Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.     

Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).     

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low‐cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE‐4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low‐cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.     

Separation of purine and pyrimidine bases by ion chromatography with direct conductivity detection

A simple, rapid and accurate method for the simultaneous determination of four purine and pyrimidine bases (cytosine, 5-methylcytosine, adenine and N6-methyladenine) has been developed. The quantitative determination of these bases was accomplished by ion chromatography (IC) with direct conductivity detection (CD) based on their ionization in acidic medium without chemical suppression. The recovery of cytosine, 5-methylcytosine, and adenine in calf thymus DNA was more than 98% (n=3) and the relative standard deviation (RSD, n=5) less than 2.4%. In a single chromatographic run, the four bases could be separated and determined in less than 10 min. The detection limits were found to be 0.05 microg/mL for cytosine, 0.08 microg/mL for 5-methylcytosine, 0.07 microg/mL for adenine, and 0.07 microg/mL for N6-methyladenine. Linear ranges were 0.2-95.1 microg/mL for cytosine (r2=0.9996), 0.3-196.6 microg/mL for 5-methylcytosine (r2=0.9994), 0.3-105.5 microg/mL for adenine (r2=0.9998), and 0.3-159.1 microg/mL for N6-methyladenine (r2=0.9999). With the proposed method, purine and pyrimidine bases could be successfully detected in calf thymus DNA. We also determined these bases in calf thymus DNA using RP-HPLC. Compared to RP-HPLC, the IC method offers advantages such as high selectivity and simple mobile phase.     

Whey proteins eluted reversed phase gradients

The behavior of bovine whey proteins in Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) systems has been investigated. The Linear Solvent Strength (LSS) model has been applied to the separation of these proteins studying how their retention time and band broadening change when different gradient parameters are modified. From our results it is deduced that the LSS model describes the behaviour of the whey proteins in RP-HPLC. Also, it seems that t_s (the retention time for non-retained solutes) depends on the size of these proteins. The good fit observed between experimental data and the equations deduced from the LSS model allows the prediction of a gradient shape that permits a rapid analysis of the above mentioned proteins.     

Determination of proteins at nanogram levels by their quenching effect on the chemiluminscence reaction between luminol and hydrogen peroxide with manganese-tetrasulfonatophthalocyanine as a new catalyst

The manganese-tetrasulfonatophthalocyanine (MnTSPc) catalyzed luminol-hydrogen peroxide chemiluminescence (CL) systems can be quenched in the presence of proteins. A highly sensitive CL quenching method has been developed for the determination of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.1-20 microg/mL for human serum albumin (HSA), 0.2-20 microg/mL for human gamma-IgG, and 0.5-50 microg/mL for the bovine serum albumin (BSA) with the corresponding detection limits were 1.9 ng/mL, 2.7 ng/mL, and 3.4 ng/mL. The method has been applied to the analysis of total proteins in human serum samples and the results were in good agreement with clinical data provided.     

荧光法研究氧氟沙星与牛乳铁蛋白的相互结合作用

用荧光光谱法研究了稀水溶液中牛乳铁蛋白与氧氟沙星分子之间的结合作用机制.以荧光猝灭法测定了该反应的结合常数K, 结合位点数n；并依据Förster偶极-偶极无辐射能量转移机制,得到氧氟沙星和牛乳铁蛋白色氨酸残基之间结合距离r和能量转移效率E；并用同步荧光技术考察了氧氟沙星对牛乳铁蛋白构象的影响；牛乳铁蛋白与氧氟沙星分子之间有较强的结合作用,而且氧氟沙星对牛乳铁蛋白的构象有一定的影响.     

Quantitative Characterization of Bovine Serum Albumin, α-Lactalbumin and β-Lactoglobulin in Commercial Whey Sample by RP-LC

A simple, sensitive and precise reverse phase liquid chromatographic method has been developed and validated for quantification of bovine serum albumin (BSA), α-lactalbumin (α-La) and β–lactoglobulin (β-Lg) that are removed from whey waste by foam fractionation method. The data is reproducible over a wide concentration range. This optimized method allowed analysis of BSA, α-La, β-Lg in a mixture within 5 min and could be applied to the analysis of a variety of commercial and laboratory whey products within a short time.     

超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白

建立了超高效液相色谱-串联质谱技术定量检测乳制品中牛乳铁蛋白含量的方法。样品经脱脂、酶解后,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）和Protein Pilot软件分析,实现了乳铁蛋白及其肽段的鉴定;通过初级局部序列搜索工具（basic local alignment search tool,BLAST）与Uniprot数据库对比分析筛选出牛乳铁蛋白的8个特征肽段;选择其中3个响应强度高、稳定性好的特征肽段进一步通过超高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）进行验证和多反应监测（MRM）定量研究,并考察了空白基质匹配的外标法和内标法对乳铁蛋白检测结果的差异。采用外标法定量时,3个肽段在各自范围内线性关系良好,检出限为0.023~0.041 mg/kg,定量限为0.077~0.137 mg/kg,平均回收率为93.8%~103.9%,日内精密度≤8.8%,日间精密度≤9.5%。该方法抗干扰能力强,灵敏度高,重复性好,适用于乳及乳制品中牛乳铁蛋白的含量测定。     

Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography

A new downstream procedure for the isolation of bovine lactoferrin (bLf), lactoperoxidase and bovine lactoferricin (LfcinB) from sweet cheese whey was developed at the laboratory scale, based on membrane adsorber technology. The procedure was upscaled later on to an industrially relevant scale for the purificationof sweet whey concentrate with a recovery yield for lactoferrin of more than 90%. Based on these results the industrial process for 1 x 10(8) kg whey per year was projected. These high-value proteins were downstreamed by using cation-exchange membrane systems (Sartobind S, Sartorius, G?ttingen, Germany). These strongly acidic membranes trap proteins in its anionic form. The dynamic loading capacity for both proteins as well as the optimal elution profiles with sodium chloride gradients were derived from laboratory experiments using membrane modules with 15-75 cm2 membrane material. Further investigations were performed with 1 m2 modules in a continuous process mode. The enzymatic preparation of LfcinB from bLf was performed by pepsin hydrolysis and the isolation of LfcinB was directly carried out from the enzymatic digest mixture. The identification of the proteins was performed with matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS). LfcinB and bLf were both tested afterwards in biological assays in order to show not only the efficiency of the downstreaming process in regard to product quantity but also to product quality (biological activity).     

A high-throughput method for the quantification of iron saturation in lactoferrin preparations

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A _280/466). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.     

Simultaneous determination of residues of dipyrone and its major metabolites in milk, bovine muscle, and porcine muscle by liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method for the determination of dipyrone (DIP) and the DIP-related residues 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA), and 4-methylaminoantipyrine (MAA) in milk, bovine muscle, and porcine muscle is presented. The analytes are extracted from the sample with methanol and defatted with hexane. The methanol extracts are then concentrated and injected into the LC system. Compounds are determined by reversed-phase LC using an Inertsil ODS-3 column with ammonium formate-acetonitrile mobile phase and MS detection using positive-ion electrospray ionization. Calibration curves were linear between 0.02 and 0.20 microg/g matrix equivalent concentration for FAA, AA, and MAA, and between 0.2 and 2.0 microg/g for DIP. The relative standard deviations for measurements by the proposed method were <11% for milk and porcine samples, with slightly greater variability for bovine samples. Average recoveries ranged from 82 to 128%, depending on the compound and matrix involved. The method detection limits of FAA, AA, and MAA were <0.02 microg/g for all matrixes tested, while those of DIP were <0.13 microg/g. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 20 samples in a regular working day.     

Analysis of phenols in water by high-performance liquid chromatography using coumarin-6-sulfonyl chloride as a fluorogenic precolumn label

A simple, sensitive and rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method is proposed for the analysis of some environmentally important phenols in water. The use of coumarin-6-sulphonyl chloride (C6SCl) as a fluorescence-labeling reagent has been investigated. The compound reacts with phenols within 20 min under mild conditions (ambient temperature, pH 9.0) to give sulphonates that can be separated by RP-HPLC employing fluorescence detection at lambda(ex) = 360 and lambda(em) = 460 nm. The optimum conditions for fluorescence, derivatization and chromatographic separation have been established and detection limits in the range 0.1-0.9 microg l(-1) were obtained for the studied compounds. The calibration curves were linear for the range 6-200 microg l(-1) for phenol, 3-200 microg l(-1) for 2-chlorophenol, 4-chlorophenol and 2,3,5-trichlorophenol and for the range of 3-100 microg l(-1) for 2,3-dichlorophenol and 3,5-dichlorophenol. The practical applicability of the method to environmental samples was demonstrated by analyzing drinking and industrial water samples spiked with the phenolic compounds.     

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.     

Fluorometric study on the interaction between lomefloxacin and bovine lactoferrin.

The binding of lomefloxacin to bovine lactoferrin (BLf) in a dilute aqueous solution was studied using fluorescence spectra. The binding constant (K) and the number of binding sites (n) were obtained by a fluorescence quenching method. The binding distance (r) and energy-transfer efficiency (E) between lomefloxacin and bovine lactoferrin were also obtained according to the mechanism of Fo?rster-type dipole-dipole nonradiative energy-transfer. The effect of lomefloxacin on the conformation of bovine lactoferrin was also analyzed by synchronous fluorescence spectroscopy. The interaction between lomefloxacin and bovine lactoferrin is strong. Lomefloxacin can affect the conformation of bovine lactoferrin to some degree.     

Determination of biotin following derivatization with 2-nitrophenylhydrazine by high-performance liquid chromatography with on-line UV detection and electrospray-ionization mass spectrometry

Currently, biotin is typically determined in Japan using a microbiological method. Such microbiological assays are sensitive, but they are not always highly specific and are also rather tedious and time-consuming. In the present study, RP-HPLC and LC-MS methods for the determination of biotin have been developed by derivatizing the carboxyl group with 2-nitrophenylhydrazine hydrochloride. 2-Nitrophenylhydrazine is used for the derivatization of carboxylic acids, and these derivatives are known to be applicable to LC-MS detection. Biotin in tablets were extracted by the addition of water and ultrasonic agitation. In order to clean up the sample solution, the filtrate was applied to an ODS cartridge and eluted with methanol. The conditions for preparing the 2-nitrophenylhydrazide derivatives were modified from a previous report for fatty acids. Good recovery rates of over 70% were obtained for the addition of 5-125 microg of biotin per formulation. The detection limit in HPLC at 400 nm was 0.6 ng per injection, with good linearity being obtained over the concentration range 0.001-0.2 microg per injection. Further, derivatives were determined by LC-MS with electrospray ionization, where the spectra indicated the molecular ions [M+H](+). The detection limit was 0.025 ng per injection in the selected ion monitoring analysis, and linearity was observed in the range of 0.6-6 ng per injection. The proposed method could be used to specifically determine the presence of biotin in relatively clean samples.     

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.     

Simultaneous anion and cation exchange chromatography of whey proteins using a customizable mixed matrix membrane

Membrane chromatography can overcome some of the limitations of packed bed column chromatography but preparation of adsorptive membranes usually involves complex and harsh chemical modifications. Mixed matrix membranes (MMMs) require only the physical incorporation of an ion exchange resin into the membrane polymer solution prior to membrane casting. An advantage of MMMs not previously exploited is that resins with differing adsorptive functionalities can be conveniently embedded within a single membrane at any desired ratio. This presents the opportunity to customize an adsorptive membrane to suit the expected protein profile of a raw feed stream e.g. bovine whey or serum. In this work, a novel mixed mode interaction MMM customized to extract all major proteins from bovine whey was synthesized in a single membrane by incorporating 42.5 wt% Lewatit MP500 anionic resin and 7.5 wt% SP Sepharose cationic resin into an ethylene vinyl alcohol base polymer casting solution. The mixed mode MMM developed was able to bind both basic and acidic proteins simultaneously from whey, with binding capacities of 7.16±2.24 mg α-lactalbumin g(-1) membrane, 11.40±0.73 mg lactoferrin (LF)g(-1) membrane, 59.21±9.90 mg β-lactoglobulin g(-1) membrane and 6.79±1.11 mg immunoglobulin Gg(-1) membrane (85 mg total protein g(-1) membrane) during batch fractionation of LF-spiked whey. A 1000 m(2) spiral-wound membrane module (200 L membrane volume, 1m(3) module volume) is predicted to be able to produce approximately 25 kg total whey protein per h.     

Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins

The separation of the four major whey proteins by sodium dodecyl sulphate (SDS)-capillary gel electrophoresis (CGE) is described. Whilst commercially purified whey proteins could be analysed using the recommended protocol, the more complex nature of an acid whey and a reconstituted whey protein concentrate (WPC) powder necessitated considerable refinement of the CGE sample buffer. Individual whey proteins in the acid whey and WPC samples were then also separated and quantitated using capillary zone electrophoresis, polyacrylamide gel electrophoresis (PAGE) and HPLC methods and the results were compared. The values obtained for -lactalbumin (-Lac) and β-lactoglobulin (β-Lg) were consistent throughout the various methods, although size-exclusion HPLC, SDS-PAGE and SDS-CGE could not separate the two β-Lg variants or the glycosylated form of -Lac from the β-Lg. There was considerable variation in the values for the bovine serum albumin and immunoglobulin determined by the different methods and it was concluded that none of the methods could satisfactorily quantitate all four whey proteins.     

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).