Authenticity issues and fraudulent practices regarding animal products are affecting consumer confidence. Verifying the description, composition, processing or origin of foods can be challenging. To explore British and Kurdish consumers’ perceptions of kebab meat products, focus groups and questionnaire surveys were applied. About 40% of participants in the UK tend to purchase fewer processed meats after the European horsemeat scandal. Issues raised by participants indicated their concerns about the declaration of species, meat content, and other ingredients incorporated in kebab and other meat products. Lack of consumer trust has been linked to authenticity issues. Reactions towards the addition of fat-replacing inulin were positive by more than half of respondents. A further study aimed to investigate the effect of commercial inulin (CI) and Jerusalem artichoke (JA) tubers as fat replacers on the eating quality and overall acceptability of kebabs. Inulin flour prepared from JA by a simple protocol presented advantages with about 10% higher cooking yield and overall acceptability when compared with CI. Levels of inulin as low as 0.5% were detected in meat products using enzymatic assay, which could be relevant to detect additives and enforce labelling requirements. The authenticity (origin and species) was investigated in fish samples from commercial markets in Erbil, Kurdistan Region of Iraq (KRI). The declared fish species was checked using DNA barcoding with Cytochrome b region. A 10 % rate of mislabelling occurred only for wild common carp (Cyprinus carpio), with 9 out of 12 discovered to be the related species goldfish (Carassius auratus), which was deemed to be accidental rather than deliberate fraud. Such occurrences were from street markets and fishmongers, while none were from supermarkets. Wild and farmed common carp samples were not discriminated by DNA barcoding. Further fingerprinting using compositional profile and nearinfrared spectroscopy (NIRS) together with chemometric analysis aimed to predict composition and discriminate between wild and farmed common carp and species identity. NIRS-predictions of composition and some macrominerals of fish have a strong correlation with the references. NIRS with chemometric analysis is promising, but were not satisfactorily accurate for micro-minerals. Even with no clear solution from principal component analysis (PCA), NIRS-PCA may contribute to discriminating sample groups, but not for authentication when used alone. Having reliable techniques for authentication of food of animal origin may discourage deliberate replacement in retail, wholesale and international trade, and may contribute to reductions in food mislabelling, therefore protecting consumers from fraudulent practices.

The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.

La globalizzazione del mercato agroalimentare ha determinato una crescente attenzione da parte dei consumatori verso i prodotti alimentari, non solo in termini di qualità e di sicurezza, ma anche di origine geografica. Infatti, il territorio d’origine ha un forte impatto sull’alimento, dovuto alle condizioni pedoclimatiche che ne determinano le caratteristiche. Poiché non esistono dei metodi analitici di routine per l’autenticazione della provenienza geografica, lo scopo del progetto di ricerca è quello di determinare l’origine geografica e l’autenticità degli alimenti mediante profiling dei composti fenolici e steroli, grazie all’applicazione di tecnologie omiche, tecniche statistiche e chemometriche. La componente fenolica e/o steroli dei campioni, viene analizzata tramite cromatografia liquida (UHPLC) accoppiata ad uno spettrometro di massa (Q-TOF-MS). I dati così ottenuti, vengono elaborati mediante statistica multivariata. L’applicazione combinata di avanzate tecnologie omiche e tecniche statistiche chemometriche ha portato come risultato l’effettiva identificazione della provenienza geografica e autenticità di numerose matrici alimentari. I dati ottenuti dimostrano che i metaboliti secondari possiedono proprietà discriminanti. L’approccio di metabolomica UHPLC/Q-TOF-MS combinato a una statistica multivariata risulta essere adeguato per identificare potenziali markers. Il lavoro attuale è focalizzato sulla ricerca di nuovi metaboliti che, insieme a fenoli e steroli possano confermare la potenza di questo approccio. / Nowadays, food traceability is a growing consumer interest worldwide. Food traceability could be considered a fundamental tool for ensuring safety and high quality of food. Food quality is based not only on the safety and integrity of food, but also on the authenticity, the genuineness of the raw material and the geographical origin. The aim of the work was to investigate the potential of untargeted metabolomics to ensure the authenticity and traceability of foods. Secondary metabolites, like polyphenols and sterols, could be conveniently used to meet this goal due to their chemical diversity and their responses to environmental stimuli. Samples were analyzed through UHPLC-ESI/QTOF-MS. The obtained data were subjected to multivariate statistical analysis. The obtained results showed that secondary metabolites can be efficiently used for authenticity and traceability purposes, with regards to cultivars and geographical origin. These information confirm the role of environmental factors in shaping the actual profile of secondary metabolites in plant foods. The markers found could be used for a target quantification method, a less expensive and less sophisticated analysis, in order to provide an efficient tool that could help to guarantee food quality on routine basis.

Qualità e sicurezza degli alimenti, inclusa la loro tracciabilità ed autenticità, è diventato ngli ultimi anni obiettivo primario per la salute e il benessere dei consumatori. Il progetto è diretto allo sviluppo e applicazione di metodiche DNA-based per la tracciabilità di specie vegetali, varietà di frumento duro e microorganismi a difesa della qualita’, salubrita’ ed autenticità della filiera grano e prodotti processati. Le attività progettuali dello studio sono finanziate da industria (Barilla S.P.A.) con il coinvolgimento di enti pubblici di ricerca (Università Cattolica Del Sacro Cuore Di Piacenza, CREA-GB di Fiorenzuola D’arda). Il progetto si articola in tre argomenti principali: WP1.Tracciabilità di specie vegetali nella filiera pasta. Tracciabilità di varietà di frumento duro nella filiera pasta A questo scopo sono state intraprese nel secondo anno di attività due azioni dirette allo sviluppo e validazione di due diverse metodiche di fingerprinting varietale. Partendo dalle direttive UPOV in materia di impiego di marcatori molecolari per la caratterizzazione varietale è stata applicata e validata su di un pool di 26 varietà di interesse per l’industria un’ analisi basata su di una combinazione di marcatori SSR (Simple Sequence Repeats) che ha consentito di identificare in maniera univoca ciascuna delle varietà in esame. Il saggio è stato trasferito ai laboratori dell’industria che lo applica attualmente nella routine per il controllo di partite di granella. A fronte della robustezza dell’analisi SSR si pongono però i lunghi tempi analitici. Per ottimizzare questo aspetto si è completata un’attività di sequenziamento parziale del genoma di 28 varietà presenti in due repliche biologiche (52 campioni) e 12 mix di DNA di due varietà (4 differenti percentuali per ogni coppia di varietà miscelate). I dati ottenuti hanno fornito circa 15.000 marcatori molecolari DArT-seq (Diversity Array Technology ) e SNP (Simple Nucleotide Polimorphisms). Dall’intero set di marcatori è stato quindi individuato, attraverso una procedura bioinformatica, un set ridotto di marcatori ad alta informatività in grado di identificare univocamente le singole varietà e di predire la presenza di altre varietà in miscela e la percentuale di contaminazione. WP2.Tracciabilità Di Microrganismi Fungini Nella Filiera Pasta Questo studio è volto al controllo e identificazione di specie microbiche patogene che possono svilupparsi lungo la filiera grano con conseguente impatto negativo sulla salubrità di granella, di semole e dei prodotti finiti. A questo scopo sono stati prodotti campioni di granella a contaminazione controllata. Si è costituita attraverso l’analisi delle sequenze Barcode una ceppoteca che comprende i maggiori patogeni fungini che possono contaminare la granella durante la crescita della pianta in campo o durante lo stoccaggio della granella. Dopo l’inoculo artificiale dei singoli ceppi in due varietà di frumento duro, sono stati raccolti, a tempi crescenti, campioni di spighe e granella. La metodica è risultata rapida e sensibile nell’identificazione di DNA fungino fin dalle prime fasi dell’infezione, quando i sintomi della malattia risultavano ancora non ancora visibili. Le informazioni di sequenza Barcoding, in prospettiva, potranno essere utilizzate per sviluppare nuovi metodi di identificazione fungina più sensibili e rapidi. WP3. Identificazione molecolare di microrganismi vivi o morti in pesto L’obiettivo di questo lavoro è stato quello di sviluppare metodiche di V-qPCR (Viability-PCR), per permettere l’identificazione, quantificazione e discriminazione cellule microbiche vive o morte in alimentiprocessati, come il pesto. Per lo studio è stato scelto il batterio patogeno B.cereus, microrganismo ubiquitario, patogeno e sporigeno di difficile identificazione soprattutto in matrici complesse. Durante questo lavoro è stato sviluppato un protocollo analitico che prevede l’estrazione del DNA batterico da pesto, matrice interferente e complessa. Parte del lavoro è stata svolta presso l’Istituto di Microbiologia dell’Università Cattolica e l’Istituto IATA CSIC Institut d’Agroquímica i Tecnologia dels Aliments in Valencia. / Food quality and safety, including food traceability and authenticity, have become crucial in the last decades. Today, molecular and genetic progress can support the agri-food industry, due to the improvement of new analytical tools. Among the available applications, DNA-based methods can detect the presence of a particular species or variety along the food supply chain, verify the genetic identity of food and feed ingredients and detecting the presence of contaminating organisms, thus becoming an essential tool to study patterns, causes, and risk factors of diseases and outbreaks. As a consequence, genetic analysis has become increasingly popular even among non-specialists and highly beneficial for consumers, agricultural farmers, governments, and the private sector (Reid, O’Donnell, and Downey 2006). In this framework, the research developed in this thesis arises by active collaboration between the private company Barilla G. & R. Fratelli S.p.A., the public research institute CREA-GB (Consiglio per la Ricerca in agricoltura e l'analisi dell Economia Agraria) and Università Cattolica del Sacro Cuore, to develop a set of DNA-based methods to improve the traceability and authenticity of plant and microbial species and durum wheat varieties applicable from farm to fork. Following these aims, the research developed in this thesis includes: 1. The optimization and validation of qPCR assay for the discrimination of plant species along the pasta production chain through the organization of a ring test involving nine Italian public and private laboratories. The results obtained in this study were published in the Journal of Cereal Science (Chapter 2); 2. The discrimination of durum wheat varieties by selecting SSRs and DarT molecular markers as reliable methods for variety fingerprinting (Chapter 3). The results confirm the sensitivity of the method and the feasibility to 7 protect the food industry from fraud and ensure the consumer a certified pasta quality; 3. The application of the Barcoding technique and the development of qPCR assay for the identification and quantification of field fungi (Fusarium, Alternaria, Michrodochium, Cochliobolus spp.) and saprophytic fungi (Aspergillus, Penicillium, Rhizopus spp) along the wheat chain (Chapter 3). The sensitivity of the method was investigated by inoculating potted durum wheat plants at full anthesis and wheat kernels (pre and postharvest trials). The DNA-based methods demonstrate a key role in pathogen detection and the application in several points of the wheat chain (e.g., for control of both locally and imported grains, for storage lots, to evaluate the environmental risk associated with grain powder for farmers and workers); 4. The optimization of Viability q-PCR (V-qPCR) for the discrimination of dead and alive Bacillus cereus, a spore-forming bacteria (Chapter 4). The results of PMAxx, combined with qPCR, have demonstrated the selective discrimination of B.cereus viable cells, with no false-positive signals determined by dead cells, a peculiar aspect of thermally treated food; 5. The comparison of two DNA extraction kits (FastDNA® SPIN Kit for Soil – MB and NucleoSpin Tissue - Macherey Nagel) by detecting B.cereus spores in basil pesto sauce, selected as a model food matrix. Despite the limit of detection (LOD) achieved (respectively 1.8x102 spores/gr by using Fast DNA TM SPIN and 2.7 x 105 spores/gr by using NucleoSpin®), the principal challenge remains the spores' DNA extraction from the complex matrix. Lastly, the results obtained during the doctoral research project were globally discussed (Chapter 5).

The aim of presented diploma thesis was to determination of authenticity of fruit baby foods for early infant feeding using molecular methods. In the experimental part, isolation kit was used for isolation of plant DNA from fruits (strawberry, apricot, raspberry, apple) and from six commercial fruit products for children. Isolated DNA was characterized and verified using PCR methods with primers specific for plant rDNA (ITS2). Specific primer pairs were designed to amplify DNA for the detection of one fruit species. Primer specificity was assessed with four fruit species. A mixture of fruit puree from the two fruits was used to determine the sensitivity of the multiplex PCR assay. Six commercial fruit products were evaluated to verify the applicability of the multiplex PCR assay. The methodology of molecular detection of fruit DNA by qPCR and multiplex qPCR (duplex) includes approaches, which enable to detect two fruits (strawberry-raspberry, apricot-apple) in one reaction and thus reduces time and money requirements.

L'authenticité et la traçabilité des aliments gagnent un intérêt croissant au cours de la dernière décennie puisque la connaissance de la provenance des aliments est considérée comme une garantie supplémentaire de leur qualité. Les consommateurs ont également des inquiétudes et des préoccupations par l'origine de la nourriture qu'ils consomment car divers produits sont sujets à l'adultération ou à la fausse dénomination. L'intérêt accru à l’égard de la protection des consommateurs et de la lutte antifraude ont entraîné un accroissement de la recherche scientifique appliquée et le développement d'outils efficaces pour contrôler l'authenticité des produits alimentaires. Entre techniques analytiques appliquées à l'authenticité et à la traçabilité des aliments, les méthodes les plus prometteuses sont basées sur les empreintes d'éléments lourds mesurées par la spectroscopie atomique. La spectrométrie de masse à multicollection à couplage à plasma induit (MC-ICP-MS) est reconnue comme la méthode optimale pour effectuer des mesures de haute précision de nombreux éléments du tableau périodique en contrôlant simultanément les rapports entre leurs isotopes stables. Cette étude présente une nouvelle stratégie analytique basée sur des isotopes stables non-traditionnels combinés avec des éléments traces déterminés par ICP-MS. Les avantages de combiner les informations de deux systèmes isotopiques, l'un traçant le sol (Sr), et l'autre traçant la pollution environnementale ambiante (Pb), ont permis d'obtenir de nouvelles informations exceptionnelles sur la traçabilité et l'authenticité des matrices alimentaires sélectionnées : vins de Bordeaux, jambons secs et thé. En utilisant des techniques analytiques complémentaires telles que les empreintes des élémentaires traditionnelles, la spécification régionale, ainsi que le traçage du processus de préparation des aliments sont possibles. Traitée par la chimiométrie, cette approche analytique constitue un nouvel outil efficace et prometteur pour détecter des fraudes alimentaires, y compris l’imitation de produits de grande valeur, l’étiquetage erroné et la substitution par des produits moins cher. / Food authenticity and traceability have received an increasing interest during the last decade since the knowledge of food provenance is regarded as an additional warranty of its quality. The world's globalization brought to the consumers is more and more concerned with the origin of the food they eat because various products are subjected to adulteration or false denomination. The augmentative interest in anti-fraud and consumer protection has led to the extension of scientific research and development of effective tools of food authenticity control. Among the analytical technics applied to food authenticity and traceability, one of the most rapidly developing and promising method is based on fingerprinting of heavy elements detected by atomic spectroscopy. The multicollection inductively coupled plasma mass spectrometry (MC-ICP-MS) is recognized as a method of choice for the high precision measurement of numerous elements of the periodic table as well as ratios of their stable isotopes. This study present a new analytical strategy based on combined non-traditional stable isotopes and trace elements determination by ICP-MS. The benefits of combining information from two isotopic systems, one tracing the soil (Sr), and the other tracing environmental ambient pollution (Pb), allowed to obtain an exceptional new information about traceability and authenticity of selected food matrixes: prestigious Bordeaux wines, dry-cured hams and tea. Using complementary analytical techniques such as traditional elemental fingerprinting, the regional specification, as well as tracing of the food preparation process are possible. When combined with chemometrics, these analytical advances constitute an efficient and promising tool to detect food frauds, including adulteration of high value products with cheaper substitutes, forgery and falsification.