Individuazione e caratterizzazione dei prodotti di degradazione ossidativa dei secoiridoidi nell'olio vergine di oliva mediante tecniche analitiche innovative

Lo scopo di questo lavoro di tesi è la caratterizzazione dei prodotti di ossidazione di diversi fenoli idrofili contenuti nell’olio vergine d’oliva come idrossitirosolo, tirosolo e la forma dialdeidica dell’acido decarbossimetil elenolico legato all’idrossitirosolo, e la loro identificazione nel prodotto durante la conservazione. L’obiettivo della ricerca è trovare degli indici analitici che possono essere usati sia come marker di “freschezza” dell’olio vergine di oliva sia nella valutazione della “shelf life” del prodotto stesso. Due sistemi di ossidazione sono stati usati per ossidare le molecole sopracitate: ossidazione enzimatica e ossidazione di Fenton. I prodotti di ossidazione sono stati identificati come chinoni, dimeri e acidi.

Nuove vie di sintesi dell'idrossitirosolo

L’idrossitirosolo (3,4-diidrossifeniletanolo) è uno degli antiossidanti naturali più potenti e con molte proprietà benefiche sull'organismo umano ed è per questo molto pregiato. In natura, l’idrossitirosolo è presente nelle olive e in quantità maggiori nelle foglie dell’olivo. L’obiettivo di questo lavoro di tesi ha riguardato lo studio esplorativo di vie di sintesi alternative per l’idrossitirosolo che partissero da reagenti a basso costo e green, attraverso la catalisi eterogenea e utilizzando acqua come solvente. Sulla base di questi requisiti abbiamo delineato un progetto per la sintesi che, partendo dal catecolo (o dal suo omologo guaiacolo), prevede l’introduzione di una catena C2 sull’anello aromatico (reazione già nota a livello industriale per la sintesi della vanillina)seguita da una riduzione one pot del derivato mandelico a dare il prodotto di interesse, l’idrossitirosolo. L’obiettivo posto è pertanto molto ambizioso e consente di ottenere l’idrossitirosolo in soli due passaggi sintetici in catalisi eterogenea a partire da reagenti green e a basso costo.

Tecniche elettroanalitiche-chemiometriche applicate alla tipizzazione dli oli monovarietali.

Il presente lavoro di tesi si propone di verificare se le tecniche elettrochimiche consentano di effettuare una classificazione del cultivar dell’olio. In particolare si vuole proporre un nuovo metodo per eseguire misure voltammetriche con un microelettrodo di platino direttamente in campioni di olio extravergine di oliva a cui sono aggiunti RTILs (Room Temperature Ionic Liquids) per rendere le matrici adeguatamente conducibili e quindi adatte per le analisi. I voltammogrammi ciclici dovranno poi essere elaborati attraverso diverse tecniche chemiometriche con l’obiettivo di discriminare i campioni sulla base della loro origine. Si studieranno quindi delle metodologie analitiche che potrebbero consentire di riconoscere l'origine delle olive usate per produrre un olio e di verificarne l'aderenza alle normative o ai disciplinari delle DOP e l'eventuale presenza di materiale estraneo. Le ricerche hanno l’obiettivo di individuare nell'olio il “fingerprint” da cui sarà possibile risalire alle varie cultivar di provenienza, identificando la composizione e l'origine dell'olio, ciò consentirà inoltre di difendere i consumatori da eventuali frodi commerciali.

Recupero di polifenoli contenuti nelle acque di vegetazione provenienti da oleifici mediante adsorbimento

I problemi ambientali legati alla tossicità delle OMW e l’impossibilità del loro utilizzo diretto come acque di irrigazione, a causa dell’elevato contenuto di composti fenolici, spingono verso lo sviluppo di un processo di separazione che permetta la decontaminazione e valorizzazione delle acque di scarto e il recupero dei composti fenolici ad alto valore aggiunto. Lo scopo del lavoro è stato quello di studiare la fattibilità di un processo in continuo per il recupero dei composti fenolici contenuti nelle acque di vegetazione di due differenti oleifici, mediante adsorbimento in fase solida su una resina polimerica.

Country-of-Origin Labeling for Processed Foods

The country-of-origin is the “nationality” of a food when it goes through customs in a foreign country, and is a “brand” when the food is for sale in a foreign market. My research on country-of-origin labeling (COOL) started from a case study on the extra virgin olive oil exported from Italy to China; the result shows that asymmetric and imperfect origin information may lead to market inefficiency, even market failure in emerging countries. Then, I used the Delphi method to conduct qualitative and systematic research on COOL; the panel of experts in food labeling and food policy was composed of 19 members in 13 countries; the most important consensus is that multiple countries of origin marking can provide accurate information about the origin of a food produced by two or more countries, avoiding misinformation for consumers. Moreover, I enhanced the research on COOL by analyzing the rules of origin and drafting a guideline for the standardization of origin marking. Finally, from the perspective of information economics I estimated the potential effect of the multiple countries of origin labeling on the business models of international trade, and analyzed the regulatory options for mandatory or voluntary COOL of main ingredients. This research provides valuable insights for the formulation of COOL policy.

The impact of grants in the agriculture sector in Albania: a counterfactual approach

The objective of this study is to measure the impact of the national subsidy scheme on the olive and fruit sector in two regions of Albania, Shkodra and Fier. From the methodological point of view, we use a non- parametric approach based on the propensity score matching. This method overcomes problem of the missing data, by creating a counterfactual scenario. In the first step, the conditional probability to participate in the program was computed. Afterwards, different matching estimators were applied to establish whether the subsidies have affected sectors performance. One of the strengths of this study stays in the data. Cross-sectional primary data was gathered through about 250 interviews.. We have not found empirical evidence of significant effects of government aid program on production. Differences in production found between beneficiaries and non-beneficiaries disappear after adjustment by the conditional probability of participating into the program. This suggests that subsidized farmers would have performed better than the subsidized households even in the absence of production grants, revealing program self-selection. On the other hand, the scheme has affected positively the farm structure increasing the area under cultivation, but yields has not increased for beneficiaries compared to non beneficiaries. These combined results shed light on the reason of the missed impact. It could be reasonable to believe that the new plantation, in particular in the case of olives, has not yet reached full production. Therefore, we have reasons to believe on positive impacts in the future. Concerning some qualitative results, the extension of area under cultivation is strongly conditioned by the small farm size. This together with a thin land market makes extremely difficult the expansion beyond farm boundaries.

New and More Sustainable Processes for the Synthesis of Phenolics: 2-phenoxyethanol and hydroxytyrosol

The research work has dealt with the study of new catalytic processes for the synthesis of fine chemicals belonging to the class of phenolics, namely 2-phenoxyethanol and hydroxytyrosol. The two synthetic procedures investigated have the advantages of being much closer to the Green Chemistry principles than those currently used industrially. In both cases, the challenge was that of finding catalysts and methods which led to the production of less waste, and used less hazardous chemicals, safer solvents, and reusable heterogeneous catalysts. In the case of 2-phenoxyethanol, the process investigated involves the use of ethylene carbonate (EC) as the reactant for phenol O-hydroxyethylation, in place of ethylene oxide. Besides being a safer reactant, the major advantage of using EC in the new synthesis is the better selectivity to the desired product achieved. Moreover, the solid catalyst based on Na-mordenite was fully recyclable. The reaction mechanism and the effect of the Si/Al ratio in the mordenite were investigated. In the case of hydroxytyrosol, which is one of the most powerful natural antioxidants, a new synthetic procedure was investigated; in fact, the method currently employed, the hydrolysis of oleuropein, an ester extracted from the waste water processing of the olive, makes use of large amounts of organic solvents (hexane, ethyl acetate), and involves several expensive steps of purification. The synthesis procedure set up involves first the reaction between catechol and 2,2-dimethoxyacetaldehyde, followed by the one-pot reduction of the intermediate to give the desired product. Both steps were optimized, in terms of catalyst used, and of reaction conditions, that allowed to reach ca 70% yield in each step. The reaction mechanism was investigated and elucidated. During a 3-month period spent at the University of Valencia (with Prof. A. Corma’s group), a process for the production of diesel additives (2,5-bis(propoxymethyl)furan) from fructose has been investigated.

Einfluss von Hilfsstoffen auf die Bioverfügbarkeit von Substanzen der BCS Klasse III

In dieser Arbeit wurde der Effekt verschiedener Hilfsstoffe auf die Permeabilität von Substanzen der BCS Klasse III untersucht. Drei pharmazeutische Hilfsstoffe wurden hinsichtlich der Möglichkeit ihres Einsatzes als Permeationsverbesserer in Arzneistoffformulierungen untersucht. Außerdem wurde die Beteiligung von Gallensalzen an der Nahrungsmittel-Interaktion von Trospium untersucht.rnEs wurden Komplexe aus Trospium und λ-Carrageen hergestellt. Eine verbesserte Permeation, die höchstwahrscheinlich durch Mukoadhäsion zustande kam, war im Ussing-Kammer-Modell sehr gut reproduzierbar. In vivo war der Effekt nur bei einigen Tieren zu sehen und es kam zu hohen Standardabweichungen.rnTrospium bildet Ionenpaare mit Gallensalzen, welche zu einer besseren Permeabilität des Wirkstoffes führten. In Gegenwart von Nahrungsfetten blieb dieser Effekt aus. Eine Beteiligung der Interaktion von Trospium und Gallensalzen am Food-Effekt kann auf Basis dieser Ergebnisse als wahrscheinlich gelten.rnIm Caco-2-Modell konnte bereits eine Verbesserung der Permeabilität von Trospium durch Zusatz von Eudragit E gezeigt werden. Nun konnte gezeigt werden, dass durch den Hilfsstoff auch in vivo in Ratten eine verbesserte Permeation erreicht werden kann.rnDie Permeationsverbesserung von Aciclovir durch Zusatz von Chitosan-HCl sollte untersucht werden. Im Caco-2-Modell kam es zu einer signifikanten Permeationsverbesserung. Im Ussing-Kammer-Modell wurde die Permeation nicht verbessert. In Loop-Studien konnte nur bei hohen Hilfsstoff-Konzentrationen eine Tendenz zur Permeationsverbesserung erkannt werden.rn

Co-frangitura oliva e nocciola: Studio del prodotto

In recent times, the choices of consumers have been more conscious and oriented to foods with health benefits. The present paper deals with the study of oil from crushing of olive and huzelnut with the aim of obtaining a “functional food”. Different samples of oil derived from the crushing of olive (O), olive with 5% of hazelnut (O5N) and olive with 10% of hazelnut (O10N), exposed to different temperatures (28 and 35°C) and times (15 and 30 minutes) of malaxation. The samples of oil were initially subjected to a qualitative assessment by the analysis of peroxide and free acidity. Following further analyses were carried out namely the determination of fatty acids and triglycerides by FAST GC-FID, the determination of tocopherols by HPLC-FLC, the analysis of sterols by GC/MS and the spectroscopic analysis with FT-MIR combined with statistical analysis with PCA and PLS. The results showed that increasing the time and temperature of malaxation there aren’t relevant significant differences (p<0,05) in the composition of fatty acids, triglycerides and tocopherols in the different oils, but there are higher extraction yields. The increase of content of hazelnut in phase of crushing causes the decrease of triglycerides C50 and C52, the increase of the class C54, total tocopherols and of total sterols as well. The samples analysed with FT-MIR spectroscopy have showed, on the contrary to conventional analytical techniques, a good discrimination between different oils despite of the similar chemical composition of olive and hazelnuts. After the PLS models were built from spectra FT-MIR in order to estimate the content of triglycerides C50, C52 and C54 and total tocopherols, with good R2 in full cross validation (R2>0,821).

Valorizzazione di acque di vegetazione provenienti da oleifici tramite recupero di polifenoli per adsorbimento

The environmental problems associated to the disposal of the olive oil wastewater (OMW, Olive Mill Wastewaters), and the difficulty in the recycling because of their polyphenolic content, led to propose the separation of their polyphenols. The recovery of polyphenols from olive mill wastewaters (OMWs) provides the double opportunity to obtain high-added value biomolecules and to reduce the phytotoxicity of the effluent. The separation can be obtained by a continuous flow extraction process of adsorption and desorption, this process is characterized by the possibility to recycling the adsorbing phase and the extraction solvent. The present work is part of a project aimed to the development of pilot scale process based on the use of the AMBERLITE XAD16 as adsorption resin. The adsorption stage is preceded by a wastewater filtration pretreatment, and a final desorption process is added to recover the polyphenols using acidified ethanol as the solvent.

Oli extra vergini di oliva prodotti da varieta autoctone dell'areale di Brisighella: Profilo sensoriale e relazioni con la frazione fenolica

Il progetto sperimentale è stato focalizzato alla valutazione qualitativa di oli extra vergini di oliva monovarietali (Nostrana di Brisighella, Ghiacciola, Don Carlo), prodotti da olive coltivate nell’areale Brisighellese analizzandoli sia per alcuni parametri chimici che per alcune caratteristiche sensoriali rilevabili in fase gustativa (attributi di amaro e piccante). Per ogni campione sono stati valutati i parametri di qualità stabiliti dal Reg. CEE n. 2568/91 e successive modifiche: acidità libera, numero di perossidi, estinzioni specifiche nell’ultravioletto, composizione in acidi grassi ed analisi sensoriale secondo il metodo del “Panel test”. Gli oli sono stati anche sottoposti alla caratterizzazione della frazione biofenolica mediante estrazione secondo metodo COI (COI/T.20/Doc. n.29) seguita da separazione cromatografica HPLC ed identificazione guidata sia da rivelatore a fotodiodi (DAD) che da spettrometro di massa (MSD). I dati ottenuti sono stati elaborati con l’ausilio di tecniche di analisi multivariata (PCA) valutando sia le correlazioni esistenti tra le variabili, che la possibilità di discriminare i campioni in gruppi omogenei per caratteristiche chimiche e/o sensoriali. Più specificatamente, si è rivolta l'attenzione alle relazioni esistenti tra la concentrazione in singole molecole a struttura fenolica e l'intensità degli attributi sensoriali positivi, amaro e piccante. In ultima analisi, si è proceduto a determinare il contenuto totale in derivati fenolici dell’idrossitirosolo e tirosolo (semplici e complessi) presenti in 20 g di olio per verificare la conformità al claim salutistico approvato dall'EFSA nel 2011 “i polifenoli dell’olio di oliva contribuiscono alla protezione dei lipidi ematici dallo stress ossidativo”, che è possibile inserire in etichetta qualora il prodotto soddisfi il limite richiesto dal Reg. UE 432/2012.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Omega-3 fatty acids and the neurovascular responses to mental stress in humans

Hypertension is the most prevalent form of cardiovascular disease (CVD) in the world, and is known to increase the risk for developing other diseases. Recently, the American Heart Association introduced a new classification of blood pressure, prehypertension (PHT). The criteria for PHT include a systolic of 120-139 mmHg and/or a diastolic blood pressure of 80-89 mmHg. It has been observed that individuals with PHT have a higher risk of developing hypertension later in life. Therefore, it is important to understand the mechanisms contributing to PHT in order to possibly prevent hypertension. Omega-3 fatty acids found in fish oils have been suggested as a means of lowering blood pressure. However, little is known on the effects of fish oil in PHT humans. Therefore we conducted two studies. In Study 1 we investigated PHT and normotensive (NT) individuals during a mental stress task. Mental stress is known to contribute to the development of hypertension. In Study 2 PHT and NT subjects were placed in an eight week double-blind placebo controlled study in which subjects consumed 9g/day of either fish oil or placebo (olive oil) in addition to their regular diets. Subjects were tested during a resting baseline (seated and supine), 5 minutes of a mental stress task, and 5 minutes of recovery both pre and post supplementation. We measured arterial pressure (AP), heart rate (HR), muscle sympathetic nerve activity (MSNA), and forearm and calf vascular responses. In Study 1 PHT demonstrated augmented AP and blunted vasodilation during mental stress, but MSNA did not change. In Study 2, fish oil did not directly influence blood pressure, MSNA or vascular responses to mental stress. However, it became clear that fish oil had an effect on some but not all subjects (both PHT and NT). Specifically, subjects who experienced a reduced blood pressure response to fish oil also demonstrated a decrease in MSNA and HR during mental stress. Collectively, the investigations in this dissertation had several novel findings. First, PHT individuals demonstrate an augmented pressor and blunted vascular response to mental stress, a response that may be contributing to the development of hypertension. Second, fish oil does not consistently lower resting blood pressure, but the interindividual responses may be related to MSNA. Third, fish oil attenuated the heart rate and MSNA responses and to mental stress in both PHT and NT. In conclusion, we found that there are both similarities and differences in the way PHT and NT individuals respond to mental stress and fish oil.

Arsenic and selenium mobilisation from organic matter treated mine spoil with and without inorganic fertilisation

Organic matter amendments are applied to contaminated soil to provide a better habitat for re-vegetation and remediation, and olive mill waste compost (OMWC) has been described as a promising material for this aim. We report here the results of an incubation experiment carried out in flooded conditions to study its influence in As and metal solubility in a trace elements contaminated soil. NPK fertilisation and especially organic amendment application resulted in increased As, Se and Cu concentrations in pore water. Independent of the amendment, dimethylarsenic acid (DMA) was the most abundant As species in solution. The application of OMWC increased pore water dissolved organic-carbon (DOC) concentrations, which may explain the observed mobilisation of As, Cu and Se; phosphate added in NPK could also be in part responsible of the mobilisation caused in As. Therefore, the application of soil amendments in mine soils may be particularly problematic in flooded systems.

Using Factorial Surveys and Stated Choice Experiments to Investigate Discriminatory Attitudes and Preferences

Empirical research on discriminatory attitudes and behaviour grapples with the social undesirability of its object. In many studies using regular survey methods, estimates are biased, and the social context of discrimination is not taken into account. Several methods have been developed, especially to deal with the first problem. In this regard, the estimation of the ‘true value’ of discriminatory attitudes is at the centre of interest. However, methodological contributions focusing on the social context of attitude communication and discriminatory behaviour, as well as the correlation between both, are rare. We present two experimental methods which address those issues: factorial surveys and stated choice experiments. In a first study, the usefulness of factorial surveys is demonstrated with data on German anti-Semitism (N=279). We show that the rate of approval with anti-Semitic statements increases if (a) respondents are told that the majority of fellows agree with such statements, (b) the term “Jews” is replaced by the term “Israelis”, and (c) reference to the Holocaust is made. Apart from the main effects of these experimental factors, significant interaction effects regarding the political attitudes and social status of respondents are observed. In a second study, a stated choice experiment on the purchase of olive oil and tomatoes was conducted in Germany (N=440). We find that respondents prefer Italian and Dutch products (control treatment) compared to Israeli and Palestinian ones (discrimination treatments). There are no significant differences between preferences for a so called ‘Peace product’ (which is produced jointly by Israelis and Palestinians) and products from Italy as well as the Netherlands. Yet, taking discriminatory attitudes (anti-Semitic and anti-Arabic attitudes) into account, a strong correlation between those attitudes and stated behaviour (purchase of Israeli, Palestinian and jointly produced products) can be found. This adds support to the hypothesis that discriminatory attitudes hold behavioural consequences.