Jeffrey Leaver

Lactosylation of milk proteins during the manufacture and storage of skim milk powders

Abstract Extensive lactosylation of milk proteins in standard skim milk powder dried against air between 185 and 90°C (inlet and outlet temperatures of the air) was detected by capillary electrophoresis. Optimisation of the drying conditions included keeping the outlet temperature low (preferably

The possible conformations of milk proteins adsorbed on oilwater interfaces

Abstract The dimensions of the milk proteins β-casein and β-lactoglobulin when adsorbed to polystyrene latices have been measured. In addition, the effect of one protein on the other in mixed adsorption systems has been measured. These studies were used as baselines to study the effect of the proteolytic enzyme, trypsin, on the adsorbed β-casein. When trypsin attacked the casein, the diameter of the latex casein complex decreased by about 20 nm. An effect of similar dimension was observed when trypsin was used to attack the β-casein in an emulsion prepared using soya oil and β-casein, indicating that the conformation of the protein in the emulsion was similar to that in the model latex system. The results on the changes in the thickness of the protein layer surrounding the latex or the emulsion can be correlated with observations of the peptides produced by the trypsin action. The decrease in the thickness of the protein layer appears to be associated with the breaking of one of two bonds in the adsorbed β-casein, which allows a possible structure of the protein on the interface to be defined.

The topography of bovine β-casein at an oil/water interface as determined from the kinetics of trypsin-catalysed hydrolysis

The topography of bovine beta-casein at a soya oil/water interface was studied by following the kinetics of the trypsin-catalysed hydrolysis. Tryptic peptides were identified from their amino acid compositions and the kinetics were compared with those obtained from beta-casein in solution. Whereas soluble beta-casein was initially hydrolysed at a number of trypsin-sensitive bonds, the hydrolysis of the protein at the interface was a more ordered event. The crucial initiating step was the cleavage of the N-terminal peptides 1-25 and 1-28 from the molecule. Hydrolysis at other trypsin-sensitive sites could then occur. This suggests that with the exception of the large hydrophilic moiety in the N-terminal region, most of the beta-casein molecule is inaccessible to the proteinase, and lies fairly flat on the oil/water interface. After removal of the N-terminal peptide, the remaining macropeptide can reorientate and other hydrophilic regions become accessible to the proteinase.

Application of a mass spectrometry sequencing technique for identifying peptides present in Cheddar cheese

Abstract Peptides were isolated from a water-soluble fraction of a commercial mature Cheddar cheese by reversed phase-high performance liquid chromatography. Characterisation of 16 peptides was performed using a matrix assisted laser desorption/ionisation time-of-flight mass spectrometer, by determining their masses and applying N-terminal sequencing using a volatile degradation reagent, trifluoroethylisothiocyanate. The experimental approach allowed the sequencing of overlapping peptide mixtures. Of the 16 peptides identified, eight came from αs1-casein, seven from β-casein and one from αs2-casein.

Variations in the binding of β-casein to oil—water interfaces detected by trypsin-catalysed hydrolysis

Abstract Trypsin-catalyzed proteolysis has been used to investigate the influence of the oil phase on the topography of interfacial β-casein in soya oil-water and tetradecane—water emulsions. In both emulsions, the initial step in the hydrolysis is the removal of the N-terminal peptide(s). The rate of this reaction is considerably faster than that of the formation of the C-terminal peptide (three-fold in soya-oil, five-fold in tetradecane), indicating that the charged N-terminal region is more accessible to the protease. As with β-casein in solution, certain of the trypsin-sensitive bonds are not readily hydrolyzed at the tetradecane—water interface. However, these bonds are hydrolyzed at the soya oil interface, suggesting that the protein possesses different conformations in the two emulsions. Surface area coverage measurements confirm that the protein covers a larger surface area on the triglyceride. The effect of the nature of the oil phase on the structure of the adsorbed protein is discussed with reference to the relative hydrophobicities of the oils.

Displacement of native and thiolated β-casein from oil—water interfaces—effect of heating, ageing and oil phase

Tetradecane- and soya oil-in-water emulsions stabilized by native and chemically thiolated β-casein were prepared using a microfluidizer. Displacement of adsorbed protein by addition of the non-ionic detergent Tween 20 after 2 and 28 days storage at 20°C, and before and after heating to 80°C for 30 min, was determined colorimetrically. Adsorbed protein was displaced more readily from the 2-day-old tetradecane emulsion than from the soya oil. Heating reduced displacement from the soya oil emulsion, but not from the tetradecane. Thiolated protein was displaced less readily than native β-casein. Storage decreased displacement of both forms of β-casein from the soya oil emulsions and heating had very little effect on the stability of the adsorbed protein in these aged emulsions.

Dimensions and Possible Structures of Milk Proteins at Oil–Water Interfaces

Publisher Summary The determination of the dimension of proteins at interfaces may allow some indications of their conformations in the adsorbed state. Thus, albumin adsorbed on a polystyrene latex appears to occupy space a little larger than its dimensions in solution, but as1- and β-caseins appear to protrude from the interface to a considerable distance, forming a layer between 10 and 15 nm in thickness around the latex particle. When considering the thicknesses of adsorbed protein layers, it is not possible to observe the build-up of such layers in emulsions as it is in the latex model systems. Nevertheless, it is possible to gain some information on the relative behavior of emulsion and latex systems by studying their behavior when the surface layer is being destroyed, for instance, by the attack of proteolytic enzymes on the adsorbed protein. It is possible to follow the proteolytic reaction chemically so that in some cases, the particular changes that occur in the layer of adsorbed protein may be defined in detail. This chapter presents a paper that deals with attempts to describe three aspects of the behavior of adsorbed protein: (i) the effect of one protein on another when they are adsorbed at a latex surface, as defined by the thickness of the adsorbed layer; (ii) a comparison between a model system based on polystyrene latex and a β-casein stabilized oil-in-water emulsion in terms of the thickness of the adsorbed protein layer; and (iii) the relationship between the specific reactions of the protease trypsin and the possible conformation of β-casein molecules at an oil-water interface.

Influence of heating regime and pH on the primary phase of renneting of whole milk

Abstract The rate and extent of the chymosin-catalysed hydrolysis of κ-casein, together with the extent of denaturation of individual and total whey proteins, has been determined in whole milk heated on a pilot plant-scale apparatus for periods of 15 s to 5 min at temperatures between 72 and 140 °C. Denaturation of about 10% of the total whey protein resulted in partial inhibition of the reaction. Additional denaturation (up to about 60% of the total whey proteins) had no further effect on the reaction until the heating conditions were sufficiently severe to cause chemical damage to the casein(s). Inhibition of the chymosin-catalysed hydrolysis milk heated at pH values between 6.2 and 8.5 and subsequently hydrolysed at pH 6.7, was greater at pH values above about 7.0. This may be due to the solubilization of micellar κ-casein which has been reported to occur under these conditions.

Chromatographic elution profiles, electrophoretic properties and free amino and sulphydryl group contents of commercial sodium caseinates

Abstract The proteins in commercial sodium caseinates and laboratory-prepared, unheated sodium caseinate were studied using anion and cation exchange FPLC, gel permeation FPLC and alkaline urea-PAGE, and free amino and sulphydryl groups were analysed. Anion and cation exchange FPLC profiles showed that the charged residues of constituent proteins in the caseinates were modified to different extents. Commercial caseinates showed an extra peak (pre-αs1-casein) on cation exchange FPLC, which eluted at a lower salt concentration than that required to elute αs1-casein; there was little pre-αs1-casein in the laboratory-prepared caseinate. Gel permeation FPLC showed that the caseinates contained different levels of high molecular weight proteins which were present at very low levels in the laboratory-prepared caseinate. Alkaline urea-PAGE gave good resolution of all proteins in the laboratory-prepared caseinate while in the commercial caseinate samples, protein bands were smeared and αs2-casein was less pronounced. The laboratory-prepared caseinate had a higher content of free amino and sulphydryl groups than the commercial caseinates.

Interactions of proteins and surfactants at oil-water interfaces : influence of a variety of physical parameters on the behaviour of milk proteins

Displacement of adsorbed β-casein from polystyrene latex and of caseinate from heated and unheated soya oil and tetradecane oil-in-water emulsions by the non-ionic detergent, Tween 20, has been studied. Protein was displaced more readily from the tetradecane emulsion than from the corresponding soya oil. Heating decreased the amount of protein displaced from the soya oil emulsion but had little effect on displacement from tetradecane. Ageing decreased the amount of protein displaced from the soya oil emulsion, but the effects of heating were less pronounced in aged soya oil emulsions. The effect of pH on protein displacement from fresh and aged caseinate-stabilised soya oil emulsions was also investigated. Protein was displaced more readily at higher pH and, as with the β-casein-stabilised soya oil emulsions, ageing decreased the displacement of adsorbed protein.