The rennet-induced clotting of para-kappa-casein revisited: inhibition experiments with pepstatin A.

The proteolysis of micellar kappa-casein by rennet was followed by SDS-polyacrylamide gel-electrophoresis and the clotting of the para-kappa-casein formed by absorbance measurements. Up to a degree of proteolysis of about 0.4 the enzyme-inhibitor pepstatin A proved able to instantaneously stop the clotting. This effect is explained by the rapid condensation of monofunctional, monomeric and polymeric particles of para-kappa-casein. At higher degrees of proteolysis pepstatin was no longer able to completely block the polymerization. This is explained by the retardation of the condensation of the monofunctionals as their size grows larger. A kinetic analysis of the enzyme-controlled stage of the clotting process predicts that the system should gel at an early degree of proteolysis of about 0.07. The actual gel points occur at considerably higher degrees of proteolysis. This suggests that the enzymic attack of the polymeric inert kappa-casein particles is not completely at random. Primary micelles of kappa-casein, however, are degraded by random attack rather than by a 'catch-and-razor' mechanism.

The influence of temperature on the flocculation rate of renneted casein micelles.

The flocculation rate constant of completely renneted casein micelles in milk ultrafiltrate was measured by Rayleigh light scattering between 20 and 35 degrees C. In this temperature range an apparent energy of activation of 103 kJ mol (+/-11 kJ mol : n = 50) was measured. At 15 degrees C clotting was not longer perceptible. The activation of the flocculation between 20 and 35 degrees C is explained not so much by the height of the energy barrier separating the clotting micelles, as by the very negative temperature coefficient of that barrier. In line with this conclusion it is suggested that renneted micelles adhere through hydrophobic bonding. The flocculation rate constant of renneted casein micelles is independent of micelle size at the four temperature levels studied.

Stable and unstable casein micelles.

In milk, casein occurs as colloidal particles with an average size of about 100 nm. These are stabilized against flocculation by an outer layer of several thousands of kappa-casein molecules. Stability of micelles is characterized by the magnitude of the Smoluchowskian flocculation rate constant, which during the renneting of milk nearly approaches the diffusion-controlled limit. The processes of the clotting of milk by rennet and the phenomena of age-thinning and age-thickening of ultra-high temperature-sterilized, concentrated milks bear interesting kinetic resemblances. Both processes are characterized by a lag phase during which viscosity decreases, followed by an explosive increase in viscosity. In the milk-clotting process, the decrease can be explained by the proteolytic action of the renneting enzyme. This strongly suggests that age-thinning and age-thickening are also caused by the action of a protease that survived the sterilization process. A quantitative check of this theory is difficult because of the apparently small amount of enzyme.

The coagulation of differently sized casein micelles by rennet.

Fractions of bovine casein micelles of different sizes were prepared by successive centrifugation steps, and dilute suspensions of the different fractions were reacted by rennet. The molecular weight increase with time after addition of rennet was measured by light scattering. After an initial lag stage, where the increase was zero, and a short intermediate stage, the molecular weight became linearly proportional to time, indicating complete conversion of the kappa-casein substrate by the enzyme. The slope of the final portion of the growth profile can be used to define values of the Smoluchowski rate constant governing the aggregation. No significant differences in this constant were found with micelles of different sizes, and the value of the rate constant suggested that the aggregation reaction of completely renneted micelles is only moderately retarded relative to the diffusion-controlled process, even at room temperature.

On enzymic clotting processes V. rate equations for the case of arbitrary rate of production of the clotting species.

The rate theory for enzyme-triggered coagulation reactions, such as the clotting of fibrin or casein, is extended to the case of an arbitrary rate of production of the clotting species. It is shown that the general expression for the growth of the weight-average molecular weight of the clotting product, MW, is given by MW = M1 [1 + ks[integral of 0tP(t)2dt]/P(t)], where M1 is the "monomer" molecular weigt, ks the smoluchowskian flocculation rate constant and P(t) the total number of monomers produced by the enzyme in t. In the purely smoluchowskian case P(t) stands for the total number of monomers at the beginning of the clotting process. Numerical examples in which the rate of enzymic production is governed by complete Michaelis-Menten kinetics, are compared to cases in which this rate equals Vmax. It is shown that after exhaustion of the substrate the system continues to coagulate in a purely smoluchowskian way. Turbidimetric experiments on the clotting of micelles of whole and kappa-casein are presented which suggest inactivation of the enzyme by non-productive binding in the flocs formed.

Casein micelles: the colloid-chemical approach.

The colloidal properties of micellar casein are reviewed. It is shown that behaviour of intact micelles is much at variance with the predictions from the Schulze-Hardy rule, and that therefore their stability cannot be explained by the principles of the DLVO theory. Towards electrolyte, micelles behave as a protein rather than a lyophobic colloid. Casein is a strong protective colloid. In the micelle, however, it does not completely cover the inorganic constituent which remains sensitive to changes in the ionic environment. The rate theory of the enzyme-induced clotting of casein micelles is summarized. It is shown that the lag phase in the clotting is due to the second order of the co-agulation reaction. Flocculation rate constants of micelles have been deduced from clotting times. Their relatively low values can be attributed to an orientational constraint. Practical consequences of the theory with respect to clot structure, gelation of sterilized products and cheese manufacture are discussed.

On enzymatic clotting processes. I. Kinetics of enzyme-triggered coagulation reactions.

The kinetics of enzymatic clotting reactions such as the clotting of blood or milk, is analyzed. The appearance of a lag phase in the clotting is shown to be due to the difference in reaction order of enzymatic production and flocculation. The weight-average particle weight of the product formed is found to be a quadratic function of the reaction time. The condition for the clotting time is t square root of ksV/2 = C, where t is the clotting time, ks the flocculation rate constant, V the maximum rate of enzymatic product formation and C a constant. In agreement with this result double-logarithmic plots of t versus enzyme dilution are always observed to be linear over a wide range of enzyme concentrations. No statistical evidence could be produced for the widely held belief that the clotting time should be inversely proportional to the enzyme concentration. Varying exponents of the latter in its relation to the clotting time are discussed in terms of von Smoluchowski's theory of the slow coagulation of colloidal particles.

On the sol-gel transition insolutions of kappa-carrageenan.

The disorder-order transition, which takes place at the gelpoint of k-carrageenan solutions was monitored by optical rotation and light scattering measurements. The coincidence of both sets of experimental data affords good evidence that the sol-gel transition is accompaned by a conformational change. Transition temperatures were observed to be linearly dependent on the logarthm of the salt concentration and this result is explained by the formation of double helices. Heats of gelation were measured by differential scanning calorimetry. It was found that the enthalpy increases with ionic strength, which was ascribed to the occurrence of a secondary process in which double helices are assembled into larger aggregates.