Appl Microbiol Biotechnol (2003) 62:331336 DOI 10.

1007/s00253-003-1309-4

ORIGINAL PAPER

H. Zorn S. Langhoff M. Scheibner R. G. Berger

Cleavage of b,b-carotene to flavor compounds by fungi

Received: 14 November 2002 / Revised: 10 January 2003 / Accepted: 10 January 2003 / Published online: 26 April 2003  Springer-Verlag 2003

Abstract More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave b,b-carotene to flavor compounds. Ten strains discolored a b,b-carotene-containing growth agar, indicating efficient degradation of b,b-carotene. Dihydroactinidiolide was formed as the sole conversion product of b,b-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of b,b-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. b-Ionone proved to be the main metabolite in each case, whereas b-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the b,b-carotene cleaving enzyme activities of M. scorodonius were partially characterized.

Introduction
Natural volatile compounds structurally derived from carotenoids are widely distributed. Due to their often low flavor thresholds, many of them are highly potent aroma compounds, and thus of great interest for the flavor and fragrance industries (Winterhalter and Rouseff 2002). Outstanding examples are the flowery smelling C13compounds b-ionone and b-damascenone, featuring
H. Zorn ()) S. Langhoff M. Scheibner R. G. Berger Zentrum Angewandte Chemie, Institut fr Lebensmittelchemie der Universitt Hannover, Wunstorfer Strae 14, 30453 Hannover, Germany e-mail: zorn@lci.uni-hannover.de Tel.: +49-511-7624583 Fax: +49-511-7624547

threshold values of 0.007 and 0.009 g l1 (in water), respectively. The sensory-active C9-, C10-, C11-, and C13-norisoprenoids naturally emerge from the oxidative cleavage of tetraterpenoid precursor carotenoids (Schreier 1995; Winterhalter 1996). Non-selective fissions of the polyene chain, induced by abiotic factors like heat or light, result in a broad spectrum of oxidized breakdown products (Beatriz et al. 1993; Handelman et al. 1991). A smaller range of products, which often display biological activity, is released by enzymatic cleavage reactions. Enzymes catalyzing the centrical or excentrical fission of specific carotenoids in vivo have been isolated and characterized in recent years from plants, insects, birds and mammals (e.g. Kiefer et al. 2001; Quin and Zeevaart 1999; Schwartz et al. 1997, 2001; Thompson et al. 2000; von Lintig and Vogt 2000; Wyss et al. 2000; Yan et al. 2001). The occurrence of norisoprenoids in natural sources is restricted to trace amounts, and extraction has turned out to be tedious and costly. Thus, biotechnological processes copying natural carotenoid degradation have been envisaged. The co-oxidation of carotenoids using mainly lipoxygenase or xanthine oxidase systems has been intensely investigated. Thereby carotenoids are oxidized by free-radical species generated from another substrate by enzymatic reactions (e.g. Wache et al. 2002; Wu and Robinson 1999). Sanchez-Contreras et al. (2000) described the conversion of lutein from marigold flowers (Tagetes erecta) to 7,8-dihydro-b-ionol, b-ionone, 7,8dihydro-b-ionone and 3-hydroxy-b-ionone by mixed cultures of Geotrichum sp. and Bacillus sp. The presence of both species was indispensable for flavor production. Here, a systematic new approach for the microbial production of carotenoid-derived flavor compounds is reported. In a broad screening, the potential of fungi and yeasts to cleave the model substrate b,b-carotene to form norisoprenoids was evaluated. Although some 100 million tons of carotenoids are biosynthesized (Britton et al. 1999) and subsequently degraded naturally every year, there are surprisingly few data regarding microbial carotenoid degradation.

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Materials and methods

General Due to the light and heat sensitivity of carotenoids, all b,bcarotene-containing solutions were prepared freshly before use. All cultivations were carried out in the complete absence of light, and standard sterile techniques were applied. Quantitative data represent means of duplicate analyses. Microorganisms The microorganisms were from the Dutch Centraalbureau voor Schimmelcultures, Baarn (CBS), from the German Sammlung fr Mikroorganismen und Zellkulturen, Braunschweig (DSM), or from the culture collection of the Agricultural University of Wageningen, Netherlands (WAG). Chemicals The constituents of standard nutrient solution were purchased from Merck (Darmstadt, Germany), b,b-carotene and b-ionone were from Fluka (Neu-Ulm, Germany). Tween 40 was from Sigma (Taufkirchen, Germany) and checked for the absence of peroxides before use. Solvents were provided by BASF (Ludwigshafen, Germany) and Baker (Deventer, Netherlands). All solvents were distilled before use. High-purity water was prepared with an E-pure water purification system (Barnstead, Dubuque, Iowa, USA). Screening on carotene-agar For preparation of b,b-carotene-containing growth agar plates, 125 mg of b,b-carotene and 12.5 g of Tween 40 were dissolved in 200 ml of dichloromethane. The solvent was distilled off under reduced pressure and the residue was dispersed into standard nutrient medium (30 g l1 glucose1 H2O; 4.5 g l1 asparagine1 H2O; 1.5 g l1 KH2PO4; 0.5 g l1 MgSO4; 3.0 g l1 yeast extract; 15 g l1 agar agar; 1 ml l1 trace element solution containing Cu, Fe, Zn, Mn, and EDTA; the pH was adjusted to 6.0 with 1 N NaOH) prior to sterilization. b,b-Carotene agar plates were inoculated with small agar pieces (~ 1 cm2), densely covered with fungal mycelium of the strain under investigation. The color of the agar plates was examined daily and b,b-carotene-degrading strains were visually detected by color fading after 314 days. Transformation of b,b-carotene in submerged cultures For preparation of pre-cultures, small mycelium-covered pieces (~ 1 cm2) from the carotene agar plates were homogenized using an Ultra-Turrax (Janke and Kunkel, Germany) in 100 ml standard nutrition solution (analogous to standard nutrient medium without agar agar). After cultivation for 13 weeks (depending on the growth rate of the respective fungus) at 24 C and 150 rpm, the cultures were homogenized using an Ultra-Turrax. Ten ml of the pre cultures were transferred into 300-ml Erlenmeyer flasks containing 100 ml of fresh standard nutrition solution. Growth of the cultures was monitored by determination of pH, glucose content, and dry mass (filtration of an aliquot of culture medium and drying at 103 C for 12 h). Twenty mg of b,b-carotene (emulsified with 200 mg of Tween 40) in 2 ml ethanol were added to each flask after 1 (yeasts) or 3 days (filamentous fungi), respectively. The cultures were incubated at 24 C and 150 rpm, and samples were analyzed daily (yeasts) or on every second to third day (filamentous fungi). Remaining b,b-carotene and metabolites thereof were extracted from the culture supernatant thrice with pentane/dichloromethane (1:1, v/v). The organic phases were combined, washed with NaHCO3 solution (saturated), dried over Na2SO4 and concentrated to 5 ml using a Vigreux column. Prior to

the extraction, a-terpineol was added as internal standard. To detect intracellular metabolites, 515 g of mycelium were disrupted in a bead mill (DYNO-Mill, W.A. Bachofen, Switzerland; 5 C; 3,200 rpm; 15 min) with 20 ml of methanol and 50 ml of a methanol/water (70:30 v/v) mixture using 80 ml of glass beads (0.5-mm diameter, Braun Biotech, Germany). After settling of the glass beads, the suspension was decanted and extracted as described above. Transformation of b,b-carotene in mycelium-free culture media Pre-cultures and experimental cultures were prepared as described above, but the fungal mycelia were separated from the growth media by centrifugation (3,300 g, 5 C, 20 min) prior to the addition of b,b-carotene. For emulsification, 10 mg of b,b-carotene and 1 g of Tween 40 were dissolved in dichloromethane. The solvent was distilled off in a rotary evaporator and the residue was taken up in 70 ml of water. At 40 C and 25 kPa, the solution was further concentrated until complete solubilization of b,b-carotene. The stock solution was obtained by diluting the emulsion to 100 ml with water. An aliquot of the stock solution (20 ml) was added to 100 ml of the cell-free growth medium. Samples were shaken for 12 h (24 C, 100 rpm) in the complete absence of light. For the chemical blanks, standard nutrition solution was used instead of culture supernatants. After extraction with pentane/dichloromethane and concentration in vacuum, biodegradation of b,b-carotene was monitored by means of high performance liquid chromatography with diode array detection (HPLC/DAD) whereas the formation of volatiles was observed by means of gas chromatography/flame ionization detector (GC/FID) and gas chromatography/mass spectrometry (GC/MS). Ultra-filtration Mycelium-free culture supernatants were concentrated ten-fold by use of ultrafiltration units (Centricon Plus-80, Millipore, USA), exclusion limit 30 kDa (3,300 g, 5 C). Kinetic investigations One hundred ml of cell-free media of Marasmius scorodonius were mixed with 1.5 mg of solubilized b,b-carotene and incubated at 28 C and 80 rpm. Samples (1.0 ml) were drawn in frequent intervals and degradation of b,b-carotene was monitored photometrically at 450 nm. Partial characterization of b,b-carotene-cleaving enzyme activities of M. scorodonius Enzyme assay One hundred l of b,b-carotene stock solution were added to 1.5 ml of enzyme-containing sample solution. Oxygen, which is needed as a co-substrate for the oxidative cleavage of carotenoids, was provided by the air-saturated sample or buffer solutions, respectively. The time-dependent decrease of absorbance was monitored at 450 nm using a thermostatable spectral photometer, equipped with a magnetic stirrer unit (Lambda 12, Perkin Elmer, Germany). After an assay time of 10 min, enzyme activity was calculat DEVt ed according to the following equation: mUmL1 Vs de 106 [U=enzyme activity catalyzing the degradation of 1 mol b,bcarotene per minute; DE=decrease of absorbance per minute; Vt=total volume in cuvette (ml); Vs=sample volume in cuvette (ml); e=extinction coefficient of b,b-carotene, l=450 nm (the extinction coefficient of b,b-carotene in aqueous emulsion was determined to be 95,000 l mol 1cm1, n=10, CV=1.7%); d=thickness of cuvette (cm)]. Applicability of the enzyme test was checked by diluting aliquots of the sample to volume with buffer solution (0.1 M citrate/ phosphate buffer, pH 5.0). A linear correlation between activity and sample amount was ascertained.

333 The temperature optimum was determined by adding 0.1 ml of b,b-carotene solution to 1.5 ml of cell-free growth medium of M. scorodonius and measuring the time-dependent decrease of absorbance at varying temperatures. For determination of the pH optimum, cell-free nutrient medium of M. scorodonius was concentrated tenfold by means of ultrafiltration. For each measurement, 0.2 ml of the concentrated retentate were diluted with 1.3 ml of buffer solutions of varying pH. One hundred l of b,b-carotene stock solution were added to each cuvette and the decrease in absorbance was monitored photometrically at 450 nm. Gas chromatography/mass spectrometry A Hewlett-Packard HP 5890 gas chromatograph equipped with an on-column injector (40 C) was directly coupled to an HP 5989 mass spectrometer. Separation of volatiles was achieved on a CP-5 CB fused silica column (30 m0.32 mm i.d., film thickness 0.25 m, Varian, Germany), using helium as carrier gas (3.2 ml min1). The temperature program was as follows: 5 min isothermal at 50 C, raised to 250 C at 4 C/min. The temperature of the ion source was 250 C, the electron energy for all EI mass spectra was 70 eV. Degradation products of b,b-carotene were identified by comparing their Kovats indices and mass spectra with published data (e.g. Hohler 1986; Krammer et al. 2002) and, if available, with authentic reference materials. Gas chromatography/flame ionization detector A Trace GC 2000 Thermoquest, USA), equipped with a splitsplitless injector (220 C) and a flame ionization detector (250 C) was applied for quantitative analyses. Separation of volatiles was achieved on a DB 5 column (30 m0.25 mm i.d., film thickness 0.25 m, J and W, Germany). Hydrogen was used as carrier gas at a flow rate of 2.1 ml min1 and the same temperature program was used as for GC/MS. High performance liquid chromatography with diode array detection For HPLC separation, a Nucleosil 1205 analytical column (Macherey and Nagel, Germany; 250 x 4 mm) with 5-m C18 reversed-phase material including a pre-column (C18, 10 x 4 mm, 5 m) was used. The mobile phase consisted of mixtures of methanol/ water (containing 10 mM ammonium acetate) (70:30 v/v) (A) and methanol/dichloromethane 80:20 v/v (B), starting with 100 % A, followed by a gradient to obtain 100 % B after 15 min, and isocratic 100 % B from 15 to 45 min at a flow rate of 0.6 ml/min. The injection volume was 20 l. Detection was carried out with an UV/ vis-photodiode array detector MD 910 (Jasco, Germany).

carotene. Cultivation of the strains on a special b,bcarotene-containing growth agar proved to be an adequate test system as positive strains could be detected visually by color fading after 12 weeks. Whilst no bleaching was observed with most of the species, ten strains discolored the agar in the region covered by mycelium, or formed a bleaching zone around the fungus, indicating efficient degradation of b,b-carotene (Table1). All of the b,bcarotene-degrading strains discovered in the screening were further investigated for their capability to cleave b,b-carotene to aroma compounds. Biotransformation of b,b-carotene in submerged cultures b,b-Carotene, emulsified with Tween 40, was added to submerged cultures of the respective fungi or yeasts. Growth of the microorganisms was not or only marginally inhibited by b,b-carotene and Tween 40, as indicated by parallel cultivations without emulsified b,b-carotene and with Tween 40 only. Non-inoculated media supplemented with emulsified b,b-carotene served as chemical blanks. To detect volatile degradation products over the entire cultivation period, complete cultures were analyzed daily (yeasts) or on every second to third day (Asco- and Basidiomycetes), respectively. Although b,b-carotene was apparently degraded, no volatile breakdown products were detected with Cryptococcus laurentii, Cyathus pallidus, Ischnoderma benzoinum, Marasmius scorodonius, Phaffia rhodozyma, and Trametes versicolor. Dihydroactinidiolide was detectable in low yields as the sole conversion product of b,b-carotene in the cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. No volatile b,b-carotene-derived products were detected under conditions with the chemical blanks and with non-supplemented cultures of the respective fungi. Biotransformation of b,b-carotene in mycelium-free culture media Discoloration of the carotene agar appeared outside the fungal mycelium (Table 1), indicating secretion of b,bcarotene-degrading extracellular enzymes and/or mediators into the surrounding growth medium. Thus, in an alternative approach, mycelium-free growth media were applied for the bioconversion of b,b-carotene. All of the strains that tested positively in the screening were cultivated as described, and the mycelia were separated from the growth media by centrifugation prior to the addition of b,b-carotene. Nearly complete degradation of

Results
Screening procedure To identify microorganisms suitable for the production of carotenoid derived flavor compounds, more than 50 fungi and yeasts (a complete list of all strains is available upon request) were screened for their capability to cleave b,b-

Table 1 Microorganisms degrading b,b-carotene when grown on b,b-carotene-containing agar plates. + Pronounced bleaching zone around the mycelium, +/ weak color fading

Cryptococcus laurentii Cyathus pallidus Ganoderma applanatum Hypomyces odoratus Ischnoderma benzoinum

CBS CBS CBS CBS CBS

318 376.80 250.61 764.68 311.29

+/ + + + +

Kuehneromyces mutabilis Marasmius scorodonius Phaffia rhodozyma Trametes suaveolens Trametes versicolor

DSM 1013 CBS 850.87 CBS 5905 DSM 5237 WAG EIK 39

+ + +/ + +

334 Table 2 Degradation of b,b-carotene by mycelium free culture supernatants of fungi and yeasts after 12 h Species Chemical blank Degradation of b,b-carotene [%] <8 Cryptococcus laurentii Cyathus pallidus Ganoderma applanatum Hypomyces odoratus Ischnoderma benzoinum Kuehneromyces mutabilis Marasmius scorodonius Phaffia rhodozyma Trametes suaveolens Trametes versicolor Yeast Basidiomycete Basidiomycete Ascomycete Basidiomycete Basidiomycete Basidiomycete Yeast Basidiomycete Basidiomycete 9 85 24 9 95 10 93 7 60 98 Volatile degradation products + + +

Table 3 Formation of volatile degradation products (in mol %) of b,b-carotene by use of mycelium-free culture supernatants of basidiomycetous fungi after 12 h Species I. benzoinum M. scorodonius T. versicolor b-Ionone 8 8 10 2-Hydroxy-2,6,6-trimethyl-cyclohexanone 5 trace 6 Dihydroactinidiolide trace 4 4 b-Cyclocitral trace trace 2

Fig. 1 Volatile compounds formed by mycelium-free culture supernatants; 1 2-Hydroxy-2,6,6-trimethyl-cyclohexanone, 2 bcyclocitral, 3 b-ionone, 4 dihydroactinidiolide

Fig. 2 Degradation of b,b-carotene by mycelium-free culture media of Marasmius scorodonius

the substrate was observed with culture media of five out of ten species after incubation for 12 h. Using myceliumfree culture media, volatile products were detected with Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor (Table 2). b-Ionone was the main metabolite in each case, whereas b-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities (Table 3, Fig. 1). The edible mushroom M. scorodonius was chosen as an example to further investigate b,b-carotene cleavage by fungal extracellular enzyme activities. To supply conclusive evidence for enzymatic processes being responsible for the cleavage of b,b-carotene, cell-free supernatants were subjected to ultrafiltration.

Fig. 3 Temperature optimum and pH dependence of b,b-carotenecleaving enzyme activities of M. scorodonius

Under the conditions described above, degradation rates using the ultrafiltration permeate mirrored the chemical blank. Nearly complete b,b-carotene degradation and formation of volatiles was observed with the respective retentates (data not shown).

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Kinetics of b,b-carotene degradation Kinetic investigations of the b,b-carotene degradation by mycelium-free culture media of M. scorodonius indicated a rapid reaction profile. About 40% of the initially added b,b-carotene had been degraded 60 min after the start of incubation (Fig. 2). Degradation of b,b-carotene with the chemical blanks (using water instead of cell-free medium) was <1% after 120 min. A fast and efficient enzyme test, modified after the method of Ben Aziz et al. (1971), was used to partly characterize the b,b-carotene degrading extracellular enzyme activities of M. scorodonius. Optimal carotene degradation occurred at 27 C. The pH optimum was 5.0 (Fig. 3).

Discussion
A systematic investigation was carried out to find microorganisms capable of cleaving carotenoids to compounds with aroma. b,b-Carotene was chosen as a model substrate, as it is readily available and its primary cleavage product, b-ionone, represents a highly valued flavor compound. Microorganisms were screened that either produce terpenoids (monoterpenes, sesquiterpenes, carotenoids) themselves de novo or are known for terpene biotransformation (e.g. Janssens et al. 1992). Furthermore, species isolated from terpene-rich substrates were included in the screening program. Out of more than 50 fungi and yeasts tested, ten strains bleached the b,bcarotene agar under conditions. Thus, even amongst species that someway or another handle terpenoid substrates, degradation of b,b-carotene seems not to be a common characteristic. The molds Fusarium moniliforme, Aspergillus flavus, and Penicillium citrinum had been reported to decrease the carotenoid content of silk (Prasad and Singh 1995), but no bleaching of the b,bcarotene agar was observed with these fungi in our study. A different chemical environment may be responsible for these apparently contradictory findings. All of the species bleaching the test agar were further examined for their ability to produce flavor compounds. In submerged cultures, dihydroactinidiolide was formed as the single degradation product of b,b-carotene by four of the ten species under investigation. The lactone dihydroactinidiolide has been described as a secondary product of the oxidative carotenoid degradation (e.g. Bosser et al. 1995). It exhibits a pleasant, hay-like odor and is supposed to be important to the flavor of black tea. Nevertheless, due to low transformation yields and extended cultivation periods, conversion of b,b-carotene in submerged cultures of fungi and yeasts did not prove to be an appropriate tool for the biotechnological production of carotenoid-derived flavor compounds. Furthermore, extraction of metabolites was complicated by the presence of substantial amounts of exo-polysaccharides. Handling of the transformation system was essentially simplified when culture supernatants were applied instead

of submerged cultures. b,b-Carotene was degraded effectively by five of the culture supernatants, with three of them showing volatile-product formation. The product spectrum, comprising b-ionone, b-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone, qualitatively corresponds largely to that observed in cooxidation experiments (e.g. Bosser and Belin 1994; Wu and Robinson 1999). b-Ionone was the main product, which is in good agreement with recent molecular mechanics calculations. Mohamed et al. (2001) showed the C9C10 double-bond in (Z)- and (E)-conformation to be a thermodynamically preferred point of attack for the oxidative cleavage of b,b-carotene. While this is the first report on the bioconversion of b,b-carotene to b-ionone by fungi, several microorganisms have been used to transform b-ionone as a substrate. Complex mixtures of metabolites, comprising oxygenated and side-chain-degraded products, were obtained (Grivel and Larroche 2001; Grivel et al. 1999; Winterhalter 1996 and references therein). Interestingly, none of the strains that produced dihydroactinidiolide in the submerged cultures formed volatiles in the cell-free conversions. Cellular enzymes probably contribute to the formation of dihydroactinidiolide in submerged cultures of G. applanatum, H. odoratus, K. mutabilis, and T. suaveolens. A cellular, membrane-bound unstable carotene dioxygenase that cleaved b,b-carotene into two molecules b-cyclocitral (C10) and crocetindial (C20) was reported for the cyanobacterium Microcystis (Jttner et al. 1985). By contrast, extracellular enzymes catalyzed the cleavage of b,b-carotene to b-ionone and further degradation products in the cell-free media of I. benzoinum, M. scorodonius, and T. versicolor. No b,b-carotene degradation products had been detected before in submerged cultures of these fungi which suggests a further metabolization of the primarily formed products. Strong indications for extracellular enzymes being responsible for the degradation of b,b-carotene were derived from ultrafiltration experiments. Whilst the enzyme containing retentates of ultrafiltration effectively catalyzed b,bcarotene degradation and formation of volatiles, no cleavage products were observed with the respective permeates. Compared to the degradation of lutein by mixed cultures of Geotrichum sp. and Bacillus sp. (<60% under optimized conditions after 48 h; Sanchez-Contreras et al. 2000), a very fast degradation of b,b-carotene (~40% after 1 h) was observed with cell-free culture supernatants of M. scorodonius. Numerous extra-cellular enzymes from basidiomycetes, namely from white-rot fungi, have been characterized on a molecular level in recent years (e.g. Mester and Field 1998; Ruiz-Duenas et al. 1999). They are well known for degrading lignin, cellulose, hemicellulose, and numerous low-molecular-weight compounds including even many xenobiotics. Nevertheless, the production of carotenoidderived flavor compounds by extracellular enzyme activities of microorganisms has not been reported. Theoretically, two molecules bearing the trimethylcyclohexene

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motif should result from the two-fold excentrical cleavage of b,b-carotene. However, maximum overall molar yields of about 25% were reached in the cell-free bioconversions. The reason for this balance gap is not yet clear. Biochemical and molecular biological investigations are currently in progress to isolate and characterize the enzymes responsible for carotenoid degradation. In preliminary studies, a temperature of 27 C and a pH of 5.0 provided optimal conditions for the b,b-carotene degradation by extracellular enzymes of M. scorodonius. These comparatively mild reaction conditions together with a fast reaction profile turn cell-free culture supernatants of selected basidiomycetes into a promising tool for the biotechnological production of norisoprenoid aroma compounds. Further research is required to gain insight into the mechanism of the cleavage reaction and the particular properties of the enzymes involved.
Acknowledgements Support of the work by the Deutsche Forschungsgemeinschaft (ZO 122/11) is gratefully acknowledged. All experiments comply with the current laws of the Federal Republic of Germany.

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