Cleavage of b,B-carotene to Flavor Compounds by Fungi

8/3/2019 Cleavage of b,B-carotene to Flavor Compounds by Fungi

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Appl Microbiol Biotechnol (2003) 62:331336DOI 10.1007/s00253-003-1309-4

O R I G I N A L P A P E R

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 ofmono-, 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 growthagar, indicating efficient degradation of b,b-carotene.Dihydroactinidiolide was formed as the sole conversionproduct ofb,b-carotene in submerged cultures of Gano-derma applanatum, Hypomyces odoratus, Kuehnero-myces mutabilis, and Trametes suaveolens. Whenmycelium-free culture supernatants from five specieswere applied for the conversions, nearly complete deg-radation ofb,b-carotene was observed after 12 h. Carot-enoid-derived volatile products were detected in themedia of Ischnoderma benzoinum, Marasmius scorodo-nius, and Trametes versicolor. b-Ionone proved to be the

main metabolite in each case, whereas b-cyclocitral,dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcy-clohexanone were formed in minor quantities. Using aphotometric bleaching test, the b,b-carotene cleavingenzyme activities of M. scorodonius were partiallycharacterized.

Introduction

Natural volatile compounds structurally derived fromcarotenoids are widely distributed. Due to their often lowflavor thresholds, many of them are highly potent aromacompounds, and thus of great interest for the flavor andfragrance industries (Winterhalter and Rouseff 2002).Outstanding examples are the flowery smelling C13-compounds b-ionone and b-damascenone, featuring

threshold values of 0.007 and 0.009 g l1 (in water),respectively. The sensory-active C9-, C10-, C11-, andC13-norisoprenoids naturally emerge from the oxidative

cleavage of tetraterpenoid precursor carotenoids (Schreier1995; Winterhalter 1996). Non-selective fissions of thepolyene chain, induced by abiotic factors like heat orlight, result in a broad spectrum of oxidized breakdownproducts (Beatriz et al. 1993; Handelman et al. 1991). Asmaller range of products, which often display biologicalactivity, is released by enzymatic cleavage reactions.Enzymes catalyzing the centrical or excentrical fission ofspecific carotenoids in vivo have been isolated andcharacterized in recent years from plants, insects, birdsand mammals (e.g. Kiefer et al. 2001; Quin and Zeevaart1999; 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 isrestricted to trace amounts, and extraction has turned outto be tedious and costly. Thus, biotechnological processescopying natural carotenoid degradation have been envis-aged. The co-oxidation of carotenoids using mainlylipoxygenase or xanthine oxidase systems has beenintensely investigated. Thereby carotenoids are oxidizedby free-radical species generated from another substrateby enzymatic reactions (e.g. Wache et al. 2002; Wu andRobinson 1999). Sanchez-Contreras et al. (2000) de-scribed the conversion of lutein from marigold flowers(Tagetes erecta) to 7,8-dihydro-b-ionol, b-ionone, 7,8-dihydro-b-ionone and 3-hydroxy-b-ionone by mixedcultures of Geotrichum sp. and Bacillus sp. The presenceof both species was indispensable for flavor production.

Here, a systematic new approach for the microbialproduction of carotenoid-derived flavor compounds isreported. In a broad screening, the potential of fungi andyeasts to cleave the model substrate b,b-carotene to formnorisoprenoids was evaluated. Although some 100 milliontons of carotenoids are biosynthesized (Britton et al.1999) and subsequently degraded naturally every year,there are surprisingly few data regarding microbialcarotenoid degradation.

H. Zorn ()) S. Langhoff M. Scheibner R. G. BergerZentrum Angewandte Chemie,Institut fr Lebensmittelchemie der Universitt Hannover,Wunstorfer Strae 14, 30453 Hannover, Germanye-mail: [email protected].: +49-511-7624583Fax: +49-511-7624547


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Materials and methods

General

Due to the light and heat sensitivity of carotenoids, all b,b-carotene-containing solutions were prepared freshly before use. Allcultivations were carried out in the complete absence of light, andstandard sterile techniques were applied. Quantitative data repres-ent means of duplicate analyses.

Microorganisms

The microorganisms were from the Dutch Centraalbureau voorSchimmelcultures, Baarn (CBS), from the German Sammlung frMikroorganismen und Zellkulturen, Braunschweig (DSM), or fromthe culture collection of the Agricultural University of Wagenin-gen, Netherlands (WAG).

Chemicals

The constituents of standard nutrient solution were purchased fromMerck (Darmstadt, Germany), b,b-carotene and b-ionone werefrom 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 weredistilled before use. High-purity water was prepared with an E-purewater purification system (Barnstead, Dubuque, Iowa, USA).

Screening on carotene-agar

For preparation of b,b-carotene-containing growth agar plates,125 mg ofb,b-carotene and 12.5 g of Tween 40 were dissolved in200 ml of dichloromethane. The solvent was distilled off underreduced pressure and the residue was dispersed into standardnutrient medium (30 g l1 glucose1 H2O; 4.5 g l

1 asparagine1H2O; 1.5 g l

1 KH2PO4; 0.5 g l1 MgSO4; 3.0 g l

1 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 withsmall agar pieces (~ 1 cm2), densely covered with fungal myceliumof the strain under investigation. The color of the agar plates wasexamined daily and b,b-carotene-degrading strains were visuallydetected by color fading after 314 days.

Transformation ofb,b-carotene in submerged cultures

For preparation of pre-cultures, small mycelium-covered pieces(~ 1 cm2) from the carotene agar plates were homogenized using anUltra-Turrax (Janke and Kunkel, Germany) in 100 ml standardnutrition solution (analogous to standard nutrient medium withoutagar agar). After cultivation for 13 weeks (depending on thegrowth rate of the respective fungus) at 24 C and 150 rpm, thecultures were homogenized using an Ultra-Turrax. Ten ml of the

pre cultures were transferred into 300-ml Erlenmeyer flaskscontaining 100 ml of fresh standard nutrition solution. Growth ofthe cultures was monitored by determination of pH, glucosecontent, and dry mass (filtration of an aliquot of culture mediumand 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 addedto 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 tothird day (filamentous fungi). Remaining b,b-carotene and metabo-lites thereof were extracted from the culture supernatant thrice withpentane/dichloromethane (1:1, v/v). The organic phases werecombined, washed with NaHCO3 solution (saturated), dried overNa2SO4 and concentrated to 5 ml using a Vigreux column. Prior to

the extraction, a-terpineol was added as internal standard. To detectintracellular metabolites, 515 g of mycelium were disrupted in abead mill (DYNO-Mill, W.A. Bachofen, Switzerland; 5 C;3,200 rpm; 15 min) with 20 ml of methanol and 50 ml of amethanol/water (70:30 v/v) mixture using 80 ml of glass beads(0.5-mm diameter, Braun Biotech, Germany). After settling of theglass beads, the suspension was decanted and extracted as describedabove.

Transformation ofb,b-carotene in mycelium-free culture media

Pre-cultures and experimental cultures were prepared as describedabove, but the fungal mycelia were separated from the growthmedia by centrifugation (3,300 g, 5 C, 20 min) prior to the additionofb,b-carotene. For emulsification, 10 mg ofb,b-carotene and 1 gof Tween 40 were dissolved in dichloromethane. The solvent wasdistilled off in a rotary evaporator and the residue was taken up in70 ml of water. At 40 C and 25 kPa, the solution was furtherconcentrated until complete solubilization of b,b-carotene. Thestock solution was obtained by diluting the emulsion to 100 ml withwater. An aliquot of the stock solution (20 ml) was added to 100 mlof the cell-free growth medium. Samples were shaken for 12 h(24 C, 100 rpm) in the complete absence of light. For the chemicalblanks, standard nutrition solution was used instead of culturesupernatants. After extraction with pentane/dichloromethane andconcentration in vacuum, biodegradation of b,b-carotene was

monitored by means of high performance liquid chromatographywith diode array detection (HPLC/DAD) whereas the formation ofvolatiles was observed by means of gas chromatography/flameionization detector (GC/FID) and gas chromatography/mass spec-trometry (GC/MS).

Ultra-filtration

Mycelium-free culture supernatants were concentrated ten-fold byuse 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 weremixed with 1.5 mg of solubilized b,b-carotene and incubated at28 C and 80 rpm. Samples (1.0 ml) were drawn in frequentintervals and degradation of b,b-carotene was monitored photo-metrically at 450 nm.

Partial characterization ofb,b-carotene-cleaving enzyme activitiesof M. scorodonius

Enzyme assay

One hundred l ofb,b-carotene stock solution were added to 1.5 mlof enzyme-containing sample solution. Oxygen, which is needed asa co-substrate for the oxidative cleavage of carotenoids, wasprovided by the air-saturated sample or buffer solutions, respec-tively. The time-dependent decrease of absorbance was monitoredat 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-

ed according to the following equation: mUmL1

DEVtVs de

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[U=enzyme activity catalyzing the degradation of 1 mol b,b-carotene per minute; DE=decrease of absorbance per minute;Vt=total volume in cuvette (ml); Vs=sample volume in cuvette (ml);e=extinction coefficient ofb,b-carotene, l=450 nm (the extinctioncoefficient ofb,b-carotene in aqueous emulsion was determined tobe 95,000 l mol 1cm1, n=10, CV=1.7%); d=thickness of cuvette(cm)]. Applicability of the enzyme test was checked by dilutingaliquots of the sample to volume with buffer solution (0.1 M citrate/phosphate buffer, pH 5.0). A linear correlation between activity andsample amount was ascertained.

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The temperature optimum was determined by adding 0.1 ml ofb,b-carotene solution to 1.5 ml of cell-free growth medium of M.scorodonius and measuring the time-dependent decrease of absor-bance at varying temperatures.

For determination of the pH optimum, cell-free nutrient mediumof M. scorodonius was concentrated tenfold by means of ultrafil-tration. For each measurement, 0.2 ml of the concentrated retentatewere diluted with 1.3 ml of buffer solutions of varying pH. Onehundred l of b,b-carotene stock solution were added to eachcuvette and the decrease in absorbance was monitored photomet-

rically at 450 nm.

Gas chromatography/mass spectrometry

A Hewlett-Packard HP 5890 gas chromatograph equipped with anon-column injector (40 C) was directly coupled to an HP 5989mass spectrometer. Separation of volatiles was achieved on a CP-5CB fused silica column (30 m0.32 mm i.d., film thickness0.25 m, Varian, Germany), using helium as carrier gas (3.2 mlmin1). The temperature program was as follows: 5 min isothermalat 50 C, raised to 250 C at 4 C/min. The temperature of the ionsource was 250 C, the electron energy for all EI mass spectra was70 eV. Degradation products of b,b-carotene were identified bycomparing their Kovats indices and mass spectra with publisheddata (e.g. Hohler 1986; Krammer et al. 2002) and, if available, withauthentic reference materials.

Gas chromatography/flame ionization detector

A Trace GC 2000 Thermoquest, USA), equipped with a split-splitless injector (220 C) and a flame ionization detector (250 C)was applied for quantitative analyses. Separation of volatiles wasachieved on a DB 5 column (30 m0.25 mm i.d., film thickness0.25 m, J and W, Germany). Hydrogen was used as carrier gas at aflow rate of 2.1 ml min1 and the same temperature program wasused as for GC/MS.

High performance liquid chromatographywith diode array detection

For HPLC separation, a Nucleosil 1205 analytical column(Macherey and Nagel, Germany; 250 x 4 mm) with 5-m C18reversed-phase material including a pre-column (C18, 10 x 4 mm, 5m) was used. The mobile phase consisted of mixtures of methanol/water (containing 10 mM ammonium acetate) (70:30 v/v) (A) andmethanol/dichloromethane 80:20 v/v (B), starting with 100 % A,followed by a gradient to obtain 100 % B after 15 min, and isocratic100 % B from 15 to 45 min at a flow rate of 0.6 ml/min. Theinjection volume was 20 l. Detection was carried out with an UV/vis-photodiode array detector MD 910 (Jasco, Germany).

Results

Screening procedure

To identify microorganisms suitable for the production ofcarotenoid derived flavor compounds, more than 50 fungiand yeasts (a complete list of all strains is available uponrequest) were screened for their capability to cleave b,b-

carotene. Cultivation of the strains on a special b,b-carotene-containing growth agar proved to be an adequatetest system as positive strains could be detected visuallyby color fading after 12 weeks. Whilst no bleaching wasobserved with most of the species, ten strains discoloredthe agar in the region covered by mycelium, or formed ableaching zone around the fungus, indicating efficientdegradation of b,b-carotene (Table1). All of the b,b-

carotene-degrading strains discovered in the screeningwere further investigated for their capability to cleaveb,b-carotene to aroma compounds.

Biotransformation ofb,b-carotene in submerged cultures

b,b-Carotene, emulsified with Tween 40, was added tosubmerged cultures of the respective fungi or yeasts.Growth of the microorganisms was not or only marginallyinhibited by b,b-carotene and Tween 40, as indicated byparallel cultivations without emulsified b,b-carotene andwith Tween 40 only. Non-inoculated media supplemented

with emulsified b,b-carotene served as chemical blanks.To detect volatile degradation products over the entirecultivation period, complete cultures were analyzed daily(yeasts) or on every second to third day (Asco- andBasidiomycetes), respectively. Although b,b-carotenewas apparently degraded, no volatile breakdown productswere detected with Cryptococcus laurentii, Cyathuspallidus, Ischnoderma benzoinum, Marasmius scorodo-nius, Phaffia rhodozyma, and Trametes versicolor. Dihy-droactinidiolide was detectable in low yields as the soleconversion product of b,b-carotene in the cultures ofGanoderma applanatum, Hypomyces odoratus, Kuehner-omyces mutabilis, and Trametes suaveolens. No volatile

b,b-carotene-derived products were detected under con-ditions with the chemical blanks and with non-supple-mented cultures of the respective fungi.

Biotransformation ofb,b-carotenein mycelium-free culture media

Discoloration of the carotene agar appeared outside thefungal mycelium (Table 1), indicating secretion of b,b-carotene-degrading extracellular enzymes and/or media-tors into the surrounding growth medium. Thus, in analternative approach, mycelium-free growth media wereapplied for the bioconversion of b,b-carotene. All of thestrains that tested positively in the screening werecultivated as described, and the mycelia were separatedfrom the growth media by centrifugation prior to theaddition ofb,b-carotene. Nearly complete degradation of

Table 1 Microorganisms de-grading b,b-carotene whengrown on b,b-carotene-contain-ing agar plates. + Pronouncedbleaching zone around the my-celium, +/ weak color fading

Cryptococcus laurentii CBS 318 +/ Kuehneromyces mutabilis DSM 1013 +Cyathus pallidus CBS 376.80 + Marasmius scorodonius CBS 850.87 +Ganoderma applanatum CBS 250.61 + Phaffia rhodozyma CBS 5905 +/ Hypomyces odoratus CBS 764.68 + Trametes suaveolens DSM 5237 + Ischnoderma benzoinum CBS 311.29 + Trametes versicolor WAG EIK 39 +

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Kinetics ofb,b-carotene degradation

Kinetic investigations of the b,b-carotene degradation bymycelium-free culture media of M. scorodonius indicateda rapid reaction profile. About 40% of the initially addedb,b-carotene had been degraded 60 min after the start ofincubation (Fig. 2). Degradation ofb,b-carotene with thechemical blanks (using water instead of cell-free medium)

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motif should result from the two-fold excentrical cleavageofb,b-carotene. However, maximum overall molar yieldsof about 25% were reached in the cell-free bioconver-sions. The reason for this balance gap is not yet clear.Biochemical and molecular biological investigations arecurrently in progress to isolate and characterize theenzymes responsible for carotenoid degradation. In pre-liminary studies, a temperature of 27 C and a pH of 5.0

provided optimal conditions for the b,b-carotene degra-dation by extracellular enzymes ofM. scorodonius. Thesecomparatively mild reaction conditions together with afast reaction profile turn cell-free culture supernatants ofselected basidiomycetes into a promising tool for thebiotechnological production of norisoprenoid aromacompounds. Further research is required to gain insightinto the mechanism of the cleavage reaction and theparticular properties of the enzymes involved.

Acknowledgements Support of the work by the DeutscheForschungsgemeinschaft (ZO 122/11) is gratefully acknowledged.All experiments comply with the current laws of the FederalRepublic of Germany.

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Cleavage of b,B-carotene to Flavor Compounds by Fungi

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