Effects of dietary protein levels on the activity of the digestive enzyme of albino and normal Apostichopus japonicus (Selenka)

We evaluated the effects of different proportions of dietary protein (5%, 10%, 15%, 20%, 25% and 30% protein) on the activity of digestive enzymes of normal and albino Apostichopus japonicus. The experimental diets were fed for 60 days, the optimal conditions for digestive enzyme activity in sea cucumbers were studied. The optimal temperature for protease was 29.3°C and the optimal pH was 1.8. The optimal temperature for amylase was 34.3°C and the optimal pH was 6.7. The optimal temperature for cellulase was 56°C and the optimal pH was 5.9. The activity of intestinal protease increased at first and then decreased as the proportion of dietary protein increased, reaching the maximum when the proportion of protein was 19.7%. The activity of protease in the intestine of normal sea cucumber was significantly lower than that of albino sea cucumber, and the activity of amylase was significantly higher than that of albino sea cucumber. This study is expected to provide a basis for further explaining the ecological difference of albino and normal A. japonicus.     

Effect of low temperature rearing, using deep-sea water, on gonadal maturation of the short-spined sea urchin, Strongylocentrotus intermedius, in Rausu, Hokkaido

It has long been hoped that sea urchins could be served to visitors to seafood restaurants, hotels, and summer festivals in Rausu, located in the world natural heritage site ??Shiretoko,?? during the summer tourist season. However, to date this has not been feasible because of the sea urchin spawning season (July?CSeptember), during which the quality of the gonads, the edible part of the sea urchin, decreases due to maturation. In this study, we examined the possibility of suppressing gonadal maturation and maintaining high-quality sea urchin gonads by low-temperature rearing using deep-sea water. Unripe sea urchins captured before the spawning season were reared under two temperature conditions from June to September. In those groups reared at ambient temperatures (2.8?C19.6?°C), gametogenesis in both sexes progressed rapidly with increased temperature, and almost all sea urchins reached full maturity by late July. In contrast, in groups reared at low temperatures (2.1?C5.1?°C), gametogenesis progressed slowly and >60?% of the sea urchins did not reach maturity even by early September. The feeding experiment also revealed that feeding with live Saccharina diabolica could increase the gonadal volume efficiently under low-temperature rearing conditions. These results demonstrate that low-temperature rearing, supplemented with feeding, is effective in suppressing gametogenesis to allow for the harvesting of high-quality sea urchins during the summer tourist season.     

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg²⁺, whereas inhibited by Zn²⁺, Ca²⁺ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO₄ ^2? and product HPO₄ ^2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V _max and K _m values were 24.45 μmol/L min and 5.76 mM, respectively.     

Some proteolytic properties of hatching liquid in the sea trout,Salmo trutta m. trutta L.

Proteolytic activity of sea trout hatching liquid was examined towards casein and azocazein as a function of pH and temperature. The optimum pH for caseinolytic and azocaseinolytic activities were 9.4, and 9.0, respectively. At alkaline pH the enzyme was activated by low concentrations of Zn²⁺ ions (10⁻⁵ M). Maximum proteolytic activity of the hatching liquid was observed at 25°C. Temperatures exceeding 30°C caused a rapid reduction in enzyme activity. Proteolytic activity observed at 10°C was approximately 50% of that observed at 25°C. In general, a pseudo-Arrhenius plot indicated a Q₁₀ of 1.6 between 6 and 25°C.     

Purification and properties of a histidine decarboxylase from Staphylococcus epidermidis TYH1 isolated from Japanese fish-miso

Histidine decarboxylase (HDC) from Staphylococcus epidermidis TYH1, a halotolerant histamine-producing bacterium isolated from Japanese fermented fish-miso, was purified to homogeneity for the first time. The enzyme was purified 182-fold from cell-free extracts by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The N-terminal amino acid sequences of two polypeptide chains of 27–30 and 7–9 kDa were highly homologous with those of α- and β-chains of other staphylococcal HDCs. The optimum pH and temperature for the enzyme were 6.0 and 60 °C, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan or ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the V _max and K _m values were 45.5 μmol histamine min^?1 mg^?1 and 1.10 mmol/L, respectively. Moreover, this enzyme was resistant to heat treatment (80 °C for 15 min) and was stable upon freezing at ?30 °C for 7 days. The very similar physiological properties of this enzyme and the almost identical N-terminal amino acid sequence to that of the HDC from S. capitis indicated that this enzyme may be evolutionally highly conserved in the genus Staphylococcus. The biophysical properties of staphylococcal HDC were elucidated using native purified enzyme.     

Characterization of acetylcholinesterase from the gut of sea cucumber Stichopus japonicus

An acetylcholinesterase was purified from the gut of sea cucumber Stichopus japonicus by anion exchange chromatography followed by gel filtration chromatography. The enzyme was purified 35.49-fold with a total yield of 7.73 %. The molecular mass of purified acetylcholinesterase was 68 kDa as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme displayed maximum activity at pH 7.5 and 35 °C with acetylthiocholine iodide as substrate. The enzyme activity appeared to be stable over pH 6.0–8.0 and up to 40 °C. It displayed an apparent Michaelis–Menten behavior in the concentration range from 0.1 to 0.8 mM with K _m values of 0.62 mM for acetylthiocholine iodide and 2.53 mM for butyrylthiocholine iodide. More than 95 % of acetylcholinesterase activity was inhibited by 1 mM eserine or 1,5-bis(4-allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), but only 19.1 % of the activity was inhibited by tetraisopropylpyrophosphoramide (iso-OMPA) at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true acetylcholinesterase. Nevertheless, the purified acetylcholinesterase exhibited insensitivity to substrate inhibition phenomenon. Its biochemical properties were compared with those reported for different species.     

Influence of water temperature on food intake,food efficiency and gross composition of juvenile sea bass,Dicentrarchus labrax

The optimum ration in relation to water temperature has been determined for juvenile sea bass (Dicentrarchus labrax). Fish were reared at 15 and 20°C. The spontaneous food intake was positively related to temperature. Four daily rations were tested at both temperatures. The optimum rations were 1.7% at 20°C and 1.2% at 15°C for fish weighing 20–30 g. The influence of temperature on growth rate, body composition and food conversion was studied. Increasing the temperature improved growth rate and gross conversion efficiency but not gross protein efficiency. The results are discussed in terms of energy utilization.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

Effect of Setting Condition on the Gel-Forming Ability of Rohu

To investigate the setting condition of the gel-forming ability of rohu, optimum setting temperature for strong and weak gels of unwashed and washed rohu gel and optimum setting time for maximum proteolytic activity were investigated. Nine setting temperatures were studied for textural properties and trichloroacetic acid (TCA)-soluble peptide contents. Both unwashed gel (UW-gel) and washed gel (W-gel) showed similar optimum setting conditions for producing a strong gel that was set at 40°C for 30 min, followed by heating at 90°C for 20 min. They displayed different optimum setting conditions for weak gel. Weak gel from the degradation of UW-gel and W-gel formed at 65 and 60°C, respectively. The occurrence of protein degradation of W-gels during setting at 60°C suggested that washing did not remove the endogenous protease, and the degradation of unwashed and washed mince was due to water-soluble protease and myofibril-bound protease, respectively. Eight setting times for maximum proteolytic activity were shown by the TCA-soluble peptide contents, accompanying the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. Both gels had similar results for setting for 120 min.     

Mortality of salmonids cultured at low temperature in sea water

Brook trout, rainbow trout, and Atlantic salmon were acclimated to sea water (30‰ S) and placed during autumn in floating cages in a concrete tide pool. The salmon (mean length ca 26 cm in mid-October) grew about 2 cm before they stopped feeding in December. The brook trout (19 cm) and rainbow trout (21 cm) grew little, if any, during December. Feeding by the rainbow trout and salmon continued until temperature fell below 1°C. The brook trout fed little, if any, during December while temperature fell from 4 to 1°C. Brook trout, particularly the mature individuals, had high mortality rates from the time they were put into sea water. Blood plasma osmolalities indicated that brook trout, but not the other species, were under severe osmotic stress in sea water. Most of the fish (all three species) died in a 4-day period during early January when the water cooled to ?0.7 to ?0.8°C. A temperature of ?0.7°C is a reasonable approximation of the lower lethal temperature for these salmonids at 30‰ salinity. Based on our observations, it is not practicable to hold the above species in sea water which is likely to get colder than 1°C because of poor growth and the risk of mortality.     

Combined effects of acute thermal and hypo-osmotic stresses on osmolality and hsp70, hsp90 and sod expression in the sea cucumber Apostichopus japonicus Selenka

The combined effects of acute temperature and salinity on osmolality, expressions of heat shock proteins mRNA (hsp70, hsp90a and hsp90b) and superoxide dismutase mRNA (sod) were investigated in the sea cucumber Apostichopus japonicus Selenka. There were 12 treatments (combinations of temperature at 16, 20, 24 and 28 °C and salinity at 22, 27 and 32 ppt). In low salinity environments, the osmolality of the sea cucumber’s coelomic fluid decreased immediately and reached osmotic balance within 6 h. The decline of osmolality after 2 h of hypo-osmotic stress was faster at high temperatures (28 °C) than that at low temperatures (16 and 20 °C). Cellular level stress was indicated by up-regulation of hsp70, hsp90s and sod mRNA, and the maximal expression of all genes occurred at 6 h after stresses. The up-regulation of hsps and sod mRNA indicated the emergence of protein denaturation and oxidative damage and also suggested an increase in energy consumption at high temperature and low salinity. These results indicated that high temperature and low salinity could change biochemical pathways and energy budgets and then potentially impair the osmoregulation of the sea cucumber. Therefore, effective ways should be taken (e.g., draining off the upper freshwater, exchanging water and adding man-made sea water) to prevent the damage to sea cucumber culture caused by low salinity induced by rainstorms, especially at high temperature.     

Physiological investigations of a neurotoxin-producing phytoflagellate, Chattonella marina (Raphidophyceae)

The effects of temperature, salinity, light intensity and pH on the growth and morphology of Chattonella marina (Subrahmanyan) Hara & Chihara were examined. Optimal growth was observed at temperatures of 20-25°C, salinities of 20-30%o, light intensities of 60-140 μE m^?2 s^_1 and pH 7.5-8.5. Growth did not occur at temperatures below 15°C or above 30°C, and at salinities below 10%o. The morphology (shape) of the cells was strongly affected by temperature. At 20°C and 25°C, the population occurred mostly in a spindle-like form, whereas at 10°C, 90% of the cells became spherical within 10 days of inoculation and stationary phase cultures consisted entirely of spherical cells. Morphology was also markedly affected at 30°C. The number of spindle-like cells was highest at 20-30%o and was less at lower salinities. Light intensity and pH did not influence morphology markedly under the range of light intensities (20-180 μE m ^?2 s-^?1) and pH (6.5-8.5) tested.     

Effect of Frozen Storage Temperature on Quality-Related Changes in Rainbow Trout (Oncorhynchus mykiss)

The effect of frozen storage temperature on quality-related parameters of rainbow trout (Oncorhynchus mykiss) muscle was studied in the interval from ?10 to ?80°C on samples stored for 1 to 18 months. The following quantities were measured: drip loss, water holding capacity and water distribution, color, lipid oxidation (thiobarbituric acid-reactive substances, TBARS), and membrane stability (enzyme activity). No effect of temperature on drip loss, water holding capacity, water distribution, or membrane stability was observed for samples stored below ?20°C, whereas storage at ?40°C or lower compared to ?30°C or higher resulted in a reduced level of secondary lipid oxidation (TBARS). No advantage was gained by using temperatures below ?40°C for frozen storage of trout regarding any of the properties investigated.     

Effect of physicochemical factors on the survival of Vibrio parahaemolyticus on fish

Various physicochemical agents were tested for their effects on the survival of Vibrio parahaemolyticus on stored fish (Labeo rohita). When contaminated fish were kept at 0° to 4°C and at ?20°C the number of Vibrio parahaemolyticus was reduced significantly at ?20°C. After heat treatment (65, 70, 75 and 80°C for 1 min) no Vibrio parahaemolyticus was found on fish subjected to 80°C. Ultra-violet radiation resulted in considerable reductions in numbers of Vibrio parahaemolyticus after exposure for 15 min and still greater reductions after 30 min.     

Temperature and dietary starch level affected protein but not starch digestibility in gilthead sea bream juveniles

A study was carried out with gilthead sea bream juveniles to assess the effect of water temperature (18 and 25°C) and dietary pregelatinized starch level (10, 20 and 30%) on digestibility of protein and starch and on the activity of proteolytic and amylolytic enzymes. ADC of pregelatinized starch was very high (>99%) irrespectively of dietary inclusion level, and it was not affected by water temperature. ADC of protein was also high (>90%) but improved at the higher water temperature. Dietary starch interacted with protein digestibility, which decreased as dietary starch level increased. Temperature affected both acid and basic protease activities, with acid protease activity being higher at 25°C and basic protease activity being higher at 18°C. However, total proteolytic activity and amylase activities were not affected by water temperature. Dietary carbohydrate exerted no effect on proteolytic or amylolitic activities. It is concluded that gilthead sea bream juveniles digest pregelatinized starch very efficiently irrespective of water temperature, due to adjustments of amylase activity to cope with temperature differences. Pregelatinized starch interacts negatively with protein digestibility, with the ADC of protein decreasing as dietary starch levels increase.     

Autolysis and Characterization of Sarcoplasmic and Myofibril Associated Proteinases of Oxeye Scad (Selar boops) Muscle

ABSTRACT

The autolytic profile of oxeye scad mince was characterized. Mince showed higher proteolytic activity than washed mince. The highest autolysis was observed at 65 and 60°C for mince and washed mince, respectively. Both mince and washed mince showed the optimum pH for autolysis at pH 9.0, and their activities decreased with increasing NaCl concentration (0–3.5%). Autolysis of washed mince was markedly inhibited by soybean trypsin inhibitor (SBTI), suggesting that myofibril-associated proteinase was serine proteinase. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase having the optimal pH and temperature of 9.0 and 60°C, respectively. The activities were stable at pH range of 8.0–11.0 at 20–40°C. The crude proteinase was inhibited by N-p-tosyl-L-lysine chloromethyl ketone, SBTI, and phenylmethanesulfonyl fluoride, suggesting the predominance of serine proteinases, especially trypsin. NaCl suppressed the activity while β-mercaptoethanol, dithiothreitol, and CaCl₂ activated the activity. Therefore, trypsin-like proteinase is a major endogenous proteinase responsible for autolysis in oxeye scad muscle. The present results can be used as scientific guidelines to predict the gel strength of surimi made from oxeye scad muscle.     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) was 1105.26 s^?1 mM^?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.     

Development of Intermediate Moisture Fish Patties from Minced Rockfish Meat (Sebastes sp.)

Abstract

An Intermediate Moisture (IM) fish patty was formulated from rockfish mince (85.7%), sorbitol (6.0%), soy protein isolate (2.0%), tapioca starch (2.0%), chicken bouillon (1.3%), refined salt (2.5%), and dried seasonings (0.5%). The mixture was dehydrated at 40 °C for 10 h and vacuum packaged. The dried product had a moisture content of 33% and a water activity of 0.82. The desorption isotherm was sigmoid in shape indicating water activity of the product at ambient temperature in the Philippines would remain stable. Seasonings were added to improve the taste and possibly the stability of the product. Sorbitol addition improved the texture, as judged by a sensory panel, as well as the rehydration index of the patties. Humectat and seasonings improved the overall acceptability of the patties by a sensory panel. Product was prepared with and without preservatives (potassium sor-bate and TBHQ), dehydrated at 40 °C for 10 h, vacuum packed and held at 38°C or ?20°C for two months. Moisture content, water activity, pH, rehydration index, TEA value, Hunter-Gardner color, texture profile, amino acid composition, sensory properties and microbial growth were determined at storage intervals. Instrumental and sensory tests showed no significant differences (p < 0.05) among samples stored with or without preservative and at ?20°C vs. 38°C.     

Gel Forming Ability and Other Properties of Eleven Underutilized Tropical Marine Fish Species

Abstract

The gel forming ability and other characteristics of the mince of 11 underutilized marine fish were studied. They were Bombay duck, silverbelly, sea catfish, silver jewfish, jewelled shad, queenfish, Spanish mackerel, hardtail, Indian tuna, tripletail and false conger eel. Mince was prepared from fillet and a portion of the mince was washed two times with cold water (5°C) containing 0.1% NaCl. Both washed and unwashed mince were ground with 3% NaCl. Ground paste was then stuffed into plastic tube and heated for one- and two-step heating. In the one-step heating, the tubes were subjected to 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. In the two-step heating, the tubes were pre-heated at 25°, 30°, 35°, 40°, 50°, 60°, 70° and 80°C for 60, 120 and 180 min. After the pre-heating, the tubes were immediately subjected to 85°C for 30 min. The gel was subjected to puncture, folding, expressible moisture and sensory tests.

Two-step heating distinctly improved the gel strength compared to the one-step heating. The improvement due to two-step heating was more at low preheating temperatures from 25-35°C. Washing improved the texture and color of all of the gels except Bombay duck and decreased the extent of gel-disintegration in silverbelly, queenfish, sea catfish and hardtail. The gels were set optimally at 35°-40°C for most species. Species variation in the disintegration of the gels was observed. Bombay duck mince produced very weak gel. Neither two-step heating nor washing could improve the gel quality of Bombay duck mince. Our data suggested that jewelled shad, queenfish, silver jewfish, sea catfish, tripletail and false conger eel could be suitable as the material for surimi.     

Preliminary studies on the growth and food consumption of the juvenile loggerhead turtle (Caretta caretta L.) in captivity

The effects of temperature and food ration on the growth of juvenile loggerhead turtle, Caretta caretta L. were examined in relation to body weight under controlled conditions in the laboratory. With increasing food ration there was increasing growth rate. The best daily growth rate at 20°C water temperature was 1.87%. Basal metabolism was reached at a daily feed rate of 1.01% body weight at 20.5°C derived from a formula for growth, namely g = Af ? B where g = 0.62f ? 0.63.