双酶法在鲨鱼肉水解液制备中的工艺改良研究

采用同时添加枯草杆菌中性蛋白酶与木瓜蛋白酶的改良酶解技术，对鲨鱼肉进行水解。正交试验结果表明，上述二酶同时水解的最适条件为：温度45℃，pH7．0，时间3h，酶浓度200U／ml（枯草杆菌中性蛋白酶及木瓜蛋白酶各100U／ml），原料：水＝1．5。蛋白质水解率达83．5％，所得水认的氨基酸含量为45．4mg/ml，其中必需氨基酸总量为19．8mg/ml。     

利用酶法制备菲律宾蛤仔抗肿瘤肽的研究

为确定酶解法提取菲律宾蛤仔(Ruditapes philippinarum)抗肿瘤活性多肽的最佳工艺条件,选用5种蛋白酶在相同的料液比、酶解时间和酶添加量、各自最适温度和pH的条件下酶解菲律宾蛤仔。通过甲醛电位滴定法和Cell counting kit-8(CCK-8)方法分别测定了菲律宾蛤仔酶解液氨基态氮含量和酶解多肽的抗肿瘤活性,分析了氨基态氮含量与A549抑制率之间的相关性。在单因素试验的基础上,以人非小细胞肺癌A549抑制率为指标,正交分析优化酶解工艺条件。结果显示,菲律宾蛤仔的5种酶解产物中,风味蛋白酶的酶解液中氨基态氮含量最高,而胃蛋白酶获得的酶解多肽抗肿瘤活性最高。胃蛋白酶酶解菲律宾蛤仔正交实验确定酶解最佳工艺条件为:料液比(w/w)1∶1,pH 2.0,酶解时间1 h,酶添加量2 000 U/g,酶解温度37℃。当设定最佳酶解工艺获得的酶解多肽浓度为20 mg/m L时,其对A549的抑制率为99.83%。研究表明,酶的种类对菲律宾蛤仔酶解液氨基态氮含量和酶解多肽的抗肿瘤活性有较大影响,菲律宾蛤仔酶解液中氨基态氮含量与A549抑制率没有直接的正相关性。     

鲢酶解物对羟自由基的清除作用

通过测定酶解物对Fenton体系产生的羟自由基的清除效果，从胰蛋白酶、木瓜蛋白酶、胃蛋白酶、枯草杆菌蛋白酶和复合蛋白酶5种酶中，筛选出木瓜蛋白酶和胰蛋白酶作为酶解鲢制备具有较高清除羟自由基活性酶解物的理想水解酶；用正交试验L9(3^4)对两种酶的水解条件进行了优化，并对最佳酶解条件下得到的酶解物进行Sephadex G-25凝胶柱分离，洗脱液分别在波长280nm处比色，测定酶解物中主要抗氧化活性肽的分子量分布。结果表明，木瓜蛋白酶在温度50℃、酶解时间15min、pH=6．5、酶质量分数1．50％、底物：水=1：2的水解条件下，酶解物对羟自由基清除效果较好，清除率为88．2％；胰蛋白酶在温度55℃、酶解时间60min、pH=8．0、酶质量分数0．25％、底物：水=1：2的水解条件下，酶解物对羟自由基清除效果较好，清除率为84．2％。木瓜蛋白酶酶解物在最大洗脱峰时有最大羟自由基清除率峰，清除率为95．1％，在最大峰处酶解物中活性肽的分子量为2．2kDa；胰蛋白酶酶解物在最大洗脱峰时也有最大羟自由基清除率峰，其清除率为89．6％，该峰处活性肽的分子量为14．2kDa。     

紫菜降血压肽酶法制备工艺的优化

摘要：降血压肽又称为血管紧张素转化酶抑制肽，是具有降血压活性的生物活性肽，对高血压患者有显著疗效。为探讨从紫菜中制备降血压肽的酶法工艺，本实验选用AS．1398中性蛋白酶、胰蛋白酶、复合酶、木瓜蛋白酶、碱性蛋白酶、风味蛋白酶等6种蛋白酶分别对紫菜进行酶解，通过对其酶解液的ACE抑制活性和水解度的比较，发现采用AS．1398中性蛋白酶酶解的产物ACE抑制活性最高。对AS．1398中性蛋白酶的酶解工艺进一步优化，确定其最佳酶解工艺为：温度50℃、pH值7．5、加酶量E／S10000U·g^-1。在此条件下，酶解4h和10h的酶解产物抑制活性最高均达70％以上，并发现紫菜酶解液的ACE抑制率与水解度的增长不成显著的线性关系。     

影响淡水鱼头酶解的因素研究

选用中性蛋白酶和碱性蛋白酶对草鱼鱼头进行水解,单因素分析和正交实验L9(34)相结合,对影响其酶解条件的因素进行了探讨.结果表明:碱性蛋白酶水解效果较中性蛋白酶好,其最佳酶解条件为酶用量0.018%、温度53℃、pH值7.6、水解5h,在此条件下酶解液中游离氨基酸可达1.25mg/ml.并分析了两种酶在最佳酶解条件下,可溶性蛋白质含量的变化.     

复合酶提取牡蛎抗氧化肽的工艺研究

以牡蛎为原料,首先从木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶中筛选出胰蛋白酶为内切酶,以水解度为指标,得到最佳的酶解工艺条件:时间4h,温度50℃,pH8.0,料水比1∶2,加酶量3％(7 500 U/g),水解度为49.50％;同时以水解度为指标,得到外切酶风味蛋白酶的最佳酶解工艺:时间5h,温度50℃,pH8.0,料水比1∶2,加酶量3％ (450 U/g),实际水解度50.95％.最后,以清除羟自由基能力和水解度为指标,探讨内切酶(胰蛋白酶)和外切酶(风味蛋白酶)不同复合酶解方式的抗氧化能力.最终确定,复合酶解制备牡蛎抗氧化肽效果最好,其酶解条件为胰蛋白酶为3％(7 500 U/g)、风味蛋白酶加酶量为3％ (450 U/g),pH8.0,时间5h,料水比1∶2,温度50℃时,水解度高达53.94％,体外清除·OH的EC50为0.56 mg/mL.     

酶法制备脱脂鱼蛋白的初步研究

采用5种酶对脱脂鲢肉进行水解制备水解鲢蛋白。脱脂鲢肉是分别采用异丙醇、正己烷、乙酸乙酯3种溶剂萃取法进行脱脂后得到。分别用木瓜蛋白酶、枯草杆菌中性蛋白酶、枯草杆菌碱性蛋白酶、胰蛋白酶和复合蛋白酶对3种脱脂鲢肉及未脱脂鲢肉进行水解。试验结果表明，乙酸乙酯对鲢肉脱脂率最高，脱脂率为96．33％。复合蛋白酶为最适酶，乙酸乙酯脱脂鱼肉为最佳底物；在最佳水僻条件下：pH6．5，温度55℃，酶量3500U／g蛋白质，底物浓度（乙酸乙酯脱脂鲢肉：水）1：5，其水解度达到最大，为44．00％。     

海参多肽的制备工艺优化及其抗氧化测定

本文对海参酶解工艺条件进行优化，提取液经超滤和冷冻干燥获得海参多肽，并测定其组成及抗氧化活性。结果显示，中性蛋白酶对海参最佳的酶解工艺为：加酶量1800U／g，酶解温度为55℃，酶解pH值为7．5，酶解时间为3h。经超滤与冷冻干燥后，多肽得率为6．9％，蛋白质含量达94．9％。且在抗氧化方面具有较好的清除羟自由基和超氧自由基能力。     

木瓜蛋白酶在制取斑鰶鱼蛋白中的应用

为确定木瓜蛋白酶酶解斑鰶鱼的条件。研究了酶解温度、酶浓度、酶解时间、加水量、pH值对斑鰶鱼氨基氮含量和总氮得率的影响。结果表明：温度小于55℃。随着酶解温度的提高、酶浓度的增加、酶解时间的延长斑鰶鱼酶解液氨基氮含量和总氮得率均增加；pH值对水解效果及氮回收率影响不大。综合考虑，最佳水解条件为：温度55℃。加酶量6．0‰。酶解时间1．5h．原料：水=1：2．5，pH值6．8。此时。氨基氮生成率达26．7％。总氮得率2．28％。     

木瓜蛋白酶对罗非鱼下脚料的水解作用

试验结果表明,木瓜蛋白酶水解罗非鱼下脚料蛋白的适宜条件为:底物浓度1∶1.5,温度60℃,加酶量1100 IU/g原料,初始pH,酶解3 h;最佳水解条件下的酶解液氨基态氮含量达1.25 g/L,蛋白质含量5.19%,氮回收率为69.88%。酶活力下降是酶解反应减缓的主要原因之一。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO₂ and ions should be more often evaluated. Plasma Na⁺ and Cl^‐ are very useful markers of acidosis, due to their respective exchange for H⁺ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.     

Are hatchery‐reared abalone naïve of predators? Comparing the behaviours of wild and hatchery‐reared northern abalone,Haliotis kamtschatkana (Jonas, 1845)

Abalone populations have declined worldwide, generating interest in enhancement using hatchery‐reared individuals. In many cases, such restoration efforts have met with limited success due to high predator‐induced mortality rates. Furthermore, the mortality rates of outplanted hatchery abalone are often considerably higher than for wild individuals. This study uses northern abalone (Haliotis kamtschatkana) as a case study to determine whether hatchery‐reared abalone behave differently than their wild counterparts. In the field, outplanted hatchery‐reared abalone were significantly less responsive than wild abalone, in terms of number of abalone responding and intensity of response, to nearby movement and to physical contact with an inert probe. Also, when encountering a cue to which all abalone responded (a seastar predator), hatchery‐reared individuals remained subdued. Anti‐predator behavioural deficits in hatchery‐reared abalone were more pronounced in 4‐year‐old individuals than in 1‐year‐old individuals, suggesting an influence of either age or amount of time spent in the hatchery environment. These behavioural differences are expected to increase the vulnerability of hatchery‐reared abalone to predators, and are likely a major cause of their elevated predator‐induced mortality when outplanted.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.     

Migratory dynamics of stream-spawning longnose gar (Lepisosteus osseus)

Abstract– Literature evidence suggests that lake-dwelling longnose gar (Lepisosteus osseus) enter tributary streams to spawn, Until the present study, the dynamics of this breeding migration had never been investigated quantitatively. During the summers of 1991 and 1992, longnose gar were captured as they entered Weaubleau Creek, Missouri, a tributary of Harry S. Truman Reservoir. The in-stream spawning migration began in early April and ended in late May, and was positively correlated with stream flow and water level, and negatively correlated with water temperature. In-stream residence times ranged from 15 to 94 days, with males exhibiting longer residence times than females. Once in-stream, longnose gar travelled as far as 10 km upstream and occupied certain pools at greater relative frequencies. Although the reason for this preferential utilization is not completely understood, it may relate to pool depth and riffle proximity. Longnose gar disperse from the spawning stream great distances, with gar captured in Weaubleau Creek being recaptured up to 48 km away. This information should provide fisheries biologists the means to consider the reproductive ecology of this species in their conservation and management decisions.     

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-¹⁴C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.