Biochemical Characterization and Comparison of Pancreatic Lipases from the Pacific Bluefin Tuna,Thunnus orientalis; Totoaba,Totoaba macdonaldi; and Striped Bass,Morone saxatilis

Lipolytic activity from pancreatic crude extracts from the Pacific bluefin tuna (PBT), Thunnus orientalis; totoaba, Totoaba macdonaldi; and striped bass, Morone saxatilis, were biochemically characterized to detect possible differences in the digestive capacity of each fish. The maximum activity was at temperatures ranging from 35 to 45 C and at pH 8.0; the activity decreased significantly at a pH lower than 7.0. Lipases from PBT and totoaba showed an increase in activity with the presence of sodium taurocholate (NaTC; 6 mM). However, no increase in activity could be observed when a mixture of bile salts or natural bile extracts was used in the reaction mixture, which was observed in the presence of NaTC. In contrast, the lipase activity from striped bass increased accordingly when NaCl was added at different levels (0–1.5 M). Calcium was not required in the reaction mixture for any of the fish species tested. We concluded that all three lipases have different physicochemical properties and ionic strength and emulsifier requirements to obtain their maximum activity. Striped bass was far too sensitive to NaCl, while totoaba and PBT were sensitive to NaTC. The differences in activity shown here suggest the possibility of studying the use of different additives on feed formulations to maximize lipase efficiency.     

Partial characterization of pyloric-duodenal lipase of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In the present study, we report the isolation and characterization of seabream Sparus aurata pyloric caeca-duodenal lipase. Optimum activity was found at pH 8.5 and salinity of 50 mM NaCl. Lipase activity was sensitive to divalent ions, and extreme pH values (4, 5, and 12), being more stable at alkaline than acid pH. Optimum temperature was found at 50°C, but lipase was stable at temperatures below 40°C. Lipase has a bile salt sodium taurocholate requirement for increased activity. Gradient PAGE electrophoresis revealed the presence of four isoforms with apparent molecular masses of 34, 50, 68, and 84 KDa, respectively. Pyloric-duodenal lipase was able to hydrolyze emulsified alimentary oils. Results confirm the presence of true lipases in Sparus aurata digestive tract.     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Digestive enzyme activity during early larval development of the Cuban gar Atractosteus tristoechus

The ontogenesis of digestive enzymes (proteases, amylases, lipases, and phosphatases) in Cuban gar Atractosteus tristoechus was determined in larvae between 5 and 18 days after hatching (DAH). Variations in specific activities of most enzymes were related to the transition from endogenous to exogenous feeding and to the transition from the larval to the juvenile stage. Alkaline protease activity was not detected until 8 DAH in contrast to acid protease activity, which was quantifiable at 5 DAH. Acid protease activity was consistently higher than alkaline protease activity, indicating the presence of a functional stomach in the early stages of larval development. The acid protease activities of larvae and adults were compared by means of zymogram analysis. Four acid protease bands were found in adults (two more than in larvae). This result is the first time that more than one band of acid proteolytic activity has been found in Lepisosteidae. High lipase activity indicated the importance of lipid utilization, particularly during yolk-sac absorption. In contrast to the other enzymes studied, amylase activity was consistently low, probably due to the strictly carnivorous diet of gar larvae and their low capacity for carbohydrate digestion. High activities of aminopeptidase and acid and alkaline phosphatases suggest intestinal absorption. This result, together with the existence of a short gut and a lower proteolytic activity in the intestine than in the stomach, suggest that most of the proteolytic activity takes place in the stomach, while the primary function of the intestine is nutrient uptake.     

Supplemental effect of bile salts to soybean meal-based diet on growth and feed utilization of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

A feeding experiment was conducted to evaluate the supplemental effect of bile salts to a defatted soybean meal-based non-fish meal diet for rainbow trout Oncorhynchus mykiss. A fish meal-based diet (FM) and two non-fish meal diets with and without bovine bile salts (SC+B and SC, respectively) were fed to fish (13 g initial weight) for 10 weeks. Fish fed diet SC showed inferior growth and feed efficiency, while bile salt supplementation improved the parameters to the same levels as fish fed diet FM. Crude fat and starch digestibility of diet SC-fed fish decreased after the 10-week feeding trial compared to the data obtained with fish that had no experience of the diet. Total biliary bile salt content and intestinal maltase activity of fish fed diet SC were the lowest among treatments, while these parameters were improved by bile salt supplementation. Morphological changes occurred in the distal intestine and liver of the diet SC group, although the histological features of fish fed diet SC+B were similar to those of fish fed diet FM. These results suggest that bile salt supplementation to a soybean meal-based diet improves the nutrient utilization by normalizing digestive processes in rainbow trout.     

温度、pH对泥鳅肠道消化酶活性的影响

以泥鳅(Misgurnus anguillicaudatus)为研究材料,测定不同温度及不同pH条件下肠道中蛋白酶、淀粉酶和脂肪酶的活性。结果表明,随温度、pH值的升高,三种酶活性的变化均表现为先增高后降低。蛋白酶、淀粉酶、脂肪酶的最适温度分别为50℃、50℃、40℃,最适pH值分别为7.0、7.0、7.5。     

Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula

A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀?×?Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.     

Ontogeny of protease, amylase and lipase in the alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus × Oreochromis aureus)

Juvenile tilapias (Oreochromis niloticus × O. aureus), with average body weights of groups I, II and III of 55.14, 122.82, and 225.68 g respectively were used to study changes in the activity of protease, amylase, and lipase in different organs and sections of the alimentary tract. Additionally, tract containing digesta, which is closer to physiological conditions, may help reveal the mechanisms of whole chemical digestion process. Another group of tilapias (mean body weight of 111.81 ± 0.14 g) was used to analyze changes in activity of protease, lipase, and amylase in response to varying pH and temperatures. The activity of enzymes in all digestive organs increased when body weight increased from 55 to 122 g, and the activity further increased when the weights increased from 122 to 225 g. The rates of increase in digestive enzyme activity were significantly greater than the rates of growth of the fish. When the activity was compared in different sections of the tract, a similar regularity was found for the three enzymes in all fish. The highest amounts of activity were seen in the foregut and the lowest in the rectum, with the middle amount of activity in the hindgut. The pH ranges for the maximum activity of tilapia protease in the stomach and intestine were 1.6–3.3 and 9.5–10.5 respectively and the optimal temperature in these organs was 55°C. The pH ranges for the maximum amylase activity in the intestine and hepatopancreas were 6–7 and 7.5 respectively, and high amylase activity in these organs was found at 25–35°C. The activity of lipase in the intestine remained unchanged at pH 6.0–9.0 and with temperatures between 25 and 35°C. These results suggest that the ability of tilapia to utilize feed varies at different growth stages and remains relatively stable with changes in environmental pH and temperature, which should be taken into account when designing feed formulas for tilapia.     

Autolysis of krill protein from North Pacific krill Euphausia pacifica during protein recovery via isoelectric solubilization/precipitation

Autolysis of North Pacific krill protein in acidic and alkaline solubilization during protein recovery via isoelectric solubilization/precipitation was investigated. Pronounced auto-degradation of myosin heavy chain and actin from krill protein was observed at alkaline pH values (pH 9.0–12.0), with maximum autolytic activity at about pH 12.0. Meanwhile, proteolytic activity in krill protein at pH 12.0 was observed at low temperatures, suggesting a possible cause for the autolysis of krill protein during the protein recovery process. Three major proteinases were detected by zymographic analysis of myofibrillar protein, with approximate molecular weights of 26 kDa (KP-1), 18 kDa (KP-2) and 17 kDa (KP-3). KP-3 was active over a pH range from pH 5.0–12.0, suggesting that it may be responsible for the autolysis of krill protein during alkaline-induced protein recovery. Further study on substrate specificity and inhibitory specificity of KP-3 showed that KP-3 is serine type proteinase.     

卵形鲳鲹消化酶活性的研究ⅢpH对幼鱼和成鱼消化酶活性的影响

研究了pH对卵形鲳鲹幼鱼和成鱼3种主要消化酶(蛋白酶、淀粉酶和脂肪酶)活力的影响。实验用鱼暂养7 d后解剖取样,在设计的10个酶反应pH值(2.2,3.2,4.2,5.2,6.2,7.2,8.0,8.6,9.6,10.6)下测定消化酶活力。结果表明:卵形鲳鲹胃内pH为强酸性,幼鱼肠内为中性偏酸性,肝、幽门盲囊和成鱼肠内为中性偏弱碱性。胃蛋白酶的最适pH值幼鱼为2.2,成鱼为3.2;肝、幽门盲囊和肠的蛋白酶活性最适pH值幼鱼为7.2,成鱼为8.0。淀粉酶活性的最适pH值幼鱼为8.0,成鱼为8.6。脂肪酶活性的最适pH值均为弱酸性,幼鱼和成鱼均为6.2。卵形鲳鲹幼鱼和成鱼3种相同消化酶活性的变化趋势相似,并以胃蛋白酶、肠淀粉酶、肠脂肪酶的活力最高。     

温度对沙塘鳢消化酶活力的影响

通过改变反应温度分析了一定pH值下沙塘鳢(Odontobutis obscura)三种主要消化酶活性的变化。结果表明:pH6.0时,沙塘鳢肝胰脏、胃、肠道淀粉酶的最适温度分别为25℃、35℃、25～30℃;肝胰脏和肠道pH7.5时,胃pH2.0时,蛋白酶的最适温度分别为40℃、35℃、35℃;pH7.5时,肝胰脏、胃、肠道脂肪酶的最适温度分别为30℃,40℃,25℃。淀粉酶和脂肪酶活性分布是肝胰脏最高,胃次之,肠道最低;蛋白酶活性分布是胃最高,肝胰脏和肠道稍低。     

温度和pH值对河鲈消化酶活性的影响

研究了离体状态下河鲈(Perca fluviatilis)体内淀粉酶、蛋白酶和脂肪酶在不同温度和pH时的活性变化。结果显示:在河鲈肝胰脏、肠道、胃3部位,淀粉酶的最适温度均为30℃,最适pH值分别为5.0、7.0、8.0;脂肪酶的最适温度均为40℃,最适pH值分别为4.0、3.0、6.0;肝胰脏、肠道蛋白酶的最适温度为50℃,胃蛋白酶的最适温度为40℃。肝胰脏、肠和胃蛋白酶的最适pH值分别6.0、8.0、2.0。在各自最适温度下,肠道中脂肪酶比活力最高,其次是肝胰脏和胃;肝胰脏淀粉酶比活力最高,其次是肠道和胃;肠道中的蛋白酶比活力最高,其次是胃和肝胰脏。     

Effects of fish oil replacement by vegetable oil blend on digestive enzymes and tissue histomorphology of European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) juveniles

The impact of replacing circa 70 % fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca—PC, AI, PI, distal intestine—DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70 % FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology.     

北太平洋金枪鱼延绳钓钓钩垂直分布及浸泡时间对渔获物的影响

为积累黄鳍金枪鱼 (Thunnus albacares) 幼鱼陆基循环水养殖基础数据,测定了不同体质量的黄鳍金枪鱼幼鱼血清、肌肉、肝脏、胃、肠道等组织的酸性磷酸酶 (ACP)、碱性磷酸酶 (AKP)、淀粉酶、脂肪酶、蛋白酶等活性。结果显示,ACP活性在各组织中由高至低为肠道>肌肉>肝脏>血清,AKP活性则为肠道>肝脏>肌肉>血清。随着体质量的增加,ACP活性在肌肉、肠道和肝脏组织中差异较小,而AKP活性在肝脏中呈逐渐上升的趋势,且相邻两组间差异显著 (P<0.05),其在肠道、肌肉中呈波动性变化;黄鳍金枪鱼幼鱼消化酶活性整体稳定,组间差异不显著 (P>0.05),仅在体质量达1 000 g时肠道胰蛋白酶活性显著下降 (P<0.05),随后小范围浮动,组间差异不显著 (P<0.05)。综上,ACP和AKP活性在黄鳍金枪鱼幼鱼组织中较高且稳定,这种稳定的高活性模式可保证机体对磷 (P) 高效分解、吸收、再利用,提供对外源物的持续免疫力;黄鳍金枪鱼在养殖期间营养需求稳定,且蛋白质需求旺盛。     

Dietary modulation of digestive enzymes by the administration of feed additives to rainbow trout,Oncorhynchus mykiss Walbaum

The aim of this study was to assess the metabolic and digestive enzyme activities in rainbow trout, Oncorhynchus mykiss Walbaum fed dietary supplements. The biometric indices and the following enzymes activities were measured: acid protease and pepsin from the stomach homogenates, alkaline phosphatase (AP) from the small intestine and the brush border membrane, total proteolytic enzymes and trypsin from the small intestine and liver/pancreas. Dietary garlic induced a higher pepsin activity in the stomach than lipopolysaccharide (LPS) and ginger. Conversely, dietary ginger and LPS showed a significant difference (P > 0.05) in acid protease activity compared to the controls. There was not any significant difference observed between treated groups and the controls for hepato‐pancreas proteolytic enzyme activity, except for trypsin. AP activity increased significantly with dietary garlic, ginger and LPS compared to the controls.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Comparative Characterization of Enzymatic Digestion from Fish and Soybean Meal from Simulated Digestive Process of Pacific Bluefin Tuna,Thunnus orientalis

The digestive process of the Pacific bluefin tuna (PBT), Thunnus orientalis, was simulated through two phases of in vitro digestion: acidic digestion with porcine pepsin, followed by alkaline digestion with pancreatic crude extract (PCE) obtained from the PBT to hydrolyze fish meal (FM) and soybean meal (SBM) as protein substrates. The crude protein from FM resulted in a lower degree of hydrolysis (73.3%) compared with SBM (79.2%). However, the resulting digested products showed that FM contained 35% more small peptides, with sizes <6.5 kDa than those from the starting material (>150 kDa). The SBM had an increase of only 1.3% in the similar peptide cut‐offs found after hydrolysis. These results suggested that FM appeared to be a better source of protein according to the amount of low‐molecular weight peptides. In addition, the proteolytic activity of PCE showed that 88.9% of its alkaline proteolytic activity corresponded to trypsin and 2.9% corresponded to chymotrypsin activity. The results shown here demonstrate that peptide sizes are important in identifying suitable protein sources for aquafeed production to reinforce the primary results obtained from the in vitro digestibility using the pH‐Stat system. These results also contribute to a better understanding of the digestibility process in aquatic organisms.     

热带印度洋大眼金枪鱼渔场时空分布与温跃层关系

为了解印度洋大眼金枪鱼(Thunnus obesus)温跃层参数适宜分布区间及季节变化,采用Argo浮标剖面温度数据重构热带印度洋各月平均温跃层特征参数,并结合印度洋金枪鱼委员会(IOTC)大眼金枪鱼延绳钓渔业数据,本文绘制了月平均温跃层特征参数和月平均CPUE的空间叠加图,用于分析热带印度洋大眼金枪鱼渔场CPUE时空分布和温跃层特征参数的关系。结果表明,热带印度洋温跃层上界深度、温度和下界深度都具有明显的季节性变化,大眼金枪鱼中心渔场分布和温跃层季节性变化有关。夏季季风期间,高CPUE渔区温跃层上界深度在30~50 m,浅于冬季的50~70 m;温跃层上界温度范围为24~30℃。在冬季季风期间,高CPUE区域对应的温跃层上界温度范围为27~30℃;从马达加斯加岛北部沿非洲大陆至索马里附近海域,温跃层下界深度在170~200 m时的渔区CPUE普遍较高;当深度超过300 m时,CPUE值均非常低。采用频次分析和经验累积分布函数计算其最适温跃层特征参数分布,得出大眼金枪鱼最适温跃层的上界、下界温度范围分别是26~29℃和13~15℃;其上界、下界深度范围分别是30~60 m和140~170 m。文章初步得出印度洋大眼金枪鱼中心渔场温跃层各特征参数的适宜分布区间及季节变化特征,为金枪鱼实际生产作业和资源管理提供理论参考。     

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.     

In vitro amino acid absorption using hydrolysed sardine muscle or soybean meal at different intestinal regions of the Pacific bluefin tuna (Thunnus orientalis)

The amino acid (AA) absorption along the intestinal tract of the Pacific bluefin tuna (Thunnus orientalis) was evaluated using two hydrolysed protein sources (fresh sardine muscle and soybean meal) with the everted intestine technique. Pork pepsin and pancreatic enzyme extract from the bluefin tuna were used to hydrolyse the protein from fresh sardine (FSH) and soybean meal (SMH) under optimal bluefin tuna fish physiological conditions. Both of the hydrolysate solutions were tested within three intestinal sections from the bluefin tuna. The everted intestinal fractions immersed in the hydrolysate solutions were sampled at different times to analyse for AA and absorption rate calculations. Fresh sardine and SMH contained greater amounts of essential amino acids (EAA) than those of non‐essential amino acids (NEAA); however, the profiles of AA absorbed showed higher absorption of NEAA in both cases. Using a similar concentration solution, the absorption rates within the intestinal fractions showed a preferential absorption in the proximal and distal regions for Arg and His when FSH was used. However, the absorption rates for Lys resulted in a decreasing proximal‐to‐distal gradient between the different intestinal regions for FSH and SMH. The possibility of a catabolic role of certain AAs in the enterocytes being able to explain the differences in absorption is discussed.