绿茶抗氧化肽的分离纯化与抗氧化活性研究

通过饱和硫酸铵盐析的方法从干绿茶中提取得到绿茶粗蛋白,粗蛋白透析除盐后用葡聚糖凝胶Sephadex G-75层析柱进行凝胶色谱法分离纯化,收集具有抗氧化活性的蛋白样品,并冷冻干燥,同时利用SDS-PAGE的方法对凝胶分离后收集的活性样品进行纯度的鉴定及相对分子质量的测定,并采用·OH清除法及O-2·清除法研究绿茶抗氧化多肽纯品的抗氧化活性。结果表明,绿茶抗氧化多肽经葡聚糖凝胶sephadex G-75层析柱后,得到很好的分离和纯化,在SDS-PAGE电泳图谱上只有1条条带,其相对分子质量为2¹5ku;绿茶抗氧化多肽对·OH具有很强的清除作用,其半清除浓度（IC50）为0.069μg/mL;对O-2·也具有较强的清除作用,其半清除浓度（IC50）为18.462μg/mL,与VC的清除作用（IC50为11.186μg/mL）相当。      

绿茶抗氧化肽的分离纯化与抗氧化活性研究

通过饱和硫酸铵盐析的方法从干绿茶中提取得到绿茶粗蛋白,粗蛋白透析除盐后用葡聚糖凝胶Sephadex G-75层析柱进行凝胶色谱法分离纯化,收集具有抗氧化活性的蛋白样品,并冷冻干燥,同时利用SDS-PAGE的方法对凝胶分离后收集的活性样品进行纯度的鉴定及相对分子质量的测定,并采用·OH清除法及O-2·清除法研究绿茶抗氧化多肽纯品的抗氧化活性。结果表明,绿茶抗氧化多肽经葡聚糖凝胶sephadex G-75层析柱后,得到很好的分离和纯化,在SDS-PAGE电泳图谱上只有1条条带,其相对分子质量为2~15ku;绿茶抗氧化多肽对·OH具有很强的清除作用,其半清除浓度(IC50)为0.069μg/mL;对O-2·也具有较强的清除作用,其半清除浓度(IC50)为18.462μg/mL,与VC的清除作用(IC50为11.186μg/mL)相当。     

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.     

脱脂米抗氧化肽的分离纯化与研究

通过碱性蛋白酶酶解脱脂米糠蛋白制得脱脂米糠抗氧化肽（DRBAP）,用DEAE-52、SephadexG-15纯化DRBAP,薄层层析鉴定其纯度。通过亚油酸体系中抗氧化能力、自由基捕获能力、金属离子螯合力等不同的实验体系研究其纯化组分的体外抗氧化活性,同时分析了其氨基酸组分和分子量分布情况。结果表明,分离纯化得到抗氧化活性较高的组分G4,较纯化前的DRBAP其对铁离子的螯合率提高了近11%,对DPPH自由基、·OH、O2-·清除率依次提高了20%、9%、16%,在亚油酸体系中吸光值下降了0.7,其主要氨基酸成分为Asp、Glu、Leu、Phe、Arg、Pro,相对分子质量小于1000u的比例达到90.83%,分子量小于500u的达到14.38%。      

鲜马奶乳清蛋白抗氧化肽分离纯化及其活性研究

分离纯化鲜马奶抗氧化活性肽并检测其抗氧化活性。以胰蛋白酶为酶源酶解鲜马奶乳清蛋白,采用3KDa、10KDa、30KDa不同分子量范围超滤膜对水解液进行分离,分子量在3~10KDa段的酶解液还原能力、DPPH清除能力、羟自由基清除能力,超氧阴离子清除能力均较高,分别为0.8949、63.19%、77.13%、70.08%。用SephadexG-50葡聚糖凝胶色谱纯化鲜马奶抗氧化活性肽并检测其抗氧化活性,得到A、B两个组分,B组分的还原能力、DPPH清除能力、羟自由基清除能力、超氧阴离子清除能力均高于A,分别为0.9814、55.28%、74.25%、56.67%。说明鲜马奶乳清蛋白酶解液具有一定的抗氧化活性,为鲜马奶抗氧化肽进一步纯化、结构鉴定及其生物活性研究提供基础。     

乳清多肽的分离纯化及体外抗氧化活性研究

采用乳酸菌发酵乳清制备乳清多肽,利用超滤和凝胶Sephadex G-25分离纯化乳清多肽,并研究了超滤后乳清多肽对羟自由基、氧自由基和DPPH自由基的清除作用。结果表明,超滤后乳清多肽主要包含两个级分,分子量分别为2 031 u和328 u,对羟自由基、氧自由基和DPPH自由基具有清除作用。     

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%。     

海洋胶原低聚肽的分离纯化及其抗氧化活性研究

通过总抗氧化能力、羟基自由基清除能力、超氧阴离子自由基清除能力及还原能力实验,对海洋胶原低聚肽的体外抗氧化活性进行考察,然后采用反相高效液相色谱（RP-HPLC）对海洋胶原低聚肽进行分离纯化,测定收集得到的11个组分的总抗氧化能力。结果表明,海洋胶原低聚肽具有一定的总抗氧化能力、羟基自由基和超氧阴离子自由基清除能力,并且具有一定的还原能力;RP-HPLC分离纯化后,5个组分的抗氧化活性显著提高,对总肽整体的抗氧化活性起到了重要的作用,并且疏水性肽组分对抗氧化活性的贡献大于亲水性肽组分。      

猪皮胶原蛋白抗氧化肽的分离纯化及体外抗氧化活性研究

采用碱性蛋白酶对猪皮胶原蛋白进行酶解,制备抗氧化肽。为了得到抗氧化活性高且纯度高的抗氧化肽,本实验采用多种体外抗氧化评价体系研究超滤获得的各分子质量段猪皮胶原蛋白抗氧化肽的体外抗氧化活性;依次采用离子交换色谱、凝胶色谱对猪皮胶原蛋白酶解液进行分离纯化。结果显示:超滤分离获得5~10ku的抗氧化肽较其他分子量范围的抗氧化肽具有较好的抗氧化活性;采用离子交换色谱、凝胶色谱两种分离方法分步分离能达到较好的分离纯化效果,离子交换色谱分离得到7个组分,其中组分P1对O-2·清除率最高,多肽浓度为0.85mg/mL时,清除率为46.30%,IC50值为1.24mg/mL;该组分经过凝胶色谱分离后得到2个组分,其中组分P1-B对O-2·清除率最高,多肽浓度为0.9mg/mL时,清除率为49.43%,IC50值为0.98mg/mL。     

苦瓜分离纯化物抗氧化活性试验的研究

通过4种不同的体系(猪油、羟自由基、亚硝酸盐、DPPH·)研究了苦瓜粗提物经H103大孔树脂纯化的各洗脱样的抗氧化活性,结果表明,20%乙醇洗脱样的抗氧化效果最佳,在其对油脂的抗氧化体系中,其抗氧化性和维生素C相当.     

ANTIOXIDATIVE ACTIVITY OF PROTEIN HYDROLYSATE FROM ROUND SCAD MUSCLE USING ALCALASE AND FLAVOURZYME

Antioxidative activity of hydrolysates from round scad (Decapterus maruadsi) muscle with degrees of hydrolysis (DH ) of 20, 40 and 60%, prepared using Alcalase (HA) or Flavourzyme (HF), was determined. At the same DH, HF exhibited a higher 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity and reducing power, but a lower Fe²⁺ chelating ability than HA. HF from isopropanol‐defatted muscle with 60% DH was extracted using different solvents, and hexane (E1), dichloromethane (E2), ethyl acetate (E3) and residual (R) fractions were obtained. Among all fractions, E2 and E3 exhibited the highest DPPH radical scavenging activity and reducing power. HF with 60% DH and E2 at 1,000 ppm exhibited antioxidant activity in linoleic oxidation and lecithin liposome systems, and the results were comparable to butylated hydroxytoluene (BHT) at 100 ppm. Therefore, type of proteinase, DH and defatting process prior to hydrolysis exerted an influence on the antioxidative activity of hydrolysates.     

PURIFICATION AND CHARACTERIZATION OF A PHYTASE FROM SPELT

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K_M 400 μmoll^?1 and k_cat 368s^?1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: I. PURIFICATION AND PROPERTIES

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-100 column chromatography. The enzyme was purified 11.8 fold resulting in a specific activity of 250.3 units/mg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K_mfor catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4.11 mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and 1-naphtol did not show any activity. Data for V_max/K_m which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu²⁺ ions bound was found to be 2 per enzyme molecule.     

PURIFICATION AND CHARACTERIZATION OF THREEISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purified to homogeneity from tomato (var. S. marzano). The isozymes were separated by affinity chromatography on Heparin-Sepharose column. They exhibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sulfate gel electrophoresis and of 35 kDa in gel-filtration chromatography in native conditions. The isoelectric points of all three isozymes were found to be higher than 9.3. The K_ms calculated for the three isozymes were different toward citrus pectin used as substrate; one had a K_m of 9.7 mM (by expressing the pectin concentration as mmoles/L of methoxy groups) and the other two had similar K_ms of 3.0 and 2.6 mM, respectively. The isozyme having the higher K_m for substrate was inhibited by citrus pectin (which had a degree of methylation of 70%) at concentrations higher than 5 mM, but no inhibition was found using a pectin with a degree of methylation of 30% at concentrations up to 13 mM (i.e. 9 mg/ml) with a K_m of 14.7 mM. Furthermore, this isozyme showed a more broad range of activity in a pH range 5–10 with respect to that exhibited by the other two isozymes. All three isozymes were found to be glycosylated, although to different extents.     

PURIFICATION AND CHARACTERIZATION OF A LIPASE FROM LACTOBACILLUS PLANTARUM 2739

A 65 kDa intracellular lipase from Lactobacillus plantarum 2739 was purified to homogeneity (482-fold, specific activity of 251 μmol/mg per min) and characterized. The purification procedure included chromatography with Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The purified lipase was optimally active at pH 7.5 and 35C; it retained about 40% of the maximum activity at pH 5.0 and 15C. The enzyme was stable at 65C (D_65C= 18.6 min) and was irreversibly inactivated at 75C for 2 min. On triglycerides, the highest activity was determined on tributyrin but trilaurin and tripalmitin were hydrolyzed also. The Km on tributyrin was 2.31 mM. β-Naphthyl esters of fatty acids from C2 to C12 were hydrolyzed with a preference for β-naphthyl butyrate. After lipolysis, the fatty acid profiles in β-monoacylglycerols of milk fat showed similarities among porcine pancreatic lipase, rennet paste and lipase from Lb. plantarum 2739, but the bacterial enzyme caused a greater hydrolysis of C10 and C12 fatty acids esterified at the Sn-2 position of glycerol. The lipase was strongly inhibited by 1 mM Nethylmaleimide and iodoacetic acid, by 10 mM Hg²⁺ and Ag⁺, and was moderately stimulated by Ca²⁺ and Mg²⁺.     

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean (Phaseolus mungo). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CM‐Sephadex and high performance liquid chromatography on POROS HS‐20. The purified protein exhibited a molecular mass of 38 kDa in SDS‐polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 µM. The partial N‐terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean (Glycine max) and potato (Solanum tuberosum), respectively.