Quantification of cow’s milk percentage in dairy products – a myth?

The substitution of ewe's and goat's milk for cheaper cow's milk is still a fraudulent practice in the dairy industry. Moreover, soy-based products (e.g., soy milk, yoghurt) have to be checked for cow's milk as they are an alternative for people suffering from an allergy against bovine milk proteins. This work reports the evaluation of different protein-based electrophoretic methods and DNA-based techniques for the qualitative detection as well as the quantitative determination of cow's milk percentage in dairy and soy milk products. Isoelectric focusing (IEF) of γ-caseins using an optimized pH gradient was appropriate not only for the detection of cow's milk, but also for an estimation of cow's milk percentage in mixed-milk cheese varieties. Urea-polyacrylamide gel electrophoresis (PAGE) proved the method of choice to detect cow's milk in soy milk products, whereas IEF and SDS-PAGE of proteins were not applicable due to false-positive results. Polymerase chain reaction (PCR) analysis was used to confirm the results of protein-based electrophoretic methods. Problems inherent in quantitative analysis of cow's milk percentage using protein-based techniques and even more using DNA-based methods were emphasized. Applicability of quantitative real-time PCR for the determination of cow's milk percentage in mixed-milk cheese was shown to be hampered by several factors (e.g., somatic cell count of milk; technological parameters influencing the final DNA concentration in ripened commercial cheese samples). The implementation of certified reference standards (of major relevant cheese groups) containing 50% cow's milk was urgently recommended to enable at least a yes/no decision in commercial mixed-milk cheese samples.     

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.     

Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.     

Rehydratable agarose gels: application to isoelectric focusing in 9 molar urea

A method is described for the preparation of rehydratable agarose gels, with specific application to the direct incorporation of 9 M urea and carrier ampholytes into rehydratable agarose gels for use in isoelectric focusing. After drying the agarose gel containing an uncharged linear polyacrylamide, one gel volume of a 9 M urea-carrier ampholyte solution is absorbed directly into the gel in 60 min, eliminating equilibration or dialysis of the gel in larger volumes of the 9 M urea-carrier ampholyte solution. Proteins with a molecular mass of 970,000 Da can be separated by isoelectric focusing in these rehydratable gels. The focused proteins can then be quantitatively transferred to nitrocellulose in less than 10 min, and any immunostaining procedure can be used to probe the blotted proteins. These agarose gels are easy to make, they rehydrate rapidly and they can be used in applications other than isoelectric focusing.     

Application of Neuhoff's optimized Coomassie brilliant blue G-250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.     

Study on the Correlation between the Protein Profile of Lupin Milk and Its Cheese Production Compared with Cow’s Milk

Australian sweet lupin, the largest legume crop grown in Western Australia, is receiving global attention from the producers of new foods. To understand the effect of protein on cheese yield, lupin milk proteins were separated from the first, second, and third filtrations by cheesecloths. However, proteins from the first and second were analyzed using two-dimensional polyacrylamide gel electrophoresis; then, the isolated proteins associated with cheese production were identified. The research also focused on identifying the optimal method of cheese production based on the coagulation process, temperature, yield, and sensory evaluation. Lupin curds from the two cultivars, Mandelup and PBA Jurien, were produced using vinegar, lemon juice, starter culture, vegetable rennet enzyme as coagulant, as well as curd generated using starter culture and vegetable rennet enzyme. Cow’s milk was used as a control. The results indicated that first-time filtration produced better extraction and higher yield of lupin proteins and cheese than the second filtration. A sensory analysis indicated that lupin cheese produced from PBA Jurien lupin milk using vinegar, 7.80% expressed as acetic acid, and ground in 45 °C water, was the most acceptable. The cheeses were examined for their protein, carbohydrates, fat, ash, and moisture contents. The concentration of protein was approximately 27.3% and 20.6%, respectively, in the cheese from PBA Jurien and Mandelup. These results suggest that lupin milk can adequately supply the proteins needed in human diets and, thus, could be used in the production of many existing products that require animal milk as an input.     

Identification of adulteration in water buffalo mozzarella and in ewe cheese by using whey proteins as biomarkers and matrix-assisted laser desorption/ionization mass spectrometry

A rapid and accurate method to identify bovine and ewe milk adulteration of fresh water buffalo mozzarella cheese by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The differentiation among mozzarella made from water buffalo milk and from mixtures of less expensive bovine and, more recently, ewe milk with water buffalo milk is achieved using whey proteins, alpha-lactalbumin and beta-lactoglobulins as molecular markers. It is worth noting that the method proposed here is, to our knowledge, the first strategy able to characterize possible fraudulent additions of ewe milk in samples of water buffalo milk devoted to the production of water buffalo mozzarella cheese. In addition, a linear relationship was found between the relative response of the molecular ion and the abundance of the analysed whey proteins. This demonstrates that this approach can be used to determine the amount of bovine or ovine milk added to water buffalo milk employed for mozzarella cheese production. Furthermore, this method also appears suitable for the analysis of ewe cheese. Hence these findings open the way to a new field for mass spectrometry in the evaluation of possible fraudulence in dairy industry production.     

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.     

Qualitative and quantitative analysis of proteins and peptides in milk products by capillary electrophoresis

Milk protein is an important component of the human diet throughout much of the world. The ability to assess the relative composition and integrity of milk proteins or peptides in dairy foods or food ingredients is important because these molecules have a profound effect on product functionality and quality. This communication describes two capillary electrophoretic methods that are useful for the analysis of proteins and casein-derived peptides in cheese and milk products. One technique, which uses a buffer containing citrate/phosphate (pH 3.3), 4 M urea, and a polymeric additive in a coated capillary, is useful for qualitative and quantitative analysis of proteins and peptides in milk, cheese, and whey products. The second method employs a citrate/phosphate buffer (pH 2.8) and a bare silica capillary, and is well suited for the analysis of small, casein-derived peptides in aqueous cheese extracts.     

Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.     

Effect of Calcium Compound Type and Dosage on the Properties of Acid Rennet Goat’s Milk Gels

The aim of this study was to determine the effect of adding calcium compounds to processed goat’s milk, and on the properties of acid rennet goat’s milk gels, which are a middle product obtained in the manufacture of acid rennet cheese. The properties of the gels directly affect the quality of acid rennet cheeses. The analysis of raw goat’s milk was carried out, then acid rennet gels were produced with the addition of six different calcium compounds (chloride, citrate, bisglycinate, gluconate, lactate, and carbonate). The dynamics of milk fermentation were performed by monitoring the pH value of milk during acidification. The pH, syneresis, color, and texture profile were determined in the formulated acid rennet gels. An organoleptic evaluation was also performed. The study demonstrated that, not only calcium chloride, but also calcium citrate, gluconate, lactate, bisglycinate, and calcium carbonate could be used in the production of goat’s milk acid rennet gels, or the middle product in the manufacture of acid rennet curd cheese from goat’s milk. Notably, the addition of citrate, bisglycinate, and calcium carbonate in doses of 20 mg Ca 100 g⁻¹ most effectively reduced syneresis compared to the control sample by 4.76% (citrate), 7.85% (bisglycinate), and 10.28% (carbonate). The hardness of the control gels ranged from 2.35 N to 2.99 N. The addition of chloride, citrate, gluconate, lactate, and calcium carbonate to the milk improved the acid rennet gel’s hardness. The addition of 20 mg Ca 100 g⁻¹ as gluconate increased the hardness the most (3.61 N). When increasing the calcium dosage in the form of all compounds, there was a tendency to increase the gel’s springiness. The addition of chloride, citrate, and bisglycinate to milk did not result in a darkening of the gel’s color. The addition of calcium compounds mostly reduced the intensity of goatish taste and odor. Calcium gluconate, in particular, reduced the goatish taste the most, a taste which is not always acceptable by the consumers.     

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.     

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.     

Flow-injection flame atomic absorption spectrometric determination of trace amounts of cadmium in solid and semisolid milk products coupling a continuous ultrasound-assisted extraction system with the online preconcentration on a chelating aminomethylphosphoric acid resin

A new sensitive and low-cost method that combines continuous acid extraction, online preconcentration, and flame atomic absorption spectrometry for cadmium determination at microg levels in solid and semisolid milk products is described. A continuous ultrasound-assisted extraction system is used to carry out the dynamic acid extraction step. The acid extract is preconcentrated online on a minicolumn packed with a chelating resin (Chelite P, with aminomethylphosphoric acid groups), and the retained cadmium is eluted with hydrochloric acid and continuously monitored by flame atomic absorption spectrometry. An experimental design (Plackett-Burman 2-6*3/16) is used to optimize the continuous extraction and the preconcentration step. The method allowed a total sampling frequency of 28 samples/h. A good precision of the whole procedure (3.0% relative standard deviation) for a cheese sample containing 0.103 +/- 0.004 microg/g Cd (dry mass), a high enrichment factor (20.5), and detection and quantification limits of 0.014 and 0.067 microg/g, respectively, for a 60 mg of sample were obtained with this methodology. The method was successfully applied to the determination of trace amounts of cadmium in solid and semisolid milk products, such as cheese and yogurt samples.     

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.     

Polyethyleneglycol methacrylate 200 as an electrophoresis matrix in hydroorganic solvents.

The properties of gels composed of poly(polyethyleneglycol methacrylate) and copolymers of polyethyleneglycol methacrylate and acrylamide were studied. These novel electrophoresis matrices are amphigels (swellable in water and organic solvents) that have unprecedented organic solvent compatibility. Hydrophobic proteins which are poorly solubilized in aqueous detergent systems (e.g., zein) are well-resolved in these gels with hydro-organic solvents. This is especially relevant for isoelectric focusing, to avoid using either ionic detergents that may interfere with the focusing, or urea that may cause carbamylation of proteins. Variations of crosslinker, buffer, and solvent systems in these gels were explored.     

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.     

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

Cow and Ewe Cheeses Made with Saffron: Characterization of Bioactive Compounds and Their Antiproliferative Effect in Cervical Adenocarcinoma (HeLa) and Breast Cancer (MDA-MB-231) Cells

Saffron is a widespread consumed spice containing many phytochemicals. It is often used in dairy technologies to enhance color and flavor of cheeses, but it is also known for its several therapeutic effects, as well as its antiproliferative and anticancer properties. In this study High Performance Liquid Chromatography was used to characterize saffron bioactive compounds in cow and ewe cheeses made with saffron, and the antiproliferative effect of the crocin-rich extracts from cheeses was investigated on different cellular lines (CaCo2, MDA-MB-231 and HeLa) by MTT assay. Crocins were observed in all cheese samples, with the total content ranging between 0.54 and 30.57 mg trans-4-GG/100 g cheese, according to the different cheese making process. Picrocrocin was detected in no cheese (probably due to its degradation during cheese making), while safranal was detected only in one ewe cheese (mainly due to its high volatility). HeLa and MDA-MB-231 cells were sensitive to treatment with crocin-rich extracts from cheeses, while no effect was observed on CaCo2 cells. The chemical environment of the food matrix seems to have a great influence on the crocin antiproliferative effect: the crocin-rich extracts from cheese with both high residual N/protein and fat contents showed increased antiproliferative effect compared to pure crocin (trans-4-GG), but cheeses from different milk species (type of fats and proteins) could also play an important role in modulating crocin’s antiproliferative effects.     

A reliable two-dimensional gel electrophoresis procedure for separating neural proteins

A facile two-dimensional gel electrophoresis procedure has been developed for the analysis of neural tissue proteins which eliminates the serious problems associated with protein insolubility at the point of sample application onto polymerized first-dimension isoelectric focusing gels. This was accomplished by combining the methods of two previously published procedures. Our procedure provides an alternative method to the complex gel systems often employed for less soluble proteins, and yields very reproducible, high resolution separations. This procedure, which is in routine use in our laboratories for the analysis of total proteins extracted from retina and brain, produces protein patterns that are easily compared using both visual and computer-assisted image analysis techniques. Presented here are the results of a set of experiments designed to identify proteins unique to retina. This procedure should be useful to investigators studying protein changes resulting from genetic mutation, development, drug treatment or disease, in neural tissue as well as in virtually all other tissues.