棉籽蛋白源ACE抑制肽的制备过程中脱盐技术的研究

综合比较了001 ×7、201 ×7离子交换树脂、MWCO100透析袋和DA201-C大孔吸附树脂对棉籽蛋白水解液的脱盐效果,分析了DA201-C树脂脱盐对棉籽蛋白水解液ACE抑制活性和氨基酸组成的影响,并对Sephadex G-25分离纯化获得ACE抑制肽各组分进行活性测定.结果表明:DA201-C大孔吸附树脂的脱盐效果最好,控制流速为3 mL/min时,脱盐率可达到94.78％,氮回收率为86.32％,脱盐后疏水性氨基酸得到了有效富集,ACE抑制率显著提高.分离纯化后的ACE抑制肽各组分中,组分Ⅲ的抑制率最高,分子质量小于1000u.     

大豆肽的离子交换色谱分离及其活性评价

采用Sephadex C-25阳离子交换剂对经过DA201-C型大孔吸附树脂脱盐、乙醇梯度洗脱(75%乙醇洗脱组分)的Alcalase水解大豆肽(DH14%)进行进一步的分离纯化,并对分离得到的各组分ACE抑制活性进行了评价。试管试验确定的SephadexC-25色谱分离条件为:上样量0.004g/mLIE,起始缓冲液为1M醋酸,上样吸附率为61.19%。经SP-Sephadex C-25分离得到6个组分,其中未吸附的3个组分ACE抑制活性低,但NaCl梯度洗脱的3个组分ACE活性均为60%左右,从纯化ACE抑制活性肽的角度考虑,分离效果比SephadexG-15差。     

大豆肽的大孔吸附树脂以及凝胶过滤色谱分离

采用DA201．C型大孔吸附树脂对Alcalase水解。DH14％的大豆肽进行脱盐和乙醇分布洗脱，不同浓度乙醇洗脱物的氨基酸组成分析结果表明：洗脱是按照疏水性递增的方式进行的。乙醇浓度为75％时洗脱下来的组分都具有最高的降血压活性，其ACE抑制率为56．52％。体外模拟实验测定结果表明：经过胃肠道酶的作用，大豆肽的ACE抑制活性仍达到49．52％。采用1M的醋酸作为洗脱液可以实现75％乙醇洗脱组分的Sephadex G-15有效分离，其中ACE抑制活性最强组分的抑制率为69．57％．IC50为0．144mg／mL。     

核桃蛋白ACE抑制肽分离纯化研究

核桃ACE抑制肽经超滤后,其ACE抑制率由76.58%增至78.43%,再经DA201-C大孔吸附树脂脱盐纯化后,脱盐率达到98.70%,ACE抑制率升高至80.73%;采用Sephadex G-15凝胶分离后,核桃ACE抑制肽被分为三个组分,其中活性最大的G2组分,其ACE抑制率达83.10%,且IC50为1.308 mg/mL,G2组分主要分布在500 Da¹80 Da,系为2180 Da,系为2⁴个氨基酸残基组成小肽。     

燕麦ACE 抑制肽的分离纯化及其活性研究

通过对3 种大孔吸附树脂的比较,选择DA201-C 树脂对燕麦ACE 抑制肽进行纯化。纯化后的燕麦肽产物的ACE 抑制率达到92.86%,利用HPLC 测得纯化后燕麦ACE 抑制肽的分子质量分布在240.10～1292.11D 之间,这部分物质在整个纯化产物中占99.82%。采用SephadexG-15 凝胶分离燕麦ACE 抑制肽得到D峰,其IC50 为0.103mg/mL,分子质量545D。采用大孔吸附树脂及凝胶层析法能够较好地分离纯化燕麦ACE 抑制肽。     

大孔树脂吸附分离蚕蛹ACE抑制肽的研究

为了找出大孔树脂纯化蚕蛹ACE抑制肽的方法。比较了5种大孔树脂在纯化蚕蛹ACE抑制肽时的吸附率和解吸率,得出DA201-C分离蚕蛹ACE抑制肽的效果最好。再进一步考察上样流速、上样浓度、分级洗脱等对DA201-C大孔树脂吸附及分离效果的影响。最终结果为75%乙醇洗脱下的多肽的ACE抑制率最高,其IC50为0.632mg/mL,并且发现疏水性与ACE抑制率呈正相关。     

发酵豆酱中ACE抑制肽的制备及分离纯化

为了获得ACE抑制肽,利用微生物发酵法以大豆为原料制成中国传统豆酱,通过研磨、溶解、超滤后获得了ACE抑制活性最高的肽段区间3～10k,并依次利用DEAE琼脂糖微球a、Sephadex G-75凝胶对该组分进行分离纯化,最后纯化获得组分DGIII的ACE抑制活性最高,达到(98.3±1.24)%,IC50为0.08mg/mL,说明豆酱具有开发高抑制率ACE食品的潜力。     

花生ACE抑制肽的分离纯化、结构鉴定及体内降血压功能研究

在水酶法提取花生油和水解蛋白的中试生产基础上,将中试生产得到的水解蛋白通过DA201-C大孔吸附树脂、葡聚糖凝胶Sephadex G-15色谱分离纯化制得花生ACE抑制肽Ⅰ,再经半制备和分析型RP-HPLC进一步分离纯化后.利用基质辅助激光解吸电离-飞行时间-飞行时间(MALDI-TOF-TOF)串联质谱对具有最高ACE抑制活性的组分进行结构鉴定,得到氨基酸序列为Pro-Gly-Arg-Val-Tyr的花生ACE抑制肽Ⅱ.按照该结构合成得到较大量的花生ACE抑制肽Ⅱ,与花生ACE抑制肽Ⅰ-起作为受试样品进行SHR大鼠体内降血压功能实验.实验证明,花生ACE抑制肽在体内确实具有降低血压的作用,与降血压药物相比其服用剂量较大,但安全性相对较高.     

澳洲坚果抗菌肽分离纯化及其肽段鉴定与分析

以液压压榨澳洲坚果粕为原料,采用碱性蛋白酶水解制备澳洲坚果多肽（macadamia nut peptide-0,MNP-0）,以对金黄色葡萄球菌与白色念珠菌的抑菌活性为跟踪指标,通过超滤、DA201-C型大孔吸附树脂、交联葡聚糖凝胶Sephadex G-25对其进行逐级分离纯化,获得抑菌活性较强的抗菌肽,并运用液相色谱串联质谱（liquid chromatography tandem mass spectrometry,LC-MS/MS）技术对其进行肽段组成与氨基酸序列分析。结果表明：超滤分离的不同分子量澳洲坚果多肽在所有多肽中所占质量分数不同,抑菌活性也有所差异。其中,澳洲坚果多肽组分MNP-4占比最高,为29.55%;抑菌活性也最强,对金黄色葡萄球菌与白色念珠菌的抑菌圈直径分别为10.01 mm与9.41 mm。大孔吸附树脂纯化多肽组分MNP-4的技术条件为75%乙醇溶液为解吸剂,静态吸附3 h,动态洗脱体积60 mL。经大孔吸附树脂与交联葡聚糖凝胶Sephadex G-25分离纯化,获得3个澳洲坚果纯化多肽（macadamia nut purified peptide,MPP）组...     

酶法提取燕麦蛋白及脱色工艺的研究

通过正交实验,确定了利用Alcalase从燕麦麸皮中提取燕麦蛋白及YD-303处理燕麦蛋白提取液脱色的工艺条件.结果表明,Alcalase提取燕麦蛋白的最佳条件为:加酶量(E/S)为3%,pH为8.5,料液比为1:22,温度为65 ℃,提取时间为180min.YD-303处理燕麦蛋白提取液脱色最优工艺为:添加量(g/mL)为1.5%.pH为3.5,温度为40℃,脱色时间为75min.     

大豆降胆固醇活性肽的初步分离纯化

研究了大豆降胆固醇活性肽的初步分离纯化,用Alcalase碱性蛋白酶水解大豆分离蛋白得到的酶水解物经过大孔吸附树脂脱盐,并用不同浓度的乙醇以疏水性大小洗脱,得出浓度为75%的乙醇洗脱物具有最高的降胆固醇活性,将此洗脱物再经过SephadexG15凝胶过滤色谱进行分子量分级,得到了部分纯化的大豆降胆固醇肽,其最高降胆固醇活性肽是分子量大多在1000以下的小肽,抑制率为81.26%。     

Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice

The yield and in vitro hypocholesterolemic activity of low-molecular weight soy protein hydrolysates were studied. The yield and cholesterol micellar solubility inhibitory rate (CMSIR) were higher for soy protein Alcalase hydrolysates (SAPH) than soy protein AS1398 hydrolysates (SNPH). The highest CMSIR of 48.6% was found with SAPH with a degree of hydrolysis (DH) of 18%. Optimal parameters for desalting SAPH with macroporous adsorption resin DA201-C were decided to be pH 4.5, SAPH dispersion to resin ration 75:100, and a loading rate 1 BV/h, at which the adsorption rate of the peptide was 89.7%. Only a 4% decrease in CMSIR was found with SPAH (DH 18%) after gastrointestinal protease digestion. In a mice feeding study for 30 d, the serum level of LDL-C + VLDL-C in the SAPH diet group with 0.5 and 2.5 g/kg BW SAPH decreased by 34% and 45% respectively compared to the mice fed only the hypercholesterolemia diet.     

Separation and purification of hypocholesterolaemic peptides from whey protein and their stability under simulated gastrointestinal digestion

In this study, hypocholesterolaemic peptides were separated from whey protein trypsin hydrolysates (WPTHs) and the inhibition of cholesterol micellar solubility (ICMS) and the stability of hypocholesterolaemic peptides after exposure to simulated gastrointestinal conditions were evaluated. Whey protein trypsin hydrolysates were concentrated by ultrafiltration and then desalted through macroporous adsorption resin DA201‐C. Sephadex G‐50 gel filtration chromatography was used to separate the hypocholesterolaemic peptides from the fraction eluted with 75% ethanol. The results suggested that the fraction obtained by gel filtration with a molecular weight (MW) ranging from 1900 Da to 3100 Da exhibited the highest hypocholesterolaemic activity. This fraction was further separated by reversed‐phase high‐performance liquid chromatography into 14 fractions; the peptides with the highest activities were obtained from the fifth peak with a MW of 2454 Da and 58.77% ICMS. The hypocholesterolaemic peptides were relatively stable when exposed to simulated gastrointestinal digestion.     

Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates

A novel hypocholesterolemic peptide was fractionated by gradient ethanol elution from a macroporous adsorption resin (MAR DA201-C), and then separated on Sephadex G-15 and RP-HPLC from a soy protein hydrolysate (SAPH DH 18%). Identification of the hypocholesterolemic peptide structure was accomplished with HPLC–MS. The peptide with the highest hypocholesterolemic activity was found in 75% ethanol fraction among the four fractions from gradient ethanol elution with MAR DA201-C. The calculated average hydrophobicity by amino acid composition of each ethanol eluted fraction suggested that the peptides could be separated in terms of hydrophobicity with MAR DA201-C. Four peaks were obtained with further fractionation on Sephadex G-15, the highest cholesterol micellar solubility inhibition rate, 81.3%, was obtained in Peak 2, corresponding to the molecular weight fraction of 300–800 Da. Fifteen main peaks were obtained with RP-HPLC fractionation, the highest cholesterol micellar solubility inhibition rate (94.3%) was in Peak 7. The amino acid sequence of this peptide was identified as WGAPSL with LC–MS and amino acid composition analysis.     

蚕豆蛋白酶解物分离纯化及降胆固醇活性

采用凝胶过滤色谱对经过大孔吸附树脂纯化的蚕豆蛋白酶解物进行分离纯化,以期得到高降胆固醇活性的酶解物组分。结果表明:单因素试验得到蚕豆蛋白酶解物凝胶过滤色谱最佳分离纯化工艺条件为Sep G-10、G-25葡聚糖凝胶为柱填充材料,10 mg/m L的酶解液上样量4 m L,洗脱剂为去离子水,洗脱流速1 m L/min。凝胶过滤色谱分离纯化后得到F1~F6 6个蚕豆蛋白酶解物组分;与10 mg/m L考来烯胺散阳性对照比较,F3、F6组分对3种胆酸盐(胆酸钠、甘氨胆钠酸、牛磺胆酸钠)抑制率均高于阳性对照,其中F6组分的相对抑制率最高,分别为(274.98±0.19)%、(140.22±0.20)%、(130.99±0.22)%。     

鱼降压肽的大孔吸附树脂分离及其活性稳定性

采用大孔吸附树脂对碱性蛋白酶水解、水解度为34.52% 的鱼降压肽进行分离。结果表明,DA201-C 型大孔吸附树脂对鱼降压肽吸附性最好,乙醇体积分数为75% 时洗脱下来的组分具有最高的ACE 抑制活性,其IC50为1.52mg/ml。鱼降压肽中灰分由分离前的9.47% 减少到分离后的2.34%,肽和蛋白质含量分别由分离前的72.81%和81.26% 增加到分离后的80.24% 和92.42%。活性稳定性分析显示,胃蛋白酶和胰酶、中性和低酸性pH 值、55℃以下温度以及1000mmol/L 以内的NaCl 浓度对鱼降压肽的ACE 抑制活性影响较小。     

κ-卡拉胶脱色方法的研究

采用次氯酸钠、过氧化氢、活性碳和大孔树脂D113-Ⅲ、D201和D303对麒麟菜κ-卡拉胶进行脱色研究。比较κ-卡拉胶脱色率、多糖保留率和凝胶强度。结果表明,次氯酸钠和过氧化氢脱色后,多糖保留率和凝胶强度较低;大孔树脂D201和D113-Ⅲ多糖脱色率很低;活性炭脱色效果较好,但脱色后多糖溶液中残留的活性炭难以清除,对多糖品质有一定影响;大孔树脂D303的脱色效果较好,脱色率达到90.73%,多糖保留率85.76%,凝胶强度1103.7g/cm2。     

Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates

Porcine haemoglobin hydrolysates were prepared through hydrolysis by Alcalase followed by Flavourzyme, and their protein compositions were analyzed using Sephadex G-50 gel filtration chromatography. The antioxidant activities, including reducing power, ferrous ion chelating ability, and DPPH radical scavenging activity, of the hydrolysates were evaluated. The results showed that the hydrolysates of haemoglobin exhibited low reducing powers, but high ferrous ion chelating abilities and DPPH radical scavenging activities. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 h and followed by 1% Flavourzyme for 6 h, had the highest ferrous ion chelating ability of 63.54% at a concentration of 5.0 mg/mL. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 hrs, had the highest DPPH radical scavenging activity of 41.94% at a concentration of 5.0 mg/mL. According to the results of protein composition analysis, we divided the hydrolysates into three groups, including high molecular weight (MW) group (Group I), medium MW group (Group II), and low MW group (Group III). The reducing power and ferrous ion chelating ability of the hydrolysates were significantly and positively correlated to the relative amount of Group I, and negatively correlated to the relative amount of Group III. This study revealed that the antioxidant activities of porcine haemoglobin hydrolysates were dependent on their protein compositions. The high MW protein fraction (Group I) was responsible for the high reducing power and ferrous ion chelating ability of the hydrolysate.