从牛初乳中提取酪蛋白、免疫球蛋白、乳白蛋白、乳铁蛋白生产工艺的研究

本文详细概述了酶法分离酪蛋白、分步超滤法分离球蛋白与乳铁蛋白及离子交换色谱法纯化乳铁蛋白的工业化生产过程。文中对分离工艺、设备选型及操作参数作了全面论述。     

牛乳中乳铁蛋白的纯化和抗菌活性研究

采用凝胶过滤层析和离子交换法层析分离纯化乳铁蛋白,通过SDS-PAGE和IEF-PAGE鉴定所得样品;同时利用抗菌实验(滤纸片法)进行样品的抗菌活性研究,并研究了乳铁蛋白分离纯化过程中抗菌活性的变化。结果表明,离子交换层析纯化乳铁蛋白的效果优于凝胶过滤层析,抗菌活性证实乳铁蛋白样品具有广谱抗菌作用,以蜡状芽孢杆菌为指示菌时,比活为3077.3AU/mg(试管法),纯化倍数提高了20.2倍。     

乳铁蛋白及其应用研究进展

综述了乳铁蛋白的理化学性质、生物学功能、分离纯化方法及其应用进展,并对乳铁蛋白的应用及开发前景进行了展望。     

乳铁蛋白分离纯化及功能特性的研究进展

乳铁蛋白作为乳中一种功能性成分,具有多种生理功能,目前被广泛应用于食品和医药等领域,具有良好的发展潜力。本文对乳铁蛋白进行了概述,分析比较了离子交换色谱法、盐析法、超滤法以及亲和色谱法等多种分离纯化方法,并简要介绍了乳铁蛋白的生理功能特性,为后续进一步研究乳铁蛋白的生产工艺和生理功能提供了理论基础。     

牛乳铁蛋白及乳铁素的生产与应用现状

牛奶中的乳铁蛋白和其水解产物乳铁素具有多种生物活性功能。综述了国外的乳铁蛋白和乳铁素生产、分离和纯化的方法,并介绍了乳铁蛋白和乳铁素作为食品保存剂、抗氧化剂、营养强化剂、免疫强化剂、免疫调节剂等应用的现状。展望了我国乳铁蛋白和乳铁素生产的前景。     

牛乳中乳铁蛋白的分离纯化

采用强阳离子交换色谱梯度洗脱法,对牛乳中的乳铁蛋白进行分离和纯化,并利用SDS-PAGE定性检测.结果表明:采用强阳离子交换色谱梯度洗脱法可较好地分离提取出乳铁蛋白,分离出的乳铁蛋白显示为单一区带,每毫升牛乳能提取乳铁蛋白0.23 mg.纯度为95%.     

乳铁蛋白分离纯化技术研究进展

对乳铁蛋白的分离纯化方法进行了归纳和总结,并介绍了近年来涌现出来的新技术、新方法,以期为低成本、高效率的乳铁蛋白工业化生产工艺开发提供参考。     

乳铁蛋白

本文概述了乳铁蛋白的基本性质、分离方法和生物活性．并对其开发利用提出展望。乳铁蛋白是一种铁结合糖蛋白，它能调节动物体的多种生理功能和影响铁的代谢，这些生物活性和铁量、盐类、ＰＨ、抗体、介质密切相关；色谱法被认为是生产高纯乳铁蛋白的有效方法．超滤法可用于食品级乳铁蛋白的分离。     

反相高效液相色谱法快速测定乳铁蛋白含量

目的建立一种反相高效液相色谱法(RP-HPLC)快速测定乳铁蛋白在乳原料及乳制品中含量的方法。方法根据酪蛋白在等电点(p H 4.6)凝聚沉淀特点,采用RP-HPLC法,用p H4.6醋酸盐缓冲液溶解分离乳铁蛋白,用C18色谱柱快速分离,用流动相A为0.1%三氟乙酸水溶液,流动相B为含有0.09%(V:V)三氟乙酸的90%(V:V)乙腈水溶液,进行梯度洗脱,检测波长214 nm,流速0.7 m L/min。结果乳铁蛋白在10~500 mg/L之间呈现良好的线性,相关系数为0.9999,平均回收率为98.0%,RSD=0.8%(n=9)。结论该方法推广性高、快速,简便、准确,可用于乳铁蛋白原料、奶粉、牛奶及保健品等中乳铁蛋白的含量检测。     

乳铁蛋白的特性、分离纯化方法及特点

乳铁蛋白是一种铁结合性糖蛋白,在初乳中含量较高,乳铁蛋白具有多种生物学功能,如抗菌性、抗氧化、抗病毒、抗癌症等。乳铁蛋白的分离纯化方法较多,就吸附色谱法、离子交换色谱法、亲和色谱法、固定化单克隆抗体法、超滤法、盐析法等进行了概述,并就其工业化生产方法进行了展望。     

Lactoferrin concentration during involution of the bovine mammary gland.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.     

Unsaturated Iron-Binding Capacity of Human Milk

The unsaturated iron-binding capacity (UIBC) and lactoferrin concentration of human milk from early stages of lactation were determined. The UIBC decreased significantly (p < 0.01) from 342 μg Fe/100 ml at the early transitional stage to 261 μg Fe/100 ml at the transitional stage and 215 μg Fe/100 ml at the mature stage. The lactoferrin concentration decreased significantly (p < 0.01) from 406 mg/100 ml at the early transitional stage to 241 mg/100 ml at the mature stage. No significant diurnal variations in UIBC or lactoferrin concentration were noted. Approximately 60% of the UIBC was retained after three different pasteurization treatments while less than 40% of the lactoferrin was retained. Seventy-five percent of the UIBC was retained after sterilization; however, lactoferrin was completely destroyed. After frozen storage, the UIBC of the initial sample increased by 37% while the lactoferrin concentration decreased by 30%. These results indicate that there is not a direct relationship between iron-binding capacity and lactoferrin concentration.     

基于核酸适配体的胶体金比色法检测奶粉中的乳铁蛋白

目的 建立一种基于核酸适配体(aptamer)胶体金(colloidal gold, AuNPs)比色法检测奶粉中乳铁蛋白(lactoferrin, LF)的方法。方法 以无标记的LF适配体为生物识别元件, AuNPs为信号分子, 利用AuNPs的光学特性构建一种可定性和定量检测LF的比色适配体传感器。结果 在最优条件下, 20 min内即可完成检测, 在5~75 nmol/L浓度范围内具有良好线性关系,相关系数(r2)为0.998, 检出限为3.6 nmol/L, 特异性较好, 并且在基质标准曲线中也表现出较好的线性关系(r2=0.987)。结论 该方法操作简单, 分析快速, 安全环保, 灵敏度较高, 有望应用于奶粉中LF的检测。     

In vitro reconstitution of antimicrobial pathogen activity by expressed recombinant bovine lactoferrin N-terminal peptide in Escherichia coli

Recombinant bovine lactoferrin N-terminal polypeptide (rbLF-N) Escherichia coli expression system was constructed and the rbLF-N antimicrobial activity was displayed by enzymatic proteolysis in this study. A 162 bp 5'-terminal fragment of bovine lactoferrin (bLF) gene from bovine liver gDNA was amplified by PCR. The DNA fragment containing exon-2 of the bLF gene was cloned into the expression vector pGEX-4T1 and the glutathione-S-transferase-rbLF-N (GST-rbLF-N) fusion protein was obtained by over-expression in Esch. coli BL21(DE3). After thrombin/pepsin digestion, the rbLF-N was released from the fusion protein. The recombinant peptide was separated and identified by SDS-PAGE, HPLC and LC-MS/MS analysis. A very strong anti-food-born microbial pathogen activity of the rbLF-N peptides was displayed through bio- and kinetic-assays in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the rbLF-N peptide for bacterial pathogens Staphylococcus aureus, Streptococcus mutans, Esch. coli and Klebsiella pneumoniae were 11.7, 11.7, 11.7, 23.4 microg and 23.4, 11.7, 11.7, 46.4 microg, respectively. This study created a new route for exploring lactoferrin peptide application in food science.     

Effect of bovine lactoferrin addition to milk in yogurt manufacturing

The aim of this work was to study the effect of milk supplementation with lactoferrin of different iron saturation on the manufacturing and characteristics of yogurt. Bovine lactoferrin was added at concentrations of 0.5, 1, and 2 mg/mL in the holo (iron saturated) and apo (without iron) forms. Some physicochemical properties, such as pH, concentration of lactic acid, and texture of supplemented yogurts, were determined throughout the shelf-life period storage (28 d) at 4°C. We also evaluated the stability of lactoferrin in supplemented yogurt throughout the storage time. The supplementation of milk with bovine lactoferrin did not greatly affect the physical properties of the yogurt, though apo-lactoferrin slightly delayed the decrease of pH. This could be attributed to the partial inhibition observed on the growth of Streptococcus thermophilus. The integrity and immunoreactive concentration of lactoferrin, determined by Western blotting and noncompetitive ELISA, respectively, remained constant throughout the shelf life of yogurt.     

Enhancement of the activity of novobiocin against Escherichia coli by lactoferrin

The activity of novobiocin against Escherichia coli ATCC 25922 and three E. coli strains that were isolated from cases of bovine mastitis was determined in timekill studies in the presence of bovine lactoferrin. Lactoferrin alone did not affect the growth of any of the strains of E. coli. A combination of 1.0 mg/ml of lactoferrin and novobiocin at 1/16x minimum inhibitory concentration (MIC) was bactericidal for E. coli ATCC 25922. When the concentration was increased to 3.0 mg/ml of lactoferrin, novobiocin was bactericidal at 1/64x MIC. Among the mastitis strains tested, 6789 and 6806 were more susceptible to killing by novobiocin than was strain 6800. Strains 6789 and 6806 were killed when treated with novobiocin concentrations of 2, 1/2, and 1/4x MIC. When these strains were also treated with lactoferrin at 3.0 mg/ml, there was a bacteriostatic effect at novobiocin concentrations of 1/8 and 1/16x MIC for strains 6789 and 6800. Strain 6806 appeared to be more susceptible to the combination of lactoferrin and novobiocin as was evidenced by a bactericidal effect over the 24-h testing period. The combination treatment with cephapirin and lactoferrin showed that there was a synergistic bactericidal effect against all of the E. coli strains tested. These studies indicate that lactoferrin can potentiate the activity of antibiotics against Gram-negative bacteria.     

Effect of ACTH and oxytocin treatment on lactoferrin and citrate in cows' milk

Six dairy cows were treated before milkings with either oxytocin (Pitocin, 20 i.u.) or ACTH (Synacthen, 150 i.u.), principally to determine their effect on the ratio of citrate: lactoferrin concentrations in the milk. With ACTH treatment, after 3 d milk yield and citrate concentration decreased significantly, lactoferrin and bovine serum albumin (BSA) concentrations increased significantly. Somatic cell counts (SCC) increased temporarily in the milk of three of the cows which previously had greater than 100 000 cells/ml. Lactoferrin yield remained fairly constant but citrate yield was significantly reduced. The citrate: lactoferrin molar ratio decreased from 1373 to 606. With oxytocin treatment, after 4 d milk yield first increased and then significantly decreased, citrate concentration decreased significantly while there were no significant changes in lactoferrin or BSA concentration or in the yield of any other milk constituents. The citrate: lactoferrin molar ratio decreased from 1621 to 1301. There were no significant changes in SCC either during treatment or 4 d after treatment but there was a significant rise at 16 d after treatment. It was concluded that in lactating cows both hormones affected citrate and lactoferrin concentrations in the direction that would improve the antibacterial properties of milk, but that this was accompanied by adverse effects on milk secretion. The extent of the change was not sufficient to be likely to produce inhibition of coliform bacteria.     

常用防腐剂对牛乳铁蛋白抗菌活性的影响

乳铁蛋白的抗菌活性常受到其他共存成分的影响。本文研究了常用防腐剂对乳铁蛋白抗菌活性的影响。在研究过程中,以大肠杆菌为实验菌株,进行了实验研究。研究表明,在牛肉膏蛋白胨培养基中,pH6.0,菌浓为106CFU/ml的条件下,乳铁蛋白对大肠杆菌的最小抑菌质量浓度为3mg/ml;苯甲酸钠、山梨酸钾、乳酸链球菌素、对羟基苯甲酸等分别与乳铁蛋白共同作用时,对乳铁蛋白的抗菌活性都有明显的影响。其中,山梨酸钾和苯甲酸钠减弱了乳铁蛋白的抗菌活性,乳酸链球菌素和对羟基苯甲酸丁酯加强了乳铁蛋白的抗菌活性。     

不同来源乳铁蛋白及乳铁蛋白素对小鼠脾淋巴细胞增殖影响的比较

以牛乳清乳铁蛋白（bovine whey lactoferrin,LFW）、牛初乳乳铁蛋白（bovine colostrum lactoferrin, LFC）、人乳铁蛋白（human lactoferrin,LFH）、牛乳铁蛋白素（bovine lactoferricin,LfcinB）和人乳铁蛋白 素（human lactoferricin,LfcinH）为研究对象,研究其对小鼠脾淋巴细胞增殖的影响并比较不同来源乳铁蛋白 （lactoferrin,LF）及乳铁蛋白素（lactoferricin,Lfcin）作用效果的差异性。采用CCK-8法检测不同来源的LF与 Lfcin作用小鼠脾淋巴细胞24、48、72 h后,及其与丝裂原共同作用对小鼠T、B淋巴细胞增殖作用的影响。结果显 示：LF与Lfcin均在48 h时对小鼠脾淋巴细胞增殖的促进效果最好,且在一定质量浓度范围内促进效果随质量浓度升 高而加强;LF与Lfcin均在一定质量浓度范围内提高刀豆蛋白A诱导的T淋巴细胞的增殖和脂多糖诱导的B淋巴细胞 的增殖。结果表明：两种牛乳铁蛋白（bovine lactoferrin,LFB）及LfcinB可替代LFH应用于婴幼儿配方产品中以提 高婴幼儿免疫能力,LFB推荐添加质量浓度为0.50～2.00 mg/mL,Lfcin推荐添加质量浓度为75～125 μg/mL。