BIOTECHNOLOGY AND BIOENGINEERING,

VOL. XVIII, PAGES 927-938 (1976)

Biosynthesis of Flavors by Penicillium roqueforti

JOHN E. KINSELLA and DAN HWANG, Department of Food

Science, Cornell University, Ithaca, N e w York 1485s

Summary
The development of the unique flavor of blue type cheese depends on the
concerted action of numerous enzymes of Penicillium roqueforti involved in
protein and lipid metabolism. Protease(s) by degrading casein modify the
texture and background flavor of the ripening cheese. Lipase by hydrolyzing
milk triglycerides provides flavorful fatty acids and precursors of methyl ketones.
The enzyme complex involved in the partial oxidation of free fatty acids and the
properties of 8-ketoacyl decarboxylase which generates the major flavor com-
ponents of blue cheese are discussed. Fermentation of P . roqueforti for the rapid
production of methyl ketones is briefly reviewed.

INTRODUCTION
Because of increasing consumption, the growing use of blue type
cheese for flavoring, and the need for improving the efficiency of
production, it is expedient to elucidate in detail the biochemistry of
flavor development in cheese made with Penicillium roqueforti.
Conventionally, blue cheeses are ripened for approximately 90 days
a t 10°C with a relative humidity of >go%. The mold grows per-
vasively through the cheese to give the typical bluish-green mottling.
During the ripening period the mold grows and sporulates; proteoly-
sis, lipolysis, amino acid, and fatty acid metabolism occurs and flavor
compounds are progressively generated. The environmental condi-
tions, i.e., humidity, sodium chloride concentration, temperature,
relative partial pressures of oxygen, and carbon dioxide, that have
been arrived at empirically, favor the enzymatic actions required to
produce the desired flavor and texture in blue cheese.

BIOCHEMICAL CHANGES
The biochemical events occurring during cheese ripening are dy-
namic, complex, and intimately interrelated. The changes are mostly
attributable to the actions of different enzymes acting in concert.
927
@ 1976 by John Wiley & Sons, Inc.
928 KINSELLA AND HWANG

For descriptive purposes, these may be divided into enzymes in

milk, those added, and those in the mold, P. roqueforti. The lipase
associated with the milk lipids may cause some lipolysis initially,
especially after homogenization.' The starter culture organisms
(Streptococcus lactis) degrade the lactose to produce mostly lactic
acid, some citric acid, and perhaps some ethanol. The lactic acid
regulates pH of the ripening cheese, the citric acid provides carbon
for the mold, and ethanol may be involved in flavor generation.
The rennin reputedly causes some proteolysis and its action may
persevere throughout the ripening. However, it is generally con-
ceded that all the important changes occurring during ripening are
almost exclusively caused by enzymes in P. roquejorti.
A controversy has existed as to whether the spores or mycelium
are the more important sources of the enzymes involved in cheese
ripening. Present evidence indicates that both mycelium and spores
possess enzymes that are important in cheese ripening.

PROTEOLYSIS
Extensive proteolysis occurs during maturation of blue cheese
(20-35% breakdown of protein). Some of this may be caused by
residual protease activity from starter organisms and from the pro-
teolytic action of rennin, but the proteolytic capacity of P. roqueforti
predominates and exceeds that of other sources.
Proteolysis by P. roqueforti is essential for the development of a
soft, smooth, fully flavored cheese. If insufficient proteolysis has
occurred, a tough, dry, crumbly cheese ensues, whereas excessive
hydrolysis results in a soft cheese with a slightly bitter aftertaste.
Proteolysis is important for the development of proper texture,
background flavor (peptides/amino acids), and for providing amino
acids which act as germination stimulants: Amino acids also enhance
methyl ketone production.2 In addition, free amino acids also buf-
fer the cheese around pH 6.5.
Maximum protease activity in mycelium and in extracellular me-
dium occurs when the mycelium has attained full g r ~ w t h . Thus,
~
proteolysis probably occurs more rapidly during the first few weeks
of cheese ripening.
Several researchers have exanlined the proteolytic capacity of P.
roqueforti and invariably have reported on the tremendous disparity
among different ~trains.~-"JSalvadori*reported that proteolysis va-
ried fivefold among 19 different strains of P. roqueforti. Niki et a1.5
showed that strains of P. roqueforti with high proteolytic activity
possessed low lipolytic activity (Fig. 1).
 BIOSYNTHESIS O F FLAVORS 929

**,
F-I,,'

1. , , , , , , .. ,
' 4 a 12 16 20
RIPENIN6 PERIOD
(WEEKS)

Fig. 1. Differences in rate of protein and lipid hydrolysis in blue cheese by 2

strains of P. Toqueforti. Strain F-1 with high lipase activity has low protease
activity. Note that proteolysis (BP-13) plateaus after 9 weeks.6 (- - - ) Lipase
activity, (-) protease activity.

The properties of both extracellular and intracellular proteases of

P . roqueforti have been reported by Niki et a1.,5Modler et a1.,loand
Gripon and Berge~-e.~Sodium chloride and free fatty acids, at con-
centrations possible for ripening cheese, depress protease activity and
may function to prevent excessive proteolysis and bitterness in blue
cheese.
LIPID METABOLISM
The manufacture of a quality blue cheese critically depends upon
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 p-ketoacids in the original milk
fat."J2
Because of the close positive correlation between free fatty acid
levels and methyl ketone formation, lipase performs a key role by
providing the free fatty acids for flavor formation. The fatty acids
are flavorful and are also precursors of the methyl ketones (Fig. 4).
In the absence of adequate lipolysis cheese, flavor is poor and very
slow to d e ~ e l o p . l ~ - ' ~
930 KINSELLA AND HWANG

During ripening, triglycerides (TG) are progressively hydrolyzed to

monoglycerides and free fatty acids. Thus, in cheese samples, the
TG decreases from 96-98% of the lipids (about 35% of cheese) in
early stages to 7 5 4 0 % of the lipids (32% of cheese) at maturity.
The extent of the breakdown is governed by lipase activity which in
turn is influenced by: the growth of the P. roqueforti strain used,
the duration of ripening, the amount of residual lipolytic activity
from the milk and microorganisms of the starter culture, the efficiency
of homogenization of the milk, the number and type of surface
organisms, pH, temperature, and sodium chloride concentration in
the cheese.
The lipase(s) native to milk causes a significant amount of hy-
drolysis as indicated (Fig. 2). However, homogenization is a critical
process in facilitating subsequent hydrolysisI6which is predominantly

0 16
- 24
RIPUIIL(LIEMS)
32 40

Fig. 2. 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 effect of homogenization (A vs.
C) on the rate and extent of lipolysis and the effect of the lipase inherent in milk
on fatty acid levels (A vs. B).14
 BIOSYNTHESIS O F FLAVORS 931

caused by lipase in P. r0quej0rti.l~ The triglycerides in the non-

homogenized raw milk are not as accessible to the lipase indicating
that the intact membrane surrounding these lipids must be ruptured
to permit the enzyme to cause hydrolysis. Homogenization also
markedly expands the total surface area of milk fat available for
lipase attack.
During the ripening of blue cheese there is a progressive build-up
in free fatty acids (Fig. 2), the composition of which reflects those of
milk fat. The release of these fatty acids is caused mainly by lipase
in P. roquejorti. Stadhouders et al.13 reported that the bacteria
which survive in the cheese have little lipolytic activity.
Lipolytic activity of various strains of the Penicillium species
varies markedly. 5 , 1 7 ~ 1 8 Stepaniak et a1.I8 grouped mold strains into
four categories possessing relative 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. roquejorti, e.g., 100:50:27:37 and 100: 120:49:50 for
butyric, caproic, caprylic, and capric acids released by strains with
high and low lipase activity, respectively. Cheese samples made
with P. roquejorti strains of high and low lipase activity contained
147.5 and 27.2 mg of fatty acids (C2-C10 inclusive), respectively.
The organoleptic quality of the cheese made with low lipase strains
of P. roquejorti was inferior. Lamberet and Lenoir17 divided 89
strains of P. caseicolum into two groups one with high and the other
with low lipolytic activity. The former were better for practical
cheese manufacture. Niki et al. compared the relative lipolytic
activity of various strains of P. roquejorti. The disparate activities
of two strains are summarized in (Fig. 3). I t is important to note
the correlation of lipase activity with the development of methyl
ketones.
LIPASE
These data show that lipase is important in developing the flavor
of blue cheese. Consequently numerous researchers have examined
the lipase(s) associated with P. roquejorti. Eitenmillerlg showed that
high lipase activity was obtained in mold grown in a medium con-
taining Casitone-proflo combination broth. ThibodeauZ0and Morris
et a1.21obtained similar results indicating that nitrate is an inadequate
nitrogen source for enzyme production by P. roquejorti and that a
source of amino acids is necessary. Lipase production by P. roque-
jorti was inhibited by lactose, glucose, or galactose but was increased
by the addition of butter fat.22 This was in contrast to other results
932 KINSELLA AND HWANG

,-
X.
,' -.. F-1
,/
,'
100 p
I
#
I
.
-. *.$'
'
I

BP-$x - --
"
c
-- -
I
I
I **
i
I W
0-
uK
.-
, I -w
-1
\0
w
II
I
I
50 s3
s

2 4 6 8 10 12 14 16 18 20
RIPENING PERIOD
(WEEK)

Fig. 3. Comparison of the relationship between the lipolytic activity and

methyl ketone formation in cheese samples made with 2 different strains of P .
roqueforti.6 (- - - -) Methyl ketones, (-) volatile free fatty acid.

showing that the addition of 1% (v/v) butter oil to the growth me-
dium decreased lipase produ~tion.1~
Penicillium roqueforti possesses an intracellular and a secretable or
extracellular lipase and the former demonstrates greater activity. 5 ~ 1 4 * 2 2
The lipase is heat labilelg and shows an optimum temperature of
32°C. 21.23

FATTY ACID METABOLISM

During ripening there is a gradual accumulation of methyl ketones
in blue cheese (Table I). The rate of release of fatty acids may limit
the production of methyl ketones because the addition of lipase or
increased levels of mold lipase enhances free fatty acid release and
methyl ketone formation in ~ h e e s e . ~ ~ - ~ ~
Of the various methyl ketones formed during ripening, 2-hepta-
none is usually the most abundant followed by 2-nonanone, 2-penta-
none, and 2-unde~anone.~'The relative concentration of these vary
with cheese samples (Table I).
These methyl ketones are derived by the partial oxidation of fatty
acids released by lip%= (Fig. 4)-
 BIOSYNTHESIS O F FLAVORS 933

Ic
/4 -OXIDATION

ETO ACYL-COAI + T H * o L A S E ~ A ~ ~ ~ - +C ~ACETYL-COA

A

6
/ THIOHYDROLASE
\
'.- - '

I B-KETO
ACID]

-I
\hME/ DECARBOXY
LASE E

Fig. 4. General outline showing the metabolism of fatt.y acids by P. rogueforti

as it occurs in blue cheese. The acyl group may contain from 4 to 16 carbons.
K C denotes the Krebs or citric acid cycle.

TABLE I
2-Alkanone Content of Blue Cheese Samples

BAlkanone pg/lO g Dry blue cheese

2-Propanone
A*
-
65
B'
54
Ca
-
75
Db
-
210
-
Fb
-
-
Go
60
-
Hc
T
2-Pentanone 360 140 410 1022 367 51 372 285
2-Hep tanone 800 380 380 1827 755 243 3845 3354
ZNonanone 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

8 A,B,C Samples: commercial samples of ripe blue cheese.40

D,E,F, samples of blue cheese ripened for 2, 3, and 4 months, respectively.60
c G,H, samples of very small batches of experimental blue cheese ripened for

2 and 3 m0nths.n

FORMATION OF 2-ALKANONES
The mechanism formation of methyl ketones from fatty acids by
molds has received much a t t e n t i ~ n . ~ Accumulated
~-~~ information
indicates that methyl ketone production from fatty acids by the
mold proceeds via the classical fatty acid P-oxidation pathway.
Gehrig and Knight,30 although unable to isolate a reconstructed
fatty acid oxidizing system, concluded that methyl ketone formation
934 KINSELLA AND HWANG

from fatty acids by the spores of P. roqueforti involved the poxidation

reaction. Lawrence31 demonstrated that 2-heptonone formed from
l-C14-octanoate was radioactive and the specific activity of the
2-heptanone was almost identical to that of 2-C14-octanoate. Methyl
ketones are produced by decarboxylation of the corresponding P-keto-
a ~ i d s . ~Apparently,
~.~~ during the conventional p-oxidation cycle in
the spores and in the mycelium of P. roqueforti, the 0-ketoacyl-CoA
formed by the dehydrogenation of the P-hydroxy acyl-CoA is de-
acylated to P-ketoacid and CoASH by a 0-ketoacyl-CoA deacylase
or thiohydrolase and the p-ketoacids are rapidly decarboxylated to
methyl ketones. Thus, these molds convert fatty acids to corre-
sponding 2-alkanones with one less carbon atom (Fig. 4). Obviously,
in these molds the 0-ketoacyl-CoA deacylase activity greatly exceeds
that of thiolase and apparently the P-ketoacid decarboxylase causes
rapid decarboxylation t o carbon dioxide and methyl ketones, thereby
preventing any accumulation of @-ketoacids. This capacity is en-
hanced when the molds have more readily oxidizable substrates sug-
gesting that any “slowdown’l in the normal poxidation pathway,
especially of the thiolase step, aids the deacylation decarboxylation
step.
Studies indicate that 0-keto-octanoyl-CoA is the preferred sub-
strate for the deacylation reaction. However, methyl ketones of
varying chain length may be formed from long chain fatty acids at
successive cycles of P - o x i d a t i ~ n . ~ ~ , ~ ~
0-Decarboxylase is present in several molds3s and Hwang and
Kinsella3 demonstrated that resting spores, germinated spores, and
mycelium in P. roqueforti actively decarboxylate P-ketolaurate to
2-undecanone. The rate of 2-undecanone formation increased as
resting spores germinate and activity was highest in mycelium
(Fig. 5 ) . Glucose stimulated the formation of 2-undecanone by rest-
ing spores but had no effect on @-ketodecarboxylaseactivity in my-
celium (Fig. 5). The enhanced methyl ketone production in germi-
nating spores is attributed to the progressive increase in activity of
@-ketoacyldecarboxylase as spores germinated.
The enzyme was isolated from the cell-free extract and it was
shown to consist of heat stable and heat labile species. The optimum
pH for the enzyme was 6.5-7.0 which corresponds to the optimum
pH observed for methyl ketone production from lauric acid, by ger-
minating spores in the P. r o q ~ e f o r t i . ~Biphasic
~ substrate saturation
curves and a discontinuous slope in the Arrhenius plot for P-ketoacyl
decarboxylase suggested the existence of two species of the enzyme.
 BIOSYNTHESIS O F FLAVORS 93.5

HYCELl UM

GERMINATED SPORE

RESTING SPORE

2 4- 6 a
INCUBITION ( H I

Fig. 5. Relative activity of P-ketoacyl decarboxylase in spores, germinating

spores, and mycelium in P. ropuejorti (substrate 8-ketolaurate). (- - -) Glucose
added; (-) no glucose.

Of various 8-ketoacid substrates, 8-ketolaurate was the most pre-

ferred substrate for mold de~arboxylase.~~
Further basic studies are warranted to explain the unique behavior
of the @-oxidationenzyme sequence in P . roqueforti since this may
be further used in developing a continuous enzymatic system for
methyl ketone production.
TABLE I1
Summary of Apparent Optimum Conditions for Generation of Methyl Ketones
from Fatty Acids by Spores and Mycelium in P . roquejorti

Conditions Spores Mycelium

Temperature 25-27°C 25°C

Age 2-3 days .50 hr culture
PH 6 (5-7) 6 (5-7)
Oxygen > 5% > 4%
Carbon dioxide > .033 > .033
Energy glucose, sugar glucose
Nitrogen alanine, proline amino acids
Fatty acids 1-.5 m M 1 mM
Preference Octanoic Octanoic
936 KINSELLA AND HWANG

All of these studies have unequivocally demonstrated that both

spores and mycelium are capable of generating methyl ketones.
Both function best under a rather similar range of environmental
conditions (Table 11). It is quite possible however that in ripening
cheese, particularly under conditions prevailing in latter stages, that
spore metabolism is favored. 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
0-ketoacid from 0-ketoacyl-CoA and the CoASH becomes available
to activate a new fatty acid and initiate another &oxidation cycle.
This redundant cycle then results in the accumulation of methyl
ketones.
The 2-alkanones are easily reduced to the corresponding 2-alkanols
by both spores and mycelium in the P. r o q ~ e f o r t i . ~ ~ This
- ~ l reaction
occurs 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 di-nucleotides (NAD, NADP)
under the reducing conditions prevailing in cheese.

FERMENTATION METHODS
Because of the demand for flavor concentrates to impart a full
blue cheese quality to many food items, e.g., salad dressings, pro-
cessed cheese, a number of processes have been described for providing
a typical blue cheese flavor concentrate by fermentati0n.~~.~~.~4,49
Knight42patented a procedure for producing blue cheese flavor by
incubating spores in P. roqueforti with lipolyzed fat. Fresh vegeta-
tive mycelium could also be employed: L i t ~ h f i e l dreviewed
~~ the
production and potential applications of blue cheese flavors by using
submerged cultures of P. roqueforti. Nelson46has described in detail
the commercial method presently used for the production of a blue
cheese flavor concentrate by batch fermentation as patented by
Watts and Nelson.43 Basically, this involves the pressurized incu-
bation, under aseptic conditions of spores (and mycelium) in P.
roqueforti with homogenized milk, lipolyzed cream, and salt (3-495)
with aeration for two to three days. The mixture is then pasteurized
(130°C for 4 sec). Though it contains about tenfold the concentra-
tion of ketones found in blue cheese, its flavor efficacy is about four-
 BIOSYNTHESIS OF FLAVORS 937

fold that of blue cheese as an ingredient in salad dressings, snacks,

and party dips.
More recently, a similar method for making a blue cheese flavor
product by aerobic culture of P . roqueforti in a medium containing
casein and milk fat was ~ a t e n t e d . ~ ’A strong blue cheese flavor was
obtained by growing P . roqueforti aerobically with agitation and
aeration in an aqueous medium containing 10% sodium caseinate
and 5y0 butterfat a t 25°C for 48 hr.
Jolly and K o s i k o ~ s kdescribed
i~~ a submerged fermentation system
consisting of whey powder and cream dispersed in water to which
spores and microbial lipase was added. After two to three days the
product was a good source of blue cheese flavor concentrate since it
contained approximately fivefold the methyl ketone concentration of
cheese.
Dwivedi and K i n ~ e l l adeveloped
~~ a laboratory scale, semicontinu-
ous fermentation system using mycelium and a continuous stream of
lipolyzed milk fat for the production of a blue cheese flavor concen-
trate. The product contained about fourfold the concentration of
flavors found in blue cheese. Further research on this system is
warranted.
Portions of the work described herein were supported by a grant from Dairy
Research Inc. to one of the authors (J.E.K.).

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938 KINSELLA AND HWANG

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Accepted for Publication March 13, 1976