Bioconversion of Citrus d-Limonene - ACS Symposium Series (ACS

Chapter 13

Bioconversion of Citrus d-Limonene 1

2

Downloaded by UNIV OF MASSACHUSETTS AMHERST on February 23, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0596.ch013

R. J. Braddock and K. R. Cadwallader 1

Istitute

of Food and Agricultural Sciences, Citrus Research and Education Center, University of Florida, 700 Experimental Station Road, Lake Alfred, FL 33850 Department of Food Science and Technology, Mississippi State University, Box 9805, 112 Herzer Building, Mississippi State, MS 39762 2

Microbiological or enzymatic conversion of terpenes to value -added compounds is increasingly important because of consumers' preference for "natural" flavors. Microbes and enzymes capable of using terpenes such as limonene as growth media and substrates have been identified and studied. One such enzyme, α-terpineol dehydratase (αTD), purified > 10-fold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4R)-(+)limonene to (4R)-(+)-α-terpineol or (4S)-(-)-limonene to (4S)-(-)α-terpineol. αTD also showed stereoselectivity, since the hydration rate of (4R)-(+)-limonene was approximately 10X the rate of hydration of (4S)-(-)-limonene. Presence of αTD is believed to be common among psuedomonads. For example, a strain of P. cepacia, closely related to P. gladioli, was found capable of converting limonene to α-terpineol. Other pseudomonads have also been isolated that perform this conversion. The terpene, (4R)-(+)-4-isopropenyl-l-methylcyclohexene ( C A Registry No. 5989275), (+)-limonene, commonly called d-limonene, is the major constituent of citrus essential oils (approximately 95% (v/v) in oils from orange and grapefruit peel). Some chemical and physical properties and material safety data for (+)-limonene have been compiled and published (7). The primary sources of (+)-limonene are in raw materials generated during processing of citrus fruit for juice. It is decanted from evaporator condensate streams during concentration of peel press liquor to molasses. It is steam distilled from emulsions during cold-pressed peel oil processing and is also recovered as a product of essential oil concentration (2). (+)-Limonene is utilized as a raw material for chemical syntheses of terpene resin adhesives and value-added flavor chemicals. Other applications include solvent uses in waterless hand cleaners, pet shampoo and degreasing agents (5). Annual world availability of (+)-limonene amounts to about 50 million kg and is primarily dependent on

0097-6156/95/0596-0142$12.00/0 © 1995 American Chemical Society In Fruit Flavors; Rouseff, Russell L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


13. BRADDOCK & CADWALLADER

Bioconversion of Citrus a-Limonene

143

the amount of oranges processed in the major citrus growing regions of Florida and Brazil (4). The perfume and flavor industries find many applications for monoterpenes derived from (+)-limonene and similar terpenes. Many are oxygenated compounds which can be chemically synthesized using various process conditions. However, such chemical processes may result in non-stereospecific, uneconomic or undesirable end-products. Since terpene flavor compounds are synthesized by a variety of microorganisms and higher plants, there is considerable interest in using biological processes to produce metabolites of value for flavoring foods. Bioconversion of terpenes includes use of plant cells in suspension to convert valencene to nootkatone (5) as well as tissue culture techniques. Knorr et al. (6) described the bioconversion of (+)-limonene to carvone by dill cultures. Such biotechnological methods applied to flavor production have been the subject of many reports and were reviewed by Schreier (7). Microbial Conversions Bacteria and fungi may differ in their metabolism of terpenes. If one considers (+)-limonene, bacteria generally metabolize by progressive oxidation starting with the 7-methyl group. Generation of small amounts of neutral products which are not further metabolized may occur. Fungi attack (+)-limonene by hydration of the double bond of the isopropenyl substituent or by epoxidation-hydrolysis of the 1,2 double bond. Some of the terpene products identified during various microbial conversions of (+)-limonene are illustrated in Figure 1. Many of these compounds, in addition to ( + )-limonene, have economic value as flavoring ingredients and odorants. Other Terpenes The subject of microbial terpene conversions has been reported in the literature by a number of authors who studied compounds other than (+)-limonene. The degradation of (+)-camphor using a pseudomonad isolated from sewage resulted in 2,5-diketocamphane and other products (8). Prema and Bhattacharyya (9) transformed a- and β-pinene with Aspergillus niger and isolated optically pure (+)verbenone and (+)-cis-verbenol. β-Pinene was also transformed by Pseudomonas pseudomonallai to camphor, borneol, α-terpineol and similar compounds (10). Transformation by Botrytis cinerea of citronellol, a significant terpene of grape must, resulted in production of several diols, depending on growth conditions (11). A patent was issued for bacterial decomposition of geraniol and citronellol from citrus processing waste streams using Pseudomonas strains (12). Limonene (+)-Limonene has been the subject for many microbial bioconversion studies, because of its abundance and chemistry. Research to improve the quality of citrus oils resulted in the discovery that bacterial growth in peel oil emulsions reduced the quantity of (+)-limonene, increasing the concentration of a-terpineol

In Fruit Flavors; Rouseff, Russell L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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FLAVORS


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Figure 1. Chemical structures (1-15) of some products resulting from the microbial transformation of limonene. (1) limonene, (2) a-terpineol, (3) carveol, (4) p-mentha-l,8-dien-4-ol, (5) p-mentha-2,8-dien-l-ol, (6) perillic acid, (7) dihydroperillic acid, (8) carvone, (9) dihydrocarvone, (10) perillyl alcohol, (11) p-menth-8-ene-l ,2-diol, (12) p-menth-8-ene-l -ol-2-one, (13) p-menth-1 -ene6,9-diol, (14) 2-hydroxy-p-menth-8-ene-7-oic acid, (15) β-isopropenyl pimelic acid.





145

(13). A bacterium capable of conversion of (+)-limonene to perillic acid was isolated (14). Cladosporium (15) as well as Diplodia and Corynespora (16) were capable of converting (+)-limonene into 1,2-diols. Bowen (17) isolated Pénicillium strains that could transform (+)-limonene to several products, including p-menthadienol, carveol, carvone and perillyl alcohol. The pathway has been defined for Pseudomonas incognita conversion of (+)-limonene to perillic acid and β-isopropenyl pimelic acid (18). The bacterium, Pseudomonas gladioli, isolated from pine bark and sap was shown to metabolize (+)-limonene as a sole carbon source, producing (+)-aterpineol and (-l-)-perillic acid (19). In this study, concentrations of (+)-limonene as high as 10% (v/v) had no apparent toxic effect on the organism's growth; however, a considerable lag period was observed. This lag phase was speculated to be a result of the organism conditioning to the growth media. Optimum growth conditions (highest growth rate and maximum cell concentration) occurred near 1% (v/v) concentration of (+)-limonene. The organism produced the limonene bioconversion products, (+)-perillic acid and (+)-a-terpineol, illustrated in Figure 2. The (+)-perillic acid, after increasing, was metabolized during extended growth of the organism; however, (+)-a-terpineol increased and maintained a constant concentration. In addition to P. gladioli, other pseudomonads have been isolated that covert limonene to α-terpineol. A strain of P. cepacia, capable of metabolizing either a- or β-pinene as sole carbon source, was isolated from soil (5). The bacterium exhibited optimum growth at 30°C and pH 5.5, and grew equally well in media containing from 1 to 5% (v/v) a- or β-pinene. Major bioconversion products from α-pinene included borneol, p-cymene, α-terpinolene, limonene, camphor, terpinen-4-ol, α-terpineol and p-cymene-8-ol. Bioconversion products from βpinene were similar except for the presence of fenchyl alcohol and absence of camphor. Precursor studies have indicated that limonene was an intermediate product, readily metabolized as sole carbon source by the bacterium (6) to form perillic acid as major product and a trace amount of α-terpineol. In addition to P. cepacia, several other soil pseudomonads give a similar product profile from limonene (7). A recent study has shown that (+)-limonene can be converted by the mold Aspergillus cellulosae in sugar media containing limonene to (+)-limonene diol, (+)-carveol, (+)-perillyl alcohol and (+)-isopiperitenone (20). A review of many microbial conversions of (+)-limonene leading to products for theflavorand perfume industries has also been published (21). Enzymatic Conversions Enzymes have a long history of being used as processing aids in the food and flavor industries. Major enzymes and uses have been reviewed (22). Enzymes capable of terpene bioconversions have been isolated from some microorganisms. An aldehyde dehydrogenase catalyzing conversion of perillyl aldehyde to the acid was isolated from a soil pseudomonad which utilized limonene as a carbon source (23). This enzyme was active towards a number of aldehydes including phellandral, benzaldehyde and acetaldehyde. A typicalflavorconversion



146 FRUIT FLAVORS





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mediated by alcohol dehydrogenase is the production of geranial from geraniol (24). A n enzyme, α-terpineol dehydratase (aTD), has been isolated from P. gladioli, partially solubilized and characterized (25). This enzyme might also be described as A-8,9-limonene hydratase. a T D stereospecifically converted (4R)(+)-limonene to (4R)-(+)-a-terpineol or (4S)-(-)-limonene to (4S)-(-)-a-terpineol. aTD also showed stereoselectivity, since the hydration rate of (4R)-(+)-limonene was approximately 10X the rate of hydration of (4S)-(-)-limonene. Kinetic studies of the formation of (+) and (-)-a-terpineol from racemic limonene were followed using chiral gas chromatography (26). The relative amounts of each enantiomer at various reaction times are presented in Table I. The relative percent concentration of (4R)-(+)-a-terpineol increased at a faster rate than (4S)-(-)-a-terpineol, indicating stereoselectivity for the (+)-limonene substrate. Table I. Relative Percent Concentration of a-Terpineol Enantiomers as a Function of Time for the Hydration of 0.2 m M Racemic Limonene by a-Terpineol Dehydratase at 25°C Relative Percent Concentration* Time (hr)

(4S)-(-)-a-terpineol

(4R)-(+)-a-terpineol

0

0.0

0.0

5

10.8

89.2

10

11.3

88.7

20

11.4

88.6

40

13.3

86.7

•Relative percent cone. = Cone, of pure α-terpineol enantiomers Total cone, of α-terpineol The aforementioned studies demonstrate the considerable potential of bioconversion processes for the transformation of inexpensive monoterpene precursors, such as limonene, into more valuable flavor and fragrance compounds. The majority of these investigations were concerned with the elucidation of pathways for limonene metabolism; however, a few of these researchers recognized the potential of using bioconversions for the generation of novel and more valuable flavor and fragrance compounds. The real advantage of a biotechnological process over a synthetic process may not necessarily be in its greater selectivity and specificity, or its ability to produce rare or novel compounds, rather the fact that it is a natural process and thus generates natural products. This is especially important currently, when consumers prefer "natural" flavors and fragrances over their "synthetic" or "artificial" counterparts.


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Acknowledgments Florida Agricultural Experiment Station Journal Series No. R-03582.


Literature Cited 1. Braddock, R.J.;Temelli, F; Cadwallader, K. R. FoodTechnol.1986, 40(11), 114. 2. Braddock, R. J.; Cadwallader, K. R. Food Technol. 1992, 46(2), 105. 3. Matthews, R.F.; Braddock, R. J. Food Technol. 1987, 41(1), 57. 4. McBride, J.J. Trans. Citrus Eng. Conf., Amer. Soc. Mech. Eng. 1990, 36, 72-80. 5. Drawert, F.; Berger, R. G.; Godelmann, R. Plant Cell Rpts. 1984, 3, 37. 6. Knorr, D.; Beaumont, M. D.; Caster, C. S. Food Technol. 1990, 44(6), 71. 7. Schreier, P. Food Rev. Int. 1989, 5(3), 289. 8. Bradshaw, W. H.; Conrad, H. E.; Covey, E.J.;Gunsalas, I. C.; Lednicer, D. J. Amer.Chem.Soc. 1959, 81, 5507. 9. Prema, B. R.; Bhattacharyya, P. K. Appl. Microbiol. 1962, 10, 524. 10. Dhavlikar, R. S.; Ehbrecht, Α.; and Albroscheit, G. DragacoRpt.1974, 3, 47. 11. Brunerie, P.; Benda, I.; Bock, G.; Schreier, P. Appl. Microbiol 1987, 27, 6. 12. Vandenbergh, P. Α.; U.S. Patent 4,593,003, June 3, 1986. 13. Murdock, D. I.; Allen, W. E. J. Food Sci. 1970, 35, 652. 14. Dhere, S. G.; Dhavlikar, R. S. Sci. Cult. 1970, 36, 292. 15. Mukherjee, Β. B.; Kraidman, G.; Hill, I. D. Appl. Microbiol. 1973, 25, 447. 16. Abraham, W. R.; Kieslich, K.; Reng, H.; Stumpf, B. Third Eur. Congr. Biotechnol.; Verlag Chemie, Deerfield Beach, FL, 1984, Vol. 1, 245. 17. Bowen, E. R. Proc. Fla. State Hort. Soc. 1975 , 88, 305. 18. Rama Devi,J;Bhattacharyya, P. K. Indian J. Biochem. Biophys. 1977, 14, 359. 19. Cadwallader, K. R.; Braddock, R. J.; Parish, M. E.; Higgins, D. P.J.Food Sci. 1989, 54, 1241. 20. Noma, Y.; Yamasaki, S.; Asakawa, Y. Phytochemistry 1992, 31(8), 2725. 21. Gabrielyan, Κ. Α.; Menyailova, I.I.; Nakhapetyan, L. A. Appl.Biochem.1992, 28(3), 241. 22. Welsh, F. W.; Murray, W. D.; Williams, R. E. Crit. Rev. Biotechnol. 1989, 9, 105. 23. Ballal, N. R.; Bhattacharyya, P. K.; Rangachari, P. N. Biochem. Biophys. Res. Commun. 1967, 29(3), 275. 24. Legoy, M. D.; Klim, H. S.; Thomas, D. Process Biochem. 1985, 20, 145. 25. Cadwallader, K. R.; Braddock, R.J.;Parish, M. E. J. Food Sci. 1992, 57, 241. 26. Cadwallader, K. R.; Braddock, R. J. In Food Science and Human Nutrition, G. Charalambous (Ed.); Elsevier B.V.: Amsterdam, The Netherlands, 1992; pp 571-584. RECEIVED October 18,

1994


Bioconversion of Citrus d-Limonene - ACS Symposium Series (ACS

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