The adsorption of chymosin and lysozyme onto emulsion droplets and their association with casein

Abstract

The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free amino acids, which act as flavour precursors. The ripening of cheese under conditioned storage is time consuming and costly. Addition of specific enzymes to the cheese milk is one of several options to accelerate ripening. A major problem then is that hardly any of these proteases end up in the cheese and most disappear with the whey stream. Entrapment of bacteria and milk fat globules into the casein matrix of the curd is due to their particle character. Immobilisation of proteases onto particles would thus result in retention of these proteins in the curd. Ideally, for reasons of acceptance, these particles should originate from the milk itself or at least be edible.In this study, in a more general approach, soya oil emulsion droplets and casein micelles, being protein aggregates, were tested as the carrier system. Chymosin and lysozyme were taken as the enzymes to be immobilized, because of their relevance to the dairy industry and because they are scientifically well-known. Moreover, their biochemical divergence make them suitable models for study.The literature provides several studies on adsorption of proteins onto interfaces. Few of these proteins are enzymes. In cases where lysozyme was studied, it was included because of its extraordinary properties as a protein and not because of its enzymatic activity. Apart from (phospho)lipases there is scarcely any literature that describes activity of enzymes adsorbed onto the oil/water or air/water interfaces. Proteins tend strongly to accumulate in interfaces and for that reason are said to be very surface active. This adsorption is accompanied by a conformational change of the three-dimensional structure of the protein that results in some unfolding, ranging from almost full stretching of the peptide chain to a more conserved conformation. Hydrophobic residues or patches of the protein, mostly buried inside the molecule, will tend to position themselves next to or even protrude partly into, the hydrophobic phase of an oil/water interface.The extent of conformational change depends on the conformational stability of the protein, which, in turn, depends on pH, temperature, ionic strength etc. Furthermore, the extent of unfolding will be dependent on the surface area available and on the time scale, and hence, on protein concentration. In a static condition, adsorption at the interface will be diffusion driven, whereas during emulsification the time of adsorption will be determined by convection and will be very much shorter. Consequently, conformational changes of proteins during emulsification should be smaller because full surface coverage may be reached before unfolding can occur. In the case of enzymes being adsorbed the emulsification process would therefore offer an opportunity to retain activity.The relation between the surface pressureΠi.e. the extent of surface tension decrease and the amount of protein adsorbed per unit surface area availableΓ, provides a possibility to relate the size of the adsorbed protein molecules, and thereby the extent of unfolding, to the surface load. As mentioned earlier the extent of unfolding should be less at a higherΓvalue. It has been calculated that lysozyme, an enzyme of high conformational stability, hardly unfolds at the air/water interface, even at low surface coverage. For the oil/water interface, however, a considerable increase in the radius of the protein molecules was observed at low surface load. At surface coverage of &gt; 1.5 mg.m -2, the radius remained more or less constant, indicating that substantial unfolding did not occur. Despite this rigidity, the enzyme had lost all of its enzymatic activity in situ and it even remained inactive after desorption.Apparently, conformational changes in the enzyme molecule do not necessarily become manifest in a larger size for the molecule. Chymosin, being an enzyme of smaller conformational stability, naturally lost all of its activity due to adsorption onto the oil/water interface. In experiments with the enzymes coadsorbed simultaneously with bovine serum albumin, or the one after the other, there was no retention of in situ activity. Chymosin also proved to be inactivated at the expanding air/water interface due to air incorporation, if this occurred e.g. during homogenization.During the cheese-making process enzymes like chymosin and lysozyme are retained in the curd. This retention must be due to association of the enzymes with the casein from milk. In order to adsorb the enzymes with retention of activity, the various casein fractions were used to make and stabilize a soya-oil emulsion, and the enzyme was subsequently allowed to associate with the casein. The extent of association of lysozyme with the casein fractions was in the orderα s -casein &gt;β-casein &gt;κ-casein. Only for theκ-casein stabilized emulsion, was lysozyme association dependent on pH within the range of pH 5.2 - 6.4 (greater for a lower pH). Furthermore, the association with the caseins was not dependent on temperature, indicating that hydrophobic interactions were not predominant. The same trends were found with the various caseins in solution, albeit that association withκ-casein hardly occurred. It should be kept in mind that casein adsorbed at an interface will expose other amino acid residues compared to its behaviour when free in solution. For that reason the association behaviour in the two systems may differ.Because the association varies between caseins the extent of association with lysozyme depended on the composition of the casein micelles (aggregates of many casein molecules and calcium phosphate, as occurring especially in milk). As expected, casein micelles containing a higher proportion ofκ-casein associated less with lysozyme. It was found that lysozyme did not lose activity due to association with casein adsorbed on soya oil droplets or free in solution. However, lysozyme activity was markedly reduced when the enzyme was associated with casein micelles. In this system lysozyme also associated with casein in the interior of the casein micelle. The apparent loss of activity was most probably due to internal diffusion limitation. The difference of association for the various systems was also reflected in the free equilibrium concentration at which the surface excess plateau value was reached. In the system of adsorbed caseins this value was reached at a free lysozyme concentration of about 3 _M, whereas for the micellar system this value was about 100 times higher.The association of chymosin with casein has been studied in the same three systems of casein adsorbed onto soya-oil emulsion droplets, caseins in solution and caseins aggregated in casein micelles. It appears that chymosin only associated with adsorbedκ-casein and not with adsorbedαs- orβ-casein. Preceding the association, the caseinomacropeptide part ofκ-casein is split off, followed immediately by the aggregation of the soya-oil emulsion droplets containing the remaining para-κ-casein. This coagulation behaviour is identical to the renneting of milk during the cheese-making process. The association characteristics for chymosin are also comparable. The association was strongly dependent on pH and ionic strength, and on chymosin and casein concentration. Theκ-casein stabilized emulsion has proven to be a good model system for studying chymosin retention in curd. The chymosin associated with para-κ-casein was shown to be still active on addedκ-casein or on a fluorescent small hexapeptide substrate. Consequently, the active centre of the enzyme is presumably not involved in the association with casein.The association of chymosin with caseins free in solution has also been studied. Only in a solution containingκ-casein will addition of chymosin result in protein flocculation and precipitation. This flocculation is due to splitting off the caseino-macropeptide part ofκ-casein and the consecutive aggregation of the fairly hydrophobic and almost electrically neutral para-κ-casein molecules. The precipitated protein fraction also contains associated chymosin, to an extent depending on conditions like pH, ionic strength and casein and chymosin concentrations. In this system time and temperature also affected the extent of chymosin association. The association decreased with increased contact time and was stronger at higher temperatures.The protein content in the supernatant after centrifugation increased not only due to dissociation of chymosin but also due to the presence of casein fragments. Apparently, the dissociation of chymosin was related to its proteolytic action. The dissociation rate increased with decreasing pH where chymosin becomes more active and less specific. The dissociation also increased with temperature for a given time of contact. However, when extrapolated to a contact time of t = 0 (i.e. when dissociation due to proteolysis has not occurred yet) the association was observed to be somewhat stronger for a higher temperature. The effect of temperature on the proteolysis-dependent dissociation, apparently was stronger than its effect on the increase of the association. Since chymosin association depends on mutual association of caseins (see below), it will also depend on the temperature dependence of the latter. Dissociation of chymosin was not found in the system of caseins adsorbed onto emulsion droplets.The addition of small amounts ofαs- orβ-casein strongly decreased the extent of association of chymosin with para-κ-casein. This effect was stronger forαs-casein than forβ-casein. It was also found that the extent of chymosin association (moles of chymosin per mole of para-κ-casein) was larger when the system was diluted or, in other words, when the casein concentration was reduced. Both phenomena can be explained by assuming that competitive association occurs between the caseins and chymosin for interaction with a para-κ-casein molecule. Chymosin is only able to associate with a para-κ-casein molecule when that is not associated with other casein molecules. Thermodynamically speaking, the extent of association of chymosin is determined by the association constants that exist between all caseins under conditions as in the system. These association constants vary with pH, ionic strength, casein concentration and temperature.The model of competitive association is further developed and applied to the association of chymosin with casein micelles of various composition. It follows that chymosin will associate less with casein micelles composed ofαs- andκ-casein than with micelles composed ofβ- andκ-caseins. Again, this behaviour can be explained by competitive association, since different association constants exist for the caseins and chymosin for association with para-κ-casein. The relations for association and dissociation found in this casein micelle system are comparable with those found with caseins in solution. The kinetic model for competitive association is only a crude approximation. It does not provide possibilities of calculating all the association constants occurring in milk from the relations found from retention of chymosin in curd.</p