biosynthesis of flavors by penicillium roqueforti

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 Penici l l ium 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; proteolysis, lipolysis, amino acid, and fatty acid metabolism occurs and flavor compounds are progressively generated. The environmental conditions, 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 proteolytic 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 medium 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.~-"J Salvadori* reported that proteolysis varied 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 OF FLAVORS 929

**, F-I,,'

1 . , , , , , , . . , ' 4 a 12 16 20

RIPENIN6 PERIOD (WEEKS)

Fig. 1. strains of P. Toqueforti. activity. activity, (-) protease activity.

Differences in rate of protein and lipid hydrolysis in blue cheese by 2 Strain F-1 with high lipase activity has low protease

Note that proteolysis (BP-13) plateaus after 9 weeks.6 (- - - ) Lipase

The properties of both extracellular and intracellular proteases of P . roqueforti have been reported by Niki et a1.,5 Modler et a1.,lo and Gripon and Berge~-e.~ Sodium chloride and free fatty acids, at concentrations 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 de~elop. l~- '~


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 7540% 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 hydrolysis as indicated (Fig. 2). However, homogenization is a critical process in facilitating subsequent hydrolysisI6 which is predominantly

- 0 16 24 32 40

RIPUIIL(LIEMS)

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


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 containing Casitone-proflo combination broth. ThibodeauZ0 and Morris et a1.21 obtained 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. roquejorti was inhibited by lactose, glucose, or galactose but was increased by the addition of butter fat.22 This was in contrast to other results


,- X. F-1 ,' ' -. / , . ,

I

I -. ' # .

I *.$' I BP-$x - -- I I I

** -- - 0-

, I I

I I

I

100 p

c i

"

W

K u.-

w \ -10 - w

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 medium 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-heptanone is usually the most abundant followed by 2-nonanone, 2-pentanone, 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) -


/4 -OXIDATION

T H * o L A S E ~ A ~ ~ ~ - C ~ A + ACETYL-COA Ic

ETO ACYL-COAI + \ / THIOHYDROLASE ' . - - '

6 I B-KETO ACID]

I- \hME/ DECARBOXY LASE E

Fig. 4. General outline showing the metabolism of fatt.y acids by P. rogueforti The acyl group may contain from 4 to 16 carbons. as it occurs in blue cheese.

KC 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 2-Pentanone 2-Hep tanone ZNonanone 2-Undecanone 2-Tridecanone

Total

A* 65

360 800 560 128

-

1940

B' 54

140 380 440 120

1146

Ca 75

410 380

1760 590

-

-

4296

Db 210

1022 1827 1816 136 100

5111

-

367 755 600 135 120

1978

Fb Go Hc - 60 T 51 372 285

243 3845 3354 176 3737 3505 56 1304 1383 77 309 845

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


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 corresponding 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 to carbon dioxide and methyl ketones, thereby preventing any accumulation of @-ketoacids. This capacity is enhanced 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 substrate 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 resting spores but had no effect on @-ketodecarboxylase activity in mycelium (Fig. 5). The enhanced methyl ketone production in germinating spores is attributed to the progressive increase in activity of @-ketoacyl decarboxylase 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 germinating 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 OF 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 (- - -) Glucose spores, and mycelium in P. ropuejorti (substrate 8-ketolaurate).

added; (-) no glucose.

Of various 8-ketoacid substrates, 8-ketolaurate was the most preferred substrate for mold de~arboxylase.~~

Further basic studies are warranted to explain the unique behavior of the @-oxidation enzyme 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 Age PH Oxygen Carbon dioxide Energy Nitrogen Fatty acids Preference

25-27°C 2-3 days 6 (5-7) > 5% > .033

glucose, sugar alanine, proline

1-.5 mM Octanoic

25°C .50 hr culture

6 (5-7) > 4% > .033

glucose amino acids

1 mM Octanoic


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 . ~ ~ - ~ l This 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

Knight42 patented 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 d ~ ~ reviewed the production and potential applications of blue cheese flavors by using submerged cultures of P. roqueforti. Nelson46 has 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 concentration of ketones found in blue cheese, its flavor efficacy is about four-


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 ~atented.~’ 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 k i ~ ~ described 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 a ~ ~ developed 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 concentrate. 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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7. J. Proks, J. Dolezalek, and Z. Pech, 14th Znt. Dairy Congr., 2, 401 (1956). 8. P. Salvadori, B. Bianchi, and V. Cavalli, Lait, 44, 129 (1965); Dairy Sci.

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39. H. W. Jackson and R. V. Hussong, J. Dairy Sci., 41,920 (1958). 40. D. F. Anderson and E. A. Day, J . Agr. Food Chem., 14, (3), 241 (1966). 41. C. Franzke and V. Thurm, Die Nahrung, 14. 279 (1970). 42. S. Knight, U.S. Patent No. 3,100,153 (1963). 43. J. C. Watt and J. H. Nelson, U.S. Patent No. 3,072,488 (1963). 44. R. F. Gehrig and S. G. Knight, Nature, 182, 1237 (1958). 45. J. H. Litchfield, Dewelop. Znd. Microbiol., 11, 341 (1969). 46. J. H. Nelson, J . Agr. Food Chem., 18, 567 (1970). 47. A. J. Luksas, U.S. Patent No. 3,720,520 (1973). 48. R. C. Jolly and F. V. Kosikowski, J . Food Sci., 40, 285 (1974). 49. B. K. Dwivedi and J. E. Kinsella, J . Food Sci., 39, 83 (1974). 50. D. P. Schwartz and 0. W. Parks, J. Dairy Sci., 46, 989 (1963).

(1970).

(1942).

press) (1976).

(1962).

Accepted for Publication March 13, 1976

biosynthesis of flavors by penicillium roqueforti

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