Professional Documents
Culture Documents
TESIS DOCTORAL
Ciudad Real, 2008
UNIVERSIDAD DE CASTILLA-LA MANCHA
Facultad de Ciencias Qumicas
Departamento de Qumica Analtica y Tecnologa de los Alimentos
VB
VB
CERTIFICA:
VB
Pgina
Papel de los compuestos voltiles en la calidad del aceite de oliva virgen extra ... 14
Contribucin de los compuestos fenlicos a la calidad del aceite de oliva virgen ... 23
1.4. Factores que influyen en la composicin voltil y fenlica del aceite de oliva virgen de
calidad ............................................................................................................................... 26
Influencia de aspectos agronmicos y pedoclimticas ............................................ 26
3.2. Planificacin experimental del riego en olivares jvenes de cubierta incompleta (Olea
3.3. Planificacin experimental del estudio de componentes minoritarios del fruto (Olea
3.4. Planificacin experimental del estudio de los componentes minoritarios del aceite de
I
ndice
Effect of cultivar and ripening on minor components in Spanish olive fruits and their
Phenolic and volatile compounds of extra virgin olive oil (Olea europaea L. Cv.
Cornicabra) with regard to fruit ripening and irrigation management . 103
II
ndice
Virgin olive oil and olive fruit minor constituents as affected by irrigation
management based on ETc, SWP and TDF in medium-density young olive orchards
Effect of malaxation conditions on phenolic and volatile profiles of olive paste and
their corresponding virgin olive oils (Olea europaea L. cv. Cornicabra) . 135
III
IV
Abreviaturas
ABREVIATURAS UTILIZADAS
3,4-DHPEA hidroxitirosol
3,4-DHPEA-AC acetato de hidroxitirosol
3,4-DHPEA-EA forma aldehdica del cido elenlico unida a hidroxitirosol
3,4-DHPEA-EDA forma dialdehdica del cido elenlico unida a hidroxitirosol
AAT alcohol acil transferasa
ADH alcohol deshidrogenasa
AOV aceite de oliva virgen
C16:0 cido palmtico
C18:0 cido esterico
C18:1 cido olico
C18:2 cido linolico
C18:3 cido linolnico
DAD detector de diodos en lnea
DO Denominacin de Origen
DOC Denominacin de Origen Controlada
DOP Denominacin de Origen Protegida
ETc evapotranspiracin del cultivo
FAO Food and Agriculture Organization
FCP perfil de libre eleccin
FID detector de ionizacin en llama
GC cromatografa de gases
GC-MS cromatografa de gases-espectrometra de masas
GC-O cromatografa de gases-olfatometra
HPL hidroperxido liasa
HPLC cromatografa lquida de alta eficacia
IM ndice de madurez
IP ndice de perxidos
LC-MS cromatografa lquida-espectrometra de masas
LOX lipoxigenasa
MDS escalado multidimensional
MOD mxima oscilacin del dimetro de tronco
MS espectrometra de masas
MUFA cidos grasos monoinsaturados
p-HPEA tirosol
p-HPEA-AC acetato de tirosol
p-HPEA-EA forma aldehdica del cido elenlico unida a tirosol
p-HPEA-EDA forma dialdehdica del cido elenlico unida a tirosol
PAL L-fenilalanina amonio liasa
PCA anlisis de componentes principales
V
Abreviaturas
VI
Publicaciones Cientficas
Salvador, M.D., Gomez-Rico, A., Inarejos A.M., Fregapane, G. Minor components with
nutritional value, antioxidant activity and organoleptic properties status through the market
period of commercial olive oil types, in Special Issue of Italian Journal of Food Science- Shelf-
life International Meeting. Ed. Chiriotti/GSICA/DOFATA. Pg. 337-342 (2006) ISSN 1120-1770
Aurora Gmez-Rico, M. Desamparados Salvador, Giuseppe Fregapane. Virgin olive oil and
olive fruit minor constituents as affected by irrigation management based on ETc, SWP and TDF
VII
Publicaciones Cientficas
in medium-density young olive orchards (Olea europaea L. cv. Cornicabra and Morisca). Food
Research International, (enviado).
VIII
1. INTRODUCCIN GENERAL
1. Introduccin General
El cultivo del olivo se encuentra localizado en los pases de la cuenca mediterrnea, que
cuenta con el 98% del total de la superficie olivarera mundial, aunque durante los ltimos aos
se ha extendido ms all de las zonas tradicionales de cultivo, como son los Estados Unidos,
Sudfrica, Sudamrica y Australia. De la misma forma, la mayor parte de la produccin mundial
de aceite de oliva se localiza en los pases mediterrneos, siendo Espaa, Italia, Grecia y los
pases del Magreb los principales productores, con un aporte medio de aproximadamente 2,5
toneladas anuales (C.O.I, 2005). En Espaa se localiza alrededor del 39% de la produccin
mundial de aceite de oliva y una cuarta parte de la superficie olivarera (C.O.I, 2005), lo que nos
da una clara idea de la gran relevancia social y econmica de este producto en nuestro pas.
De hecho, el consumo de este aceite vegetal constituye la principal fuente de lpidos utilizada
en la dieta mediterrnea, aunque ste se ha visto incrementado durante los ltimos aos en
pases fuera de este rea, debido por una parte a sus caractersticas sensoriales nicas y por
otro lado, a que recientes investigaciones han demostrado que el consumo habitual de aceite
de oliva virgen supone un aporte continuo de compuestos antioxidantes, descritos como
sustancias protectoras contra enfermedades neuro-degenerativas, problemas cardiovasculares
y algunos tipos de tumores (Visioli et al., 1995; Owen et al., 2000b; Rocca et al., 2002, etc.).
Para mantener o incluso incrementar el consumo de este producto a nivel mundial es necesario
ampliar los mercados, para lo cual se hace necesario proporcionar a los consumidores, no slo
aceites de oliva virgen de alta calidad, sino tambin informacin nutricional relevante sobre los
beneficios del consumo de este aceite respecto del resto de aceites vegetales. Para ello, el
nfasis ya realizado sobre los beneficios proporcionados por el aceite de oliva virgen debido a
su alta composicin en cidos grasos monoinsaturados, necesita ser reforzado con informacin
3
1. Introduccin General
El principal factor que determina el grado de preferencia del aceite de oliva virgen por parte de
los consumidores es la calidad sensorial del mismo, y que en este producto depende
bsicamente de las caractersticas organolpticas que son percibidas por el consumidor como
un conjunto de sensaciones valoradas a travs del olfato y el gusto (Morales et al., 2000).
Existen multitud de compuestos orgnicos que determinan las caractersticas organolpticas
del aceite de oliva virgen, siendo los compuestos fenlicos y voltiles los principales
responsables de las mismas, y que son retenidos en l debido a que durante su proceso de
elaboracin solamente se utilizan procedimientos mecnicos, sin posteriores operaciones de
refinado.
Los compuestos voltiles son los principales responsables del aroma del aceite de oliva virgen
y estn relacionados con las notas aromticas frescas y verdes propias de la materia prima de
la que procede. Por otro lado, los compuestos fenlicos han sido relacionados con el sabor del
aceite, en particular con los atributos sensoriales positivos de amargo y picante (Morales et
al., 2000; Angerosa et al., 2000c), adems de presentar un importante carcter antioxidante y
ser considerados compuestos bio-activos, lo que los hace tambin responsables, entre otros,
de la estabilidad oxidativa y del valor nutricional del producto (Shahidi et al., 1992; Servili et al.,
1996). Por lo tanto, la presencia final de estos compuestos minoritarios en el aceite de oliva
virgen representan en gran medida su aceptacin sobre otras grasas vegetales comestibles
presentes en el mercado.
4
1. Introduccin General
Por todo ello, en el presente trabajo de investigacin se plantea el estudio de distintos factores
agronmicos y tecnolgicos que afectan al contenido y composicin de estos componentes
minoritarios del aceite de oliva virgen, como son el cultivar de origen, el grado de maduracin
del fruto, las tcnicas de riego aplicadas en el olivar, as como la influencia de determinadas
variables durante el proceso tecnolgico de batido de la pasta de aceituna.
5
1. Introduccin General
El principal objetivo del control analtico de los aceites vegetales comestibles establecido segn
la reglamentacin internacional del Codex Alimentarius y la Comisin Europea es el de
determinar la calidad, la pureza y la posible presencia de contaminantes, y est principalmente
orientada hacia la clasificacin de los aceites comestibles en diversas categoras comerciales.
De esta forma, la evaluacin de la calidad y la pureza de los aceites de oliva virgen se lleva a
cabo mediante la medida de una serie de parmetros analticos orientados principalmente
hacia la deteccin de posibles fraudes, adulteraciones o degradacin en el producto. Y por
tanto, los lmites establecidos por la reglamentacin vigente estn orientados bsicamente
hacia la clasificacin del aceite de oliva dentro de la categora comercial correspondiente, sin
atender explcitamente los atributos de calidad positivos y especficos de este apreciado
producto.
De hecho, los criterios de calidad y pureza de los distintos tipos de aceite de oliva establecidos
por la Reglamentacin CEE 2568/91 y sus posteriores modificaciones (virgen extra, virgen,
lampante,...) estn basados principalmente en parmetros qumicos como son el grado de
acidez, el ndice de perxidos, las caractersticas de absorcin en el UV, adems de la
valoracin sensorial, entre otros parmetros de pureza. An as, la cuantificacin de la mayora
de estos parmetros qumicos en muestras de aceite de oliva, que han sido obtenidos a partir
de frutos en buen estado y mediante procesos de extraccin adecuados, no excede los lmites
mximos establecidos por la reglamentacin vigente, siendo la evaluacin sensorial del
producto la herramienta ms importante para determinar la calidad final del producto y que
permite en muchos casos establecer el precio del aceite de oliva.
Asimismo, se debe destacar que son las caractersticas organolpticas del producto las que
determinan el grado de preferencia de los consumidores, ya que la eleccin de los alimentos
que consumimos se basa primordialmente en los atributos que percibimos a travs de nuestros
sentidos. Los atributos sensoriales que consideramos principalmente en los alimentos son la
apariencia, el color, la textura, la forma y el tamao, la viscosidad, las sensaciones tctiles y
kinestsicas, el olor y el sabor, adems de sensaciones quimioestsicas, como una respuesta
combinada de la sensacin cutnea, trmica y de los estmulos dolorosos producidos por
determinadas sustancias qumicas irritantes (Angerosa, 2000a).
En el caso del aceite de oliva virgen, como alimento lquido que es, el nmero de atributos que
determinan su calidad sensorial y que son valorados por los consumidores quedan
bsicamente reducidos al color, olor y sabor, adems de ciertas sensaciones quimioestsicas,
como el picor y la astringencia tpicas de este producto (Morales et al., 2000).
6
1. Introduccin General
A lo largo de las dos ltimas dcadas, numerosas investigaciones han tratado de identificar
cualitativa y cuantitativamente los compuestos orgnicos voltiles y no-voltiles responsables
del flavor del aceite de oliva virgen y relacionarlos con los atributos sensoriales que percibe el
ser humano, sobre todo aplicando procedimientos estadsticos multivariantes, con lo que se ha
conseguido relacionar determinados compuestos con las notas sensoriales que generan
(Morales et al., 1993; Morales et al., 1995; Aparicio et al., 1996; Andrewes et al., 2003).
Algunos estudios han destacado la estrecha relacin existente entre determinados compuestos
fenlicos, principalmente los derivados secoiridoideos del hidroxitirosol y tirosol, con la
intensidad del atributo amargo del aceite de oliva virgen, el cual se considera la percepcin
gustativa ms importante del producto, adems de ser responsables de las sensaciones
7
1. Introduccin General
La evaluacin sensorial del aceite de oliva virgen est ampliamente descrita en los
Reglamentos CEE 2568/91 y CE 640/2008. Para llevar a cabo la valoracin de los atributos
sensoriales del aceite de oliva virgen se ha generado un vocabulario especfico que permite
describir el flavor de este producto, basado en la metodologa propuesta por el COI en 1987.
Por una parte, se distinguen los atributos positivos: frutado, amargo y picante, definidos
como:
8
1. Introduccin General
Por otro lado, tambin se han descrito una serie de atributos negativos, con el fin de describir
los posibles defectos que se pueden encontrar en los aceites de oliva de baja calidad, y que se
deben a la presencia de ciertos compuestos voltiles indeseables que pueden aparecer si el
aceite se ha obtenido a partir de frutos daados y/o almacenados durante largo tiempo a
temperatura ambiente y altas humedades relativas, o bien si se ha producido un procesado
deficiente de la materia prima o a un almacenamiento inadecuado del producto final. Estos
atributos sensoriales negativos se encuentran ampliamente descritos en el Reglamento CE
640/2008 y se denominan con los siguientes descriptores: atrojado/borras, moho-humedad,
avinado-avinagrado/cido-agrio, metlico, rancio, cocido o quemado, heno-madera, basto,
lubricante, alpechn, salmuera, esparto, tierra, gusano, pepino y madera hmeda.
La primera aplicacin del anlisis sensorial a este producto fue introducida por el COI en 1987
y posteriormente se incorpor en el Reglamento de la Comunidad Econmica Europea (CEE)
2568/91, con la finalidad de detectar posibles defectos sensoriales y clasificar los aceites de
oliva virgen en diversas categoras comerciales (Virgen extra, Virgen, Virgen corriente y
Virgen lampante), en funcin de la intensidad de los defectos y atributos sensoriales positivos.
El mtodo empleaba una escala estructurada de 0 a 5, en la cual el 0 indicaba ausencia total
y el punto 5 indicaba percepcin extrema, adems el catador deba proporcionar una
puntuacin total del aceite en una escala de 9 puntos. En este reglamento se indican, segn las
directrices establecidas por el COI, las caractersticas de la sala de cata y las cabinas que se
deben utilizar. Tambin se describe en detalle las pautas a seguir en el proceso de seleccin y
entrenamiento de los catadores, adems de las normas y condiciones necesarias para la
realizacin del ensayo sensorial, el empleo de una copa normalizada para la degustacin y una
descripcin detallada de las caractersticas generales en las que debe efectuarse la valoracin
sensorial.
9
1. Introduccin General
defectos que se pueden encontrar en el aceite y del atributo frutado obtenidas por el panel de
cata integrado como mnimo por ocho catadores expertos. Remarcar que con la nueva norma,
el formulario a emplear mantiene la misma estructura, a excepcin de la incorporacin de una
casilla en la que el catador seala si el atributo frutado presenta un carcter de verde o
maduro y el agrupamiento de los defectos atrojado y borras dentro de la misma percepcin
olfativa.
En los casos en los que el anlisis sensorial del aceite de oliva virgen tiene por finalidad una
diferenciacin en el producto, como es el caso de aceites acogidos a una Denominacin de
Origen Protegida (DOP) o Controlada (DOC), cuya clasificacin est ligada a factores
geogrficos y varietales, el uso de la ficha de cata empleada actualmente en la norma CE
640/2008 no ofrece una informacin completa sobre el perfil que incluya posibles notas que
pueden describirlo y caracterizarlo. Para llevar a cabo esta evaluacin sensorial el COI ha
adoptado en la norma COI/T.20/Doc n 22 (Noviembre de 2005) un mtodo especfico para la
valoracin organolptica de un aceite de oliva virgen extra que opta a una denominacin de
origen (DO). La norma propone, basado en un mtodo de perfil, la seleccin de descriptores
caractersticos de los productos a partir de una amplia lista en los que se incluyen sensaciones
olfativas evaluadas por va nasal directa o retronasal como almendra, manzana, hierba, tomate,
alcachofa, verde, etc., sensaciones gustativas como amargo y dulce, la persistencia de la
sensacin percibida por va retronasal y sensaciones tctiles como la fluidez y el picante.
La fraccin voltil del aceite de oliva virgen est compuesta principalmente por numerosos
aldehdos, alcoholes, steres, cetonas e hidrocarburos, la mayora de los cuales han sido
identificados mediante tcnicas de CG-MS (Angerosa, 2000a).
Por otro lado, la fraccin voltil de los aceites de oliva que presentan defectos sensoriales se
caracteriza por la menor concentracin o ausencia total de los productos de la ruta LOX, as
como por la elevada presencia de aldehdos insaturados de siete a once tomos de carbono
10
1. Introduccin General
(C7-C11) (Solinas et al., 1987a y 1988), dienales de seis a diez tomos de carbono (C6-C10)
(Aparicio et al., 2000), compuestos carbonlicos de ocho tomos de carbono (C8) (Angerosa et
al., 1999b) o aldehdos y alcoholes ramificados de cinco tomos de carbono (Angerosa et al.,
1996a), todos ellos caracterizados por poseer umbrales de deteccin bajos y por ser
responsables de los atributos sensoriales negativos.
Aunque existen una serie de voltiles presentes en el aceite de oliva virgen que se encuentran
inicialmente de forma natural en el fruto intacto, los principales compuestos responsables del
aroma de este producto son metabolitos secundarios, cuya formacin comienza
inmediatamente despus de la ruptura del fruto y contina durante el batido de la pasta de
aceituna, debido a la actividad de diversas enzimas que actan en presencia de oxgeno
(Tressl et al., 1973; Olias et al., 1993) y que utilizan como principales sustratos los cidos
grasos y algunos aminocidos, como la leucina, la isoleucina y la valina.
Las principales rutas bioqumicas implicadas en la sntesis de los compuestos voltiles del
aceite de oliva virgen de calidad son las que se resumen a continuacin:
11
1. Introduccin General
12
1. Introduccin General
alcohol aciltransferasa (AAT). Esta enzima no acta sobre alcoholes de cadena corta,
como el metanol y el etanol, mostrando una actividad baja sobre el butanol y el 3-
metilbutanol (Sanchez et al., 2000), por lo que la falta de actividad de esta enzima sobre
los alcoholes de cadena corta explica le escasez de los acetatos de hexilo y hexenilo
que se observa en el aroma del aceite de oliva virgen (Morales et al., 1995), a pesar de
la alta concentracin relativa de los correspondientes sustratos (hexan-1-ol, Z-3-hexen-
1-ol y E-2-hexen-1ol) (Salas, 2004). An as los mayores valores de actividad
enzimtica se han obtenido al utilizar como sustratos el hexan-1-ol y el Z-3-hexen-1-ol
(Salas, 2004).
- Existe una rama adicional en la ruta LOX que tiene como sustrato especfico el
hidroperxido -13 del cido linolnico, a travs de la cual se generan dmeros de
penteno y alcoholes C5, como el 1-penten-3-ol y el 2-penten-1-ol. La posterior actividad
de la ADH podra ser la responsable de la formacin de los correspondientes aldehdos
C5 que tambin se han encontrado en el aroma del aceite de oliva virgen (Angerosa et
al., 1998a).
ACETATO DE
CIDO HEXANAL HEXAN-1-OL
HEXILO
LINOLEICO ADH AAT
CIDO
LINOLNICO Z-3-HEXENAL
RADICAL
13-ALCOXI
ACETATO DE
Z-3-HEXEN-1-OL Z-3-HEXENILO
RADICAL ADH AAT
PENTENO
DMERO 2-PENTEN-1-OL
PENTENO 1-PENTEN-3-OL
2-PENTENAL
1-PENTEN-3-ONA
13
1. Introduccin General
Ciertos voltiles presentes en el perfil aromtico del aceite de oliva virgen proceden de la
estructura ramificada de aminocidos, como la valina, leucina e isoleucina, generando
respectivamente los aldehdos ramificados, 2-metilpropanal, 3-metilbutanal y 2-metilbutanal, a
travs de una serie de transformaciones bioqumicas (Wyllie et al., 1995). La posterior
actuacin de la alcohol deshidrogenasa y la alcohol aciltransferasa supone la formacin de los
correspondientes alcoholes y steres ramificados (Van de Hijden et al., 1996; Wyllie et al.,
1996).
Todos los componentes de la fraccin voltil del aceite de oliva virgen no presentan la misma
influencia en la percepcin sensorial del aroma del producto, ya que, aunque todos estos
compuestos presentan unas caractersticas comunes, como son una masa molecular relativa
baja (por debajo de los 300 Da), un carcter polar y ser suficientemente liposoluble, lo cual
permite por un lado su difusin en el mucus que recubre las clulas epiteliales olfativas y su
posterior disolucin en la membrana lipdica que rodea los receptores proteicos, la capacidad
olorosa depende adems de otros factores, como son:
14
1. Introduccin General
extra, as como los umbrales de deteccin determinados por diversos autores y recogidos en
bibliografa.
En estos trabajos se puede observar que para un mismo compuesto voltil se han determinado
diferentes umbrales de deteccin, lo cual puede deberse por una parte a que la matriz oleosa
empleada en la determinacin del mismo ha sido diferente, utilizndose aceite de girasol
refinado (Aparicio et al., 1998; Reiners et al., 1998), aceite vegetal refinado (Aparicio et al.,
2002), aceite de oliva refinado (Morales et al., 2005) o parafina. Tambin se conoce el hecho
de que las sustancias proteicas, carbohidratos u otros componentes minoritarios pueden alterar
la intensidad aromtica de las sustancias voltiles debido a la formacin de complejos
intramoleculares o a procesos de adsorcin (Jung et al., 2000). Y por ltimo, se debe tener
tambin en cuenta que la determinacin de este valor umbral depende en gran parte de la
sensibilidad y entrenamiento de los catadores y que puede ser variable entre distintos sujetos.
Otro hecho que destaca es que en ocasiones las notas aromticas descritas por los catadores
para un determinado compuesto voltil puedan ser ligera o bastante diferentes, como ocurre en
el caso del hexan-1-ol (ver Tabla 1.1). Angerosa et al. (2002) argumenta que puede deberse a
la dificultad de conciliar las sensaciones percibidas por los distintos paneles sensoriales, a
pesar de utilizar un mismo vocabulario consensuado.
No obstante, queda suficientemente aceptado que los aldehdos y alcoholes C6 son los
principales responsables de las notas sensoriales verdes y herbceas perceptibles en los
aceites de oliva virgen obtenidos a partir de frutos verdes o en envero, mientras que los steres
C6 estn ligados a las sensaciones frutadas y florales del producto. Por otro lado, los
compuestos C5 tambin contribuyen de forma positiva al flavor del aceite de oliva virgen, al
estar relacionados con las notas sensoriales verdes, adems de estar involucrados en la
percepcin del atributo picante, como es el caso del pentan-1-ol y 1-penten-3-ona, sustancias
voltiles capaces de generar sensaciones punzantes, speras y astringentes.
Como se ha dicho previamente (apartado 1.1), han sido numerosos los estudios que han
tratado de relacionar los compuestos voltiles presentes en el aroma del aceite de oliva virgen
con los atributos sensoriales que percibe el ser humano. A pesar del importante desarrollo de la
instrumentacin de hoy da, las tcnicas analticas todava no son capaces de evaluar las
interacciones y los sinergismos que derivan de la combinacin e interaccin de las diferentes
sustancias voltiles entre s y con los receptores sensoriales localizados en orificios nasales y
boca. No obstante es posible relacionar determinados compuestos voltiles con las distintas
notas sensoriales evaluadas por grupos de catadores entrenados, gracias al empleo de
diferentes procedimientos estadsticos aplicados a los valores proporcionados por los equipos
instrumentales y los paneles de cata.
15
1. Introduccin General
Umbral deteccin
Compuesto Nota aromtica Referencias
(g/Kg de aceite)
Hexanal Verde-dulce 75 Aparicio & Luna, 2002
Manzana verde, 80 Morales et al., 2005
herbceo
Hexan-1-ol Fruta, banana, 400 Aparicio & Morales,1998
csped recin cortado
Indeseable 400 Aparicio & Luna, 2002
Acetato hexilo Verde, frutado, dulce 1040 Aparicio & Luna, 2002
Morales et al., 1997
E-2-hexenal Verde, manzana 424 Reiners & Grosch, 1998
Verde, almendra 420 Morales et al., 2005
amarga
Verde astringente 1125 Aparicio & Luna, 2002
Z-3-hexenal Verde 3 Aparicio & Luna, 2002
Hoja 1,7 Reiners & Grosch, 1998
E-2-hexen-1-ol Csped, hojas 5000 Morales et al., 2005
Verde, herbceo, 8000 Aparicio & Morales, 1998
dulce
Z-2-hexen-1-ol Fruta verde ------ Aparicio & Morales, 1998
Z-3-hexen-1-ol Verde 6000 Aparicio & Luna, 2002
Hoja 1100 Reiners & Grosch, 1998
E-3-hexen-1-ol Verde 1500 Morales et al., 1997
Acetato Z-3-hexenilo Verde, frutado 750 Aparicio & Luna, 2002
Banana, floral 200 Reiners & Grosch, 1998
E-2-pentenal Verde, manzana 300 Morales et al., 2005
Verde, almendra 300 Aparicio & Luna, 2002
amarga
Z-2-pentenal Verde ------ Morales et al., 1997
Pentan-1-ol Picante ------ Morales et al., 1997
Z-2-penten-1-ol Banana ------ Morales et al., 1997
1-penten-3-ona Verde, punzante 50 Aparicio & Luna, 2002
Verde, picante 0,73 Reiners & Grosch, 1998
1-penten-3-ol Tierra hmeda ------ Morales et al., 1997
* Fuentes utilizadas: Angerosa et al. (2004) y Kalua et al. (2007).
16
1. Introduccin General
De esta forma, Angerosa et al. (2000c) utilizaron el Anlisis de Regresin Lineal para relacionar
los compuestos C6 y C5 de la ruta LOX con los atributos sensoriales evaluados por un panel
de jueces entrenados, obteniendo valores de R2 no demasiado elevados para las distintas
notas aromticas verdes evaluadas (entre 0,21 y 0,65). An as, se encontraron ciertas
relaciones interesantes, como el hecho de que el hexanal presenta un papel esencial en la
formacin de la mayora de los atributos sensoriales verdes, mientras que el E-2-hexenal
participa en las notas olfativas descritas bajo los trminos hierba, banana y almendra, el E-2-
hexen-1-ol se asocia con las notas florales, frutadas y tomate, y el acetato de Z-3-hexenilo es el
mayor contribuyente en las notas banana y cscara de nuez.
De la misma forma Morales et al. (1995) utilizaron el Escalado Multidimensional (MDS) para
establecer las posibles inter e intra-relaciones entre los compuestos voltiles del aceite de oliva
virgen y los atributos sensoriales evaluados por seis paneles de catadores distintos, dos de
ellos integrados por jueces expertos y el resto formado por consumidores habituales y
potenciales entrenados para el ensayo. Al aplicar la tcnica de MDS las diversas notas
sensoriales se clasificaron en siete grupos de diferentes percepciones sensoriales -dulce,
frutado, fruta madura, fruta sobre-madura, indeseable, amargo-picante y verde- dentro de las
cuales se clasificaron los distintos compuestos voltiles presentes en el aceite de oliva virgen,
de forma que los agrupamientos de sustancias aromticas obtenidos deberan explicar cada
una de las percepciones sensoriales definidas. De esta forma, el E-2-hexenal se incluye en la
percepcin amargo-picante, debido a sus notas sensoriales de almendra amarga, al igual que
el E-3-hexen-1-ol. Compuestos como Z-3-hexenal, E-2-hexen-1-ol, Z-3-hexen-1-ol, Z-2-penten-
1-ol y los steres C6 forman parte de la sensacin de verde, mientras que el hexanal y el 1-
penten-3-ona se localizan en la percepcin de dulce-frutado.
17
1. Introduccin General
Cabe destacar la elaboracin de la denominada Rueda Sensorial para el aceite de oliva virgen
extra llevada a cabo por Aparicio et al. (1995), en donde se representa el flavor global de un
aceite a travs de siete zonas sensoriales verde, amargo-picante, indeseable, aceituna
madura, fruta madura, frutado y dulce- mediante la utilizacin de datos sensoriales obtenidos a
partir del QDA y tcnicas estadsticas multivariantes, como el PCA. La asignacin de
coordenadas dentro de la rueda sensorial para cada compuesto voltil permite obtener
informacin de las caractersticas sensoriales de estos componentes orgnicos en base a las
propiedades sensoriales del sector de la rueda del que forman parte. As, compuestos como el
Z-3-hexen-1-ol, acetato de hexilo y acetato de Z-3-hexenilo fueron localizados en el sector
verde, enfatizando la contribucin de estos voltiles a la percepcin de las notas sensoriales
verdes, mientras que el hexanal y 1-penten-3-ona se incluyeron en el dulce y voltiles como el
E-2-hexen-1-ol, 1-penten-3-ol y hexan-1-ol formaron parte de la zona de indeseable.
La mayor parte de los compuestos fenlicos presentes en el aceite de oliva virgen estn
presentes tambin en el fruto o bien son derivados de stos, transfirindose al aceite durante
las distintas etapas del proceso de extraccin (Servili et al., 1999a).
18
1. Introduccin General
Los compuestos fenlicos presentes en el fruto del olivo son reconocidos, entre otros aspectos,
por su actividad antimicrobiana (Fleming et al., 1973; Tranter et al., 1993; Aziz et al., 1998), por
su papel preventivo en las infecciones por Dacus oleae (Lo Scalzo et al., 1994) y por su
actividad inhibidora de enzimas celulolticas, tanto endgenas como exgenas (Hereda et al.,
1990), constituyendo, por tanto, una parte importante del sistema qumico de defensa de la
aceituna.
El fruto del olivo presenta un elevado contenido de fenoles, los cuales llegan a constituir hasta
el 3% del peso fresco de la pulpa (Raina, 1995), siendo los cidos fenlicos, alcoholes
fenlicos, flavonoides y secoiridoideos los principales grupos fenlicos presentes.
19
1. Introduccin General
Ruta del cido siqumico: ruta responsable de la formacin de los aminocidos aromticos L-
fenilalanina y L-tirosina. La gliclisis no oxidativa de la glucosa origina el fosfoenol piruvato y la
eritrosa-4-fosfato, compuestos que se unen mediante una serie de reacciones dando lugar al
cido siqumico, precursor biosinttico de los aminocidos aromticos. El posterior conjunto de
reacciones implicadas en la formacin del cido p-cumrico a partir de L-fenilalanina, es lo que
se conoce como metabolismo general fenilpropanoide. Las enzimas que catalizan las
reacciones parciales de esta ruta son: L-fenialanina amonio liasa (PAL), cinamato-4-hidroxilasa
(C4H) y hidroxicinamato CoA ligasa.
L-TIROSINA
Ac. SINPNICO Ac. FERLICO Ac. CAFEICO Ac. p-CUMRICO Ac. CINMICO
C4H
H.CoA ligasa
Ac. o-CUMRICO
FLAVONOIDES
LIGNINAS
LIGNANOS
20
1. Introduccin General
Ruta del cido mevalnico: ruta responsable de la sntesis de los compuestos secoiridoideos
del fruto y forma parte del metabolismo secundario de los terpenos. Damtoft et al. (1993)
propusieron la ruta biosinttica esquematizada en la Figura 1.3 para la formacin de los
secoiridoideos ligustrsido y oleuropena a partir del cido mevalnico.
H 3C OH OH
OH OH
OH
c. mevalnico
CHO COOH
O O O
OH OH OH
HO O
O O O
O
O O O
HO
O O O
HO CO OMe O COOMe
HO
O O
Figura 1.3. Ruta del cido mevalnico. Propuesta por Damtoft et al. (1993) para la biosntesis de los
secoiridoideos oleuropena y ligustrsido presentes en la aceituna.
(Fuente utilizada: Ryan et al., 2002).
21
1. Introduccin General
Los cidos fenlicos, similares a los encontrados en la aceituna, fueron los primeros
compuestos fenlicos identificados en el aceite de oliva virgen. stas sustancias junto a los
alcoholes fenlicos (hidroxitirosol y tirosol) y las flavonas (luteolina y apigenina), procedentes
de la hidrlisis de sus correspondientes glucsidos presentes en la pulpa y que se produce
durante el proceso de extraccin del aceite, se encuentran en concentraciones muy bajas en el
aceite de oliva virgen (Vzquez-Roncero et al., 1976; Solinas et al., 1981; Brenes et al., 1999).
Sin embargo, los compuestos fenlicos mayoritarios en el aceite de oliva virgen son derivados
secoiridoideos del hidroxitirosol y tirosol, los cuales proceden de la hidrlisis enzimtica de la
oleuropena, la demetiloleuropena y el ligustrsido, proceso que se inicia con la molienda del
fruto y continua durante el batido de la pasta de aceituna debido a la accin de la enzima -
glucosidasa. Los principales secoiridoideos del aceite son las formas dialdehdica y aldehdica
del cido elenlico unido al hidroxitirosol, denominadas generalmente como 3,4-DHPEA-EDA y
3,4-DHPEA-EA, respectivamente, y sus formas anlogas del cido elenlico unido al tirosol: p-
HPEA-EDA y p-HPEA-EA, respectivamente. La identificacin de la estructura de los derivados
3,4-DHPEA-EDA, 3,4-DHPEA-EA y p-HPEA-EDA fue llevada a cabo en 1993 por Montedoro et
al. mediante RMN y posteriormente confirmada por otros autores (Angerosa et al., 1995;
Angerosa et al., 1996b; Owen et al., 2000a). En 1995, Angerosa et al. determin la estructura
del p-HPEA-EA mediante GC-MS, la cual fue confirmada posteriormente con RMN por Mateos
et al. (2001). Aunque, an en la actualidad, se desconocen los mecanismos bioqumicos que
generan la presencia de estos compuestos secoiridoideos en el aceite de oliva virgen, se ha
propuesto la ruta de biosntesis esquematizada en la Figura 1.4.
22
1. Introduccin General
R R R
O
O COOMe O O O O
HO HO COOMe HO COOMe
- Glu
I II
O
-glucosidasa
O O
OGlu
OH O
R R
O O O O
HO COOMe HO COOMe
III
OH O
O O
Metilesterasa - CH3
R
R
O O
HO O O
HO COOH
IV
O
O
O
O
Figura 1.4. Mecanismo propuesto para la biosntesis de los derivados secoiridoideos presentes
en el aceite de oliva virgen. I: R=H, ligustrsido; R=OH, oleuropena. II: R=H, p-HPEA-EA; R=OH, 3,4-
DHPEA-EA. III y IV: R=H, p-HPEA-EDA carboximetilada y decarboximetilada; R=OH, 3,4-DHPEA-EDA
carboximetilada y decarboximetilada, respectivamente.
(Fuente utilizada: Servili et al., 2004).
Los compuestos fenlicos estn relacionados con las notas sensoriales amargas del aceite de
oliva virgen, as como con las sensaciones quimioestsicas de astringencia y picor
caractersticos de este producto.
Varios estudios han puesto de manifiesto la relacin existente entre los atributos sensoriales
positivos amargo y picante y el contenido fenlico total del aceite. En 1992, Gutirrez-
Rosales et al. observaron una elevada correlacin entre la intensidad de amargor del aceite de
oliva virgen y la absorbancia a 225 nm de sus extractos fenlicos correspondientes, parmetro
denominado por los autores como K225.
23
1. Introduccin General
Aunque, an en la actualidad, no se han definido claramente las relaciones existentes entre los
compuestos fenlicos individuales y las caractersticas sensoriales del aceite de oliva virgen,
algunos estudios han puesto en evidencia tanto la falta de correlacin existente entre los cidos
fenlicos y el amargor del aceite de oliva virgen, as como el importante impacto sensorial de
los derivados secoiridoideos presentes en este producto (Uccella et al., 2001).
De esta forma, se han descrito la elevada correlacin estadstica entre las formas 3,4-DHPEA-
EDA, 3,4-DHPEA-EA y p-HPEA-EDA y la intensidad de amargor del aceite de oliva virgen
(Garca et al., 2001; Kiritsakis et al., 1998b; Gutirrez-Rosales et al., 2003; Tovar et al., 2001).
Por otro lado, en un intento de determinar las propiedades sensoriales de los compuestos
fenlicos de forma individual, Gutirrez et al. (2000) fraccionaron los fenoles de un aceite de
oliva virgen utilizando HPLC preparativa encontrando cuatro picos mayoritarios. Tras su
separacin y aislamiento, los extractos fueron presentados a un panel de cata y fueron
descritos dos de ellos como ligeramente amargos, otro de ellos como fuertemente amargo, y
uno como picante. Los picos aislados no fueron identificados, aunque tras el anlisis HPLC de
la fraccin ms amarga se encontr que a su vez estaba constituida por al menos tres picos
distintos. Un estudio ms exhaustivo realizado por Andrewes et al. (2003) puso de manifiesto,
tras el aislamiento e identificacin parcial de diversas fracciones fenlicas a travs de sus
caractersticas de absorcin en el UV y por MS, y posterior presentacin ante un panel de cata,
que aunque todas las fracciones fenlicas estudiadas fueron descritas como amargas y
picantes, el p-HPEA-EDA, identificado mediante MS y RMN, es el principal contribuyente a las
notas sensoriales picantes de los aceites de oliva virgen, al producir una sensacin ardiente en
la parte posterior de la garganta, mientras que la forma 3,4-DHPEA-EDA slo caus una
pequea sensacin picante en los catadores.
Por otro lado tambin es conocida desde hace tiempo la capacidad antioxidante de los
compuestos fenlicos presentes en el aceite de oliva virgen, ya que son numerosos los autores
que han relacionado el contenido de compuestos fenlicos con su estabilidad oxidativa,
evaluada mediante el test de capacidad de absorcin del radical oxgeno (ORAC), como con la
vida til del producto, evaluada mediante ensayos acelerados de estabilidad oxidativa, como
son el mtodo Rancimat o el mtodo del oxgeno activo (AOM) (Gutirrez et al., 1977;
Gutfinger, 1981; Montedoro et al., 1992a; Baldioli et al., 1996; Litridou et al., 1997; Salvador et
al., 1999; Ninfali et al., 2002).
24
1. Introduccin General
la estabilidad por resonancia de los radicales fenoxilo a los que dan lugar, que se encuentra
condicionada por la presencia de sustituyentes donadores de electrones en el anillo aromtico
y por el tamao de los mismos (Scott, 1965).
Existen varios estudios sobre la capacidad antioxidante individual que presentan determinados
compuestos fenlicos del aceite de oliva virgen, como es el caso del hidroxitirosol, tirosol y
cidos fenlicos, como el cafeico, p-cumrico, ferlico o vanlico, cuya actividad antioxidante ha
sido estudiada tras su adicin a aceite de oliva refinado o de girasol, observndose el gran
poder antioxidante del hidroxitirosol (Chimi et al., 1991; Papadopoulos et al., 1991).
Asimismo, la capacidad antioxidante de los compuestos fenlicos del aceite de oliva virgen, y
en especial del hidroxitirosol, ha sido relacionada durante los ltimos aos con una efectiva
actividad protectora frente a enfermedades cardiovasculares, neuro-degenerativas y algunos
tumores, debido a que estas sustancias son capaces de inhibir ciertas especies reactivas de
oxgeno (ROS) responsables del estrs oxidativo que se produce en todas estas
enfermedades. As por ejemplo, estudios in vivo han demostrado que la ingesta habitual de
aceite de oliva virgen reduce el riesgo relativo estimado de cncer de pecho (Martn-Moreno et
al., 1994), pncreas (Soler et al., 1998), coln y recto (Stoneham et al., 2000), entre otros.
Mientras que, otras investigaciones in vitro han establecido la capacidad protectora del
hidroxitirosol frente a los cambios producidos por los peroxinitrilos en las bases del DNA
(Dejana et al., 1999), su actividad inhibidora de los procesos de agregacin plaquetaria (Petroni
et al., 1995), as como su capacidad protectora de los eritrocitos, las lipoprotenas de baja
densidad y los fosfolpidos de los liposomas frente a procesos oxidativos (Aeschbach et al.,
1994; Visioli et al., 1995; Manna et al., 1999).
25
1. Introduccin General
Dentro de los derivados secoiridoideos presentes en el aceite de oliva virgen, resulta de gran
inters destacar el reciente re-descubrimiento del p-DHPEA-EDA como un potente agente
antiinflamatorio no-esteroideo, similar al ibuprofeno, y denominado por los autores con el
nombre de oleocantal (Beauchamp et al., 2005).
El perfil fenlico y voltil del aceite de oliva virgen, as como el resto de componentes
minoritarios y mayoritarios del mismo, depende de una serie de interacciones entre factores
genticos, ambientales y tecnolgicos que influyen tanto en el desarrollo y maduracin del
fruto, como en el posterior procesado del mismo (Montedoro et al., 1991).
26
1. Introduccin General
medida que madura el fruto, como es el caso de la ADH (Salas et al., 1998) y la LOX (Salas et
al., 1999c), lo cual apoya la disminucin en la formacin de estos compuestos voltiles que han
observado numerosos autores, a medida que aumenta el grado de maduracin de las
aceitunas (Morales et al., 1996; Aparicio et al., 1998). An as, la evolucin de los distintos
compuestos C6 es variable; por ejemplo, se ha descrito como el E-2-hexenal aumenta hasta
una concentracin mxima, lo cual sucede cuando el color del fruto pasa de verde-amarillento
a prpura, momento a partir del cual su contenido en el aceite de oliva virgen comienza a
disminuir (Solinas et al., 1987b), y mientras que el contenido de la mayora de los alcoholes C6
disminuye con la maduracin del fruto, la concentracin de hexan-1-ol aumenta en algunas
variedades (Benincasa et al., 2003).
Por otro lado, la disponibilidad de agua por parte del fruto durante su desarrollo, ha sido
descrito como uno de los factores agronmicos ms importantes en la posterior composicin
fenlica del aceite de oliva virgen obtenido. Varios autores han determinado un menor
contenido fenlico en los aceites obtenidos a partir de frutos desarrollados bajo diversas
tcnicas de riego, y que por tanto, no han sufrido condiciones de estrs hdrico apreciable,
encontrando una relacin inversamente proporcional entre las dosis de agua aplicadas en el
olivar y la presencia de sustancias fenlicas en los aceites (DAndria et al., 1996; Motilva et al.,
1999; Motilva et al., 2000; Tovar et al., 2001; Romero et al., 2002; Servili et al., 2007). Este
hecho podra estar relacionado con la menor concentracin fenlica observada en los frutos
cuando se aplica riego al olivar (Patumi et al., 1999; Tovar et al., 2002b), y tambin ha sido
27
1. Introduccin General
An as, no todas los compuestos fenlicos presentes en el aceite de oliva virgen se ven
afectados de igual forma por la disponibilidad de agua durante el desarrollo del fruto, as es el
contenido de los derivados secoiridoideos 3,4-DHPEA-EDA, 3,4-DHPEA-EA y p-HPEA-EDA el
que ms disminuye al descender el estrs hdrico de los frutos (Tovar et al., 2001; Romero et
al., 2002; Servili et al. 2007).
Varios autores han apuntado la importancia de las distintas fases de extraccin del aceite de
oliva virgen sobre la composicin minoritaria del mismo y los cambios observados en estos
compuestos.
As, el uso de molinos metlicos permite una mayor ruptura de los tejidos vegetales dando
lugar a una mejor extraccin de las sustancias fenlicas en comparacin con los molinos de
piedra (Caponio el al., 2003), obtenindose de este modo aceites ms amargos y con una
mayor estabilidad oxidativa (Di Giovacchino et al., 2002). De igual forma, Servili et al. (2002b)
describieron tambin cambios en la composicin voltil de este producto en funcin del tipo de
molino utilizado, de forma que la cantidad total de voltiles era significativamente superior en
aceites obtenidos a partir de molinos de piedra, sobretodo en lo que respecta al contenido de
Z-3-hexen-1-ol, hexanal y E-2-hexenal. Este ltimo compuesto lleg casi a triplicarse frente al
aceite obtenido utilizando un molino metlico de discos. La razn de ello, es que la temperatura
que puede alcanzar la pasta de aceituna despus de su molturacin en un molino metlico es
10 C superior a la de un molino de piedra, con el consecuente descenso en la actividad de las
enzimas de la ruta LOX (Salas et al., 1998 y 1999b).
En el caso de los molinos metlicos, tambin se ha descrito que el tamao de la luz de malla
del tamiz es un importante factor a valorar, ya que cuanto menor es el dimetro del tamiz,
28
1. Introduccin General
mejor es la extraccin de las sustancias polares presentes en los tejidos vegetales, como son
los compuestos fenlicos (Di Giovacchino et al., 2002).
En lo que respecta al posterior proceso de batido, esta etapa permite no slo la ruptura de las
emulsiones entre la fase acuosa y oleosa presentes en la pasta de aceituna y la consiguiente
agregacin fsica de las partculas de aceite, sino que adems tengan lugar numerosos
procesos bioqumicos que afectan a la calidad final del producto obtenido. De hecho, las
condiciones utilizadas durante esta etapa afectan tanto al rendimiento graso obtenido en el
proceso de extraccin, como a la composicin minoritaria del aceite de oliva virgen.
El tiempo y la temperatura de batido son las principales variables a controlar durante esta
etapa, de forma que es posible cambiar potencialmente la composicin voltil y fenlica del
aceite de oliva virgen obtenido y, por consiguiente, sus caractersticas sensoriales. As, un
incremento de la temperatura de batido generalmente implica un mayor contenido fenlico en
los aceites (Di Giovacchino et al., 1991; Ranalli et al., 2001), debido al aumento que se produce
en los coeficientes de particin de estos compuestos entre la fase acuosa y oleosa de la pasta
de aceituna, y que supone el incremento de la solubilidad relativa en la fase oleosa (Rodis et
al., 2002). Por el contrario, en la composicin voltil del aceite obtenido disminuye el contenido
de Z-3-hexen-1-ol y steres C6, mientras que aumenta el hexan-1-ol y E-2-hexen-1-ol, adems
de favorecerse la acumulacin de 2-metilbutanal y 3-metilbutanal, a causa de la activacin de
la ruta de conversin de los aminocidos (Morales et al., 1999; Angerosa et al., 2001).
Por otro lado, los tiempos de batido largos favorecen la degradacin oxidativa, ya sea qumica
o enzimtica, de los compuestos fenlicos, obtenindose aceites de oliva virgen con un menor
contenido en las formas 3,4-DHPEA-EDA, 3,4-DHPEA-EA y p-HPEA-EDA (Lercker et al., 1999;
Angerosa et al., 2001; Di Giovacchino et al., 2002), aunque se ha puesto de manifiesto que una
reduccin en la disponibilidad de oxgeno, disminuye la actividad de la polifenoloxidasa y
peroxidasa, aumentando el contenido fenlico del aceite (Servili et al., 1999). Sin embargo, el
29
1. Introduccin General
El proceso de separacin slido-lquido que permite extraer el aceite de oliva virgen de la pasta
de aceituna puede realizarse de diferentes formas. En la eleccin de uno u otro sistema se
debe considerar que la composicin fenlica y voltil del aceite puede verse afectada. La
utilizacin de los sistemas de centrifugacin de tres fases implica la adicin de agua en el
proceso, aproximadamente en torno a 40-60 L por 100 Kg de pasta, para conseguir la correcta
separacin de las mismas, ejerciendo un efecto de lavado sobre el aceite, que puede modificar
el equilibrio de particin y arrastrar parte de las sustancias polares, como los compuestos
fenlicos y sustancias voltiles. Algunos estudios han descrito que los aceites obtenidos
empleando sistemas de extraccin por presin o equipos de percolacin para la separacin
slido-lquido son ms ricos en fenoles y en voltiles C6, como Z-3-hexen-1-ol, hexan-1-ol y E-
2-hexen-1-ol, que los obtenidos por sistemas de centrifugacin en tres fases (Nergiz et al.,
1991; Di Giovacchino et al., 1994; Di Giovacchino et al., 1995).
Como mejora tecnolgica para la elaboracin del aceite de oliva virgen, en la dcada de los
noventa se introdujo un nuevo sistema de centrifugacin en dos fases que no necesitaba de la
adicin de agua a la pasta de aceituna para efectuar la separacin slido-lquido. Como caba
esperar, varios estudios han descrito que los aceites obtenidos mediante este sistema
muestran un mayor contenido medio en fenoles y voltiles C6, como E-2-hexenal y alcoholes
C6, respecto a los obtenidos por centrifugacin de tres fases, aunque no se encontraron
diferencias estadsticamente significativas (Angerosa et al., 1996b y 2000b; Ranalli et al., 1996;
Salvador et al., 1998; Di Giovacchino et al., 1994 y 2001).
30
2. OBJETIVOS
2. Objetivos
El objetivo general de la Tesis Doctoral ha sido el estudio de la influencia que distintos factores
agronmicos y tecnolgicos ejercen sobre los componentes minoritarios del aceite de oliva
virgen, y muy especialmente sobre la composicin fenlica y voltil, estrechamente relacionada
con la calidad de este producto y que por tanto representan en gran medida el grado de
preferencia de este aceite frente a otras grasas vegetales comestibles presentes en el
mercado.
33
2. Objetivos
34
3. MATERIAL Y MTODOS
3. Material y Mtodos
Este ensayo fue llevado a cabo durante las campaas olivareras 2003/04 y 2004/05 en una
parcela propiedad de la Conserjera de Agricultura y Medio Ambiente, ubicada a 3 Km de la
Escuela en Capacitacin de Almodvar del Campo (Ciudad Real). En ella est ubicado un
olivar adulto de marco tradicional (50 aos, marco 12 x 12 m2) de la variedad Cornicabra (Olea
europaea L.), con una densidad de 70 olivos por hectrea y una superficie total de 46080 m2. El
clima de la zona es continental-mediterrneo con una precipitacin media anual de 404 mm,
mayoritariamente distribuida fuera de la estacin veraniega. Durante 2003 y 2004 la
precipitacin registrada fue de 426 y 484 mm, respectivamente, siendo de 41 y 138 mm desde
mayo a septiembre, poca que coincide con el periodo de riego en la parcela.
La aplicacin de los tratamientos de riego que constituyen este ensayo comenz en 2001,
encontrndose la parcela originariamente en condiciones de secano. Las estrategias
estudiadas fueron las siguientes:
(1) Secano, sin aporte de riego. Actu como control, con el fin de comparar los datos obtenidos
con los restantes tratamientos.
(2) Riego Deficitario Regulado o, en ingls, Regulated Deficit Irrigation (RDI). El lmite legal
de riego permitido en numerosas zonas olivareras espaolas es de 100 mm, por ello para este
tratamiento se estableci como objetivo dar un riego durante ambas campaas inferior a 75
mm, en funcin de la climatologa y el estado fenolgico de los olivos, aunque en cada
campaa se evaluaron dos estrategias de riego distintas. Durante el 2003, el agua fue aplicada
durante toda la estacin de riego con una distribucin de 28 mm en mayo y junio, 21 mm en
julio y agosto y 7 mm en septiembre (RDI-1); mientras que en 2004, el agua fue aplicada
solamente a partir de agosto, cuando comienza la acumulacin de aceite en el fruto (RDI-2).
(3) FAO. Este tratamiento tuvo como objetivo aplicar dosis de riego equivalentes al 100% de la
demanda evapotranspirativa del olivo, es decir, reponer el 100% de las prdidas hdricas del
cultivo en funcin de la evapotranspiracin del cultivo (ETc), la cual fue estimada segn la
metodologa propuesta por la FAO (Food and Agriculture Organization). De esta forma las
necesidades hdricas de los olivos se determinaron restando la precipitacin efectiva (41 mm
en 2003 y 138 mm en 2004) del valor de evapotranspiracin del cultivo (ETc), calculado este
ltimo trmino segn la siguiente ecuacin (Doorenbos y Pruitt, 1977);
ETc = Kc x ETo x Kr
37
3. Material y Mtodos
(4) 125 FAO. La dosis de riego aplicada en este tratamiento fue incrementada un 25% respecto
al tratamiento FAO, ya que en condiciones de baja cobertura del terreno, como es el caso de
este olivar de baja densidad (70 olivos/Ha), las necesidades hdricas reales del olivar pueden
ser ligeramente superiores a las calculadas mediante la metodologa FAO.
Figura 3.1. Diseo estadstico utilizado en el olivar experimental Cornicabra de marco tradicional.
Secano; RDI; FAO; 125 FAO; , olivo borde.
38
3. Material y Mtodos
Las dosis de agua aplicadas en la campaa 2003 en cada tratamiento de riego fueron: 56 mm
en RDI-1, 148 mm en FAO y 206 mm en 125 FAO. Para 2004: 60 mm en RDI-2, 124 mm en
FAO y 154 mm en 125 FAO. Con la finalidad de describir el estado hdrico de los olivos
sometidos a los distintos tratamientos de riego, se presentan en la Tabla 3.1 los valores de las
integrales de estrs hdrico (S) (MPa x da, segn Myers, 1998), calculadas a partir de los
datos de potencial hdrico del tronco obtenidos a medioda, y el valor potencial hdrico mnimo
registrado durante cada campaa.
Tabla 3.1. Dosis de agua aplicada en cada tratamiento y distintos parmetros representativos
del estrs hdrico de los olivos Cornicabra durante 2003 y 2004.
2003
SECANO 0,0 332 -4,1 (mediados de septiembre)
RDI-1 56,0 316 -4,1 (mediados de septiembre)
FAO 148,0 218 -2,3 (mediados de septiembre)
125 FAO 206,0 172 -1,8 (mediados de septiembre)
2004
SECANO 0,0 269 -4,1 (mediados de octubre)
RDI-2 60,0 223 -2,9 (inicios de agosto)
FAO 124,0 176 -2,2 (finales de septiembre)
125 FAO 154,0 159 -1,7 (mediados de octubre)
* Datos proporcionados por el Dr. Alfonso Moriana (C.M.A. El Chaparrillo, Ciudad Real).
39
3. Material y Mtodos
La recoleccin de las aceitunas en las dos campaas olivareras fue llevada a cabo mediante el
sistema manual de ordeo desde noviembre hasta finales de diciembre. A excepcin del quinto
y ltimo muestreo realizado en la primera campaa, que se llev a cabo a mediados de enero
utilizando un vibrador; sistema mecnico que agita los pies del olivo produciendo el
desprendimiento de las aceitunas.
Una vez recogidos los frutos, se procedi a su molturacin inmediata en la Planta Piloto de
Tecnologa de Alimentos (UCLM), emplendose el sistema de extraccin a escala de
laboratorio Abencor (Abengoa S.A., Sevilla, Espaa) (ver epgrafe 3.5). El aceite se almacen
en oscuridad en botellas de vidrio color topacio a 4 C y sin espacio de cabeza hasta su
anlisis.
Este ensayo experimental fue llevado a cabo durante las campaas olivareras 2005/06,
2006/07 y 2007/08. Se utilizaron dos parcelas experimentales de las variedades Cornicabra y
Morisca. El olivar Cornicabra estaba situado en la Finca La Entresierra (Ciudad Real) y es
propiedad de la Consejera de Agricultura de la Junta de Comunidades de Castilla-La Mancha.
Fue plantado en 1999, con una superficie total de 3,2 hectreas y un marco de plantacin de
4,75 x 7,00 m2. El olivar de la variedad Morisca, situado en la Finca La Orden (Badajoz) y
propiedad de la Consejera de Agricultura y Medio Ambiente de la Junta de Extremadura, fue
plantado en 1998, con una superficie total de 1,0 hectrea y un marco de plantacin de 6,00 x
4,00 m2. Ambas zonas presentan un clima continental-mediterrneo, aunque Badajoz muestra
influencias atlnticas, debido a su proximidad a la costa portuguesa. La media anual de
precipitaciones es de 404 mm (Ciudad Real) y 475 mm (Badajoz), distribuidas
mayoritariamente fuera de la estacin veraniega. Las precipitaciones registradas en Ciudad
Real fueron 172 y 412 mm durante 2005 y 2006, respectivamente, y 324 y 199 mm a lo largo
de 2006 y 2007 en Badajoz. Las lluvias durante el perodo de riego (desde mayo a septiembre)
fueron alrededor de 27 y 95 mm en Ciudad Real (ao 2005 y 2006, respectivamente), y 40 y
102 mm en Badajoz (ao 2006 y 2007, respectivamente). A pesar de que las lluvias en este
perodo en 2006 (Ciudad Real) y 2007 (Badajoz) fueron anormalmente altas, ambos olivares
sufrieron los periodos de sequa propios de la estacin veraniega.
Las estrategias de riego bajo estudio empleadas en ambas parcelas fueron las siguientes:
(1) Tratamiento de riego basado en la metodologa FAO. Fue utilizado como control, con el fin
de evaluar la eficacia del resto de las estrategias evaluadas. Este mtodo suministr dosis de
40
3. Material y Mtodos
riego equivalentes al 100% de la evapotranspiracin del cultivo (100% ETc), con el fin de
reponer completamente el agua extrada del terreno por parte de los olivos. La
evapotranspiracin del cultivo se calcul, tal y como se ha explicado previamente en el
apartado 3.1., mediante la ecuacin ETc = ETo x Kc x Kr (Doorenbos y Pruitt, 1974).
(2) Estrategia de riego basada en las Fluctuaciones del Dimetro del Tronco o, en ingls,
Trunk Diameter Fluctuations (TDF). Las dosis de agua aplicadas dependieron de las medidas
de la fluctuacin diaria del dimetro de tronco, realizadas con dendrmetros (modelo DF 2.5;
Solartron Metrology, West Sussex, UK), que tomaron datos con una frecuencia de 30 segundos
y calcularon los valores medios cada 15 minutos. Los ciclos de TDF permitieron obtener dos
ndices: la Tasa de Crecimiento de Tronco (TCT), calculado como la pendiente obtenida a partir
de los mximos diarios monitorizados y la Mxima Oscilacin del Dimetro de Tronco (MOD),
calculado como la diferencia entre el valor mximo y mnimo monitorizado cada da (Goldhamer
and Fereres, 2001). En funcin de los trabajos de Moriana y Fereres (2003) se establecieron
tres fases para la programacin de la estrategia de riego y en cada una se adoptaron
referencias diferentes:
(a) Desde brotacin hasta valor mximo de TCT igual a 0,15 mm/da. Se utiliz una ecuacin
que relaciona la TCT con la temperatura mnima (Tmin);
(b) Desde que se obtuvieron valores de TCT iguales a 0,15 mm/da hasta principios de
Septiembre. Se aplic el algoritmo de la Figura 3.2.
(c) Desde Septiembre hasta las primeras lluvias de Otoo. Se us la siguiente ecuacin, que
relaciona la TCT con la temperatura mxima (Tmax);
Las ecuaciones 1 y 2 fueron obtenidas a partir de los datos de TCT de Moriana y Fereres
(2003) en relacin con la temperatura. El crecimiento del tronco, representado por la TCT, es
un factor ms sensible al estrs hdrico del olivo que la MOD, por esa razn este ltimo
parmetro no se emple en las fases a y c. La primera aplicacin de riego se realiz cuando el
valor de la medida TCT indic estrs hdrico, es decir una TCT por debajo del valor marcado
para la primera fase (0,15 mm/da) y la cantidad de agua aplicada en este primer riego fue
estimada por el clculo de la ETc entre las dos ltimas fechas de medida. En los riegos
sucesivos se increment o disminuy un 10% la cantidad de agua aplicada en la fecha anterior
segn la TCT se encontraba por debajo o por encima del umbral establecido.
41
3. Material y Mtodos
NO
Disminuir 10% TCTi < TCTu
dosis de agua
SI
Aumentar 10% SI
dosis de agua MODi < MODu
NO
a=a+1
a=0
SI
a=1
NO TCTu = TCTi
SI
a=0 TCTi > TCTi-1
NO
SI NO
NO
TCTi = TCTi-1
SI
Mantener la dosis
a=4
de agua
Figura 3.2. Algoritmo de decisin para la fase b del tratamiento TDF. Los subndices u indican
valores umbrales y los i valores actuales.
Desarrollado por el Dr. Alfonso Moriana (C.M.A. El Chaparrillo, Ciudad Real).
42
3. Material y Mtodos
Como se ha dicho anteriormente, el crecimiento del olivo, representado por el parmetro TCT,
se considera un factor ms sensible al estrs hdrico que el MOD, por lo que si la TCT medida
era superior a la TCTu se consideraba que el tratamiento estaba bien regado y se disminua un
10% la aportacin de agua. En el caso contrario, se comparaba el valor de MOD, de forma que
si ste era superior al esperado se incrementaba un 10% el riego, ya que se supona que ste
era insuficiente. Si la MOD medida no era superior al esperado caba la posibilidad de que este
parmetro no fuera sensible al estrs hdrico o que los valores de TCT empleados como umbral
fueran excesivamente altos. Si era la primera vez que ocurra esto (a=1) se incrementaba el
riego un 10% para comprobar si haba respuesta de los indicadores. Si no era as y este
suceso haba ocurrido en otras ocasiones (a>1), se comparaba la TCT obtenida en el periodo
anterior con el actual; (i) si la TCT actual era mayor, implicaba que haba habido una respuesta
al incremento del riego y se incrementaba de nuevo otro 10%, (ii) si el valor de TCT actual era
inferior que en el periodo anterior se supona que exista un dficit hdrico en el olivo y se
incrementaba la dosis de riego un 10%, (iii) si el valor de TCT actual era igual al medido en el
perodo anterior, podra deberse a que el umbral empleado fuera mayor al umbral que le
correspondera realmente al olivo, ya sea por edad o por carga de fruto, de forma que en este
caso no habra variacin en el aporte de agua y cuando los valores de TCT se estabilizasen en
torno a ese valor (a=4) se usara como nuevo valor umbral (ver Figura 3.2).
(3) Programaciones de riego basados en el Potencial Hdrico del Tronco o, en ingls, Stem
Water Potential (SWP). Las programaciones de riego se realizaron segn los valores del
potencial hdrico del tronco medidos con una cmara de presin (Soil Moisture Equip., Santa
Barbara, CA, USA), utilizando hojas cercanas al tronco y cubiertas con papel de aluminio al
menos una hora antes de efectuarse la medida (13:00 horas). Las dosis de agua aplicadas a
los olivos durante el periodo de riego permitieron mantener las medidas del estado hdrico de
los olivos en dos umbrales diferentes fijados al inicio del ensayo; -1,2 MPa (SWP -1,2), el cual
no supuso ninguna condicin de estrs hdrico en el rbol, y -2,0 MPa (SWP -2,0) que implic
condiciones hdricas ligeramente deficitarias. Las medidas de SWP se efectuaron dos veces
por semana sobre doce olivos de referencia, de forma que el valor medio de las doce medidas
se utilizaba para comparar con los umbrales establecidos previamente, inicindose la
aplicacin de riego cuando la medida de potencial fuera inferior a dichos umbrales y tomando
como dosis de agua inicial los valores de ETc obtenidos entre las dos ultimas fechas de
medida. La sistemtica de riego seguida en las fechas sucesivas supuso un incremento o un
descenso de la dosis de riego de un 10%, en funcin de si el SWP medido estaba por encima o
por debajo de los umbrales establecidos (en valores superiores a 10%).
Ambas parcelas utilizaron un diseo estadstico de bloques al azar con tres repeticiones de
cada tratamiento de riego, lo que supuso un total de doce bloques distintos. Cada bloque
elemental estuvo compuesto por cuatro filas de cuatro olivos cada una, de los que se tomaron
43
3. Material y Mtodos
para los muestreos los cuatro olivos centrales de las filas intermedias. El ensayo se encontraba
rodeado por filas de olivos en regado que actuaban de bordes. Las Figuras 3.3 y 3.4
representan de forma esquemtica el diseo experimental utilizado en los olivares Cornicabra y
Morisca de cubierta incompleta utilizados para la realizacin del ensayo.
Figura 3.3. Diseo estadstico utilizado en el olivar Cornicabra de cubierta incompleta. Localizado
en la finca La Entresierra (Ciudad Real). SWP -2,0; SWP -1,2; FAO; TDF; , olivo borde.
Figura 3.4. Diseo estadstico utilizado en el olivar Morisca de cubierta incompleta. Localizado en
la finca La Orden (Badajoz). SWP -2,0; SWP -1,2; FAO; TDF; , olivo borde.
44
3. Material y Mtodos
pozo presente en cada una de las parcelas y dispona de un programador de riego y una
bomba inyectora de fertilizantes. A excepcin de las estrategias de riego descritas utilizadas, el
resto de las prcticas agronmicas y fitosanitarias empleadas en los olivares fueron similares.
Las dosis de agua aplicadas en cada una de las estrategias de riego a lo largo del ensayo
estn recogidas a modo de resumen en la Tabla 3.2. Con la finalidad de describir de manera
ms completa el estado hdrico de los olivos de las variedades Cornicabra y Morisca utilizados
en cada tratamiento, esta tabla tambin muestra las integrales de estrs hdrico estacional (S)
y las integrales de estrs hdrico correspondiente al periodo DOY 229-277 (S229-277) (MPa x
da, segn Myers, 1998), obtenidos a partir de los datos de potencial hdrico del tronco medidos
a medioda, adems del potencial hdrico mnimo registrado en cada campaa.
Tabla 3.2. Dosis de agua aplicada en cada tratamiento y distintos parmetros representativos
del estrs hdrico de los olivos durante 2005, 2006 y 2007.
CORNICABRA 05
FAO 125,0 94,6 26,0 -1,69 (mediados de agosto)
TDF 70,1 98,7 28,8 -1,71 (mediados de agosto)
SWP -1.2 179,5 75,0 19,2 -1,47 (inicios de sept.)
SWP -2.0 28,9 161,6 43,1 -2,10 (mediados de sept.)
CORNICABRA 06
FAO 92,7 138,3 53,6 -1,75 (mediados de agosto)
-2,10 (mediados de agosto
TDF 51,5 172,1 81,5
y septiembre)
SWP -1.2 101,2 141,4 54,2 -1,82 (mediados de agosto)
-2,17 (mediados de agosto
SWP -2.0 25,1 199,8 84,9
y septiembre)
MORISCA 06
-1,91 (inicios de agosto y
FAO 408,9 197,6 82,3
mediados de septiembre)
TDF 704,1 129,3 38,9 -1,28 (inicios de agosto)
SWP -1.2 388,6 185,8 63,7 -1,71 (inicios de agosto)
-2,71 (inicios de agosto y
SWP -2.0 184,2 328,9 119,6
septiembre)
MORISCA 07
FAO 297,3 185,4 44,9 -1,73 (mediados de sept.)
TDF 350,2 163,8 48,5 -2,43 (inicios de sept.)
SWP -1.2 304,8 165,1 46,3 -1,64 (mediados de sept.)
SWP -2.0 193,1 267,2 90,7 -2,97 (inicios de sept.)
* Datos proporcionados por el Dr. Alfonso Moriana (C.M.A. El Chaparrillo, Ciudad Real) y la Dra. Henar
Prieto (Centro de Investigacin Finca La Orden, Badajoz).
45
3. Material y Mtodos
Durante las campaas olivareras 2005/06, 2006/07 y 2007/08 se realizaron dos muestreos a
distintos grados de maduracin del fruto en cada uno de los doce bloques en los que se
dividan ambas parcelas (3 bloques x 4 estrategias de riego). Los muestreos se efectuaron
basndose en el grado de maduracin del fruto, definido por la pigmentacin de la piel de las
aceitunas, correspondindose con los colores pintona (al final del envero), morada y negra.
La recoleccin de las aceitunas en las tres campaas olivareras fue llevada a cabo mediante el
sistema manual de ordeo desde mediados de octubre hasta mediados de noviembre.
Una vez recogidos los frutos se procedi a su molturacin inmediata mediante el sistema de
extraccin a escala de laboratorio Abencor (Abengoa S.A., Sevilla, Espaa) (ver epgrafe 3.5).
El aceite obtenido se almacen en oscuridad en botellas de vidrio color topacio a 4 C y sin
espacio de cabeza hasta su anlisis.
Este estudio fue llevado a cabo durante la campaa olivarera 2005/06, las muestras necesarias
para el mismo se obtuvieron de un olivar experimental situado en Almodvar del Campo
(Ciudad Real) y propiedad del C.M.A El Chaparrillo, Servicio de Investigacin y Tecnologa
Agraria. El clima de la zona es continental-mediterrneo; con una precipitacin media anual de
404 mm, mayoritariamente distribuida fuera de la estacin veraniega. El olivar estaba formado
por olivos de seis aos de edad (marco 4,5 x 6,0 m2) de seis variedades espaolas distintas:
Arbequina, Cornicabra, Morisca, Picolimn, Picudo y Picual. Todo el olivar se encontraba en
condiciones de no laboreo y sin ninguna aplicacin de riego; la maleza era controlada mediante
herbicidas post-surgimiento. Destacar que el hecho de obtener todas las muestras del ensayo a
partir de un mismo olivar experimental, permiti estudiar el efecto de las variables variedad e
ndice de madurez sobre los componentes minoritarios de frutos desarrollados en el mismo
rea geogrfica, bajo la mismas condiciones agronmicas y pedoclimticas.
46
3. Material y Mtodos
Se realizaron tres muestreos distintos a lo largo de la maduracin del fruto en todas las
variedades del olivar. El grado de maduracin se determin en funcin de la pigmentacin de la
piel, correspondindose con los colores verde, pintona y negra. La recogida del fruto se realiz
mediante la tcnica manual de ordeo, tomndose dos muestras representativas de cada
variedad para cada etapa de maduracin, las cuales eran inmediatamente congeladas
mediante nitrgeno lquido y posteriormente almacenadas a -70 C hasta la realizacin de los
anlisis correspondientes.
Se realizaron dos muestreos adicionales con el fin de obtener muestras de aceite de oliva
virgen de cada variedad a partir de frutos inmaduros (pigmentacin de la piel verde-amarillenta
y pintona) y maduros (pigmentacin de la piel morada y negra). Las muestras de aceite se
obtuvieron mediante el sistema de molturacin Abencor (Abengoa S.A., Sevilla, Spain), que
como se ha comentado previamente permite la extraccin de aceite de oliva virgen a escala de
laboratorio (epgrafe 3.5). El aceite obtenido se almacen en oscuridad en botellas de vidrio
color topacio a 4 C y sin espacio de cabeza hasta su anlisis.
El estudio del efecto que determinadas variables tecnolgicas durante el batido de la pasta de
aceituna tienen sobre la composicin minoritaria del aceite de oliva virgen, se llev a cabo
durante la campaa olivarera 2005/06, utilizando dos lotes de frutos distintos (Olea europaea L.
Cornicabra cv.) de aproximadamente 1600 Kg cada uno de ellos, con un ndice de madurez de
4,5 y 4,7 (ver apartado 3.6), respectivamente.
La Almazara Experimental utilizada reproduce a escala piloto las instalaciones de una almazara
industrial de grandes proporciones, de hecho se encuentra dividida fsicamente en tres zonas
diferenciadas; (a) zona de recepcin y lavado de las aceitunas, (b) zona de molturacin, batido
47
3. Material y Mtodos
y centrifugacin de las pastas de aceituna y (c) zona de almacenamiento del aceite de oliva
virgen obtenido.
(a) La zona de recepcin de la materia prima est formada por una tolva de recepcin con una
capacidad de 400 Kg y que est conectada mediante una cinta transportadora ascendente a
una deshojadora, que permite la eliminacin de los restos vegetales de menor peso que las
aceitunas, tales como hojas y pequeas ramitas. A
continuacin, se encuentra la lavadora (modelo I10)
conectada a la red pblica de aguas y que permite una
limpieza profunda de los frutos, eliminando polvo, barro
o restos de mayor peso que las aceitunas que pueden
acompaar a la materia prima, como son pequeas
piedras procedentes de las redes donde caen las
aceitunas tras su recogida bien por vareo, o por la
accin de los vibradores mecnicos.
(b) En la zona de molturacin se encuentra un molino de martillos de tipo vertical, que presenta
una potencia de 11 KW. La etapa de batido se lleva a cabo en una batidora de dos cuerpos
(modelo 7+7, potencia; 1,5 KW), con una capacidad total de 500 Kg de pasta de aceituna, est
provista de aspas o brazos de acero inoxidable que permiten el movimiento de la pasta y de
sondas de temperatura que permiten el control de temperatura de la misma durante el batido.
La separacin slido-lquido se realiza mediante un decanter o centrfuga horizontal (modelo
Baby I, potencia; 7,5 KW), con una capacidad de trabajo de 200 Kg/h de pasta de aceituna, que
es trasegada a este equipo desde la batidora por la accin de una bomba de trasiego (modelo
p40-p40, potencia; 0,55 KW). La fase lquida obtenida es recogida en un depsito de acero
inoxidable, equipado en su parte superior con un vibrofiltro que elimina los posibles restos
slidos del producto. Posteriormente se procede a la separacin de la fase oleosa de las aguas
de vegetacin del fruto, para ello se utiliza una centrfuga vertical constituida por 60 platos
(modelo Cucciolo, potencia; 1,85 KW), equipada con una bomba autoaspirante (modelo Hydra,
potencia; 0,25 KW), que lleva el aceite desde el depsito hasta la centrfuga.
48
3. Material y Mtodos
Las variables tecnolgicas bajo estudio en este trabajo experimental han sido la temperatura y
el tiempo de batido de la pasta de aceituna y, como se ha explicado previamente, el estudio de
su efecto sobre la composicin minoritaria del aceite de oliva virgen.
Las muestras de aceite de oliva virgen obtenidas fueron recogidas a la salida de la centrfuga
horizontal, filtradas a travs de papel jarabe y almacenadas en oscuridad en botellas de vidrio
topacio a 10C hasta la realizacin de los correspondientes anlisis.
49
3. Material y Mtodos
20 C 24 C 28 C 35 C 40 C
15 min
30 min ,
45 min
60 min ,
75 min
90 min ,
, Pruebas realizadas en la almazara experimental; , Pruebas realizadas con sistema Abencor.
El molino de martillos est construido de acero inoxidable y gira a una velocidad de 3000 r.p.m.
La termobatidora consta de un bao de agua en su parte inferior, cuya temperatura est
regulada mediante un termostato, y en la parte superior un sistema de paletas de acero
inoxidable que gira a 50 r.p.m. y que permite el batido simultneo de hasta ocho muestras de
pasta de aceituna. La centrfuga de pastas es de tipo cesta, est construida de acero
inoxidable y gira a 3500 r.p.m.
El procedimiento experimental que se utiliza para la obtencin del aceite de oliva virgen
mediante este sistema consiste en molturar las aceitunas sanas, una vez limpias, lavadas y
secas, en el molino de martillos. Posteriormente se homogeneiza la pasta obtenida y se
procede al batido de la misma en cazos de acero inoxidable, en cada uno de los cuales se
pesan 700 g de pasta, durante 20 minutos a 30 C. A continuacin se aaden 100 ml de agua
caliente y se contina batiendo 10 minutos ms. En el caso de pastas difciles se aade
microtalco natural en una cantidad adecuada (1,5-2,0 %) para romper la emulsin que pudiera
formarse.
50
3. Material y Mtodos
Una vez separadas las aguas de vegetacin, el aceite de oliva virgen se recoge y se filtra a
travs de papel jarabe. El aceite filtrado se recoge en una botella de vidrio de color topacio y
se almacena sin espacio de cabeza en nevera a 4 C hasta el momento de su utilizacin.
ndice de madurez.
Para la determinacin del grado de maduracin de las aceitunas se utiliza la escala propuesta
por la Estacin experimental Venta del Llano en Menjbar (Uceda y Fras, 1975), que consiste
en tomar del conjunto de frutos unas 100 aceitunas de forma aleatoria y clasificarlas en siete
grupos diferentes en funcin del color de piel y pulpa de las mismas, segn las caractersticas
que se exponen a continuacin:
Grupo 0.- Piel verde intensa.
Grupo 1.- Piel verde amarillenta.
Grupo 2.- Piel verde con manchas rojizas (inicio del envero).
Grupo 3.- Piel rojiza o morada (terminacin del envero).
Grupo 4.- Piel negra con pulpa blanca.
Grupo 5.- Piel negra con menos de la mitad de la pulpa morada.
Grupo 6.- Piel negra con la mitad o ms de la pulpa morada.
Grupo 7.- Piel negra con toda su pulpa morada.
91 , 5 V
Rendimiento (%) =
P
siendo V, el volumen de aceite obtenido (ml) y P, la cantidad de pasta de aceituna utilizada (g).
51
3. Material y Mtodos
100 P ( mt + g ) P ( mt )
RGMS (%) =
P ( ms )
siendo P(mt+g) el peso del matraz con la grasa extrada (g), P(mt) el peso del matraz seco y
vaco y P(ms) el peso de muestra seca empleado en la extraccin (g).
La obtencin del extracto fenlico se realiza homogenizando la muestra de pulpa (4,0 g) con
una mezcla de metanol:agua (80:20 v/v, 40 ml) con el homogeneizador Ultraturrax (14000 rpm,
2 min). La suspensin obtenida se agita durante 20 minutos a una velocidad de 250 rpm sobre
un bao de hielo, para ser posteriormente centrifugada durante 10 minutos a una velocidad de
5000 rpm y una temperatura de 4 C. La fase hidrometanlica se recupera y se hace pasar a
travs de un filtro de jeringa de nylon (0,45 m) para su posterior anlisis cromatogrfico.
Para realizar la separacin de los compuestos fenlicos se utiliza un equipo HPLC Agilent serie
1100 con detector ultravioleta-visible de diodos (DAD). La columna utilizada es una Zorbax SB-
C18 con tamao de partcula de 5 m y dimensiones 250 x 4,6 mm (Agilent Technologies,
USA). La temperatura de anlisis es de 30 C, y el volumen de inyeccin es de 20 l. La
separacin de los fenoles se logra mediante una elucin en gradiente con una mezcla de agua-
cido actico 95:5 (v/v), metanol y acetonitrilo, cuyo esquema se recoge en la Tabla 3.4.
Los cromatogramas se adquieren a diferentes longitudes de onda; 280, 340 y 520 nm. El
hidroxitirosol, oleuropena, demetiloleuropena se cuantifican a 280 nm, los antocianos a 520
nm y el verbascsido, los flavonoles y flavonas a 340 nm. La cuantificacin se realiza por
interpolacin del rea de cada pico en rectas de calibrado de cinco puntos obtenidas en base a
la disolucin de las sustancias estndar correspondientes (Sigma Chemical Co. y
52
3. Material y Mtodos
Tiempo (min) H2O-cido actico (%) Metanol (%) Acetonitrilo (%) Flujo (ml/min)
0 95,0 2,5 2,5 1,0
50 34,0 33,0 33,0 1,0
52 0,0 100,0 0,0 1,0
65 0,0 100,0 0,0 1,0
68 95,0 2,5 2,5 0,6
72 95,0 2,5 2,5 1,0
La Tabla 3.5 recoge los valores de absorcin en el UV-Vis, as como la relacin masa/carga
(m/z) del in molecular y los fragmentos caractersticos obtenidos para cada uno de los
compuestos fenlicos identificados en la pulpa de aceituna.
Tabla 3.5. Caractersticas UV-Vis y relacin m/z de los compuestos fenlicos identificados en la
pulpa de aceituna.
53
3. Material y Mtodos
mAU 3
250
280 nm
200
150
100 1
50
2
0
5 10 15 20 25 30 35 min
mAU
80 340 nm 4 5
60
6 8
40
20 7
0
-20
5 10 15 20 25 30 35 min
mAU
520 nm 10
30
20
9
10
0
5 10 15 20 25 30 35 min
Figura 3.5. Cromatograma de los compuestos fenlicos de la pulpa del fruto variedad Cornicabra.
1, 3,4-DHPEA (hidroxitirosol); 2, demetiloleuropena; 3, oleuropena; 4, quercetn-3-rutinsido; 5,
verbascsido; 6, luteoln-7-glucsido; 7, quercetn-3-ramnsido; 8, apigenn-7-glucsido; 9, cianidn-3-
glucsido; 10, cianidn-3-rutinsido.
Grado de acidez.
El grado de acidez de un aceite es el contenido en cidos grasos libres presentes, expresados
en tanto por ciento en peso de cido oleico, el mayoritario del aceite de oliva. La determinacin
se realiza mediante valoracin de la muestra, previamente disuelta en ter etlico-etanol 96
(1:1 v/v), con una disolucin etanlica de potasa de concentracin exactamente conocida (0,1
0,5 N) utilizando fenolftalena como indicador (CEE 2568/91).
El grado de acidez o acidez libre del aceite de oliva se calcula a partir de la siguiente expresin:
282V N
Grado de acidez =
10 P
54
3. Material y Mtodos
ndice de perxidos.
El ndice de perxidos es el mtodo qumico ms comn para determinar el grado de deterioro
oxidativo de las grasas y aceites. Los resultados se expresan como miliequivalentes de oxgeno
activo por kilogramo de aceite que producen la oxidacin del yoduro a yodo en unas
condiciones determinadas (CEE 2568/91). En la reaccin, que tiene lugar en medio cido, se
libera un mol de yodo por cada mol de oxgeno peroxdico presente en la muestra de aceite. El
yodo liberado se valora con una disolucin de tiosulfato sdico de concentracin exactamente
conocida (0,002 0,01 N) utilizando almidn como indicador.
Paralelamente se debe realizar una prueba en blanco, sin aceite, para conocer el estado de los
reactivos y la limpieza del material empleado en la determinacin analtica.
(V Vo) N 1000
ndice de perxidos =
P
E
K =
C e
55
3. Material y Mtodos
A670 V f
Clorofilas = 10000
E1% P
A472 V f
Carotenoides = 10000
E1% P
siendo A670 y A472 la absorbancia medida a 670 y 472 nm respectivamente, Vf el volumen final
de ciclohexano, E1% el coeficiente de extincin de clorofilas y carotenoides, igual a 613 y 2000,
respectivamente, y P el peso de la muestra (g).
El protocolo seguido para la formacin de los steres metlicos en fro es una modificacin del
mtodo A.O.C.S. Ch 1-91 correspondiente al Reglamento Europeo EU 796/2002. El
procedimiento consiste en disolver en hexano (3 ml) una muestra de aceite (0,5 g). A
continuacin se aaden 0,5 ml de KOH metanlica 2 N, se agita enrgicamente durante 1
minuto y se deja reposar hasta la perfecta separacin de las fases. La disolucin sobrenadante
que contiene los steres metlicos de los cidos grasos es posteriormente inyectada en el
cromatgrafo de gases.
Para la separacin de los steres metlicos de los cidos grasos se ha seguido el mtodo
recogido en el Anexo XA del Reglamento CEE 2568/91. En la determinacin se utiliz un
cromatgrafo de gases Agilent serie 6890 equipado con un detector de ionizacin de llama
(FID). La columna capilar empleada en la separacin ha sido una SGL-1000 (Sugelabor,
56
3. Material y Mtodos
El contenido de cada uno de los steres metlicos de los cidos grasos (AGi) se expresa como
porcentaje del total, calculndose de acuerdo a la siguiente expresin:
Ai
AGi = 100
AT
pA
1 6 7
160
140
120
100
5
80
60
2
40
8
9
20 10
34 11
0
2 4 6 8 10 12 14 16 min
Figura 3.6 Cromatograma de los steres metlicos de los cidos grasos de un aceite de oliva
virgen Cornicabra. 1, cido palmtico (C16:0); 2, cido palmitoleico (C16:1); 3, cido margrico (C17:0); 4,
cido margaroleico (C17:1); 5, cido esterico (C18:0); 6, cido oleico (C18:1); 7, cido linoleico (C18:2); 8,
cido linolnico (C18:3); 9, cido araqudico (C20:0); 10, cido gadoleico (C20:1); 11, cido behnico (C22:2).
57
3. Material y Mtodos
cuantificacin se ha elegido el mtodo del patrn interno, utilizndose en este caso el cido
sirngico (Sigma Chemical Co.).
Para la obtencin del extracto fenlico se toman 2,5 g de muestra, se aaden 250 l de una
disolucin (15 ppm) de patrn interno en metanol y se lleva a un evaporador rotativo a vaco
(Bchi R-114) con el fin de eliminar el disolvente. Posteriormente, el aceite se disuelve en 6 ml
de hexano y se hace pasar por una columna de extraccin en fase slida, con un volumen de 3
ml y un relleno de 0,5 g de fase diol (Supelco Inc., USA), que previamente ha sido
acondicionada con 6 ml de metanol y 6 ml de hexano. A continuacin, la columna SPE se lava
con 6 ml de hexano y 4 ml de hexano-acetato de etilo 85:15 (v/v) para eliminar los posibles
restos de grasa y otros compuestos ligeramente polares que pudieran contaminar el extracto.
La elucin del extracto fenlico se realiza por paso a travs de la columna de tres volmenes
de 5 ml de metanol que se recogen en un solo matraz. A continuacin, el extracto fenlico se
lleva a sequedad en el evaporador rotativo a vaco a una temperatura no superior a 35 C. Una
vez concentrado hasta sequedad, el residuo se disuelve en 250 l de metanol-agua 50:50 (v/v),
para su posterior anlisis cromatogrfico.
La separacin y deteccin de los fenoles del aceite de oliva se realiza en el mismo equipo
HPLC y en condiciones similares a las utilizadas con la pulpa de aceituna. Los cromatogramas
se adquieren a las longitudes de onda de 240, 280 y 335 nm, para la cuantificacin se utilizan
solamente los datos obtenidos a 280 nm, mientras que el resto se usan con fines cualitativos.
Para la cuantificacin se recurre al empleo del patrn interno, en este caso cido sirngico, y
por tanto la concentracin de los compuestos (Ci) se expresa en miligramos de cido sirngico
por kilogramo de aceite, calculndose de acuerdo con la siguiente expresin:
C PI Ai
Ci =
API
donde CPI es la concentracin del patrn interno en el aceite (mg/kg) y Ai y API son las reas del
compuesto fenlico de inters y el patrn interno respectivamente.
C F = C i FR
58
3. Material y Mtodos
Tabla 3.6. Factores de respuesta* respecto al cido sirngico de los principales fenoles
presentes en el aceite de oliva virgen.
mAU
13
600
8
500
400
1 10
300
14
200
2
12
100 P.I. 11
3 4 5 6 7 9
0
5 10 15 20 25 30 35 40 45 min
Figura 3.7. Cromatograma de los compuestos fenlicos de un aceite de oliva virgen Cornicabra. 1,
3,4-DHPEA (hidroxitirosol); 2, p-HPEA (tirosol); 3, cido vanlico; P.I., cido sirngico; 4, vainillina; 5,
cido p-cumrico; 6, 3,4-DHPEA-AC; 7, cido ferlico; 8, 3,4-DHPEA-EDA
decarboximetilada+carboximetilada; 9, p-HPEA-AC; 10, p-HPEA-EDA; 11, pinorresinol; 12, 1-
acetoxipinorresinol+cido t-cinmico; 13, 3,4-DHPEA-EA; 14, p-HPEA-EA.
59
3. Material y Mtodos
Para la obtencin del extracto se toma 1,0 g de muestra, se aaden 250 l de una disolucin
(200 ppm) de patrn interno en metanol y se lleva a un evaporador rotativo a vaco para
eliminar el disolvente. Posteriormente, el aceite se disuelve en 2 ml de hexano y se pasa a un
tubo de centrfuga (15 ml), al que se adicionan 5 ml de acetonitrilo y se agita con ayuda de un
vrtex durante 15 s para favorecer la extraccin del oleocantal, a continuacin se centrifuga
(4000 rpm, 5 min) para separar el solvente de la fase oleosa. Este procedimiento se repite tres
veces, y el solvente del extracto obtenido se elimina mediante flujo de N2. Una vez concentrado
hasta sequedad, el residuo se disuelve en 1 ml de metanol-agua 50:50 (v/v), y se adiciona 1 ml
de hexano con el fin de eliminar las posibles restos de aceite, este proceso se repite tres veces.
El tubo se centrifuga y se recupera la fase metanlica para su posterior anlisis cromatogrfico.
Para realizar la separacin del oleocantal se utiliza un equipo HPLC Agilent serie 1100
equipado con un detector ultravioleta-visible de diodos (DAD). La columna empleada en la
separacin es una Zorbax SB-C18 con tamao de partcula de 5 m y dimensiones 250 x 4,6
mm (Agilent Technologies, USA). La temperatura de anlisis es de 25 C, y el volumen de
inyeccin es de 20 l. La separacin de los fenoles se logra mediante una elucin en gradiente
con una mezcla de acetonitrilo y agua, cuyo esquema se recoge en la Tabla 3.7. Los
cromatogramas se adquieren a la longitud de onda de 280 nm y a modo de ejemplo se
presenta uno de ellos en la Figura 3.8.
Para la cuantificacin se recurre al empleo del patrn interno, en este caso 3,5-dimetoxifenol, y
por tanto la concentracin de oleocantal (Co) se expresa en miligramos de 3,5-dimetoxifenol por
kilogramo de aceite, calculndose de acuerdo con la siguiente expresin:
60
3. Material y Mtodos
C PI AO
CO =
API
donde CPI es la concentracin del patrn interno en el aceite (mg/kg) y AO y API son las reas
del oleocantal y el patrn interno respectivamente.
mAU
40
30
20
Oleocantal
Forma cis Forma trans
10
0 5 10 15 20 25 30 min
A225 V
K225 =
100 P
siendo P el peso de la muestra (g), A225 la absorbancia del extracto medido a 225 nm y V el
volumen de extracto (ml).
61
3. Material y Mtodos
El equipo empleado para la separacin y cuantificacin del -tocoferol ha sido un HPLC Agilent
serie 1100 acoplado a un detector de fluorescencia Thermo Finnigan modelo FL3000 y una
columna (250 x 4,6 mm) de relleno Lichrosorb Si-60 de 5 m (Sugerlabor, Espaa). La fase
mvil utilizada es una mezcla de n-hexano-isopropanol 98,5:1,5 (v/v) a un flujo de 1 ml/min, y
un volumen de inyeccin de 20 l. Para la deteccin del - tocoferol mediante fluorescencia se
emplea una longitud de onda de excitacin de 290 nm y una longitud de onda de emisin de
330 nm (Figura 3.9).
mAU
225
- tocoferol
200
175
150
125
100
75
- tocoferol - tocoferol
50
1 2 3 4 5 6 7 8 min
CD V
C T =
P
62
3. Material y Mtodos
El aparato consta de un bloque calefactor, que puede alcanzar temperaturas de hasta 220 C.
Para acelerar el proceso de oxidacin se introduce un flujo de aire a travs del aceite. Este aire,
a la vez que proporciona el oxgeno necesario para la oxidacin, arrastra los compuestos
voltiles formados durante el proceso y los conduce a un frasco borboteador que contiene agua
y una clula que mide constantemente la conductividad del agua. Cuando se inicia la
descomposicin de los hidroperxidos, se desprenden una serie de compuestos voltiles, que
son retenidos en el agua desionizada dando lugar a un aumento de la conductividad. El punto
de inflexin de la curva de conductividad, denominada curva de oxidacin, define el periodo de
induccin del proceso de oxidacin de la muestra, y se muestra a modo de ejemplo en la
Figura 3.10.
Las condiciones de trabajo empleadas para el aceite de oliva virgen de este trabajo de
investigacin son una temperatura de 120 C y un caudal de aire de 20 l/h, segn el mtodo
propuesto por Labli et al. (1986).
63
3. Material y Mtodos
El procedimiento analtico consiste en pesar una muestra de aceite de oliva virgen (1,5 g) a la
que posteriormente se aade el patrn interno disuelto en aceite de oliva refinado hasta una
concentracin de 1,5 g/g en un vial de 10 ml, sellado mediante un septum de silicona. El
proceso SPME se realiza mediante la exposicin de una fibra de 2 cm de longitud de
DVB/Carboxeno/PDMS (50/30 m, Supelco Inc., USA) durante 30 min en el espacio de cabeza
de la muestra situada en un bloque calefactor a 40 C. Posteriormente la fibra se retrae dentro
de la aguja e inmediatamente se transfiere y desorbe durante un minuto en el puerto de
inyeccin de un cromatgrafo de gases Agilent serie 6890 equipado con un detector de
ionizacin de llama (FID). La separacin de los compuestos voltiles se realiza mediante una
columna capilar Supelcowax-10 (30 m x 0,25 mm, Supelco Inc., USA) recubierta interiormente
de una pelcula de 0,25 m de espesor de 100% polietilenglicol. Como gas portador se utiliza
helio, con una presin en cabeza de columna de 10 psi. Durante el anlisis la temperatura del
puerto de inyeccin y del detector se mantienen a 260 C y 280 C, respectivamente y el
gradiente de temperatura utilizado en el horno es el siguiente: temperatura inicial 35 C durante
10 minutos, rampa de 3 C por minuto hasta 160 C y posteriormente 15 C por minuto hasta
200 C, temperatura final que se mantiene durante 5 minutos.
Para la cuantificacin se recurre al empleo del patrn interno, en este caso 4-metil-2-pentanol,
y por tanto la concentracin de los compuestos (Ci) se expresa en miligramos de 4-metil-2-
pentanol por kilogramo de aceite, calculndose de acuerdo con la siguiente expresin:
C PI Ai
Ci =
API
en donde CPI es la concentracin del patrn interno en el aceite (mg/kg) y Ai y API son las reas
del compuesto voltil de inters y el patrn interno, respectivamente.
Los compuestos voltiles se identificaron por comparacin de los tiempos de retencin y los
espectros de masas de las correspondientes sustancias estndar (Sigma Chemical Co.)
adicionadas a aceites de oliva refinado. El equipo utilizado fue MS Agilent serie 5975C
equipado con un detector de ionizacin por impacto electrnico (IE+) y acoplado a un GC
Agilent serie 6850, con columna capilar DB-Wax (30 m x 0,25 mm, J&W Scientific, USA)
recubierta interiormente de una pelcula de 0,25 m de espesor de 100% polietilenglicol. Como
64
3. Material y Mtodos
gas portador se utiliza helio, con una presin en cabeza de columna de 5 psi. La temperatura
de la lnea de transferencia se mantiene a 280 C, y la de la fuente de ionizacin y el
cuadrupolo, a 230 C y 150 C, respectivamente, con un voltaje en el electromultiplicador igual
a 941 eV. La Tabla 3.8 recoge los valores de la relacin masa/carga (m/z) de los iones
caractersticos obtenidos en los principales compuestos voltiles presentes en el aceite de oliva
virgen.
Tabla 3.8. Valores m/z de los iones caractersticos obtenidos para los principales compuestos
voltiles presentes en el aceite de oliva virgen.
TR Iones MS
Pico CAS Number PM Compuesto identificado
(min) (m/z)
1 13,1 27, 57 1629-58-9 84 1-penten-3-ona
2 17,0 56, 72, 82 66-25-1 100 hexanal
3 22,3 29, 57 616-25-1 86 1-penten-3-ol
4 25,8 55, 69, 83, 98 6728-26-3 98 E-2-hexenal
5 28,6 56, 61, 69, 84 142-92-7 144 acetato de hexilo
6 31,1 57, 68 1576-95-0 86 Z-2-penten-1-ol
7 31,3 43, 55, 67, 82 3681-71-8 142 acetato de Z-3-hexenilo
8 32,9 56, 69, 84 111-27-3 102 hexan-1-ol
9 34,5 55, 67, 82 928-96-1 100 Z-3-hexen-1-ol
10 35,6 57,67, 82 928-95-0 100 E-2-hexen-1-ol
pA 4
80
70
60
50
P.I.
40
2
30 9
1
6
20 8
3
10 10
5 7
0
5 10 15 20 25 30 35 min
Figura 3.11. Cromatograma de los compuestos voltiles de un aceite de oliva virgen Cornicabra.
1, 1-penten-3-ona; 2, hexanal; 3, 1-penten-3-ol; P.I., 4-metil-2-pentanol; 4, E-2-hexenal; 5, acetato de
hexilo; 6, Z-2-penten-1-ol; 7, acetato de Z-3-hexenilo; 8, hexan-1-ol; 9, Z-3-hexen-1-ol; 10, E-2-hexen-1-
ol.
65
3. Material y Mtodos
Valoracin organolptica.
El anlisis sensorial de las muestras de aceite de oliva virgen de este trabajo de investigacin
se ha realizado siguiendo el protocolo descrito en el Anexo XII del Reglamento CE n 796/2002.
Participaron entre 10 y 12 catadores adecuadamente seleccionados y entrenados
pertenecientes al Panel Analtico de la Denominacin de Origen de los Montes de Toledo y
reconocido por el Consejo Olecola Internacional (COI).
Todos los anlisis y tratamientos estadsticos de resultados que aparecen a lo largo de esta
memoria han sido realizados aplicando el programa informtico SPSS (SPSS Inc., Chicago,
USA).
Estadstica descriptiva.
Este tratamiento se ha empleado para presentar los datos experimentales resumidos en las
tablas de la memoria, a travs de los parmetros estadsticos media y desviacin tpica, que
representan la dispersin de los valores observados.
Anlisis de la varianza.
El anlisis de la varianza estudia los efectos de uno o ms factores sobre la variacin de los
valores medios dentro de uno o ms grupos de datos, determinando si existen o no diferencias
estadsticamente significativas entre los mismos. El nivel de la significacin estadstica se
establece a partir del valor de F que proporciona este anlisis. Para determinar entre qu
grupos existen diferencias significativas se ha empleado el test de Duncan.
66
3. Material y Mtodos
Anlisis discriminante.
El anlisis discriminante es una tcnica multivariante de clasificacin supervisada, cuyo objetivo
es encontrar funciones o reglas de clasificacin que permitan la diferenciacin entre
poblaciones. Cuando en este anlisis las variables independientes se van seleccionando de
forma escalonada, de manera que en cada paso interviene la variable que ms contribuye a la
separacin entre los grupos, la tcnica se denomina anlisis discriminante por pasos sucesivos.
67
4. RESULTADOS EXPERIMENTALES
Y DISCUSIN
4.1. Resultados Experimentales
Del trabajo de investigacin realizado durante la ejecucin de esta Tesis Doctoral han derivado
las siguientes publicaciones cientficas:
ART. II: Aurora Gmez-Rico, M. Desamparados Salvador, Alfonso Moriana, David Prez,
Nicols Olmedilla, Francisco Ribas, Giuseppe Fregapane. 2007. Influence of different
irrigation strategies in a traditional Cornicabra cv. olive orchard on virgin olive oil
composition and quality, Food Chemistry, 100, 568-578.
71
4.1. Resultados Experimentales
Tras el anlisis del efecto de esta variable agronmica sobre la composicin fenlica de la
drupa, se ha determinado que el olesido oleuropena es el compuesto fenlico ms abundante
presente en los frutos de las diversas variedades estudiadas, siendo su contenido ampliamente
variable entre las mismas, al estar comprendido entre 2200 y 11600 mg/Kg, valores
cuantificados en los frutos verdes de las variedades Arbequina y Cornicabra, respectivamente
(ver ART. I; Tabla 1). Por otro lado, el olesido demetiloleuropena se ha detectado nicamente
en los frutos Arbequina, doblando su concentracin en la drupa a lo largo de la maduracin de
la misma y alcanzando valores de 2000 mg/Kg en el fruto maduro (I.M 4,0).
72
4.1. Resultados Experimentales
por los frutos de Picudo, que presentaron una alta concentracin de hidroxitirosol y un tercer
grupo formado por Arbequina, Morisca y Picolimn, que mostraron los mayores niveles de
verbascsido y un contenido similar en flavonoides (ART. I; Tabla 1).
Del mismo modo, en el anlisis de los compuestos fenlicos y voltiles de los correspondientes
aceites de oliva virgen elaborados a partir de las variedades estudiadas, se obtuvieron perfiles
de composicin cualitativa semejante, aunque muy diferentes desde un punto de vista
cuantitativo. As, mientras que los derivados secoiridoideos del hidroxitirosol y tirosol son los
principales fenoles presentes en todos los aceites, su distribucin vara entre las distintas
variedades, siendo los derivados del hidroxitirosol los fenoles complejos ms abundantes en las
variedades Arbequina, Cornicabra, Picolimn y Picual (principalmente la forma 3,4-DHPEA-
EDA), con valores comprendidos entre 105,0 mg/Kg (en Morisca con I.M 3,0) y 1113,2 mg/Kg
(en Cornicabra con I.M 2,5), mientras que los secoiridoideos del tirosol se detectaron en mayor
concentracin que sus anlogos del hidroxitirosol en los aceites Morisca y Picudo
(principalmente la forma p-HPEA-EDA), con concentraciones entre 54,8 mg/Kg (Picolimn, I.M
3,0) y 769,6 mg/Kg (Picudo, I.M 0,8) (ART. I; Tabla 2).
En lo que respecta al contenido voltil, la fraccin aldehdica C6, constituida por E-hexenal y
hexanal, ha predominado en todos los aceites obtenidos en esta experimentacin, siendo
adems los compuestos voltiles que han permitido la mejor discriminacin entre los aceites en
funcin de la variedad de procedencia, especialmente el contenido de E-2-hexenal, el cual ha
variado desde 20,55 mg/Kg, como valor medio, en los aceites Arbequina hasta 3,15 mg/Kg en
la variedad Cornicabra. Otro aspecto a destacar es la presencia de steres C6 en el aroma de
los aceites, la cual est claramente establecida en la variedad Morisca, y especialmente en
Arbequina y Picual (ART. I; Tabla 3).
Por otro lado, puesto que los compuestos fenlicos presentes en un AOV son derivados de los
olesidos y lignanos de la aceituna, se ha relacionado el contenido oleosdico de las drupas
con la composicin secoiridoidea cuantitativa descrita en los correspondientes aceites del
ensayo, observndose un ratio considerablemente heterogneo entre las variedades
estudiadas (aproximadamente 2,3 para Picudo, 4,5-5,5 para Cornicabra y Morisca, 7,0-8,5 para
Arbequina y Picual y 28 para Picolimn) (ART. I; Figura 2). Lo cual permite establecer que la
variedad Picolimn, con un contenido de oleuropena relativamente alto en su fruto (4760
mg/Kg de media entre I.M 2,5 y 4,0) proporcion el menor contenido secoiridoideo en su aceite
(160 mg/Kg, I.M 3,0; ratio 29,8), mientras que Picudo, con un nivel bajo de oleuropena (2305
mg/Kg, I.M 2,5), present un contenido relativamente alto de fenoles complejos en su aceite
(1040 mg/Kg, I.M 2,5; ratio 2,3).
73
4.1. Resultados Experimentales
Otra de las variables agronmicas bajo estudio ha sido el grado de maduracin del fruto y su
influencia tanto en la composicin fenlica de la drupa, como en los perfiles fenlicos y voltiles
del aceite de oliva virgen de calidad. Para ello, se han utilizados las drupas y aceites de oliva
virgen obtenidos a partir de las variedades Arbequina, Cornicabra, Morisca, Picolimn, Picudo y
Picual a distintos estados de maduracin, como se ha comentado previamente. Adems, con
el objetivo de realizar un estudio ms exhaustivo de esta variable agronmica y para evaluar su
efecto sobre la variedad Cornicabra, mayoritaria en la regin de Castilla-La Mancha, se
prepararon un mayor nmero de muestras de aceite de esta variedad a partir de frutos con un
I.M entre 1,5-2,0 hasta 5,0-5,5, sometidos a distintas condiciones de irrigacin (secano, riego
deficitario regulado (RDI), FAO y 125 FAO; sobre este aspecto se discutir en profundidad en el
siguiente apartado).
74
4.1. Resultados Experimentales
Sobre la evolucin de los compuestos voltiles C6 presentes en los aceites de las variedades
Morisca, Picolimn y Picudo obtenidos en estados de maduracin temprana (I.M1 0,8-1,5 y I.M2
2,3-3,0), se ha observado un incremento del contenido de E-2-hexenal, hexan-1-ol y Z-3-hexen-
1-ol, especialmente considerable en el caso del AOV Picudo (I.M1 0,8 y I.M2 2,3) en donde
estos voltiles aumentaron un 47%, 162% y 55%, respectivamente. Por otro lado, en los
aceites Arbequina, Cornicabra y Picual obtenidos a partir de frutos en estados de maduracin
algo superiores (I.M1 2,0-2,5 y I.M2 3,5-4,0), el incremento de estos voltiles fue menos
perceptible e incluso se pudo observar un ligero descenso de los aldehdos C6 (ART. I; Tabla
3). De la misma forma que en los compuestos fenlicos, la evolucin de los compuestos
voltiles de la ruta LOX a lo largo de la maduracin del fruto, se ha estudiado de forma ms
exhaustiva en la variedad Cornicabra, obtenindose un descenso de los aldehdos C6 y los
voltiles C5, como 1-penten-3-ona y 1-penten-3-ol, desde los estados inmaduros a sobre-
maduros, especialmente notable en el caso del E-2-hexenal, cuya concentracin disminuy
entre un 40% (en aceites elaborados con frutos de secano) y un 60% (en aceites obtenidos con
el tratamiento de riego FAO) durante la maduracin del fruto (campaa 2003/04; ART. III;
Figura 2 y Tabla 2). En lo referente a los alcoholes C6, se observ un considerable descenso
del E-2-hexen-1-ol, mientras que la presencia de hexan-1-ol y Z-3-hexen-1-ol aument de
forma significativa durante la maduracin del fruto. Por ejemplo el contenido de hexan-1-ol se
increment desde 0,04 mg/Kg hasta 0,14 mg/Kg y el de Z-3-hexen-1-ol desde 0,17 mg/Kg
hasta 0,35 mg/Kg (AOV FAO) (campaa 2003/04; ART. III; Tabla 2).
La aplicacin del Anlisis Discriminante sobre el contenido fenlico y voltil de los aceites de
oliva virgen Cornicabra obtenidos a lo largo de la maduracin de la aceituna, permiti clasificar
correctamente el 85% de los mismos en funcin del ndice de madurez de los frutos de los que
procedan mediante el empleo de las variables hexan-1-ol, E-2-hexen-1-ol, 3,4-DHPEA-EDA y
p-HPEA-EDA (ART. III; Figura 3), lo que pone de manifiesto la gran influencia de esta variable
agronmica en la composicin minoritaria del producto obtenido.
75
4.1. Resultados Experimentales
El nivel de estrs hdrico de los olivos est relacionado directamente con el perfil
fenlico secoiridoideo del aceite de oliva virgen e inversamente con su contenido en
voltiles C6
En lo relativo al anlisis del efecto que diversas estrategias de riego aplicadas en el olivar
ejercen sobre la calidad y composicin del aceite de oliva virgen obtenido, es importante
destacar que para llevar a cabo este estudio se han realizado dos ensayos diferentes;
(i) El primero de ellos se ha llevado a cabo en un olivar adulto Cornicabra de marco tradicional
(50 aos) originariamente en condiciones de secano, en donde se aplicaron distintas
metodologas de irrigacin basadas en el coeficiente de evapotranspiracin del cultivo (ETc) y
que suponan la aplicacin de dosis de riego equivalentes o superiores a este coeficiente, como
es el 100% ETc (o mtodo FAO) o el 125% ETc (125 FAO), adems de dos estrategias distintas
de riego deficitario regulado (regulated deficit irrigation, RDI) en las cuales las dosis de agua se
administraron bien a lo largo de toda la estacin de riego (RDI-1), o bien a partir del mes de
agosto, cuando comienza la acumulacin de aceite en el fruto (RDI-2).
(ii) El segundo ensayo se ha realizado en dos olivares jvenes de cubierta incompleta de las
variedades Morisca y Cornicabra, plantados en 1998 y 1999 respectivamente, en los cuales se
evaluaron dos programaciones de riego basadas en medidas del estado hdrico del olivo in situ,
como son el potencial hdrico del tronco (stem water potencial, SWP, con umbrales definidos a -
2,0 MPa (SWP -2,0) y -1,2 MPa (SWP -1,2), y las fluctuaciones del dimetro del tronco (trunk
diameter fluctuations, TDF), como posibles alternativas a la estrategia de riego en el olivar
basada en el clculo del ETc, como es el 100% ETc (o mtodo FAO).
En primer lugar, resaltar el aumento significativo de la productividad del olivar que supone la
aplicacin de riego en comparacin con el cultivo de secano, cifrndose aproximadamente en
un 35%. Se observ como la produccin media obtenida en el olivar adulto Cornicabra a lo
largo de las campaas olivareras consecutivas entre 2001 y 2004 aumentaba desde 39,2 Kg de
aceituna por olivo en condiciones de secano hasta valores medios de 51,8-52,7 Kg de aceituna
por olivo en las reas de regado (ART. II; Tabla 1).
76
4.1. Resultados Experimentales
En lo referente a la influencia del riego sobre la composicin fenlica de los AOV, en ambos
ensayos se ha descartado cualquier tipo de influencia de este factor agronmico sobre el
contenido de fenoles simples en el aceite, como es el caso del hidroxitirosol, tirosol y los cidos
fenlicos, ya que no se observaron diferencias estadsticas relacionadas con los diversas
estrategias de riego ensayadas. As por ejemplo, el contenido de hidroxitirosol en los AOV
Cornicabra obtenidos en el olivar tradicional adulto durante la campaa 2003/04, varo entre
1,46 mg/Kg (RDI-1; I.M 1,8) y 3,31 mg/Kg (125 FAO; I.M 4,9), y el de tirosol entre 1,62 mg/Kg
(secano; I.M 2,7) y 4,89 mg/Kg (FAO; I.M 5,5), mientras que la cantidad de cidos fenlicos
detectada fue muy pequea -vainillina (<0,22 mg/Kg), cido vanlico (<0,26 mg/Kg), cido p-
cumrico (<0,25 mg/Kg) y cido ferlico (<0,21 mg/Kg)- independientemente de la estrategia de
riego utilizada.
Por el contrario, los niveles de los derivados secoiridoideos presentes en los aceites de oliva
virgen, tanto de la variedad Cornicabra como Morisca, dependieron ampliamente de las
tcnicas de riego utilizadas en los olivares. Se ha observado que el estado hdrico del olivo
afecta en mayor medida al contenido de derivados secoiridoideos del hidroxitirosol presentes
en el AOV que a los derivados del tirosol, ya que condiciones de estrs hdrico elevadas
suponen un mayor incremento en la concentracin de los primeros (ART. III; Figura 1 y ART.
IV; Figura 2). De hecho, son las formas complejas 3,4-DHPEA-EDA, 3,4-DHPEA-EA y p-HPEA-
EDA los compuestos fenlicos ms afectados por el estado hdrico de los olivos, de manera
que la concentracin de los mismos es significativamente superior en aquellos aceites
procedentes de frutos en condiciones de mayor dficit hdrico, como es el caso de las
condiciones de secano en el olivar adulto o el riego SWP -2,0 en el olivar joven. As, por
ejemplo, en los aceites Cornicabra obtenidos en el olivar adulto, el contenido de 3,4-DHPEA-
EDA disminuy alrededor de un 40% al comparar la estrategia FAO con las condiciones de
secano (campaa 2003/04; I.M 4,0), mientras que los niveles de 3,4-DHPEA-EA y p-HPEA-
EDA descendieron alrededor del 35% y 25%, respectivamente, al comparar los mismos AOV
(ART. III; Tabla 1). Igualmente, en los aceites Cornicabra obtenidos en el olivar joven, la
concentracin de 3,4-DHPEA-EDA, 3,4-DHPEA-EA y p-HPEA-EDA disminuyeron alrededor del
40%, 50% y 25%, respectivamente, al comparar los AOV obtenidos mediante las estrategias
FAO y SWP -2,0 (campaa 2005/06; I.M 4,0) (ART. IV; Tabla 3).
77
4.1. Resultados Experimentales
125 FAO) en la campaa 2004/05 (ART. II; Tabla 4), segn los datos proporcionados por el
panel de cata oficial de la Denominacin de Origen Protegida Montes de Toledo. Tambin se
ha procedido a determinar el amargor de los aceites obtenidos en ambos ensayos mediante la
medida instrumental del amargor (ndice K225), obtenindose valores acordes con el contenido
fenlico de los mismos y siendo los aceites procedentes de las condiciones hdricas ms
deficitarias - secano y SWP -2,0 - los que presentaron los mayores valores de K225. Por
ejemplo, en los aceites Cornicabra (I.M 4,0) obtenidos en el olivar tradicional adulto los
valores de K225 variaron desde 0,77 (AOV secano) a 0,49 (AOV 125 FAO) en la campaa
2003/04 y desde 0,59 (AOV secano) a 0,36 (AOV 125 FAO) en la campaa 2004/05 (ART. II;
Tabla 4), e igualmente sucedi en los aceites procedentes del ensayo en olivares jvenes
(campaa 2006/07; I.M 3,0-3,5), para los cuales se registraron valores de K225 entre 0,78
(SWP -2,0) y 0,64 (SWP -1,2; AOV Cornicabra) y entre 0,27 (SWP -2,0) y 0,09 (SWP -1,2; AOV
Morisca) (ART. IV; Tabla 4).
Por otro lado, se ha determinado en ambos ensayos, que los compuestos voltiles del AOV
ms afectados por las tcnicas de irrigacin son el E-2-hexenal, hexanal, Z-3-hexen-1-ol y
hexan-1-ol, mostrando una relacin inversa respecto al nivel de estrs hdrico del olivo, de
manera que el contenido en voltiles C6 de los aceites procedentes de los olivos con mayor
dficit hdrico (secano o SWP -2,0) ha sido claramente inferior a aquellos obtenidos mediante el
resto de tcnicas de irrigacin, donde los olivos presentaron un menor estrs hdrico. As por
ejemplo, el contenido en voltiles C6 de los aceites Cornicabra del ensayo en el olivar
tradicional adulto se increment un 85% (campaa 2003/04; I.M 4,0) y un 56% (campaa
2004/05; I.M 4,0) al comparar los AOV obtenidos en condiciones de secano y mediante la
metodologa de riego FAO (ART. III; Tabla 2). Del mismo modo los aceites del ensayo
efectuado en el olivar joven obtenidos con el mtodo FAO presentaron un contenido en
voltiles C6 alrededor de un 35% (AOV Cornicabra) y un 18% (AOV Morisca) superior respecto
de los obtenidos con la estrategia de riego SWP -2,0 (campaa 2006/07; I.M 3,0-3,5) (ART.
IV; Tabla 5).
78
4.1. Resultados Experimentales
Las variables del proceso de elaboracin del aceite de oliva bajo estudio en este trabajo
experimental han sido la temperatura y el tiempo utilizados durante la etapa tecnolgica de
batido de la pasta de aceituna. Se realizaron diversos ensayos en Almazara Experimental
utilizndose diferentes temperaturas (20 C, 28 C y 40 C) y tiempos (30 min, 60 min y 90 min)
de batido y procesndose dos lotes de aceitunas Cornicabra diferentes (lote I y lote II). Adems
se llevaron a cabo pruebas adicionales con el sistema de extraccin a escala de laboratorio
Abencor, emplendose los mismos lotes de aceituna, pero ampliando el rango de temperatura-
tiempo estudiados a 20, 24, 28, 35 y 40 C y 15, 30, 45, 60, 75 y 90 minutos. Los detalles
especficos de las pruebas experimentales realizadas estn ampliamente recogidos en la
seccin de Material y Mtodos.
79
4.1. Resultados Experimentales
La evolucin del perfil fenlico y voltil registrado en los aceites obtenidos con el sistema de
molturacin Abencor ha sido similar a los cambios observados en los aceites de la Almazara
Experimental. El aumento de la temperatura de batido de 20 C a 40 C implica un incremento
de los derivados secoiridoideos del hidroxitirosol y del tirosol del 120% y 65%, respectivamente,
mientras que la prolongacin del tiempo de batido hasta 90 minutos supuso un descenso del
45% en los secoiridoideos del hidroxitirosol y un aumento del 50% en el nivel de los
secoiridideos del tirosol, como valor medio entre los lotes I y II (ART. V; Figuras 3a y 3b). De la
misma forma, el contenido en voltiles C6 de los aceites disminuy al aumentar la temperatura
de batido de 20 C a 40 C, debido principalmente a un considerable descenso en el contenido
80
4.1. Resultados Experimentales
de la fraccin aldehdica C6 (alrededor de un 45% como media de los dos lotes estudiados),
mientras que tiempos ms largos de batido suponen en esta misma fraccin voltil incrementos
del 37% (ART. V; Figuras 6a y 6b).
A continuacin se presentan los artculos cientficos derivados de esta Tesis Doctoral. En ellos
se describe y explica de forma detallada los resultados que se han generado tras la labor
investigadora realizada.
81
4.1. Resultados Experimentales
82
Food Research International 41 (2008) 433440
a r t i c l e i n f o a b s t r a c t
Article history: Phenolics and volatiles are the compounds mainly responsible for the desirable avour of extra virgin
Received 7 October 2007 olive oils and therefore to a large extent determine the degree of consumer preference for this highly
Accepted 24 February 2008 regarded product. The effect of both (i) the nature of the cultivar and (ii) the degree of ripening of the
olive fruit on the biophenolic and volatile proles of six different Spanish varieties (Arbequina, Cornic-
abra, Morisca, Picolimn, Picudo and Picual) and their corresponding virgin olive oils was determined in
Keywords: this study. A clear and statistically signicant difference was observed for the oleuropein content, the
Olive cultivar
main phenolic component found in the olive varieties studied. Demethyloleuropein was only found in
Ripening
Virgin olive oil
the Arbequina variety and its content doubled during the ripening process. Verbascoside steadily
Phenols increased throughout fruit maturation and cyanidin 3-O-rutinoside was the most abundant anthocya-
Volatiles nin in all the varieties studied. Within the same cultivar a relationship between the oleosides content in
the fruit and the presence of secoiridoids in the virgin olive oils was observed; however, the ratio
between biophenols content in the olive fruit and in the virgin olive oil varied signicantly for each
of the cultivars studied (ranging from 2.3 for Picudo and 28 for Picolimon). The major volatile compo-
nent was the C6 aldehyde fraction whose content varied greatly between the different varieties
studied: E-2-hexenal content ranged from 20.5 mg of internal standard (4-methyl-2-pentanol) per kg
of oil in the Arbequina variety to 3.1 mg/kg for Cornicabra; the amount of hexanal ranged from
1.75 mg/kg in Morisca to 0.70 mg/kg for Picual samples.
2008 Elsevier Ltd. All rights reserved.
1. Introduction The phenolic and volatile contents and proles of virgin olive
oils depend on both (I) the initial composition characteristics of
Extra virgin olive oil is obtained from healthy olive fruits by the olive fruits employed for processing and (II) the technological
mechanical processes only and is therefore a vegetable oil ready factors used during the oil mill process, in particular milling and
for direct human consumption. The ne sensory characteristics of malaxation conditions. The chemical and biochemical (especially
this fruit juice, which possesses a unique aroma and taste, are enzyme activity) composition of olives rely on some agronomical
mainly due to the presence of minor components, chiey volatile factors, e.g. olive cultivar (Esti, Cinquanta, & La Notte, 1998; Romani,
and phenolic compounds (Angerosa, 2002; Aparicio & Luna, Mulinacci, Pinelli, Vincieri, & Cimato, 1999), the ripening stage of
2002). Volatiles are mainly responsible for the aroma of virgin olive the fruit (Amiot, Fleuriet, & Macheix, 1989), pedoclimatic condi-
oil, especially for the green sensory notes of high-quality extra vir- tions (Vinha et al., 2005) and irrigation management (Patumi
gin olive oils, whereas compounds with a phenolic structure affect et al., 2002; Tovar, Romero, Girona, & Motilva, 2002). The charac-
both the taste, in particular the positive bitterness organoleptic teristics and quality of the olive fruits entering the oil mill is there-
attribute, and the oxidative stability of virgin olive oil (Angerosa, fore a key factor, and probably the most important variable,
Mostallino, Basti, & Vito, 2000). Phenolics and volatiles are the involved in the quality assurance of the nal product.
compounds mainly responsible for the desirable avour of extra Several studies have already been carried out with the aim of
virgin olive oils and therefore to a large extent determine the describing the differences found between the phenolic proles of
degree of consumer preference for this highly regarded product. different olive cultivars (Esti et al., 1998; Romani et al., 1999; Vin-
ha et al., 2005), as well as their evolution throughout the ripening
process (Amiot, Fleuriet, & Macheix, 1986; Morell, Vuorela,
* Corresponding author. Romero, Motilva, & Heinonen, 2005; Servili, Baldioli, Selvaggini,
E-mail address: amparo.salvador@uclm.es (M.D. Salvador). Macchioni, & Montedoro, 1999). Moreover, phenolic and volatile
0963-9969/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2008.02.003
434 A. Gmez-Rico et al. / Food Research International 41 (2008) 433440
compounds have likewise been used to discriminate between mat- 2.4. Analytical determinations in olive fruit
uration stages, geographical origin and variety (Angerosa, Basti, &
Vito, 1999; Aparicio & Morales, 1998; Vichi, Pizzale, Conte, Buxade- 2.4.1. Phenolic compounds
ras, & Lopez-Tamames, 2003). A sample of olive pulp (4.0 g) was homogenised with a mix-
However, there have been few (Kalua, Allen, Bedgood, Bishop, & ture of methanol:water (80:20 v/v) (40 ml) during 2 min with
Prenzler, 2005) research projects focusing on phenols and volatiles an Ultraturrax homogenizer (14,000 rpm). The suspension ob-
in both olives and virgin olive oils. In fact, one of the main novelties tained was shaken (20 min, 150 rpm, <4 C in darkness), and then
of this research is to combine these two issues studying the olive centrifuged (10 min, 5000 rpm and 4 C). The hydromethanolic
fruit minor components as directly related to the corresponding phase was recovered and ltered with a 0.45 lm nylon syringe
virgin olive oil in order to get a deeper and clearer idea of the effect lter. The phenolic fraction extracted was analyzed by high-per-
of cultivar and ripening on the quality of the nal product. More- formance liquid chromatography (HPLC) using an Agilent Tech-
over, in this study all the olive varieties have been grown in the nologies 1100 series system equipped with an automatic
same olive orchard, with therefore identical agronomical and injector, a column oven and a diode array UV detector. A Spheri-
pedoclimatic conditions. sorb S3 ODS2 column (250 4.6 id mm, 5 lm particle size)
The aim of this work was, therefore, to determine the effect of (Waters Co., Milford, Massachusetts, USA), maintained at 30 C,
both (i) the nature of the cultivar and (ii) the ripening stage of was used with an injection volume of 20 ll and a ow rate of
the olive fruit on the biophenolic prole of six different Spanish 1.0 ml/min. The mobile phase consisted of a mixture of water/
varieties: Arbequina, Cornicabra, Morisca, Picolimn, Picudo and acetic acid (95:5 v/v) (solvent A), methanol (B) and acetonitrile
Picual, and their corresponding virgin olive oils. The volatile com- (C): from 95% (A)2.5% (B)2.5% (C) to 34% (A)33% (B)33% (C)
position of the virgin olive oils obtained at different maturation in 50 min. Chromatograms were recorded at 280 nm, 340 nm
stages was likewise studied, the ultimate goal being to enhance and 520 nm. Hydroxytyrosol, oleuropein and demethyloleuropein
knowledge with regard to the most relevant minor components were quantied at 280 nm, anthocyanins at 520 nm and verbas-
of olive fruits and their virgin olive oils which could be used as cul- coside and avonoids at 340 nm. Phenolic compound quantica-
tivar markers and to improve the quality of virgin olive oil. tion was achieved using a ve-point calibration curve on the
basis of the corresponding standard substances with the excep-
tion of hydroxytyrosol which was quantied as tyrosol, and
2. Material and methods demethyloleuropein, cyanidin 3-glucoside and cyanidin 3-rutino-
side as oleuropein.
2.1. Experimental olive orchard Identication of colourless phenolic compounds was carried out
by comparison of their retention times, UVVisible characteristics
The study was carried out during the 2005/06 olive season in an and MS spectra with their standard substances. In the case of the
olive orchard maintained by C.M.A. El Chaparrillo, Servicio de Inves- anthocyanins and demethyloleuropein (a colourless phenol),
tigacin y Tecnologa Agraria, located in Almodovar del Campo, Ciu- these were tentatively identied by their UVVisible characteris-
dad Real, Spain (398N, 38W, altitude 640 m). The climate of the tics and MS spectra. The mass detector used was a LCQ Deca XP
area is Mediterranean; the average annual rainfall was 404 mm, Plus (Thermo Electron Corporation, Waltham, MA, USA) equipped
mostly distributed outside of a 4-month summer drought period. with an electrospray ionisation system. Nitrogen was used as
The olive orchard was composed of six-year-old olive trees of six nebulizing gas at a ow rate of 14 (arbitrary units). The temper-
different Spanish varieties: Arbequina, Cornicabra, Morisca, Pic- ature and voltage of the capillary were 250 C and 4.50 kV,
olimn, Picudo and Picual. The soil at the experimental orchard respectively. Data were acquired in the negative ionisation
was a clay loam (depth 1.6 m). The orchard was managed under mode. Fragmentation experiments were performed using helium
no irrigation and no tillage conditions; weeds were controlled with as the collision gas with collision energy between 30% and
post-emergence herbicides. 40%.
2.5.2. Volatile compounds (adapted from Vichi et al., 2003) 3. Results and discussion
Solid-phase microextraction (SPME) followed by GC were used
to analyze the volatile compounds in the virgin olive oil samples 3.1. Olive fruit biophenols
studied. Olive oil (1.5 g) spiked with 4-methyl-2-pentanol (as
internal standard) to a concentration of 1.5 lg/g was placed in Concentrations, expressed as mg/kg of fresh weight, of the major
a 10 ml vial tted with silicone septum. The SPME sampling biophenolic compounds found in the different olive fruit varieties
was performed by exposing the DVB/Carboxen/PDMS ber (50/ studied at three different ripening stages are reported in Table 1.
30 lm, 2 cm long from Supelco) for 30 min in the headspace of The main phenolic components found in the olive fruits studied
the sample maintained at 40 C and then retracted into the was oleuropein, as previously reported (Amiot et al., 1986,1989;
needle and immediately transferred and desorbed for 1 min in Servili et al., 1999). The content of this oleoside decreased signi-
the injection port of a gas chromatoghaph equipped with an cantly during the course of fruit ripening especially between
FID. Compounds were resolved on a Supelcowax-10 column the spotted and black drupes in almost all the varieties studied.
(30 m 0.25 mm 0.25 lm, Supelco Inc., Bellefonte, PA) under For example in the Arbequina cultivar the levels of oleuropein
the following conditions: injection port temperature 260 C; decreased from 2230 mg/kg to 60 mg/kg (3% of its initial content)
helium ow 0.8 ml/min; oven temperature ramp: 35 C for during fruit ripening and from 11600 mg/kg to 6340 mg/kg for
10 min, 3 C/min up to 160 C and then 15 C/min up to 200 C the Cornicabra variety (55% of initial content). However, it is
(maintained for 5 min). Volatile compounds were tentatively important to note that oleuropein content increased until reaching
identied by comparison with standard substances added to its maximum level in the spotted fruits in the Picual and Picolimn
the rened oils. cultivars and then decreased signicantly in black olives, as also re-
ported by Amiot et al. (1986), Ryan, Robards, and Lavee (1999) and
All experiments and analytical determinations were carried out
Morell, Romero, and Motilva (2004). This behaviour could be due
at least in duplicate.
to the turnover of the phenolic moieties into new conjugates; on
2.5.3. Statistical analysis the other hand, the steady decrease in oleuropein contents may
Analysis of ANOVA and discriminant analysis were performed be due to its extensive degradation (Ryan, Antolovich, Prenzler,
using SPSS 14.0 statistical software (SPSS Inc., Chicago, IL). Dun- Robards, & Lavee, 2002). In the case of the Morisca variety, the
cans test (p 6 0.05) was used to discriminate among the mean levels of oleuropein increased along the fruit maturation continuum,
values. although this rise was not statistically signicant. This trend was
Table 1
Content of major olive fruit biophenolic (mg/kg of fresh weight) with regards to olive cultivar and fruit ripening stage
Different letters within a compound (ad) indicate signicant differences (p < 0.05) with respect to olive cultivar at each ripening stage.
Last column of each variety indicates signicant differences (p < 0.05, ) with respect to maturation for each cultivar; NS, not signicant.
436 A. Gmez-Rico et al. / Food Research International 41 (2008) 433440
Function 2
The phenol demethyloleuropein was only found in the Arbequ-
ina variety and its content doubled during the course of fruit ripen-
ing to the point that at the black stage it was the major phenolic
2
compound in these fruits, while no trace was found in the other
varieties studied. This phenolic compound, which is most likely a
degradation product of the oleuropein as some researches claim 3 2
(Amiot et al., 1989; Servili et al., 1999), could also be treated as a 0
6
cultivar marker. This nding is in agreement with Amiot et al.
4
(1989) who found demethyloleuropein in only two of eleven 1
French varieties (Cailletier cv. and L11 cv.) and with Esti et al. -2
(1998) who found this compound in only two out of eight Italian
varieties (Coratina cv. and Leccino cv.). -5.0 -2.5 0.0 2.5 5.0 7.5
The concentration of the simple phenol hydroxytyrosol in-
Function 1
creased as the fruit ripened although no signicant statistical dif-
ferences were found in these increases for nearly any of the Fig. 1. Discriminant functions plot of olive fruits from the different cultivar studied,
varieties studied. The level of this phenolic acid probably increased classied according to their phenolic prole. Variables: Oleuropein, Hydroxytyrosol,
as the result of the degradation of the oleuropein during fruit rip- apigenin 7-O-glucoside, cyanidin 3-O-rutinoside. s (open circle), Arbequina;
ening due to the increased activity of some hydrolytic enzymes j (solid square), Picual; 4 (open up-triangle), Morisca; $ (open down-triangle),
Picolimn; (solid diamond), Picudo; h (open square), Cornicabra.
during maturation (Amiot et al., 1989; Esti et al., 1998), in partic-
ular glycosidases, like b-glucosidase which hydrolyze the oleurop-
ein to oleuropein aglycon and 3,4-DHPEA-EDA (Limiroli et al.,
1995; Montedoro et al., 1993). similar, allowing for the statistical discrimination of the Cornicabra
Verbascoside, the main hydroxycinnamic derivative in olives, and Picual cultivars from the other varieties (Fig. 1).
steadily increased along the fruit maturation continuum in all Anthocyanins are principally responsible for the black colour of
the varieties studied, ranging from 90% in the case of the Arbequ- over-ripe fruits, this ripening stage also being characterized by a
ina variety and up to 250% for the Picolimn variety (Table 1). signicant decrease in chlorophyll content. Table 1 shows the val-
Moreover, an inverse relationship between oleuropein and ver- ues of the two cyanidin glycosides identied. For all the varieties
bascoside content was found, the Arbequina variety exhibiting studied, cyanidin 3-O-rutinoside was the most abundant anthocy-
the highest verbascoside content (between 15% and 22% of the anin ranging from 1050 mg/kg for the Morisca fruits and 3240 mg/
total phenols depending on fruit ripening) and the lowest level kg for the Cornicabra variety at the black ripening stage. Similar re-
of oleuropein (between 50% and 1%), whereas the varieties rich- sults were reported by Romani et al. (1999) and Vinha et al. (2005)
est in oleuropein (between 90% and 50% of the total) such as Cor- who found higher values for cyanidin 3-O-rutinoside than for
nicabra, Picual and Picolimn had the lowest verbascoside cyanidin 3-O-glucoside in some Italian and Portuguese cultivars.
content (between 0.5% and 4%). These trends were also observed The results of the Anova and Principal Component Analyses
by Amiot et al. (1989), Servili et al. (1999) and Vinha et al. were applied to the Discriminant Analysis to more accurately de-
(2005), the rst authors having suggested a metabolic relation- scribe the differences observed in the phenol prole of the fruits
ship between oleuropein and verbascoside based on the partial of the six Spanish varieties studied. The most useful variables for
degradation of the oleuropein molecule that could be responsible the classication of the olive fruit samples according to variety
for the formation of verbascoside since the latter is not detected were oleuropein, apigenin 7-O-glucoside, hydroxytyrosol and
in young fruits. cyanidin 3-O-rutinoside (Fig. 1). The rst two discriminant func-
In this study, four colourless avonoids were also identied and tions accounted for 93.9% of the variance (82.1% and 11.8% respec-
quantied. In all of the varieties studied, rutin and luteolin 7-O- tively), yielding an excellent classication (100%) of olive fruit
glucoside were the major avonoids. A clear increase in the con- samples from the different cultivars.
centrations of these compounds during fruit ripening was observed As depicted in Fig. 1, the olive fruit samples studied can be bro-
and was statistically signicant for the Morisca (an increase of ken down into three different groups in relation to their phenolic
160% for rutin and 200% for luteolin 7-O-glucoside) and Picolimn prole. The rst group is constituted by the Cornicabra (number
varieties (270% and 170%, respectively) (Table 1). A similar trend 6 in the gure) and Picual (number 2) cultivars, whose oleuropein,
was observed by Esti et al. (1998) for some Italian cultivars like avonoids and anthocyanin composition was very similar. The sec-
Coratina and Leccino among others. In contrast, clear differences ond group contains the Picudo (5) variety, characterized by a high-
were not found in the rutin content of the six varieties used in this er content of hydroxytyrosol, and the third group was formed by
assay with the exception of the unripe Cornicabra and Picudo fruits the Arbequina (1), Morisca (3) and Picolimn (4) olive fruits, which
whose content was higher than the rest of cultivars. However, lute- have similar avonoid content and the highest verbascoside levels.
olin 7-O-glucoside did permit discrimination between the Picual
and the other cultivars drupes since its content was signicantly 3.2. Virgin olive oil minor components
higher at the three different ripening stages employed. Quercetin
3-O-rutinoside and apigenin 7-O-glucoside content in the olive 3.2.1. Phenolic compounds
fruits was very low except for the Cornicabra cultivar. However, Virgin olive oil biophenols are mainly derivatives of the oleo-
apigenin 7-O-glucoside content for the six cultivars was very dis- sides and lignans contained in olive fruit. Table 2 reports the
A. Gmez-Rico et al. / Food Research International 41 (2008) 433440 437
Table 2
Content of major virgin olive oil phenols (mg/kg) with regards to olive cultivar and fruit ripening stage
Different letters within a compound (af) indicate signicant differences (p < 0.05) with respect to olive cultivar at each ripening stage.
Last column of each variety indicates signicant differences (p < 0.05, ) with respect to maturation for each cultivar; NS, not signicant.
7000 7000
ARBEQUINA Oleuropein CORNICABRA
RI = 3.0 - 3.5 RI = 4.0
6000 6000
Phenolic Content (mg/Kg)
Phenolic Content (mg/Kg)
5000 5000
Oleuropein +
2000 Demethyloleuropein 2000
Total phenols
Hydroxytyrosol
Verbascoside secoiridoids
1000 1000
Hydroxytyrosol Tyrosol
Verbascoside
secoiridoids secoiriodoids
Htyr Tyrosol Htyr
Total phenols
secoiriodoids
Htyr Tyr Htyr Tyr
0 0
Olive fruit Virgin olive oil Olive fruit Virgin olive oil
7000 7000
PICOLIMON PICUDO
RI = 3.0 - 3.5 RI = 2.5
6000 6000
Phenolic Content (mg/Kg)
Phenolic Content (mg/Kg)
2000 2000
Fig. 2. Comparison between the biophenol prole in olive fruit and their corresponding virgin olive oils.
438 A. Gmez-Rico et al. / Food Research International 41 (2008) 433440
concentrations of the main phenolic compounds expressed as mg Fig. 2 depicts the oleoside, verbascoside and hydroxytyrosol
per kg of VOO samples obtained at two different ripening stages content of Arbequina, Cornicabra, Picolimon and Picudo fruits
from the six varieties studied. and values of total phenols, hydroxytyrosol, tyrosol and their
The secoiridoid derivatives of hydroxytyrosol and tyrosol were secoiridoid derivatives in the corresponding virgin olive oils ob-
the major phenolic fraction in all the varieties studied, but their tained. Within the same cultivar a relationship between oleoside
distribution varied in the different cultivars. In fact, the secoiridoid (oleuropein plus demethyloleuropein, the latter only found in
derivatives of hydroxytyrosol were the most important complex Arbequina) content in the fruit and the presence of secoiridoids
phenols for Arbequina, Cornicabra, Picolimn and Picual VOO, in virgin olive oils was observed and therefore an increase in
especially 3,4-DHPEA-EDA whose content ranged from 105.0 mg/ the oleuropein content in the drupe always led to a proportional
kg to 1113.2 mg/kg, whereas the tyrosol secoiridoids were the ma- increase in the corresponding total phenol concentration in virgin
jor ones found in Morisca and Picudo VOO (mainly p-HPEA-EDA) olive oil. In contrast, the ratio between oleoside content in the
with values ranging from 54.8 mg/kg to 769.6 mg/kg. This fact is olive fruit and secoiridoid derivatives in the virgin olive oil was
relevant, since hydroxytyrosol and its complex derivative forms quite different for each of the cultivars studied (approximately
are known to possess much greater antioxidant activity and sen- 2.3 for Picudo, 4.55.5 for Cornicabra and Morisca, 7.08.5 for
sory inuence than the tyrosol group (Baldioli, Servili, Peretti, & Arbequina and Picual and 28 for Picolimon, calculated as an aver-
Montedoro, 1996; Gennaro, Piciola Bocca, Modesti, Masella, & Coni, age value for unripe and ripe olives). This means that the Picol-
1998). imon variety with a relatively high oleuropein content (4760 mg/
On the other hand, a signicant decrease in these complex phe- kg on average between RI 2.5 and 4.0) in the olive fruit exhibited
nols in the different cultivars studied was observed as the fruit rip- the lowest secoiridoid content in the virgin olive oil (160 mg/kg,
ened except for the Morisca variety which showed an increase in RI 3.0; ratio 29.8). However Picudo, with a low oleuropein con-
the secoiridoid forms of hydroxytyrosol during ripening from tent in the drupe (2305 mg/kg, RI 2.5), showed a relatively high
111.4 mg/kg to 157.8 mg/kg, this behaviour probably having to content of complex phenols in virgin olive oil (1040 mg/kg, RI
do with the increase of oleuropein values observed as the Morisca 2.5; ratio 2.3). This is probably due to the varying enzyme levels
olive fruits ripened. of each olive cultivar.
As regards simple phenol content, VOO hydroxytyrosol (ranging
from 0.4 mg/kg up to 5.0 mg/kg in the different varieties) and tyro- 3.2.2. Volatile prole
sol values (1.229.8 mg/kg) were apparently not affected by the The concentrations of C6 and C5 volatile compounds from the
nature of the cultivar (Table 2), with the exception of the higher lipoxygenase (LOX) pathway, expressed as mg of the internal stan-
values in the Picudo variety. This behaviour was similar to that ob- dard (IS) used (4-methyl-2-pentanol) per kg of oil in VOO samples
served for the rest of the minor simple phenols identied, which for the six cultivars studied are reported in Table 3. These com-
were present in small amounts p-coumaric acid (ranging from pounds, responsible for the positive green sensory notes in VOO,
0.3 mg/kg up to 1.1 mg/kg in the different cultivars), vanilic acid are produced through the LOX pathway which takes place during
(0.20.5 mg/kg), pinoresinol (4.513.0 mg/kg), ferulic acid (0.5 crushing of the olive fruit and olive paste malaxation and are incor-
5.0 mg/kg) with the exception of the higher ferulic acid content porated into the oily phase (Sanchez & Harwood, 2002). The differ-
in the Arbequina cultivar (ranging from 25.0 mg/kg up to ent activities of the enzymes inuence the biogenesis of the
33.7 mg/kg). volatiles (Scheier, 1984) and explain the typical proles of the
Table 3
Content of virgin olive oil volatiles (mg/kg of IS) with regards to olive cultivar and fruit ripening stage
Different letters within a compound (af) indicate signicant differences (p < 0.05) with respect to olive cultivar at each ripening stage.
Last column of each variety indicates signicant differences (p < 0.05, ) with respect to maturation for each cultivar; NS, not signicant.
A. Gmez-Rico et al. / Food Research International 41 (2008) 433440 439
Tovar, M. J., Romero, M. P., Girona, J., & Motilva, M. J. (2002). L-Phenylalanine Vichi, S., Pizzale, L., Conte, L. S., Buxaderas, S., & Lopez-Tamames, E. (2003).
ammonia-lyase activity and concentration of phenolics in developing fruit of Solid-phase microextraction in the analysis of virgin olive oil volatile
olive tree (Olea europaea L. cv. Arbequina) grown under different irrigation fraction: characterization of virgin olive oils from two distinct geographical
regimes. Journal of Science and Food Agriculture, 82, 892898. areas of northern Italy. Journal of Agriculture and Food Chemistry, 51,
Vichi, S., Castellote, A. I., Pizzale, L., Conte, L. S., Buxaderas, S., & Lopez-Tamames, 65726577.
E. (2003). Analysis of virgin olive oil volatile compounds by headspace solid- Vinha, A. F., Ferreres, F., Silva, B. M., Valentao, P., Goncalves, A., Pereira, J. A., Oliveira,
phase microextraction coupled to gas chromatography with mass B., Seabra, R. M., & Andrade, P. B. (2005). Phenolic proles of Portuguese olive
spectrometric and ame ionisation detection. Journal of Chromatography A, fruits (Olea europaea L.): Inuences of cultivar and geographical origin. Food
983, 1933. Chemistry, 89, 561568.
Food
Chemistry
Food Chemistry 100 (2007) 568578
www.elsevier.com/locate/foodchem
Received 1 August 2005; received in revised form 30 September 2005; accepted 30 September 2005
Abstract
The olive tree is generally grown under rain-fed conditions. However, since the yield response to irrigation, even with low amounts of
water, is great there is increasing interest in irrigated agriculture. The main goal of this study was therefore to optimize sustainable irri-
gation conditions in the Cornicabra olive cultivar grown in Castilla-La Mancha, a region where the aquifers are over-exploited, and to
study the eect of dierent irrigation strategies on the composition and quality of Cornicabra virgin olive oil. Dierent irrigation treat-
ments, based on regulated decit irrigation (RDI), 100% ETc, 125% ETc, and rain-fed as control, were applied to a traditional olive orch-
ard (cv Cornicabra) in a randomized complete-block design with four replications. The average olive production of the trees grown under
rain-fed conditions was much lower, about 35%, than that obtained by applying the dierent irrigation treatments studied, between
which practically no dierence were observed. The total phenol content, which aected the sensory bitterness in the oils, decreased sig-
nicantly as the amount of supplied water increased. This is very relevant, as high levels of phenols, typical of Cornicabra virgin olive
oils, may decrease consumer preference. Notably, one of the RDI strategies produced olive oil similar in composition and quality to that
obtained by 100% ETc but with reduced water usage.
2005 Elsevier Ltd. All rights reserved.
0308-8146/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.09.075
A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578 569
point of view of both the production and quality of olive orchard were identical, with the exception of the amount
fruit, since high-quality olive oil cannot be obtained from of water applied.
olive fruit suering from a high degree of water stress.
Nevertheless, a satisfactory compromise between the 2.2. Irrigation treatments
amount of water applied and the improvement in the pro-
duction and quality of the olive crop must be fully Four treatments were applied two years before the com-
investigated. mencement of this assay: rain-fed (RF), regulated decit
There is scarce information available on the inuence of irrigation (RDI), FAO and 125 FAO. Rain-fed treatment
irrigation on olive tree growth and production and on the was used as the control to compare the results obtained
composition and quality of the virgin olive oil obtained, with the three irrigation treatments studied. In the FAO
especially in the case of the Cornicabra variety. Some treatment, the water requirements were obtained using
recent research has shown dierences in the chemical methodology proposed by the Food and Agriculture Orga-
makeup and sensory characteristics of virgin olive oil from nization of the United Nations, by subtracting the eective
irrigated and rain-fed olive trees (Aparicio & Luna, 2002). precipitation (41 mm in 2003 and 138 mm in 2004) from
The chemical components most inuenced by irrigation are the crop evapotranspiration (ETc), this latter term being
the phenolic compounds, which aect both the oxidative calculated using the eective crop coecient (Kc), the refer-
stability and the sensory characteristics, especially the bit- ence crop evapotranspiration (ETo; 822 mm in 2003 and
terness attribute, showing in both cases an inverse relation- 801 mm in 2004) obtained from an agronomic weather sta-
ship with the amount of water applied to the olive trees tion and a reductor coecient (Kr) that depended on the
(DAndria, Morelli, Martuccio, Fontanazza, & Patumi, size of the tree (ETc = Kc ETo Kr; Doorenbos & Pruitt,
1996; Motilva, Romero, Alegre, & Girona, 1999; Motilva, 1977). In 125 FAO treatment, an irrigation dosage 25%
Tovar, Romero, Alegre, & Girona, 2000; Tovar, Romero, higher than the FAO treatment was applied. As for the reg-
& Motilva, 2001). This aspect is important in olive cultivars ulated decit irrigation (RDI), a maximum amount of
that produce virgin olive oils with high bitterness and pun- 75 mm of water was established since, in many Spanish irri-
gency, such as, the Cornicabra variety in Castilla-La Man- gated olive areas, there is a legal limitation of 100 mm, and
cha, and therefore just the right level of irrigation could two dierent strategies were evaluated. In 2003, water was
enhance its sensory characteristics. applied throughout the entire season with dierent rates of
The main goal of this study was therefore to optimize application (10% FAO in May and June, 4% FAO in July
sustainable irrigation conditions in the Cornicabra olive and August and 18% FAO in September); however, in
cultivar grown in Castilla-La Mancha, a region where aqui- 2004, based on the results obtained during the previous
fers are over-exploited, and to study the eect of dierent crop season, water was applied only from the beginning
irrigation strategies on the composition and quality of Cor- of August, when the oil formation starts in the fruit, with
nicabra virgin olive oil. Dierent irrigation treatments the purpose of investigating which RDI treatment is more
(based on 100% crop evapotranspiration, ETc, also known eective in reaching similar olive production and olive oil
as the FAO method, 125% ETc, two dierent regulated def- quality to that obtained by the FAO method but consider-
icit irrigation strategies and rain-fed) were applied to a tra- ably reducing the total amount of water applied. In all irri-
ditional olive orchard (cv Cornicabra) planted at 70 trees gation treatments, olive trees were irrigated daily with eight
per hectare in a randomized complete-block design with compensating drippers (4 l/h) placed around the trees.
four replications. The total water applied in 2003 for the dierent irriga-
tion treatments was: 56 mm for RDI, 148 mm for FAO
2. Materials and methods and 206 mm for 125 FAO; and in 2004: 60 mm for RDI,
124 mm for FAO and 154 mm for 125 FAO. In order to
2.1. Experimental olive orchard fully describe the dierent irrigation strategies used, the
water stress integrals (MPa day; as dened by Myers,
The study was carried out during the 2003/2004 and 1998), calculated from the midday steam water potential
2004/2005 olive crop seasons in an experimental olive orch- data, and the minimum potential values are reported.
ard of Cornicabra cv. maintained by Conserjera de Agri- These values during 2003 were: 332 MPa d and 4.1 MPa
cultura y Medio Ambiente (Department of Agriculture (observed in the middle of September) for RF; 316
and the Environment), located in Almodovar del Campo MPa d, 4.1 MPa (middle of September) for RDI;
(Ciudad Real, Spain). About three hundred and twenty 218 MPa d, 2.3 MPa (middle of September) for FAO;
50-year-old trees, spaced 12 12 m2, were used in a ran- 172 MPa d, 1.8 MPa (middle of September) for 125
domised complete block design with four dierent treat- FAO. The following experimental data were observed in
ments and four replications. Each experimental unit 2004: 269 MPa d and 4.1 MPa (middle of October) for
consisted of 4 3 trees, where only the central ones were RF; 223 MPa d, 2.9 MPa (beginning of August)
used for sampling. The experimental olive orchard was for RDI; 176 MPa d, 2.2 MPa (end of September) for
enclosed by two outer rows of irrigated olives. All of the FAO; 159 MPa d, 1.7 MPa (middle of October) for 125
agronomical treatments applied to the experimental olive FAO.
570 A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578
2.3. Olive and olive oil samples added to a sample of virgin olive oil (2.5 g) and the solvent
was evaporated with a rotary evaporator at 35 C under
Olive fruit samples from rain-fed and irrigation treat- vacuum. The oil was then dissolved in 6 ml of hexane
ments trees were harvested throughout ripening, from and a diol-bonded phase cartridge (Supelco Co., Belle-
immature stage to normal harvest period for the Cornic- fonte, USA) was used to extract the phenolic fraction.
abra variety. Five and three samplings were gathered in The cartridge was conditioned with methanol (6 ml) and
2003/2004 and 2004/2005, respectively; the samples were hexane (6 ml), the oil solution was then applied, and the
collected by hand from the beginning of November to SPE column was washed with hexane (2 3 ml) and with
the end of December, whereas the fth sampling col- hexane/ethyl acetate (85:15, v/v; 4 ml). Finally, the phenols
lected from the 2003/2004 crop was collected by a were eluted with methanol (15 ml) and the solvent was
mechanical shaker at the beginning of January. The olive removed with a rotary evaporator at 35 C under vacuum
fruit sampling of the dierent irrigation treatments was to dryness. The phenolic residue was dissolved in metha-
not always carried out on the same date, with a view nol/water (1:1 v/v; 250 ll).
to obtaining a more homogeneous pool of samples HPLC analysis was performed using an Agilent Tech-
between the irrigation treatments studied. Four represen- nologies 1100 series system equipped with an automatic
tative subsamples from each treatment (four subsam- injector, a column oven and a diode array UV detector.
ples four treatments) were picked at each sampling A Spherisorb S3 ODS2 column (250 4.6 id mm, 5 lm
and brought to the laboratory for oil extraction. Virgin particle size) (Waters Co., Milford, Massachusetts, USA)
olive oil samples of Cornicabra variety were then was used, maintained at 30 C, with an injection volume
obtained using the Abencor method and analysed for of 20 ll and a ow rate of 1.0 ml/min. Mobile phase was
this study. a mixture of water/acetic acid (95:5 v/v) (solvent A), meth-
anol (B) and acetonitrile (C): from 95% (A) 2.5% (B)
2.4. Analytical determinations in olive fruits 2.5% (C) to 34% (A) 33% (B) 33% (C) in 50 min. Phe-
nolic compounds were quantied at 280 nm using syringic
Ripeness index. The olive ripeness index was determined acid as internal standard and the response factors deter-
according to the method proposed by the International mined as by Mateos et al. (2001).
Olive Oil Council (IOOC, 1984), based on the evaluation Tocopherols were evaluated following the AOCS Method
of the olive skin and pulp colours. Ripeness index values Ce 8-89. A solution of oil in hexane was analysed on an Agi-
range from 0 (100% intense green skin) to 7 (100% purple lent Technologies HPLC (1100 series) on a silica gel Lichro-
esh and black skin). sorb Si-60 column (particle size 5 lm, 250 mm 4.6 mm i.d.;
Industrial oil yield. An Abencor system was used to Sugerlabor, Madrid, Spain) which was eluted with hexane/2-
extract the virgin olive oil. The oil obtained was separated propanol (98.5:1.5) at a ow rate of 1 ml/min. A uorescence
by decanting and the amount measured. The industrial oil detector (Thermo-Finnigan FL3000) was used with excita-
yield was expressed as a percentage of fresh olive paste tion and emission wavelength set at 290 and 330 nm.
weight (Martnez, Munoz, Alba, & Lanzon, 1975). Samples Oxidative stability was evaluated by the Rancimat
were ltered and stored at 4 C in darkness using amber method (Laubli & Bruttel, 1986). Stability was expressed
glass bottles without headspace until analysis. as the induction time (hours) measured with the Rancimat
Water and oil content. The water content of olive paste 679 apparatus (Metrohm, Switzerland).
was determined by desiccation according to the UNE Span- Fatty acid composition was determined following the
ish standard method. The fat content was determined by European Regulations EEC 2568/91 and subsequent
Soxhlet extraction and was expressed as a percentage of amendments, corresponding to the AOCS method Ch 2-
dry olive paste weight (UNE Spanish Standard 55032:1973). 91. To determine fatty acid composition, the methyl-esters
were prepared by vigorous shaking of a solution of oil in
2.5. Analytical determinations in virgin olive oil hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potas-
sium hydroxide and analysed by GC with a FID detector.
All reagents used were of analytical, HPLC or spectro- A fused silica column (50 m length 0.25 mm i.d.) coated
scopic grade, and were supplied by Merck (Darmstadt, with SGL-1000 phase (0.25 lm thickness; Sugerlabor,
Germany). Spain) was used. The carrier gas was helium, at a ow
Free acidity, given as % of oleic acid, peroxide value through the column of 1 ml/min. The injector and detector
(PV) expressed as milliequivalents of active oxygen per temperatures were set at 250 C and the oven temperature
kilogramme of oil (meq O2/kg), and K232 and K270 extinc- at 210 C. The injection volume was 1 ll.
tion coecients calculated from absorption at 232 and Sensory evaluation was done by an International Olive
270 nm, were measured following the analytical methods Oil Council recognized Panel of assessors from the Pro-
described in European Regulation EEC 2568/91 and subse- tected Designation of Origin Montes de Toledo (Toledo,
quent amendments. Spain) and the University of Castilla-La Mancha according
For phenolic compounds a solution of the internal stan- to Annex XII of Regulation EC 796/2002 (amending ECC
dard (250 ll of 15 mg/L of syringic acid in methanol) was 2568/91).
A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578 571
Bitterness index (K225) was determined by the method low fruit load behaviour of the olive trees in successive crop
described by Gutierrez-Rosales, Perdiguero, Gutierrez, seasons, especially in the case of the RF conditions.
and Olas (1992), which consists of the extraction of the bit-
ter components from a sample of 1.0 0.01 g of oil dis- 3.2. Characteristics and composition of the olive fruit
solved in 4 ml of hexane passed through a C18 column
(Bakerbond spe, J.T. Baker, Phillipsburg, NJ, USA) previ- Table 2 lists the olive fruit characteristics and composi-
ously activated with methanol and washed with hexane. tion, as aected by the dierent irrigation treatments stud-
After elution, 10 ml of hexane was passed to eliminate ied and the ripeness index of the fruits for the two crop
the oil residues and then the retained compounds were seasons studied (2003/2004 and 2004/2005).
eluted with methanol/water (1:1) to 25 ml. The absorbance The olive fruit sampling of the dierent irrigation condi-
of the extract was measured at 225 nm against methanol/ tions was not always carried out on the same date, with a
water (1:1) in a 1-cm cuvette. view to obtaining a more homogeneous pool of samples
Chlorophyll and carotenoid compounds (mg/kg) were between the irrigation treatments studied. For this reason
determined at 472 and 670 nm in cyclohexane using specic a discussion of the statistically signicant dierence in the
extinction values, by the method described by Mnguez- ripeness index of the olive fruit, among the dierent irriga-
Mosquera, Rejano, Gandul, Sanchez, and Garrido (1991). tion treatments studied, cannot be performed. Neverthe-
All experiments and analytical determinations were car- less, taking into account the harvesting date of the
ried out at least in duplicate. dierent olive sampling (data not shown), it should be
noted that, with the exception of the last sampling, the
2.6. Statistical Analysis olive fruits of the FAO irrigation treatment generally had
a higher ripeness index than those of rain-fed conditions.
Statistical analyses were performed using SPSS 11 statis- For the 125 FAO treatment, corresponding to the higher
tical software (SPSS Inc. Chicago, IL). amount of water applied, this tendency was not observed,
due to the greater olive production obtained with this treat-
3. Results and discussion ment in the crop season 2003/2004 (Table 1), given that, as
olive production rises, fruit ripening slows down.
3.1. Production of the olive grove In crop season 2003/2004 the fresh fruit weight and the
pulp/pit ratio of the olive fruit were consistently higher in
The olive production data of the experimental olive the irrigation treatments than in rain-fed conditions which
orchard studied, expressed as weight of fruits per olive tree contributed to the higher production yield observed in the
throughout the 2001/2002 to 2004/2005 crop seasons for irrigated olive orchard (Table 2). Similar results were
the dierent irrigation treatments studied, rain-fed (RF), observed by Lavee, Nashef, Wodner, and Harshemesh
regulated decit irrigation (RDI), FAO (Food and Agricul- (1990), Pastor et al. (1999), Patumi et al. (1999), Patumi
ture Organization methodology, based on the crop evapo- et al. (2002), Moriana, Orgaz, Fereres, and Pastor (2003)
transpiration, ETc), and 125% FAO, are listed in Table 1. among other researchers. The weight of the fruit in the
The average olive production of the trees grown under 125 FAO irrigation treatment was slightly lower than in
rain-fed conditions (39.2 kg/tree) was much lower, about FAO and RDI, probably due, as previously mentioned,
35%, compared with that obtained applying the dierent to the fact that, in the crop season 2003/2004, the produc-
irrigation treatments studied (from 51.8 to 52.7 kg/tree), tion of 125 FAO was greater (about 80 kg per tree) than in
between which practically no dierences were observed. the FAO and RDI treatments (65 and 60 kg per tree,
This observation agrees with the results obtained by respectively) and, as the olive tree production increases,
Patumi et al. (1999) and Pastor et al. (1999), who reported the size of the fruit diminishes (Lavee & Wodner, 2004).
a rise in olive production using irrigation, but no statisti- In the 2004/2005 season, this behaviour, in terms of the
cally signicant dierence between the irrigation doses weight of the fruit was not observed, probably due to the
used. The reported data also show the typical high and relatively high fruit damage produced by an olive y attack
which was not detected in the previous crop season (Table
2).
The fruit damage observed in the 2004/2005 crop (and
Table 1
that aected mainly the irrigated olive trees), as well as
Olive production in the dierent irrigation treatments studied
varying weather conditions, meant that a number of statis-
Crop Season Olive Production (kg/tree)
tically signicant dierences observed in the previous crop
Rain-fed Decit irrigation FAO 125 FAO season could not be fully conrmed at the following har-
2001/2002 47.4 54.3 62.0 50.2 vesting. As is well known, this is one of the most relevant
2002/2003 27.0 58.3 52.5 52.4 limitations in experimental agronomical studies in which
2003/2004 62.3 61.1 67.2 80.1
it is generally necessary to monitor the evolution of one
2004/2005 20.1 35.5 28.9 24.4
crop for several years running to reach a general conclusion
Mean 39.2 52.3 52.7 51.8 on the eect of the factors studied.
572 A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578
Table 2
Olive fruit characteristics and composition, as aected by the dierent irrigation treatments studied and the ripeness index of the fruits
Ripeness index Fresh wt. (g/olive) Pulp/pit ratio Fruit damage (%) Water content (%) Oil content Soxhlet (%)
2003/2004
Rain-fed 1.5 0.5a,w 2.14 0.23a,w 3.49 nd 50.4 1.6a,w 39.5 6.8a,w
Decit irrigation 1.8 0.6ab,w 2.65 0.96a,w 4.50 nd 53.9 2.3b,x 39.8 5.8a,w
FAO 2.5 0.4b,w 2.93 0.45a,w 4.30 nd 52.2 1.9ab,y 42.2 4.5a,w
125 FAO 2.0 0.3ab,v 2.56 0.45a,w 3.82 nd 52.0 2.3ab,x 39.1 3.9a,w
Rain-fed 2.7 0.3a,x 2.13 0.19a,w 3.60 nd 47.9 2.9a,w 42.9 6.0a,wx
Decit irrigation 2.8 0.4a,x 2.67 1.02a,w 4.05 nd 49.8 3.4a,wx 42.3 2.2a,w
FAO 3.1 0.3a,x 2.80 0.49a,w 4.20 nd 49.5 1.8a,xy 43.5 4.1a,w
125 FAO 2.8 0.2a,w 2.42 0.51a,w 3.62 nd 50.2 4.5a,wx 42.9 3.6a,w
Rain-fed 3.2 0.3a,xy 2.14 0.22a,w 3.69 nd 47.3 3.0a,w 43.8 4.9a,wx
Decit irrigation 3.5 0.4a,xy 2.55 0.88a,w 4.19 nd 51.1 1.9a,wx 44.3 2.6a,w
FAO 3.6 0.4a,xy 2.81 0.54a,w 4.38 nd 49.5 2.0a,xy 46.7 1.3a,w
125 FAO 3.4 0.3a,x 2.42 0.41a,w 3.76 nd 48.2 3.5a,wx 43.5 5.1a,w
Rain-fed 3.7 0.2a,y 2.11 0.25a,w 3.75 nd 46.5 4.0a,w 46.2 5.1a,wx
Decit irrigation 3.8 0.3a,y 2.27 0.64a,w 3.80 nd 48.2 3.9a,x 46.0 4.6a,w
FAO 4.0 0.4a,y 2.72 0.38a,w 4.25 nd 47.8 2.5a,wx 47.4 3.4a,w
125 FAO 3.9 0.1a,y 2.39 0.35a,w 3.74 nd 45.8 2.8a,w 43.1 4.8a,w
Rain-fed 5.7 0.4b,z 2.52 0.19a,x 4.12 nd 47.3 3.0a,w 49.2 4.5a,x
Decit irrigation 5.4 0.5ab,z 2.84 0.87a,w 4.60 nd 49.4 3.5a,wx 42.8 8.5a,w
FAO 5.5 0.3ab,z 2.95 0.43a,w 4.57 nd 46.1 1.6a,w 46.1 2.6a,w
125 FAO 4.9 0.1a,z 2.62 0.44a,w 4.12 nd 48.2 1.2a,wx 43.2 4.0a,w
2004/2005
Rain-fed 2.8 0.2b,w 2.44 0.10a,w 5.5 0.0a,w 50.3 1.1b,x 42.9 0.5a,w
Decit irrigation 2.3 0.1a,w 2.72 0.21b,w 25.8 0.1b,w 50.8 0.2b,x 40.1 1.8a,w
FAO 2.5 0.2ab,w 2.56 0.00a,w 17.8 0.0b,w 46.7 0.6a,x 40.2 0.1a,w
125 FAO 2.4 0.1a,w 2.47 0.11a,w 30.8 0.1b,wx 49.8 0.9b,x 40.1 2.7a,w
Rain-fed 3.4 0.0a,x 2.57 0.28b,w 4.0 0.0a,w 48.7 1.2a,x 46.7 0.4a,w
Decit irrigation 3.4 0.0a,x 2.55 0.17b,w 35.0 0.1c,x 50.1 1.4a,x 42.9 4.2a,w
FAO 3.4 0.0a,x 2.37 0.08a,w 17.0 0.0b,w 49.6 0.6a,y 48.7 0.0a,x
125 FAO 3.5 0.1a,x 2.66 0.37b,w 36.0 0.0c,x 47.7 0.7a,x 42.2 2.6a,w
Rain-fed 4.1 0.1a,y 2.40 0.05a,w 4.5 0.0a,w 43.2 1.3a,w 43.8 3.4a,w
Decit irrigation 4.2 0.0a,y 2.39 0.14a,w 23.5 0.0b,w 43.5 0.7a,w 43.6 3.1a,w
FAO 4.2 0.0a,y 2.39 0.07a,w 22.5 0.0b,w 44.0 0.3a,w 47.3 1.5a,x
125 FAO 4.2 0.0a,y 2.15 0.14a,w 25.5 0.0b,w 42.1 1.0a,w 40.6 1.4a,w
Dierent letters within a column (ac) indicate signicant dierences (p < 0.05) with respect to irrigation treatment in each sampling. Dierent letters
within a column (wy) indicate signicant dierences (p < 0.05) with respect to ripeness index for each treatment. nd, not detected.
Moreover, in the discussion of the experimental results end of the ripeness index (greater than 3.54.0) these
observed in the two crop seasons studied, it is important increases were modest (similar to rain-fed conditions) or
to note that a dierent RDI strategy was employed each even slightly less in terms of the values observed (as in the irri-
year (see details in Section 2), with the purpose of investi- gation treatments), similar to results previously reported by
gating which RDI treatment was more eective in attaining Salvador, Aranda, and Fregapane (2001) for the same olive
similar olive production and olive oil quality to that variety. The irrigation treatment apparently did not aect
obtained by the FAO method but considerably reducing the oil accumulation in the Cornicabra fruit since no statisti-
the total amount of water applied to the olive grove. cally signicant dierences in the oil yield were observed in
Although the mean value of the water content of the the present study. In contrast, Lavee and Wodner (1991),
olive fruit (Table 2) was generally slightly lower under Motilva et al. (2000) did observe a slight delay in oil accumu-
RF conditions than under irrigation, especially in the crop lation in fruits from non-irrigated olive trees as a conse-
season 2003/2004, practically no statistically signicant dif- quence of hydric stress at the end of the summer season.
ferences were observed. Apparently, the evolution of the
fruit water content was not aected by the ripeness index. 3.3. Virgin olive oil quality and composition
Similar results were also reported by Motilva et al. (2000).
The industrial oil content, determined by the Abencor 3.3.1. Quality indices
method, and the Soxhlet fat yield of the olive fruit generally The observed free acidity ranging from 0.09% to 0.20%,
increased during ripening (Table 2). However, at the higher and peroxide value, from 1.7 to 3.4 meqO2 kg 1, of the dif-
A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578 573
ferent types of virgin olive oils studied in this assay in the index in all treatments. Statistically signicant dierences
crop season 2003/2004 (Table 3) were considerably lower were obtained in K232 and K270 between oils from rain-
than the upper limit of 0.8% as oleic acid and fed conditions and the dierent irrigation treatments stud-
20 meqO2 kg 1, respectively, established by EU legislation ied. These indices were always higher in RF and decreased
for extra virgin olive oil. Moreover, these two quality indi- by increasing the amount of the water employed in the irri-
ces were not inuenced by irrigation, since no statistically gation. This eect is probably caused by the interference of
signicant dierences in oil from rain-fed and irrigation the phenolic compounds content, which absorbs in the UV
treatments in the crop season 2003/2004 were obtained. region in these analytical determinations. In fact, the
This was also observed by Tovar et al. (2001) in virgin olive observed eect of irrigation on UV characteristics could
oils from Arbequina cultivar, by Dettori and Russo (1993) not be conrmed in the 2004/2005 crop in which the pheno-
in Leccino, Nociara and Ogliarola Salentina cultivars and lic compounds were less aected by the amount of water
Patumi et al. (1999) in Nocellara del Belice and Ascolana applied.
Tenera cultivars. All the virgin olive oils obtained using the dierent irri-
On the contrary, in crop 2004/2005, a statistical dier- gation treatments of the trees studied were classied as
ence for free acidity and peroxide value was indeed extra virgin oil by mean of the organoleptic evaluation
obtained between RF and the irrigation treatments, due carried out by an IOOC (International Olive Oil Council)
to the higher degree of fruit damage as a consequence of recognized olive oil taster panel, as shown in Table 4.
the olive y attack (Table 3). Nevertheless, the values of Sensory attributes aected by irrigation were bitter-
free acidity and the peroxide value of the olive oil obtained ness, pungency and fruitiness, according to what
from partially damaged fruit were not high from an olive has previously been described for other olive cultivars
oil quality point of view: a maximum acidity of 0.4% and (Salas, Pastor, Castro, & Vega, 1997; Tovar et al., 2001;
a 5.4 peroxide value were observed. Tovar, Romero, Alegre, Girona, & Motilva, 2002). As is
In both crop seasons, a slight increase in free acidity was known, the intensity of sensory pungency, and especially
generally observed during the ripening of the olive fruit. bitterness, are related to the phenol content in the olive
Spectrophotometric absorption characteristics in the oil, which, as expected, was higher in oils obtained under
UV region at 270 and 232 nm decreased at later ripeness rain-fed conditions. In all cases, a slight decrease in the
Table 3
Virgin olive oil quality indices, as aected by the dierent irrigation treatments studied and the ripeness index of the fruits
Ripeness index Free acidity (%) Peroxide value (meqO2/kg) K232 K270
2003/2004
Rain-fed 2.7 0.3a,x 0.10 0.01a,w 2.9 0.3ab,w 1.90 0.05b,xy 0.18 0.00b,y
Decit irrigation 2.8 0.4a,x 0.10 0.03a,w 2.4 0.6a,w 1.74 0.08a,xy 0.16 0.02ab,x
FAO 3.1 0.3a,x 0.10 0.03a,w 2.5 0.2a,wx 1.67 0.06a,y 0.14 0.01a,x
125 FAO 2.8 0.2a,w 0.10 0.02a,w 3.4 0.8b,w 1.64 0.11a,xy 0.14 0.02a,xy
Rain-fed 3.7 0.2a,y 0.10 0.01a,w 2.4 0.2ab,w 1.84 0.02b,x 0.16 0.01b,x
Decit irrigation 3.8 0.3a,y 0.09 0.01a,w 2.4 0.5ab,w 1.65 0.10a,wx 0.13 0.02a,w
FAO 4.0 0.4a,y 0.10 0.02a,w 2.0 0.3a,w 1.54 0.05a,wx 0.11 0.01a,w
125 FAO 3.9 0.1a,y 0.12 0.01a,w 2.8 0.4b,w 1.54 0.09a,wx 0.12 0.01a,wx
Rain-fed 5.7 0.4b,z 0.14 0.02ab,x 2.6 1.2a,w 1.68 0.04c,w 0.15 0.01b,w
Decit irrigation 5.4 0.5ab,z 0.11 0.02a,w 1.7 0.5a,w 1.56 0.04b,w 0.12 0.01a,w
FAO 5.5 0.3ab,z 0.18 0.07ab,x 2.3 0.4a,wx 1.47 0.03a,w 0.11 0.00a,w
125 FAO 4.9 0.1a,z 0.20 0.06b,x 2.6 0.5a,w 1.45 0.08a,w 0.11 0.01a,w
2004/2005
Rain-fed 2.8 0.2b,w 0.14 0.01a,w 2.7 0.3a,w 1.67 0.14a,w 0.15 0.01a,w
Decit irrigation 2.3 0.1a,w 0.23 0.02b,w 3.4 0.5a,w 1.73 0.03a,x 0.15 0.00ab,w
FAO 2.5 0.2ab,w 0.25 0.01b,w 3.1 0.1a,w 1.74 0.00a,y 0.16 0.00ab,x
125 FAO 2.4 0.1a,w 0.25 0.02b,w 5.4 0.1b,w 1.73 0.02a,y 0.17 0.00b,y
Rain-fed 3.4 0.0a,x 0.15 0.01a,w 2.7 0.2a,w 1.59 0.11a,w 0.13 0.01a,w
Decit irrigation 3.4 0.0a,x 0.31 0.09b,w 3.6 0.6ab,w 1.63 0.01a,w 0.14 0.02a,w
FAO 3.4 0.0a,x 0.28 0.05b,w 3.7 0.1b,x 1.63 0.01a,x 0.14 0.00a,wx
125 FAO 3.5 0.1a,x 0.32 0.07b,w 3.4 0.0ab,w 1.53 0.01a,x 0.13 0.00a,x
Rain-fed 4.1 0.1a,y 0.17 0.03a,w 2.2 0.0a,w 1.62 0.17a,w 0.13 0.02a,w
Decit irrigation 4.2 0.0a,y 0.32 0.03b,w 4.1 0.8ab,w 1.64 0.03a,w 0.13 0.00a,w
FAO 4.2 0.0a,y 0.31 0.00b,w 3.9 0.0ab,y 1.58 0.02a,w 0.13 0.01a,w
125 FAO 4.2 0.0a,y 0.38 0.01b,w 4.4 1.1b,w 1.43 0.00a,w 0.11 0.01a,w
Dierent letters within a column (ac) indicate signicant dierences (p < 0.05) with respect to irrigation treatment in each sampling. Dierent letters
within a column (wy) indicate signicant dierences (p < 0.05) with respect to ripeness index for each treatment.
574 A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578
Table 4
Virgin olive oil organoleptic evaluation, as aected by the dierent irrigation treatments studied and the ripeness index of the fruits
Ripeness index Grade Sensory attributes K225
Fruity Bitterness Pungency
2003/2004
Rain-fed 2.7 0.3a,x Extra virgin 6.1 0.2b,x 8.2 0.4b,w 7.8 0.3a,w 0.78 0.01c,x
Decit irrigation 2.8 0.4a,x Extra virgin 6.2 0.5b,w 8.0 0.3b,wx 8.0 0.3a,wx 0.67 0.07b,x
FAO 3.1 0.3a,x Extra virgin 5.5 0.1b,w 7.7 0.5ab,w 7.7 0.3a,w 0.66 0.04b,yz
125 FAO 2.8 0.2a,w Extra virgin 4.9 0.3a,w 7.2 0.3a,wx 7.6 0.3a,w 0.56 0.07a,xy
Rain-fed 3.7 0.2a,y Extra virgin 5.4 0.2ab,w 8.5 0.2c,w 8.4 0.2ab,w 0.77 0.01c,x
Decit irrigation 3.8 0.3a,y Extra virgin 6.2 0.3b,w 8.3 0.2c,x 8.5 0.1b,w 0.64 0.07b,wx
FAO 4.0 0.4a,y Extra virgin 5.5 0.2ab,w 7.7 0.3b,w 8.0 0.2ab,w 0.56 0.04ab,x
125 FAO 3.9 0.1a,y Extra virgin 5.3 0.2a,w 6.9 0.4a,w 8.0 0.2a,w 0.49 0.08a,wx
Rain-fed 5.7 0.4b,z Extra virgin 5.4 0.3ab,wx 8.3 0.2a,w 8.1 0.2ab,w 0.66 0.05c,w
Decit irrigation 5.4 0.5ab,z Extra virgin 6.2 0.1b,w 7.5 0.5a,w 7.7 0.1a,x 0.57 0.03b,w
FAO 5.5 0.3ab,z Extra virgin 5.0 0.3a,w 7.7 0.3a,w 7.9 0.2a,w 0.46 0.03a,w
125 FAO 4.9 0.1a,z Extra virgin 6.0 0.1b,x 8.0 0.3a,x 8.0 0.2b,w 0.41 0.09a,w
2004/2005
Rain-fed 2.8 0.2b,w Extra virgin 6.4 0.3a,w 7.6 0.2a,w 8.0 0.3a,w 0.66 0.09a,w
Decit irrigation 2.3 0.1a,w Extra virgin 6.0 0.2a,x 7.4 0.5a,w 7.9 0.6a,wx 0.70 0.00a,x
FAO 2.5 0.2ab,w Extra virgin 5.6 0.4a,w 6.7 0.3a,w 7.5 0.2a,w 0.67 0.00a,x
125 FAO 2.4 0.1a,w Extra virgin 5.2 0.6a,w 7.3 0.2a,w 7.7 0.3a,w 0.60 0.00a,y
Rain-fed 3.4 0.0a,x Extra virgin 5.5 0.2ab,w 7.0 0.5a,w 7.4 0.5a,w 0.60 0.08a,w
Decit irrigation 3.4 0.0a,x Extra virgin 4.9 0.4a,w 7.0 0.3a,w 7.1 0.3a,w 0.60 0.03a,x
FAO 3.4 0.0a,x Extra virgin 5.5 0.2ab,w 6.9 0.4a,w 7.6 0.2a,w 0.59 0.03a,x
125 FAO 3.5 0.1a,x Extra virgin 5.5 0.5b,w 6.6 0.3a,w 7.4 0.2a,w 0.50 0.02a,x
Rain-fed 4.1 0.1a,y Extra virgin 6.0 0.4a,w 7.4 0.3ab,w 7.6 0.4a,w 0.59 0.12b,w
Decit irrigation 4.2 0.0a,y Extra virgin 5.4 0.3a,wx 7.6 0.2b,w 8.2 0.2a,x 0.57 0.01b,w
FAO 4.2 0.0a,y Extra virgin 5.4 0.3a,w 7.4 0.3b,w 7.5 0.3a,w 0.54 0.01ab,w
125 FAO 4.2 0.0a,y Extra virgin 5.9 0.2a,w 6.6 0.4a,w 7.4 0.3a,w 0.36 0.04a,w
Dierent letters within a column (ac) indicate signicant dierences (p < 0.05) with respect to irrigation treatment in each sampling. Dierent letters
within a column (wy) indicate signicant dierences (p < 0.05) with respect to ripeness index for each treatment.
intensity of these positive attributes was observed, more 3.3.2. Fatty acid composition
marked in the case of bitterness, by increasing the amount The eect of irrigation and ripening on the main fatty
of water delivered through irrigation. This observation is acid composition of the dierent types of virgin olive oils
very relevant from the olive quality and marketing point is shown in Table 5.
of view since, although bitterness is a positive sensory attri- In both crop seasons studied, and in all irrigation treat-
bute in virgin olive oil, a high level of bitterness could cause ments studied, the palmitic acid content slightly decreased
consumers to reject the oil. A high level of bitterness is a as fruit ripened, i.e., from 10.4% down to 9.1% and from
unique characteristic of the Cornicabra variety virgin olive 11.4% to 9.7%, respectively, for RF and FAO irrigation
oils sensory prole, and therefore the use of irrigation treatments, whereas oleic and linoleic acids showed an
could produce a desirable descent in the intensity of this opposite trend, i.e., the oleic acid content varied from
attribute and hence increase consumer preference. 78.4% to 79.5% and the linoleic acid from 3.7% to 4.6%
However, in the 2004/2005 crop season no statistically under the FAO conditions. The increase in oleic acid con-
signicant dierences were obtained in the positive sensory tent is due to the triacylglycerols active biosynthesis which
attributes, including bitterness, between the olive oils takes place throughout fruit ripening, involving a fall in the
obtained under rain-fed and irrigation conditions. relative percentage of the oils palmitic acid content. On the
Olive oil bitterness can also be measured by the instru- other hand, the increase in linoleic acid content is due to
mental K225 parameter called bitterness index (Gutierrez- the transformation of oleic acid into linoleic acid by the
Rosales et al., 1992). In the 2003/2004 crop, a dramatic oleate desaturase activity which is active during triacylglyc-
decrease in the bitterness index was observed as the water erol biosynthesis (Sanchez & Harwood, 2002). The content
dose applied to olive trees increased (Table 4), varying of the other fatty acids remained practically unchanged
from 0.77 to 0.49, respectively, for RF and 125 FAO for during fruit ripening.
the sampling close to a ripeness index of 4.0. However, in In the 2003/2004 crop, rain-fed olive oils always showed
the 2004/2005 crop, no statistically signicant dierences a statistically signicant higher content of oleic acid,
were obtained. whereas olive oils from irrigated trees had higher contents
A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578 575
Table 5
Virgin olive oil main fatty acid composition, as aected by the dierent irrigation treatments studied and the ripeness index of the fruits
Ripeness index C16:0 (%) C18:1 (%) C18:2 (%)
2003/2004
Rain-fed 2.7 0.3a,x 10.4 0.4a,x 80.2 0.4b,x 3.5 0.3a,w
Decit irrigation 2.8 0.4a,x 11.1 1.0ab,xy 78.2 1.9a,w 4.2 0.5b,w
FAO 3.1 0.3a,x 11.4 0.3ab,y 78.4 0.2a,wx 3.9 0.1ab,wx
125 FAO 2.8 0.2a,w 11.8 0.3b,y 77.9 0.3a,wx 3.8 0.1ab,wx
Rain-fed 3.7 0.2a,y 9.7 0.6a,wx 80.8 0.5c,y 3.7 0.2a,w
Decit irrigation 3.8 0.3a,y 10.0 0.8ab,wx 79.6 1.2b,w 4.4 0.3b,w
FAO 4.0 0.4a,y 10.6 0.2bc,x 78.7 0.3ab,x 4.3 0.4b,yz
125 FAO 3.9 0.1a,y 11.0 0.4c,x 78.2 0.5a,x 4.2 0.5b,y
Rain-fed 5.7 0.4b,z 9.1 0.8a,w 81.1 0.4c,y 4.1 0.5a,w
Decit irrigation 5.4 0.5ab,z 9.4 0.8ab,w 79.7 1.2b,w 4.7 0.3b,w
FAO 5.5 0.3ab,z 9.7 0.4ab,w 79.5 0.6b,y 4.6 0.3ab,z
125 FAO 4.9 0.1a,z 10.2 0.2b,w 78.4 0.3a,x 4.8 0.2b,z
2004/2005
Rain-fed 2.8 0.2b,w 11.0 0.2a,x 78.2 0.2a,w 4.3 0.1c,w
Decit irrigation 2.3 0.1a,w 11.7 0.0b,x 78.8 0.1a,w 3.3 0.1a,w
FAO 2.5 0.2ab,w 11.5 0.0b,y 78.3 0.2a,w 3.7 0.1b,w
125 FAO 2.4 0.1a,w 11.6 0.1b,y 78.7 0.3a,w 3.5 0.1ab,w
Rain-fed 3.4 0.0a,x 10.3 0.4a,wx 78.9 0.5a,w 4.4 0.0c,wx
Decit irrigation 3.4 0.0a,x 10.9 0.0b,w 79.1 0.1a,w 3.7 0.0a,x
FAO 3.4 0.0a,x 10.7 0.0ab,x 78.8 0.2a,w 4.0 0.1b,w
125 FAO 3.5 0.1a,x 10.8 0.1ab,x 78.9 0.1a,w 3.9 0.1ab,x
Rain-fed 4.1 0.1a,y 9.9 0.2a,w 78.8 0.2a,w 4.9 0.2b,x
Decit irrigation 4.2 0.0a,y 10.7 0.2b,w 79.0 0.2a,w 4.2 0.1a,y
FAO 4.2 0.0a,y 10.3 0.0ab,w 78.7 0.2a,w 4.6 0.1b,x
125 FAO 4.2 0.0a,y 10.5 0.0b,w 78.7 0.1a,w 4.6 0.0b,y
Dierent letters within a column (ac) indicate signicant dierences (p < 0.05) with respect to irrigation treatment in each sampling. Dierent letters
within a column (wy) indicate signicant dierences (p < 0.05) with respect to ripeness index for each treatment.
palmitic and linoleic acids. As a consequence, the unsatu- 650 mg/kg. Panelli, Famiani, Servili, and Montedoro
rated/saturated and MUFA/PUFA ratios were signi- (1989), Salas et al. (1997), Patumi et al. (1999, 2002)
cantly higher in oils obtained in rain-fed conditions, in observed similar behaviour for other olive cultivars, such
line with the results obtained by Salas et al. (1997). How- as Picual, Nocellara del Belice, Kalamata and Ascolana
ever, these changes are very slight and do not have any Tenera. In the 2004/2005 crop, although the total phenol
nutritional relevance. content in the olive oil samples was lower, a trend similar
to that of the previous crop season with the water applied
3.3.3. Natural antioxidants content was observed (Table 6). Moreover, the regression lines of
The values of the a-tocopherol and total phenol content RDI and FAO treatments were closer (data not shown),
and the oxidative stability of the oils from the dierent showing that the RDI strategy employed in the second crop
treatments studied are shown in Table 6. season, produced an olive oil whose composition, speci-
The a-tocopherol content decreased slightly during rip- cally regarding the phenolic compounds, was more similar
ening, whereas insignicant dierences in its concentration to that of FAO than in the previous year, and therefore
were observed between the irrigation treatments studied. these RDI conditions are apparently more ecient.
Fig. 1 illustrates the evolution of the total phenol con- As far as the ripening of the olive fruit is concerned, the
tent of oils in the four irrigation treatments studied mean concentration of phenolic compounds in the olive
throughout fruit maturation in the 2003/2004 crop season. oils greatly decreased in both crop seasons studied, as pre-
The total phenol content of the oils was signicantly viously described in the same olive variety (Salvador et al.,
aected by the irrigation such that, as the water dose 2001; Salvador, Aranda, Gomez-Alonso, & Fregapane,
applied to olive trees increased, the amount of the phenolic 2003).
compounds in the virgin olive oil obtained decreased signif- The observed dierences in phenol concentration in the
icantly (Fig. 1 and Table 6). For example, in crop 2003/ oils could be a consequence of the dierent water stress
2004, in the case of rain-fed virgin olive oil samples, the level of olives from rain-fed to irrigation conditions that
total phenol content decreased from 1700 to 900 mg/kg involve changes in the activity of enzymes responsible for
through fruit ripening, whereas for olive oil samples under phenolic compound synthesis, such as L-phenylalanine
FAO treatment, the phenol content decreased from 1080 to ammonia-lyase whose activity is greater under higher water
576 A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578
Table 6
Virgin olive oil antioxidants content and oxidative stability, as aected by the dierent irrigation treatments studied and the ripeness index of the fruits
Ripeness index a-Tocopherol (mg/kg) Total phenols (mg/kg) Oxidative stability (h)
2003/2004
Rain-fed 1.5 0.5a,w 283 64a,x 1719 130c,y
Decit irrigation 1.8 0.6ab,w 284 59a,w 1354 42b,y
FAO 2.5 0.4b,w 222 25a,w 1076 122a,z
125 FAO 2.0 0.3ab,v 273 33a,x 968 254a,y
Rain-fed 2.7 0.3a,x 235 43a,wx 1380 62c,x 38.3 0.5d,x
Decit irrigation 2.8 0.4a,x 259 35a,w 1084 146b,x 34.0 0.4c,w
FAO 3.1 0.3a,x 212 25a,w 998 85b,yz 31.1 1.3b,x
125 FAO 2.8 0.2a,w 254 26a,wx 805 125a,xy 27.1 0.1a,w
Rain-fed 3.2 0.3a,xy 226 41ab,wx 1294 64c,x
Decit irrigation 3.5 0.4a,xy 252 29b,w 946 40b,x
FAO 3.6 0.4a,xy 201 25a,w 868 78b,xy
125 FAO 3.4 0.3a,x 237 8ab,wx 699 139a,wx
Rain-fed 3.7 0.2a,y 225 47a,wx 1364 107c,x 38.4 0.5d,x
Decit irrigation 3.8 0.3a,y 242 44a,w 1004 160b,x 31.9 1.3c,w
FAO 4.0 0.4a,y 204 21a,w 824 56ab,x 30.1 1.3b,x
125 FAO 3.9 0.1a,y 227 25a,w 651 124a,wx 24.6 0.4a,w
Rain-fed 5.7 0.4b,z 193 32a,w 905 189b,w 34.4 0.3b,w
Decit irrigation 5.4 0.5ab,z 226 31a,w 757 12ab,w 28.5 3.2ab,w
FAO 5.5 0.3ab,z 202 11a,w 654 108a,w 24.3 0.5a,x
125 FAO 4.9 0.1a,z 233 19a,w 536 124a,w 22.2 3.4a,w
2004/2005
Rain-fed 2.8 0.2b,w 298 17b,w 1019 216a,w 29.8 2.2a,w
Decit irrigation 2.3 0.1a,w 250 7a,w 905 10a,x 32.5 2.0a,w
FAO 2.5 0.2ab,w 272 10ab,x 877 11a,x 30.3 0.9a,x
125 FAO 2.4 0.1a,w 271 19ab,w 724 38a,x 28.7 1.0a,x
Rain-fed 3.4 0.0a,x 280 14b,w 921 183b,w 29.7 2.5a,w
Decit irrigation 3.4 0.0a,x 238 11a,w 691 11ab,w 28.2 1.6a,w
FAO 3.4 0.0a,x 263 2b,wx 724 57ab,w 28.5 1.6a,wx
125 FAO 3.5 0.1a,x 238 3a,w 551 14a,wx 25.5 0.1a,x
Rain-fed 4.1 0.1a,y 269 12b,w 818 224b,w 27.2 3.4b,w
Decit irrigation 4.2 0.0a,y 226 3a,w 739 51b,w 27.4 1.1b,w
FAO 4.2 0.0a,y 241 8ab,x 679 19b,w 23.9 1.8ab,w
125 FAO 4.2 0.0a,y 241 17ab,w 423 102a,w 18.3 3.6a,w
Dierent letters within a column (ad) indicate signicant dierences (p < 0.05) with respect to irrigation treatment in each sampling. Dierent letters
within a column (wz) indicate signicant dierences (p < 0.05) with respect to ripeness index for each treatment.
stress conditions (Patumi et al., 1999; Tovar, Romero, & were diminished to only a few ppm, as previously reported
Girona, 2002). for the same variety (Salvador et al., 2001).
As was previously mentioned, the concentration of phe-
nolic compounds aects the sensory bitterness attribute 3.4. Discriminant analysis
with the benecial and important consequences earlier dis-
cussed in the case of the Cornicabra olive oil variety, as Results obtained from Anova and principal component
well as oxidative stability. In terms of the latter, the analyses were applied to stepwise discriminant analysis
observed decrease in the oxidative stability does not aect showing that total phenol content, oleic acid, linoleic acid
the Cornicabra virgin olive oil shelf-life or quality since this and K232 were the most useful variables for classication
is a very stable and phenol-rich olive oil variety, but could of the virgin olive oils from the dierent treatments stud-
signicantly reduce the shelf-life of other varieties, such as ied. The rst two discriminant functions of the statistical
Arbequina, due to its naturally poor phenol content. analysis explained 96% of the variance (84% and 12%,
respectively) for both crop seasons studied. The plotting
3.3.4. Chlorophyll and carotenoid pigments of the discriminant functions is shown in Fig. 2, which
These contents of the oils was not inuenced by irriga- shows that virgin olive oils obtained using rain-fed condi-
tion (data not shown). However, as expected, an important tions were clearly separated from those obtained using the
decrease in pigment content during fruit ripening was dierent irrigation treatments studied. Virgin olive oils
observed, since at later stages of fruit ripening pigments from the FAO treatment were midway between those of
A. Gomez-Rico et al. / Food Chemistry 100 (2007) 568578 577
Acknowledgement
References
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7130 J. Agric. Food Chem. 2006, 54, 71307136
This study investigated the effect of both the degree of ripening of the olive fruit and irrigation
managementsrain-fed, two different regulated deficit irrigations (RDI), the method proposed by
the Food and Agriculture Organization of the United Nations (known as FAO), and 125 FAO (125%
FAO)son the phenolic and volatile composition of Cornicabra virgin olive oils obtained during two
crop seasons. Secoiridoid phenolic derivatives greatly decreased upon increase of both irrigation
and ripening, for example, the 3,4-DHPEA-EDA content decreased from 770 to 450 mg/kg through
fruit ripening under rain-fed conditions and from 676 to 388 mg/kg from rain-fed conditions to FAO
irrigation treatment (at a ripeness index of approximately 4). Moreover, secoiridoid derivatives of
hydroxytyrosol decreased more than those of tyrosol. The levels of major volatile components
decreased in the course of ripening but were higher in irrigated olive oils: for example, the E-2-
hexenal content ranged between 4.2 and 2.6 mg/kg (expressed as 4-methyl-2-pentanol) over fruit
maturation under rain-fed conditions and between 8.0 and 3.5 mg/kg under FAO scheduling. It is
important to note that where water was applied only from the beginning of August (RDI-2), when oil
begins to accumulate in the fruit, the resulting virgin olive oil presented a phenol and volatile profile
similar to those of the FAO and 125 FAO methods, but with a considerable reduction in the amount
of water supplied to the olive orchard.
KEYWORDS: Virgin olive oil; phenols; volatiles; ripening; irrigation; Olea europaea L. cv. Cornicabra
INTRODUCTION factors, for example, the olive cultivar, the degree of ripening
Extra virgin olive oil (EVOO) is obtained from healthy olive of the olive fruit, the irrigation management, and the extraction
fruits by mechanical processes only and is the most important process, in particular the milling and malaxation conditions and
vegetable oil ready for direct human consumption. The fine the type of centrifugation system employed. For example, the
sensory characteristics of this fruit oil, which possesses unique level of phenolic compounds and volatiles in the olive oil
aroma and taste, are mainly due to the presence of minor decreases in the course of maturation of the olive fruits (5-8).
components, chiefly volatile and phenolic compounds (1, 2). In addition, some researchers have reported that of the chemical
Volatiles are mainly responsible for the aroma of virgin olive components of virgin olive oil, phenolic compounds were the
oil, especially for the green sensory notes of high-quality virgin most influenced by irrigation, and their concentration is in
olive oils, whereas compounds with a phenolic structure affect inverse proportion to the amount of water applied to the olive
both the taste, in particular the positive bitterness organoleptic trees (9-12). Nevertheless, to date there is no detailed informa-
attribute, and the oxidative stability of the virgin olive oil (3, tion available on the influence of irrigation and fruit ripening
4). Phenolics and volatiles are therefore the compounds chiefly on the volatile composition of virgin olive oil, especially in the
responsible for the flavor of EVOOs and to a large extent case of the Cornicabra variety, which is the second most
determine the degree of consumer preference for this highly important variety in Spain (13).
appreciated product. The aim of this work was to determine the effect of both (i)
It is known that the amount of these minor components in the degree of ripening of the olive fruit and (ii) five different
virgin olive oil depends on agronomical and technological types of irrigation management on virgin olive oil volatile and
phenolic composition. EVOOs (Olea europaea L. cv. Corni-
* Corresponding author (e-mail giuseppe.fregapane@uclm.es; fax +34 cabra) obtained during the 2003/2004 and 2004/2005 crop
926 295318).
Departamento de Qumica Analtica y Tecnologa de los Alimentos. seasons were used. The ultimate goals are to enhance knowledge
Instituto Regional de Investigacion Cientfica Aplicada. with regard to the composition and quality of virgin olive oil
10.1021/jf060798r CCC: $33.50 2006 American Chemical Society
Published on Web 08/25/2006
VOO Phenolic and Volatile Compounds J. Agric. Food Chem., Vol. 54, No. 19, 2006 7131
and to define, if possible, (i) the optimum harvesting period was then dissolved in 6 mL of hexane, and a diol-bonded phase cartridge
and (ii) sustainable irrigation conditions in the Cornicabra olive (Supelco Co., Bellefonte, PA) was used to extract the phenolic fraction.
cultivar grown in Castilla-La Mancha, a region where aquifers The cartridge was conditioned first with methanol (6 mL) and then
are overexploited. with hexane (6 mL). The oil solution was then applied, and the solid-
phase extraction (SPE) column was washed with hexane (2 3 mL)
and with hexane/ethyl acetate (85:15 v/v; 4 mL). Finally, the phenols
MATERIALS AND METHODS were eluted with methanol (15 mL) and the solvent was removed with
Experimental Olive Orchard. The study was conducted during two a rotary evaporator at 35 C under vacuum until dry. The phenolic
consecutive crop seasons (2003/2004 and 2004/2005) in an experimental residue was dissolved in methanol/water (1:1 v/v; 250 L).
olive (O. europaea L.) orchard of cv. Cornicabra maintained by the HPLC analysis was performed using an Agilent Technologies 1100
Consejera de Agricultura (Department of Agriculture) of Castilla-La series system equipped with an automatic injector, a column oven, and
Mancha, located in Almodovar del Campo (Ciudad Real, Spain). About a diode array UV detector. A Spherisorb S3 ODS2 column (250 4.6
320 50-year-old trees, spaced 12 12 m2, were used in a randomized i.d. mm, 5 m particle size) (Waters Corp., Milford, MA) was used,
complete block design with four different treatments and four replica- maintained at 30 C, with an injection volume of 20 L and a flow
tions. Each experimental unit consisted of 4 3 trees, of which only rate of 1.0 mL/min. The mobile phase was a mixture of water/acetic
the central ones were used for sampling. The experimental olive orchard acid (95:5 v/v) (solvent A), methanol (B), and acetonitrile (C): from
was surrounded by two outer rows of irrigated olives. All of the 95% A-2.5% B-2.5% C to 34% A-33% B-33% C in 50 min.
agronomical treatments applied to the experimental olive orchard were Phenolic compounds were quantified at 280 nm using syringic acid as
identical, with the exception of irrigation practice. internal standard and the response factors determined by Mateos et al.
Irrigation Treatments. Four treatments were applied 2 years before (17).
the commencement of this assay: rain-fed conditions (RF), regulated GC Analysis of Volatile Compounds [Adapted from Vichi et al. (18)].
deficit irrigation (RDI), FAO, and 125 FAO. Rain-fed conditions were Solid-phase microextraction (SPME) followed by GC was used to
used as a control to compare the results obtained with the irrigation analyze the volatile compounds in the virgin olive oil samples studied.
treatments studied. In the FAO treatment the water requirements were Olive oil (1.5 g) spiked with 4-methyl-2-pentanol (as internal standard)
calculated using a methodology based on the crop evapotranspiration to a concentration of 1.5 g/g was placed in a 10 mL vial fitted with
(ETc) proposed by the United Nations Food and Agriculture Organiza- a silicone septum. The SPME sampling was performed by exposing
tion (14). In 125 FAO treatments, a total irrigation dosage 25% higher the DVB/Carboxen/PDMS fiber (50/30 m, 2 cm long from Supelco
than the FAO treatment was applied. Inc.) for 30 min in the headspace of the sample maintained at 40 C;
For regulated deficit irrigation (RDI), a maximum of 75 mm of water it was then retracted into the needle and immediately transferred and
was established because in many Spanish irrigated olive areas there is desorbed for 1 min in the injection port of an Agilent 6890 series gas
a legal limitation of 100 mm. Two different strategies were evaluated. chromatoghaph equipped with a flame ionization detector (FID).
In 2003 (RDI-1), water was applied throughout the entire season with Compounds were separated on a Supelcowax-10 column (30 m 0.25
different rates of application (10% FAO in May and June, 4% FAO in mm 0.25 m, Supelco Inc.) under the following conditions: injection
July and August, and 18% FAO in September), whereas in 2004 (RDI- port temperature, 260 C; helium flow, 0.8 mL/min; oven temperature
2), on the basis of the results obtained during the previous crop season, ramp, 35 C for 10 min, 3 C/min to 160 C and then 15 C/min to
water was applied only from the beginning of August, when the oil 200 C (maintained for 5 min). Volatile compounds were tentatively
starts to form in the fruit, for the purpose of investigating which RDI identified by comparison with standard substances (Sigma Aldrich)
treatment is more effective in achieving olive production and olive oil added to the refined oils.
quality similar to that obtained by the FAO method while considerably The analytical determinations were carried out at least in duplicate.
reducing the total amount of water applied. In all irrigation treatments, Statistical Analysis. Analysis of ANOVA and discriminant analysis
olive trees were irrigated daily (4 L/h) using eight compensating drippers were performed using SPSS 13 statistical software (SPSS Inc., Chicago,
placed around the trees. IL). Duncans test (p e 0.05) was used to discriminate among the mean
The total water applied in 2003/2004 for the different irrigation values.
treatments was 56 mm for RDI-1, 148 mm for FAO, and 206 mm for
125 FAO; in 2004/2005 these levels were 60 mm for RDI-2, 124 mm RESULTS AND DISCUSSION
for FAO, and 154 mm for 125 FAO. More detailed data on the irrigation
management have been previously reported (12). The influence of fruit ripening and irrigation management
Olive Oil Samples. Olive fruit samples from rain-fed and irrigated on (i) the production of the olive grove, which was significantly
trees were harvested throughout ripening at various ripeness indices lower under rain-fed conditions than under irrigation (35%),
(RI), from the immature stage (1.5 < RI < 2.0) to the normal harvest (ii) the characteristics and composition of the olive fruit, and
period for the Cornicabra variety (RI 5.5). The olive ripeness index (iii) the quality indices and major components of virgin olive
was determined according to the method proposed by the International oil has been analyzed and discussed elsewhere (12).
Olive Oil Council (IOOC) (15), based on the evaluation of the olive Phenolic Compounds. The concentrations of the major phe-
skin and pulp colors. RI values range from 0 (100% intense green skin)
nolic compounds found in virgin olive oil (VOO) obtained
to 7 (100% purple flesh and black skin). Five and three samplings were
gathered in 2003/2004 and 2004/2005, respectively; the samples were throughout ripening from olives subjected to the different irri-
collected by hand from the beginning of November to the end of gation treatments are reported in Table 1. As previously reported
December, whereas the fifth sampling from the 2003/2004 crop was by our research group, the main phenolic compounds in Corni-
collected by a mechanical shaker at the beginning of January. Four cabra monovarietal VOO are the dialdehydic form of elenolic
representative subsamples from each treatment (4 subsamples 4 acid linked to hydroxytyrosol and tyrosol (3,4-DHPEA-EDA
treatments) were picked at each sampling and brought to the laboratory and p-HPEA-EDA), oleuropein aglycon (3,4-DHPEA-EA), and
for oil extraction. Virgin olive oil samples of Cornicabra variety were ligstroside aglycon (p-HPEA-EA). The main simple phenols are
then obtained using the Abencor analyzer (Abengoa S.A., Sevilla, tyrosol (p-HPEA) and hydroxytyrosol (3,4-DHPEA) (19).
Spain); this system reproduces at laboratory scale the industrial process
The VOO hydroxytyrosol contents (ranging from 1.34 to 3.30
through three basic elements: hammer mill, thermomixer, and centri-
fuge (16). The oil obtained was separated by decanting and stored in
mg/kg, expressed as the 10th and 90th percentiles of the data
amber glass bottles at 4 C in darkness without headspace until analysis. distribution, respectively) and tyrosol (1.12-4.92 mg/kg) were
Methods. HPLC Analysis of Phenolic Compounds. A solution of apparently not affected by the water doses applied or by the
the internal standard (250 L of 15 mg/kg of syringic acid in methanol) degree of ripening of the fruit, because practically no statistically
was added to a sample of virgin olive oil (2.5 g), and the solvent was significant differences were observed (Table 1). This behavior
evaporated with a rotary evaporator at 35 C under vacuum. The oil was similar to that observed for the rest of the minor simple
7132 J. Agric. Food Chem., Vol. 54, No. 19, 2006 Gomez-Rico et al.
Table 1. Levels of Major Virgin Olive Oil Phenolic Compounds (Milligrams per Kilogram) with Regard to Fruit Ripening Stage and Irrigation
Managementa
ripeness
index 3,4-DHPEA 3,4-DHPEA-EDA 3,4-DHPEA-EA p-HPEA p-HPEA-EDA p-HPEA-EA total phenol
2003/2004
rain-fed 1.5 0.5a,w 2.80 0.56b,w 770 49c,y 301 41b,x 1.86 0.59a,w 498 47c,z 138 20b,y 1719 130c,y
RDI-1 1.8 0.6ab,w 1.46 0.25a,w 637 83b,x 180 21a,y 1.89 1.00a,w 440 65bc,y 88 18a,y 1354 42b,y
FAO 2.5 0.4b,w 1.55 0.79a,w 451 31a,y 150 26a,w 2.76 1.22a,w 451 31ab,z 79 23a,x 1076 122a,z
125 FAO 2.0 0.3ab,v 1.46 0.41a,w 433 120a,x 129 33a,w 2.26 0.72a,w 336 81a,y 61 20a,y 968 254a,y
rain-fed 2.7 0.3a,x 2.28 0.42a,w 651 42c,x 245 23c,x 1.62 0.66a,w 378 29c,y 94 12b,x 1380 62c,x
RDI-1 2.8 0.4a,x 2.75 1.99a,w 542 145bc,wx 163 26b,x 2.56 2.52a,w 307 11ab,x 61 10a,x 1084 146b,x
FAO 3.1 0.3a,x 1.91 0.62a,wx 442 13ab,xy 144 22ab,w 3.10 1.10a,w 335 42bc,yz 66 17a,wx 998 85b,yz
125 FAO 2.8 0.2a,w 1.79 0.22a,w 372 85a,wx 113 24a,w 2.41 1.17a,w 264 25a,x 47 7a,xy 805 125a,xy
rain-fed 3.2 0.3a,xy 2.44 0.74a,w 642 48c,x 240 31c,x 1.63 0.73a,w 323 15c,x 77 10b,x 1294 64c,x
RDI-1 3.5 0.4a,xy 1.82 0.51a,w 482 69b,wx 143 16b,x 2.40 1.69ab,w 260 14b,x 49 9a,wx 946 40b,x
FAO 3.6 0.4a,xy 2.13 0.29a,wx 399 24ab,xy 129 13ab,w 3.79 1.35b,w 276 46b,xy 51 10a,w 868 78b,xy
125 FAO 3.4 0.3a,x 1.97 0.12a,w 338 91a,wx 99 27a,w 2.51 0.72ab,w 215 23a,wx 36 8a,wx 699 139a,wx
rain-fed 3.7 0.2a,y 2.14 0.51a,w 675 51c,xy 258 36c,x 1.68 0.85a,w 335 29c,xy 85 15c,x 1364 107c,x
RDI-1 3.8 0.3a,y 2.38 0.71a,w 522 133b,wx 161 25b,x 2.17 1.37ab,w 259 20b,x 53 9b,wx 1004 160b,x
FAO 4.0 0.4a,y 2.22 0.62a,wx 388 39a,x 123 17ab,w 4.05 1.23b,w 255 28b,x 47 8ab,w 824 56ab,x
125 FAO 3.9 0.1a,y 2.87 0.37a,x 310 86a,wx 102 22a,w 3.54 1.47ab,w 192 15a,w 35 4a,wx 651 124a,wx
rain-fed 5.7 0.4b,z 2.44 1.19a,w 446 126b,w 168 53b,w 2.25 1.88a,w 228 21c,w 52 10b,w 905 189b,w
RDI-1 5.4 0.5ab,z 2.53 1.13a,w 384 41ab,w 123 5ab,w 2.34 1.02a,w 200 27bc,w 40 8ab,w 757 12ab,w
FAO 5.5 0.3ab,z 3.22 1.47a,x 302 55a,w 128 41ab,w 4.89 2.51a,w 168 38ab,w 42 13ab,w 654 108a,w
125 FAO 4.9 0.1a,z 3.31 1.05a,x 255 77a,w 90 17a,w 3.87 1.88a,w 151 30a,w 28 4a,w 536 124a,w
2004/2005
rain-fed 2.8 0.2b,w 0.99 0.04a,w 519 95b,w 148 46a,w 1.01 0.01a,w 289 58a,w 53 16a,w 1019 216a,w
RDI-2 2.3 0.1a,w 2.09 0.26ab,w 429 11ab,x 142 3a,x 1.88 0.12b,w 266 5a,x 54 3a,x 904 9a,x
FAO 2.5 0.2ab,w 2.71 0.94b,w 401 13ab,x 152 9a,w 2.12 0.09bc,w 253 33a,x 57 1a,y 877 10a,x
125 FAO 2.4 0.1a,w 2.43 0.17b,w 327 14a,x 122 4a,w 2.43 0.16c,w 215 14a,x 44 4a,w 724 38a,x
rain-fed 3.4 0.0a,x 1.09 0.34a,w 478 54c,w 136 50a,w 1.24 0.48a,w 252 54b,w 45 21a,w 921 183b,w
RDI-2 3.4 0.0a,x 8.23 0.99c,x 319 2ab,w 117 2a,w 4.92 1.02c,x 187 8ab,w 45 0a,wx 691 11ab,w
FAO 3.4 0.0a,x 2.97 1.27ab,w 354 42b,wx 125 12a,w 2.43 0.84ab,w 192 3ab,wx 39 1a,w 724 57ab,w
125 FAO 3.5 0.1a,x 3.78 0.38b,w 240 8a,wx 94 15a,w 4.26 0.22bc,w 166 14a,wx 34 4a,w 551 14a,wx
rain-fed 4.1 0.1a,y 1.39 0.15a,w 396 81b,w 145 67a,w 1.79 0.19a,w 229 48b,w 39 25a,w 818 224b,w
RDI-2 4.2 0.0a,y 2.89 1.76a,w 365 23b,w 130 7a,wx 2.97 0.11a,w 186 17ab,w 42 4a,w 738 51b,w
FAO 4.2 0.0a,y 3.84 1.38a,w 307 3b,w 136 14a,w 3.79 0.69a,w 172 5ab,w 46 3a,x 679 18b,w
125 FAO 4.2 0.0a,y 6.97 3.26a,w 160 54a,w 88 15a,w 7.34 3.99a,w 119 36a,w 32 5a,w 422 102a,w
a Different letters (ac) within a column indicate significant differences (p < 0.05) with respect to irrigation treatment in each sampling. Different letters (wy) within a
column indicate significant differences (p < 0.05) with respect to ripeness index for each irrigation treatment.
phenols identified (data not shown), which were present in very 1). It is important to note that the differences in phenol contents
small amountssvanillin (<0.22 mg/kg), vanillic acid (<0.26 between the oils from FAO and the second regulated deficit
mg/kg), p-coumaric acid (<0.25 mg/kg), and ferulic acid (<0.21 irrigation (RDI-2) strategy were not statistically significant; this
mg/kg)sexcept for pinoresinol (<4.40 mg/kg), the content of indicates that the RDI scheduling employed in the second crop
which was higher. season (RDI-2), when water was applied from the beginning
In contrast, there was a considerable difference in the of August only, produced a VOO with a phenolic composition
concentrations of secoiridoid derivatives of hydroxytyrosol and more similar to that of oil from FAO-treated olives than to that
tyrosol observed in the VOO in the course of fruit ripening and from olives grown under RDI-1 water scheduling of the previous
under the various irrigation treatments studied. In fact, the year. In this view one of the main goals of the study was
compounds most affected by irrigation scheduling of the olive attained. Indeed, the phenolic and volatile composition related
grove and by ripening of the fruit were the complex phenol to the quality of VOO comparable to FAO management was
chemical forms, the levels of which decreased significantly in achieved with less demand in water supply.
the VOO during ripening and as the water supplied increased. A high level of bitterness, which as well-known is related to
For example, in the 2003/2004 crop the 3,4-DHPEA-EDA the phenol content (6), is a peculiar characteristic of the sensory
content decreased from 770 to 450 mg/kg and the 3,4-DHPEA- profile of VOOs of the Cornicabra variety (8). Nevertheless,
EA diminished from 300 to 170 mg/kg in the course of fruit an excessive level of this positive organoleptic attribute could
ripening in the rain-fed (RF) VOO samples, whereas from RF cause consumers to reject the product (8). Thus, to meet product
conditions to FAO irrigation, at a RI of 4.0, the 3,4-DHPEA- quality and marketing needs, the use of irrigation could produce
EDA content decreased from 676 to 388 mg/kg and the 3,4- a desirable reduction in the intensity of bitterness and, conse-
DHPEA-EA from 258 to 123 mg/kg (Table 1). Tovar et al. quently, improvement in consumer preference may be achieved.
(11) observed similar behavior for the Arbequina cultivar, for A slight but statistically significant decrease in the intensity of
which the levels of secoiridoids diminished as the irrigation dose bitterness was indeed observed in the organoleptic evaluation
of olive trees increased. of the VOO obtained in this study (12), which was carried out
As far as the 2004/2005 crop season is concerned, although by an olive oil taster panel certified by the IOOC.
the levels of complex phenols in the VOO samples were lower, The observed differences in VOO phenol composition could
the trend was similar to that of the previous crop season (Table be a consequence of the different water stress levels of the olive
VOO Phenolic and Volatile Compounds J. Agric. Food Chem., Vol. 54, No. 19, 2006 7133
Figure 1. Evolution of hydroxytyrosol and its complex secoiridoid forms and of tyrosol and its derivatives in the course of fruit ripening as affected by
irrigation management in crop season 2003/2004: (O) rain-fed; (2) regulated deficit irrigation, RDI-1; (0) FAO; ([) 125 FAO.
trees under rain-fed conditions and under the experimental when fruit skin color turned from yellow-green to purple;
irrigation conditions, which prompts changes in the activity of beyond that point the volatile content decreased.
the enzymes responsible for biosynthesis of the phenolic With respect to the evolution of C6 alcohols, there was a
compounds, such as L-phenylalanine ammonia-lyase, which is significant decrease in E-2-hexen-1-ol content, whereas hexan-
more active under higher water stress conditions (20, 21). 1-ol and Z-3-hexen-1-ol increased slightly during fruit ripening.
The complex phenols were not affected in the same way by It is worth noting that this observed increase was statistically
irrigation, because the secoiridoid derivatives of hydroxytyrosol significant in VOO from olives under FAO and 125 FAO
decreased more than those of tyrosol, as clearly shown in Figure treatments, which received the highest irrigation doses. A similar
1. This is very important because hydroxytyrosol and its trend was observed in the crop season 2004/2005 (Table 2).
complex derivative forms are known to possess much greater The moderate increase in the hexan-1-ol and Z-3-hexen-1-ol
antioxidant activity and organoleptic influence than the tyrosol content has not been observed by other researchers in other VOO
group (22, 23). These results were similar to those reported by varieties, such as cvs. Picual and Coratina (5, 26), probably due
Tovar et al. (11, 24) for the Arbequina variety. to the different activities of alcohol dehydrogenase (ADH),
Volatile Compounds. Table 2 reports the concentrations of which is genetically determined in each cultivar (28). With
C6 volatile compounds from the lipoxygenase (LOX) pathway, regard to C6 esters such as hexyl acetate and Z-3-hexyl acetate,
expressed as milligrams of internal standard (4-methyl-2- these were present in very small amounts in Cornicabra VOO,
pentanol) per kilogram of oil, in VOO samples taken throughout indicating that there was also little activity of the alcohol acyl
the course of fruit ripening according to the different irrigation transferase (AAT).
treatments, for the two crop seasons studied. In both crop seasons, the volatile compounds most affected
In all of the Cornicabra VOO samples analyzed, the major by the irrigation were E-2-hexenal, Z-3-hexen-1-ol, and hexan-
volatile component was the C6 aldehyde fraction, the content 1-ol, in the sense that the increase in the water applied to the
of which decreased as ripening progressed. For example, the olive trees produced an increase in these volatiles, mainly in
E-2-hexenal content ranged between 4.2 and 2.6 mg/kg of IS oils from fruits whose ripeness index was >2.5-3.0.
over fruit maturation for VOOs under RF conditions and An additional branch of the LOX pathway is active on the
between 8.0 and 3.5 mg/kg for those under FAO scheduling; linolenic acid substrate, leading to the production of C5 volatile
the amount of hexanal was lower and varied between 1.10 and compounds, which are also present in the VOO aroma (4, 5).
0.45 mg/kg over fruit ripening under RF conditions and between Reasonable amounts of 1-penten-3-one and 1-penten-3-ol were
0.90 and 0.50 mg/kg under FAO treatment. These compounds, found in Cornicabra VOO, and their contents ranged between
which are responsible for the positive green sensory notes in 1.40 and 0.80 mg/kg of IS for 1-penten-3-one and between 0.30
VOO, are produced through the LOX pathway that takes place and 0.16 mg/kg for 1-penten-3-ol over the course of fruit
during crushing of the olive fruit and olive paste malaxation maturation (Table 2). On the other hand, these volatiles were
and are incorporated into the oily phase (25). apparently not affected by the irrigation strategies studied.
Figure 2 depicts the evolution of the main volatile compounds RDI-1 resulted in a VOO with a volatile composition similar
found in Cornicabra VOO in the course of fruit ripening as to those from FAO treatment. On the other hand, the total
affected by RF and FAO irrigation conditions in crop season volatile levels in VOOs produced under RDI-2 conditions were
2003/2004. As already mentioned, the decrease of C6 aldehydes higher than in VOOs produced under FAO conditions and
was steady from the unripe to over-ripe stages, especially that similar to the levels found in VOOs produced under 125 FAO
of E-2-hexenal, which is the major volatile compound in various conditions. It is therefore very important to note that the VOOs
VOO cultivars. Its concentration diminished by about 40% in produced by the second RDI strategy were richer in volatile
oils from RF conditions and 60% in oils from FAO treatment compounds than those produced under FAO conditions, but with
throughout fruit maturation (RI between 2.0 and 5.0) (Figure a considerable reduction in the total amount of water used in
2 and Table 2). Aparicio et al. (26) reported similar behavior, the olive grove.
but other researches (5, 27) have shown that during olive Discriminant Analysis. The results of ANOVA and principal
ripening the amount of volatile compounds, especially E-2- component analyses (PCA) were applied to the discriminant
hexenal, increased to a maximum concentration, which occurred analysis to better describe the differences observed in the phenol
7134 J. Agric. Food Chem., Vol. 54, No. 19, 2006 Gomez-Rico et al.
Table 2. Levels of Major Virgin Olive Oil Volatile Compounds (Milligrams per Kilogram of IS) with Regard to Fruit Ripening Stage and Irrigation
Managementa
Z-3-
ripeness hexyl Z-3-hexen- E-2-hexen- hexenyl 1-penten- 1-penten-
index hexanal hexan-1-ol acetate E-2-hexenal 1-ol 2-ol acetate 3-one 3-ol
2003/2004
rain-fed 1.5 0.5a,w 1.10 0.16ab,z 0.02 0.00a,w <0.01 4.18 1.72a,y 0.11 0.03a,w 0.11 0.02a,x <0.01 1.14 0.17a,z 0.34 0.02a,y
RDI-1 1.8 0.6ab,w 1.38 0.28b,y 0.03 0.02a,w <0.01 6.46 1.19ab,x 0.15 0.03a,w 0.14 0.00b,y <0.01 1.51 0.27b,y 0.34 0.08a,y
FAO 2.5 0.4b,w 0.90 0.14a,x 0.04 0.00a,w <0.01 7.74 2.21ab,x 0.17 0.04a,w 0.12 0.02ab,y <0.01 1.06 0.16a,y 0.26 0.02a,y
125 FAO 2.0 0.3ab,v 0.91 0.09a,x 0.03 0.01a,w <0.01 9.74 2.14b,y 0.12 0.04a,w 0.12 0.00ab,y <0.01 1.07 0.07a,x 0.25 0.05a,w
rain-fed 2.7 0.3a,x 0.68 0.03a,y 0.03 0.01a,wx <0.01 3.76 0.10a,xy 0.11 0.05a,w 0.06 0.00a,w <0.01 0.95 0.07a,y 0.29 0.02a,x
RDI-1 2.8 0.4a,x 0.90 0.16b,x 0.06 0.01ab,wx <0.01 5.69 1.25ab,x 0.18 0.05a,w 0.08 0.00b,x <0.01 1.18 0.18a,x 0.27 0.02a,xy
FAO 3.1 0.3a,x 0.68 0.06a,w 0.05 0.01ab,w <0.01 5.82 1.84ab,wx 0.23 0.06ab,w 0.08 0.00b,x <0.01 0.98 0.07a,xy 0.24 0.01a,xy
125 FAO 2.8 0.2a,w 0.68 0.04a,w 0.08 0.02b,wx <0.01 7.21 2.27b,xy 0.39 0.12b,y 0.09 0.01b,x <0.01 0.98 0.11a,wx 0.26 0.03a,w
rain-fed 3.2 0.3a,xy 0.62 0.06a,xy 0.05 0.01a,x <0.01 3.25 0.35a,wx 0.08 0.01a,w 0.05 0.00a,w <0.01 0.81 0.07a,xy 0.26 0.03a,x
RDI-1 3.5 0.4a,xy 0.75 0.11a,wx 0.08 0.04ab,wx <0.01 4.71 0.99ab,wx 0.15 0.03b,w 0.06 0.01a,wx <0.01 0.97 0.07a,x 0.23 0.04a,wx
FAO 3.6 0.4a,xy 0.66 0.09a,w 0.08 0.03ab,wx <0.01 4.02 1.19ab,w 0.19 0.05bc,w 0.05 0.01a,wx <0.01 0.90 0.11a,xy 0.22 0.01a,x
125 FAO 3.4 0.3a,x 0.73 0.03a,w 0.12 0.01b,xy <0.01 5.48 0.83b,wx 0.23 0.03c,wx 0.06 0.01a,w <0.01 0.92 0.10a,w 0.23 0.03a,w
rain-fed 3.7 0.2a,y 0.52 0.05a,wx 0.04 0.02a,wx <0.01 1.90 0.40a,w 0.11 0.09a,w 0.05 0.01a,w <0.01 0.72 0.08a,x 0.24 0.02a,x
RDI-1 3.8 0.3a,y 0.67 0.09b,wx 0.12 0.07a,x <0.01 3.40 1.10ab,w 0.27 0.19ab,w 0.06 0.00a,w <0.01 0.91 0.10b,x 0.26 0.03a,xy
FAO 4.0 0.4a,y 0.58 0.07ab,w 0.11 0.03a,xy <0.01 3.70 1.35ab,w 0.36 0.19ab,w 0.06 0.01a,wx <0.01 0.82 0.07ab,x 0.22 0.02a,x
125 FAO 3.9 0.1a,y 0.73 0.08b,w 0.21 0.06b,z <0.01 5.14 0.74b,wx 0.42 0.08b,y 0.06 0.01a,w <0.01 0.87 0.07ab,w 0.24 0.03a,w
rain-fed 5.7 0.4b,z 0.44 0.06a,w 0.08 0.03a,x <0.01 2.46 0.68a,wx 0.23 0.04a,x 0.05 0.02a,w <0.01 0.48 0.05a,w 0.15 0.02a,w
RDI-1 5.4 0.5ab,z 0.50 0.09a,w 0.12 0.01ab,x <0.01 3.10 0.77a,w 0.32 0.05ab,w 0.06 0.00a,w <0.01 0.57 0.12a,w 0.16 0.02a,w
FAO 5.5 0.3ab,z 0.52 0.15a,w 0.14 0.03ab,y <0.01 3.09 1.79a,w 0.35 0.07b,w 0.05 0.01a,w <0.01 0.56 0.08a,w 0.16 0.01a,w
125 FAO 4.9 0.1a,z 0.71 0.08b,w 0.17 0.03b,yz <0.01 3.91 0.76b,w 0.35 0.04b,xy 0.06 0.01a,w <0.01 0.82 0.04b,w 0.20 0.03b,w
2004/2005
rain-fed 2.8 0.2b,w 0.83 0.07a,x 0.05 0.01a,w <0.01 3.50 0.38a,x 0.08 0.02a,w 0.09 0.00a,x <0.01 1.10 0.11a,x 0.28 0.01b,w
RDI-2 2.3 0.1a,w 0.74 0.06a,x 0.06 0.00a,w <0.01 5.45 0.67ab,x 0.14 0.00b,w 0.08 0.01a,w <0.01 1.17 0.05a,y 0.24 0.01a,w
FAO 2.5 0.2ab,w 0.73 0.12a,x 0.05 0.00a,w <0.01 4.73 1.47ab,x 0.12 0.00b,w 0.08 0.00a,x <0.01 1.07 0.02a,y 0.26 0.01ab,w
125 FAO 2.4 0.1a,w 0.83 0.06a,x 0.06 0.01a,w <0.01 6.38 0.12b,x 0.14 0.04ab,w 0.09 0.00a,x <0.01 1.21 0.00a,x 0.27 0.00ab,wx
rain-fed 3.4 0.0a,x 0.56 0.11a,wx 0.03 0.01a,w <0.01 1.92 0.88a,w 0.18 0.07a,wx 0.07 0.01a,wx <0.01 0.89 0.11a,wx 0.26 0.02a,w
RDI-2 3.4 0.0a,x 0.73 0.08a,x 0.13 0.02b,wx <0.01 4.80 0.54b,x 0.38 0.13a,w 0.08 0.03a,w <0.01 0.99 0.03a,x 0.23 0.00a,w
FAO 3.4 0.0a,x 0.58 0.02a,wx 0.07 0.00b,wx <0.01 3.17 0.30ab,x 0.32 0.02a,x 0.06 0.00a,wx <0.01 0.86 0.01a,x 0.26 0.00a,w
125 FAO 3.5 0.1a,x 0.72 0.08a,wx 0.19 0.10b,x <0.01 4.43 0.50b,w 0.32 0.04a,w 0.06 0.00a,w <0.01 0.87 0.12a,w 0.21 0.03a,w
rain-fed 4.1 0.1a,y 0.36 0.15a,w 0.10 0.00a,x <0.01 1.40 0.74a,w 0.28 0.02a,x 0.04 0.01a,w <0.01 0.58 0.20a,w 0.21 0.03a,w
RDI-2 4.2 0.0a,y 0.45 0.02a,w 0.24 0.07b,x <0.01 2.64 0.00ab,w 0.83 0.17a,x 0.06 0.01b,w <0.01 0.63 0.02a,w 0.21 0.01a,w
FAO 4.2 0.0a,y 0.43 0.00a,w 0.20 0.07b,x <0.01 2.09 0.20ab,w 0.64 0.28a,x 0.04 0.00ab,w <0.01 0.69 0.01a,w 0.27 0.01b,w
125 FAO 4.2 0.0a,y 0.57 0.06a,w 0.23 0.06b,x <0.01 3.14 0.53b,w 0.55 0.09a,x 0.07 0.00c,w <0.01 0.86 0.03a,w 0.28 0.01b,x
a Different letters (ac) within a column indicate significant differences (p < 0.05) with respect to irrigation treatment in each sampling. Different letters (wy) within a
column indicate significant differences (p < 0.05) with respect to ripeness index for each irrigation treatment.
Figure 2. Evolution of main volatile compounds in virgin olive oils under rain-fed conditions and FAO irrigation scheduling in the course of fruit ripening
in crop season 2003/2004: (O) E-2-hexenal; (9) hexanal; (]), Z-3-hexen-1-ol; (2) hexan-1-ol; ([) E-2-hexen-1-ol.
and volatile compound profile according to the ripening stage The first two discriminant functions explained 99.9% of the
and the different experimental irrigation treatments. variance (97.9 and 2.0%, respectively), yielding a good clas-
The most useful variables for classification of the VOO sification (85%) of VOO oil samples obtained from unripe to
samples according to the stage of ripening of the fruit in both over-ripe fruits.
crop seasons 2003/2004 and 2004/2005 were hexanol, E-2- On the basis of this result and, in particular, the pattern of
hexen-1-ol, 3,4-DHPEA-EDA, and p-HPEA-EDA (Figure 3). evolution of phenolic and volatile compositions throughout fruit
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(16) Martnez, J. M.; Munoz, E.; Alba, J.; Lanzon, A. Informe sobre
(27) Solinas, M.; Maesilio, V.; Angerosa, F. Evoluzione di alcuni
utilizacion del analizador de rendimiento Abencor. Grasas
componenti dellaroma degli oli vergini di oliva in relazione del
Aceites 1975, 26, 379-385.
grado de maturazione delle olive. RiV. Ital. Sostanze Grasse 1987,
(17) Mateos, R.; Espartero, J. L.; Trujillo, M.; Ros, J. J.; Leon-
Camacho, M.; Alcudia, F.; Cert, A. Determination of phenols, 64, 475.
flavones and lignans in virgin olive oil by solid-phase extraction (28) Angerosa, F.; Basti, C.; Vito, R. Virgin olive oil volatile
and high-performance liquid chromatography with diode array compounds from lipoxigenase pathway and characterization of
ultraviolet detection. J. Agric. Food Chem. 2001, 49, 2185- some Italian cultivars. J. Agric. Food Chem. 1999, 47, 836-
2192. 839.
(18) Vichi, S.; Castellote, A. I.; Pizzale, L.; Conte, L. S.; Buxaderas,
S.; Lopez-Tamames, E. Analysis of virgin olive oil volatile
Received for review March 22, 2006. Revised manuscript received July
compounds by headspace solid-phase microextraction coupled
to gas chromatography with mass spectrometric and flame 5, 2006. Accepted July 20, 2006. This research project was supported
ionisation detection. J. Chromatogr. A 2003, 983, 19-33. by the Conserjera de Ciencia y Tecnologa de la Junta de Comunidades
(19) Gomez-Alonso, S.; Salvador, M. D.; Fregapane, G. Phenolic de Castilla-La Mancha (Project PBI-03-015).
compounds profile of Cornicabra virgin olive oil. J. Agric. Food
Chem. 2002, 50, 6812-6817. JF060798R
Artculo IV
Virgin olive oil and olive fruit minor constituents as affected by irrigation
management based on ETc, SWP and TDF in medium-density young olive orchards
(Olea europaea L. cv. Cornicabra and Morisca)
--------------------------------------------------------------------------------------------------------------------------------
ABSTRACT
This study investigated the effect of different irrigation scheduling, based on the measurements of
the water status of olive tree, like the stem water potential (SWP) and the trunk diameter
fluctuations (TDF), as compared to the FAO methodology, with respect to the virgin olive oil and
olive drupe minor constituents composition in two different experimental olive orchards (Olea
europaea L. Cornicabra cv. and Morisca cv.). No clear relationships between the water stress
integral, both seasonal and obtained from DOY 229-277 (period from day 229 to day 277 of the
year), and the oleuropein content in the drupes were found. Nevertheless, a good agreement
between the content of this biophenol and the minimum SWP of the olive trees, measured from
the beginning of August to the end of the irrigation season, were found (r2 = 0.88-0.95 and r2 =
0.90-0.95 in Cornicabra and Morisca cultivars, respectively). A lower minimum water potential
corresponds therefore with a higher biophenols content in the drupe and consequently with a
superior phenolic content in VOO. In both cultivars, the volatile compounds most affected by the
water status of olive trees were hexanal, E-2-hexenal and hexan-1-ol, showing an inverse
relationship with the water stress integral observed in the trees. Furthermore, it was observed that
the irrigation schedule based on the SWP measurement, with a threshold about -1.2 MPa (SWP -
1.2), provided VOO in both cultivars with a similar phenolic and volatile composition with respect
to those obtained with the FAO treatment.
KEYWORDS: Virgin olive oil; olive fruit; phenols; volatiles; irrigation; water status measurement.
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INTRODUCTION
The olive tree is an arboreous crop mainly located in the Mediterranean countries, which
cultivation has been recently extended outside this traditional area in countries like United States
(California), South America, South Africa and Australia, since it is a crop with a great economic
and social relevance. Almost all these new orchards employ different irrigation techniques in order
to accelerate the rate of growth of the olive trees, diminish the characteristic alternate-bearing
pattern of this crop, and increase fruit yields per hectare and consequently the oil production
(Beede & Goldhammer, 1994; Moriana, Orgaz, Fereres & Pastor, 2003).
The methodology generally used to determine the water requirements of the olive trees has been
proposed by the Food and Agriculture Organization (FAO) and it is based on the crop
evapotranspiration (ETc) (Doorenbos & Pruitt, 1974). Nevertheless, the great water demand of the
FAO procedure recently have promoted the study of diverse regulated deficit irrigation (RDI)
scheduling in the olive orchards in order to diminish and achieve a proper use of the water
applied, without diminish the yields obtained under fully irrigation conditions, either setting inferior
water doses or using different rates of the ETc along all the growing season or according to the
phenological stage of the crop (Moriana et al., 2003; Moriana, Prez-Lpez, Gmez-Rico,
Salvador, Olmedilla, Ribas & Fregapane, 2007; Tognetti, dAndria & Morelli, 2005). However, the
ETc parameter estimation is subjected to great uncertainties related to meteorology and olive crop
characteristics (load level, crop age, vegetative stage of olive tree, etc.) (Fereres, Goldhamer &
Parsons, 2003; Naor, 2003), so that the use of irrigation scheduling based on the direct
measurements of olive tree water status seem to be a proper alternative to determine the
irrigation doses to be applied in the orchard. Two interesting measurements effectively related to
the water status of the plant, and already used in some fruit tree species and more recently in
olive orchards, are the Stem Water Potential (SWP) and the Trunk Diameter Fluctuations (TDF)
(Moriana & Fereres, 2002; Gucci, Lodolini & Rapaport, 2007).
However, it is very relevant to remark that any type of irrigation management employed in the
olive orchard must take into account the ultimate effect on the quality and composition of the
virgin olive oil obtained, especially with respect to its content in minor compounds, like phenols
and volatiles, which are chiefly responsible of the unique aroma and taste of this vegetable oil
(Angerosa, 2002; Aparicio & Luna, 2002).
Several research works have already been carried out in order to study the effect of irrigation
techniques on the minor composition of virgin olive oils and reporting a direct relationship between
the water stress of olive trees and the phenolic compounds of the final product obtained,
especially with the secoiridoid derivatives of hydroxytyrosol (Tovar, Romero & Motilva, 2001;
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Romero, Tovar, Girona & Motilva, 2002; Gmez-Rico, Salvador, La Greca & Fregapane, 2006;
Servili, Esposto, Lodolini, Selvaggini, Taticchi, Urbani, Montedoro, Serravalle & Gucci, 2007),
furthermore, some changes in the volatile profile of the oils have been related to different irrigation
schedules applied to Cornicabra cv. and Leccino cv. orchards (Gmez-Rico et al., 2006; Servili et
al. 2007, respectively), so the fine sensory characteristics of virgin olive oil are clearly affected by
the water status of the olive trees (Berenguer, Vossen, Grattan, Connell & Polito, 2006; Gmez-
Rico, Salvador, Moriana, Prez, Olmedilla, Ribas & Fregapane, 2007; Servili et al., 2007).
Nevertheless, up to date there is only a few detailed information about the effect of different
irrigation strategies based on the measurements of the water status in the olive tree itself, like the
SWP and TDF, on the major and minor composition of virgin olive oils (Servili et al., 2007).
The ultimate goal and the main innovation of this study is therefore (i) to test different irrigation
scheduling applied using the measurements of the water status of olive tree, like the stem water
potential (SWP) and the trunk diameter fluctuations (TDF), as compared to the FAO methodology,
with respect to their influence on virgin olive oil and olive drupe composition and (ii) to compare
the possible advantages attached to irrigation on these two unlike varieties. In this assay two
experimental olive orchards of Spanish olive varieties (Olea europaea L. Cornicabra cv. and
Morisca cv.) were used.
Experimental olive orchards. The study was carried out during three consecutives olive crop
seasons (2005/06, 2006/07 and 2007/08) in two experimental olive (Olea europaea L.) orchards
of Cornicabra and Morisca cultivars. The Cornicabra cv. orchard, maintained by the Consejera de
Agricultura of Castilla-La Mancha, was located in Ciudad Real (Spain), while Morisca cv. orchard,
maintained by the Conserjera de Agricultura y Medio Ambiente of Junta de Extremadura, was
sited in Badajoz (Spain). Both areas show a Mediterranean continental climate, although Badajoz
experiences a mild Atlantic influence, due to the proximity of the Portuguese coast; the average
annual rainfall is just 404 mm (Ciudad Real) and 475 mm (Badajoz), mostly distributed outside of
a 4-month (June-September) summer drought period. The total rainfall in Ciudad Real was 172
and 412 mm during 2005 and 2006, respectively, and 324 and 199 mm along 2006 and 2007 in
Badajoz. The rainfall from May to September (the irrigation season) was 27 and 95 mm in Ciudad
Real, for each season, and 40 and 102 mm in Badajoz. Although the rainfall during the irrigation
season in 2006 (Ciudad Real) and 2007 (Badajoz) was uncommonly high, both orchards
experienced summer drought. Cornicabra cv. orchard (3.2 ha) was planted in 1999 with olive
trees spaced 4.75m x 7.0m, whereas Morisca cv. orchard (1 ha) was planted in 1998 with trees
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spaced 6.0m x 4.0m. All of the agronomical treatments applied to the experimental olive orchards
were identical, with the exception of the irrigation management. A randomised complete block
design was used with three blocks that each received the irrigation treatments denominated FAO,
TDF and SWP.
FAO - Irrigation scheduling establish according to the FAO methodology. This method applied the
estimated evapotranspiration (100%ETc) based on the fully replenishing soil water extraction. ETc
was calculated using the method proposed by the Food and Agriculture Organization (ETc = ETo x
Kc x Kr; Doorenbos & Pruitt, 1974), and employing the crop coefficient (Kc) suggested for olive
trees growing under the conditions reigning in Cordoba (Spain) (Orgaz & Fereres, 1997), with
correction for the canopy size (Kr) (Fereres & Goldhamer, 1990). The reference
evapotranspiration (ETo) was estimated using the PenmanMonteith equation using daily data
from a nearby automatic weather station.
TDF - Irrigation treatment in relation to the trunk diameter fluctuations (TDF), which were
measured with linear variable differential transformers (LVDT) (model DF 2.5; Solartron
Metrology, West Sussex, UK). Measurements were taken on each experimental tree every 30 s
and the datalogger (model CR21X, Campbell Sci., Logan, USA) was programmed to calculate 15
min means. The daily TDF cycles provides two indices; maximum daily shrinkage (MDS),
calculated as the difference between the maximum daily diameter (MXTD) and the minimum daily
diameter (MNTD) (Goldhamer & Fereres, 2001) and trunk growth rate (TGR), calculated as the
slope of MXTD daily records. These two indices were used to estimate the irrigation doses
according to the results obtained by Moriana et al. (2002).
SWP - Two different irrigation managements based on the measurements of the stem water
potential (SWP). SWP was determined with a pressure chamber (Soil Moisture Equip., Santa
Barbara, CA, USA) using fully expanded leaves on branches near the trunk which were covered
with aluminium foil at least 1 hour before removal at midday (13:00 h civil time). The water doses
applied to the olive trees during the irrigation seasons allowed to preserve this water status
measurement in two different thresholds; -1.2 MPa (SWP -1.2), which supposed any water deficit
conditions in olive trees, and -2.0 MPa (SWP -2.0) which involved medium water stress.
The total water applied for the different irrigation treatments during the three crop seasons in both
varieties is reported in Table 1. In order to fully describe the different irrigation strategies used
and water status of Cornicabra and Morisca olive trees, the seasonal water stress integrals (S)
and the water stress integrals from DOY 229-277 (S229-277) (MPa x day; as defined by Myers,
1998), calculated from the midday stem water potential data, and the minimum potential values
observed are also reported.
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Table 1. Irrigation doses employed and water status in Cornicabra and Morisca olive trees.
Water stress Water stress DOY
Water doses Minimum potential value
seasonal integral 229-277 integral
(mm) (MPa)
(MPa x day) (MPa x day)
CORNICABRA
2005
FAO 125.0 94.6 26.0 -1.69 (middle August)
TDF 70.1 98.7 28.8 -1.71 (middle August)
-1.47 (beginning
SWP -1.2 179.5 75.0 19.2
September)
SWP -2.0 28.9 161.6 43.1 -2.10 (middle September)
CORNICABRA
2006
FAO 92.7 138.3 53.6 -1.75 (middle August)
-2.10 (middle August and
TDF 51.5 172.1 81.5
middle September)
SWP -1.2 101.2 141.4 54.2 -1.82 (middle August)
-2.17 (middle August and
SWP -2.0 25.1 199.8 84.9
middle September)
MORISCA 2006
-1.91 (beginning August
FAO 408.9 197.6 82.3
and middle September)
TDF 704.1 129.3 38.9 -1.28 (beginning August)
SWP -1.2 388.6 185.8 63.7 -1.71 (beginning August)
-2.71 (beginning August
SWP -2.0 184.2 328.9 119.6
and beginning September)
MORISCA 2007
FAO 297.3 185.4 44.9 -1.73 (middle September)
-2.43 (beginning
TDF 350.2 163.8 48.5
September)
SWP -1.2 304.8 165.1 46.3 -1.64 (middle September)
-2.97 (beginning
SWP -2.0 193.1 267.2 90.7
September)
Olive fruit and olive oil samples. Olive fruit samples from the different irrigation treatments were
harvested at two ripening indexes (RI13.5 and RI24.5) for both varieties studied (Cornicabra cv.
and Morisca cv.). The olive ripeness index was determined according to the method proposed by
the International Olive Oil Council (IOOC, 1984), based on the evaluation of the olive skin and
pulp colours. Three representative subsamples from each treatment (3 subsamples x 4
treatments) were picked at each sampling and brought to the laboratory for oil extraction,
moreover 100 g of olive fruit from each subsample were frozen using liquid N2 and stored at
70C for the analysis of biophenols in olive fruits. Virgin olive oil samples of Cornicabra and
Morisca variety were obtained using the Abencor analyzer (Abengoa S.A., Sevilla, Spain), this
system reproduces at laboratory scale the industrial process through three basic elements;
hammer mill, thermo-mixer and centrifuge. The oil obtained was separated by decanting and
stored in amber glass bottles at 4C in darkness without headspace until analysis.
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Analytical determinations in olive fruits. All reagents used were of analytical, HPLC or
spectroscopic grade, and were supplied by Merck (Darmstadt, Germany).
Biophenols. A sample of olive pulp (4.0 g) was homogenized with a mixture of methanol:water
(80:20 v/v) (40 mL) during 2 min with an Ultraturrax homogenizer (14000 rpm). The suspension
obtained was shaken (20 min, 150 rpm, <4C in darkness), and then centrifuged (10 min, 5000
rpm and 4C). The hydromethanolic phase was recovered and filtered with a 0.45 m nylon
syringe filter. The phenolic fraction extracted was analysed by high-performance liquid
chromatography (HPLC) using an Agilent Technologies 1100 series system equipped with an
automatic injector, a column oven and a diode array UV detector. A Zorbax SB-C18 column (250
x 4.6 id mm, 5 m particle size) (Agilent Technologies, USA), maintained at 30 C, was used with
an injection volume of 20 l and a flow rate of 1.0 ml/min. The mobile phase consisted of a
mixture of water/acetic acid (95:5 v/v) (solvent A), methanol (B) and acetonitrile (C): from 95%
(A)-2.5% (B)- 2.5% (C) to 34% (A)-33% (B)-33% (C) in 50 min. Chromatograms were recorded at
280 nm, 340 nm and 520 nm. Hydroxytyrosol, oleuropein and demethyloleuropein were quantified
at 280 nm, anthocyanins at 520 nm and verbascoside and flavonoids at 340 nm. Phenolic
compound quantification was achieved using a five-point calibration curve on the basis of the
corresponding standard substances with the exception of hydroxytyrosol which was quantified as
tyrosol, and demethyloleuropein and anthocyanins as oleuropein. Identification of colourless
phenolic compounds was carried out by comparison of their retention times, UV-Visible
characteristics and MS spectra with their standard substances. In the case of the anthocyanins
and demethyloleuropein, these were tentatively identified by their UV-Visible characteristics and
MS spectra. The mass detector used was a LCQ Deca XP Plus (Thermo Electron Corporation,
Waltham, MA, USA) equipped with an electrospray ionisation system. Nitrogen was used as
nebulizing gas at a flow rate of 14 (arbitrary units). The temperature and voltage of the capillary
were 250 C and 4.50 kV, respectively. Data were acquired in the negative ionisation mode.
Fragmentation experiments were performed using helium as the collision gas with collision energy
between 30-40%.
Fatty acid composition (European Regulations EEC 2568/91 and subsequent amendments,
corresponding to the AOCS method Ch 2-91). To determine fatty acid composition, the methyl-
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esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml
of 2N methanolic potassium hydroxide and analysed by GC with a FID detector. A fused silica
column (50 m length x 0.25 mm i.d.) coated with SGL-1000 phase (0.25 m thickness;
Sugerlabor, Spain) was used. The carrier gas was helium, at a flow through the column of 1
ml/min. The injector and detector temperature was set at 250C and the oven temperature at
210C. The injection volume was 1 l.
Oxidative stability. This was evaluated by the Rancimat method (Labli & Bruttel, 1986). Stability
was expressed as the induction time (hours) measured with the Rancimat 679 apparatus
(Metrohm, Switzerland).
Phenolic compounds. A solution of the internal standard (250 l of 15 mg/Kg of syringic acid in
methanol) was added to a sample of virgin olive oil (2.5 g) and the solvent was evaporated with a
rotary evaporator at 35 C under vacuum. The oil was then dissolved in 6 ml of hexane and a diol-
bonded phase cartridge (Supelco Co., Bellefonte, PA) was used to extract the phenolic fraction.
The cartridge was conditioned first with methanol (6 ml) and then with hexane (6 ml). The oil
solution was then applied, and the solid phase extraction (SPE) column was washed with hexane
(2 x 3 ml) and with hexane/ethyl acetate (85:15, v/v; 4 ml). Finally, the phenols were eluted with
methanol (15 ml) and the solvent was removed with a rotary evaporator at 35 C under vacuum
until dry. The phenolic residue was dissolved in methanol/water (1:1 v/v; 250 l).
Chromatographic conditions were the same as those used with the olive pulp phenolic fraction.
Phenolic compounds were quantified at 280 nm using syringic acid as internal standard and the
response factors determined by Mateos, Espartero, Trujillo, Ros, Len-Camacho, Alcudia & Cert
(2001).
Tocopherols were evaluated following the AOCS Method Ce 8-89. A solution of oil in hexane was
analysed on an Agilent Technologies HPLC (1100 series) on a silica gel Lichrosorb Si-60 column
(particle size 5 m, 250 mm x 4.6 mm i.d.; Sugerlabor, Madrid, Spain) which was eluted with
hexane/2-propanol (98.5:1.5) at a flow rate of 1 ml/min. A fluorescence detector (Thermo-Finnigan
FL3000) was used with excitation and emission wavelength set at 290 and 330 nm.
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Volatile compounds adapted from Vichi, Castellote, Pizzale, Conte, Buxaderas & Lopez-
Tamames, 2003). Solid phase microextraction (SPME) followed by GC were used to analyze the
volatile compounds in the virgin olive oil samples studied. 1.5 g of olive oil spiked with 4-methyl-2-
pentanol (as internal standard) to a concentration of 1.5 g/g was placed in a 10 mL vial fitted with
a silicone septum. The SPME sampling was performed by exposing the DVB/Carboxen/PDMS
fiber (50/30 m, 2 cm long from Supelco Inc., Bellefonte, PA) for 30 min in the headspace of the
sample maintained at 40C; it was then retracted into the needle and immediately transferred and
desorbed for 1 min in the injection port of an Agilent 6890 Series gas chromatograph equipped
with a flame ionization detector (FID). Compounds were separated on a Supelcowax-10 column
(30 m x 0.25 mm x 0.25 m, Supelco Inc., Bellefonte, PA) under the following conditions: injection
port temperature 260C; helium flow 0.8 mL/min; oven temperature ramp: 35C for 10 min,
3C/min up to 160C and then 15C/min up to 200C (maintained for 5 min). Volatile compounds
were identified by comparison of retention times and mass chromatograms of standard
substances (Sigma Aldrich) added to refined olive oil. The equipment used was an Agilent 5975C
Series mass spectrometer (Agilent Technologies, USA) equipped with an electron ionisation (EI+)
detector and coupled to an Agilent 6850 Series gas chromatograph, the capillary column used
was a DB-Wax (30 m x 0.25 mm x 0.25 m, J&W Scientific, USA). Helium was employed as
carrier gas at a flow rate of 0.8 mL/min. The transfer line temperature was 280 C and the
temperature of the ionisation source and the quadrupole were 230 C and 150 C, respectively,
with an electromultiplier voltage of +941 eV.
Statistical Analysis. Analysis of ANOVA analysis was performed using SPSS 15 statistical
software (SPSS Inc., Chicago, IL). Duncans test (p 0.05) was used to discriminate among the
mean values.
The influence of the different irrigation management proposed in this study, based on the Trunk
Diameter Fluctuations (TDF) and the Stem Water Potential (SWP) measurements, as compared
with the FAO methodology on agronomical aspects like the growth rate and the production of the
olive groves of Cornicabra and Morisca cultivars are currently being deeply analyzed and will be
discussed elsewhere.
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First of all, it is worth to remark the great differences found between Cornicabra and Morisca
varieties respect to their fatty acid composition, especially in the oleic acid content, ranging from
77.5-80.3% and 55.8-59.0% in Cornicabra and Morisca VOO, respectively, and linoleic acid,
which content varied from 3.0-3.3% and 18.5-22.5% in Cornicabra and Morisca VOO,
respectively. As a consequence, the MUFA/PUFA ratios obtained in Cornicabra VOO were about
7.4 times higher than in Morisca VOO (Table 2). The potential oxidative susceptibility is therefore
higher in the Morisca VOO (lower oxidative stability) than other VOO, like Cornicabra or Picual
varieties, a part from the natural antioxidant content of each variety.
With respect to the effect produced in the fatty acid composition by the irrigation practices applied
in both olive orchards, these were very weak and moreover did not show similar trends in the
different successive crop seasons studied (complete data not shown). Moreover, these not
statistically significant changes in the fatty acid composition do not possess any relevance on the
nutritional value. In a similar way, the MUFA/PUFA and SFA/UFA ratios were practically
unaffected by the different irrigation scheduling studied. Berenguer et al. (2006) observed similar
trends in Arbequina VOO obtained from a young super-high-density orchard, where different
regulated deficit irrigation (based on several %ETc) were applied, moreover Inglese, Barobe &
Gullo (1996) and Patumi, dAndria, Fontanazza, Morelli, Giori & Sorrentino (1999) found that the
fatty acid composition of different Italian varieties was affected mainly by cultivar factors and not
by irrigation practices. Other researches found that irrigation implied an increase in palmitic and
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linoleic acids and a decrease in oleic and linolenic acid content in VOO but significant differences
were observed only between rainfed and the rest of the irrigation treatments studied (Salas,
Pastor, Castro & Vega, 1997; Gmez-Rico et al., 2007).
Table 2. Virgin olive oil main fatty acid composition as affected by the irrigation scheduling
studied.
2005/06 R.I. C16:0 (%) C18:0 (%) C18:1 (%) C18:2 (%) C18:3 (%) MUFA/PUFA UFA/SFA
b a a a a a a a
FAO 3.7 0.2 11.6 0.2 3.1 0.3 79.4 0.6 3.1 0.1 0.6 0.0 21.3 0.8 5.4 0.2
a a a a a b a a
TDF 3.6 0.2 12.8 0.9 2.8 0.0 78.1 0.6 3.3 0.1 0.7 0.0 19.9 0.7 5.2 0.3
a a a a a a,b a a
SWP -1.2 3.6 0.3 11.4 0.7 2.9 0.2 79.5 0.9 3.3 0.0 0.7 0.0 20.2 0.1 5.6 0.4
a a a a a b a a
SWP -2.0 3.6 0.3 12.6 0.8 2.9 0.5 78.2 1.1 3.3 0.0 0.7 0.0 20.3 0.9 5.0 0.4
MORISCA cv.
2006/07
b a b b a a b b,c
FAO 3.5 0.1 14.9 0.1 3.6 0.0 59.0 0.3 18.7 0.3 1.1 0.0 3.1 0.1 4.2 0.0
a b b a b b a a
TDF 3.1 0.1 15.6 0.2 3.5 0.2 55.9 2.0 20.8 1.5 1.2 0.0 2.6 0.3 4.0 0.1
b b a a b b a a,b
SWP -1.2 3.5 0.2 15.5 0.2 3.4 0.2 56.2 0.6 20.9 0.3 1.2 0.0 2.6 0.1 4.1 0.1
b a a b b a a,b c
SWP -2.0 3.5 0.2 14.9 0.2 3.3 0.1 58.3 0.3 19.7 0.7 1.1 0.0 2.8 0.2 4.3 0.1
Different letters within a column (a-c) indicate significant differences (p<0.05) with respect to irrigation
treatment in each sampling.
R.I: ripeness index.
No clear relationships between the water stress integral, both seasonal and obtained from DOY
229-277, and the oleuropein content in the drupes were found. Nevertheless, a good agreement
between the content of this biophenol and the minimum stem water potential of the olive trees,
measured from the beginning of August to the end of the irrigation season, were obtained (r2 =
0.88-0.95 and r2 = 0.90-0.95 in Cornicabra and Morisca cultivars, respectively; Table 1 and
Figure 1): therefore, a lower minimum water potential correspond with a higher biophenols
content. In fact, in 2006 crop season, in which the minimum potential values reported in
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Cornicabra olive trees under the different irrigation schedules were similar (varied between -1.75
MPa and -2.17 MPa; Table 1), no significant differences in oleuropein content in drupes were
found. Furthermore, in 2007 crop season, the minimum potential values in Morisca olive trees
were -1.64 MPa (SWP -1.2 strategy), -1.73 MPa (FAO treatment), -2.43 MPa (TDF strategy) and -
2.97 MPa (SWP -2.0 strategy), and the oleuropein content in olive drupes were similar in FAO-
SWP -1.2 strategies and TDF-SWP-2.0 irrigation schedules.
14000
12000
Oleuropein content (mg/Kg)
10000
8000
6000
3000
2000
1000
0
2005 2006 2006 2007
Cornicabra drupe Morisca drupe
Several research works (Patumi et al., 1999; Tovar, Romero, Girona & Motilva, 2002) have
reported that different water status of olive trees under different irrigation strategies, implied
changes in the activity of enzymes responsible for phenolic compounds synthesis in drupes, such
as L-Phenylalanine ammonia-lyase (PAL), whose activity is greater under higher water stress
conditions, leading superior phenolic contents in olive flesh and therefore in the virgin olive oils
obtained. However, according to the results of this study, the activity of PAL would be probably
affected by critical drop water status that could happen during the phenological stage III of the
olive fruit, and not only by the total seasonal water status of the olive tree.
The total phenol content and the secoiridoid forms of hydroxytyrosol and tyrosol in Cornicabra and
Morisca VOO from the different irrigation strategies and crop seasons studied are depicted in
Figure 2.
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1250 1250
CORNICABRA 2005 CORNICABRA 2006
Phenolic content (mg/Kg)
1000 1000
750 750
500 500
250 250
0 0
Total phenols Secoiridoids of Secoiridoids of Total phenols Secoiridoids of Secoiridoids of
hydroxytyrosol tyrosol hydroxytyrosol tyrosol
450 450
MORISCA 2006 MORISCA 2007
Phenolic content (mg/Kg)
300 300
150 150
0 0
Total phenols Secoiridoids of Secoiridoids of Total phenols Secoiridoids of Secoiridoids of
hydroxytyrosol tyrosol hydroxytyrosol tyrosol
Figure 2. Total phenols and complex secoiridoid forms of hydroxytyrosol and tyrosol
contents in Cornicabra and Morisca VOO as affected by the irrigation management.
, FAO; , TDF; , SWP -1.2; , SWP -2.0
Each box includes the content within the 10th and 90th percentiles of the data distribution (RI 3.0-3.5).
The major phenolic fraction in both varieties were the secoiridoids derivatives of hydroxytyrosol
and tyrosol. As expected, within the same variety a relationship between oleuropein concentration
in the fruit and the presence of these compounds in virgin olive oils (Gmez-Rico, Fregapane &
Salvador, 2008) was observed and therefore an increase in the oleuropein content in the drupe
led to a proportional increase in the total phenol concentration in virgin olive oil. As a
consequence, the content in these complex phenols was significantly higher in Cornicabra VOO
than in Morisca VOO. On top of this, the different water status of olive trees affected more the
content of secoiridoid derivatives of hydroxytyrosol in VOO than those of tyrosol, since higher
water stress conditions lead to superior increases in the first ones (fig. 2), as previously reported
(Gmez-Rico et al., 2006).
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Table 3 reports the concentrations of the main phenolic compounds (mg/Kg) found in VOO
obtained from Cornicabra and Morisca olives subjected to the different irrigation schedules.
Table 3. Virgin olive oil main individual phenolic compounds (mg/Kg) as affected by the
irrigation strategies studied.
CORNICABRA cv.
3,4-DHPEA- 3,4-DHPEA-
2005/06 R.I. 3,4-DHPEA
EDA EA
p-HPEA p-HPEA-EDA p-HPEA-EA
a a a a a a,b a
FAO 3.1 0.4 2.20 1.85 350.4 59.6 138.2 17.4 3.77 1.33 343.4 36.4 55.6 10.8
a a a a a a,b a,b
TDF 3.0 0.2 1.04 0.14 365.4 27.1 147.3 14.3 2.83 0.80 342.5 22.3 60.3 14.6
a a a a a a a
SWP -1.2 3.0 0.3 0.94 0.06 310.1 12.4 124.4 4.4 2.50 0.06 328.4 7.3 51.7 3.3
a a b b a b b
SWP -2.0 3.0 0.3 1.08 0.05 477.6 65.9 199.1 27.1 2.37 0.34 395.7 32.8 79.8 11.4
b a a,b a,b a a,b a
FAO 3.7 0.2 0.98 0.77 150.1 70.6 82.5 28.7 3.12 1.21 218.4 34.7 40.5 8.8
a a a,b a,b a a,b a,b
TDF 3.6 0.2 0.94 0.42 175.2 64.4 86.9 22.8 2.81 0.43 248.1 49.3 51.4 11.3
a a a a a a a
SWP -1.2 3.6 0.3 0.43 0.02 96.8 1.4 59.9 1.7 1.96 0.06 182.0 0.5 35.9 1.9
a a b b a b b
SWP -2.0 3.6 0.3 0.79 0.27 259.3 83.1 123.3 32.3 2.28 0.38 295.1 52.2 65.3 12.3
2006/07
a a a a a a a
FAO 3.0 0.2 1.06 0.41 400.9 32.7 163.6 23.7 4.06 1.59 370.5 29.3 77.7 0.3
a b a a,b a a a
TDF 3.0 0.1 1.28 0.16 392.1 12.1 183.6 5.8 4.03 0.11 366.8 34.4 79.2 13.7
a a a a a a a
SWP -1.2 3.1 0.1 0.94 0.03 369.6 13.2 155.3 22.0 3.72 0.01 395.0 22.0 81.315.2
a b a b a a a
SWP -2.0 3.1 0.2 1.73 0.09 433.9 43.0 215.4 16.5 4.82 0.58 409.6 39.2 109.8 20.9
MORISCA cv.
3,4-DHPEA- 3,4-DHPEA-
2006/07 R.I. 3,4-DHPEA
EDA EA
p-HPEA p-HPEA-EDA p-HPEA-EA
b a a a b a a
FAO 3.5 0.1 0.59 0.36 9.8 8.0 18.9 7.0 2.54 0.05 20.6 5.8 3.6 0.7
a a a a b a a
TDF 3.1 0.1 0.62 0.08 5.31 2.3 13.8 1.5 2.56 0.46 16.8 2.1 3.5 0.1
b a a a a a a
SWP -1.2 3.5 0.2 0.46 0.09 7.5 1.4 15.9 0.7 1.77 0.14 22.0 1.1 4.1 0.3
b a b b a b b
SWP -2.0 3.5 0.2 0.82 0.32 120.1 14.8 35.2 2.1 1.81 0.33 124.1 25.8 7.8 1.0
a a a a a a a
FAO 4.2 0.1 0.38 0.17 4.4 3.5 15.3 4.7 2.47 0.15 17.6 3.4 3.3 0.8
a a a a a a a
TDF 4.3 0.1 0.21 0.07 1.4 0.3 10.6 1.6 2.22 0.37 13.0 1.6 3.2 .3
a a a a a a a
SWP -1.2 4.1 0.2 0.23 0.09 1.5 0.1 10.3 0.1 3.25 1.08 15.8 0.9 3.1 0.2
b b b b a b b
SWP -2.0 4.7 0.1 1.73 0.86 87.5 56.8 26.7 9.8 2.83 0.69 114.5 60.7 6.9 2.0
2007/08
a a a,b a,b a a a
FAO 2.9 0.1 1.03 0.13 50.6 22.3 24.9 2.6 2.82 0.56 61.2 19.3 3.2 0.7
a a a,b a,b a a a
TDF 3.4 0.6 0.99 0.15 87.3 17.2 33.8 6.7 3.66 2.09 92.5 11.7 3.2 0.3
a a a a a a a
SWP -1.2 3.10.1 0.74 0.11 42.2 13.3 23.8 6.6 2.670.12 66.2 27.5 2.8 0.3
a a b b a a a
SWP -2.0 3.0 0.1 0.78 0.20 96.7 9.8 37.9 9.0 2.13 0.08 95.7 2.6 3.5 0.4
a a a a a a a
FAO 4.1 0.1 0.72 0.21 43.4 11.8 25.8 0.8 2.72 0.30 59.1 11.8 2.8 0.0
a a b a a b a
TDF 4.0 0.1 1.41 0.89 80.5 18.5 31.6 4.7 4.04 2.02 94.7 11.2 3.4 0.1
a a a,b a a a,b a
SWP -1.2 4.2 0.1 0.79 0.40 51.3 28.6 22.9 6.5 3.55 0.30 65.1 20.8 3.1 0.1
a a b a a a,b a
SWP -2.0 4.1 0.3 0.87 0.26 83.3 21.0 26.2 4.5 1.82 0.41 86.1 16.2 3.1 0.7
Different letters within a column (a-c) indicate significant differences (p<0.05) with respect to irrigation
treatment in each sampling.
R.I: ripeness index.
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The phenolic compounds most affected by the irrigation scheduling employed in the olive
orchards were the 3,4-DHPEA-EDA, 3,4-DHPEA-EA and p-HPEA-EDA, which concentrations
were significantly higher in the VOO from the more stressed treatment (SWP -2.0), while no clear
statistical significant differences were observed between FAO, SWP -1.2 and TDF schedules
along the successive crop seasons in Cornicabra and Morisca cultivars.
As previously reported in the case of the oleuropein in the drupes, the total water doses employed
in the olive trees under the different irrigation strategies, and consequently the different water
status of the plant (Table 1), apparently were not clearly related neither to the oleoside content of
the drupes nor to the phenolic composition of virgin olive oils (Figure 1 and Figure 2). On the
contrary, the main falls in the water status of the trees, expressed as the minimum potential
values (Table 1), were the main factor responsible of the final phenolic content reported in drupes
and virgin olive oils.
For example, in 2006/07 season (RI 3.0), the content of 3,4-DHPEA-EDA and p-HPEA-EDA
were about 400.9 mg/Kg and 370.5 mg/Kg, respectively, in Cornicabra VOO under FAO treatment
(water dose: 92.7 mm; S: 138.3 MPa x day), whereas in VOO from TDF strategy (water dose:
51.5 mm; S: 172.1 MPa x day) were similar, 392.1 mg/Kg and 366.8 mg/Kg, though the irrigation
dose was almost halved and the water stress integrals were significantly different (see Table 1).
On the other hand, in 2007 season (RI 3.5), the content of the same phenolic compounds in
Morisca VOO from FAO schedule (water dose: 297 mm; S: 185.4 MPa x day) were 50.6 mg/Kg
and 61.2 mg/Kg, respectively, while in TDF virgin olive oils (water dose: 350 mm; S: 163.8 MPa
x day) were significantly higher - 87.3 mg/Kg and 92.5 mg/Kg - despite the irrigation doses and
the water stress integrals were almost the same (Table 1).
Recently, Tovar et al. (2001), Gmez-Rico et al. (2006) and Servili et al. (2007) reported in
Arbequina, Cornicabra and Leccino cultivars, respectively, a higher decrease in the levels of 3,4-
DHPEA-EDA, 3,4-DHPEA-EA and p-HPEA-EDA as the water stress of olive trees fell off, finding
clearly significant differences between the VOO from the most stressed treatments and those
from medium-highly irrigated treatments, depending on the irrigation schedule used. However, in
this study, the effect reported on phenolic composition were lower, this fact can be probably due
to the different strategies employed in the olive orchards, which involved not very dissimilar water
status in olive trees, with the exception of the SWP -2.0 strategy and the differences observed in
the minimum water potential value reported in FDT, FAO and SWP -1.2 in Cornicabra and
Morisca orchards (Table 1).
As concerned the simple phenols found in virgin olive oils, the hydroxytyrosol contents (ranging
from 0.42 mg/Kg up to 3.72 mg/Kg in Cornicabra VOO and from 0.13 mg/Kg up to 2.45 mg/Kg in
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Morisca VOO, expressed as the 10th and 90th percentiles of the data distribution respectively)
and tyrosol (1.94 - 5.31 mg/Kg in Cornicabra and 1.49 - 5.80 mg/Kg in Morisca oils) were
apparently not affected by the irrigation strategies studied, since no statistically significant
differences were observed (Table 3). This behavior was similar to that observed for the rest of the
minor simple phenols identified (data not shown), which were present in very small amounts
vanillin (<0.14 mg/Kg and <0.94 mg/Kg in Cornicabra and Morisca VOO, respectively), vanillic
acid (<0.32 mg/Kg and <0.28 mg/Kg), p-coumaric acid (<0.29 mg/Kg and <0.91 mg/Kg) except
for ferulic acid (ranging from 12.2 up to 31.4 mg/Kg in Morisca and <1.28 mg/Kg in Cornicabra,
respectively) and pinoresinol (4.5 - 6.8 mg/Kg in Morisca and 5.6 - 11.5 mg/Kg in Cornicabra),
which contents were higher.
As previously reported, the complex phenols have been not only related with the oxidative stability
(Baldioli, Servili, Peretti & Montedoro, 1996; Gennaro, Piciola Bocca, Modesti, Masella & Coni,
1998), but also with the sensory properties of the virgin olive oil, especially with the positive
sensory attribute bitterness and pungency (Morales & Tsimidou, 2000). The oxidative stability,
expressed in hours, and the bitterness of the virgin olive oils of the study, measured by the
instrumental K225 parameter (Gutierrez-Rosales et al., 1992), are reported in Table 4.
Table 4. Virgin olive oil oxidative stability (OS), K225 and -tocopherol content as affected
by the irrigation strategies studied.
CORNICABRA cv.
2005/06 2006/07
-tocopherol -tocopherol
OS (h) K225 OS (h) K225
(mg/Kg) (mg/Kg)
FAO 30.4 2.0a 0.65 0.02a 317.1 5.7a 32.9 1.2a 0.67 0.06a 259.6 25.5a
TDF 30.9 1.2a 0.68 0.03a 295.2 23.4a 32.7 0.5a 0.69 0.02a 278.9 18.9a,b
SWP -1.2 29.2 0.3a 0.65 0.02a 309.6 6.4a 31.7 1.3a 0.64 0.03a 344.4 17.5c
SWP -2.0 35.9 2.2b 0.74 0.05b 315.9 34.1a 36.3 1.4b 0.78 0.01b 321.4 15.8b,c
MORISCA cv.
2006/07 2007/08
-tocopherol -tocopherol
OS (h) K225 OS (h) K225
(mg/Kg) (mg/Kg)
FAO 3.8 0.3a 0.12 0.02b 436.1 18.6a 5.0 0.4a 0.17 0.03a 592.9 10.9a
TDF 2.8 0.1a 0.09 0.02a 467.8 7.4b 5.6 0.5a,b 0.19 0.03a 639.7 32.1a
SWP -1.2 2.9 0.1a 0.09 0.01a 459.1 8.3a,b 4.3 0.7a 0.15 0.03a 616.3 30.8a
SWP -2.0 5.5 1.0b 0.27 0.02c 476.4 13.7b 5.8 0.3b 0.21 0.00a 647.1 11.9a
Different letters within a column (a-b) indicate significant differences (p<0.05) with respect to irrigation
treatment in first sampling (RI 3.0-3.5).
R.I: ripeness index.
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As expected, according to the large differences found between the phenolic composition of
Cornicabra and Morisca VOO, a greater oxidative stability in Cornicabra VOO (ranging from 29.2
up to 36.3 hours in the different irrigation strategies) than in Morisca ones (from 2.9 up to 5.8
hours) was observed. The same trend was observed for the bitterness index (directly related to
the K225), which was about 3 times higher in Cornicabra than in Morisca oils from SWP -2.0
treatment, and about 4.4, 4.5 and 5.4 times higher in Cornicabra oils from TDF, FAO and SWP -
1.2 strategies, respectively, respecting the Morisca ones. As observed in the phenolic
composition, clearly significant differences were only reported between the VOO from the most
stressed treatment (SWP -2.0) and the rest ones (Table 4).
The bitterness decrease due to the irrigation technique used, though very slightly in the oils used
in this study, is very relevant from a marketing point of view, so high levels of bitterness are
rejected by the consumers and therefore the descent of this positive attribute could be desirable in
rich-phenol virgin olive oil, like the Cornicabra variety. On the contrary, probably this factor could
affect negatively Morisca VOOs sensory profile, so due to their natural low phenolic content, a
descent of these compounds would imply to obtain virgin olive oils too mild and flat, and moreover
the implied decrease in the oxidative stability could reduced significantly the shelf-life of the
product, since due to its high content in polyunsaturated fatty acids the oxidation processes could
be accelerated.
Tocopherols.
Changes observed in -tocopherol content, practically the only tocopherol present in VOO, in
from both varieties (Cornicabra and Morisca cv.), as affected by the irrigation strategies and along
the successive crop seasons studied are reported in Table 4.
The small differences found in the -tocopherol content in both olive orchards were not
statistically significant. Gmez-Rico et al. (2007) reported similar observations in virgin olive oils
obtained from different irrigation treatments applied in a Cornicabra cv. low-density orchard. On
the other hand, according to Tovar et al. (2001) and Tovar, Romero, Alegre, Girona & Motilva
(2002) contradictorily results were found, although the irrigation strategies applied in these cases
were not similar, and anyway the observed changes were not noteworthy to vary the VOO
oxidative stability.
As well known seasonal and cultivar are the main factors affecting the -tocopherol content in
VOO. In this case, the content of this antioxidant was 50-70% higher in Morisca than Cornicabra
and depended on the crop season as well.
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Volatile compounds.
Figure 3 shows the distribution of C6 aldehydes, alcohols, esters and C5 volatiles in the VOO of
the study, whereas Table 5 reports the concentrations these individual volatile compounds from
the lipoxygenase (LOX) pathway, expressed as mg of internal standard (4-methyl-2-pentanol) per
kg of oil, in Cornicabra and Morisca VOO samples according to the different irrigation techniques
and the successive crop seasons studied,
These compounds are related with the green, fruity and floral sensory notes found in high-
quality virgin olive oils and are produced through the LOX enzymatic pathway mainly during the
crushing of the fruit and incorporated to the oily phase during the kneading stage of the olive
paste (Sanchez & Harwood, 2002).
C5 volatiles
C6 esters
C6 alcohols
C6 aldehydes
CORNICABRA 2005 CORNICABRA 2006
0,0 0,5 1,0 1,5 3,0 4,5 6,0 7,5 9,0 0,0 0,5 1,0 1,5 3,0 4,5 6,0 7,5 9,0
Volatile content (ppm IS) Volatile content (ppm IS)
C5 volatiles
C6 esters
C6 alcohols
C6 aldehydes
MORISCA 2006 MORISCA 2007
0,0 1,0 2,0 3,0 6,0 9,0 12,0 15,0 0,0 1,0 2,0 3,0 6,0 9,0 12,0 15,0
Volatile content (ppm IS) Volatile content (ppm IS)
Figure 3. C6 and C5 (LOX pathway) volatiles content in Cornicabra and Morisca VOO as
affected by the irrigation management.
, FAO; , TDF; , SWP -1.2; , SWP -2.0
Each box includes the content within the 10th and 90th percentiles of the data distribution (RI 3.0-3.5).
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The volatile composition observed in virgin olive oils from the two studied cultivars was quite
different (Figure 3), since, as known, the aromatic profile depend mainly on the enzymatic
activities which are genetically determined in each cultivar (Angerosa, Basti & Vito, 1999).
Table 5. Virgin olive oil volatiles (ppm of IS) from the LOX pathway (C5 and C6) as affected
by the irrigation strategies studied.
CORNICABRA cv.
Z-3-
Hexyl Z-3-hexen- E-2-hexen- 1-penten- 1-penten-
2005/06 Hexanal Hexan-1-ol
acet.
E-2-hexenal
1-ol 1-ol
hexenyl
3-ol 3-one
acetate
a a b a a a a
FAO 0.360.13 0.140.05 <0.01 5.85 1.49 0.840.47 0.070.00 <0.01 0.150.04 0.260.10
a a b a a b a
TDF 0.470.05 0.150.05 <0.01 6.57 1.41 0.770.41 0.080.01 <0.01 0.210.03 0.390.10
a a b a a a a
SWP -1.2 0.370.12 0.170.04 <0.01 6.00 1.91 0.680.46 0.070.00 <0.01 0.150.05 0.270.12
a a a a a b a
SWP -2.0 0.330.07 0.090.03 <0.01 4.14 0.95 0.580.39 0.070.02 <0.01 0.260.05 0.440.05
a b b a a a a
FAO 0.360.03 0.230.10 <0.01 5.04 0.67 0.330.16 0.050.02 <0.01 0.110.05 0.190.07
a b b a a a a
TDF 0.310.14 0.310.04 <0.01 4.72 0.76 0.470.29 0.060.01 <0.01 0.140.05 0.230.10
a b b a a a a
SWP -1.2 0.280.05 0.340.12 <0.01 3.94 0.19 0.320.17 0.050.00 <0.01 0.110.03 0.200.08
a a a a a a a
SWP -2.0 0.230.06 0.150.03 <0.01 3.56 0.18 0.230.15 0.070.03 <0.01 0.150.02 0.250.03
2006/07
b b a,b a a a a
FAO 0.290.06 0.020.00 <0.01 2.57 0.58 0.050.00 0.050.03 <0.01 0.150.08 0.100.07
a b a a b b b
TDF 0.140.05 0.020.00 <0.01 2.07 0.32 0.070.03 0.080.02 <0.01 0.350.05 0.370.09
b b b a a a a
SWP -1.2 0.240.01 0.030.00 <0.01 3.48 0.57 0.060.00 0.060.01 <0.01 0.190.04 0.150.05
a a a a b b b
SWP -2.0 0.140.02 0.010.00 <0.01 1.91 0.46 0.070.02 0.100.00 <0.01 0.330.01 0.410.11
MORISCA cv.
Z-3-
Hexyl E-2- Z-3-hexen- E-2-hexen- 1-penten- 1-penten-
2006/07 Hexanal Hexan-1-ol
acet. hexenal 1-ol 1-ol
hexenyl
3-ol 3-one
acetate
a b b b a a a a c
FAO 1.590.16 0.330.08 0.010.00 8.140.95 0.130.01 0.120.00 0.030.01 0.220.01 0.290.05
a a b b c a b a a
TDF 1.930.31 0.250.05 0.020.00 8.750.57 0.490.05 0.450.38 0.060.01 0.220.02 0.230.07
a b c c c a c a a
SWP -1.2 1.790.16 0.370.08 0.040.00 11.72.37 0.470.02 0.470.33 0.100.01 0.200.01 0.170.03
a a a a b a b b c
SWP -2.0 1.520.15 0.230.01 0.020.00 6.510.48 0.380.05 0.200.01 0.070.01 0.300.01 0.320.02
a a b b a a b a a
FAO 1.920.15 1.150.24 0.170.05 6.750.42 0.750.17 0.140.07 0.310.03 0.180.01 0.260.03
b b b a b a c a a
TDF 2.660.65 1.700.32 0.220.05 5.230.46 1.740.78 0.280.20 0.630.09 0.140.05 0.200.09
b a b c a a b a a
SWP -1.2 3.190.20 1.040.22 0.270.05 7.470.56 0.650.06 0.360.18 0.400.04 0.160.02 0.170.04
a a a c a a a a a
SWP -2.0 1.550.09 0.860.09 0.060.04 8.340.35 0.930.54 0.250.12 0.200.3 0.200.06 0.260.08
2007/08
b a a c a a a a b
FAO 2.140.27 0.610.01 0.060.01 9.761.39 0.350.01 0.040.06 0.110.01 0.200.01 0.580.07
b a a a,b a b a a a
TDF 1.610.32 0.650.11 0.050.03 5.971.15 0.380.06 0.160.01 0.070.03 0.200.01 0.470.13
b b a b,c b a a a b
SWP -1.2 1.920.26 1.070.21 0.050.01 8.040.96 0.580.21 0.070.02 0.120.03 0.200.01 0.720.07
a a a a a a b a a
SWP -2.0 1.3 0.18 0.450.17 0.090.02 4.372.19 0.310.02 0.060.05 0.240.03 0.220.05 0.490.03
a a b a a a a a a
FAO 2.130.36 1.170.47 0.130.01 5.561.42 0.900.43 0.150.03 0.340.11 0.180.01 0.640.06
a a b a a a a a a
TDF 1.850.17 0.900.47 0.120.04 4.430.72 0.670.50 0.120.02 0.260.06 0.180.02 0.640.06
a a b a a a a a a
SWP -1.2 2.180.37 0.980.30 0.110.02 4.961.12 0.580.18 0.120.01 0.190.04 0.200.01 0.720.15
a a a a a a a a a
SWP -2.0 1.850.05 0.840.05 0.070.00 3.531.25 1.010.25 0.120.02 0.220.00 0.170.01 0.680.07
Different letters within a column (a-c) indicate significant differences (p<0.05) with respect to irrigation
treatment in each sampling.
R.I (ripeness index) are similar to those from the first column of Table 3.
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The C6 aldehydes fraction were the major volatile components in both varieties, being
significantly superior in the Morisca VOO (Figure 3), especially the hexanal content (related to
apple, green, cut grass sensory notes), which average content depending on irrigation treatment
and crop season ranged from 0.14 to 0.47 mg/Kg in Cornicabra VOO and from 1.33 to 3.19
mg/Kg in Morisca samples (Table 5). In a similar way, the C6 alcohols concentration was higher
in Morisca oils, specially the hexan-1-ol (fruity, grassy and soft sensory notes), which content
varied between 0.02-0.34 mg/Kg in Cornicabra VOO, and between 0.23-1.70 mg/Kg in Morisca
oils (Tab. 5). As regards C6 esters such as hexyl-acetate and Z-3-hexenyl-acetate (also related
with sweet, fruity and green leaves notes), these were present in very small amounts in
Cornicabra VOO, indicating a little activity of the alcohol acyl transferase (AAT) in this cultivar,
contrariwise the virgin olive oils from Morisca variety contained much higher quantities of these
volatiles. Hence, according to the different volatile profiles reported, virgin olive oils from Morisca
cultivar are characterized by significantly higher levels of green and fruity notes than Cornicabra
ones.
Although the volatile composition in each variety was also affected by the seasonal factor, there
were similar and clear changes in volatiles that can be attributed to the different irrigation
practices studied. Hence, in both cultivars, the volatile compounds most affected by the water
status of olive trees were mainly hexanal, E-2-hexenal and hexan-1-ol, showing an inverse
relationship with the water stress integral observed in the plants (Table 1), thus the volatile
composition of VOO from the SWP -2.0 treatment was clearly inferior to VOO obtained under the
other irrigation strategies which showed similar water status in many occasions, with the
exception of 2006/07 season, when water status of Cornicabra olive trees from SWP -2.0 and
TDF schedules were similar (S (S229-277): 199.8 (84.9) and 172.1 (81.5) MPa x day, respectively
for both irrigation treatments) and therefore the volatile profiles of these virgin olive oils were also
alike. Similar trends in C6 aldehydes and alcohols were reported by Gmez-Rico et al. (2006) and
Servili et al. (2007) in Cornicabra cv. and Leccino cv. under different irrigation strategies. Hence,
independently of other aspects, such as olive cultivar and growing season, VOO obtained from
olive trees under low water stress conditions are characterized by higher green sensory notes
than those from rainfed or stressful situations; being a helpful factor in olive cultivars usually
typified by a low content in volatiles from the LOX pathway, like the Cornicabra VOO.
The main C5 volatile compounds found in both varieties and generated by an additional branch of
the LOX pathway from the linolenic acid (Angerosa, Mostallino, Basti & Vito, 2000), were the 1-
penten-3-ol and 1-penten-3-one, which are also related with sweet, green and wet earth odour
notes in virgin olive oils. Similar values for 1-penten-3-ol and 1-penten-3-one were reported in
both cultivars, ranging between 0.11-0.35 mg/Kg and 0.10-0.44 mg/Kg, respectively, in
Cornicabra VOO along the successive growing seasons and between 0.14-0.30 mg/Kg and 0.17-
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0.72 mg/Kg, respectively, in Morisca VOO (Table 5). Their content was affected mainly by the
seasonal and ripening factors, whereas no clear significantly differences were attributed to the
irrigation practices.
Conclusions
According to the results obtained in this study, where similar irrigation strategies were used in two
different Spanish olive cultivars (Cornicabra cv. and Morisca cv.) during two crop seasons, it can
be concluded that the composition and quality of the olive drupe and of the virgin olive oils,
especially related to phenolic and volatile profiles, depend greatly not only on the water status of
the young olive trees and, moreover, of critical decreases in the water potential of the plant which
probably can be occurred during the phenological stage III, when begins the oil accumulation in
the fruit.
It was further observed that the irrigation schedule based on the stem water potential measure,
with a threshold around -1.2 MPa (SWP -1.2), provided VOO in both cultivars with a similar
phenolic and volatile composition with respect to those obtained with the FAO treatment, since the
water status measured in olive trees were alike. The use of this type of measurement can be
proposed, as an alternative of the ETc parameter, for the scheduling of irrigation treatments. On
the contrary, the treatment based on the daily trunk diameter fluctuations (TDF) provided VOO
with a minor composition similar to FAO or SWP -2.0 strategies, depending on the water status
generated in the olive trees in the successive growing seasons and conflicting results were
reported between Cornicabra and Morisca cultivars; thereby, volatile and phenolic profiles of TDF
Cornicabra VOO were similar to FAO in 2005 and alike to SWP -2.0 in 2006, since the water
stress generated in Cornicabra trees was quite different in both seasons (S229-277: 28.8 and 81.5
MPa x day, respectively). In the same way, Morisca VOO from TDF treatment had a minor
composition comparable to FAO or SWP -1.2 in 2006 and more similar to SWP -2.0 in 2007,
despite the water status reached in both periods was alike (S229-277: 38.9 and 48.5 MPa x day,
respectively), but during 2007, great punctual decreases in the water potential of the trees, similar
to SWP -2.0, were produced.
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983, 19-33.
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Effect of malaxation conditions on phenolic and volatile profiles of olive paste and
their corresponding virgin olive oils (Olea europaea L. cv. Cornicabra)
---------------------------------------------------------------------------------------------------------------------------------
ABSTRACT
Malaxation of the olive paste must be considered much more than a simple physical separation,
since a complex bioprocess takes place, which is very relevant to the final product quality and
composition. A combined study of the effect of the kneading temperature and time on the minor
composition of the olive paste and its corresponding virgin olive oil, processed in an experimental
oil mill (Pieralisi, Fattoria) with a working capacity of 200 kg/h, is reported. A great fall in the
oleuropein content in the olive paste with respect to the initial content in the olive fruit (between
91.5% and 96.7%) was observed, which supposed its almost total degradation during the
crushing operation. The major phenolic compound found in the olive paste during kneading was
the 3,4-DHPEA-EDA (between 64% and 76% of the initial total phenols content) which greatly
decreased during malaxation (from 7100 to 2990 mg/Kg). Moreover, while the oleuropein and the
secoiridoid derivatives of hydroxytyrosol values in olive paste decreased increasing the
temperature and the kneading time (e.g. close to 40 C and 90 minutes), an opposite trend was
observed in their corresponding VOO, with a significant increase in the secoiridoids of
hydroxytyrosol and tyrosol as the malaxation temperature raised. The content of C6 volatiles in
olive paste was great at temperatures close to 20 C, especially in the case of the aldehyde E-2-
hexenal (1.36 ppm of IS), while a fall in the C6 aldehydes in VOO as the malaxation temperature
increased was observed, especially in the E-2-hexenal content (about a 30%). In contrast, a
significant increase in C6 aldehydes of the oils from the oil mill plant as the malaxation time
increased from 30 to 90 minutes, chiefly the E-2-hexenal content (about a 70%) was observed.
KEYWORDS: Virgin olive oil; olive paste; phenols; volatiles; malaxation temperature; malaxation
time.
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INTRODUCTION
As well known the unique sensory profile of extra virgin olive oil is due to the presence of some
minor components, chiefly phenolic and volatile compounds (1, 2), which remain in this oil thanks
to the use of mechanical processes only for its extraction from the fruit of the olive tree (Olea
europaea L.). The fresh and green aroma sensory notes of high quality virgin olive oils are mainly
due to the presence of volatiles formed by the lipoxygenase (LOX) pathway from free
polyunsaturated fatty acids (3), whereas phenolic compounds affect both the taste, in particular
the positive bitterness and pungency organoleptic attributes, and the oxidative stability of the
virgin olive oil (4). Phenolics and volatiles are therefore the compounds largely responsible for
the flavor of extra virgin olive oils and to a large extent determine the degree of consumer
preference for this highly appreciated product.
The phenolic and volatile contents and profiles of virgin olive oils depend on both (I) the initial
composition characteristics of the olive fruits employed during processing and (II) the
technological factors used during the oil mill process, in particular milling and malaxation
conditions.
Several researches have pointed out the importance of the different virgin olive oil processing
stages in the minor composition and changes observed in the final product. Thus the use of
mechanical crushers led to a higher phenol extraction from the vegetable tissues than the
traditional stone mills (5) and therefore oils with higher antioxidant capacity are obtained (6).
Servili et al. (7) also reported changes in the volatile composition and pigment content of the oil in
terms of the type of mill employed.
Moreover, it is important to remark the great importance of the malaxation process of the olive
paste; indeed kneading must be considered much more than a simple physical separation, since
a complex bioprocess takes place, which is very relevant to the final product quality and
composition. Indeed malaxation conditions have proved to affect not only the oil yield but in
particular the composition and quality of the final virgin olive oil. For example higher kneading
temperature generally improves phenols content (8) and as a consequence, oxidative stability
and bitter taste improve (9), whereas volatile content usually decreases as the kneading
temperature increases (8). On the contrary kneading time apparently affects negatively the
phenols content, while volatile profile improves its presence in the oil (6, 10-13).
However, there have been only a few (14) research projects studying at the same time phenols
and volatiles affected by both malaxation temperature and time conditions. In fact, one of the
main novelty of this work is to combine these two issues in the study of the minor composition of
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the olive paste as directly related to the minor compounds of the virgin olive oil obtained in an
experimental oil mill.
The aim of this study was therefore to improve the scientific and technological knowledge on the
variables involved in the kneading operation that affects the minor components of the Cornicabra
virgin olive. The ultimate goal is to define the optimum kneading conditions in the Cornicabra
variety grown in Castilla-La Mancha to be applied by the industrial oil mills of our region. Indeed,
proper combined effect of temperature and time conditions during malaxation should be applied
to enhance in particular the sensory characteristics and therefore the consumers preference of
this product. To this end, a continuous experimental virgin olive oil mill plant (Pieralisi, Fattoria)
was employed in this study. The olive paste was kneaded at different temperatures (20-40 C)
and times (30-90 min) both in the pilot plant and in a laboratory scale equipment (Abencor),
obtaining a wide matrix of results that cover the range of the real malaxation conditions used by
the industry.
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Water and oil content. The water content of olive fruit was determined by desiccation according
to the UNE Spanish standard method. The fat content was determined by Soxhlet extraction and
was expressed as a percentage of dry olive paste weight (15).
Phenolic compounds. A sample of olive pulp, olive paste or olive pomace (4.0 g) was
homogenised with a mixture of methanol:water (80:20 v/v) (40 ml) during 2 min with an
Ultraturrax homogenizer (14000 rpm). The suspension obtained was shaken (20 min, 150 rpm,
<4 C in darkness), and then centrifuged (10 min, 5000 rpm and 4 C). The hydromethanolic
phase was recovered and filtered with a 0.45 m nylon syringe filter.
The phenolic fraction extracted was analysed by high-performance liquid chromatography
(HPLC) using an Agilent Technologies 1100 series system equipped with an automatic injector, a
column oven and a diode array UV detector. A ZORBAX SB-C18 column (250 x 4.6 id mm, 5 m
particle size) (Agilent Technologies, USA), maintained at 30 C, was used with an injection
volume of 20 l and a flow rate of 1.0 ml/min. Mobile phase was a mixture of water/acetic acid
(95:5 v/v) (solvent A), methanol (B) and acetonitrile (C): from 95% (A)-2.5% (B)- 2.5% (C) to 34%
(A)-33% (B)-33% (C) in 50 min. Chromatograms were recorded at 280 nm, 340 nm and 520 nm.
The hydroxytyrosol, oleuropein, demethyloleuropein from olive fruit and secoiridoids of
hydroxytyrosol and tyrosol from the olive paste and pomace were quantified at 280 nm, the
anthocyanins at 520 nm and the verbascoside and flavonoids at 340 nm. Phenolic compounds
quantification was achieved by a minimum of five point calibration curve on the basis of the
corresponding standard substances, with the exception of hydroxytyrosol which was quantified
as tyrosol, and demethyloleuropein, anthocyanins from olive fruit and secoiridoids of
hydroxytyrosol and tyrosol from the olive paste and pomace as oleuropein.
The identification of non-coloured phenolic compounds was carried out by comparison of their
retention times, UV-Visible characteristics and MS spectra with their standard substances. In the
case of the anthocyanins and demethyloleuropein, these were tentatively identified by their UV-
Visible characteristics and MS spectra. The mass detector used was a LCQ Deca XP Plus
(Thermo Electron Corporation, Waltham, MA, USA) equipped with an electrospray ionisation
system. Nitrogen was used as nebulizing gas at a flow rate of 14 units. The temperature and
voltage of the capillary were 250 C and 4.50 kV, respectively. Data were acquired in the
negative ionisation mode. Fragmentation experiments were performed using helium as the
collision gas with collision energy between 30-40%.
Volatile compounds adapted from Vichi et al. (16). Solid phase microextraction (SPME)
followed by GC-FID were used to analyze the volatiles compounds in the olive paste samples
studied. 1.5 g of olive paste was placed in a 10 ml vial fitted with silicone septum. The SPME
sampling was performed by exposing the DVB/Carboxen/PDMS fiber (50/30 m, 2 cm long from
Supelco Inc., Bellefonte, USA) for 30 min in the headspace of the sample maintained at 40 C
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and then retracted into the needle and immediately transferred and desorbed for 5 min in the
injection port of a gas chromatograph equipped with an FID. Compounds were resolved on a
Supelcowax-10 column (30 m x 0.25 mm x 0.25 m, Supelco Inc., Bellefonte, USA) under the
following conditions: injection port temperature 260 C; helium flow 0.8 ml/min; oven temperature
ramp: 35 C for 10 min, 3 C/min up to 160 C and then 15 C/min up to 200 C (maintained for 5
min). Volatile compounds were identified by comparison of retention times and mass spectra of
standard substances (Sigma Aldrich) added to refined olive oil. The equipment used was an
Agilent 5975C Series mass spectrometer (Agilent Technologies, USA) equipped with an electron
ionisation (EI+) detector and coupled to an Agilent 6850 Series gas chromatograph, the capillary
column used was a DB-Wax (30 m x 0.25 mm x 0.25 m, J&W Scientific, USA). Helium was
employed as carrier gas at a flow rate of 0.8 ml/min. The transfer line temperature was 280 C
and the temperature of the ionisation source and the quadrupole were 230 C and 150 C,
respectively, with an electromultiplier voltage of +941 eV. Their quantification was achieved by a
minimum of five point calibration curve on the basis of the corresponding standard substances.
Phenolic compounds. A solution of the internal standard (250 l of 15 mg/kg of syringic acid in
methanol) was added to a sample of virgin olive oil (2.5 g) and the solvent was evaporated with a
rotary evaporator at 35 C under vacuum. The oil was then dissolved in 6 ml of hexane and a
diol-bonded phase cartridge (Supelco Co., Bellefonte, USA) was used to extract the phenolic
fraction. The cartridge was conditioned with methanol (6 ml) and hexane (6 ml), the oil solution
was then applied, and the SPE column was washed with hexane (2 x 3 ml) and with hexane/ethyl
acetate (85:15, v/v; 4 ml). Finally, the phenols were eluted with methanol (15 ml) and the solvent
was removed with a rotary evaporator at 35 C under vacuum until dryness. The phenolic residue
was dissolved in methanol/water (1:1 v/v; 250 l) and analysed by HPLC.
Chromatographic conditions were the same as those used with the olive pulp and olive pomace
phenolic fractions. Phenolic compounds were quantified at 280 nm using syringic acid as internal
standard and the response factors determined by Mateos et al. (17).
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tube was centrifuged at 4000 rpm for 5 min to separate the solvent from the oil phase, and the
solvent extract was collected in another centrifuge tube. Each sample was extracted three times
in this way, and the solvent of the combined extract was removed with a N2 stream. The residue
of the extract was dissolved with 1 ml of methanol:water (1/1, v/v). Hexane (1 ml) was added to
the solution to wash away any remaining oil, this procedure was carried out three times. The tube
was centrifuged and the methanolic phase was recovered.
The fraction extracted was analysed by HPLC using an Agilent Technologies 1100 series system
equipped with an automatic injector, a column oven and a diode array UV detector. A ZORBAX
SB-C18 column (250 x 4.6 id mm, 5 m particle size) (Agilent Technologies, USA), maintained at
25 C, was used with an injection volume of 20 l and a flow rate of 1.0 ml/min. Mobile phase
was a mixture of acetonitrile (A) and water (B): from 25% (A)- 75% (B) for 35 min to 80% (A)-
20% (B) in 0.01 min during 10 min and returned to the initial conditions in 0.01 min. Oleocanthal
was quantified at 280 nm using 3,5-dimethoxyphenol as internal standard.
Volatile compounds. Solid phase microextraction (SPME) followed by GC-FID were used to
analyze the volatile compounds in the virgin olive oil samples studied. 1,5 g of olive oil was
placed in a 10 ml vial fitted with silicone septum. The SPME sampling and CG-FID conditions, as
well as identification and quantification of volatiles, were the same as those used with the olive
paste volatile fraction.
All experiments and analytical determinations were carried out at least in duplicate.
Statistical Analysis. Statistical analyses were performed using SPSS 15 statistical software
(SPSS Inc., Chicago, IL).
Furthermore, Table 1 also reports the phenolic composition of the olive paste obtained just after
the crushing stage; showing the greater fall detected in the oleuropein content in the olive paste
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with respect to the original content in the olive fruit (between 91.5% and 96.7%), which supposed
almost its total degradation during the crushing stage (t=0 in Tab. 1). This was probably due by
the activity of the -glucosidase enzyme, so it is accepted its activation during the crushing step
and moreover this activity is confirmed by the presence in the olive paste of the different
secoiridoid derivatives.
Table 1. Olive fruit and olive paste initial composition of the two Cornicabra cv. batches
employed.
BATCH I BATCH II
The values of the individual phenols of the olive paste from the different kneading conditions
studied in the oil mill plant are shown in Table 2 and 3.
As expected phenolic compounds found in the olive paste were a mixture of the phenols found in
the olive fruit of origin and in final virgin olive oil. Relating to secoiridoid compounds, it is
important to note the fairly decrease of the oleuropein during the malaxation process (from 790
mg/Kg to 270 mg/Kg, as average between batch I and II; Tab. 2 and 3), surely due by the action
of the -glucosidase, which degradation activity continued along the kneading of olive paste, a
similar trend was reported by Artajo et al. (23). The major phenolic compound found in the olive
paste during kneading phase was the dialdehydic form of elenolic acid linked to hydroxytyrosol
(3,4-DHPEA-EDA), which content was between 64% and 76% of the initial total phenols and
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greatly decreased during malaxation (from 7100 mg/Kg to 2990 mg/Kg, as average between
batch I and II; Tab. 2 and 3), probably due to the activity of oxidative enzymes, like the
polyphenoloxidase (PPO), peroxidase (POD) or lipoxygenase (LOX) (24). In contrast, the other
secoiridoid derivatives determined in the olive paste showed values much lower (among 5.4-
9.5% of the initial total phenols for the 3,4-DHPEA-EA, 5.9-13.8% for the p-HPEA-EDA and 0.4-
0.7% for the p-HPEA-EA) and did not clearly change along kneading stage, excepting the slightly
decrease of the 3,4-DHPEA-EA.
OLIVE PASTE
20 C 28 C 40C
P25 P50 P75 P25 P50 P75 P25 P50 P75
Phenolic
alcohol
c,w c,x c,y b,w b,x b,y a,w a,x a,y
I 212 1 233 2 248 3 181 1 189 3 201 3 159 2 172 8 182 3
3,4-DHPEA c,w c,x c,x a,w a,x b,y b,x b,x a,w
II 225 2 241 2 242 2 146 1 186 3 237 3 196 3 197 1 172 2
I nd nd nd nd nd nd nd nd nd
p-HPEA
II nd nd nd nd nd nd nd nd nd
Secoiridoids
c,y c,x b,w a,x b,w b,w b,x a,w a,w
I 584 9 408 9 372 9 418 4 383 7 371 6 468 9 316 5 323 2
Oleuropein c,y c,x b,w a,w a,w a,w b,x b,w b,w
II 421 8 393 6 375 7 312 8 293 8 288 9 386 4 315 7 307 3
I nd nd nd nd nd nd nd nd nd
Ligstroside
II nd nd nd nd nd nd nd nd nd
Secoiridoid Der.
b,y b,x b,w c,y c,x c,w a,y a,w a,x
I 55758 51807 45806 619314 531010 47105 44309 42567 43218
3,4-DHPEA-EDA b,y c,x c,w a,y a,x b,w a,x b,y a,w
II 53938 47928 39516 35685 34587 33397 35986 39666 30539
b,w b,x c,w a,x a,x a,w c,y a,w b,x
I 513 4 526 3 512 5 468 2 461 2 445 4 574 2 465 5 493 3
3,4-DHPEA-EA c,y c,x c,w a,w a,x b,y b,y b,x a,w
II 470 5 432 6 423 5 345 5 382 1 400 3 413 5 395 4 367 3
a,w a,x a,x b,w b,w b,w b,w c,x c,y
I 433 3 516 3 519 9 582 4 582 9 593 7 574 3 674 4 721 5
p-HPEA-EDA a,x a,x a,w a,w b,x b,x b,w c,x c,w
II 456 6 450 2 445 5 474 8 526 7 528 9 703 7 732 9 710 9
b,w b,x a,x c,x b,w a,w a,w a,x b,y
I 28 1 42 1 38 1 43 2 40 3 40 1 16 2 24 2 45 1
p-HPEA-EA b,x b,x a,w a,w a,w a,x c,x c,x a,w
II 44 1 39 1 30 1 26 2 29 2 33 2 50 2 45 2 31 1
b,w b,w a,w a,w a,w a,x a,w a,x a,x
I 123 3 126 2 125 3 112 3 118 7 123 5 115 3 121 2 127 3
Verbascoside a,w a,w a,w a,w a,w a,w a,w a,w a,w
II 55 2 57 3 58 2 56 2 58 4 56 3 55 4 57 1 58 2
Flavonoids
c,w c,w c,w b,w b,w b,w a,w a,w a,w
I 141 2 139 2 139 2 112 2 111 3 109 2 102 2 99 1 97 1
Rutin b,x c,x c,w a,w b,w b,w a,x a,w a,w
II 135 1 138 2 126 2 105 3 108 2 106 3 108 2 100 1 94 2
a,w a,w a,x b,w b,w b,x b,w b,w a,w
I 97 3 101 1 108 2 108 2 111 3 116 3 107 3 107 2 111 2
Luteolin-7-O-G a,w a,x a,x b,x b,w a,w c,w c,w b,w
II 89 1 95 2 96 1 102 2 100 2 98 1 116 9 111 2 114 3
a,w a,w a,w a,w a,w a,w a,w a,w a,w
I 56 1 58 2 54 2 55 4 56 3 57 3 57 2 53 1 50 4
Apigenin-7-O-G a,w a,w a,w a,x a,x a,w a,w a,w a,w
II 55 2 57 1 60 1 60 2 58 2 50 3 59 2 57 1 58 2
Different letters within a column (a-c) indicate significant differences (p<0.05) with respect to malaxation
temperature in olive paste.
Different letters within a column (w-y) indicate significant differences (p<0.05) with respect to olive paste at
different stages of malaxation process (P25, P50 and P75).
nd, not detected.
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OLIVE PASTE
30 min 60 min 90 min
P25 P50 P75 P25 P50 P75 P25 P50 P75
Phenolic
alcohol
b,w a,w a,x c,w b,x c,y a,w b,x b,x
I 165 1 163 2 176 2 181 1 189 3 201 3 156 2 186 3 181 2
3,4-DHPEA b,w c,w a,x a,w b,x a,y c,x a,w b,y
II 211 2 210 2 234 2 146 1 186 3 237 3 224 2 175 3 258 3
I nd nd nd nd nd nd nd nd nd
p-HPEA
II nd nd nd nd nd nd nd nd nd
Secoiridoids
b,y c,x c,w a,x a,w b,w a,x b,x a,w
I 11875 714 8 471 9 418 4 383 7 371 6 406 9 407 3 318 2
Oleuropein b,y c,x b,w a,w b,w b,w a,y a,x a,w
II 392 7 335 7 260 7 312 8 293 8 288 9 299 2 245 3 227 3
I nd nd nd nd nd nd nd nd nd
Ligstroside
II nd nd nd nd nd nd nd nd nd
Secoiridoid Der.
c,y c,x c,w b,y a,x b,w a,y b,x a,w
I 82016 78169 63798 619314 531010 47105 55968 54869 39058
3,4-DHPEA-EDA c,y c,x c,w b,y b,x b,w a,y a,x a,w
II 60108 47498 41879 35685 34587 33397 33795 26849 20835
c,w c,w c,x b,x b,x b,w a,x a,x a,w
I 488 5 487 3 503 4 468 2 461 2 445 4 442 4 448 2 355 4
3,4-DHPEA-EA b,x c,y c,w a,w b,x b,y c,y a,w a,x
II 442 3 472 7 424 2 345 5 382 1 400 3 457 2 310 2 363 5
a,w a,x a,y b,w b,w b,w c,x c,x c,w
I 476 5 501 9 560 5 582 4 582 9 593 7 678 7 660 9 637 9
p-HPEA-EDA b,w a,x a,y a,w a,x a,x b,w b,x b,x
II 498 6 515 7 524 2 474 8 526 7 528 9 523 9 581 4 589 9
b,y c,x c,w a,x a,w b,w a,w b,x a,w
I 66 1 50 2 46 1 43 2 40 3 40 1 40 1 44 1 37 2
p-HPEA-EA b,w c,w c,w a,w b,w b,x a,w a,w a,x
II 40 1 39 1 40 1 26 2 29 2 33 2 24 1 21 2 29 2
a,w a,w a,w a,w a,w a,x a,w a,w a,w
I 115 3 119 2 121 2 112 3 118 7 123 5 113 2 115 3 119 2
Verbascoside a,w a,w a,w a,w a,w a,w a,w a,w a,w
II 55 2 56 1 59 2 56 2 58 4 56 3 56 1 55 2 58 1
Flavonoids
b,y b,x c,w a,w a,w b,w a,x a,x a,w
I 150 2 141 2 127 2 112 2 111 3 109 2 108 2 109 2 101 2
Rutin b,x c,y a,w a,w b,w a,w a,x a,w a,x
II 111 2 120 2 106 2 105 3 108 2 106 3 108 2 103 2 108 2
b,w b,x a,x a,w a,w a,x b,w b,x a,x
I 115 2 118 2 118 1 108 2 111 3 116 3 112 2 115 2 116 2
Luteolin-7-O-G a,w b,y b,x b,x a,w a,w b,w a,w b,x
II 94 3 111 1 105 2 102 2 100 2 98 1 101 1 100 1 108 2
a,w a,w a,x a,w a,w a,w a,w a,w a,w
I 50 3 52 2 57 1 55 4 56 3 57 3 53 2 55 2 56 1
Apigenin-7-O-G a,x a,x a,w a,x a,x a,w a,w a,w a,w
II 59 1 57 3 51 2 60 2 58 2 50 3 55 3 54 2 552
Different letters within a column (a-c) indicate significant differences (p<0.05) with respect to malaxation
time in olive paste.
Different letters within a column (w-y) indicate significant differences (p<0.05) with respect to olive paste at
different stages of malaxation process.
nd, not detected.
Figure 1 clearly depicts these trends along the malaxation process, being the secoiridoids of
hydroxytyrosol, especially the 3,4-DHPEA-EDA, the phenolic compounds in olive paste most
affected by time during the kneading stage. On the contrary, statistical significant increases were
reported for the phenolic alcohol 3,4-DHPEA (tab. 2) along the malaxation process, due to the
hydrolysis of the secoiridoids derivatives, whereas the presence of p-HPEA was not detected in
the olive paste. It is important to remark the well-known hydrophilic character of these simple
phenols and their secoiridoids derivatives, which was clearly observed by the higher values found
in the wet pomace, specially for the 3,4-DHPEA-EDA (data not shown). Concerning the
concentration of verbascoside and flavonoids, like rutin, luteolin-7-O-glucoside and apigenin-7-O-
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glucoside, did not vary with time along the malaxation process and remained in the wet pomace
(data not shown), thus indicate the retention of this complex compounds in the solid phases, alike
results were described by Artajo et al. (23).
8000
Phenol Content (mg/Kg of oleuropein)
6000
4000
2000
1000
750
500
250
0
20 40 60 80 100
Olive Paste Kneading Time (min)
As for as the combined effect of kneading temperature and time on the phenolic composition of
olive paste, it could be seen that oleuropein and the secoiridoid derivatives of hydroxytyrosol
(3,4-DHPEA-EDA and 3,4-DHPEA-EA) values decreased at temperature close to 40 C and time
nearby 90 minutes, whereas p-HPEA-EDA content showed an opposite trend (Table 2 and 3);
these details confirm the considerable oxidative degradation, as chemical as enzymatic, of
compounds with o-diphenolic structure. Since, it is known that LOX acts catalysing the formation
of hydroperoxides and could be implied in the indirect oxidation of the o-diphenols (11), which are
characterised by a higher antioxidant capacity. On the other hand, PPO from the olive fruit
possesses a higher affinity by the o-diphenols than monophenols, which are practically not
affected by this enzyme (25), insomuch, it was active on the secoiridoids derivatives of
hydroxytyrosol, yielding the decrease of these compounds as the malaxation time and
temperature of the olive paste increased. In contrast, the verbascoside and flavonoids content
were not visibly affected by the processing temperature and time.
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Artculo V
2,5 2,5
2a 2b
2,0 2,0
Normalized values
Normalized values
1,5 1,5
1,0 1,0
0,5 0,5
0,0 0,0
20 30 40 30 60 90
Figure 2. Oil mill plant VOO phenolic profile as affected by the malaxation temperature (2a)
and time (2b).
, hydroxytyrosol; , secoiridoids of hydroxytyrosol; S, tyrosol; U, secoiridoids of tyrosol; {,
total phenols
Similar behaviours in the phenol content were observed in virgin olive oil samples obtained at
different malaxation temperatures with the laboratory scale Abencor system (see Figure 3a),
concerning to the significant increase of the secoiridoids derivatives with the temperature, mainly
3,4-DHPEA-EDA (300% as average between batch I and II) and p-HPEA-EDA (110%, as
average), however other works carried out in small laboratory set ups (10, 28) reported a
decrease in the phenol content of the oil as the kneading temperature of the olive paste
increased.
On the other hand, as the malaxation time increased, it was observed a tiny diminish of the
derivatives secoiridoids of hydroxytyrosol content of about a 5%, whereas the content of
secoiridoids of tyrosol increased between 15 and 20% in the oil mill plant (Figure 2b), these
trends were similar to those observed in the olive paste. Similar results were found by Di
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Artculo V
Giovacchino et al. (6), Angerosa et al. (10) and Lercker et al. (12). Moreover, these tendencies
were also observed in the oils from the Abencor system, although the decrease in the derivatives
of hydroxytyrosol and the increase in those from the tyrosol were somehow superior, 45% and
50%, respectively, as average between batch I and II (Figure 3b).
2,5 2,5
3a 3b
2,0 2,0
Normalized values
Normalized values
1,5 1,5
1,0 1,0
0,5 0,5
0,0 0,0
20 25 30 35 40 15 30 45 60 75
Kneading Temperature (C) Kneading Time (min)
Figure 3. Abencor VOO phenolic profile as affected by the malaxation temperature (3a)
and time (3b).
, hydroxytyrosol; , secoiridoids of hydroxytyrosol; S, tyrosol; U, secoiridoids of tyrosol; {,
total phenols
As well known, the phenolic compounds affect the sensory properties of the VOO, hence the
intensity of sensory pungency is mainly related to p-HPEA-EDA content (29), moreover, recently
have been discover the ibuprofen-like activity of the deacetoxy-ligstroside aglycone, re-called
oleocanthal (30); thus, it was of special interest to analyse the evolution of this bioactive
compound in VOO as affected by kneading conditions. Values about 3.0 times higher in VOO
from the oil mill plant by rising malaxation temperature from 20 C to 40 C and time from 30 min
to 90 min (Figure 4a and 4b) were observed, similar trends were noted in the oils obtained with
the Abencor system.
On the other hand, other secoiridoids, especially 3,4-DHPEA-EDA and 3,4-DHPEA-EA, are
associated to the sensory intensity of the bitterness attribute in the virgin olive oil (31), and
consequently high levels of these compounds may produce an excessive bitterness that can
imply a lower preference of the product. Therefore, the changes reported in the phenolic
composition in virgin olive oils depending on the kneading conditions employed, could be very
important from the nutritional and sensory point of view in biophenol-rich olive oil varieties, like
Cornicabra cultivar, since the control of these technological variables could provide an adequate
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Artculo V
level of phenolic substances in order to improve the nutritional value of the product and, at the
same time, the intensity of these positive attributes.
0 0
4a 4b
Figure 4. Changes in oleocanthal (mg/Kg of IS) in virgin olive oils from the oil mill plant
(4a) and Abencor system (4b), as affected by the malaxation temperature and time.
, cis-oleocanthal; U, trans-oleocanthal; z, total oleocanthal (cis+trans)
As expected, in the olive paste a slight decrease of the C6 aldehydes, mainly hexanal (related to
apple and green fruity attributes) and E-2-hexenal (responsible of almond and green sensory
notes), after crushing of the olive fruits and the first kneading stage was observed (see time 0
and P25), which is due to their transformation in their respective C6 alcohols, hexan-1-ol, Z-3-
hexen-1-ol and E-2-hexen-1-ol (related to fruity, green, grassy and sweet notes). On the other
hand, the content of these volatiles in the olive paste were not varied significantly along the
kneading process (time: see P25, P50 and P75), which could indicate a higher activity levels of
the LOX pathway enzymes only during the beginning of this stage. As regards C6 esters, such as
hexyl-acetate and Z-3-hexyl-acetate (with sweet, fruity and green leaves sensory notes), were
present in very small amounts in olive paste and consecutively in Cornicabra virgin olive oil,
indicating that there was also little activity of the enzyme alcohol acyl transferase (AAT), as
previously reported for this variety (32).
147
148
Artculo V
Table 4. Volatile LOX content in olive paste in relation to malaxation temperature (kneading time 60 min).
OLIVE PASTE
20 C 28 C 40C
t=0
P25 P50 P75 P25 P50 P75 P25 P50 P75
b,w b,w c,x a,w a,w b,x a,w a,w a,x
I 0.32 0.02 0.190.01 0.160.02 0.260.03 0.060.01 0.040.02 0.100.01 0.050.01 0.050.02 0.070.01
Hexanal b,x b,w b,w b,w b,w c,w a,w a,w a,w
II 0.22 0.01 0.160.02 0.130.01 0.100.02 0.140.01 0.120.01 0.140.01 0.020.01 0.010.01 0.020.01
b,w b,w b,w b,w c,w b,w a,w a,w a,w
I 0.35 0.01 0.440.02 0.430.01 0.460.01 0.460.01 0.470.02 0.460.01 0.400.02 0.400.01 0.390.01
Hexan-1-ol b,w b,w a,w a,w a,w a,w c,w b,w b,w
II 0.49 0.01 0.570.01 0.580.01 0.570.02 0.530.02 0.540.01 0.540.01 0.610.01 0.590.02 0.610.03
I <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Hexyl acetate
II <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
c,x c,w c,y b,y b,w b,x a,w a,w a,w
I 0.65 0.01 0.630.01 0.580.01 0.710.02 0.590.01 0.500.03 0.560.01 0.450.01 0.440.02 0.460.01
C6 from C18:2 c,x c,x b,w b,w b,w b,w a,w a,w a,w
II 0.71 0.01 0.720.02 0.700.01 0.660.01 0.670.01 0.660.01 0.670.02 0.630.01 0.590.03 0.630.01
OLIVE PASTE
30 min 60 min 90 min
t=0
P25 P50 P75 P25 P50 P75 P25 P50 P75
a,w a,w a,x a,w a,w a,x b,w b,w b,w
I 0.32 0.02 0.050.01 0.050.01 0.110.01 0.060.01 0.040.02 0.100.01 0.090.01 0.080.01 0.130.01
Hexanal a,w b,x a,x b,w a,w b,w a,w a,w a,x
II 0.22 0.01 0.110.01 0.130.01 0.140.01 0.140.01 0.120.01 0.140.01 0.120.01 0.110.01 0.150.01
b,w b,w b,w b,w b,w a,w a,w a,w a,w
I 0.35 0.01 0.490.02 0.480.01 0.480.01 0.460.01 0.470.02 0.460.01 0.440.02 0.440.01 0.450.01
Hexan-1-ol b,w b,w b,w a,w a,w a,w b,w a,w c,x
II 0.49 0.01 0.550.01 0.570.02 0.550.01 0.530.02 0.540.01 0.540.01 0.550.01 0.550.01 0.580.01
I <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Hexyl acetate
II <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
a,w a,w a,x b,y a,w a,x a,w a,w a,x
I 0.65 0.01 0.540.01 0.530.01 0.590.02 0.590.01 0.500.03 0.560.01 0.530.01 0.520.01 0.580.02
C6 from C18:2 a,w b,x b,x a,w a,w a,w a,x a,w b,x
II 0.71 0.01 0.670.01 0.700.01 0.690.01 0.670.01 0.660.01 0.670.02 0.670.01 0.640.01 0.700.01
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Artculo V
As for the effect of malaxation temperature on the C6 volatile compounds of olive paste, their
concentration was great at temperatures close to 20 C, especially in the case of the aldehydes
E-2-hexenal (1.36 ppm of IS, as average between batch I and II) and hexanal (0.18 ppm of IS, as
average between batch I and II), so low temperatures permitted a higher activity of enzymes from
LOX pathway, especially for the hydroperoxide lyase (HPL) which have the major activity at 15
C and decrease largely at temperatures up 35 C (33), while almost any difference was
observed in C6 alcohols (Table 4). On the other hand there were not found practically significant
differences in the C6 volatiles content in olive paste with respect to malaxation time (between 30
to 90 minutes, at 28 C, Table 5), except for the slightly increase in E-2-hexenal content.
1,75 1,75
1,50
5a 5b
1,50
Normalized values
Normalized values
1,25 1,25
1,00 1,00
0,75 0,75
0,50 0,50
0,25 0,25
20 30 40 30 60 90
Kneading Temperature (C) Kneading Time (min)
Figure 5. Oil mill plant VOO volatile profile as affected by the malaxation temperature (5a)
and time (5b).
, C6 aldehydes; , C6 alcohols; {, LOX volatiles (C6 volatiles + C5 volatiles)
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Artculo V
In contrast, it was monitored a significant increase in C6 aldehydes of the oils as the malaxation
time increased, chiefly the E-2-hexenal content which rose about a 70% between 30 and 90
minutes in the oil mill plant, as average between batch I and II, these findings agree with
Angerosa et al. (10) and Ranalli et al. (11), while no clear trend in the content of C6 alcohols was
observed (Figure 5b).
Similar trend in the volatile content in virgin olive oil samples obtained at different malaxation
temperatures and times with the laboratory scale Abencor system were reported (Figure 6a and
6b), specially concerning the E-2-hexenal and hexanal variations, which decreased about 48%
and 32% respectively, as average between batch I and II, as the kneading temperature rose from
20 C up to 40 C.
1,75 1,75
1,50
6a 6b
1,50
Normalized values
1,25
Normalized values
1,25
1,00 1,00
0,75 0,75
0,50 0,50
0,25 0,25
20 25 30 35 40 15 30 45 60 75
Kneading Temperature (C) Kneading Time (min)
Figure 6. Abencor VOO volatile profile as affected by the malaxation temperature (6a) and
time (6b).
, C6 aldehydes; , C6 alcohols; {, LOX volatiles (C6 volatiles + C5 volatiles)
The selection of optimal kneading conditions for the Cornicabra cultivar forces to establish a
suitable compromise between the industrial yield obtained and the sensory quality of the virgin
olive oil, that depends mainly on the phenolic and volatile profiles. Hence, in terms of the results
attained in this assay, the best malaxation conditions may be a temperature below 28 C and a
time upper than 60 minutes, since the phenolic content of the virgin olive oil obtained would
improve the sensory properties of this variety through a desirable decrease of bitterness, and
nonetheless the logical diminution in the oxidative stability does not affect the Cornicabra virgin
olive oil shelf-life, as this is a very stable and phenol rich olive oil variety. Moreover, the volatile
profile from the LOX pathway is favoured by these kneading conditions and the green odour
notes of the appreciated product would increase.
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compounds in Olea europaea L. Scientia Horticulturae, 2002, 92, 147-176.
(23) Artajo, L.S.; Romero, M.P.; Suarez, M.; Motilva, M.J. Partition of phenolic compounds during the virgin
olive oil industrial extraction process. Eur. Food Research Technol., 2006, 225, 617-625.
(24) Servili, M.; Baldioli, M.; Begliomini, A.; Selvaggini, R.; Montedoro, G.F. The phenolic and volatile
compounds of virgin olive oil: relationship with the endogenous oxidoreductases during the mechanical
oil extraction process. In Flavour and Fragrance Chemistry; Proceedings of the Phytochemical Society
of Europe, Campobasso, Italy, January 13-16 2000; Kluwer Academic: Dordrecht, The Netherlands,
2000; pp 163-173.
(25) Toscano, G.; Colarieti, M.L.; Greco, G. Oxidative polymerisation of phenols by a phenol oxidase from
green olives. Enzyme Microbial Technol., 2003, 33, 47-54.
(26) Rodis P.S.; Karathanos, V.T.; Mantzavinou, A. Partitioning of olive oil antioxidants between oil and
water phases. J. Agric. Food Chem., 2002, 50, 596-601.
(27) Di Giovacchino, L. Lestrazione dellolio con la centrifugazione diretta delle paste di oliva. Nota I:
influenza della gramolazione. Riv. Ital. Sostanze Grasse, 1991, 68, 314-420.
(28) Servili, M.; Baldioli, G.; Montedoro, G. Phenolic composition of virgin olive oil in relationship to some
chemical and physical aspects of malaxation. Acta Horticulturae, 1994, 356, 331-336.
(29) Andrewes, P.; Busch, J.; De Joode, T.; Groenewegen, A.; Alexandre, H. Sensory properties of virgin
olive oil polyphenols: Identification of deacetoxy-ligstroside aglycone as a key contributor to pungency.
J. Agric. Food Chem., 2003, 51, 1415-1420.
(30) Beauchamp, G. K.; Keast, R. S. J.; Morel, D.; Lin, J.; Pika, J.; Han, Q.; Lee, C.-H.; Smith, A. B.;
Breslin, P. A. S. Ibuprofen-like activity in extra virgin olive oil: Enzymes in an inflammation pathway are
inhibited by oleocanthal, a component of olive oil. Nature, 2005, 437, 45-46.
(31) Gutirrez-Rosales, F.; Rios, J.J.; Gomez-Rey, M.L. Main polyphenols in the bitter taste of virgin olive
oil. Structural confirmation by on-line high-performance liquid chromatography electrospray ionization
mass spectrometry. J. Agric. Food Chem., 2003, 51, 6021-6025.
(32) Gmez-Rico, A.; Salvador, M.D.; La Greca, M.; Fregapane, G. Phenolic and volatile compounds of
extra virgin olive oil (Olea europaea L. Cv. Cornicabra) with regard to fruit ripening and irrigation
management. J. Agric. Food Chem., 2006, 54, 7130-7136.
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(33) Salas, J.J.; Sanchez, J. The decrease of virgin olive oil flavour produced by high malaxation
temperature is due to inactivation of hydroperoxide lyase. J. Agric. Food Chem., 1999, 47, 809-812.
(34) Angerosa, F.; dAlessandro, N.; Basti, C.; Vito, R. Biogeneration of volatile compounds in virgin olive
oil: Their evolution in relation to malaxation time. J. Agric. Food Chem., 1998, 46, 2940-2944.
154
4.2. Discusin General
Una de las mayores novedades de este estudio ha sido poder estudiar simultneamente tanto
la composicin minoritaria de los frutos, as como la de sus correspondientes aceites de oliva
virgen, con el fin de determinar la presencia de posibles marcadores qumicos varietales.
Por una parte, el anlisis del perfil fenlico de las aceitunas ha permitido la excelente
clasificacin de las mismas segn la variedad de procedencia (100% de los casos agrupados
correctamente tras la aplicacin del Anlisis Discriminante). Alguno de los aspectos de especial
inters ha sido la composicin oleosdica de los frutos, que adems de ser la ms abundante
en las drupas, ha resultado ser tambin la que ha presentado mayor variabilidad entre las
distintas variedades en estudio, destacando el hecho de que la demetiloleuropena solo fue
detectada en los frutos de la variedad Arbequina y aumentando su concentracin
significativamente a lo largo del proceso de maduracin. Este aspecto permite por un lado la
confirmacin de que este compuesto fenlico es un producto de degradacin de la oleuropena
(Amiot et al., 1989; Servili et al., 1999) y por otro lado, su carcter de marcador varietal, ya que
investigadores como Amiot et al. (1989) y Esti et al. (1998) slo detectaron demetiloleuropena
en dos de once variedades francesas (Cailletier cv. y L11 cv.) y en dos de ocho variedades
italianas (Coratina cv. y Leccino cv.). Sobre el contenido de verbascsido de los frutos y su
relacin inversa con la concentracin de oleuropena, tendencia ya observada por varios
autores (Amiot et al., 1989; Servili et al., 1999 y Vinha et al., 2005), podra corroborar la
existencia de una ruta metablica entre estos dos compuestos fenlicos, ya sugerida por Amiot
et al. (1989) y basada en la degradacin parcial de la molcula de oleuropena para originar el
verbascsido, ya que ste ltimo no es detectado en frutos muy verdes.
155
4.2. Discusin General
Por otra parte, la presencia mayoritaria de los derivados secoiridoideos del hidroxitirosol y
tirosol en todas las muestras de AOV estudiadas, pone de manifiesto la importancia de la ruta
de degradacin de los olesidos del fruto en la gnesis de los compuestos fenlicos del aceite,
la cual se inicia por la actividad de la enzima -glucosidasa tras la rotura de los tejidos de la
drupa. Otro hecho a destacar es la relacin tan heterognea observada entre el contenido de
olesidos de la drupa (oleuropena ms demetiloleuropena, este ltimo encontrado slo en
Arbequina) y los compuestos secoiridoideos del AOV en cada una de las variedades espaolas
estudiadas, con ratios desde 2,3 para el caso de Picudo, de 4,5 a 5,5 para Cornicabra y
Morisca y de 7,0 a 8,5 para Arbequina y Picual y como valor mximo observado 28 para la
variedad Picolimn, todos ellos calculados como valores medios entre los frutos maduros e
inmaduros. Todos estos datos avalan que probablemente el contenido de derivados
secoiridoideos de un AOV depende principalmente de su contenido enzimtico responsable de
la degradacin de los olesidos, el cual presenta un marcado carcter varietal.
De la misma forma, el nivel de actividad de las enzimas de la ruta de la lipoxigenasa (LOX), que
se encuentra estrechamente asociado a factores genticos (Angerosa et al., 1999), ha
determinado considerablemente la biognesis de los compuestos voltiles C6 y C5 presentes
en los AOV monovarietales del ensayo realizado. De esta forma se obtienen perfiles aromticos
totalmente distintos desde un punto de vista cuantitativo, lo que indica que los distintos aceites
se caracterizan por intensidades de notas verdes y frutadas muy distintas entre si;
destacando el hecho de que el contenido de E-2-hexenal -asociado a notas aromticas verde,
manzana y almendra amarga- ha permitido la diferenciacin de los distintos AOV en funcin de
la variedad de procedencia, lo que corrobora los resultados ya descritos por Aparicio & Luna
(2002) que sugirieron la diferenciacin de aceites monovarietales en funcin de la
concentracin de este voltil. Es importante resaltar tambin el nivel de actividad tan
heterogneo observado en la enzima alcohol acil transferasa (AAT) en las distintas variedades
estudiadas, ya que la presencia de steres C6 -acetato de Z-3-hexenilo y acetato de hexilo,
responsables de las sensaciones olfativas verde, frutado, floral y dulce- slo ha sido detectada
apreciablemente en las variedades Morisca, y especialmente, en Arbequina y Picual.
Se debe resaltar que a pesar de que la composicin voltil y fenlica de un aceite de oliva
virgen est esencialmente determinada por factores genticos propios de la variedad de
procedencia, el perfil de estos compuestos minoritarios tambin puede modularse en funcin de
la actividad de las enzimas presentes en el fruto a travs de una serie de parmetros externos,
156
4.2. Discusin General
como son el grado de maduracin y la disponibilidad de agua durante el desarrollo del fruto, y
las condiciones utilizadas en el proceso de extraccin de su aceite de oliva virgen, entre otros.
Tal y como se ha podido apreciar en las publicaciones cientficas presentadas, el resto de los
ensayos realizados en el trabajo experimental de esta Tesis Doctoral han estado
principalmente orientados hacia el estudio de la influencia de estas variables sobre la
composicin minoritaria de este producto.
Asimismo es bien conocido el hecho de que las caractersticas organolpticas tan apreciadas
en el aceite de oliva virgen se deben fundamentalmente al contenido fenlico y voltil del
mismo, por lo que la finalidad ltima de esta investigacin ha sido tambin poder definir y
seleccionar las variables que ms influyen sobre ellos con el propsito de obtener perfiles
fenlicos y voltiles deseables, con el consiguiente incremento de la calidad sensorial del
producto y el aumento, por tanto, de la preferencia de este aceite por parte de los
consumidores respecto del resto de los aceites vegetales presentes en el mercado.
Antes de continuar con la discusin de los resultados, destacar que la razn que ha llevado a
que estos ensayos se hayan realizado fundamentalmente con frutos Cornicabra, se debe a que
el cultivo de esta variedad, localizado mayoritariamente en las provincias de Toledo y Ciudad
Real, supone el 15% de la superficie nacional dedicada al olivo, y representa una aportacin a
la produccin nacional de aceite de oliva del 7% (MAPA, 2001), lo que ofrece una idea de la
gran importancia social y econmica de este cultivo en la regin de Castilla-La Mancha.
Tambin es importante destacar que el aceite de oliva virgen Cornicabra se caracteriza por una
elevada estabilidad oxidativa y un intenso amargor, ambos como consecuencia de su gran
contenido fenlico natural. Este hecho implica que aunque el atributo amargo est valorado
positivamente en este producto, intensidades excesivas del mismo llegan a producir el rechazo
de los consumidores. Por todo ello resulta ser de gran importancia en este aceite monovarietal
la investigacin de las variables que favorezcan la obtencin de aceites de oliva virgen
Cornicabra de gran calidad, pero que permitan adems modular su perfil fenlico segn las
preferencias de los consumidores, lo que supondr el aumento de su competitividad en el
mercado actual.
157
4.2. Discusin General
Con los ensayos efectuados se ha confirmado como la maduracin del fruto implica un
considerable descenso del contenido en derivados secoiridoideos del hidroxitirosol y tirosol
presentes en el AOV obtenido, debido seguramente a la disminucin observada en la cantidad
de precursores fenlicos presentes en la aceituna, como es el caso de la oleuropena, que ha
sido observada en la mayora de los variedades bajo estudio (ART. I; Tabla 1), aspecto que
tambin ha sido descrito por varios autores (Amiot et al., 1986; Ryan et al., 1999; Brenes et al.,
1999; Uceda et al., 1999; Morell et al., 2004). Es importante destacar que en variedades como
Picual y Picolimn se ha observado un incremento del contenido en oleuropena en los frutos
en estado de envero para posteriormente disminuir significativamente en los frutos sobre-
maduros, comportamiento que puede ser debido a la transformacin de sustancias fenlicas en
nuevos productos conjugados, mientras que el posterior descenso se debera al considerable
proceso de degradacin que sufren estos compuestos (Ryan et al., 2002).
158
4.2. Discusin General
Por otro lado, la evolucin de los voltiles C6 y C5 presentes en las muestras de AOV
Cornicabra obtenidas con diferente grado de madurez de los frutos: desde I.M = 1,5-2,0 hasta
I.M = 5,0-5,5, ha puesto de manifiesto un descenso constante y significativo en el contenido de
aldehdos C6 y voltiles C5, como 1-penten-3-ol y 1-penten-3-ona. Por el contrario el contenido
de alcoholes C6, como Z-3-hexen-1-ol y hexan-1-ol, ha aumentado a lo largo del proceso de
maduracin del fruto (ART. III; Figura 2 y Tabla 2), y aunque algunos autores no han
observado estos incrementos en otras variedades de aceituna, como Picual, Arbequina o
Koroneiki (Morales et al., 1996; Aparicio et al., 1998), Benincasa et al. (2003) s detectaron
aumentos similares en la variedad Coratina, lo cual vuelve a poner de manifiesto la importante
dependencia de carcter varietal de la actividad de las enzimas de la ruta LOX.
Por ltimo indicar que uno de los objetivos previstos de este estudio era establecer el estado de
maduracin recomendable para el procesamiento de los frutos Cornicabra con el fin de obtener
un aceite de oliva virgen de mxima calidad. Se ha determinado una influencia mnima del
grado de madurez de los frutos sobre los ndices de calidad normalizados que se han estudiado
(grado de acidez, ndice de perxidos y absorcin en el ultravioleta), con la excepcin de un
ligero descenso en la intensidad de los atributos sensoriales frutado y amargo tras realizar el
anlisis sensorial de las muestras, clasificndose todas ellas dentro de la categora comercial
virgen extra. A la vista de estos resultados se ha considerado conveniente establecer el ndice
de madurez ptimo en esta variedad en base al patrn de evolucin de su perfil de compuestos
fenlicos y voltiles, sugirindose por lo tanto un estado de maduracin ptimo para valores de
I.M entre 3,0 y 4,0. De hecho, a ndices de madurez ms elevados superiores a 4,5-5,0 el
contenido de los voltiles mayoritarios de la ruta LOX (la fraccin aldehdica C6) presentes en
el aceite es demasiado bajo y por tanto las notas aromticas verdes tan apreciadas en este
producto pueden ser poco perceptibles, lo cual reduce su grado de preferencia por parte del
consumidor. Adems, la obtencin de aceites de oliva virgen a partir de frutos sobre-maduros
supone una disminucin, posiblemente excesiva, de los fenoles complejos que no resultara
beneficioso para este producto, ya que aunque en principio un descenso en la intensidad del
sabor amargo resultara deseable en este aceite monovarietal, tambin implicara un importante
descenso de su estabilidad oxidativa y su valor nutricional.
159
4.2. Discusin General
Dentro del estudio de la composicin minoritaria del aceite de oliva virgen llevado a cabo en
esta Tesis se ha evaluado la influencia que la actual tendencia de aplicacin de riego en el
olivar posee sobre el perfil minoritario de los aceites obtenidos en estas condiciones. Por ello se
han estudiado distintas estrategias de riego con el objetivo general de determinar el efecto que
la disponibilidad de agua en la plantacin tiene sobre su productividad y, muy especialmente,
sobre la composicin y calidad del aceite resultante.
As, el ensayo realizado en un olivar tradicional adulto Cornicabra efectuado en las campaas
2003/04 y 2004/05 ha puesto de manifiesto un aumento significativo de la productividad de los
olivos bajo condiciones de riego respecto a los de secano (alrededor de un 35%), y aunque el
contenido graso de las aceitunas no se ha visto afectado por las tcnicas de irrigacin, el
obtener una mayor produccin frutcola implica claramente un incremento de la produccin
global de aceite por hectrea de olivar, con resultados similares a los descritos por Pastor et al.
(1999) en un olivar tradicional Picual y por Patumi et al. (1999) en varios olivares intensivos de
diversas variedades italianas. Por tanto la aplicacin de riego, incluso en zonas donde las
fuentes de agua son limitadas, se convierte en una tcnica agronmica aconsejable desde un
punto de vista social y econmico.
160
4.2. Discusin General
Los perfiles voltiles y fenlicos del aceite de oliva virgen dependen tanto del estado
hdrico registrado en el olivo, como de la programacin de riego utilizada; as una
estrategia de riego deficitario regulado, centrada durante la etapa fenolgica III del
desarrollo del fruto, permite la obtencin de aceites con una composicin fenlica y
voltil comparable a los obtenidos con la metodologa de riego FAO
La metodologa ms utilizada para determinar los requerimientos hdricos de los olivos ha sido
propuesta por la Organizacin de Alimentacin y Agricultura (FAO) y est basada en el
coeficiente de evapotranspiracin del cultivo (ETc), desarrollada por Doorenbos y Pruitt en
1974. Este mtodo implica la aplicacin del 100% de las prdidas hdricas del olivo (100% ETc)
y ha sido ampliamente recomendada para el riego de los olivares, aunque hay que destacar las
grandes dosis de agua que en ocasiones se requieren. Es importante destacar que en muchas
zonas olivareras de Espaa existe un lmite mximo legal de riego de 100 mm, por lo que la
aplicacin de esta metodologa no siempre resulta factible. Por todo ello se han desarrollado las
denominadas estrategias de riego deficitario regulado (regulated deficit irrigation, RDI), que
tienen como finalidad hacer un uso ms adecuado del agua mediante la utilizacin de dosis de
riego inferiores, bien durante toda la estacin de irrigacin o de acuerdo al estado fenolgico
del cultivo (Moriana et al., 2003 y 2007; Tognetti et al., 2005).
Por esta razn se evaluaron dos estrategias de RDI diferentes en un olivar tradicional adulto
Cornicabra, que inicialmente se encontraba en condiciones de secano. La primera estrategia
denominada RDI-1 consista en la aplicacin de las dosis de agua durante toda la estacin de
riego y la segunda, denominada RDI-2, slo desde principios de agosto, momento en el que
comienza la acumulacin de aceite en el fruto (estado fenolgico III), con el fin de determinar
cual de ellas permita obtener aceites de oliva virgen de una calidad y composicin
comparables a la metodologa FAO o a la estrategia de irrigacin excesiva 125 FAO, con dosis
de riego equivalentes a 125% del valor del parmetro ETc.
161
4.2. Discusin General
Con respecto a la composicin en cidos grasos, los resultados obtenidos han mostrado que
los aceites obtenidos a partir de olivos en condiciones de secano presentan un contenido
ligeramente superior en el cido graso C18:1, mientras que por el contrario los aceites de las
tcnicas de irrigacin se caracterizan por niveles mayores de C16:0 y C18:2, aunque hay que
destacar que estos cambios son muy leves respecto a la composicin acdica global del aceite,
por lo que no presentan ninguna relevancia nutricional. Salas et al. (1997) tambin detectaron
ligeros cambios en la composicin en cidos grasos de aceites Picual atribuibles a la aplicacin
de riego en el olivar.
Hay que destacar de forma explcita que han sido los derivados secoiridoideos del hidroxitirosol
presentes en el AOV los fenoles complejos ms afectados por el estado hdrico del olivo,
disminuyendo de forma ms acusada que los secoiridoideos del tirosol. Este aspecto resulta de
gran inters, ya que es bien conocida la elevada capacidad antioxidante e influencia sobre la
percepcin del atributo sensorial amargo del AOV que poseen el hidroxitirosol y sus
secoiridoideos, frente al tirosol y sus derivados, que han demostrado una escasa actividad
antioxidante y una mayor influencia sobre el atributo sensorial picante (Baldioli et al., 1996;
Mateos, 2002; Andrewes et al., 2003; Carrasco-Pancorbo et al., 2005). Este hecho justifica las
diferencias registradas tanto en las caractersticas organolpticas como en la estabilidad
oxidativa Rancimat de los aceites de oliva virgen del ensayo, ambas significativamente
superiores en las muestras procedentes del cultivo en condiciones de secano.
162
4.2. Discusin General
Por otro lado, es importante destacar que hasta el momento de la realizacin de este ensayo
no existan datos en la bibliografa del efecto del riego sobre la composicin voltil del aceite de
oliva virgen, siendo ste punto de especial inters por su influencia en las caractersticas
organolpticas del producto y una de las principales novedades de este estudio. De manera
que el anlisis de estos componentes minoritarios ha permitido establecer la existencia de una
relacin directa entre el estado hdrico del olivo y la cantidad de aldehdos C6 (especialmente
E-2-hexenal) y alcoholes C6 (como Z-3-hexen-1-ol y hexan-1-ol) presentes en el AOV; siendo
su contenido significativamente superior en los aceites Cornicabra obtenidos a partir de frutos
que no han sufrido condiciones de estrs hdrico apreciables, lo que indica probablemente que
la actividad de las enzimas de la ruta LOX, especialmente las enzimas hidroperxido liasa
(HPL) y alcohol deshidrogenasa (ADH), tambin resulta afectada por disponibilidad de agua
durante el desarrollo de las aceitunas.
Por tanto, se ha puesto de manifiesto que las tcnicas de irrigacin permiten acentuar las notas
sensoriales verdes y frutadas del aceite de oliva virgen, disminuyendo simultneamente la
intensidad de atributo sensorial amargo, siendo por tanto, una prctica agronmica
aconsejable en AOV caracterizados bien por un bajo contenido en voltiles C6 o por una
elevada concentracin fenlica, como es el caso de la variedad Cornicabra.
Asimismo, este estudio sobre la influencia del riego en el olivar sobre la calidad y composicin
del aceite de oliva virgen ha demostrado como los perfiles voltiles y fenlicos de este producto
se encuentran afectados no slo por el estado hdrico estacional registrado en el olivo, sino en
particular por la programacin de riego utilizada; as la utilizacin de la estrategia RDI-2, donde
las dosis de irrigacin comenzaron a principios de agosto (etapa fenolgica III del desarrollo del
fruto), ha dado lugar a una composicin fenlica y voltil en los aceites comparable a los
obtenidos con las estrategias de riego FAO y 125 FAO, pero con una considerable reduccin
de la cantidad de agua suministrada en el olivar, siendo ste uno de los objetivos marcados
inicialmente en el estudio. Este aspecto resulta de gran inters en los olivares de Castilla-La
Mancha, regin que como se ha comentado previamente presenta unos recursos hdricos
escasos. A la vista de los resultados descritos en este ensayo, la tcnica de irrigacin ms
apropiada para los olivos tradicionales de esta regin es un riego deficitario regulado, estrategia
que permite un uso ms racional y adecuado del agua disponible, adems aparentemente
basta con centrar la aplicacin de riego durante la etapa fenolgica III del desarrollo del fruto
para permitir la recuperacin del estado hdrico del olivo y conseguir situaciones similares a las
obtenidas con la metodologa recomendada por la FAO.
163
4.2. Discusin General
Una programacin de riego basada en la medida del potencial hdrico del tronco, con
un umbral alrededor de -1,2 MPa, ha dado lugar a aceites de oliva virgen monovarietales
con una calidad y composicin minoritaria similar a la metodologa FAO, por lo que
resulta ser una alternativa factible al clculo del parmetro ETc para planificar el riego en
un olivar joven de cubierta incompleta
A diferencia del ensayo de riego llevado a cabo en el olivar adulto Cornicabra, la ejecucin del
segundo ensayo de irrigacin presentado en esta memoria se ha basado en el hecho de que
varios autores (Fereres et al., 2003; Naor, 2003) han descrito recientemente que la estimacin
matemtica del parmetro ETc, en el que se fundamenta la metodologa FAO, se encuentra
sujeta a numerosas incertidumbres relacionadas con cuestiones meteorolgicas y agronmicas
como el nivel de carga del olivo, edad del cultivo, estado vegetativo del rbol, etc., por lo que el
uso de programaciones de irrigacin basadas en medidas del estado hdrico del olivo in situ
parece ser una opcin ms apropiada a este coeficiente agronmico para determinar las dosis
de agua a aplicar en el olivar. Dos de los ndices que permiten conocer el estado hdrico de la
planta, y que han sido utilizados recientemente en olivares, son el potencial hdrico del tronco
(stem water potential, SWP) y las fluctuaciones del dimetro del tronco (trunk diameter
fluctuations, TDF) (Moriana et al., 2002; Gucci et al., 2007).
Comentar en primer lugar que los resultados experimentales obtenidos en este ensayo de
irrigacin han confirmado algunos aspectos observados en las pruebas con el olivar tradicional
adulto, como es la ausencia de prcticamente cualquier relacin significativa entre el estado
hdrico del olivo y los valores de los ndices de calidad normalizados evaluados en el aceite de
oliva virgen obtenido (grado de acidez, ndice de perxidos y caractersticas de absorcin en el
UV) o con el contenido en tocoferoles del mismo, as como los pequeos cambios producidos
por la aplicacin de riego en el olivar sobre la composicin de cidos grasos global del
producto.
164
4.2. Discusin General
Adems, resulta interesante destacar una estrecha relacin entre el contenido en oleuropena
de las drupas, y por consiguiente en los niveles de derivados secoiridoideos presentes en los
aceites del ensayo, con los valores de potencial hdrico mnimo registrados en cada
programacin de riego durante la etapa fenolgica III del desarrollo del fruto. De tal forma que
posiblemente la actividad de las enzimas responsables de la biosntesis de precursores
fenlicos en la aceituna, como la enzima PAL, est determinada no slo por el estado hdrico
estacional de los olivos, sino tambin por posibles descensos significativos en el potencial
hdrico del rbol desde que comienza la acumulacin de aceite en el fruto (etapa fenolgica III).
A la vista de los resultados presentados en los ensayos de irrigacin realizados en los olivares,
e independientemente de factores como la variedad y el grado de maduracin de la
aceituna, se puede decir que el perfil fenlico y voltil de un aceite de oliva virgen depende
considerablemente de la disponibilidad de agua durante el desarrollo del fruto, siendo
especialmente decisivo para la actividad de las enzimas responsables de la biognesis de
compuestos fenlicos y voltiles el estado hdrico del olivo durante la etapa fenolgica III, que
abarca tanto la acumulacin de aceite en el fruto como el proceso de maduracin del mismo.
Por otro lado, y de acuerdo al propsito general planteado en este ensayo de irrigacin, se
puede proponer la programacin de riego basada en la medida del potencial hdrico del tronco,
con un umbral de -1,2 MPa (SWP -1,2), como una alternativa factible al clculo del parmetro
ETc para realizar la planificacin de las estrategias de irrigacin en un olivar joven de cubierta
incompleta, puesto que el estado hdrico registrado en los olivos FAO y SWP -1,2 ha sido
semejante y por tanto, los AOV monovarietales obtenidos mediante ambas tcnicas ofrecieron
una calidad y composicin minoritaria similar. Por el contrario, la utilizacin de la estrategia de
irrigacin basada en las fluctuaciones del dimetro del tronco (TDF), ha generado niveles
hdricos considerablemente variables entre las campaas sucesivas estudiadas, presentando
en ocasiones valores similares al tratamiento FAO o a la estrategia moderadamente estresada
SWP -2,0, por lo que su utilizacin a priori en el desarrollo de planificaciones de riego parece
ser algo imprecisa y por tanto su uso como posible opcin al clculo del parmetro ETc debera
continuar bajo estudio.
165
4.2. Discusin General
Otro aspecto estudiado en esta Tesis ha sido el efecto que diversas variables durante la etapa
tecnolgica de batido ejercen sobre la composicin minoritaria del aceite de oliva virgen
Cornicabra.
El tiempo y temperatura de batido son las principales variables a controlar durante esta etapa,
de forma que es posible variar potencialmente la composicin voltil y fenlica del aceite de
oliva virgen obtenido y, por consiguiente, sus caractersticas sensoriales. De hecho, una de las
principales innovaciones de este estudio es la de combinar estas dos variables en el anlisis
166
4.2. Discusin General
del perfil fenlico y voltil de la pasta de aceituna y los correspondientes aceites de oliva virgen
obtenidos en una Almazara Experimental, con una capacidad de trabajo de 200 Kg/h. El
principal propsito de este trabajo se ha dirigido, no slo a ampliar el conocimiento tecnolgico
y cientfico existente sobre los parmetros implicados en esta operacin mecnica, sino
tambin en determinar unas condiciones de batido favorables para la variedad Cornicabra
mayoritaria en Castilla-La Mancha, de manera que puedan ser aplicadas por las almazaras
industriales de la regin para optimizar la composicin en compuestos minoritarios y por tanto
las caractersticas sensoriales de este aceite de oliva virgen.
Por otra parte, el principal compuesto fenlico cuantificado en la pasta de aceituna es la forma
3,4-DHPEA-EDA, cuyo contenido representa entre el 64% y el 76% de los fenoles totales
presentes y disminuye significativamente a lo largo de la etapa de batido, posiblemente debido
a la degradacin oxidativa llevada a cabo por enzimas como polifenoloxidasa (PPO),
peroxidasa (POD) o lipoxigenasa (LOX) (Servili et al., 2000). En cambio, los niveles de
derivados secoiridoideos del tirosol hallados en las pastas de aceituna representan un pequeo
porcentaje del contenido fenlico total, sin observarse ningn cambio o evolucin evidente en
su contenido a lo largo de la etapa. Respecto a la presencia de los alcoholes fenlicos
hidroxitirosol y tirosol, mencionar que solamente fue detectado en concentraciones
significativas el hidroxitirosol, cuyo contenido aument de forma apreciable a lo largo del
proceso de batido, lo que confirma la formacin de estos fenoles simples como productos de
degradacin de los derivados secoiridoideos. El carcter hidroflico de estos compuestos
fenlicos queda obviamente patente al cuantificarse su presencia en el orujo hmedo obtenido
tras la centrifugacin de las pastas, ya que la mayor parte de estas sustancias fueron retenidas
en este sub-producto.
167
4.2. Discusin General
Por otro lado, comentar que el contenido de oleuropena y secoiridoideos del hidroxitirosol
detectado en las pastas de aceituna obtenidas en los ensayos de batido han disminuido
significativamente al aumentar la temperatura de trabajo desde 20 C a 40 C y el tiempo desde
30 minutos a 90 minutos, mientras que la forma p-HPEA-EDA ha mostrado un comportamiento
opuesto; aspectos que confirman la degradacin oxidativa, ya sea qumica o enzimtica, de los
compuestos con estructura o-difenlica, puesto que por una parte la actividad de la enzima
LOX cataliza la formacin de hidroperxidos y podra estar implicada en la oxidacin indirecta
de los o-difenoles (Ranalli et al., 2003), ya que es sobradamente conocida la capacidad
antioxidante de estos compuestos, frente al tirosol y sus derivados que han demostrado una
escasa o inexistente actividad antioxidante (Mateos, 2002; Carrasco-Pancorbo et al., 2005). De
igual forma, el trabajo realizado por Toscano et al. (2003) ya haba demostrado que la enzima
PPO presente en las aceitunas posea una mayor afinidad por los sustratos con estructura o-
difenlica que por los monofenoles, que apenas se ven afectados por esta enzima, lo cual
tambin explica el descenso observado en los derivados secoiridoideos del hidroxitirosol
presentes en la pasta de aceituna.
168
4.2. Discusin General
proceso de batido, lo que supone la posibilidad tanto de aumentar el valor biolgico de este
producto, como de adecuar los niveles de los atributos positivos amargo y picante del AOV a
las necesidades del mercado, aspecto especialmente importante en la variedad Cornicabra,
que tal y como se ha comentado previamente se caracteriza por un elevado amargor.
De igual forma que los compuestos fenlicos, tras la molienda de los frutos, el contenido de
compuestos voltiles del aceite de oliva virgen depende principalmente de los fenmenos de
particin de estos componentes minoritarios entre las fases acuosa y oleosa de la pasta de
aceituna y que se producen durante el proceso de batido (Angerosa et al., 1998). Por esta
razn, el contenido en voltiles C6 cuantificado en los AOV del ensayo al variar la temperatura
o tiempo de batido mostr una evolucin similar a la composicin voltil de la pasta de aceituna
de la que proceda. As, el incremento de la temperatura de trabajo de 20 C a 40 C supuso
una disminucin de la fraccin aldehdica C6, en especial del E-2-hexenal con una cada del
30%, mientras que los tiempos de batido prolongado favorecieron la presencia de este mismo
compuesto, aumentado su concentracin alrededor de un 70% entre tiempos de 30 y 90
minutos de batido.
169
4.2. Discusin General
variables de trabajo para incrementar la intensidad de las notas sensoriales verdes y frutadas
tan apreciadas en este producto.
Adems de los ensayos realizados en Almazara Experimental, con el objetivo de ampliar los
rangos de tiempo-temperatura de batido estudiados se utiliz un sistema de procesado a
escala de laboratorio (Abencor). Con la ampliacin de estas condiciones se pone de manifiesto
que en todos los casos se obtuvieron tendencias similares de evolucin voltil y fenlica a las
observadas en los aceites de oliva virgen obtenidos en almazara, siendo en algunos casos ms
o menos acusada. Aunque, en varios estudios llevados a cabo en plantas de extraccin
pequeas, como son los trabajos descritos por Servili et al. (1994) o Angerosa et al. (2001), se
ha observado un descenso significativo en la concentracin de derivados secoiridoideos del
hidroxitirosol de los AOV al incrementar la temperatura de batido, lo cual puede atribuirse a una
actividad enzimtica oxidativa excepcional que supone la degradacin de estos compuestos al
favorecerse el contacto del O2 atmosfrico con la pasta de aceituna, fenmeno que es ms
evidente al procesar poca cantidad de muestra. En nuestro estudio con el sistema de
laboratorio Abencor, donde se ha procesado alrededor de 1 Kg de fruto, no se ha observado
esta tendencia.
Por ltimo, para poder establecer unas condiciones de batido ptimas en la variedad
Cornicabra se deben tener en cuenta varios factores, como son el rendimiento de aceite
obtenido durante el proceso de extraccin y la calidad sensorial del aceite de oliva virgen, que
depende principalmente como se ha comentado en numerosas ocasiones de los perfiles
fenlicos y voltiles. Los resultados del estudio realizado sugieren que las condiciones de
batido ptimas en esta variedad de aceituna seran temperaturas por debajo de 28 C y
tiempos superiores a 60 minutos, ya que el reducido contenido fenlico obtenido mejorara las
propiedades sensoriales de este aceite de oliva virgen a travs de una disminucin en la
intensidad del amargor percibido por los consumidores, sin afectar este aspecto de forma
significativa a la vida til del producto, ya que el elevado contenido fenlico natural de esta
variedad asegura la estabilidad oxidativa de este aceite. Adems, estas condiciones de batido
favorecen la presencia de compuestos voltiles de la ruta LOX y consecuentemente las notas
sensoriales verdes podran incrementarse en el producto.
Se puede concluir con que los resultados obtenidos en esta memoria de investigacin han
permitido ampliar el conocimiento existente sobre la influencia que ciertos factores agronmicos
y tecnolgicos ejercen sobre la composicin minoritaria tanto del fruto de partida, como de la
pasta de aceituna y sobretodo del aceite de oliva virgen resultante, favoreciendo especialmente
el conocimiento de la variedad espaola Cornicabra, producto de gran repercusin econmica
en Castilla-La Mancha y en Espaa. Asimismo, se ha puesto en evidencia como estos factores
170
4.2. Discusin General
pueden ser utilizados a fin de incrementar la calidad sensorial del aceite de oliva virgen y la
presencia de componentes de inters biolgico, al permitir la modulacin de los perfiles
fenlicos y voltiles del mismo, permitiendo de esta forma adecuar los atributos sensoriales de
este aceite vegetal a las exigencias de los consumidores y generando productos con una
mayor competitividad en el mercado actual.
171
5. CONCLUSIONES
5. Conclusiones
III. Los niveles de E-2-hexenal detectados en el aroma de diversos aceites de oliva virgen
monovarietales ha permitido su diferenciacin en base a la variedad de procedencia. Adems,
la presencia de steres C6 -acetato de Z-3-hexenilo y acetato de hexilo- slo se ha cuantificado
de forma notable en las variedades Morisca, Arbequina y Picual especialmente, confirmando
que la actividad de las enzimas de la ruta de la lipoxigenasa (LOX) se encuentra
estrechamente asociada a factores genticos.
IV. La maduracin del fruto implica un notable descenso en la fraccin aldehdica C6 del aroma
del aceite de oliva virgen, adems de una disminucin de su contenido en derivados
secoiridoideos del hidroxitirosol y tirosol, a causa de la menor cantidad de precursores fenlicos
presentes en la aceituna, como es el caso de la oleuropena.
175
5. Conclusiones
VIII. El nivel de estrs hdrico registrado en los olivos a lo largo de las distintas campaas
olivareras est relacionado directamente con el contenido en fenoles complejos del aceite de
oliva virgen obtenido, afectando de manera especial a los derivados secoiridoideos del
hidroxitirosol (3,4-DHPEA-EDA y 3,4-DHPEA-EA), e inversamente con la cantidad de aldehdos
C6 y alcoholes C6 (como Z-3-hexen-1-ol y hexan-1-ol) cuantificados en el producto.
IX. Se ha establecido una estrecha relacin entre el contenido en oleuropena de las drupas, y
los niveles de derivados secoiridoideos presentes en los aceites, con los valores de potencial
hdrico mnimos en los olivos jvenes de las variedades Cornicabra y Morisca, durante la etapa
fenolgica III del desarrollo del fruto.
X. Se ha puesto de manifiesto que el riego permite acentuar las notas sensoriales verdes y
frutadas del aceite de oliva virgen, disminuyendo la intensidad del atributo sensorial amargo,
siendo por tanto esta prctica agronmica aconsejable en aceites caracterizados bien por un
bajo contenido en voltiles C6 o por una elevada concentracin fenlica, como es el caso de la
variedad Cornicabra. Por el contrario, esta tcnica podra afectar negativamente a aceites de
variedades caracterizadas por un bajo contenido fenlico natural, como Morisca o Arbequina,
ya que podra suponer no slo la obtencin de aceites con escasa intensidad en los atributos
amargo y picante, sino tambin un descenso excesivo en su estabilidad oxidativa que
reducira considerablemente la vida til del producto.
XI. La estrategia de riego deficitario regulado utilizada en el olivar adulto de Cornicabra durante
la campaa 2004/05 (RDI-2), en donde la aplicacin de riego se ha centrado en la etapa
fenolgica III del desarrollo del fruto, adems de lograr un uso ms racional y adecuado del
agua disponible, permite recuperar el estado hdrico del olivo y alcanzar situaciones similares a
las obtenidas con la metodologa de riego recomendada por la FAO.
XII. La programacin de riego basada en la medida del potencial hdrico del tronco, con un
umbral de -1,2 MPa (SWP -1,2), se puede proponer como una alternativa factible al clculo del
parmetro ETc en el que se basa la metodologa de riego FAO, para realizar la planificacin de
las estrategias de irrigacin en un olivar joven de cubierta incompleta. Por el contrario, la
utilizacin de estrategias de irrigacin basadas en las fluctuaciones del dimetro del tronco
(TDF), genera niveles hdricos variables, por lo que su utilizacin a priori en el desarrollo de
planificaciones de riego es imprecisa y por tanto su uso debera continuar bajo estudio.
176
5. Conclusiones
177
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