Formation of Flavor Compounds in Cheese

Formation of Flavor Compounds in Cheese
P. F. FOX AND J. M. WALLACE
Department of Food Chemistry

University College
Cork, Ireland
I. Introduction
A. Ripening Agents in Cheese
11. Glycolysis
A. Metabolism of Lactose by Lactic Acid Bacteria
B. Metabolism of Lactic Acid in Cheese
C. Effect of Lactose Concentration on Cheese Quality
D. Citrate Metabolism
111. Lipolysis
A. Lipases in Cheese
B. Catabolism of Free Fatty Acids
IV. Proteolysis
A. Contribution of Individual Proteolytic Agents
B. Proteolysis in Cheese
C. Free Amino Acids in Cheese
V. Catabolism of Amino Acids
A. Decarboxylation and Production of Amines
B. Deamination/Formation of Ammonia and Neutral or Acidic Compounds
C. Transamination, Strecker Degradation, and Production of Aldehydes
D. Catabolism of Sulfur Amino Acids
E. Catabolism of Phenylalanine, Tyrosine, and Tryptophan
F. Other Important Flavor Compounds from Amino Acid Catabolism
G. Reactions of Free Amino Acids with Other Compounds in Cheese
H. Nonprotein Amino Acids
VI. Chemistry of Cheese Off-Flavors
References
I. Introduction
The quality of cheese is determined by its flavor (taste and aroma),

rheological properties (commonly referred to as body and texture, in-
cluding such parameters as hardness, cohesiveness, sliceability, melta-
bility, and stretchability), and visual appearance (e.g., intensity and
uniformness of color, presence or absence of eyes or slits, closeness,
uniformity of mould growth, or smear, if relevant). The relative impor-
tance of these three quality attributes varies with the cheese variety, and
they are interrelated, at least to some extent. The flavor impact of cheese
17
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18 P. F. FOX AND J. M. WALLACE
is very strongly influenced by its rheological properties, but, unfortu-

nately, these attributes are rarely studied in a single investigation.
The flavor of cheese has been the subject of scientific investigation
for nearly a century. Initially, it was believed that cheese flavor might
be due to a single compound, but it soon became apparent that cheese
flavor is due to the correct balance and concentration of a wide range
of sapid and aromatic compounds that led to the “Component Balance
Theory” (Mulder, 1952; Kosikowski and Mocquot, 1958). The develop-
ment of gas chromatography (GC) in the 1950s and its interfacing with
mass spectrometry (MS)permitted detailed and sensitive analysis of the
volatile compounds in cheese believed to be responsible for its aroma
and to be major contributors to its overall flavor. Nonvolatile com-
pounds, for example, acids, amino acids, and small peptides, also make
significant contributions to cheese flavor, especially its taste. Paper,
thin-layer, and ion-exchange chromatography were initially used to
study the nonvolatile flavor compounds in cheese, but have been super-
seded by the much more powerful technique of reverse-phase high-per-
formance liquid chromatography (RP-HPLC).An extensive literature on
various aspects of cheese flavor has accumulated and has been reviewed
extensively: 57 reviews on various aspects of cheese flavor are cited by
Imhof and Bosset (1994). Two general reviews have been compiled by
Urbach (1993, 1995).
Techniques for studying the volatile fraction of cheese have been
reviewed by Imhof and Bosset (1994). More than 200 volatile com-
pounds have been recovered from Cheddar cheese (Aishima and Nakai,
1987; Maarse and Visscher, 1989). The principal compounds are listed
in Table I. Most of the water-soluble nonvolatile compounds are pro-
duced by glycolysis and especially proteolysis; methods for monitoring
these have been reviewed by Fox et al. (1995b) and McSweeney and Fox
(1997). About 200 peptides, some of which may have an impact on
flavor, have been identified in Cheddar cheese (Singh et a]., 1995, 1997,
unpublished data; Mooney and Fox, 1997). The water-soluble fraction
of cheese contains considerable concentrations of free amino acids that
probably contribute to the background flavor of cheese and serve as
substrates for various flavor-generatingreactions.
All cheese varieties that have been studied in sufficient detail contain
essentially the same range of sapid compounds. Their flavors differ
owing to the absolute and relative concentrations of these compounds.
In spite of very considerable progress on identification and quantitation
of volatile and nonvolatile flavor compounds in cheese, it is not yet
possible to fully describe cheese flavor in chemical terms. It is much
FORMATION OF FLAVOR COMPOUNDS IN CHEESE 19
TABLE I
COMPOUNDS THATHAVEBEENIDENTIFIEXI IN CHEDDAR CHEESE'
VOLATILE FLAVOR
Acetaldehyde Dimethyl sulfide Methyl acetate

Acetoin Dimethyl disulfide 2-Methylbutanol
Acetone Dimethyl trisulfide 3-Methylbutanol
Acetophenone S-Dodecalatone 3-Methyl-2-butanone
P-angelicalatone Ethanol 3-Methylbutyric acid
1,2-Butanediol Ethyl acetate 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 &Pentanediol
n-Butyl butyrate n-Hexanoic acid Z-Pentanol
n-Butyric acid n-Hexanol Pentan-2-one
Carbon dioxide 2-Hexanone n-Propanol
p-Cresol Hexanethiol Propanal
y-Decalactone 2-Hexenal Propenal
&-Decalactone Isobutanol n-Propyl butyrate
n-Decanoic acid Isohexanal Tetrahydro furan
Diacetyl Methanethiol Thiophene-2-aldehyde
Diethyl ether Methional 2 -'hi decanone
2-Undecanone
'Adapted from Urbach (1993).
easier to chemically define flavor defects that are usually due to a single
compound or a class of compounds.
Cheese flavor is normally assessed by subjective sensory methods. A
key task here is establishment or recognition of the flavor characteristic
of a variety. No stable reference exists, and personal preferences can be
very important. Another major task is the development of a vocabulary
of terms with which to describe cheese flavor. Very interesting work on
this subject has been published by McEwan et al. (1989) and Muir and
Hunter (1992).For various reasons, there has been a longstanding desire
to develop objective chemical and physical methods for describing and
assessing cheese flavor.
In this review we do not intend to review the literature on cheese
flavor or the methodology used but to describe the biochemical path-
ways for the formation of known sapid and aromatic compounds in
cheese, compounds believed to contribute to its flavor. Particular atten-
tion is focused on the catabolism of amino acids.
A. RIPENINGAGENTS
IN CHEESE
Cheese curd is bland, and its characteristic taste and aroma, as well
as texture, develop during ripening. The ripening of cheese involves a
series of reactions catalyzed by:
1. Living microorganisms (primary and secondary starters, and non-
starter microorganisms).
2. Enzymes secreted by these microorganisms or released from their
cells after death and lysis, and enzymes indigenous in milk or
added to the milk or curd, especially the coagulant.
3. Chemical reactions, principally involving interactions between and
modification of the products of biological and enzymatic reactions.
The principal sapid compounds in cheese are produced through gly-
colysis, lipolysis, and proteolysis, which are now fairly well estab-
lished. The secondary reactions involved in the evolution of cheese
flavor are less well characterized, but several have been elucidated.
II. Glycolysis
A. METABOLISM OF LACTOSEBY LACTICACIDBACTERIA
Most (-98%) of the lactose in milk is removed in the whey as lactose
or lactic acid, and that retained in the cheese curd is metabolized to
lactate, partly during curd manufacture and partly during the early
stages of ripening, normally by the starter.
The pathway for lactose metabolism is characteristic of the type of
starter used (see Cogan and Hill, 1993). Lactococcus lactis ssp. lactis
and Lc. lactis ssp. cremoris metabolize lactose to L(+) lactic acid. The
glucose moiety is metabolized via the Embden-Meyerhof (EM) path-
way, while galactose is metabolized via the tagatose pathway. In cheeses
where a thermophilic starter is used, lactose is absorbed by S. thermo-
philus (which grows first as the curd cools) and hydrolyzed by P-galac-
tosidase; the glucose moiety is metabolized via the EM pathway to
L-lactate. S. thermophilus is unable to metabolize galactose, which it
secretes. When the curd has cooled sufficiently, Lactobacillus spp. grow.
The galactose-positive lactobacilli convert galactose via the Leloir path-
way to Glu-6-P, which is then metabolized to DL-laCtate via the EM
pathway. However, many strains of Lb. delbruekii ssp. lactis and Lb.
delbruekii ssp. bulgaricus are galactose-negative.
Lactic acid makes a major contribution to the flavor of acid-coagu-
lated cheeses and contributes significantly to the flavor of young ren-
net-coagulated cheeses. As the sapid compound present at the highest

concentration, it probably makes some contribution to the flavor of all
cheeses. However, the principal effect of the lactic acid fermentation on
cheese quality is indirect: Cheddar cheese with a pH > 5.4 (which
usually also has a high moisture content) has a high probability of
developing off-flavors, while low pH cheese is crumbly (see Fox et al.,
1990). A high pH also favors survival or growth of pathogenic and
food-poisoning microorganisms.
Starters for some cheeses include Leuconostoc spp., which isomerize
the galactose moiety of lactose via the Leloir pathway to Glu-6-P and
then metabolize it, and the glucose moiety of lactose via the phosphoke-
tolase pathway to lactic acid, ethanol, and CO,.
The lactose in brine- or dry-surface-salted cheeses is usually com-
pletely metabolized by the starter before salting, but in Cheddar-type
cheeses, which are salted by mixing milled curd with dry salt, the salt
concentration may rapidly reach a level that is inhibitory to the starter
and residual lactose is then metabolized by salt-tolerant nonstarter
lactic acid bacteria (usually mesophilic lactobacilli, but perhaps also
micrococci and pediococci), some of which may be heterofermentative,
producing lactic acid, ethanol, and CO,.
Ethanol may contribute directly to cheese flavor or may be esterified
with various fatty acids. The principal esters produced are ethyl hex-
anoate and ethyl octanoate, which are mainly responsible for the fruity
off-flavors sometimes encountered in cheese. The CO, may be sufficient
to cause pinholes and cracks in the cheese, generally considered unde-
sirable in Cheddar-type cheese.
B. METABOLISM
OF LACTICACIDIN CHEESE
Young cheese contains 1.0-1.5% lactic acid, the fate of which de-
pends on the variety (see Fox et al., 1990).
In Cheddar, Dutch, and similar varieties, L-lactate is isomerized to
DL-lactateby nonstarter lactic acid bacteria (NSLAB; Thomas and Crow,
1983). Racemization of L-lactate is not significant from the flavor view-
point, but calcium D-lactate,which is less soluble than Ca-L-lactate, may
crystallize in cheese, causing undesirable white specks, especially on
cut surfaces (see Fox et al., 1990).
Lactate can be oxidized by NSLAB in Cheddar, and probably in
similar cheeses, to acetate and COz (see Fox et al., 1996). This reaction
depends on the availability of 0,, which is determined by the size of
the cheese and the oxygen permeability of the packaging material
(Thomas, 1987). Acetate, which can also be produced by starter bacteria
from lactose (Thomas et al., 1985), or citrate (see below), or from amino
acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is
usually present at fairly high concentrations in Cheddar and is consid-
ered to contribute to its flavor, although high concentrations may cause
off-flavors (see Aston and Dulley, 1982). Thus, the oxidation of lactate
to acetate may contribute to Cheddar flavor.
The fermentation of lactose and lactate in Swiss-type cheeses has
been described comprehensively by Turner et al. (1983). Typically,
Emmental cheese contains about 0.4 and 1.2% D- and L-lactate, respec-
tively, at 1 4 days, at which time the sugars have been metabolized
completely. On transfer to a warm room, propionibacteria grow and
preferentially metabolize L-lactate to propionate, acetate and CO,:
SCH3CHOHCOOH + ZCH,CH,COOH + CHzCOOH + COZ + HZO

Citric acid Propionic acid Acetic acid
The COz generated is responsible for eye development, a characteristic

feature of these varieties. Acetate, and especially propionate, contribute
to the flavor of these cheeses.
The metabolism of lactate is very extensive in surface mold-ripened
varieties, for example, Camembert, Brie, and Carre de 1’Est (see
Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987). After
manufacture, these cheeses contain 1.O% lactic acid, produced mainly
or exclusively by the mesophilic starter. Secondary organisms quickly
colonize and dominate the surface of these cheeses, principally Penicil-
lium caseicolum, and to a lesser extent Geotrichum candidum and
yeasts, and perhaps Brevibacterium linens. G. candidum and f? caseico-
lum rapidly metabolize lactate to C 0 2 and H,O, causing an increase in
pH. Deacidification occurs initially at the surface, resulting in a pH
gradient from the surface to the center and causing lactate to diffuse
outward. When the lactate has been exhausted, E! caseicolum metabo-
lizes proteins, producing NH3, which diffuses inward, further increas-
ing the pH; the pH of mature Camembert may be 7.5 at the surface and
6.5 at the center. The concentration of calcium phosphate at the surface
exceeds its solubility at the higher pH and precipitates as a layer of
CaJPO,), on the surface, thereby causing a calcium phosphate gradient
within the cheese. Reduction of the calcium phosphate concentration
in the interior helps to soften the body of the cheese. The elevated pH
favors the action of plasmin, which together with residual coagulant, is
responsible for proteolysis in this cheese rather than proteinases se-
creted by the surface microorganisms, which diffuse into the cheese to
only a very limited extent, although the products of their action on
surface proteins may diffuse into the cheese. The combined action of
increased pH, loss of calcium, and proteolysis are necessary for the
characteristic softening of the body of Brie and Camembert. The meta-
bolism of lactate and the concomitant increase in pH probably influence
the flavor of these cheeses, which is rather mild and creamy, but their
flavor is dominated by the products of other reactions.
The ripening of surface smear-ripened cheeses, for example, Lim-
burger, Munster, Tilsit, and Trappist, is characterized by a very complex
surface microflora that includes Brevibacterium linens, Br. ammonia-
genes, Arthrobacter spp., Coryniforms spp., G. candidum, and various
yeasts. The surface of these cheeses is colonized first by yeasts, which
catabolize lactic acid, causing an increase in pH. Deacidification is
essential for the growth of Brevibacterium spp., which do not grow
below pH 5.8, but probably has little direct effect on the flavor of
smear-ripened cheeses, which is dominated by the catabolic activity of
Brevibacterium spp.
A common defect in many cheeses arises from the metabolism of
lactate (or glucose) by Clostridium spp. to butyrate, H2, and CO, (see
Fox et al., 1996). This reaction leads to late gas blowing and off-flavors
in many cheese varieties unless precautions (e.g., good hygiene, addi-
tion of NaN03 or lysozyme, bactofugation, or microfiltration) are taken.
c. EFFECTOF LACTOSECONCENTRATION ON CHEESE QUALITY

Although the concentration of lactose in milk decreases with advanc-
ing lactation, its concentration in bulked factory supplies from cows on
a staggered calving pattern is essentially constant. However, when syn-
chronized calving is practiced ( e g , New Zealand, Ireland, and Austra-
lia), substantial seasonal changes occur in the concentration of lactose
in milk and, consequently, in fresh cheese curd. Variations in the
concentration of lactose in cheese curd probably affect the final pH of
the cheese, which affects cheese texture, enzyme activity, and perhaps
the nonstarter microflora. Cheese flavor is likely to vary owing to
variations in the concentration of lactic acid and catabolites therefrom,
and due to variations in the metabolic activity of the cheese microflora.
The concentration of lactose in cheese curd is affected by certain
features of the manufacturing process. As far as we know, the concen-
tration of lactose in cheese curd is not increased intentionally for any
variety, but it is advertently increased in curd made from milk concen-
trated by ultrafiltration (UF),and the concentration of lactose may have
to be reduced to an appropriate level by diafiltration. However, the
concentration of lactose in the curd for several varieties, including
1.5
1.o
0.5
0.0
0 4 8 12 16 20 24 28 32 36
Time, weeks
FIG.1. Metabolism of lactose in Cheddar cheese curds during ripening: control (O),
lactose-enriched cheeses: 1 ( A ) , 2 (A), whey-replaced cheese (01,
and washed-curd
cheese ( 0 ) .(Modified from Waldron, 1997.)
Gouda and Edam, is reduced by replacing part of the whey by warm

water. This process, which was probably introduced as a simple method
for cooking curds on farmsteads lacking jacketed cheese vats, effectively
controls the pH of the cheese. Curds are washed in the washed-curd
variant of Cheddar cheese and perhaps in the production of low-fat
cheese (to increase moisture content).
The effect of variations in the concentration of lactose in cheese curd
on the quality of mature cheese has received very little attention. In an
attempt to vary the concentration of lactose in Cheddar cheese curd,
Huffman and Kristoffersen (1984) added lactose to the curd-whey
after cutting the coagulum, but because there is a strong outflow
of whey from the curd at that stage due to syneresis of the curd, the
increase in lactose concentration within the curd was quite small. In
a study by Waldron (1997), the lactose content of Cheddar cheese curd
was reduced by removing 35-45% of the whey shortly after cutting
the coagulum and replacing it with an equal volume of warm water
4.6
0 4 8 12 16 20 24 28 32 36
Time, weeks
FIG. 2. Changes in the pH of lactose-modified Cheddar cheeses during ripening:
control (0),lactose-enriched cheeses 1 ( A ) , 2 (A),whey-replaced cheese (01and
, washed-
curd cheese (). (Modified from Waldron, 1997.)
or by washing the curd after milling, or increased by adding lactose to

the cheese milk up to a level of -8%. The concentration of lactose in
the curd at milling ranged from 0.6 to 2.5%. Changes in the concentra-
tion of lactose in the cheese during ripening and the pH of the cheese
are shown in Figs. 1 and 2.
The lactose in the two types of washed-curd cheese was completely
metabolized within -2 weeks, but it persisted in the high-lactose
cheeses throughout ripening. Not surprisingly, the pH of the cheeses
was inversely proportional to the concentration of lactose in the curd.
The pH of high-lactose cheeses continued to decrease (to -4.8) through-
out ripening, whereas in the washed-curd cheeses the pH increased
once the lactose had been exhausted, as is common for many varieties
of cheese, probably due to proteolysis and production of NH,. Flavor
development was substantially faster in the high-lactose cheeses than
in the washed-curd cheeses, although it was considered to be rather
harsh, perhaps due to the low pH. The flavor of the low-lactose cheeses
was clean and mild. The rate of growth and final number of NSLAB
were not affected by the concentration of lactose, suggesting that

NSLAB do not depend on lactose as a growth substrate. They probably
utilize amino acids or are physically constrained within the curd, or
colony size is restricted by local production of toxic substances. The
types of NSLAB in the various cheeses may have varied, but this was
not studied.
The results of this study suggest that the concentration of lactose in
cheese curd has a substantial effect on the quality of Cheddar and
probably other cheeses. Replacing some of the whey with water or
washing the cheese curd might be considered when a mild, clean flavor
is desired. Normal variations in the lactose content of milk from mixed-
calving herds are probably not significant but may have a substantial
effect when synchronized calving is practiced. The effect may be very
substantial when UF-concentrated milk is used due to low levels of curd
syneresis. Diafiltration of the cheese milk or washing of the curd would
appear to be desirable. Cheese made from milk with a high content of
fat and casein may have a reduced lactose content, as may cheese
produced from milk, the protein content of which is increased by
adding UF retentate.
The foregoing discussion indicates that the metabolism of lactose and
lactate in cheese is well understood. In quantitative terms, these
changes are among the principal metabolic events in most cheese varie-
ties. However, compared to other biochemical changes during cheese
ripening, conversion of lactose to lactate may have relatively little direct
effect on the flavor of mature cheese, but since it determines its pH, it
is of major significance in regulating the various biochemical reactions
that occur during ripening. The isomerization of lactate probably has
little impact on cheese flavor, but its conversion to propionate and/or
acetate is probably significant, and, when it occurs, the metabolism of
lactate to butyrate has a major negative effect on cheese quality.
D . CITRATE
METABOLISM
Bovine milk contains -1.8 g liter-' citrate (-8 mM), about 95% of
which is soluble and lost in the whey. Citrate is not metabolized by Lc.
lactis or Lc. cremoris, but it is metabolized by Lc. lactis ssp. lactis biovar
diacetylactis and Leuconostoc spp. with the production of diacetyl and
GO2 (for reviews, see Cogan, 1985; Cogan and Daly, 1987; Cogan and
Hill, 1993). It is not metabolized by S . thermophilus or by thermophilic
lactobacilli (Hickey et al., 1983), but several species of mesophilic
lactobacilli metabolize citrate with the production of diacetyl and for-
mate (Fryer, 1970).
Production of COz from citrate is responsible for the characteristic

eyes in Dutch-type cheese, and for undesirable openness and floating
curd in Cheddar and Cottage cheese, respectively. Diacetyl is very
significant for the aroma-flavor of cottage cheese, Quarg, and many
fermented milks, and also contributes to the flavor of Dutch-type
cheeses. Cheddar contains 0.2 to 0.5% (wt/wt) citrate, which is meta-
bolized at a rate dependent on the NSLAB microflora. Diacetyl occurs
at a considerable concentration in Cheddar and is generally regarded as
a significant contributor to its flavor. Acetate from citrate may also
contribute to cheese flavor.
Diacetyl can be converted to acetoin, 2,s-butylene glycol, and 2-bu-
tanone. The latter is considered to be a major component of Cheddar
cheese flavor (see, e.g., Dimos et al., 1996).
I I I . Lipolysis
The fat fraction of cheese has a major effect on cheese texture and is
important for the perception and development of cheese flavor. Poor
development of flavor and texture in low-fat cheeses is a considerable
technological problem that limits the market for such products. In
addition to serving as a direct (fatty acids) or indirect (methyl ketones,
lactones, esters) source of flavor compounds in cheese, fat serves as a
solvent for sapid compounds produced from other constituents. It has
been reported that a natural milk-fat emulsion yields better cheese than
milk fat emulsified in skim milk, suggesting that the fat-water interface
plays an important role in the development of cheese flavor; perhaps
enzymes in the natural fat globule membrane (see Andrews et al., 19921
are important in flavor development.
The fat in cheese can undergo degradation via lipolysis (enzymatic)
or oxidation (chemical]. The degree of lipid oxidation in cheese is
limited, probably due to the low redox potential of cheese and the
presence of natural antioxidants. Many highly flavored compounds are
produced via lipid oxidation, but the possible contribution of lipid
oxidation to cheese flavor/off-flavor has been largely ignored.
Fatty acids released upon lipolysis contribute directly to cheese fla-
vor, especially in hard Italian and mold-ripened varieties and, probably,
to a lesser extent in other varieties, especially when extra-mature,
provided they are properly balanced by products of proteolysis and
other reactions. However, extensive lipolysis is considered undesirable
in most cheese varieties: Cheddar, Gouda and Swiss-type cheeses con-
taining even a moderate level of free fatty acids (FFAs) would be
TABLE I1
TYPICAL. OF FREE
CUNCENTRATIONS FATTY
Acros (FFAs) IN SOME VARIETFES
CHEESE
Variety FFA (mg1kg-I) Variety FFA (mg/kg-’)
Sapsago 21 1 Gjetost 1658

Edam 356 Provolone 2118
Mozzarella 363 Brick 2150
Colby 550 Limburger 4187
Camembert 681 Goat’s milk 4558
Port Salut 700 Parmesan 4993
Monterey Jack 736 Romano 6754
Cheddar 1028 Blue-mould (US) 32230
Roquefort 32453
~~ ~~~ ~~
Date from Woo et a1 (1984)and Woo and Lindsay (1984).
considered rancid. The total concentrations of FFAs in some major

cheese varieties are summarized in Table 11.
A. LIPASESIN CHEESE
Lipases in cheese originate from milk, rennet paste (some varieties),
starter, adjunct starter or nonstarter bacteria, or exogenous sources.
Milk contains a well-characterized lipoprotein lipase (LPL) that lib-
erates fatty acids from the sn-1 and 3 positions of mono-, di-, and
triglycerides and the 1 position of glycerophospholipids (Olivecrona
and Bengtsson-Olivecrona, 1991; Olivecrona et al., 1992). In milk fat,
highly flavored short-chain acids that are esterified predominantly at
the sn-3 position and hence even low LPL activity can have a significant
effect on flavor. LPL is associated mainly with the casein micelles and
is incorporated into cheese. LPL probably causes significant lipolysis in
raw milk cheese but probably contributes little in pasteurized milk
cheese.
Good-quality rennet extracts are free of lipolytic activity, but the
rennet paste used in the manufacture of some Italian varieties (e.g.,
Romano, Provolone) contains a potent lipase-pregastric esterase
(PGE)-that catalyses the extensive lipolysis responsible for the “pic-
cante” flavor characteristic of such varieties. The literature on PGE, also
called lingual or oral lipase, was comprehensively reviewed by Nelson
et al. (1977) and updated by Fox and Stepaniak (1993).
PGE is highly specific for short-chain acids esterified at the sn-3

position. Slight differences in specificity between calf, lamb, and kid
PGEs permit the manufacture of Italian cheeses with slightly different
flavor characteristics. Most other lipases are unsuitable for the manu-
facture of Italian cheeses because of incorrect specificity, but it has been
claimed that certain fungal lipases may be acceptable alternatives (see
Fox and Stepaniak, 1993). The use of PGE to accelerate the ripening of
other cheese varieties was discussed by Fox (1988-89).
C ~ Sare weakly lipolytic, but in the absence of strongly
L Q C ~ O C O Cspp.
lipolytic microorganisms they probably make a significant contribution
to the low level of lipolysis in Cheddar and Dutch varieties. The intra-
cellular esterase-lipase of two Lactococcus strains has been isolated and
characterized (Holland and Coolbear, 1996; Chick et al., 1997). Little is
known about the genetics of these enzymes. Isolation of lipase/esterase-
negative variants of Lactococcus would permit the significance of these
enzymes in cheese ripening to be assessed.
Both mesophilic and thermophilic lactobacilli possess (mainly) intra-
cellular esterolytic-lipolytic activity (Khalid et al., 1990; Gobbetti et al.,
1996). The esterolytic-lipolytic activity in cell homogenates of a num-
ber of lactobacilli was characterized by El-Soda et al. (1986), and an
intracellular lipase and an intracellular esterase from Lb. plantarum
were purified and characterized by Gobbetti et al. (1997a,b).
Micrococcus spp., which constitute part of the nonstarter microflora
of cheese, especially the surface microflora, produce lipases that may
contribute to lipolysis during ripening (Bhowmik and Marth, 1990a,b).
The nonstarter microflora of cheese may also include Pediococcus spp.
that are weakly esterolytic and lipolytic (Tzanetakis and Litopoulou-
Tzanetaki, 1989; Bhowmik and Marth, 1989).
Propionibacterium shermanii possesses a lipase(s) (Paulsen et al.,
1980) that contributes to lipolysis in Swiss varieties. An intracellular
lipase of I! shermanii was partially characterized by Oterholm et al.
(1970). Brevibacterium linens, a major component of the surface of
smear-ripened cheeses, possesses intracellular lipases and esterases
(Smhaug and Ordal, 1974; Foissy, 1974). The intracellular esterase of
Br. linens ATCC 91 74 has also been purified and characterized (Rattray
and Fox, 1997a).
Very extensive lipolysis occurs in mold-ripened cheese, particularly
blue mold varieties in which up to 25% of the total FFAs may be
liberated (see Gripon, 1987, 1993). However, the impact of FFAs on the
flavor of blue mold-ripened cheeses is less than for hard Italian varie-
ties, possibly due to neutralization as the pH increases during ripening
and the dominant influence of methyl ketones on the flavor of blue
30 I? F. FOX AND 1. M. WALLACE
mold cheeses. Lipolysis in mold-ripened varieties is due primarily to

the lipases of Penicillium roqueforti or I! camemberti, which secrete
potent extracellular lipases that are well characterized (see Kinsella and
Hwang, 1976; Gripon, 1987, 1993).
Psychrotrophs, which dominate the microflora of refrigerated milk,
are potentially an important source of potent lipases in cheese if their
numbers exceed lo7 ml-I (Cousins et al., 1977). Many psychrotrophic
lipases are heat stable and may cause rancidity in cheese over a long
ripening time (Mottar, 1989; Smhaug and Stepaniak, 1997). Unlike
psychrotrophic proteinases, which are largely lost in the whey, psy-
chrotrophic lipases adsorb onto the fat globules and are concentrated in
the cheese.
B. CATABOLISM OF FREEFATTY
ACIDS
The flavor and aroma of blue mold cheese are dominated by saturated
alkan-2-ones (n-methyl ketones). A homologous series of alkan-Zones
with odd-numbered carbon chains from C3 to CI7, and some with
even-numbered carbon chains, occurs in these cheeses: heptan-2-one,
nonan-&one, and undecan-2-one are dominant during most of the rip-
ening period (Dartley and Kinsella, 1971). Some reduction of alkan-2-
ones to alkan-2-01s occurs during ripening, which has an undesirable
effect on flavor.
The metabolism of fatty acids in cheese by Penicillium spp. involves
four main steps: (1)release of fatty acids by lipases, (2) oxidation to
a-ketoacids, (3) decarboxylation to alkan-2-ones with one less carbon
atom, and (4) reduction of alkan-Zones to the corresponding alkan-2-01;
step four is reversible under aerobic conditions. Alkan-2-ones can also
be formed by mold from the ketoacids naturally present at low concen-
trations in milk fat (-1%of total fatty acids) or by the oxidation of
monounsaturated acids. The rate of alkan-Zone production in cheese is
affected by temperature, pH, the physiological state of the mold, and the
concentration of fatty acids (Adda et al., 1982). Both resting spores and
mycelia are capable of producing alkan-Zones at a rate that does not
depend directly on the concentrations of FFA precursors. Indeed, high
concentrations of FFAs are toxic to I! roqueforti (Fan et al., 1976).
Lactones are formed by the intramolecular esterification of hy-
droxyacids through the loss of water to form a ring structure. a- and
0-lactones are very reactive and unstable, but y- and S-lactones are stable
and occur in cheese. Lactones possess a strong aroma, which, although
not cheese-like, may contribute to overall cheese flavor. y- and S-lac-
tones may be formed spontaneously from the corresponding y- and

6-hydroxyacids following their release from triglycerides; their concen-
tration in cheese should therefore correlate with the extent of lipolysis.
Lactones may also be formed from ketoacids after reduction (Wong et
al., 1975). The formation and significance of lactones in cheese have
received relatively little attention.
IV. Proteolysis
Proteolysis is the most complex and perhaps the most important

biochemical event during the maturation of most cheese varieties. The
extent of proteolysis varies from very limited (e.g., Mozzarella] to very
extensive (e.g., blue mold varieties), and the products range from large
polypeptides, comparable in size to intact caseins, through a range of
medium and small peptides, to free amino acids. Proteolysis is mainly
responsible for softening of the texture of cheese during the early stages
of ripening and influences the development of cheese flavor via the
formation of amino acids and peptides that make a direct, although
probably limited, contribution to flavor. Amino acids also serve as
substrates for the formation of numerous flavor compounds. Proteolysis
also affects the mouthfeel of cheese and the release of flavor compounds
from cheese during mastication.
Various methods have been developed for quantifying the extent and
pattern of proteolysis in cheese. They are reviewed by Fox et al. (1995b)
and McSweeney and Fox (1997).
A. CONTRIBUTION OF INDIVIDUAL PROTEOLYTIC AGENTS

Proteolytic enzymes in cheese originate from milk, coagulant, starter,
secondary starter, and nonstarter microorganisms. Information on the
relative importance of individual proteolytic agents has been obtained
from studies on cheeses with controlled microflora, in which the coagu-
lant, plasmin, starter, and nonstarter bacteria, in various combinations,
have been excluded or inactivated. These studies, which have been
summarized by Fox et (11.(19931, indicate that rennet and plasmin are
mainly responsible for the initial hydrolysis of as1-and p-caseins, re-
spectively, and for the production of most of the water- or pH 4.6-soluble
N in cheese.
Although the lactic acid bacteria (Lactococcus, Lactobacillus, Strep-
tococcus) are weakly proteolytic, they possess a very comprehensive
proteolytic system that has been studied extensively and reviewed (see
Fox and McSweeney, 1996; Kunji et al., 1996). The proteolytic system
of Lactococcus has been particularly well studied. They possess a cell
wall-associated proteinase, several intracellular proteinases, at least two
intracellular oligoendopeptidases (PepO, PepF), at least three amino-
peptidases (PepN, PepA, PepC), a dipeptidylaminopeptidase (PepX), a
tripeptidase, a general dipeptidase, and proline-specific dipeptidases.
However, Lactococcus spp. and most Lactobacillus spp. lack a car-
boxypeptidase. They also possess a number of peptide and amino acid
transport systems. This comprehensive proteolytic system is necessary
to enable the lactic acid bacteria to grow to the requisite high numbers
( 1 0 ~ - 1 0cfu/ml)
~~ in milk, which contains only low levels of small
peptides and free amino acids. The proteolytic system of Lactobacillus
spp. has been studied less thoroughly than that of Lactococcus, but the
two systems appear to be generally similar.
The cell wall-associated proteinase contributes to the formation of
small peptides in cheese, probably by hydrolyzing larger peptides pro-
duced from a,,-casein by chymosin or from p-casein by plasmin. The
aminopeptidases, dipeptidases, and tripeptidases, which are intracellu-
lar, are responsible for the release of free amino acids after the starter
cells have died and lysed. The proteolytic system of NSLAB in Cheddar,
and probably in similar cheeses, appears to supplement the proteolytic
action of the starter, producing generally similar peptides and amino
acids (Lynch et al., 1997).
The proteinases and peptidases of P roqueforti and l? comemberfi
make a major contribution to proteolysis in mold-ripened cheeses (see
Gripon, 1993). Br. linens secretes an extracellular proteinase and an
aminopeptidase and possesses a number of intracellular peptidases,
which may be released on cell lysis (see Rattray et al., 1995; Rattray and
Fox, 1997b). These enzymes probably contribute to proteolysis, includ-
ing the production of free amino acids, in the surface of smear-ripened
cheeses. Propionibacterium shermanii, the characteristic secondary mi-
croorganism of Swiss-type cheeses, is weakly proteolytic but has high
peptidase activity, especially proline-specific peptidases, and contrib-
utes significantly to proteolysis in Swiss cheese.
The proteolytic specificity of chymosin, plasmin, and the cell wall-
associated proteinase of several lactococcal strains on a,,-, as2-,p-, and
K-caseins has been determined (see Fox et al., 1995a, 1996; Fox and
McSweeney, 1996). The specificity of the extracellular proteinases of €?
roqueforti, €? camemberti, and Br. linens on as1-and p-caseins has been
determined (see Gripon, 1993; Rattray et al., 1996, 1997).
B. PROTEOLYSIS IN CHEESE
The extent and type of proteolysis in a number of the principal cheese
varieties has been characterized (see Fox and McSweeney, 1996). Pro-
teolysis in Cheddar is very well characterized and can be summarized
as shown in Figs. 3, 4, and 5. a,,-casein is completely hydrolyzed
by chymosin, typically within about 3 months, at the Phe2,-Phez4 bond.
The larger peptide, a,,-CN f24-199, is further hydrolyzed extensively
by chymosin at the Leulol-Lys,oz bond and to a lesser extent at Phe3,-
Gly33, Leug8-Leu,,, and Leulog-Glullo, probably not in sequence. Some
of the Lyslo3-Tyrlo4 and Lyslo5-Vallos bonds are also hydrolyzed by
plasmin. The large C-terminal peptides, a,,-CN f24-199,102-199,110-
199, 99-199, 33-199, 104-199, and 106-199, are detectable in the
water-insoluble fraction (Fig. 6). The complementary N-terminal pep-
tides (e.g., a,,-CN f24-101, 24-98, 24-109) have not yet been identified
in the water-insoluble fraction, but since these are highly phosphory-
lated, they may be in the water-soluble extract. The large peptides,
which have not yet been characterized, are also hydrolyzed by lactococ-
cal CEP and possibly by lactococcal PepO and several small peptides
produced therefrom and are present in the UF permeate or retentate of
the water-soluble extract (WSE) (Fig. 3).
The peptide a,,-CN fl-23 is hydrolyzed rapidly by the lactococcal
CEP at bonds Glng-Glyl0, Gln13-Glu14, Glu14-Val15, and Leul6-Asnl7,
and probably at other sites, depending on the specificity of the CEP. The
peptides aSl-CNh-9,1-13, and 1-14 accumulate and dominate the UF
permeate or 70% ethanol-soluble fraction of the water-soluble extract of
Cheddar. According to Exterkate and Alting (1995), these peptides do
not affect the flavor of cheese. The complementary C-terminal peptides
(i.e., a,,-CN f10-23, f14-23, f15-23, and f17-23) have been identified
in the UF permeate, and some of them have been partially hydrolyzed
by an aminopeptidase, probably PepN, releasing amino acids (Fig. 3).
Peptides from the N-terminal region of a,,-CN f24-199 are also hy-
drolyzed by lactococcal CEP, or possibly PepO or PepF. The N-terminals
of some of these peptides do not correspond to known CEP cleavage
sites, suggesting the action of aminopeptidase, PepO, PepF, or NSLAB
enzymes.
In Cheddar and most other cheeses, p-casein is much more resistant
to hydrolysis than a,,-casein: only about 50% is hydrolyzed within 6
months. It is hydrolyzed mainly by plasmin, at Lys28-Lys,g, LyslOs-
Glnlos,and Lyslo7-Glulo8,yielding yl-,f-, and f-caseins (P-CN f29-209,
106-209, and 108-209, respectively), which are clearly evident in the
W
ip
-19 1
93 -lY
14- 7
DF Rctcotatc
I* -35
2s-u
IS-35 116-124 IU -am
U
-
n 115-121
15-Y
110- !’
14 -
3
4
14 -
1
, Cleavage sites uf crll-euvelupc pruteinas uf starter L.Clueoccur rpp.
IW57
-
-
Zb-31 Y3 ?
-
1-9
I
I
1)
17-7
IJ 2J-m
16-31
y
-?
4l-?
*(-?
1.1
IBS
- !’
?
DF Pernienle
18 - ? Jl-?
Water-hsoluble Fraction
2, .......... ................. - 199
.................. .- . - ~ . -. . ~ . . 1-
21 .- ~ _ _ , lu.-.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 199

33 ......... , lu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IIO.-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
- IY,
- - I99
”......._.....
.. , 121 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I*y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
199
I99
7y .......... 1% ?
FIG.3. ~ - C a s e i n - d e r i v e dpeptides isolated from the water-insoluble (----I, the diafiltrate (DF), retentate (-1, and the DF
permeate (-1 of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et ol., 1997, and Mooney, 1997.)
I 7
DF Permeate 191- 197
175- lU2
176-1 ZOC-207
61-71 Cleavage sites of Plasmin

61-70
DF retentate
2s -=
2
7
- 41
FIG.4. aS2-Casein-derivedpeptides isolated from the DF retentate (-), and the DF permeate (-) of the water-soluble fraction
of mature Cheddar cheese. (Data from Singh et a]., 1997.)
51 93 w
51 93 Q,
51 92
n-n
7
3
-
n
I In Zm
109
. . W
FIG.5. p-Casein-derived peptides isolated from the water-insoluble fiaction (----I, the DF retentate (-1, and the D F permeate
[-) of the water-soluble fraction of a mature Cheddar cheese. (Modified from Singh et al., 1997, and Mooney, 1997.)
FIG.6. Urea polyacrylamide gel electrophoretogram of sodium caseinate (C) and the
water-insoluble nitrogen fraction of Cheddar cheese after manufacture (slot 2) and ripen-
ing for 1, 2, 3, 4, 5, 6 , 8, 10, 1 2 , 14, 16, 18, and 20 weeks (slots 3-14, respectively).
Peptides isolated from the 20-week sample were identified as follows: 1. P-CN f106-149,
2. P-CN flO6-128, 3. P-CN flO6-209 (y),P-CN f29-209 (r'),5. P-CN flO8-209 (y),6. 0-CN,
7. P-CN f1-187/192 (P-I-CN),8. P-CN fI-*, 9. asI-CN f99-199,lO. CX~I-CN f80-*, 11. asl-CN,
12. as1-CN f102-199, 13. asl-CN f24-199 (USi-I-CN), 14. as1-CN 33-*, 15. asl-CN f121-
199, 16. asl-CN fl29-199, 17. CXSI-CNf60-*, 18. asl-CN f110-199, 19. USI-CN f70-156.
* = peptide not identified completely. (Modified from Mooney, 1997.)
water-insoluble fraction of Cheddar and most other varieties (Fig. 7),

and proteose peptone 8 fast (P-CN fl-ZS), 8 slow (P-CN f29-105/107),
and 5 (P-CN fl-105/107), which are present in the UF retentate or 70%
ethanol-insoluble fraction of the WSE.
Most of the peptides that have been identified in the UF retentate (or
70% ethanol-insoluble fraction) of the WSE are produced from p-casein
by the action of lactococcal CEP, probably on proteose peptones rather
than on intact p-casein, since none of the identified peptides contain a
plasmin cleavage site (Fig. 5). The N-terminal of many of the identified
peptides corresponds to the carboxyl residue of a plasmin or CEP
cleavage site, but several appear to be 1 , 2 , or 3 residues short, indicating
FIG.7 . Urea-PAGE of the water-insoluble fraction of a selection of cheese varieties.

Lane 1 = Na-caseinate; lane 2 = Cheddar 1; lane 3 = Cheddar 2; lane 4 = Cheddar 3; lane
5 = Emmental 1; lane 6 = Emmental 2; lane 7 = Emmental 3; lane 7 = Maasdam; lane 9
= Jarlsberg; lane 10 = Edam 1; lane 11 = Edam 2; lane 1 2 = Edam 3; lane 13 = Gouda 1;
lane 14 = Gouda 2; lane 15 = Gouda 3. (Modified from McGoldrick, 1996.)
aminopeptidase activity. The C-terminal of several peptides does not

correspond to reported CEP cleavage sites, suggesting carboxypeptidase
activity (which has not been reported in Lactococcus spp.) or unknown
CEP cleavage sites, or the action of other proteinases, for example, from
NSLAB or oligopeptidases (PepO, PepF).
Thus, the large water-insoluble peptides in Cheddar are the C-termi-
nal segments of asl-casein produced mainly by chymosin or of p-casein
(y-CNs)produced by plasmin. Para-K-casein (K-CN fl-105) is very resis-
tant to proteolysis and is not hydrolyzed during ripening. The concen-
tration of a,,-casein appears to decrease during ripening, but its fate is
unclear. No large peptide derived from a,,-casein has yet been reported,
and only eight small as2-derived peptides have been identified in the
WSE (Fig. 4). Perhaps a,,-CN, which represents only -10% of total
casein, is hydrolyzed rather nonspecifically to several peptides, none of
which are present at a high concentration, and hence have been over-
looked.
The small water-soluble peptides appear to be produced from chy-
mosin- or plasmin-produced peptides by lactococcal CEP and perhaps
endopeptidases. Some of the small peptides are hydrolyzed by ami-
nopeptidases.
Although NSLAB dominate the viable microflora in Cheddar for most
of the ripening period, they appear principally to supplement the pep-
tidolytic activity of the starter, especially in the production of amino
acids (Lynch et al., 1997). The accelerated maturation of cheeses with
added lactobacilli appears to be closely related to the concentration of
free amino acids (Lloyd et al., 1980; Hickey et ~ l . 1983;
, Ardo and
Pettersson, 1988; Ardo et al., 1989; Puchades et al., 1989; Broome et al.,
1990; Lee et al., 1990a,b;Lynch et al., 1997). Although most lactobacilli
were found to increase the concentration of amino acids and the inten-
sity of cheese flavor, the flavor was found to be atypical when cheese
milk was inoculated with Lb. helveticus (Lloyd et al., 1980), Lb. brevis
(Puchades et al., 1989),or Lb. casei MIL2A (Broome et al., 1990).
Proteolysis in Cheddar- and Dutch-type cheeses, and in many other
varieties, is generally similar (Fig. 7). The high-cooked varieties, for
example, Emmental and Parmesan, differ, partly because chymosin is
extensively or totally inactivated during cooking while plasmin activity
is increased, and partly because the thermophilic starter lactobacilli are
more peptidolytic than Loctococcus spp. In addition, Swiss-type
cheeses contain Propionibacterium, which has very high proline-spe-
cific peptidase activity. Commercially mature Emmental undergoes
relatively little primary proteolysis: only -50% of the aSl-casein is
hydrolyzed at Phe,,-Phe,, (by chymosin) and/or Lys103-Tyr104 and
Lyslo5-Vallo, (by plasmin); the Leulol-Lysloz bond is not hydrolyzed.
Although primary proteolysis is relatively limited in Parmesan, very
extensive peptidolysis occurs; most of the WSE occurs as very small
peptides or free amino acids.
c. FREEAMINOACIDSIN CHEESE
The final products of proteolysis are free amino acids, the concentra-
tions of which have been used as indices of ripening for many years
(Harper and Swanson, 1949; Kosikowski, 1951; Law, 1981; Hickey et al.,
1983; Wood et al., 1985; Amantea et al., 1986; Puchades et al., 1989;
Wilkinson, 1993; Resmini et al., 1993). Resmini et a]. (1993) reported
that the concentration of free amino acids in Grana Padano cheese
correlated with flavor intensity but not with ripening time. However,
others reported that amino acids appeared to have no effect on cheese
flavor (Dacre, 1953; Mabbit et al., 1955; Law, 1966; Law and Sharpe,
1977). As discussed in Section IV.B, ripening and flavor development
appear to be accelerated when high levels of free amino acids are
produced early in the ripening process.
Although catabolic products of amino acid degradation are thought
to be mainly responsible for Cheddar flavor, small peptides and free
amino acids contribute to the flavor of most cheese varieties (Urbach,
1995), for example, “brothy,” “nutty,” and “sweet” tastes (Biede and
Hammond, 1979). The flavor characteristics and flavor threshold of
free amino acids are summarized in Table 111. High levels of proline
contribute to sweetness in Swiss cheese (Reinbold, 1973; Langsrud and
Reinbold, 1973; Lloyd et al., 1978, 1980) and to a sweet Swiss cheese-
like flavor in experimental Cheddar (Lloyd et al., 1980). Arginine
has been associated with an unpleasant bittersweet taste (Puchades
et al., 1989).Free amino acids can also contribute to bitterness in cheese
(Ney, 1971).
About 20 to 30% of the total N in a mature Cheddar cheese is soluble
in water, while about 2 to 5 % is soluble in 5% phosphotungstic acid
(PTA). The levels of peptides and free amino acids soluble in 5% PTA
in cheese are considered to be reliable indicators of the rate of flavor
development (Aston et al., 1983; Pham and Nakai, 1984; Amantea et al.,
1986; Ardo and Pettersson, 1988); however, the composition of the
amino acid fraction and the relative proportions of individual amino
acids are thought to be most important for the development of a typical
characteristic flavor (Broome et al., 1990). The release of specific amino
acids, particularly Glu, Met, and Leu, coincides with the development
of Cheddar cheese flavor (Broome et a]., 1990; Marsili, 1985). Leu and
Met are considered to be major contributors to cheesy flavor in the
water-soluble extract of Cheddar (Kowalewska el al., 1985; Marsili,
1985; Aston and Creamer, 1986).
The concentration of free amino acids in cheese at any stage of
ripening is the net result of the liberation of amino acids from casein
and their transformation to catabolic products. The concentration of
individual amino acids in a number of cheese varieties are compiled in
Table IV. Comparison of free amino acid profiles in different varieties is
limited for a number of reasons. Cheeses are ripened at different rates,
TABLE I11
TASTEDESCFXPTORAND
THRESHOLD
VALUESOF AMINOACIDS'
Con-
Taste centra-
Q" threshold tion'in Per- Taste
Amino (cal (mg Cheddar cep-
acid mol-'I 100 ml-') (wg g-'1 tiand Sweet Salt Sour Bitter Umami
Gly 0 130 371 ***

Ser -300 150 1210 **I
Thr 400 260 649 *** *

His 500 20 436 ***
Asp 0 3 606 **
Glu 0 5 5075 *** **
A% 750 50 1737 ***
Ala 500 60 337 ***
Met 1300 30 869 ***
LYs 1500 50 2330 ** **
Val 1500 40 2022 ***
Leu 1800 190 4610 ***
Pro 2600 300 389 *** ***
Phe 2500 90 2400 ***
Tyr 2300 607 ***
Ile 2950 90 466 ***
3li.p 3400 90 - ***
"Adapted from O'Callaghan (1994).

bHydrophobicity values of Bigelow and Channon (1976).
Wilkinson (1992); 6-month-old Cheddar; total concentration = 24.1 mg g-' cheese.
dAmino acids in Cheddar are deemed to be perceived if their concentration in a water extract (50 g
cheese containing 37% moisture to 100 ml water) is greater than their threshold concentration.
even within a single variety. The age of the cheeses in Table IV vary, and
in some cases are unknown. Different analytical techniques lead to
variations in results. The rate of amino acid degradation differs between
and, to a lesser extent, within varieties due to different manufacturing
and ripening conditions and variations in cheese microflora. Without
monitoring protein-bound as well as free amino acids, no conclusions
as to the exact concentrations of amino acids released may be drawn
since some of the free amino acids may have been degraded prior to
analysis.
The relative proportions of individual amino acids appear to be
similar in many varieties. Leu, Glu, and Lys are the principal amino
acids in Cheddar (Wood et al., 1985). Glu has been described as an
important contributor to Cheddar flavor by many authors (Harper and
Wang, 1981; Marsili, 1985; Nsar and Younis, 1986; Broome et a]., 1990).
TABLE IV
(mg kg-' CHEESE) OF FREEAMINOACIDS
CONCENTRATION AND THEIR RELATIVI?.PROPORTIONS
(% TOTAL FREE AMINO ACIDS) I N DWI%RENT CHEESE VARIETIES
Chnese
[Ripening time]
(:hedrlm (1) Cheddar (2) HnrrgArd (5) Emmental (6)

I8 niol 19 mol Edam (3) Gouda (4) [.I4wkl [fi mol
Amino mg/ '&of mg/ % of mg/ % of mg/ %of mg/ Ohnf mg/ % of
acid kg FAA kg FAA kg FAA kg FAA k FAA kg FAA
CYS 44.7 029 - - ~ - - -

Asp 1532 8 9.94 - 0.65 20.5 0.9 91.7 1.4 1.4 1fifi.S 0.9
Thr - - 1.11 144.5 6.0 272.9 4.2 2.3 688.5 3.7
SW 41fi 3 2.7 - 1.85 71.1 3.0 167 3 2.fi 2.7 548.9 2.9
GlU :i144 1 20.39 - 6.21 351.7 1 2 3! 992.1 15.1 14.3 26no.s 14.3
Pro 336.2 2.18 - i5y.n 54 345.6 5.3 4.8 2535.2 13.5
GlY 30fi.n~ 1.99 - 2.32 34.ti 1 2 108.G 1.6 2.7 430.8 23

Ala 3Sfi.2 2.31 - 3.67 68.6 24 iun.9 2.8 2.7 568.2 3.0
Val 1096.4 7.11 - 1 5 26 167.4 6.8 47fi.s 7.3 7.0 1561.5 8.4
Met 434.8 2.82 - 8.14 60.3 2.3 176.1 2.7 1.n 502.7 27
1Ie - ~
8 46 48 1 2.4 200.5 3.1 2.3 1051 1 5.G
i.PI1 2774.1 17.99 ~ 26.90 428.0 17.5 1056.9 16.2 14 3 17949 9.6
'Tyr 464.1 3.01 - 89.2 3.4 165.3 2.5 2.3 2859 1.5
I'hn 1472.6 9.55 - 7.56 291.9 11.6 645.9 9.9 56 1279.1 fi.8
IIis - - ~
49.7 1.7 143.6 2.2 3.8 RGR.2 4.6
LYS 1127.2 7.31 ~ 11 00 245.5 9.1 fi71.4 10.3 12 LJ 22198 11.9
Arg 1096.4 7.11 ~ 0 00 130.5 4.2 167.3 2.6 Asrr 11 7 192 0.1
Gln 7.7
Total 258'~ 05211 18616
'The concentrations of amino acids were either given by, or calculated from tho results of 1: Wood et
a]. (19851; 2: Puchades ot a]. (1989); 3,4,6: Antila and Antila (1968);5: Ardo and Gripon (1995).
High levels of Asp and Met were also observed by these authors.
Comparatively low levels of Glu (as a percentage of total FAAs) were
found in Cheddar by Puchades et al. (1989), who found that Met
represented >8% of total FAAs; in contrast, Wood et ~ l(1985). found
that Met was only 2.8% of FAAs. According to Wood et al. (19851, -10%
of total FAAs in Cheddar is Asp, while Puchades et al. (1989) reported
a value of only 0.65%.Large variations within single varieties have been
reported for Emmental, Gruyere, Parmagiano Reggiano, Camembert,
Stilton, Danablue, Cabrales, and Mahon (Table IV). There are, however,
clear similarities within, and differences between, different cheese va-
rieties.
High concentrations of free amino acids in Parmesan are thought to
be due to high numbers of lactobacilli and a long ripening time. High
TABLE IV
continued
Cheese
(Ripening time1
Grana (101 Parmigino (111 Parinigino (121

Gruyere (7) Gruyhrr (HI Appenznller (9) Padano Reggiano Reggiano
[60samples [mean of
10-20 wkl 60 cheeses1
Amino mgi 70of mgl B of mgi %of mgl *A of mgl %of mgl “A of
acid kg FAA kg FAA kg FAA kg FAA kg FAA kg FAA
CYS - - - - 115.0 0.3
ASP 328.9 1.7 834.8 2.6 774 6 23 3.1 3241.9 4.3 2231.9 fi.3
Thr 7R1.2 4.0 1017.2 3.1 1317.1 2.4 3.2 4033.3 5.3 1031.3 3.2
Snr 446.1 2.2 979.4 3.0 253.2 0.76 4.4 4459.5 5.9 1580.5 5.0
GI” 2963.5 15.2 6415.9 19.7 6823.4 20.42 18.0 14489.0 19.0 7580.2 21.4
Pro 2817.3 14.4 3546.9 10.9 3299.3 9.87 9.5 - - 3649.6 10.3
Gly 493.0 2.5 716.0 2.2 74Y.O 2.2 2.4 2115.6 2.8 667.3 1.9
Ala 597.6 3.1 865.1 2.65 925.65 2.8 2.7 2260.2 3.0 1028.2 2.9
Val 1817.9 8.3 2381.5 7.3 3594.4 10.8 7.2 6011.9 7.9 2330.9 6.6
Met 532.1 2.7 868.3 2.7 922.0 2.8 2.5 2351.5 3.1 970.7 2.7
Ile 1189.2 6.1 1901.8 5.97 1YY6.3 6.0 6.1 5205.2 6.H 1762.5 5.0
Leu 1778.7 9.1 3765.6 12.5 4176.1 12.5 9.1 7290.4 9.6 2905.6 9.1
TYr 321.3 1.6 992.9 2.7 884.2 2.7 2.7 2054.7 2.7 1963.7 5.5
Phe 1366 7 7.0 2839.4 7.6 2548.7 7.6 5.1 4314.9 5.7 1753.0 4.9
His 680.6 3.4 1276.8 3.2 1081.3 3.2 3.3 - - 1034.5 2.9
LYS 2227.8 11.3 4038.7 13.7 4573.7 13.7 12.2 10091 13.3 2876.9 8.1
21.3 0.1 158.5 0 0 0 - 791.4 1.0 977.1 2.8
‘4%
Tulal 19342 32598 33419 76100 34459
The concentrations of amino acids were either given by, or calculated from the results of 7: Antila
and Antila (1968); 8: Lavanchy and Buhlmann (1983);9: Lavanchy et 01. (1979); 10: Resmini et al.
(1993); 11: Resmini et al. (1988); 12:Mariani et a1. (1993).
concentrations of free amino acids are also present in mold-ripened and

Swiss cheeses. Of the cheeses tabulated, Edam and Gouda contain the
lowest levels of free amino acids. However, an Edam-type cheese made
from goat’s milk contained high levels of FAAs, as did another goat’s
milk cheese, Rumelia (Baltajieva et al., 1985).
Resmini et al. (1993) attempted to “fingerprint” cheeses using chemo-
metric models based on free amino acid profiles. These models allowed
Parmigiano Reggiano, Grana Padano, and Fontina to be distinguished
from each other and from similar cheeses. In another comparative study,
Engels and Visser (1994) found that the amino acid profiles for Cheddar,
Edam, Gouda, Gruyere, Maasdam, Parmesan, and Proosdij cheeses were
44 €? F. FOX AND J. M. WALLACE
TABLE IV
continued
Cheese
[Kipening time]
Danish 1131 Camnm- 114) Stil- (15) Stik- (16) Stil- (17) Dana- (18)
Camembert hrt ton ton tnn blue
170 days] IMarketl [Market]
Amino mgl YO of rngl % uf mgl %of mgf Ohof mgl %of rngl "h of
cys - fi.4 0.1 - ~ ~

- 1020 10.4 -
Asp - 5.9 99.7 4.0 5390 28 626.77 2.2 340 3.5 1098.1 3.9
Thr - 2.5 134.8 6.9 751.6 4.0 837 5 3.5 1811 1.8 1526.6 5.4
Ser - 3.2 56.2 27 761.0 4.0 621.4 2.6 57u 58 896.6 3.2
Glu - 22.5 566.6 20.5 1750.0 8,2 4238.4 175 1090 11 1 in302 6.5
Pro - 3.1 192.3 6.6 1446.3 7.6 1267.2 5.2 510 52 332.0 12
G~Y - 1.2 36.9 1.3 216.4 1.1 650.6 2.7 140 1.4 485.2 1.7
Ale - 1,8 93.8 3.3 876.9 4G 793.8 3.3 350 357 13194 47
VAI - 5.8 169.1 5.7 1664.6 8.8 2687.4 in 7 fiiu 6.22 3353.~~ 11s
Met - 3.6 65.6 3.1 603.6 3.2 929.7 3.8 410 4.2 1679.R 5.9
Ile - 5.8 134.2 4.2 848.4 4.5 1716.3 7.1 310 3.2 2434.G 8.6
LCU - 11.8 317.6 11.5 3015.9 15.9 2920.3 12.0 1220 12.5 3708.6 13.1
TLr - 5.3 160.1 5.9 1495.6 7.9 1733.2 7.1 260 2.7 2340.9 17.3
PllB - 6.4 241.8 8.7 1776.5 9.4 2386.2 9.8 610 6.2 2fi89.9 9.5
His ~ 2.6 175.1 6.6 6337.7 3.4 463.7 1.9 490 5 996.0 3.5
4 s ~ 93 209.1 6.5 1975.8 10.4 2456.7 10.1 1560 15.9 2760.9 9.7
*u ~ 1.4 36.2 1.5 616.9 3.3 279.2 1.2 130 1.3 919.3 3.2
Tulal 2790 18980 24275 YB00 28375
The concentrations of amino acids were either given by, or calculated from the results of 14: Antila
and Antila (1968); 13,17:Ismail and Hansen (1972); 14: Antila and Antila (1968); 15,16,18:Madkor et
a]. (1987); 17:Ismail and Hansen (1072); 17: Zarmpoutis (1995).
similar, with Glu, Leu, and Phe being the principal amino acids; Val,
Pro and Lys were also quite abundant in these varieties. Maasdam,
Cheddar, Gouda, and Edam contained similar concentrations of free
amino acids in the UF permeate (MW < 500 Da), but higher concentra-
tions were found in Gruyere, Proosdij, and Parmesan cheeses. The
authors concluded that flavor and the concentration of free amino acids
could not be correlated since Cheddar, Maasdam, Gouda, and Edam
have very different flavors, although the concentration and relative
proportions of free amino acids were generally similar.
Although the concentration of free amino acids depends on the
cheese variety and the dairy plant where the cheese is manufactured,
they generally increase during ripening, with the exception of Arg, the
TABLE IV
continued
Cheese
[Ripening time]
Dana- 1191 Dana- (20) Gorgon- (21) Cashel (22) Chetwynd (23) Gamonedo (24)
blue blue zola (Blue1 (Blue) (Blue)
-____ 18.3 mol
~ ~ _
-
190 days1
_
Amino mg/ %of mgl %of mg/ % of mg/ %of mg/ %of mg/ % of
CYS 1160 9.48 - - 1380 5.3 740 13.7 590 10.2 -

ASP 300 2.5 - 2.9 1020 3.9 140 2.6 190 3.3 3.7
Thr 190 1.6 - 3.8 530 2.0 80 1.5 100 17 6.4+gly
Ser 1020 8.3 - 3.5 1570 6.0 330 6. 1 390 68 4.2
Glu 1730 14.2 - 16.1 3940 15.1 620 11.5 850 14.7 18.5
Pro 530 4.3 - 10.3 2320 1.2 160 3.0 160 2.77
G~Y 160 1.3 - 1.6 390 1.5 70 1.3 100 1.7 +Thr
Ala 340 2.R - 5.8 1140 4.4 120 2.2 150 2.6 6.2
Val 610 5.0 - 6.1 2220 8.5 350 6.5 360 6.2 7.8
Met 500 4.1 - 37 760 3.0 180 33 200 3.5 2.5
Ile 300 2.5 - 6.5 1300 5.0 2 70 3.1 220 3.8 6.1
Leu 1530 12.5 - 10.1 2910 11.2 690 12.8 650 11.3 11.5
5 r 520 4.3 ~ 3.9 650 3.3 290 5.4 270 4.7 7.4
Phe 680 5.6 - 6.0 1590 6.1 320 5.9 310 5.4 5.9
His filO 5.0 ~ 1.5 800 3.1 240 4.4 270 4.7 3.5
LYS 1540 12.6 - 7.5 3050 11.7 650 12.0 960 16.6 4.5
510 4.2 - 1.5 280 1.1 260 4.8 - 10
Told 12.230 26070 5410 5770
The concentrations of amino acids were either given by, or calculated from the results of 19,21,
22, 23: Zarmpoutis (1995): 20: Isrnail and Hansen (1972); 24: Gonzalez de Llano et 01. (1991).
levels of which are reported to decrease during the later stages of

ripening (Puchades et a]., 1989; Broome et a]., 1991; Wilkinson, 1993).
V. Catabolism of Amino Acids

The general catabolism of free amino acids is summarized in Fig. 8
(Hemme et al., 1982).
A. DECARBOXYLATION
AND PRODUCTION OF AMINES
Amines, including biogenic mines, are produced in cheese by enzy-

matic decarboxylation of free amino acids (Joosten and Stadhouders,
46 P. F. FOX AND J. M.WALLACE
TABLE IV
continued
Cheese
[Ripening time]
Cabrales (251 Cabrales @ti) Maribo (281 Maribo (291 Havarti (30)
(BhlC) (Blue) Danbu (27) (rindless) (rind) [Smear]
[4 rnol 14 mol 18.3 mu] [8.4 mo] In.4 mol
Amino mgl %of rngl '% of mgl of

'Yo ing/ Ohof mgl % of mg/ Oh of
Cyr - - - -
ASP 2800 0 ti.7 2880.2 5.0 1.9 1.1 1.7 1.7
Thr 4190.0 13.6 3039 fi 52 27 1.5 3.0 2.0
Ser 1230 0 4.0 790.5 1.4 22 1.0 2.6 2.3
Glu 4140.0 13.4 7604.9 13.1 21.0 11.3 19.2 21.2
PR, 4.7 3.1 4.1 7.ti
Gly 590.0 1.9 1041.n 1.8 1.R 1.7 1.7 1.8

Aln 1970 0 6.4 1893.6 3.3 2.8 5.6 2.8 2.n
Val 2040.0 6.8 4504.1 78 90 10.0 8.7 78
Mct 11R0.0 38 2696.3 9G 3.4 4.3 3.4 33
110 1310.0 4.2 4124.1 71 6.2 5.9 5.8 5.0
LOU 2640.0 8.6 7525.2 13.0 14.0 17.8 1ti.0 13 2
TY~ 540.0 18 333Y.8 5.8 1.7 1.7 1 6 33

I'hn 1730.0 5G 4406.0 7.6 7.9 8.3 R.0 7.6
llis 1140.0 3.7 1403.3 2.4 1.Y 2.0 13 1.5
1.ys 3270.0 10.6 71335.5 13.2 8.8 6.8 7.4 R.8
AX 870.0 2.R 281.9 0.5 0.1 0.4 0.2 0.1

Total 30890 57897
The concentrations of amino acids were either given by, or calculated from the results of 25:
Gonzalez de Llano et al. (1988);26: Tuckey and Sahasrabudhe (1958);27,28,29,30: Ismail and Hansen
(1'372).
1987). Although amines are thought to be important contributors to

cheese flavor, some may cause bitterness (Ney, 1971). Amines are po-
tentially toxic to humans due to their ability to react with nitrogen
oxides, forming carcinogenic nitrosamines, and their ability to induce
symptoms of hypo- or hypertension in certain individuals (Rice et al.,
1978). Concentrations of amines in Cheddar are generally too low to
cause adverse effects (Joosten and van Boekel, 1988; Flynn, 1992).
The principal amines in most cheeses are tyramine and histamine,
produced by decarboxylation of Tyr and His (Fig. 9) (Fox et al., 1995a).
The concentrations of tyramine and histamine in cheeses inoculated
with lactobacilli were twice as high as in control cheeses, indicating
that decarboxylases of lactobacilli play a major role in their production
TABLE IV
continued
Cheese
[Ripening time]
Kesti (36)
Lim- (31) Roma- (33) Hovi (341 Kreivi (35) Wlsit mil
burger Brick (32) dour (Gervais) (Tilsit) Kummeln~satc)
110 wkl I10 wkl
~
Amino mg/ % of mg/ % of mg/ % of mg/ Yn of mg/ % of mg/ % of

CYS 450 3.5 - - - - - -
ASP 450 3.5 430 7.2 1ofi.n 2.0 7.5 3.4 96.5 1.2 114.6 1.8
Thr 600 4.7 in0 3.0 75.1 1.4 6.3 2.6 457.4 5.9 375.6 5.1
Ser 290 2.3 210 3.5 61.6 1.2 6.7 3.0 276.0 3.5 242.2 3.5
Glu 2580 20.1 600 10.1 519.0 9.7 53.2 23.8 117R.7 15.2 782.9 13.1
Pro 320 2.5 244.6 4.6 14.0 6.4 534.3 6.4 368.5 5.6
Gly 380 3.0 500 8.4 111.2 2.1 3.0 1.3 131.1 1.6 113.2 1.7
Ala 890 6.9 460 7.7 171.7 3.2 6.2 2.8 213.0 2.5 180.2 2.7
Val lain 14.1 190 3.2 610.6 11.4 6.1 2.7 608.9 7.4 491.0 7.5
Met 1100 8.6 480 8.0 230.0 4.3 3.1 1.3 229.4 2.8 192.5 2.9
Ile - 333.9 6.2 5.6 2.2 288.0 3.2 215.9 3.3
Leu 1900 14.8 lion in4 1039.1 19.4 16.6 7.3 1161.7 15.6 919.8 13.8
TYr R90 6.9 420 7.0 a3 o 1.6 7.1 3.2 327.2 4.0 257.8 3.9
Phe 100 0.8 520 87 468.1 8.8 13.0 5.7 796.1 10.0 658.2 10.0
His 260 2.0 223.6 4.2 5.9 2.6 575.1 7.2 148.9 2.3
LYs 370 29 726 8 13.6 24.0 10.7 974.8 11.8 748.0 11.5
Arg - 34 n 0.6 11.9 5.3 161.8 2.1 235.2 3.7

Total 12818 4624 224.2 799n 6461
The concentrations of amino acids were either given by, or calculated from the results of 31, 32:
Tuckey and Sahasrabudhe (1958); 33,34, 35, 36: Antila and Antila (1968).
(Broome et al., 1990). The concentration of tyramine in cheese often

exceeds that of histamine, with lesser amounts of phenylethylamine
(from Phe), tryptamine (from Trp), cadaverine (from Lys), and putricine
(from ornithine) (Tokita and Hosono, 1968); the relative proportion of
amines depends on the cheese variety and the nonstarter microflora.
Lactobacilli appear to be responsible for the production of tyramine,
histamine, and putricine in Gouda cheeses. In normal Gouda and
Maasdam produced under hygienic conditions from pasteurized milk,
concentrations of biogenic amines were low, indicating the inability of
starter cells to produce them (Joosten and Stadhouders, 1987). A similar
conclusion applies to Edam and Emmental (Antila ef al., 1984). Entero-
cocci and coliforms in Gouda are capable of producing biogenic amines
(Gripon et al., 1991);Br. linens is capable of producing tyramine, hista-
48 P. F. FOX AND 1. M. WALLACE
TABLE IV
continued
Cheese
[Ripening time)
Trap- (37) Edam- (42)

pist- Ras (38) Kashkaval (39) Kopanisti (40) Rumelia ( 4 1 ) type
type (Egyptian) (Egyptian) (Greek) (goat's) (goat's)
135 days] [4 mol [4 mol 146 days]
Amino mgl % uf mgi % uf mgi % of rngl % uf rngi % vf rngi Yo of

acid kg FAA kg I'AA kg I'AA kg FAA kg FAA kg FAA
Cye 12.4 0.4 - - ~ ~ ~
Asp 130.0 3.8 18.1 0.5 182.5 7.7 56.3 0.6 530.8 1.6 1217.3 4.2
Tlir 44.4 1.3 103.0 2.9 109.4 4.6 462.1 5.0 1642.4 4.9 1403.8 4.0
Ser 143.1 4.2 99.9 28 41.7 1.8 181.1 2.0 2146.3 64 10615 64
Gl" 784.2 22.8 700.0 I'J 8 401.0 16.9 211.0 2.3 71322 21.2 59159 212
Pro m8.n 5.5 167.0 4.7 173.7 7.3 815.3 8.8 18978 57 1719.0 5.7
Gly 59.2 1.7 83.3 2.4 67.7 2.8 286.6 3.1 12778 3.8 1244.7 3.8
Ald 204.0 5.1) 279.0 7.g 191.1 8.1 1022.8 11.1 1221.0 3.6 970 1 36
Val 290.6 8.5 307.0 8.7 1043 44 1280.4 14.0 30742 92 26543 92
Me1 32.3 09 2530 7.2 66.5 2.8 500.4 54 11670 35 936 8 3.5
Ile 12'J 7 38 261.0 7.4 81.5 34 1026.6 11 1 2077.0 6.2 1524.2 G.2
LCU 400.8 14.3 345.0 9.8 2600 11.0 2033.1 22.1 4014.7 12.0 3281.2 12.0
Tyr 57 n 17 75.3 2 1 130.9 55 25.1 0.3 521.0 1.n 38S.5 1.6
Phe 270 3 7 9 315.0 8'1 295 0 12.4 11as.9 12 0 1850.6 5.5 1475.7 5.5
His 74 fi 22 87.1 2.5 62.2 2.6 98.4 1.1 863.5 2.6 1202.5 2.6
LYS 480.6 140 1590 4.5 126.1 5.3 106.3 1.2 3921.0 11.6 4103.4 11.6
Arg 46 4 14 279.0 7.9 80.3 3.4 - - 254.4 0.8 216.5 0.8
Total 3439 3530 2380 m n 33594 29450
l'hc concentrations of amino acids were either given by, or calculated from the results of 37:Ades
and Cone (1969); 38:Omar (1984); 39: Omar and El-Zayat (1986); 40: Kaminarides et aJ. (1990); 41:
Baltajieva et al. (1985);42:Ramos et aJ. (1987).
mine, monomethylamine (Fig. 10a), monoethylamine (Fig. lob), tri-

methylamine (Fig. lOc), dimethylamine (Fig. lod), cadaverine (Fig.
loe), monopropylamine (Fig. 100, dipropylamine (Fig. log), tripropy-
lamine (Fig. loh), triethylamine (Fig. l O i ) , and piperidine (Fig, loj) in
model systems (Tokita and Hosono, 1968; Hosono and Tokita, 1969).
Simple decarboxylation can explain the formation of most amines
found in cheese, but there is no readily available explanation for the
formation of secondary and tertiary amines and n-butylamine in cheese
(Adda et a]., 1982).
No relationship has been found between the concentration of free
amino acids and the production of amines in cheese (Smith, 1981),
probably due to differences in the rate of decarboxylation of individual
TABLE IV
continued
Cheese
[Ripening time1
Mahon (46) Mahon (47)

Turunmss (43) Sir0 (44) Loustari (45) (tradi- (Indus-
(Finnish) (Finnish) (Port Salut) tional) trial)
Amino rngi % of rngi %of rngi % of rngi % of rngi % of

acid kg I'AA kg FAA kg FAA kg FAA kg FAA
CYS
- - - - -
ASP 53.3 0.9 70.3 1.3 113.2 1.9 60 1.3
Thr 349.2 7.3 354.6 6.5 185.6 8.7 5.7+gly 5,5+gly
Ser 139.2 3.0 186.8 2.9 +Ser tSer 5.5 3.8
Clu 740.5 159 51G.G 95 942.0 17.2 17.2 15.3
Pro 253.3 6.1 237.9 4.4 176.1 4.4 9.7 9.0
tily 63.4 14 110.7 1.U 73.1 1.4 +Thr +Thr
Ala 110.8 2.0 181.1 3.3 176.2 2.8 3.4 3.2
Val 372.1 8.4 420.4 7.7 420.2 7.6 13.8 12.7
Met 125.6 2.8 197.9 3.6 157.7 3.1 0 2.9
Ile 123.8 2.4 lfi4 9 3.0 218.7 3.6 9.1 18.8
Leu 707.2 16.6 704 4 12.9 800.1 15.5 -
5 r 242.0 1.9 164.7 3.0 161.1 3.1 - -
Phe 500.1 11.7 600.9 11.0 567.5 11.3 22.2 20.3
His 63.3 0.8 114.1 2.1 100.4 1.8 - -
Lys 434.8 7.8 542.1 9.9 527.8 96 -
A% 95.5 2.4 352.6 6.5 51.4 1 5 0 74 1.0
Total 4733 5453 5076
The concentrations of amino acids were either given by, or calculated from the
results of 43,44,45:
Antila and Antila (1968);46,47:Polo et al. (1985).
amino acids or in the rate of deamination of resulting amines (Polo et

a]., 1985).
B. DEAMINATION-FORMATION AND NEUTRAL

OF AMMONIA
OR ACIDICCOMPOUNDS
Deamination of free amino acids leads to the production of ammonia

and a-keto acids (Hemme et al., 1982). Bassett and Harper (1956)
identified a number of a-keto acids, including pyruvic and p-hydroxy-
phenyl pyruvic acids, in cheese. However, more recent studies have not
identified a-keto acids, perhaps due to their instability when measured
50 P. E FOX AND J. M. WALLACE
Amino acids
Transamination Oxidative I I
deamination
\
Amines Amino acids a-keto acids
I _i,,
Aldehydes
Phenols
v
Indole
I
Other sulphur compounds
Alcohols Acids
FIG.8. Catabolism of free amino acids in cheese. (Modified from Hemme et al., 1982.)
H~N-CH-COOH H,N- JH,

Tryptophan T ptamine
FIG.9. Production of biogenic amines from tyyosine and tryptophan.
(a) (C)
NH, CH,
I I
CH, N
C$ C'H,
Monomethyl amine Trimethylamine
(d) (0
H,N
CH,
I
N -CH, CH, CH,
H
I
Dimethylamine Monopropylamine
(g) (9
cY 7' CH
CH,
I
CH,
I
I N- CHI-CH,
NH
I I
CH,
I
/:
CH,
' CH,
CH,
Dipropylamine Triethylamine
ti)
0 HI
Pipiradine
FIG.10. Amines that have been identified in cheese (Tokita and Hosono, 1968; Hosono
and Tokita, 1969).
by GLC (Urbach, personal communication). Ammonia is an important

constituent in many cheeses, such as Camembert, Gruyere, and Comte
(Fox et a]., 1995a).
G. candidurn is capable of deamination, and Br. linens strains possess
very active deaminases and produce large quantities of ammonia via the
catabolism of serine, and to a lesser extent of glutamine, asparagine, and
COOH
COOH OH I
I I
Isovaleric acid Isobutyricacid Acetic acid Propionic acid

FIG. 11. Volatile fatty acids produced by amino acid catabolism.
RCOOOH RCH + C Q
II II
0 0
0
Strecker aldehyde
FIG.12. Strecker degradation pathway. (Modified from Urbach, 1995.)
threonine (Hemme et al., 1982). Ammonia can also be formed by oxida-

tive deamination of amines, forming aldehydes (Hemme et al., 1982) or
volatile fatty acids. Isovalerate (Fig. l l a ) , isobutyrate (Fig. I l b ) , acetate
(Fig. l l c ) , and propionate (Fig. I l d ) are produced by oxidative deami-
nation of isoleucine, valine, glycine-alanine-serine, and threonine,
respectively.
0
II
0 CH
0 II I
CH CH,- CH
I
x
I
CH, CH3
Phenylacetaldehyde Isobutanal 3-methylbutanal
0
II
CH
I
3-methylthiopropmal Methional
FIG.13. Aldehydes produced by Strecker degradation of amino acids.
c. TRANSAMINATION,
STRECKER DEGRADATION,
AND PRODUCTION
OF ALDEHYDES
Production of aldehydes from free amino acids can result from decar-
boxylation, deamination, transamination (Fox et al., 1995a), or via
Strecker degradation (Keeney and Day, 1957; Dunn and Lindsay, 1985;
Urbach, 1995) (Fig. 12). Enzymatically catalyzed transamination of a
free amino acid results in the formation of an intermediary imide that
is subsequently degraded by decarboxylation or the Strecker reaction,
resulting in the formation of an aldehyde (Keeney and Day, 1957; Polo
et al., 1985). Phenylacetaldehyde (Fig. 13a), isobutanal (Fig. 13b), 3-
methylbutanal (Fig. 13c), and 3-methylthiopropanal (Fig. 13d) and me-
thional (Fig. 13e) can be formed by this mechanism from Phe, Leu-Ile,
Val, and Met, respectively (Adda et al., 1982). Transamination also leads
to the production of other amino acids (Fox et ai.,1995a), such as Glu
from Phe (as in Section V.E.1) (Lee and Desmazeaud, 1985)
Interconversion of amino acids has been demonstrated in Tallegio and
other Italian cheeses by using radiotracers (Cicchi et al., 1979), giving
rise to various metabolites such as a-ketoglutaric and pyruvic acids that

could participate in further interconversion reactions (Polo et al., 1985).
Aldehydes are thought to contribute to the flavor of many cheese varie-
ties. In Parmesan, where they accounted for 2% of the total volatiles,
their production is assumed to be via Strecker degradation (Barbeiri ef
al., 1994). The low pH of Parmesan during ripening is thought to be
responsible for the decarboxylation of amino acids to amines that are
subsequently oxidized to aldehydes when there is an increase in pH
during the later stages of ripening (Belitz and Grosch, 1987).
When the concentration of Strecker-derived compounds exceeds a
certain threshold in Cheddar, unclean flavors develop (Dunn and
Lindsay, 1985).Poor flavors were noted when the concentrations of two
Strecker aldehydes (3-methylbutanal/2-methylbutanal or 2-methylpro-
panal) exceeded 200 pg/kg in Cheddar (Dunn and Lindsay, 1985).
Isovaleraldehyde, 2-methylbutanal, isobutanal, and methional were
formed by Strecker degradation of Leu-Ile, Val, Phe, and Met, respec-
tively, in aqueous extracts of cheese and cultures of cheese microorgan-
isms (Griffith and Hammond, 1989). Dunn and Lindsay (1985) found no
correlation between the concentrations of Strecker-derived compounds
and concentrations of individual free amino acids in Cheddar cheese.
D. CATABOLISM OF SULFUR AMINOACIDS

The catabolic products of sulfur amino acids have been implicated as
major contributors to Cheddar cheese flavor (Manning, 1974; Manning
et al., 1976; Adda et al., 1982; Dunn and Lindsay, 1985; Kristoffersen,
1985; Barlow et al., 1989; Gripon et al., 1991; Alting et al., 1995)
(Fig. 14).
Certain smear surface-ripened cheeses exhibit flavors described as
“cabbagey,” “garlic,” or “putrid” (Adda eta]., 1978; Hemme et al., 1982;
Ferchichi et al., 1985; Manning and Nursten, 1985). These flavors are
attributable to sulfur compounds derived from methionine and perhaps
to a lesser extent from cysteine. Sulfur compounds in cheese (e.g.,
methanethiol (CH,SH), hydrogen sulfide (H2S), dimethylsulfide
(CH3SCH3), dimethyldisulfide (CH3SSCH3), dimethyltrisulfide
(CH3SSSCH3),and carbonyl sulfide (O=C=S)) are thought to interact
with each other and with other compounds in cheese, generating typical
cheese flavors (Kim and Olson, 1989). Sulfur compounds have been
identified in many cheese varieties, but their importance in smear
surface-ripened cheeses appears to be accentuated by their high concen-
trations at the surface (Ferchichi et al., 1985; Gripon et al., 1991).
a 3
Homoalanine Homocy steine
CH,SH a-keto-thiornethy1
Methanethiol butyrate dernethiolase
Sulphur derivatives
FIG.14. Catabolism of methionine. (Modified from Hemme et al., 1982.)
(b)
L-Methionine a-Ketoglutarate L-Cy steine

FIG.15. Chemical structures of methionine, a-ketoglutaric acid, and cysteine.
The principal microorganisms in the smear of smear-ripened cheeses

are coryneform bacteria, particularly strains of Brevibacterium linens.
Sharpe et al. (1976) were the first to report the ability of Br. linens to
produce methanethiol enzymatically. Br. linens is also found on the
surface of many other cheeses, such as Camembert (Ferchichi et a].,
1985; Hemme and Richard, 1986) or Gruyere (Law, 1981), the distinct
flavors of which have been attributed to the formation of sulfur com-
pounds, particularly methanethiol, at the surface (Manning and Price,
1982).
The enzyme demethiolase, involved in methanethiol production, is
highly dependent on the availability of Met (Fig. 15a) (Ferchichi et al.,
1987) and the ability of cells to transport it into the cytoplasm (Forss,
1979; Ferchichi et af., 1985). The production of methanethiol involves
a-ketoglutarate (Fig. 15b), which is used by the bacteria as a carbon
source, as an intermediate (Hemme and Richard, 1986). Uptake of Met
into the cells is strongly inhibited by high concentrations of Cys (Fig.
15c) (Ferchichi et al., 1987), as is its conversion to methanethiol
(Urbach, 1995). Some authors (Adda et a]., 1982) claim that the enzyme
has a preference for free methionine, but others (Ferchichi et a!., 1986b)
have demonstrated higher activity on Met in the dipeptides L-Ala-L-Met
and L-Met-L-Ala than on free Met.
Methionine and methanethiol may affect characteristics of the cheese
other than flavor. The production of a red-orange pigment by Br. linens
appears to depend on the concentrations of methanethiol and dissolved
O2 (Ferchichi et a]., 1986a). Methionine inhibits the germination of
certain mold spores; the active compound is thought to be methanethiol

(Beattie and Torney, 1986).
Manning et al. (1976) correlated the concentration of methanethiol
with the flavor of Cheddar cheese but pointed out that this finding may
only apply to experimental cheeses manufactured in their laboratory.
Other authors (see Aston and Dulley, 1982) have since found that
the concentration of methanethiol is a poor indicator of flavor, particu-
larly in Cheddar cheeses subjected to accelerated ripening (Aston et
a]., 1983).
In cheeses such as Cheddar, which lack a surface microflora, flavor is
produced by starter and nonstarter bacteria and their enzymes (Urbach,
1995). Since coryneforms do not occur in Cheddar, the production of
methanethiol was thought to be a chemical process (Urbach, 1995).
Certain NSLAB can produce methanethiol enzymatically from methion-
ine, but the enzymes involved are thought to be very unstable in ripen-
ing cheese and are therefore unlikely to influence the concentration of
methanethiol during ripening (Law and Sharpe, 1978). In cheese varie-
ties such as Cheddar, the role of microorganisms in amino acid catabo-
lism may be indirect. Key chemicals, such as methanethiol, may be
produced chemically in the cheese as a result of starter-induced condi-
tions (low pH and Eh) rather than enzymatically (Law, 1981; Kim and
Olson, 1989). However, Urbach (1995) proposed that the secondary
flora, particularly in Cheddar and Emmental, is likely to be more im-
portant than chemical reactions for the formation of sulfur compounds.
This hypothesis was supported by Alting et al. (1995), who isolated an
enzyme (similar to cystathionine P-lyase) from L. lactis ssp. cremoris
B78 that was capable of producing methanethiol from sulfur amino
acids under cheese ripening conditions (pH 5.2-5.4 and 4% NaCl).
Engels and Visser (1996) produced typical Gouda flavor by adding
methionine to cell-free extracts of this strain.
Fermentation of lactose during manufacture reduces the pH to -5.1
and the redox potential from about +300 mV to -130 mV or lower (Law
and Sharpe, 1977; Adda et al., 1982). In Swiss cheese, in which the
highly reductive propionic acid fermentation results in a further de-
crease in Eh, the concentration of active sulfhydryl groups is twice that
in Cheddar (Kristoffersen, 1973). Law and Sharpe (1977) found low
levels of sulfur compounds in starter-free Cheddar cheeses (chemically
acidified with GDL) and attributed this to the high redox potential of
the GDL cheeses. Manning (1979a), who artificially reduced the Eh of
GDL-acidified cheese using dithiotreitol and glutathione, demonstrated
the nonenzymatic formation of methanethiol. Although the reducing
conditions were created artificially, Manning concluded that the reduc-
ing conditions produced by starter bacteria would be adequate to cause

the same effect. Ponce-Trevino et al. (1987, 1988), who manufactured
chemically and microbially acidified cheese slurries, found that the
production of H2S,carbonyl sulfide, methanethiol, and dimethyl sulfide
was highest in cheese slurries with added starters. When starter cells
were inactivated by antibiotics, no H2S or (CH3)2Swas produced. When
the Eh of chemically acidified slurries was reduced by using NADH,
O=C=S and CH3SH were produced but H2S and (CH3)2Swere not.
Samples (1985) commented that, while a negative redox potential may
be necessary for the production of sulfur compounds in cheese, it is not
sufficient. He felt that glutathione, which is stored in the cells of certain
lactococcal strains (Fernandes and Steele, 1993), plays an important
role. A low Eh does, however, have a stabilizing effect on sulfur com-
pounds (Law et al., 1976a,b).
Although the exact pathway for the nonenzymatic formation of
CH,SH has not been established, a mechanism proposed by Manning
(1979a,b) is generally accepted by most investigators (Law, 1981; Adda
et al., 1982; Hemme et al., 1982; Barlow et a]., 1989; Fox et al., 1995a).
The proposed pathway involves the release of H2S from cysteine in the
presence of a reducing agent, followed by the reaction of H2S with
methionine, in which the C-S bond is cleaved, with the release of
CH3SH.
This proposed pathway is supported by the fact that H2S is released
initially in the curd prior to the formation of CHoSH(Manning, 1979a,b;
Adda et a]., 1982). Green and Manning (1982) showed that low concen-
trations of H2S in cheese during the early stages of ripening coincided
with the production of low levels of CH3SH. This appears to indicate
that H2S is essential for the formation of CH3SH. However, Cys is an
inhibitor of CH3SH production and the release of H2S from Cys may
simply remove the inhibitor for the enzymatic production of CH3SH
(Urbach, 1995).
The thiol-producing enzyme system is thought to be inactivated by
high heat treatment of milk (Samples, 1985), and indigenous milk
enzymes may be responsible for the production of CH3SH in cheese
(Urbach, 1995). The production of both CH3SH and H,S was increased
by the addition of reduced glutathione to cheese (Samples, 1985), and
glutamyltranspeptidase, which acts by desulfurating glutathione, is
thought to be partially responsible for the formation of H2S, and possi-
bly of CH3SH. Other enzyme(s) are also involved (Samples, 1985; Ur-
bach, 1995). SH groups are a limiting factor in the formation of H,S
(Samples 1985). Since there are few cysteine-cystine residues in casein,
it seems likely that at least some of the sulfur groups arise from the
action of y-glutamyltranspeptidase on glutathione, which is introduced

into the cheese in starter cells (Fernandes and Steele, 1993; Wiederholt
and Steele, 1994; Folkertsma and Fox, 1996).
The formation of H2S is thought to be influenced by Eh and the
concentration of glutathione. The presence of H2S in fresh curd indi-
cates that some may also be formed during pasteurization of cheese milk
(Law et al., 1976a). Concentrations of H2S in cheeses with added
NSLAB showed that some lactobacilli are capable of desulfurating Cys
and producing H2S, while Group D streptococci, staphylococci, micro-
cocci, and pediococci are not (Sharpe and Franklin, 1962; Law, 1981).
While the concentration of H2S in cheese increased during ripening,
Aston et al. (1983) found no correlation between its concentration (or
that of other sulfur compounds) and flavor development. Similar obser-
vations were reported by Lawrence (1963) and Manning (1978). On the
other hand, Kristoffersen and Nelson (1955)found that cheeses with the
highest concentration of H2Sreceived the highest flavor scores. In later
studies (Kristoffersen and Gould, 1960; Kristoffersen, 1967), fluctua-
tions in H2S were observed throughout ripening, and the authors sug-
gested that the ratio of H2S to short-chain fatty acids was important for
flavor development. Barlow et al. (1989) found a high correlation be-
tween flavor and the concentration of H2S, particularly when the value
for H2S was combined with either the concentration of water-soluble
nitrogen or lactic acid. The authors concluded that these parameters
(H2S + WSN/lactate) were better predictors of the way in which young
cheeses would mature than their flavor or composition at an early age.
A notable feature of Parmesan cheese is that, although a number of
sulfur compounds have been identified, this cheese is characterized by
low concentrations of H2S and CH3SH (Barbeiri et al., 1994). This may
be due to inactivation of indigenous milk enzymes by the relatively high
cooking temperature during manufacture (Barbeiri et al., 1994).
Dimethylsulfide (DMS), dimethyldisulfide (DMDS), and dimethyl-
trisulfide (DMTS) are thought to be important contributors to cheese
flavor (Manning et al., 1976; Barbeiri et al., 1994). DMS is a component
of Swiss cheese flavor and is a metabolite of propionibacteria (Langler
et al., 1966; Adda e f al., 1982). DMS levels remained constant in
Cheddar cheese for up to 6 months of ripening and decreased thereafter
(Aston et al., 1983).Manning et al. (1976) proposed that the concentra-
tion of DMS is seasonal and originates in the cheese milk rather than
from the catabolism of sulfur amino acids. Variability of the concentra-
tions of DMS and H2S throughout ripening rendered these two com-
pounds of little value as indices of ripening (Aston and Douglas, 1983).
DMDS is formed as an end-product of Strecker degradation (Belitz
and Grosch, 1987). Strecker degradation involves a reaction between an
CH,
I
S
I
CH,
I
CH,
I
---
Streckex Degradation S
I
FH2
CH,
I
S N - CH - O H o= CH
Methionine Methional
FIG.16. Structure of methional.
amino acid and a diketone, resulting in the formation of an aldehyde

with one carbon atom less than the original amino acid (Aston and
Dulley, 1982; Keeney and Day, 1957; Urbach, 1995) (Fig. 12). 3-Methyl-
propanal is formed in the first step of this reaction. DMDS has been
identified in Parmesan (Barbeiri et al., 1994), Cheddar (Aston et al.,
1983; Barlow et al., 1989), and surface-ripened cheeses (Jollivet et al.,
1992). In Cheddar it was found to correlate fairly well with flavor scores
(Barlow et al., 1989).
DMTS is another potent aroma compound and has been associated
with the aroma of cooked cabbage, broccoli, or cauliflower. It has been
identified in Parmesan (Barbeiri et a]., 1994) and Cheddar (McGugan,
1975; Wood, 1989).
Although DMDS is formed by Strecker degradation of methionine, the
principal product of this reaction is methional (P-methylmercaptopro-
pionaldehyde) (Keeney and Day, 1957; Aston and Dulley, 1982) (Fig.
16). Methional contributes positively to cheese only when present at
low concentrations; a sweet corn-like aroma is detectable at high levels
(Barbeiri et al., 1994). Methional is present in the volatile fraction of
many cheeses, including Cheddar (Wijensundera and Urbach, 1993).
Dunn and Lindsay (1985) noted that methional plays a role in flavor
suppression in bland curds and proposed that this compound may play
a critical role in masking off-flavors in cheese. Methional can also be
produced enzymatically by many strains of lactic acid bacteria in en-
riched media (Tracey and Britz, 1989).
Several other sulfur compounds have been identified in cheese vola-
tiles. Thioesters, produced by esterification of methanethiol and acetic
or propionic acid, have a cheesy aroma (Dumont and Adda, 1978).
S-methylthioacetate (Fig. 17a) can be produced under certain condi-
tions by selected strains of Br. linens (Ferchichi et al., 1986a,b) and has
-N 0
II
II FH
S-Methylthioacdate Benzothiazole 2-methyl propanal

FIG.17. Chemical structures of some volatile compounds derived from methionine.
CH,SH +
Methanethiol + Formaldehyde-> Bis(methy1thio)methane

FIG.18. Production of bis(methylthio)methae by condensation of methanethiol with
formaldehyde.
been found in Limburger cheese (Parliment et al., 1982). The mecha-

nism of thioester formation is unknown, but it is thought to be enzy-
matically controlled as the presence of micrococci was required for their
formation in model systems (Adda et al., 1982).
A reported component of Parmesan aroma is the methionine-related
benzothiazole (Meinhart and Scheier, 1986; Barbeiri et a]., 1994) (Fig.
17b). No mechanism for its formation has been reported. A sulfur-con-
taining aldehyde, 3-methylpropanal (Fig. 17c), presumably formed by
transamination of methionine, followed by decarboxylation of an inter-
mediate imide, has been identified in Cheddar (Adda et al., 1982) and
Parmesan (Barbeiri et al., 1994) and may coiitribute to the aroma of
these varieties. Methylthiopropionate, which has a cheesy aroma, has
been found in Cheddar (Cuer et al., 1979).
Bis(methy1thio)methane (Fig. 18),an important component in Cam-
embert flavor, can be produced by the condensation of methanethiol
with formaldehyde (Dumont et al., 1976) (Fig. 18).
The concentration of carbonyl sulfide increases throughout ripening
and is less variable than other sulfur compounds (Aston and Douglas,
CH, CH, 0
I
CHZ
I
c=o I
I I
CHZ
I
OH CH, COOH
Phenylmethanol Phenylethanol Phenylpropanone Methylphenyl
hydroxyacetic acid
CH,
I
O=CH OH-CH-CH2 -COOH
Phenylacetaldehyde Pheny lpyruvic Phenylethanol acetic

acid acid
FIG.19. Some phenylalanine-derived catabolites that have been identified in cheese.
1983; Aston et al., 1983). It has not been established whether the
concentration of carbonyl sulfide correlates with cheese flavor.
E. CATABOLISM OF PHENYLALANINE, TYROSINE,

AND TRYPTOPHAN
1, Phenylalanine
Phe is released at high concentrations, particularly during the early
stages of cheese ripening. Initial cleavage of the Phe,,,-Metlo6 bond of
K-casein by rennet and of Phe,,-Phe,, of aSl-casein during early ripen-
ing provides terminal Phe, which could be easily released by bacterial
exopeptidases (Dunn and Lindsay, 1985).
A number of flavor compounds arising from Phe have been identified
in cheese (Adda et al., 1982; Dunn and Lindsay, 1985; Lee and Des-
mazeaud, 1985) and in model systems (Jollivet et al., 19921, that is,
phenylmethanol (Fig. 19a), phenylethanol (Fig. 19b), phenylpropanone
(Fig. I ~ c ) methylphenyl
, hydroxyacetate (Fig. 19d), phenylacetalde-
hyde (Fig. 19e), phenylpyruvate (Fig. 19f), and phenylethanol acetate
(Fig. 19g). L-G~u(0.93 moll and phenylpyruvate (1.0 mol) were pro-
Phenylalanine + 2-0xyglutaric acid + Phenylpyruvic acid + Glutamic acid

FIG.20. Transamination reaction with the production of glutamic acid from pheny-
lalanine.
duced when Phe acted as amino group donor and 2-oxyglutarate as

acceptor in a transamination reaction (Lee and Desmazeaud, 1985)
(Fig. 20).
Br. linens rapidly catabolyzes Phe and other aromatic amino acids and
can utilize them as nitrogen sources. The enzymes involved in the
initial breakdown of Phe are aromatic amino acid amino transferase,
L-amino acid oxidase, L-phenylalanine ammonia lyase, L-aromatic
amino acid decarboxylase, and phenylalanine dehydrogenase. Many of
these enzymes have been identified in Br. linens (Jollivet et al., 1992,
1994), but their activity appears to be strain-dependent (Lee and Rich-
ard, 1984). Removal of the amino group from Phe by Br. linens is
thought to be by oxidative deamination. A general L-amino acid oxidase
is considered to be responsible for the ability of coryneform bacteria to
utilize amino acids for nitrogen (Coudert and Vandcastelle, 1975). Sur-
face yeasts in Camembert are thought to possess at least a transaminase
and a decarboxylase active on Phe (Roger et al., 1988). In Cheddar (and
other varieties with only primary starters),Phe is thought to be degraded
by Strecker degradation (Dunn and Lindsay, 1985).
The typical aroma of Camembert has been attributed to phenylethanol
(Fig. 19b) produced by surface yeasts (Adda et al., 1982) involving
transamination, decarboxylation, and reduction reactions (Dunn and
Lindsay, 1985). Yeasts have been implicated as the main producers of
phenylethanol in mold/smear-ripened cheeses (Roger et al., 1988; Jol-
livet et al., 1992); however, Br. linens degrades phenylethanol further
(Jollivet et al., 1992).
Phenylacetaldehyde (Fig. 19e) is considered to be an important flavor
compound in many cheeses (Dunn and Lindsay, 1985; Jollivet et d.,
1992). In Cheddar, phenylethanol and phenylacetaldehyde can be pro-
duced by Strecker degradation of Phe (Dunn and Lindsay, 1985). Con-

centrations of phenylethanol in good- and poor-quality Cheddar cheeses
(-100 kg/kg) were comparable, but the concentration of phenylacetal-
dehyde ranged horn 4 0 to 400 pg/kg, the highest levels being present
in poor-quality cheese (Dunn and Lindsay, 1985).
Both phenylethanol and phenylacetaldehyde have rosy-floral aromas
and have been associated with off-flavors produced by Brewer’s yeast
(Saccharomyces cervisiae) in beer (Sentheshanmuganathan, 1960). Un-
clean rose-like flavors observed in some poor-quality Cheddar have
therefore been attributed to the presence of these compounds (Dunn and
Lindsay, 1985). High concentrations of phenylacetaldehyde also con-
tribute astringent, bitter, and stinging sensations to Cheddar flavor. The
concentrations of these compounds were highest and their effects great-
est in young and mid-ripened cheeses. In mature Cheddar cheese with
more intense flavors, the effects of phenylethanol and phenylacetalde-
hyde are diluted, and off-flavors are either combined with, or masked
by, other aroma compounds (Dunn and Lindsay, 1985).
An important component of Camembert flavor is hydroxyphenyl-
acetic acid, thought to be formed via initial deamination of phenylalan-
ine followed by hydroxylation (Simonart and Mayaudon, 1956).
Phenylethanolacetate (Fig. 19g)from Phe catabolism has been identified
in many cheeses (Jollivet et al., 1992).Although phenylpropanone (Fig.
19c) is present in cheese (Jollivet et al., 1992), the mechanism for its
formation has not been established.
2. Tyrosine and Tryptophan
Tyrosine serves as a precursor for three compounds in cheese:
tyramine, formed by decarboxylation, and p-cresol (Fig. 21a) and phe-
nol (Fig. 21b) by atypical Strecker degradation (Elsden et a]., 1976).
Phenol and indole, pyruvate, and ammonia can be produced by the
action of C-C lyases on Tyr and Trp (Hemme et al., 1982). Tyrosol (Fig.
21c) and tryptophol (Fig. 21d), two classical Strecker-type alcohols,
have been reported to impart slightly bitter flavors to beer (Dunn and
Lindsay, 1985);however, they have not been identified in cheese.
p-Cresol and phenol have strong flavor potencies. While phenol (100-
225 mg k g * ) appears to have no detrimental effect on Cheddar flavor,
p-cresol has been implicated in putrid “utensil” aromas (Dunn and
Lindsay, 1985) when concentrations exceed 100 mg kg’ in Cheddar.
Lactobacilli in Gouda and Vacherin cheeses are thought to be responsi-
ble for producing p-cresol (Badings et a]., 1968; Dumont et al., 1974).
The concentration of phenol is unusually high in Limburger (Parliment
eta]., 1982).
YCOH H&H,
p-Cresol Phenol Tyrosol Tryptophol
FIG.21. Aroma compounds produced by the degradation of tyrosine and tryptophan.
F. OTHER IMPORTANT FLAVOR

COMPOUNDS FROM AMINO
ACIDCATABOLISM
Other compounds, thought to be important in cheese flavor, produced
by Strecker degradation include benzaldehyde (Fig. 22a) and acetophe-
none (Fig. 22b) from Phe, 2-acetylthiazole (Fig. 22c) from Cys, and
methylglyoxal alkypyrollizines from Lys. Amines can be acetylated,
yielding to N-isobutyl acetamides, which have been identified regularly
in Camembert (Dumont and Adda, 1978).
Aldehydes are usually reduced as soon as they are formed (Polo et
al., 1985) with the concomitant production of alcohols: isobutanal
(Leu-Ile) is reduced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to
3-methylbutanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol
(Fig. 22f), and 3-methylthiopropanal (Met) to 3-methylthiopropanol
(Fig. Zag) (Adda et al., 1982).
2,5-Dimethylpyrazine is produced in significant quantities for flavor
perception in model systems containing Br. linens. This compound is
present in many cheeses and can give a nutty, toasted note to the flavor.
It could be produced by degradation of Thr to amino-acetone, the
condensation of two amino acetone molecules forming one molecule of
2,5-dimethylpyrazine (Jollivet et al., 1992). Isobutanal (Leu-Ile) is re-
duced to isobutanol (Fig. 22d), 3-methylbutanal (Val) to 3-methylbu-
tanol (Fig. 22e), phenylacetaldehyde (Phe) to phenylethanol (Fig. 22f),
and 3-methylthiopropanal (Met) to 3-methylthiopropanol (Fig. 22g)
(Adda et al., 1982).
G. REACTIONSOF FREEAMINOACIDSWITH OTHER

COMPOUNDS IN CHEESE
Cheesy flavors have been noted when free amino acids react with
other molecules in the cheese. Addition of calcium or magnesium to
0 0
II II
Benzaldehyde
sYc-cH3
L-’c-cH3
Acetophenone Acetylthiazole
(0
OH
OH
I I
CK
x
CH3 CH,
Isobutanol 3-methylbutanol Phenylethanol

(g)
OH
1
CH-CH,SH
I
YK
CH3
Frc. 22. Some minor amino acid catabolic products found in cheese.
cas-amino acids enhanced their sweetness (Biede and Hammond, 1979),

suggesting that Ca (or Mg) amino acid complexes may contribute
to the sweet flavor of Swiss cheeses. A number of mono- and dicar-
bonyls are produced from free fatty acids in cheese (Reps et a]., 1987),
particularly by lactobacilli. Kowalewska et al. (1985) showed that much
of the flavor in the nonvolatile water-soluble fraction was generated
from amino acid-carbonyl reactions. Glyoxal, methylglyoxal, dihy-
droxyacetone, and ethanal were prominent among the carbonyls, and
Val, Leu-Ile, Met, Cys, Phe, Pro, and Lys were the principal amino acids
that react chemically with them (Griffith and Hammond, 1989). Al-
though the exact pathways for carbonyl-amino acid reactions are not
known, Griffith and Hammond (1989) proposed a pathway for the
+ CH3
I
c= 0
I
H
Cysteine Methylglyoxal
COOH
I II
-
KC- CH-N =CH- C-CH3 + CO,
I
T
SH I
KC- C K - W C H - C-CH, + CO,
I
c=0 SH
2- Acdylthiazole
FIG.23. Production of 2-acetylthiazole by reaction of cysteine with methylglyoxal;
chemical pathway proposed by Griffith and Hammond (1989).
reaction of Cys with methylglyoxal (Fig. 23). The reaction of Lys with
glyoxal or dihydroxyacetone can give rise to 6-valerolactam (Fig. 24a)
or 2-acetyl pyrroline (Fig. 24b). Proline can react with ethanal, methyl-
glyoxal, or dihydroxyacetone to produce 2-acetyl-1-pyrroline (Fig. 24c),
2-methyl benzaldehyde (Fig. 24d), and isomers of 2,3-dihydropyroliz-
ine, respectively (Griffith and Hammond, 1989). These compounds
-(2)-
H
I
+ c= 0
I
c=o
-
I
H
Ly sine + Glyoxal 6 -valerolactam (2-peperodine)

0)
‘i“.
CHZ H
I I
CHZ
I + I
CHZ c=o CCH,
‘iHz
I I
H
H2N- CH - COOH
COOH
Proline + Ethanal- 2- acdyl-1-pyrrolhe

FIG.24. Compounds formed by the reaction of amino acids with carbonyls. (Modified
from Griffith and Hammond, 1989.)
were identified in aqueous extracts of cheese (Kowaleska et al., 1985)

and Lb. bulgaricus cultures (Kowalewska et al., 1985; Reps et al., 1987).
COOH
;,H
""\/ \/" + R;3
-
CH,-C-C,
H
YC - CH,
Proline + Methylglyoxal 2 Methyl b d d e h y d e
I
I (f 1
I
I
I
I
yH3 I 7H3
I
S I S
I I
I
I
CH, I CH,
I I I
li' y".
I
II
0 yH2 I
CH,C&C-NH-CH COOH - I CH,- C- NH-CH-COOH
I
I
I
I
I
N-Propionyl methionine I N-acetyl methionine
I
I
I
I
I
N-Ropionyl leucine I N-Propionylphenylalanine
FIG24d-h.
Roudot-Algaron et al. (1993) isolated N-propionylmethionine (Fig. 24e),

N-acetylmethionine (Fig. 24f), N-propionylleucine (Fig. 24g), and N-
propionylphenylalanine (Fig. 24h) from the water-soluble fraction of
Gruyere de Comte cheese: the first compound was cheesy, while the
others had bitter flavors.
H. NONPROTEIN
AMINOACIDS
A number of nonprotein amino acids have been found in most cheese
varieties, the principal being y-aminobutyric acid, formed by decarboxy-
lation of Glu (Kaminarides et al., 1990) (Fig. 25a) and ornithine by
arginase activity on Arg (Fig. 25b) (Broome et al., 1991). y-Aminobutyric
is present at high concentrations in mold-ripened cheeses (Ismail and
Hansen, 1972; Gripon et al., 1991). In Kopanisti cheese, y-aminobutyric
acid levels increased 120-fold during ripening with a concomitant de-
crease in the concentration of Glu (Kaminarides et al., 1990). a-Ami-
nobutyric acid has also been identified in many cheese varieties:
Cabrales (Gonzalo de Llano et al., 1987; Ramos et al., 1987), Stilton
(Madkor et al., 1987), Kopanisti (Kaminarides et al., 1990), and
Gamonedo (Gonzalez de Llano et a]., 1991). However, its concentration
was generally lower than that of y-aminobutyric acid.
Enzymatic hydrolysis of the guanidino group of Arg leads to the
formation of ornithine or citrulline (Hemme et al., 1982). Decreases in
the concentration of Arg, particularly in the later stages of ripening,
have been reported by a number of workers (Puchades et a]., 1989;
Broome et a]., 1991). Certain strains of L. cremoris are capable of
degrading arginine to a limited extent (Broome et a]., 1990). Low corre-
lations between nonprotein amino acids and ripening time have been
reported (Resmini et a]., 1969; Gonzalez de Llano et a]., 1991).
VI. Chemistry of Cheese Off-Flavors

In addition to the characteristic desirable flavor of cheese, cheese
frequently suffers from specific flavor defects. While normal desirable
flavor has been difficult to define in chemical terms, the specific causes
of many of the principal defects have been established more or less
definitively and are described below.
Bitterness in cheese results from the accumulation of hydrophobic
short peptides that can originate from both asl-and p-caseins. Certain
sequences in the caseins are particularly hydrophobic and when ex-
cised by proteinases can lead to bitterness. The action of chymosin has
decarboxylation
Glutamkacid * y -amino butyric acid
(Kaminarides et al., 1990)
(b)
W-CH - COOH
Arginine > Ornithine + Urea

Arginase
(Broome ad.,1991)
FIG.25. Formation of nonprotein amino acids.
been implicated in the formation of bitter peptides in cheese (see

Lemieux and Simard, 1991, 1992), and thus factors that affect the
retention and activity of rennet in the curd may influence the develop-
ment of bitterness. Lawrence et al. (1972) pointed out the importance
of both starter strain and rennet type and suggested that the major role
of rennet in the development of bitterness may be the production of long
peptides that are subsequently degraded to small bitter peptides by
starter proteinases. These authors found a higher concentration of free
amino acids in cheeses made with nonbitter starters, suggesting greater
peptidase activity in these strains. Certain strains of starter and Penicil-

lium spp. are associated with the development of bitterness (see Adda
et nl., 1982), and it would appear that bitterness in cheese results from
the action of chymosin on casein with the release of bitter peptides.
These peptides accumulate in bitter cheese due to the inability of
“bitter” starters to hydrolyze them to nonbitter peptides due to a defi-
ciency in peptidase activity (Stadhouders and Hup, 1975), or perhaps
by degradation by bacterial enzymes of peptides that otherwise would
be too large to be perceived as bitter (Lemieux and Simard, 1991). The
development of bitter taste is common in low-fat cheeses (Banks et a).,
1992). In full-fat cheese, a certain proportion of bitter peptides, being
hydrophobic, probably partition into the fat phase, where they are less
likely to be perceived as being bitter. The literature concerning bitter-
ness in dairy products has been reviewed by Lemieux and Simard
(1991, 1992).
The majority of studies in which bitter peptides were identified were
conducted in model systems consisting of isolated casein incubated
with various enzymes. Bitter peptides have been identified in hydro-
lyzates of a,,-, as2-,and P-caseins. Para-K-casein is a potential source of
bitter peptides (Visser, 1981), although in the few studies in which the
hydrolysis of para-lc-casein was investigated it was found to be resistant
to proteolysis. Bitter a,,-casein-derived peptides identified in hydro-
lyzates include f22-43 and f45-50 (see Sullivan and Jago, 1972), f23-34,
f9l-100, and f145-151 (Hill and van Leeuwen, 1974). LeBars and
Gripon (1989) considered that three short peptides produced from the
C-terminal region of a,,-casein by plasmin (f198-207, f182-207, and
f189-207) may be bitter. Bitter peptides found in hydrolyzates of p-ca-
sein include f203-208, fl95-209, f201-209 (see Sullivan and Jago,
1972), and f53-79 (Clegg et al., 1974). A number of bitter peptides
isolated hom cheese or casein hydrolyzates are summarized in Table V.
A number of authors have described the isolation of bitter peptides
from cheese (e.g., Harwalkar and Elliott, 1971; Visser, 1977), but there
is some disagreement as to the source of these peptides (Visser, 1981).
However, unlike peptides produced from a,,-casein, the initial peptides
produced from p-casein by chymosin are probably bitter, and thus the
limited hydrolysis of this protein may contribute to bitterness. The
concentration of NaCl has a major effect on the hydrolysis of p-casein
by chymosin in solution (Fox and Walley, 1971) and in cheese (Kelly,
1993), and thus may be a factor in the development of bitterness. The
formation of p-casein f193-209 (the primary product of chymosin action
on p-casein and potentially bitter) was inhibited by increasing NaCl
FORMATION O F FLAVOR COMPOUNDS IN CHEESE 73
TABLE V
ISOLATED
BITTERPEPTIDES FROM CHEESE‘
Hydrophobicity
Cheese Origin Sequence Q, cal residue-’
Cheddar aS,-CN fl4-17 E.V.L.N 1162.5

a,l-CN fl7-21 N.E.N.L.L 1074.0
a,,-CN f26-32 A.P.F.P.E.V.F 1930.0
CX,~-CN26-33 A.P.F.P.E.V.F.G 1688.8
8-CN f46-67 Q.D.K.I.H.P.F.A.Q.T.Q.S.L.
V.Y.P.F.P.G.P.1.P 1580.5
P-CN f46-84 Q.D.K.I.H.P.F.A.Q.T.Q.S.L.
V.Y.P.F.P.G.P.I.P.N.S.L.P.Q.N.
1.P.P.L.T.Q.T.P.V.V.V 1508.5
P-CN f193-209 Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P.
1.I.V 1762.4
Gouda P-CN f84-89 V.P.P.F.L.Q 1983.3
P-CN f193-207 Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P.1 1686.7
ondh P-CN fl93-208 Y.Q.Q.P.V.L.G.P.V.R.GP.F.P.I.1 1766.9
P-CN fl93-209 Y.Q.Q.P.V.L.G.P.V.R.G.P.F.P.
1.I.V 1762.4
Alpkase aSl-CN fl98-199 L.W 2710.0
Butterkase P-CN f61-69 P.F.P.G.P.1.P.N.S 1792.2
“Adapted from Lemieux and Simard (1992).
concentrations (Kelly, 1993). The primary action of plasmin on p-casein

probably does not produce bitter peptides. The fate of as2-casein in
cheese is unclear, but plasmin can release potentially bitter peptides
from this protein in solution (LeBars and Gripon, 1989). Guigoz and
Solms (1974) found the bitter dipeptide, asl-CN fl98-199 (Leu-Trp), in
Alpkese cheese. Stepaniak and Fox (1995) demonstrated the production
of this peptide from asl-CN fl65-199 by a lactococcal endopeptidase
(PepO).
In addition to peptides, a number of other compounds can contribute
to bitterness in cheese, including amino acids, amines, amides, substi-
tuted amides, long-chain ketones, and some monoglycerides (Adda et
d.,
1982).
Rancidity, which is occasionally encountered in cheese, is due to
excessive or unbalanced lipolysis caused by lipases-esterases from
starter or NSLAB, psychrotrophs in the cheese milk or indigenous milk
lipoprotein lipase. As discussed in Section II.C, a high concentration of

lactic acid, perhaps arising from the retention of an excessive level of
lactose in the cheese curd, imparts an acidic, harsh taste to cheese.
Graders frequently use the term “over-acid” to describe a flavor defect
in overripe cheese. The precise cause of this defect is not known, but it
is probably due to excessive or unbalanced proteolysis rather than to a
high concentration of lactic acid,
The principal compounds responsible for fruitiness in Cheddar are
ethyl butyrate and ethyl hexanoate, formed by esterification of FFAs
with ethanol. Production of ethanol appears to be the limiting factor, as
FFAs are present in cheese at relatively high concentrations (Bills et al.,
1965). Ethyl esters are present at low concentrations in nonfruity
cheeses and thus the fruity defect occurs as a result of increased pro-
duction of ethanol or its precursors.
The origin of “unclean” and related flavors in Cheddar was investi-
gated by Dunn and Lindsay (1985), who quantified a number of
Strecker-type compounds, including phenylacetaldehyde, phenetha-
nol, 3-methylbutanol, 2-methylpropanol, phenol, and p-cresol. Pheny-
lacetaldehyde concentrations were elevated in cheeses with an “un-
clean rosy” off-flavor, and the addition of this compound to
clean-flavored mild Cheddar reproduced this defect. At higher concen-
trations (>400 pg/kg), phenylacetaldehyde imparted astringent, bitter,
and stinging flavors to cheese. Concentrations of phenethanol were
similar in most of the cheeses studied (-100 pg/kg). p-Cresol was found
to impart a putrid “utensil”-type flavor when present in high concen-
trations (>lo0 pg/kg-l). Dunn and Lindsay (1985) also discussed the
potential of short-chain fatty acids to potentiate the flavor impact of
p-cresol. Branched-chain Strecker-type aldehydes (3-methylbutanal, 2-
methylbutanal, and 2-methylpropanal) did not cause flavor defects
when added at concentrations below 200 pg/kg to clean-flavored
cheese. Phenol (100-225 pg/kg-l) appeared to have no detrimental effect
on cheese flavor and indeed enhanced the sharpness of Cheddar flavor
(Dunn and Lindsay, 1985). Dunn and Lindsay (1985) added quinine to
cheese to simulate the bitterness often associated with the development
of Strecker-type compounds. It enhanced the flavor impact of these
compounds.
ACKNOWLEDGMENTS
The authors would like to express their thanks to Ms. Anne Cahalane
for her assistance in preparing the manuscript.
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Formation of Flavor Compounds in Cheese

P. F. FOX AND J. M. WALLACE

Department of Food Chemistry

The quality of cheese is determined by its flavor (taste and aroma),

is very strongly influenced by its rheological properties, but, unfortu-

Acetaldehyde Dimethyl sulfide Methyl acetate

'Adapted from Urbach (1993).

net-coagulated cheeses. As the sapid compound present at the highest

SCH3CHOHCOOH + ZCH,CH,COOH + CHzCOOH + COZ + HZO

The COz generated is responsible for eye development, a characteristic

c. EFFECTOF LACTOSECONCENTRATION ON CHEESE QUALITY

Gouda and Edam, is reduced by replacing part of the whey by warm

or by washing the curd after milling, or increased by adding lactose to

were not affected by the concentration of lactose, suggesting that

Production of COz from citrate is responsible for the characteristic

Variety FFA (mg1kg-I) Variety FFA (mg/kg-’)

Sapsago 21 1 Gjetost 1658

Date from Woo et a1 (1984)and Woo and Lindsay (1984).

considered rancid. The total concentrations of FFAs in some major

PGE is highly specific for short-chain acids esterified at the sn-3

mold cheeses. Lipolysis in mold-ripened varieties is due primarily to

tones may be formed spontaneously from the corresponding y- and

Proteolysis is the most complex and perhaps the most important

A. CONTRIBUTION OF INDIVIDUAL PROTEOLYTIC AGENTS

21 .- ~ _ _ , lu.-.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 199

61-71 Cleavage sites of Plasmin

water-insoluble fraction of Cheddar and most other varieties (Fig. 7),

FIG.7 . Urea-PAGE of the water-insoluble fraction of a selection of cheese varieties.

aminopeptidase activity. The C-terminal of several peptides does not

Gly 0 130 371 ***

Thr 400 260 649 *** *

"Adapted from O'Callaghan (1994).

(:hedrlm (1) Cheddar (2) HnrrgArd (5) Emmental (6)

CYS 44.7 029 - - ~ - - -

GlY 30fi.n~ 1.99 - 2.32 34.ti 1 2 108.G 1.6 2.7 430.8 23

Grana (101 Parmigino (111 Parinigino (121

CYS - - - - 115.0 0.3

concentrations of free amino acids are also present in mold-ripened and

cys - fi.4 0.1 - ~ ~

CYS 1160 9.48 - - 1380 5.3 740 13.7 590 10.2 -

levels of which are reported to decrease during the later stages of

V. Catabolism of Amino Acids

Amines, including biogenic mines, are produced in cheese by enzy-

Amino mgl %of rngl '% of mgl of

Gly 590.0 1.9 1041.n 1.8 1.R 1.7 1.7 1.8

TY~ 540.0 18 333Y.8 5.8 1.7 1.7 1 6 33

AX 870.0 2.R 281.9 0.5 0.1 0.4 0.2 0.1

1987). Although amines are thought to be important contributors to

Amino mg/ % of mg/ % of mg/ % of mg/ Yn of mg/ % of mg/ % of

CYS 450 3.5 - - - - - -

Arg - 34 n 0.6 11.9 5.3 161.8 2.1 235.2 3.7

(Broome et al., 1990). The concentration of tyramine in cheese often

Trap- (37) Edam- (42)

Amino mgl % uf mgi % uf mgi % of rngl % uf rngi % vf rngi Yo of

Cye 12.4 0.4 - - ~ ~ ~

mine, monomethylamine (Fig. 10a), monoethylamine (Fig. lob), tri-

Mahon (46) Mahon (47)

Amino rngi % of rngi %of rngi % of rngi % of rngi % of

amino acids or in the rate of deamination of resulting amines (Polo et

B. DEAMINATION-FORMATION AND NEUTRAL

Deamination of free amino acids leads to the production of ammonia

H~N-CH-COOH H,N- JH,

Monomethyl amine Trimethylamine

by GLC (Urbach, personal communication). Ammonia is an important