Investigation of the enzymatic coagulation of casein micelles

Kenneth Walter Ruettimann, Purdue University

Abstract

The enzymatic coagulation of casein micelles is a key step in the cheesemaking process. In this reaction, a enzyme hydrolyzes the $\kappa$-casein which resides on the surface of the protein agglomerates known as casein micelles. The micelles are destabilized and coagulate, eventually forming a gel which undergoes further processing. In order to design more efficient processes based on sound engineering principles, the details of the enzymatic coagulation reaction must be known. The hydrolysis kinetics were studied using gel electrophoresis to follow the formation of para-$\kappa$-casein and the turbidimetric behavior of hydrolyzed milk was studied using stopped-flow turbidity. First order rate constants were determined and related to the turbidimetric response using a simple clotting assay. It was found that the micelles do not coagulate until approximately 90% hydrolysis. Micelles prepared from reconstituted skim milk were characterized using photon correlation spectroscopy and laser doppler electrophoresis and were found to have properties similar to micelles obtained from fresh milk. The results of the measurement of zeta potentials during enzymatic hydrolysis supported the hairy micelle structural model. Dark field microscopy was developed as a novel technique for observing the casein micelle coagulation reaction, in-situ, allowing micelle interactions and the process of floc assembly to be directly visually observed. Dark field observations were useful in determining the coagulation behavior of the micelles under various environmental conditions. The dark field method was extended using image analysis for quantitative analysis of casein micelle floc structures. The fractal dimensions of the micelle flocs, determined from image analysis measurements, were found to he most sensitive to changes in the enzyme concentration. Computer simulations of the coagulation reaction were developed and explored. A coagulation model based on the time-dependent development of active surface sites resulted in three reaction regimes: diffusion-limited aggregation, site-directed aggregation, and reaction-limited aggregation. The change in the fractal dimension of the computer models was consistent with experimental determinations.

Degree

Ph.D.

Advisors

Tsao, Purdue University.

Subject Area

Chemical engineering|Food science

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