Colloidal gels from proteins and peptides - Physical Gels from Biological and Synthetic Polymers - page 266

κ -casein, the glycomacropeptide (commonly abbreviated GMP), mainly located at the

outside of the casein micelles, protrudes from the casein micelle surface into the
solution. This is the species that helps stabilize the casein micelles by the steric
mechanism (Walstra, 1999 ).
Flocculation of casein micelles and subsequent gel formation can then be induced
either at neutral pH and temperatures above 20°C by the addition of a proteolytic enzyme
(rennet), which acts speci

cally to cleave off the glycomacropeptides, or by lowering the

pH. After acidi cation of a milk dispersion to pH 4.6 (the isoelectric pH of the casein

particles), a physically stable suspension of casein particles is produced at low temper-
atures. Gelation results from the aggregation of these casein particles at temperatures
above 10°C, but these particles have a complex structure due to the association of
numerous different casein molecules.

9.3.2 Gelation kinetics

κ -casein is a substrate for chymosin and plays the most important role in the gelation of

casein micelles. The substrate speci

city of chymosin is very high, and it attacks only one

speci -casein. Gel formation of casein micelles consists of three steps: (i)

cleavage induced by chymosin upon the

c bond in κ

κ

-casein within micelles, (ii) aggregation of the

κ

destroyed -caseins and (iii) consolidation by the aggregation of micelles. The various

stages are identi ed by turbidity, and by yield stress measurements when the gel forms

(Walstra et al., 1985 ). Of these stages, only the

first step, the proteolytic cleavage, can be
monitored totally independently of the others by following the release of the glycoma-
cropeptide, or the formation of para-

-casein. The aggregation reaction of the destabi-
lized micelles is a consequence of this proteolysis, but its rate cannot be separated easily
from that of the proteolysis reaction, and aggregation overlaps the proteolysis reaction
( Figure 9.6 ; Horne and Banks, 2004 ).
The early aggregation phase can be followed readily by turbidity or light scattering
(Payens et al., 1977 ; Dalgleish et al., 1981 ), whereas gel formation and development is
most easily monitored in the laboratory by rheometry (Tokita et al., 1982 ).
The shear modulus appears suddenly at a so-called latent time and increases with
reaction time. This latent time increases with decreasing enzyme concentration, while the
curvature of the gelation curve, which re

κ

ects the rate constant of the gelation process,
depends on the concentration of enzyme. However, gelation curves seem to converge to
the same value of the shear modulus at an in

final value of the
elastic modulus G of a casein gel is independent of enzyme concentration. Niki and
co-workers (Niki et al., 1994a , 1994b ) prepared casein micelles of various sizes from
skim milk using differential centrifugation, and the observed gelation curve as a function
of time, after rennet was added to the casein micelle solutions, was apparently well
approximated as a

nite reaction time, i.e. the

first-order reaction.
Measurement of non-protein nitrogen (NPN) liberated from casein micelles has been
conventionally used to follow the progress of the enzymatic phase of milk-clotting.
Hooydonk and Olieman ( 1982 ) estimated the amount of the glycomacropeptide (GMP)
using an HPLC system, and measured the extent of enzymatic reaction after the addition