Methyl Ketone Formation by Penicillium camemberti in Model Systems

Methyl Ketone Formation by Penicillium camemberti in Model Systems

Methyl Ketone Formation by Penici//ium camembert/in Model Systems J, O K U M U R A and J. E. K I N S E L L A Institute of Food Science Cornell Univers...

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Methyl Ketone Formation by Penici//ium camembert/in Model Systems J, O K U M U R A and J. E. K I N S E L L A Institute of Food Science Cornell University Ithaca, NY 14853 ABSTRACT

heptanone is usually the most prevalent ketone (10, 11, 12, 22). Because of the burgeoning developments in ultrafiltration, its practical potential for making soft cheeses, and the keen interest in accelerating the development of ripened cheese flavor, more basic information concerning the biochemistry of cheese flavor formation is needed (11, 12). In this work, using a simple system, we studied the rate of synthesis of carbonyls and methyl ketones by Penicillium camemberti from milk lipids, sodium oleate, and sodium laurate.

Penicilliurn camernberti incubated in a

model system containing milk lipids (45 raM) caused hydrolysis of the lipids with subsequent oxidation of the free fatty acids to carbonyl compounds of which approximately 60% was composed of methyl ketones, mainly of 2-nonanone and 2-undecanone. The addition of lipase greatly enhanced carbonyl production by the mycelium. Incubation in the presence of sodium laurate (45 mM) reduced mycelium growth, but extensive synthesis of undecanone occurred. The myceIium was more tolerant to exogenous oleic acid and produced mostly heptanone and :aonanone.

MATERIALS AND METHODS

Lyophilized spores of Penicillium camemberti (Thorn 4845) obtained from American Type Culture Collection were hydrated and cultured on potato dextrose agar slants. The resultant mycelium was inoculated aseptically into sterile Czapek solution containing all the necessary nutrients (19) and incubated at 27°C for 48 h with shaking (130 rpm) in an Orbit shaker (Labline Instruments, 1L). The mycelium was harvested aseptically by filtration with sterile cheesecloth and then used for the experimental incubation systems. To determine the capacity of the mycelium to generate met,hyl ketones, in a series of experiments P. camemberti was incubated with milk fat, sodium oleate, and sodium laurate. Homogenized milk (500 ml) was diluted with 1 liter of phosphate buffer (.15 M) containing 15 ml of Czapek's solution and 45 g sucrose and sterilized at 85°C for 30 mix. The concentration of milk lipids was 45 mM, and the pH was 6.8. For incubation, 53 mg of mycelium was inoculated into 50 ml of the diluted milk in an Erlenmeyer (125 ml) flask, plugged with a cotton plug, and incubated at 27°C in the shaker for specified periods. To determine if free fatty acid concentrations affected methyl ketone production, lipase (.5 ml of a 1%

INTRODUCTION

The important classes of flavor compounds in Camembert cheese, i.e., methyl ketones, fatty acids, alkanols, esters derived from milk tipids, and amines, sulfur compounds, short chain aldehydes, and amino acids derived by hydrolysis of proteins, are formed via enzymatic activities associated with the mold Penicillium camemberti (4, 5, 8, 10, 13, 15, 16, 17, 18, 20, 21, 22). However, factors affecting amounts and types of compounds formed, i.e., the final flavor profile, have not been determined. Methyl ketones are major flavor components and 2-pentanone, 2-heptanone, 2-nonanone, and 2-undecanone, which may amount to .6, 3.0, 6.7, and 1.2/1moles/100 g cheese, are the major homologues (5, 8, 17). Significant disparities in total and relative concentrations of the alkanones have been reported. Nonanone occurs most abundantly in Camembert, which in contrast to Roquefort cheese where 2-

Received March 2, 1984. 1985 J Dairy Sci 68:11--15

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OKUMURA AND KINSELLA

aqueous solution of Steapsin, Nutritional Biochem Corp.) was added to the diluted milk; then the mycelium was added and the mixture incubated as has been described. To assess the capacity of mycetium to synthesize methyl ketones from free fatty acids as compared to milk lipids, mycelium (53 mg) was incubated at 27°C in Czapeks medium (50 ml) made in phosphate buffer, pH 6.8, containing dissolved sodium laurate or oleate (~45 mM). Analyses

Following incubation, flasks were cooled (8°C) and contents quickly filtered to recover the mycelium, which then was washed, dried, and weighed. The cooled filtrates were diluted to 100 ml with distilled water. Samples (10 ml) in triplicate were reacted immediately with 20 ml of dinitrophenylhydrazine (DNPH) reagent, shaken vigorously for 30 min, and after 3 h the DNPH-derived carbonyls were extracted thrice into hexane (1, 22). Total carbonyls were quantified spectrophotometrically at 340 nm with a molar extinction coefficient of 22,500 (1). Free fatty acids in the filtrate were determined by the method of Shipe et al. (24). Individual methyl ketones were quantified by high pressure liquid chromatography (HPLC). A standard Waters Associates (Model 45)/ (HPLC) system with a Model 450 variable wavelength detector was used. Reverse-phase chromatography used Licbrosorb RP-18, 10/am packed in a column (250 mm × 4 mm). The methyl ketone-DNPH derivatives were extracted from the hexane into 20 ml acetonitrile. This was concentrated, and samples were injected into the HPLC, which had been calibrated by standard mixtures of methyl ketone DNPH derivatives. The methyl ketones were eIuted sequentially by a gradient solvent system of acetonitrile and water. Of the initial concentration of acetonitrile, 75% was increased linearly to 95% during the initial 15 min of the elution. Flow rate was 2 ml/min, and the detector was set at 360 nm. The detector response was calibrated against known concentrations of standard DNPH derivatives. Recoveries also were determined by subjecting standard mixtures of C5, C7, C9, C l l methyl ketones to the same extraction, derivatization, and analytical procedures. Journal of Dairy Science Vol. 68, No. t, 1985

RESULTS AND DISCUSSION

The state of lipids in the culture medium had a marked effect on growth of mycelium (Figure 1). Growth was rapid in the milk system ; however, addition of lipase and culturing with unesterified fatty acids significantly impaired growth. Laurie acid was particularly inhibitory. These data are consistent with data showing that the growth of mycelium of P. roqueforti was suppressed by free fatty acids ( F F A ) (6, 7). Analysis of the milk system containing steapsin revealed a dramatic increase of F F A (Table 1). There was a slight increase of F F A in the control milk system up to 48 h and then a doubling in F F A by 80 h. This was consistent with knowledge that Penicillium sp. contain and secrete lipase(s) into the culture medium, particularly those containing milk proteins and, of course, during cheese ripening F F A concentrations increase (11, 12, 14). The high concentrations of F F A in the culture system containing steapsin impaired growth of mycelium, although after 50 h mycelium growth increased, and there was a concomitant drop of F F A concentration. This may have reflected

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TABLE l. Changes in free fatty acid concentration in milk system during incubation with Penicillium camemberti in absence and presence of lipase. Free fatty acid Time of !incubation

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adaptation of the mycelium to F F A with aging as for P. r o q u e f o r t i (6, 7) and an increased ability to esterify the F F A into triglycerides (23). Despite the lower concentrations of mycelium, carbonyl production was significantly greater in the milk system containing steapsin (Figure 2). This may have reflected greater substrate availability for oxidation; effects of higher concentrations of F F A in causing dissociation of fl-ketoacyl residues from fl-

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oxidative enzymes and the availability of greater amounts of short chain fatty acids that preferentially are converted to methyl ketones (10, 11, 12). The preferential conversion of lauric acid, even by low concentrations of mycelium (Figure 1), is consistent with the latter suggestion. The eventual decrease of total carbonyls in milk systems may have been due to conversion of methyl ketones to secondary alcohols (11, 12), which occur abundantly in Camembert cheese (5, 17). Methyl ketones accounted for approximately 60% of the total carbonyls in each system. As in Camembert cheese, nonanone was the principal ketone in the milk system (Figure 3). The milk system containing lipase contained much higher individual methyl ketones. The 2-nonanone tended to be in highest concentration up to 50 h after which 2-undecanone predominated. This may have reflected the initial preference of the enzymes for shorter chain fatty acids as observed earlier. Despite the report that high concentrations of F F A can depress methyl ketone formation (9), in the milk system containing lipase that had very high F F A , P. c a m e m b e r t i produced the largest amounts of methyl ketones. Conceivably, in the milk system partitioning of the F F A into the lipid phase effectively reduced the concentration of F F A or their soaps in the aqueous phase, thereby minimizing the actual concentration available to the mycelium. Heptanone and nonanone were the principal ketones synthesized in the system containing oleic acid, although the total concentrations were less than those obtained in the milk system containing lipase. Lauric acid, which Journal of Dairy Science Vol. 68, No. 1, 1985

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OKUMURA AND KINSELLA

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Figure 3. Patterns of individual methyl ketones produced following incubation of Penicillium camemberti with milk system (A), milk system with steapsin (B), and Cpazak's medium containing sodium oleate (O) and laurate (L). C5-C 11 denote 2-pentanone, 2-heptanone, 2-nonanone, 2-undecanone, and 2-tridecanone, respectively.

initially i n h i b i t e d m y c e l i u m g r o w t h (Figure 1), was m e t a b o l i z e d progressively, a n d u n d e c a n o n e was t h e p r i n c i p a l m e t h y l k e t o n e p r o d u c t . This was similar t o p a t t e r n s o b t a i n e d w i t h P. roqueforti i n c u b a t e d w i t h lauric acid (2, 3). Penicillium camemberti m y c e l i u m b e h a v e s similarly to P. roqueforti w h e n c u l t u r e d in d e f i n e d systems, a n d thus, it s h o u l d be possible to develop s y s t e m a t i c a l l y an o p t i m u m inc u b a t i o n s y s t e m for d e v e l o p i n g a flavor conc e n t r a t e a n d accelerating d e v e l o p m e n t o f C a m e m b e r t cheese flavor. REFERENCES

1 Dartey, C. K., and J. E. Kinsella. 1971. Rate of formation of methyl ketones during blue cheese ripening. J. Agric. Food Chem. 19:771. Journal of Dairy Science Vol. 68, No. 1, 1985

2 Dartey, C. K., and J. E. Kinsella. 1973. Oxidation of sodium UJ4C palmitate into carbonyl compounds by Penicillium roqueforti spores. J. Agric. Food Chem. 21:721. 3 Dartey, C. K., and J. E. Kinsella. 1973. Metabolism of U-t4C lauric acid to methyl ketones by the spores of Penicillium roqueforti. J. Agric. Food Chem. 21:933. 4 Dumont, J. P., S. Roger, and J. Adda. 1976. Autres composes mineurs mis en evidence. Lair 56:595. 5 Dumont, J. P., S. Roger, P. Cere, and J. Adda. 1974. Etu de des composes neutres volatiles presents dans le Camembert. Lair 54:501. 6 Dwivedi, B. K., and J. E. Kinsella. 1974. Continuous production of blue-type cheese flavor by submerged fermentation of Penicillium roqueforti. J. Food Sci. 39:620. 7 Dwivedi, B. K., and J. E. Kinsella. 1974. Carbonyl production from lipolyzed milk fat by the continuous mycelial culture of Penicillium roqueforti. J. Fond Sci. 39:83.

PEN1CILLIUM CAMEMBERTI F L A V O R 8 Groux, M., and M. Moinas. 1974. La flaveur des fromages. II. Etude comparative de la fraction volatile neutre de divers fromages. Lait 54:44. 9 Hawke, J. C. 1966. The formation and metabolism of methyl ketones and related c o m p o u n d s : review. J. Dairy Res. 33:225. 10 Hwang, D. H., and Y. J. Lee, and J. E. Kinsella. 1976. 3-ketoacyl Decar-boxylase activity in spores and m y c e l i u m of Penicillium roqueforti. Int. J. Biochem. 7:165. 11 Kinsella, J. E., and D. Hwang. 1976. Biosynthesis of flavor by Penicillium roqueforti. Biotechnol. Bioeng. 18(7):927. 12 Kinsella, J. E., and D. Hwang. 1976. Enzymes of Penicillium involved in biosynthesis of cheese flavor. Crit. Rev. Food Sci. Nutr. 4:191:228. 13 Lamberet, G. 1970. Aptitude o f Penicillium caseicolum species for lipolysis. XVItI. Int. Dairy Congr. 1E, 140. 14 Lamberet, G., and J. Lenoir. 1972. Ability o f Penicillium caseicolum to produce lipolytic enzymes. Lait 52:175. 15 Lenoir, J., C. Choisy, and B. Auberger. 1970. Aptitude of Penicillium caseicolurn for proteolysis. XVIII. Int, Dairy Congr. 1E, 141. 16 Matsuoka, H,, and T. Tsugo. 1963. Studies on the ripening of the semi-soft white m o u l d cheese ripened by Penicillium caseicolum. I. Flavor c o m p o u n d s in the semi-soft white m o u l d cheeseIdentification of the volatile carbonyl c o m p o u n d s .

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J. Agric. Chem. Soc. Jpn. 37:332. 17 Moinas, M., M. Groux, and I. Horman. 1973. La flaveur des flaveur des fromages I Une methodologie nouvelle d'isolement de constituants volatiles application an R o q u e f o r t et an Camembert. Lait 53:601. 18 Moinas, M., M. Groux, and I. Horman. 1975. La flaveur des fromages III. Mise en evidence de quelques constituants mineurs de l'arome du Camembert. Lait 55:414. 19 Myers, E., and S. G. Knight. 1958. Studies on the nutrition of Penicillium roqueforti. Appl. Microbiol. 6:174. 20 Ney, K. H., and I.P.G. Wirotama. 1971. Aliphatic m o n s a m i n e in deutschen u n d franzosischen Camembert. Z. Lebensm. Unters. Forsch. 146:343. 21 Ney, K. H., and I.P.G. Wirotama. 1973. Unsabstituted alphatic monocarboxylic acids and a-keto acids in C a m e m b e r t cheese. Z. Lebensm. Unters. Forsch. 152:32. 22 Schwartz, D. P., and O. N. Parks. 1963. Methyl ketones in C a m e m b e r t cheese. J. Dairy Sci. 46:1136. 23 Shimp, J., and J. E. Kinsella. 1977. Lipids of Penicillium roqueforti. Influence of culture temperature and age on u n s a t u r a t e d fatty acids. J. Agric. Food Chem. 25:793. 24 Shipe, W. F., A. F. Senyk, and K. Fountain. 1980. Modified copper soap solvent extraction m e t h o d for measuring free fatty acids in milk. J. Dairy Sci. 63:193.

Journal o f Dairy Science Vol. 68, No. 1, 1985