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.

The aims of this research study is to explore the opportunity to set up Performance Objectives (POs) parameters for specific risks in RTE products to propose for food industries and food authorities. In fact, even if microbiological criteria for Salmonella and Listeria monocytogenes Ready-to-Eat (RTE) products are included in the European Regulation, these parameters are not risk based and no microbiological criteria for Bacillus cereus in RTE products is present. For these reasons the behaviour of Salmonella enterica in RTE mixed salad, the microbiological characteristics in RTE spelt salad, and the definition of POs for Bacillus cereus and Listeria monocytogenes in RTE spelt salad has been assessed. Based on the data produced can be drawn the following conclusions: 1. A rapid growth of Salmonella enterica may occurr in mixed ingredient salads, and strict temperature control during the production chain of the product is critical. 2. Spelt salad is characterized by the presence of high number of Lactic Acid Bacteria. Listeria spp. and Enterobacteriaceae, on the contrary, did not grow during the shlef life, probably due to the relevant metabolic activity of LAB. 3. The use of spelt and cheese compliant with the suggested POs might significantly reduce the incidence of foodborne intoxications due to Bacillus cereus and Listeria monocytogenes and the proportions of recalls, causing huge economic losses for food companies commercializing RTE products. 4. The approach to calculate the POs values and reported in my work can be easily adapted to different food/risk combination as well as to any changes in the formulation of the same food products. 5. The optimized sampling plans in term of number of samples to collect can be derive in order to verify the compliance to POs values selected.

Seafood is known for its valuable and healthy nutrients; however scientific studies demonstrated the unavoidable presence of contaminants in fish and shellfish. Authorities strongly recommend fish consumption, especially to sensible groups of population (pregnant or nursing women, infants and children), and ask the scientific community for novel and coherent data to establish clear guidelines for consumers. There is also a serious need to extend the monitoring to emerging chemical pollutants, other than methylmercury, dioxins and polychlorinated biphenyls, to better understand the level of seafood contamination. An innovative UHPLC-MS/MS method for the quantification of glycine betaine (GB) in Tapes philippinarum was developed and validated, taking the 2002/657 European Decision as guideline; GB is an endogenous osmolyte abundant in molluscs, and represent a vital methylating agent in humans. Moreover, a previously developed and validated UHPLC-MS/MS method was used to conduct a preliminary monitoring of the presence of the two main perfluorinated contaminants, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), in six fish species from Italian markets (Dicentrarchus labrax, Merluccius merluccius, Mugil cephalus, Pleuronectes platessa, Scomber scombrus and Sparus aurata). The daily contribution of seafood to PFOS and PFOA dietary intake in Italian population was calculated and data were compared to the corresponding Tolerable Daily Intakes established by EFSA in 2008. Results revealed that the risk of contamination related to fish consumption is unlikely, but suggested the need to conduct further surveys focused on certain species from different sampling site. Subsequently, a mono-specie monitoring was carried out in 140 farmed and wild subjects of Dicentrarchus labrax, from 14 different locations in the Mediterranean area. The obtained results showed that wild subjects are sensibly more contaminated than farmed ones, and that levels of fish contamination are strongly influenced by their geographical origin and the proximity to polluted areas.

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The quality of fish products is indispensably linked to the freshness of the raw material modulated by appropriate manipulation and storage conditions, specially the storage temperature after catch. The purpose of the research presented in this thesis, which was largely conducted in the context of a research project funded by Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF), concerned the evaluation of the freshness of farmed and wild fish species, in relation to different storage conditions, under ice (0°C) or at refrigeration temperature (4°C). Several specimens of different species, bogue (Boops boops), red mullet (Mullus barbatus), sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), during storage, under the different temperature conditions adopted, have been examined. The assessed control parameters were physical (texture, through the use of a dynamometer; visual quality using a computer vision system (CVS)), chemical (through footprint metabolomics 1H-NMR) and sensory (Quality Index Method (QIM). Microbiological determinations were also carried out on the species of hake (Merluccius merluccius). In general obtained results confirmed that the temperature of manipulation/conservation is a key factor in maintaining fish freshness. NMR spectroscopy showed to be able to quantify and evaluate the kinetics for unselected compounds during fish degradation, even a posteriori. This can be suitable for the development of new parameters related to quality and freshness. The development of physical methods, particularly the image analysis performed by computer vision system (CVS), for the evaluation of fish degradation, is very promising. Among CVS parameters, skin colour, presence and distribution of gill mucus, and eye shape modification evidenced a high sensibility for the estimation of fish quality loss, as a function of the adopted storage conditions. Particularly the eye concavity index detected on fish eye showed a high positive correlation with total QIM score. / La qualità dei prodotti ittici è indispensabilmente legata alla freschezza della materia prima modulata, dalla manipolazione appropriata dalle condizioni di conservazione, specialmente la temperatura di stoccaggio dopo cattura. Lo scopo della ricerca presentata in questa tesi, che è stata condotta in gran parte nel contesto di un progetto di ricerca finanziato dal Ministero Italiano delle Politiche Agricole, Alimentari e Forestali (MIPAAF), riguarda la valutazione della freschezza di specie ittiche d'allevamento e selvatiche in relazione alle diverse condizioni di stoccaggio (campioni conservati sotto ghiaccio (0°C) o a temperatura di refrigerazione (4°C)). Vari esemplari di specie diverse, quali boga (Boops boops), triglia di fango (Mullus barbatus), orata (Sparus aurata) e spigola (Dicentrarchus labrax)), sono stati esaminati durante lo stoccaggio nelle condizioni adottate. Come parametri di controllo sono state effettuate valutazioni di tipo fisico (texture, attraverso l'uso di un dinamometro; qualità visiva utilizzando un sistema di visione del computer (CVS)), chimico (attraverso (1H-NMR)) e sensoriale (attraverso il quality index method (QIM)). Sulla specie nasello (Merluccius merluccius) sono state effettuate anche determinazioni microbiologiche. L’applicazione di queste tecniche è stata finalizzata alla valutazione delle modificazioni qualitative del prodotto. In generale, i risultati hanno mostrato che la temperatura di manipolazione/conservazione è un fattore chiave per il mantenimento della freschezza del pesce. Spettroscopia NMR ha mostrato di essere in grado di quantificare e valutare la cinetica di composti selezionati durante la degradazione pesce, anche a posteriori. Questo può essere adatto per lo sviluppo di nuovi parametri relativi alla qualità e freschezza. Dalla ricerca è emerso come lo sviluppo di metodi fisici per la valutazione della freschezza del pesce, presenti un elevato potenziale per lo sviluppo di un indice qualitativo strumentale correlato sia con gli indici chimici che sensoriali, al fine di integrare/sostituire i metodi sensoriali attualmente utilizzati.

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Mycotoxins are heterogeneous chemical compounds characterized by a low molecular weight and synthesized by the secondary metabolism of different molds. Fumonisins are water-soluble mycotoxins produced by Fusarium species spoiling corn and derived produc ts. These mycotoxins can be a health hazard when consuming contaminated cereals, but they can reach humans also indirectly through the consumption of food products derived from animals fed with contaminated feed. Fumonisins have been associated with several animal and human diseases: they are suspected risk factors for esophageal and liver cancers, neural tube defects and cardiovascular problems. Improved methods are needed to accurately assess fumonisins concentrations in food of vegetable and animal origin, in order to prevent acute and chronic human exposure. The aim of the present work was to evaluate the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis from foods and matrices of animal origin. Different methods for the identification and quantification of fumonisins and related products have been developed and validated to determine fumonisin B1 in milk, fumonisin B1, fumonisin B2 and their complete hydrolyzed products (HFB1 and HFB2) in pig liver and fumonisins B1 and B2 in complete and complementary dry dog food. The experimental procedures have been carefully studied, considering matrices features, number and type of molecules to detect. Therefore, several extraction, clean up and separation techniques were tested in order to obtain the better conditions of sample processing. The fit for purpose sample preparation, matched with high mass spectrometry sensibility and specificity, have allowed to achieve good results in any tested animal matrices. Hence, the developed methods were validated and have shown a high accuracy, sensibility and precision, fulfilling performance requirements of Decision 2002/657/EC and of European Project Standard, Measuring and Testing (SMT). In any developed method, the analytes were identified and quantified even at very low concentrations : the limits of quantification resulted lower than other similar works, performed with different detectors. These methods were applied to some commercial samples and to some samples collected for research projects in the Department of Veterinary Public Health and Animal Pathology (DVPHAP) of University of Bologna. Although the disclosed data must be considered completely preliminary and without statistical significance, they emphasize the presence of mycotoxins in animal products. The outcomes obtained from the processed samples (bovine milk, pig liver and dry dog food) suggest the efficacy of these methods also on other food matrices, confirming the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis. Moreover the results underline the need to set up a large scale monitoring in order to evaluate the presence of fumonisins in food of animal origin for human consumption.

Listeria monocytogenes è un batterio patogeno responsabile di una malattia potenzialmente molto grave per l’uomo. L’infezione avviene soprattutto tramite l’ingestione di alimenti di origine animale contaminati, e può propagarsi per via transplacentare al feto. Il potenziale patogeno di L. monocytogenes è dovuto soprattutto a caratteristici fattori di virulenza con i quali alcuni ceppi sono in grado di attaccare la cellula dell’organismo ospite potendo aderire, invadere, moltiplicare e propagare alle cellule adiacenti. Il presente studio è rivolto al rilevamento tramite reazione polimerasica a catena (PCR) di alcuni fattori di virulenza di ceppi di L. monocytogenes isolati da campioni prelevati presso macelli suini, mediante l’identificazione dei geni responsabili della sintesi delle proteine di superficie che intervengono nel processo patogenetico, allo scopo di valutare la potenziale pericolosità di quelli isolati sia sulle carcasse, sia dal contenuto intestinale.

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