Food Chemistry 141 (2013) 2952–2959

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Food Chemistry

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Formation of complex natural flavours by biotransformation of applepomace with basidiomycetes

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.05.116

⇑ Corresponding author. Tel.: +49 (0) 641 99 34 900; fax: +49 (0) 641 99 34 909.E-mail address: holger.zorn@lcb.chemie.uni-giessen.de (H. Zorn).

Andrea K. Bosse, Marco A. Fraatz, Holger Zorn ⇑Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology, Heinrich-Buff-Ring 58, 35392 Giessen, Germany

a r t i c l e i n f o

Article history:Received 2 April 2013Received in revised form 23 May 2013Accepted 27 May 2013Available online 5 June 2013

Keywords:BasidiomycetesApple pomaceBiotransformationTyromyces chioneus3-Phenylpropanal

a b s t r a c t

Altogether 30 different basidiomycetes were grown submerged in liquid culture media using seven dif-ferent by-products of the food industry as the only carbon source. Seven fungus/substrate combinationsrevealed interesting flavour profiles. Culture supernatants of Tyromyces chioneus grown on apple pomacewere extracted, and the aroma compounds were analysed by gas chromatography–olfactometry (GC–O).Potent odorants were identified by aroma extract dilution analysis (AEDA), calculation of the odour activ-ity values (OAV), and proven by confection of an aroma model. 3-Phenylpropanal, 3-phenyl-1-propanol,and benzyl alcohol were identified as potent aroma biotransformation products. Headspace solid-phasemicroextraction gas chromatography mass spectrometry (HS-SPME–GC–MS) experiments showed that3-phenylpropanal, 3-phenyl-1-propanol, benzyl alcohol, methyl 3-phenylpropionate, methyl 2-phenylac-etate, cinnamaldehyde and methyl cinnamate were produced during the cultivation period of eight days.By means of labelling experiments, (E)-cinnamic acid was identified as the precursor of 3-phenylpropanaland 3-phenyl-1-propanol. Basidiomycetes were able to biotransform food by-products to pleasant com-plex flavour mixtures.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The industrial demand for natural flavours is constantlyincreasing (Guentert, 2007). Concurrently, the volume of availableby-products of the food industry is growing, as food products aremore and more processed and ready-made. Many by-products con-tain valuable components, such as lipids or amino acids, whichmay act as biogenetic precursors of flavour compounds. Article3.2(c) of the Regulation (EC), No 1334/2008 considers a ‘‘naturalflavouring substance’’ as ‘‘a flavouring substance obtained byappropriate physical, enzymatic or microbiological processes frommaterial of vegetable, animal or microbiological origin, either inthe raw state or after processing for human consumption by oneor more of the traditional food preparation processes listed in An-nex II. Natural flavouring substances correspond to the substancesthat are naturally present and have been identified in nature’’. Fer-mentation of by-products of the food industry with traditionalstarter cultures is not very promising, as e.g., yeasts of the genusSaccharomyces form many, but barely complex and intense flavourcompounds (Carrau et al., 2008).

Basidiomycetes, which comprise almost all edible mushrooms,possess a unique extracellular enzyme system (the so-called secre-tome) and have already been shown to produce bioflavours de novo

as well as by biotransformation (Bouws, Wattenberg, & Zorn, 2008;Fraatz & Zorn, 2010). De-novo-produced flavour compounds ofbasidiomycetes are chemically identical to flavour substances iso-lated from plants. Pleurotus sapidus for example was able to trans-form the sesquiterpene hydrocarbon valencene into the grapefruitflavour nootkatone (Fraatz et al., 2009).

In 2010, 69.6 million tons of apples were produced worldwide(Food and Agriculture Organization, 2012). 25-30% Were pro-cessed to juice, cider, and frozen or dried products. Apple juiceconcentrate represents the major processed product, with a totalrecovery of 70-75% in the industrial process. Therefore, 25-30% ofapple pomace remains as a side-stream (Bhushan, Kalia, Sharma,Singh, & Ahuja, 2008). In Germany, approximately 800 kt of ap-ples are processed to apple juice annually, and 200 kt of applepomace accrue (Binnig, 2001). These by-products do not onlycause disposal costs, but also are a major environmental problem(Bhushan et al., 2008). Currently, they are used as feed (50 kt), forthe production of biogas (50 kt), and for the isolation of pectin(100 kt) (Binnig, 2001).

Apple pomace has also been suggested for the extraction of die-tary fibres, proteins, natural antioxidants, biopolymers and pig-ments (Bhushan et al., 2008). Furthermore, it may be employedfor the production of enzymes, organic acids, ethanol, single cellproteins (Bhushan et al., 2008), edible mushrooms, feeds (Vendrus-colo, Albuquerque, Streit, Esposito, & Ninow, 2008), and aromacompounds (Bramorski, Soccol, Christen, & Revah, 1998).

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A.K. Bosse et al. / Food Chemistry 141 (2013) 2952–2959 2953

In the present study, the ability of basidiomycetes to transformby-products of the food industry to complex flavour mixtures wasinvestigated. The well-known concept of fermentative flavour pro-duction by traditional food biotechnology was translated to thecultivation of the biochemically complex basidiomycetes. Cultureswere extracted by liquid/liquid extraction as well as by headspacesolid-phase microextraction coupled to gas chromatography massspectrometry (HS-SPME–GC/MS). The flavour profiles of theliquid/liquid extracts were investigated by aroma extract dilutionanalysis (AEDA), and the odour activity values (OAVs) werecalculated.

2. Materials and methods

2.1. Microorganisms

The fungi were obtained from the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Brunswick, Germany)and the Centraalbureau voor Schimmelcultures (CBS, Utrecht, TheNetherlands).

2.2. Substrates

The by-products apple pomace, broken waffle, broken cake, co-coa shells, cocoa powder, coffee grounds, and wine pomace (Gewü-rztraminer) were received from different industrial partners andwere used as substrates for submerged cultivation.

2.3. Chemicals

Solvents were purchased from Fisher Scientific (Schwerte, Ger-many) and Carl Roth (Karlsruhe, Germany). All solvents were dis-tilled before use. High-purity water was prepared with alaboratory water system (Sartorius, Goettingen, Germany). Sodiumchloride and benzyl alcohol were obtained from AppliChem(Darmstadt, Germany). 3-Phenylpropanol, 3-phenylpropanal,BME Vitamins 100� solution, benzaldehyde, (E)-D7-cinnamic acid,a-farnesene and (E)-D7-cinnamic acid were purchased from Sig-ma–Aldrich (Taufkirchen, Germany), 1H-pyrrole-2-carboxalde-hyde and (E)-cinnamic acid from Acros Organics (Geel, Belgium),and acetic acid, sodium sulfate and thymol from Carl Roth. Allchemicals were of analytical grade.

2.4. Submerged cultures

Apple pomace, broken waffle, broken cake, cocoa shells, cocoapowder, coffee ground and wine pomace (Gewürztraminer) wereused as substrates for the submerged cultures and were stored at�20 �C prior to usage. Cocoa powder, cocoa shells and wine pom-ace were used untreated. Apple pomace, broken waffle, brokencake and coffee ground were freeze dried (�25 �C, 0.63 mbar,4 days, moisture content

Fig. 1. Flow chart of the screening of fungi and by-products for flavour production.

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OAV: Odor activity valuesc: concentration of the odorant [lg L�1]a:odour threshold in water [lg L�1]

2.9. Aroma model of the flavour extracts

As a matrix for the recombination experiments pentane/diethylether (1/1.12 v/v) was selected. The recombinate was composedaccording to the determined concentrations of the odorants benz-aldehyde, 3-phenylpropanal, 3-phenylpropanol, and benzyl alco-hol. One millilitre of the aroma model was transferred intoamber glass vials with a screw-cap, containing a piece of scentlessfilter paper. This model was compared to the flavour extracts ob-tained by the biotransformation of apple pomace by T. chioneusand evaluated by 5 skilled persons. The intensity was determinedas described in Section 2.5.

2.10. Determination of odour threshold values

Odour threshold values were determined in water. 3-Phenyl-propanal and 3-phenylpropanol were dissolved in ethanol andthen diluted with water. The water/ethanol mixture was testedfor lack of odour. The samples were analysed in order of increasingconcentrations in 1:1 dilution steps, and the odour threshold of thetest groups was calculated from the quadratic mean of the individ-ual odour thresholds according to § 64 (Amtliche Methoden 1999,2007) LFGB methods volume I (L) L 00.09–7 and L 00.09–9.

2.11. Capillary gas chromatography–mass spectrometry (GC/MS)

2.11.1. GC–MS after liquid/liquid extractionHigh resolution GC–MS was conducted on an Agilent 7890A gas

chromatograph equipped with an Agilent 5975C MSD triple-axisdetector. Agilent Technologies J&W Scientific HP-Innowax(0.25 lm film thickness, 30 m � 0.25 mm i.d.) and Agilent Technol-ogies DB-5MS columns (30 m � 0.25 mm i.d., 0.25 lm film thick-ness), and a split/splitless injector (250 �C, split 1:10) were used.The following temperature programs were employed: 40 �C

(3 min), 5 �C min�1 to 240 �C (12 min) (polar column), and 40 �C(3 min), 5 �C min�1 to 240 �C (12 min), 20 �C min�1 to 300 �C(7 min) (non-polar column).

Helium was used as carrier gas (1.2 mL min�1, constant flow).The ion source was set to 230 �C, the interface to 250 �C, and thequadrupole to 150 �C. The electron impact energy was 70 eV. Sys-tem software, data management, and analysis were controlled byChemStation (E.02.00.493).

Compounds were identified by comparison of their mass spec-tra with those of authentic standards and to the database NIST2008 MS LIB. The concentrations were estimated by the internalstandard method, using thymol as internal standard. The responsefactor (Rf) was calculated for each analyte according to the follow-ing equation:

Rf ¼ AIS �mamIS � Aa

ð2Þ

where A = peak area, m = mass (lg), IS = internal standard,a = analyte.

2.11.2. HS-SPME–GC–MSHS-SPME analyses were performed with a split/splitless injector

(250 �C, splitless time: 5 min) using an Agilent Technologies J&WScientific HP-Innowax column (30 m � 0.25 mm i.d., 0.25 lm filmthickness) under the conditions described above. Compounds wereidentified by comparison of their mass spectra with those of thedatabase NIST 2008 MS LIB and were confirmed by calculation oftheir respective Kováts indices.

3. Results and discussion

3.1. Screening

In a broad screening, 30 different basidiomycetes were grownon seven different by-products of the food industry (Fig. 1). Alto-gether, seven fungus/substrate combinations imparted interestingflavour impressions (Fig. 1). Due to the intense and unique flavour,

Fig. 2. (I) GC/FID/O chromatogram of the biotransformation of apple pomace by T. chioneus (day 4) with FID (black) and ODP signal (red); ⁄internal standard and (II) potentodorants (flavor dilution chromatogram) formed during the biotransformation. (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

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the cultures of T. chioneus grown on freeze-dried apple pomace assubstrate were chosen for further analysis.

After four days, these cultures emitted an odour reminiscent ofstewed fruit, sweetish and plum purée. The olfactory impressionsof the corresponding blanks of T. chioneus and of lyophilised applepomace differed significantly from those of the biotransformations.The blank cultures of T. chioneus smelled sweetish and musty,while the substrate blanks smelled like apples and freshly-squeezed apple pomace.

3.2. Flavour analysis

Fourteen flavour compounds were detected by means of GC–Oin the liquid/liquid extracts in two independent runs (Fig. 2). Sevencompounds were identified by comparison of Kováts indices andmass spectra with those in the database (NIST 2008 MS LIB) and

confirmed by authentic standards on a polar and a non-polar GCcolumn. FD-factors were determined by AEDA (Fig. 2; Table 1).3-Phenylpropanal (FD-factor 128; OAV 94) was identified as themost potent odorant (Table 2). The odour of 3-phenylpropanalhas been described as ‘‘green and flowery’’ (Ranson & Belitz,1992). Apart from 3-phenyl-1-propanol (FD-factor 8, OAV 30),benzyl alcohol (FD-factor 32, OAV 17) contributed significantly tothe overall flavour of the cultures. Benzyl alcohol exhibits a floweryand fruity flavour (Fang & Qian, 2005). The influence of acetic acidwith an OAV smaller than one was negligible. 3-Phenylpropanal,benzyl alcohol, and 3-phenyl-1-propanol were formed bybiotransformation and shaped the sweetish, plum-purée-likeflavour. While 3-phenyl-1-propanol and benzyl alcohol are wellknown basidiomycetes biotransformation products, 3-phenylprop-anal has not been reported as a flavour compound formed bybasidiomycetes yet. The basidiomycete Bjerkandera adusta

Table 1Volatile flavour compounds of the biotransformation of apple pomace by T. chioneus with FD-factors and Kováts indices (KI) in increasing elution order on an HP-Innowax column.

No. Compounda Odor descriptionb KI GC-OHP-Innowax

KI GC-MSHP-Innowax

KI GC–MS DB5-MS

FD-factor

1 n.i. Sweetish fruity 912 22 Acetic acid Vinegar 1447 1465

Fig. 3. (A) Formation of phenylpropanoic and benzoic volatile compounds in the biotransformation of apple pomace by T. chioneus during the cultivation period of 8 days. (B)Formation of minor compounds.

A.K. Bosse et al. / Food Chemistry 141 (2013) 2952–2959 2957

Furthermore, methyl 3-phenylpropionate, methyl 2-phenylacetate,cinnamaldehyde, and methyl cinnamate were detected. Methyl 3-phenylpropionate was found in cooked pine mushrooms andmethyl 2-phenylacetate in the hydrodistillates of the basidiomy-cete Gloeophyllum odoratum (Cho, Choi, & Kim, 2006; Rösecke &König, 2000). Cinnamaldehyde contributed to the pungent andspicy flavour of the medicinal mushroom Hericium erinaceus (pompom mushroom) (Abraham & Berger, 1994). In the culture brothsof Lenzites frabea, Poria subvermispora, Hirschioporus pergamenus,Poria subacida, and Vararia effuscata, up to 250 lg L-1 methyl cinna-mate were found, depending on the media composition (Gallois,Gross, Langlois, Spinnler, & Brunerie, 1990).

3.4. Cinnamic acid as a precursor of 3-phenylpropanal and3-phenylpropanol

To identify the precursor and to elucidate the biogenetic path-way of 3-phenylpropanal and 3-phenyl-1-propanol, (E)-cinnamicacid and (E)-D7-cinnamic acid (1 mM) were added to the culturemedia of T. chioneus. The culture supernatants were extracted bymeans of liquid/liquid extraction and analysed by GC–MS. Themass spectra were compared to the mass spectra of authenticstandards.

The addition of (E)-cinnamic acid led to a ten times increasedformation of 3-phenylpropanal and 3-phenyl-1-propanol (Fig. 4).Additionally, 2-phenyl-1-ethanol, maltol and cinnamaldehydewere detected in these cultures. In oven-dried apple pomace,�180 mg kg�1 cinnamic acid was detected (Bai, Yue, Yuan, &Zhang, 2010). This represents a sufficient amount to explain theformation of the C6–C3 biotransformation products. To elucidatethe potential role of L-phenylalanine as an additional precursor of3-phenylpropanal and 3-phenyl-1-propanol, 10 mM of l-phenylal-

anine were added to the culture media of T. chioneus. In contrast tothe addition of (E)-cinnamic acid, no enhanced formation of 3-phe-nylpropanal and 3-phenyl-1-propanol was observed in the corre-sponding cultures.

The mass spectrum of 3-phenylpropanal after supplementationwith (E)-D7-cinnamic acid showed an intensive molecular ions(M+�) at m/z 140 and 141 compared to the non-labelled referencecompound with an M+� at m/z 134 (Fig. 4). The ratio of analyte Ato the labelled analyte Ad6+d7 was 40/757, which represented a pro-portion of labelled 3-phenylpropanal of 95%. This confirmed thereduction of cinnamic acid to 3-phenylpropanal. The massspectrum of 3-phenyl-1-propanol after supplementation with(E)-D7-cinnamic acid revealed an intense M+� at m/z 142 and 143.compared to the non-labelled molecule with an M+� at m/z 136.The ratio of A to Ad6+d7 was 5/294, which indicated 98% labelled3-phenyl-1-propanol. Likewise, 3-phenyl-1-propanol was thebiotransformation product of cinnamic acid.

The carboxyl group of (E)-cinnamic acid was reduced to cinna-maldehyde. The Ca–Cb double bound was hydrogenated, and thealdehyde was subsequently reduced to the corresponding alcohol.All of the metabolites of the postulated pathway were detected inthe cultures supplemented with (E)-cinnamic acid. A similar bio-synthetic pathway from cinnamic acid via cinnamaldehyde andcinnamic alcohol to 3-phenyl-1-propanol was presumed in thewhite rot mushroom Schizophyllum commune (Nimura et al.,2010).

This reaction pathway is commonly catalysed by two classes ofenzymes: aldehyde oxidoreductases and alcohol dehydrogenases(Van den Ban et al., 1999). The reduction of aromatic acids andthe release of aldehydes by the breakdown of lignin has alreadybeen reported (Hage, Schoemaker, & Field, 1999). Numerous whiterot mushrooms are able to reduce aryl acids to the corresponding

Fig. 4. (I) Concentrations of the biotransformation products of apple pomace by T.chioneus supplemented with (E)-cinnamic acid compared to the cultures withoutaddition of cinnamic acid, and (II) mass spectrum of 3-phenylpropanal aftersupplementation of (E)-D7-cinnamic acid (1 mM) as a precursor to the biotransfor-mation of apple pomace by T. chioneus (B) in comparison to the mass spectrum ofthe non-labelled standard 3-phenylpropanal (A).

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aldehydes and alcohols. The reduction of the aldehyde to the alco-hol is catalysed by the NADPH-dependent enzyme aryl alcoholdehydrogenase. The aryl alcohols serve as substrates for the extra-cellular enzyme aryl alcohol oxidase (AAO), which produces theH2O2 required for peroxidase activity (Hage et al., 1999).

Oxidative enzymes of white rot mushrooms have been studiedintensely. On the other hand, only little is known about reductiveenzymes. Up to now, a number of quinone reductases wereidentified, like 1,4-benzoquinone reductase, which reduces qui-nones to hydroquinones by a ping-pong steady state (Brock & Gold,1996). A xylose reductase was identified in the fungus Cryptococcusflavus, which can reduce d-xylose NADPH dependent to xylitol(Mayr, Petschacher, & Nidetzky, 2003). A membrane-associatedactivity of an aromatic nitroreductase was found in extracts ofPhanerochaete chrysosporium. The extracts reduced the nitrogroups of 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotolu-ene, 1-chloro-2,4-dinitrobenzene, and 2,4-dichloro-1-nitrobenzene(Rieble, Joshi, & Gold, 1994).

An aldehyde oxidoreductase has not been isolated from basidi-omycetes yet. Li and Rosazza (1997) purified and characterized a163 ± 3.8 kDa aldehyde oxidoreductase from the gram-negativebacterium Nocardia sp. NRRL 5646, which was able to reduce arylcarboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and poly-aromatic ring carboxylic acids to the corresponding aldehydes.

4. Conclusions

A pleasant flavour mixture reminiscent of stewed fruit andplum purée was generated by the biotransformation of apple pom-ace using submerged cultures of the basidiomycete T. chioneus

after four days. Fourteen flavour compounds were detected byGC–O in the liquid/liquid extracts of the culture media. Seven com-pounds were identified (acetic acid, benzaldehyde, a-farnesene, 3-phenylpropanal, benzyl alcohol, 1H-pyrrole-2-carboxaldehyde and3-phenyl-1-propanol). The biotransformation products 3-phenyl-propanal, 3-phenyl-1-propanol, and benzyl alcohol were identifiedas the most potent biotransformation products. HS-SPME–GC–MSanalyses of the culture broths over the cultivation period of eightdays revealed that a-farnesene, benzaldehyde and linalool werepartially degraded, while 3-phenylpropanal, 3-phenyl-1-propanol,benzyl alcohol, methyl 3-phenylpropionate, methyl 2-phenylace-tate, cinnamaldehyde, and methyl cinnamate were formed. (E)-Cinnamic acid was identified as the precursor of 3-phenylpropanaland 3-phenyl-1-propanol. Basidiomycetes successfully trans-formed by-products of the food industry to complex and highlyinteresting natural flavour mixtures.

Acknowledgements

The authors thank the FEI (Forschungskreis der Ernährungsin-dustrie e.V.) and AiF (Arbeitsgemeinschaft industrieller Fors-chungsvereinigungen ‘‘Otto von Guericke’’) for funding theresearch project AiF 299 ZN and Katja Fast for her support in theolfactometric evaluation. Part of the work was supported by theexcellence initiative of the Hessian Ministry of Science and Artwhich encompasses a generous grant for the LOEWE research focus‘integrative fungal research’’.

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Formation of complex natural flavours by biotransformation of apple pomace with basidiomycetes1 Introduction2 Materials and methods2.1 Microorganisms2.2 Substrates2.3 Chemicals2.4 Submerged cultures2.5 Sensory evaluation2.6 Sample preparation2.6.1 Liquid/liquid extraction2.6.2 Headspace solid-phase microextraction (HS-SPME)

2.7 Capillary gas chromatography–olfactometry (GC–O)2.8 Aroma extract dilution analysis (AEDA) and odour activity values (OAV)2.9 Aroma model of the flavour extracts2.10 Determination of odour threshold values2.11 Capillary gas chromatography–mass spectrometry (GC/MS)2.11.1 GC–MS after liquid/liquid extraction2.11.2 HS-SPME–GC–MS

3 Results and discussion3.1 Screening3.2 Flavour analysis3.3 Kinetics of flavour formation3.4 Cinnamic acid as a precursor of 3-phenylpropanal and 3-phenylpropanol

4 ConclusionsAcknowledgementsReferences