牛初乳中乳铁蛋白的分离和纯化

利用超滤、盐析和层析三种方法,采用单因素实验和正交实验对酸牛乳清中的乳铁蛋白进行分离和纯化,得到盐析的最佳条件为:先以50%硫酸铵除去杂蛋白,然后在30°C,pH8.5,80%硫酸铵进行盐析。用CM-SephadexC-100进行层析,进样量为2mL,洗脱液pH为7.4时可以得到乳铁蛋白纯品。SDS-PAGE凝胶电泳结果表明,牛初乳中乳铁蛋白的纯度为92%,分子量大约为78000±2000Da。     

Anticancer activity of lactoferrin isolated from caprine colostrum on human cancer cell lines

In the present study, lactoferrin was purified from caprine colostrum and its anticancer effects were investigated on various cancer cell lines, including lung, colon, cervix, stomach and breast cancer cells. The caprine lactoferrin (CLF) was purified by ultrafiltration and sequential chromatography using a CM-Sephadex C-50-120 ion-exchange column and affinity chromatography on Hi-Trap^TM Heparin HP. The purified CLF exhibited antiproliferative effects on the five tested cancer cell lines in a dose-dependent manner, with 20–30% survival as compared to the control. Interestingly, the most intense inhibition of cell growth was observed on the ZR-75-1 breast cancer cells (IC₅₀ = 27.5 µg/mL). Our findings suggest, for the first time, that purified CLF from caprine colostrum may constitute a novel anticancer agent for the food industry.     

Complex coacervates obtained from lactoferrin and gum arabic: Formation and characterization

Proteins and polysaccharides are the most frequently used hydrocolloids in the food industry, and their interaction can provide products such as complexes coacervates, which can be used as ingredients and biomaterials or in microencapsulation systems. In the present work, the interaction between lactoferrin (0.1, 0.2, 0.3, 0.5 and 1% w/w) and gum arabic (0.1% w/w) with various concentrations of NaCl (0, 0.01, 0.25, 0.3 and 0.5 mol/L) and at various pH values (from 1.0 to 12.0) was studied. The pH for the formation (higher turbidity) of the insoluble complex coacervates (pH_Ø1) varied according to the amount of NaCl used in the system (pH 3.5 to 5.3); these values are below the isoelectric point of lactoferrin (8.0), at which the protein is more positively charged, generating electrostatic binding. At a pH of approximately 2.0, this bond weakens, leading to the solubilization of precipitates, resulting in a sudden decrease in the turbidity (pH_Ø2). Samples containing a lower concentration of lactoferrin (0.1, 0.2 and 0.3% w/w) showed greater turbidity and consequently a higher formation of precipitates or aggregates. Even these samples, which contained a salt concentration of 0.3 mol/L, showed higher turbidity and displacement points of pH_Ø1 and pH_Ø2. The zeta potential and particle size values were used to study the influence of the pH, ionic strength and temperature on the interaction between the biopolymers. It was observed that the formation of macromolecules occurred between the isoelectric point of the protein (8.0) and the pKa of the polysaccharide (2.0), and a certain salt concentration (0.25 mol/L) led to larger particle sizes. It was observed that, at pH 7.0, a concentration of 0.1% gum arabic was able to stabilize the denaturation of the protein in solutions containing 0.1% lactoferrin, resulting in a constant particle size at all temperatures studied.     

Comparison of lactoferrin content in colostrum between different cattle breeds

Lactoferrin content of colostrum obtained from cows within 24 h after parturition was measured using a single radial immunodiffusion test and was compared among cows of two dairy breeds (Holstein-Friesian, Jersey) and two beef breeds (Japanese Black and Japanese Brown). Average lactoferrin content in colostrum of dairy breeds was 2 mg/ml and in colostrum of beef breeds was .5 mg/ml. Lactoferrin content of colostrum due to lactation number was also different among breeds. In dairy breeds, multiparous cows had lactoferrin content two to three times higher than that of primiparous cows; beef breeds showed no obvious differences between lactation years. Lactoferrin content also varied considerably within breed. In beef breeds, half the cows had values of nearly zero. Transferrin content in colostrum was fairly constant (.9 mg/ml) and was not as variable among and within breeds. There was no correlation between lactoferrin and transferrin contents in colostrum. Examination of cows lacking lactoferrin suggested that transferrin plays an important role as an iron carrier from a cow to her newborn calf.     

One-step isolation of lactoferrin using immobilized monoclonal antibodies

Immunoaffinity columns made with monoclonal antibodies to either human or bovine lactoferrins were prepared to isolate human lactoferrin or bovine lactoferrin from milks by a single chromatographic step. Recoveries of human lactoferrin and bovine lactoferrin were 98 and 97%, respectively. The human lactoferrin recovered from defatted human colostrum was 98% pure with 93% iron-binding capacity. Amount of recovered bovine lactoferrin, as well as purity and iron-binding capacity, varied widely depending on the source of bovine milks and pretreatments (particularly pasteurization temperature). The best source to isolate bovine lactoferrin was raw skim milk yielding a protein 97% pure and with a 99% iron-binding capacity. Thus, immunoaffinity chromatography provides an effective one-pass isolation of highly pure human or bovine lactoferrin with reasonable recovery and iron-binding capacity.     

牛初乳sIgA的制备与检测

牛初乳sIgA与人初乳sIgA具有相同的免疫源性,对提高机体的免疫力有着重要的作用。采用盐析、超滤、凝胶过滤层析和离子交换层析的方法进行分离sIgA,并用HPLC进行检测,制得的sIgA纯度为75.38%,收率约为54%,本研究为实现牛初乳sIgA的工业化生产提供了有益的探索。     

Determination of lactoferrin in bovine milk,colostrum and infant formulas by optical biosensor analysis

An automated, rapid, sensitive and label-free biosensor-based immunoassay for lactoferrin in bovine milk utilising surface plasmon resonance (SPR) optical detection is described. Lactoferrin content is estimated from its specific interaction with an anti-bovine lactoferrin antibody immobilised on the sensor surface in a direct- binding assay format. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilisation, flow-rate, contact time and regeneration have been defined and non-specific binding considerations evaluated. Performance parameters include a working range of 0–1000 ng mL⁻¹, a method-detection limit of 19.9 μg mL⁻¹ in undiluted milk, overall instrument response RSD_R of 3.50%, a mean inter-assay RSD_R of 10.8% for milk and surface stability over ca 500 samples. The technique was applied to the measurement of lactoferrin content of consumer milks, colostrum and infant formulas, and temporal change during early bovine lactation.     

豌豆粉丝厂废水中蛋白质的提取和性质研究

本论文以豌豆粉丝厂废水中的蛋白为原料提取豌豆分离蛋白,通过对提取工艺进行研究,确定了在pH12.0,固液比1∶20(g/mL),30℃下提取30min的提取条件,提取率达到62.25%。氨基酸分析结果表明,所提豌豆分离蛋白(PPI)氨基酸没有遭到破坏,营养价值较高。通过对PPI功能性质研究发现,除了吸水吸油性外,PPI的乳化性、乳化稳定性、起泡性都和大豆分离蛋白(SPI)接近。由此可见,PPI可以作为SPI的替代品应用于食品,具有很大的实用价值。采用DSC研究了PPI的热力学性质,在87.67℃(7S)和104.28℃(11S)有两个峰,这可能是在碱溶酸沉过程中,蛋白结构发生了一定变化造成的。      

虫花菌菌丝体糖蛋白的分离、纯化及其性质研究

目的　研究虫花菌[Isaria farinosa(Dicks)Fri]菌丝体糖蛋白的分子性质和免疫活性。方法　糖蛋白经热水提取、离子交换柱层析、乙醇分级沉淀、DEAE Biogel A柱层析及SephadexG 50柱层析手段分离纯化。用聚丙烯酰胺凝胶电泳和Sepharose6B柱层析证明其均一性。用柱层析法测定其分子量。用纸层析和气相层析分析确定其单糖组成。用红外光谱和核磁共振图谱推测其结构连接特性。动物实验研究其免疫活性。结果　分离得到一种糖蛋白PCAⅠ,为单一均匀的糖蛋白,分子量55.6万,糖含量74.2%,蛋白质含量25.6%。单糖组成为甘露糖和半乳糖,其摩尔比为1:1。糖链为高度分支的半乳甘露聚糖。PCAⅠ可明显提高小鼠单核巨噬细胞吞噬能力,并可使免疫器官脾脏明显增重。结论　PCAⅠ是虫花菌菌丝体的一种生物活性大分子成分。     

Different effects of the methods of administering lactoferrin,IGF-1 and IgG from bovine colostrum to rabbits based on the cytokine communication network

Lactoferrin, IGF-1 and IgG are three well-known bioactive proteins found in bovine colostrum. We studied the effects of these three active ingredients on the levels of eight cytokines in serum and on the cellular communication networks in vivo by oral administration, intraperitoneal injection and intravenous injection. The results suggested that bioactive ingredients, especially biomacromolecules, can induce different effects, even opposite effects, on the immune system depending on the route of administration. These effects are related to both the functions of the bioactive ingredient in vivo and to its specific receptor binding in the gastrointestinal mucosa that activates different signalling pathways.     

Buffalo alpha S0-casein: isolation and characterization.

A method for the preparation of alpha S0-casein, and for the preparation of alpha S1-casein fractions 1 and 2 from buffalo casein are described. The amino acid composition and phosphorus content of alpha S0-casein are given and compared with those of other alphaS-caseins.     

牛初乳中sIgA的分离纯化

为进一步开发利用牛初乳中生物活性物质sIgA,有必要建立快速分离纯化sIgA的方法。利用凝胶层析和亲和层析等技术进行分离sIgA。采用非还原SDS-PAGE和HPLC进行检测。结果表明:分离纯化的sIgA纯度为97.52%,质量浓度为5.03 g/L。成功建立了纯化牛初乳中sIgA的方法,为实现sIgA的规模化生产提供参考。     

Separation of lactoferrin-a and -b from bovine colostrum

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.     

Improved isolation of bioactive components of bovine colostrum using cross‐flow microfiltration

Bovine colostrum contains bioactive components such as growth factors, immunoglobulins and antimicrobial factors. As conventional heat treatment methods inactivate these valuable components, cross‐flow microfiltration (MF) seems to be a promising option for the processing of bovine colostrum. A series of cross‐flow MF experiments with tubular ceramic membranes of various pore sizes and geometries were conducted. MF with pore sizes of 0.8 and 1.4 μm resulted in a 5.4‐ and 3.5‐log reduction of the microbial content, respectively. Applying 0.14‐ and 0.2‐μm membranes lead to a permeate that was almost free from micro‐organisms and casein. However, the maximum transmission of whey protein into the permeate was only 33%.     

传统米酒中产酸菌的分离筛选及其发酵性能的研究

从云南及黑龙江地区的传统米酒中分离了41株乳酸菌,通过产酸能力、产乙醛和双乙酰能力及耐酒精能力试验,筛选出产酸能力强的菌株WT2和产乙醛和双乙酰能力强的菌株IW9作为发酵菌株发酵米酒。     

黑乳参多糖的分离和组成鉴定

通过木瓜蛋白酶酶解方法从黑乳参中提取粗多糖,再经H2O2脱色、乙酸钾除蛋白、柱层析后等程序后得到精制的酸性黏多糖。琼脂糖凝胶电泳试验表明该多糖为单一成分,经GPC法测定重均分子量为76008Da。黑乳参黏多糖中硫酸基、葡萄糖醛酸、氨基半乳糖、岩藻糖含量分别为28.08%、20.71%、15.38%、12.48%。     

Exopolysaccharide-producing lactic acid bacteria strains from traditional Thai fermented foods: isolation, identification and exopolysaccharide characterization

Lactic Acid Bacteria (LAB) isolated from various traditional Thai fermented foods were screened for exopolysaccharides (EPS) production. From 104 isolates, two rod-shaped and five coccal-shaped LAB were able to produce EPS from sucrose on solid media. However, only the cocci were capable of producing EPS in liquid media and these were identified as Pediococcus pentosaceus. Pediococcus pentosaceus strains AP-1 and AP-3 produced EPS in high yield. In liquid media containing sucrose as carbon source, the amount of EPS produced by AP-1 and AP-3 strains was 6.0 and 2.5 g/L, respectively. The isolated and purified EPSs were chemically characterized. On the basis of sugar composition, methylation analysis and nuclear magnetic resonance spectroscopy, both the EPSs were shown to belong to the same dextran class. In particular, both EPSs differed from linear dextran by branching through 3,6-di-Osubstituted alpha-D-glucopyranosyl residues. The EPS from P. pentosaceus AP-3 was characterized by a relatively higher degree of branching and by a higher molecular weight than that from P. pentosaceus AP-1.     

Highly-hydrophobic nanofiber mat for efficient colonic delivery of lactoferrin: Preparation,characterization and release mechanism

Lactoferrin (Lf) is a bioactive protein with varied biological effects. To improve its anti-digestive stability in oral administration, a novel nanocarrier with high hydrophobicity for colonic delivery of Lf was creatively developed by modified coaxial electrospinning. First, a suitable biocompatible solvent, acetic acid, was screened as the mono-solvent for ES100 electrospinning, creating highly-hydrophobic ES100 nanofiber mat (contact angle = 133.8^o). Then, Lf-loaded W/O emulsion was prepared as the core fluid to ensure the successful coaxial electrospinning and generate Lf encapsulated core/shell nanofiber mat (ES@Lf). Lf was demonstrated maintaining structural integrity and anti-colon cancer activity during encapsulation and oral delivery. In vitro assay indicated 92.3% Lf was sustainably released in colon, and its release followed a complex mechanism in which the erosion was dominant. Instead of pH-dependent erosion, the synergistic action through gut microbiota adhesion and their metabolites, especially short-chain fatty acids, was illustrated for disintegration of ES@Lf nanofiber for the first time.