鲤鱼抗菌精蛋白的分离纯化及鉴定

以鲤鱼鱼白为原料,采用SephadexG-50凝胶过滤和CMSepharoseCL-6B离子交换层析成功地分离、纯化得到一种具有抑菌活性的蛋白质。经反相高效液相色谱、Tricine-SDS-聚丙烯酰胺凝胶不连续电泳鉴定及氨基酸组成成分分析,表明该蛋白质为单一组分,分子量为15320Da,其碱性氨基酸总量为40.76%。     

菜籽饼粕中多肽的提取纯化及抗肿瘤活性

从菜籽饼粕中分离纯化多肽,并验证其抗肿瘤活性.采用超声-微波协同萃取法获得菜籽饼粕总蛋白,通过碱性蛋白酶水解提取菜籽饼粕总多肽,连用分子排阻色谱和反相高效液相色谱纯化获得菜籽饼粕多肽(RMP).通过对Hela细胞生长的抑制作用评价RMP的抗肿瘤活性.结果 显示:RMP由8个氨基酸组成,序列为Asp-Val-Phe-Va...     

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.     

植物乳杆菌JLA-9产细菌素的分离纯化

将来源于东北传统发酵酸菜中的植物乳杆菌JLA-9产的细菌素进行分离纯化,首先利用饱和度为80%的硫酸铵溶液对发酵上清液进行沉淀粗分离。复溶后利用Sephadex LH-20进行凝胶层析纯化,之后采用Hitrap QFF进一步进行离子交换层析纯化,利用反相高效液相色谱(Reversed-Phase High Performance Liquid Chromatography,RP-HPLC)C_(18)柱进行最终纯化,得到单一活性抑菌组分,说明细菌素得到基本纯化,经基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF/MS)确定该细菌素的分子质量约为0.9 kDa左右。采用琼脂扩散法测定了细菌素对常见的食源性致病菌和腐败菌的抑菌效果,结果显示该细菌素具有较好的抑制作用。     

凝结芽孢杆菌XZQ-16抗菌脂肽分离鉴定及抗菌特性

对凝结芽孢杆菌(Bacillus coagulans)XZQ-16脂肽分离鉴定并探讨其抗腐败菌特性.通过酸沉、硅胶柱层析和Sephadex LH-20层析对脂肽分离纯化,利用傅里叶红外光谱(FT-IR)和反相高效液相色谱(RP-HPLC)对脂肽分析鉴定.结果表明,硅胶柱层析得到三个流分具有明显抗菌活性;Sephadex...     

乳酸菌活性物质LPX-600的分离纯化及其性质的研究

经硫酸铵沉淀、阳离子交换层析、凝胶过滤、反相高效液相色谱等分离纯化过程,从类植物乳杆菌(Lactobacillus paraplantarum LPX-600)的发酵液中分离出1种活性物质LPX600。LPX600在酸性条件下活性较高,对热稳定,对蛋白酶K、胃蛋白酶、木瓜蛋白酶不敏感,并且对沙门氏菌、变形杆菌、金黄色葡萄球菌、粘性沙雷氏菌等食品致病菌有很强的抑菌活性。     

米糠蛋白类阿片拮抗肽的分离纯化

采用体积排阻色谱和反相高效液相(RP-HPLC)对米糠蛋白酶解物进行分离纯化,得到一种具有较高活性的类阿片拮抗肽,体积排阻高效液相色谱(SE-HPLC)测定它的相对分子质量为546Da。     

发酵液中牛肉风味肽分离纯化方法的比较研究

使用反相高效液相色谱（RP-HPLC）对重组酵母发酵液中一种牛肉风味肽（BMP）的稳定性进行了研究，并分别运用D301离子交换层析和Sephadex G-25凝胶过滤层析对其进行了分离纯化，最后利用RP-HPLC对目标肽进行分析检测。结果表明，在pH值为7．4条件下，发酵液中的目标肽于室温下放置0h-8h后仍呈现良好的稳定性。分离纯化结果显示，Sephadex G-25凝胶过滤层析的分离纯化效果优于D301离子交换层析，更适合于分离复杂体系发酵液中的风味肽。     

榆干离褶伞菌丝体中溶栓酶的分离纯化

采用离子交换层析、凝胶过滤层析及快速蛋白液相色谱等蛋白质分离纯化技术,从榆干离褶伞菌丝体中分离纯化了一种溶栓酶。实验结果表明,其分子量约为50ku,比活性为750.6Unit/mg,活性得率为12.5%。榆干离褶伞很有潜力成为治疗血栓的新的药物来源。     

草鱼蛋白源抗氧化肽的分离及鉴定

利用超滤、离子交换色谱、高效逆流色谱以及凝胶过滤色谱等系列分离纯化技术从草鱼蛋白酶解产物中分离纯化抗氧化活性肽,分离过程中发现相对分子质量1～3kD 的组分抗氧化活性最强,且碱性肽组分的抗氧化活性强于酸性或中性肽组分、疏水性肽组分的抗氧化活性强于亲水性肽组分。最终借助反相高效液相色谱在线连接的电喷雾质谱结合氨基酸分析鉴定出一种抗氧化肽,一级结构序列为Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val,相对分子质量为966.3D。     

具有ACE抑制活性的大豆肽的RP-HPLC分离和结构鉴定

在优化RP-HPLC洗脱条件的基础上，对经大孔吸附树脂DA201-C、凝胶过滤色谱Sephadex G-15分离得到的ACE抑制活性大豆肽组分进行进一步的纯化。在分离得到的11个组分中，峰9的ACE抑制活性最高，在0．2mg／mL．的浓度下，其ACE抑制率达到96．92％。分析型RP—HPLC纯化组分9得到三个组分，其中组分9-Ⅰ具有最高的ACE抑制活性，为98．46％，其含量约占组分9的50％以上。经过LC-MS序列分析，降血压组分9-Ⅰ的分子质量为772．4，有三种可能的结构：VISTGAEP、VLSTGAEP和ANSAGTVGP。     

HPLC-ESI-MS对珠蛋白酶解液中的血管紧张素I转换酶抑制肽的分离鉴定

本实验利用ESI-MS/MS对反相高效液相色谱分离的具有血管紧张素I转换酶(ACE)抑制活性的珠蛋白小肽的结构进行鉴定。结果表明:此肽的序列为Val-Val-Tyr-Pro-Trp-Thr(VVYPWT),位于猪的血红蛋白β链的34-39氨基酸序列片断,它对ACE有很好的抑制活性,其IC50为6.02μmol/L。     

Intervarietal comparison of proteolysis in commercial cheese

 Proteolysis in different varieties of cheese, i.e. Cheddar, British, Dutch and Swiss types and Italian varieties, was compared by polyacrylamide gel electrophoresis (PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). Urea-PAGE of the water-insoluble fraction (WISF) of cheese appeared to be unable to distinguish between Cheddar and Dutch-type cheeses, whereas it could be used to distinguish Emmental from Parmesan and both of these from Cheddar and Dutch types. Urea-PAGE of the 70%-ethanol-insoluble fraction of the water-soluble extract (WSE) showed large inter-varietal differences but there were also some intra-varietal differences. RP-HPLC of the 70%-ethanol-soluble and -insoluble fractions of the WSEs of cheeses was more effective than urea-PAGE of the WISFs or of the 70%-ethanol-insoluble fraction of the WSEs of cheeses when classifying cheese according to variety. However, analysis of a larger number of cheese samples is required to verify this result. One of the problems with using either urea-PAGE or RP-HPLC to identify cheese is that the characteristics analysed by these techniques change as ripening progresses. Received: 22 September 1997 / Revised version: 8 June 1998     

Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate

This study sought to purify and identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from whey protein hydrolysed by trypsin. The peptide’s amino acid sequence, as well as the molecular mechanism of the interactions between the peptide and the ACE, were also studied. Using ultrafiltration, the hydrolysate was separated into three fractions. The fraction with molecular weight of <6 kDa had the greatest ACE inhibitory activity and was further separated by size exclusion chromatography on Sephadex G-25 and G-10 columns. Reverse-phase high performance liquid chromatography (RP-HPLC) was used to separate the most active fraction. The amino acid sequence of the peptide with the greatest ACE inhibitory characteristics was confirmed as Leu–Leu (LL). The molecular mechanisms, position, type, and energy of the LL/ACE interaction were investigated by using flexible molecule docking technology.     

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.     

Characterization of cheese proteolysis by capillary electrophoresis and reverse-phase HPLC analyses of peptides

 The possibilities of reverse-phase high performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE) when used for separation of cheese peptides are discussed. A CE method using a coated capillary column and a low pH buffer was developed to analyze the water-soluble fraction of a 6-month-old cow’s milk cheese. The CE patterns were compared with the chromatograms obtained by RP-HPLC using a C18 column and a gradient of acetonitrile in water. The CE method gave shorter analysis times but RP-HPLC provided lower coefficients of variation of the retention times and better detection limits. In addition, the elution behavior of peptides in CE strongly depended on the sample matrix. The results show that both techniques provide complementary information for the analysis of cheese peptides. Received: 10 June 1997 / Revised version: 20 October 1997     

Identification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide (ACEIP) from silkworm pupa

The angiotensin I-converting enzyme inhibitory peptide (ACEIP) was isolated and characterized from silkworm pupae and purified using Sephadex G-25 gel filtration. The structure and physicochemical properties of pupa ACEIP were analyzed. The α-P₃ fraction exhibited the most potent ACE inhibitory activity. After purification via semi-preparative reverse-phase HPLC (RP-HPLC) and HPLC, the α-P₃-6-b component was revealed to have the highest ACE inhibitory activity (IC₅₀=28.3 μg/mL). Edman degradation revealed a Val-Glu-Ile-Ser amino acid sequence in which novel active sequences were identified. Physicochemical property testing showed that purified pupa ACEIP exhibits good solubility, heat resistance, and acid resistance that all indicate ACEIP derived from silkworm pupa is an excellent food-derived ACEIP.     

Fractionation, separation, and identification of antioxidative peptides in potato protein hydrolysate that enhance oxidative stability of soybean oil emulsions

Abstract: The objective of the study was to identify the active peptides responsible for the antioxidant activity of potato protein hydrolysate (PPH). PPH was fractionated using ammonium sulfate precipitation; the efficacy of different fractions for scavenging 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^{+ •}) radicals and inhibiting lipid oxidation (hexanal, TBARS) in soybean oil-in-water emulsions was investigated. Of all fractions, the fraction precipitated by 50% saturated ammonium sulfate (P50) exhibited the strongest ABTS^{+ •} scavenging activity and antioxidant activity. Active peptides based on the ABTS^{+ •} scavenging assay were isolated and purified by RP-HPLC and ultra performance liquid chromatography. Amino acid sequencing by tandem mass spectrometry identified Ser-Ser-Glu-Phe-Thr-Tyr and Ile-Tyr-Leu-Gly-Gln in P50 to be the dominant peptides that matched the sequences in metallocarboxypeptidase inhibitor and lipoxygenase 1, respectively.     

Improved methodology for the characterisation of transgenic Bt-11 maize cultivars using RP-HPLC profiles of albumin,globulin, prolamin,and glutelin protein fractions and chemometric analysis

A clear differentiation between Bt-11 transgenic and isogenic non-transgenic maize cultivars has successfully achieved analysing the perfusion and monolithic RP-HPLC profiles of the albumin, globulin, prolamin, and glutelin maize fractions together with a discriminant analysis. Significant differences between transgenic and isogenic non-transgenic cultivars were observed in the chromatograms obtained from any of the four protein fractions. The application of linear discriminant analysis to the area percentages corresponding to every peak detected in every protein fraction was successfully employed for the classification of transgenic Bt maize lines obtaining a global percentage of correct classification of 100%. For perfusion RP-HPLC, the variables with more discriminant power and prediction capability were the following peaks: peaks 1 and 3 for albumins, peak 2 for globulins, peaks 3 and 6 for prolamins, and peaks 7 and 8 for glutelins. In the case of monolithic RP-HPLC, the variables were peaks 2 and 3 for albumins, peak 5 for globulins, peaks 5, 6, and 7 of prolamins, and peak 10 for glutelins.     

The influence of ripening temperature and sampling site on the proteolysis in Reggianito Argentino cheese

The effects of elevated ripening temperature and sampling site on proteolysis in Reggianito Argentino cheese were evaluated. Cheeses ripened at 12 or 18 °C and 85% relative humidity were analysed at 2, 4 and 6 months in 2 sampling zones (central and external). Samples were analysed to determine the physicochemical and proteolysis parameters through the urea-PAGE of the urea-soluble fraction, RP-HPLC analysis of the water-soluble fraction at pH 4.6, and the free amino acid analysis. Proteolysis was significantly affected by ripening temperature and sampling site. Urea-PAGE analysis showed that elevated temperature increased the degradation of α_s1- and β-casein. The degradation of α_s1-casein was larger in the central zone than in the external one, while β-casein degradation was similar in both zones. The majority peaks detected by RP-HPLC of the water-soluble fraction at pH 4.6 and free amino acids were significantly affected by ripening temperature and sampling site. Glu, His, Val, Leu, and Lys had the higher concentrations. Principal component analysis showed useful groupings when results from chromatograms were studied. In conclusion, the results obtained not only are useful to characterise the ripening of an Argentinean hard cheese, but also to evaluate the effect of an increase of ripening temperature on Reggianito Argentino cheese proteolysis.