ADSA Foundation Scholar Award
Formation and Physical Properties of Milk Protein Gels
J. A. Lucey1
Department of Food Science,
University of Wisconsin-Madison,
1605 Linden Drive, Madison, WI 53706
Address for correspondence:
Dr John A. Lucey
Department of Food Science,
University of Wisconsin-Madison,
1605 Linden Drive,
Madison, WI 53706-1565
Tel. No. + 1 608 265 1195
Fax No. + 1 608 262 6872
E-mail address: email@example.com
Gelation of milk proteins is the crucial first step in both cheese and yogurt
manufacture. Several types of milk gels are discussed with an emphasis on recent
developments in our understanding of how these gels are formed and some of their key
physical properties. Areas discussed include the latest dual-binding model for casein
micelles; some recent developments in rennet-induced gelation; review of the methods
that have been used to monitor milk coagulation; and a discussion of some of the possible
causes for the wheying-off defect in yogurts. Casein micelles are the primary building
blocks of casein-based gels, however, there is continuing controversy about its structure.
The latest model proposed for the formation of casein micelles is the dual-binding model
proposed by Horne, which suggests that casein micelles are formed as a result of two
binding mechanisms namely hydrophobic attraction and colloidal calcium phosphate
(CCP) bridging. Most previous models for the casein micelle have treated milk gelation
from the viewpoint of simple particle destabilization and aggregation but they have not
been able to explain several unusual rheological properties of milk gels. Although there
have been many techniques used to monitor the milk gelation process over the past few
decades, only a few appear attractive as possible in-vat coagulation sensors. Another
important aspect of milk gels is the defect in yogurts called wheying-off, which is the
appearance of whey on the gel surface. The factors responsible for its occurrence are still
unclear but they have been investigated in model acid gel systems.
(Key words: casein micelle, rheology, rennet coagulation, yogurt)
Abbreviation key: CCP = colloidal calcium phosphate, G* = complex modulus, GDL =
glucono-δ-lactone, NIR = near infrared, RCT = rennet coagulation time, G´ = storage
modulus, tan δ = loss tangent
Milk protein gels are irreversible, in contrast to many other food gels (Green,
1980). Milk gels are usually classified as particle gels although it is now recognized that
they are not simple particle gels as the internal structure of the casein particle plays an
important role in the rheological properties of milk gels (Horne, 1998). Milk gels have an
enormous economic importance as gelation is a critical first step in both cheesemaking
and yogurt manufacture, which are two very popular food products. Gelation can be
induced by enzyme (rennet) action, acidification and/or heat treatment of milk. In milk
gels, the overall visual appearance, microstructure and rheological properties are
important physical attributes, which contribute to the overall sensory perception and
functionality of these products. Natural cheeses are made by the use of rennet to
coagulate milk but starter culture is usually added first to produce acid for the entire
cheesemaking process. Subsequent acid production dramatically alters the rheological,
mechanical and textural properties of the initial rennet-induced gel (Lucey et al., 2000,
2001). The casein particles also undergo rearrangement, fusion and syneresis in the
process of forming cheese curd. Casein-based gels are inherently dynamic in nature and
the rearrangement processes involved in the syneresis of rennet-induced gels have been
studied extensively by the Wageningen group (van Dijk, 1982; van den Bijgaart, 1987;
van Vliet et al., 1991; Walstra, 1993; Mellema, 2000). However, many of the physio-
chemical events that transform the initial rennet-induced gel into cheese curd remain
The New Horne Model for the Structure of the Casein Micelle
The basic building blocks of milk gels (those based on casein) are the casein
micelles. Caseins constitute approximately 80% of the protein in bovine milk, with four
main types (αs1-, αs2-, β-, and κ-caseins) in combination with appreciable quantities of
micellar or colloidal calcium phosphate (CCP) nanoclusters in the form of aggregates
called casein micelles (Holt, 1992). At least three types of models for the structure of
casein micelles have been proposed; one type of model proposes that the micelle core is
divided into discrete subunits (submicelles) with distinctly different properties from an
outside “hairy” layer (Schmidt, 1982; Walstra, 1990); another model suggests that the
internal substructure resembles a mineralized, entangled or cross-linked web of chains of
casein molecules (Holt, 1992); the most recent model proposes a dual-binding
(polycondensation-type) mechanism for gel assembly (Horne, 1998).
Originally, it was assumed that any subunits were held together (“cemented”) in
the micelle solely by bridges of CCP (Schmidt, 1982) but it is now clear that a number of
factors are responsible for maintaining the integrity of micelles. The formation of CCP is
an effective means of “burying” or “storing” a considerable amount of Ca and phosphate
within casein micelles (which is critical for milk to achieve its primary role of providing
nutrition to the young calf). It is now recognized that CCP can be removed without
(apparently) disrupting the micelle if milk is acidified at temperatures < 20°C (Dalgleish
and Law, 1988; Lucey et al., 1997a). This suggests that CCP does not cement the micelle
together but rather it helps to control and modulate the effects of Ca and charged groups
on caseins. It is also clear that hydrophobic and hydrogen bonding are important for
micelle integrity since the addition of urea disrupts the micelle structure without having
to dissolve CCP (McGann and Fox, 1974). Evidence often used to support the
submicellar model includes electron microscopic images of the casein micelle having a
raspberry-like structure (Schmidt, 1982) as well as neutron and x-ray scattering
measurements that have been interpreted as indicating that there is an internal
(sub)structure within the micelle. As pointed out by Holt (1992) and Horne (1998), all
that in reality one can conclude from the various scattering studies is that, the internal
structure of the casein micelle is heterogeneous, composed of regions of high and low
scattering power. In addition, it has been suggested by Holt that the CCP nanoclusters
could be contributing to this variation in scattering intensity. Another major failing of the
earlier micelle models was their lack of a plausible mechanism for assembly, growth and
more importantly termination of growth (Horne, 1998). All such elements appear to be in
place in the new dual-binding model that was recently proposed by Horne (1998, 2002).
The dual-binding model for the assembly and structure of the casein micelle
proposed by Horne, is a polycondensation or polymerization model that envisages two
crosslinking routes for assembly of the micelle. These are cross-linking of individual
caseins through hydrophobic regions of the caseins and bridging involving CCP. The
formation (and integrity) of the micelle is viewed as being controlled by a balance
between attractive and repulsive forces in casein micelles, i.e., localized excess of
hydrophobic attraction over electrostatic repulsion. Another feature of the model is that
since κ-casein can only hydrophobically bond with other caseins (since it does not bind
much Ca as it lacks sufficient phosphoserine residues) its acts as a chain terminator. A
schematic representation of this model is shown in Figure 1. This model appears to
successfully accommodate the responses of casein micelles to changes in pH,
temperature, urea addition or removal of calcium phosphate by EDTA (Horne, 1998,
2002). The surface position of κ-casein can also be easily explained by its role as a chain
terminator, which also provides a plausible mechanism for controlling the growth of the
casein aggregate. This new model may also help us to understand some of the rheological
behaviour of milk gels and this aspect is discussed later in this article.
Rennet Coagulation and Rennet-Induced Gels
Rennet coagulation of milk may be divided into primary (enzymic hydrolysis) and
secondary (aggregation) stages although these stages normally overlap during
cheesemaking. During the primary stage, κ-casein is cleaved by rennet resulting in a
reduction in both the net negative charge and steric repulsion, such that rennet-altered
micelles become susceptible to aggregation (Zoon et al., 1988; Walstra, 1990). When
milk is clotted under normal conditions of pH and protein content, the viscosity does not
increase until the enzymatic phase is mostly complete, and it is approx. 60% of the
(visual) rennet coagulation time (RCT) (Green et al., 1978). Small linear chains of
micelles initially form and these continue to aggregate to form clumps, clusters and
eventually a system-spanning network.
The enzymatic stage is now well understood and there have been several excellent
reviews (e.g., Dalgleish, 1987; Hyslop, 2001). In contrast to the enzymatic phase of
rennet coagulation, there have been fewer studies on the rheological properties of the
rennet-induced gels. However, more attention has been paid to this important aspect in
the last 10-15 years (e.g., Zoon et al., 1998). Some of the important physical properties of
both rennet and acid-induced milk gels are shown in Table 1. Yogurt gels have higher
storage modulus (G´) values mainly because they are made from heated milk. Yogurt
gels have a lower fracture strain and higher levels of spontaneous whey separation.
Rennet-induced gels rapidly synerese if disturbed by cutting or by wetting the gel surface,
in contrast to most acid-induced gels (Walstra, 1993).
Fermentation-produced chymosin is widely used in the US (possibly > 90% of
coagulant used) and in many countries around the world. However, there has been recent
work on plant coagulants such as those extracted from the flowers of the Cynara plant
(Esteves et al., 2001a, b). These coagulants are widely used in artisan cheesemaking in
some countries like Portugal but there may be growing interest in plant-derived or
organically produced coagulants in the future. The coagulants extracted from Cynara
plants appear to have similar enzyme specificities compared with chymosin although they
are slightly more proteolytic. The process of rennet gel assembly and the detailed
rheological properties of the resultant gels were generally similar to that of chymosin
(Esteves et al., 2001a, b). The lack of commercial quantities of this (new) type of
coagulant will limit interest in assessing its cheesemaking properties (i.e., yield, flavor,
texture) in the US for the present time. It is still widely considered that chymosin is the
ideal coagulant for cheesemaking. With the great progress in biotechnology and genetic
engineering it may be possible one day to develop a coagulant that has some attributes
that were superior to chymosin (i.e. gave better yield, less propensity to produce bitter
peptides, sharper and faster cheese flavor development, etc).
As more groups are studying the rheological properties of milk gels, it is also
becoming more important to standardize the conditions used to disperse or re-hydrate the
milk powder, as low heat NDM is the most common substrate. Too short a time may
result in incomplete re-hydration of the milk while too long a holding time at a high
temperature may also allow plasmin hydrolysis of caseins (e.g., Esteves et al., 2001b). No
standard conditions have been agreed on for the rheology testing, in contrast to existing
standards for quantifying milk-clotting activity (IDF, 1997).
Methods for Monitoring Milk Coagulation
There have been several reviews that have discussed the advantages and
disadvantages of various instrument and devices that have been used to monitor milk
coagulation (Thomasow and Voss, 1977; van Hooydonk and van den Berg, 1988; Fox et
al., 2000; O’Callaghan et al., 2000). An attempt is made in Table 2 to collect a
chronological list of some of the different techniques that have been used to monitor milk
coagulation. Only methods that attempt to monitor the gelation process are listed, i.e.
single-point tests, such as penetrometers, are not reported. A very wide range of physical
and chemical methods has been used and the popularity of studies on milk coagulation is
due to the great economic importance of this gelation process. Many of the techniques
that have been used are non-destructive but a common disadvantage is they may provide
only limited information on the rheological properties of the gel. Since many of the
techniques try to monitor milk non-destructively (so that milk gelation proceeds without
any disturbances) it can be hard to predict the resistance to cutting from the (low
deformation) measured property. Many of the techniques have been used as research
tools to understand the influence of variables, such as Ca and temperature, on the gelation
properties; while many others were attempts to develop on-line or in-vat sensors to
determine the “optimum” cutting time. Very few of these techniques are commercially
used in cheese factories, which continue to operate on standard time schedules, or on
experienced cheesemakers who manually determine the optimum coagulum firmness for
cutting. Some of the possible difficulties in developing an acceptable and (industrially)
popular instrument for an in-vat application are listed in Table 3.
An important aspect that is critical for the successful operation of any on-line or
in-vat coagulation sensor is having a suitable process control system that would be able to
make changes that alter the coagulation time and be able to make those changes quickly.
Newer process control systems that are capable of introducing rapid changes in the make-
procedure to maintain the moisture content of the cheese within tight limits, are now
operating in Australia (Jeff Mayes, personal communication). On-line, near-infrared
(NIR) systems are already commonly used to standardize the composition of cheesemilk
in large factories so controlling the coagulation process seems an obvious next step in
gaining more control over the cheesemaking process. Neutral network have been used to
try to predict the pH of cheese from manufacturing inputs (e.g. pH at cutting) (Paquet et
al., 2000) and this is another example of trying to improve our control of the
A successful milk coagulation sensor would make it possible for control systems
in the future to be able to make adjustments (e.g., add more/less CaCl2 or rennet) to
control and standardize the coagulation process. Changing the cutting time is probably
not an attractive option for large modern cheese factories, as this would be difficult for
scheduling processing runs. In an attempt to better control the coagulation process,
especially in countries with seasonal milk supplies, such as New Zealand, a different
approach has been taken. Low-concentration-factor UF is commonly used to standardize
the protein content of cheesemilk, which is one of the main causes of variation in
coagulation properties. This is probably less of an issue in countries, such as the US,
where there is less milk compositional variation.
Another challenge, as well as an opportunity, for in-vat sensors, is the current
interest in using high milk solids in the cheese vat by adding milk concentrated by UF to
whole or partly-skimmed milk. The increased casein content of the fortified cheesemilk
results in shorter coagulation times and faster rates of curd firming. Thus, cheesemakers
have to modify their traditional make-procedures to cope with the higher solids and an in-
vat sensor may help them in controlling this process. One problem is that if higher solids
milks are not cut at the correct time, then the gel becomes excessively firm and tearing of
the gel can occur resulting in increased fat and casein losses.
Whey Separation in Yogurt and Other Acid-induced Milk Gels
Yogurt is formed by the slow fermentation of lactose to lactic acid by the
thermophilic starter bacteria, Streptococcus salivarius subsp. thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus. In set-style yogurt, gels are formed
(undisturbed) in the retail pot. Stirred-type yogurt is made by breaking the set gel before
mixing with fruit and filling into retail containers (Tamime and Robinson, 1999). Yogurt
is a fast growing dairy product in the US with sales up 10.7% (to $2.1 billion) in 2000
compared with 1999 (Berry, 2001). Lowfat yogurt (i.e., products with fat contents
between 0.5 to 2%) is the major segment of this market with ~55% of total sales (Berry,
2001). It is generally believed yogurts can only be made by giving milk a very severe
heat treatment and performing the fermentation at very high temperatures, such as 45°C.
Several recent studies suggest that we may need to re-adjust some of the current
processing conditions if we want to control wheying-off, which is the one of the biggest
physical defects in yogurt manufacture.
Whey or serum separation, which is also called wheying-off, is the appearance of
whey on the surface of a gel and it is a common defect during storage of fermented milk
products like yogurts. Manufacturers try to prevent whey separation by increasing the
total solids content of milk, subjecting the milk to a severe heat treatment or by adding
stabilizers (e.g., gelatin, pectin, starches, whey protein concentrate, or various gums).
Spontaneous syneresis is contraction of a gel without the application of any external
forces (e.g., centrifugation) and is related to instability of the gel network (i.e., large scale
rearrangements) resulting in the loss of the ability to entrap all the serum phase (Lucey et
al., 1998a). Most of the yogurts sold in the US market are the stirred-type and they have
been made with added sugar and various types of fruits. In stirred-type yogurt, a
combination of higher solids content, and the addition of both fruit and stabilizers provide
the manufacturer with several options to control the texture and physical properties of the
yogurt. However, wheying-off remains a problem in several different brands of yogurt
currently in the US market.
However, a better understanding of the causes of physical defects, such as
wheying-off, may allow yogurt manufacturers to reduce the amount of fortification of
milk that is currently required and to use less stabilizers. The lowfat and nonfat yogurts
that are common in the US (~90% of total sales) also make it more challenging to control
texture as the presence of homogenized fat contributes to the structure of yogurt. Another
issue to consider is that consumers are demanding more “natural” products that use less
or no additives/stabilizers. Fermented milk products already have a positive health image
and with increasing emphasis on probiotic products, there is a growing need to be able to
produce fermented products that do not whey-off during storage without using stabilizers
(Lucey and Singh, 2001).
One of the difficulties in studying the whey separation defect was the absence of a
published or accepted method to quantify or measure it. A simple, empirical method to
quantify spontaneous whey separation in acid milk gels was recently published (Lucey et
al., 1998a). This procedure involved forming the gels in several types of containers and
measuring the surface whey that is produced in the gels. There have been many previous
studies that have tried to estimate the sensitivity of yogurts to undergo syneresis. These
approaches fall into two main categories; they involve measuring the quantity of whey
expelled from yogurt as a result of high-speed centrifugation or which drains through a
screen (Harwalkar and Kalab, 1983, 1986; Guirguis et al., 1984; Dannenberg and
Kessler, 1988). The drainage of whey from a broken gel distributed over a screen
measures whey separation when a very large surface area is available and is more
relevant to products such as cottage cheese or casein, where whey is separated from curd
by screen drainage, than to set yogurt. High-speed centrifugation measures water holding
capacity and gel rigidity under relatively high forces. Therefore, these whey expulsion
methods are not relevant to the spontaneous whey separation defect that may occur in
some “set” gels (Lucey et al., 1998a).
In recent work, it has been found that conditions such as fast rates of acidification
at high incubation temperatures, gave high levels of whey separation compared with gels
made from unheated milk that were incubated at low temperatures and where the rate of
acidification was slow, e.g., when bacterial cultures were used instead of the acidogen,
glucono-δ-lactone (GDL) (Table 4) (Lucey et al., 1998c; Lucey, 2001). Several of these
approaches are practiced in the yogurt industry (i.e., fast acidification at high
temperatures). Fortunately, the yogurt industry is now dominated by the stirred-type
product, which allows manufacturers to add various stabilizers to try to prevent wheying-
off. An important question is why do some gels exhibit spontaneous whey separation?
This defect is associated with an unstable network that has a strong tendency to undergo
further rearrangement of the network structure resulting in the loss in its ability to entrap
all the moisture that was originally trapped in the gel. But what causes an unstable
Casein gels are dynamic by nature. Excessive rearrangements of particles making
up the gel network before and during gelation have been implicated as being responsible
for whey separation and several rheological conditions that appeared to indicate the
possible occurrence of this defect have been identified (Lucey, 2001). These rheological
parameters include the following: the dynamic moduli, which indicate the strength and
number of bonds in the network, the yield stress and strain, which determine the
susceptibility of the strands to breakage, and loss tangent (tan δ), with higher values
favouring relaxation of bonds (van Vliet et al., 1991; Lucey and Singh, 1997).
A surprising conclusion from recent studies is the severe heat treatment of milk
does not prevent whey separation as is commonly believed and it may even increase
wheying-off, at least in model GDL-induced milk gels (Lucey et al., 1998a). The
tendency to exhibit whey separation in acid gels made from heated milk has been related
to a low fracture strain and an increase in tan δ (observed at both high and low
frequencies) during the gelation process (Lucey, 2001).
Milk used for yogurt manufacture is normally subjected to an extensive heat
treatment. Heat treatment of milk at a temperature above 70°C causes denaturation of
whey proteins, some of which associate with casein micelles, involving κ-casein, via
hydrophobic interactions and intermolecular disulphide bonds (Haque and Kinsella,
1988; Singh, 1995). The detailed mechanism by which heating affects the rheological
properties of acid milk gels has been described recently (van Vliet and Keetels, 1995;
Lucey et al., 1997b, 1998b). These denatured whey proteins associated with casein
micelles help cross-link casein particles in the gel network resulting in an increase in the
G´ and an increase in the pH at gelation (Lucey et al., 1998b). It now seems that although
heat treatment increases the rigidity of yogurt gels (which is an important textural
attribute) it is not very effective at preventing wheying-off that occurs in milk incubated
at very high temperatures. More work is needed on the control of this defect especially in
cultured system as they can behave very differently to model (GDL-induced) acid gels.
Processing and fermentation conditions also influence starter growth and
metabolism, which in turn alters the flavour and sensory attributes of the final product. In
yogurts with added sugar and fruit, the original flavour is not as important as in plain
yogurt but nevertheless excessive acidity or any other objectionable flavours may
influence the acceptability of the product to the consumers. This is another factor to
consider before changing yogurt processing conditions.
Can the Horne Model for the Structure of the Casein Micelle Help Explain Some
Properties of Milk Gelation?
It appears that the Horne model can be used to help explain certain unusual
rheological behavior that is observed during the gelation of milk proteins. Horne (2001)
reported that in acid-induced milk gels made under certain incubation conditions (i.e.,
high incubation temperatures or high levels of acidulant) there was a peak in the complex
modulus (G*) profile just after gelation had occurred. In his study milk was acidified
using GDL as an acidulant. The occurrence of this peak was explained as being due to
solublization of CCP within and between casein micelles, which caused a weakening of
the network structure.
Lucey et al. (1998b) have observed a similar phenomenon in several different
types of milk gels and they have employed another rheological parameter; tan δ, to
monitor this phenomenon. The tan δ parameter is sensitive to any shift in the slopes of
the dynamic (storage and loss) moduli. The rheological properties of acid gels made from
unheated and heated milk are shown in Figure 2. Although no maximum or peak was
seen in the G´ profile of heated milk, there was a clear maximum in tan δ. The maximum
in tan δ was interpreted as a partial loosening of bonds within and between casein
molecules in these gel networks due to the solubilization of CCP and the release of Ca2+
at pH values < 5.8. The solubilization of CCP appears to alter the balance between
viscous and elastic components in the network. At lower pH values (≤ 5.0) there is a
decrease in the net negative charge on casein, which results in increased electrostatic
attraction between casein molecules and particles. Consequently, tan δ decreased, which
indicated that the gel had a more elastic character. Simple particle gel models (e.g., de
Kruif, 1997) do not anticipate that the internal structural components of casein micelle
play such a major role in gel properties. The Horne model suggests that acidification of
milk solubilizes CCP, which loosens the bridging between caseins and this disturbs the
delicate balance of attraction and repulsion that governs micellar integrity. The loss of
CCP weakens the internal structure of the micelles but the gel network remains intact due
to the presence of other interactions (e.g. hydrophobic and electrostatic). This behaviour
in not only important in acid milk gels but it may also occur in milk gels that are formed
by a combination of rennet and acid, i.e., combined gels (Lucey et al., 2000, 2001;
Most natural cheese varieties use rennet to form the initial coagulum but
acidification also occurs in these gels due to the action of the starter culture. In these
combined gels, the initial gel is initiated by rennet action but the rheological properties
are drastically altered by subsequent acid production (Figure 3). Combined milk gel made
using GDL as the acidulant (Lucey et al., 2000) exhibited similar rheological properties
to those made with starter culture. Initially, G´ increases steadily after gelation until pH ~
5.5 when it sharply decreases due to solubilization of CCP, concomitantly tan δ increases
and goes through a maximum. At low pH values (pH < 5.0) G´ starts to increase and tan δ
decreases due to increased electrostatic interactions as the pH of the system approaches
the isoelectric point of casein. We can again interpret the unusual changes in the
rheological properties as being caused by an alteration in the balance of attractive and
repulsive forces that govern micelle integrity.
Areas for Future Study
The enzymatic (hydrolysis) stage of rennet coagulation is well understood but the
gel formation, and rearrangements that occur in the network are still not very well
understood. Understanding the changes that occur in the structural properties of milk gels
is vitally important in the ability to control syneresis and ultimately, cheese texture. On-
line or in-vat coagulation sensors would provide a useful means of improved process
control but there remains several important issues that need to be successfully addressed
to achieve this objective. Theories that facilitate modeling of milk protein gelation are
needed that should be able to provide kinetic parameters of the aggregation process and
not just be limited to predicting the (initial) aggregation time. Any useful theory should
also yield insights into the rheological properties of the network. The factors that control
whey separation in cultured milk systems need further investigation although these have
been studied in model (GDL)-induced acid gels. The reasons for the differences in the
physical properties of model acid gels made with GDL and those produced via
fermentation should also be investigated, as this is one of the major difficulties in
translating recent progress made with model systems into “real” yogurt manufacturing
The author wants to thank David Horne for sharing various pre-publication materials on
his new dual-binding model for the casein micelle. Thanks also to useful comments on
this topic by David Horne, Norm Olson, Mark Johnson and Selvarani Govindasamy-
Lucey. The author is grateful for the financial support for this research by the Wisconsin
Center for Dairy Research, Wisconsin Milk Marketing Board, and USDA Hatch project
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Figure 1. A schematic diagram of the Horne model for the casein micelle (Horne, 1998)
(re-drawn with permission from Elsevier Publishers) (not yet requested……..).
Figure 2. Rheological properties of a model cheese gel system containing both rennet
(~7.5 ml per 100 kg) and acid (Cheddar starter culture added: ~16 g per 100 kg). The
following parameters were measured at 32°C as a function of time: (●) storage modulus,
G’; (▲) loss tangent and (□) pH. A frequency of 0.1 Hz and an applied strain of 1% was
used in the rheometer which was operated as described previously (Lucey et al., 2000).
Figure 3. Effect of heat treatment on the rheological properties of a yogurt gel made at
40°C from unheated milk (circles) or milk heated at 82°C for 30 min (triangles). Solid
and open symbols are the storage modulus and loss tangent, respectively. pH (□) was also
measured as a function of time.
αS2-CN αS1-CN β-CN κ-CN CCP
Storage modulus, G' (Pa)
0 100 200 300 400
250 0.65 7.0
Storage modulus, G' (Pa)
100 0.45 5.5
0 100 200 300 400 500
Table 1. A comparison of some of the physical properties of rennet- and acid-induced
milk gels (from various sources)
Rennet gels1 Acid gels2
Storage modulus3, Pa 85-95 200-220
Loss tangent, maximum value4 ~ 0.35 0.50-0.54
Fracture stress5, Pa 80-95 90-100
Fracture strain5, (-) ~ 1.50 0.65-0.75
Permeability, × 10-13 m2 ~ 2.5 ~ 1.5
Surface whey separation6, % ~ 0.1 ~ 1.1
Measured 6 h after rennet addition to milk at 32°C
Measured at pH 4.6 in yogurt gels made from milk heated at 82°C for 30 min and
incubated at 40°C with 2% starter culture.
Storage modulus measured at 0.1 Hz.
This is the maximum value attained by the loss tangent during the gelation phase;
yogurts exhibit a clear peak while rennet gels do not.
Fracture stress and strain were determined by the low constant shear rate technique of
Lucey et al. (1997b)
Whey separation was measured as the amount of spontaneous surface whey produced in
flasks as described by Lucey et al. (1998a)
Table 2. A chronological list of some of the techniques that have been used to monitor coagulation of milk (only techniques
that can be used to continuously monitor the properties of the gels are mentioned)
Technique or device Principle and Comments Reference
Cheesemakers finger The cheesemakers makes a slight cut in the coagulum with a finger or knife Traditional practice
or knife test and lifts the curd to see if there is a clean break and if clear whey is exuded
which indicates the coagulum is ready for cutting. This also provides an
indication of the firmness of the gel.
Viscosity Measurement of the viscosity changes in milk during aggregation using Holter (1932), Scott Blair
various types of viscometers (e.g., capillary, rotational). Mainly used for & Oosthuizen (1961)
the early stages of aggregation.
Visual coagulation or Visual observation of the aggregation of milk in a rotating test tube to give Berridge (1952)
the Berridge method a rennet coagulation time (RCT).
Electron microscopy Images from electron microscopy can be used to indicate the extent of Hostettler et al. (1955),
aggregation and coagulation Green et al. (1978)
Turbidity Can use both undiluted and diluted samples as well as several different Claesson and Nitschmann
measurements wavelengths (1957)
Manometry: U-Tube Milk is exposed to a slight over-pressure in a U-tube and the firmness of Scott Blair and Burnett
the gel is read-off a capillary tube. (1958)
Torsionmeter Milk is subjected to torsional forces and the response of the gel is Burnett and Scott Blair
monitored by a variety of different types of sensors. (1963)
Blood clot-timer A rotor stirred the milk and as soon as the milk clots a drop of the mixture de Man and Batra (1964)
becomes deposited on a set of electrodes, which closes an electrical circuit.
Thrombelastography A commercial version of this technique was sold as the Formagraph. Frentz (1965), Marcais
or lactodynamography Movement of a suspended cylinder or loop, which is immersed in milk (1965), McMahon and
samples, is monitored as the sample holder is oscillated or rotated. Brown (1982)
Helipath Viscometry A T-shaped wire attached to the viscometer cuts a helical path through the Richardson et al. (1971)
coagulum while measuring viscosity.
Instron Universal The viscous drag on a ball or probe suspended in a milk sample is Steinsholt (1973)
Testing Machine: monitored continuously with a load cell.
falling ball viscometry
Electrical conductivity Conductivity measurements are performed during milk coagulation. Tsouli et al. (1975)
Manometry: pressure Several different types; most involved hydraulically oscillating diaphragms Vanderheiden (1976)
transmitting systems and the pressure transmitted through the milk to a sensor is recorded.
Diffuse reflectance Reflectance was originally measured with a color difference meter; newer Hardy and Fanni (1981),
studies use a fiber optic probe as a light source and several different λ are Ustunol et al. (1991)
used. In-cheese vat sensors of this type have been installed.
Dynamic light Usually operated in the photon correlation spectroscopy (PCS) mode. Laser Walstra et al. (1981)
scattering (DLS) light sources are used and the time-averaged intensity of the light scattered
is measured. This exploits the rapid fluctuations in intensity that arises as a
result of the random diffusional motion of the scattering particles. Highly
diluted milk must be used (to avoid multiple scattering).
Vibrational viscometry Several types of instruments have been used and the setup usually involves Marshall et al. (1982),
a stainless steel probe that usually vibrates longitudinally (e.g., Ultra- O’Callaghan et al. (2000)
Viscoson or Nametre) or torsionally (e.g., Rheoswing).
Oscillatory rheometry Non-destructive, uses a rheometer and applies a sinusoidally oscillating Tokita et al. (1982),
waves to apply stress (usually) to the sample and measure the responses of Bohlin et al. (1984)
the sample to the applied stress. Provides detailed rheological information
on the gelation process.
Near infrared (NIR) Various commercial instruments are available, e.g. Gelograph. Uses light Anon (1983),
transmission or reflectance measured in the NIR range for coagulating O’Callaghan et al. (2000)
milk. No moving parts.
Thermal conductivity A constant thermal energy is supplied by a heating (hot) wire and the Hori (1985)
or hot wire method temperature of the sample monitored by a thermocouple. Increasing
kinematic viscosity of the sample during clotting results in an increase in
the ∆T between the hot wire and the probe. In-cheese vat sensors of this
type have been installed.
Vatimer Stainless steel sensing probes are oscillated vertically and the force on the Richardson et al. (1985)
probes is monitored by strain gauges.
Instron Universal A rectangular gauze is oscillated up and down. The mechanical resistance Van Hooydonk et al.
Testing Machine: exerted on the gauze by the gel is monitored continuously with a load cell. (1986)
Refractometry Changes in the refractive index of milk during coagulation can be Korolczuk et al. (1988)
Diffusing Wave Similar to DLS but multiple back scattering is an essential component of Horne and Davidson
Spectroscopy (DWS) this technique. Molecular mobility of scatters decreases as milk starts to (1990)
aggregate and this results in an increase in the relaxation time, which can
be related to particle size. Undiluted milk can be used.
Dark-field microscopy Dark-field illumination increases image contrast compared to standard light Ruettimann and Ladisch
microscopy and coagulation was observed in situ. (1991)
Ultrasound: High Frequencies > 1 MHz are used and the absorption and velocity variations in Benguigui et al. (1994)
frequency the ultrasound wave, as it passed through milk, are interpreted in terms of
various types of moduli.
Electroacoustics An alternating electric field is applied to the sample and this causes the Wade and Beattie (1998)
casein particles to oscillate and generate pressure disturbances in the
surrounding liquid. This generates sound waves and the phase lag between
the applied signal and resulting response gives information on particle size.
Fluorescence Emission fluorescence spectra of tryptophan residues are monitored during Herbert et al. (1999)
spectroscopy milk coagulation.
Ultrasound: Low Frequencies in the range 50-100 kHz are used in an ultrasonic device, Nassar et al. (2001)
frequency changes in the time-of-flight for the ultrasound wave are determined
between two sensors and this data is used to probe gel formation
Table 3. Some of the difficulties that have been encountered in trying to get a successful
in-vat coagulation device
Difficulties in cleaning the device (in-place)
Some devices need to be removed during the cutting and stirring operations
Robustness of the device in a commercial setting
Need a rapid and accurate prediction of the cutting point and an indication of the rate of
firming after cutting would be useful (which would help in the optimization of the
stirring and healing processes which becomes more important in milk with higher
Some responses are sensitive to the protein and fat content of the cheesemilk (may need
to be calibrated when there is a different milk composition)
Optimum parameters at cutting selected by a device may only be valid for particular
cheesemaking conditions (e.g., certain cheese vat design, cutting cycle, cheese type,
Questions concerning what physical properties the device is actually measuring and how
that is related to the “cuttability” and “rate of firming” of the coagulum
Some techniques extrapolate the predicted cutting point from some initial readings and
this may be more difficult with changes in milk composition (e.g., cheesemilk with
high protein contents)
Many factories do not have fast process control systems to quickly alter coagulation
conditions if the previous vat was slow to coagulate
Larger plants are under enormous time pressures and do not want to have to make
continuous changes in the cutting time as it upsets their scheduling (minimizing
coagulation differences is one of the reasons most of them now standardize the
protein content of cheesemilk)
Cheese yield does not seem to be very sensitive to small changes in the curd firmness at
cutting (at least for non-fortified milks)
Table 4. Possible conditions that may contribute to wheying-off in yogurt (the extent of
wheying-off will depend on the actual combinations of these conditions) (adapted from
High incubation temperatures (e.g. 45°C compared with 40°C)
Rapid rate of acidification (e.g. glucono-δ-lactone instead of starter cultures)
Excessive heat treatment of milk (e.g. ≥ 85°C for 30 min of batch heating) in
conjunction with high incubation temperatures
Low total solids content (especially protein but fat is also important as the milk is
Low acid production (e.g. pH 4.9 compared with 4.6)
Containers with sloping walls or containers not stored in the upright position (causes
stresses on the gel which may come away from the sides and shrink)
Agitation during gelation (e.g. any disturbances while the gel is still weak)