鳕鱼多肽的抗氧化活性及其分离纯化

研究了鳕鱼多肽的抗氧化活性及分离纯化.为获得高纯度的抗氧化肽,将鳕鱼多肽依次通过超滤、凝胶过滤层析、阴离子交换层析(Q-FF)和反相高效液相色谱层析(RP-HPLC)进行分离纯化.结果表明:鳕鱼多肽具有很强的清除羟自由基的能力、清除DPPH的能力和还原能力;经Q-FF柱层析分离得到了B1、B2和B3 3个组分,B2经RP-HPLC检测显示为单一峰,获得了纯度较高的鳕鱼多肽,这为鳕鱼多肽的开发利用提供了科学理论依据.     

黄粉虫抗氧化活性肽的分离纯化研究

为制备具有抗氧化活性的黄粉虫生物活性肽,采用双酶水解黄粉虫蛋白粉,对酶解产物进行葡聚糖SephadexG-25 凝胶柱层析和阳阴离子交换柱层析分离纯化,并对其各组分分子量分布及其抗氧化性进行研究,对抗氧化活性最佳的肽段进行氨基酸组成分析。结果显示,纯化得到的抗氧化多肽对羟自由基和超氧阴离子自由基具有很强的清除作用,清除率可分别达到74.20% 和87.32%。     

乳清蛋白钙螯合肽的分离及结构性质表征

目的:酶解乳清蛋白,通过连续色谱手段分离出强钙螯合活性的肽,再通过结构表征研究其钙螯合性质。方法:酶法水解制备乳清蛋白多肽,采用DEAE-650M阴离子交换色谱、Sephadex G-25凝胶过滤色谱、C18反向高效液相色谱(RP-HPLC)分离乳清蛋白酶解产物,得到特异性的钙螯合肽。采用核磁共振、X射线衍射、TGDSC、Zeta电位结构表征分析钙螯合性质。结果:乳清蛋白多肽经分离纯化得到具有强钙螯合力的肽,命名为WPH-13。其钙螯合活性为63.49μg/mg,结构鉴定发现钙离子可能被一个或多个WPH-13包裹在中央,主要结合位点是羰基氧和氨基氮。与钙结合后p H稳定性和热稳定性提高,抗氧化活性加强。结论:从乳清蛋白中分离得到的钙螯合肽具有较高的钙螯合活性。通过结构性质鉴定研究螯合物的作用机理,为第3代补钙剂的生产应用奠定基础。     

酪蛋白磷酸肽锌螯合肽的分离及结构性质表征

酪蛋白磷酸肽(Casein Phosphopeptides,CPPs)是一类具有二价金属元素螯合活性的多肽集,而定向获得特异性营养元素螯合活性的酪蛋白磷酸肽具有理论和实际意义。采用Q强阴离子交换色谱和高效反相制备色谱(RP-HPLC)连续色谱方案分离碱性蛋白酶酶解得到的CPPs,获得锌高螯合活性的CPPs,并运用氨基酸组成、紫外光谱、红外光谱、固体核磁共振~(13)C谱、~1H谱、~(31)P谱表征分析酪蛋白磷酸肽螯合锌(CPP-Zn)的螯合性质。结果表明:CPPs经连续色谱分离可得具有高锌螯合活性的肽,其锌螯合活性可达88.68μg/mg。螯合前后CPPs的结构及氨基酸组成发生了明显变化,其中的磷酸基团参与了螯合,推测其主要结合位点是—COOH、—NH_2及P—OH中的—OH。     

鱼蛋白酶水解物亚铁螯合修饰物抑菌特性及机理研究

目的:探讨鱼蛋白酶水解物亚铁螯合修饰物的抑菌特性及机理,为带鱼下脚料和其它低值鱼高值化以及多肽亚铁螯合物抑菌剂的应用奠定理论基础。方法:采用葡聚糖凝胶、SDS-PAGE聚丙烯酰胺凝胶电泳等方法分离纯化并鉴定;以牛津杯双层平板法测定抑菌特性;通过透射电镜观测螯合物对大肠杆菌形态的影响。结果:分离到1种具有较高抑菌活性的纯化组分,相对分子质量约26 kDa;其对多种常见微生物,包括大肠杆菌、金黄色葡萄球菌等均有较强的抑制作用。红外光谱检测表明亚铁离子与多肽形成环状共轭结构;多肽亚铁螯合物通过形成跨膜的离子通道,使菌体内容物外泄,最终导致菌体死亡;铁含量与多肽亚铁螯合物抑菌活性有关。结论:多肽亚铁螯合物抑菌机理是通过在微生物细胞表面形成跨膜离子通道,从而具有抑菌功效。推测螯合物可竞争性结合微生物生长所需铁元素,且螯合环结构对其抑菌活性也有贡献。     

西兰花多肽的提取及其抗氧化活性分析

采用丙酮沉淀法提取西兰花花蕾粗多肽,并利用凝胶过滤层析对提取物进行分离纯化,制备西兰花多肽组分;采用超氧阴离子、DPPH自由基的清除及还原力测定实验研究多肽组分的抗氧化活性。结果表明:利用丙酮沉淀法、凝胶过滤层析等方法可分离获得3个组分,其中组分II在0.01 mg/m L和0.1 mg/m L浓度时,其超氧阴离子自由基清除率分别为23.38%、45.19%,高于同浓度维生素C;还原力分别为同浓度维生素C的1.37倍和1.6倍,表现出了良好的抗氧化活性,为进一步开展活性研究及西兰花多肽功能产品开发提供了参考。     

线纹海马活性多肽的体外抗氧化活性评价

近年来,随着人们对海马研究的深入,海马作为制备功能食品的原料受到越来越多的关注。通过研究洗脱流速、上样浓度及上样量对Sephadex G25树脂分离效果的影响,选择出最佳分离条件。利用Sephadex G25凝胶层析柱在最佳纯化条件下分离出不同分子量段的线纹海马混合肽,并对这些不同分子量组分体外抗氧化活性进行评价,以探究线纹海马多肽的抗氧化活性与分子量的关系。结果表明:0.4 mL/min的洗脱流速,0.10 mL的上样量,25 mg/mL的上样浓度作为Sephadex G25凝胶层析的最佳纯化条件。在此最佳纯化条件下分离出4个分子量段的多肽组分,通过测定各组分对DPPH自由基、超氧阴离子自由基和羟自由基清除率后发现,0.43 kDa~2.78 kDa的线纹海马多肽组分比其他分子量范围的组分有更好的抗氧化活性。     

鲍鱼内脏胶原ACE抑制肽纯化及结构初探

以超滤所得鲍鱼内脏胶原血管紧张素转化酶(Angioemin-I Converting Enzyme,ACE)抑制肽(分子量小于3000 u)为研究对象,通过DEAE-52纤维素柱层析分离后用葡聚糖凝胶G-25柱层析对鲍鱼内脏胶原ACE抑制肽进行纯化,得到ACE抑制活性较高的肽。采用红外光谱技术对所得组分的二级结构进行了初步的研究。结果表明:鲍鱼内脏胶原ACE抑制肽经DEAE-52纤维素阴离子交换层析分离,Na Cl溶液(0.1、0.3、0.6 mol/L)单一浓度和梯度洗脱均得到一个组分,梯度洗脱的多肽得率为82.7%,该组分的ACE抑制率为81.23%;再过葡聚糖凝胶G-25分子筛柱,用蒸馏水洗脱,所得的洗脱曲线为单一对称峰,ACE抑制率为82.12%。通过傅里叶红外光谱分析得出胶原ACE抑制肽中具有β-折叠和α-螺旋结构。初步认为,β-折叠和α-螺旋结构对鲍鱼内脏胶原多肽的ACE抑制活性起到重要作用。     

猪骨免疫活性肽的分离纯化

为了从猪骨中分离纯化出免疫活性肽,使用超滤、sephadex G-25凝胶层析、SP-sephadex C-25离子交换层析等手段对鲜猪骨的Alcalase碱性蛋白酶的酶解液进行分离纯化,采用MTT法测定各分离产物对小鼠脾淋巴细胞的增殖活性。结果显示:猪骨酶解液经超滤分离获得的分子质量小于2 ku的组分对小鼠脾淋巴细胞增殖率最高;对分子质量小于2 ku的组分采用凝胶过滤层析,对层析后活性最高的组分再进行离子交换层析,最终得到的组分质量浓度为100μg/m L时,对小鼠脾淋巴细胞的增殖率为114.30%。     

羊骨多肽亚铁螯合物的制备工艺优化及结构表征

为探究羊骨多肽亚铁螯合物的最优制备工艺及结构的表征,本试验采用羊骨粉为原料,制备出羊骨多肽,并超滤分离得出螯合效果最好的肽段为0～3 KDa,再与FeCl_2进行螯合,得到羊骨多肽亚铁螯合物。以螯合率为指标,通过单因素与响应面试验,得到最优条件为反应的肽铁质量比为3∶1,底物浓度为30 mg/mL,pH为5.2,螯合时间为30 min,螯合温度为35℃,最终螯合率为78.00%。以羊骨多肽为参照,试验结果表明,定性检测确定了螯合物的形成,紫外光谱显示亚铁离子与多肽作用,红外光谱表明螯合位点为氨基,羟基和C=O,扫描电镜可看出羊骨多肽与亚铁离子发生了吸附作用,结构紧密。通过氨基酸分析可得出,氨基酸组成符合胶原多肽的成分特征,螯合后天冬氨酸与谷氨酸含量增加显著。     

Purification and characterization of a dipeptidase from Lactobacillus delbrueckii subsp. bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu²⁺, and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50°C. Reducing agents, like dithiothreitol and β-mercaptoethanol, increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co²⁺ and Mn²⁺. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K_m-values for both substrates were about 0·56 and 1·23 mM, with turnover numbers of 870 and 480 s⁻¹, respectively.     

Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates

A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purification of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chromatography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combination of high His contents, around 20–30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption.     

Purification of an Arabinofuranosidase and Two β‐Xylopyranosidases from Germinated Wheat

Arabinosidase and β‐xylosidase activities were detected in germinated wheat grain, and both increased over seven days of germination, under malting conditions. Arabinosidase was partially purified by anion exchange chromatography, chromatofocusing and gel filtration chromatography. The pH optimum of the partially purified enzyme was 4.2 and the K_M was 1.90 mM p‐NP‐Ara. Edman degradation, MALDI‐TOF mass spectrometry and nano‐ESI mass spectrometry were used to identify the two major proteins in the partially purified arabinosidase mixture. The two proteins were a β‐amylase with an amino acid sequence partially homologous to a barley β‐amylase, and three wheat serine protease inhibitors. Further purification, by affinity chromatography and hydrophobic interaction chromatography, removed the identified contaminating proteins. At this point an 80 kDa protein was detected by SDS‐PAGE. No identity could be assigned to this protein by MALDI‐TOF mass spectrometry by reference to electronic protein databases. Similarly, the β‐xylosidase was partially purified by anion exchange chromatography followed by chromatofocusing and gel filtration chromatography. The first step separated the mixture into two distinct fractions with K_M values of 3.35 and 4.01 mM p‐NP‐Xyl and pH optima of 4.5. The latter fraction also displayed xylanase activity against RBB‐xylan.     

水晶冰菜总黄酮提取工艺优化、结构表征及组成成分分析

本研究优化了超声辅助法提取水晶冰菜总黄酮的条件并对其结构和组成成分进行了深入分析.以得率为指标,在单因素实验基础上,对料液比、浸提时间、温度和功率四个因素进行正交试验,得出最优提取条件;在此条件下采用D101大孔树脂纯化水晶冰菜总黄酮,通过紫外可见光谱(UV-Vis)、傅立叶变换红外光谱(FT-IR)对纯化前后总黄酮的...     

巴西坚果过敏原Ber e 1的分离纯化及表征鉴定

以巴西坚果仁为原料,通过粉碎、脱脂、浸提、阴离子交换层析纯化过敏原蛋白Ber e 1;利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、液相色谱-串联质谱联用、Western blot等方法对其进行鉴定,并通过圆二光谱仪和紫外分光光度计表征其二、三级结构。结果表明：纯化获得的巴西坚果过敏原Ber e 1,单轮制备量可达20 mg以上,且纯度大于95%,其蛋白质高级结构未被破坏,能够被巴西坚果过敏患者的血清准确识别。该纯化技术路线简单、对设备要求低且高效,为巴西坚果过敏原Ber e 1的相关研究奠定了一定的基础。     

酶解大豆蛋白抗氧化肽的分离纯化与鉴定

对前期优化的碱性蛋白酶最优酶解条件下的大豆蛋白抗氧化肽进行进一步分离、纯化及鉴定。采用高效强阴离子交换柱（HiTrap Q HP）、快速弱阴离子交换柱（HiTrap DEAE-FF）和制备液相色谱方法,对酶解液进行分离纯化,获得高纯度的抗氧化肽SHP-1。通过液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry,LC-MS/MS）测定抗氧化肽SHP-1的分子质量,为741.41 Da;采用氨基酸分析仪对抗氧化肽SHP-1的组成进行分析,表明抗氧化肽SHP-1是由5 种氨基酸组成。通过LC-MS/MS对抗氧化肽SHP-1氨基序列进行解析并与大豆蛋白数据库比对,确定抗氧化肽SHP-1氨基酸序列为IPPGVPY,来自于大豆球蛋白G4亚基。     

Iron-chelating activity of chickpea protein hydrolysate peptides

Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption.     

鱼鳔类肝素的分离纯化与结构鉴定

本研究以鱼鳔为原料,采用酶法提取类肝素化合物,采用阴离子交换树脂吸附、氯化钠溶液梯度洗脱和醇沉法进行分离纯化;并进一步分析其结构特征,分别通过紫外光谱法、高效凝胶色谱分析其纯度和分子质量,醋酸纤维素薄膜电泳初步鉴定其种类,柱前衍生-高效液相色谱法分析其单糖组成,傅里叶变换红外光谱分析其官能团结构,酶裂解-质谱/质谱法测定其二糖组成,核磁共振鉴定其一级结构。结果显示：鱼鳔类肝素的得率为（2.21±0.03） mg/g,纯度较高,类肝素含量为（85.79±0.63）%,分子质量约为84 000 u;单糖组成为葡萄糖醛酸（glucuronic acid,GlcA）和N-乙酰基半乳糖胺（N-acetylgalactosamine,GalNAc）,同时含有少量的艾杜糖醛酸、半乳糖;电泳位移与硫酸软骨素相似,且具有羧基、乙酰氨基、硫酸基轴向伸缩等特征吸收峰。最终确定鱼鳔类肝素主要组成是由重复的二糖单位[→4GlcUA β1→3GalNAc(4S)β1→]构成的硫酸软骨素A。     

Purification and identification of antioxidant peptide from black pomfret, <Emphasis Type="Italic">Parastromateus niger</Emphasis> (Bloch, 1975) viscera protein hydrolysate

To utilize fish waste, black pomfret, Parastromateus niger viscera was analysed for its proximate and amino acid composition followed by hydrolysis using various proteases to extract antioxidant peptide. Antioxidant activities of the crude hydrolysate was evaluated using DPPH (54%), metal chelating (78.6%) at a concentration of 1 mg/mL, whereas the reducing power assay was done with different concentration (0.5–2.5 mg/mL) and the activity also increased with increasing concentration (0.021–0.068). Furthermore, the hydrolysate was purified by diethylaminoethyl (DEAE) ion-exchange and Sephadex G-25 gel filtration chromatography. Finally, the purified peptide had a mass of 701.9 Da, and the amino acid sequence was identified as Ala-Met-Thr-Gly-Leu-Glu-Ala using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Moreover, the protection ability of the peptide toward hydroxyl radical-induced oxidative DNA damage and inhibiting lipid peroxidation was evaluated and compared with natural antioxidant α-tocopherol.     

Purification of Amylase from Honey

The major amylase in honey was concentrated by ultrafiltration, isolated by ultracentrifugation and gel filtration, and purified by ion‐exchange chromatography. The amylase activity was in the flow‐through fraction of the anion‐exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzyme fraction from the anion‐exchange chromatography was loaded onto a cation‐exchange column, and the amylase activity was eluted as a single band at 50 mM NaCl. The purification factor after this step was 531‐fold. The purified enzyme was an a‐amylase, as determined by thin‐layer chromatography, with a molecular weight of 57000 Da according to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The results supported the concept that amylase in honey had a high degree of similarity with bee amylase.