The Mediterranean diet is rich in healthy substances such as fibres, vitamins and phenols. Often these molecules are lost during food processing. Olive oil milling waste waters, brans, grape skins are some of the most relevant agri-food by-products in the Mediterranean countries. These wastes are still rich in extremely valuable molecules, such as phenolic antioxidants, that have several interesting health promoting properties. Using innovative environmental friendly technologies based in the rational use of enzymatic treatment is possible to obtain from agri-food by-products new ingredients containing antioxidants that can be used as functional ingredients in order to produce fortified foods. These foods, having health protecting/promoting properties, on top of the traditional nutritional properties, are attracting consumer’s attentions due to the increasing awareness on health protection through prevention. The use of these new ingredients in different food preparation was studied in order to evaluate the effects that the food processing might have on the antioxidant fraction, the effect of these ingredient on foods appearances as well as the impact in terms of taste and scent, crucial feature for the acceptability of the final product. Using these new ingredients was possible to produce antioxidant bred, pasta, cheese, cookies and ice-cream. These food products retains very well the antioxidant properties conferred by the added ingredients despite the very different treatments that were performed. The food obtained had a good palatability and in some cases the final product had also a good success on the market.

Il formaggio sta dimostrando possedere, oltre alle caratteristiche nutrizionali classiche, degli aspetti nutrizionali innovati derivanti dalle proprietà di peptidi bioattivi contenuti nella frazione proteica caseinica del formaggio e rilasciati in seguito a proteolisi, ed ancora dalle proprietà di acidi grassi insaturi, quale l’acido linoleico coniugato. L’obiettivo di tale studio di dottorato è stato indagare su questi aspetti nutrizionali innovativi in formaggi tipici italiani quali Grana Padano e TrentinGrana, che pur essendo due formaggi molto simili tra loro, differiscono per l’uso della molecola antibatterica lisozima solo per la produzione del Grana Padano. Simulazioni in vitro della digestione gastrointestinale, nei campioni dei due formaggi a diversi tempi di stagionatura, ha dimostrato che esiste una relazione positiva tra tempo di stagionatura e digeribilità del calcio nel Grana Padano ,quando vengono considerati nella correlazione i risultati dei campioni con stagionatura superiore ai 24 mesi. A tempi di stagionatura inferiori a 24 mesi, i risultati di digeribilità del calcio del Grana Padano sono dispersi come appare nei campioni di TrentinGrana analizzati. Inoltre l’analisi RP-HPLC della distribuzione molecolare degli oligopeptidi dei campioni dei due formaggi, dimostra che la frazione peptidica coinvolta nel legame del calcio e quindi nel suo assorbimento, è quella compresa tra 1000 e 1500 D, e che l’analisi Seldi ha rilevato essere quella dei peptidi attivi fosfocaseinici. Inoltre differenze tra i due formaggi con e senza lisozima, appaiono solo per campioni tra 15 e 20 mesi di stagionatura, in cui campioni di formaggio senza lisozima appaiono più idrolizzati di quelli che lo contengono. Quindi dai risultati ottenuti appare che le differenze del profilo peptidico apportate dal lisozima non modificano le proprietà dei fosfopeptidi di assorbimento del calcio, forse influenzate da altri fattori che intervengono nella produzione del formaggio. L’attività ACE-inibitoria di abbassamento della pressione arteriosa esercitata da peptidi bioattivi è stata testata nei due formaggi, dimostrando che né il tempo di stagionatura e il grado di proteolisi, né il lisozima sono correlati all’attività ACE-inibitoria. Ed infine per l’importante ruolo che ha il minerale calcio nella dieta, indici di digeribilità del minerale in diversi alimenti sono stati considerati, in modo da poter formulare in maniera corretta diete alimentari coprendo il fabbisogno giornaliero con l’adeguato apporto energetico. I formaggi hanno dimostrato possedere indici di digeribilità del calcio superiori ad altri alimenti vegetali o a base di soia. In particolare il valore di digeribilità del Calcio del Grana Padano calcolato in vitro ha confermato il valore ottenuto in vivo , pari a 80%. / This study aimed to investigate about the not common known nutritional aspects of cheeses, which derive from their chemical components. In fact, in addition to the supply of macronutrient, cheeses are gaining interest as a source of bioactive peptides, of conjugated linoleic acid or for the new insight in the metabolic role of calcium. In vitro simulation of human gastrointestinal digestion revealed that cheeses have an higher digestibility of calcium than other foods, because of their casein-derived bioactive phosphopetides (CPPs) content has the ability to carry calcium minerals and avoid calcium precipitation, making it available for intestinal absorption. The in vitro calcium digestibility was calculated for different foods- cheeses, soya based foods and vegetables- to correct cover calcium requirements with an equilibrate energy intake. Calcium digestibility was also assessed in different ripened time Italian locally produced, semi fat, hard cheeses, Grana Padano and TrentinGrana. The main difference between them is the use or not of lysozyme during manufacturing. In Grana Padano samples, produced using lysozyme, there is a positive relationship between aging and dCa (r2 = 0.27; P<0.05) when sample > 24 months aged are considered (Grana Padano dCa results of samples less 24 months aged are quite widespread) , while in TrentinGrana, produced without the use of lysozyme, no significant correlation has been detected. RP-HPLC distribution analysis of oligopeptides molecular weight of these cheese showed that the only difference between them is that cheeses without lyzozyme, aged between 15 and 20 months, are more hydrolyzed than the same ripened time Grana Padano samples. Moreover the fraction of oligopeptides involved in calcium binding ranges between 1000 and 1500 D. SELDI analysis confirmed CPPs presence in this range. Therefore changes in cheese peptidic profiles probably caused by the use of lysozyme do not influence calcium digestibility because according to this study there is not a connection between change in peptidic profile and calcium digestibility results. The difference in calcium digestibility in Grana Padano samples aged over 24 month results should be probably ascribed also to the influence of other factors occurring during cheese manufacturing. Moreover, ACE-inhibitory activity of bioactive peptides was tested on in vitro digested Grana Padano and TrentinGrana samples with different ripening times. Correlation was not found between ACE-inhibitory activity and proteolysis level in different ripened time samples nor the lysozyme influence in releasing ACE-inhibitory bioactive peptides.

Grape (Vitis vinifera L.) is among the most cultivated plants in the world. Its origin traces back to the Neolithic era, when the first human communities started to domesticate wild Vitis sylvestris L. grapes to produce wines. Domestication modified Vitis vinifera to assume characteristics imparted from the humans, selecting desired traits (e.g. specific aromas), and excluding the undesired ones. This process made this species very different from all the other wild grape species existing around the world, including its progenitor, Vitis sylvestris. Metabolomics is a field of the sciences that comparatively studies the whole metabolite set of two (or more) groups of samples, to point out the chemical diversity and infer on the variability in the metabolic pathways between the groups. Crude metabolomics observation can be often used for hypotheses generation, which need to be confirmed by further experiments. In my case, starting from the grape metabolome project (Mattivi et al. unpublished data), I had the opportunity to put hands on a huge dataset built on the berries of over 100 Vitis vinifera grape varieties, tens of grape interspecific hybrids and few wild grape species analyzed per four years; all included in a single experiment. Starting from this data handling, I designed specific experiments to confirm the hypotheses generated from the observation of the data, to improve compound identification, to give statistical meaning to the differences, to localize the metabolites in the berries and extrapolate further information on the variability existing among the grape genus. The hypotheses formulated were two: 1) several glyco-conjugated volatiles can be detected, identified and quantified in untargeted reverses-phase liquid chromatography-mass spectrometry; 2) The chemical difference between Vitis vinifera and wild grape berries is wider than reported in literature. Furthermore, handling a huge dataset of chemical standards injected under the same conditions of the sample set, I also formulated a third hypothesis: 3) metabolites with similar chemical structures are more likely to generate similar signals in LC-MS, therefore the combined use of the signals can predict the more likely chemical structure of unknown markers. In the first study (chapter 5), the signals putatively corresponding to glycoconjugated volatiles have been first enclosed in a specific portion of the temporal and spectrometric space of the LC-HRMS chromatograms, then they have been subjected to MS/MS analysis and lastly their putative identity have been confirmed through peak intensity correlation between the signals measured in LC-HRMS and GC-MS. In the second study (chapter 6), a multivariate regression model has been built between LC-HRMS signals and the substructures composing the molecular structure of the compounds and its accuracy and efficacy in substructure prediction have been demonstrated. In the third study (chapter 7), I comparatively studied some wild grapes versus some Vitis vinifera varieties separating the basic components of the grape berry (skin, flesh and seeds), with the aim to identify all the detected metabolites that differentiate the two groups, which determine a difference in quality between the wild versus domesticated grapes, especially regarding wine production.

In the recent years, the consumer choices have been focused on health-promoting plant-based food and their preferences are oriented towards regional foodstuff from local productions. Therefore, an important factor for vegetables grown Trentino-Alto Adige (Italy) is to point out the added value of alpine farming to evaluate the nutritional values of farming products. Omics technologies (e.g. genomics, transcriptomics, proteomics and metabolomics) are aimed at investigating the assessment of different pools of molecules and how they are translated into the structure, function, and dynamics of a biological system or systems in order to provide a comprehensive characterization of a specific organism. Research use the omics techniques to exhaustively understand the functionality of food components. Several sophisticated chromatographic methods, spectroscopic techniques and chemometric tools are applied to give an insight into a comprehensive overview of the intrinsic quality, typicality and regionality of specific plant-based foods in the present PhD thesis: apples and potatoes. The quality of these foods is evaluated by quantifying the secondary metabolites to investigate their nutraceutical values. The aim of this PhD project is to use several analytical techniques (LC-MS, UV-VIS) that are capable of comprehensively characterizing the food metabolome with particular emphasis on those components with high nutritional values. The data analysis and data handling of omics data requires advanced bioinformatic, statistical, and chemometric tools. Potatoes and apples are chosen as target matrices for these studies for their relevance in the local economy and for the peculiar chemical composition of particular interest for their health-promoting proprieties. The information is acquired using several sophisticated chromatographic and spectroscopic techniques, such as ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC– MS/MS) and UV/VIS. It is integrated to chemometric approaches (principal component analysis (PCA), partial least square regression (PLS), and data fusion) to achieve a comprehensive targeted chemical characterization. The sampling procedures gathers, in the case of the potatoes study, reference cultivars that may be found in the common retailers of Trentino/Alto-Adige and different production areas, the apples of 22 cultivars were harvest from the fields of the Laimburg Research Centre (Vadena, Italy) to guaranty comparability of the obtained data. Our results may be used as solid foundation for a reliable evaluation of apples and potatoes healthy "potential" value based on cutting-edge techniques, which are capable of providing comprehensive data regarding the alpine food quality parameters with high efficiency and reliability

Gli isomeri del CLA (Acido Linoleico Coniugato) potrebbero avere effetti benefici sulla salute in relazione al raggiungimento di un determinato intake, ma, per quanto ci è dato sapere, non esiste letteratura in Italia riguardante l'effettiva quantità di CLA negli alimenti e al relativo intake nell'uomo. Quindi è stata condotta una quantificazione degli isomeri c9,t11 e t10,c12 negli alimenti disponibili sul mercato italiano, seguita da una stima dell'intake di questi isomeri in una coorte della popolazione italiana attraverso un diario alimentare. Lo step successivo del progetto di ricerca, dovuto al livello molto basso di CLA trovato negli alimenti, è stato di investigare i fattori genetici che influenzano il CLA nella carne, per poter aumentare la concentrazione finale di CLA per il consumatore finale. Quindi gli obiettivi del lavoro sono stati: la stima dell'effettivo intake di CLA in Italia attraverso una completa analisi della concentrazione di c9,t11 e t10,c12 nei prodotti lattiero-caseari e carnei comunemente disponibili, e una valutazione dei fattori genetici coinvolti nella sintesi di CLA nella carne. / CLA could exert some healthy properties in relation to a needed daily intake, but, at our knowledge, no literature in Italy is currently available on the effective CLA amount in foods and on the relative intake in humans. Thus a quantification of c9,t11 and t10,c12 CLA isomers in foods available in Italy large retail-scale trade was performed, followed by an estimation of the isomers intake in a cohort of the Italian population by a food diary. Background 6 The following step of the research project, due to the very low concentration of CLA found in foods, investigated genetic factors affecting CLA in meat, in order to enhance its concentration for final consumers. Thus the aims of the present work were to estimate the effective CLA intake in Italy through a complete analisys of c9,t11 and t10,c12 concentrations in dairy and meat products commonly purchasable, and an evaluation on the genetic factors involved in CLA synthesis in meat.

L’alimentazione proteica risulta di fondamentale importanza per l’ottimizzazione della produttività e per il benessere degli animali di interesse zootecnico. L’identificazione di fonti vegetali alternative e/o complementari a quelle attualmente utilizzate (soia in primis) nel razionamento degli animali si rende necessaria per motivi economici, ambientali e legislativi, cercando di evitare, tuttavia, penalizzazioni sull’appetibilità della razione e sulla risposta produttiva. La ricerca condotta ha riguardato lo studio della composizione nutrizionale di semi di cece (Cicer arietinum) e lupino azzurro (Lupinus angustifolius), con approfondimenti sul contenuto di composti nutrizionalmente attivi e/o antinutrizionali, la valutazione della fermentescibilità ruminale mediante tecniche in vitro, anche alla luce di possibili interazioni genetiche (varietà e linee genetiche) e colturali (epoca e densità di semina). I risultati ottenuti hanno permesso di comprendere eventuali limiti di utilizzo di tali leguminose per l’alimentazione degli animali, in particolar modo dei ruminanti, e di attribuire le fonti di variabilità per la massimizzazione produttiva e qualitativa. / Ruminants protein nutrition is an essential item for optimizing the productivity and welfare of animals. The identification of pulses sources as alternative or complementary to those currently used (primarily soya) in the ration of ruminants is necessary for economic, environmental and legislative reasons, trying to avoid, however, penalties on palatability and productive response. The research focused on the study of the nutritional composition of seeds of chickpea (Cicer arietinum) and blue lupine (Lupinus angustifolius), with insights on the content of nutritionally active compounds and / or antinutritionals, the evaluation of the ruminal fermentability using in vitro techniques, also considering possible genetic interactions (varieties and genetic lines) and farming practices (time and density of sowing). The results of the present study suggest that these pulses could be a potentially good feedstuff contributing at the same time to the energy and protein balance of animal diet. Moreover, the presence of active compounds like tannins at levels that should not be regarded as anti-nutritional, can be useful in ruminant feeding due to the moderating activities exerted by those compounds toward the ruminal degradation of proteins.

Lo scopo della tesi è di caratterizzare i rischi per la salute umana, che derivano dall’inquinamento delle acque (acque sotterranee) e dal cibo (riso) tramite l’applicazione e lo sviluppo di nuove tecniche analitiche. La tecnica “diffusive gradients in thin films (DGT)” con resina a base di ferrihydrite, è stata studiata per l'accumulo delle forme inorganiche (As(III) e As(V)) e organiche (DMA(V) e MMA(V)) di Arsenico in matrici acquose. I DGT sono stati applicati su campioni di acque sotterranee, raccolti in sei diverse città del Nord Italia, dove la concentrazione di As è molto alta. Antimonio (Sb) è associato all’As in diversi studi perché le proprietà fisiche e chimiche di questi due elementi sono simili, ed esso è stato recentemente riconosciuto come contaminante dell'acqua. In questa tesi per la prima volta sono state riportate le prestazioni dettagliate dei DGT con ferrihydrite, impiegati in soluzioni acquose di Sb(III) e Sb(V). L’analisi delle forme chimiche di Sb(III) e Sb(V) in campioni acquosi è stata realizzata con l’applicazione della diluizione isotopica. In generale il riso, a differenza di altri prodotti alimentari di origine terrestre, contiene quantità significative di Arsenico inorganico. L’analisi della speciazione di As è stata realizzata in 70 campioni di riso italiano, rappresentativo di diversi tipi di coltivazione. Le forme più abbondanti nel riso erano As(III) e DMA(V). Inoltre è stato interessante studiare la localizzazione dell’As nel chicco di riso, sottoposto a diversi processi di lavorazione (risone, riso integrale e lavorato con o senza parboiling), perché sia la distribuzione che la presenza delle diverse forme chimiche nel chicco sono fattori chiave che controllano la biodisponibilità del contaminante. La distribuzione dell’ As nei chicchi di riso di due varietà (Gladio e Ronaldo) sottoposti a diversi processi, è stato determinato con Laser Ablation ICP-MS. In realtà il contenuto di As era più alto nei chicchi di riso non-parboiled rispetto a quello parboiled. La relazione tra intensità dell’As e le varie parti del chicco di riso ha rivelato che i livelli di As diminuisco dalla parte esterna verso la posizione centrale, mentre i valori di As sembrano essere simili tra la parte media e interna dei non parboiled. / The aim of this thesis was to explore new analytical techniques as well as to carry out further characterisations of human health risks, which derive from water pollution, in particular groundwater, and food, in particular rice. The prevention of water pollution is an environmental aspect, that includes monitoring of both natural enrichment and outside pollution with routine analysis but also with new techniques, e.g. the application of passive sampling techniques and advanced technologies. The diffusive gradients in thin films (DGT) technique with ferrihydrite adsorbent, has been investigated for the accumulation of different species of Arsenic (As), like Inorganic Species (arsenite and arsenate) and Organic Arsenic (dimethylarsinic and monomethylarsenate) in aqueous matrix. To evaluate the performance of DGT method for accumulation of arsenic species, after deployment in synthetic solutions, DGT devices were carried out on groundwaters collected in six different towns in the North of Italy, where the As concentration is very high. Recently, health effects at arsenic exposures have been observed in areas where levels of inorganic As in drinking water are not excessive. Antimony (Sb) is associated to As in several studies because the physical and chemical properties of these two elements are similar, and it has been recently recognized as water contaminant. In this thesis I reported for the first time detailed performance characteristics of the Fe-oxide gel associated to DGT devices deployed in known aqueous solutions of trivalent and pentavalent Sb. Speciation analysis of Sb(III) and Sb(V) in aqueous samples was performed through extraction and on-line determination of isotope dilution concentration after a chromatographic separation. Generally rice, unlike food products of terrestrial origin, contains significant amounts of inorganic arsenic. Recently some Government Organizations (e.g. EFSA) debated the possibility to set an upper limit for total and inorganic arsenic in rice. Arsenic speciation was realized in 70 Italian rice samples from different representative cultivation conditions. The most abundant forms in rice were As(III) and DMA(V). After that, it was fundamental to investigate the localization of As in rice grains in different processes (raw, brown and milled rice with or without parboiling technique), because both speciation and distribution throughout the grain are key factors controlling bioavailability of the contaminant. The As distribution in rice grains of two varieties (Gladio and Ronaldo) from different processes, was determined by LA-ICP-MS. The distribution of As varied between the various parts of the grains (exterior, medium and interior part). During parboiling, the partial boiling of food as the first step in the cooking process, arsenic might have released from the grain to the boiling water. Thus, parboiling of rice grain may reduce the magnitude of arsenic intake in human body. Actually the As content was higher in non-parboiled rice grain than in parboiled rice. The relationship between As intensities and the different parts of rice grain revealed that As levels decreased from the external part towards the middle position, and then the intensity values seem to be similar between medium and internal part in non parboiled products.

La tesi è divisa in quattro manoscritti. Lo scopo è lo studio di tre aspetti, come casi-studio indipendenti, all’interno della gestione della catena alimentare dai mangimi al latte. Il primo manoscritto riguarda la fortificazione del latte bovino con iodio e selenio allo scopo di migliorare la qualità del latte e dei suoi derivati; il secondo riguarda la contaminzione dei mangimi con melamina come fattore di rischio per l’industria lattiero-casearia e la salute pubblica. Gli ultimi due lavori sono corollari alla qualità del latte derivante dallo iodio: ovvero un confronto analitico tra procedure di determinazione dello iodio in latte crudo, e uno studio di speciazione dello iodio inorganico nel latte crudo fortificato. Il primo lavoro offre informazioni per meglio comprendere i fattori coinvolti nel miglioramento della concentrazione dello iodio e del selenio nel latte a seguito di una fortificazione della dieta animale. Riguardo la melamina, lo studio conferma il passaggio di essa dai mangimi al latte e la sua rapida escrezione anche a livelli bassi di ingestione da parte dell’animale; uno scarso passaggio dal latte al formaggio. Il terzo lavoro mostra che, per analisi dello iodio con ICP-MS, l’estrazione dell’elemento con soluzione d’ammoniaca è una valida alternativa al metodo ufficiale con TMAH. La speciazione dello iodio inorganico nel latte scremato è un’operazione possibile utilizzando delle diluizioni con soluzione d’ammoniaca (quarto manoscritto). / The thesis is divided into 4 manuscripts. The aim is to study three aspects, as independent case studies, within the quality management of feed-for-food system. The first manuscript is about the fortification of feeds with iodine and selenium to improve the quality of cow’s milk and derivatives; the second one investigates the contamination of feeds with melamine as risk factor for dairy industries and human health. The last two works are corollaries to the iodine-linked quality of milk: i.e. a comparison between analytical procedures for the determination of iodine in raw milk, and a speciation study of iodine in fortified milk. The first work helps to better understand factors involved in improvement of milk iodine and selenium concentration and CO following supplementation with inorganic sources. Regarding melamine, the study confirmed the pathway for its transmission from feed to milk and its rapid excretion, also at a very low level of melamine ingestion by cows; and a low transfer from milk to cheese. The third study shows that the ammonia extraction of iodine is a valid alternative to the official method with TMAH for iodine determination in milk, by using ICP-MS. The speciation of inorganic iodine in skimmed milk is feasible by using ammonia dilution (fourth manuscript).