毛细管电泳法对乳及乳制品中乳源蛋白的研究

采用毛细管电泳方法对原料乳、市售鲜奶、不同厂家的巴氏灭菌乳、不同厂家和产地超高温灭菌乳(UHT)、调味乳、乳酸饮料、复原乳、酸奶、奶粉中蛋白成分进行检测。选择聚乙烯醇涂层毛细管,采用柠檬酸缓冲体系,在紫外检测214nm、分离电压20kV条件下对乳及乳制品中的α一乳白蛋白(α-La)、β一乳球蛋白(β-Lg)、α-酪蛋白(α-CN)、β-酪蛋白(β-CN)和k-酪蛋白(k-CN)进行分离测定。结果表明:五种蛋白的含量在原料乳(巴氏灭菌乳、市售鲜奶)、UHT乳、酸奶、调味乳、乳酸饮料、复原乳中依次降低,而UHT乳含量随保质期的增加而减少,奶粉中蛋白质含量因其适应人群而有差异。乳及乳制品中蛋白质的含量与其存在形式、产地及加工工艺相关。     

Determination of major metal cations in milk by capillary zone electrophoresis

A reliable and rapid capillary zone electrophoresis method for the determination of the major of cations in milk samples was developed. Sample preparation consisted of dilution, acidification to pH 4.0 with 1 mol L⁻¹ acetic acid and filtration. The complete separation of K⁺, Ca²⁺, Na⁺ and Mg²⁺ could be achieved in 4 min with a simple electrolyte composed of 10 mmol L⁻¹ imidazole and 1 mol L⁻¹ acetic acid (pH 3.6). The running voltage was 25 kV at 25 °C. Indirect UV detection was achieved at 185 nm. Detection limits ranged from 0.06 to 0.57 mg L⁻¹ and quantification limits ranged from 0.16 to 0.62 mg L⁻¹. Precision data showed relative standard deviations (RSD%) lower than 4.1% for relative migration time and 2.4% for milk concentrations, respectively. Recoveries of cations in samples analysed ranged from 97.7% to 101.1%. Thirty samples of milk were analysed, obtaining mean values of 1.46, 1.19, 0.505 and 0.126 g L⁻¹ for K⁺, Ca²⁺, Na⁺ and Mg²⁺, respectively.     

3种毛细管电泳方法分析牛乳蛋白的比较

对比毛细管凝胶电泳(CGE)、毛细管未涂层管区带电泳(CZE)和涂层管区带电泳(CZE)在牛乳蛋白分离和定量分析,结果表明在分离条件、分离效果和定量特征上,毛细管涂层区带电泳优于其他两种电泳。应用涂层毛细管区带电泳对不同阶段婴幼儿配方乳粉的蛋白组成进行测定,分离效果较好。     

Analysis of cyclopiazonic acid in milk by capillary electrophoresis

A Micellar Electrokinetic Capillary Chromatography (MEKC) method to detect cyclopiazonic acid (CPA) in milk and compare its quantifying efficiency to the Reverse Phase Liquid Chromatography (RPLC) method was evaluated. Alkaline milk samples were defatted, then acidified before being twice liquid–liquid extracted with chloroform. Bare fused-silica capillary-extended light path with 50 μm i.d. and Nova-pak C18-column, were used for the CPA separation in MEKC and RPLC respectively. The analytical response was linear from 40 ppb to 100 ppm CPA in MEKC (correlation coefficient, r=0.99995). Recoveries of spiked CPA in milk were 78–81% over the range of 20 ppb to 500 ppb in MEKC and 71–80% in the range of 50 ppb to 500 ppb in RPLC. The detectable limit of CPA by MEKC was 0.27×10⁻⁰⁷ pg ml⁻¹. Capillary electrophoresis (MEKC) is a better and rapid method for CPA detection in milk.     

高效毛细管电泳测定液态乳中乳糖

针对常用乳糖测定方法存在的局限性和不便性,结合高效毛细管电泳(HPCE)法简单快速、高效低耗的特点,探索出了高效毛细管电泳仪配合紫外检测器测定乳糖的实验条件:熔融石英毛细管(50μm×34 cm);缓冲液为50 mmol/L硼砂/NaOH,pH值为10.0;电泳为10 kV,温度为60℃;检测波长为195 nm;进样为5000 Pa;时间3 s。此方法操作简便、准确度高、精密度好。该方法的测定结果与高效液相色谱法的结果较一致,样品中其他糖类对该方法不存在干扰,克服了莱因-埃农氏法测定乳糖不具专一性的缺点。     

Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R² = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%.     

Rapid determination of sodium in milk and milk products by capillary zone electrophoresis

A capillary zone electrophoresis method for the determination of Na in milk and milk products was developed and compared with an International Organization for Standardization/International Dairy Federation standard method that is based on flame atomic absorption spectrometry. The adoption of a background electrolyte consisting of 10 mM imidazole adjusted to pH 3.75 by the addition of oxalic acid allowed baseline separation of Na from other milk cations and from Li ion, which was adopted as an internal standard. Method validation was performed and the results for linearity, precision, limit of detection, limit of quantification, and recovery are presented. The procedure was tested on commercial milk samples differing in fat content (whole, semiskimmed, and skimmed) and processing conditions (pasteurization, UHT sterilization, and microfiltration). The reliability of the method was confirmed for different varieties of cheese and other milk products. The method enables the routine measurement of Na content by a rapid and accurate capillary zone electrophoresis procedure.     

Detection of rennet whey solids in UHT milk by capillary electrophoresis

The fraudulent presence of rennet whey solids in UHT milk was studied by capillary electrophoresis (CE). Commercial UHT samples of different origins, genuine milk samples and milk samples adulterated with rennet whey were analysed. Linear discriminant functions using ratios of peak areas of caseinmacropeptide (CMP) and two other CMP-like degradation products were defined. The interference of proteolysis in the detection was estimated in samples adulterated on purpose with rennet whey, UHT treated in a pilot plant, and stored at 10, 20 and 30°C for up to 150 days. The application of the classification functions obtained allowed the detection of rennet whey solids added to milk. Only interferences due to very severe proteolysis, occurring after very long storage periods and/or at storage temperatures above room temperature, were observed.     

A study of polymorphism in milk proteins from local and imported dairy sheep in Australia by capillary electrophoresis

Casein and whey protein fractions of milk obtained from 47 ewes of five breeds or crossbreeds (Awassi, Merino, East Friesian×Merino, Awassi×Merino and Awassi×East Friesian) were analysed by capillary electrophoresis (CE). The experiments were performed on a Beckman P/ACE™ system 5510 with an uncoated fused-silica capillary and a low pH buffer containing urea and a polymeric additive. The four major caseins (α_s1-, α_s2-, β- and κ-casein) in an acid precipitate were well separated, as were the two whey proteins, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg). The electromigration of the proteins was in the order of α-La, β-Lg, α_s2-CN, α_s1-CN, κ-CN and β-CN. The milk samples were composed of the same variant of α-La and two different genotypes (A and B) of β-Lg while the β-Lg AB genotype was evident in the milk of some animals. The α_s1-CN fractions displayed considerable heterogeneity with at least 4 different peaks, representing 4 different variants. A fifth peak, corresponding to the Welsh variant (or α_s1-CN D), was present in 90% of the ewes’ milk samples. The κ-CN fraction was resolved as a single peak, while the β-CN revealed significant heterogeneity with 3 variants. It appears that the presence of the α_s1-CN Welsh variant in Merino ewe and its crosses with Awassi and East Friesian ewes adversely affected milk composition and yield.     

Fast analysis of proteins in wines by capillary gel electrophoresis

Wine proteins play an important role in different characteristics of wine (e.g., aroma and body, foaming in sparkling wines). They can also cause a number of technological problems during vinification and may be responsible for the appearance of turbidity in bottled wine. These important features of proteins in wine have made necessary the development of new and fast analytical methods that can provide deeper knowledge about these biopolymers. However, separation and characterization of wine proteins is difficult and time-consuming mainly due to their low concentration and large number of interfering compounds. Besides, long sample preparation protocols can bring about protein decomposition. This paper proposes a new and fast method for carrying out the analysis of the protein fraction of wines. The procedure consists of direct treatment of wine using a centrifugal filter device (CFD), denaturation of the proteinaceous fraction with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and subsequent CGE analysis of SDS-proteins. Results on the molecular weight (Mw) and relative quantity of proteins of wines are attained in about 1 h with this procedure. The method is applied to analyze different wines from Canary Islands. To our knowledge, this is the first report of separation of wine proteins according to their Mw by CGE.     

Antibacterial and antiviral activity of camel milk protective proteins.

Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.     

采用毛细管电泳技术快速检测牛乳中外源蛋白成分

牛乳中非乳源性蛋白成分的检测对于保障牛乳质量安全具有重要意义。介绍了毛细管凝胶电泳技术在牛乳中外源蛋白成分快速检测上的应用。通过优化条件,建立牛乳毛细管凝胶电泳方法,检测到大豆水解蛋白标志峰,分别测定纯大豆水解蛋白溶液及牛乳-大豆水解蛋白混合溶液中的水解蛋白标志峰峰面积-浓度线性关系,并将毛细管电泳与SDS-PAGE垂直板电泳及蛋白芯片电泳进行比较,以验证毛细管电泳结果,并比较不同方法优劣性。     

Determination of melamine in milk and milk-containing products by capillary zone electrophoresis

For determination of melamine methods of reversed phase and ion-pair high-performance liquid chromatography, and also immune-enzyme analysis are used. The purpose of our work was working out of a technique of determination melamine in milk and lactiferous products method CZE. As a result of researches determination conditions melamine in foodstuff by method CZE (sensitivity from 0.8-1.0 mg/l) are developed, and also conditions of its isolation from objects of research are adapted.     

利用毛细管电泳法测定乳品中乳铁蛋白方法的研究

将样品经样品缓冲液处理,毛细管柱选择为直径50μm、有效长度400mm的涂层中性毛细管柱;柱温为40℃;缓冲溶液为柠檬酸缓冲液(pH为3.0±0.2);工作电压为21kV;检测波长为214nm;进样时间为5s,进行检测。结果乳铁蛋白平均回收率为75%～91%;相对标准偏差(RSD)为1.1%～2.8%;最低检出限为0.003mg/mL。该方法简便,准确,灵敏度高,是快速检测乳品中乳铁蛋白含量的最适合的分析方法。      

3种电泳方法定量分析牛奶蛋白的比较

通过实验比较了毛细管区带电泳（CZE），毛细管凝胶电泳（CGE）及聚丙烯酰胺平板凝胶电泳（SDS-PAGE）在分离、定量牛奶蛋白上的效果比较。结果表明，在分离的效果及重现性上，CZE，尤其是用带有聚乙烯醇（PVA）涂层的毛细管，酸性的电泳缓冲液和低离子强度的样品缓冲液要优于其他方法，可用于快速分离、定量牛奶蛋白。     

Determination of lactose in sugar-free milk powder by capillary electrophoresis with electrochemical detection

Lactose in three sugar-free milk powder samples were satisfactorily determined by capillary electrophoresis with electrochemical detection using a 300 μm copper disk electrode as the working electrode in 0.1 mol/l NaOH medium. There was acceptable linearity (0.9969) between the peak current and concentration of lactose in the range from 5.0×10⁻⁶ to 5.0×10⁻³ g/ml with the detection limit (S/N=3) of 1×10⁻⁷g/ml. The proposed method was successfully applied to analyze the actual milk powder samples.     

一种利用毛细管电泳间接测定生鲜乳中添加大豆分离蛋白的方法

运用高效区带毛细管电泳法以pH2.7浓度配制为15mmol/L柠檬酸-20mmol/L柠檬酸三钠作电泳缓冲液同时测定了生鲜乳蛋白和大豆分离蛋白。结果显示β-CN峰面积和迁移时间的相对标准偏差(RSD)分别为3.01%和0.62%,κ-CN峰面积和迁移时间的RSD分别为2.03%和0.49%,大豆分离蛋白峰面积和迁移时间的RSD分别小于5.12%和0.48%,均满足定性和定量分析的要求。建立了利用蛋白峰面积比例关系间接测定生鲜乳中大豆分离蛋白的分析方法,其中β-CN和κ-CN检测限分别为0.17mg/L和0.23mg/L,回收率为99.37%和99.23%,大豆分离蛋白检测限和回收率分别为5.73mg/L和87.44%.     

Characterization of galactomannans by capillary electrophoresis

Capillary electrophoresis (CE) was utilized in the characterization of various galactomannans. Standards of gums were extracted with 50% CH₃CN to remove the residual proteins from the gum matrix. Separation buffers were optimized with respect to pH, buffer concentration and presence of sodium dodecyl sulphate, yielding protein profiles from which the desired information could be obtained. Examples are given of the profiles generated by various gums and gum blends to aid in the verification of component presence, and to demonstrate levels of adulteration detectable under the buffer conditions used.     

Analysis of malts by capillary electrophoresis

A range of malts, as well as their high‐ and low‐molecular‐mass fractions, has been examined by capillary electrophoresis in phosphate buffer, pH 2.5, and in carbonate buffer, pH 9.5, and the results have been compared with those for roasted barley and for caramels. The malts fall into two categories: (i) the lightly roasted malts, where the high‐molecular‐mass coloured fraction is negatively charged at pH 9.5 and positively charged at pH 2.5; and (ii) the highly roasted malts (and the roasted barley), where the high‐molecular‐mass fraction migrates close to the electro‐osmotic flow at both pH 9.5 and 2.5, implying that it carries little or no charge. The former category shows migration patterns similar to Class III caramels, whereas the latter migrates differently from Class I, III and IV caramels as well as from the former. Capillary electrophoresis therefore has considerable potential for differentiating between malts and between malts and caramels and roasted barley. © 2002 Society of Chemical Industry