Coagulation of Milk: Processes and Characteristics: of The International Dairy Federation

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Coagulation of
Milk: Processes and
Characteristics
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CONTENTS
PRICE : 24 Euro 420/2007
Coagulation of Milk: Processes and Characteristics

Foreword 1
Summary 2
1. Introduction 3
2. Coagulation 4
3. Characteristics of Coagulated Milk
Proteins 13
4. Changes in Coagulated Dairy Products
During Further Processing and Storage 13
5. Verification of Coagulation 16
6. References 18
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Bulletin of the International Dairy Federation 420/2007 Coagulation of Milk: Processes and Characteristics
Coagulation of
milk processes and
characteristics
Foreword
The review of the milk coagulation process was initially a work item of the former IDF Standing
Committee on Standards for Cheese (SCSC). The SCSC was disbanded in May 2004 and its work
items were integrated into a new IDF Standing Committee on Standards of Identity (SCSID).
IDF National Committees adopted new work on the subject of “Investigations into Methods which
can be used for the Recognition of Coagulation of Protein in Cheese” as part of the SCSC work
programme in November 2002. The result of the work was confirmed as the establishment of a
scientifically based position for publication in the Bulletin of IDF, taking into account :
(a) the various known (and possible future) mechanisms of coagulation, including a discussion of
their complexity;
(b) any commonalities between these mechanisms;
(c) possible means by which coagulation might be demonstrated and problems associated with
these techniques (e.g. proteolysis) in the specific case of cheese.
After endorsement by the SCSID, the paper was also reviewed by the members of the Standing
Committee on Dairy Science and Technology.
This review is not intended to provide a legal opinion on the interpretation of current international
dairy standards, but it will provide the reader with references illustrating and facilitating the un-
derstanding of the complexity and diversity of the milk coagulation reaction as encountered in the
manufacture of dairy products.
The IDF is most grateful to all experts involved for their valuable input and contribution to this
work, and particularly to Keith Johnston (NZ), drafting leader of the SCSID, for his relentless ef-
fort in coordinating the work. Acknowledgments are also due to Lyndon Taylor (AU), IDF Action
Team leader for the initial work in this area, to Roderick P. W. Williams (Food Science Australia)
for his 2003 review entitled ‘Coagulation and Dairy Products: Definition and Characteristics’, which
formed the framework of the IDF document, and to the members of the SCDST for reviewing the
final draft.
Christian Robert
Director General
August 2007

Summary
There are a number of dairy definitions and dairy standards that use the term ‘coagulation’ with-
out further defining the term or defining a method that can be used to objectively determine that
coagulation has taken place.
The objective of this document is to provide the reader with sufficient references to gain an
understanding of the complexity and diversity of the milk coagulation reaction as encountered
in the manufacture of dairy products. However, the content of this document does by no means
provide a legal opinion on the interpretation of the current international dairy standards.
As a term, ‘coagulation’ does not have a single or simple definition. Coagulation of milk pro-
teins can be initiated by a number of different actions or agents. These agents include rennets
and other proteolytic enzymes, changes in pH, exposure to high or low temperatures, alterations
in solvent properties and cross-linking agents. The coagulation processes are complex and each
type involves contributions from a number of different interactions, such as covalent, hydropho-
bic, ionic, van der Waals’, calcium mediated and/or hydrogen bonding in character. Many of these
forces or interactions are also involved in maintaining the structures present in normal milk.
In the manufacture and storage of cheese and other dairy products, there is a wide range
of post-coagulation events. These events contribute to a change in the physical and chemical
structure of the initial coagulum and form the basis for the diversity of cheeses. Post-coagulation
events are highly complex and can vary widely both between and within particular cheese types.
The tendency of many biochemical changes that occur following coagulation is to diminish bond-
ing within the cheese protein network.
Post-coagulation changes that take place during the processing and storage of cheese are ex-
plored to underline the difficulties that might be encountered in definitively demonstrating that
coagulation has taken place at an earlier stage.
This document does suggest that, regardless of the means, coagulation is the result of desta-
bilisation of the protein micelles in some form, and that the insoluble, destabilised micelles form
structures that are dependent, to some extent, upon processing conditions following coagula-
tion.
The complexity of the coagulation reaction and the myriad of post-coagulation processes make
it difficult to identify one method that can be used universally to determine if coagulation has
taken place. However, the reader is provided with a choice of procedures that can be used, singly
or in combination, to view and/or characterise dairy structures and the results may be used to
support the argument that coagulation has occurred.

1. Introduction
The process of ‘coagulation’ has traditionally been described as the key process in the transfor-
mation of milk into a range of solid or semi-solid dairy products such as cheese.
Although the sequence of events required to manufacture cheese of a specified type can be
relatively well defined, all of the chemical changes that occur through the process are yet to be
fully understood. A further level of complexity is introduced when the diversity of cheese types
and other ‘coagulated’ products is considered.
There are a number of dairy definitions and dairy standards that use the term ‘coagulation’
without further defining the term or defining a method that can be used to objectively deter-
mine that coagulation has taken place; for example, in cheese once it has been made and ma-
tured. Indeed, it became obvious during the development of this document that there is no one
universal method that can be used to ‘measure’ or establish that coagulation has occurred.
Consequently, the objective of this document is to provide the reader with sufficient published
information and references to be able to gain an understanding of coagulation as encountered
in the manufacture of a range of dairy products. It is not intended that this document should
be a definitive, scientific review of the various mechanisms of coagulation; neither should it be
considered as a review of the various process parameters that may influence that reaction.
Nor does the content of this document provide a legal opinion on the interpretation of the
current international dairy standards.
The document explores the post-coagulation changes that take place during the processing
and storage of cheese to demonstrate the difficulties that might be encountered in definitively
demonstrating that coagulation has taken place at an earlier stage. In addition, a number
of microscopic and rheological techniques are briefly reviewed to provide the reader with a
choice of procedures that can be used, singly or in combination, to view and/or characterise
dairy structures and the results may be used to support the argument that coagulation has oc-
curred.

2. Coagulation
As stated by Horne, “Unravelling the mechanism of the coagulation of casein micelles, by what-
ever route, is a complex topic” (Horne 1992).
A great part of the complexity arises from the complex nature of milk and its various compo-
nents. As a colloidal system, milk consists of various particles, some containing proteins and
minerals and others containing fats, suspended in a serum that also contains proteins, minerals
and many other components.
For the following discussions, the structure of the principal protein component, the casein mi-
celle, is of particular significance. A number of models for the structure of casein micelles have
been suggested (e.g. Schmidt 1982, Walstra 1990, Holt 1992, Horne 1998, Walstra 1999, Dal-
gleish et al 2004). Although opinions differ, it is considered that the structure of casein micelles
is governed by the self-associating forces between caseins, a balance of attractive hydrophobic
and repulsive electrostatic forces, and calcium-mediated interactions (Horne 1998).
One feature common to the latest models is the characteristic ‘hairy’ layer provided at the
micelle surface by the C-terminal half of κ-casein. This layer creates a barrier against aggrega-
tion (Holt and Horne 1996). This characteristic is key to the current thinking on most of the
discussion of the forms of coagulation in milk.
Typical manufacturing processes involve the application of heat, acid and/or rennet (or other
proteolytic enzymes) either alone or in combination to coagulate milk and form products. For
example, some acid development is required in the formation of typical renneted cheeses such
as Cheddar. Cottage cheeses are described as acid coagulated although a small amount of ren-
net is sometimes added. Ricotta is made by coagulating whey by heat followed by chemical
acidification (Fox et al 2000). Yoghurt is another product that relies on the action of both heat
and acid. Other processes that cause milk proteins to precipitate, coagulate or gel have been
described in the literature and these are reviewed.
2.1. Definitions of Coagulation

Initially, it is useful to consider what coagulation can mean depending on the context and pur-
pose. For example, three typical sources define coagulation, as follows.
Dictionary: Coagulation n. “the process by which a liquid changes into a semi-solid mass”
Concise Oxford Dictionary, 8th edition (Allen 1991).
Science: Coagulation of proteins: “…the protein becomes denatured…it then becomes in-
soluble and either remains in suspension or is precipitated as a clot or a curd” Penguin Diction-
ary of Science (Isaacs and Uvarov 1979).
Food Science: “Random aggregation reactions with denaturation where protein-protein in-
teractions dominate over protein-solvent interactions”… leading to the formation of a coarse
coagulum (Cheftel et al 1985).
Nevertheless, coagulation in dairy products does not necessarily imply denaturation of pro-
teins.
The language used in these definitions ranges from a basic generic description to an attempt
to describe the change in specific scientific language. However, the net effect described by any
of these definitions is essentially the same; solid insoluble material that develops into some
type of structure is formed.
There are other terms in use for more or less similar events, such as precipitation, floccula-
tion and gelation.
Cheftel et al (1985) define precipitation as a general term covering all aggregation reactions
that lead to a total or partial loss of solubility, flocculation as a process of random interactions
and aggregation reactions in the absence of denaturation and gelation as a process in which
denatured molecules aggregate to form an ordered network.
Walstra (2003) provides one of the clearest and more comprehensive differentiations be-
tween the terms aggregation, flocculation and coagulation. According to this author, aggrega-
tion is the most general of the three terms. Two particles are said to be aggregated if they stay
together for a much longer time than they would in the absence of colloidal interaction forces.

Flocculation is used to denote weak (reversible) aggregation, and coagulation is used to denote
strong (irreversible) aggregation — as for instance in the case of enzymatic milk coagulation.
In practical terms, separating any of these three will generally be a matter of opinion. What
constitutes ‘order’ to one person under one set of criteria is unlikely to apply in all cases. Simi-
larly, the term ‘denaturation’ can be subject to interpretation.
In dairy manufacturing, the primary coagulated products are the cheeses and fermented
milk products such as yoghurt. However, in dairy science, the term ‘coagulation’ has been used
to encompass a range of phenomena that occur in manufacturing or experimental procedures
where proteins aggregate to form a precipitate, coagulum or gel. Typical examples of these are
given in Table 1 and these processes are discussed in greater detail below.
Table 1. Coagulation of Milk Proteins by Various Agents

Agents Main Proteins Mechanisms of Chemical Forces Involved
Affected Aggregation/Gel Formation
Heat Caseins and Protein unfolding, -SH/SS inter- Covalent, hydrophobic, ionic,
whey proteins change, interaction of whey pro- and calcium-mediated bonding.
teins with casein micelles, modi- Disulphide bond formation
fication of steric stabilisation of
casein micelles
Rennet (and Caseins Loss of steric and Hydrophobic, ionic and calcium-
proteolytic electrostatic stabilisation of casein mediated bonding
enzymes) micelles due to enzymatic hydroly-
sis of к-casein
Acid (starter/ Caseins Loss of electrostatic stabilisation Hydrophobic, ionic and calcium-
chemical) due to charge neutralisation. Col- mediated bonding
loidal calcium phosphate deminer-
alisation
Ethanol Caseins Loss of steric stability due to dehy- Hydrophobic, ionic and calcium-
dration, calcium-mediated interac- mediated bonding
tions
Charged Caseins and whey Specific interactions, thermody- Specific polysaccharide/protein
polysaccharide proteins namic incompatibility interactions, hydrophobic, ionic
and calcium-mediated bonding
Low tempera- Caseins and whey Loss of steric stabilisation due to Hydrophobic, ionic and calcium-
ture proteins dehydration effect mediated bonding
Other en- Caseins and whey Chemical cross-linking Covalent bonding, plus others
zymes proteins depending on product
2.2. Traditional Processes of Coagulation

2.2.1. Heat Coagulation
The term ‘coagulation’ has been applied to the precipitation of milk proteins that occurs on pro-
longed heating for a considerable length of time, e.g. Rogers et al (1921), and the use of the
term continues, e.g. O’Connell and Fox (2000). A considerable amount of scientific effort has
been expended in attempting to understand the phenomena and the area has been reviewed
frequently in reference to the heat stability of milk. Singh and Creamer (1992) provide a re-
cent review and the IDF publication of Fox (1995) covers many of the heat-induced changes in
milk.
Milk is heat treated in the production of many dairy products and heat-induced aggregation
is a key to the structure of coagulated products such as yoghurt (Tamime and Robinson 1985)
and some cheese types such as Ricotta and Mascarpone (Fox et al 2000).
One key effect of heat is the formation of complexes between β-lactoglobulin and κ-casein
through the formation of hydrophobic interactions and the formation of intermolecular covalent,
disulphide bonds (Sawyer 1969, Haque and Kinsella 1988, Singh et al 1995). This causes a
modification of the properties of the micelle surface, affecting the way in which κ-casein pro-

vides steric stabilisation.

A large proportion of the work in this area has been done on concentrated milks, because of
the greater susceptibility of sterilised milk products such as evaporated milk. The effects seen
are highly pH dependent. Based on electron microscope studies and other work, it has been
suggested that, depending on the pH, one of three different forms of coagulation can take place
(Singh et al 1989, 1995).
At lower pH (less than 6.5), the aggregates appear to be formed from casein micelles linked
by some bridging material and hence it is considered that the coagulation is mediated by dena-
tured whey proteins and ionic interactions. At pH values between about 6.5 and 6.9, the mi-
celles appear to fuse or join together tightly. Other measurements have indicated that, at this
pH range, there is a significant loss of κ-casein from the micelles into the serum phase and so
it is proposed that the coagulation in this pH range is primarily a consequence of destabilisation
of the κ-casein-depleted micelles. The coagulation occurs through calcium-mediated aggrega-
tions between the micelles and calcium phosphate precipitation (Singh and Creamer 1992).
Above pH 7.0, the micelles appear to be largely dispersed and the coagulum is formed from
a gelled matrix of whey proteins and dissociated caseins. Solubility studies by Nieuwenhuijse
et al (1991) have shown that the coagula formed at different pH conditions are soluble under
different conditions, suggesting that both ionic and covalent bonding are involved but that dif-
ferent types of bonds are more significant at different pH values.
As well as interacting with casein micelles, the whey proteins can also self-aggregate on heat-
ing. A general sequence of events during the aggregation of β-lactoglobulin has been described
by Sawyer and co-workers as: (a) dissociation of the native dimer into monomers, (b) loosen-
ing of the monomer, (c) unfolding of the protein and (d) formation of aggregates by disulphide
interchange/cross-linking and non-covalent interactions (Sawyer et al 2002). A wide range of
aggregates can be formed. In pure β-lactoglobulin systems, heat-induced aggregates of be-
tween five and more than 100 monomer units have been observed (Schokker et al 1999).
β-Lactoglobulin also forms aggregates with α-lactalbumin on heating (Corredig and Dalgleish
1999). Given sufficient preheating, whey protein preparations become susceptible to precipi-
tation at low pH (pH 4.6 and lower) (O’Kennedy and Kelly 2000) and hence contribute to the
coagulum in heat- and acid-formed gels such as Ricotta and Mascarpone. They also become
susceptible to aggregation through calcium-mediated binding combined with the suppression of
electrostatic repulsion if the ionic strength of the solution is made sufficiently high (Bryant and
McClements 1998). When used as a supplement to milk solids for the production of yoghurt,
the gels become firmer with a reduced tendency to whey drainage (Puvanenthiran et al 2002).
A wide range of investigations has also been undertaken to investigate the use of whey pro-
teins as specialist ingredients in their own right to produce or modify gelled products such as
heat-set gels (Foegeding et al 2002) or as modified ingredients to produce cold-setting gels
(Bryant and McClements 1998).
2.2.2. Enzymatic Coagulation

The use of rennet to coagulate casein to form a curd that is then further processed into cheese
is a traditional process that forms the initial step in the manufacture of a wide variety of cheese
types. Rennet is, by definition, an extract of the abomasum (the fourth ‘true’ stomach) of ru-
minants and contains different ratios of chymosin and pepsin, depending on the feeding regime
and the age of the ruminant. Extracts from the abomasa of young milk-fed calves (calf rennet)
are dominated by chymosin, whereas extracts from older ruminants (adult bovine rennet) are
dominated by pepsin (Andrén 2002). Typically, when discussing the action of rennet, most au-
thors are discussing the action of chymosin although other enzymes (coagulants) can achieve
similar results. Some examples of these are discussed below.

Rennet coagulation occurs in two main phases.
Figure 1. Coagulation of milk (Fox et al 2000).
Initially, the casein micelles of milk are modified by a limited proteolysis of their κ-casein
component; typically, cleavage occurs at the bond between the Phe105 and Met106 amino acid
residues (Fig. 1). This causes the release of the charged, glycosylated portion of the κ-casein
molecule (glycomacropeptide) from the micelles and a reduction in micelle diameter (Walstra
et al 1981). The loss of this section of the κ-casein protein results in a reduction in the zeta
potential of the micelles and a loss of the steric stabilisation that it provides.
In the second phase of rennet coagulation (Fig. 1), which begins when about 60–85% of the
total κ-casein has been hydrolysed, the destabilised micelles are aggregated by a combination
of van der Waals’, hydrophobic, calcium-mediated and specific ion-pair interactions (Dalgleish
1993). At this stage, a protein network forms and traps both fat and moisture.
However, the nature of the actual reactions involved in the second phase of rennet coagula-
tion is not fully understood (Dalgleish 1993, Lucey 2002). Some authors ambiguously differ-
entiate between concepts, such as aggregation and gel assembly. In this way, Green (1984)
considered the curd assembly to be the later stage of aggregation but analysed it separately
from aggregation. Fox and Mulvihill (1990) also treated the gel assembly separately from the
secondary phase of coagulation. Several authors claim the existence of a third milk coagula-
tion stage, gel firming (Storry and Ford 1982, McMahon and Brown 1984, Carlson et al 1987a),
where further cross-linking takes place to increase gel strength. McMahon and Brown (1990)
gave a continuous description of the process: aggregation; formation of a network of casein
particles at the gelation point; continuation of casein micelle incorporation into the gel network
and rearrangements of chains of casein; fusion and consolidation of micelles into thick strands
with whey expulsion.
Most authors do not differentiate clearly between aggregation and curd firming steps because
these processes are not well known at the molecular level. In fact, there is an important lack
of knowledge regarding the molecular sequence and the nature of the cross-linking process,
especially after the primary network formation. One of the reasons, which could be argued, is
that the results derived from different devices normally used to study the kinetics of aggrega-
tion and curd firming (viscosity, rigidity modulus and light scattering) show more sensitivity to
one of the different stages of coagulation, which should considerably modify the type of kinetics
observed. For example, viscosity increases exponentially between the beginning of aggregation
and the onset of visual coagulation (Fox and Mulvihill 1990). Most devices normally used to
measure the increase in curd firmness do not have the sensitivity to measure the small change
of the modulus at the gelation point (McMahon and Brown 1990). As a consequence, the ma-
jority of the mathematical models developed to describe gel assembly during milk coagulation
generally focus on either aggregation or curd firming processes. Nevertheless, Carlson et al
(1987b, 1987c) analysed separately the kinetic aspects of both casein micelle aggregation and
curd firming. Castillo et al (2003) studied milk coagulation using light backscatter and attrib-
uted its increase after the inflection point of the light backscatter ratio profile to the overlapping

of casein micelle aggregation (disappearance of single micelles in solution to form doublets) and
curd firming reactions (development of gel firmness as a result of disappearance of cross-link-
ing sites in the preliminary network by further incorporation of casein micelles or strands, or by
the formation of new links between micelles). A kinetic model that combines a second order
reaction model to describe aggregation reactions and a first order reaction model to describe
firming process reactions was proposed.
Post-coagulation events further influence the formation of the protein network and the final
curd structure. Consequently, the way in which the curd network is transformed into the fi-
nal cheese structure for a particular variety of cheese depends on a series of post-coagulation
events.
The usual characteristic measurement of the process (the rennet coagulation time) is influ-
enced by a number of factors. As little aggregation occurs below 20˚C, it has been suggested
that hydrophobic bonding has a role (Lucey 2002) as have other factors that have been mod-
elled by Mellema et al (1999). However, as a too low Ca2+ ion concentration can also inhibit
aggregation, charge neutralisation or specific ion-binding effects are also implicated (Dalgleish
1983).
The pH is also a contributing factor as, in practice, many renneted milk products are also
acidified by the action of starter cultures or direct acidification. Rennet coagulation occurs
faster and the strength of the coagulum increases more rapidly when the pH is lower (Zoon et
al 1989). Factors involved in this effect include the reduction in the electrostatic repulsion be-
tween micelles by charge neutralisation, leading to the ability for flocculation to occur when less
of the κ-casein has been hydrolysed, and the increase in rennet activity at lower pH (Walstra
1993). Reduced pH also increases the association between chymosin and para-κ-casein (Lars-
son et al 1997, de Roos et al 1998). Some of these factors are also common to the discussion
on acid coagulation.
The other major factors that affect enzymatic coagulation include protein concentration, ren-
net concentration, addition of CaCl2 and coagulation temperature.
Coagulation by Other Proteolytic Enzymes
As described above, traditional preparations of rennet are generally associated with the enzyme
chymosin, but a range of other enzymes may be used to bring about a similar coagulation
event. For a review of rennets and coagulants, see Andrén (2002).
Often-called recombinant chymosin or fermentation-derived chymosin, produced by microor-
ganisms into which the bovine gene responsible for chymosin expression has been inserted, is
a common alternative to calf rennet.
Pepsin, an acid proteinase from bovine and chicken sources, and acid proteinases from the
microbial sources Rhizomucor miehei (e.g. Rennilase), R. pusillus (e.g. Emporase), psychro-
trophic bacteria (de Greef Trial et al 2003) and Cryphonectria parasitica (e.g. Surecurd) are
given as alternatives to traditional rennets by Fox and McSweeney (1998). It should be noted
that, in addition to the highly susceptible Phe105–Met106 bond, other peptide bonds of κ-casein
can be split by these proteolytic enzymes, leading to a shorter or longer casein macropeptide
(Manso and López-Fandiňo 2004).
Other examples from the literature are enzymes extracted from flowers (Sousa and Malcata
2002), used in traditional cheeses in Spain and Portugal, or leaves (Lo Piero et al 2002), which
can bring about similar coagulation events. Enzymes extracted from fruits (such as papaïn from
pineapple) can also induce coagulation. These enzymes tend to have additional proteolytic ac-
tivity towards the other caseins. This can alter the final characteristics of the coagulum formed
because of a reduction in the amount of intact protein available (Esteves et al 2001).
In general, the mechanisms by which the coagula are formed are considered to be similar
to the processes involved in coagulation by chymosin (Esteves et al 2001). However, it is also
recognised that the use of enzymes other than chymosin that are generally less specific in their
proteolytic action can lead to unwanted flavours and bitterness in long storage cheeses such
as Cheddar and decrease yield. Therefore, the choice of coagulating enzyme depends not only

on the enzyme’s ability to initiate the coagulation reaction but also on the type of cheese being
produced.
2.2.3. Acid Coagulation

As a part of various cheese making processes, and as the basis for the production of yoghurts,
the acid coagulation of milk has been an area that has attracted considerable interest. Two
recent reviews are those of Lucey and Singh (1997) and Horne (1999). The effect of acidifica-
tion is altered by a number of factors, notably the heating of the milk, but addition of calcium,
alteration of the kinetics of acidification and alteration of the casein to whey protein ratio have
also been investigated.
In manufacturing, acid is produced by the action of starter cultures; however, some prod-
ucts are acidified directly (Fox and McSweeney 1998). When unheated milk is acidified, the
calcium phosphate that resides within the micelles is dissolved (Pyne and McGann 1960) and,
depending on the temperature, caseins are also released from the micelles into the milk serum
(Dalgleish and Law 1988).
Aggregation and coagulation of the caseins, both micellar and serum based, occur as pH 4.6
is approached. A particulate structure is produced (Horne 1999) in which clusters and chains of
casein particles (the remnants of the original micelles) form loose three-dimensional networks
(Kalab et al 1976). Although particles remain, the loss of calcium phosphate and some casein
implies that they are quite different in character from the original micelles (Lucey and Singh
1997).
Hydrophobic interactions are involved both in maintaining the structure of the remnant mi-
celles as the pH falls and in the creation of the final coagulum, as the liberation of the caseins
from the micelles during acidification is enhanced by low temperatures (Dalgleish and Law
1988) but the formation of the final aggregate is inhibited by low temperatures (Roefs et al
1985).
In products such as yoghurt (Lucey and Singh 1997), the milk is normally heated before fer-
mentation. As described above, a significant effect of the heating is to cause covalent linkages
between κ-casein and β-lactoglobulin and the aggregation of whey proteins. Thus the coagu-
lated gel in these products involves the formation of covalent linkages in addition to the prevail-
ing hydrophobic, calcium-mediated and reduced electrostatic repulsion contributions provided
by the reduction in pH.
Coagulation can be obtained by using previously cited processes in combination, such as heat
and acid or enzymatic and acid. For example, high heat milk coagulates at pH 5.1–5.7 and the
gel characteristics are dependent on CaCl2 or other salts or chelating agent addition (Goddard
and Augustin 1995). Alternatively, milk adjusted to pH 5.2–5.9 coagulates when heated at
100°C for 1 h (Surel and Famelart 2003).
Acid coagulation as practised in yoghurt making is, for example, a sequential utilisation of
heat and acidification. Coagulation in cheese making is also a mixed process as acidification
and enzymatic coagulation are applied together. In some cases, acidification and enzymatic
kinetics can be performed simultaneously (Noel et al 1991, Lucey et al 2000, Tranchant et al
2001) or a partial enzymatic hydrolysis can be applied prior to acidification (Gastaldi et al 2003,
Niki et al 2003).
2.3. Non-traditional Processes of Coagulation

2.3.1. Ethanol Coagulation
The testing of milk based on the addition of ethanol and the precipitation of milk proteins to
predict a range of properties (e.g. early detection of decreased colloidal stability of milk due to
different reasons such as acidification) had its origins about 1890 whereas the term coagulation
was applied to the phenomenon at least as early as 1923 (Sommer and Binney 1923). It is still
being used in publications (e.g. Czerniewicz et al 1999).
The literature on the effect of ethanol on milk has recently been reviewed thoroughly by
Horne (1992). The primary effect of high levels of added ethanol appears to be the removal

or reduction of the steric stabilisation provided by the κ-casein ‘hairy layer’. The collapse of
the surface layer has been attributed to a loss of solvent quality, caused by a reduction in the
dielectric constant of the system (O’Connell et al 2001). The net negative charge on the ca-
seins is also reduced and calcium phosphate may precipitate (O’Connell et al 2001). The criti-
cal ethanol concentration that induces coagulation is also influenced by factors such as the pH,
ionic strength and calcium concentration (Horne 1992).
As in the case of rennet coagulation, once the stabilisation has been removed, the micelles
are susceptible to aggregation which may be calcium mediated. The main proteins precipitated
are the caseins (Czerniewicz et al 1999) whereas whey proteins such as β-lactoglobulin are
partially precipitated.
2.3.2. Low Temperature Coagulation

The precipitation of milk proteins following frozen storage was observed in the 1930s and was
the subject of considerable research at the time of the Second World War.
Among the early workers (e.g. Webb and Hall 1935, Babcock et al 1946), the effect was
generally discussed in terms of destabilisation, precipitation or insolubility, whereas Doan and
Warren (1947) used the term flocculation. This term was also employed by Muir when he re-
viewed this area (Muir 1984). Regardless of the terminology used, the characteristics of the
phenomenon are consistent with some of those that occur during coagulation. Milk that has
been stored at low temperatures — the effect being greatest between 8 and 12˚C below zero,
according to Koschak et al (1981) — is found to develop a sediment of precipitated protein when
thawed. The time taken to form the precipitate varies, with the milk being stable for short pe-
riods, depending on the storage temperature. The initial precipitate can be re-dissolved but the
precipitate becomes progressively harder to re-disperse as the storage time progresses.
Chemical analyses have shown that the precipitate is predominantly casein (Wildasin and
Doan 1951) and electron microscope studies have demonstrated the presence of aggregated
casein micelles (Saito et al 1963). Various studies (Wells and Leeder 1963, Chen and Yamauchi
1971) have shown that the precipitation is associated with a reduction in the concentrations of
calcium, inorganic phosphorus and citrate in the soluble phase. Muir (1984) therefore suggest-
ed the general hypothesis that the caseins are aggregated by calcium phosphate cross-links.
Other factors that have been shown to have a role in the precipitation of the proteins are: the
concentration of the milk; the level of lactose and the extent of lactose crystallisation; the use of
calcium chelating agents such as polyphosphate (Doan and Warren 1947) or oxalate (Wildasin
and Doan 1951); and the addition (Doan and Warren 1947) or removal (Haller and Bell 1950)
of calcium. In general, these observations all point towards a calcium-phosphate-mediated
aggregation of casein micelles that is initiated by a freeze-concentration of the salt and casein
components of the milk.
2.3.3. Proteolytic Coagulation of Whey Proteins

Although the traditional proteolysis by rennet does not affect the whey proteins, gelation of
isolated whey proteins has been induced through the use of proteinases (Sato et al 1995, Otte
et al 1999). The mechanism by which this process occurs is still under investigation. Presently,
it is considered that partial proteolysis of specific sections of the whey proteins creates a reduc-
tion in electrostatic repulsion, allowing attractive forces to predominate resulting in the forma-
tion of a gel. The gels formed are stronger if the whey proteins have been pre-denatured and
aggregated by heating, which increases the level of disulphide covalent linkages within the gel
and increases the size of the gel-forming aggregates (Otte et al 1999). The discussion below
regarding the plastein reaction and caseins may have some bearing on this form of aggregation
of the whey proteins.
2.3.4. Coagulation by Polysaccharides

A range of precipitation/flocculation/coagulation events can occur when milk or solutions of milk
proteins are mixed with polysaccharide ingredients. When mixed, these systems can follow
three basic trends in behaviour: (a) co-solubility, in which the solutions remain homogeneous;
10
(b) incompatibility, where precipitation occurs and the solution separates into protein-rich/
polysaccharide-poor and protein-poor/polysaccharide-rich phases; (c) complexation, where the
protein and the polysaccharide precipitate together (de Kruif and Tuinier 2001).
Effects of this type have been described between milk and carrageenans (Langendorff et al
1997, Puvanenthiran et al 2001) and between milk and pectins (Maroziene and de Kruif 2000).
Incompatibility reactions between caseins and carboxymethyl cellulose, sodium alginate, gum
arabic, dextran sulphate, amylopectin, dextran and Ficoll under experimental conditions have
been described (Grinberg and Tolstoguzov 1997).
Incompatibility involves the promotion of attractive forces between the micelles (de Kruif and
Tuinier 2001). A number of models have been suggested. At this stage, it seems to be simplest
to consider that the increase in effective concentration of the proteins (Ledward 1994) caused
by excluded volume interactions of the polysaccharide overcomes the steric stabilisation effect
provided by the κ-casein, leading to hydrophobic, electrostatic or calcium-mediated aggrega-
tion of the micelles.
Complexation involves the formation of linkages between the polysaccharide and the milk
proteins which then self-associate to form large complexes. For example, the formation of
specific complexes between κ-carrageenans and the region of κ-casein between residues 97
and 102 has been suggested (Snoeren et al 1975). Electrostatic interaction and calcium-me-
diated binding have been suggested as the likely forces involved in their formation (Dalgleish
and Morris 1988). The further aggregation of these micelle–carrageenan complexes into gels
or networks is then mediated by ionic interactions between carrageenans (Puvanenthiran et al
2001).
Superimposed over these trends is the possibility for gelation mediated by a number of fac-
tors such as pH (Maroziene and de Kruif 2000), temperature (Langendorff et al 1997) or ionic
composition (Puvanenthiran et al 2001).
2.3.5. Coagulation by Enzymatic Cross-linking

The use of the cross-linking enzyme transglutaminase has been described as an adjunct to
the manufacture of a range of dairy products. Examples from the literature are: Quarg with
a creamier and softer consistency than traditionally manufactured products (Lorenzen et al
2002a); yoghurt with higher gel strength and reduced syneresis (Lorenzen et al 2002b); Cream
cheese that does not require the use of additional stabilisers and incorporates whey proteins
(Xiao-Qing Han et al 2002); powdered milk (Imm et al 2000, Miwa et al 2002) and cheese
(Budtz 1997, Soeda et al 1999, Xiao-Qing Han and Spradlin 2001).
Transglutaminase cross-links proteins by catalysing the formation of covalent bonds between
a γ-carboxyamide group of a glutamine residue within one protein and an amine group such as
an e-amino group of a lysine residue in another protein (Sharma et al 2001) and consequently
its action can be identified by the presence of the ε-(γ-glutamyl)lysine dipeptide (Sharma et al
2001).
However, at the natural pH of milk (approximately pH 6.7), treatment with transglutaminase
alone is not sufficient to form a gel (Schorsch et al 2000). Schorsch et al (2000) suggested that
this is because the κ-casein layer on the surface of the micelles keeps the micelles sufficiently
separated to prevent linkages from being formed. However, in transglutaminase-treated un-
heated milks, the levels of monomeric κ- and β-caseins and α-lactalbumin are reduced, suggest-
ing that these proteins are involved in covalent linkages, whereas, in milk that has been heated
and then enzyme treated, the extent of reactivity of β-lactoglobulin is increased (Sharma et
al 2001). Lorenzen (2000) demonstrated that a combination of heating and transglutaminase
treatment can severely inhibit renneting. He ascribed this to a ‘sealing’ effect of β-lactoglobulin
cross-linked on to the surface of the micelles. This is consistent with the general lengthening of
the rennet coagulation time caused by heating alone, which is also ascribed to whey protein at
the surface of the micelle inhibiting the action of rennet (Fox and McSweeney 1998).
The majority of the processes cited above use traditional practice in combination with trans-
glutaminase treatment, either simultaneously or as a pre-treatment. From a structural point of
view, the key difference is the additional component of covalent bonding. This leads to altera-
tions in composition of the products, e.g. increased incorporation of whey proteins (Xaio-Qing
11
Han and Spradlin 2001) and/or marked changes in structure (Schorsch et al 2000).
2.3.6. The Plastein Reaction

A further specialised form of aggregation known as the plastein reaction has also been ap-
plied to milk proteins such as caseins and whey proteins (Sukan and Andrews 1982, Budtz and
Nielsen 1996). This reaction occurs when proteins have been extensively digested, and their
hydrolysates concentrated and then reacted further with more proteolytic enzymes (Brownsell
et al 2001). The reaction is considered to be complex with multiple pathways (Brownsell et al
2001). Although it has been considered that the reaction could include the formation of novel
polypeptide structures by the covalent linkage of peptides by condensation and/or transpepti-
dation, recent studies by Stevenson et al (1999) and Brownsell et al (2001) have concluded
that these reactions are not significant in casein hydrolysates and that the aggregation occurs
through physical processes.
2.3.7. High Pressure Coagulation.

High pressure processing of dairy protein systems can influence coagulation through many of
the mechanisms described earlier. In particular, high pressure processing is known to cause
reductions in casein micelle size, release of colloidal calcium phosphate, denaturation of whey
proteins and complex formation between β-lactoglobulin and casein, and in general both elec-
trostatic and hydrophobic interactions may be influenced by pressure (Huppertz et al 2002).
Whey proteins denature and lose solubility under pressure, and pressure-induced denatura-
tion of β-lactoglobulin increases with temperature and decreases with acidification.
Whey proteins self-aggregate under pressure, although the complexes formed are different
from those formed under heat. The sensitivities of specific whey proteins to denaturation are
different under pressure compared with under heat. Both hydrophobic and disulphide-linked
aggregates of β-lactoglobulin and α-lactalbumin form under pressure, and pressure-induced
whey protein aggregates are susceptible to precipitation over an acid pH range.
Yoghurt made from pressure-processed milk coagulates at a higher pH than yoghurt made
from heat-treated milk and the casein micelles are more densely packed in the coagulum. Gel
firmness increases with pressure, probably because of the increased surface area for interaction
of the smaller micelles.
Rennet gels formed from pressure-processed milks will incorporate some whey proteins (par-
ticularly β-lactoglobulin). Such gel networks may be denser, more fine stranded and more
continuous than those formed from unprocessed milks. Gel firmness can be increased. Hy-
drolysis of κ-casein can be impeded in pressure-treated milks by interactions with denatured
β-lactoglobulin.
Acidification of milk can cause coagulation under pressure and the coagulum structure varies
considerably with both pressure and time.
12
3. Characteristics of Coagulated Milk Proteins

To date, although there have been many studies investigating the various coagulation proc-
esses, there have been only a limited number of studies investigating the chemical characteris-
tics of the milk coagulum itself. As described above, many of the factors involved are common
to several of the different forms of coagulation. For example, calcium-mediated linkages are
implicated in coagulation brought about by heat, rennet, acid and ethanol and most probably
other forms as well. The pattern is further complicated in that many of the forces that are in-
volved in bringing about coagulation (e.g. hydrophobic, hydrogen bonding, calcium-mediated
and electrostatic forces) are also involved in maintaining the structure of casein micelles in nor-
mal milk (Horne 1998). Thus, they can be present both before and after coagulation although
the balance of forces and the specific proteins involved may have changed.
Some attempts have been made to characterise the interactions present within coagula using
solubility with specific dissociating agents. Lefebvre-Cases et al (1998) made a comparison of
experimental rennet and acid-coagulated gels and found that the predominant forces in rennet
gels were hydrophobic and calcium mediated in character, whereas bonding in acid gels relied
more on hydrophobic, hydrogen bonding and electrostatic interactions.
Nieuwenhuijse et al (1991) showed that the coagula formed by heat involved both ionic and
covalent bonding but that the balance of the different forces could vary depending on the pH
conditions. Gagnaire et al (2002) investigated the forces present in Emmental cheese and con-
cluded that calcium cross-linking, electrostatic interactions and, to a lesser degree, hydrogen
bonds all contributed to the formation and stability of the cheese matrix.
Marchesseau and Cuq (1995) and Marchesseau et al (1997) used similar techniques to study
processed cheese and found that, in addition to ionic interactions, hydrogen bonding and hy-
drophobic interactions were significant in these products, particularly at higher cooking tem-
peratures. Their later study also highlighted that small changes in pH and ionic strength during
processing could introduce large changes in the final product characteristics.
4. Changes in Coagulated Dairy Products During Further

Processing and Storage
Many different post-coagulation procedures, which help to create variety and diversity in dairy
products, occur in manufacture. The variety is very large. In 1981, the IDF catalogue of
cheeses listed 510 different products (Burkhalter 1981), and Tamime and Robinson (1985)
listed more than 45 variants of yoghurt. For cheese, simplification of this diversity through the
development of classification systems has been attempted by several authors but a universal
system has not yet been accepted (Fox et al 2000). For this document, a number of typical
processes and events are examined. Generally, these come under the headings of processing
events or ripening events.
4.1. Processing Events

In cheese manufacture, typical post-coagulation processing involves cutting of the curd, whey
separation, acidification, special operations such as cheddaring or stretching, salting, moulding
and pressing (Fox et al 2000). Although acidification is mentioned here as a post-coagulation
process, its potential to contribute to coagulation, as described above, should not be neglect-
ed.
These processes occur to different extents depending on the type of cheese. For example,
Cheddar and many so-called ‘hard’ cheeses undergo renneting at about 30˚C, cutting of the
coagulum into small pieces and cooking at about 40˚C, whey drainage, the cheddaring process
(in which the curds are textured) followed by dry milling and salt addition once sufficient acidity
has developed and, finally, pressing (Fox et al 2000). In Pasta filata cheeses such as Mozza-
rella-type or Pizza cheese, textured curds are further modified by being kneaded, stretched and
folded while immersed in hot water (approximately 70˚C) to develop the desired texture.
13
These processes create the characteristic macroscopic and microscopic textures of these
products that have been observed by light (Fox et al 2000), confocal (Auty et al 2001) or elec-
tron (Kalab 1993, Oberg et al 1993) microscopy.
In contrast, surface-mould-ripened cheeses, such as Camembert types, are cut but not
cooked and the curd is placed directly in moulds, where whey drainage occurs. The soft tex-
ture of these cheeses is created by the action of the surface moulds, which is discussed below.
Internal-mould-ripened cheeses, such as the Blue veined cheeses, may or may not be cooked
(Fox et al 2000) but are inoculated with moulds and processing and storage conditions are
utilised to maximise mould growth. Acid-coagulated cheeses (Cream cheese, Cottage cheese,
Quarg etc) are acidified to induce coagulation and then processed simply by draining the whey
and packing. Industrially, processes such as centrifugal separation or ultrafiltration are used
and, in some cases, other additions such as hydrocolloid stabilisers are made and these are
incorporated with additional mixing and sometimes homogenisation (Fox et al 2000). Heat/
acid-set cheeses such as Ricotta and Mascarpone involve heating followed by acidification to
establish the curd rather than a simple collection of the curd and drainage in moulds. Whereas
‘in-container’ set-style yoghurt gels receive little processing after coagulation by acidification,
stirred-style yoghurts are stirred, mixed and packaged after coagulation and are often mixed
with a range of other ingredients (Tamime and Robinson 1985).
The physical/mechanical actions involved in the processing of these products have a major
role in the development of the morphology of the products. The stretching process in Mozzarel-
la-type cheese manufacture produces longitudinal strands of protein within the product (Oberg
et al 1993) and elongated pools of fat (Guinee et al 1999). This gives the product a ‘grain’, so
that the patterns observed when cutting with the grain (longitudinal section) are quite different
from those seen when cutting across the grain (transverse section) (Guinee et al 1999).
Some orientation of fat has also been described in Cheddar-type cheeses by Auty et al
(2001), who attributed it to the pressing stage of manufacture. Fox et al (2000) also suggested
that the coalescence of fat globules leading to the formation of fat pools and free fat occurs in
all cheeses as a post-coagulation event. The salt content of Mozzarella-type cheeses has also
been shown to influence their free oil content (Kindstedt et al 1992, Rowney et al 2000). These
changes affect the macro- and microscopic structure of the protein, and the structure is no
longer the same as when coagulation first took place.
4.2. Ripening Events

In contrast to the processes described above, maturation or ripening of cheeses and dairy prod-
ucts alters the chemical characteristics of the chemical components of the products as well as
their physical characteristics.
With respect to protein coagulation, the key factors are proteolysis, the chemical breakdown
of the protein network and pH change. Proteolysis involves the actions of residual rennet, pro-
teinases secreted by microorganisms in the product and/or endogenous milk enzymes (Visser
1998). The proteins are initially broken down into large fragments which are then further de-
graded into small peptides. In studies of Cheddar-type cheeses, it has been shown that the
initial proteolysis is generally caused by rennet, but that the production of small peptides and
free amino acids is due primarily to the action of enzymes from starter bacteria. Proteolytic
activity varies between types and strains of starter bacteria.
The amount of rennet that remains active in the cheese varies from type to type. Fox et al
(2000) suggested that almost none of the original added rennet is retained in high cook cheeses
such as the Mozzarella, Parmesan and Emmental types, whereas about 6% remains active in
Cheddar types and about 20% in Camembert types.
The indigenous milk enzyme plasmin is a significant contributor to proteolysis in the high
cook cheeses, where the added rennet is inactivated by heating (Fox and McSweeney 1998).
Plasmin catalyses the breakdown of β-casein to γ-caseins in a wide range of cheeses (Fox et
14
al 2000). Enzymes from a wide range of starter and non-starter bacteria also contribute to
the diversity of proteolysis. For example, more than 60 different organisms are listed by Fox
et al (2000, Table 11-3). The rate and the pattern of proteolysis vary greatly both within and
between different cheese types. They vary depending on the age of the cheese and the level
of added salt (Fox et al 2000), and may also vary from place to place within a cheese sample
(Sousa et al 2001).
Proteolysis in cheese has been followed by analysing either the water-soluble fraction (which
increases as proteolysis occurs) or the water-insoluble fraction (which decreases). A study of
proteolysis in Cheddar cheese by Gouldsworthy et al (1999) demonstrated that, over 246 days
of maturation, almost all of the αs1-casein and 60–70% of the β-casein became water soluble,
whereas there was little change in the levels of para-κ-casein. The proteolysis process results
in a complex mix of protein fragments in cheese. For example, Mooney et al (1998) confirmed
the presence of 23 different water-insoluble peptides in 20-week-old Cheddar arising from the
breakdown of αs1- and β-casein. Studies on Mozzarella cheese (Farkye et al 1991) showed that
9.66% of the total nitrogen was soluble in water after 14 days of maturation at 4˚C (up from
4.07% in the fresh cheese). Tsuda et al (1994) found that the proportion of water-soluble ni-
trogen in Camembert-type cheese increased from 1.66% in the fresh cheese to 20.03% after
aging at 4˚C for 28 days. The potential for rapid proteolysis in Camembert-type cheeses is
emphasised by the identification of 48 small peptides in the whey from Camembert cheese ob-
tained at the demoulding stage (i.e. 20 h following coagulation) by Boutrou et al (2001). These
peptides were derived from all four caseins (αs1 , as2-, β- and κ-casein).
The pH of different cheese types alters with time as the cheese matures. The most signifi-
cant changes are seen in surface-mould-ripened cheeses (i.e. the Camembert type) because of
the metabolism of first lactate and then proteins to ammonia, which can then diffuse into the
cheese (Fox et al 2000). This effect sets up a gradient of pH between the surface and the core
of the cheese, leading to migration of calcium phosphate to the surface of the cheese, where it
becomes insoluble and precipitates as Ca3(PO4)2. This lowers the concentration of soluble cal-
cium and it has been proposed by Karahadian and Lindsay (1987) that this reduction in calcium
leads to the characteristic softening of the surface-mould-ripened cheeses through the removal
of calcium cross-links formed between proteins in the cheese matrix.
The pH of the internal-mould-ripened cheeses (i.e. the Blue cheese type) is also modified
in this way whereas surface yeasts can also modify the pH of selected varieties (Gobbetti et al
1997). Changes in pH of Cheddar-type cheeses were observed by Fenelon and Guinee (2000),
with the pH of reduced-fat cheeses increasing during maturation but with the pH of full-fat
cheeses falling over 120 days.
The pH of the cheese can also influence the apparent extent of proteolysis in cheese depend-
ing on the method used, because extractability of the different proteolysis products is also
affected by the pH (Sousa et al 2001). It has also been suggested that higher pH leads to
increased activation of plasmin, leading to higher levels of plasmin-mediated proteolysis (Rich-
ardson and Elston 1984).
Proteolysis also occurs in yoghurt (Laws and Marshall 2001). In this case, the proteolytic en-
zymes are secreted by the cultures used to ferment the yoghurt and the proteolytic activity var-
ies between types and strains of the starter bacteria (Shihata and Shah 2000). As in the case
of cheese, the pattern of proteolysis is complex. For example, Schieber and Bruckner (2000)
reported the identification of 30 different peptides derived from the proteolytic breakdown of
αs1-, β- and κ-caseins. Proteolytic activity can lead to the alteration of the structure of yoghurt
on aging, leading to rheological changes (Afonso and Maia 1999).
15
5. Verification of Coagulation
Coagulation is the conversion or transformation of the liquid milk to a range of solid or semi-
solid dairy products.
Clearly, there are a number of processes by which coagulation can take place and, just as
clearly, there are a number of processes that take place after coagulation that have an impact
on both the composition and the texture of the final coagulated product. For example, in cheese
making, post-coagulation processes include cooking, moisture loss, development of acidity,
draining (curd separation), cheddaring, milling, salting, pressing, brining, stretching and prote-
olysis during (long) storage at various temperatures. Variations in these post-coagulation proc-
esses and their use in varying combinations, in part, determine the variety of cheese made.
This document does suggest that, regardless of the means, coagulation does result from
destabilisation of the micelles in some form, and that the insoluble, destabilised micelles then
form structures that are dependent, to some extent, on the processing conditions following
coagulation.
However, the complexity of the coagulation reaction and the myriad of post-coagulation proc-
esses make it difficult to identify one method that can be used universally to determine if co-
agulation has taken place.
Nevertheless, there are a number of methods and procedures that can be used to view and/or
characterise dairy structures and the results may be used to support the argument that coagu-
lation has occurred.
5.1. Microscopic Methods

There are a number of ‘visual’ techniques that can be used to show the structural elements of
dairy foods. The type of technique to be used will depend on the product composition, because
some techniques are more proficient at illustrating the structure than others. The technique se-
lected will also depend on the scope of the study and the degree of detail required. Listed below
are the more ‘common’ methods for evaluating food structure; however, it should also be noted
that there are other more complex ways of determining structure that are not covered here.
5.1.1. Low Magnification/Low Resolution Techniques

These techniques will give a general view of the fat and protein structure and the overall ho-
mogeneity of the product. The approximate limit of resolution is 0.2 µm, which is affected by
the sample type and the preparation method.
Confocal Laser Scanning Microscope (Fluorescent Technique)
The confocal laser scanning microscope (CLSM) offers the same magnification range as a stand-
ard light microscope, but with the added benefit of specifically labelling (by fluorescent dyes)
different structures of interest such as fat and protein. Clear images of the structure can be
obtained from a relatively ‘thick section’. The confocal pinhole screens out all the information
above and below the focal plane, resulting in clearer images — a benefit not available on a tradi-
tional light microscope. It is a rapid and relatively non-invasive method of evaluating food-type
structures. It is also able to scan and store a series of images that can then be processed to
give a three-dimensional image of the structure being examined.
Examples of where this technique has been used to view food structure can be found in Lucey
et al (1998) and Auty et al (2001).
‘Double-dye’ Method (Histochemical Staining Technique)
The ‘double-dye’ method is another way of viewing structure by a standard light microscope
(under brightfield illumination). A relatively thin (5–10 µm) fresh (frozen) specimen is sub-
jected to a comprehensive staining procedure. The protein is stained by a Schiff base reaction
16
followed by a simple ‘association’ staining of the fat. As the sample undergoes fairly rigorous
preparation, it is not clear how much of the procedure affects the ‘natural’ structure of the prod-
uct. An example of this technique can be found in the work of Naganawa et al (2002).
Resin-embedded Samples
Samples that have been prepared for transmission electron microscopy (TEM) (fixed, stained
and embedded) can be sectioned to approximately 1 µm and stained with Toluidine Blue and
then viewed using a light microscope under brightfield illumination. Although the magnification
range is the same as for the confocal technique, this technique can provide more information
about the structure of some products. A disadvantage with this technique is that sample prepa-
ration (for TEM) is time consuming and invasive and the technique is mainly used only when
samples are to be examined using TEM, or when a CLSM is not available. An example of this
technique can be found in the work of Kalab (1981).
5.1.2. Medium Magnification/High Resolution Technique

Scanning Electron Microscopy (SEM)
This technique is primarily used for the identification of crystalline material (SEM-energy dis-
persive spectroscopy (EDS)) in ‘gel-type’ dairy products. The sample preparation results in the
removal of most of the fat. It is a technique that is more useful for viewing the surface morphol-
ogy of products such as powders. The resolution is approximately 20 nm and is affected by the
sample type and the preparation method.
5.1.3. High Magnification/High Resolution Technique

Transmission Electron Microscopy (TEM)
TEM involves the fixing, staining and embedding of the sample so that 80 nm thick sections can
be sliced using an ultramicrotome. TEM can provide intricate detail of the protein network of
products such as cheese. It is used to determine the homogeneity of the protein phase. In-
formation about the fat globule membrane can also be obtained, but overall a CLSM or a light
microscope allows for a more accurate estimation of fat globule size and distribution. The reso-
lution is 0.2 nm and, as with SEM, is affected by the sample type and the preparation method.
5.2. Rheological Methods

The rheological properties of a material can be measured by compression testing and/or small
amplitude oscillatory rheometry (SAOR). Rheological and fracture properties derived from
these tests show how liquid-like or how solid-like a material is (i.e. its viscous and/or elastic
characteristics).
A commonly used type of rheological test to characterise solid-like dairy products (e.g.
cheese) is to apply a constant strain rate (partly related to the speed of compression) during
compression and to measure stress (van Vliet et al 1991). Specific parts of this derived stress
versus strain curve can correlate with sensory properties including rigidity, firmness, toughness
and longness (Zoon 1991). The addition of a tension stroke after this compression enables the
estimation of adhesiveness (Zoon 1991).
A commonly used type of rheological test to characterise liquid-like dairy products (e.g. yo-
ghurt) is to apply a constant strain rate (partly related to the speed of rotation) in shear mode
and to measure viscosity or shear stress. Viscosity can correlate with sensory properties such
as thickness in the mouth (Hill 1991).
Dairy products including cheese are, to a first approximation, viscoelastic materials exhibit-
ing both viscous (like a liquid) and elastic (like a solid) properties, depending on the time scale
of the applied deformation (Walstra and Peleg 1991). Therefore, the viscoelasticity (degree of
liquid-like-ness or solid-like-ness) is sometimes measured, typically varying applied stress or
strain sinusoidally and measuring strain or stress respectively (van Vliet et al 1991).
17
Although the above methods can be used to define and differentiate the structure of the co-
agulated product, the results by themselves do not necessarily demonstrate that coagulation
has occurred. What is needed, in addition, is an appreciation of the processing techniques that
provide for the diverse range of coagulum properties and an appreciation of the influence of
post-coagulation events.
6. References
Afonso I M & Maia J M (1999)
Rheological monitoring of structure evolution and development in stirred yoghurt. Journal of
Food Engineering, 42, 183–190.
Allen R E ed. (1991)

Concise Oxford Dictionary, 8th edn, Oxford University Press, Oxford, p. 215.
Andrén A (2002)
Rennets and coagulants. In Encyclopedia of Dairy Sciences, Roginski H, Fuquay J W and Fox P
F eds, Academic Press, London, pp. 281–286.
Auty M A E, Twomey M, Guinee T P & Mulvihill D M (2001)

Development and application of confocal scanning laser microscopy methods for studying the
distribution of fat and protein in selected dairy products. Journal of Dairy Research, 68, 417–
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28
COAGULATION OF MILK: PROCESSES AND CHARACTERISTICS
ABSTRACT
Review of the milk coagulation reaction as encountered in the manufac-

ture of dairy products, covering definitions, traditional and non-tradition-
al processes of coagulation, characteristics of coagulated milk proteins,
changes in coagulated dairy products during processing and storage (in-
cluding ripening), methods of analysis to verify coagulation, and refer-
ences.
Keywords: acid, casein, cheese, CLSM, coagulation, covalent, cross-link-

ing, dairy, enzymes, ethanol, flocculation, gelation, hydrophobic, ionic,
methods, micelle, milk, plastein, polysaccharides, precipitation, protein,
proteolysis, proteolytic, rennet, rheological, ripening, SEM, TEM, Van der
Waals
28 pp - English only
Bulletin N°420/2007 - 24 Euro (electronic) - Date: 2007

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litre........................................................... Not liter unless the author is American
ml, mg,.................................................. Space between number and ml, mg,...
skimmilk................................................. One word if adjective, two words if substantive
sulfuric, sulfite, sulfate............................... Not sulphuric, sulphite, sulphate (as agreed by IUPAC)
AOAC International............................. Not AOACI
programme............................................ Not program unless a) author is American or b) computer program
milk and milk product........................ rather than “milk and dairy product” - Normally some latitude can be allowed in non scientific texts
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Decimal comma................................... in Standards (only) in both languages (as agreed by ISO)
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litre........................................................... ø l
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Coagulation of Milk: Processes and Characteristics: of The International Dairy Federation

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Coagulation of Milk: Processes and Characteristics: of The International Dairy Federation

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Bulletin

of the International Dairy Federation

Bulletin of the International Dairy Federation 420/2007

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Bulletin of the International Dairy Federation

Coagulation of Milk: Processes and Characteristics

2.1. Definitions of Coagulation

Table 1. Coagulation of Milk Proteins by Various Agents

2.2. Traditional Processes of Coagulation

vides steric stabilisation.

2.2.2. Enzymatic Coagulation

Rennet coagulation occurs in two main phases.

Figure 1. Coagulation of milk (Fox et al 2000).

Coagulation by Other Proteolytic Enzymes

2.2.3. Acid Coagulation

2.3. Non-traditional Processes of Coagulation

2.3.2. Low Temperature Coagulation

2.3.3. Proteolytic Coagulation of Whey Proteins

2.3.4. Coagulation by Polysaccharides

2.3.5. Coagulation by Enzymatic Cross-linking

2.3.6. The Plastein Reaction

2.3.7. High Pressure Coagulation.

3. Characteristics of Coagulated Milk Proteins

4. Changes in Coagulated Dairy Products During Further

4.1. Processing Events

4.2. Ripening Events

5.1. Microscopic Methods

5.1.1. Low Magnification/Low Resolution Techniques

Confocal Laser Scanning Microscope (Fluorescent Technique)

‘Double-dye’ Method (Histochemical Staining Technique)

5.1.2. Medium Magnification/High Resolution Technique

5.1.3. High Magnification/High Resolution Technique

5.2. Rheological Methods

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