牛初乳中乳铁蛋白的分离及纯化

乳铁蛋白(Lactoferrin,简称Lf)作为一种糖蛋白,主要存在于哺乳动物的各种外分泌物中,如乳汁、眼泪、唾液等,乳汁尤其是初乳中Lf的含量很高犤1犦。本文通过盐析法和有机溶剂沉淀法从牛初乳中分离出乳铁蛋白,并应用酶联免疫法鉴定各步骤中分离的乳铁蛋白,获得的乳铁蛋白的相对分子量为75,000Da。     

乳铁蛋白的最新研究进展

乳铁蛋白（Lactoferrin,简称Lf)主要存在于哺乳动物的各种外分泌物中，乳中的含量最高。Lf不仅具有广谱的力性质，而且还能够增强机体的抗病毒，抗癌能力和免疫能力，防治感染性疾病，作为一种新型的具有抗菌性质的蛋白质。Lf有望作为新一代功能性疗效食品的功能组分。本文主要综述了乳铁蛋白的基本性质，功能特性及其一些最新的研究进展，并对牛初乳中乳铁蛋白的开发利用进行了展望。     

牛初乳中主要生物活性物质开发的最新进展

综述了近年来牛初乳资源的开发利用状况 ,重点介绍了几种主要功能性物质如免疫球蛋白 (IgG) ,乳铁蛋白 (Lf)及类胰岛素生长因子 (IGF)的分离纯化技术 ,并对牛初乳的综合利用前景进行展望     

主要业务部门 主要研究方向——食品研究组

乳铁蛋白是一种多功能蛋白质，具有广谱的抗菌作用，增强机体的免疫力。通过盐析法和有机溶剂沉淀法，从牛初乳中分离出乳铁蛋白，此过程中还可得到球蛋白及酪蛋白两种副产品。     

主要业务部门 主要研究方向——食品研究组

一．乳铁叠自的提取工艺 乳铁蛋白是一种多功能蛋白质．具有广谱的抗菌作用．增强机体的免疫力。通过盐析法和有机溶剂沉淀法．从牛初乳中分离出乳铁蛋白．此过程中还可得到球蛋白及酪蛋白两种副产品。     

牛初乳中乳铁蛋白的分离纯化与免疫功能检测

牛初乳经离心、盐析后获得乳铁蛋白粗提物，通过CM-Sephadex C50离子交换色谱和Heparin-Sepharose CL-6B亲和色谱纯化获得纯度分别为76．98％和92．75％的乳铁蛋白，经SDS-PAGE检测乳铁蛋白存在分子质量分别为76000Da和78000Da的两种异构体，免疫电泳说明提纯样品与乳铁蛋白标准品具有相同的免疫原性。小白鼠喂养和免疫实验说明乳铁蛋白可提高机体免疫功能。     

添加乳铁蛋白对延长巴氏奶贮存时间的影响研究

本文对添加乳铁蛋白延长巴氏奶贮存时间进行了研究。乳铁蛋白是从牛初乳中分离出来的一种功能性成分，具有广谱抗菌、抗病毒感染作用，同时还能促进人体对铁的吸收。因此，将乳铁蛋白添加到巴氏奶中，不仅可以增强其抗菌能力，延长保质期，同时还能够提高产品的营养价值。在不同温度下(恒温37℃、常温23℃、冷藏4℃)，在巴氏奶中添加不同浓度(0．025、0．05、0．1mg／ml)的乳铁蛋白，通过对巴氏奶添加乳铁蛋白和不添加乳铁蛋白在不同温度贮存时微生物的变化的比较，观察其微生物生长变化情况；结果表明，添加乳铁蛋白具有延长巴氏消毒奶保藏期的作用。     

乳及乳制品中乳铁蛋白含量的电泳迁移率检测

凝胶电泳迁移率(EMSA)是研究蛋白质和DNA分子相互作用的经典分子生物学方法。本研究根据乳铁蛋白(Lactoferrin,Lf)和DNA分子的特异性结合,基于Lf-DNA复合物和DNA分子迁移率差异,建立了一种聚丙烯酰胺凝胶电泳(PAGE)检测乳铁蛋白的方法。实验中以FAM荧光基团标记DNA分子,根据凝胶成像图中Lf-DNA复合物荧光条带的存在与否、荧光条带灰度值的大小,来判断样品中乳铁蛋白是否存在、含量多少,从而实现乳铁蛋白的定性与定量检测。结果表明,该方法具有良好的特异性及准确性,经过简单稀释后便可直接应用于乳及乳制品中乳铁蛋白的准确定量检测。     

乳铁蛋白与纳米脂质体的相互作用及对磷脂膜结构的影响

为探明纳米脂质体(N-lip)与食品蛋白的相互作用，采用薄膜分散法制备N-lip。以乳铁蛋白(Lf)为模型蛋白，评价不同蛋白浓度和滴加方式下形成的纳米脂质体/乳铁蛋白(N-lip/Lf)复合物的粒径、Zeta-电位和多分散系数，并用隐形率(Fs)来估计N-lip与Lf的结合程度。利用透射电子显微镜比较乳铁蛋白作用前、后的形貌变化，采用光谱法和X射线衍射分析Lf与N-lip的相互作用。结果表明:在磷脂质量浓度为10.0 mg/mL、磷脂与胆固醇质量比为10 ∶ 1、磷脂与Tween-80的质量比为4 ∶ 1、蛋白质量浓度为0.1 mg/mL时，形成的N-lip/Lf复合物平均粒径为(100.80±0.141) nm，电位为(-54.9±2.276) mV，具有较好的分散度。透射电子显微镜观察可见，Lf在N-lip边缘形成浅色包裹层。Lf与N-lip的相互作用促进了Lf中色氨酸和酪氨酸残基所处环境的疏水性增强，Lf的α-螺旋结构含量增加；Lf可以通过氢键、范德华力和疏水作用与N-lip磷脂双分子层膜发生相互作用，吸附到磷脂膜表面。研究结果可为脂质体与食品蛋白的互作及脂质体在食品工业中的潜在应用提供理论依据。     

高效毛细管电泳法快速测定乳铁蛋白原料的纯度北大核心CSCD

建立了动态涂层毛细管电泳快速检测乳铁蛋白含量的方法。并运用该方法对不同乳铁蛋白原料的纯度进行了检测。经检测4种乳铁蛋白原料的纯度分别为乳铁蛋白A91.05%,乳铁蛋白B93.62%,乳铁蛋白C94.38%,乳铁蛋白D75.81%;相对标准偏差(RSD)分别为1.0%,0.8%,1.2%,0.9%。并与供应商所提供的原料纯度进行比较,讨论了造成二者之间略微差异的原因。结果表明,该动态涂层方法能够有效抑制毛细管内壁对乳铁蛋白的吸附,对于乳铁蛋白的检测及纯度的验证十分有效,为此类蛋白的检测提供了一种手段。     

膜分离牛初乳中乳铁蛋白的工艺参数研究

研究采用膜分离技术分离牛初乳中乳铁蛋白的工艺条件,并摸索出膜污染的解决办法。以牛初乳为原料,采用单因素试验和二次通用旋转组合设计法优化膜分离乳铁蛋白的工艺条件,并进行响应面分析,比较水法、碱法和水法结合碱法清洗膜的方法。结果显示:提取乳铁蛋白的最适温度38℃、操作压力0.19 MPa、pH 6.9。在此条件下得到膜通量53.2 L/m2.h,膜效能0.79 g/m2.min。膜清洗采用水洗10 min,0.5 mol/L NaOH与4%NaClO混合液清洗15 min,膜通量恢复率可以达到95%以上。     

Composition and clinical use of bovine colostrums

In recent years, a large number of the researches giving fuller picture about structure and properties of bovine colostrums (BC), that allows to apply it at various diseases in clinical practice was carried out in the world. The critical analysis of the modern literature showed bovine colostrums is rich with immunoglobulins, antimicrobials and growth factors in comparison with nature milk. The positive effect of supplementation of bovine colostrums in diarrhea in persons with immune-deficiency syndromes, for treatment NSAID-induced gastrointestinal disturbances, at postoperative responses and in treatment of upper respiratory infection is supposed.     

高压脉冲电场加工牛初乳的研究

探索了利用高压脉冲电场处理牛初乳的可行性,对以高 压脉冲电场不同加工参数处理的牛初乳样品进行主要微 生物(细菌总数、大肠菌群、霉菌和酵母)的检测,并检测 其中功能性成分IgG含量,与未经处理的牛初乳中IgG 含量对比,证明了用高压脉冲电场加工技术处理牛初乳 的可行性,并确定了较适合的工艺条件。     

牛初乳胰岛素样生长因子的稳定性研究

研究了牛初乳类胰岛素生长因子在不同温度、pH值、添加保护剂条件下对其降糖活性影响,并利用Balb/c3T3成纤维细胞糖代谢测定了IGFs降糖活性。结果表明:细胞培养液的葡萄糖浓度降到对照组的60%,与胰岛素相比同样具有明显降糖活性。IGFs在pH值2～8范围内活性稳定,在温度为4℃时活性最大,30%甘露醇能有效提高IGFs降糖活性。     

Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis

The objective of the present study was to evaluate the therapeutic potential of bovine lactoferrin or lactoferricin in combination with penicillin G against Staphylococcus aureus. Minimal inhibitory concentrations of lactoferrin, lactoferricin, penicillin, and combinations of lactoferrin or lactoferricin with penicillin were determined for 15 S. aureus strains including several strains resistant to beta-lactam antibiotics. The fractional inhibitory concentration index indicated a synergistic effect between lactoferrin and penicillin. Combination of lactoferrin with penicillin increased the inhibitory activity of penicillin by two- to fourfold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of lactoferrin by penicillin was 16- to 64-fold. The addition of iron to the medium containing a combination of penicillin and lactoferrin had no effect on growth inhibition. Electron microscopy revealed that concentration below the minimal inhibitory concentrations of penicillin induced important ultrastructure alterations, which were further enhanced by the presence of lactoferrin. When S. aureus cells were grown in the presence of a combination of penicillin and lactoferrin, changes in the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin, were observed. These data suggest that bovine lactoferrin or lactoferricin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to antibiotics.     

Lactoferrin research,technology and applications

Lactoferrin is an iron-binding glycoprotein present in milk as well as other exocrine secretions and neutrophil granules in mammals. Lactoferrin is considered to be an important host defense molecule and has a diverse range of physiological functions such as antimicrobial/antiviral activities, immunomodulatory activity, and antioxidant activity. During the past decade, it has become evident that oral administration of lactoferrin exerts several beneficial effects on the health of humans and animals, including anti-infective, anticancer, and anti-inflammatory effects. This has enlarged the application potential of lactoferrin as a food additive. The technology of producing bovine lactoferrin on a factory scale was established over 20 years ago. Bovine lactoferrin is purified by cation-exchange chromatography from bovine skim milk or whey, and is commercially available from several suppliers. Recombinant human lactoferrin is produced by Aspergillus niger, transgenic cows, and rice, and its efficacy is being evaluated. In this article, we review basic research and technological aspects of the application of lactoferrin.     

Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin.

The antibacterial properties of enzymatic hydrolysates of bovine lactoferrin were examined to determine whether active peptides are produced from this protein. Hydrolysates prepared by cleavage of lactoferrin with porcine pepsin, cod pepsin, or acid protease from Penicillium duponti showed strong activity against Escherichia coli O111, whereas hydrolysates produced by trypsin, papain, or other neutral proteases were much less active. Low molecular weight peptides generated by porcine pepsin cleavage of lactoferrin showed broad-spectrum antibacterial activity, inhibiting the growth of a number of Gram-negative and Gram-positive species, including strains that were resistant to native lactoferrin. The antibacterial potency of the hydrolysate was at least eightfold greater than that of undigested lactoferrin with all strains tested. The active peptides retained their activity in the presence of added iron, unlike native lactoferrin. The effect of the hydrolysate was bactericidal as indicated by a rapid loss of viability of E. coli O111. The lactoferrin hydrolysate described in the present study has commercial value as a natural preservative agent for use in foods and cosmetics, and as a functional component of new clinical foods for prevention or treatment of gastrointestinal disease.