金枪鱼(Katsuwonus pelamis)碎肉蛋白降压肽的酶解制备及活性研究

以血管紧张素转换酶(ACE)抑制率为指标,通过正交试验L9(34)确定碱性蛋白酶(Alcalase)对金枪鱼(Katsuwonus pelamis)碎肉蛋白的最佳酶解条件,利用D101大孔树脂建立了酶解物的脱盐工艺,利用超滤、葡聚糖凝胶(G-25)色谱和反相-高效液相色谱(RP-HPLC)分离降压肽,并确定其纯度,利用氨基酸序列分析和ESI-MS鉴定纯化多肽的结构。结果表明,碱性蛋白酶酶解金枪鱼碎肉蛋白制备降压肽的最佳酶解条件是:pH 9.5,酶用量1.5%,酶解温度50°C,酶解时间5h;D101大孔吸附树脂脱盐工艺条件为:上样浓度10 mg/mL、上样流速1.5 BV/h、解吸剂为75%乙醇;酶解物经超滤和Sephadex G-25分离获得一个纯度较高的四肽,经氨基酸序列分析和ESI-MS鉴定结构为Phe-GlyGly-Val(FGGV),ESI-MS检测给出分子离子峰m/z 379.50([M+H]+)。利用碱性蛋白酶酶解并经超滤和色谱技术制备的金枪鱼碎肉蛋白降压肽具有良好的ACE抑制活性,可作为降压药物、保健食品或添加剂进行开发。     

酶解蓝圆鲹蛋白制备降血压肽的工艺研究

以蓝圆(Decapterus maruadsi)鱼肉蛋白为原料,选用6种蛋白酶在各自适宜的条件下酶解制备鲹降血压肽,通过检测酶解产物对血管紧张素转化酶(ACE)的体外抑制活性,筛选出木瓜蛋白酶为制备蓝圆降血压肽的最佳用酶。考察了不同水解度的酶解产物对ACE抑制效果,当水解度为13.7%时,产物对ACE的最高抑制活性可达67.4％。在此基础上,分别了考察酶解时间、初始酶用量、初始底物浓度、酶解温度和 pH 值对酶解工艺的影响,鲹得到制备蓝圆降血压肽的适宜酶解工艺为：初始酶用量鲹为7000 U/g,初始底物浓度为25 g/L,酶解温度为45℃, pH为7.0,酶解240 min。采用超滤技术对酶解产物活性组分进行分离富集,发现产物中高活性部分均可富集于10 kDa和5kDa渗透液中,且高活性组分分子量主要集中在5 kDa以下,该组分对ACE的活性抑制率为88.47%。     

双酶分步水解金枪鱼(Eleotridae)碎肉制备高F值酶解液的工艺研究

以金枪鱼碎肉为原料, 采用双酶分步水解法制备高F值酶解液, 通过Box-Behnken试验设计, 分别确定两步酶解的最佳条件, 酶解液经活性炭静态吸附去除游离芳香族氨基酸, 对脱芳后的酶解液进行氨基酸组成分析并测定F值。结果表明, 胃蛋白酶为第一步水解用酶, 酶解的最佳工艺条件为酶用量650U/g, 料水比1∶7(g/mL), 温度35.9℃; 风味蛋白酶为第二步水解用酶, 酶解的最佳工艺条件为酶用量50700U/g, pH 6.51, 温度51℃, 最终水解度达到36.87%±0.54%; 酶解液在pH 3.0, 温度35℃条件下, 经5%(质量体积分数)的活性炭吸附时间3h后, 脱芳率达到63.18%, F值为30.33, 符合高F值肽的要求。     

黄海西部沿岸冷水在夏季南黄海西部底层冷水形成和季节演变过程中作用的化学水文学分析

以金枪鱼碎肉为原料, 采用双酶分步水解法制备高F值酶解液, 通过Box-Behnken试验设计, 分别确定两步酶解的最佳条件, 酶解液经活性炭静态吸附去除游离芳香族氨基酸, 对脱芳后的酶解液进行氨基酸组成分析并测定F值。结果表明, 胃蛋白酶为第一步水解用酶, 酶解的最佳工艺条件为酶用量650U/g, 料水比1∶7(g/mL), 温度35.9℃; 风味蛋白酶为第二步水解用酶, 酶解的最佳工艺条件为酶用量50700U/g, pH 6.51, 温度51℃, 最终水解度达到36.87%±0.54%; 酶解液在pH 3.0, 温度35℃条件下, 经5%(质量体积分数)的活性炭吸附时间3h后, 脱芳率达到63.18%, F值为30.33, 符合高F值肽的要求。     

酶解马氏珠母贝肉制备降血压肽工艺条件优化

以马氏珠母贝（Pinctada martensii）肉为原料,选用6种蛋白酶在各自适宜的条件下进行酶解,采用HPLC法分析酶解产物对血管肾张素转换酶（ACE）的抑制活性（IP）,筛选出碱性蛋白酶为其制备的最佳酶种类,并确定了水解度为21％时,产物具有较好的抑制活性。在此基础上,分别考察了加酶量,底物浓度,酶解温度及pH对水解度的影响。结果表明：当加酶量为0．175g／L,底物浓度为35g／L,温度为45℃,pH为9．5,水解时间240min为酶解最适宜工艺条件。通过SDS-聚丙烯酰胺凝胶电泳测定分子质量分布,表明酶解产物中主要为14．4ku以下的多肽分子。     

响应面法优化香港巨牡蛎蛋白酶解工艺的研究

以水解度为指标,比较了6种蛋白酶对香港巨牡蛎(Crassostrea hongkongensis)的水解效果。在单因素考察实验的基础上,采用Box-Behnken模型响应面设计,建立香港巨牡蛎蛋白酶解的二次回归模型,确定最佳的水解条件。实验结果表明,海产品专用蛋白酶对香港巨牡蛎蛋白的水解效果优于其他蛋白酶,该酶的最佳水解条件为:酶与底物比为5.93%,酶解时间为4.07 h,酶解温度为54.6℃。在此条件下,海产品专用蛋白酶酶解香港巨牡蛎蛋白的水解度达到25.73%±2.39%。本研究为香港巨牡蛎的开发和利用提供了理论依据。     

响应面法优化罗非鱼(Oreochromis niloticus)鱼排蛋白分段酶解工艺

采用生物蛋白酶酶解罗非鱼鱼排蛋白,以水解度(DH)为指标,进行了单酶、复合酶酶解效果的比较分析,并利用响应面分析方法(RSM)对酶解参数进行优化。结果表明,采用风味蛋白酶和木瓜蛋白酶双酶组合分段酶解,固定总酶添加量为3.0%、双酶复合比2:1、酶解时间3h、分段酶解时间比0.6:1,酶解温度56℃,分段酶解pH为7.0和6.2时,酶解效果最佳,罗非鱼鱼排蛋白水解度达到32.49%,酶解产物中游离氨基酸含量达32.84mg/g鱼排,占罗非鱼鱼排总氨基酸含量的25.43%,其中呈味氨基酸含量达18.48%,必需氨基酸含量达67.96%。     

鹌鹑蛋蛋清蛋白酶解工艺条件的研究

以鹌鹑蛋蛋清蛋白作为原料,探讨酶解鹌鹑蛋蛋清蛋白生成小分子活性肽的加工工艺。采用单因素和响应面分析试验,优化出蛋清蛋白酶解的最佳工艺条件为:选用胰蛋白酶,固液比1∶3,加酶量187.5 U/g底物,水解时间5 h,pH 8.07,酶解温度51.76℃,水解度可达到91.37%,且得到的酶解液风味良好。采用高效液相色谱法分析酶解液中游离氨基酸的组成及含量,结果表明:酶解液含有7种人体必需氨基酸,其中赖氨酸含量达16.22%,是1种天然优质的水解制品,可精制成富含多种氨基酸的蛋白溶液,应用于新型产业的开发。     

牙鲆孵化酶的分离纯化及其部分生物化学性质研究

利用凝胶过滤柱层析和阴离子交换柱层析技术,从牙鲆（Paralichthys olivaceus）胚胎孵化液中分离纯化出了大小约为34．8ku的牙鲆孵化酶。该酶卵膜裂解的最适反应温度为35℃,最适pH为7．0;对底物酪蛋白的米氏常数Km值为1．53mmol／L。该酶对丝氨酸蛋白酶和胰蛋白酶的特异性抑制剂非常敏感,而对其他蛋白酶抑制剂不敏感,表明该酶极可能是一种丝氨酸蛋白酶类型的胰蛋白酶。此外,该酶可浓度依赖性地被EDTA所抑制,被Cu^2＋所强烈抑制,被Ca^2＋和Mg^2＋所激活,对Zn^2＋则不敏感,表明该酶很可能是一种金属蛋白酶。     

溶菌酶对海洋生物高分子壳聚糖的降解研究

用粘度法和吸光度法 ,研究了溶菌酶非专一性降解壳聚糖过程中温度、pH、时间、酶浓度、底物浓度对溶菌酶降解壳聚糖反应速度的影响 ,确定了以壳聚糖为底物的溶菌酶的一些催化特性 :温度为60℃ ,pH值为4.0时 ,达到最大酶反应速度。0.5～4.0h内酶反应速度恒定 ;酶浓度在0.5～4U/ml范围内 ,酶反应速度与酶浓度成线性关系。米氏常数Km 为9.03×10-4 mol/L。     

黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

本文旨在研究黄鳍金枪鱼(Thunnus albacares)碎肉中4种不同蛋白酶酶解物的3k Da超滤组分对RAW264.7细胞的免疫活性。以RAW264.7细胞为实验模型, 采用噻唑蓝比色法检测细胞增殖活性, 中性红吞噬实验检测细胞吞噬能力, Griess试剂检测细胞培养上清液中NO含量, 并分析酶解物氨基酸组成和分子量分布。实验结果表明, 胰蛋白酶酶解物在12.5~200μg·mL^-1范围内无明显细胞毒性, 能增强RAW264.7细胞吞噬能力, 浓度为400μg·mL^-1时达到最大值132.50%(P<0.01), 浓度在50~200μg·mL^-1范围内能促进巨噬细胞NO释放(P<0.01), 且NO释放量与酶解液浓度存在一定的量效关系; 胰蛋白酶酶解物中必需氨基酸(essential amino acid, EAA)含量最高, 为51.09%, 其碱性氨基酸组氨酸、赖氨酸和精氨酸的含量分别为11.59%、11.00%和6.90%, 均高于其他蛋白酶酶解物, 并且分子量小于1k Da的小肽含量高达97.48%。研究结果表明, 4种蛋白酶酶解物中, 黄鳍金枪鱼碎肉胰蛋白酶酶解物的3k Da组分具有较明显的免疫活性, 这为其作为免疫活性肽的开发利用提供了理论依据。     

低分子质量褐藻寡糖的高效酶法制备及其抗氧化活性评价

褐藻寡糖具有多种生理活性,本研究通过酶解与分级条件的优化,获得了低分子质量褐藻寡糖的酶法制备工艺,并在体外模型上评价了不同分子质量组分的抗氧化活性。结果表明,优化后的褐藻寡糖制备条件为:底物浓度1.20%,酶底比4.35 U/mg,酶解时间4 h。进一步采用乙醇沉淀和超滤分离后,获得了重均分子质量分别为0.84、1.40、2.25和34.56 k Da的4种组分,其得率分别为50.82%、5.15%、11.48%和12.00%。各组分均具有一定的还原能力,可有效清除DPPH和羟自由基,其中低分子质量组分A的抗氧化活性最为显著,其清除羟自由基的能力与维生素C相近。     

动物蛋白酶解物与无机氮、磷源的组合对小球藻(Chlorella vulgaris)生长和生化指标的影响

采用三因素不完全正交设计方法,通过对细胞密度、干重、叶绿素和可溶性蛋白质含量的测定.确定了N、P源与蛋白酶解物之间的最佳组合,研究碳酸氢铵作为N源、磷酸二氢钠作为P源与动物蛋白酶解物之间的组合对小球藻生长和生化指标的影响.结果表明:(1)动物蛋白酶解物与N、P源间存在互作效应--影响小球藻细胞密度、叶绿素的主要因素分别是P浓度、蛋白酶解物浓度;而N浓度及蛋白酶解物浓度同时对小球藻的蛋白质含量起主要作用;(2)同时添加N 50mg/L、P 4mg/L、蛋白酶解物12ml/L能显著提高小球藻生物量,促进叶绿素和藻体蛋白质的合成(P<0.05).     

大西洋热带海域长鳍金枪鱼渔场预报模型的比较

为提高大西洋热带海域长鳍金枪鱼(Thunnus alalunga)渔场预报的准确率,对K最近邻(k nearest neighbor,KNN)、逻辑斯蒂回归(logistic regression,LR)、决策与分类树(classfication and regression tree,CART)、梯度提升决策树(gr...     

Application of fluorescence spectroscopic techniques and probes to the detection of biopolymer degradation in natural environments

The activities and substrate specificities of extracellular enzymes in natural systems are not well understood, despite their critical role in microbial remineralization of organic carbon. These enzymes initiate organic carbon degradation by selectively hydrolyzing high molecular weight substrates to lower molecular weight products which can be transported into cells. A set of single- and dual-labeled fluorescent polysaccharides was synthesized and characterized to explore a variety of approaches for measuring enzymatic hydrolysis of biopolymers via photophysical techniques, focusing particularly on rapid and robust optical techniques which are amenable to field measurements in remote locales. A shotgun-labeling approach yielded dual-labeled probes that exhibited substantial donor fluorophore quenching. The photophysical response of these probes to hydrolysis via purified enzymes was investigated in the lab, and fluorescence polarization proved to be a rapid and reliable technique for monitoring probe hydrolysis. Initial field results were also obtained from hydrolysis experiments in sediment porewaters. Because polarization measurements are rapid and simple, this approach could be used to follow the extracellular enzymatic hydrolysis of a wide range of biopolymers which fuel microbial metabolism.     

青鳞鱼下脚料水解蛋白的制取及其营养评价

以青鳞鱼（Harengula zunast)下脚料为原料，采用碱萃取，蛋白酶水解，浓缩，喷雾干燥等手段，制得水解鱼蛋白。研究了碱萃取，蛋白酶水解条件及不同沉淀方法对水解鱼蛋白沉淀效果的影响，并对水解鱼蛋白制品营养价值进行了评价，结果表明，下脚料水解鱼蛋白制品含粗蛋白72．9％，氨基酸总量67．6％，其中必需氨基酸占44．6％，氨基酸分为96，苏氨酸为其限制性氨基酸（1973年FAO／WHO标准），无机盐钙，镁，铁，锌，铜，磷含量丰富，以有机溶剂（异丙醇，乙醇)为沉淀剂沉淀水解蛋白时，粗蛋白损失率约为30％（干基计）。     

基于深度卷积嵌入式聚类（DCEC）的海洋环境特征提取对渔情预报模型的改进研究

In order to improve the forecasting ability of the fishery forecast model for the longline bigeye tuna (Thunnus obesus), we proposed a marine environment feature extraction method based on deep convolutional embedded clustering (DCEC), combined with generalized additive model (GAM) for forecasting the longline bigeye tuna fishing grounds in the Southwest Indian Ocean. We used the MODIS-Aqua and MODIS-Terra sea surface temperature (SST) three-level inversion image data (in days) from January to December in 2018 at 0.041 6°×0.041 6° to construct a DCEC model, determined the optimal number of clusters based on the Davies-Bouldi index (DBI), and extracted the category feature value (F_M) of each month’s sea surface temperature (SST); we used monthly 1°×1° bigeye tuna longline fishery data from January to December in 2018 generated from the Indian Ocean Tuna Commission (IOTC), and calculated the catch per unit effort (CPUE); we matched the monthly category feature value F_M and the monthly average value of Chl a concentration with the CPUE data to construct an improved GAM; we matched the monthly average SST, the monthly average Chl a concentration and CPUE data to build a basic GAM; we used the joint hypothesis test (F test) to verify the influence of model explanatory variables; we used akaike information criterion (AIC), mean square error (MSE), and draw the frequency distribution diagrams and box diagrams of measured and predicted values, etc., to analysis the improvement effect of the improved GAM compared to the basic GAM. The results showed that: (1) the category feature value (F_M) extracted based on the DCEC model could better reflect the temporal and spatial dynamic characteristics of SST in the Southwest Indian Ocean, and was related with the climatic conditions, monsoon conditions, and hydrological characteristics in the Southwest Indian Ocean; (2) the factor interpretation of F_M was higher than that of the monthly average SST in GAM, which means F_M had more significant impact on the CPUE of bigeye tuna. The high catch rate was concentrated in the areas where the F_M category was 2, 10, 24 with intersections between the warm and cold currents; (3) the AIC of the improved GAM was reduced by 9.17% than that of the basic GAM and MSE of the improved GAM was reduced by 26.7% than that of the basic GAM; the frequency distribution of the CPUE logarithmic value predicted by the improved GAM was closer to the normal distribution, and the high frequency distribution interval was closer to that of the measured value; the scatter plot showed that the CPUE predicted by the improved GAM had a significant correlation with the measured CPUE, with r equaled to 0.60. This study proves the effectiveness of the DCEC model in extracting marine environmental features, and can provide a reference for the further study on the bigeye tuna fishery forecast.     

Peptide decomposition by extracellular hydrolysis in coastal seawater and salt marsh sediment

Extracellular peptide hydrolysis rates were measured in seawater and sediment from Flax Pond salt marsh using peptide analogs (LYA-peptides) as substrates. This technique allows the direct measurement of specific hydrolysis products and thus provides insights into enzymatic hydrolysis pathways. In sediments, hydrolysis rate constants of LYA-peptides varied seasonally and with depth. Highest activity was found in spring and summer, and most cores exhibited a subsurface maximum. Calculations using the concentrations of chemically-measured peptides suggested that extracellular hydrolysis of peptides is faster than the rate of free amino acids uptake. However, not all peptides may be available for enzymatic hydrolysis. In both seawater and sediment, extracellular hydrolysis of peptides of up to 8 amino acids yielded smaller peptides and free amino acids. Hydrolysis rates depended on size of the peptide substrate, although a clear relationship with number of amino acid constituents was not evident. Peptides containing >2 amino acids were hydrolyzed 10–400 times faster than dipeptides or the fluorogenic substrate Leucine-MCA. Thus, dipeptidases are either uncommon in nature, or hydrolysis is carried out by nonspecific hydrolases that with a low affinity for dipeptides. This is also suggested by the presence of a lag time before dipeptide hydrolysis begins, and the absence of dipeptide hydrolysis in 0.2-μm-filtered. One implication of this finding is that measurements of hydrolysis rates using substrates like Leu-MCA may not accurately predict the magnitude of hydrolysis rates of macromolecules in the marine environment. Even though dipeptide hydrolysis is slow compared to that of larger peptides, LYA-dipeptides are preferentially produced from the hydrolysis of larger substrates. LYA-dipeptides do not penetrate cell membranes of microorganisms because of their size, but natural dipeptides are smaller and can be transported across the cell membrane. Since dipeptides do not appear to accumulate in natural waters, they must be rapidly removed by microorganisms.