Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context

The ability to quantify the casein content by an exact and cost-effective approach represents an issue of crucial importance in the dairy industry as the natural variations in milk protein concentration can markedly affect the yield of the cheesemaking processes, thus causing a direct and significant economic impact on the producers. In this work, the separation and quantification of α_s1-, α_s2-, κ- and β-casein was carried out by direct RP-HPLC analysis of milk. The identification of each casein was established by electrospray ionization mass spectrometry. The data show that this method is able to effectively separate the bovine casein fractions, it provides simplified analytical conditions (with special regard to mobile phase composition and gradient profile) and faster separation while ensuring adequate precision to achieve reliable quantifications in milk samples from dairy production.     

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.     

Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS

Two major milk whey proteins, β-lactoglobulin and α-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for β-lactoglobulin and α-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for β-lactoglobulin and α-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk.     

Separation range and separation efficiency in high-speed gel filtration on TSK-GEL SW columns

Separation ranges for gel filtration on TSK-GEL SW columns, G2000SW, G3000SW and G4000SW, were measured for globular protein, dextran and polyethylene glycol. It was possible to separate globular proteins of molecular weights from 5000 to 7,000,000, dextrans of molecular weights from 1000 to 500,000 and polyethylene glycols of molecular weights from 500 to 250,000 by using these three columns of different pore sizes. The column having the highest separation efficiency for globular proteins was also determined. The highest separation efficiency for molecular weights of less than 30,000, 30,000–500,000 and greater than 500,000 was exhibited by G2000SW, G3000SW and G4000SW, respectively.     

高效液相色谱法分离测定牦牛乳中的8种蛋白质

建立了同时分离和测定牦牛乳中4种酪蛋白和4种乳清蛋白的反相高效液相色谱方法。脱脂牦牛乳经分散剂处理后,采用C4色谱柱(250 mm×4.6 mm,300,5μm i.d.)进行分离,以0.1%的三氟乙酸水溶液和0.1%的三氟乙酸乙腈溶液为流动相,流速为0.8 mL/min,梯度洗脱,二极管阵列检测器(DAD)在220nm波长下检测,外标法定量。结果表明,牦牛乳中8种主要蛋白质在40 min内完全分离,在各自的线性范围内呈良好线性,除α-乳白蛋白外,其余7种蛋白的相关系数均大于0.99。8种蛋白质的回收率为86%～103%,相对标准偏差(RSDs)为1.7%～8.7%;检出限(LODs)为10.7～39.2 mg/L,定量下限(LOQs)为35.7～130.7 mg/L。该方法的准确度和精密度均较高,能够满足实际检测的要求。     

Thermal stability of whey proteins studied by differential scanning calorimetry

The thermal stability of the bovine whey proteins.; β-lactoglobulin (β-1g), α-lactalbumin (α-1a) and serum albumin (BSA) was studied individually and in mixtures in the temperature range 25–140°C by differential scanning calorimetry. The thermal denaturation temperature (T_D) and the transition enthalpies (ΔH_app) were determined at different pH-values (3.0–10.0) in simulated milk ultrafil-trate (SMUF).β-Lg was, except at pH 9.0 and 10.0, the most thermostable protein at all pH-values. At acidic pH-values BSA was the least thermostable. At alkaline pH-values, however, α-la had lower thermal stability than BSA. α-La exhibited double peak behaviour at acidic pH-values and ΔH_app was dependent on Ca-content. Mixtures of the proteins were studied at pH 4.0, 5.0 and 6.6. In general, when mixed, the proteins seemed to denaturate independently of each other.     

Efficient separation of homologous α-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography

Transgenic bovine milk could be a rich source of recombinant human proteins. However, the co-presence of bovine and human homologous proteins can be a challenge for product purification. In this study, the average surface hydrophobicity and electric potential of human α-lactalbumin (HLA) and bovine α-lactalbumin (BLA) were analyzed and compared through the exposure area calculation of different amino acids. Based on the analysis, calcium independent hydrophobic interaction chromatography was selected for separation of recombinant human α-lactalbumin (rHLA) from BLA in transgenic bovine milk. The operating conditions for the best separation of two proteins were predicted by fluorescence data. Three commercially available HIC resins (Butyl Sepharose 4 FF, Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF) were compared. The transgenic milk was skimmed and treated by pH adjustment to remove a large quantity of casein protein. The supernatant was loaded on the hydrophobic interaction chromatographic matrix. The correct elution fraction was further treated with gel filtration chromatography. The overall recovery of rHLA was up to 67.1% with the purity greater than 95%. Circular dichroism spectroscopy (CD) and mass spectrogram (MS) confirmed the native state and glycosylated form of the purified rHLA.     

Separation of Proteins in Human Milk

Abstract

A separation of proteins in human milk whey using high performance ion exchange chromatography is reported. Three chromatographic peaks were identified on the basis of retention times as bile salt-stimulated lipase (BSSL), lactoferrin, and α-lactalbumin. Implications for the purification of BSSL are discussed and suggestions for future purification procedures are made.     

Evaluation of the thermal history of bovine milk from the lactosylation of whey proteins: an investigation by liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (RP-HPLC–ESI-MS) has been used for analysis of the native and lactosylated forms of the main whey proteins, α-lactalbumin and β-lactoglobulins A and B, in commercial bovine milk samples after different thermal treatment (pasteurisation and ultra high-temperature, UHT, treatment), of different lipid content, and of different brands, to find markers of the thermal history of the milk. A new quantification strategy was developed, based on peak-area integration after multiple ion current extraction and considering all the ions detectable in the multi-charge ESI mass spectrum for each type of protein. Validation of the procedure for native forms was first accomplished by calibration with model solutions. Linearity was always good. Sensitivity was different for α-lactalbumin and β-lactoglobulins; the signal was stronger for the latter with only a slight difference between variants A and B of β-lactoglobulins. Application of the quantification approach to pasteurised and UHT milk samples showed that the distributions of the three proteins and of their three main forms (native, and mono and bi-lactosylated) in whey extracts can be used as statistically robust discriminatory properties for recognition of commercial thermal treatment of milk. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Dedicated to Professor P. G. Zambonin on the occasion of his 72nd birthday.     

Separation of casein micelles from whey proteins by high shear microfiltration of skim milk using rotating ceramic membranes and organic membranes in a rotating disk module

This paper investigates the microfiltration of skim milk in order to separate caseins micelles from two whey proteins, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), using a modified dynamic filtration pilot (MSD) consisting in 6 ceramic 9-cm diameter membrane disks of 0.2 μm pores, rotating around a shaft inside cylindrical housing. A comparison was made with another dynamic filtration module consisting in a disk rotating near a fixed PVDF 15.5 cm diameter membrane with 0.15 μm pores. Maximum permeate fluxes were 120 L h⁻¹ m⁻² with the MSD module at 1930 rpm and at 40 °C, and 210 L h⁻¹ m⁻² at 2500 rpm and 45 °C, with the rotating disk module. Casein rejection was around 99% at high speed for both membranes. α-La transmission decreased with increasing transmembrane pressure (TMP) from 75% to 60% for ceramic membranes and from 25% to 10% for the PVDF one. β-Lg transmissions were lower, ranging from 23% to 15% for ceramic membranes and from 20% to 5% for the PVDF one. In a concentration test with the PVDF membrane at 2000 rpm, the flux decayed from 200 L h⁻¹ m⁻² at initial concentration to 80 L h⁻¹ m⁻² at VRR = 3.2 and 22.1% of the initial α-La mass was recovered in the permeate, against 8.1% for β-Lg. Permeate fluxes in the mass transfer limited regime (J_lim) of the MSD and rotating disk module operated at various speeds were well correlated by the equation J_lim = 17.13 V_av where V_av denoted the disk azimuthal velocity averaged over the membrane area. Measurements of J_lim, taken from Ref. [G. Samuelsson, P. Dejlmek, G. Tragardh, M. Paulsson, Minimizing whey protein retention in crossflow microfiltration of skim milk. Int. Dairy J. 7 (1997) 237–242] during MF of skim milk using tubular ceramic membranes at velocities from 1.5 to 8 m s⁻¹ with permeate co-current recirculation were found to obey the same correlation.     

Analysis of major ovine milk proteins by reversed-phase high-performance liquid chromatography and flow injection analysis with electrospray ionization mass spectrometry

Ovine milk proteins were analyzed both by coupling HPLC and electrospray ionization mass spectrometry (ESI-MS) and by flow injection analysis and ESI-MS detection after separation and collection of fractions from gel permeation chromatography. These methods resolved the four ovine caseins and whey proteins and made it possible to study the complexity of these proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. The experimental molecular masses of ewe milk proteins were: 19 373 for κ-casein 3P; 25 616 for _s2-casein 10P; 23 411 for _s1-casein C-8P; 23 750 for β-casein 5P; 18 170 and 18 148 for β-lactoglobulins A and B; 14 152 for -lactalbumin A and 66 322 for serum albumin.     

Variation of the calcium content in skim milk by diafiltration and ion exchange – Effects on permeation rate and structure of deposited layers in the RO

The effect of different calcium levels on the permeation rate and on the structure of deposits formed in reverse osmosis (RO) was studied as a function of the transmembrane pressure difference. An almost complete removal of the soluble calcium together with the other low molecular constituents by diafiltration significantly changed the deposited protein layer compared with untreated skim milk. By determination of the flow resistance of the deposited layer Δp_L it was concluded that by the accumulation of individual micellar casein particles a loose structure was formed. The removal of the total calcium, i.e. both soluble and the calcium bound in the casein micelles, by ion exchange resulted in the disintegration of the micelle structure, causing a very dense deposited protein layer consisting of casein submicelles and a significant reduction of the permeation rate compared with untreated skim milk.     

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.     

Proteolytic Activities of Kiwifruit Actinidin (Actinidia deliciosa cv. Hayward) on Different Fibrous and Globular Proteins: A Comparative Study of Actinidin with Papain

Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.     

Immobilization of Amylases on Silica Support to Study Breakdown Products of Potato Starch by HPLC

Abstract

Immobilization of α- and β-amylases on epoxypropylsilanized PartiSphere-5 was achieved. Hydrolysis of 2% potato starch solution yielded limit dextrin on α-amylase bound column while a mixture of limit dextrin, maltose and glucose was obtained from β-amylase bound column. The β-amylase bound column converted limit dextrin from α-amylase column into glucose.     

Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major they protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.     

Rapid and sensitive determination of protein by light-scattering technique with Eriochrome Blue Black R

Based on the interaction between Eriochrome Blue Black R (EBBR) and proteins, which causes a strong light-scattering signal with the maximum scattering peak located at 398 nm, a simple, rapid, sensitive and selective method is developed for the determination of proteins by the light-scattering technique using a common spectrofluoremeter. Under proper experimental conditions, the protein determination can be performed in the range of 0.1-25, 0.1-20 and 0.25-25 μg ml⁻¹ for bovine serum albumin (BSA), human serum albumin (HSA) and human immunoglobulin G (IgG), respectively. The detection limit, calculated as 3 times the S.D. of nine blank measurements, are 33 μg l⁻¹ for BSA, 25 μg l⁻¹ for HSA and 38 μg l⁻¹ for IgG. Moreover, there is no significant difference among the scattering signals yielded by HSA, IgG and BSA, and almost no interference of many amino acids and metal ions. The method has been satisfactorily applied to the direct determination of the total protein in human serum, saliva and urine samples. The results obtained from the studies on the binding characteristics of EBBR to BSA indicated that an electrostatic force existed in the binding system, and the binding constant (K) and the number of the binding sites (n) at 25 °C are 1.69×10⁵ l mol⁻¹ and 0.946, respectively.     

Fractionation of glycoproteins according to lectin affinity and molecular size using a high-performance liquid chromatography system with sequentially coupled columns

The lectin phytohaemagglutinin was coupled to porous silica (10 micron) and used as adsorbent in a high-performance liquid affinity column sequentially coupled to a TSK-G 3000SW gel permeation column. This system was used for high-speed separation/analysis of human serum glycoproteins according to their lectin affinity and molecular size. Serum samples could be resolved in at least six different peaks representing glycoproteins exhibiting different molecular weights but with a carbohydrate content compatible with the specificity of phytohaemagglutinin.     

Isolation and analysis of peptidic fragments of alpha-gliadin using reversed-phase high-performance liquid chromatography

Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.