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。     

贻贝蛋白酶解降血压肽的降压活性及相对分子质量与氨基酸组成

以贻贝（Mytilus coruscus）为原料制取降血压肽，采用均匀设计试验探讨了不同酶解条件对其ACE( angiotensin converting enzyme) 抑制活性的影响，利用高效液相色谱分析了其相对分子质量分布，以氨基酸自动分析仪分析其氨基酸和游离氨基酸组成；并利用自发性高血压大鼠（SHR)饲喂试验检测其降血压活性。结果表明，对于P1酶来说, 酶量从25~175 IU• mL-1，酶解时间从2~7 h，随着酶解程度的提高，其ACE抑制率下降；其最高的ACE抑制率为88.08%，对应的最佳酶解条件为：酶量25 IU•mL-1，酶解时间2 h，pH 7.0，酶解温度60 ℃。此外发现降血压肽的相对分子质量在1.6 ku以下，其中10肽以下的短肽约占50.4%，证实了降血压肽主要以分子量较小的短肽组成。降血压肽游离氨基含量为8.8 g•（100g）-1，水解氨基酸总量为59.6 g•（100g）-1，疏水性氨基酸含量为24.86 g•（100g）-1，氨基酸组成比例平衡，必需氨基酸含量丰富,其中含量最高的氨基酸是谷氨酸,占8.57%。此外以3.0 g•kg-1体重的降血压肽样品剂量饲喂SHR，在饲喂后2～6 h降压效果显著，平均降低幅度为18～28 mmHg（P＝0.05）。研究表明以贻贝等水产蛋白为原料可开发具有高ACE抑制活性和显著降血压作用的降血压肽。     

四种药物对斑马鱼急性毒性试验

为探究消毒液和农药对斑马鱼的毒性与安全评价,首次选用高锰酸钾、甲醛、精碘和卡螨死四种试剂进行急性毒性试验,以24～96h半致死浓度（LC50）判断斑马鱼对四种药物的敏感性。结果表明,24、48、72、96h的LC50,高锰酸钾分别：勾1．15、1．12、1．12、1．11mg／L;甲醛为48、45、45、40mg／L;精碘分别为7．8×10^-3、5．8×10^-3、5．6×10^-3、5．3×10^-3mg／L;卡螨死分别为1．95×10^-7、1．23×10^-7、0．65×10^-7、0．62×10^-7mg／L。高锰酸钾、甲醛、精碘和卡螨死的安全质量浓度（SC）分别为1．07、11．8、0．96×10^-3和1．48×10^-6mg/L。参照我国化学物质对鱼类毒性分级标准,判定高锰酸钾对斑马鱼急性毒性为II级,甲醛为Ⅲ级,精碘和卡螨死对斑马鱼急性毒性为I级。     

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。     

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)。     

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。     

虾头自溶产物中ACE抑制肽的分离鉴定

虾头在一定的条件下发生自溶作用,其所含蛋白质以肽和氨基酸等形式释放出来,有些肽产物具有ACE抑制活性.实验采用8000、5000和3000 u的超滤膜分级分离虾头自溶产物,活性检测结果表明,ACE抑制肽主要分布在3000 u超滤组分中;3000 u超滤组分进一步经Sephadex G-25葡聚糖凝胶层析、SP Sephadex C-25离子交换层析及Sephadex G-15葡聚糖凝胶层析纯化,ACE抑制活性提高将近8倍(IC50 =0.19 mg/mL);Sephadex G-15葡聚糖凝胶层析收集的高活性成分再经两次RP-HPLC纯化,分离纯化得到两条ACE抑制肽,质谱分析推测其氨基酸序列为Tyr-Pro和Leu-Pro/Ile-Pro,分子量分别为279和229 u.     

国外简讯

从大马哈鱼中提取降血压活性肽日本北海道食品加工中心研究成功从大马哈鱼头部提取降血压的保健药品与食品。提取方法是将大马哈鱼头洗净后捣碎、加蛋白酶磨成糜浆状，使蛋白质转化成活性肽，其分子量达３００。此种活性肽具有生理活性，能抑制血压上升因子—高血压蛋白Ⅰ...     

褐藻酸酶的制备及对海带细胞解离的应用研究

褐藻中富含褐藻酸,应用酶解法制备褐藻单细胞和原生质体时,高活性的褐藻酸酶是所用工具酶中的主要组分。文中介绍了用硫酸铵二级盐析和透析袋脱盐从鲍鱼的消化腺中提取褐藻酸酶的方法,测定酶活力及蛋白含量,检测该褐藻酸酶对海带细胞的解离效果。结果显示,该褐藻酸酶比活力为12 U/mg,酶活力单位为34 U/m L,得率为1.4%。用含此褐藻酸酶的酶解液解离海带细胞,酶解3~6 h内可得到游离单细胞及原生质体的数量在3×105~6×105个/g。该褐藻酸酶解离海带细胞效果显著,且本方法简单、快捷且经济。     

牡蛎酶解液的抗氧化活性

检测了牡蛎(Crassotera gigas)木瓜蛋白酶和中性蛋白酶酶解液的抗氧化活性。由Sephadex G-15葡聚糖凝胶柱层析分析牡蛎酶解液,得到具抗氧化活性组分的分子量分布。分子量为1191 D和826 D左右的木瓜蛋白酶酶解活性肽组分的自由羟基清除活性最强,当其质量浓度分别为0.184 mg/mL和0.673 mg/mL时,体外自由羟基清除活性分别为53.6%和66.5%;分子量分别为1074 D和735 D左右的中性蛋白酶酶解活性肽的自由羟基清除活性最强,当其质量浓度分别为0.166 mg/mL和0.830 mg/mL时,体外自由羟基清除活性分别57.6%和70.5%。HPLC分析结果表明,木瓜蛋白酶与中性蛋白酶酶解的活性肽组分分别由几种抗氧化肽组成。抗氧化活性最强的木瓜蛋白酶与中性蛋白酶酶解的肽组分在质量浓度为2.5 mg/mL时,活性分别可达83.6%和80.8%。用牡蛎原浆与各酶解液灌胃昆明小白鼠。研究结果表明,用木瓜蛋白酶与中性蛋白酶酶解原液及经超滤纯化的酶解液对小白鼠实施灌胃,其肝脏组织的SOD活力都显著提高(P<0.05);灌胃经超滤纯化的木瓜蛋白酶和中性蛋白酶酶解液,小白鼠肝脏组织GSH-PX活力、GSH含量显著提高(P<0.05),MDA含量显著降低(P<0.05)。     

Optimization of Hydrolysis Conditions for Production of Angiotensin-Converting Enzyme Inhibitory Peptides from Basa Fish Skin Using Response Surface Methodology

ABSTRACT

Basa fish skin free from fat and noncollagenous proteins was hydrolyzed by different proteinases to produce angiotensin-converting enzyme (ACE) inhibitory peptides. Protamex hydrolysates were found to have the highest ACE inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions of Protamex—including enzyme-to-substrate ratio, hydrolysis time, and solid (fish skin)-to-liquid (water for hydrolysis)—at a temperature of 50°C and a pH of 7.0. The mathematical model demonstrated a good fit with the experimental data. An enzyme-to-substrate ratio of 6,108 U/g, a hydrolysis time of 1.24 h, and a solid-to-liquid ratio of 1:8.05 g/mL were found to be the optimal conditions to achieve the highest value of ACE inhibitory activity. The IC₅₀ of the hydrolysates for ACE inhibitory activity was 1.417 mg/mL under these conditions.     

Recovery of Bioactive Peptides and Omega-3 Fatty Acids-Containing Phospholipids from Squid Processing By-Product Hydrolysate

ABSTRACT

The present study examined whether bioactive peptides and omega-3 fatty acids-containing phopholipids could be recovered from squid processing by-product (SPB) hydrolysate. The hydrolysate was produced at 55°C for varying times with endogenous proteases and centrifuged to yield peptides-containing supernatant and phospholipids-containing precipitate. The supernatant showed angiotensin I-converting enzyme (ACE) inhibitory activity with half maximal inhibitory concentration (IC₅₀) values decreasing with hydrolysis time from 2.11 mg (0 h) to 1.71 mg (1 h), 1.38 mg (1.5 h), and 1.34 mg (2 h). Two-hour squid hydrolysate was further fractionated to isolate the most active fraction whose molecular weight was found to be below 10 kDa with IC₅₀ of 0.32 mg. The phospholipids-containing precipitate (45.6 g/100 g oil) was analyzed for a fatty acid profile, with the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being 16.86 and 29.24 g/100 g oil, respectively. The ACE inhibition by hydrolysate peptides and omega-3 fatty acid recovery support the nutraceutical potential of the SPB hydrolysate.     

Physicochemical Properties and Angiotensin I Converting Enzyme Inhibitory Peptides of Freshwater Fish Skin Collagens

ABSTRACT

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Identification of oyster-derived hypotensive peptide acting as angiotensin-I-converting enzyme inhibitor

Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting 9 weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle, and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real effector of this penta-peptide in vivo.     

Optimization of Hydrolysis Conditions for the Production of the Angiotensin-I Converting Enzyme Inhibitory Peptides from Sea Cucumber Collagen Hydrolysates

The effects of different hydrolysis conditions on the angiotensin-I converting enzyme (ACE) inhibitory properties of giant red sea cucumber (Parastichopus californicus) collagen hydrolysates were investigated. Optimal conditions predicted by central composite rotatable design (CCDR) modeling for producing ACE inhibitory peptides were found to be 54.9°C, 1.76 h and an enzyme to substrate (E/S) ratio of 0.064. Compared with experiments performed under these optimal conditions, the predicted degree of hydrolysis (DH) and ACE inhibitory activities had error rates of 3 to 6%. Under optimum conditions, the molecular weights of collagen hydrolysates were less than 6.5 kDa. Lineweaver-Burk plots suggest that the collagen hydrolysates acted as competitive inhibitors with an inhibition constant (Ki) value of 0.706 mg/mL.     

Identification of angiotensin I-converting enzyme inhibitory peptides derived from salmon muscle and their antihypertensive effect

ABSTRACT:   The salmon peptide digested from salmon muscle showed a strong inhibitory activity against the angiotensin I-converting enzyme (ACE). The antihypertensive effect of the salmon peptide on spontaneously hypertensive rats (SHR) was examined. After the single intravenous administration of the salmon peptide at a dose of 30 mg/kg body weight, the systolic blood pressure (SBP) was significantly reduced against the control. Further, a double-blind, placebo-controlled, parallel-group study determined the efficacy of the salmon peptide in mild hypertensive subjects. The SBP, after a 1.0 g of salmon peptide intake, was significantly reduced at 4 weeks after the intake, and 2 weeks after the intake finished, compared to the value before ingestion. Bioassay-guided separation of the salmon peptide, using a combination of column chromatographic techniques, led to the identification of 20 active di- and tri-peptides, including Ile-Val-Phe and Phe-Ile-Ala as two new ACE inhibitory tripeptides. Ile-Trp had the strongest ACE inhibitory activity (IC₅₀ = 1.2 μM) in vitro , and contributed 5.2% to the total ACE inhibitory activity. The salmon peptide and Ile-Trp showed a digestive resistance by in vitro assay, which mimicked the digestive organ, and had no affinity for factors related to blood pressure regulation, except for the ACE inhibitory activity.     

Antihypertensive effect of Narezushi, a fermented mackerel product, on spontaneously hypertensive rats

ABSTRACT:   During the fermentation of mackerel to narezushi , the concentration of peptides required to inhibit 50% of the ACE activity in the assay media (IC₅₀), as an index of the angiotensin  I-converting enzyme (ACE) inhibitory activity, was remarkably decreased with a rapid increase in peptide contents. Systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR) decreased between 2 and 4 h after the single oral administration of greater than 10 mg peptide/kg narezushi extract, and recovered to the initial level by 8 h thereafter. The SBP decreased at seven successive daily doses of 10 mg/kg of narezushi extract and then recovered to the initial level 5 days after stopping a total of 10 daily administrations. The extract was administered to five-week-old SHR rats for 70 days and SBP decreased 21 days after starting and continued for 28 days after the end of administration. The peptide-rich fraction from narezushi extract had a powerful antihypertensive effect, whereas the other fraction had a similar, but weak effect.     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.