乳铁蛋白和乳铁素的抗菌活性比较

牛乳铁蛋白是从牛乳中提取出来的一种铁结合性糖蛋白，牛乳铁素是从牛乳铁蛋白N-端水解下来的25个氨摹酸残荩。它们具有多种乍物学功能，其中的广谱抗菌性尤为引人注目。本实验以牛初乳中提取的乳铁蛋白及其水解产物乳铁素为研究对象，选取大肠杆菌为实验菌株，进行铁饱和乳铁蛋白和缺铁性乳铁蛋白、乳铁素对大肠杆菌生长抑制的比较研究。研究结果表明：铁饱和乳铁蛋白、缺铁性乳铁蛋白和乳铁索的最小抑菌浓度分别为6mg／ml、3mg／ml和15μg／ml，乳铁素的最小杀菌浓度为30μg／ml。乳铁蛋白水解后，经纯化获得的乳铁素，其抗菌能力较缺铁性乳铁蛋白增加200倍，较铁饱和乳铁蛋白增加400倍。     

乳铁蛋白的生理功能

叙述了乳铁蛋白具有促铁吸收,调节免疫,抑菌,抗病毒,阻断氧自由基,避免刺激肠胃和促进双歧杆菌生长等功能,上述功能在乳品（特别是婴幼儿食品）加工中具有广泛的应用。     

酶联免疫法测定牛初乳中乳铁蛋白含量

应用酶联免疫法制定牛初乳中乳铁蛋白的含量,最小检出量为０．５ｎｇ／ｍｌＬＦ,浅性范围为０．８ｎｇ／ｍｌ至１００ｎｇ／ｍｌ。标准曲线性方程为Ｙ＝－２２．７２Ｘ＋２１．３８,相关系数γ为０．９９６。结果较为理想,这种对ＬＦ的检测方法在国内未见报道。     

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。     

重组人乳铁蛋白抗氧化活性研究

目的考察重组人乳铁蛋白自身及其在奶粉中的抗氧化活性。方法通过二苯代苦味肼基(DPPH)自由基抑制率、羟基自由基抑制率、超氧阴离子自由基抑制率、还原能力和抑制大鼠肝脏体外脂质过氧化等试验评价重组人乳铁蛋白和牛乳铁蛋白的体外抗氧化性能。结果重组人乳铁蛋白和牛乳铁蛋白的浓度与DPPH自由基抑制率、抑制羟基自由基能力、抑制超氧阴离子自由基能力和还原能力之间呈明显剂量-效应关系,且差异有统计学意义(P0.05);样本和浓度之间没有交互作用,重组人乳铁蛋白和牛乳铁蛋白在不同浓度下所得值的趋势相同。重组人乳铁蛋白和牛乳铁蛋白在对大鼠肝组织匀浆抗氧化性影响差异有统计学意义(P0.05),不同浓度乳铁蛋白之间差异有统计学意义(P0.05)。结论重组人乳铁蛋白和牛乳铁蛋白具有一定的抗氧化活性,可以添加到保健品或者化妆品中作为天然抗氧化剂。     

乳铁蛋白铁吸收效果的研究

对乳铁蛋白和硫酸亚铁中铁的吸收进行了研究。结果表明,乳铁蛋白比硫酸亚铁更容易吸收。     

乳铁蛋白及乳铁素对嗜酸乳杆菌生长影响的研究

本实验主要针对乳铁蛋白和乳铁素对嗜酸乳杆菌的生长影响进行研究。结果表明,在37℃恒温培养下,添加一定浓度的乳铁蛋白或乳铁素,可促进嗜酸乳杆菌生长繁殖。乳铁蛋白的最适添加浓度为2.5mg/ml,乳铁素的最适添加浓度为0.15mg/ml,培养26h后活菌数分别达到6.7×108CFU/ml和8.0×108CFU/ml。     

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。      

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。     

乳铁蛋白基因工程菌培养条件研究

对乳铁蛋白基因工程菌发酵条件进行了初步研究,在廉价高效的原则下对培养基组分进行了选择性优化,同时对几个主要的发酵参数进行了优化。     

Identification of milk proteins enhancing the antimicrobial activity of lactoferrin and lactoferricin

Lactoferrin (LF) is known as an iron-binding antimicrobial protein present in exocrine secretions such as milk and releases the potent antimicrobial peptide lactoferricin (LFcin) by hydrolysis with pepsin. The antimicrobial activity of LF and LFcin has been studied well; however, their cooperative action with other milk proteins remains to be elucidated. In this study, we identified milk proteins enhancing the antimicrobial activity of bovine LF and LFcin against gram-negative bacteria, gram-positive bacteria, and fungi. As the target fraction, we isolated a minor milk protein fraction around 15 kDa, which was identified as bovine RNase 5 (angiogenin-1), RNase 4, and angiogenin-2 by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. As these proteins are collectively known as the RNase A family, we referred to the target protein fraction as milk RNase of 15 kDa (MR15). The number of colony-forming units of Escherichia coli and other pathogenic microorganisms with the addition of MR15 to LF (MR15:LF ratio = 16:1,000) was dramatically lowered than that with LF alone. On the other hand, MR15 itself did not show any reductions in the number of colony-forming units at the concentrations tested. Similarly, the antimicrobial activities of LFcin against various microorganisms were significantly enhanced by the addition of MR15. These results suggest that LF and MR15 may be concomitantly acting antimicrobial agents in milk.     

Osteogenic effects of the peptide fraction derived from pepsin-hydrolyzed bovine lactoferrin

Osteoporosis is a common disease that frequently occurs in the older population, particularly in postmenopausal women. It severely compromises the health of the older population, and the drugs commonly used to treat osteoporosis have a variety of adverse effects. Lactoferrin (LF) is a protein present in milk that has recently been found to exhibit osteogenic activity. Lactoferrin is nontoxic and harmless, suggesting that it may have excellent biocompatibility and tolerability after human consumption. Oral consumption of LF in an ovariectomized rat model has been found to ameliorate osteoporosis. However, the mechanism underlying this effect remains to be clarified. In this study, bovine LF (bLF) was first hydrolyzed by pepsin for 1 h, and the hydrolyzed mixture was freeze-dried and collected. The hydrolyzed mixture was then separated into 5 components (E1–E5), of which E3 had the greatest effect in promoting proliferation of osteoblasts (MC3T3-E1). Component E3 was further isolated into 21 components with preparative reversed phase HPLC, and the E3-15 component had maximal bioactivity. With HPLC-mass spectrometry and peptide sequencing, E3-15 was identified to contain amino acids 97 to 208 from the bLF N terminus. Then, E3-15 was divided into 6 different peptide segments (P1–P6), and the corresponding segments were generated by solid-phase synthesis. Only the P1 peptide (amino acids 97–122 from the N terminus of bLF) significantly promoted osteoblast proliferation. The bioactivity of P1 toward osteoblast cells and alkaline phosphatase activity were tested as a function of P1 concentration, and a nonlinear effect was observed.     

乳铁蛋白活性多肽的抑菌性能

概述了乳铁蛋白活性多肽的结构、制备方法、抑菌谱、抑菌机理及抑菌活性的影响因素.     

Antilisterial activity of dromedary lactoferrin peptic hydrolysates

The aim of this study was to explore the antibacterial peptides derived from dromedary lactoferrin (LFc). The LFc was purified from colostrum using a batch procedure with a cation exchange chromatography support and was hydrolyzed with pepsin to generate peptic digest. This peptic digest was fractionated by cation exchange chromatography, and the antilisterial activity of LFc, peptic digest, and obtained fractions was investigated using the bioscreen method. The growth of Listeria innocua ATCC 33090 and LRGIA 01 strains was not inhibited by LFc and its hydrolysates. Two fractions of dromedary lactoferrin peptic hydrolysate were active against both strains. A tandem mass spectroscopy analysis revealed that the 2 active fractions comprised at least 227 different peptides. Among these peptides, 9 found in the first fraction had at least 50% similarity with 10 known antimicrobial peptides (following sequence alignments with the antimicrobial peptide database from the University of Nebraska Medical Center, Omaha). Whereas 9 of these peptides presented homology with honeybee, frog, or amphibian peptides, the 10th peptide, F₁₅₂SASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY₁₉₂ (specifically found in 1 separated fraction), exibited 54% homology with a synthetic antibacterial peptide (AP00481) derived from human lactoferrin named kaliocin-1. Similarly, the second fraction contained 1 peptide similar to lactoferrampin B, an antibacterial peptide derived from bovine milk. This result suggests that peptic hydrolysis of LFc releases more active antimicrobial peptides than their protein source and thus provides an opportunity for their potential use to improve food safety by inhibiting undesirable and spoilage bacteria.     

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.     

鲁西黄牛乳铁蛋白N-末端多肽基因的表达与活性检测

从黄牛的肝脏中提取总的DNA，根Genebank牛乳铁蛋白的cDNA序列，设计特异性引物，采用RT-PCR进行扩增，将扩增片段克隆于PGEM－Teasy　vector中。测序结果表明，该片断是乳铁蛋白基因的cDNA序列，编码50个氨基酸的150bp的多肽片断，通过BamH1和ＥcoR1双酶酶切该片断，并连接到表达载体PGEX-4T1中，转化大肠杆菌BL21，进行诱导表达，蛋白电泳显示，表达成功；经纯化、胃蛋白酶酶解后，具有抗菌生物活性。     

牛乳中乳铁蛋白的分离纯化研究

牛乳经酸沉淀除酪蛋白后,通过超滤、离子交换法分离纯化得到乳铁蛋白,利用考马斯亮蓝、SDS-PAGE电泳定性定量检测。结果表明:每毫升牛乳能提取乳铁蛋白0.22mg,纯度为95%。