血管紧张素转换酶（ACE）的提取与活性验证

在从紫菜中提取制备降血压肽的研究中,为筛选能够抑制血管紧张素转换酶（ACE）活性的功能肽,需要大量ACE进行体外检测。但ACE成本昂贵,本文利用硫酸铵分级盐析法提取猪肺组织中的ACE,测其酶活力为15．45×10^-3U／mg,比活力为33．80×10^-3U／mg。通过卡托普利来验证其抑制效果,抑制率达49．15％。采用本实验室初步制得的紫菜降血压肽对自制ACE和商品ACE的活性进行抑制,测定IC50发现该抑制剂对2种ACE的抑制效果差异不大,且活性较为稳定,因此所提取的ACE粗酶液可直接用于降血压肽的体外检测实验。     

中国毛虾ACE抑制肽的初步研究

对以中国毛虾为原料酶法制备具有抑制血管紧张素转换酶(ACE)活性的酶解产物的方法作了探讨。以体外活性(ACE抑制率)为指标,通过正交试验确定胃蛋白酶的最佳酶解条件为pH2.4、温度41℃、酶量900U·g-1底物、底物浓度8%;酶解产物的IC50为0.65mg·mL-1,再分别利用SephadexG 25和SephadexG 15对其进行进一步分离提纯,IC50降至0.084mg·mL-1和0.046mg·mL-1,活性组分中的疏水性氨基酸含量增高,最终活性产物的分子量分布在700～1900。     

血管紧张素转换酶抑制肽的分离

采用截留分子量分别为30 ku、10 ku、6 ku、3 ku的中空纤维超滤膜对扇贝酶解产物进行分级分离,并结合凝胶柱层析分离技术测定了各分离组分的分子量分布,同时还测定了各分离组分对血管紧张素转换酶(ACE)的抑制活性。实验结果表明,不同截留分子量超滤膜可以实现酶解产物按其分子量大小的分级分离,不同分离组分的ACE抑制力活性差异较大,总趋势是截留分子量相对较小的超滤膜透过液的ACE抑制活性较强。其中,截留分子量为3 ku超滤膜的透过液具有最强的ACE抑制活性,其ACE抑制率I(%)=96.17%,半抑制浓度IC_(50)= 0.078 mg·mL~(-1)。通过对截留分子量为3 ku超滤膜的透过液依次经过Sephadex G-25及Sephadex G-15凝胶柱层析分离纯化后,分离出了ACE抑制活性最强的2个组分活性肽,其平均分子量分别为1 300 u和900 u左右,其IC_(50)分别为0.026 mg·mL~(-1)和0.012 mg·mL~(-1)。     

罗非鱼肉双酶分步控制酶解制备血管紧张素转化酶抑制肽的研究

本实验以罗非鱼肉为材料，利用Trypsin、Alcalase2．4L、Pratamex两两复合后，分步控制酶解制备血管紧张素转化酶抑制肽（ACEIP）。以ACE抑制率（Ⅰ值）、氨基酸态氨含量（Cn值）、和TCA可溶性蛋白（短肽）含量（Cp值）为指标对酶解过程进行分析，说明双酶分步控制酶解过程以及产物的特点，指出以单酶控制酶解为基础的双酶分步控制酶解法优于单酶控制酶解法。Trypsin与Alcalase2．4I．、Trypsin与Protamex及Alcalase2．4L与Protamex双酶分步控制酶解产物经稀释40倍后对ACE的最大抑制率分别可达到82．9％、82．9％和91．91％，具有较好的抑制效果。     

22种海水鱼中ACEI的制备及其化学修饰

以取自中国黄海的22种鱼类为研究对象，测定其鱼肉组织上清液的酶解物对血管紧张素转化酶（ACE）的抑制作用。从6种商业用酶和1种新型海洋低温碱性蛋白酶（MLAP）中，筛选出MLAP作为酶解制备ACEI的理想水解酶。以该酶水解上述22种海水鱼鱼肉组织上清液，并将水解液经Sephadex G-25凝胶层析和DEAE Sepharose阴离子交换层析，得到ACEI纯品。测定各类ACEI体外抑制ACE的活性发现，这22种海水鱼中以缇鱼（Engraulis japonicus）所产ACEI活性最高，其余鱼类无显著差异。以缇鱼制备血管紧张素转化酶抑制剂（ACEI-22），并对ACEI-22作进一步研究，结果发现，ACEI-22对自发性高血压大鼠（SHR）有降压效果，但不显著；ACEI-22经聚乙二醇化学修饰后，修饰率为68％，且给药SHR后，大鼠收缩压显著降低，与相同条件下同剂量的卡托普利的降压效果相当。故聚乙二醇修饰的ACEI-22可有效地降低SHR的血压。     

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果=Angiotensin I converting enzyme inhibitory activity of shrimp meat hydrolysate by endogenous enzymes from shrimp head and its antihypertensive effects on spontaneously hypertensive rats［刊,中］/洪鹏志（广东海洋大学食品科技学院,湛江524088）,曹文红,郝更新,章超桦//水产学报,—2007,31(1).-76～83 在pH 7.35、温度57.2 ℃、水解时间4 h、虾肉/虾头为1∶1,底物浓度为20%（W/V）的条件下,利用虾头的内源酶酶解虾肉（南美白对虾）,制备了具有血管紧张素转化酶（ACE）抑制活性的酶解产物,其对ACE的抑制率为41.9%（2 mg·mL-1）。通过凝胶过滤层析测定出酶解产物的分子量分布较广,在7.4×104至29.7之间。分子量在2.7×103以下的凝胶层析组分占酶解产物氨基酸总量的83.1%。收集的高活性组分分子量在959～338之间,其IC50为0.32 mg·mL-1苯丙氨酸、疏水氨基酸（亮氨酸、异亮氨酸和缬氨酸）和芳香族氨基酸（脯氨酸、酪氨酸和色氨酸）占氨基酸总量的42.33%。体内试验的结果显示,南美白对虾的虾肉虾头内源酶酶解产物经灌喂原发性高血压老鼠后显示较强的降血压活性。 图4表2参13 关键词：南美白对虾;内源酶;血管紧张素转化酶抑制活性;抗高血压作用 E-mail:zhangch@gdou.edu.cn     

酶解罗非鱼鱼皮胶制备降血压肽的研究

为探讨罗非鱼鱼皮胶制备降血压肽的酶解工艺,研究选用Neutrase中性蛋白酶、Alcalase碱性内切蛋白酶、胰蛋白酶、木瓜蛋白酶和复合蛋白酶分别对罗非鱼鱼皮胶进行酶解,对其酶解液的水解度进行比较。结果表明复合蛋白酶的酶解效果最好。对复合蛋白酶的酶解工艺进行单因素优化,确定其最佳酶解工艺为:温度50℃、pH值7.0、料液比1∶5、酶量为底物量的1.5%。在此条件下水解6h时,水解度为60%,酶解产物对血管紧张素转化酶(ACE)的抑制率最高,达到78.96%。     

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。     

3942中性蛋白酶酶解中国毛虾蛋白制备血管紧张素转移酶抑制肽

采用3942中性蛋白酶酶解中国毛虾(Acetes chinensis)蛋白，制备具有抑制血管紧张素转移酶(ACE)活性的多肽。采取正交实验研究料水比、酶量、反应温度、反应时间4个因素对酶解中国毛虾蛋白产物的ACE抑制活性的影响。结果显示，在料水比1：3(体积比)，酶量0．5％，温度45℃，时间8h的水解条件下得到的酶解产物F7的ACE抑制活性最高，其抑制率达到92．86％。用层析柱Sephadex G-25、SP-Sephadex C-50对F7进行分离纯化，选取ACE抑制活性最高的峰进一步用反相高压液相色谱进行纯化，得到单一组分FⅠ。经飞行时间质谱和碰撞诱导裂解分析确定，FⅠ为新型的ACE抑制肽Ser-Pro，IC50值为272μmol／L。     

急性操作胁迫对刀鲚应激反应相关神经内分泌因子的影响

为深入了解刀鲚应激反应中相关神经内分泌因子作用的分子机理,采用手工捕捉的方式对刀鲚进行了急性操作胁迫。通过放射免疫法和化学发光法测定刀鲚应激反应后头肾和血浆皮质醇含量的变化,结果显示,刀鲚胁迫刺激后血浆皮质醇含量极显著性升高,血浆皮质醇浓度平均升高56.48%,头肾皮质醇含量显著性升高,头肾皮质醇浓度平均升高49.68%,表明急性操作胁迫确实引起刀鲚的应激反应。通过同源克隆的方法获得刀鲚促肾上腺皮质激素释放激素(CRH)、硬骨鱼紧张肽(UI)、阿黑皮素原(POMC)基因的部分序列,并应用实时荧光定量PCR(RT-qPCR)方法检测上述神经内分泌因子mRNA的表达变化,结果显示,CRH基因的表达水平极显著性下降,POMC基因的表达水平显著性下降,UI基因的表达水平有下降趋势但不显著。上述结果显示,皮质醇、CRH、UI和POMC等神经内分泌因子通过鱼类下丘脑—脑垂体—肾间腺轴参与刀鲚应激反应的调节,为进一步了解刀鲚胁迫应答的作用机理,实现对其有效调控打下基础。     

Angiotensin Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory,and Antioxidant Activities of a Blue Mussel (Mytilus edulis) Meat Protein Extract and Its Hydrolysates

ABSTRACT

Protein extracted from mussel processing coproducts was hydrolyzed with four different food-grade enzyme preparations and assessed for angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory and oxygen radical antioxidant capacity (ORAC) activities. All hydrolysates tested showed higher activity than the intact protein. ACE and DPP-IV IC₅₀ values in the range 1.13–3.34 and 0.33–2.43 mg mL^?1, respectively, and ORAC values in the range 66.26–121.56 µmol trolox equivalents g^–1 were obtained. These results suggest that some of the mussel meat protein hydrolysates may have potential as functional food ingredients for the management of diseases such as type II diabetes and hypertension.     

Angiotensin-converting enzyme I inhibitory activity of phlorotannins from Ecklonia stolonifera

ABSTRACT:   As part of our study of the isolation of antihypertensive agents derived from natural marine products, the bioactivity of 10 edible Korean seaweeds were screened by angiotensin converting enzyme (ACE) inhibitory and peroxynitrite assays. Among the crude extracts of selected seaweeds, including five Phaeophyta ( Ecklonia stolonifera , E. cava , Pelvetia siliquosa , Hizikia fusiforme , and Undaria pinnatifida ), four Rhodophyta ( Gigartina tenella , Gelidium amansii , Chondria crassicaulis , and Porphyra tenera ) and one Chlorophyta ( Capsosiphon fulvescens ), the ethanol extracts of E. stolonifera , E. cava , P. siliquosa , U. pinnatifida , and G. tenella exhibited significant inhibitory properties against ACE at more than 50% inhibition at a concentration of 163.93 µg/mL. Phloroglucinol 1 , eckstolonol 2 , eckol 3 , phlorofucofuroeckol A 4 , and dieckol 5 had been isolated previously, and triphlorethol-A 6 and fucosterol 7 were isolated for the first time from E. stolonifera. Also, the ACE inhibitory and peroxynitrite scavenging properties of phlorotannins 1–6 were evaluated, along with fucosterol 7 obtained from E. stolonifera . Among profound peroxynitrite scavenging compounds 1–6 , phlorotannins 3 , 4 and 5 were also determined to manifest marked inhibitory activity against ACE, with 50% inhibition concentration (IC₅₀) values of 70.82 ± 0.25, 12.74 ± 0.15, and 34.25 ± 3.56 µM, respectively.     

Characterization of Collagen Enzymatic Hydrolysates Derived from Lizardfish (Synodus fuscus) Scales

Acid soluble collagen was extracted from the scales of lizardfish and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. After 8 h of collagen hydrolysis, hydrolysates had an estimated degree of hydrolysis (DH) from 4 ± 0.05 to 25 ± 0.63% (p < 0.05). All hydrolysates had angiotensin converting enzyme inhibitory and antioxidant activity. These activity levels showed little change after treatment with gastrointestinal proteases. Results indicated that the lizardfish by-products may be improved by enzymatic treatment with acid-soluble collagen from lizardfish scales.     

Potential Precursor of Angiotensin-I Converting Enzyme (ACE) Inhibitory Activity and Structural Properties of Peptide from Peptic Hydrolysate of Cutlassfish Muscle

ABSTRACT

Peptic hydrolysates were prepared by digesting the cutlassfish muscle protein using pepsin for 1, 3, and 6 h, and their inhibitory activity against angiotensin-I converting enzyme (ACE) was studied. The ACE-inhibitory effect of the peptic hydrolysate of cutlassfish muscle generated at the 3 h time point exhibited the strongest activity. After identifying the optimal hydrolysate, the active peptide was isolated by ultrafiltration, gel permeation, and high performance liquid chromatography (HPLC). The resulting purified peptide was characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and was identified to be a 496.44 Da pentapeptide (Phe-Ser-Gly-Gly-Glu). The ACE-inhibitory activity of the active peptide exhibited an IC₅₀ value of 0.033 ± 0.003 mg/ml. A molecular docking program was used to simulate the interaction between the peptide and ACE, which revealed that the inhibitory effect was mainly due to the hydrogen bonds between ACE and the peptide. Based on the ACE-inhibitory properties and the molecular docking study of the resulting active peptide, we demonstrated an increase in nitric oxide (NO) production in a dose-dependent manner. In conclusion, cutlassfish protein hydrolysate and the resulting active peptide could be used as active ingredients in functional food as anti-hypertensive agents.     

饲用酶制剂在水产动物中应用的最新研究进展

酶制剂作为一类安全、高效的绿色饲料添加剂,在畜禽类饲料中已得到广泛应用.酶制剂不仅可以在一定程度上缓解国内饲料资源的不足,还可以促进绿色养殖环境的发展.由于酶制剂受热敏性条件制约的原因,其在水产饲料中的应用率较低,但随着一些设备的应用,如多道真空后喷涂等,使液体酶制剂在水产饲料中的广泛应用成为可能.本研究就酶制剂在水产...     

中国主要水产品出口国的技术性贸易壁垒浅析

中国水产品出口主要集中在日本、美国、欧盟和韩国四个市场上,近年来,这四大市场大约占到了85%左右。同时,这些国家基本上都对进口水产品制定了严格的技术性标准,我国的水产品不可避免地要受到这些国家的技术性贸易壁垒的限制。文章介绍了我国遭受的技术性贸易壁垒的总体状况和日本、美国、欧盟以及韩国的技术性贸易壁垒概况,同时也介绍了这些国家对我国水产品贸易造成的影响。