Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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and could inhibit starters if present at sufficiently high

concentrations.

0046 Nonadherence to the time/temperature regimes of

the cheesemaking process can also affect acidifica-

tion, e.g., overscalding by 1–2



C in Cheddar cheese-

making would cause significant slowing of the rate of

acidification for a culture containing predominantly

Lc. lactis ssp. cremoris strains.

0047 Phage is probably the most important destructive

agent in cheesemaking. Phage is found in milk and

can lie dormant (lysogenic state) within some wild

type and starter LABs. These are viruses with a

hollow protein head containing DNA and a hollow

protein tail with a plate assembly at the end. Phage

attack LAB by attaching themselves to the bacterial

surface by means of the plate assembly, injecting their

DNA through the tail and into the host cell; once

inside, their DNA directs the host to produce more

phage particles, and, when mature (30–50 min), lysin

is produced to dissolve the bacterial cell wall and

release the new phage; up to 200 particles can be

produced in one such cycle (in the same time span,

one LAB cell would have divided to give two cells).

Phage are present in most, if not all, cheesemaking

plants, and it is only through the use of good manu-

facturing practices and hygiene control, together

with the selection of suitable LAB, that phage is

maintained at low, noninvasive levels. Culture tech-

niques aimed at reducing/eliminating phage prob-

lems, which have already been touched on in this

article, include:

0048 selection of naturally resistant strains;

0049 genetic modification (e.g., plasmid transfer),

where legally permitted;

0050 blending several phage resistant/unrelated strains;

0051 use of culture rotation, three or four blends (a

different blend used on different days);

0052 inclusion of Sc. thermophilus in mesophilic blends

(used in Cheddar technology).

See also: Lactic Acid Bacteria; Starter Cultures; Yeasts

Further Reading

Cogan TM and Hill C (1993) Cheese starter cultures. In:

Fox PF (ed.) Cheese Chemistry Physics and Microbiol-

ogy, vol. 1, pp. 193–255. London: Chapman & Hall.

Davies FL and Law BA (eds) (1984) Advances in the Micro-

biology and Biochemistry of Cheese and Fermented

Milk. Barking, UK: Elsevier Applied Science.

Gasson MJ and de Vos WM (eds) (1994) Genetics and

Biotechnology of Lactic Acid Bacteria. London: Chap-

man & Hall.

Salminen S and von Wright A (eds) (1993) Lactic Acid

Bacteria. New York: Marcel Dekker.

Stanley G (1998) Microbiology of fermented milk products.

In: Early R (ed.) The Technology of Dairy Products,

pp. 2–80. London: Chapman & Hall.

Tamime AY (1983) Microbiology of starter cultures. In:

Robinson RK (ed.) Dairy Microbiology, vol. 2, pp.

113–156. Barking, UK: Applied Science.

Chemistry of Gel Formation

J M Banks and D S Horne, Hannah Research

Institute, Ayr, UK

Background

0001The basic ingredient for cheese is milk, usually from

cows, sheep, or goats, although the milk of yak, rein-

deer, camels, mares, and buffaloes is also used in

local, indigenous products in some regions of the

world. In nature, milk is produced by the mother

mammal to feed her newborn offspring; milk, there-

fore, has to provide all elements essential for their

growth and nourishment, namely protein, fat, carbo-

hydrate, minerals, and water, but in proportions rele-

vant to the needs of the animal species. Recognizing

the nutritional benefits, man initially sought to pre-

serve this fundamental food for the lean times of low

milk availability. Cheese manufacture, which retains

the majority of the protein, fat, and some of the

minerals, provides one such outcome of this exercise

of his ingenuity, with a wide range of products to suit

all tastes and desires in the modern portfolio of the

cheesemaker’s art.

0002Cheesemaking capitalizes on the curdling of milk.

Curds can be produced by either acidification of

milk to pH 4.6 or the use of a protease in conjunction

with acidification to pH 6.4. The former method is

used to produce fresh high-moisture cheeses, e.g.,

cottage cheese, and the curd is formed by the isoelec-

tric precipitation of milk casein. The latter approach

is the basis for producing hard ripened cheeses, such

as Cheddar, Emmental, or Parmesan, which have a

low moisture content. Gel formation is initiated by

proteolysis of casein, and the destabilized casein ag-

gregates to form a gel. The gel produced at the higher

pH using a protease differs markedly from that pro-

duced by acid alone. The gel is more elastic, and in the

presence of increasing acidity and the application

of heat, the gel shrinks and expels moisture by a

process termed syneresis. To understand the process

of curd formation, we must look more closely at the

individual components of milk to discern their role,

1056 CHEESES/Chemistry of Gel Formation

particularly the fat and protein, and also the agents

used in proteolysis.

Fat

0003 Fat in milk exists as small globules surrounded by me

mbrane proteins and in a size range dependent on

breed, lactational status, and diet of the cow. The

fat in milk contributes to flavor development in

mold-ripened cheeses with a strong lipolytic flavor,

e.g., blue cheeses. In cheeses such as Cheddar or

Gouda, in which proteolysis is dominant during

maturation, the fat acts as a solvent for flavor com-

pounds. The fat globules are trapped in the protein

network created during curd formation; hence, their

size and the network mesh size interact in determin-

ing overall yield of cheese. Otherwise, the fat globules

play no active role in determining curd properties.

Protein

0004 Two types of protein are found in milk: the globular

whey proteins, which are soluble in the serum phase,

and the caseins, which exist in a stable colloidal

suspension of aggregates known as casein micelles.

Cheesemaking exploits the destabilization mechan-

isms nature has built into this colloidal system

by using the natural enzyme, chymosin, originally

extracted from the stomach of the calf, to hydrolyze

the k-casein and induce the destabilization of the

micelle to form the gel, which goes forward through

to the finished cheese. Nowadays, a number of alter-

natives to the calf stomach extract of chymosin are

commercially available for cheese manufacture.

These include microbial rennets and cloned chymosin

produced using yeast or fungi as the host organism.

Casein Micelle Structure

0005 The physicochemical properties of the different

casein proteins dictate how the casein micelle is as-

sembled, and they govern the response of the micelle

to destabilizing influences. The caseins and casein

micelles have been studied extensively over a long

period. Several different models of micelle assembly

have emerged. Criticisms have been leveled at most of

these models, though a more recent dual-binding

model answers most of these points and plausibly

explains micellar assembly and stability.

0006 In bovine milk, the casein micelles have an average

molecular weight of *10

Da and a mean diameter

of *100 nm (range 50–600 nm). The micelles are

very open, highly hydrated structures with a typical

hydration value of 2–3 g of H

O per gram of protein,

depending on the method of measurement. They

contain almost all of the casein protein and a large

fraction of the mineral calcium phosphate found in

milk. The casein proteins are four distinct gene prod-

ucts, designated a

-, b-, a

-, and k-casein, and are

present in a ratio of approximately 40:35:10:12 in

cows’ milk. Two posttranslational modifications of

the proteins newly synthesized in the mammary

gland, have a major impact on their physicochemical,

functional, and assembly properties. These reactions

are glycosylation and phosphorylation. In bovine

milk, only k-casein is found glycosylated, with several

threonine and occasionally serine residues in the

hydrophilic C-terminal end of the k-casein molecule

carrying relatively short sugar chains. These chains

increase the hydrodynamic bulk and hydrophilic

character of this end of the k-casein molecule.

0007The second posttranslational reaction is phos-

phorylation. All of the caseins are phosphorylated at

serine, or rarely threonine, to varying extents. The

phosphorylation reaction requires a particular se-

quence template, —Ser —X —A —, where X is any

amino acid, and A is Glu, SerP, or rarely Asp. The

pattern of serine residues along the molecular se-

quences of a

-, a

-, and b-casein ensures that most

of the phosphorylated residues are found in clusters in

these molecules. k-Casein is unique amongst the case-

ins in the absence of phosphoseryl clusters. Most

k-casein molecules carry only one phosphoseryl resi-

due. Some have two or three, but all are singlets and,

again, are located in the hydrophilic C-terminal

region. The presence or absence of these phosphoseryl

clusters has important consequences for micellar

assembly and structure.

0008The caseins are not globular proteins locked into a

secondary conformational structure and are not truly

random coil polymers. They have been described as

rheomorphic proteins, indicating that the conforma-

tional structure adopted is dictated by, and responsive

to, the molecular environment. The caseins have little

a-helical structure, no denaturation temperature, and

a high hydrodynamic volume.

0009When categorized as nonpolar hydrophobic or

polar/charged hydrophilic groups, the amino acids

of the caseins show distinct segregation into different

regions of the molecular chains. Each casein possesses

its own pattern (Figure 1), and this helps rationalize

their selfassociation behavior and the conformations

adopted at a hydrophobic interface. Together with

the phosphoseryl clusters in the hydrophilic loops/

tails of a

-, a

-, and b-caseins, these sequence

patterns allow modeling of the micellar assembly as

the polymerization process described below.

0010In the dual-binding model, two distinct forms of

bonding take part in micellar assembly and growth.

These are cross-linking through hydrophobic regions

CHEESES/Chemistry of Gel Formation 1057

of the caseins or through phosphoseryl cluster

bridging across calcium phosphate nanoclusters,

small mineral crystallites the precipitation of which

is regulated by the presence of the caseins. These

cross-linking mechanisms are depicted in Figure 2.

Each casein molecule effectively functions as a block

copolymer, as detailed in Figure 1, with each block

possessing different and possibly multiple functional-

ity for the two distinct cross-linked paths, which can

be traced through the model micellar network.

Thus, a

-casein can chain-polymerize through the

hydrophobic blocks, B, giving a worm-like chain ap-

pearance, as highlighted in Figure 2. Further hydro-

phobic blocks can also link to these duplexes to create

branches, but limitations on growth are likely be-

cause of electrostatic repulsion from the neighboring

contiguous hydrophilic regions of these molecules.

That is unless, as postulated for the casein micelle,

the chains also polymerize through the negatively

charged phosphoseryl cluster cross-linking to a posi-

tively charged calcium phosphate nanocluster, with

the potential for multiple protein binding to each

nanocluster allowing the second different pathway

to be extended. b-Casein, with only two blocks, the

phosphoseryl cluster (P) and the hydrophobic region

(B), can form links into the network through both

and thus facilitate chain growth through these. a

Casein is envisaged in this model as having two of

each block (Figure 1), two phosphate clusters, and

two hydrophobic regions. It is only a small fraction

of total casein, but by being able to sustain growth

through all its blocks, it is likely to be bound tightly

into the network. k-Casein is the most important of

the caseins in this dual-binding model of micellar

assembly. It can link into the growing chains

through its hydrophobic N-terminal block, but its

carboxy-terminal block, denoted C in Figure 1,

has no phosphoserine cluster and therefore cannot

extend the polymer chain through a nanocluster

link. Chain and network growth are therefore termin-

ated here. The occurrence of this event on all the

growing chains leaves the casein micelle network

with a surface layer of k-casein molecules, limiting

micellar growth and ensuring the stability and integ-

rity of the system.

0011Such a surface location for k-casein is demanded by

experimental evidence linking micellar size and casein

composition. Casein micelles can be fractionated

from the initial wide size distribution found in milk

by subjecting the milk to successive centrifugation

steps of increasing speed and duration. A short, low-

speed centrifugation sediments the largest micelles.

Subjecting the supernatant to a higher-speed, longer-

duration stage produces a second pellet of slightly

smaller micelles and a supernatant for the next

stage, and so on until a clear supernatant is obtained.

The end result is a series of pellets, containing largely

monodisperse slices of the micellar size distribution.

The pellets can be analyzed for protein and mineral

composition. Their resuspension in milk ultrafiltrate

allows measurement of their size by dynamic light

scattering. Such experiments demonstrate that the

proportion of a

- and a

-caseins is constant with

micelle size and that k-casein is inversely proportional

to size. For an object such as a sphere, the surface-

to-volume ratio is inversely proportional to the radius,

-casein

β-casein

κ-casein

-casein

P Hydrophilic with phosphate center, cross-links through CaP.

B Hydrophobic, cross-links with ⱖ one other hydrophobic region

C Hydrophilic, NO phosphate center, no cross-link to CaP

fig0001 Figure 1 Schematic description of segregation of hydrophobic/

hydrophilic residues in the caseins. Hydrophobic regions (B) are

represented by the bar structures, which do not imply any form of

rigidity; hydrophilic loop regions containing phosphoserine

clusters are denoted P and the hydrophilic carboxy peptide of

k-casein by the letter C.

CaP

B-B

fig0002 Figure 2 Part of the polymer network built up in the casein

micelle according to the dual-binding model. Bridging of casein

molecules across calcium phosphate nanoclusters, cross-linking

of caseins via hydrophobic interactions, and chain termination by

k-casein are all illustrated. The hydrophobic pathway follows

along the straight bars; the nanocluster cross-link points are

indicated by the starred circles. Other chains can attach at

these points to extend the network or terminate it in the case of

k-casein attaching to a hydrophobic region.

1058 CHEESES/Chemistry of Gel Formation

so such results imply that the k-casein component

resides predominantly on the micellar surface where

it controls micellar surface area and hence micellar

size. Model and experiment are fully consistent in the

role and location of k-casein, which also readily ex-

plains the destabilization of the micelle system on

proteolysis of the k-casein by chymosin. This prote-

olysis of the k-casein is termed the primary enzymatic

phase of gel formation, and it is followed by a sec-

ondary nonenzymic aggregation stage.

Primary Enzymatic Phase of Curd

Formation

0012 The k-casein molecules provide a steric stabilizing

layer with their hydrophilic C-terminal peptides pro-

truding into the aqueous phase. Gel formation is a

direct consequence of the proteolysis of the k-casein

molecules, the casein being cleaved specifically at the

Phe

105

— Met

106

bond, releasing the hydrophilic

peptide, termed the caseinomacropeptide, into the

serum phase, leaving the N-terminal region bound

into the micelle network, and thus destabilizing the

micelle.

Secondary Aggregation Stage of Gel

Formation

0013The gradual loss of the caseinomacropeptide is ac-

companied by a decrease in the micellar zeta potential

from 20 to 10 mV. The overall process of de-

stabilization and aggregation to a gel network is

depicted schematically in Figure 3. Approximately

80% of the k-casein hairs need to be cut before sig-

nificant aggregation to define a coagulation time be-

comes detectable. During this lag phase, the lower

levels of k-casein proteolysis are accompanied by a

measurable decrease in micellar hydrodynamic size,

as the hairs are lost.

0014During the development of the gel, the micelle con-

tinues to behave as if it were a hard sphere that had

become sticky or adhesive with the loss of the stabiliz-

ing k-casein hairs. The gel is therefore initially a net-

work of aggregated particles. As such, it can be

predicted that the gel stiffness should be inversely pro-

portional to the cube of the micelle size and directly

proportional to protein concentration. Such behavior

has been observed experimentally in gels made from

micellar fractions, pelleted as described above.

0015This mechanistic model also helps to explain the

influence of the genetic variant of k-casein on the

cheesemaking properties of milk. The C-terminal se-

quences of the major genetic variants, denoted A and

B, of k-casein are listed in Figure 4. k-Casein B differs

from k-casein A only at two residues with an isoleu-

cine substituted for a threonine, a potential glycosyla-

tion site, at position 136 and an alanine substituted

for an aspartic acid residue at position 148. Milk

from cows which are homozygous for the k-B variant

have a shorter coagulation time, show a faster rate of

gel firming and give a stiffer gel than that from k-AA

cows. All of these changes mean that higher cheese

yields are recorded for k-BB milks. The genetic modi-

fications reduce the net charge of the hydrophilic

Pro-His-Leu-Ser-Phe-Met-Ala-lle-Pro-Pro-Lys-Lys-Asn-Gln-Asp-LysThr-Glu-lle-Pro-

Thr-lle-Asn-Thr-lle-Ala-Ser-Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr- -Glu-Ala-Val-Glu-

Thr-Val-Gln-Val-Thr-Ser-hr-la-Val.OH

101

121

141 Ala (Variant B)

lle (Variant B)

Thr (Variant A)

Asp (Variant A)

105 106

Ser-Thr-Val-Ala-Thr-Leu-Glu- -SerP-Pro-Glu-Val-lle-Glu-Ser-Pro-Pro-Glu-lle-Asn-

161 149

fig0004 Figure 4 Primary sequences of the major genetic variants of k-casein. Only the C-terminal region is given, since modifications are

found only in this section of the molecule.

Initial attack proteolysis almost

complete

Gelation

under way

fig0003 Figure 3 Depiction of the various stages involved in curd

formation following proteolysis of k-casein by chymosin.

CHEESES/Chemistry of Gel Formation 1059

C-terminal peptide, making it a less efficient steric

stabilizer, leading to a shorter coagulation time and

a faster rate of gel firming. The firmer gels are more

difficult to explain because the modified hydrophilic

peptide is lost to the serum phase and plays no further

role in curd network formation and growth. Even

more remarkable is that the increase in cheese yield

is due to a greater efficiency in fat retention in these

curds. The explanation is that k-BB milk also has a

higher content of k-casein. This means they have

smaller micelles on average, and the increased gel

firmness results from this difference, in line with the

predictions of the particle gel model, and not from the

genetic modifications themselves.

Coagulants and their Activities in Curd

Manufacture

0016 Proteases capable of initiating the required proteoly-

sis of k-casein, which is the first step in gel formation,

are aspartic proteinases (EC 3.4.23) and originate

from a number of sources. Milk-clotting enzymes

were historically derived by extraction from the stom-

ach of ruminants, and calf rennet is used widely in

commercial cheese manufacture today. Rennets can

be extracted from other species such as sheep, goat,

and pig, and generally, the clotting activity of these

rennets is best suited to milk of the species from

which the rennet is derived. The active clotting

enzyme in these rennets is chymosin, and the propor-

tion of chymosin in rennet extracts varies with the age

of the animal at slaughter. Calf rennet extract con-

tains approximately 90% chymosin with a small

amount of pepsin, whereas adult bovine rennet may

contain only 10% chymosin and 90% pepsin. Alter-

native sources of rennet have been sought to sustain

the growth in commercial cheese manufacture world-

wide. This has led to the development of milk-clotting

agents from microbial sources. While the term rennet

is applied to clotting extracts derived from ruminants,

clotting enzymes from nonruminant sources are

termed coagulants.

0017 Commercially produced microbial coagulants for

cheese manufacture are produced by fungal fermen-

tation. The most widely used microbial coagulants on

the market today are produced from Rhizomucor

miehei, but Rhizomucor pusillus and Cryphonectria

parasitica are also used.

0018 Since 1991, pure chymosin coagulants produced by

fermentation of a genetically modified organism have

been available for commercial use. These are termed

fermentation-produced chymosins and are produced

from either of two genetically modified host or-

ganisms, the yeast, Kluyveromyces lactis or the

fungus Aspergillus niger. Following fermentation,

the chymosin extract is treated with acid which des-

troys any residual DNA or RNA from the host organ-

ism.

Gel Formation with Rennets and

Coagulants

0019The formation of a rennet coagulum in curd manu-

facture takes place in two stages as described above.

The primary phase is enzyme-mediated and involves

the hydrolysis of micellar k-casein at the Phe

105

—

Met

106

. This bond is also hydrolyzed by the aspartic

proteases of the microbial coagulants produced

by Rhizomucor miehei and Rhizomucor pusillus.

However, with Cryphonectria parasitica, the

Leu

104

—Phe

105

is hydrolyzed preferentially.

0020Approximately 6% of the chymosin added during

cheese manufacture is retained in curd. For the micro-

bial coagulants, retention levels are approximately

2–3%. The level of retention of chymosin is influ-

enced by the pH of the curd at cut, and the greater

the acidity at cut, the greater the retention of chymo-

sin in curd. Retention of microbial coagulants is not

pH-dependent. The temperature profile during cheese

manufacture also affects chymosin activity in curd

during ripening. The high scald temperature used in

the manufacture of Swiss-type cheeses inactivates

residual chymosin activity in curd.

0021The rennet used in cheese manufacture should have

a high ratio of milk clotting to nonspecific proteolytic

activity to maximize cheese yield. While hydrolysis of

the Phe

105

—Met

106

is essential for clotting, other

nonspecific proteolysis processes in the cheese vat

would result in reduced cheese yield owing to a

weak coagulum and high losses of fat and protein in

the whey. Fermentation-produced chymosin and calf

rennet have the lowest level of nonspecific protease

activity, whereas increasing levels of nonspecific pro-

tease are seen in bovine rennet, Rhizomucor miehei,

Rhizomucor pusillus, and C. parasitica coagulants.

0022Differences in the specificity of proteases in coagu-

lants and their overall proteolytic activity may impact

on cheese flavor development, and in producing par-

ticular types of cheese, there may have to be a com-

promise between yield and flavor potential of the

coagulant. Residual chymosin activity in Cheddar

has been researched extensively and characterized.

Chymosin retained in curd rapidly hydrolyzes a

casein at the Phe

—Phe

bond producing a

-casein

(f1–23) and a

-casein (f24–199). This results in

softening of the cheese texture in the early stages

of ripening. In mature Cheddar (> 6 months), this

hydrolysis is complete. During the early stages of

ripening, the concentration of a

-casein (f24–199)

increases initially, but as ripening progresses, it is

1060 CHEESES/Chemistry of Gel Formation

further hydrolyzed by chymosin, predominantly at

the Leu

101

—Lys

102

and, to a lesser extent, at the

Phe

—Gly

and Leu

109

—Glu

110

. Peptides resulting

from these activities including the a

-casein (f24–

199), a

-casein (f33–199), a

-casein (f102–199),

and a

-casein (110–199) have been identified in

Cheddar. Further hydrolysis of the a

-casein peptides

is achieved by action of the indigenous milk protease,

plasmin, and the lactococcal cell envelope-associated

proteinase from the starter culture, and these activ-

ities result in the formation of small peptides and

amino acids, which may be further catabolized to

generate flavor components. The specificity of rennet

substitutes on the caseins during maturation has not

been characterized but is known to differ from that

resulting from chymosin action.

Factors Influencing Curd Formation with

Rennet

0023 Factors influencing curd formation include rennet

concentration, pH, temperature, and ionic calcium.

Each has the potential to influence either the primary

enzymatic phase or the secondary phase of aggrega-

tion, but some influence both.

0024 Increasing rennet concentration results in an

increase in the rate of the primary enzymatic phase.

The secondary aggregation phase of renneting also

proceeds more rapidly at higher rennet concentra-

tions. Generally, the quantity of rennet is selected to

ensure adequate gel formation within 20–40 min

during commercial cheese manufacture.

0025 Rennet coagulation of milk is pH-dependent, with

optimum activity for hydrolysis of the Phe

105

—

Met

106

in k-casein at pH 6.0. Decreasing the natural

pH of milk from 6.7 to 6.0 results in solubilization of

calcium phosphate and a decrease in the charge on the

casein micelles, which also encourages gel formation.

The net effect of a decrease in pH is therefore an

increase in both the primary enzymatic and secondary

aggregation phase of coagulation.

0026 The higher the temperature at renneting, the

greater is the rate of aggregation. At a temperature

of less than 15



C, the primary enzymatic phase pro-

ceeds normally, whereas the aggregation phase does

not proceed at a low temperature. Also, research

studies indicate that gel firmness falls off above 40



0027 Natural seasonal deficiencies in the calcium levels

in milk can be overcome by the addition of calcium

chloride, which causes a slight decrease in pH, which

will promote the primary enzymic phase of renneting,

while the secondary stage of aggregation is enhanced

by calcium addition. Processing treatments applied to

milk, e.g., excessive cold storage or high heat treat-

ment of milk, can inhibit gel formation. These effects

can be overcome by the addition of calcium chloride

or reduction of the pH of milk.

0028Although a great deal of research effort has been

directed into methods for the determination of

optimum gel strength during cheese manufacture, in

normal circumstances, there is a window extending to

10 min during which time the coagulum can be cut

without any detrimental effects on cheese yield. How-

ever, the manufacture of cheese from abnormally

weak gels, produced for example from milk deficient

in calcium or with a high proportion of the k-AA

casein variant, results in reduced yield. The appropri-

ate time for cutting the curd is dependent on the

manufacturing plant, and the time of cutting must

be adjusted to allow for seasonal variations in milk

composition.

See also: Calcium: Physiology; Casein and Caseinates:

Methods of Manufacture; Uses in the Food Industry;

Cheeses: Starter Cultures Employed in Cheese-making;

Enzymes: Uses in Food Processing; Milk: Analysis;

Cheeses: Dutch-type Cheeses

Further Reading

Dalgleish DG (1992) The enzymatic coagulation of milk.

In: Fox PF (ed.) Advanced Dairy Chemistry 1. Proteins,

2nd edn. pp. 579–619. London: Elsevier Applied

Science.

DeKruif CG (1999) Casein micelle interactions. Inter-

national Dairy Journal 9: 183–188.

Farrell HM Jr. (1988) Physical equilibria: proteins. In: Wong

N (ed.) Fundamentals of Dairy Chemistry, pp. 461–510.

New York: Van Nostrand Reinbold.

Fox PF and McSweeney P (1997) Rennets: their role in milk

coagulation and cheese ripening. In: Law BA (ed.)

Microbiology and Biochemistry of Cheese and Fer-

mented Milk, pp. 1–40. London: Blackie Academic &

Professional.

Harboe M and Budtz P (1999) The production, action and

application of rennets and coagulants. The formation

of cheese curd. In: Law BA (ed.) Technology of Cheese-

making, pp. 66–92. Sheffield, UK: Sheffield Academic

Press.

Holt C (1992) Structure and stability of the bovine casein

micelle. In: Anfinsen CB, Edsall JD, Richards FR and

Eisenberg DS (eds) Advances in Protein Chemistry, Vol.

43, pp. 63–151. San Diego, CA: Academic Press.

Holt C and Horne DS (1996) The hairy casein micelle:

evolution of the concept and its implications for dairy

technology. Netherlands Milk and Dairy Journal 50:

85–111.

Horne DS (1992) Ethanol stability. In: Fox PF (ed.)

Advanced Dairy Chemistry 1. Proteins, 2nd edn, pp.

657–689. London: Elsevier Applied Science.

Horne DS (1998) Casein interactions: casting light on the

black boxes, the structure of dairy products. Inter-

national Dairy Journal 8: 171–177.

CHEESES/Chemistry of Gel Formation 1061

Lomholt SB and Qvist KB (1999) The formation of cheese

curd. In: Law BA (ed.) Technology of Cheesemaking,

pp. 33–65. Sheffield, UK: Sheffield Academic Press.

Sousa MJ, Ardo Y and McSweeney PLH (2001) Advances

in the study of proteolysis during cheese ripening. Inter-

national Dairy Journal 11: 327–345.

Chemistry and Microbiology of

Maturation

N Y Farkye, California Polytechnic State University,

San Luis Obispo, CA, USA

Cheese Maturation (Ripening)

0001 Most rennet-coagulated cheeses are ripened for at

least 4 weeks before consumption. When manufac-

tured from raw (unpasteurized milk), the cheese must

be ripened for at least 60 days at not less than 1.7



before consumption. Cheese ripening involves several

complex and dynamic biochemical processes that

result in textural and flavor changes, unique for

each variety. Notably, a ‘green,’ rubbery, tough cheese

with a bland taste is converted into a ‘mature,’ firm,

elastic or soft-body cheese with a characteristic flavor.

0002 The major biochemical processes that occur during

ripening include metabolism of lactose/lactate, prote-

olysis, and lipolysis. The biochemical changes are

results of the activities of residual rennet, enzymes

from starter, starter adjuncts and nonstarter microor-

ganisms, indigenous milk enzymes, and exogenous

enzymes added to the milk or cheese for specific

functions. The composition of cheese curd (especially

moisture, salt, and pH) and ripening conditions (tem-

perature and humidity) affect enzyme activity and,

consequently, the rate and extent of ripening.

Rennets

0003 The term ‘rennet’ was originally used to describe the

milk-clotting enzyme preparation from calf stomach,

which contains the active digestive enzyme called

chymosin (rennin). At present, the term ‘rennet’ is

used broadly to describe milk-clotting enzymes. Com-

mercial milk-clotting enzymes currently available are

listed in Table 1.

0004 Chicken pepsin and papain are used in some coun-

tries for cheese-making. Also, rennets from kid and

lamb stomachs are available. Rennet action during

cheese-making results in the conversion of milk to

cheese curd. The principal reaction that causes coagu-

lation of milk is the cleavage of Phe

105

–Met

106

bond

in the milk protein, k-casein. A portion (0–30%) of

the rennet used in cheese-making is retained in the

cheese, depending on the type. The amount of rennet

retained depends on the pH of the milk at setting, the

type and amount used, and its ability to survive

cooking temperatures used in cheese-making. The

retention of chymosin, bovine pepsin, or porcine

pepsin in cheese increases as the pH of milk is reduced

at the time of rennet addition (setting). However, the

pH of milk at setting does not affect the amount of

Mucor miehei, Mucor pusillus,orCryophonectria

parasitica proteinases retained in the cheese curd.

Starter and Nonstarter Microorganisms

0005The term, ‘starter’ refers to a culture of lactic acid

bacteria used for acid production, through the fer-

mentation of lactose, during cheese-making. Table 2

contains a list of starter and nonstarter microorgan-

isms important in the ripening of major cheeses.

0006Mesophilic starters (Lactococcus lactis ssp. cre-

moris (formerly Streptococcus cremoris)orLc. lactis

ssp. lactis (formerly S. lactis)) are important in the

ripening of Cheddar and Dutch-type (Gouda, Edam)

cheeses. Thermophilic starters (Streptococcus ther-

mophilus (formerly S. salivarius ssp. thermophilus)

or Lactobacillus delbruekii ssp. bulgaricus (formerly

Lb. bulgaricus)) are important in the ripening of

Emmental-type (Swiss, Gruye

re) and Italian-type

(Romano, Parmesan, and Provolone) cheeses.

0007Other lactic acid bacteria (Leuconostoc spp.,

citrate

Lc. lactis ssp. lactis (formerly Lc. lactis ssp.

lactis biovar. diacetylactis or S. diacetylactis)orLb.

helveticus) play a role in the ripening of Dutch- and

Emmental-type cheeses. Microorganisms, other than

lactic acid bacteria, significant to cheese ripening

include Propionibacterium shermanii for Emmental-

type cheese, molds (Penicillium roqueforti, P. glau-

cum) for blue-veined varieties, P. camemberti for

tbl0001Table 1 Commercial milk-clotting enzymes and their sources

Commercial name Systematic name Source

Calf rennet Chymosin Calf stomach

Bovine rennet Pepsin A Adult bovine

stomach

Porcine pepsin Pepsin A Pig stomach

Microbial rennet Mucorpepsin Mucor pusillus Lindt

Microbial rennet Mucorpepsin Mucor miehei

Microbial rennet Enthothiapepsin Cryophonectria

(Endothia)

parasitica

Fermentation-produced

rennet

Chymosin Escherichia coli K12

Kluyveromyces

lactis

Aspergillus niger

1062 CHEESES/Chemistry and Microbiology of Maturation

Camembert and Brie, and Brevibacterium linens for

cheeses such as Limburger, Brick, and Port Salut.

0008 Probably all cheese varieties contain nonstarter

lactic acid bacteria (NSLAB), such as lactobacilli

(Lb. casei, Lb. plantarum, Lb. brevis), pediococci

(P. pentosaceus), and micrococci. Some of the bio-

chemical processes that occur during cheese ripening

are due to the activities of NSLAB.

Microbiological Changes

0009 Starter bacteria multiply from about 10

–10

colony-

forming units (cfu) per milliliter milk to 10

cfu per

gram of fresh cheese curd. There is a decline in starter

population as ripening progresses. The rate of decline

varies among cheeses and is dependent on the elim-

ination of lactose (the primary source of energy),

inhibition by salt, and/or autolysis. Starter activity is

inhibited when the salt-in-the-moisture (S/M) of

cheese exceeds 5%.

0010 In Cheddar and Gouda cheese, the starter popula-

tion declines to < 10

cfu g

1

within the first few

weeks, whereas in varieties like Provolone and Par-

mesan, cell densities remain high (> 10

cfu g

1

), even

after 12 months of ripening.

0011In Emmental cheese, the maximum cell density of

the lactic streptococci (10

cfu g

1

) at the periphery

and 10

cfu g

1

in the center) is reached after about

3 h of pressing. The lactic acid produced by the stre-

ptococci stimulates the growth of lactobacilli, which

reach cell densities of about 10

cfu g

1

after 10–20 h

of pressing. The populations of both streptococci and

lactobacilli decline after pressing and brining. When

cheese is placed in a warm room (20–25



C and 80–

85% relative humidity, RH), the propionibacteria

multiply rapidly, reaching cell densities of about

cfu g

1

in the center of the cheese in 3 weeks.

0012The growth of starter bacteria is inhibited in blue-

veined cheeses because of the high salt content (10%

S/M). However, growth of P. roqueforti is induced

after the cheese is pierced with needles to allow air

into its interior, causing blue veining. Ripening of

blue-veined cheeses occurs at 10



C and 96% RH,

and growth of P. roqueforti reaches a maximum in

90 days.

0013In many varieties, growth during NSLAB occurs of

ripening. The cell densities of nonstarter lactobacilli

reach 10

cfu g

1

in 10 weeks. Also, there is growth of

Pediococcus, sp. and cell densities of Micrococcus sp.

reach about 100 cfu g

1

during the same period.

tbl0002 Table 2 Microorganisms of importance in cheese ripening

Cheese category

and examples

Moisture content

(% max)

Starter Secondary starter orothermicroorganism

Hard grating

Parmesan 32 Lactobacillus delbrueckii ssp. bulgaricus Pediococcus sp.

Asiago 32 Streptococcus thermophilus Micrococcus sp.

Romano 34 Propionibacterium sp.

Hard 39 Lc. lactis ssp. cremoris Lb. casei, Lb. brevis, Lb. plantarum, Pediococcus sp.

Micrococcus sp.

Cheddar Lc. lactis ssp. lactis

;

S. thermophilus

Gruye

re Lb. delbruekii ssp. bulgaricus

Lb. helveticus

Semisoft 41 Propionibacterium shermanii

Emmental (Swiss)

45 Lc. lactis ssp. cremoris

Gouda 45 Lc. lactis ssp. lactis

Edam Citrate

Lc. lactis ssp. lactis

Leuconostoc sp.

Surface-ripened by bacteria and yeast

Limburger 50 Lc. lactis ssp. cremoris Yeast

Brick Lc. lactis ssp. lactis Brevibacterium linens

Port Salut Artherobacter spp.

Corynebacterium spp.

Internally ripened by mold growth

Blue 46 Lc. lactis subsp. cremoris Penicillium roqueforti

Roquefort 45 Lc. lactis ssp. lactis Yeast, micrococci

Gorgonzola 42 Citrate

Lc. lactis ssp. lactis

Stilton 42 Leuconostoc sp.

Soft ripened 50–80

Brie Lc. lactis ssp. cremoris P. camemberti, P. c a s e i c o l u m , P. candidum, B. linens

Camembert Lc. lactis ssp. lactis

CHEESES/Chemistry and Microbiology of Maturation 1063

Biochemical Changes

Metabolism of Lactose, Lactate, and Citrate

0014 Lactic acid bacteria use either the phosphoenolpyru-

vate-dependent phosphotransferase or the lactose

permease system to transport lactose into the cell. In

lactococci, lactose is phosphorylated to lactose-P and

transported into the cell, where it is hydrolyzed by

phospho-b-galactosidase to glucose and galactose-

6-phosphate. The glucose is fermented via the

Embden–Meyerhof–Parnas (glycolytic) pathway to

produce lactic acid. The galactose-6-phosphate is fer-

mented via the tagatose phosphate pathway to lactic

acid. In S. thermophilus and Lb. bulgaricus, lactose is

not phosphorylated before it is transported into the

cell. In the cell, it is hydrolyzed by b-galactosidase (or

lactase) to glucose and galactose. The glucose moiety

is converted to glucose-6-phosphate and fermented

via the glycolytic pathway. The galactose moiety is

converted to glucose-6-phosphate via the Leloir path-

way.

0015 Citrate is metabolized by citrate

L. lactis ssp.

lactis and Leuconostoc species. Both organisms have

specific pH-dependent permeases to transport citrate

across the cell membrane into the cell, where it is

converted into acetate, pyruvate, and CO

. Pyruvate

is converted into other products such as diacetyl,

which is an important aroma compound in dairy

products.

0016 The concentration of lactose in fresh cheese (1 day

old) ranges from < 0.1% in Dutch- and Emmental-

type cheeses to about 1% in Cheddar. At S/M levels

< 5%, starter and NSLAB metabolize residual lactose

to lactate, which may be produced in the l(þ)ord()

isomeric form, depending on the organism (Table 3).

0017 Lactic acid (1.5%, in Cheddar) reduces the initial

pH of cheese to < 5.3, and is a source of energy for

some microorganisms, thereby serving as a precursor

of flavor compounds. l(þ)-Lactate is metabolized

by propionibacteria, in the pH range 5.0–5.3, to

propionic acid, acetic acid, and CO

(eqn. (1)) in

Emmental-type cheese. Some of the CO

produced

accumulates in the cheese and forms holes, which

are commonly called ‘eyes.’

3CH

-CHOH-COOH 2CH

-CH

-COOH

Lactic acid Propionic acid

Acetic acid (1)

+ CH

-COOH + CO

+ H

In Cheddar cheese, NSLAB are capable of metaboliz-

ing lactose to produce ethanol and formic acid, both

of which are undesirable in large quantities. The

NSLAB may also oxidize lactate to acetate and

. Pediococci and lactobacilli convert l(þ)-lactate

to d()-lactate, resulting in a racemic mixture of both

isomers after 6 months of ripening. d()-Lactate

forms an insoluble calcium salt that may crystallize

in cheese and appear as undesirable white specks on

cut surfaces. The ingestion of high levels of d()-

lactate by humans causes metabolic disturbances.

Lb. casei and Lb. plantarum oxidize citrate to acetate

and CO

, resulting in a gradual increase in acetic acid

during Cheddar cheese ripening.

0018In Dutch-type cheeses, pyruvate, produced from

citrate metabolism, is oxidized to diacetyl and CO

(eqn. (2)) by citrate

L. lactis ssp. lactis and Leuco-

nostoc species.

2CH

-CO-COOH + O

-CO-CO-CH

Pyruvic acid Diacetyl

+ 2CO

+ H

O. (2)

The catabolism of lactate and the formation of alka-

line N-compounds (due to proteolysis) result in an

overall increase in the pH of some varieties during

ripening. After 6 months of ripening, the pH of

Emmental-type cheese increases from *5.3 to

*5.9, and that of Camembert and Blue increases

from *4.8 to *7. The pH of Cheddar cheese, how-

ever, increases only slightly (0.1 pH unit), because the

concentration of lactic acid remains high (1.2–1.9%),

even after 12 months of ripening.

Proteolysis

0019Of the major milk proteins, a

-, a

-, and b-caseins

predominate in cheese. Proteolysis involves the break-

down of these proteins and polypeptides by residual

rennet, indigenous milk proteases, and/or protei-

nases/peptidases of starter and nonstarter microor-

ganisms. Figure 1 shows the effects of different

rennets on the release of water-soluble nitrogen in

cheese. In general, the lowest level of proteolysis

tbl0003 Table 3 Isomer of lactic acid produced by lactic acid bacteria

Lactate isomer Organism

L(þ) Lc. lactis ssp. cremoris

Lc. lactis ssp. lactis

Citrate

Lc. lactis ssp. Lactis

Leuconostoc spp.

S. thermophilus

D() Lb. delbruekii ssp. bulgaricus

Lb. lactis

DL Lb. helveticus

Lb. plantarum

Lb. brevis

Lb. casei

P. pentosaceus

1064 CHEESES/Chemistry and Microbiology of Maturation

occurs in cheese made with porcine pepsin, whereas

the most extensive proteolysis occurs in cheese made

with microbial rennets.

0020 Chymosin hydrolyzes the Phe

–Phe

(and Phe

–

Val

) bond of a

-casein to produce a

–I[a

–CN

(f24/25–199)] peptide. The hydrolysis of this bond is

probably the most important reaction responsible for

the initial softening of cheese. Subsequent degrad-

ation of the a

–I casein by rennet occurs during

ripening. The peptide, a

–CN(f1–24/25), is rapidly

hydrolyzed by starter cell wall-associated proteinases.

0021Proteolysis of b-casein is less extensive than that of

-casein in cheeses made with chymosin, bovine

pepsin, or porcine pepsin, but more extensive break-

down of b-casein occurs in cheeses made with prote-

ases from M. miehei, M. pusillus, and C. parasitica.

0022Plasmin, an indigenous milk proteinase, hydrolyzes

all the caseins except k-casein. Specifically, plasmin

hydrolyzes b-casein to g-caseins [b-CN(f29–209,

106–209, and 108–209)] and proteose peptones.

The activity of plasmin is high in cheeses like Romano

and Emmental, for which high cooking temperatures

are used during manufacture, and in which the coagu-

lant is denatured.

0023Starter and nonstarter bacteria have very limited

proteolytic abilities towards whole caseins in cheese,

although proteinase-positive strains of lactococci

have cell envelop associated serine proteinases that

hydrolyze caseins. Starter lactococci also contain a

variety of peptidases (Table 4) that hydrolyze oligo-

peptides and di- and tripeptides resulting from the

action of rennet and/or indigenous milk proteinases

on caseins to amino acids essential for the develop-

ment of flavor. The most common free amino acids in

cheese include glutamic acid, methionine, asparagine,

histidine, alanine, valine, phenylalanine, leucine,

and lysine. In Emmental-type cheese, proline is also

present.

0024Proteolytic enzymes from Penicillium species and

Brevibacterium species are major contributors to pro-

teolysis in mold-ripened cheeses. Depending on the

13579

Age of cheese (months)

Water-soluble nitrogen (%)

Porcine pepsin

Bovine pepsin

Chymosin

MM proteinase

MP proteinase

fig0001 Figure 1 Water-soluble nitrogen (percentage of total nitrogen)

during ripening of Cheddar cheese made with identical milk-

clotting activities of different enzymes. MM, Mucor miehei; MP,

Mucor pusillus.

tbl0004 Table 4 Peptidases in Lactococci

Enzyme Othername/abbreviation

Specificity

Endopeptidases PepO Hydrolyzes glucagon

LEPI Hydrolyzes a

-CN(f1–23)

General aminopeptidases AMP Broad specificity. Hydrolyze several di-, tri-, and tetrapeptides

Aminopeptidase A PepA Hydrolyzes N-terminal Glu- and Asp residues

Glutamyl aminopeptidase A GAP N-terminal Glu- and Asp-containing peptides

Aminopeptidase C PepC Active on Lys, Phe, His, Glu, and Leu

Aminopeptidase N PepN Substrates with N-terminal Lys, Arg and Leu

X-prolyl-dipeptidyl aminopeptidase PepX (PepXP) Hydrolyzes with N-terminal X–Pro in X–Pro–Y-containing peptides

Pyrrolidonyl carboxyl peptidase PCP Removes N-terminal pyroglutamate residues from peptides

Aminopeptidase PepP Hydrolyzes N-terminal residues from X–Pro–Pro–Y

General aminopeptidase PepT Hydrolyzes Pro–Gly–Gly

Dipeptidase DIP Hydrolyzes dipeptides

Prolidase PRD Hydrolyzes dipeptides containing C-terminal proline (X–Pro)

Tripeptidases TRP Cleaves N-terminal amino acids. Do not hydrolyze tripeptides

containing Pro as second amino acid

Proline iminopeptidase PIP Hydrolyzes di- and tripeptides containing N-terminal Pro residues

The abbreviation ‘Pep’ is used for peptidases that have been genetically characterized. Others denote peptidases that have not been genetically

characterized.

CHEESES/Chemistry and Microbiology of Maturation 1065