Μαρία Χαλαμπαλάκη, Επίκουρη Καθηγήτρια, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Αλέξιος-Λέανδρος Σκαλτσούνης, Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστρικό Πανεπιστήμιο Αθηνών
Εμμανουήλ Μικρός, Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστρικό Πανεπιστήμιο Αθηνών
Stephan Schwarzinger, Department of Biopolymers and Research Centre for Bio-Macromolecules, University of Bayreuth
Σοφία Μητάκου, Καθηγήτρια, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Πέτρος Α. Ταραντίλης, Καθηγητής, Τμήμα Επιστήμης Τροφίμων και Διατροφής του Ανθρώπου
Ιωάννης Κωστάκης, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
The field of nutrition regarding humankind has undergone numerous major pattern-shifts over the last centuries. Studies have turned their attention towards exploring the impact of nutrition on human health, from one’s well-being to heritable changes in expressed genetic landscapes. Bioactive compounds contained in food commodities, mainly of natural origin, have been greatly associated with plausible positive attributes in human health. Products of primary or secondary metabolism in plant goods belong to chemical classes like polyphenols, terpenes, sterols, carotenoids, minerals, stanols, alkaloids, coumarins, flavonoids and fatty acids. A broad spectrum of health-promoting effects has been linked with them, i.e. cardioprotective, antioxidative, anti-cancer, antibacterial, neuroprotective, immunomodulatory and anti-inflammatory.
The introduction of new technologies and methods in the food industry has enabled the development of high throughput methodologies for the quality assessment of such commodities. The expectancy of consumers over certain characteristics such as organoleptic and nutritional, accompanied by resulting benefits and certain desirable traits that could justify the added value paid by the customer are novel additions to the term “Food Quality”. This also entails forms and area of production or botanical origin of the consumed goods. Products of the olive tree, with the primary focus on olive oil and table olives, have attracted the attention of the scientific world, especially since the issued health claims regarding the formers chemical composition linked with positive impacts in human health.
In this context, olive oils from the top-3 producing countries worldwide, Spain, Italy and Greece, were selected and studied primarily in this thesis. Acknowledging the importance of sample preparation methods prior to analysis, the first attempt in this thesis was the comparison and optimization of already existing and new methods, and especially the ones suggested by the International Olive Council (IOC). Specifically, extraction (recovery of compounds) is one of the most critical decision-making steps in the analysis of foods since it defines qualitatively and quantitatively the compounds to be analyzed. Moreover, the analytical determination of OO biophenols was investigated since the already existing ones show several limitations and, most importantly, the HPLC-UV based method suggested by IOC to this day. Thus, different extraction and HPLC-UV based analytical procedures for the recovery of the total phenolic fraction (TPF) and the determination of their biophenol content (BC) were compared and new ones were proposed. Furthermore, single compounds, like tyrosol (Tyr), hydroxytyrosol (HT), oleacein (OLEA) and oleocanthal (OLEO), which are the most relevant to the EFSA claim, were quantified using the IOC and newly suggested HPLC-DAD-based analytical methods, as well as approaches such as the hydrolysis of biophenols All qualitative and quantitative results (TPF, BC, Tyr, HT, OLEO, OLEA) were compared and evaluated towards the optimization of the entire procedure and determined in the selected commercial EVOO samples. NMR spectroscopy was employed for its robustness and quantitative traits in the determination of oleacein’s and oleocanthal’s levels in analyzed samples with different developed methods. Results from these different methodologies were compared for the first time in olive oil analysis.
Metabolomics, an integrated approach that incorporates advanced analytical instrumentation, such as NMR magnets and high-resolution mass spectrometers, with the power of bioinformatics has enabled the accumulation and interpretation of large sets of data. Ultimately, this has led to the mapping of food products, among them olive oil, based on designation of origin, production procedure or botanical derivation, and the discovery of new bioactive compounds or markers that attach unique traits to the quality of a product. In this study, two different sets of samples were subjected to NMR-based metabolic profiling. Greek and European olive oils (commercial samples from Greece, Spain, Italy, other EU countries and blends) were investigated for tentative markers that would aid in the determination of their geographical, or even botanical origin in the case of Greek samples. The outcomes of the analysis of the two samples sets were integrated in a statistical manner. To our knowledge such a study in OO from top producing countries using NMR metabolomics has not been carried out to this day.
Furthermore, as we insisted in strengthening the identification confidence of metabolites, a serious bottleneck in foodomics, Statistical Total Correlation Spectroscopy (STOCSY) was employed. STOCSY is a strong statistical tool that takes advantage of the multi-co-linearity of the variables’ intensity in a set of spectra to generate pseudo-NMR spectra, which display the correlation of intensities of the various peaks across the whole sample. Initially introduced for human metabolomics and with only a few applications existing in foodomics, it was used for the first time in EVOO analysis. This tool, in combination with recorded 2D spectra and a widespread literature search, has led to an ongoing assignment of compounds in the 1H NMR spectrum of the total extract of olive oils.
Finally, NMR and HRMS metabolomic platforms were utilized for the investigation of Greek edible olive drupes. Table olives, despite the high levels of HT, are neglected compared to olive oil. Thus, in the present study the main Greek types of edible olives originating from different regions of the country, different cultivars and alternate debittering methods were investigated. STOCSY, 2D spectra and literature employed for NMR along with various dereplication tools in the case if HRMS, has led to an unprecedented characterization of their chemical profiles. Chemometrics was applied to both datasets, while discrimination models allowed the identification of certain compounds-markers that were specific to geographical origin, cultivar or debittering method.
Overall, the combination of different approaches in the study of olive oils and table olives comprises a systematic attempt to expand our knowledge regarding their chemical composition and fraud and mislabeling. We aspire that our approaches and methods, aside from the olive field, will be useful for the analysis of other foods and foodstuffs. Metabolomics applications in this field, also known as “Foodomics” pose as an area of the future that will help overcome some of the challenges that it is faced with. Novel methodologies are turning out to be a necessity, in this day and age, under the global effort to assure food origin and quality control. NMR spectroscopy and MS approaches could certainly lift this weight for the years to come.