中国毛虾酶解产物的分子量分布及ACE抑制活性

利用胃蛋白酶酶解中国毛虾制备具有血管紧张素转化酶抑制活性的酶解产物,并采用凝胶色谱法探讨酶解产物的分子量分布。结果表明:在45℃、pH2.5、酶量2%(w/w)、酶解4h、底物浓度20%(w/v)条件下,中国毛虾酶解产物的ACE抑制率高达85.9%;Sephadex G-15葡聚糖凝胶色谱图上出现5个吸收峰,其中高ACE抑制活性组分的分子量在1320～311之间,IC50为0.77mg/mL。     

Purification and identification of an ACE inhibitory peptide from the peptic hydrolysate of Acetes chinensis and its antihypertensive effects in spontaneously hypertensive rats

Acetes chinensis is a marine shrimp found in the coastal waters of China. The shrimp was hydrolysed by pepsin to prepare hydrolysates with angiotensin I‐converting enzyme (ACE) inhibitory activity. The hydrolysate with the highest ACE inhibitory activity resulted from a 3–5 h incubation at 45 °C and pH 2.5 with pepsin. Gel filtration and RP‐HPLC were used to separate ACE inhibitory peptides from the hydrolysate. The gel filtration fraction of the hydrolysate with a molecular weight range from 1320 Da to 311 Da exerted the highest ACE inhibition activity. This fraction was separated by RP‐HPLC into fifteen fractions, of which fraction F9 showed 92.7% of the ACE inhibition activity. Its peptide sequence was determined to be Leu–His–Pro. It showed a potent antihypertensive activity in spontaneously hypertensive rats. The results suggested that this peptide may be a potent ACE inhibitor which might be developed into a healthy food to lower blood pressure.     

Angiotensin I-converting enzyme inhibitory activity of Acetes chinensis peptic hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Peptic hydrolysate with angiotensin I-converting enzyme (ACE) inhibitory activity was prepared from Acetes chinensis . The 3 kDa ultrafiltration filtrates (UF-IV) of the desalted hydrolysate exerted a potent ACE inhibitory activity with IC50 being 0.67 mg mL⁻¹. The fractions with molecular weight located between 611 and 240 Da achieved a total percentage of 63.6% in the UF-IV, with a mean peptide length of about 5.6–2.1. Gastrointestinal digestions were simulated on the hydrolysate and UF-IV and addressed their effect on the ACE inhibitory activity. The ACE inhibitory activity of the UF-IV could survive or be maintained in active form by simulated gastrointestinal digestion. Furthermore, the UF-IV exerted potent antihypertensive effect after oral administrations were given to spontaneously hypertensive rats (SHR) at a dose of 900 mg kg⁻¹ body weight. In conclusion, the UF-IV could serve as useful antihypertensive peptides in the prevention and treatment of hypertension and other associated disorders.     

酶法制备沙漠果蛋白ACE抑制肽工艺的研究

在单酶筛选和单因素实验的基础上,采用响应面分析法优化沙漠果蛋白血管紧张素转换酶抑制肽(ACEIP)酶解制备的技术条件,主要研究了酶解时间、酶解温度、初始p H及底物浓度四个因素对酶解工艺条件的影响。以ACE抑制率为响应值,确定碱性蛋白酶的最佳酶解条件为:酶解时间114 min,酶解温度57℃,初始p H10,底物浓度3.24%,酶添加量2000 U/g,此时ACEIP抑制率为69.83%。本研究可为沙漠果蛋白的高效利用奠定基础。      

Optimising the free radical scavenging activity of shrimp protein hydrolysate produced with alcalase using response surface methodology

Response surface methodology (RSM) was used to optimise the hydrolysis parameters of Acetes chinensis by Alcalase 2.4L in order to obtain a hydrolysate with potent radical scavenging activity. The parameters were temperature, pH and enzyme concentration/substrate concentration (E/S) ratio with degree of hydrolysis (DH) being the response. The results showed that the optimum condition was: temperature at 57 °C, pH at 8.0, E/S ratio at 2.6AU 100 g⁻¹ shrimp, hydrolysis time 3 h. The DH was 26.32%, the hydroxyl radical scavenging activity was up to 88.12% and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was 35.61%. The gel column filtration chromatography by a Sephadex G-25 column yielded five fractions. The molecular weight of the most potent free radical scavenging activity fraction ranged from 915 to 207 Da, its IC₅₀ for hydroxyl radical was 0.03 mg mL⁻¹, and IC₅₀ for DPPH radical was 8.86 mg mL⁻¹.     

响应面法优化龙须菜蛋白酶解工艺及酶解液的抗氧化活性

为获得高抗氧化活性的龙须菜蛋白酶酶解液,运用响应面(RSM)分析方法对龙须菜蛋白酶解工艺条件进行优化。经单酶筛选,在单因素实验基础上,以亚铁离子螯合率和DPPH自由基清除率为主要指标,水解度为辅助指标,研究酶解时间、p H、酶解温度、底物质量浓度、加酶量对龙须菜蛋白酶解液抗氧化活性和水解度的影响。结果表明:在所选的5种蛋白酶中,复合蛋白酶是龙须菜蛋白酶解的最适用酶;酶解液抗氧化活性的最优酶解条件为酶解时间8.1 h、酶解温度50.1℃、p H7.0、底物浓度10 g/L、加酶量0.1%(0.15 AU/g)。在此条件下,酶解液的亚铁离子螯合率为74.61%,DPPH自由基清除率为47.66%,水解度为17.58%。对比常用抗氧化剂,酶解液在亚铁离子螯合能力方面显著高于0.01%维生素C和0.01%BHA(p<0.05),而在DPPH自由基清除能力和还原能力方面,酶解液低于0.01%维生素C和0.01%BHA(p<0.05)。优化后的龙须菜蛋白酶酶解液具有一定的抗氧化活性。      

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Preparation and purification of angiotensin‐converting enzyme inhibitory peptides from hydrolysate of shrimp (Litopenaeus vannamei) shell waste

Angiotensin I‐converting enzyme (ACE) inhibitory peptides from the shrimp shell waste (SSW) were isolated using different proteases. The orthogonal test results showed alcalase hydrolysates with ACE inhibitory activity of 67.07% under the optimal hydrolysis conditions of 60 °C hydrolysis temperature, pH = 9.5, 25 g L^?1 substrate and 4000 U g^?1 of enzyme, whereas neutral protease hydrolysates had an ACE inhibitory activity of 84.04% under the hydrolysis temperature of 50 °C at pH = 7.0 with 25 g L^?1 of substrate and in the presence of 2000 U g^?1 of enzyme. Neutral protease was more suitable for the production of ACE inhibitory peptides from SSW, where peptides with MW <5 kDa were recommended. The results of this study indicated that peptides obtained from SSW are as beneficial as antihypertension compounds in the functional food resources.     

Effect of enzyme type and hydrolysis conditions on the in vitro angiotensin I‐converting enzyme inhibitory activity and ash content of hydrolysed whey protein isolate

Removal of salts from protein hydrolysate mixture on large scale is very difficult and relatively inefficient. Selecting practical proteinase system and hydrolysis conditions for the production of whey protein isolate (WPI) enzymatic hydrolysates with high angiotensin I‐converting enzyme (ACE) inhibitory activity and low ash content is very useful. The effect of alcalase, neutrase, trypsin and their combined system, i.e. alcalase‐neutrase and trypsin‐neutrase, under two different hydrolysis conditions, i.e. pH‐controlled and pH‐spontaneous drop, on the formation of ACE‐inhibitory peptides and the characteristics of WPI hydrolysate was investigated. Results showed that the ACE‐inhibitory activity of WPI hydrolysate obtained with alcalase was significantly higher than that of its trypsin or neutrase hydrolysate obtained at the same hydrolysis time by both pH‐controlled and pH‐spontaneous drop method (P < 0.05). The WPI hydrolysate obtained after 3 h incubation with alcalase plus 2 h with neutrase under pH‐spontaneous drop condition possessed the highest ACE‐inhibitory activity of 54.30% and the lowest ash content of 2.95%. This is practical as a functional ingredient in the food industry because of its high ACE‐inhibitory capability, commercial availability in large supply of alcalase and neutrase and no needing for additional desalting process.     

Influence of degree of hydrolysis on functional properties and angiotensin I‐converting enzyme‐inhibitory activity of protein hydrolysates from cuttlefish (Sepia officinalis) by‐products

BACKGROUND: In Tunisia the cuttlefish‐processing industry generates large amounts of solid wastes. These wastes, which may represent 35% of the original material and constitute an important source of proteins, are discarded without any attempt at recovery. This paper describes some functional properties and the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of protein hydrolysates prepared by hydrolysis of cuttlefish (Sepia officinalis) by‐products with crude enzyme extract from Bacillus licheniformis NH1. RESULTS: Cuttlefish by‐product protein hydrolysates (CPHs) with different degrees of hydrolysis (DH 5, 10 and 13.5%) were prepared. All CPHs contained 750–790 g kg^?1 proteins. Solubility, emulsifying capacity and water‐holding capacity increased while fat absorption and foaming capacity decreased with increasing DH. All hydrolysates showed greater fat absorption than the water‐soluble fraction from undigested cuttlefish by‐product proteins and casein. CPHs were also analysed for their ACE‐inhibitory activity. CPH3 (DH 13.5%) displayed the highest ACE inhibition (79%), with an IC₅₀ value of 1 mg mL^?1. CONCLUSION: Hydrolysis of cuttlefish by‐product proteins with alkaline proteases from B. licheniformis resulted in a product with excellent solubility over a wide pH range and high ACE‐inhibitory activity. This study suggests that CPHs could be utilised to develop functional foods for prevention of hypertension. Copyright © 2010 Society of Chemical Industry     

Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests

In vitro gastrointestinal digestion of pea and whey protein produced high angiotensin I converting enzyme (ACE) inhibitory activity with IC₅₀ values of 0.070 and 0.041 mg protein ml^?1 respectively. Ultrafiltration/centrifugation using a membrane with a molecular weight cut‐off of 3000 Da decreased the IC₅₀ value to 0.055 mg protein ml^?1 for pea permeate and 0.014 mg protein ml^?1 for whey permeate. Further fractionation by reverse phase HPLC gave IC₅₀ values as low as 0.016 mg protein ml^?1 for pea and 0.003 mg protein ml^?1 for whey. Consequently, these purification steps enriched the ACE inhibitory activity of the pea digest more than four times and that of the whey digest more than 13 times. HPLC profiles after digestion and ultrafiltration indicate that high ACE inhibitory activity is due to short and more hydrophobic peptides. The results also suggest that potent ACE inhibitory peptides were present alongside low active peptides in whey hydrolysate, while all peptides had more or less the same ACE inhibitory activity in pea hydrolysate. In addition, the hydrolysates and enriched fractions will resist in vivo gastrointestinal digestion after oral administration. Hence these ACE inhibitory peptides, as part of functional foods, can play significant roles in the prevention and treatment of hypertension. Copyright © 2004 Society of Chemical Industry     

响应曲面优化菟丝子多糖提取工艺及抗氧化活性研究

采用响应曲面优化法对菟丝子多糖提取中的料液比、提取温度和提取时间进行优化,确定各因素最佳的水平组合,并对菟丝子多糖抗氧化活性进行研究。结果表明,在料液比1∶25（g∶mL）、提取温度70 ℃、提取时间90 min条件下,菟丝子多糖提取物中多糖的含量为7.67%,实际测得菟丝子多糖提取产率与理论预测值相对误差较小。抗氧化活性试验表明,菟丝子多糖能够保护羟基自由基（OH·）所产生的氧化损伤,对OH·有一定的清除能力,菟丝子多糖的质量浓度为4.0 mg/mL,最大清除率为18.3%。     

Preparation and identification of angiotensin I-converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure

Sweet potato protein hydrolysates (SPPH) with angiotensin I-converting enzyme (ACE) inhibitory activity were prepared by papain, pepsin and alcalase under high hydrostatic pressure (HHP, 100–300 MPa). HHP significantly increased degree of hydrolysis (DH), nitrogen recovery (NR) and molecular weight (MW) <3 kDa fractions contents of SPPH by all three enzymes (P < 0.05). MW < 3 kDa peptide fractions from SPPH by alcalase under 100 MPa showed the highest ACE inhibitory activity (IC₅₀ value 32.24 µg mL⁻¹), and was subjected to purification and identification by semi-preparative RP-HPLC and LC-MS/MS. Fifty-four peptides ranged from 501.28 to 1958.88 Da with 5–18 amino acids were identified and matched sporamin A and B sequences. Five identified peptides with sequences of VSAIW, AIWGA, FVIKP, VVMPSTF and FHDPMLR displayed good ACE inhibitory activity with the contribution of Val, Trp, Phe and Arg. Thus, SPPH by enzymatic hydrolysis under HHP can be potentially used in functional food.     

响应面法优化鸡胸肉肌原纤维蛋白酶法水解工艺条件

以鸡胸肉为原料,提取肌原纤维蛋白,测定其蛋白质浓度及氨基酸组成;以水解度为测定指标,利用响应面法优化该肌原纤维蛋白酶解工艺条件,并考察其产物肽分子量分布。结果表明:提取的肌原纤维蛋白浓度为59.27%;该蛋白氨基酸种类齐全,组成比例均衡,必需氨基酸含量高达40.90%;碱性蛋白酶是肌原纤维蛋白酶解的最佳用酶;在单因素实验基础上,经Box-Behnken实验优化得到碱性蛋白酶水解肌原纤维蛋白最佳工艺条件为:加酶量3500 U/g、p H7.8、温度44℃,此条件下经6 h酶解,水解度达33.17%;肽分子量分布分析知,最佳水解条件下酶解液中<1000 u的小肽含量高达61.5%,说明碱性蛋白酶能较高程度的水解肌原纤维蛋白。      

Application of surface response methodology to optimize hydrolysis of wheat gluten and characterization of selected hydrolysate fractions

BACKGROUND: The aim of this work was to optimize the hydrolysis experimental conditions to obtain wheat gluten hydrolysates and to characterize fractions from hydrolysates with different hydrolysis degrees in order to develop protein functional ingredients. Surface response methodology was used to analyze the effect of reaction factors on the degree of hydrolysis to assess the conditions for maximum fungal protease enzyme activity. Hydrolysates having three different trichloroacetic acid indices (TCAI) were prepared. Soluble fractions at pH 4, 6.5 and 9 from these hydrolysates were characterized by electrophoresis, reverse‐phase high‐performance liquid chromatography, free amino group content and peptide chain length. RESULTS: Temperature and pH ranges for highest enzyme activity at 2.5 h were 54–58 °C and 4.2–4.4, respectively. Hydrolysate fraction composition differs according to the hydrolysis degree and extracting pH, the difference being more pronounced at low TCAI. Hydrolysate having 32% TCAI is composed of peptides whose size is lower than 18.5 kDa, with an average peptide chain length of 14 amino acid residues. CONCLUSION: The combination of hydrolysis degree and pH of extraction allows fractions of different peptide composition to be obtained, which could be taken into account when trying to find a defined composition related to determined functional characteristics. Copyright © 2008 Society of Chemical Industry     

响应面法优化核桃降压肽的酶解工艺研究

本研究以低温脱脂核桃粕为原料,采用碱溶酸沉提蛋白后,分别用碱性蛋白酶、中性蛋白酶、木瓜蛋白酶和风味蛋白酶制备核桃多肽,以血管紧张素转化酶（Angiotensin-I-Converting Enzyme,ACE）抑制率和水解度为指标,选出酶解效果最好的酶,并且对其底物质量浓度、加酶量、酶解温度、酶解时间和p H进行单因素实验,在此基础上采用响应面实验优化其制备核桃降压肽的最佳水解工艺。结果表明:在底物质量浓度30g/L、加酶量8000U/g、酶解温度57℃和p H8.6的条件下水解3h,ACE抑制率可达64.32%,此时酶解液的水解度为21.57%。      

Optimisation of hydrolysis conditions for the production of the angiotensin-I converting enzyme (ACE) inhibitory peptides from whey protein using response surface methodology

In the present research, the effect of process conditions on the angiotensin-I converting enzyme (ACE) inhibitory activity of whey protein concentrate hydrolysed with crude proteinases preparation from L. helveticus LB13 was investigated systematically using response surface methodology. It was shown that ACE inhibitory activity of the whey hydrolysates could be controlled by regulation of three process conditions (hydrolysis temperature, pH and enzyme to substrate (E/S) ratio). Hydrolysis conditions for optimal ACE inhibition were defined using a response surface model. E/S ratio at 0.60, pH at 9.18 and temperature at 38.9 °C were found to be the optimal conditions to obtain high ACE inhibitory activity close to 92.2% and DH of the whey protein was 18.8%.     

响应面法优化复合酶提取香椿叶总膳食纤维的工艺参数

以香椿叶为试材,采用单因素实验和响应面分析法对复合酶(纤维素酶∶淀粉酶=1∶1)提取香椿叶总膳食纤维(TDF)的工艺参数进行了优化。结果表明:在复合酶添加量0.5%,酶解时间2.0h,酶解温度61℃,酶解pH6.1时,香椿叶TDF提取率最高可达74.01%;所得TDF杂质含量低,生理活性好;持水力和膨胀力分别为6.50g/g和4.50mL/g。      

酶法制备鱼降血压肽的工艺优化及其组分分析

为了开发鲬鱼蛋白,以体外血管紧张素转换酶(ACE)抑制率为指标,通过适宜酶酶解鲬鱼蛋白质制备降血压活性肽。根据供试酶的水解进程筛选适宜水解酶,在单因素试验的基础上,采用响应面分析法优化水解条件,凝胶过滤层析法测定活性肽相对分子质量分布。结果表明,Alcalase是供试酶中最适的水解酶,最优水解条件为:温度48.9℃,pH7.93,加酶量为32396.6 U/g·pro,固液比1:5 g/mL,水解时间2.5 h。在此条件下,酶解液的ACE抑制率IC₅₀=0.269 mg/mL;酶解物经Sephadex G-15凝胶过滤层析得到三个组分,相对分子质量分别为635、258、67,其中的2号组分(C=0.74 mg/mL) ACE抑制活性相对较高,达到89.09%。