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Enzymes of penicillium roqueforti involved in the

biosynthesis of cheese flavor
a b c
John E. Kinsella , Dan H. Hwang & Basant Dwivedi
a
Associate Professor of Food Science , Cornell University , Ithaca, New York
b
Research Associate of Food Science , Cornell University , Ithaca, New York
c
Vice President of Research and Quality Control and Development , Estee Candy Company ,
Parsippany, New Jersey
Published online: 29 Sep 2009.

To cite this article: John E. Kinsella , Dan H. Hwang & Basant Dwivedi (1976) Enzymes of penicillium roqueforti
involved in the biosynthesis of cheese flavor, C R C Critical Reviews in Food Science and Nutrition, 8:2, 191-228, DOI:
10.1080/10408397609527222

To link to this article: http://dx.doi.org/10.1080/10408397609527222

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 ENZYMES OF PENICILLIUM ROQUEFORTI INVOLVED IN THE BIOSYNTHESIS
OF CHEESE FLAVOR

Authors: John E. Kinsella

Dong H. Hwang
Department of Food Science
Cornell University
Ithaca, New York

Referee: Basant Dwivedi

Estee Candy Company
Parsippany, New Jersey
Downloaded by [New York University] at 13:24 09 February 2015

INTRODUCTION and develop methods for accelerating cheese

The consumption of mold-ripened cheese, parti-
cularly blue cheese, has increased from 13 million
pounds in 1960 to approximately 25 million
pounds in 1976. Obviously, there is a growing
market for mold-ripened cheeses. Much of this
cheese is consumed directly, but increasing
quantities are being used as flavorant for salad
dressings, mayonnaise, snack foods, and bakery
items. Because of the growing use of the cheese for
its specific flavor impact rather than its overall
quality, there is an interest in examining and
developing methods for the production of a
quality blue cheese flavor concentrate.
In commercial production, blue cheese is made
from raw or pasteurized homogenized milk using
appropriate strains of Penicillium roqueforti. The
process takes from 70 to 100 days at 8°C and
high relative humidity, (around 95%) to develop
optimum flavor. This traditional method results in
a constant large inventory of ripening cheeses that
requires large maturing rooms with constant
cooling and humidification and relatively intensive
and skilled labor to ensure balanced ripening and
final quality.
There is a real need to improve the efficiency
ripening. However, because of the unique and
complex nature of the flavor and texture of blue
cheese, a thorough knowledge of the composition
of the flavors and an intimate understanding of the
biochemistry responsible for their development
should precede useful attempts to accelerate their
formation.
To achieve this objective, much basic infor-
mation concerning the physiology and bio-
chemistry of the mold P. roqueforti, must be
collected and organized. Considerable information
is available concerning the behavior of the mold P.
roqueforti in the manufacture of blue cheese.
Therefore, we will first review its involvement in
the cheese-ripening process and then review some
of the enzymes involved in flavor and texture
development. Finally, we will summarize the
available information on the use of P. roqueforti
cultures for the production of blue cheese-type
flavor concentrates.
CHEESE MANUFACTURE
A comprehensive review of the manufacture of
blue-veined cheeses was published by Aarnes.1
Bakalor6 summarized the information regarding

November 1976 191

 the techniques and chemistry of blue cheese
manufacture (up to 1962). Blue cheese is usually
made from high-quality pasteurized, homogenized
milk containing 3.5% milk fat. In some cases
cream (40% fat) may be homogenized separately
to facilitate initial lipolysis, and then added back to
the pasteurized skim milk. Iipase may be added
after pasteurization to enhance free fatty acid
p r o d u c t i o n . 6 ' 6 3 ' 6 4 ' 7 3 ' 1 1 8 ' 1 1 9 A starter culture
containing 1 to 3% Streptococcus lactis, is added
to the milk at 30°C. This organism provides
enzymes which metabolize the lactose of milk to
organic acids, mostly lactic acid2 * thereby causing
reduction of pH to around 4.7. Limited
proteolysis may also occur. This acidification
facilitates subsequent processing, ensures proper
curd firmness and aids whey drainage and it
favorably influences the ripening process. When
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pH is 4.8 (acidity of 0.5 to 0.1%) rennet is added.
The major proteins of milk are caseins (phos-
phoproteins), which are colloidally distributed in
milk as polydisperse micellar aggregates in asso-
ciation with calcium and are stabilized against
aggregation by K-casein.24 The enzyme rennin (as
low as 0.2 ppm) destabilizes this protein system by
cleaving the K-casein resulting in aggregation of
the protein micelles which fall out of solution and
gradually form a continuous curd.2 * This step is
aided by the low pH. Because of the cost of
rennet, alternate proteolytic enzymes are being
sought for this application.
After curdling for approximately 75 min, the
curd is cut and the whey drained off. Lower
temperatures, i.e., less than 30°C ensure slower
curdling so that a softer looser curd is obtained;
stirring also prevents excessive matting of the
proteins. After drainage, mold powder (about 5
mg/100 g curd), i.e., spores of Penicillium roque-
forti, are dusted on the curd and 1.5% sodium
chloride is added. The curd is mixed and placed in
hoops which are about 7 in. in diameter. The curd
is then drained at 20°C and turned occasionally to
allow relatively loose matting. After 24 hr the curd
is removed from the hoops and salt is rubbed on
the surface for 6 to 10 days at 15°C or the cheese
may be immersed in a saline solution.1 i 9 The salt
toughens the outer surface and penetrates the
cheese to give a final concentration of 4.5% by
weight or an approximate concentration of 10% in
the aqueous phase. Salt performs several important
functions. It selectively permits the growth of P.
roqueforti while inhibiting growth of bacteria,
192 Critical Reviews in Food Science and Nutrition
yeasts and other molds (except on the cheese
surface where Bacterium linens and B. erythro-
genes may grow). It influences the activity of
enzymes of P. roqueforti, e.g., Upases and pro-
teases, by preventing excessive hydrolysis. It
specifically inhibits protease(s) and prevents the
build-up of soluble peptides and amino acids
which could cause bitterness. It impedes excessive
matting of curd and contributes to the perceived
flavor of the ripened cheese.
After salting the cheese loaf which may receive
a coat of wax, it is pierced with numerous holes.
The cheese loaves are then placed on edge and
ripened for about 90 days in rooms at 10°C and
96% relative humidity. The cheese is pierced to
facilitate mold growth and penetration of
mycelium into the body of the cheese. The
numerous holes allow egress of carbon dioxide and
entry of air into cheese interior. Overall, these
perforations which influence mold growth ensure
more uniform ripening and flavor development. A
surface slime containing yeast and bacteria may
develop on nonwaxed cheese and this occasionally
must be scraped off to prevent excessive surface
proteolysis. After 90 days at 10°C the cheese may
be wrapped in foil and allowed to mature further
at7°C.
During the ripening period the mold grows and
sporulates; proteolysis, amino acid metabolism,
lipolysis, and fatty acid oxidation occur, and
flavor development progresses. The pH of the
cheese gradually increases from 4.7 to 6.5 as lactic
acid is metabolized. The cheese develops the
characteristic bluish-green mottling which appears
as veins along the channels of the punched holes.
The cheese is ripened at a low temperature to
obtain the proper balance of lipid oxidation and
proteolysis which ensures good flavor and texture.
Dolezalek and Hoza 17 reported that by holding
cheese at 15°C much more methyl ketones were
formed and the relative concentrations were
different, i.e., relatively higher concentrations of
2-undecanone were obtained at 15°C.
MOLD PROPERTIES
Penicillium roqueforti is aerobic, but it can
successfully tolerate low oxygen and high carbon
dioxide concentrations. Growth of mycelium is
retarded at low oxygen levels and at very high
concentrations of carbon dioxide. The capacity to
tolerate low oxygen and high carbon dioxide
 concentrations facilitates the exclusive growth of
P. roqueforti in blue cheese.38
Vegetative growth of mold is pervasive in the
cheese during the initial 3 to 7 weeks of ripening.
This is because of the availability of nutrients from
the cheese and lack of competition from other
organisms. Sporulation becomes apparent after 10
to 14 days and continues throughout ripening.
Little information is available concerning the
dynamic factors which regulate the rates of
mycelial growth and extent of sporulation.
In cultures, spores germinate within 15 to 20 hr
at 20°C, and conidia formation begins 2 to 3 days
later. Spore formation occurs facilely at low
oxygen levels; thus, spore formation may be
favored in the latter stages of cheese ripening.90
Sodium chloride (1 to 5%) impedes germination.1
Thus, several changing factors may be involved in
regulating mold growth and sporulation, for
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example, depletion of monosaccharides, prevailing
salt concentration, gradual increase in pH, gradual
accumulation of carbon dioxide, the p CO 2 / p O2
ratio, and accumulation of certain metabolites
(i.e., fatty acids, methyl ketones, amino acids).
These factors may impede mycelial growth and
stimulate sporulation.
A particular ratio of mycelium to spores is
optimal for proper ripening. Although this ratio
has not been established, it is obvious that the
conventional conditions of ripening which have
been arrived at empirically are best for the
practical production of a quality product. Thus,
the environmental and endogenous conditions of
blue cheese ripening are normally conducive to
optimum mold growth and sporulation. However,
little is known of the requirements, kinetics, and
associated biochemical changes that occur during
germination, growth, and sporulation of P. roque-
forti.22'23
The growth of mold has a marked effect on
cheese quality, and its growth must be carefully
controlled. This is normally achieved in practice
by salting, by puncturing the pressed cheese and
by regulating the temperature and the relative
humidity during. ripening. If the cheese is too
porous or loose the mold tends to overgrow and
vice versa. In cheeses with excessive mold growth,
much greater amounts of methyl ketones are
generated. Also, evidence indicates that greater
amounts of alcohols are produced in these cheeses,
imparting a musty flavor.
Different strains of P. roqueforti vary markedly
in their capacity to produce a quality blue cheese.
The intensity of flavor in different blue cheese
samples has been attributed to variation in
lipolytic activity and growth rate of the mold. 127
Graham3 9 reported that only three out of eight
strains of P. roqueforti consistently produced
cheeses of good flavor and acceptable quality. A
good correlation was observed between the
organoleptic quality and the content of carbonyls
plus volatile fatty acids in several cheese samples.
Several authors have reported on the variability
in the enzymatic activity (lipolysis and pro-
teolysis) of different strains of P. roqueforti.
Stepaniak and Habaj, 139 Sato et al., 1 2 7 and Niki
et al. 1 0 6 have demonstrated the negative cor-
relation between proteolysis and lipolysis in
different strains of P. roqueforti. Mold strains vary
tremendously in lipolytic activity, with fivefold
differences often observed.1 7 >3 9 >'2 7
BIOCHEMICAL CHANGES
The biochemical events that occur during
cheese ripening are dynamic, complex, and inti-
mately interrelated and the changes are mostly
attributed to different enzymes acting together.
For descriptive purposes, these may be classified
into enzymes inherent in the milk, those added,
and those of the mold P. roqueforti. The lipase
associated with the milk lipids may initially cause
some lipolysis, especially after homogenization
(see Figure 7). 1 1 8 The starter culture organisms
(Streptococcus lactis) degrade the lactose to pro-
duce mostly lactic acid, some citric acid and
perhaps some ethanol. The lactic acid regulates the
pH levels during cheese ripening. The citric acid
provides carbon for the mold and ethanol may be
involved in flavor generation.
The rennin reputedly causes some marginal
proteolysis, and its action may persevere through-
out the ripening period. However, it is generally
conceded that all the important changes that occur
during ripening are almost exclusively effected by
enzymes of P. roqueforti.
Controversy has long existed as to whether the
spores or the mycelium are the more important
enzyme sources involved in cheese ripening.
Present evidence indicates that both the mycelium
and the spores possess enzymes that are important
in cheese ripening.4 7
PROTEOLYSIS
Extensive proteolysis (20 to 30% breakdown of
proteins) occurs during maturation of blue cheese
(Figure 1). Some of this may be caused by residual
November 1976 193

no
a 30
1 5. Amino acids — enhance methyl ketone production
20
| 7. Amino acids — precursors of amines
10
1 i
16 21 32
RIPENING (WEEKS)
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FIGURE 1. The progressive proteolysis during the ripen-
ing of blue cheese samples. A represents the average rate
of tyrosine release for three cheese samples. B, C, and D
indicate the different rates of proteolysis in cheese
samples made with different strains of Penicillium
roqueforti. (Data adapted from Morris et a l . " and Sato
etal. 127 )
protease activity from starter organisms and from
the proteolytic action of rennin but the pro-
teolytic capacity of Penicillium roqueforti is
predominant. Proteolysis by P. roqueforti is
essential for the development of a soft, smooth,
full-flavored cheese. If insufficient proteolysis
occurs a tough, dry, crumbly cheese ensues,
whereas excessive hydrolysis results in a soft
cheese with a slightly bitter aftertaste.
Proteolysis is necessary and important for the
development of proper texture, for background
flavor (peptides and amino acids), for provision of
amino acids which act as precursors of flavors
(aldehydes, alcohols, and esters), and as germina-
tion stimulants (Table 1). Amino acids also
enhance methyl ketone production. 80 ' 81 The
importance of proteolysis in the ripening and
organoleptic quality of blue cheese was not fully
realized until quite recently. It was discovered that
imitation flavors devoid of proteolytic products,
(i.e., peptides, amino acids, and aldehydes
generated from free amino acids), did not fully
resemble the authentic flavor of a quality cheese.
Protease activity in mycelium (and in extra-
cellular medium) is maximum when the mycelium
attains full growth.4 ° Thus, proteolysis probably
occurs more rapidly during the initial few weeks of
194 Critical Reviews in Food Science and Nutrition
TABLE 1
Effects of Protease/Proteolysis
1. Texture — softens, moistens curd
2. Flavor — peptides, amino acids
3. Amino acids - flavor precursors, aldehydes, alcohols,
esters
4. Amino acids - stimulate germination and growth
6. Amino acids - buffers
cheese ripening when mycelia growth is extensive.
Lindquist and Storgards87 reported that both a-
and 0-casein are extensively hydrolyzed during
ripening of cheese with P. roqueforti. The break-
down of protein is progressive during the ripening
(Figure 1), and in several cheese samples 20 to
40% of the casein is converted to soluble non-
protein nitrogenous compounds including amino
acids (10 to 15%) and ammonia. The gradual
build-up in amino acids was studied by Sato et
al. 1 2 7 and Morris et al. 96 Jolly and Kosikowski63
reported that soluble nitrogenous compounds
comprised about 25% of the total N in ripened
blue cheese and 34% of cheese- made with added
lipase. Amino acid levels in cheese samples may
range from 280 to 900 mg/10 g dry cheese.72
The free amino acid levels in samples of Danish
blue cheese ranged from 5 to 10% of the total
nitrogen; while most amino acids were present,
their distribution differed from that of casein.5 7
Peruffo et al. 11 s reported that the intracellular
protease activity in Penicillium strains from
Gorgonzola cheese was markedly inhibited by free
amino acids when grown in Czapeks medium;
however, repression was less when the mold was
grown on an intact casein curd.
Bolcato et al.9 determined that the intracellular
protease activity of Penicillium was subject to
end-product repression by amino acids in the
medium, thus by increasing amino acid concentra-
tion in media to 2.5%, 90% inhibition was
obtained. Extracellular protease activity tended to
increase with amino acids. This may represent
another of the dynamic feedback control systems
operating during blue cheese ripening.
The gradual accumulation of low molecular
weight peptides and amino acids during cheese
ripening is markedly retarded when the salt content
of the cheese is increased.6 9 Thus, the addition of
salt helps control excessive proteolysis in blue
cheese. Several researchers have examined the pro-
 teolytic capacity of P. roqueforti and invariably
have reported on the tremendous disparity among
different strains.3 2 ' 4 0 ' 1 0 6 ' 1 2 1 ' 1 2 6 ' 1 2 7 Salvador!
et al. 12 s reported that proteolysis varied as- much
as fivefold among 19 different strains of P.
roqueforti tested. Interestingly, the strains seemed
to differ inversely with regard to proteolytic and
lipolytic capacities. Niki et al. 106 significantly
showed that strains of P. roqueforti with high
proteolytic activity possessed low lipolytic activity
and vice versa (Figure 2).
Czulak12 attributed a marked bitterness in
cheese to the accumulation of peptides. Pelissier et
al. 1 1 4 associated the bitterness with the excessive
protease activity of certain strains of P. roqueforti
Niki et al. 1 0 6 reported that strains with marked
proteolytic capacity which hydrolyzed over 50%
of the protein produced soft cheeses with a pasty
texture and an unpleasant bitter taste, whereas the
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seemed to produce better quality cheeses in a
much shorter period. The latter strains (F-l in
Figure 2) were suggested for cheese making to
assure more uniform quality. These findings
suggest that flavors generated via lipolysis are
ostensibly the more important determinants in
blue cheese flavor. Strains with high proteolytic
activity cause more general and complete
proteolysis with a resultant build-up of amino
acids. These may directly contribute to off flavors
and act as precursors of various aldehydes via
Strecker degradation-type reactions and of amines
via decarboxylation. Accumulation of these
beyond a minimum level is undesirable from both
an organoleptic and a physiological standpoint as
discussed below. Because of the marked disparity
in proteolytic activity, various suitable strains of P.
roqueforti should be carefully selected and
frequently evaluated.

strains with low protease and high lipase activities The precise role of other microorganisms in

LIPASE ACTIVITY
• PROTEASE ACTIVITY

250
F-l /

200

<
a
o
to
o

£ 50 100

o
CO

8 12 16 20
RIPENING PERIOD
(WEEKS)
FIGURE 2. Differences in rate of protein and lipid hydrolysis in blue cheese by
two strains of Penicillium roqueforti as indicated by increase in soluble nitrogen
and free fatty acids, respectively. Strain F-l with high lipase activity has low
protease activity. (Data adapted from Niki et al. 106 )
November 1976 195
 relation to the proteolysis in blue cheese has not 1.2 x
been determined. ImamuraS3 and Knoop and PROTFASE OF P. ROQUEFORTI
Peters74 contend that Streptococcus and Lacto-
bacillus species may be helpful because products
of their metabolism initially stimulate mold
growth, perhaps by generating some free amino
acids which stimulate spore germination. Morris
and Jezeski,95 demonstrated that slimed blue 0.8
cheese developed finer flavor and body than
unslimed blue cheese, indicating that micro- MYCELIUM
organisms other than P. roqueforti may be active
in the flavor production of blue cheese, possibly
through their lipolytic and/or proteolytic activity.
Hartley and 'Jezeski46 isolated five groups of
bacteria from the slime of blue cheese. The flora
of the slime was greatly affected by ripening 0.4
temperatures, manufacturing techniques, type of
seeding, and methods of handling during curing.
Downloaded by [New York University] at 13:24 09 February 2015

Kanauchi et al.6 6 '*7 isolated 34 strains of micro-

cocci and 28 strains of Brevibacteriaceae from the
surface slime and the interior of the blue-veined
cheese. It is generally believed that the slime
organisms are mostly proteolytic in their effect on
cheese. The salt concentration generally limits the
growth of bacteria after the initial few days,
although some proteolytic strains (Bacterium
linens, and B. erythrogenes) survive on the surface
of the cheese.69
Because of the importance of proteolysis in the
ripening and flavor development of blue cheese a
number of researchers have examined some
properties of the protease from P. roqueforti.
Protease activity in Penicillium varies with strain,
conditions of culture, age of mycelium, and the
presence of amino acids, salt, etc.9 3 The protease
activity of different species and strains of
Penicillium used in manufacture of cheese also
varies with available substrate. The inclusion of
hydrolyzed casein in a test culture medium
resulted in a three- to fourfold enhancement of
proteolytic activity.85 These preparations also
possessed a higher extracellular protease activity.
Actively growing P. roqueforti secretes proteolytic
enzyme(s) into culture media. 4 1 ' 5 3 ' 9 3 ' 9 4 ' 1 0 6
This enzyme has been referred to as extracellular
protease, while the protease isolated from the
mycelium is called intracellular protease. These
appear to be different enzymes.
Intracellular protease isolated from mycelium
has a relatively broad activity range extending
from pH 3 to pH 6 . 9 3 ' 9 4 ' 1 0 6 The extracellular-
protease has a narrower optimum (Figure 3)
6
PH
FIGURE 3. Effect of pH on the activity of the
intracellular (mycelium) and extracellular protease from
Penicillium roqueforti. (Data from Modler et aL9 3 >'4 )
between pH 5 and 6, but functions over a pH
range of 4.5 to 7 indicating its potential func-
tioning in cheese. These proteases are rather
nonspecific or general showing an optimum pH
that varies with substrate. Gripon and Bergere40,
Gripon and Hermier41 and Modler et al. 9 3 ' 9 4
reported pH optima of 5.5, and 4.2, and 3.0 for
extracellular protease action on casein, hemo-
globin and bovine serum albumin, respectively
(Figure 4). Gripon and Bergere40 found that a
protease preparation from culture media showed
two pH optima for casein hydrolysis viz pH 3.0
and 6.0. The optimum temperature for protease
activity is 35 to 43°C (Figure 5) and inactivation is
rapid at 45°C or above (Figure 6). Activity of the
p r o t e a s e ( s ) d r o p off rapidly below
30°C.4 ° ' 4 ' ' 9 3 >94 Thus, during the cheese ripening
process the low temperature (10°C) obviously
slows proteolysis and thereby retards excessive
accumulation of peptides and amino acids.
Sodium chloride tends to inhibit the extra-
cellular protease, whereas calcium chloride tends
to stimulate it somewhat.4 * The activity of

196 Critical Reviews in Food Science and Nutrition

 PROTEASE PROTEASE
ioo-
100

50-

50-
10 20 30 10 50 60
TEMPERATURE (C)

FIGURE 6. The thermal stability of the extracellular

protease of Penicillium roqueforti at various tempera-
tures. Substrate, 2.5% casein in 0.1 M phosphate buffer
pH 6.0; preincubation time, 3 hr; reaction time, 3 hr.
(From Modler, H. W., Brunner, J. R., and Stine, C. M.,/.
Downloaded by [New York University] at 13:24 09 February 2015

Dairy Sci., 57, 523 and 530, 1974. With permission.)

2 3 1 5 6 7
PH
protease from Penicillium was progressively
FIGURE 4. Differences in pH optima of extracellular depressed by sodium chloride above 0.5%. 69
protease from Penicillium roqueforti as affected by
substrate, Le., 1% bovine serum albumin (BSA) and 2.5% Imamuras 3 reported that fatty acids inhibited the
casein. (Data adapted from Gripon and Bergere.40) protease from P. roqueforti It has not been
determined whether or not such an inhibition
prevails in cheese. Conceivably, the very high
concentrations of free fatty acids (see Table 3)
further control or prevent excessive proteolysis.
100 PROTEASE The extracellular protease(s) facilely degrade a
wide array of proteins including a- and j3-
casein. 93 ' 94 Of the various substrates tested,
Gripon and Hermier41 reported that protamine
and histones were the preferred substrates for
protease of P. roqueforti (Table 2). Thus, it
appears that these proteases are broad-spectrum-
type proteolytic enzymes.
Little information is available on the
V
mechanism of action of these proteases.
Sulfhydryl blocking agents had no effect; the
serine protease inhibitor phenylmethyl sulfonyl-
flouride had no inhibitory effect. However, diazo-
acetoglycine methyl ester inhibited proteolysis by
90%. 94 This would indicate the involvement of a
free carboxylic group at the active site of the
enzyme.
Overall, proteolysis is obviously important in
20 30 50 60 the manufacture of a quality blue cheese. From
TEMPERATURE (C) the above fragmentary data the activity of
protease(s) is modulated by complex interactions
FIGURE 5. Effect of temperature on the activity of the
extracellular protease from Penicillium roqueforti. (From between the strain of P. roqueforti used, rate of
Modler, H. W., Brunner, J. R., and Stine, C. M., J. Dairy mold growth, and environmental conditions such
Sci.. 57, 523 and 530, 1974. With permission.) as salt, temperature, pH, fatty acids, and free

November 1976 197

 TABLE 2
Relative Hydrolytic Acti-
vity of Extracellular Pro-
tease of Penicillium roque-
forti on Various Substrates8
Relative
Substrate activity
Casein 100
Hemoglobin 38
Keratin 00
Fibrin 7 indicating that the intact membrane surrounding
Gelatin 3 these lipids must be ruptured to permit the
Collagen 0
2
Elastin
Protamine 320
Histone 220
a
Data from Gripon and
Hermier.4'
Downloaded by [New York University] at 13:24 09 February 2015
amino acids. Basic studies are needed to syste-
matically elucidate and quantify these interrela-
tionships.
LIPID METABOLISM
The manufacture of a quality blue cheese
critically depends on the metabolism of the lipid
substrate in cheese. The unique and dominating
flavor in these mold-ripened cheeses is caused by
methyl ketones which are predominantly derived
via partial oxidation of free fatty acids in the
cheese. A small amount (~40 mg/kg) of methyl
ketones may be derived from the original jS-
ketoacids in the milk fat. 1 0 9 ' 1 1 0
Because of the close positive correlation
between free fatty acid levels and methyl ketone
formation, lipase performs a key primary role by
providing the free fatty acids for flavor formation.
The fatty acids are flavorful per se and are also
precursors of the methyl ketones (Figure 16). In
the absence of adequate lipolysis cheese flavor is
poor and develops s l o w l y . 9 6 ' 9 7 ' 1 0 5 - 1 3 6 ' 1 3 7
Triglycerides are progressively hydrolyzed to
monoglycerides and free fatty acids during cheese
ripening. Thus, in cheese samples the triglycerides
(TG) decrease from 96 to 98% of lipids (about
35% of cheese) in early stages to 75 to 80% of the
lipids (32% of cheese) at maturity. The extent of
breakdown is governed by lipase activity, growth
of the Penicillium roqueforti strain used, duration
of ripening; amount of residual lipolytic activity
from the milk and microorganisms of the starter
198 Critical Reviews in Food Science and Nutrition
culture; efficiency of homogenization of the milk,
number and type of surface organisms, (if any),
pH, temperature, and sodium chloride concentra-
tion in the cheese.
The lipase(s) which are native to milk cause a
significant amount of hydrolysis (as indicated in
Figure 7). However, homogenization is a critical
process in facilitating subsequent hydrolysis43
which is predominantly caused by lipase of P.
roqueforti96 The triglycerides in the nonhomo-
genized raw milk are not as accessible to the lipase,
enzyme to cause hydrolysis. Homogenization also
markedly expands the total surface area of milk
fat available for lipase attack. Pasteurization of the
milk essentially destroys the milk lipase activity.
The importance of lipase is evidenced by the
positive effect of adding lipase to the milk with
the starter culture or with the mycelial
spores. 7 3 ' 1 1 6 ' 1 1 7 Jolly and Kosikowski 63 ' 64
studied the effect of added Upases from several
sources. Of fourteen lipase preparations (animal
and microbial) tested, only one (a lipase from
Aspergillus oryzae) significantly enhanced and
accelerated the maturation of experimental blue
cheese (Figure 8). Other lipase preparations, e.g.,
gastric lipase, had an intermediate impact. The
cheese containing fungal lipase attained acceptable
maturity within 40 to 70 days, whereas the
control (normal) cheese did not attain the same
status for over 90 days. This approach warrants
further study for enhancing flavor development
and accelerating ripening.
The addition of lipases to pasteurized,
homogenized milk used in the manufacturing of
blue cheese results in enhanced lipolysis and
formation of methyl ketones. However, in many
cases this is accompanied by development of bitter
flavors which is attributed to protease activity in
the lipase preparations or may be due to abnormal
proteolysis by the P. roqueforti producing bitter
peptides. This exemplifies an undesirable result of
upsetting the normal interrelationships prevailing
between enzymes and their products during the
cheese ripening.
During blue cheese ripening there is a progres-
sive build-up of free fatty acids (Figure 7); the
composition of these acids resemble those of milk
fat (Table 3). The release of these acids is caused
mainly by lipase of P. roqueforti, although some
researchers have suggested that other micro-
organisms may be involved. The presence of some
 120

90
A - HOMOGENIZED
B - PASTEURIZED-HOMOGENIZED
C - UNTREATED

2 60
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30

a—Q I
16 21 32 10
RIPENING WEEKS

FIGURE 7. Progressive lipolysis as indicated by free fatty acid accumulation in

blue cheese made from milk that had been homogenized (A) pasteurized and
homogenized (B), and untreated (C). Note the dramatic effect of homogenization
(A vs. C) on rate and extent of lipolysis and the effect of the lipase inherent in
milk on fatty acid levels (A vs. B). (From Morris, H. A., Jezeski, J. J.,Combs, W.
B., and Kuramoto, S., J. Dairy Set, 46, 1, 1963. With permission.)

yeasts may enhance flavor development (e.g.,
Candida lipolytica) by providing more free fatty
acids which may accelerate and improve
ripening.1 * ! » ' ' 2 However, Stadhouders et al. 1 3 6
reported that the bacteria which survive in the
cheese have little lipolytic acitivity.
The lipolytic activity of different strains of
Penicillium species varies markedly. 7 6 ' 1 0 6 ' 1 3 9
Stepaniak et al. 1 3 9 grouped mold strains into four
categories with relative lipase activities of
100:63:44:14 in order of decreasing lipase
activity. The proportions of the various volatile
fatty acids released from milk fat emulsions varied
with different strains of P. roqueforti, e.g.
100:50:27:37 and 100:120:49:50 for butyric,
caproic, caprylic and capric acid released by strains
with high and low lipase activity respectively.
Cheese samples made with P. roqueforti strains of
high and low lipase activity contained 147.5 and
27.2 mg of fatty acids (C2 to CIO inclusive),
respectively. The organoleptic quality of the
cheese made with low lipase strains of P.
roqueforti was inferior. Lamberet and Lenoir76
divided 89 strains of P. caseicolum into two
groups, one with high and one with low lipolytic
activity, i.e., 7.4 and 4.1 units, respectively; the
former were more desirable for cheese manu-
facture. Niki et al. 1 0 6 compared the relative
lipolytic activity of various strains of P. roqueforti.
The disparate activities of two strains are sum-
marized in Figure 9. lipase activity correlated
with development of methyl ketones. Sato et

November 1976 199

 TABLE 3

Content of Unesterified Fatty Acids in

Samples of Commercial Blue Cheese
mg/10 g dry cheesea

Fatty
10 acid A B C

C2 28.2 14.2 6.8

C4 25.4 11.2 50.4
C6 17.6 7.4 29.4
C8 15.6 5.8 24.8
CIO 28.2 10.2 40.6
C12 33.8 12.4 49.2
C14 124.0 40.8 186.4
C16 264.2 97.0 406.2
C18 97.6 32.2 128.0
C18:l 286.8 96.4 364.0
IS 30 15 60 75 90 C18:2 26.6 . 5.4 32.2
MATURATION (DAYS)
C18:3 27.2 5.6 26.4
FIGURE 8. The effect of the addition of a fungal lipase a
Data adapted: From Anderson and
on the rate of flavor development in experimental samples
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Day."a
of blue cheese. (Data adapted from Jolly and
KosikowskL")

METHYL KETONES
VOLATILE FREE FATTY ACIDS

250 -

200 -
(9

UJ
z
o

100 -

2 H 6 8 10 12 V\ 16 18 20
RIPENING PERIOD (WEEKS)

FIGURE 9. Comparison of the relationship between the lipolytic activity and

methyl ketone formation in cheese samples made with two different strains of
Pencillium roqueforti. (Data adapted from Niki et a l . 1 0 6 )

200 Critical Reviews in Food Science and Nutrition

 al. 1 2 7 also reported marked differences in lipoly-
tic activity of different strains of P. roqueforti as
evidenced by large variations in free fatty acid
levels in blue cheese samples during ripening
(Figure 10). The methyl ketone concentrations
paralleled these patterns.
Lipase
These data conclusively show that lipase is an
important enzyme in developing the flavor of blue
cheese. Consequently numerous researchers have
examined the lipase(s) associated with Penicillium
roqueforti. Various factors affect lipase pro-
duction by P. roqueforti in experimental cultures
but the optimum conditions for lipase production
are generally similar to those for the ripening of P.
roqueforti type cheeses.
Downloaded by [New York University] at 13:24 09 February 2015
significant amount of lipase, whereas mold grown
in a medium containing Casitone broth produced
high lipase activity. Thibodeau and Macy 143 and
Morris and Jezeski 95 had previously obtained
similar results on a Czapek medium. This indicated
that nitrate, the only nitrogen source in Czapek
medium, is an inadequate nitrogen source for
enzyme production by P. roqueforti and that a
source of amino acids is necessary. Imamura and
Kataoka54 showed that lipase production by P.
roqueforti was inhibited by lactose, glucose or
galactose; however, it was increased by the
addition of butterfat. The increased production of
lipase by the addition of butterfat is in contrast to
the result obtained by Eitenmiller et al. 20 They
reported that the addition of 1% (v/v) butter oil or
corn oil to the growth medium caused a decrease

Eitenmiller et al. 20 showed that the mold in lipase production.

grown in a Czapeck broth did not produce any In whey culture, lipase production is greatest at

N) A
a 3J
\n
in

/
3;

th 2
f aB

1 1 / A- """" -AC

i
30 60 90 (DAYS)
RIPENING

FIGURE 10. Differences in the free fatty acid levels in cheese samples made with
four strains denoted A, B, C, and D of Penicillium roqueforti. (Data adapted from
Satoetal. 1 2 7 )
November 1976 201
 neutral or alkaline pH at 20°C. 106 However, 1-1 LIPASE OF P. ROQUEFORTI
Eitenmiller et al. 20 obtained greatest lipase
production at pH 5.5 and 27°C. Imamura and
Kataoka 54 ' 55 found that production of intra-
cellular lipase increased with time at 7°C while at
25°C the intracellular lipase could not be found MYCELIUM
after 28 days in culture. On the other hand, the
production of extracellular lipase increased with
time at the latter temperature.
The lipase activity and, possibly, its con- 52-
centration, in P. roqueforti vary with strain, as
discussed above. The mold apparently possesses an EXTRACELLULAR
intracellular and a secretable or extracellular
lipase, and the former demonstrates greater
activity. 5 4 ) S 6 ' 9 5 ' 1 0 6 Whether or not the secreted
enzyme is identical to the mycelial lipase is not
known; however, their similar responses to pH is
indicative. Niki et al. 1 0 6 reported two pH optima
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PH
(6.0 and 7.5) for both the intra- and extracellular
8
lipase (Figure 11) indicating the possible presence
of two discrete enzymes in both preparations. Two FIGURE 11. Effect of pH on the activities of intra-
•types of Upases were also isolated from P. cellular (mycelium) and extracellular lipases from Peru-
cyclopium.ss Oi et al. 1 0 7 isolated two types of cillium roqueforti. (Data adapted from Niki et al.1 0 < )
extracellular lipases and found that they were
mutually convertible under appropriate con-
ditions, i.e., in the presence of sodium deoxy-
cholate, ethylenediaminetetraacetate and
parachloromercuribenzoate. Enzymes treated with
hydrogen peroxide or sodium borohydride caused
the opposite interconversion. Eitenmiller et al. 20
and Fodor and Charl2 s reported that the pH
optimum for the lipase is at pH 8.0, whereas
Morris and Jezeski9 s reported that the pH
optimum varies between pH 6.0 and 7.8,
depending on the type of enzyme and substrate
employed.
The optimum temperature of the lipase from P.
roqueforti activity was 30 to 35°C 9 5 ' 1 3 3 whereas
Eitenmiller20 reported it at 37°C. These apparent t 50C ,
variations in the optimum condition for the
20 40 60
activity of lipase, may well be due to the
TIME (M)
difference in growth condition, fungal strain, and
analytical method used. Obviously, the lipase FIGURE 12. Kinetics of thermal inactivation of lipase
system in the mold is quite active at low isolated from Penicillium roqueforti. (Data adapted from
temperatures, e.g., 10°C. Eitenmiller etal. 2 0 )
The enzyme is quite heat labile (Figure 12). At
37°C and 40°C the lipase of P. roqueforti lost 65 75 and 90% of the activity is still maintained after
and 90% of its activity in 1 hr. 20 The enzyme is 6 months when kept in the form of a filtrate from
fairly stable at low temperatures and may be held crushed fungus cells. 133
frozen for long periods of time without loss of Shipe 133 observed a stimulatory effect of
activity. Conversely, in dry mycelium the lipase CaCl2 on the lipases of Aspergillus niger and P.
loses activity within 2 months at 2 to 3°C, while roqueforti. Eitenmiller et al. 20 observed that

202 Critical Reviews in Food Science and Nutrition

 MnCl2 and MgCl2 at a concentration of 10~2 and
10" 3 , respectively, stimulate the activity of P.
roqueforti lipase. However, they did not observe
the stimulatory effect by CaCl2. Morris and
Jezeski9S and Imamura and Kataokas s >s 6 showed
that NaCl (above 3%) lowers both intra- and
extracellular lipase activity.
Several investigators reported that P. roqueforti
lipase apparently preferentially hydrolyzes short-
chain fatty acids from butter fat. 148 Shipe 133
reported that the intracellular lipase of P.
roqueforti splits different triglycerides in the
following order: C4>C 6 >C8>C3. Eitenmiller et
al. 20 obtained similar results using various tri-
glycerides (Figure 13). Triolein was the poorest
substrate among the triglycerides (tripro-
pionin, tributyrin, tricaprylin and tricaprin) tested.
Salvadori and Salvadori126 and Stepaniak and
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Habaj 139 observed that lipase from different
strains of P. roqueforti showed different substrate
specificity. Because of the greater toxicity of
long-chain fatty acids to the mold3 9 a few authors
have suggested that the apparent preferential
hydrolysis of short-chain acids by the lipase is a
protective mechanism of the mold. However, this
is speculative. The observed specificity may not be
valid because of the peculiar physical nature of the
systems used. The dangers of misinterpreting data
concerning lipase specificity have been discussed in
detail by Jensen.60 From the copious data avail-
able it is obvious that the lipase from P. roqueforti
is capable of hydrolyzing the wide array of
triglycerides which is found in milk fat.
The rate of release of free fatty acids may be a
limiting factor in methyl ketone production by P.
roqueforti in blue cheese. This is consistent with
the results obtained using strains of mold with
different lipase activities127 and also with data
obtained following the addition of various lipase
preparations, 7 3 ' 1 1 1 ' 1 1 2 ' 1 1 6 - 1 1 7 ' 1 4 8
The capacity of the mold to oxidize fatty acids
to methyl ketones and carbon dioxide apparently
exceeds the lipolytic capacity of most strains.
Evidence from in vitro studies indicates that free
fatty acids are toxic to the mycelium and actually
inhibit methyl ketone formation. Lawrence81 ' 8 2
observed that high concentrations of triglycerides
were readily oxidized to methyl ketones by spores
of P. roqueforti, while the equivalent concen-
trations of free fatty acid were only slowly
oxidized or were inhibitory. Since negligible
amounts of free fatty acids accumulated during
TRIGLYCERIDES
1001
CIO
u
<
50-
C3
C18
FIGURE 13. Relative rate of hydrolysis of triglycerides
by lipase isolated from Penidllium roqueforti Legend:
C3, C4, C8, CIO, C18 denote triproprionin, tributyrin,
tricaprylin, tricaprin and triolein, respectively. (Data
adapted from Eitenmiller et al.2 °)
the incubation with triglycerides it seemed that if
lipolysis occurred, its rate did not exceed their rate
of oxidation and therefore, inhibition of respira-
tion by free fatty acids was avoided. Lawrence and
Hawke84 showed that free fatty acids above 1
mmol/mg of mycelium in an aqueous culture
media impaired methyl ketone formation, whereas
in a medium containing lipolyzed fat, inhibition
was only observed at much higher free fatty acid
levels. 18 '' 9 Thus, the high concentrations of fatty
acids in cheese (Figure 7) may riot be inhibitory to
the mold because they are mostly associated with
the lipid phase rather than being in contact with
the mycelium. Therefore, in cheese, the very high
lipase activity by releasing abundant fatty acids
permits methyl ketone to progress at a high rate
while inhibition may be minimized by the
solubility of the fatty acids in the fat.
Fatty Acid Metabolism
During ripening there is a gradual accumulation
of methyl ketones in blue cheese (Figures 14 and
15). The levels observed in these samples were very
November 1976 203

30
3 formation of methyl ketones by the mold might
i microorganisms. Girolami and Knight 37 suggested
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10
o FORMATION O F 2-ALKANONES
-ftTHYL KETONES
6 10
WEEKS OF RIPENING
FIGURE 14. The progressive accumulation of methyl
ketones and total caibonyls (aldehydes and ketones) in
experimental samples of blue cheese. (Data adapted from
Dartey and Kinsella.13)
high because small experimental samples of cheese
were analyzed during maturation. Normally, lower
levels occur in cheese (Table 4). Usually when
small experimental samples are used there is more
copious growth of mold which probably accounts
for the higher concentrations.
Of t h e various methyl ketones found
2-heptanone is usually the most abundant
followed by 2-nonanone, 2-pentanone and
2-undecanone (Figure 15). The relative con-
centration of these varies with cheese sample.
These methyl ketones are derived by the partial
oxidation of fatty acids released by lipase (Figure
16).
Starkle 138 initially reported on the ability of
growing cultures of fungi to convert fatty acids to
methyl ketones. It has since been well established
that several fungi oxidize short-chain fatty acids to
the corresponding methyl ketone with one less
carbon atom.14'ls'19'2"I'34-37>'"'5()'73-Sfr-
84,i4o-i42,i44 Gehrig and Knight 3S ' 36
204 Critical Reviews in Food Science and Nutrition
Lawrence 80 ' 81 and Dartey and Kinsella 14>ls
reported that washed spore suspensions of Penicil-
lium roqueforti produced 2-alkanones when
incubated with fatty acids.
The majority of molds from Penicillium and
Aspergillus genera and some related molds produce
methyl ketones as metabolic by-products.2 7 ' 3 4 ' 47
The physiological role of this conversion in overall
mold metabolism is unclear. Stokoe 140 postulated
that the abnormal oxidation of fatty acids to
methyl ketones may represent a detoxifying
mechanism, and Franke et al. 28 found that the
also function by inhibiting other competitive
that methyl ketones were also inhibitory to P.
roqueforti and could possibly prevent overgrowth
of the mold during cheese ripening.
The mechanism of methyl ketone formation
from fatty acids by molds has received much
attention.34"3 7
>4 7 >8 0 " 8 4 > 9 8 > 1 3 8 , 1 * 0 , 1 4 2 , 1 4 4
Accumulated information indicates that methyl
11
ketone production from fatty acids by the mold
proceeds via the classical fatty acid j3-oxidation
pathway. Thaler and Eisenlohr 141 showed that
Penicillium glaucum produced methyl ketones not
only from saturated fatty acids but also from the
acyl intermediates of 0-oxidation, i.e. a,|3-
unsaturated fatty acids and 0-hydroxy fatty acids.
Although unable to isolate a reconstructable fatty
acid oxidizing system, Gehrig and Knight 3 5 ' 3 6
concluded that methyl ketone formation from
fatty acids by the spores of P. roqueforti involves
the ^-oxidation reaction. Lawrence81 demon-
strated that 2-heptonone formed from 1-C14-
octanoate by the spore of P. roqueforti was not
radioactive, whereas that from 2-C14-octanoate
was radioactive, and the specific activity of the
2-heptanone was almost identical to that of
2-C14-octanoate. These data and experiments with
enzyme preparations from various fungi showed
that methyl ketones were produced by decarboxy-
lation of /3-ketoacids.27 These observations
provide evidence for the functioning of 0-
oxidation in the formation of methyl ketones
from fatty acids. Thus, apparently during the
conventional /3-oxidation cycle in the spores and
mycelium of P. roqueforti, the 0-ketoacyl-CoA
formed by the dehydrogenation of the 0-hydroxy
and acyl-CoA, is deacylated to 0-ketoacid and CoASH
Downloaded by [New York University] at 13:24 09 February 2015

FIGURE 15. Rate of accumulation of different methyl ketones during the

ripening of experimental samples of blue cheese. C5 through C13 denotes
(C5)2-Pentanone, (C7)2-heptanone, (C9)2-nonanone, (Cll)2-undecanone and
(C13)2-tridecanone.

TABLE 4

2-Alkanone Content of Blue Cheese Samples

fig/10 g dry blue cheese

2-Alkanone A a
B a
C a
Db Eb Fb Gc Hc

2-Propanone 65 54 75 210 _ 60 T
2-Pentanone 360 140 410 1022 367 51 372 285
2-Heptanone 800 380 380 1827 755 243 3845 3354
2-Nonanone 560 440 1760 1816 600 176 3737 3505
2-Undecanone 128 120 590 136 135 56 1304 1383
2-Tridecanone - - - 100 120 77 309 845

Total 1940 1146 4296 5111 1978 603 9627 9372

a
Commerical samples of ripe blue cheese.4
b
Samples D, E, and F of blue cheese ripened for 2, 3 and 4 months, respec-
tively. 12 '
c
Samples G and H of very small batches of experimental blue cheese ripened for
2 and 3 months, respectively.'3

November 1976 205

 LlPIDS
1
LlPASE
FATTY ACIDS
/B-OXIDATION
, / THIOLASE ACYL-COA + ACETYL-COA
KETO ACYL-COA ^ Uc
N
,' C02
THIOHYDROLASE ""
-KETO ACID
2-ALKANOL
\SDECARBOXYLASE
s REDUCTASE
^ 2-ALKANONE
FIGURE 16. General outline of pathway showing the
metabolism of fatty acids by Penicillium roqueforti as
occurs in blue cheese. The acyl group may contain from
Downloaded by [New York University] at 13:24 09 February 2015
4 to 16 carbons; KC denotes the Krebs or citric acid
cycle. Broken line denotes possible inhibition of thiolase
by acyl-CoA, thereby facilitating (3-ketoacid production
for the decarboxylase.
by a /3-ketoacyl-CoA deacylase or thiohydrolase,
instead of being split by the thiolase into acetyl-
CoA and an acyl-CoA with two less carbons. Thus
for some unexplained reason these molds (both
spores and mycelia) convert fatty acids especially
those of short and medium chain length, to
corresponding 2-alkanones with one less carbon
atom (Figure 16). Obviously in these molds the
/3-ketoacyl-CoA deacylase activity greatly exceeds
that of thiolase, and the /3-ketoacid decarboxylase
apparently causes rapid decarboxylation to carbon
dioxide and methyl ketones thereby preventing
any accumulation of /3-ketoacids. This latter step
may actually be the facilitating if not the driving
force responsible for this peculiar behavior of
molds in producing methyl ketones. This capacity
is enhanced when the molds have more readily
oxidizable substrates83 suggesting that inhibition
of the normal /3-oxidation pathway (especially of
the thiolase step) may aid the deacylation and
decarboxylation steps.
All studies to date indicate that |3-ketooctanoyl-
CoA is the preferred substrate for the deacylation
reaction. Since many studies have been conducted
using octanoic acid, it is generally held that all
methyl ketones are derived from the cor-
responding fatty acids in the initial cycle of the
/3-oxidation scheme. However, methyl ketones of
varying chain length may be formed from long-
206 Critical Reviews in Food Science and Nutrition
chain fatty acids at successive cycles of /3-
oxidation. 14>is
Because of the involvement of the /3-oxidation
enzymes in methyl ketone formation We
attempted to isolate this complex from P.
roqueforti mycelium with a view to elucidating the
mechanism(s) responsible for methyl ketone pro-
duction and possibly exploiting the system to
generate methyl ketones in a continuous reactor.
However, to date, because the conventional
methods required to disrupt the mycelial cell wall
are too harsh and destructive, it has been
impossible to isolate an active fatty acid )3-
oxidation complex from this mold. The use of
hydrolytic enzymes to digest the mycelial cell
walls may solve this problem.51 ' s 2
Because of its possible involvement as a
facilitory step in the formation of methyl ketones,
we examined the /3-decarboxylase activity in P.
roqueforti. This enzyme is very active in several
molds. 28 Hwang et al. 52 demonstrated that
resting spores, germinating spores, and mycelium
of P. roqueforti actively decarboxylate /3-keto-
laurate to 2-undecanone. The rate of 2-
undecanone formation increases as resting spores
germinate, and activity is highest in mycelium
(Figure 17). Glucose stimulated the formation of
2-undecanone by resting spores; however, it had no
effect on /3-ketodecarboxylase activity in
mycelium (Figure 17). The enhanced methyl
ketone production in germinating spores is attri-
buted to the progressive increase in activity of
/3-ketoacyl decarboxylase as spores germinated.
The enzyme was isolated from the cell-free
extract. By various heat treatments it was shown
to consist of heat-stable and heat-labile species
(Figure 18). Optimum pH for the enzyme was 6.5
to 7.0 (Figure 19), which corresponds to the
optimum pH observed for methyl ketone product-
tion from lauric acid by germinating spores of P.
roqueforti.14'15 Biphasic substrate saturation
curves (Figure 20) and a discontinuous slope in
Arrhenius plot for /3-ketoacyl decarboxylase
further indicated the existence of two species of
this enzyme.52 Of the various /3-ketoacid sub-
strates tested, Franzke and Thurm 3 0 ' 3 1 showed
that j3-ketolaurate was the most preferential sub-
strate for mold /3-ketoacid decarboxylase. Futher
basic studies are warranted to explain the unique
behavior of the /3-oxidation enzyme sequence in P.
roqueforti as this may be exploitable in developing
a continuous enzymatic system for methyl ketone
production.
 + GLUCOSE
180
GLUCOSE

MYCELIUM

I 90 - GERMINATED SPORE

RESTING SPORE
Downloaded by [New York University] at 13:24 09 February 2015

2 W. 6
INCUBATION (H)

FIGURE 17. Relative specific activity of 0-ketododecanoyI decarboxylase in

spores, germinating spores and mycelium of Penicillium roqueforti in the presence
and absence of glucose.

300

200

| O-o
^ IOO

10 20 10
PREINCUBATION TIME (M) PK

FIGURE 18. Thermal stability of 0-ketoacylde- FIGURE 19. The effect of pH on the formation of-
carboxylase from mycelium of Penicillium roqueforti at 2-undecanone from P-ketododecanoic acid by (3-ketoacyl
different temperatures. decarboxylase from Penicillium roqueforti.

November 1976 207

 9"

6-
CD —
z: ui
<£ I-
«_> o
LLJ a.
<=> a-

5 3- V>

1-

B KETOLAURATE
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FIGURE 20. Typical biphasic substrate saturation curve displayed by /Mcetoacyl

decarboxylase from Penicillium roqueforti. This property may enable the enzyme
to function over a wide range of substrate concentrations.

TABLE 5

Relative Concentrations of 2-Alkanones and 2-Alkanols in Commercial Blue Cheese Samples

jxg/10 g dry blue cheese

A B C D
Chain
length 2-Alkanone 2-Alkanol 2-Alkanone 2-Alkanol 2-Alkanone 2-Alkanol 2-Alkanone 2-Alkanol

C5 370 12 144 6 418 38 175 27

C7 . 815 126 380 164 1436 182 380 69
C9 560 78 446 74 1766 46 640 34

Data A, B, and C calculated from Anderson and Day.* Data D calculated from Svenson and Ottestad.15 *

The 2-alkanones produced by decarboxylation RELATIVE METABOLIC ACTIVITY

are easily reduced to the corresponding 2-alkanols
by both spores and mycelium of P. roqueforti.4'
3 o ,31 ,s 9 j ^ j j r e a c t j o n o c c u r s rather rapidly and it
has been proposed as a mechanism for minimizing
the toxic effect of methyl ketones. However, it
may also be a method of regenerating oxidized
nucleotides (NAD and NADP) under the reducing
conditions prevailing in cheese. While the concen-
tration of alcohols is variable, it is normally much
lower than that of the corresponding methyl
ketones (Table 5).
OF SPORES AND MYCELIUM
With regard to the important biochemical
changes which occur during, cheese ripening a
recurring question concerns the activities of spores
and mycelium and their contribution to the
quality of the final product. It is quite impossible
to determine this in cheese, but a number of
researchers have examined the capacity of spores
and mycelia to metabolize lipid substrates. Infor-
mation concerning the factors which affect the

208 Critical Reviews in Food Science and Nutrition

 metabolic activity of spores and mycelium is very
important in developing culture systems for pro-
ducing blue cheese flavors.
Because of the involvement of both spores and
mycelia in the metabolism of lipids during cheese
ripening, several characteristics of their metabolic
activities are reviewed below and related events
which occur in blue cheese during ripening.
Spore Metabolism
Several aspects of methyl ketone production
from fatty acids by PenicilUum roqueforti spores
have been studied. 1 4 ' 1 5 - 2 2 ' 2 3 ' 3 5 - 7 3 ' 8 0 ' 8 1 The
changing morphology and biochemical properties
of the spores during germination have been studied
in our laboratory. 23 ' 51 The spores require a
source of energy from both carbon (sugar) and
nitrogen (amino acids) for germination.80"84
Downloaded by [New York University] at 13:24 09 February 2015
When placed in such a medium, the spores swell by
imbibition of water and after 12 to 15 hours at
26°C and pH 6.5, germ tubes emerge and grow at a
rate of 20 /xm/hr for about 40 hr (Figure 21) and
then transform into mycelium.22 During this
period there is a marked increase in oxygen
consumption accompanied by successive synthesis
of nucleic acids, protein, complex carbohydrates,
and lipid material.
Electron microscopic examination revealed that
100
80
GERMINATION
§60
20
8 24
GERMINATION TIME (HR.)
FIGURE 21. The germination rate (percent) and changes in dry weight and
length of germ tube during germination of spores of PenicilUum roqueforti. (From
Fan, Y. S., Hwang, D., and Kinsella, J. E., J. Agric. Food Chem., 24, 443, 1976.
With permission.)
the spores possessed thick cell walls with four
distinct layers.2 3 >51 However, the newly emerging
germ tube possesses a much thinner cell wall with
only a single layer, while the mature mycelium has
a much thicker cell wall with three apparent
layers. This may account for the apparent superior
metabolic activity of spores compared to
mycelium, i.e., spores with incipient emerging
germ tubes may be very permeable to oxidizable
substrates. Earlier, Lawrence 80 ' 81 suggested that
permeability is a major controlling factor in mold
metabolism. The possession of a thick cell wall
may also explain the greater resistance of spores to
the toxic effects of fatty acids (which will be
discussed below).
It had been claimed that only spores form
methyl ketones 73 or that they are superior to
mycelium in the formation of methyl ketones.
Therefore, Fan et al. 23 studied the relative rates of
2-heptanone formation from octanoic acid by
spores at progressive stages of germination. Under
short incubation conditions, mycelium was much
more active; however, with prolonged incubation
(i.e., 8 hr) the "resting" spores were also very
active (Figure 22). Under certain conditions,
mycelia derived from a known number of spores
had a greater capacity to form 2-heptanone from
octanoic acid, than the same number of spores in
600 - 300
480 - 240
360£_|l80
240 £ - 120
120 - 60
40
November 1976 209
 3.0 A RESTING SPORE
B SWOLLEN SPORE
C GERM-TUBE EMERGENCE STAGE
D MAXIMUM GERMINATION STAGE
E GERM-TUBE ELONGATION STAGE
F MYCELIUM
*ui 2.0 F F
Di
E
E E
m
z
o D A
z
I-
mm
B
O-
A
1.0

BC
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AB C D BC
L j_
2 1 6 8
INCUBATION TIME (HR)

FIGURE 22. The relative capacity of equal numbers of spores at different stages of physio-
logical development to convert octanoic acid to 2-heptanone. Equal numbers of spores were
preincubated for 0, 8, 12, 16, 30, and 48 hr to obtain the stages denoted by A, B, C, D, E, and
F, respectively, and then were incubated with octanoic acid (1 mM) for the specified incubation
periods. (From Fan, Y. S., Hwang, D., and Kinsella, J. E.,/. Agric Food Chem., 24,443,1976.
With permission.)

resting state. However, on an equal dry-weight

basis (with the exception of the immediate post-
emergence phase) spores and mycelia demon-
strated comparable rates of fatty acid oxidation.
However, this could be varied with pH and 200
particularly with substrate concentration, i.e., at
fatty acid levels greater than 2 mAf the mycelium
was inhibited, whereas the spores continued to I GLUCOSE (80MM)
generate methyl ketones. 14 j l s >81 >82 •- +
In a series of studies Lawrence82 and Dartey OCTANOIC ACID (IMM)
and Kinsella 14 ' 1S examined the ability of spore 13 100
suspensions to metabolize fatty acids. Using spores
in culture media Lawrence 80 ' 81 showed that
oxygen uptake closely reflects metabolic activity
and is markedly stimulated by glucose or casamino / /OCTANOIC ACID QMN)
acids. Oxidation of octanoic acid is concurrent
with oxygen uptake (Figure 23). Lawrence 80 ' 81
did not observe the germ tube emergence during
this period. Several amino acids (33 mM) 7 (H)
(casamino acids, alanine, serine, and proline) and
sugars (85 mAf) (glucose, xylose, sucrose, and FIGURE 23. Relative rates of oxidation of octanoic
acid by spores of Penicittium roqueforti in absence and
galactose) gave an approximately fourfold increase presence of glucose (0.5 X 10' spores per millimeter
in heptanone formation from octanoic acid by phosphate buffer pH 6.5, 27°C). (Data adapted from
spores (Figures 23 and 24). Lawrence.* *)

210 Critical Reviews in Food Science and Nutrition

 10
o
3 6
UJ
O
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10 20
FIGURE 24. The relative rates of formation of methyl ketones from palmitic acid (5 mM) by spore suspensions (1 x 10'
spores) of Penicillium roqueforti in phosphate buffer (A), with 20 mM glucose (B), with 20 mM proline (C) and with 10
mM each of glucose and proline (D). Incubation temperature 30°C, pH 6.5.
Certain substrates readily oxidizable by spores
stimulate germination and ketone produc-
tion. 4 7 ' 8 3 Lawrence 80 ' 81 claimed that substances
stimulating oxidation functioned by increasing the
transport of octanoic acid into the spores. He 81
observed that as spores aged their ability to
oxidize octanoic acid to methyl ketone decreased
(Figure 25). In all cases there was an initial lag
phase before spores formed 2-alkanone from
octanoic acid. This may be related to the increased
thickening and toughening of the spore cell wall
with aging, resulting in decreased permeability.
This lag period was affected by the concentration
of fatty acid being longer at higher levels of fatty
acid (Figure 26). The optimum spore to acid
concentration was 2 X 109 spores with 3 mM fatty
acid at pH 5 to 6. The inclusion of glucose,
sucrose, or amino acids reduced the lag phase and
stimulated the generation of ketones. 1 4 > l s ' 8 °' 8 1
30 A5 HR.
The optimum pH for ketone formation varied
with concentration and type of fatty acid. At an
octanoate concentration of 1 mAf optimum pH
was 5.5 to 6, and it was observed that pH
optimum increased as fatty acid concentration was
increased,81 i.e., 1, 3 and 20 mM of octanoic acid
gave maximum yields of 2-heptanone at pH 5.8,
6.4, and 6.8, respectively. Incidentally, this may
coincide with events occurring in cheese produc-
tion where the pH increases during ripening and
there is a concurrent increase in fatty acid levels.
Thus, metabolism of fatty acids by spores is
facilitated. Optimum temperature for ketone for-
mation was 25 to 28°C (Figure 27). Dartey and
Kinsella I 4 ' l s observed a 20-fold increase in
methyl ketone formation as the temperature of
incubation was dropped from 37 to 30°C. Thus,
the low temperatures prevailing during cheese
ripening may limit the rate of ketone production
November 1976 211
 AGE OF SPORES AT
HARVEST
15 4 DAYS

O 10

I
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2 4 HR.
INCUBATION TIME

FIGURE 25. The effect of age of spores of Penicillium roqueforti on rate of

oxidation of octanoic acid. (Data adapted from Lawrence.81)

by spores and may affect the patterns of ketones
produced, as indicated by Dolezalek and Hoza. 17
Lawrence18 and Dartey and Kinsella14
reported that formation of methyl ketones from
medium chain length fatty acids (C8 through C12)
was considerably increased when small amounts of
metabolic CO2 were retained in the culture.
Although not all fatty acids have been tested, it
seems that spores can oxidize all fatty acids with 4
to 16 c a r b o n s . 1 4 ' 1 5 ' 2 7 " 3 1 ' 3 5 ' 8 1 However,
octanoic acid is the preferred substrate, with
activity falling off for long- and short-chain fatty
acids (Figure 28). Oxidation of long-chain fatty
acids is very limited and detectable by using
radioactively labeled acids and high concentrations
of spores.
Spores are much more resistant than mycelia to
the inhibitory effects of fatty acids. Hence, in
cheese where the free fatty acids may attain
concentrations of 150 to 600 mg/10 g (Table 3), it
is conceivable that the spores play a very signifi-
cant role in the generation of methyl ketones.
Thaler and Eisenlohr141 and Lawrence82
reported that spores could also utilize triglycerides
and generate methyl ketones. This conversion was
stimulated by sugars and amino acids.82 It is
possible that germinating spores were the active
entities in these studies. Thus, the spores (with
emerging germ tubes) probably secreted lipase
which hydrolyzed the triglycerides, and the
resultant fatty acids were then converted to
methyl ketones in normal fashion.
Studies of fatty acid absorption and
metabolism by spores at progressive stages of
germination with concurrent examination of
spores by scanning and electron microscopy
should be helpful in correlating metabolic activity
with morphological characteristics of the spores.s 1
As spores germinate, they demonstrate an
enhanced capacity to reduce exogenous
2-heptanone to 2-heptanol (Figure 29). This
capacity has been observed in spores and
mycelia. 2 3 ' 3 0 ' 3 1 ' 8 1
Mycelium Metabolism
Mycelium of Penicillium roqueforti possesses
the ability to hydrolyze triglycerides and to
o x i d i z e free fatty acids to methyl
ketones. 1 9 ' 3 0 ' 3 1 ' 5 2 ' 8 4 Lawrence and Hawke84
and Dwivedi and Kinsella 18 ' 19 carefully obtained
mycelial cultures devoid of spores and demon-
strated their ability to metabolize fatty acids.
Vinje and Ghosh 144 and Lawrence and Hawke84
showed that P. roqueforti mycelia harvested after

212 Critical Reviews in Food Science and Nutrition

 SPORE FATTY ACID CONCN 2.0

/3MM

jj

13
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3
Q
XI
r
• 1.0
HI
/ /
2-HEPT ANOH
o

/ IMM

1 /

/ 18 24 30 36 CO
INCUBATION TEMPERATURE
FIGURE 27. Relative rato of production of 2-heptanone from octanoic acid by
spores of Penkilllum roqueforti incubated at different temperatures. (Adapted
from Lawrence."1)
10 15
INCUBATION TIME

z FIGURE 26. Effect of concentration of octanoic acid on lag phase and rate of
2-heptanone formation by spores of Penicillium roqueforti (Data adapted from
Lawrence.")
Downloaded by [New York University] at 13:24 09 February 2015

10 HR.

FIGURE 28. Relative rates of generation of carbonyls following the incubation

of fatty acids (2 mM) with spores (1 X 1O10) of Penicillium roqueforti in
phosphate buffer (0.1 mM, pH 6.5) containing 5 mM glucose. C6 to C16 denotes
octanoic through palmitic acid, respectively.

48 to 60 hr showed the greatest oxygen uptake
and methyl ketone formation. Mycelia grown for
less than 30 hr showed diminished oxidation of
fatty acids.
Dwivedi and Kinsella19 showed that the lag
phase for fatty acid metabolism by fresh P.
roqueforti mycelium is longer compared to aged
mycelium, especially in the case of long chain
fatty acids. Also, mycelia that had been cultured
for over 48 hr were more tolerant to fatty acids
and showed a superior ability to metabolize fatty
acids to methyl ketones as compared to fresh
mycelia (Table 6). This may reflect an adaptation
by the mold, an increase in enzymes of the
/3-oxidation scheme, or changes in permeability of
the cell wall, making the aged mold less sensitive
to the toxic effects of fatty acids. As mycelium
ages its capacity to oxidize fatty acids is enhanced.
The presence of carbon dioxide apparently stimu-
lates production of methyl ketones by mycelia in
culture. 84
Mycelium can oxidize fatty acids over a wide
pH range, but is most efficient at pH 5 to 7.
Lawrence and Hawke84 showed that pH optima
changed significantly with concentration of fatty
acids. Thus, optima of pH 5 and 6 were observed
at concentrations of 0.3 and 8 mM of octanoic
acid per 10 mg mycelium, respectively. Dwivedi
and Kinsella19 reported similar trends, i.e.,
optimum pH for methyl ketone production
increased as concentration of fatty acids increased.
This is pertinent to the changes which occur in
cheese during ripening.
When pH is increased, the relative rate of indiv-
idual methyl ketone production is decreased84 (Fig-
ure 30). These data may be significant in
relation to the pattern of methyl ketones formed
in blue cheese during blue cheese ripening, i.e., at
lower pH values 2-propanone and 2-pentanone
accumulated but as the pH was increased, longer
chain 2-alkanones of longer chain length were
generated. As blue cheese ripens the pH slowly
increases from pH 5.5 to about pH 6.5. Thus,
relatively greater amounts of 2-propanone and
2-pentanone may initially appear, whereas with
progressive ripening, greater amounts of
2-heptanone, 2-nonanone and 2-undecanone
should be formed. However this pattern has not

214 Critical Reviews in Food Science and Nutrition

 SPORE GERMINATION STAGE

I 3.0
2k HR
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1 • >\ 6 8
INCUBATION TIME (HR)

FIGURE 29. Progressive reduction of 2-heptanone to 2-heptanol during incuba-

tion of resting and germinating spores of PeniciUium roquefortL (From Fan, Y. S.,
Hwang, D., and Kinsella, J. E., J. Agric. Food Chem., 24, 443, 1976. With
permission.)

been analyzed. According to Lawrence and
Hawke84 the optimum fatty acid concentration
was 1 jitmole/mg dry mycelium at pH 6.5. The
optimum substrate concentration varies with age
of mycelium, presence of lipids, molecular weight
of t h e f a t t y acid and pH of the
media. 1 8 ' 1 9 - 3 0 ' 3 1 ' 8 4
Exposure of washed mycelia to low levels of
fatty acids markedly stimulates their subsequent
capacity to generate methyl ketones.84 Mycelia of
P. roqueforti can adapt to higher concentrations of
fatty acids, as indicated by a lag phase in in vitro
cultures. The extent of lag phase increases with
relative concentration of fatty acids and age of
mycelia. 19 ' 84
The studies of Lawrence and Hawke84 showing
the marked inhibitory effects of fatty acids to
metabolic activity of P. roqueforti were done in an
aqueous system. However, the relevance to blue
cheese was questionable since the concentration of
free fatty acid in blue cheese may average 50 mM,
i.e., potentially very toxic levels. The presence of
bulk lipid material effectively reduces the concen-
tration of free fatty acids in the aqueous phase
which may reduce the toxicity of these fatty acids
to the mycelium.19 Dwivedi and Kinsella19
showed that, after a lag period, cultured mycelium
generated methyl ketones from very high levels of
fatty acids, although the overall yield was low.
High concentrations of fatty acids tend to
impede the complete oxidation of all fatty acids,
although methyl ketone production proceeds. This
would indicate that fatty acids may inhibit the
thiolase enzyme of ^-oxidation scheme, whereas
they do not affect the ketoacyl-CoA deacylase nor
the (3-ketoacid decarboxylase (Figure 16). Thus in
November 1976 215
 TABLE 6

The Effect of Free Fatty Acid Concentration on Methyl Ketone Production from Lipolyzed Milk Fat by Aged and Fresh
Mycelium of P. roqueforti

Methyl ketone concentration (jimol/mg mycelium)

Fatty acid level (Mmol/mg) Fatty acid level (Mmol/mg)

Time of aged mycelium fresh mycelium
incubation Methyl
(hr) ketone 13 26 39 52 13 26 39 52

12 cs 0.06 0.02 Tr Tr Tr Tr Tr Tr
c, 0.29 0.09 Tr Tr 0.11 0.07 Tr Tr
c, 0.51 0.09 Tr Tr 0.20 0.06 Tr Tr
c,, 0.08 Tr Tr Tr Tr Tr Tr Tr
18 c5 0.14 0.10 Tr Tr Tr Tr Tr Tr
C
7 0.31 0.41 0.16 0.11 0.15 0.60 0.17 Tr
c, 0.53 0.45 0.19 0.10 0.30 0.59 0.19 Tr
c,, 0.18 Tr Tr Tr Tr Tr Tr Tr
24 c5 0.17 0.24 0.14 0.11 0.05 0.21 Tr Tr
c7 0.30 0.54 0.59 0.45 0.21 0.68 0.67 Tr
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c, 0.51 0.84 0.97 0.43 0.46 0.96 0.60 Tr

ctl 0.20 0.10 Tr Tr 0.07 0.12 Tr Tr
48 cs 0.12 0.29 0.20 0.31 0.06 0.25 0.29 0.58
c, 0.22 0.55 1.14 1.22 0.23 0.64 1.06 1.38
c, 0.38 0.85 1.82 1.57 0.39 1.13 1.56 1.14
c,, 0.15 0.14 0.53 Tr 0.11 0.17 Tr Tr

Note: Tr = trace amount

Data from Dwivedi and Kinsella. 1 9

the presence of ample fatty acids methyl ketones
are the principal products. However, when the
concentration of free fatty acids to mycelium is
very low, complete oxidation to CO2 results.84
This latter behavior is very pertinent to blue
cheese ripening, i.e., strains of P. roqueforti with
poor lipase activity form much lower levels of
methyl ketones, conceivably because the low
concentration of fatty acids are completely
oxidized, whereas strains with very active lipase
generate proportionally more ketones because free
fatty acids are abundant. This idea is corroborated
by the studies of Sato et al. 12 7 on the lipolytic
capacity of different strains of P. roqueforti
(Figures 9 and 10) and by the increased produc-
tion of methyl ketones obtained following the
addition of lipase to milk for blue cheese manufac-
ture. 6 3 ' 7 3
At pH 5 to 7 mycelium shows a preference for
short and medium chain fatty acids (1 mM):
octanoic>hexanoic>decanoic>butanoic=
dodecanoic acid. At this concentration
2-heptanone, 2-pentanone, and 2-nonanone are the
principal ketones formed. Fatty acid specificity
varies with pH (Figure 30). Franzke and
Thurm3 ° ' 3 ' reported relative rates of oxidation of
100, 70, 50, and 20 for octanoic, hexanoic,
dodecanoic and decanoic acid, respectively.
Negligible oxidation of longer chain fatty acids
(C14 to C16) occurred at this concentration and
pH range.84 However, high concentrations of
mycelium are capable of oxidizing myristic acid.s 2
All of these studies unequivocally demonstrate
that both spores and mycelium are capable of
generating methyl ketones. Both function best
under a rather similar range of conditions (Table
7). However, it is quite possible that spore
metabolism is favored in ripening cheese,
particularly under the conditions prevailing in the
latter stages. Thus spores can continue to produce
methyl ketones in the presence of high fatty acid
concentrations and at relatively high carbon
dioxide levels. The carbon dioxide may actually
enhance ketone formation by inhibiting the
normal oxidation of acetyl-CoA via the citric acid
cycle, thereby causing a "back-up" of the fatty
acid oxidation pathway. Thus, the deacylase
generates /3-ketoacid from |3-ketoacyl-CoA, and the

216 Critical Reviews in Food Science and Nutrition

 C7

C5 C7
o
S3
§
Q_

fCM

C9

I
C3 C9
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C3 i Cll

PH 5.2 PH 6.8
FIGURE 30. The effect of pH on the relative capacity of mycelium of
Penicillium roqueforti to form methyl ketones from the corresponding fatty acids
C4 through C12 inclusive. (Adapted from data of Lawrence and Hawke. 84 )

TABLE 7 accelerating flavor development in blue cheese.

Thus, the addition of Upases have been widely
Summary of Apparent Optimum Conditions for advocated and some success has been reported. 50 '
Generation of Methyl Ketones from Fatty Acids by
Spores and Mycelium of P. roqueforti 6 3,64,73,116,117 However, in several instances
undesirable and bitter flavors are generated. This
Conditions Spores Mycelium may be traced to the use of crude enzymes;
conceivably, if more purified lipase preparations
Temperature 25-27°C 25° C were employed at lower levels a wholly beneficial
Age 2-3 days 50 hr
pH 6 (5-7) 6 (5-7)
result might be obtained. This approach needs
Oxygen 5% 4% further investigation.
Carbon dioxide 0.033 0.033 Krezlewicz et aL7S reported that the addition
Energy Glucose, sugar Glucose of certain amino aicds (particularly sodium
Nitrogen Alanine, proline Amino acids glutamate) to cheese curd accelerated ripening.
Fatty Acids 1-5 n * imM Flavor development can be accelerated by ripening
Preference Octanoic Octanoic
the cheese as an open granular curd rather than as
the normal hooped loaf. This approach was
CoASH becomes available to activate a new fatty studied in detail by Hedrick48 and Blakeley.8
acid and initiate another /3-oxidation cycle. This Instead of hooping the curd, Hedrick ripened the
redundant cycle then results in the accumulation particulate or open curd and reduced the ripening
of methyl ketones. period from 90 to 14 days. This expedited
uniform mold growth, salting, and flavor develop-
ment. Blakeley found that fatty acids increased
ACCELERATED RIPENING
rapidly in this loose curd cheese within 7 days and
during subsequent storage exceeded the levels
A number of reports describe methods for normally found in cheese. The concentration of
November 1976 217
 methyl ketones was lower than that found in TABLE 8
conventionally ripened blue cheese; in contrast to
normal cheese (where 2-heptanone is dominant) Classes of Flavor Compounds in Blue-Type Cheese
2-nonanone was the major ketone in this loose Volatile Nonvolatile
curd cheese. This cheese product may be useful as
a blue cheese flavorant in salad dressings and in Methyl ketones Organic acids
processed blue cheese spreads. However, its use as Fatty acids (short-chain) Fatty acids Gong-chain)
a blue cheese per se may be limited because 2-Alkanols Fatty acid soaps
Aldehydes Alcohols
excessive mold growth frequently occurs and this Amines Amino acids
results in the development of a musty, moldy Esters Secondary amines
flavor. Further research is needed concerning Sulfides Peptides
practical methods for accelerated ripening. Lactones

COMPOSITION O F BLUE CHEESE methyl ketones per 10 g, with a preponderance of

2-heptanone, 2-nonanone and 2-pentanone.
The proximate composition of commercial blue 2-Heptanone and 2-nonanone are usually the
cheese is 40 to 42, 30 to 33, 20 to 23 and 5 to 7% major component of the methyl ketones, although
moisture, lipids, nitrogenous materials and ash, blue cheese samples vary tremendously in
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respectively. The lipids are composed of 75 to
90%, triglycerides 5 to 8%, mono- and diglycerides
5 to 12%, free fatty acids and approximately 1%
polar lipids. The relative concentration of each of
the lipid classes depends on the extent of lipolysis
which, as discussed earlier, can vary greatly from
sample to sample.
Of the nitrogenous materials, 65 to 75% is
protein and the remainder is composed of 25 to
33% soluble compounds consisting of peptides,
free amino acids and ammonia. Ismail and
Hansens 7 reported that amino acids comprise 5 to
10% of the nitrogenous material in Danish blue
cheese, and Kosikowski and Dahlberg72 reported
that amino acids range from 2.8 to 9 g/100 g dry
blue cheese. All the common amino acids are
present. Schormuller12 8 reviewed the extensive
research on the accumulation of amino acids in
cheese and discussed the importance of their
relative concentrations and their degradation on
flavor and cheese quality.
Flavors in Blue-type Cheese
The classes of chemicals involved in the flavor
of mold-ripened cheeses (e.g., Blue, Roquefort,
Stilton, and Camembert) are listed in Table 8. The
homologous series of methyl ketones, particularly
2-heptanone and 2-nonanone are the predominant
flavor compounds responsible for the typical
flavor of these c h e e s e s . 3 ' 4 ' 1 3 ' 3 5 ' 1 0 0 ' 1 0 1 ' 1 1 3 ' 1 3 8
The total concentration of ketones varies
widely in different blue cheese samples, i.e., from
1 to 10 mg/10 g dry cheese (Table 4). A
good-quality blue cheese contains 3 to 5 mg of
c o m p o s i t i o n . 4 ' 9 2 ' 1 2 9 Anderson and Day4
reported that blue cheese samples lacking
2-heptanone and 2-nonanone possessed poor
flavor. Dartey and Kinsella13 found that
2-heptanone and 2-nonanone were the pre-
ponderant ketones at progressive stages of blue
cheese ripening. The two ketones increased in
concentration to a maximum at about the 70th
day of ripening and decreased slightly thereafter.
The level of free fatty acids ranges from 2 to 7
g/100 g cheese (Table 3). The more volatile fatty
acids (C2 through C10 inclusive) are the second
major flavor components imparting sharp notes to
blue cheese. 4 ' 1 '
Nelson9 9 alluded to the contribution of fatty
acids and their soaps to the authentic flavor of
blue cheese. In addition to their flavor/taste
impact, the soaps may also affect texture and
smoothness of the cheese.
Several branched-chain fatty acids have been
identified in blue and other mold-ripened
cheeses. 3 ' 100 Numerous a-ketoacids derived from
free amino acids presumably by deamination have
been identified in blue cheese, but their role in the
flavor of these cheeses has not been elucidated. 100
Since blue cheese contains over 30% milk fat
the homologous series of 4- and 5-hydroxy
alkanoic acids must occur in blue cheese; these
acids may generate several lactones via de-
hydration. 30 ' 71 Actually, both y and 5 lactones
have been isolated from the cheese.6 2 A maximum
concentration of 3 mg lactones per 100 g cheese is
possible with a fat content of 30%.
In addition to the methyl ketones and fatty

218 Critical Reviews in Food Science and Nutrition

 acids, several other classes of compounds which
are associated with and affect the flavor of blue
cheese have been identified. 3 ' 4 ' 42 ' 100 The
2-alkanols (Table 5) which are found in greater
quantities in aged cheese have flavor notes similar
to the corresponding ketones although they may
impart a musty, moldy flavor at higher levels.
Numerous primary alcohols have been identified,
including methanol, ethanol, 2-methylbutanol,
3-methylbutanol, 1-pentanol, and 2-phenyl-
ethanol. 3 ' 4 ' 1 0 0 These alcohols particularly the
methylbutanols, and their esters, may impart a
fruity, nutty flavor to cheese. They are probably
formed by reduction of aldehydes derived from
free amino acids by oxidative deamination (Table
9). Even at low dilutions, phenylethanol has an
odor reminiscent of yeast and may, like the other
alcohols, impart the necessary background flavors
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to blue cheese.
Oct-l-en-3-ol has been found in Camembert
cheese and is desirable in blue cheese flavor at
around 10 p p m . 3 ' 4 2 ' 1 0 2 ' 1 0 4 This compound is
probably formed by the oxidation of linoleic acid
which occurs abundantly in the mycelium and
spores of P. roqueforti.22'132 Kaminski et al. 65
reported that oct-l-en-3-ol and oct-l-en-2-ol were
produced in varying but significant amounts by
Penidllium species. At high concentrations, they
possess a musty, mushroomy flavor.
Small quantities of aldehydes including
alkanals, alkenals, and alkadienals occur in blue
cheese. l5 2 Ethanal, propional, 3-methylbutanal,
isobutanal, pentanal, hexanal, nonanal, 1-octenal,
a n d p h e n y l a c e t a l d e h y d e have been
reported. 3 ' 4 ' 100 The importance of these on the
flavor impact of blue cheese has not yet been
determined. These are probably formed by
deamination and oxidation of corresponding
amino acids (Figure 31) and presumably the mold
TABLE 9
Alcohols Formed from Free Amino Acids by Oxidative Decarboxylation, Deamination and Reduction
Amino acid
CH2-NH,-COOH Glycine
CH, - C H NH2 -COOH Alanine
(CH,), CH-CH NH 2 COOH Valine
( C H j ) . , - C H - C H , - C H NH,COOH Leucine
CH 3 CH, - C H - C H NH, COOH Isoleucine
I |1
CHj
CH6 H 5 CH, - C H NH, COOH Phenylalanine
possesses the enzymes involved in these reactions.
Numerous methyl, ethyl, isopropyl, and some
butyl esters of organic and alkanoic acids have
been described in cheese. 3 ' 4 ' 1 0 0 ' 1 0 2 These
possess fruity flavors and may participate to a
limited extent in blue cheese flavor by minimizing
the sharpness and bitterness caused by fatty acids
and amines, respectively.
Mold-ripened cheeses, e.g., blue and Roquefort,
contain significant quantities of organic acids such
as acetic, 30; lactic, 300; pyruvic, 15; malic, 30;
and isobutyric, 110 mg/100 g cheese, respectively.
Succinic, benzoic, hydroxybenzoic, p-
hydroxybenzoic, and p-hydroxyphenylacetic acids
have also been identified. 128 Their concentra-
tions, particularly lactic acid, may affect pH and
thereby affect the quantities of dissociated fatty
acids in the cheese which may have an impact on
flavor. In simulated blue cheese flavors, the in-
clusion of both lactic and/or succinic acid is
required to provide the needed background flavor
notes.
The involvement of free amino acids and
peptides in the overall flavor of blue cheese is
likely although it has not been experimentally
ascertained. The inclusion of methionine, lysine
and sodium glutamate in blue cheese flavors
imparts a more intensive blue cheese taste.' ° 3 >l 0 4
Depending on their composition, amino acids may
impart sweet or bitter aftertastes. Thus, glycine,
alanine, proline, and threonine possess a sweetish
taste. The aliphatic hydrophobic amino acids and
cystine are bitter, whereas glutamic acid salt has a
brothy taste. 103 Several hydrophobic peptides,
especially those with N-terminal leucine, are
bitter.5
Several amines have been identified in mold-
ripened cheese. Volatile amines (ranging from 0.64
to 1.3 mg/10 g cheese) composed of methyl,
Alcohol
CH 3 OH Methanol
CH 3 CH,OH Ethanol
( C H 3 ) , - C H - C H , OH 2-Methylpropanol
(CH 3 ), - C H - C H , - C H , OH 3-MethyIbutanol
CH,-CH,-CH-CH,OH 2-Methylbntanol
CH 3
C,H5-CH,CH,OH Phenylethanol
November 1976 219
 Crk-CH-CH-COOH -> CH3-CH-CH0
CH3 NH 2 CH3
VALINE 2-METHYLPROPANAL
CH3-CH-CH2-CH-COOH -CH 3 -CH-CH 2 -CH0
I
CH 3 NH 2 CH3
LEUCINE 3-METHYLBUTANAL
FIGURE 31. Production of aldehydes by deamination
and oxidation of amino acids.
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ethyl-, butyl-, isobutyl-, isopentyl-, dimethyl-,
diethyl-, dipropyl-, dibutyl-, and hexylamine have
been described in Penicillium-ripened
c h e e s e s . 1 0 0 ' 1 3 s Secondary amines, i.e.,
dimethylamine, occur in blue and Camembert
cheese at 360 and 180 n mol/10 g, re-
spectively.108 Nitrosamine was absent from blue
cheese but present in Camembert at 25 n mol/10 g
of cheese.
The concentration of nonvolatile amines in
mold-ripened cheese is quite variable and ranges
from 0.5 to 3.5 mg/10 g cheese. Blue cheese
samples contained 3.6, 23, and 11 mg of tyramine,
histamine, and tryptamine, respectively, per 10 g
cheese, and roquefort contained 6.5 mg of
tyramine per 10 g cheese. 3 ' 6 8 ' 1 4 6 However, much
higher concentrations of these amines have been
determined in blue cheese, e.g., up to 30 mg
tyramine per 10 g. 10
These volatile and nonvolatile amines are
probably derived by decarboxylation of free
amino acids (Figure 32) under conditions of low
oxygen tension where oxidative deamination is
retarded. Under similar conditions, secondary
alcohols may also accumulate. These amines are
probably important in the background flavor notes
of blue cheese; however, their actual contribution
has not been experimentally quantified.
FLAVOR CONCENTRATES
Presently there is a demand for a flavor
concentrate which could be used to impart a full
blue cheese quality to many food items e.g., salad
220 Critical Reviews in Food Science and Nutrition
dressings, processed cheese. Therefore, a number
of processes have been described for providing a
typical blue cheese flavor concentrate by
fermentation or by chemical formulation.
Fermentation Methods
Several researchers have described methods for
the production of blue cheese flavor by
f e r m e n t a t i o n . 7 3 ' 1 4 7 Knight73 patented a
procedure for producing blue cheese flavor by
incubating spores of Penicillium roqueforti with
lipolyzed fat. Fresh vegetative mycelium could
also be employed.3 s > 3 6 Litchfield88 reviewed the
production and potential applications of blue
cheese flavors by using submerged cultures of P.
roqueforti.
Nelson" has described in detail the com-
mercial method presently used for the production
of a blue cheese flavor concentrate by batch
fermentation as patented by Watt and Nelson.14 7
Basically, this involves the pressurized incubation
(under aseptic conditions) of spores and mycelium
of P. roqueforti with homogenized milk, lipolyzed
cream and 3 to 4% salt with aeration for 2 to 3
days. The mixture is then pasteurized at 130°C for
4 sec. Although it contains about tenfold the
concentration of ketones found in blue cheese, its
flavor efficacy is about fourfold that of blue
cheese as an ingredient in salad dressings, snacks,
and party dips.
More recently, Luksas89 has patented a similar
method for making a blue cheese flavor product
by aerobic culture of P. roqueforti in a medium
containing casein and milk fat. A strong blue
cheese flavor was produced by growing P.
roqueforti aerobically with agitation and aeration
for 48 hr in an aqueous medium containing 10%
sodium caseinate and 5% butterfat at 25°C.
Jolly and Kosikowski64 described a submerged
fermentation system consisting of 10 to 20 g of
whey powder and 10 to 15 g of cream dispersed in
80 g of water to which 15 mg of spores and 10 mg
of microbial lipase were added. This was incubated
with constant aeration for 2 to 3 days. The
product, as a freeze-dried powder, was a good
source of blue cheese flavor concentrate as it
contained approximately fivefold the methyl
ketone concentration of cheese. Coconut fat
served successfully as a lipid substrate. When
blended with milk proteins, it provided a
proteinaceous food material with a strong blue
cheese flavor.
 CH3-CH-CH2-CH-C00H
CH3 NH2
LEUCINE
C6H5-CH2-CH-C00H
I
PHENYLALANINE
H0C6H5-CH2-CH-C00H
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TYROSINE
FIGURE 32. Formation of amines by decarboxylation of free amino acids as
might occur in blue cheese. These reactions are favored under conditions of low
oxygen tension.
Dwivedi and Kinsella 18 ' 19 developed a
laboratory scale semicontinuous fermentation
system using mycelium and a continuous stream of
lipolyzed milk fat for the production of a blue
cheese flavor concentrate. The product contained
about fourfold the concentration of flavors found
in blue cheese.
Flavor Formulation
From their extensive analytical data Anderson
and Day4 attempted, with some success, to
formulate a synthetic blue cheese flavor. Using a
cottage cheese curd and cream or butterfat base
containing salt, they added the methyl ketones,
alcohols and acetic through myristic acids in
concentrations found in cheese (Table 10). The
resulting product had a very bitter soapy flavor.
By lowering fatty acids to approximately 50 to
60% of their normal concentrations and omitting
CIO, C12, and C14, the "soapiness" was avoided
and a reasonable facsimile was obtained. By
including phenylethanol, ethyl butyrate and
methyl esters of hexanoic and octanoic acid, a less
harsh, cleaner flavor was obtained. A pH range of
4.7 to 5.6 did not affect the perceived flavor. As
indicated in Table 10, the concentration of fatty
acids was lower than that found in cheese. This
->. CH3-CH-CH2-CH2-NH2
CH3
ISOBUTYLAMINE
C6H5-CH2-CH2-NH2
PHENYLETHYLAMINE
H0C5H5-CH2-CH2-NH2
TYRAMINE
may be explained by the knowledge that in blue
cheese many of the free fatty acids (particularly
the longer chain fatty acids which are omitted
from the synthetic flavor) are associated with the
fat; hence, their impact on flavor is mollified. The
concentration of methyl ketones and secondary
alcohols is higher than found in normal cheese,
although it is not in excess of the levels reported
for some cheese samples. The above formulation
lacks the authentic background flavor of ripened
blue cheese. This is probably because of
differences in texture and mouth-feel and the
absence of the flavor contribution by products of
proteolysis. Nevertheless, this mixture is effective
in salad dressings. The addition of some peptized
casein might impart a fuller flavor to this mixture.
Moines et al.9 x assembled a blue cheese flavor
formula containing 0.8% 2-heptanone, 18%
2-nonanone, 14% 2-heptanol, 0.2% phenol, 63%
propionic acid, 27-oct-l-en-3-ol and 2% methyl
cinnamate. When 200 ppm was added to a cheese
fondue, it acquired a smooth blue cheese-like
flavor.
Several proprietary imitation blue cheese flavors
are now commercially available. Many of these are
made of partially hydrolyzed lipased milk fat to
which varying amounts of methyl ketones are
November 1976 221
 TABLE 10

Formulation of Simulated Blue Cheese Flavot

Concentration (mg/kg)

Average Flavor Average Flavor

Compounds in cheese mixture Compounds in cheese mixture

Acetic acid 825 550 Acetone 3.0 6

Butyric acid 1500 950 2-Penatone 15 30
Hexanoic acid 900 600 2-Heptanone 35 70
Octanoic acid 770 515 2-Nonanone 33 65
Decanoic acid 2000 — 2-Undecanone 8 17
Dodecanoic acid 3000 _ 2-Pentanol 0.5 1.0
Others 33 X 103 _ 2-Heptanol 6.0 12.0
Phenylethanol TR 2.0 2-Nonanol 3.5 7.0
Ethyl butyrate TR 1.5 Methyl hexanoate - 6.0
Methyl octanoate — 6.0
a
Data from Anderson and Day.4
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TABLE 11

Formula for Blue Cheese Flavor

Compound mg/kg Compound mg/kg

2-Pentanone 8 Acetic acid 8
2-Heptanone 65 Butyric acid 6
2-Nonanone 116 Isobutyric acid 2
2-Undecanone 10 Pentanoic acid 3
2-Tridecanone 2 3-Methylbutanoic acid 3
Hexanoic acid 265
2-Pentanola 6 Heptanoic acid 5.4
2-Heptanola 1 Octanoic acid 89
2-Nonanola 10 Nonanoic acid 4.5
l-Octen-3-ol 22 Decanoic acid 133
Glyoxylic acida 71
Ethanala 5 Pyruvic acida 20
Propanala 2 2-Oxo-3-Mebutanoic acida 17
Butanala 2 2-Oxo-4-Mebutanoic acida 43
Pentanala 2 2-Oxo-Butanedioic acida 51
Phenylethanala 2 2-Oxo-Pentanedioic acida 42
Methional 0.1
Ethyl butanoatea 0.5
5-6-Decalactone 0.7 Ethyl hexanoatea 0.5
5-6-Dodecalactone 0.7 Ethyl octanoatea 0.5
Indole 0.1 Cinnamic acid 0.03
a
Denotes optional ingredients depending upon application. Me denotes methyl.
From data of Ney et al.1 0 4

added. The flavor quality of most of these
preparations could be improved by using improved
formulae.
Ney et al. 1 0 4 formulated an authentic blue
cheese flavor consisting of a more complex
mixture of compounds (Table 11). The addition of
l-octen-3-ol to the acids and ketones markedly
improved the overall impact and masked the
chemical notes imparted by the other components.
As indicated, several of the chemicals are optional
and these may be included for specific appli-
cations. The addition of monosodium glutamate,
lysine and methionine to attain a final con-
centration of 0.3 to 0.8% in the flavored food

222 Critical Reviews in Food Science and Nutrition

 intensified the blue cheese taste. At levels of 0.02
to 0.3% succinic acid further improved the final
flavor.
NUTRITIONAL VALUE OF
BLUE CHEESE
Limited information is available concerning the
nutritional value of blue cheese. Poznanski et
al. 1 2 0 reported that the net protein utilization
(NPU) values for Roquefort and Camembert
cheese fed to rats were 62 and 56, respectively,
compared to 72 for Edam cheese.
Abdel-Kader et al.2 reported toxic materials
associated with the nonsaponifiable lipid
compounds in mold-ripened cheese, specifically
Roquefort. These workers reported that rats
receiving casein based diets supplemented with
nonsaponifiable material from Roquefort cheese
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demonstrated protein efficiency ratio (PER) values
from 1.5 to 3.2. However, heart, liver, and
epithelium tissues of rats receiving the nonsa-
ponifiable matter revealed marked degenerative
and necrotic damage. The nature of the deleterious
factor(s) was not further determined. The report
of Abdel-Kader et al. needs to be rechecked
experimentally. The nonsaponifiable materials of
blue cheese contain methyl ketones, primary and
secondary alcohols, cholesterol, and unidentified
flourescent compounds.
The metabolism of the methyl ketones ingested
with blue cheese has not been specifically studied.
Forney and Markovetz26 have reviewed the
available information on methyl ketones in regard
to their biological importance and general
metabolism. Most of the existing information
concerns the metabolism of acetone which can be
converted to acetic, lactic, and pyruvic acid in
mammalian systems. Information on the
catabolism of long chain aliphatic methyl ketones
is very limited. Leibman86 reported that methyl
ketones are reduced to corresponding secondary
alcohols in rat liver. However, knowledge of their
subsequent metabolism is lacking.
Methyl ketones at concentrations above 80 [M
(about 18 /ig/ml) were significantly cytotoxic
when included in culture medium of HeLa
cells. 149 This effect may have been caused by
inhibition of the aldehyde reductase enzyme
system, leading to depletion of long-chain alcohols
which are required for the synthesis of alkoxy
glycerolipids. These lipids, especially the
phosphoglyceride components, are integral
components of mitochondria and microsomes of
cardiac muscles. Hence, it should prove interesting
to examine the effects of dietary methyl ketones
typical of those occurring in blue cheese on
cardiac lipids and cardiac function in experimental
animals.
Extracts of blue cheese and cultures of
Penicillium roqueforti contain organic solvent-
soluble flourescent compounds. Although it is
possible that species oi Penicillium may produce
aflatoxins, 150 Shih 1 3 1 failed to detect any
aflatoxins in samples of blue and Roquefort cheese
following specific analyses for these compounds.
Furthermore, although species of Penicillium can
produce the mycotoxin patulin, 1 2 3 ' 1 2 4 there are
no reports of P. roqueforti producing this
compound.
Cultures of P. roqueforti can produce alkaloids
isofumigaclavine and roquefortine and these
alkaloids were isolated from several samples of
blue cheese. 153 The toxicological significance of
these has not been determined.
The amines in cheese are of some significance
because of their biological activity, e.g., tyramine
is a pressor amine that may produce hypertensive
crises in patients treated with monoamine oxidase
inhibitors. 7 ' 4 9 ' 1 3 0 Amines from cheese may also
cause migraine in susceptible persons, i.e., those
with deficiencies of monamine oxidase. 10 ' 45
However, with these exceptions, it is generally
conceded that consumption of normal levels of
commercial blue-type cheeses will not cause any
discomfort or toxicity problems with regard to
these amines. 146
Rudman et al. 1 2 4 examined the ammonia
content of foods and reported that blue cheese
had the highest level, i.e., 4% of the total nitrogen
in the cheese. They reported that one serving of
blue cheese caused post-prandial hyperammonemia
in 32 cirrhotic patients with a history of
encephalopathy. Mammals can tolerate only small
concentrations of ammonia, and Visek 145 has
detailed the numerous potential deleterious and
toxic effects of excessive ammonia in animal cells.
Additional research is necessary to quantify the
average ammonia concentration in commercial
blue cheese, elucidate the factors which affect its
production during cheese ripening and determine
if it is physiologically significant to the consumers
of blue-type cheeses.
NEEDED RESEARCH
As cited in the foregoing text, several aspects of
the biochemistry of Penicillium roqueforti remain
November 1976 223
 to be described in order to improve the production
of blue cheese and flavor of blue cheese and to
determine their nutritional value and safety. The
unique ability of P. roqueforti to produce methyl
ketones during oxidation of fatty acids is of both
fundamental and applied interest. This phen-
omenon may be related to the selective perme-
ability of the mycelium wall and of the mito-
chondria to fatty acids. Thus, a practical method is
needed to isolate intact functional mitochondria
from growing mycelium. The use of digestive
enzymes for this purpose may prove successful.5!
Differences in permeability of the cell wall of
spores and mycelium may account for their
differential ability to metabolize fatty acids. 23
Thus, detailed knowledge of the ultrastructure of
the cell wall should be useful, particularly if it can
be related to biochemical events such as fatty acid
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The cellular location of methyl ketone
production and the mechanism of secretion of ke-
tones from the mycelium warrants study, as do the
properties of the enzymes involved in the
deamination and decarboxylation of free amino
acids. Data from research designed to elucidate the
biology and biochemistry of P. roqueforti can be
applied to improve the production and quality of
blue cheese and may be exploited in the design of
successful systems for the production of blue
cheese flavors.
ACKNOWLEDGMENT
The contributions of Dr. S. Fan and Jim Shimp
are acknowledged. Part of the research reported
herein was supported by a Grant-in-Aid from

absorption. Dairy Research, Inc.

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