical package (SPSS Inc., on all of them. Ethyl butanoate, ethyl-3-methylbutanoate,
IL, USA) and Statgraphics Centurion, version 15.0 (Stat- ethyl hexanoate, diethyl pentanedioate, and diethyl succi-
point Inc., USA) for Windows XP. Analysis of variance nate showed higher values for those wines aged in wood,
(ANOVA) was applied to establish the possible signicant whereas ethyl esters with higher molecular weight (ethyl
differences among the samples for each volatile compound. octanoate, ethyl decanoate, ethyl dodecanoate, ethyl hex-
Furthermore, cluster analysis (CA), factor analysis (FA), adecanoate, and ethyl octadecanoate) exhibited higher
and linear discriminant analysis (LDA) were carried out values for wines aging in stainless steel (data not shown).
with the aim of highlighting the similarity of the samples Camara et al. [28] found great decreases in fatty acids ethyl
and the main contributors to the variance found among esters (C6C16) and acetates, and high increases in ethyl
them. esters of diprotic acids such as diethyl succinate for
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Eur Food Res Technol (2013) 237:905922 909
Madeira wines aged in oak wood during 25 years. In this During the course of aging of the wine, the ethyl ester
sense, Alves et al. [19] observed that ethyl octanoate, composition changes due to shifts toward chemical equilib-
diethyl succinate, and cis-oak lactone could be used as rium. The hydrolysis and esterication reactions that can affect
discriminating variables for the correct differentiation this type of volatile compounds are very common during this
among Madeira wines according to wine type and time of period [28] and depend on several factors such as wine pH,
aging, observing the same tendency that Camara et al. [28] temperature, alcoholic degree, and type of acid. Therefore, all
observed for ethyl octanoate, ethyl decanoate, and diethyl these factors should be taken into account in order to explain the
succinate. variations in the ethyl ester content observed in this study.
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910 Eur Food Res Technol (2013) 237:905922
Table 2 Identication and quantication parameters for volatile compounds identied in the commercial aromatized vinegar samples
Number LRI Compound m/z Identication
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Eur Food Res Technol (2013) 237:905922 911
Table 2 continued
Number LRI Compound m/z Identication
In the case of the factor fermentation type, in general, whereas decanoic and short-chain fatty acids exhibited
higher values were found for those wines of the Experi- signicant increases during a high period of aging in wood.
ments D2, D3, and D5. Ethyl esters are produced during
alcoholic fermentation from the reactions of ethanol and Acetates Regarding acetates, n-butyl acetate, 4-methyl-2-
acyl-coenzyme A derivates [29], and their concentrations pentanol acetate, and ethyl-2-phenyl acetate showed sig-
in the wine after the process of alcoholic fermentation nicant decreases as the aging time increased. Similar
depend on the specic conditions under which this process behavior was observed by Camara et al. [28] in Madeira
was carried out (temperature, yeast strain, etc.). The wines during a prolonged period of aging in oak wood. The
experiments with the highest values for ethyl esters were compounds, produced from enzymatic reactions, are
conducted under the addition of nutrients (Experiment D2), gradually hydrolyzed toward their corresponding acids and
pellicular maceration (Experiment D3), and pellicular alcohols during the storage of the wine.
maceration and fermentation with S. bayanus yeast About the factor fermentation type, Experiments D1
(Experiment D5), factors that have provoked a higher and D2 revealed a higher content in acetates. Both exper-
content of this type of compounds in the wine. Selli et al. iments were carried out at room temperature by S. cere-
[16] found that the skin contact treatment for musts from visiae and without maceration. The factor aging type
Muscat grapes increased the total free aroma compounds, was the least signicant for acetates with no clear inuence
especially terpenes and some higher alcohols. Aleixandre on these ones.
et al. [30] concluded that static clarication of must from
Muscat grapes with pectolytic enzymes produced wines Terpenes and terpenols Sanchez-Palomo et al. [25] found
with larger content of higher alcohols, esters, and terpenic that compounds such as linalool, geraniol, and nerol were
compounds. Pineiro et al. [17] observed that the vinica- the major compounds in pulp and skins of this grape
tion of several grape varieties with maceration provoked a variety, being the responsible for the typical oral aroma of
higher content in ethyl esters of lactic, acetic, and succinic Muscat grapes and contributing to the aroma of their wines.
acids. Ugliano et al. [31] observed that S. bayanus exhib- Fenoll et al. [35] found that the most abundant free volatile
ited a higher ability to release volatile compounds from compounds in Muscat grape during ripening, with signi-
glycosidic precursors under optimal conditions (pH 5 and cant increases during grape development, were some ter-
30 C) than S. cerevisiae, and that these differences were penic monoalcohols (linalool, geraniol, citronellol, nerol).
less signicant when the yeast strains were used to conduct In the group of glycosidically bound volatile compounds,
alcoholic fermentations in a chemically dened grape some terpenols together with other volatile compounds
juice-like medium containing glycosides extracted from such as trans and cis furan linalool oxides, benzyl alcohol,
grape juice. Other authors had also obtained contradictory and 2-phenylethanol were found [35].
results in studies about the activity of yeast glycosidase Concerning terpenes and terpenols, as can be seen in
enzymes during winemaking [3234]. Table 3, the most signicant factor was time of aging.
Terpenols such as linalool, a-terpineol, b-citronellol, nerol,
Acids In relation to acids, their contents were signicant and geraniol showed signicant decreases as the time of
inuenced by the three factors considered in this study. aging increased, whereas their oxides increased. This fact
About aging type, the lowest values were found for is in concordance with that observed by Karagiannis et al.
those wines aged in oak casks. The factor time of aging [27] in aged mistelles from Muscat grapes, with the
inuenced positively on the content of these volatile exception of a-terpineol, whose concentration increased.
compounds. Camara et al. [28] observed signicant Marais and van Wyk [36] observed that some terpenic
decreases in the content of octanoic and hexanoic acids, monoalcohols (linalool, nerol, and geraniol) during the
123
Table 3 Means (relative peak area) and SD for volatile compounds of each experiment
912
Volatile compounds D1 D2 D3 D4 D5 p value p value p value
fermentation aging aging
123
time type
Ethyl esters
Ethyl butanoate 0.0000* 0.0000* 0.0000*
Initial 0.180 0.083 0.296 0.158 0.302 0.032 0.149 0.028 0.635 0.064
Final 0.245 0.212 0.818 0.863 0.631 0.062 0.227 0.136 1.71 0.03
Ethyl 3-methylbutanoate 0.0000* 0.0000* 0.0000*
Initial 0.051 0.023 0.064 0.014 0.059 0.039 0.045 0.010 0.077 0.021
Final 0.070 0.046 0.166 0.110 0.097 0.017 0.072 0.042 0.143 0.039
Ethyl hexanoate 0.0000* 0.0090* 0.0000*
Initial 1.63 1.16 2.78 2.37 3.25 0.39 1.10 0.09 0.79 0.87
Final 2.30 1.93 6.99 7.54 5.69 0.36 1.26 0.04 1.16 0.39
Diethyl pentanedioate 0.0000* 0.0000* 0.0000*
Initial 0.014 0.005 0.026 0.005 0.040 0.026 0.015 0.006 0.034 0.011
Final 0.028 0.011 0.080 0.044 0.061 0.003 0.029 0.014 0.061 0.026
Ethyl octanoate 0.0000* 0.0000* 0.0000*
Initial 2.50 0.62 3.30 0.69 0.942 3.91 2.09 0.35 1.38 1.88
Final 3.13 0.58 6.23 0.70 1.93 0.42 2.88 0.22 2.19 3.91
Ethyl decanoate 0.0000* 0.0000* 0.0000*
Initial 0.863 0.408 1.33 0.694 0.120 0.121 0.436 0.159 0.244 0.489
Final 2.13 1.04 4.80 1.24 0.234 0.365 1.00 0.31 0.790 2.52
Diethyl succinate 0.0000* 0.0000* 0.0000*
Initial 0.950 0.403 1.35 0.340 1.83 1.27 0.945 0.472 2.30 0.517
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Final 1.89 0.90 4.48 2.68 3.53 0.26 1.97 1.23 4.35 1.27
Ethyl dodecanoate 0.0000* 0.0000* 0.0000*
Initial 0.028 0.028 0.111 0.111 0.014 0.087 0.021 0.007 0.011 0.014
Final 0.091 0.071 0.288 0.166 0.016 0.034 0.034 0.014 0.053 0.087
Ethyl tetradecanoate 0.0138 0.0000* 0.4404
Initial 0.026 0.021 0.050 0.019 0.026 0.011 0.024 0.011 0.026 0.013
Final 0.046 0.033 0.029 0.014 0.010 0.026 0.041 0.022 0.084 0.011
Ethyl hexadecanoate 0.2729 0.1607 0.0065
Initial 0.093 0.089 0.062 0.023 0.072 0.040 0.072 0.044 0.079 0.051
Final 0.084 0.052 0.089 0.033 0.024 0.113 0.095 0.024 0.273 0.040
Ethyl octadecanoate 0.0007* 0.0786 0.0000*
Initial 0.024 0.023 0.019 0.011 0.012 0.012 0.017 0.012 0.016 0.005
Final 0.017 0.011 0.023 0.006 0.004 0.030 0.010 0.002 0.028 0.012
Eur Food Res Technol (2013) 237:905922
Table 3 continued
Volatile compounds D1 D2 D3 D4 D5 p value p value p value
fermentation aging aging
time type
Acids
2-ethylhexanoic acid 0.0000* 0.0000* 0.0000*
Initial 0.038 0.006 0.045 0.004 0.031 0.024 0.042 0.003 0.010 0.019
Final 0.024 0.006 0.048 0.015 0.019 0.004 0.033 0.005 0.003 0.024
Octanoic acid 0.0000* 0.0000* 0.0000*
Initial 0.348 0.068 0.362 0.064 0.085 0.311 0.306 0.051 0.238 0.202
Final 0.410 0.164 0.466 0.027 0.113 0.074 0.408 0.122 0.244 0.311
Eur Food Res Technol (2013) 237:905922
Final 0.335 0.224 0.251 0.109 0.038 0.008 0.231 0.065 0.295 0.100
Acetates
n-butyl acetate 0.0000* 0.0000* 0.0000*
Initial 0.009 0.001 0.009 0.000 0.005 0.002 0.005 0.001 0.007 0.001
Final 0.005 0.004 0.013 0.011 0.007 0.001 0.004 0.003 0.009 0.002
4-methyl-2-pentanol acetate 0.0051* 0.0000* 0.4759
Initial 0.013 0.010 0.006 0.005 0.007 0.000 0.022 0.002 0.027 0.000
Final 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.010 0.000
Isoamyl acetate 0.0000* 0.0000* 0.0307
Initial 0.497 0.084 0.622 0.075 0.149 0.562 0.260 0.007 0.291 0.159
Final 0.432 0.054 0.987 0.192 0.310 0.109 0.351 0.126 0.443 0.562
Phenylethyl acetate 0.0000* 0.0000* 0.0000*
913
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Table 3 continued
914
Volatile compounds D1 D2 D3 D4 D5 p value p value p value
fermentation aging aging
123
time type
Initial 0.341 0.064 0.385 0.003 0.478 0.429 0.441 0.001 0.337 0.394
Final 0.436 0.049 0.822 0.216 0.574 0.082 0.569 0.011 0.456 0.429
Ethyl 2-phenyl acetate 0.0000* 0.0000* 0.0000*
Initial 0.900 0.133 1.01 0.239 0.155 0.621 0.488 0.049 0.511 0.182
Final 0.489 0.195 0.881 0.002 0.158 0.152 0.265 0.008 0.262 0.621
Terpenes and terpenols
Nerol oxide 0.0000* 0.0000* 0.0000*
Initial 0.063 0.018 0.055 0.008 0.070 0.076 0.069 0.003 0.086 0.075
Final 0.105 0.029 0.109 0.002 0.092 0.026 0.125 0.019 0.118 0.076
Linalool oxide 0.0000* 0.0000* 0.0788
Initial 0.004 0.001 0.003 0.000 0.006 0.003 0.005 0.000 0.005 0.001
Final 0.004 0.000 0.004 0.000 0.006 0.001 0.003 0.002 0.007 0.003
Linalool 0.0000* 0.0016* 0.0000*
Initial 0.459 0.085 0.388 0.035 0.424 0.375 0.426 0.003 0.397 0.329
Final 0.357 0.079 0.638 0.108 0.381 0.110 0.474 0.009 0.378 0.375
4-terpineol 0.0347 0.0071* 0.1862
Initial 0.055 0.053 0.071 0.098 0.003 0.001 0.009 0.009 0.011 0.002
Final 0.003 0.000 0.003 0.001 0.003 0.036 0.003 0.000 0.004 0.001
a-terpineol 0.0170 0.0014* 0.0312
Initial 0.214 0.041 0.203 0.092 0.210 0.126 0.200 0.026 0.198 0.129
Final 0.166 0.041 0.179 0.001 0.179 0.048 0.202 0.020 0.071 0.126
personal copy
Initial 0.033 0.052 0.002 0.001 0.001 0.000 0.002 0.001 0.018 0.000
Final 0.001 0.000 0.002 0.001 0.001 0.065 0.002 0.002 0.003 0.000
Carvacrol 0.5621 0.0500 0.0097*
Initial 0.017 0.028 0.002 0.001 0.002 0.000 0.001 0.001 0.003 0.000
Final 0.001 0.000 0.003 0.003 0.001 0.034 0.001 0.000 0.003 0.000
b-myrcene 0.3967 0.0000* 0.0002*
Initial 0.009 0.007 0.005 0.004 0.010 0.001 0.008 0.004 0.006 0.001
Eur Food Res Technol (2013) 237:905922
Final 0.003 0.002 0.006 0.005 0.002 0.010 0.003 0.002 0.001 0.001
Dehydroxylinalool oxide I 0.0000* 0.0000* 0.0170
Initial 0.018 0.002 0.015 0.002 0.019 0.013 0.017 0.001 0.042 0.022
Final 0.032 0.007 0.028 0.010 0.037 0.000 0.037 0.006 0.077 0.013
Dehydroxylinalool oxide II 0.0000* 0.0000* 0.0000*
Initial 0.022 0.009 0.026 0.001 0.018 0.009 0.022 0.002 0.030 0.027
Final 0.029 0.012 0.027 0.014 0.015 0.003 0.040 0.002 0.047 0.009
Aldehydes, acetones and alcohols
Hexanal 0.0000* 0.0000* 0.0705
Initial 0.009 0.001 0.009 0.002 0.008 0.003 0.008 0.001 0.010 0.002
Final 0.005 0.000 0.010 0.005 0.007 0.002 0.004 0.001 0.010 0.003
2-hexenal 0.1346 0.0002* 0.4616
Initial 0.372 0.167 0.307 0.136 0.450 0.186 0.496 0.106 0.640 0.402
Final 0.594 0.069 1.91 1.65 1.18 0.235 0.663 0.094 0.886 0.186
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Table 3 continued
916
Volatile compounds D1 D2 D3 D4 D5 p value p value p value
fermentation aging aging
123
time type
Initial 0.072 0.027 0.069 0.028 0.104 0.021 0.072 0.019 0.091 0.032
Final 0.086 0.032 0.149 0.119 0.193 0.035 0.085 0.040 0.215 0.021
3-methyl-2-butanol 0.0015* 0.0000* 0.1049
Initial 0.737 0.111 0.718 0.430 0.709 0.523 0.772 0.033 1.00 0.665
Final 0.979 0.186 2.37 1.63 1.91 0.124 1.03 0.085 1.13 0.523
2-phenylethanol 0.0000* 0.5980 0.0083*
Initial 0.925 0.125 1.06 0.162 1.26 0.778 0.959 0.011 1.43 0.652
Final 0.860 0.031 1.47 0.370 1.19 0.156 0.969 0.047 1.53 0.778
Miscellaneous
Phenol 0.0235 0.0505 0.4117
Initial 0.033 0.002 0.034 0.002 0.026 0.014 0.032 0.007 0.033 0.019
Final 0.036 0.012 0.044 0.024 0.024 0.001 0.028 0.001 0.044 0.014
Eugenol 0.0000* 0.0000* 0.0000*
Initial 0.012 0.006 0.008 0.005 0.019 0.003 0.014 0.008 0.031 0.001
Final 0.013 0.010 0.025 0.030 0.033 0.008 0.022 0.023 0.055 0.003
4-ethylphenol 0.0001* 0.7190 0.0000*
Initial 0.005 0.004 0.005 0.004 0.003 0.001 0.003 0.002 0.005 0.000
Final 0.003 0.003 0.006 0.007 0.006 0.002 0.004 0.005 0.007 0.001
2-furaldehyde 0.0000* 0.0000* 0.0000*
Initial 0.058 0.023 0.051 0.016 0.082 0.005 0.055 0.014 0.082 0.009
Final 0.059 0.042 0.058 0.051 0.113 0.017 0.098 0.085 0.180 0.005
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aging may be transformed into a-terpineol and other terp- Aldehydes, alcohols, and ketones Sanchez-Palomo et al.
enes such as linalool oxide with higher aroma thresholds. [41] identied, in musts from Muscat grapes, aldehydes
However, Loscos et al. [37] observed that the concentration and alcohols of six carbon atoms, monoterpenes, poly-
of a-terpineol increased signicantly after a week of oxygenated terpenes, and benzoic compounds such as
accelerated wine aging (50 C), and then, its concentration benzaldehyde and 2-phenylethanol. Acids and volatile
fell until the end of the aging process. As could be compounds resulting from reactions favoured by high
expected, the factor temperature is a crucial parameter that temperatures such as 2-furfuraldehyde and b-damascenone
has to be taken into account during the storage. These were also found in these musts [41].
authors explained these facts taking into account the initial In our studies, certain alcohols and aldehydes (1-hexa-
conversion of terpenols such as linalool into a-terpineol nol, hexanal, benzaldehyde, 2-phenylethanol) showed
and the subsequent conversion of this one into terpin increases for those wines obtained after maceration
hydrate. Observing the data correspond to Experiments D3 (Experiments D3 and D5). Selli et al. [16] found signicant
and D5 in comparison with the remaining ones, for which increases in the content of terpenes and some alcohols
the aging in cask was the only one studied (Table 3), it can (hexanol and 2-phenylethanol) in studies of skin contact
be observed that the decreases in terpenic compounds were treatment for musts from Muscat grapes.
higher for those wines aged in oak casks where the con- Some authors have found signicant increases for higher
tinuous micro-oxygenation favoured through the pores of alcohols during aging [28]. In our experiments, 3-methyl-2-
the wood provoked a higher oxidation of these compounds. butanol and 1-hexanol exhibited signicant increases dur-
About the factor fermentation type, the use of pel- ing aging. For aldehydes, benzaldehyde exhibited a clear
licular maceration with pectolytic enzymes (Experiments rising tendency during aging, with higher values for those
D3 and D5) did not clearly increase the content in this type wines aged in wood. Loscos et al. [37], in studies of
of compounds. The glycosidase activity of these enzymes accelerated aging (50 C and 9 weeks), and Pereira et al.
depends on the medium pH and the content in glucose and [42], in liqueur wines aged in casks, observed this same
glucolactone [38] with a clear inhibition of this activity as tendency for benzaldehyde.
the content in these compounds increases.
Selli et al. [16] found in studies of skin contact for Miscellaneous Among the volatile compounds that have
musts from Muscat grapes that terpenes increased slightly, demonstrated a clear increase in wines during their aging in
especially a-terpineol and b-citronellol. Aleixandre et al. oak wood, some authors [18, 28] have found 2-furaldehyde
[30] found clear increases in the content of linalool, and 5-methyl-2-furaldehyde. Both of them are formed by
a-terpineol, b-citronellol, nerol but not geraniol in assays the degradation of carbohydrates during the toasting of the
of maceration of Muscat musts with pectolytic enzymes. barrel and then are transferred to the wine during its aging.
In our case, only b-citronellol and nerol showed a clear In wines without aging in wood, both of them can provide
higher content for that wine from the Experiment D3, from the dehydration of sugars during the process of drying
macerated with pectolytic enzymes and fermented with [43].
S. cerevisiae. Eugenol, 2-furaldehyde, and 5-methyl-2-furaldehyde
In relation to the yeast strain employed, only geraniol demonstrated signicant increases during the aging, with
exhibited an obvious higher content for those wines fer- the highest values found for those wines aged in wood
mented with S. bayanus. Gamero et al. [39] observed in (Table 3, Experiments D3 and D5). 2-furaldehyde and
Muscat wines fermented by Saccharomyces species and 5-methyl-2-furaldehyde have been found in musts from
hybrid yeast that the amount of geraniol decreased in most Pedro Ximenez grapes after their sun-drying [43]. 2-fur-
wines with respect to the initial must except for S. bayanus furaldehyde is produced when pentoses (xylose) are heated
wines, whereas the a-terpineol amount was higher in wines and 5-methyl-2-furfuraldehyde arises from the rhamnose.
fermented by S. cerevisiae and hybrid yeasts. The amount Therefore, aging in wood and the use of a drying chamber
of linalool observed by Gamero et al. [39] was similar in all could explain the content found for these compounds in the
wines. No relationship between b-D-glucosidase activity wines studied. Eugenol, present in oak wood without
and terpenes prole of wines obtained after fermentation toasting and whose concentration augments with the
with Saccharomyce species and hybrids was found by these toasting temperature during the barrel making, is trans-
authors. However, Henschke et al. [40] established that the ferred to the wine during its aging [44].
use of S. bayanus strains can produce wines with different In relation to 1,1,6-trimethyl-1,2-dihydronaphthalene
sensory proles. In this study [40], wines made with S. (TDN), its content was higher for those wines aged in wood
bayanus contained more glycerol, acetaldehyde and suc- for a year. The presence of this compound in wines is
cinic acid, and less acetic acid and ethyl acetate than those principally related to the maturation process by carotenoid
made with S. cerevisiae. degradation [45]. Parameters such as temperature, sunlight,
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918 Eur Food Res Technol (2013) 237:905922
and storage time have a positive inuence on the content of use of nutrients or pellicular maceration conditioned their
this compound. Silva et al. [46] found a high concentration later evolution in oak cask.
for this compound in a forced aging study. The groups obtained demonstrate that the volatile
compounds studied possess sufcient explanatory power to
Cluster analysis (CA) detect the type of aging process and differentiate two yeast
strains.
The data matrix (volatile compounds) was subjected to a Thus, in order to obtain suitable classication rules for
hierarchical agglomerative cluster analysis of cases, taking assigning categories to samples, a supervised learning
the squared Euclidean distance as metric and the Ward pattern recognition method was applied. A forward step-
method as amalgamation rule. The dendrogram obtained is wise linear discriminate analysis (LDA) was carried out.
shown in Fig. 2.
Three main clusters can be appreciated: one cluster for Linear discriminant analysis (LDA)
the samples from Experiments D2 and D3 aged in oak
wood; another one for the samples from Experiments D1, LDA assumes an a priori knowledge of the number of
D4, and D5 aged in oak wood; and nally, a third one for classes and the sample class memberships. This analysis
the samples from the experiments submitted to aging in was performed according to the Wilks0 lambda statistic
stainless steel and aged with chips. Some initial samples [47] in order to choose the descriptors that best distinguish
(S0, S1, and S2) from Experiment D1 are grouped together the different classes. The so-called leave-one-out method
with those ones from Experiments D2 and D3. As can be has been employed [48]. Three possible differentiation
seen, in relation to the rst ones, those samples aged in criteria have been considered: fermentation type (Experi-
wood, a clear differentiation between wines fermented with ments from D1 to D5), time of aging (S0, S1, S2, S3, S4,
S. bayanus (Experiments D4 and D5) and those fermented and S5), and aging type (aging in oak wood, aging with
with S. cerevisiae (Experiments D2 and D3) was obtained chips, and aging in stainless steel).
(Fig. 2). Clusters were formed especially according to the The variables included in the discriminant functions
aging type (aging in wood and in stainless steel) and to the obtained according to fermentation type mainly belon-
yeast strain employed. Taking into account that only the ged to four chemical families (ethyl esters, terpenes, fur-
initial samples from Experiment D1 (S. cerevisiae without anic compounds, and acetates). For the rst function, ethyl
nutrients and pellicular maceration) aged in wood were butanoate, diethyl pentanedioate, 2-phenylethanol (with
grouped together with those ones fermented with the same negative sign) and 5-methyl-2-furfuraldehyde, and deca-
yeast strain, it could be concluded that, for these wines, the noic acid (with positive sign) were the volatile compounds
200
Distance
160
120
80
40
0
S5D2S
S4D2S
S3D2S
S2D2S
S1D2S
S5D1CH
S5D1S
S4D1S
S4D1CH
S3D1S
S1D1CH
S3D4S
S0D2S
S2D1S
S2D1S
S2D4S
S0D4S
S4D4S
S1D4S
S1D1S
S2D1CH
S0D1CH
S5D4C
S5D4C
S5D1C
S3D4C
S4D1C
S4D4C
S4D4C
S3D4C
S3D1C
S5D5C
S4D5C
S3D5C
S2D5C
S1D5C
S0D5C
S5D4S
S2D4C
S2D4C
S1D4C
S1D4C
S4D1C
S5D1C
S0D4C
S0D4C
S0D1S
S3D1C
S2D1C
S5D1C
S5D2C
S3D2C
S4D2C
S1D1C
S2D1C
S1D1C
S2D2C
S4D3C
S4D3C
S3D3C
S2D3C
S1D3C
S0D3C
S1D2C
S0D1C
S0D1C
Chips and stainless steel Oak wood. Experiments 1, 4 and 5 Oak wood. Experiments 2 and 3
123
Author's personal copy
Eur Food Res Technol (2013) 237:905922 919
Function 2
highest standardized coefcients. The scatterplot of the
1
samples onto the plane dened by the rst two discriminant
functions for fermentation type is shown in Fig. 3. As -1
can be seen in Fig. 3, clear separations among the samples
of different vinication experiments were obtained. Sam- -3
2
positive sign and diethyl succinate with negative sign) for
the rst function, and nerolidol and nerol oxide with -1
positive sign and negative sign, this last one, for the rst
and the second functions, respectively. The scatterplot of -4
the samples onto the plane dened by the rst two dis-
criminant functions for aging type is shown in Fig. 4. As -7
-8 -4 0 4 8
can be seen, samples from different aging type are Function 1
undoubtedly separated with a 99.24 % of correct classi-
cation in the check process by the leave-one-out method. Fig. 5 Volatile compounds. Linear discriminant analysis (LDA).
With regard to the criterion time of aging, mainly Scatterplot of the samples on the plane dened by the rst two
discriminant functions for the factor aging time. , S0; , S1; ,
terpenes, ethyl esters and acetates were the variables
S2; , S3; , S4; , S5
included in the two rst discriminant functions: nerol
2
phenylethyl acetate with negative sign. The scatterplot of
the samples onto the plane dened by the rst two dis-
-2 criminant functions is shown in Fig. 5. As can be observed,
Function 1 separates undoubtedly those samples with short
-6 aging (S0, S1, and S2, with negative values for this func-
tion) from those aged during a long time (S3, S4, and S5).
-10 A 98.48 % of correct classication was obtained in the
-21 -16 -11 -6 -1 4 9
Function 1 check process by the leave-one-out method.
123
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920 Eur Food Res Technol (2013) 237:905922
1,0 4
CITRIC D1S
FRUITY D1C
0,8 3 D1CH
D2S
D2C
FLORAL 2 D3C
0,6
D4S
D4C
Factor 2
Factor 2
1 D5C
0,4
DRIED FRUITS
AROMATIC INTENSITY
GENERAL IMPRESSION
0
0,2
HONEY
-1
WINE CHARACTER
0,0
SWEET
OFF-ODOUR OAK -2
-0,2
-0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 -3
-2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0
Factor 1
Factor 1
Fig. 6 Sensory analysis. Factor analysis (FA). Loadings of the
different descriptors considered on the plane dened by the rst two Fig. 7 Sensory analysis. Factor analysis. Scatterplot of the samples
factors extracted on the plane dened by the rst two factors
performed. As a previous stage, those descriptors with very sensorial characteristics. From a sensorial point of view,
low intensities (minor to 1) were excluded, i.e., herba- wines D1C and D2C, placed in the rst quadrant (Fig. 7),
ceous, young wood/sawmill, and dairy. showed more complex aromatic proles with higher
When FA was carried out, two major factors were intensities in aging and varietal aromas.
extracted, which explained 57 % of the total variability. In summary, from the results obtained, it can be
According to Fig. 6, which represents the loadings of the observed an important inuence of the three factors con-
different descriptors on the Factors 1 and 2, Factor 1 sidered, fermentation type, aging type, and aging time, on
(which accounts for 42.2 % of the variance) was correlated volatile compounds and sensorial characteristics of the
positively with general impression (loading value: 0.82). wines studied. Taking into account the global volatile
The sweet notes (especially sweet0.86and ripe fruit prole, the factor aging type has demonstrated the
0.71), a higher aromatic intensity (0.89), and the oak highest inuence on the volatile content of the wines
note due to the aging (0.71), also seem to contribute to this (Fig. 2), with wines from Experiments D4 and D5 (fer-
factor. Factor 2 (14.7 % of the explained variance) pre- mented with S. bayanus) and aged in oak wood clearly
sented high loadings for fruit (0.86), citric (0.89), and oral differentiated from those fermented with S. cerevisiae and
notes (0.73); therefore, it seems that Factor 2 could be aged in wood or stainless steel.
related to primary and variety aromas, which are fully Sensorially, wines fermented with S. Cerevisiae and
expressed after the fermentation process. aged in wood were the most valued ones.
When the samples were placed in the new plane dened
by F1F2 (Fig. 7), some comments could be done if we Conict of interest None.
take into account the interpretation of the dimensions of the
Compliance with ethics requirements This article does not con-
factors extracted. On the one hand, according to the load- tain any studies with human or animal subjects.
ings of F1, the wines located to the right (D1C, D2C, D3C,
and some of the D4C set) are the most appreciated. The
presence of both aging and sweet notes, and also a higher
aromatic intensity, can be related to its preference. On the References
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123
Ruiz-Bejarano et al. Ethyl carbamate in sweet Sherry wines 1
Abstract
Background and Aims: Ethyl carbamate (EC) is a known carcinogen that has been found in wines. The aims of this
study were: (i) to study the effect of fermentation under different enological conditions on the EC concentration in
sweet Sherry wines made from the two grape cultivars, Muscat and Pedro Ximnez, during three consecutive
vintages (20082010); and (ii) to investigate the evolution of EC concentration during ageing in oak and in stainless
steel.
Methods and Results: Before and during ageing, the wines obtained were periodically sampled, and EC content
was determined by HPLC with uorescence detection. Mean values of EC ranged from 6.16 g/L for samples after
alcoholic fermentation to 120 g/L for samples after 28 months of ageing in oak. Data were submitted for ANOVA
and cluster analysis. It has been observed that the various factors considered (vinication conditions, ageing type,
ageing time, grape cultivar and vintage) have signicant inuence on the EC concentration of the wines. The use of
Saccharomyces bayanus instead of Saccharomyces cerevisiae reduced the EC concentration signicantly. Both the use of
nutrients and skin contact with pectolytic enzymes increased EC concentration. Ageing increased the EC content for
all vinication conditions.
Conclusions: Ageing was the most signicant factor affecting EC concentration of the wines. Wines made from
Muscat, fermented with S. cerevisiae and aged in oak cask for up to 28 months presented the highest EC concentration.
Signicance of the Study: The ageing of sweet Sherry wines in oak results in a concentration of EC higher than
that from ageing of similar wines in stainless steel.
Keywords: ethyl carbamate, oak cask, stainless steel, sweet Sherry wine, yeast strain
doi: 10.1111/ajgw.12147
2015 Australian Society of Viticulture and Oenology Inc.
2 Ethyl carbamate in sweet Sherry wines Australian Journal of Grape and Wine Research 2015
in those fermented with S. montrachet. Balcerek and Szopa different enological conditions, over three consecutive vintages
(2006) found that the EC of plum mashes fermented with (20082010).
S. bayanus was lower than that of mashes fermented with other
strains of selected wine yeasts. Materials and methods
No signicant difference was found by Uthurry et al. (2006)
in the EC content of wines fermented by S. bayanus and Chemicals and reagents
S. cerevisiae at 25 and 30C and at pH 3.2 and 3.8. In these same Hydrochloric acid (37%) and absolute ethanol were obtained
studies, however, it was observed that the urea concentration from Panreac Quimica (Barcelona, Spain). Methanol (HPLC
was higher for those wines fermented by S. cerevisiae at 30C and grade) was supplied by Fisher Scientic (Loughborough,
pH 3.8, suggesting that this strain of yeast has a greater capacity England) and 1-propanol (analytical quality) was obtained from
for yielding urea and, consequently, for producing EC. Merck (Darmstadt, Germany). Ethyl carbamate and
Several preventive actions have been recommended by the 9-xanthydrol (analytical grade and a minimum purity of 98%)
US Food and Drug Administration to reduce EC in wine; these were purchased from TCI Europe (Zwyndrecht, Belgium) and
actions include control of vineyard fertilisation techniques, Aldrich (Madrid, Spain), respectively. Ultrapure water was
adjustment of nutrient content in grape musts, use of suitable obtained from a Milli-Q system (Millipore Corporation,
yeast and lactic acid bacteria strains and application of urease Bedford, MA, USA).
(Butzke and Bisson 1997, Coulon et al. 2006).
It has also been demonstrated that physicochemical condi- Samples
tions during the ageing and storage of wines noticeably inu- Grapes of the cvs Muscat and Pedro Ximnez from vintages
ence the formation of EC (Kodama et al. 1994). Larcher et al. 2008, 2009 and 2010 were obtained from a local winery in the
(2013) found that storage temperature was crucial for the for- Jerez-Xrs-Sherry DO (Denomination of Origin) region. About
mation of EC in white wines stored at 12 and 40C. Those wines 2000 kg of ripe bunches of each cultivar were dried using a
with a urea concentration higher than 20 mg/L and exposed to climatic chamber (Ibercex, Madrid, Spain) for 5 days at 40C
40C had an EC concentration that ranged from 15 to 30 g/L in and relative humidity of 10%. The bunches were distributed
less than 5 days. Similar results were obtained by Koch-Wilk uniformly inside the chamber forming a single layer. Water loss
et al. (2006) in fruit wines stored in the dark at cellar tempera- was monitored daily, and at the end of the drying process, it was
ture (610C) or in daylight at room temperature (2030C). In about 35%; after drying, grapes were 45Brix.
this study, these latter conditions signicantly increased the EC Grapes were de-stemmed, crushed and pressed with a ver-
concentration. tical press. The highest pressure in each of the three cycles was
In Andalusia, Spain, sweet wines are obtained from grapes 30 MPa. For those experiments with skin contact (pellicular
dried by direct exposure to the sun. These wines are elabo- maceration), grapes were crushed without de-stemming and
rated in Malaga, Montilla-Moriles and Jerez by a traditional then submitted to maceration. In the next stage, the pH of the
method, using the grape cvs Muscat and Pedro Ximnez. This must was adjusted to 3.5 with tartaric acid (Agrovin, Alcazar de
traditional process is frequently affected adversely by the San Juan, Spain). The concentration of total sulfur dioxide was
attack of insects and by the growth of fungi, which can give also set at 120 mg/L by adding potassium metabisulte
rise to the formation of toxins in the wine (Valero et al. 2008, (Agrovin). Five experimental conditions (in duplicate) of
Ruiz et al. 2010). In this process, the grapes attain a sugar vinication were tested Table 1):
concentration of more than 300 g/L and are crushed and
Experiment E1 the must was fermented with a S. cerevisiae
pressed in a vertical press. Subsequently, the musts, with high
yeast (40 g/hL, Lalvin D254, Lallemand, Edwardstown, SA,
sugar content and thus difcult to ferment, are partially forti-
Australia) at room temperature (below 30C).
ed with ethanol to ensure that the wine will contain at least
Experiment E2 the must was fermented as in E1, but with
1518% alcohol.
the addition of yeast nutrients (10 g/hL diammonium phos-
In the production of fortied wines, the fortication may
phate) (Actimax Plus+, Agrovin).
aggravate the problem of urea excretion by yeast. Urea is often
Experiment E3 the fermentation conditions were as in E1;
formed during the early and middle stages of fermentation, with
however, the crushed grapes were subject to a pre-
subsequent generations of yeast utilising it during the later
fermentation cold maceration for 24 h at 4C using 3 g/hL of
stages. If the fermentation process is stopped early by fortica-
pectolytic enzymes (Enozym Arome, Agrovin); after which,
tion, wines with higher urea concentration may be obtained
the macerated grapes were pressed in a vertical press under
(Daudt et al. 1992). In the production of sweet Sherry wines,
the same conditions previously mentioned.
after fermentation and fortication, the young sweet wine is,
Experiment E4 must was fermented at low temperature
generally, aged in oak following a system of dynamic oxidative
(below 10C) with S. bayanus (40 g/hL, Uvaferm 43,
ageing (Casas 1985).
Lallemand).
In recent years, a possible alternative to this traditional
Experiment E5 the crushed grapes were subject to a pre-
sun-drying technique has appeared. It is being used for the
fermentation cold maceration for 24 h at 4C using 3 g/hL of
drying of horticultural products and consists of the removal of
pectolytic enzymes (Enozym Arome, Agrovin); after which,
water from the grapes by means of forced convection with hot
the macerated grapes were pressed in a vertical press under
air in drying chambers (Vega-Mercado et al. 2001). This drying
the same conditions previously mentioned; the must was
system avoids the problems mentioned above involving insects
fermented with S. bayanus at room temperature.
and fungi, which generate a high level of ochratoxin A and
cause a signicant loss of product during the sun drying (Ruiz When the sugar concentration of the fermenting must was
et al. 2010). about 90100 g/L, the fermentation was stopped by adding
The objective of our work was to study the evolution, under alcohol up to 1718% (v/v). Then, the sweet wines obtained
two types of ageing (oak and stainless steel), of the EC concen- were aged in 30-L medium-toasted oak casks (samples E1C to
tration of sweet Sherry wines obtained from the cvs Muscat and E5C) and in 10-L stainless steel vessels (samples E1S, E2S and
Pedro Ximnez dried in drying chambers and fermented under E4S). During this ageing period, wines were stored in the same
Table 1. Experimental design for determining the effect of must handling, fermentation conditions and ageing on the ethyl carbamate
concentration of wine.
rt, room temperature (<30C), lt, low temperature (<10C). Addition of 10 g/hL diammonium phosphate. Must held at 4C for 24 h with the addition of pectic
enzyme preparation.
room at approximately 20C. Sampling commenced at the end and equilibrated with 39% Milli-Q water for 2 min in order to
of fermentation (S0d) and continued periodically for 28 months prepare the system for the next sample. The volume of sample
(S20d, 20 days; S40d, 40 days; S2, 2 months; S4, 4 months; S7, injected was 60 L. A Waters 2475 scanning uorescence detec-
7 months; S8, 8 months; S12, 12 months; S18, 18 months and tor (FLD) was used. The wavelengths of excitation and emission
S28, 28 months). Samples were stored at 4C until they were were 233 and 600 nm, respectively. The data acquisition time
analysed. for each of the samples was set at 37 min; the software
employed for the recording and processing of the data was
Analysis of ethyl carbamate Empower Pro 2002 (Waters Corporation).
The method chosen for the determination of EC in wines, A calibration curve was prepared with a standard solution of
described by Madrera and Valles (2009), is based on an initial EC at a concentration ranging from 10 to 350 g/L. All solutions
derivatisation followed by separation by HPLC with uores- were prepared in duplicate, in a 17% v/v hydro-alcoholic
cence detection. This methodology was originally applied to matrix and were treated in the same way as samples.
cider spirits, so some modications were made. The calibration curve obtained was linear over the range,
with a regression coefcient of 0.999. The limit of detection
[three times the relative standard deviation (RSD) of the ana-
Sample derivatisation. The stability of xanthylEC was lytical blank values calculated from the calibration curve] and
tested in alcoholic solutions of variable alcohol concentration. A limit of quantitation (10 times the RSD of the analytical blank
solution of 250 g/L of EC was prepared in a matrix of synthetic values calculated from the calibration curve) were established at
wine: Milli-Q water with 3 g/L tartaric acid and an alcohol 2.69 and 8.98 g/L, respectively.
concentration of 17, 20 and 40% v/v, was analysed at intervals
from 30 min to 11 h and 30 min after the reaction with
9-xanthydrol started.
Statistical treatments
Statistical analyses were carried out with Statgraphics Centu-
After modifying and adapting the methodology to the
rion, version 15.0 (Statpoint Technologies, Warrenton, VA,
wine samples studied in this work, a solution of 0.02 mol
USA) for Windows XP. The possible signicant difference among
9-xanthydrol in 1-propanol and 1.5 mol HCl in ethanol was
samples according to the different factors considered in this
prepared. Wine samples were diluted in ethanol in a ratio 83:17
study was established by ANOVA. Furthermore, cluster analysis
(v/v) and ltered through a 0.45-m lter syringe (Millipore).
(CA) was carried out with the aim of highlighting the similarity
Diluted sample (1 mL), 0.2 mL of xanthydrol solution and
of the samples and the main contributors to the variance found
0.1 mL of 1.5 mol HCl were added to a chromatographic vial
among them.
and mixed. After waiting for 30 min to ensure the reaction was
complete, the mixture was injected into the HPLC system.
Results and discussion
Analysis by HPLC scanning uorescence detector. The Inuence of alcohol concentration on the determination of EC in
HPLC system used was a Waters Alliance 2695 (Waters Cor- sweet wines
poration, Milford, MA, USA) with a 110A C18 Gemini reverse Several studies have been undertaken of the effect of pH and
phase column of 250 mm length 3.0 mm i.d., particle size alcohol concentration on the stability of xanthylEC. Madrera
of 5 m (Phenomenex, Torrance, CA, USA) and a security and Valles (2009) found that the analytical signal decreased on
guard Gemini C18 4 2.0-mm cartridge. The column tempera- average by 1% for each increase in alcohol concentration within
ture was maintained at 35C. Chromatographic conditions, the studied range (from 20 to 65%). Ajtony et al. (2013),
gradient and ow, injection volume and temperature applied observed that xanthyl-EC was pH dependent, with low pH
to the chromatographic column (from 30 to 40C) were values favouring its decomposition. We have examined the
optimised to obtain the best resolution of xanthylEC (data inuence of variable alcohol concentration on the stability of
not shown). xanthylEC, using a strong HCl medium (pH value at about
The binary system phases were Milli-Q water and methanol 0.94). Our results were consistent with those described by
with a ow rate of 0.4 mL/min. The gradient used was as Madrera and Valles (2009). Matrix wine with 17 and 20% (v/v)
follows: 0 min, 39% Milli-Q water; 25 min, 39% Milli-Q water; ethanol showed a greater response than wine with 40% (v/v)
and 26 min, 0% Milli-Q water. Finally, the column was washed due to the inuence of the alcoholic strength on nucleophilic
with 100% methanol for 10 min at a ow rate of 0.8 mL/min substitution (data not shown). The xanthylEC compound,
Table 2. Effect of ageing 2008 vintage Muscat wines in stainless steel tanks and in oak casks.
0 Days 10.2 2.1 11.0 3.2 40.8 1.7 n.d. 13.2 0.7
20 Days 19.5 6.1 30.3 8.6 48.6 1.2 23.9 1.4 17.2 1.2
40 Days 22.2 7.1 43.7 20.2 57.0 0.3 34.1 6.6 25.8 0.3
2 Months 26.2 8.1 50.3 13.6 85.7 2.9 38.9 3.6 24.7 0.9
4 Months 38.8 11.5 71.1 17.0 103 3 57.6 7.0 37.9 1.2
7 Months 51.9 22.2 74.1 18.7 109 2 44.3 25.8 45.2 0.7
8 Months 59.4 18.1 86.1 21.3 123 1 73.0 14.1 57.9 0.3
12 Months 67.2 18.8 100 19 146 2 60.5 27.7 63.7 0.3
18 Months 74.6 18.7 121 10 182 1 94.7 15.7 67.0 1.1
28 Months 96.0 33.8 147 16 194 6 106 9 125 2
Aged in stainless steel tanks and oak casks, n = 8. Only aged in oak casks, n = 4. Values are means standard deviation. E1, must
fermented at room temperature with Saccharomyces cerevisiae; E2, must fermented as in E1 but with addition of 10 g/L diammonium
phosphate; E3, must subjected to skin contact for 24 h at 4C with added pectic enzyme preparation, pressed and fermented as in E1; E4,
must fermented with S. bayanus at <10C; E5, must treated as in E3 and fermented with S. bayanus at room temperature. n.d., not
detected.
ANOVA. For each row, different letters show a signicant difference at P < 0.01 (Tukeys test). Initial: S0d; 20 days: S20d; 40 days: S40d; 2 months: S2; 4 months: S4; 7 months: S7; 8 months: S8; 12 months: S12; 18 months: S18; 28
months: S28. E1, must fermented at room temperature with Saccharomyces cerevisiae; E2, must fermented as in E1, but with an addition of 10 g/L diammonium phosphate; E3, must subjected to skin contact for 24 h at 4C with added
Table 3. Mean ethyl carbamate concentration and standard deviation found for each factor considered (vintage, grape cultivar, ageing time, ageing type and vinication conditions). For each factor, all
119 37f
wines obtained from the cvs Muscat and Pedro Ximnez fermented
under different enological conditions, over three consecutive vin-
28
tages (20082010), and aged in oak casks and stainless steel tanks
for 28 months.
97.7 35.7e.f
Factors F value P value
47.8 32.5a
Type of vinication 77.21 0.0000*
Type of ageing 54.23 0.0000*
pectic enzyme preparation, pressed and fermented as in E1; E4, must fermented with S. bayanus at <10C; E5, must treated as in E3 and fermented with S. bayanus at room temperature.
E5
12
43.6 33.9d
33.7 24.3b
E4
47.3 30.1b
(P < 0.01).
The results obtained from the study of comparison of means
E3
using the Tukeys test (P < 0.01) for all signicant factors are
4
80.5 39.0b
54.4 44.9a
50.6 30.2a
57.4 42.7a
(Days)
E1
The year 2010 exhibited a signicantly lower EC concentra- between the EC concentration for these two experiments
tion than that of 2008 and 2009. Martnez-Pinilla et al. (2013) reveals the important inuence of the yeast strain on the for-
observed a clear inuence of the vintage factor on the amino mation of EC.
acid concentration for several red varietal wines, and obtained a Experiments E2 and E1 were fermented under the same
clear separation of wines by vintage according to this concen- conditions, but for E2, which had a higher EC concentration,
tration. Climatic conditions appear to have a clear effect on nutrients were added. Several previous studies have demon-
amino acid concentration of grape must (Ortega-Heras et al. strated that the addition of diammonium phosphate has a sig-
2014), and therefore a signicant inuence on EC concentra- nicant inuence on the EC concentration of the wine obtained
tion of wine. In the Jerez-Xrs-Sherry DO, the total precipita- (Henschke and Ough 1991, Shinohara and Yanagida 1999,
tion from January to October for the vintages 2008, 2009 and Adams and Van Vuuren 2010).
2010 was 343, 275 and 601 L/m2, respectively (Ministerio de Results obtained from the vinication under different con-
Agricultura, Alimentacin y Medioambiente 2014). For Verdejo ditions, as described for each experiment, show that the use of
grapes, Ortega-Heras et al. (2014) observed that a warmer and S. bayanus instead of S. cerevisiae signicantly reduced the EC
drier year, with lower rainfall, produced grapes with higher concentration, and that both the use of nutrients and skin
amino acid concentration. Therefore, the notably higher pre- contact with pectolytic enzymes increased EC concentration.
cipitation for the vintage 2010 could explain the lower EC Finally, the type of ageing inuences the EC concentration,
concentration found for the wines of this vintage; however, a with signicantly higher values for EC obtained for those
more thorough study needs to be made in order to conrm this wines aged in oak casks (Tables 3,4). This signicant difference
observation. could be explained by possible microbiological modications,
When the inuence of the type of vinication (experiments) favoured under the low aeration that takes place through the
is considered, it can be seen (Tables 2,3) that the lowest EC pores of the oak cask (Blouin and Peynaud 2005). This would
concentration corresponds to E4, whereas the highest value increase some of the precursors of EC for those wines aged in
corresponds to E3. Experiment E4, fermented with S. bayanus, wood (Liu et al. 1994). Depending on the bacterial strain,
was the only experiment that was carried out at low tempera- citrulline and carbamyl phosphate can be formed from arginine
ture (<10C) without the addition of nutrients and pectolytic and their subsequent reaction with ethanol, during a specic
enzymes. Ough et al. (1991) observed that the employment of period of time and under a given temperature, leads to the
low temperature during alcoholic fermentation produced wines formation of EC. In relation to this, the organic acid content for
with a lower urea concentration; and the lower capacity of all wines was measured according to Guilln et al. (1998).
S. bayanus for EC production has been previously observed by Data for the organic acid concentration (malic acid, lactic
several authors (Daudt et al. 1995, Balcerek and Szopa 2006, acid and acetic acid) were submitted to ANOVA considering
Uthurry et al. 2006). In addition, several studies have demon- type of ageing as the independent variable. For all of these
strated that the application of pectolytic enzymes in the process acids, a signicant difference was found between ageing in oak
of skin contact modies the composition of musts, increasing cask and ageing in stainless steel. Lower acetic and lactic acid
the concentration of amino acids (Kishkovskii et al. 1974, values were found for samples aged in stainless steel (data not
Aldave 1992, Bosso and Ponzetto 1994). That nding is in shown). A previous study on the volatile content of some of
agreement with the high EC concentration found for experi- these wines showed that some ethyl esters had signicantly
ment E3, macerated with pectolytic enzymes and fermented higher values for those wines aged in oak casks (Ruiz-Bejarano
with S. cerevisiae at room temperature (<30C). Experiment E3 et al. 2013). This suggests that possible microbiological activity
(S. cerevisiae) and experiment E5 (S. bayanus) were fermented has taken place during ageing in oak. However, further studies
under the same vinication conditions. The difference found should be made in order to clarify this point.
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