Precipitation Of Casein Research Articles

Interaction with Al and Zn induces structure formation and aggregation in natively unfolded caseins

Caseins are phosphoproteins that form the principal protein component of milk, their chief function being the transport of inorganic calcium and phosphate to the neonates. The four major members of the casein family are α s1-, α s2- (together referred to as α s-casein), β- and κ-casein, each having a characteristic high negative net charge as well as high hydrophobicity and preferring extended conformational states in solution. We have investigated the influence of the polyvalent metal cations Zn(II) and Al(III) on the structure of bovine caseins, using fluorescence and circular dichroic (CD) spectroscopy and light scattering. Changes in Trp and ANS fluorescence parameters (blue shifts of the emission maxima and enhancement of fluorescence intensity) and in the far-UV CD spectra of the caseins caused by the presence of both metals suggest that conformational changes are induced in them by low concentrations (20–40 μM) of the metal cations. These changes lead to formation of solvent-accessible hydrophobic clusters or cavities that, in turn, cause self-association and precipitation of caseins at higher concentration of the metals. These conclusions are supported by increased binding of ThT to the caseins, as well as enhancement of light scattering intensity, observed in presence of Al(III). The chaperonic property of α s-casein, which enables it to inhibit thermal aggregation of alcohol dehydrogenase, is shown to be partially destroyed by Zn(II)-induced structural alterations, due possibly to loss of flexibility of the natively unfolded casein chains.

Oxford Catalysts expands laboratory

The determination of casein in milk by infrared spectrometry still suffers from deficiencies specific to the spectrometers used. Measurements with filtometers are based on the difference in the absorbance before and after casein precipitation. Computerised Fourier Transform Infrared (FT-IR) spectrometry allows the use of more spectral information but robust casein calibrations were achieved only with high numbers of natural calibration samples. This is expensive due to the costly reference analyses needed for calibration. To reduce effort and costs of casein calibrations we studied the usefulness of commercial calibration milks (designed for fat, protein, and lactose calibration) for casein calibrations. We tested calibration models with and without natural milk samples and assessed the validation results according to International Organization for Standardization and International Dairy Federation standards. We found the combined use of commercial designed samples and fresh natural raw milk samples a very promising approach to the partial least squares casein calibration of FT-IR spectrometers. Measurement uncertainties for casein as low as 0.020 g/100 g for the bias and 0.047 g/100 g for the standard error of prediction (on 25 validation samples of high variability) were achieved with a calibration of 38 samples. This accuracy complies with the requirements of the International Dairy Federation standard 141C:2000 for the infrared analysis of milk.

HYDROLYSIS OF MAJOR DAIRY PROTEINS BY LACTIC ACID BACTERIA FROM BULGARIAN YOGURTS

Twenty-one Lactobacillus strains isolated from three types of Balkan homemade yogurts were grown on sodium caseinate, β-casein or whey proteins, and the proteolysis was followed by electrophoresis and reversed-phase high-performance liquid chromatography. The best conditions allowing obtaining proteolysis without casein precipitation are 0.8% casein in 50-mM phosphate buffer. The strains tested showed a relatively high proteolytic activity despite the limited conditions for bacterial growth. Within 72 and 96 h of incubation, 80–90% of β-casein was consumed. They showed also a proteolytic activity toward α-lactalbumin (ALA), being able to reduce its concentration between 5 and 55%, depending on the strains used. The capacity of the strains to hydrolyze β-lactoglobulin was lower as compared with hydrolysis of ALA. Hydrolysis of casein by all strains produced peptides with an antibacterial effect against Escherichia coli. Consequently, to obtain a maximal hydrolysis of the dairy proteins seconded by appearance of antimicrobial peptides, a combination of strains with different beneficial properties to be used as starters was proposed. PRACTICAL APPLICATIONS Some of the 21 Lactobacillus strains isolated from three types of Balkan homemade yogurts may be used to proteolyze milk proteins in order to produce peptides with an antibacterial effect against Escherichia coli. To obtain a maximal hydrolysis of the dairy proteins seconded by appearance of antimicrobial peptides, a combination of strains with different beneficial properties to be used as starters should be determined. During fermentation process, milk proteins are acidified by the production of lactic acid and are hydrolyzed by proteases and peptidases from bacteria. This proteolysis is followed by a reduction of the number of epitopes and consequently by a decrease in allergenicity of hydrolyzed proteins. For these reasons, starters as Lactobacillus strains with beneficial properties able to reduce the allergenicity of fermented milk products are of great interest for the dairy industry.

The effects of seasonal climate changes in the caatinga on tannin levels in Myracrodruon urundeuva (Engl.) Fr. All. and Anadenanthera colubrina (Vell.) Brenan

The influence of seasonal climatic changes on tannin concentrations was examined in two caatinga (semi-arid) plant species (Myracrodruon urundeuva (Engl.) Fr. All. and Anadenanthera colubrina (Vell.) Brenan). The Folin-Ciocalteau method was used to quantify total phenols, and the casein precipitation method was used for tannins. In general, there is a close relationship between tannin levels and rainfall, although species seem to adopt different strategies of tannin compound production in response to periods of drought and rainfall in the caatinga.

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.

Characterization of Casein Fractions from Algerian Dromedary (Camelus dromedarius) Milk

To characterize casein fractions in Algerian dromedary’s milk, samples from two breeds, Larbaa and Targui, were analyzed using electrophoretic and chromatographic techniques. Analysis performed by isoelectric focusing showed heterogeneity within the same breed and between the breeds. Some samples featured a reduced beta-lactoglobulin content and one sample from Larbaa breed showed the lack of bands in the most acidic region, where kappa-casein focuses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this sample allowed identifying the alpha -, alpha and beta-casein fractions only. High s1 s2 performance liquid chromatography analysis of precipitated casein showed three main peaks. In order to characterize them, electrospray ionization-mass spectrometry was exploited. Their measured molecular masses were 24,760, 22,060 and 24,970, corresponding to alpha -, alpha and beta-casein. Analytical s1 s2 results suggested the absence of kappa-casein in this sample. Nevertheless, investigations at DNA level are necessary in order to better characterize kappa-casein locus in Larbaa breed and to define this species’s milk peculiar properties.

Characteristic aroma components of rennet casein.

Rennet casein, produced by enzymatic (rennet) precipitation of casein from pasteurized skim milk, is used in both industrial (technical) and food applications. The flavor of rennet casein powder is an important quality parameter; however, the product often contains an odor described as like that of animal/wet dog. Two commercial rennet casein powders were evaluated to determine the compounds responsible for the typical odor. Aroma extracts were prepared by high-vacuum distillation of direct solvent (ether) extracts and analyzed by gas chromatography-olfactometry (GCO), aroma extract dilution analysis (AEDA), and GC-mass spectrometry (MS). Odorants detected by GCO were typical of those previously reported in skim milk powders and consisted mainly of short-chain volatile acids, phenolic compounds, lactones, and furanones. Results of AEDA indicated o-aminoacetophenone to be a potent odorant; however, sensory descriptive sensory analysis of model aroma systems revealed that the typical odor of rennet casein was principally caused by hexanoic acid, indole, guaiacol, and p-cresol.

Dynamics of isoelectric precipitation of casein using sulfuric acid

AbstractThe acid precipitation of casein from skim milk is an interesting and complex operation, as it involves several mechanistic steps occurring simultaneously. Research has focused so far on the influence of static variables such as temperature and final pH on the curd properties. Here the dynamics of the process was investigated. From a characteristic times analysis it was concluded that the most important mechanistic steps in the acid precipitation of casein were acid mixing, aggregation/breakup, and transport in/out of the precipitate particles. Experiments in a fed batch setup using sulfuric acid as the precipitant showed how the precipitation influenced the pH profile and demonstrated the large influence of process variables, such as the acid addition rate, the mixing intensity, and aging time on particle‐size distributions and the release of minerals from the casein.

Dynamics of precipitation of casein with carbon dioxide

Abstract Carbon dioxide is a green alternative for mineral acids (e.g., sulphuric acid) in protein precipitation. The precipitation using a gaseous precipitant differs from the conventional precipitation in the way and the rate at which the precipitant can be applied to the aqueous solution. In this paper, the consequences of using carbon dioxide were investigated for the precipitation of the milk protein casein. Product properties, such as particle size, solids content and calcium release were investigated in a batch system at various mixing conditions and gas addition rates. In addition, mass transfer coefficients were determined from pH response data. The experiments revealed a strong influence of stirring rate and gas flow rate on the particle size. The main effect of varying the gas flow was through the change of acidification rate, much alike precipitation with sulphuric acid. At high acidification rates, the particle size of casein precipitated with carbon dioxide was smaller than with sulphuric acid.

Evaluation of Designed Calibration Samples for Casein Calibration in Fourier Transform Infrared Analysis of Milk

The determination of casein in milk by infrared spectrometry still suffers from deficiencies specific to the spectrometers used. Measurements with filtometers are based on the difference in the absorbance before and after casein precipitation. Computerised Fourier Transform Infrared (FT-IR) spectrometry allows the use of more spectral information but robust casein calibrations were achieved only with high numbers of natural calibration samples. This is expensive due to the costly reference analyses needed for calibration. To reduce effort and costs of casein calibrations we studied the usefulness of commercial calibration milks (designed for fat, protein, and lactose calibration) for casein calibrations. We tested calibration models with and without natural milk samples and assessed the validation results according to International Organization for Standardization and International Dairy Federation standards. We found the combined use of commercial designed samples and fresh natural raw milk samples a very promising approach to the partial least squares casein calibration of FT-IR spectrometers. Measurement uncertainties for casein as low as 0.020 g/100 g for the bias and 0.047 g/100 g for the standard error of prediction (on 25 validation samples of high variability) were achieved with a calibration of 38 samples. This accuracy complies with the requirements of the International Dairy Federation standard 141C:2000 for the infrared analysis of milk.

Improvement of a method for the measurement of lactoperoxidase activity in milk

Abstract Lactoperoxidase (LPO) is one of the two most used indigenous enzymes to evaluate milk heat treatment, alkaline phosphatase being the other. In the present work, LPO activity was measured using Clarifying Reagent® to facilitate spectrophotometric measurement in milk. Satisfying repeatability was obtained with a CV of 5.2% (n=33). Results obtained by this method correlated well with the classic Rothenfusser method (R2=0.97; n=20) and also with another method using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate diammonium salt) (R2=0.98; n=21). This very easy rapid colorimetric method avoided preliminary casein precipitation and filtration steps. It has been shown to be suitable for routine characterisation of heat treatment of milk.

Composition and calcium status of acid whey from pasteurized, UHT-treated and in-bottle sterilized milk.

Whey extracts were obtained from pasteurized, UHT-treated and in-bottle sterilized milks. After acidic precipitation of casein the concentration of protein, NPN, lactose, lipid, calcium, magnesium and potassium was determined. Among the parameters examined, protein content was significantly reduced in the whey extracts from UHT-treated and in-bottle sterilized milks compared with that from pasteurized milk, while lactose content was increased. Calcium extracted in whey was at least 80% of total calcium of the milk. The total calcium to protein ratio of whey was increased as a function of the thermal treatment of milk, while ionic calcium was about 50% of total calcium in all whey extracts. In vitro protein digestibility was found to be significantly lower in whey from UHT-treated and in-bottle sterilized milks than in that from pasteurized milk. Parallel estimation of the percentage of ionic calcium and of the solubility of proteins in the pH range 2-10 indicated that calcium was not involved in the pH-dependent solubility of proteins extracted in the whey, the extent of solubility being essentially a function of the thermal treatment of milk. The results suggest that calcium was not responsible for the formation of soluble protein macroaggregates with impaired digestibility that are present in whey from milk subjected to heat treatment of increasing intensity.

Electrochemical Sensor Detecting Free Sulfhydryl Groups: Evaluation of Milk Heat Treatment

We describe a new and rapid method for the evaluation of reactive sulfhydryl groups in whey proteins obtained after precipitation of casein by acetic acid at pH 4.6. The procedure is based on the use of a wire tungsten electrode operating at −0.2V versus saturated calomel electrode in flow injection analysis. The method was applied to raw milks and to commercial pasteurized and UHT milks. Results showed that the tungsten electrode constituted a robust amperometric sensor that could be used to differentiate milks that underwent different heat treatments. The decrease of thiol content in the whey proteins from samples was in agreement with the whey protein content found by HPLC. The procedure is suitable for on-line quality control of heat-treated milks.

Partitioning of divalent metal ions in aqueous casein-containing systems of variable mineral compositions

The influence of the salt composition of casein-water-Cd(NO3)2 and defatted milk-Cd(NO3)2 systems on the partitioning of Cd2+ during acidic precipitation of casein was studied. The conditions that minimize the content of the toxicant ion in the isolated protein (pH and salt composition) were determined.

Effect of membrane permselectivity on the fouling of cationic membranes during skim milk electroacidification

A previous study on skim milk demonstrated that bipolar-membrane electroacidification (BMEA) is a technology that can be used to produce isoelectric precipitation of casein from milk. However, a deposit, suspected to be calcium hydroxide, was observed on the cation-exchange membrane (CEM) side in contact with the base. Also, the performance of the process decreased as well as the lifetime of the membrane. The aim of the present study was therefore to evaluate two types of CEM with different permselectivities in terms of electrodialysis efficiency and membrane parameters. BMEA allowed the separation of high purity bovine milk casein (about 97% protein), and the permselectivity of both membranes tested does not influence the purity of the isolates produced. However, the migration of cations through the cationic membrane was found to be influenced by the permselectivity leading to isolates with different ash contents. The CSV™ membrane slowed down the migration of cations in comparison with CMX™ membranes. The membrane fouling of cation-exchange membrane was identified as precipitating calcium and magnesium hydroxides. Moreover, the fouling formed at the outer layer surface of the CEM was demonstrated to be reversible for both membranes while the fouling of the CSV™ membrane inner layer was irreversible.

Orthokinetic flocculation of caseinate-stabilized emulsions: influence of calcium concentration, shear rate, and protein content.

Calcium-induced flocculation of caseinate-stabilized soybean oil-in-water emulsions in conditions of Couette flow was studied. A concentrated emulsion (20% oil, 0.5-2.0% sodium caseinate in 20 mM imidazole, pH 7) was diluted 20 times in buffer containing concentrations of CaCl(2) between 9 and 17 mM and sheared at rates between 335 and 1340 s(-)(1). The average particle size (d(43)), measured by integrated light scattering, increased in a sigmoidal manner with shearing time. An increased shear rate resulted in an increased flocculation rate, because of the increased number of collisions between particles, but a decreased value of the maximum d(43), because higher shear rates increasingly disrupted the flocs. The flocculation rate was increased by increasing the calcium concentration, indicating an increased collision efficiency. The orthokinetic stability of the emulsions was increased with increased protein content, and it is postulated that the increased surface coverage and hydrodynamic thickness of the adsorbed protein layer increased steric repulsion between droplets, so that higher calcium concentrations were necessary to induce sufficient conformational change of the proteins to allow flocculation. At high caseinate concentrations, calcium may also induce precipitation of unadsorbed caseins from the serum to the oil-water interface, thereby increasing steric repulsion and hence increasing orthokinetic stability.

Isoelectric Precipitation of Casein Using High-Pressure CO2

High-pressure CO2 was used as a recyclable acid for the isoelectric precipitation of casein. Measurement of the pH during the high-pressure process allowed the comparison of the process with conven...

Bipolar membrane electroacidification to produce bovine milk casein isolate.

Bipolar membrane electroacidification (BMEA) has been developed previously (Bazinet et al., Report for the Canadian Electricity Association 9326 U 987, 1996; Bazinet et al., J. Agric. Food Chem. 1997, 45, 2419-2425, 3788-3794) and has been used for isoelectric precipitation of soybean proteins. The purpose of this study was to validate the feasibility of BMEA for the precipitation of milk casein and to investigate the effect of flow rate. High-purity isolates containing 1.23 and 2.00% ash and 85.4 and 91.6% total protein were obtained with flow rates of 0.2 and 1.2 gal/min. The molecular composition profiles of the isolates obtained by HPLC showed that only caseins were precipitated. However, except for protein precipitation curves, the flow rate did not influence the final composition and purity of the isolates. These results showed that BMEA is a new alternative process for the production of high-purity bovine milk casein isolate.

Measurement of the solubility of carbon dioxide in milk at high pressures

The solubility of CO 2 in milk was studied at temperatures of 25, 38 and 50°C and at pressures up to 6.9 MPa using a static method with an accuracy of 5%. These data are important for the design and control of processes which use CO 2 for precipitation of casein from milk. The solubility of CO 2 in milk was found to increase with pressure and decrease with increasing temperature — a trend similar to that observed for the solubility of CO 2 in water. At 25°C, the solubility of CO 2 in milk was less than the solubility in water over the entire pressure range studied. Precipitation of casein was not observed. At 38°C and 50°C and pressures greater than 5.5 MPa, complete precipitation of casein from milk was observed, and the solubility of CO 2 in milk was approximately the same as that in water. This indicates that casein destabilization is accompanied by an increase in water available for dissolution of CO 2.

Determination of protein and casein in milk by fourth derivative UV spectrophotometry

The aim was to develop a simple and rapid method for determination of milk protein in general, and casein content in particular, in milk without involving preparation procedures such as skimming and casein precipitation. Fresh raw milk samples were diluted with 6 M guanidine-HCl buffer before spectroscopic scanning. The total protein and casein contents of 45 milk samples determined by this method were not statistically significantly different from the values obtained by the reference method based on Kjeldahl determination of N ( p < 0.05). By using fourth derivative UV spectrophotometers, the determination of total protein and casein content in milk can be achieved directly in a single run. Treatments of milk such as heating, homogenization, preservation or hydrolysis with protease had no influence on the accuracy of protein determination in milk by this method. The accuracy of determination with 0.03% total protein and 0.04% casein for the reference method could not be achieved under the condition used, due to the effect of high dilution rate.