10 Chemistry and biochemistry of cheese and

fermented milks

10.1 Introduction

Cheese is a very varied group of dairy products, produced mainly in Europe,

North and South America, Australia and New Zealand and to a lesser
extent in North Africa and the Middle East, where it originated during the
Agricultural Revolution, 6000-8000 years ago. Cheese production and con-
sumption, which vary widely between countries and regions (Appendices
10A and lOB), is increasing in traditional producing countries (2-4% p.a.
for several years) and is spreading to new areas. On a global scale, 30% of
all milk is used for cheese; the proportion is about 40% in North America
and about 50% in the European Union.
Although traditional cheeses have a rather high fat content, they are rich
sources of protein and in most cases of calcium and phosphorus and have
anticarigenic properties; some typical compositional data are presented in
Table 10.1. Cheese is the classical example of a convenience food: it can be
used as the main course in a meal, as a dessert or snack, as a sandwich filler,
food ingredient or condiment.
There are at least 1000 named cheese varieties, most of which have very
limited production. The principal families are Cheddar, Dutch, Swiss and
Pasta filata (e.g. Mozzarella), which together account for about 80% of total
cheese production. All varieties can be classified into three superfamilies
based on the method used to coagulate the milk, i.e. rennet coagulation
(representing about 75% of total production), isoelectric (acid) coagulation
and a combination of heat and acid (which represents a very minor
group).
Production of cheese curd is essentially a concentration process in which
the milkfat and casein are concentrated about tenfold while the whey
proteins, lactose and soluble salts are removed in the whey. The acid-
coagulated and acid/heat-coagulated cheeses are normally consumed fresh
but the vast majority of rennet-coagulated cheeses are ripened (matured) for
a period ranging from 3 weeks to more than 2 years, during which numerous
microbiological, biochemical, chemical and physical changes occur, resulting
in characteristic flavour, aroma and texture. The biochemistry of cheese
ripening is very complex and is not yet completely understood.
380 DAIRY CHEMISTRY AND BIOCHEMISTRY

Table 10.1 Composition of selected cheeses (per 100 g)

Water Protein Fat Cholesterol Energy

Cheese type (9) (g) (g) (mg) (kJ)
Brie 48.6 19.3 26.9 100 1323
Caerphilly 41.8 23.2 31.3 90 1554
Camern bert 50.7 20.9 23.1 15 1232
Cheddar 36.0 25.5 34.4 100 1708
Cheshire 40.6 24.0 31.4 90 1571
Cottage 79.1 13.8 3.9 13 413
Cream cheese 45.5 3.1 47.4 95 1807
Danish blue 45.3 20.1 29.6 75 1437
Edam 43.8 26.0 25.4 80 1382
Emmental 35.7 28.7 29.7 90 1587
Feta 56.5 15.6 20.2 70 1037
Fromage frais 77.9 6.8 7.1 25 469
Gouda 40.1 24.0 31.0 100 1555
Gruyere 35.0 21.2 33.3 100 1695
Mozzarella 49.8 25.1 21.0 65 1204
Parmesan 18.4 39.4 32.1 100 1880
Ricotta 72.1 9.4 11.0 50 599
Roquefort 41.3 19.7 32.9 90 1552
Stilton 38.6 22.7 35.5 105 1701

10.2 Rennet-coagulated cheeses

The production of rennet-coagulated cheeses can, for convenience, be

divided into two phases: (1) conversion of milk to curds and ( 2 ) ripening of
the curds.

10.2.1 Preparation and treatment of cheesemilk

The milk for most cheese varieties is subjected to one or more pre-
treatments (Table 10.2). The concentrations of fat and casein and the ratio
of these components are two very important parameters affecting cheese
quality. While the concentrations of these components in cheese are deter-
mined and controlled by the manufacturing protocol, their ratio is regulated
by adjusting the composition of the cheesemilk. This is usually done by
adjusting the fat content by blending whole and skimmed milk in propor-
tions needed to give the desired fat :casein ratio in the finished cheese, e.g.
1.0:0.7 for Cheddar or Gouda. It should be remembered that about 10% of
the fat in milk is lost in the whey while only about 5% of the casein is lost
(unavoidably, see section 10.2.2).
With the recent commercial availability of ultrafiltration, it has become
possible to increase the concentration of casein, thus levelling out seasonal
variations in milk composition and consequently in gel characteristics and
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 381

Table 10.2 Pre-treatment of cheese milk

Standardization of fat: protein ratio

Addition of skim milk
Removal of some fat
Addition of ultrafiltration retentate
Addition of CaCI,
Adjustment of pH (e.g. by gluconic acid-6-lactone)
Removal or killing of contaminating bacteria
Thermization (e.g. 65°C x 15 s)
Pasteurization (e.g. 72°C x 15 s)
Bactofugation
Microfiltration

cheese quality. The capacity of a given plant is also increased by pre-

concentrating milk by ultrafiltration.
The pH and the concentration of calcium in milk also vary, with
consequential effects on the properties of renneted milk gels. The addition
of CaCl, to cheesemilk (0.02%) is widely practised and adjustment and
standardization of milk pH by using the acidogen, gluconic acid-d-lactone
(GDL), is recommended and commercially practised on a limited scale.
Although raw milk is still widely used for cheese manufacture, e.g.
Parmigiano-Reggiano (Italy), Emmental (Switzerland), Comte and Beaufort
(France) and many less well known varieties, both on a factory and
farmhouse scale, most Cheddar and Dutch-type cheeses are produced from
pasteurized milk (HTST; c. 72°C x 15 s). Pasteurization is used primarily to
kill pathogenic and spoilage bacteria. However, desirable indigenous bac-
teria are also killed by pasteurization and it is generally agreed that cheese
made from pasteurized milk ripens more slowly and develops a less intense
flavour than raw milk cheese, apparently because certain, as yet unidentified,
indigenous bacteria are absent. At present, some countries require that all
cheese milk should be pasteurized or the cheese aged for at least 60days
(during which time pathogenic bacteria die off). A global requirement for
pasteurization of cheesemilk has been recommended but would create
restrictions for international trade in cheese, especially for many of those
with ‘Appellation d’Origine Protegee’ status. Research is under way to
identify the important indigenous microorganisms in raw milk cheese for use
as inoculants for pasteurized milk. While recognizing that pasteurization is
very important in ensuring safe cheese, pH (below about 5.2) and water
activity ( a w ,which is controlled by addition of NaCl) are also critical safety
hurdles.
Milk may be thermized (c. 65°C x 15s) on receipt at the factory to
reduce bacterial load, especially psychrotrophs, which are heat labile. Since
thermization does not kill pathogens, thermized milk is usually fully
pasteurized before cheesemaking.
382 DAIRY CHEMISTRY AND BIOCHEMISTRY

Clostridium tyrobutyricum (an anaerobic spore-former) causes late gas

blowing (through the production of H, and CO,) and off-flavours (butanoic
acid) in many hard ripened cheeses; Cheddar-type cheeses are major
exceptions. Contamination of cheese milk with clostridial spores can be
avoided or kept to a very low level by good hygienic practices (soil and
silage are the principal sources of clostridia) but they are usually prevented
from growing through the use of sodium nitrate (NaNO,) or, less frequently,
lysozyme, and/or removed by bactofugation (centrifugation) or microfiltra-
tion.

10.2.2 Conversion of milk to cheese curd

Typically, five steps, or groups of steps, are involved in the conversion of
milk to cheese curd: coagulation, acidification, syneresis (expulsion of whey),
moulding/shaping and salting. These steps, which partly overlap, enable the
cheesemaker to control the composition of cheese, which, in turn, has a
major influence on cheese ripening and quality.

Enzymatic coagulation of milk. The enzymatic coagulation of milk involves

modification of the casein micelles via limited proteolysis by selected
proteinases, called rennets, followed by calcium-induced aggregation of the
rennet-altered micelles:

Rennet
Casein ____.* Para-casein + Macropeptides
Ca?', - 30°C
Gel

If present, the fat globules are occluded in the gel but do not participate in
the formation of a gel matrix.
As discussed in Chapter 4, the casein micelles are stabilized by ti-casein,
which represents 12-15% of the total casein and is located mainly on the
surface of the micelles such that its hydrophobic N-terminal region reacts
hydrophobically with the calcium-sensitive clsl-, cts2- and 0-caseins while its
hydrophilic C-terminal region protrudes into the surrounding aqueous
environment, stabilizing the micelles by a negative surface charge and steric
stabilization.
Following its isolation in 1956, it was found that ti-casein is the only
casein hydrolysed during the rennet coagulation of milk and that it was
hydrolysed specifically at the Phe,,,-Met,,, bond, producing para-lc-
casein (K-CN fl- 105) and macropeptides (f106- 169; also called glycomac-
ropeptides since they contain most or all of the sugar groups attached to
ti-casein) (Figure 10.1). The hydrophilic macropeptides diffuse into the
surrounding medium while the para-#-casein remains attached to the
 CHEMISTRY AND BIOCHEMISTRYOF CHEESE AND FERMENTED MILKS 383

1
Fyro Glu-Glu-Gln-Asn-Gln-Glu-GIn-Pro-Ile-Arg-Cys-GIu-Lys-Asp-GIu-Arg-Phe-Phe-Ser-Asp-

21
Lys-Ile-Ala-Lys-Tyr-lle-Pro-lle-GIn-Tyr-Val-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-
41
Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr-
61
Tyr-AIa-Lys-Pro-Ala-Ala-Val-Arg-Ser-Pto-Ala-G1n-lle-Leu-Gln-Trp-GIn-Val-Leu-Ser-
81
Asn-Thr-Val-Pro-Ala-L n-Ala-Gln-Pro-Thr-Thr-Met-Ala-Arg-His-Pro-His-
ys-Ser-Cys-G1
101 105 106
Pro-His-Leu-Ser-Ph~et-Ala-lle-Pro-Pro-Lys-Ly~-Asn-Gln-As~-~ys-~r-Glu-IIe-Pro-
121 Ile (Variant B)
Thr-He-Asn-Thr-Ile-Ala-Ser-Gly-Glu-Pro-Thr-
Ser-Thr-Pro-Thr - -Glu-Ala-Val-Glu-
Thr (VariantA)
141 Ala (Variant8)
Ser-Thr-Val-Ala-Thr-Leu-Glu--SerP - Pro-Glu-Val-lle-Glu-Ser-Pro-Pro-G1u-Ile-Asn-
Asp (VariantA)
161 169
Thr-Val-GIn-Val-Thr-Ser-Thr-Ala-Val.OH
Figure 10.1 Amino acid sequence of K-casein, showing the principal chymosin cleavage site (I);
oligosaccharides are attached at some or all of the threonine residues shown in italics.

micelle core (the macropeptides represent c. 30% of Ic-casein, i.e. 4-5% of

total casein; this unavoidable loss must be considered when calculating the
yield of cheese). Removal of the macropeptides from the surface of the casein
micelles reduces their zeta potential from about -20 to -1OmV and
removes the steric stabilizing layer. The proteolysis of ic-casein is referred to
as the primary (first) phase of rennet-coagulation.
When about 85% of the total ic-casein in milk has been hydrolysed, the
collojdal stability of the micelles is reduced to such an extent that they
coagulate at temperatures greater than about 20°C (c. 30°C is used in
cheesemaking), an event referred to as the secondary phase of rennet
coagulation. Calcium ions are essential for the coagulation of rennet-altered
micelles (although the binding of Ca2+ by casein is not affected by
renneting).
The Phe,,,-Met,,, bond of ic-casein is several orders of magnitude more
sensitive to rennets than any other bond in the casein system. The reason(s)
for this unique sensitivity has not been fully established but work on
synthetic peptides that mimic the sequence of Ic-casein around this bond has
provided valuable information. The Phe and Met residues themselves are
not essential, e.g. both Phe,,, and Met,,, can be replaced or modified
without drastically changing the sensitivity of the bond - in human, porcine
and rodent Ic-caseins, Met,,, is replaced by Ile or Leu, and the proteinase
from Cryphonectria parasitica (section 10.2.2.2), hydrolyses the bond
Ser,,,-Phe,,, rather than Phe,,,-Met,,,. The smallest Ic-casein-like pept-
384 DAIRY CHEMISTRY AND BIOCHEMISTRY

Table 10.3 Kinetic parameters for hydroloysis of K-casein peptides by chymosin at pH 4.7
(compiled from Visser et al., 1976; Visser, Slangen and van Rooijen, 1987)

k,,,
Peptide Sequence (s- I )

S.F.M.A.I. 104-108 0.33 8.50 0.038

S.F.M.A.I.P. 104-109 1.05 9.20 0.1 14
S.F.M.A.I.P.P. 104-1 10 1.57 6.80 0.231
S.F.M.A.I.P.P.K. 104- I 1 1 0.75 3.20 0.239
L.S.F.M.A.I. 103-108 18.3 0.85 21.6
L.S.F.M.A.I.P. 103-109 38.1 0.69 55.1
L.S.F.M.A.I.P.P. 103-110 43.3 0.41 105.1
L.S.F.M.A.I.P.P.K. 103- 111 33.6 0.43 78.3
L.S.F.M.A.I.P.P.K.K. 103- 112 30.2 0.46 65.3
H.L.S.F.M.A.1 102-108 16.0 0.52 30.8
P.H.L.S.F.M.A.1 101- 108 33.5 0.34 100.2
H.P.H.P.H.L.S.F.M.A.I.P.P.K. 98- 111 66.2 0.026 2509
98-111" 46.2" 0.029" 1621"
k--Caseinb 2-20 0.001-0.005 200-2000
L.S.F.(NO,)Nle A.L.OMe 12.0 0.95 12.7

"pH 6.6.
bpH 4.6.

ide hydrolysed by chymosin is Ser.Phe.Met.Ala.Ile (K-CNfl04- 108); extend-

ing this peptide from its C and/or N terminus increases its susceptibility to
chymosin (i.e. increases kcat/K,,,);the peptide K-CN f98-111 is as good a
substrate for chymosin as whole K-casein (Table 10.3). Ser,,, appears to be
essential for cleavage of the Phe,,,-Met,,, bond by chymosin, and the
hydrophobic residues, Leu,,,, Ala,,, and Ilelo8 are also important.

Rennets. The traditional rennets used to coagulate milk for most cheese
varieties are prepared from the stomachs of young calves, lambs or kids by
extraction with NaCl (c. 15%) brines. The principal proteinase in such
rennets is chymosin; about 10% of the milk-clotting activity of calf rennet is
due to pepsin. As the animal ages, the secretion of chymosin declines while
that of pepsin increases; in addition to pepsin, cattle appear to secrete a
chymosin-like enzyme throughout life.
Like pepsin, chymosin is an aspartyl (acid) proteinase, i.e. it has two

-
essential aspartyl residues in its active site which is located in a cleft in the
globular molecule (molecular mass 36 kDa) (Figure 10.2). Its pH opti-
mum for general proteolysis is about 4, in comparison with about 2 for
pepsins from monogastric animals. Its general proteolytic activity is low
relative to its milk-clotting activity and it has moderately high specificity for
bulky hydrophobic residues at the PI and Pi positions of the scissile bond.
Its physiological function appears to be to coagulate milk in the stomach of
the neonate, thereby increasing the efficiency of digestion, by retarding
discharge into the intestine, rather than general proteolysis.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 385

Figure 10.2 Schematic representation of the tertiary structure of an aspartyl proteinase,

showing the cleft which contains the active site; arrows indicate p structures and cylinders the
%-helices(from Foltmann, 1987).

Due to increasing world production of cheese and the declining supply

of young calf stomachs (referred to as vells), the supply of calf rennet has
been inadequate for many years. This has led to a search for suitable
substitutes. Many proteinases are capable of coagulating milk but most are
too proteolytic relative to their milk-clotting activity, leading to a decrease
in cheese yield (due to excessive non-specific proteolysis in the cheese vat
and loss of peptides in the whey) and defects in the flavour and texture of
the ripened cheese, due to excessive or incorrect proteolysis. Only six
proteinases are used commercially as rennet substitutes: porcine, bovine and
chicken pepsins and the acid proteinases from Rhizomucor miehei, R. pusillus
and Cryphonectria parasitica. Chicken pepsin is quite proteolytic and is used
widely only in Israel (for religious reasons). Porcine pepsin enjoyed limited
success about 30years ago, usually in admixtures with calf rennet, but it is
very sensitive to denaturation at pH values above 6 and may be denatured
extensively during cheesemaking, leading to impaired proteolysis during
ripening; it is now rarely used as a rennet substitute. Bovine pepsin is quite
effective and many commercial calf rennets contain up to 50% bovine
pepsin. Rhizomucor miehei proteinase, the most widely used microbial
rennet, gives generally satisfactory results. Cryphonectria parasitica pro-
teinase is, in general, the least suitable of the commercial microbial rennet
substitutes and is used only in high-cooked cheeses in which extensive
denaturation of the coagulant occurs, e.g. Swiss-type cheeses.
The gene for calf chymosin has been cloned in Kluyveromyces marxianus
var. lactis, Aspergillus niger and E. coli. Microbial (cloned) chymosins have
386 DAIRY CHEMISTRY AND BIOCHEMISTRY

given excellent results in cheesemaking trials on various varieties and are

now widely used commercially, although they are not permitted in some
countries. Significantly, they are accepted for use in vegetarian cheeses. The
gene for R. miehei proteinase has been cloned in A . oryzae; the resultant
product, Marzyme GM, is commercially available (Texel, Stockport, UK)
and is reported to be a very effective coagulant.

Coagulation of rennet-altered micelles. When c. 85% of the total u-casein

has been hydrolysed, the micelles begin to aggregate progressively into a gel
network. Gelation is indicated by a rapid increase in viscosity ( q ) (Figure
10.3). Coagulation commences at a lower degree of hydrolysis of rc-casein if
the temperature is increased, the pH reduced or the Ca2+ concentration
increased.

0 20 40 M) RO
Iof visunlly ohxrvcd dolling time

Figure 10.3 Schematic representation of the rennet coagulation of milk. (a) Casein micelles with
intact ti-casein layer being attacked by chymosin (C); (b) rnicelles partially denuded of ti-casein;
(c) extensively denuded micelles in the process of aggregation; (d) release of macropeptides (+)
and changes in relative viscosity (B)during the course of rennet coagulation.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 387

The actual reactions leading to coagulation are not known. Ca2+ are
essential but Ca-binding by caseins does not change on renneting. Colloidal
calcium phosphate (CCP) is also essential: reducing the CCP concentration
by more than 20% prevents coagulation. Perhaps, hydrophobic interactions,
which become dominant when the surface charge and steric stabilization are
reduced on hydrolysis of K-casein, are responsible for coagulation (the
coagulum is soluble in urea). The adverse influence of moderately high ionic
strength on coagulation suggests that electrostatic interactions are also
involved. It is claimed that pH has no effect on the secondary stage of rennet
coagulation, which is perhaps surprising since micellar charge is reduced by
lowering the pH and should facilitate coagulation. Coagulation is very
temperature-sensitive and does not occur below about 18"C, above which
the temperature coefficient, Qlo,is approximately 16.

Factors that afect rennet coagulation. The effect of various compositional

and environmental factors on the primary and secondary phases of rennet
coagulation and on the overall coagulation process are summarized in
Figure 10.4.
No coagulation occurs below 20"C, due mainly to the very high tempera-
ture coefficient of the secondary phase. At higher temperatures (above
55-60"C, depending on pH and enzyme) the rennet is denatured. Rennet
coagulation is prolonged or prevented by preheating milk at temperatures
above about 70°C (depending on the length of exposure). The effect is due
to the interaction of /3-lactoglobulin with K-casein via sulphydryl-disulphide
interchange reactions; both the primary and, especially, the secondary phase
of coagulation are adversely affected.

Measurement of rennet coagulation time. A number of principles are used

to measure the rennet coagulability of milk or the activity of rennets; most
measure actual coagulation, i.e. combined first and second stages, but some
specifically monitor the hydrolysis of K-casein. The most commonly used
methods are described below.
The simplest method is to measure the time elapsed between the addition
of a measured amount of diluted rennet to a sample of milk in a tempera-
ture-controlled water-bath at, e.g. 30°C. If the coagulating activity of a
rennet preparation is to be determined, a 'reference' milk, e.g. low-heat milk
powder reconstituted in 0.01% CaCl,, and perhaps adjusted to a certain pH,
e.g. 6.5, should be used. A standard method has been published (IDF, 1992)
and a reference milk may be obtained from Institut National de la
Recherche Agronomique, Poligny, France. If the coagulability of a particu-
lar milk is to be determined, the pH may or may not be adjusted to a
standard value. The coagulation point may be determined by placing the
milk sample in a bottle or tube which is rotated in a water-bath (Figure
10.5); the fluid milk forms a film on the inside of the rotating bottle/tube but
388 DAIRY CHEMISTRY AND BIOCHEMISTRY

Factor First phase Second phase Overall effect,

Temperature + ++ a
PH +++ b
Ca +++ C
Pre-heating ++ ++++ d
Rennet concentration ++++ e
Protein concentration + ++++ f

tez

20 40 60 6.4
0
PH
C

0
Ca C 65

1 /Rennet % Protein

Figure 10.4 Principal factors affecting the rennet coagulation time (RCT) of milk.

flocs of protein form in the film on coagulation. Several types of apparatus

using this principle have been described.
As shown in Figure 10.3, the viscosity of milk increases sharply when
milk coagulates and may be used to determine the coagulation point. Any
type of viscometer may, theoretically, be used but several dedicated pieces
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 389

Milk sample

Figure 10.5 Apparatus for visual determination of the rennet coagulation time of milk.

of apparatus have been developed. The most popular of these, although with
limited use, is the Formograph (Foss Electric, Denmark), a diagram of
which is shown in Figure 10.6a. Samples of milk to be analysed are placed
in small beakers which are placed in cavities in an electrically heated metal
block. Rennet is added and the loop-shaped pendulum of the instrument
placed in the milk. The metal block is moved back and forth, creating a
‘drag’ on the pendulum in the milk. The arm to which the pendulum is
attached contains a mirror from which a flashing light is reflected on to
photosensitive paper, creating a mark. While the milk is fluid, the viscosity
is low and the drag on the pendulum is slight and it scarcely moves from
its normal position; hence a single straight line appears on the paper. As the
milk coagulates, the viscosity increases and the pendulum is dragged out of
position, resulting in bifurcation of the trace. The rate and extent to which
the arms of the trace move apart is an indicator of the strength (firmness)
of the gel. A typical trace is shown in Figure 10.6b. A low value of r indicates
a short rennet coagulation time while high values of a3, and k,, indicate a
milk with good gel-forming properties.
A recently developed, and apparently industrially useful, apparatus is the
hot wire sensor. A diagram of the original assay cell is shown in Figure
10.7a. A sample of milk is placed in a cylindrical vessel containing a wire of
uniform dimensions. A current is passed through the wire, generating heat
which is dissipated readily while the milk is liquid. As the milk coagulates,
generated heat is no longer readily dissipated and the temperature of the
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 391

I 1)ata acquisition

*----
Enzymatic
reaction
c Non-enzymatic
-----------coagulation

Time after rennet addition

.
Figure 10.7 (a) Hot wire sensor for objectively measuring the rennet coagulation of milk. (b)
Changes in the temperature of the hot wire during the course of the rennet coagulation of milk.

automation and cutting of the gel at a consistent strength, which is

important for maximizing cheese yield.
The primary phase of rennet action may be monitored by measuring the
formation of either product, i.e. para-lc-casein or the GMP. Para-lc-casein
may be measured by SDS-polyacrylamide gel electrophoresis (PAGE),
392 DAIRY CHEMISTRY AND BIOCHEMISTRY

20
Time (min)
Figure 10.8 Schematic representation of hydrolysis and gel formation in renneted milk;
H = hydrolysis of K-casein; V = changes in the viscosity of renneted milk (second stage of
coagulation), G = changes in the viscoelastic modulus (gel formation).

which is slow and cumbersome, or by ion-exchange high performance liquid

chromatography (HPLC). The GMP is soluble in TCA (2-12% depending
on its carbohydrate content) and can be quantified by the Kjeldahl method
or more specifically by determining the concentration of N-acetylneuraminic
acid or by reversed phase HPLC (RP-HPLC).
The activity of rennets can be easily determined using chromogenic
peptide substrates, a number of which are available.

Gel strength (curd tension). The gel network continues to develop for a
considerable period after visible coagulation (Figure 10.8). The strength of
the gel formed, which is very important from the viewpoints of syneresis
(and hence moisture control) and cheese yield, is affected by several factors
- the principal ones are summarized in Figure 10.9.
The strength of a renneted milk gel can be measured by several types of
viscometers and penetrometers. As discussed on p. 389, the Formograph
gives a measure of the gel strength but the data can not be readily converted
to rheological terms. Penetrometers give valuable information but are
single-point determinations. Dynamic rheometers are particularly useful,
allowing the buildup of the gel network to be studied.

Syneresis. Renneted milk gels are quite stable if undisturbed but synerese
(contract), following first-order kinetics, when cut or broken. By controlling
the extent of syneresis, the cheesemaker can control the moisture content of
cheese curd and hence the rate and extent of ripening and the stability of
the cheese - the higher the moisture content, the faster the cheese will ripen
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 393

Gel strength -
Figure 10.9 Principal factors that affect the strength of renneted milk gels (curd tension); pH
(O),calcium concentration (O),protein concentration (O),preheat treatment ( x ).

45’C
40’C
3sc
30’c

t Time after cutting

pH 6.3

pH 6.4

pH 6.5

pH 6.6

t Time after cutting

Figure 10.10 Effect of temperature (a) and pH (b) on the rate and extent of syneresis in
cut/broken renneted milk gels.
394 DAIRY CHEMISTRY AND BIOCHEMISTRY

but the lower its stability. Syneresis is promoted by:

0 cutting the curd finely, e.g. Emmental (fine cut) versus Camembert (large
cut);
0 low pH (Figure 10.1Ob);
0 calcium ions;
0 increasing the cooking temperature (Camembert, c. 30°C; Gouda, c. 36°C;
Cheddar, c. 38°C; Emmental or Parmesan, 52-55OC) (Figure 10.1Oa);
0 stirring the curd during cooking;
0 fat retards syneresis, while increasing the protein content (up to a point)
improves it; at high protein concentrations, the gel is too firm and does
not synerese (e.g. U F retentate).
Gels prepared from heated milk synerese poorly (assuming that the milk
does coagulate). Such reduced syneresis properties are desirable for fer-
mented milk products, e.g. yoghurt (milk for which is severly heated, e.g.
90°C x 10min) but are undesirable for cheese.
Good analytical methods for monitoring syneresis are lacking. Principles
that have been exploited include: dilution of an added marker, e.g. a dye,
which must not adsorb on to or diffuse into the curd particles, measurement
of the electrical conductivity or moisture content of the curd or by
measuring the volume of whey released (probably the most commonly used
method although only one-point values are obtained).

10.2.3 Acidification
Acid production is a key feature in the manufacture of all cheese varieties -
the pH decreases to about 5 (k0.3, depending on variety) within 5-20h, at
a rate depending on the variety (Figure 10.11). Acidification is normally
achieved via the bacterial fermentation of lactose to lactic acid, although an
acidogen, usually gluconic acid-6-lactone, alone or in combination with
acid, may be used in some cases, e.g. Mozzarella.
Traditionally, cheesemakers relied on the indigenous microflora of milk
for lactose fermentation, as is still the case for several minor artisanal
varieties. However, since the indigenous microflora varies, so does the rate
of acidification and hence the quality of the cheese; the indigenous micro-
flora is largely destroyed by pasteurization. ‘Slop-back’ or whey cultures
(starters; the use of whey from today’s cheesemaking as an inoculum for
tomorrow’s milk) have probably been used for a very long time and are still
used commercially, e.g. for such famous cheese as Parmigiano-Reggiano and
Comte. However, selected ‘pure’ cultures have been used for Cheddar and
Dutch-type cheeses for at least 80 years and have become progressively more
refined over the years. Single-strain cultures were introduced in New
Zealand in the 1930s as part of a bacteriophage control programme.
Selected phage-unrelated strains are now widely used for Cheddar cheese;
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 395

2 5
Time (h)
Figure 10.11 pH profile of Cheddar during cheese manufacture.

although selected by a different protocol, highly selected cultures are also

used for Dutch and Swiss-type cheeses.
Members of three genera are used as cheese starters. For cheeses that are
cooked to a temperature below about 39"C, species of Lactococcus, usually
Lc. lactis ssp. cremoris, are used, i.e. for Cheddar, Dutch, Blue, surface mould
and surface-smear families. For high-cooked varieties, a thermophilic Lac-
tobacillus culture is used, either alone (e.g. Parmesan) or with Streptococcus
saliuarius ssp. therrnophilus (e.g. most Swiss varieties and Mozzarella).
Leuconostoc spp. are included in the starter for some cheese varieties, e.g.
Dutch types; the function is to produce diacetyl and CO, from citrate rather
than acid production.
The selection, propagation and use of starters will not be discussed here.
The interested reader is referred to Cogan and Hill (1993).
The primary function of cheese starter cultures is to produce lactic acid
at a predictable and dependable rate. The metabolism of lactose is sum-
marized in Figure 10.12. Most cheese starters are homofermentative, i.e.
produce only lactic acid, usually the L-isomer; Leuconostoc species are
heteroferrnentative. The products of lactic acid bacteria are summarized in
Table 10.4.
Acid production plays several major roles in cheese manufacture:

0 Controls or prevents the growth of spoilage and pathogenic bacteria.

0 Affects coagulant activity during coagulation and the retention of active
coagulant in the curd.
396 DAIRY CHEMISTRY AND BIOCHEMISTRY

LQcrococcr Some lacrobocrlii kuronosrocs

ood srreprococci

EXTERNAL Lactose Lactose Lactose

ENVIRONMENT
CELL WALL
MEMBRANE I’MF

CYTOI’LASM

Laciore-P
i
Lactose
J.
Laclose
I

Glucose Lactose
t
Glucose

p 4 - t
Galactose-6-P Glucose-6-P
ADI’ - KT1’- LlDP t +
Galactose-I-P
GlUCoSe-I-P
AUI’ Glucore.6.P

J 1 {C::::
k”,
Tagatose.6-P

ADI’
Fructose-6-P

t
6-Phosphogluconate

K,::
t
Ribulose-5-P co2
Tagatose.1.6.blP Fructose-1’6-hiP
$.
Xylulose.5-P

11-
Dthydroxyacetone-P Glyceraldehyde-3-P t
p,

v
Acetyl-P

:::,yp
1.3-Diphosphoglycerate

24
3-Phosphoglycerate
CuASH

$
ACETY L-CoA

t
?-Phosphoglycerate CnASH
I
ACETYLALDEHYDE

K
Phosphoenolpyruvate NADH

‘\I I’
A,.. 2
Fyruvare l\AIl’
Ethanol

Lactate
Tagatose Glycolytic Leloir Phosphoketolase
pathway pathway pathway pathway

Figure 10.12 Metabolism of lactose by lactic acid bacteria; many Lactobacillus species/strains
can not metabolize galactose (from Cogan and Hill, 1993).

Solubilizes of colloidal calcium phosphate and thereby affects cheese

texture; rapid acid production leads to a low level of calcium in the cheese
and a crumbly texture (e.g. Cheshire) and vice versa (e.g. Emmental).
Promotes syneresis and hence influences cheese composition.
Influences the activity of enzymes during ripening, and hence affects
cheese quality.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE A N D FERMENTED MILKS 397

Table 10.4 Salient features of lactose metabolism in starter culture organisms (from Cogan and
Hill, 1993)
~

Cleavageb Products
Organism Transport” enzyme Pathway‘ (mol mol- lactose)

Lactococcus spp. PTS ppgal GLY 4 L-Lactate

Leuconostoc spp. ? Bgal PK +
2 D-Lactate 2 ethanol + 2C0,
Str. salicarius PMF GLY 2 L-Lactated
subsp. thermophilus
Lb. delbrueckii PMF? /?gal GLY 2 D-Lactated
subsp. lactis
Lb. delbrueckii PMF? jgal GLY 2 D-Lactated
subsp. bulgarrcus
Lb. helveticus PMF? Pgal GLY 4 L- (mainly) + D-lactate

OPTS, phosphotransferase system; PMF, proton motive force.

*ppgal, phospho-8-galactosidase; pgal, 8-galactosidase.
‘GLY, glycolysis; PK. phosphoketolase.
dThese species metabolize only the glucose moiety of lactose.

The primary starter performs several functions in addition to acid

production, especially reduction of the redox potential (Eh, from about
+250mV in milk to - 150mV in cheese), and, most importantly, plays a
major, probably essential, role in the biochemistry of cheese ripening. Many
strains produce bacteriocins which control the growth of contaminating
micro-organisms.
The ripening of many varieties is characterized by the action, not of the
primary starter, but of other micro-organisms, which we will refer to as a
secondary culture. Examples are Propionibacterium in Swiss-type cheeses,
Penicillium rogueforti in Blue cheeses, Penicillium camemberti in surface
mould-ripened cheeses, e.g. Camembert and Brie, Breuibacterium linens and
yeasts in surface smear-ripened cheese, Lactococcus lactis ssp. lactis biovar
diacetylactis and Leuconostoc spp. in Dutch-type cheeses. The specific
function of these micro-organsims will be discussed in section 10.2.7 on
ripening. Traditionally, a secondary culture was not used in Cheddar-type
cheeses but there is much current interest in the use of cultures of selected
bacteria, usually mesophilic Lactobacillus spp. or lactose-negative Lactococ-
cus spp., for Cheddar cheese with the objective of intensifying or modifying
flavour or accelerating ripening; such cultures are frequently referred to as
‘adjunct cultures’.

10.2.4 Moulding and shaping

When the desired pH and moisture content have been achieved, the curds
are separated from the whey and placed in moulds of traditional shape and
size to drain and form a continuous mass; high-moisture curds form a
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 399

Salt plays a number of important roles in cheese:

0 It is the principal factor affecting the water activity of young cheeses and
has a major effect on the growth and survival of bacteria and the activity
of enzymes in cheese, and hence affects and controls the biochemistry of
cheese ripening.

Pasteurized milk (31°C)

Starter CaCI, (0.02%. w/v)
(1-296, wlv, Lactococcus lacris ssp.cremnris R~~~~~(1:15000)
and/or Lactococcus lactis ssp. lactis)
f
Coagulum

Cutting (approx. 6 mm cubes)

1
Cooking (increasing temperature from 3OoC
to 37-39°C over approx. 30 min;
hold for approx. 60 min)

Whey drainage

Cheddaring

Milling (when curd pH = 5.2,approx.)

Dry salting (2%. w/w. approx.)

Moulding a h pressing

(a) Ripening (0.5-2years at 6-8'C)

Figure 10.14 Protocols for the manufacture of (a) Cheddar, (b) Gouda, (c) Emmental and
(d) Parmigiano-Reggiano.
400 DAIRY CHEMISTRY AND BIOCHEMISTRY

Pasteurized milk (31°C)

Lactococcus lactis ssp. cremoris
Starters (0.7%) Leuconostoc
Lactococcus lactis ssp. lactis biovar diacetylactis

v
Rennet addition (0.022%0)

1
Edam, 0.015% CaCI,
Gouda, 0.06% CaCI,

Cut (approx. 25 mi,)

Stir (for approx. 20 min)

i
NaNO, (0.015%) drain 1/3 of whey and replace
by warm water

Cook (35°C)

Stir (at 35°C for 20 min)

1
Whey drainage

Moulding and pressing

Brining (20% NaCI, 0.5% CaC1,)

(b)
Ripening (12OC for 3 months)

Figure 10.14 (Continued).

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 401

Rawlpasteunxd milk (3 l0C)

Streptococcus salivarius ssp. thermophilus (0.1%)

Starters Lactobacillus helveticus (0.1%)
Propionibacterium freudenreichi ssp.shermunii
(0.025~
Rennet addition (19 m1/1W 1 )

Cut (approx. 30 mm)

Stir (for approx. 30 min)

Cook (increasing temperature to 53-55OC

over 30 to 40 min)

Stir (at 53-55OC for 30 to 60 min

until whey pH = 6.3 to 6.4)

Curd scparation

Moulding

Brining

t 1-2 weeks at 10 to 15°C

3-7 weeks at 20 to 23°C
4-12 weeks at 5°C
Figure 10.14 (Continued).
402 DAIRY CHEMISTRY AND BIOCHEMISTRY

Low-fat milk (2%). 32OC

Starters
0.75% Lh. bulgoricus

1 lncuhate at 32OC for 30 min

Rennet addition

Cutting (appmx. 3 mm pieces)

Agitiate curds gently (30 min)

Cooking (55OC lh)

Draining and Dipping

Pressing

Brining (after 3 days)

(d)
1 246 NACIfor 14-15 days

Ripening (15°C for 10-24 months)

Figure 10.14 (Continued).

0 Salting promotes syneresis and hence reduces the moisture content of

cheese; about 2 kg of water are lost for each kilogram of salt absorbed.
0 It has a positive effect on flavour.
0 Cheese contributes to dietary sodium, high levels of which have undesir-
able nutritional consequences, e.g. hypertension and osteoporosis.

10.2.6 Manufacturing protocols for some cheese varieties

The manufacturing protocols for the various cheese varieties differ in detail
but many elements are common to many varieties. The protocols for the
principal varieties are summarized in Figures 10.14a-d.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 403

10.2.7 Cheese ripening

While rennet-coagulated cheese curd may be consumed immediately after
manufacture (and a little is), it is rather flavourless and rubbery. Conse-
quently, rennet-coagulated cheeses are ripened (matured) for a period
ranging from about 3 weeks for Mozzarella to more than 2 years for
Parmesan and extra-mature Cheddar. During this period, a very complex
series of biological, biochemical and chemical reactions occur through which
the characteristic flavour compounds are produced and the texture altered.
Four, and in some cheeses five or perhaps six, agents are responsible for
these changes:
1. The cheese milk. As discussed in Chapter 8, milk contains about 60
indigenous enzymes, many of which are associated with the fat globules
or casein micelles and are therefore incorporated into the cheese curd; the
soluble enzymes are largely removed in the whey. Many of the indigenous
enzymes are quite heat stable and survive HTST pasteurization; at least
three of these (plasmin, acid phosphatase and xanthine oxidase) are
active in cheese and contribute to cheese ripening; some indigenous lipase
may also survive pasteurization. The contribution of other indigenous
enzymes to cheese ripening is not known.
2. Coagulant. Most of the coagulant is lost in the whey but some is retained
in the curd. Approximately 6 % of added chymosin is normally retained
in Cheddar and similar varieties, including Dutch types; the amount of
rennet retained increases as the pH at whey drainage is reduced. As much
as 20% of added chymosin is retained in high-moisture, low-pH cheese,
e.g. Camembert. Only about 3% of microbial rennet substitutes is
retained in the curd and the level retained is independent of pH.
Porcine pepsin is very sensitive to denaturation at pH 6.7 but becomes
more stable as the pH is reduced.
The coagulant is major contributor to proteolysis in most cheese
varieties, notable exceptions being high-cooked varieties, e.g. Emmental
and Parmesan, in which the coagulant is extensively or totally denatured
during curd manufacture.
A good-quality rennet extract is free of lipolytic activity but a rennet
paste is used in the manufacture of some Italian varieties, e.g. Romano
and Provolone. Rennet paste contains a lipase, referred to as pre-gastric
esterase (PGE), which makes a major contribution to lipolysis in, and to
the characteristic flavour of, these cheeses. Rennet paste is considered
unhygienic and therefore semi-purified PGE may be added to rennet
extract for such cheeses (Chapter 8).
3. Starter bacteria. The starter culture reaches maximum numbers at the
end of the manufacturing phase. Their numbers then decline at a rate
depending on the strain, typically by 2 log cycles within 1 month. At least
some of the non-viable cells lyse at a rate dependent on the strain. As far
as is known, the only extracellular enzyme in Lactococcus, Lactobacillus
404 DAIRY CHEMISTRY AND BIOCHEMISTRY

and Streptococcus is a proteinase which is attached to the cell membrane

and protrudes through the cell wall; all peptidases, esterases and phos-
phatases are intracellular and therefore cell lysis is essential before they
can contribute to ripening.
4. Non-starter bacteria. Cheese made from pasteurized, high-quality milk in
modern factories using enclosed automated equipment contains very few
non-starter bacteria ( < 50 cfu g- ') at one day but these multiply to
107-108cfug-' within about 2 months (at a rate depending on, especial-
ly, temperature). Since the starter population declines during this period,
non-starter bacteria dominate the microflora of cheese during the later
stages of ripening.
Properly made cheese is quite a hostile environment for bacteria due
to a low pH, moderate-to-high salt in the moisture phase, anaerobic
conditions (except at the surface), lack of a fermentable carbohydrate and
the production of bacteriocins by the starter. Consequently, cheese is a
very selective environment and its internal non-starter microflora is
dominated by lactic acid bacteria, especially mesophilic lactobacilli, and
perhaps some Micrococcus and Pediococcus.
5. Secondary and adjunct cultures. As discussed in section 10.2.3, many
cheese varieties are characterized by the growth of secondary micro-
organisms which have strong metabolic activity and dominate the ripen-
ing and characteristics of these cheeses.
6. Other exogenous enzymes. An exogenous lipase is added to milk for a
few varieties, e.g. pre-gastric lipase (in rennet paste) for Romano or
Provolone cheese. In recent years, there has been considerable academic
and commercial interest in adding exogenous proteinases (in addition to
the coagulant) and/or peptidases to accelerate ripening. The enzymes
may be added to the milk or curd in various forms, e.g. free, microencap-
sulated or in attenuated cells.

The contribution of these agents, individually or in various combinations,

has been assessed in model cheese systems from which one or more of the
agents was excluded or eliminated, e.g. by using an acidogen rather than
starter for acidification or manufacturing cheese in a sterile environment to
eliminate non-starter lactic acid bacteria (NSLAB). Such model systems
have given very useful information on the biochemistry of ripening.
During ripening, three primary biochemical events occur, glycolysis,
lipolysis and proteolysis. The products of these primary reactions undergo
numerous modifications and interactions. The primary reactions are fairly
well characterized but the secondary changes in most varieties are more or
less unknown. An overview of the principal biochemical changes follows.

Glycolysis. Most (about 98%) of the lactose in cheese-milk is removed in

the whey as lactose or lactic acid. However, fresh cheese curd contains 1-2%
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 405

lactose which is normally metabolized to L-lactic acid by the Lactococcus

starter within a day for most varieties or a few weeks for Cheddar. In most
varieties, the L-lactate is racemized to DL-lactate by NSLAB within about 3
months and a small amount is oxidized to acetic acid at a rate dependent
on the oxygen content of the cheese and hence on the permeability of the
packaging material.
In cheese varieties made using Streptococcus salvarius ssp. thermophilus
and Lactobacillus spp. as starter, e.g. Swiss types and Mozzarella, the
metabolism of lactose is more complex than in cheese in which a
Lactococcus starter is used. In these cheeses, the curd is cooked to 52-55"C,
which is above the growth temperature for both components of the starter;
as the curd cools, the Streptococcus, which is the more heat-tolerant of the
two starters, begins to grow, utilizing the glucose moiety of lactose, with the
production of L-lactic acid, but not galactose, which accumulates in the
curd. When the curd has cooled sufficiently, the Lactobacillus spp. grow,
and, if a galactose-positive species/strain is used, it metabolizes galactose,
producing DL-lactate (Figure 10.15). If a galactose-negative strain of
Lactobaciilus is used, galactose accumulates in the curd and can participate
in Maillard browning, especially during heating, which is undesirable,
especially in Pizza cheese.
Swiss-type cheeses are ripened at about 22°C for a period to encourage
the growth of Propionibacterium spp. which use lactic acid as an energy

2
h
0
Y
c
M

.
0
0
v
M
.-C
- I
2
c
a,
S

0
0 10 20 I0 20 30 411
Time (h) Time (days)
Figure 10.15 Metabolism of lactose, glucose, galactose, D- and L-lactic acid in Emmental
cheese. Cheese transferred to hot room (22-24°C) at 14 days. 0, D-lactate; 0,acetate;
H, galactose; 0,L-lactate; +, glucose; 0,lactose; A,propionate.
406 DAIRY CHEMISTRY AND BIOCHEMISTRY

P.
CH,.CH,-CH,-COOH
Bulyrate
2p, 7 NAD*

0.4 Acslyl-CoA

Aceiyl-P

ACCIY~~

~CHI-CO-COOH 2CHyHCOH-COOH
ATP CH,-CH~-CH~-CO-CoA Pymvale Lactate
t)utyryl.coA

NAD
NAI)Hi 1
CHI-CH =CH-CO-CoA
Cmtonyl-CoA
t
ZCHKO-CoA
AcetyI-CoA

+
NAD' NADH?

Figure 10.16 Metabolism of glucose or lactic acid by Clostridium tyrobutyricurn with the
production of butyric acid, CO, and hydrogen gas.

source, producing propionic acid, acetic acid and CO, (Figure 10.15):

3CH3CHOHCOOH + 2CH,CH,COOH + CH3COOH + CO, + H,O

Lactic acid Propionic acid Acetic acid

Propionic and acetic acids probably contribute to the flavour of Swiss-

type cheeses, while the CO, is responsible for their large characteristic eyes.
Lactic acid may be metabolized by Clostridium tyrobutyricum to butyric
acid, CO, and hydrogen (Figure 10.16); butyric acid is responsible for
off-flavours and the CO, and H, for late gas blowing. Clostridia are
controlled by good hygienic practices, addition of nitrate or lysozyme,
bactofugation or microfiltration. The principal sources of clostridia are soil
and silage.
In surface mould-ripened cheeses, e.g. Camembert and Brie, Penicillium
camemberti, growing on the surface, metabolizes lactic acid as an energy
source, causing the pH to increase. Lactic acid diffuses from the centre to
the surface, where it is catabolized. Ammonia produced by deamination of
amino acids contributes to the increase in pH which reaches about 7.5 at
the surface and 6.5 at the centre of the cheese. Ripening of Camembert and
Brie is characterized by softening (liquefaction) of the texture from the
surface towards the centre. Softening is due to the increase in pH, proteolysis
and diffusion of calcium phosphate to the surface, where it precipitates due
to the high pH. These events are summarized in Figure 10.17.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 407

Soluble CaPOJlactate
* Lactate metabolized
Concentration Gradient ca3(po4)'2

5 precipitated

.- (Lower)
pH gradient
(Higher)

Ammonium ion (Higher)

* 'Ammonia
concentration gradient produced

i Cheese
exterior with
Cross-sectionalview surface microflora

Figure 10.17 Schematic representation of the gradients of calcium, phosphate, lactic acid, pH
and ammonia in ripening of Camembert cheese.

In surface smear-ripened cheeses, e.g. Munster, Limburger, Tilsit, Trapist,

the surface of the cheese is colonized first by yeasts which catabolize lactic
acid, causing the pH to increase, and then by Breuibucterium linens, the
characteristic micro-organism of the surface smear but which does not grow
below pH 5.8, and various other micro-organisms, including Micrococcus,
Arthrobacter and coryneform bacteria.

Lipolysis. Some lipolysis occurs in all cheeses; the resulting fatty acids
contribute to cheese flavour. In most varieties, lipolysis is rather limited
(Table 10.5) and is caused mainly by the limited lipolytic activity of the
starter and non-starter lactic acid bacteria, perhaps with a contribution from
indigenous milk lipase, especially in cheese made from raw milk.
Extensive lipolysis occurs in two families of cheese in which fatty acids
and/or their degradation products are major contributors to flavour, i.e.
certain Italian varieties (e.g. Romano and Provolone) and the Blue cheeses.
Rennet paste, which contains pre-gastric esterase (PGE) rather than rennet
extract, is used in the manufacture of these Italian cheeses. PGE is highly
specific for the fatty acids on the sn-3 position of glycerol, which, in the case
of milk lipids, are predominantly highly flavoured short-chain fatty acids
(butanoic to decanoic). These acids are principally responsible for the
characteristic piquant flavour of these Italian cheeses.
408 DAIRY CHEMISTRY AND BIOCHEMISTRY

Table 10.5 Free fatty acids in a selection of cheese varieties (Woo and Lindsay, 1984; Woo,
Kollodge and Lindsay, 1984)

Variety FFA (mg kg-') Variety FFA (mg kg-')

Sapsago 21 1 Gjetost 1658
Edam 356 Provolone 2118
Mozzarella 363 Brick 2150
Colby 550 Limburger 4187
Camembert 68 1 Goats' milk 4558
Port Salut 700 Parmesan 4993
Moneterey Jack 736 Romano 6743
Cheddar 1028 Roquefort 32453
Gruyere 1481 Blue (US) 32230

Blue cheeses undergo very extensive lipolysis during ripening; up to 25%

of all fatty acids may be released. The principal lipase in Blue cheese is that
produced by Penicillium roqueforti, with minor contributions from indigen-
ous milk lipase and the lipases of starter and non-starter lactic acid bacteria.
The free fatty acids contribute directly to the flavour of Blue cheeses but,
more importantly, they undergo partial fl-oxidation to alkan-2-ones (methyl
, ,o
ketones; ( R X - C H , ) through the catabolic activity of the mould (Figure
10.18). A homologous series of alkan-2-ones from C, to C,, is formed
(corresponding to the fatty acids from C, to CI8), but heptanone and
nonanone predominate; typical concentrations are shown in Table 10.6.The
characteristic peppery flavour of Blue cheeses is due to alkan-2-ones. Under
anaerobic conditions, some of the alkan-2-ones may be reduced to the
corresponding alkan-2-01s (secondary alcohols), which cause off-flavours.

Proteolysis. Proteolysis is the most complex, and perhaps the most impor-
tant, of the three primary biochemical events in the ripening of most cheese
varieties. In internal, bacterially ripened cheeses, e.g. Cheddar, Dutch and
Swiss varieties, it is mainly responsible for the textural changes that occur
during ripening, i.e. conversion of the tough rubbery texture of fresh curd to
the smooth, pliable body of mature cheese. Small peptides and free amino
acids contribute directly to cheese flavour and amino acids serve as
substrates in several flavour-generating reactions, e.g. decarboxylation,
deamination and desulphuration. Amino acids may also react chemically
with carbonyls via the Maillard reaction and Strecker degradation, with the
production of a great diversity of sapid compounds (Chapter 2). Excessive
amounts of hydrophobic peptides may be produced under certain circum-
stances and may lead to bitterness which some consumers find very
objectional; however, at an appropriate concentration, and when properly
balanced by other compounds, bitter peptides probably contribute positive-
ly to cheese flavour.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 409

Saturated fatty acids (CJ

CoA-SH
P-Oxitliltitin. -2H2+ H20

CoA-SH
Thiohydrdilsu

+ P-Keto acid
Keio acyl CoA
CoA.SH

Acalyl CoA
i
+ Acyl CoA (C2,J

Methyl ketone (C,,.]) + C O ,

! Rcduciasc

Secondary alcolinl (C,,.])

Figure 10.18 P-Oxidation of fatty acids to methyl ketones by Penicilliwm roqueforti and
subsequent reduction to secondary alcohols.

Table 10.6 Typical concentrations of alkan-2-ones in Blue cheese (from Kinsella and Hwang,
1976)

pg per 10 g dry Blue cheese

2-A1kanone A" Ba C" Db Eb Fb G' H'

2-Propanone 65 54 75 210 - 0 60 Td
2-Pentanone 360 140 410 1022 367 51 372 285
2-Heptanone 800 380 380 1827 755 243 3845 3354
2-Nonanone 560 440 1760 1816 600 176 3737 3505
2-Undecanone 128 120 590 136 135 56 1304 1383
2-Tridecanone - - - 100 120 77 309 945
Total 1940 1146 4296 5111 1978 603 9627 9372

"Commercial samples of ripe Blue cheese.

bSamples D, E and F of Blue cheese ripened for 2, 3 and 4 months, respectively.
'Samples G and H of very small batches of experimental Blue cheese ripened for 2 and 3
months, respectively.
*Trace.

The level of proteolysis in cheese varies from limited (e.g. Mozzarella)

through moderate (e.g. Cheddar and Gouda) to very extensive (e.g. Blue
cheeses). The products of proteolysis range from very large polypeptides,
only a little smaller than the parent caseins, to amino acids which may, in
turn, be catabolized to a very diverse range of sapid compounds, including
amines, acids and sulphur compounds.
410 DAIRY CHEMISTRY AND BIOCHEMISTRY

Depending on the depth of information required, proteolysis in cheese is

assessed by a wide range of techniques. Electrophoresis, usually urea-
PAGE, is particularly appropriate for monitoring primary proteolysis, i.e.
proteolysis of the caseins and the resulting large polypeptides. Quantifying
the formation of peptides and amino acids soluble in water, at pH 4.6,in
TCA, ethanol or phosphotungstic acid, or the measurement of free amino
groups by reaction with ninhydrin, o-phthaldialdehyde, trinitrobenzene or
fluorescarnine, is suitable for monitoring secondary proteolysis. Reversed
phase HPLC is especially useful for fingerprinting the small peptide profile
in cheese and is now widely used. High-performance ion-exchange or size
exclusion chromatography are also effective but are less widely used.
Proteolysis has not yet been fully characterized in any cheese variety but
considerable progress has been made for Cheddar and, as far as is known,
generally similar results apply to other low-cook, internal bacterially
ripened cheeses (e.g. Dutch types). Proteolysis in Cheddar will be sum-
marized as an example of these types of cheese.
Urea-PAGE shows that a,,-casein is completely hydrolysed in Cheddar
within 3-4 months (Figure 10.19). It is hydrolysed by chymosin, initially at
Phe,,-Phe,, and later at Leu,,,-Lys,,,, and to a lesser extent at Phe,,-
Gly,,, Leu,,-Lys,, and Leu,,,-Glu, Although p-casein in solution is
readily hydrolysed by chymosin, in cheese /3-casein is very resistant to
chymosin but is hydrolysed slowly (c. 50% at 6 months) by plasmin at
Lys,,-Lys,,, Lys,,,-His/Gln,,, and Lys,,,-Glu,,,, producing yl, y z - and
y3-caseins, respectively, and the corresponding proteose-peptones (PP5,
PP8 slow and PP8 fast; Chapter 4). Chymosin and, to lesser extent, plasmin

C I 2 3 4 5 6 7 8 9 1011121314

Figure 10.19 Urea-polyacrylamide gel electrophoretograms of Cheddar cheese after ripening

for 0, 1, 2, 3, 4,6, 8, 10, 12, 14, 16, 18 or 20weeks (lanes 1-14); C, sodium caseinate. (Supplied
by S. Mooney.)
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 41 1

Figure 10.20 Formation of water-soluble nitrogen (WSN) in: (A) Cheddar cheese with a
controlled microflora (free of non-starter bacteria); (B) controlled microflora chemically-
acidified (starter-free) cheese; (C) controlled microflora, rennet-free cheese; (D) controlled
microflora, rennet-free, starter-free cheese.

10

.10*
h

2
00

10

10

10
I D S P I 1 2 3 4 6 8
Ripening (weeks)

Figure 10.21 Changes in the population of starter cells in cheese made using different single
strain starters. I, Inoculation; D, whey drainage; S, salting; P, after pressing.
412 DAIRY CHEMISTRY AND BIOCHEMISTRY

CASEINS, CASEIN-DERIVED PEPTIDES

I
PEPTIDES

Pepo
J.
pyro-GI u-Lys-Ala-Glx-Gly-Pro-Leu-Leu-Leu-Pro-His-Phe
PCP PIP

i
pyro-Glu.Lyr-Ala-Glx-Gly-Pro-Leu~Leu~Leu
JL 4.
Pro-His-Phe

pTN
Lys- Ala-Glx-Gly-Pro-Leu-Leu-Leu
PepN

*lfGix-Oly-PR1-Leu-Leu.Leu

7"
Glx-GIy-Pro-Leu-Leu-Leu
PepX
i
Gly-Pro-Leu-Leu-Leu

prJ :L
Gly-Pro
DIP
4
Leu-Leu

Figure 10.22 Schematic representation of the hydrolysis of casein (a) by lactococcal cell
envelope proteinase (CEP), and (b) degradation of an hypothetical dodecapeptide by the
combined action of lactococcal peptidases: oligopeptidase (PepO), various aminopeptidases
(PCP, PepN, PepA, PepX), tripeptidase (TRP), prolidase (PRD) and dipeptidase (DIP).

are mainly responsible for primary proteolysis, i.e. the formation of water
(or pH 4.6)-soluble N, as summarized in Figure 10.20.
Although in v i m , the cell wall-associated proteinase of the Lactococcus
starters is quite active on /?-casein (and that from some strains on a,,-casein
also), in cheese, they appear to act mainly on casein-derived peptides,
produced by chymosin from a,,-casein or by plasmin from /?-casein.
The starter cells begin to die off at the end of curd manufacture (Figure
10.21); the dead cells may lyse and release their intracellular endopeptidases
(Pep 0, Pep F), arninopeptidases (including Pep N, Pep A, Pep C, Pep X),
tripeptidases and dipeptidases (including proline-specific peptidases) which
produce a range of free amino acids (Figure 10.22). About 150 peptides have
 93 -106
85-92

85 - 93-?

95 DF retentate
-
1'5
1

26 35 75- !'
25-
15
25 -39
- 34
35
75-?

75- ?
115-124
115-121
15-30 75- ?
110- ?
24 -34
70- 76
24-29
70-?
Cleavage sites of cellenvelope pmteinase of starter Lactococcus spp.
1W57

Figure 10.23 Water-insoluble and water-soluble peptides derived from cc,,-casein (A), a,,-casein (B) or /I-casein (C) isolated from Cheddar cheese;
DF = diafiltration. The principal chymosin, plasmin and lactococcal cellenvelope proteinase cleavage sites are indicated by arrows (data from T.K. Singh
and S. Mooney, unpublished).
 DF permeate 191 ~~ 197

175- 182
176--? 204-207

Cleavage sites of cell envelope proteinase of Ladococas spp.

178/79
182183 197/98
79’80 88!89 115!16 137’38 150’51 166167 17W5 186/87:88 203:4

1 11 .1 5. 1 1 l J U 5- 207
tt
21/22
t
114/15
t
149/50/51
t t t
lslm 188189197m
2-b25
61- 71 Cleavage sites of plasmin
61-70
DF retentate

Figure 10.23 (Continued).

 aL-u
a6
16 LS
C6 LZ
+Ol
S8 - -m
a
-
6
9 2s-st
801-L6 V8-6V
C6 69
-m -1‘s
i--01
4-L
-
WI 09
% LS
Z6 LS
416 DAIRY CHEMISTRY AND BIOCHEMISTRY

3000

1 AM2
GI IIC25

2000
.-a
CJ

.3

-z
2
1000

. . .
A q T h r Ser Glu Pro Gly Ala Cys Val Met Ile Leu T y r Phe His Lys Arg
Amino acid
Figure 10.24 Concentration of individual amino acids in 60-day-old Cheddar cheese, made
with a single-strain starter Lactococcus lactis ssp. cremoris AM,, Gll/C25 or HP (from
Wilkinson, 1992).

been isolated from the water-soluble fraction of Cheddar, and characterized

(Figure 10.23). These show that both lactococcal proteinase and exopep-
tidase contribute to proteolysis in cheese. The proteinases and peptidases of
the NSLAB (mainly mesophilic lactobacilli) appear to contribute little to
proteolysis in Cheddar, except in the production of amino acids.
The principal amino acids in Cheddar are shown in Figure 10.24.

10.2.8 Cheese Jla vow

Although interest in cheese flavour dates from the beginning of this century,
very little progress was made until the development of gas liquid chromato-
graphy (GC) in the late 1950s, and especially the coupling of G C and mass
spectrometry (MS). More than 200 volatile compounds have been identified
in cheese by GC-MS (principal compounds are listed in Table 10.7). The
volatile fraction of cheese may be obtained by taking a sample of headspace
but the concentration of many compounds is too low, even for modern
GC-MS techniques. The volatiles may be concentrated by solvent extrac-
tion or distillation. In the former, a large solvent peak may mask important
constituents while the latter may generate artefacts, even at moderately low
temperatures. Trapping of volatiles, e.g. on adsorbants or in cold traps, is
probably the most satisfactory method for concentration.
The taste of cheese is concentrated in the water-soluble fraction (peptides,
amino acids, organic acids, amines, NaCl) while the aroma is mainly in the
volatile fraction. Initially, it was believed that cheese flavour was due to one
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 417
Table 10.7 Volatile compounds which have been identified in Cheedar cheese (modified from
Urbach, 1993)

Acetaldehyde Dimethyl disulfide 2-Methylbutanol

Acetoin Dimethyl trisulfide 3-Methylbutanol
Acetone &Dodecalactone 3-Methyl-2-butanone
Acetophenone Ethanol 3-Methylbutyric acid
1.2-Butanediol Ethyl butanol 2-Nonanone
n-Butanol 2-Ethyl butanol 6-Octalactone
2-Butanol Ethyl butyrate n-Octanoic acid
Butanone Ethyl hexanoate 2-Octanol
n-Butyl acetate 2-Heptanone 2,4-Pentanediol
2-Butyl acetate n-Hexanal n-Pentanoic acid
n-Butyl butyrate n-Hexanoic acid 2-Pentanol
n-Butyric acid n-Hexanol Pentan-2-one
Carbon dioxide 2-Hexanone n-Propanol
p-Cresol Hexanethiol Propanal
y-Decalactone 2-Hexenal Propenal
6-Decalactone Isobutanol n-Propyl butyrate
n-Decanoic acid Isohexanal Tetrahydrofuran
Diacetyl Methanethiol Thiophen-2-aldehyde
Diethyl ether Methional 2-Tridecanone
Dimethyl sulfide Methyl acetate 2-Undecanone
418 DAIRY CHEMISTRY A N D BIOCHEMISTRY

or a small number of compounds, but it was soon realized that all cheeses
contained essentially the same sapid compounds. Recognition of this led to
the component balance theory, i.e. cheese flavour is due to the concentration
and balance of a range of compounds. Although considerable information
on the flavour compounds in several cheese varieties has been accumulated,
it is not possible to fully describe the flavour of any variety, with the possible
exception of Blue cheeses, the flavour of which is dominated by alkan-2-
ones.
Many cheeses contain the same or similar compounds but at different
concentrations and proportions; chromatograms of some cheese varieties
are shown in Figure 10.25. The principal classes of components present are
aldehydes, ketones, acids, amines, lactones, esters, hydrocarbons and sul-
phur compounds; the latter, e.g. H,S, methanethiol (CH,SH), dimethyl
sulphide (H,C-S-CH,) and dimethyl disulphide (H,C-S-S-CH,), are con-
sidered to be particularly important in Cheddar cheese. The biogenesis of
flavour compounds has been reviewed by Fox et al. (1993, 1996a) and FOX,
Singh and McSweeney (1995).

10.2.9 Accelerated ripening of cheese

Since the ripening of cheese, especially low moisture varieties, is a slow
process, it is expensive in terms of controlled atmosphere storage and stocks.
Ripening is also unpredictable. Hence, there are economic and technological
incentives to accelerate ripening, while retaining or improving characteristic
flavour and texture.
The principal approaches used to accelerate cheese ripening are:
I. Elevated ripening temperatures, especially for Cheddar which is now
usually ripened at 6-8°C; most other varieties are ripened at a higher
temperature, e.g. around 14°C for Dutch types or 20-22°C for Swiss
types and Parmesan, and hence there is little or no scope for increasing
the ripening temperature.
2. Exogenous enzymes, usually proteinases and/or peptidases. For several
reasons, this approach has had limited success, except for enzyme-
modified cheeses (EMC). These are usually high-moisture products which
are used as ingredients for processed cheese, cheese spreads, cheese dips
or cheese flavourings.
3. Attenuated lactic acid bacteria, e.g. freeze-shocked, heat-shocked or
lactose-negative mutants.
4. Adjunct starters, especially mesophilic lactobacilli.
5. Use of fast-lysing starters which die and release their intracellular
enzymes rapidly.
6. Genetically modified starters which super-produce certain enzymes; un-
fortunately, the key enzymes are not yet known.
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 419

The lack of definitive information on the key flavour-generating reactions

in cheese is hampering efforts to accelerate ripening, which are, at present,
empirical. Considerable in-depth information on the biochemistry of cheese
ripening is now becoming available which will facilitate the genetic engin-
eering of starter cultures with improved cheesemaking properties. Acceler-
ation of cheese ripening has been reviewed by Fox et al. (1996b).

10.3 Acid-coagulated cheeses

On acidification to pH 4.6, the caseins coagulate, which is the principle used

to manufacture of a family of cheeses which represent about 25% of total
cheese consumption and are the principal cheeses in some countries (Appen-
dix 10B). Acidification is traditionally and usually achieved by in situ
fermentation of lactose by a Lactococcus starter but direct acidification by
acid or acidogen (gluconic acid-b-lactone) is also practised. The principal

Quarg-type Qucso Blanco

-Skim milk Quarg
-Full l i t Quarg Ricotta
-TVWO~
Mascarponc
Fromage t'tais
Ldhneh
Lahanch
Fresh cliccse preparaiiiins
Crcam cheese-type
-douhle/singlc Crcnm cliccsc
-Petit Suisse Ricottonc
-Neufchatcl
Cottage cheese-type
-Low/lht Cotiagc chccse Brown 'cheese'
-Bakers cliccsc -Mysost
-Gudhrandsalosi
-Ek\r: Gcisoat
-Floieo~t
Figure 10.26 Examples of acid-coagulated or heat-acid coagulated or whey-based cheese
varieties (from Fox et d.,
1996a).
420 DAIRY CHEMISTRY AND BIOCHEMISTRY

Standardized milk

I
Pretreatment
-Pasteurization,
-Homogenization.
-Partial acidification

J
1 -
Cooling
22-3OoC

Incu ation
(quiescent)
Starter (- 15%)

t
Gelled acidified milk
(PH 4.6)

Separation

-1
(Dehydration)

Whey/permeate C(rd - Cold pack ---t Product: Quarg

i Fromuge frnis
Cottage chcese.
Pasteurization.
Hydrocolloid and
Condiment addition
and/or Homogenization

Other
t
Hot, treated curd -- Hot pack +Prodoct: Crcam-
Fresh cheeses chccsc:
Cream, andor
Yoghurt andfor
Condiments
7 1
Heat, blend
homogenize
Other

Hot dlend Hot pack Fresh cheese

preparations
Figure 10.27 Protocol for the manufacture of fresh acid-coagulated cheese (from Fox et a/.,
1996a).

families of acid-coagulated cheeses are illustrated in Figure 10.26 and a

typical manufacturing protocol is shown in Figure 10.27.
Acid-coagulated cheeses are usually produced from skim milk and are
consumed fresh. Major varieties include quarg, (American) cottage cheese,
cream cheese and petit suisse. These cheeses may be consumed in salads, as
 CHEMISTRY AND BIOCHEMISTRYOF CHEESE AND FERMENTED MILKS 421

food ingredients and serve as the base for a rapidly expanding group of
dairy products, i.e. fromage frais-type products.
The casein may also be coagulated at a pH above 4.6, e.g. about 5.2, by
using a higher temperature, e.g. 80-90°C. This principle is used to manufac-
ture another family of cheeses, which include Ricotta (and variants thereof),
Anari, and some types of Queso Blanco. These cheeses may be made
exclusively from whey but usually from a blend of milk and whey and are
usually used as a food ingredient, e.g. in lasagne or ravioli.

10.4 Processed cheese products

Processed cheese is produced by blending shredded natural cheese of the

same or different varieties and at different degrees of maturity with emulsify-
ing agents and heating the blend under vacuum with constant agitation until
a homogeneous mass is obtained. Other dairy and non-dairy ingredients
may be included in the blend. The possibility of producing processed cheese
was first assessed in 1895; emulsifying salts were not used and the product
was not successful. The first sucessful product, in which emulsifying salts
were used, was introduced in Europe in 1912 and in the USA in 1917 by
Kraft. Since then, the market for processed cheese has increased and the
range of products expanded.
Although established consumers may regard processed cheeses as inferior
products compared to natural cheeses, they have numerous advantages
compared to the latter:
1. A certain amount of cheese which would otherwise be difficult or
impossible to commercialize may be used, e.g. cheese with deformations,
cheese trimmings or cheese after removal of localized mould.
2. A blend of cheese varieties and non-cheese components may be used,
making it possible to produce processed cheeses differing in consistency,
flavour, shape and size.
3. They have good storage stability at moderate temperatures, thus reduc-
ing the cost of storage and transport.
4. They are more stable than natural cheeses during storage, which results
in less wastage, a feature that may be especially important in remote
areas and in households with a low level of cheese consumption.
5. They are amenable to imaginative packing in various conveniently sized
units.
6. They are suitable for sandwiches and fast food outlets.
7. They are attractive to children who generally do not like or appreciate
the stronger flavour of natural cheeses.
Today, a wide range of processed cheese products is available, varying in
composition and flavour (Table 10.8).
Table 10.8 Compositional specifications and permitted ingredients in pasteurized processed cheese products" (modified from Fox ef al., 1996a)

Fat in
Moisture Fat dry matter
Product (Yo, w/w) (%, w/w) (Yo, w/w) Ingredients
Pasteurized blended cheese Q 43 - 247 Cheese; cream, anhydrous milk fat, dehydrated cream (in quantities
such that the fat derived from them is less than 5 % (w/w) in
finished product); water; salt; food-grade colours, spices and flavours;
mould inhibitors (sorbic acid, potassium/sodium sorbate, and/or
sodium/calcium propionates), at levels g0.2% (w/w) finished
product
Pasteurized processed cheese Q 43 247 As for pasteurized blended cheese, but with the following extra
optional ingredients: emulsifying salts (sodium phosphates, sodium
citrates; 3% (w/w) of finished product), food-grade organic
acids (e.g. lactic, acetic or citric) at levels such that pH of finished
product is 3 5.3
Pasteurized processed cheese 3 23 ~

As for pasteurized blended cheese, but with the following extra

foods optional ingredients (milk, skim milk, buttermilk, cheese whey, whey
proteins - in wet or dehydrated forms)
Pasteurized processed cheese 40-60 3 20 ~

As for pasteurized blended cheese, but with the following extra

spreads optional ingredients: food-grade hydrocolloids (e.g. carob bean gum,
guar gum, xanthan gums, gelatin, carboxymethylcellulose, and/or
carageenan) at levels 40.8% (w/w) of finished products; food-grade
sweetening agents (e.g. sugar, dextrose, corn syrup, glucose
syrup, hydrolysed lactose)

"Minimum temperatures and times specified for processing are 65.5"C for 30 s.
CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 423

Selection of natural cheese and other ingicdients

Blending

Shredding

Addition of cmulsifying agcnt

Thermal processing

Homogcnisadon (optional)

PJcking

Cooling

storage
Figure 10.28 Protocol for the manufacture of processed cheese.
424 DAIRY CHEMISTRY A N D BIOCHEMISTRY

10.4.1 Processing protocol

The typical protocol for the manufacture of processed cheese is outlined in
Figure 10.28.
The important criteria for selecting cheese are type, flavour, maturity,
consistency, texture and pH. The selection is determined by the type of
processed cheese to be produced and by cost factors.
A great diversity of non-cheese ingredients may be used in the manufac-
ture of processed cheese (Figure 10.29).
Emulsifying salts are critical in the manufacture of processed cheese with
desirable properties. The most commonly used salts are orthophosphates,
polyphosphates and citrates but several other agents are used (Tables 10.9
and 10.10). Emulsifying salts are not emulsifiers in the strict sense, since they
are not surface active. Their essential role in processed cheese is to
supplement the emulsifying properties of cheese proteins. This is accom-
plished by sequestering calcium, solubilizing, dispersing, hydrating and
swelling the proteins and adjusting and stabilizing the pH.
The actual blend of ingredients used and the processing parameters
depend on the type of processed cheese to be produced; typical parameters
are summarized in Table 10.11.
One of the major advantages of processed cheese is the flexibility of the
finished form, which facilitates usage. The texture may vary from firm and
sliceable to soft and spreadable. These cheeses may be presented as large
blocks (5-10kg), suitable for industrial catering, smaller blocks, e.g. 0.5 kg,

Shredded natural cheese

\
/ Melting salts
Glycerides MUSCLE FOOD INGREDIENTS

Ham
Salami
Fish
Skim-milk powder
Whey powder
Whey protein concentrate
Coprecipttates

-
Previously processed cheese

VEGETABLES AND SPICES I

HIGH FAT INGREDIENTS PROCESS CHEESE BLENDJ /
Celery
Mushrooms
Mustard

Tomatoes

COLOURING AGENTS

VOURING AGENTS Locust bean gum

Pectin
Starch

Figure 10.29 Examples of non-cheese ingredients used in processed cheese (from Caric and
Kalab, 1987).
Table 10.9 Properties of emulsifying salts for processed cheese products (from Caric and Kalab, 1987)

Solubility pH value
Group Emulsifying salt Formula at 20°C (%) (I % solution)
Citrates Trisodium citrate 2Na3C,H,0,. 1H,O High 6.23-6.26
Orthophosphates Monosodium phosphate NaH2P0,.2H,0 40 4.0-4.2
Disodium phosphate Na,HPO,. 12H,O 18 8.9-9.1
Pyrophosphates Disodium pyrophosphate Na2H2P20, 10.7 4.0-4.5
Trisodium pyrophosphate Na,HP,O, .YH,O 32.0 6.7-7.5
Tetrasodium pyrophosphate Na,P,O,. 1 0 H 2 0 10-12 10.2-10.4
Polyphosphates Pentasodium tripolyphosphate Na5P3010 14-15 9.3-9.5
Sodium tetrapolyphosphate Na,P'tO,, 14- 15 9.0-9.5
Sodium hexametaphosphate (Graham's salt) Nan+,PnOJn+,(n = 10-25) Very high 6.0-7.5
Aluminium phosphates Sodium aluminium phosphate NaH ,,AI,(P04),.4H,0 -
8.0
426 DAIRY CHEMISTRY AND BIOCHEMISTRY

Table 10.10 General properties of emulsifying salts in relation to cheese processing (from Fox
et al., 1996a,b)

Orthophos- Pyrophos- Polypho-

Property Citrates phates phates sphates Aluminium

Ion exchange Low Low Moderate High-very Low

(calcium high
sequesterization)
Buffering action High High Moderate Low-very -
in the pH range low
5.3-6.0
para-Caseinate Low Low High Very high -
dispersion
Emulsification Low Low Very high Very high Very low
(n = 3-10)
-low
Bacteriostatic Nil Low High High-very -
high

Table 10.11 Chemical, mechanical and thermal parameters as regulating factors in the cheese
processing procedures (from Caric and Kalab, 1993)

Process conditions Processed cheese block Processed cheese slice Processed cheese spread

Raw material
a. Average of cheese Young to medium ripe, Predominantly young Combination of young,
predominantly young medium ripe, overipe
b. Water-insoluble 75-90% 80-90% 60-75%
N as a % of
total N
c. Structure Predominantly long Long Short to long
Emulsifying salt Structure-building, Structure-building, Creaming, e.g. low and
not creaming, e.g. not creaming, e.g. medium molecular weight
high molecular weight phosphate/citrate polyphosphate
polyphosphate, citrate mixtures
Water addition 10-25% (all at once) 5- 15% (all at once) 20-45% (in portions)
Temperature 80-85’C 78-85°C 85-98°C (150’C)
Duration of 4-8 4-6 8-15
processing (min)
PH 5.4-5.7 5.6-5.9 5.6-6.0
Agitation Slow Slow Rapid
Reworked cheese 0-0.2% 0 5-20%
Milk powder or 0 0
whey powder
5-12%
Homogenization None None Advantageous
Filling (min) 5-15 As fast as possible 10-30
Cooling Slowly (10-12 h) Very rapid Rapidly (15-30 min) in
at room temperature cool air
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTEDMILKS 427

for household use, small unit packs, e.g. 25-50g, or slices which are
particularly suited for industrial catering and fast food outlets.

10.5 Cheese analogues

Cheese analogues represent a new range of cheese-like products which

probably contain no cheese. The most important of these are Mozzarella
(Pizza) cheese analogues which are produced from rennet casein, fat or oil
(usually vegetable) and emulsifying salts. The function of emulsifying salts is
essentially similar to those in processed cheese, i.e. to solubilize the proteins.
The manufacturing protocol is usually similar to that used for processed
cheese, bearing in mind that the protein is dried rennet casein rather than a
blend of cheeses (Figure 10.30).
The main attributes required of cheese analogues used in pizzas are
meltability and stretchability; flavour is provided by other ingredients of the

Process 1 Process 2

Rennet casein 1
Emulsifyingsalt
Other ingredients
Emulsifying salt
Oil/fat I
Water Water
I
Heat 70-80°C Heat 70-80°C
High shear mixing Lnw shear mixing

Casein hydrated Casein hydrated and

emulsion formed

additional water
c
I Mixing continued

Emulsion forms

Analogue cheese product

Figure 10.30 Typical protocols for the manufacture of cheese analogue from rennet casein.
428 DAIRY CHEMISTRY AND BIOCHEMISTRY

pizza, e.g. tomato paste, sausage, peppers, spices, anchovies, etc. It may be
possible to produce analogues of other cheeses by adding biochemically or
chemically generated cheese flavours. Apart from the use of some casein
(rennet or acid) in processed cheese blends, cheese analogues, other than
Mozzarella, are not widely used at present. As discussed in section 10.2.8,
the flavour and texture of natural cheeses are very complex and cannot be
simulated readily. The usual approach is to accelerate the ripening of
natural cheese (section 10.2.9), although this approach has enjoyed limited
success to date.

10.6 Cultured milks

Acidified (cultured) milk products may very well be the oldest dairy
products. If removed aseptically from a healthy udder, milk is essentially
sterile but, in practice, milk becomes contaminated by various bacteria,
including lactic acid bacteria (LAB) during milking. During storage, these
contaminants grow at rates dependent on the temperature. LAB probably
dominate the microflora of uncooled milk expressed by hand. Since LAB
are well suited for growth in milk, they grow rapidly at ambient tempera-
ture, metabolizing lactose to lactic acid and reducing the pH of the milk to
the isoelectric point of caseins (about pH 4.6), at which they form a gel
under quiescent conditions, thus producing cultured milks. Such products
have existed since the domestication of dairy animals and some form of
cultured milk is produced throughout the world; the principal products are

Table 10.12 Some typical examples of starter cultures employed in the manufacture of
fermented milks (from Robinson and Tamime, 1993)

Type of culture Product Micro-organisms involved

Mesophilic Taetrnojolk Lactococcus lactis subsp. lactis

Folkjolk Lactococcus lactis subsp. lactis biovar. diacetylactis
Leuconostoc mesenteroides subsp. cremoris
Ymer Lc. h i s subsp. cremoris
Lc. lactis subsp. lactis biovar. diacetylactis
Kefir Kefir grains - thermophilic lactobacilli and
Kluyoeromyces marxianus
Typical fermentation temperature 20-22'C
Thermophilic Yoghurt Streptococcus saloarius subsp. thermophilus
Lactobacillus delbrueckii subsp. bulgaricus
Yakult Lactobacillus casei subsp. casei
Acidophilus milk Lactobacillus acidophilus
A/B milk Lb. acidophilus
Bifdobacterium bifidum
A/B yoghurt As above plus yoghurt culture
Typical fermentation temperatures 37-42°C
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 429

listed in Table 10.12 (Tamime and Robinson, 1985); yoghurt in its various
forms, is probably the most important type but consumption varies widely
(Table 1.6).
The production of fermented milks no longer depends on acid production
by the indigenous microflora. Instead, the milk is inoculated with a carefully
selected culture of LAB and for some products with LAB plus lactose-
fermenting yeasts (Table 10.12). The principal function of LAB is to produce
acid at an appropriate rate via the pathways summarized in Figure 10.12.
The yoghurt fermentation is essentially homofermentative but the character-
istic flavour of cultured buttermilk is due mainly to diacetyl which is
produced from citrate by Lactococccus lactis ssp. lactis biovar diacetylactis,
which is included in the culture for this product (Figure 10.31).
Kefir and Koumiss contain about 1 and 6% ethanol, respectively, which
is produced by lactose-fermenting yeasts, usually Kluyveromyces marxianus.
The ethanol modifies the flavour of the products and the CO, produced in
the fermentation affects both their flavour and texture. Koumiss, which is
produced traditionally from mares’ milk, mainly in Russia and surrounding
areas of Asia, is not in fact coagulated.
The technology of fermented milks will not be discussed in detail and the
interested reader is referred to Tamime and Robinson (1985), Tamime and
Marshall (1997) and Marshall and Tamime (1997). A flow diagram of the
manufacturing protocol of yoghurt is presented in Figure 10.32. Depending
on the product, the milk used may be full-fat, partially skimmed or fully
skimmed. If it contains fat, the milk is homogenized at 10-20 MPa to
prevent creaming during fermentation. For yoghurt, the milk is usually
supplemented with skim-milk powder to improve gel characteristics. Acid
milk gels are quite stable if left undisturbed but if stirred or shaken, they
synerese, expressing whey, which is undesirable. The tendency to synerese is
reduced by heating the milk at, for example, 90°C x 10min or
120°C x 2min. Heating causes denaturation of whey proteins, especially
P-lactoglobulin, and their interaction with the casein micelles via K--casein.
The whey protein-coated micelles form a finer (smaller whey pockets) gel
than that formed from unheated or HTST pasteurized milk, with less
tendency to synerese.
In some countries, it is common practice to add sucrose to the milk for
yoghurt, to reduce the acid taste. It is also very common practice to add
fruit pulp, fruit essence or other flavouring, e.g. chocolate, to yoghurt, either
to the milk (set yoghurt) or to the yoghurt after fermentation (stirred
yoghurt).
In the manufacture of Labneh and other Middle Eastern fermented milks,
the fermented product is concentrated by removing part of the serum
(whey). This was done traditionally by stirring the yoghurt and transferring
it to muslin bags to partially drain. Concentration can now be achieved by
ultrafiltration, before, but preferably after, fermentation.
430 DAIRY CHEMISTRY A N D BIOCHEMISTRY
m
i
m
m
i:
3:
G
5z
+
-
I
x
9
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTEDMILKS 431

Preparation of the basic mix

I
Homogenization

.1
Cooling

Incubation *
in retail cartons lncuhation in hulk

Cooling to <4'C Agitition to hlrak

coagulum

I
Set yoghurt

Adjition ol'huit

1
Packaging
I
J
Cooling to < S T

Stirivd yoghuri

Figure 10.32 Protocol for the manufacture of yoghurt. *, Sucrose and/or fruit (fruit flavours)
may be added at this point. (From Robinson and Tamime, 1993.)

Fermented milk products exhibit thixotropic rheological properties, i.e.

the viscosity (resistance to flow) decreases as the rate of shear increases; a
typical relationship is shown in Figure 10.33. The rheological properties are
major parameters of quality and are controlled by varying the total solids
content of the milk, the heat treatment and homogenization of the milk and
the use of hydrocolloids, e.g. gelatin or carageenan.
432 DAIRY CHEMISTRY AND BIOCHEMISTRY

Shear rate -
Figure 10.33 Representation of shear stress as a function of shear rate for yoghurt displaying
rheological hysteresis.

Fermented milk products developed by chance but the increased storage

stability and desirable organoleptic properties of such products were soon
appreciated. Special therapeutic properties of yoghurt were claimed by
Metchnikoff in 1910 and have been a controversial subject since. It is now
generally accepted that fermented milk products have nutritional benefits
above those of their gross chemical constituents. It has been documented
that some Lactobacillus spp., and in particular Bifidobacterium spp., con-
tained in yoghurt can colonize the large intestine, reduce its pH and control
the growth of undesirable micro-organisms. Some of these bacteria also
produce probiotics. Yoghurts containing such cultures, often referred to as
bioyoghurt, are enjoying considerable commercial success. Legislation in
many countries specifies a minimum number of viable micro-organisms in
yoghurt.

References

Bosset, J.O. and Gauch, R. (1993) Comparison of the volatile flavour compounds of six
European 'AOC' cheeses by using a new dynamic headspace GC-MS method. Znt. Dairy J.,
3, 359-77.
Caric, M. and Kalab, M. (1993) Processed cheese products, in Cheese: Chemistry, Physics and
Microbiology, 2nd edn. Vol. 2 (ed. P.F. Fox), Elsevier Applied Science, London, pp. 467-505.
Cogan, T.M. and Hill, C. (1993) Cheese starter cultures, in Cheese: Physics, Chemistry and
Microbiology, 2nd edn, Vol. 1 (ed. P.F. Fox), Chapman & Hall, London, pp. 193-255.
F A 0 (1994) Yearbook-Production, Vol. 48, Food and Agriculture Organization, Rome.
Foltmann, B. (1987) General and molecular aspects of rennets, in Cheese: Chemistry, Physics
and Microbiology, Vol. 1 (ed. P.F. Fox), Elsevier Applied Science, London, pp. 33-61.
Fox, P.F., Singh, T.K. and McSweeney, P.L.H. (1995) Biogenesis of flavour compounds in
cheese, in Chetnistry of Structure-Function Relationships in Cheese, (eds E.L. Malin and M.H.
Tunick), Plenum Press, New York, pp. 59-98.
Fox, P.F., Law, J., McSweeney, P.L.H. and Wallace, J. (1993) Biochemistry of cheese ripening,
in Cheese: Chemistry, Physics and Microbiology, Vol. 2: General Aspects, (ed. P.F. Fox),
Chapman & Hall, London, pp. 389-483.
Fox, P.F., OConnor, T.P., McSweeney, P.L.H. et a/. (1996a) Cheese: physical, biochemical and
nutritional aspects. Adv. Food Nutr. Res., 39, 163-328.
Fox, P.F., Wallace, J.M., Morgan, S. et a / . (1996b) Acceleration of cheese ripening. Antonie van
Leeuu'enhoek, 70, 271-7.
IDF (1992) Bovine Rennets. Determination of Total Milk-clotting Activity, Provisional Standard
157, International Dairy Federation, Brussels.
I D F (1995) Consumption Statisticsfor Milk and Milk Product, Bulletin 301, International Dairy
Federation, Brussels.
Kinsella, J.E. and Hwang, D.H. (1976) Enzymes of Penicillium roqueforti involved in the
biosynthesis of cheese flavour. C R C Crit. Rev. Food Sci. Nutr., 8, 191-228.
Marshall, V.M.E. and Tamime. A.Y. (1997) Physiology and biochemistry of fermented milks,
in Microbiology and Biochemistry of Cheese and Fermented Milk, 2nd edn (ed. B.A. Law),
Blackie Academic & Professional, London, pp. 153-92.
Robinson, R.K. and Tamime, A.Y. (1993) Manufacture of yoghurt and other fermented milks,
in Modern Dairy Technology, 2nd edn, Vol. 2 (ed. R.K. Robinson), Elsevier Applied Science,
London, pp. 1-48.
Tamime, A.Y. and Marshall, V.M.E. (1997) Microbiology and technology of fermented milks,
in Microbiology and Biochemistry of Cheese and Fermented Milk, 2nd edn (ed. B.A. Law),
Blackie Academic & Professional, London, pp. 57- 152.
Tamime, A.Y. and Robinson, R.K. (1985) Yoghurt Science and Technology, Pergamon Press,
Oxford.
Urbach, G. (1993) Relations between cheese flavour and chemical composition. Int. Dairy J.,
3, 3899-422.
Visser, S., Slangen, C.J. and van Rooijen, P.J. (1987) Peptide substrates for chymosin (rennin).
Biochem. J., 244, 553-558.
Visser, S., van Rooijen, P.J., Schattenkerk, C., and Kerling, K.E.T. (1976) Peptide substrates for
chymosin (rennin). Kinetic studies with peptides of different chain length including parts of
the sequence 101-112 of bovine K-casein. Biochim. Biophys. Acta, 438, 265-72.
Wilkinson, M.G. (1992) Studies on the Acceleration of Cheddar Cheese Ripening, Ph.D. Thesis,
National University of Ireland, Cork.
Woo, A.H. and Lindsay, R.C. (1984) Concentrations of major free fatty acids and flavour
development in Italian cheese varieties. J . Dairy Sci., 67, 960-8.
Woo, A.H., Kollodge, S. and Lindsay, R.C. (1984) Quantification of major free fatty acids in
several cheese varieties. J . Dairy Sci., 67, 874-8.

Suggested reading

Berger, W., Klostermeyer, H., Merkenich, K. and Uhlmann, G. (1989) Die Schmelzkiiseherstel-
lung, BenckiserKnapsack GmbH, Ladenburg.
Brown, R.J. and Ernstrom, C.A. (1985) Milk-clotting enzymes and cheese chemistry, Part 1,
Milk clotting enzymes, in Fundamentals of Dairy Chemistry, 3rd edn (ed. N.P. Wong), van
Nostrand Reinhold, New York, pp. 609-33.
Davies, F.L. and Law, B.A. (eds) (1984) Advances in the Microbiology and Biochemistry of
Cheese and Fermented Milk, Elsevier Applied Science Publishers, London.
Eck, A. (ed.) (1984) Le Fromage, Diffusion Lavoisier, Paris.
Fox, P.F. (ed.) (1993) Cheese: Chemistry, Physics and Microbiology, 2nd edn, Vols 1 and 2,
Chapman & Hall, London.
434 DAIRY CHEMISTRY AND BIOCHEMISTRY

Frank, J.F. and Marth, E.H. (1988) Fermentations, in Fundamentals of Dairy Chemistry, 3rd
edn (ed. N.P. Wong), van Nostrand Reinhold, New York, pp. 655-738.
Johnson, M.E. (1988) Milk-clotting enzymes and cheese chemistry, Part 2, Cheese chemistry,
in Fundamentals ofDairy Chemistry, 3rd edn (ed. N.P. Wong), van Nostrand Reinhold, New
York, pp. 634-54.
Kosikowski, F.V. (1982) Cheese and Fermented Milk Foods, 2nd edn, F.V. Kosikowski and
Associates, Brooktondale, NY.
Law, B.A. (ed.) (1997). Advances in the Microbiology and Biochemistry of Cheese and Fermented
Milk, Blackie Academic and Professional, London.
Malin, E.L. and Tunick, M.H. (eds) (1995) Chemistry of Structure-Function Relationships in
Cheese, Plenum Press, New York.
Robinson, R.K. (ed.) (1995) Cheese and Fermented Milks, Chapman & Hall, London.
Scott, R. (ed.) (1986) Cheesemaking Practice, 2nd edn, Elsevier Applied Science Publishers,
London.
Tamime, A.Y. and Robinson, R.K. (1985) Yoghurt Science and Technology, Pergamon Press,
Oxford.
Waldburg, M. (ed.) (1986) Handbuch der Kiise: Kase der Welt von A a Z ; Eine Enzyklopadie,
Volkswirtschaftlicher Verlag GmbH. Kempten, Germany.
Zehren, V.L. and Nusbaum, D.D. (eds) (1992) Process Cheese, Cheese Reporter Publishing
Company, Inc., WI.

Appendices

Appendix IOA World cheese production, 1994 (FAO, 1994)

(See facing page)
 CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 435
Cheese production Cheese production
Country (tonnes) Country (tonnes)

World 14 880 089 Jordan 4612

Africa 495 298 Japan 98 000
Algeria 1045 Kazakhstan 93 000
Angola 1007 Kyrgyzstan 25 000
Botswana 1498 Lebanon I4 744
Egypt 333 950 Mongolia 1764
Eritrea 216 Myanmar 27 622
Ethiopia 4600 Oman 41 1
Kenya 210 Syria 78638
Mauritania 1664 Tajikistan 16000
Morocco 6947 Turkey 139 177
Namibia 70 Turkmenistan 7000
Niger 12064 Uzbekistan 46 000
Nigeria 7022 Yemen 9155
South Africa 38 000 Europe 7075 705
Sudan 72 479 Albania I5 400
Tanzania 1200 Austria 109 600
Tunisia 7060 Belarus 109 000
Zambia 1069 Belgium- Luxembourg 68 000
Zimbabwe 5197 Bosnia- Hercegovina I3 500
North and Central Bulgaria 66 000
America 3861 921 Croatia 16701
Canada 305 100 Czech Republic 117449
Costa Rica 5960 Denmark 288 100
Cuba 14 600 Estonia 23 000
Dominican Republic 2500 Finland 92 193
El Salvador 2580 France 1 562 496
Guatemala 11700 Germany 1371 174
Honduras 8310 Greece 210300
Mexico 116360 Hungary 77 496
Nicaragua 5318 Iceland 2050
Panama 4500 Ireland 91 250
USA 3 385 000 Italy 919373
South America 613 158 Latvia I8000
Argentina 330000 Lithuania 4000
BoI i vi a 6738 Macedonia, FYR of 8100
Brasil 60 150 Malta 83
Chile 44 599 Moldova Republic 30 500
Colombia 51 000 Netherlands 647 640
Ecuador 6288 Norway 80 300
Peru 19983 Poland 296 200
Uruguay 20 400 Portugal 64 400
Venezuela 74 000 Romania 51 204
Asia 873 151 Russian Federation 708 000
Afghanistan 15600 Slovakia 42 202
Armenia 14750 Slovenia 10000
Azerbaijan 43 000 Spain 159 000
Bangladesh 1000 Sweden I38 854
Bhutan 202 1 Switzerland 134 640
China 164 646 United Kingdom 362 OOO
Cyprus 5600 Ukraine 308 770
Georgia 54 600 Yugoslavia. FR 60 000
Iran 200089 Oceania 423 625
Iraq 24733 Australia 233 635
Israel 85 944 New Zealand 190000
The following countries are included in F A 0 (1994) but no data for cheese production are available: Burkina
Faso, Burundi, Chad, Madagascar, Guinea, Rwanda, Senegal, Somalia, Swaziland, Jamaica, Trinidad and
Tobago, Suriname, India, Indonesia, Republic of Korea, Malaysia, Nepal, Pakistan, Philippines, Saudi
Arabia, Sri Lanka, Thailand, United Arab Emirates and Fiji.
436 DAIRY CHEMISTRY AND BIOCHEMISTRY

Appendix IOB Consumption of cheese (kgper caput, 1993) (IDF, 1995)

Ripened Fresh and cottage

Country cheese" cheese Total

France 15.5 7.3 22.8

Italy 13.4 6.7 20.1
Belgium 15.1 4.7 19.8
Germany 10.5 8.0 18.5
Iceland 11.9 5.2 17.1
Switzerland 13.6 2.8 16.4
Sweden 15.5 0.9 16.4
Netherlands 14.1 1.7 15.8
Denmark 14.5 0.9 15.4
Finland 12.0 2.3 14.3
Norway 14.0 0.2 14.2
Canada 12.4 0.9 13.3
USA 11.9 1.3 13.2
Austria 7.5 3.9 11.4
Estonia 4.4 5.6 10.0
Australia 8.7 0.8 9.5

United Kingdom 8.3 ___ 8.3

Spain 8.1 ___ 8.1
New Zealand 8.1 ~ 8.1
Hungary 4.6 3.3 7.9

Irelandb 5.6 ~ 5.6

South Africa 1.5 0.1 1.6

Japan 1.4 ~ 1.4

"Including processed cheese.

bData for Ireland 1991.