New challenges in analytical determination of olive oil polyphenols. Potential use as markers linked to pedoclimatic, agronomic and technological conditions

Resumen

Virgin olive oil (VOO) is the main source of fat in the Mediterranean region. Its consumption was found to be associated with low incidence of chronic diseases such as atherosclerosis, cancer, obesity and diabetes. The healthy properties of this oil were originally attributed to its high content of monounsaturated fatty acids. However, recent studies have demonstrated that the minor fractions, mainly polyphenols, also make a major contribution to healthy VOO properties. In addition, phenolic compounds are also associated with the oxidative stability and organoleptic quality of VOO. The present doctoral thesis entitled ¿New challenges in analytical determination of olive oil polyphenols. Potential use as markers linked to pedoclimatic, agronomic and technological conditions¿, deals with the analysis of phenolic compounds in VOOs and the effect of different factors on their concentration, such as pedoclimatic, agronomic and technological conditions of production. For this purpose, the thesis is divided into two sections: introduction and experimental. The INTRODUCTION includes important information about VOO composition, oxidative stability, organoleptic and health properties of VOO polyphenols, factors affecting their content in VOO, and analytical procedures for the qualitative and quantitative characterization of this important fraction. The EXPERIMENTAL SECTION, RESULTS AND DISCUSSIONS are presented in three sections according to the different topics under study: SECTION I focuses on the study of the effect of geographical area of cultivation and agronomic practices on VOO phenolic composition. This section is divided into two chapters: Chapter 1 concerns the phenolic characterization and geographical classification of commercial Arbequina VOOs produced in southern Catalonia. The aim of the study was to explore the phenolic profile of Arbequina VOO as one of the main Spanish olive varieties, and to look for possible differences in the phenolic composition among the geographical area under study. For this purpose, VOO samples were classified into three groups (group 1, group 2 and group 3) according to their geographical origin, which were demarcated taking into account the edaphological characteristics and orography. A total of 32 olive oil samples were obtained from different mills. Then, phenolic compounds were extracted from the samples using solid-phase extraction, and the resulting extracts were analysed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-TOF/MS). This study was carried out in collaboration with la Unitat de Recerca Biomèdica (URB-CRB) de la Universitat Rovira i Virgili (Reus). In chapter 2, the changes in chemical composition, principally phenolic compounds, of Algerian Chemlal VOO with regard to irrigation and harvest time were studied. First, the olive grove was divided into two parts: one was under rain-fed conditions (non-irrigated) and the other was irrigated with 100% of crop evapotranspiration. Olive oil samples were obtained from irrigated and non-irrigated olives on three harvest dates for posterior analysis. Phenolic compounds were extracted using liquid-liquid extraction, and analysed by HPLC-ESI-TOF/MS in an effort to obtain detailed information about the phenolic behaviour under the effect of the studied factors. The work included in this chapter was carried out in collaboration with the Department of Agronomic Sciences of Tizi-Ouzou University in Algeria. SECTION II is divided into three chapters and examines the VOO production process, and the best conditions for obtaining VOO with high phenolic content. In chapter 3, the first part provides an overview of the different steps involved in the VOO elaboration process including olive harvesting time, crushing, malaxation, centrifugation, storage and filtration, and their effect on its phenolic composition. The objective was to establish the best conditions for obtaining VOO rich in phenolic compounds taking into account the reported data in the literature. The second part provides an overview of the different phenolic families characterized in olive oil by-products, and the possible use of these by-products as a potential alternative source of bioactive compounds. The work included in this chapter was the result of a collaboration with the Olive Center of California University, United States. In chapter 4, the effect of harvest dates corresponding to different olive ripening stages on Algerian Azeradj olive oil quality was studied. To carry out the study, olive fruits from the Azeradj variety were manually collected on different dates (D1, 03 November 2013; D2, 27 November 2013; and D3, 21 December 2013) from trees cultivated in the same area (Haizar), in north¿central Algeria. After that, olive oil samples were made on a laboratory scale using the Abencor system for their posterior analysis. This study is the first one available in which polyphenols of the Azeradj VOO variety have been characterized by HPLC-ESI-TOF/MS. The work included in this chapter was carried out in collaboration with the Department of Agronomic Sciences of Tizi-Ouzou University in Algeria. Chapter 5 includes the monitoring of VOO moisture and phenolic compound content during the industrial filtration process. To achieve this, a conventional filtration process was performed in duplicate using two lots (lot 1 and lot 2), for a total amount of 45,000 kg of VOO each. The VOOs were from the main Spanish olive varieties (Hojiblanca, Manzanilla, Picual and Arbequina). Cloudy VOOs were filtered using Vitacel L-90 and Filtracel EFC-950 as filter aids together with a filtration tank. The moisture content was determined in unfiltered and filtered VOOs. In addition, the individual phenolic compounds were qualitatively and quantitatively characterized by HPLC-ESI-TOF/MS. This work was carried out in collaboration with Oleoestepa Company S.C.A, with the University of Bologna (Italy) and with University of Campinas (Brazil). SECTION III is divided into two chapters and focuses on the analytical procedures for the determination of VOO phenolic composition, and the different limitations observed in the methods proposed until now. In chapter 6, the first part provides an overview of the current extraction and analytical approaches for the qualitative and quantitative characterization of phenolic compounds in VOO as well as the advantages and disadvantages of each approach. Liquid-liquid extraction, solid-phase extraction, liquid chromatography, gas chromatography, capillary electrophoresis, UV-Vis and mass spectrometry detectors were the analytical techniques reviewed in this first part. In the second part, the main current problems in the analysis of VOO phenolic compounds were discussed in order to take them into account in future studies aimed at olive oil phenolic characterization. In chapter 7, a new approach has been developed for correcting the effect that moisture reduction after VOO filtration exerts on the apparent increase in the secoiridoid content by using an internal standard during extraction. The objective of the study was to resolve one of the major problems faced in the analysis of VOO phenolic compounds. Firstly, two main Spanish varieties (Picual and Hojiblanca) were submitted to industrial filtration of VOOs. Afterwards, the moisture content was determined in unfiltered and filtered VOOs, and liquid¿liquid extraction of phenolic compounds was performed using different internal standards. The resulting extracts were analysed by HPLC-ESI-TOF/MS, in order to gain maximum information concerning the phenolic profiles of the samples under study. This research was carried out in collaboration with the University of Bologna (Italy), University of Campinas (Brazil) and Aceites Maeva Company S.L.