Removal Of Casein Research Articles

Whey proteins from camel's milk have higher in vitro wound-healing effect than whey proteins from cow's milk.

The effectiveness of whey obtained by either enzyme (sweet) or acid treatment on wound healing remains unclear. This study investigated the effectiveness of camel and bovine whey prepared enzymatically (CSW and BSW) or by pH reduction (CAW and BAW). After removing the cream from milk, HCl or rennet was used to remove casein, resulting in acid or sweet whey, respectively, followed by lactose removal using dialysis. Casein removal was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Wound-healing activity was measured in vitro on HT-29 cells by scratch assay. All four whey samples (0-1000 mg L-1) were applied on the cells, and the closure of the cell-free scratched areas was monitored for 48 h. All whey samples increased the cell migration significantly (P < 0.05) to help close the cell-free areas as an indication of wound healing compared to the negative control. However, the closure amounts between the highest dose (1000 mg L-1) and the control were not significantly different (P > 0.05). Acid whey samples significantly (P < 0.05) elevated the closure speed compared to the sweet whey samples. The highest closure percentage (64.69%) was achieved after treatment with 10 mg L-1 CAW for 48 h. Between the sweet whey samples, BSW was significantly (P < 0.05) more effective in closing the cell-free zone compared to CSW. This study investigated the wound-healing potential of camel and bovine whey in vitro by comparing their effects on HT-29 cell migration. CAW showed the greatest activity and may find uses as a treatment agent in wound healing. © 2024 Society of Chemical Industry.

Molecular insights on the binding of chlortetracycline to bovine casein and its effect on the thermostability of chlortetracycline

Bovine casein was selected as a model protein to evaluate the impact of food matrix on the thermal degradation of antibiotics. Fluorescence quenching and isothermal titration calorimetry experiments revealed that chlortetracycline (CTC) could spontaneously bind to casein via hydrogen bonding and hydrophobic interactions. The amino acid residues forming the binding pocket were further identified using molecular docking, while saturation transfer difference NMR deciphered that the binding of CTC engages its -N(CH3)2 group. Moreover, the degradation behavior of free CTC versus that bound in casein-CTC complex was compared during thermal treatment. Compared with free CTC, a lower first-order rate constant was observed in the presence of casein. Removal of casein shortened the half-life of CTC by at least 48.1% at low concentrations. Elucidating that the formation of protein-antibiotic complexes alters the amenability of antibiotics to degradative reactions, which could help eliminate residual antibiotics and guarantee the safety of dairy products.

Production of whey protein as nutritional valuable foods

Whey is a low calorie milk serum remaining after casein removal in cheese manufacturing. Composition and properties of whey depend on the type of cheese production process, type and the quality of the used milk. Whey mostly consists of lactose, proteins, vitamins, minerals, enzymes, hormones and growth factors. Functional and nutritional roles of whey components meet the requirements for dietary food. Whey proteins are liquid fractions of 20% milk proteins, which remain in whey after casein removal. The increase in the temperature of milk processing leads to denaturation of proteins and a decrease in their content. The most common whey proteins are albumin and globulin. Separation and fractionation of whey proteins may be carried out as precipitation, combined with acid treatment, high temperature treatment or centrifugation (centry-whey process), and as a membrane process (usually ultra- or microfiltration followed by a diafiltration and spray drying). Whey proteins can also be separated based on their molecular mass, charge and hydrophobicity. It is a well-known fact that whey proteins possess admirable nutritional and functional components, such as essential amino acids, bioactive peptides, antioxidants and immunopotentiators. Research of bioactive compounds and nutrients pushed whey protein to the forefront of the functional food sector, therefore, the implementation of these proteins in the diet and supplements has the potential to address many metabolic imbalance-caused diseases, and it seems imperative to harness their potential.

A Raman-spectroscopy-based approach for detection and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages at low titer in raw milk.

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~ 107pfu/mL) and have lower titers (102-103pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (102pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60min) and high coefficient of determination (R2) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).

Separation of immunoglobulins from colostrum using methods based on salting-out techniques

This study aimed to obtain a purified IgG fraction from bovine colostrum using salting-out precipitation methods as base techniques. As the first step after skimming of colostrum, isoelectric precipitation or rennet coagulation were used for casein removal. IgG concentrations in raw material and final products were determined using the ELISA method. SDS-PAGE electrophoresis was used for purity determination of the final fraction of IgG. According to the obtained results, the combination of precipitation, gel filtration, and cross-flow filtration techniques enables to separate the IgG fraction with purity up to 90% and yield up to 91%. Acid whey has a higher yield in comparison with sweet whey and both ammonium sulphate and sodium sulphate have similar effectivity for precipitation of IgG. The final concentrate of IgG can be used as a standalone product or as an additive to other food supplements.

Casein precipitation by acid and rennet coagulation of buttermilk: Impact of pH and temperature on the isolation of milk fat globule membrane proteins

Milk fat globule membrane (MFGM) proteins make up only 2–3% of the total protein content in buttermilk. Prior isolation of buttermilk MFGM material is therefore required for further investigation of its functional properties and application in food systems. Rennet induced coagulation has been applied to remove caseins, but 30–70% of MFGM proteins are entrapped in the casein curd under classical cheese making conditions. Therefore, factors influencing the rennet as well as acid induced coagulation of buttermilk with regard to a reduction of MFGM protein losses into the casein curd, i.e., pH, temperature and pre-heat treatment of buttermilk, were examined. Complete casein removal from buttermilk was reached using both coagulation methods, whereas retention of MFGM proteins in the serum phase was higher during renneting. Rennet induced coagulation at higher pH and lower temperatures resulted in weaker gel structures, which increased the yield of MFGM proteins in the buttermilk whey.

Pilot-scale purification of α-lactalbumin from enriched whey protein concentrate by anion-exchange chromatography and ultrafiltration

A method based on anion exchange chromatography (AIEX) in combination with ultrafiltration (UF) was developed to obtain kilogram-scale amounts of bovine α-lactalbumin (α-La) of high purity from α-La-enriched whey protein concentrate (αWPC). Initially, α-La was successfully purified, at laboratory scale, from a 10% solution of αWPC. Removal of casein and denatured whey protein by acid precipitation resulted in a α-La purity of 78%. A further purification by AIEX eluting with sodium acetate (NaAc) buffer led to a final purity and recovery of 100 and 85%, respectively. Based on this, the process was simplified and optimized for pilot-scale purification by selective binding of β-lactoglobulin (β-Lg). Batchwise expanded bed AIEX using a column with 5 L of Q Sepharose matrix was performed. Pure α-La was obtained in the run through buffer. In the end, 1100 g of holo form α-La was obtained with a purity of 97.4% in protein and a recovery of 80%, calculated based on the protein detectable in reversed phase high-performance liquid chromatography (RP-HPLC). Thus, by combining AIEX and UF, heating was avoided allowing production of α-La in kilo-scale with high purity and recovery from αWPC with a minimum volume of buffer solutions.

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

Determination of Immunoglobulin G in Bovine Colostrum and Milk Powders, and in Dietary Supplements of Bovine Origin by Protein G Affinity Liquid Chromatography: Collaborative Study

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

A Methodical Approach to Ultra-Scale-Down of Process Sequences: Application to Casein Removal from the Milk of Transgenic Animals

Scale-down models of individual operations are widely used in biopharmaceutical process development to obtain information about the performance of production-scale equipment on the basis of inexpensive and efficient laboratory-scale tests, for the purposes of validation or optimization or characterization studies. We have investigated the ability of scale-down models of whole process sequences to provide reliable information for process scale-up from laboratory- to pilot-scales of operation. Using the example of the recovery of a protein from transgenic milk, we have conducted an a priori scale-down analysis of a projected pilot-scale process sequence. A systematic approach was developed to ensure that all critical aspects of process behavior were included in the scale-down model, resulting in the creation of an accurate and reliable scale-down representation of the pilot-scale process. The data from scale-down process trials conducted at 70 and 200 mL scales of operation served to highlight crucial factors determining process performance, and proved reliable in predicting the performance of the pilot-scale process over a scaling factor of 1000.

Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations

Transgenic animal bioreactors can be engineered to make gram per liter quantities of complex recombinant glycoproteins in milk. However, little is known about the limitations in post-translational processing that occurs for very complex proteins and how this impacts the task of purification. We report on the purification of recombinant factor IX (rFIX) from the milk of transgenic pigs having an expression level of 2–3 g rFIX/(l −1 h −1), an expression level that is about 20-fold higher than previously reported. This purification process efficiently recovers highly active rFIX and shows that even complex mixtures like pig milk, which contains 60 g/l total endogenous milk protein and multiple subpopulations of rFIX, can be processed using conventional, non-immunoaffinity chromatographic methods. Without prior removal of caseins, heparin-affinity chromatography was used to first purify the total population of rFIX at greater than 90% yield. After the total population was isolated, the biologically active and inactive subpopulations were fractionated by high-resolution anion exchange chromatography using an ammonium acetate elution. Capillary isoelectric focusing of the active and inactive rFIX fractions demonstrated that the active subpopulations are the most acidic.

Model process for removal of caseins from milk of transgenic animals.

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.

Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.

Decreased growth of Streptococcus uberis in milk from mammary glands of cows challenged with the same mastitis pathogen.

Milk samples from mammary glands challenged with Streptococcus uberis and from unchallenged mammary glands were selected for analyses of bacterial growth, antibody response, and lactoperoxidase activity. All challenged mammary glands became infected with isolation of S. uberis and elevated somatic cell counts in milk during the first week after challenge. In vitro growth of the homologous challenge strain and a heterologous strain of S. uberis was significantly lower in milk from challenged mammary glands than in milk from control mammary glands at 3, 5, and 7 days after challenge. Removal of casein significantly reduced bacterial growth. In general, antibodies specific to S. uberis started to increase at day 3 post-challenge and were higher in milk from challenged mammary glands than in milk from control mammary glands. There was also a marked increase in total IgG in milk from challenged mammary glands. Growth of S. uberis increased following heat treatment at 56 degrees C of pooled milk or whey samples from challenged mammary glands. Growth of S. uberis correlated negatively with the specific antibody response to the bacteria (P < 0.001). Lactoperoxidase activity varied among cows and among different samples over time and did not appear to contribute to decreased growth of S. uberis. These results suggest that decreased growth of S. uberis in milk from challenged mammary glands in comparison to milk from control mammary glands could result from the interaction of antibodies with complement components.

Effects of Different Skim Milk Fractions on Activity of Cow Milk Purified Lipoprotein Lipase

Hydrolysis of a long-chain triglyceride substrate by purified cow milk lipoprotein lipase, in the absence of blood serum, was changed by adding heated skim milks. Preincubation of the substrate with small amounts of several skim milks increased (+ 50 to 300%) lipoprotein lipase activity. By contrast, greater amounts of skim milk increased further or decreased lipoprotein lipase activity (+ 300 to −90%). The inhibitory effect was due to substrate surface protection.Results obtained after removal of casein (by isoelectric precipitation or by ultracentrifugation) and of whey proteins (by heat treatment) showed that the effect of skim milk was due to the proteose-peptone fraction. Fractionation of the proteose-peptone fraction into component 5-enriched fraction and component 3-enriched fractions suggested that the activating effect was due partially to proteose-peptone component 5, and the inhibitory effect resulted from component 3. These effects were over-come by blood serum.

Improved Assay Procedure for Determination of Milk-Clotting Enzymes

An assay technique for quantifying milk-clotting enzymes is described. By the technique, enzyme concentrations as small as 2×10−5 rennet units/ml can be determined accurately in milk solutions in as little as 2h. The technique can be used to assay for enzymes in milk and whey solutions without prior removal of casein. The technique should find application in detecting enzymes in a variety of systems commonly encountered in studies of milk and cheese products.

Isolation of bovine milk fat globule membrane material from cream without prior removal of caseins and whey proteins

SummaryBovine milk fat globule membrane (MFGM) material was isolated from cream by a new technique which did not involve removal of caseins and whey proteins before destabilization of the fat globules. These components were removed by centrifugation of the membrane material extract through a concentrated sucrose solution (52·5% (w/v) sucrose in 10 mM-Tris HCl buffer, pH 7·5). Membranes collected at the sample–sucrose interface, while whey proteins remained in the supernatant and caseins migrated into the concentrated sucrose solution. The yield of membrane material using this procedure was 25% less than that from conventional methods. This reduced yield was due mainly to lower levels of lipid material, in particular triglyceride. Electrophoretic analysis showed that the polypeptide composition of ‘unwashed’ membrane material was similar to that of conventionally prepared MFGM. This method is particularly suitable for the isolation of membrane material from milks and dairy products in which the fat globule stability is reduced or unknown.

Single Radial Immunodiffusion Analysis for Quantitation of Colostral Immunoglobulin Concentration

Relative accuracy of the single radial immunodiffusion technique to measure immunoglobulin concentration of colostral preparations (whey, whole, or fat-free) has been assesed. Fresh colostrum samples were analyzed for major constituents. Gammaglobulin as a standard was compared to total immunoglobulin concentration derived from single radial immundiffusion analysis of colostral preparations with no differences except between standard and whey. Differences were in part from either enhancement or interference of immunoglobulin diffusion by colostral constituents. Removal of casein and fat during whey preparation caused a concentrating effect upon immunoglobulin constituents resulting in exaggerated precipitin rings. Whey has produced unreliable results; therefore, whole colostrum is recommended for single radial immunodiffusion analysis.

Purification and Properties of Galactosyltransferase from Human Colostrum

The galactosyltransferase (Uridine diphosphate-D-galactose: D-glucose 1-galactosyltransferase EC 2.4.1.22) was purified from human colostrum by chromatography on DEAE-cellulose, cellulose phosphate, Sephadex G-100, and hydroxylapatite after removal of caseins by centrifugation. The final preparation showed two forms of protein on polyacrylamide disc gel electrophoresis, and both of them exhibited galactosyltransferase activity. The molecular weights of the two forms of the protein were estimated as 44,000 to 45,000 and 55,000 to 57,000 by polyacrylamide disc gel electrophoresis containing sodium dodecyl sulfate. General properties of galactosyltransferase were investigated.

Galactothermin, a reversibly heat-precipitable protein of human milk at neutral pH.

1. A protein, aggregating at body temperature and solubilizing when cooled, was isolated from fresh human milk at neutral pH and studied for some of its physical, chemical and immunological properties. The name ;galactothermin' is proposed for this protein. 2. Isolation and purification of galactothermin involved casein removal from skim milk at pH4.64 followed by centrifugal fractionation of residual protein-containing solutions repeatedly heated and cooled between 40 degrees C and 0 degrees C at pH7.3. 3. The molecular weight by ultracentrifugal analysis and the minimum molecular weight by sum of amino acid residues were 11400 and 14000 respectively. The sedimentation coefficient s(25,w) was 1.05S and the diffusion coefficient was 7.15x10(-7). Reversible aggregation is favoured by increase in protein concentration, ionic strength, temperature, time and approach to the isoionic point of 7.27 from either acidic or alkaline conditions. 4. Among the amino acid residues, proline predominates and non-polar species account for two-thirds of the total. Cysteine and cystine are absent. Analysis of galactothermin showed it to be essentially free of hexose, sialic acid, calcium and phosphate. 5. Galactothermin is antigenic in the rabbit as evidenced by the passive cutaneous anaphylaxis test. Single precipitin lines are produced in immunodiffusion tests. 6. By electrophoresis in polyacrylamide gel at pH4.0 only one sharp band is produced.