Impact of genetic background, maturation, and infection by Colletotrichum acutatum on the phytochemical composition of olives and olive oils: implementation of faster infrared screening tools
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2016-05-12
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A oliveira (Olea europaea L.) é uma das culturas mais importantes dos países do Mediterrâneo, cujo cultivo tem vindo a sofrer um incremento ao longo dos últimos anos, particularmente devido às diversas propriedades benéficas e sensoriais que os seus produtos (azeitona e azeite) apresentam. Estas propriedades relacionam-se com a composição nutricional e não nutricional das azeitonas, e dependem de vários fatores de pré-colheita (condições agro-ambientais, cultivar e estado de maturação, entre outros) e pós-colheita (condições de armazenamento). Uma vez que durante o processo de extração de azeite não são adicionados quaisquer químicos, a composição nutricional e fitoquímica mantém-se praticamente inalterável. Para além dos fatores anteriormente referidos, a oliveira é ainda por vezes afetada por diferentes doenças, que podem reduzir o rendimento e a qualidade final dos seus produtos, nomeadamente em termos de metabolitos secundários. A antracnose da oliveira (também denominada por doença da gafa) é provocada pelos fungos Colletotrichum acutatum e C. gloeosporioides e, em condições propícias de humidade e temperatura, pode levar a elevadas perdas de produção. Além disso, a incidência da antracnose depende das condições ambientais, da virulência dos isolados e da suscetibilidade das cultivares. Os objetivos deste trabalho consistiram em determinar a composição fenólica de diferentes cultivares de azeitona que diferem em termos de suscetibilidade ao C. acutatum (‘Galega Vulgar’ (altamente suscetível), ‘Cobrançosa’ (moderadamente suscetível) e ‘Picual’ tolerante), quer durante o processo de maturação, quer durante o processo de infeção pelo fungo. Para além da determinação da composição fenólica por métodos clássicos, e uma vez que estes requerem o uso de reagentes e solventes dispendiosos, e o pré-tratamento das amostras, foram ainda aplicados os métodos vibracionais espectroscópicos, por transformada de Fourier e Raman. Além disso, a expressão do gene OePAL foi determinada a fim de tentar relacioná-la com a atividade da enzima fenilalanina amónia-liase (PAL) e o conteúdo em compostos fenólicos. Para atingir estes objetivos, as três cultivares acima referidas foram avaliadas ao longo dos processos de maturação e infeção em termos de composição fenólica e atividade antioxidante dos frutos e azeites. Estas análises, juntamente com a determinação do conteúdo em metais e dos parâmetros de qualidade dos azeites (acidez livre e índice de peróxidos) e a aplicação de métodos espectroscópicos e análise multivariada foram utilizados de forma a discriminar os azeites das diferentes cultivares. Para além deste estudo, foi ainda avaliado o efeito do ataque do fungo C. acutatum na expressão do gene OePAL, na atividade da enzima PAL e no conteúdo e perfil fenólico das azeitonas. Para tal, azeitonas não infetadas (controlo) e infetadas às 0, 16, 48, 144 e 360 horas após a infeção foram colhidas e analisadas. Além disso, sabendo que a cutícula é o tecido da azeitona com o qual o fungo estabelece o primeiro contacto durante o processo de infeção, a mesma foi analisada através da técnica de espectroscopia de infravermelho por transformada de Fourier (FTIR) a fim de discriminar não só as cultivares, mas também os diversos tempos de infeção, e adicionalmente avaliar esta abordagem como ferramenta auxiliar para aferição das diferentes suscetibilidades ao fungo, apresentadas pelas distintas cultivares. Os resultados referentes ao estudo da maturação demonstraram uma diminuição do conteúdo em compostos fenólicos, independentemente da cultivar estudada. Além disso, as condições climáticas demonstraram ter um papel importante nesta composição. Relativamente aos métodos espectroscópicos (FTIR e Raman) e clássicos utilizados para a determinação do conteúdo fenólico, juntamente com análise multivariada, foi possível, não só criar modelos de previsão para a determinação da composição fenólica e dos parâmetros de qualidade dos azeites, mas também discriminar as diferentes cultivares e estados de maturação. O mesmo foi conseguido através da análise do conteúdo em metais das amostras de azeites. Relativamente ao estudo do efeito do ataque do C. acutatum nas azeitonas, verificou-se um aumento da produção do conteúdo em compostos fenólicos, principalmente nas azeitonas da cultivar Galega Vulgar, descrita como suscetível a este fungo. Além disso, a expressão do gene OePAL e a atividade da enzima PAL foram induzidas pelo ataque do fungo, nomeadamente para as cultivares Galega Vulgar e Picual, que demonstraram expressão e atividade máxima às 16 horas de infeção. Em suma, este trabalho permitiu obter um maior conhecimento àcerca do efeito da maturação e da infeção pelo C. acutatum no conteúdo e perfil fenólico de azeites e azeitonas. Além disso, o uso de métodos espectroscópicos vibracionais, tais como o FTIR e Raman, articulados com a aplicação de análises quimiométricas, permitiu descrever de uma forma mais rápida e eficiente a discriminação de diferentes cultivares, estados de maturação e infeção.
The olive tree (Olea europaea L.) is one of the most important crops in the Mediterranean Basin, which cultivation has increased over the last decades, encouraged by the diverse health benefits associated to olive oils consumption and the sensorial characteristics. However, the health benefits associated to dietary olive oils are closely dependent on the nutritional and non-nutritional composition of olive fruits, which are influenced by several pre-harvest (agro-environmental conditions, cultivar, and ripening stage) and post-harvest (storage conditions) factors. Once initialized the olive fruits processing towards obtaining olive oils, the nutritional and phytochemical compounds remain almost unaltered, keeping the final products all the characteristics and constituents described in olives. In addition to the above mentioned factors, the olive tree can also be affected by diverse diseases, conditioning yield and final product quality. This fact is of special relevance regarding the content in (poly)phenols. Among the currently identified diseases affecting olive tree, olive antrachnose, caused by the fungus Colletotrichum acutatum and C. gloeosporioides, is one of the most relevant constraint, which severity is dependent on the weather conditions, pathogen virulence, and cultivar susceptibility. The objective of this work was to evaluate the phenolic composition in diverse maturation stages and infection degree in three different olive cultivars characterized by distinct susceptibility to C. acutatum (‘Galega Vulgar’ (highly susceptible), ‘Cobrançosa’ (moderately susceptible), and ‘Picual’ tolerant). The assessment of olives and olive oils on the phenolic composition was developed by classical spectrophotometric and chromatographic methods, which were complemented by spectroscopical approaches, such as Fourier- Transform Infrared spectroscopy (FTIR) and Raman. Furthermore, the expression of OePAL gene, was explored to understand the relationship between OePAL expression, phenylalanine ammonia-lyase (PAL) activity, and the (poly)phenolic content. To achieve these goals, the three olive cultivars referred above were investigated during the fruit maturation and infection process, regarding the phenolic composition and radical scavenging capacity of olive fruits and oils. The information recorded on these characteristics together with metal content, free acidity, and the peroxide value, resorting to spectroscopical methods combined with multivariate analysis was aimed to discriminate olive oils obtained from different cultivars. Besides the basic information on olives and olive oils composition, olive fruits were also assessed on the susceptibility to the infection by C. acutatum. For this purpose, the olives were infected in the olive grove. Non-infected controls and olives infected for 0, 16, 48, 144, and 360 hours after infection (hai) were harvested and assessed concerning the expression level of OePAL gene, PAL activity, and phenolic profile and content. These determinations allowed to study the correlation between OePAL gene expression, PAL activity, and phenolic content. Since cuticle constitutes the first contact point of the fungus during infection, this tissue was analysed by FTIR combined with Attenuated Total Reflectance (ATR) accessory to discriminate cultivars and infection times, besides the susceptibility/response to the infection. The major results obtained allowed to state that during the maturation process, the content on phenolic compounds decreased independently of the cultivar, whilst the climatic conditions appeared as a critical factor for the (poly)phenolic composition. The use of spectroscopical (FTIR and Raman) and classical methods for determining phenolic compounds and quality parameters of olive oils, combined with Partial Least Square regression allowed to discriminate the cultivar and the distinct maturation stages, and to create models for predicting the chemical characteristics of this food matrix during the maturation process. Besides the discrimination based in these analyses, the evaluation of the metal content was also applied alongside Partial Least Square regression, allowing to discriminate the campaign and the cultivar. Apart from the evaluation of the basic chemical and phytochemical composition and the mineral content of olives and olive oils in different cultivars and throughout the ripening stage, it was determined the effect of C. acutatum on the level of secondary metabolites (phenolics). These increased throughout the infection as a response to the fungal attack, especially concerning ‘Galega Vulgar’, which has been described as the most susceptible cultivar. Furthermore, OePAL gene expression and PAL activity were boosted by the fungal infection, mainly in cultivars Galega Vulgar and Picual, which showed the highest gene expression level and PAL activity at 16 hours after infection. As overall conclusion, this work allowed to gain a further insight in the effect of maturation and C. acutatum infection processes on the phenolic profile and (poly)phenolic content of olives and olive oils. In addition, the application of FTIR methods combined with chemometric analyses allowed to describe a faster spectroscopical method for the discrimination of different olive cultivars, maturation stages and infection incidence.
The olive tree (Olea europaea L.) is one of the most important crops in the Mediterranean Basin, which cultivation has increased over the last decades, encouraged by the diverse health benefits associated to olive oils consumption and the sensorial characteristics. However, the health benefits associated to dietary olive oils are closely dependent on the nutritional and non-nutritional composition of olive fruits, which are influenced by several pre-harvest (agro-environmental conditions, cultivar, and ripening stage) and post-harvest (storage conditions) factors. Once initialized the olive fruits processing towards obtaining olive oils, the nutritional and phytochemical compounds remain almost unaltered, keeping the final products all the characteristics and constituents described in olives. In addition to the above mentioned factors, the olive tree can also be affected by diverse diseases, conditioning yield and final product quality. This fact is of special relevance regarding the content in (poly)phenols. Among the currently identified diseases affecting olive tree, olive antrachnose, caused by the fungus Colletotrichum acutatum and C. gloeosporioides, is one of the most relevant constraint, which severity is dependent on the weather conditions, pathogen virulence, and cultivar susceptibility. The objective of this work was to evaluate the phenolic composition in diverse maturation stages and infection degree in three different olive cultivars characterized by distinct susceptibility to C. acutatum (‘Galega Vulgar’ (highly susceptible), ‘Cobrançosa’ (moderately susceptible), and ‘Picual’ tolerant). The assessment of olives and olive oils on the phenolic composition was developed by classical spectrophotometric and chromatographic methods, which were complemented by spectroscopical approaches, such as Fourier- Transform Infrared spectroscopy (FTIR) and Raman. Furthermore, the expression of OePAL gene, was explored to understand the relationship between OePAL expression, phenylalanine ammonia-lyase (PAL) activity, and the (poly)phenolic content. To achieve these goals, the three olive cultivars referred above were investigated during the fruit maturation and infection process, regarding the phenolic composition and radical scavenging capacity of olive fruits and oils. The information recorded on these characteristics together with metal content, free acidity, and the peroxide value, resorting to spectroscopical methods combined with multivariate analysis was aimed to discriminate olive oils obtained from different cultivars. Besides the basic information on olives and olive oils composition, olive fruits were also assessed on the susceptibility to the infection by C. acutatum. For this purpose, the olives were infected in the olive grove. Non-infected controls and olives infected for 0, 16, 48, 144, and 360 hours after infection (hai) were harvested and assessed concerning the expression level of OePAL gene, PAL activity, and phenolic profile and content. These determinations allowed to study the correlation between OePAL gene expression, PAL activity, and phenolic content. Since cuticle constitutes the first contact point of the fungus during infection, this tissue was analysed by FTIR combined with Attenuated Total Reflectance (ATR) accessory to discriminate cultivars and infection times, besides the susceptibility/response to the infection. The major results obtained allowed to state that during the maturation process, the content on phenolic compounds decreased independently of the cultivar, whilst the climatic conditions appeared as a critical factor for the (poly)phenolic composition. The use of spectroscopical (FTIR and Raman) and classical methods for determining phenolic compounds and quality parameters of olive oils, combined with Partial Least Square regression allowed to discriminate the cultivar and the distinct maturation stages, and to create models for predicting the chemical characteristics of this food matrix during the maturation process. Besides the discrimination based in these analyses, the evaluation of the metal content was also applied alongside Partial Least Square regression, allowing to discriminate the campaign and the cultivar. Apart from the evaluation of the basic chemical and phytochemical composition and the mineral content of olives and olive oils in different cultivars and throughout the ripening stage, it was determined the effect of C. acutatum on the level of secondary metabolites (phenolics). These increased throughout the infection as a response to the fungal attack, especially concerning ‘Galega Vulgar’, which has been described as the most susceptible cultivar. Furthermore, OePAL gene expression and PAL activity were boosted by the fungal infection, mainly in cultivars Galega Vulgar and Picual, which showed the highest gene expression level and PAL activity at 16 hours after infection. As overall conclusion, this work allowed to gain a further insight in the effect of maturation and C. acutatum infection processes on the phenolic profile and (poly)phenolic content of olives and olive oils. In addition, the application of FTIR methods combined with chemometric analyses allowed to describe a faster spectroscopical method for the discrimination of different olive cultivars, maturation stages and infection incidence.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Olea europaea , Maturação , Polifenóis , Espectroscopia de infravermelhos