T.F. Martínez

Characterization of α-amylase activity in five species of Mediterranean sparid fishes (Sparidae, Teleostei)

Abstract In the present study, amylase activity existing in the gut of different species of sparid fish was measured and characterized. The study was conducted on five species of Mediterranean sparids, (F. Sparidae), some of which coexist in the same coastal waters; Pagrus pagrus , Pagellus erytrhinus , P. bogaraveo , Boops boops and Diplodus annularis . Main differences were found in the optimal pH for amylase activity as well as in sensitivity to temperature, with resistance to heating very low in B. boops and very high in D. annularis . PAGE zymograms revealed the presence of two isoforms in all the species, while the analysis of extracts using IEF showed a range from one to three active bands. It is suggested that differences in amylase activity are related to variations in feeding habits of the studied fish, and could also represent an ecological advantage for those species showing a more diversified amylase equipment.

Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.

Improved detection of amylase activity by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with copolymerized starch

An improved method, based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

Characterization of digestive carbohydrase activity in the gilthead seabream (Sparus aurata)

The presence of digestive carbohydrases was determined in seabream intestines, and the main activity identified was amylase. Optimum activity for this enzyme was found at pH 8.0 at 40 °C. Amylase activity was highly sensitive to extreme pH, and temperatures exceeding 50 °C. The use of SDS–PAGE zymograms allowed identification of amylase in the form of a high molecular mass fraction exceeding 100 kDa. Results confirm the existence of a well developed amylase equipment in this species which supports the possibility of increasing the amount of carbohydrates in the formulation of its commercial feeds.

Freezing and Freezing-Thawing Cycles on Textural and Biochemical Changes of Meagre (Argyrosomus regius, L) Fillets During Further Cold Storage

The effects of freezing and freezing-thawing cycles during cold storage have been studied in meagre (Argyrosomus regius) fillets. Fillets were subjected to three conservation protocols: fresh, freezing at –20°C, and repeated freezing-thawing cycles. Fresh fillets were stored (4°C, 15 days), and the same protocol was followed for freezing and freezing-thawing after the freezing period. Freezing and freezing-thawing fillets were softer and presented lower water holding capacity than fresh, mostly attributable to collagen solubilization, and partial myofibrillar protein degradation. Cold storage (4°C) during 15 days caused softening in fresh and frozen fillets attributable to myofibrillar protein hydrolysis. Freezing-thawing cycles increased proteolysis, this leading to unacceptable softening even from early stages of further cold storage, and this was also revealed by SDS-PAGE.

Influence of pre-slaughtering feed restriction on muscle characteristics of farmed sea bass (Dicentrarchus labrax L.) during cold storage

BACKGROUND This paper deals with the consequences of dietary restriction or complete starvation before slaughtering on the biochemical and textural characteristics of sea bass muscle. RESULTS Results showed that only severe feed restriction influenced negatively total body and individual organ weights, and these animals showed lower condition factor as well. Neither moderate feed restriction (up to 50% of the standard ration) kept for 30 days nor total starvation up to 12 days caused significant effects on fish weight and fillet yield. Muscle lipid content was lower in feed-restricted fish, although this parameter was not altered by starvation time. Differences between the two feeding strategies studied were observed in muscle textural and biochemical parameters, and the results point to an influence of the nutritional status on the post-mortem evolution of collagen and myofibrillar proteins, although firmness was not modified. CONCLUSIONS Moderate feed restriction prior to slaughtering could be advisable in sea bass culture, given that no detrimental effects on fish quality or fish performance were noticed, whereas substantial amounts of feed can be saved.

Effects of vacuum and modified atmosphere on textural parameters and structural proteins of cultured meagre (Argyrosomus regius) fillets.

The influence of two preservation strategies (vacuum package and modified atmosphere package) on the post-mortem changes of textural parameters, pH, water holding capacity, sarcoplasmic and myofibrillar proteins, and collagen content of meagre (Argyrosomus regius) fillets was studied. Fillets were stored in a cold room in aerobic (control, C), vacuum (V) and modified atmosphere (MA) package. Samples were withdrawn at six sampling points throughout 15-day storage, and post-mortem changes were assessed. The textural parameters were significantly enhanced in V and MA compared to C. Both V and MA treatments reduced the intensity of a group of myofibrillar protein fractions (140–195 kDa) and increased insoluble collagen compared to C. Consequently, the post-mortem flesh softening in C was attributed to increased proteolysis in both intracellular and extracellular structural proteins. The preservation of the textural and biochemical characteristics of meagre fillets subjected to V and MA treatments makes these two treatments highly recommendable for the commercialization of meagre fillets.

Feed pellets containing chitosan nanoparticles as plasmid DNA oral delivery system for fish: In vivo assessment in gilthead sea bream (Sparus aurata) juveniles

&NA; The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMV&bgr;) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme &bgr;‐galactosidase (&bgr;‐gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme &bgr;‐gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against &bgr;‐gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (&bgr;‐gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on‐farm mass immunization purposes, especially with regard to DNA‐based vaccines and small size fish, in which i. m. administration remains unfeasible. Graphical abstract Figure. No Caption available. HighlightsOral administration is the ideal route for mass vaccination in fish.Plasmid‐based vaccines are readily inactivated within the digestive tract of fish.If protected in adequate nanoparticles, plasmids can resist feed processing.Feed‐delivered plasmids exhibited remarkable in vivo biological activity.Feed pellets enable non‐disturbing, efficient oral delivery of plasmid DNA.