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Fishing products are characterized for being highly perishable; therefore, preservation methods are used to retain freshness and quality and extend shelf life. One of the factors that most impact the loss of freshness, quality, and shelf life is storage temperature. This study evaluated the effect of storage temperature (0 ºC and 5 °C) on the quality and shelf life of tilapia (Oreochromis niloticus) muscle during 20 days of storage. Adenosine 5’-triphosphate (ATP) and related compounds, K-value, pH, color, texture, water holding capacity (WHC), total volatile bases (TVB-N), and total count of mesophilic microorganisms were monitored. Both time and storage temperature had a significant effect (P < 0.05) on the K-value, TVB-N, and total count of mesophilic microorganisms. The overall results of this study indicated that the edible quality of tilapia muscle was affected by storage temperature, observing a shelf life of 16 days at 0°C and 8 days at 5 ºC.

Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.

El objetivo de la presente investigación fue evaluar el efecto de la temperatura de almacenamiento (0 y 5ºC) sobre los principales metabolitos y parámetros relacionados con el rigor mortis del músculo de tilapia (Oreochromis niloticus). Se evaluó el índice de rigor (IR), adenosina 5´-trifosfato (ATP), glucógeno, ácido láctico, pH y textura. El análisis del IR se realizó durante 266 h de almacenamiento a las dos temperaturas, mientras que el resto de los parámetros se monitorearon al tiempo 0 y a las 48 h de almacenamiento en ambas temperaturas. Se encontró que tanto la temperatura como el tiempo de almacenamiento presentaron un efecto significativo sobre el IR, siendo más pronunciado a 5°C en comparación al de 0°C.ABSTRACTThe aim of this research was to evaluate the effect of storage temperature (0 and 5°C) on the main metabolites and related parameters to rigor mortis in muscle of tilapia (Oreochromis niloticus). The rigor index (IR), adenosine 5’-triphosphate (ATP), glycogen, lactic acid, pH and texture were evaluated. The IR analysis was performed during 266 hours of storage at the two temperatures, while the rest of the parameters were monitored at 0 and at 48 hours of storage at both temperatures. It was found that both temperature and storage time, had a significant effect on IR, being more pronounced at 5°C compared to 0°C.

El presente trabajo de investigación determinó el efecto de la temperatura estable y oscilante del agua de mar a tres diferentes niveles (21, 24 y 28°C), sobre la condición fisiológica de la almeja mano de león Nodipecten subnodosus. Para ello, se evaluaron los siguientes indicadores fisiológicos: contenido de adenosina trifosfato (ATP), glucógeno y carbohidratos totales, así como índice de rendimiento muscular (IRM) y carga energética adenilada (CEA), los cuales se determinaron en el músculo abductor de organismos extraídos directamente de la laguna y después de la aplicación de los tratamientos. Se encontró que, en el experimento, las concentraciones de ATP, contenido de glucógeno, carbohidratos totales, e IRM disminuyeron significantemente (P

Nanoparticles based on chitosan modified with epigallocatechin gallate (EGCG) were synthetized by nanoprecipitation (EGCG-g-chitosan-P). Chitosan was modified by free-radical-induced grafting, which was verified by Fourier transform infrared (FTIR). Furthermore, the morphology, particle size, polydispersity index, and zeta potential of the nanoparticles were investigated. The grafting degree of EGCG, reactive oxygen species (ROS) production, antibacterial and antioxidant activities of EGCG-g-chitosan-P were evaluated and compared with those of pure EGCG and chitosan nanoparticles (Chitosan-P). FTIR results confirmed the modification of the chitosan with EGCG. The EGCG-g-chitosan-P showed spherical shapes and smoother surfaces than those of Chitosan-P. EGCG content of the grafted chitosan nanoparticles was 330 μg/g. Minimal inhibitory concentration (MIC) of EGCG-g-chitosan-P (15.6 μg/mL) was lower than Chitosan-P (31.2 μg/mL) and EGCG (500 μg/mL) against Pseudomonas fluorescens (p &lt, 0.05). Additionally, EGCG-g-chitosan-P and Chitosan-P presented higher Staphylococcus aureus growth inhibition (100%) than EGCG at the lowest concentration tested. The nanoparticles produced an increase of ROS (p &lt, 0.05) in both bacterial species assayed. Furthermore, EGCG-g-chitosan-P exhibited higher antioxidant activity than that of Chitosan-P (p &lt, 0.05) in 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric-reducing antioxidant power assays. Based on the above results, EGCG-g-chitosan-P shows the potential for food packaging and biomedical applications.

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

The antibacterial activity of low-density polyethylene/adhesive resin (10%)/epigallocatechin gallate (0.03, 0.5, 5, and 10%) extrusion cast films were evaluated against Staphylococcus aureus (gram positive) and Pseudomonas sp. (gram negative) via direct contact and in solid and liquid culture media. The epigallocatechin gallate concentration in the active films was established per the in vitro antibacterial analysis of pure epigallocatechin gallate against S. aureus and Pseudomonas sp. The epigallocatechin gallate migration profile and concentration required to inhibit bacterial growth in broth were determined. In addition, the effects of epigallocatechin gallate and adhesive resin on the mechanical, color, and thermal film properties were investigated. The results indicate that pure epigallocatechin gallate inhibited the growth of both bacteria. However, only the films with 10 wt% epigallocatechin gallate (with and without adhesive resin) induced morphological changes and inhibited the growth of S. aureus (p

Vibrio parahaemolyticus (Vp), a typical microorganism inhabiting marine ecosystems, uses pathogenic virulence molecules such as hemolysins to cause bacterial infections of both human and marine animals. The thermolabile hemolysin VpTLH lyses human erythrocytes by a phospholipase B/A2 enzymatic activity in egg-yolk lecithin. However, few studies have been characterized the biochemical properties and the use of VpTLH as a molecular target for natural compounds as an alternative to control Vp infection. Here, we evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. The highest enzymatic activity was at pH 8 and 50 °C, and it was inactivated by 20 min at 60 °C with Tm = 50.9 °C. Additionally, the flavonoids quercetin, epigallocatechin gallate, and morin inhibited the VpTLH activity with IC50 values of 4.5 µM, 6.3 µM, and 9.9 µM, respectively; while phenolics acids were not effective inhibitors for this enzyme. Boltzmann and Arrhenius equation analysis indicate that VpTLH is a thermolabile enzyme. The inhibition of both enzymatic and hemolytic activities by flavonoids agrees with molecular docking, suggesting that flavonoids could interact with the active site’s amino acids. Future research is necessary to evaluate the antibacterial activity of flavonoids against Vp in vivo.

The antimicrobial activity of films made of poly(l-lactic acid)/poly(l-lactic acid) grafted maleic anhydride(copolymer)/epigallocatechin gallate(EGCG) blend was determined. The effect of epigallocatechin gallate incorporation (0.03, 0.5, 5, and 10 wt%) as a natural antibacterial was determined by direct contact, solid and liquid culture media. The film antimicrobial activity was evaluated against two bacteria (gram-negative: Pseudomonas spp.; gram-positive: Staphylococcus aureus). The copolymer was prepared and characterized by Fourier transform infrared analysis and Molau test. Furthermore, the degree of grafting was determined. The epigallocatechin gallate migration profile through the films were determined and the minimum epigallocatechin gallate concentration in films required to show antibacterial activity was evaluated. The results showed that only the films with 10 wt% epigallocatechin gallate significantly affected ( p

Response surface methodology was employed to optimize the immobilization yield of acidic proteases from Monterey sardine (Sardinops sagax caeurelea) using partially deacetylated chitin as immobiliz...

The products of fishing are highly perishable foods. Thus, it is necessary to seek viable alternatives that help to preserve product freshness and quality and to increase its shelf life. One of these alternatives is the use of extracts with antimicrobial activity obtained from plants, such as tomato, which has been observed to present compounds with antimicrobial activity. The objective of this work was to determine the effect of an extract obtained from the tomato plant and incorporated into an edible chitosan coating on the quality and shelf life of the sierra fish fillet stored on ice for 15 days. For the latter, an extract was prepared with dehydrated tomato plants, which was incorporated by immersion alone or in combination with an edible chitosan-based coating in sierra fish fillets. The following treatments were applied: C (Chitosan), TPE (Tomato Plant Extract), TPE-C (TPE-Chitosan), and control (without chitosan and extract). Color, pH, WHC, ATP-related compounds, and K value were monitored during 15 days of storage on ice. Likewise, the total count of mesophiles was determined. The results indicated that treatments C, TPE, and TPE-C were significantly different (p<0.05) from lot control in terms of the concentration of ATP-related compounds, K value, pH, and total mesophilic count. We concluded that application of the extract alone or in combination with chitosan allows for the improvement of the quality and shelf life of the sierra fish fillet.

Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

The effect of two commercialin vivotransport methods (cardboard boxes and ixtle sacks) on the physiological condition of Japanese oyster (Crassostrea gigas) was evaluated. Total carbohydrates, glycogen, adenosine 5′-triphosphate (ATP) and related products, adenylate energy charge (AEC), and pH of transported oysters in simulated conditions were determined. The results showed that the ATP initial concentration was low from the beginning of the experiment, and AEC decreased in both transport methods. With respect to the total carbohydrates and glycogen, the samples maintained in cardboard box and ixtle sack decreased during transport, respectively. Similarly, significant changes in pH were observed for both methods. Our results showed that physiologically the bestin vivotransporting method for Japanese oyster is in cardboard boxes.

Postmortem changes in blue shrimp (Litopenaeus stylirostris) muscle were studied on the basis of biochemical, chemical, physical, and microbiological changes during an 18 day storage period at 0°C. Adenosine 5′-triphosphate (ATP) content, breakdown products, K-value, pH, trimethylamine nitrogen (TMA-N), total volatile basic nitrogen (TVB-N), water holding capacity (WHC), color, and texture (shear force) changes were examined. Also, total mesophilic and psychrophilic bacterial counts were measured. K-value increased linearly (r2 = 0.98) from an initial value of 1.37 ± 0.59 to 59.42 ± 6.05% at Day 18. Spoilage indicators TVB-N and TMA-N increased from 29.56 ± 1.33 and 0.69 ± 0.25 to 39.04 and 2.04 ± 0.59 mg of N/100 g at Day 18, respectively; meanwhile, the total viable counts of mesophilic and psychrophilic bacteria increased from 3.48 ± 0.44 and 2.61 ± 0.29 log CFU/g to 6.27 ± 0.21 and 7.14 ± 0.39 log CFU/g, respectively, which indicated spoilage at the end of the storage period. The pH, texture, WHC, and...

Trypsin from the viscera of sierra (Scomberomorus sierra) was purified by affinity chromatography on Sepharose-4B coupled to soybean trypsin inhibitor and characterized with respect to its purity, sensitivity to temperature, pH and inhibition. Trypsin was purified from sierra viscera with 11.9-fold and 29.7% yield. The enzyme had a molecular weight of 25.4 kDa estimated by SDS-PAGE and two possible trypsin isoforms were observed in activity gels. Trypsin activity was strongly inhibited by soybean trypsin inhibitor and porcine trypsin inhibitor, showing a partial inhibition by a serine protease inhibitor. The optimal activity of the enzyme was observed at pH 9 and 60C with n-α-benzoyl-dl-arginine-p-nitroanilide as a substrate. The enzyme maintained more than 50% of its activity in temperatures up to 50C and within the pH range of 8–10 for a period of up to 2 h. Practical Applications The potential applications of purified trypsin from tropical sierra fish viscera are those that are already in use for trypsin, such as an aid for hydrolysis of food proteins, as an aid in reducing viscosity of stickwater from fish processing plants, and in obtaining bioactive fractions from protein hydrolisates. Specific uses of this particular trypsin from tropical sierra could be those that comply with the optimum conditions at 60C and pH 9–10 or stability described in the results.

The effect of season and storage at three temperatures (0, 5 and 10 °C) was evaluated on the main metabolites and parameters related to rigor mortis . Adenosine 5′-triphosphate (ATP), lactic acid, octopine, pH and sarcomere length in the adductor muscle of lion's paw scallop Nodipecten subnodosus were determined. Seasonality influenced the initial values of ATP, octopine and pH, however, no effect was noticed during storage, since a similar effect for the three temperatures evaluated in the four seasons was observed. A lower ATP concentration was found during storage at 0 °C than that found in muscle stored at 5 and 10 °C. According to the analyses of lactic acid, octopine and pH, the muscle acidification during rigor mortis was independent of lactic acid production. The greater sarcomere lengths for all seasons were in agreement with the highest ATP concentrations, corresponding to the samples stored at 5 and 10 °C. Therefore, according to the level of myofibrillar contraction, rigor mortis was lower at 5 and 10 °C storage in the four seasons. Hence, it is concluded that the seasonality did not show a significant impact on metabolite and parameter values related to rigor mortis during storage, and that 5 °C is the best storage temperature for the adductor muscles during the initial stage of rigor mortis and then reduce it to 0 °C, if it is focused to the fresh market.

In this work, we report the molecular characterisation of trypsin I (Try I) from Monterey sardine (Sardinops sagax caerulea). Aspects such as thermodynamic activation parameters, molecular model and cDNA-deduced amino acid sequence allow a more in depth understanding of its activity at low temperatures. The analysis of the thermodynamic activation parameters suggests that this molecule is a cold-adapted protease. From the molecular cloning, we deduced the amino acid sequence and predicted a theoretical structural model of sardine Try I with a classical trypsin fold. Cold-adaptation of this enzyme probably comes from amino acid replacement of key residues to improve flexibility at low temperature, thus increasing kcat. The cold-adaptation of sardine Try I opens a wide range of biotechnological applications for this protease and also it is interesting from the structure function relationship point of view of serine protease proteins.

Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess an enormous digestive tract representing about 10% of fish weight. Thus, the aim of this study was to partially characterize proteases from their digestive tracts. Azocasein digestion of the crude extract of intestine at different pH values and temperatures revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C. Incubation assays of the crude extract with inhibitors such as phenyl methyl sulfonyl fluoride, N-α-p-tosyl-l-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin and chymotrypsin are the main alkaline proteinases present. Zymography showed that the crude extract of Pterygoplichthysdisjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116 kDa. Trypsin and chymotrypsin were inhibited by the following ions in decreasing order: Hg2+, Fe2+, Cu2+, Li+, Mg2+, K+, while Mn2+, and Ca2+ had no effect. Activities decreased continuously as the NaCl concentration increased from 0 to 30%. These results constitute important background information for future studies and for the potential biotechnological use of the crude digestive extract from this invasive species.

Ray fish were caught, filleted, and stored in ice. Fillets were analysed for 18 days to determine the chemical, biochemical and physical changes and their relation to the muscle eating quality. Trimethylamine (TMA-N), total volatile bases (TVB-N), ATP content and breakdown products, K value, pH, texture, water-holding capacity (WHC) and colour changes were monitored. At the beginning of the study, the ray fish muscle showed a low concentration of ATP and a high value of inosine 5′-monophosphate (IMP). Regarding to the signs of freshness and deterioration, K value presented an exponential increase (r2 = 0.95) with an initial value of 4.7% and a final value of 47.5%. Furthermore, the TBV-N and TMA-N significantly increased (P

The most important cephalopod resource in the northwestern area of Mexico is the jumbo squid whose postmortem biochemical behavior has been studied in detail. Adenosine triphosphate (ATP) degradation in this organism is different than the other species because of the fast degradation of adenosine monophosphate (AMP) metabolite in mantle. In this research, AMP deaminase from jumbo squid mantle was partially characterized. The enzyme showed an optimum behavior at 50 °C, the enzyme lost more than 90% of its activity within 15 min from 55 to 60 °C, and the enzyme remained stable for 30 min from 10 to 50 °C. It was also stable within the pH range of 3.0–5.0 and exhibited an optimum activity at pH 4.5. Enzyme was strongly activated by Mg+2 and weakly activated by Ca+2. ATP was an excellent activator even in a low concentration, while adenosine diphosphate (ADP) did it at higher concentrations. These results suggest that enzyme might be regulated by the adenylate energy charge.

The biochemical, chemical and physical postmortem changes and their relation to the cazon muscle eating quality were studied during an 18 day storage period at 0 °C (ice). Content of ATP and breakdown products, K value, pH, trimethylamine (TMA-N), total volatile bases (TVB-N), water-holding capacity (WHC), colour and texture changes were examined. At the beginning of the study, the cazon muscle showed a low concentration of ATP and a high value of IMP. Regarding to the signs of freshness and deterioration, K value presented a linear increase (r2 = 0.97) with an initial value of 1.05% and a final value of 58.9%. The TBV-N and TMA-N significantly increased (P < 0.05). As for the physical analysis whereas the pH and the WHC changed (P < 0.05); texture was not affected (P < 0.05). The overall results of this study indicated that the edible quality of cazon fish muscle was maintained during at least 18 days of ice storage.

The enzyme 5′-nucleotidase of jumbo squid ( Dosidicus gigas ) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V max of 1.16 μM/min/mg of protein, K m of 1.49 mM, K cat of 3.48 μM of P ι s −1 and K cat / K m relation of 356.52 ((mol/L) −1 s −1 ). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K m of 6.34 mM, V max of 0.19 μM/min/mg of protein a K cat of 0.3388 mol of P ι s −1 and K cat / K m relation of 53.44 ((mol/L) −1 s −1 ). The low K m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.

Chymotrypsin II from the viscera of Monterey sardine was characterized as an isoform of chymotrypsin I previously characterized from the same source and compared with bovine chymotrypsin. Chymotrypsin II had a molecular weight of 25,500 Da, similar to bovine chymotrypsin. The isoform identity as chymotrypsin was established by its catalytic specificity on the specific substrates succinyl- l -ala-ala-pro- l -phenylalanine-p-nitroanilide and benzoyl- l -tyrosine ethyl ester, showing higher specific activity than bovine chymotrypsin. Both enzymes showed maximal activity at pH 8.0, chymotrypsin II being stable at alkaline pH while bovine chymotrypsin was stable at acid and alkaline pH. Optimum temperature was 45 °C for chymotrypsin II and 55 °C for bovine chymotrypsin. Both enzymes were inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, and partially by N-toluenesulfonyl- l -phenylalanine chloromethyl-ketone. This is valid only in specific conditions of this work. Km and kcat for chymotrypsin II were 0.048 mM and 4.8 s−1, and 0.09 mM and 1.9 s−1 for bovine chymotrypsin. Catalytic efficiency of chymotrypsin II was 4.8-fold higher than bovine chymotrypsin.

SUMMARY T-cell immune response plays an important role in controlling Giardia lamblia infections. Little is known about the G. lamblia-specific antigens that stimulate a cell-mediated immune response. The aim of the present study was to identify T-cell stimulating G. lamblia antigens. For this purpose, we generated a group of Giardia-specific T-cell hybridomas (2F9, 4D5, 6D10, 8B9, 9B10, 10F7 and 10G5). Hybridomas were screened for reactivity with G. lamblia protein extract by the CTLL bioassay. These T-cell hybridomas did not exhibit any significant activation either in the absence of G. lamblia protein extract or in the presence of irrelevant antigen (hen white egg lysozyme). To further characterize the T-cell hybridomas generated, we selected three hybridomas (10G5, 4D5 and 9B10). Giardia lamblia proteins of 90–110, 65–77 and 40–64 kDa showed T-cell stimulating activity for the hybridomas 10G5, 4D5 and 9B10, respectively, in a concentration-dependent manner. Protein extract obtained from different G. lamblia strains (GS/M-83-H7, WB C6 and a clinical isolate (YJJ)) stimulated all T-cell hybridomas, indicating that T-cell-stimulating antigens are expressed among different G. lamblia strains. In conclusion, we identified T-cell stimulating G. lamblia antigens by using Giardia-specific T-cell hybridomas. To our knowledge, these hybridomas are the first-described T-cell hybridomas specific for G. lamblia.

The spiny lobster, Panulirus interruptus, is a valuable crustacean and most of its production is marketed live. In the northern fisheries of Baja California, Mexico, the animals are exposed to short-term emersion at different air temperatures during the live marketing procedures. This work evaluates the effect of short-term emersion (up to 10 h) at two air temperatures (7ºC and 20ºC) and of a 2-h recovery period on ATP, its related metabolites and some muscle quality parameters. Survival was 100% after all emersion times. No significant differences were found either in the adenylic energetic charge or in the levels of ATP and its related metabolites. The highest concentrations were found for AMP, IMP and INO, while ATP had the lowest levels. Muscle quality parameters such as water holding capacity and shear force remained similar to the control values. Although pH decreased slightly with emersion, it was very close to the control values after the 2-h recovery period. The overall results suggest that P. interruptus may perform physiological compensatory work after 10 h emersion even at 20ºC without significantly affecting the muscle quality.

Postmortem biochemical changes of sierra fish muscle kept in ice over a 15-day storage period were investigated. The experimental sample (ES), fish sample handled under controlled postcapture conditions and the commercial sample (CS), handled by domestic fishermen, were thoroughly compared. The K value increased linearly for both samples; however, the slope for the CS sample was 1.5-fold higher than that for the ES. K values after 15 days on ice were 82% and 43% for CS and ES, respectively. Trimethylamine and total volatile bases values remained below critical limits for both samples. Experimental data indicated that commercial postcapture practices previous to full icing of the sample had significant effect on adenosine 5′-triphosphate pattern degradation and freshness loss. It is highly recommended for domestic commercial fishermen and fish retailers to properly ice sierra fish immediately after capture to keep an edible quality for at least 15 days.

The effect of refrigerated 48h transport and 4 days storage on the quality and shelf life of the whole lion's paw scallop Nodipecten subnodosus gonad was evaluated. Proximal composition, adenosine 5´triphosphate (ATP) and related products, K-value, total volatile bases (TVB-N), trimethylamine (TMA-N), pH, fatty acid profile and microbiological analyses were quantified. Gonad holds a significant composition of essential fatty acids while levels of gonadal ATP were initially low; moreover, K-value of the gonad remained constant. With respect to TVB-N and TMA-N, only the former exceeded allowed limits. The pH level showed no significant variation during storage and, despite the high level of TVB-N, according to the TMA-N as well as microbiological analyses it was demonstrated innocuity after 4 days under the transportation and storage conditions utilized. Avaliou-se o efeito do transporte em refrigeração por 48 horas e quatro dias de armazenamento sobre a qualidade e vida de prateleira da gônada do bivalve pata de leão, Nodipecten subnodosus. Determinou-se a composição centesimal, a adenosina 5'trifosfato (ATP) e afins, o índice K, bases voláteis totais (TVB-N), trimetilamina (TMA-N), pH, perfil de ácidos graxos e análise microbiológica. A Gônada apresentou uma importante composição de ácidos graxos essenciais e baixos níveis iniciais de ATP, enquanto o índice K manteve-se constante. Quanto a TVB -N e TMA- N, apenas as primeiras ultrapassaram os limites admissíveis. Os valores de pH não mostraram nenhuma mudança significativa durante o armazenamento e, apesar dos altos níveis de TVB -N, de acordo com a análise quantitativa e microbiológica TMA- N, a segurança do produto foi demonstrada após quatro dias sob as condições de transporte e armazenamento utilizado.

Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl- l -ala-ala-pro- l -pheilalanine- p -nitroanilide and benzoyl- l -tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor N -toluenesulfonyl- l -phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50 °C for the hydrolysis of SAAPNA. The Michaelis–Menten constant was 0.074 mM with a catalysis constant of 18.6 seg −1 , and catalytic efficiency of 252 seg −1 mM −1 . Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents.

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

Postmortem changes of Finescale triggerjish muscle during 20 days of storage at OC were evaluated by monitoring changes in chemical, biochemical and microbiological parameters. The K value increased linearly fi-om an initial value of 3.5% to 43.3% at day 20. pHfluctuatedfiom 6.38 to 6.55. TMA-Nand TYB-N remained below critical limits with final values of 1.18 mg TMA-NAOO g muscle and 29.7 mg TVB-N/lOO g muscle, respectively, suggesting a low microbial activity. Composition of muscle protein fractions was not aflected during the storage period; however, an initial high level of alkali-soluble protein was detected. The overall results indicated that triggerfish has excellent shelf-life on ice.

Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67–91 % range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin–chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.

Recovery of valuable compounds from by-products generated from seafood processing represents an opportunity to produce value-added products. Sardine acidic proteases were immobilized on partially deacetylated chitin through ionic linkages, using sodium tripolyphosphate as a support activating agent. Maximal enzyme activities of free and immobilized proteases were detected at pH 2, at which the immobilized form had higher stability than the free enzymes. Optimum temperatures for free and immobilized proteases were 40 and 50C, respectively, while thermal stability of immobilized proteases was greater than that of free proteases. Reusability studies showed that after a second catalytic cycle, the immobilized enzyme maintained about 40% of the initial activity. The immobilization process enhanced the long term storage stability of sardine proteases compared to free enzymes. Results suggest that the immobilized enzyme could be used as a biotechnological aid for food processing when low pH and temperature around 50C are needed. Practical Applications Fish waste constitutes a serious environmental problem due to high disposal costs. Besides, this waste is commonly discarded or used for low value processes. Therefore, this study proposes the immobilization of sardine (Sardinops sagax caerulea) acidic proteases from fish viscera waste, on a partially deacetylated chitin support extracted from shrimp (Penaeus spp.) head. This research suggests that seafood protease immobilization onto chitinous supports presents an opportunity to improve the stability of these enzymes and consequently enhance potential application of these valuable biocatalysts as biotechnological aid, mainly for food processing and other value added products.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

A 4 × 3 factorial study was conducted to evaluate the effect of four experimental diets (a control diet and a 0, 50, and 100% fish meal replacement diet) and the period of time Nile tilapia, Oreochromis niloticus, were fed those diets (0, 20, and 40 days) on the alkaline proteolytic activity of the animals’ digestive tract, as well as their potential interaction. Significant differences (at P < 0.05) and a significant interaction were observed among dietary treatments for the alkaline proteolytic activity of tilapia after 40 days of feeding. This study confirmed that, under these experimental conditions, a 50% fish meal replacement formulation elicited the highest alkaline proteolytic activity in the digestive tract of tilapia, which resulted in the highest final weight and specific growth rate (SGR), but further research is needed to establish the relative contribution of the alkaline proteases to the overall proteolytic activity of this omnivorous fish species.

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.