Antimicrobial Properties of Ethylene Vinyl Alcohol/Epsilon-Polylysine Films and Their Application in Surimi Preservation

[EN] Polymer films based on ethylene vinyl copolymers (EVOH) containing a 29 % (EVOH 29) and a 44 % molar percentage of ethylene (EVOH 44), and incorporating epsilon-polylysine (EPL) at 0 %, 1 %, 5 % and 10 % were successfully made by casting. The optical properties and the amount of EPL released from the films to phosphate buffer at pH 7.5 were evaluated, films showing great transparency and those of EVOH 29 copolymer releasing a greater amount of EPL. The antimicrobial properties of the resulting films were tested in vitro against different foodborne microorganisms and in vivo in surimi sticks. With regard to the antimicrobial capacity tested in vitro in liquid medium at 37 A degrees C and 4 A degrees C against Listeria monocytogenes and Escherichia coli over a period of 72 h, films showed a considerable growth inhibitory effect against both pathogens, more notably against L. monocytogenes, and being EVOH 29 more effective than EVOH 44 films. At 37 A degrees C, total growth inhibition was observed for EVOH 29 films incorporating 10 % EPL against both microorganisms whereas the copolymer EVOH 44 did show total inhibition against L. monocytogenes and the growth of E. coli was reduced by 6.64 log units. At 4 A degrees C, no film was able to inhibit completely bacterial growth. Scanning electron microscopy micrographs showed corrugated cell surfaces with blisters and bubbles, and collapse of the cells appearing shorter and more compact after treatment with EPL. Finally, the films were successfully used to increase the shelf life of surimi sticks. The results show the films developed have a great potential for active food packaging applications.The authors acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness, projects AGL2012-39920-C03-01, and fellowship funding for V. M.-G.Muriel-Galet, V.; Lopez-Carballo, G.; Gavara Clemente, R.; Hernández-Muñoz, P. (2014). 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Shelf-life and Molecular Traceability of Fish Product

The subject of my PhD course regards two aspects of fish products quality and characterization. The first one deals with the evaluation of the effects of natural antioxidants (ellagic acid and L-ascorbic acid) in enhancing fresh sole (Solea solea) shelf life.\ud Quality assurance in the fish sector involves monitoring and documenting defined quality criteria as required by regulations (European Community, 1996), product specifications and consumer demands. Fresh fish products are commonly sold at storage temperature. However, many undesirable changes can occur in the products during storage, due to microbial growth and fish spoilage, affording evident economic loss. From this point of view it is important to develop systems which help to prolong fish shelf life. In this perspective, we have studied the effects of ellagic acid (EA) and L-ascorbic acid (LAA)/\ud sodium ascorbate (SA), either alone or in combination, on the microbiological quality of fresh finfish Solea solea during storage at 0 °C. The results showed that addition of EA (0.03%) alone or in combination with L-AA (1.71%)/SA (1.98%) significantly delayed the proliferation of aerobic plate counts, psychrotrophic counts and Pseudomonas bacteria\ud extending the product shelf life up to 10 days, versus 8 days for control. During the storage, the pH increase is slowed down when the fish samples are subjected to the\ud treatment with preservative agents. The effect of lowering the pH increase is particularly pronounced when a combination of EA and L-AA/SA is used. Therefore, EA alone or in combination with L-AA and SA can be successfully utilized to reduce the microbial growth, extending the shelf life of fish during storage at 0 °C.\ud The second aspect of fish products quality and characterization studied in this PhD course deals with the identification of “species-specific genetic markers” of red mullet (M. barbatus) as useful tools for “the commercial designation of species”.\ud Consumers demand for a deeper knowledge of the quality and the origin of food is increasing significantly. The UE with the adoption of Reg. (EC) 2065/2001, laying down detailed rules for the application of Reg. (EC) 104/2000, wanted to give a strong push to the " traceability of fish products" through the labelling systems. European Council Regulation (EC) 104/2000, on the common organization of the markets in fishery and aquaculture products, specifies that these products may not be offered for retail use unless they are labelled with “the commercial name of the species”, “the production method” and “the capture zone”. Particularly, in Italy "the commercial designation" must respect the list\ud present in the enclosure A of the D.M. 27-03-02 and in the following modifications and integrations of the D.M. 25/07/2005. Therefore, the development of analytical methods for species identification is necessary to detect and avoid wilful, as well as unintentional substitution of different fish and shellfish species and to enforce labelling regulations (Mackie et al., 1999).\ud The main aim of this study was the development of a PCR-based assay for the identification of red mullet (M. barbatus) and striped red mullet (M. surmuletus), and was\ud projected on the locus 16S rDNA gene fragment of the 3 Mullid genera (Mullus, Upeneus and Pseudopeneus), widespread in the Mediterranean Sea.\ud The second aim was the investigation of the possible polymorphisms able to detect different populations of red mullet (M. barbatus) in the Mediterranean Sea.\ud The PCR-assay developed can be adequately used for “species identification” of red mullet (M. barbatus) and striped red mullet (M. surmuletus). This approach is rapid and offers the potential to detect fraudulent or unintentional mislabeling of seafood, but can be particularly indicated for application to “processed products”.\ud The randomness of genetic differences among samples indicated that the Adriatic red mullet stock probably belongs to single population unit, according to Garoia et. al., 2004.\u

Valutazione degli effetti quali- e quantitativi di diverse tecniche estrattive sulla frazione lipidica della Scapharca inaequivalvis

La Scapharca inaequivalvis è un’importante fonte alimentare soprattutto di EPA e DHA, acidi grassi appartenenti alla serie omega-3. L’utilizzo a livello industriale della Scapharca inaequivalvis ai fini estrattivi degli acidi grassi omega-3, è di sicuro interesse commerciale considerato il basso costo della materia prima che può quindi garantire un deciso incremento dei margini industriali ed un maggior valore aggiunto dei prodotti finiti. Nello studio è stata valutata l’efficacia di diversi metodi e diversi solventi nell’estrazione della frazione lipidica, dalla Scapharca inaequivalvis. Sono state inoltre determinate le composizioni degli acidi grassi dei vari estratti al fine di valutare il migliore processo estrattivo in relazione al contenuto di acidi grassi polinsaturi delle serie omega-3 e omega-6

Inhibition of microbiological activity during sole (Solea solea L.) chilled storage by applying ellagic and ascorbic acids

Fresh fish products are commonly sold at storage temperature. However, many undesirable changes can occur in the products during storage, due to microbial growth and fish spoilage, affording evident economic loss. The effects of ellagic acid (EA) and L-ascorbic acid (L-AA)/sodium ascorbate (SA), either alone or in combination, on the microbiological quality of fresh finfish Solea solea during storage at 0°C, were investigated. The results showed that addition of EA (0.03%) alone or in combination with L-AA (1.71%)/SA (1.98%) significantly delayed the proliferation of aerobic plate counts, psychrotrophic counts and Pseudomonas bacteria extending the product shelf life up to 10 days, versus 8 days for control. During the storage, the pH increase is slowed down when the fish samples are subjected to the treatment with preservative agents. The effect of lowering the pH increase is particularly pronounced when a combination of EA and L-AA/SA is used. Therefore, EA alone or in combination with L-AA and SA can be successfully utilized to reduce the microbial growth, extending the shelf life of fish during storage at 0°C

Valutazione della contaminazione da olio minerale in specie ittiche del Mare Adriatico

La contaminazione da parte di oli minerali è un problema che interessa moltissime matrici alimentari dato il numero molto vasto di applicazioni che questi derivati del petrolio possono avere. Una matrice poco studiata da questo punto di vista, sono i prodotti ittici, nonostante il loro elevato valore nutritivo ed il loro importante ruolo nella dieta mediterranea. Pertanto, abbiamo voluto portare un contributo in questo settore avviando un’approfondita ricerca. Gli alcani (ramificati, ciclici e lineari), che rappresentano i principali costituenti degli oli minerali, sono stati quantificati in diverse specie ittiche del mare Adriatico (alici, sgombri, sarde, cefali, triglie). Dopo saponificazione dell’estratto esanico, la frazione di alcani è stata isolata dalla frazione insaponificabile attraverso SPE ed in seguito analizzata attraverso GC-FID. Il contenuto di alcani di origine minerale è risultato non rilevabile nella maggior parte dei campioni di tessuto analizzati, mentre gli organi sono risultati contenere livelli di olio minerale molto più elevati. La metodologia utilizzata ed i risultati ottenuti vengono presentati

EVALUATION OF MINERAL OIL CONTAMINATION IN FISH SPECIES FROM ADRIATIC SEA

Mineral oil contamination concerns potentially any food due to the wide number of applications of these petroleum derivatives.1 Despite its important role in Mediterranean diet and its high nutritional value, fish is a food matrix which has been little investigated from this point of view.2 Thus, our study was aimed at developing a research in this field. Alkanes (branched, cyclic and linear), being the main constituents of mineral oils, were quantified in the tissue and organs of several fish species from Adriatic Sea (anchovies, red mullets, mackerels, sardines, flathead mullets). After saponification of the hexane extract, alkanes fraction has been isolated from insaponifiable matter by SPE3 and then analyzed by GC-FID. Organs contained much higher values of contamination with respect to muscular tissues which resulted in most of the cases containing undetectable amount of mineral oil (lower than 2 mg kg-1. Among the 5 samples of organs analyzed, 2 contained significant levels of mineral oil: 66 mg kg-1 in red mullet extract and 37 mg kg-1 in flathead mullet extract. Contaminating mineral oils were medium and low viscosity oils.4 Thus, since the contamination regards only the nonedible portion, the results are not alarming from the food safety point of view, even if they evidence a certain level of environmental pollution from petroleum derivatives

Comparison of Methods for Lipid Extraction from clam Scapharca inaequivalvis

Clam Scapharca inaequivalvis is a bivalve mollusk of Indo-Pacific origin, currently living also in the Adriatic Sea. Despite its content of valuable nutrients,1 it is not much appreciated as food in Mediterranean area and thus fishermen must pick it out from the clam Camelea gallina.2 Some of its precious nutrients are polyunsaturated fatty acids (PUFA) having a well-known beneficial influence on the vascular system. Considering this mollusk as a possible source of PUFA, our study was aimed at evaluating the efficiency of several methods with respect to the extraction of total lipids and the individual classes of fatty acids (saturated, monounsaturated and polyunsaturated). Conventional methods as Folch, Bligh and Dyer, and Soxhlet extraction with different solvents were tested, but also accelerated solvent extraction (ASE) and the use of low toxicity-solvents were evaluated. The highest yield of total lipid extract was 7.0 % (duplicate averaged, in relation to dry sample), obtained using ASE with chloroform/methanol, followed by a washing step with water. The next-highest yield (6.1 %) was afforded by Folch method, while the lowest yields were afforded by extraction with hexane/2-propanol and by Soxhlet extraction with hexane (2.3 %). The estimated percent total fatty acids in the total lipid extract ranged from 19.6 % (Folch method) to 39.4 % (Soxhlet extraction with hexane). The estimated percent PUFA in the total fatty acids ranged from 10.8 % (Soxhlet with hexane) to 22.5 % (ASE with chloroform/methanol). References 1) Piretti, M.V.; Taioli, F.; Pagliuca, G. Comparative Biochemistry and Physiology 1987, 88B, 1201-1208. 2) Pierantozzi, C.; Zambuchini, B.; Giosia, L.; Faini, V.; D‘Angelo, N.; Spina, M.; Ventura, A.; Buttafoco, S.; Sturba, M.; Quagliarini, C. Progress in Nutrition 2005, 7, 1-9

COMPARISON OF METHODS FOR THE EXTRACTION OF LIPIDS FROM CLAM SCAPHARCA INAEQUIVALVIS

Clam Scapharca Inaequivalvis is a mollusk of Indo-Pacific origin, currently living also in the Adriatic Sea, where it is considered undesirable since fishermen must pick it out from the clam Camelea Gallina.1 A possible valorization of this product is to use it as source of polyunsaturated fatty acids (PUFA) having a well known beneficial influence on the vascular system2. Thus, our study aimed at evaluating the efficiency of several extraction methods toward the total lipids and toward the individual fatty acids classes (saturated, monounsaturated and polyunsaturated). Conventional methods as Folch procedure3, Bligh & Dyer4, Soxhlet extraction were tested, but also accelerated solvent extraction (ASE) and the use of solvents with low toxicity were evaluated. The highest yields of total lipid extract were obtained using ASE with CHCl3/MeOH and Folch procedure, giving respectively 1.5 and 1.3 % expressed as average on the fresh sample, while the lowest yields were afforded by hexane/i-PrOH and Soxhlet with hexane giving 0.5 % of extract. The fatty acids composition of each lipid extract is being currently investigated. Results and methodology will be presented and discussed

Uso della PCR nella diagnosi di Mullus barbatus e M. surmuletus

L’UE con l’adozione del Reg. (CE) 2065/2001, recante applicazione Reg. (CE) 104/2000, ha voluto dare una forte spinta alla “rintracciabilità dei prodotti della pesca” mediante il sistema di etichettatura che obbliga il commerciante a fornire le seguenti informazioni sulle caratteristiche dei prodotti ittici messi in vendita: “il metodo di produzione”, “la zona di cattura” e la “denominazione commerciale della specie”, cioè il nome comune o scientifico del prodotto. Per l’Italia quindi “la denominazione commerciale” deve rispettare l’elenco riportato nell’allegato A del D.M. 27-03-02 e successive modifiche e integrazioni del D.M. 25/07/2005. In questo studio si presentano i risultati per l’identificazione di specie di Mullus barbatus e di Mullus surmuletus, ottenuti mediante un sistema di Polymerase Chain Reaction (PCR) multiplex progettato sul locus 16S rRNA dei 3 generi di Mullidi (Mullus, Upeneus e Pseudopeneus), più diffusi nel mediterraneo

Comparison of Eleven Extraction Methods for Quantitative Evaluation of Total Lipids and Fatty Acids in the Clam Anadara inaequivalvis (Bruguière)

The clam Anadara inaequivalvis (Bruguière) is a bivalve mollusc now populating several areas of the Mediterranean Sea seafloor where it competes with other molluscs harvested as food. In fact, even though Anadara inaequivalvis has valuable nutritional properties, it has no commercial value in European countries. The aim of this study was to evaluate quantitatively and qualitatively fatty acid content in this bivalve mollusc and to compare 11 extraction methods: conventional procedures such as Folch, Bligh and Dyer, and Soxhlet with different solvents, as well as accelerated solvent extraction (ASE) and methods using low-toxicity solvents as substitutes for chloroform (CHCl3) and methanol (MeOH). The best method was ASE CHCl3-MeOH, affording yields of total lipids, total fatty acids, and polyunsaturated fatty acids (PUFAs) significantly higher than any other method. The estimated range of total lipids was 2.29%–6.99% (on the dry sample), and the range of fatty acids was 0.74%–1.78%. Yields of polyunsaturated fatty acids (PUFAs) ranged from 0.10% to 0.40%. The solvent mixture hexane–isopropanol as substitute for CHCl3-MeOH did not afford good results. PUFAs were 22.5% in the total fatty acids and the ratio between PUFAs n-3 and PUFA n-6 was 2.74:1