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Questa tesi è dedicata alla qualità dell'alimento ittico in tre delle sue possibili accezioni. Dopo aver spiegato il complicato rapporto del consumatore con gli alimenti ittici e come l'Unione Europea abbia cercato di fare chiarezza al riguardo, gli argomenti di discussione saranno: Autenticazione d'origine La polpa di 160 esemplari di spigola (Dicentrachus labrax), suddivisi tra selvatici, allevati intensivamente e allevati estensivamente, provenienti dall'Italia e dall'estero per un totale di 18 fonti indagate, è stati analizzata individualmente per caratterizzarne la componente lipidica, isotopica e minerale e verificare le potenzialità di queste informazioni ai fini della autenticazione di origine in senso lato. Stima della Freshness Quality Numerosi lotti di seppia (Sepia officinalis), nasello (Merluccius merluccius) e triglia di fango (Mullus barbatus) sono stati sottoposti a due possibili modalità di stoccaggio sotto ghiaccio fondente, per indagare come, nell’arco della loro vita commerciale, ne evolvessero importanti connotati chimici (cataboliti dell’ATP e loro rapporti), fisici (proprietà dielettriche dei tessuti) e sensoriali (Quality Index Methods specie-specifici. Studio del profilo nutrizionale La componente lipidica di numerosi lotti di mazzancolla (Penaeus kerathurus), canocchia (Squilla mantis) e seppia (Sepia officinalis) è stata caratterizzata allo stato crudo e dopo cottura secondo tecniche “dedicate” per stabilire il contributo di queste matrici come fonte di acidi grassi polinsaturi della serie omega 3 e per pervenire alla determinazione dei loro coefficienti di ritenzione vera. / This dissertation revolves around finfish and shellfish quality according to three quite different approaches, preceded by an in-depth study of the complicated relationship between Seafood and the Consumer. Origin authentication The flesh from 160 specimens of seabass (Dicentrarchus labrax), either caught in the wild or reared at different density and coming from 18 possible sources, was individually analyzed as to lipid content and composition, macro- and micro-elemental concentrations, as well as nitrogen and carbon stable isotopic abundances. The aim was to verify if and to what extent each homogeneous set of variables was able to discriminate among seabass origin, latu sensu. Freshness quality evaluation Three complete storage experiments were conducted using several batches of European common cuttlefish (Sepia officinalis), European hake (Merluccius merluccius) and red mullet (Mullus barbatus). Specimens were stored under melting ice according to two different theses until the end of their shelf life, during which chemical determinations (i.e. ATP and related compounds, as well as pertinent ratios), physical measurements (i.e. dielectric properties) and sensory assessments (species-specific Quality Index Methods) were made, in order to ascertain which trait evolved much in the way the sensory traits did, as the leading parameters Nutritional profiling Several batches of caramote prawns (Penaeus kerathurus), mantis shrimps (Squilla mantis) and European common cuttlefish (Sepia officinalis) were analyzed as to their lipid content and composition both at the raw state and upon cooking following a specific method (steam cooking, pan frying in a non-stick pan and pressure cooking, in the very same order), the aim of this research being twofold: to determine the fatty acid content and composition of these low-fat species and to gain knowledge of the true retention coefficients of these nutrients.

Salmonella and Campylobacter are common causes of human gastroenteritis. Their epidemiology is complex and a multi-tiered approach to control is needed, taking into account the different reservoirs, pathways and risk factors. In this thesis, trends in human gastroenteritis and food-borne outbreak notifications in Italy were explored. Moreover, the improved sensitivity of two recently-implemented regional surveillance systems in Lombardy and Piedmont was evidenced, providing a basis for improving notification at the national level. Trends in human Salmonella serovars were explored: serovars Enteritidis and Infantis decreased, Typhimurium remained stable and 4,[5],12:i:-, Derby and Napoli increased, suggesting that sources of infection have changed over time. Attribution analysis identified pigs as the main source of human salmonellosis in Italy, accounting for 43–60% of infections, followed by Gallus gallus (18–34%). Attributions to pigs and Gallus gallus showed increasing and decreasing trends, respectively. Potential bias and sampling issues related to the use of non-local/non-recent multilocus sequence typing (MLST) data in Campylobacter jejuni/coli source attribution using the Asymmetric Island (AI) model were investigated. As MLST data become increasingly dissimilar with increasing geographical/temporal distance, attributions to sources not sampled close to human cases can be underestimated. A combined case-control and source attribution analysis was developed to investigate risk factors for human Campylobacter jejuni/coli infection of chicken, ruminant, environmental, pet and exotic origin in The Netherlands. Most infections (~87%) were attributed to chicken and cattle. Individuals infected from different reservoirs had different associated risk factors: chicken consumption increased the risk for chicken-attributed infections; animal contact, barbecuing, tripe consumption, and never/seldom chicken consumption increased that for ruminant-attributed infections; game consumption and attending swimming pools increased that for environment-attributed infections; and dog ownership increased that for environment- and pet-attributed infections. Person-to-person contacts around holiday periods were risk factors for infections with exotic strains, putatively introduced by returning travellers.

La presenza di Escherichia coli produttori di verocitotossine (VTEC o STEC) rappresenta una tra le più importanti cause di malattia alimentare attualmente presenti in Europa. La sua presenza negli allevamenti di animali destinati alla produzione di alimenti rappresenta un importante rischio per la salute del consumatore. In conseguenza di comuni contaminazioni che si realizzano nel corso della macellazione, della mungitura i VTEC possono essere presenti nelle carni e nel latte e rappresentano un grave rischio se la preparazione per il consumo o i processi di lavorazione non comportano trattamenti in grado d’inattivarli (es. carni crude o poco cotte, latte non pastorizzato, formaggi freschi a latte crudo). La contaminazione dei campi coltivati conseguente alla dispersione di letame o attraverso acque contaminate può veicolare questi stipiti che sono normalmente albergati nell’intestino di ruminanti (domestici e selvatici) e anche prodotti vegetali consumati crudi, succhi e perfino sementi sono stati implicati in gravi episodi di malattia con gravi manifestazioni enteriche e complicazioni in grado di causare quadri patologici gravi e anche la morte. Stipiti di VTEC patogeni ingeriti con gli alimenti possono causare sintomi gastroenterici, con diarrea acquosa o emorragica (nel 50% dei casi), crampi addominali, febbre lieve e in una percentuale più bassa nausea e vomito. In alcuni casi (circa 5-10%) l’infezione gastroenterica si complica con manifestazioni tossiemiche caratterizzate da Sindrome Emolitico Uremica (SEU o HUS) con anemia emolitica, insufficienza renale grave e coinvolgimento neurologico o con una porpora trombotica trombocitopenica. Il tasso di mortalità dei pazienti che presentano l’infezione da E. coli è inferiore all’1%. I dati forniti dall’ECDC sulle infezioni alimentari nel periodo 2006-2010 hanno evidenziato un trend in leggero aumento del numero di infezioni a partire dal 2007. L’obiettivo degli studi condotti è quello di valutare la prevalenza ed il comportamento dei VTEC per una analisi del rischio più approfondita. / The presence of verotoxigenic Escherichia coli (VTEC or STEC) is one of the most important causes of disease food currently present in Europe. Its presence in the breeding of animals for food production is a major risk to the health of the consumer. As a result of common influences that occur during slaughter and milking VTEC can be found in meat and milk and pose a serious risk if the preparation for consumption or processing do not involve treatments in the degree of inactivation (eg. raw meat or undercooked, unpasteurized milk, soft cheeses made from raw milk). The contamination of fields resulting from the dispersion of manure or through contaminated water can carry these strains that are normally present in intestine of ruminants (domestic and wild) and also vegetables eaten raw, juices and even seeds have been implicated in serious cases of enteric disease with severe manifestations and complications can cause serious pathological and even death. VTEC strains ingested with food can cause gastrointestinal symptoms as watery or hemorrhagic diarrhea, abdominal cramps, mild fever and a lower percentage nausea and vomiting. In some cases (about 5-10%) infection is complicated by gastrointestinal manifestations characterized by Hemolytic Uremic Syndrome (HUS or HUS) with hemolytic anemia, severe renal and neurological involvement or with thrombotic thrombocytopenic purpura. The mortality rate of patients with infection with E. coli is less than 1%. The data provided by the ECDC on food-borne infections in the period 2006-2010 showed a trend of slight increase in the number of infections from 2007. The aim of these studies was to evaluate the prevalence and behavior of VTEC for a more in-depth risk analysis.

Perfluoroalkylated substances are a group of chemicals that have been largely employed during the last 60 years in several applications, widely spreading and accumulating in the environment due to their extreme resistance to degradation. As a consequence, they have been found also in various types of food as well as in drinking water, proving that they can easily reach humans through the diet. The available information concerning their adverse effects on health has recently increased the interest towards these contaminants and highlighted the importance of investigating all the potential sources of human exposure, among which diet was proved to be the most relevant. This need has been underlined by the European Union through Recommendation 2010/161/EU: in this document, Member States were called to monitor their presence of in food, producing accurate estimations of human exposure. The purpose of the research presented in this thesis, which is the result of a partnership between an Italian and a French laboratory, was to develop reliable tools for the analysis of these pollutants in food, to be used for generating data on potentially contaminated matrices. An efficient method based on liquid chromatography-mass spectrometry for the detection of 16 different perfluorinated compounds in milk has been validated in accordance with current European regulation guidelines (2002/657/EC) and is currently under evaluation for ISO 17025 accreditation. The proposed technique was applied to cow, powder and human breast milk samples from Italy and France to produce a preliminary monitoring on the presence of these contaminants. In accordance with the above mentioned European Recommendation, this project led also to the development of a promising technique for the quantification of some precursors of these substances in fish. This method showed extremely satisfying performances in terms of linearity and limits of detection, and will be useful for future surveys.

Le sostanze perfluoralchiliche (PFAS), composti fluorurati ampiamente utilizzati negli ultimi anni in diverse applicazioni industriali e commerciali, sono ritrovati diffusamente nell’ambiente e in diverse specie animali. Recentemente i PFAS hanno destato preoccupazione anche per la salute umana. Il rischio di esposizione è principalmente legato alla dieta (i prodotti ittici sembrano essere gli alimenti più contaminati). Lo scopo di questo lavoro è stato quello di valutare la presenza del perfluorottanosulfonato (PFOS) e dell’acido perfluorottanoico (PFOA), in diverse matrici alimentari: latte vaccino commercialmente disponibile in Italia, latte materno italiano, diverse specie di pesce commercialmente disponibili in Italia e 140 branzini di diverse aree (principalmente Mediterraneo). I campioni di latte sono stati trattati con estrazione liquido-liquido seguita da due fasi di purificazione mediante cartucce SPE prima dell’iniezione nell’UPLC-MS/MS. L’analisi del latte vaccino ha evidenziato una contaminazione diffusa di PFOS, ma a basse concentrazioni (fino a 97 ng/L), mentre il PFOA è stato ritrovato raramente. In questo studio, in grado di individuare anche i livelli delle ultra-tracce, sono state osservate nel latte materno concentrazioni di 15-288 ng/L per il PFOS e di 24-241 ng/LPFOA. Le concentrazioni e le frequenze più alte, per entrambi i PFAS, sono stati ritrovate in campioni di latte forniti da donne primipare, suggerendo un rischio di esposizione per i primogeniti. Il metodo utilizzato per i campioni di pesce era basato su un’estrazione con solvente organico seguita da due fasi di purificazione: una con i sali e una con fase solida dispersiva. L’estratto, analizzato in UPLC-MS/MS, ha confermato la contaminazione di questa matrice a livelli significativi, ma anche l’alta variabilità delle concentrazioni misurate. Il monitoraggio monospecie ha mostrato una contaminazione rilevante (PFOS 11,1- > 10000 ng/L; PFOA < 9-487 ng/L), soprattutto nei branzini pescati, rispetto a quelli allevati. / Perfluoroalkyl substances (PFASs) are fully-fluorinated compounds widely employed for the production of different industrial and consumer applications. They have been frequently found in the environment and several animal species. These emerging contaminants have recently become of relevant concern for human health. The risk of exposure to PFASs is mainly related to diet; fish and other seafood seem to be the most contaminated foods. The aim of this work was to monitor the presence of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the two most important and investigated PFASs, in various food matrices: cow milk commercially available in Italy, Italian breast milk, different species of fish commercially available in Italy and 140 sea basses collected from different areas (mainly Mediterranean Sea). Milk samples were treated with liquid-liquid extraction followed by two purification steps through SPE cartridges before UPLC-MS/MS analysis. The analysis of cow milk demonstrated that a contamination by PFOS was often present, but at low concentrations (up to 97 ng/L), and PFOA was rarely found. This study, capable to detect these contaminants at ultra-trace levels, has found concentrations for human milk in the range 15-288 ng/L for PFOS and 24-241 ng/L for PFOA. The highest concentrations and frequencies of both PFAS were found in milk samples provided by primiparous women, suggesting the higher risk of exposure for first-borns. The method used for fish samples was based on extraction with organic solvent followed by two purification steps: one with salts and one with dispersive solid phase. The extract analyzed by UPLC-MS/MS confirmed the contamination of this matrix at significant levels, but also the high variability of measured concentrations. The single species monitoring showed a relevant contamination (PFOS 11,1- > 10000 ng/L and PFOA < 9- 487 ng/L) in particular in fished basses compared to the reared ones.

La presenza di micotossine nelle materie prime desta grande preoccupazione a causa delle importanti implicazioni nella sicurezza di alimenti e mangimi. Lo scopo di questo lavoro è stato quello di mettere a punto e validare una metodica analitica rapida e semplice, in cromatografia liquida ad ultra prestazione accoppiata a spettrometria di massa-tandem (UPLC-MS/MS), per la determinazione simultanea di differenti micotossine: aflatossine (B1, B2, G1, G2), ocratossina A, fumonisine (B1, B2), deossinivalenolo e zearalenone in matrici biologiche. Il metodo sviluppato per l’analisi di campioni di mangime secco per cani ha mostrato prestazioni adeguate ed è stato applicato a 49 campioni reperibili in commercio, al fine di valutare la sua efficacia e di ottenere alcuni dati preliminari sulla contaminazione da micotossine in alimenti per cani disponibili sul mercato italiano. Lo studio ha evidenziato una percentuale alta di campioni positivi, contenenti principalmente fumonisine, deossinivalenolo e ocratossina A; tutti i tenori si sono dimostrati inferiori al limite di legge previsto (Racc. CE 576/2006). Una seconda metodica è stata messa a punto e validata per l’identificazione e la quantificazione micotossine in campioni di formaggio; per questa matrice è stata inserita anche l’aflatossina M1, specifica dei prodotti lattiero - caseari. Le differenti proprietà chimico-fisiche degli analiti e la complessità della matrice hanno implicato alcune difficoltà nello sviluppo della metodica. Tuttavia, il metodo validato si è mostrato rapido, semplice ed affidabile ed è stato applicato a diversi tipi di formaggi per verificarne la versatilità. I risultati preliminari hanno mostrato l’assenza di contaminazione da parte delle micotossine in oggetto. Entrambi i metodi si sono dimostrati utili per il monitoraggio di contaminanti in matrici complesse ad oggi ancora poco studiate. / The occurrence of mycotoxins contamination in various crops is of major concern as it has major implications for food and feed safety. The aim of the study was the set up and the validation of fast and simple methods by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) for the simultaneous determination of different mycotoxins: aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (FB1, FB2), deoxynivalenol and zearalenone in biological matrices. A method was developed for the analysis of samples of dried pet-food; the method showed appropriate performances and was applied to 49 commercial samples in order to test its efficacy and gain some preliminary data about mycotoxins contamination in dog food available in the Italian market. The results have shown a quite high percentage of positive samples, mainly contaminated by fumonisin, deoxynivalenol and ochratoxin A. All the observed values were below the limits specified by regulations (Rec. CE 2006/576). A second method was set up and validated for the identification and quantification of mycotoxins in cheese samples; for this matrix it was included also the aflatoxin M1, which is specific of dairy products. Different chemical-physical properties of the analytes and the complex composition of the matrix led to some difficulties to the development of the method. Finally, the method validated has proved to be rapid, simple and reliable and was applied to different types of cheeses to prove its versatility. Preliminary results have shown the absence of all mycotoxins detected. Both methods are useful tools for the monitoring of mycotoxins contamination in unconventional matrices, not subjected to wide studies until now.

Foodborne diseases impact human health and economies worldwide in terms of health care and productivity loss. Prevention is necessary and methods to detect, isolate and quantify foodborne pathogens play a fundamental role, changing continuously to face microorganisms and food production evolution. Official methods are mainly based on microorganisms growth in different media and their isolation on selective agars followed by confirmation of presumptive colonies through biochemical and serological test. A complete identification requires form 7 to 10 days. Over the last decades, new molecular techniques based on antibodies and nucleic acids allow a more accurate typing and a faster detection and quantification. The present thesis aims to apply molecular techniques to improve official methods performances regarding two pathogens: Shiga-like Toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In 2011, a new strain of STEC belonging to the serogroup O104 provoked a large outbreak. Therefore, the development of a method to detect and isolate STEC O104 is demanded. The first objective of this work is the detection, isolation and identification of STEC O104 in sprouts artificially contaminated. Multiplex PCR assays and antibodies anti-O104 incorporated in reagents for immunomagnetic separation and latex agglutination were employed. Contamination levels of less than 1 CFU/g were detected. Multiplex PCR assays permitted a rapid screening of enriched food samples and identification of isolated colonies. Immunomagnetic separation and latex agglutination allowed a high sensitivity and rapid identification of O104 antigen, respectively. The development of a rapid method to detect and quantify Listeria monocytogenes, a high-risk pathogen, is the second objective. Detection of 1 CFU/ml and quantification of 10–1,000 CFU/ml in raw milk were achieved by a sample pretreatment step and quantitative PCR in about 3h. L. monocytogenes growth in raw milk was also evaluated.

La produzione normativa degli ultimi decenni in campo alimentare permette di evidenziare come sussista una sempre crescente attenzione verso la scienza da parte del diritto e la costante tendenza della dottrina a ripensare, alla luce delle evoluzioni registrate, il legame esistente tra ambiti distinti ma legati tra loro in modo indissolubile. In una globale progressione della sensibilità del legislatore europeo verso le esigenze di garanzia del corretto funzionamento del mercato nonché di una crescente tutela del consumatore e della sua sicurezza, la normativa in materia di nuovi alimenti ha cercato di trovare un equilibrio tra la spinta innovatrice che, da anni, interessa l’intero settore alimentare e le plurime necessità di operatori e consumatori che richiedono prodotti dalle caratteristiche sempre più definite e idonee a soddisfare un ampio ventaglio di preferenze. Assumendo che “la capacità della tecnica è la potenza effettiva di realizzare indefinitamente scopi e di soddisfare indefinitamente bisogni” ben si comprende come quello dei novel food risulti essere un caso paradigmatico di incontro tra scienza, bisogni del mercato e regole giuridiche e come si sia esteso l’interesse sul tema, portando a porre interrogativi sempre più complessi anche in relazione alle modalità di regolamentazione delle novità in campo scientifico. / The last decades' lawmaking in the food field highlights the increasing attention towards science and got lawyers rethinking about the link existing between law and science. In a progression of the European legislator's sensitivity to guarantee the market as well as the food consumers and their safety, the EU legislation on novel foods has tried to balance food innovation and the multiple needs of operators and consumers who require products with increasingly more defined characteristics and suitable to satisfy a wide range of preferences. If "the capacity of the technique is the effective power to achieve goals and to satisfy needs indefinitely" is easy to understand how the novel food turns out to be a paradigmatic case of an encounter between science, market needs and juridical rules. Furthermore, the increasing interest in the food sector raises the question of which methods of regulation of scientific innovations apply.

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.