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Feb 11, 2017 - ABSTRACT: The basidiomycete Pleurotus sapidus (PSA) was grown in submerged cultures with molasses as substrate for the production of ...
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Submerged Cultivation of Pleurotus sapidus with Molasses: Aroma Dilution Analyses by Means of Solid Phase Microextraction and Stir Bar Sorptive Extraction Tobias Trapp,† Martina Zajul,† Jenny Ahlborn,† Alexander Stephan,†,§ Holger Zorn,† and Marco Alexander Fraatz*,† †

Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Heinrich-Buff-Ring 17, 35392 Giessen, Germany § VAN HEES GmbH, Kurt-van-Hees-Strasse 1, 65396 Walluf, Germany S Supporting Information *

ABSTRACT: The basidiomycete Pleurotus sapidus (PSA) was grown in submerged cultures with molasses as substrate for the production of mycelium as a protein source for food applications. The volatilomes of the substrate, the submerged culture, and the mycelia were analyzed by gas chromatography-tandem mass spectrometry-olfactometry. For compound identification, aroma dilution analyses by means of headspace solid phase microextraction and stir bar sorptive extraction were performed via variation of the split vent flow rate. Among the most potent odorants formed by PSA were arylic compounds (e.g., p-anisaldehyde), unsaturated carbonyls (e.g., 1-octen-3-one, (E)-2-octenal, (E,E)-2,4-decadienal), and cyclic monoterpenoids (e.g., 3,9-epoxy-pmenth-1-ene, 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one). Several compounds from the latter group were described for the first time in Pleurotus spp. After separation of the mycelia from the medium, the aroma compounds were mainly enriched in the culture supernatant. The sensory analysis of the mycelium correlated well with the instrumental results. KEYWORDS: Pleurotus sapidus, submerged cultivation, headspace solid phase microextraction, stir bar sorptive extraction, olfactometry, aroma dilution analysis

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INTRODUCTION As the world population is expected to significantly increase, it is a global challenge to ensure a sufficient food and, especially, protein supply.1 From that, the necessity arises to develop affordable alternative protein sources suitable for human consumption. Edible basidiomycetes with their unique nutritional properties represent promising vegan candidates for this purpose. Submerged cultivation is a highly efficient and well controllable technology for growing fungi.2 Various industrial applications based on submerged-grown fungi have already been established, such as the production of secondary metabolites for food or pharmaceutical purposes.2,3 From a nutritional point of view, the fast production of biomass is a highly beneficial aspect of submerged cultivation.4 As a rich source of protein, the obtained basidiomycetous mycelium is a promising new functional ingredient for food applications, such as vegan meat substitutes.5,6 A limiting factor of submerged cultivation is the cost of the substrate. By using side-streams from the food industry (e.g., pomace or molasses) as substrates, the costs may be reduced because basidiomycetes are able to upcycle them.7 This goes along with the striving of researchers and food manufactures to lower the ecological footprint by producing less waste and using resources more efficiently.8 Previously, selected basidiomycetes cultivated with various industrial side-streams were screened in regard to growth and aroma production.7 Among these combinations, Pleurotus sapidus (PSA) showed quick biomass production when cultivated © 2017 American Chemical Society

with isomaltulose molasses (IM). IM is a side-stream of the sugar industry, which is currently still disposed of due to the lack of economically viable applications. The thus obtained mycelium already possessed acceptable color and structural properties. P. sapidusa close relative of the oyster mushroom (Pleurotus ostreaus)has been intensively investigated for various biotechnological applications. For instance, PSA converts a multitude of terpenes via oxyfunctionalization and is thus able to produce high-value aroma compounds, such as nootkatone and carvone.9−12 However, to the best of our knowledge, an overall aroma profile analysis of P. sapidus has not yet been published. As the basidiomycetous mycelium is intended to be used in meat substitutes, analysis of the formed aroma compounds is highly relevant. The present study thus focused on the identification of odor-active compounds formed by PSA cultivated with IM as substrate and the determination of the most relevant aroma compounds. For this purpose, aroma dilution analysis (ADA) procedures by means of headspace solid phase microextraction (HS-SPME) and direct immersion stir bar sorptive extraction (SBSE) were developed and compared. To the best Special Issue: 11th Wartburg Symposium on Flavor Chemistry and Biology Received: Revised: Accepted: Published: 2393

November 25, 2016 February 6, 2017 February 11, 2017 February 11, 2017 DOI: 10.1021/acs.jafc.6b05292 J. Agric. Food Chem. 2018, 66, 2393−2402

Article

Journal of Agricultural and Food Chemistry

ments solution (FeCl3·6H2O (80 mg/L), ZnSO4·7H2O (90 mg/L), MnSO4·H2O (30 mg/L), CuSO4·5H2O (5 mg/L), EDTA (400 mg/L)). For the main culture, 200 mL of the autoclaved main culture medium (pH 6.0), 20 mL of separately autoclaved isomaltulose molasses (pH 6.0), and 20 mL of the preculture were added and homogenized (UltraTurrax; 30 s, 10,000 rpm). The main culture was grown at 24 °C and 150 rpm in the dark for 6 days and stored at −20 °C afterward. Preparation of Culture and Substrate Samples. The submerged cultures were analyzed before separation and after being separated into a liquid phase (culture medium) and a solid phase (mycelium). For analysis of the nonseparated submerged cultures, CaCl2 was added (20%, w/w). After homogenization with an Ultra-Turrax (5 s, 10,000 rpm), the material was transferred into the respective vials (see below) and immediately frozen (−20 °C) until extraction (“homogenized submerged culture”). To separate culture medium and mycelium, the submerged culture was centrifuged for 5 min at 3000g and 4 °C. The liquid culture medium was removed and used for analysis after the addition of CaCl2 (20%, w/w). The culture medium was replaced by the same amount of distilled water (4 °C) to resuspend the mycelium. The suspension was centrifuged under the same conditions, and the washing water was discarded afterward. The washing was repeated twice. Afterward, the mycelium was ground with a pestle and mortar under liquid nitrogen. The disrupted mycelium was resuspended in an aqueous solution of CaCl2 (20%, w/w). The dry matter content was adjusted to that of the homogenized submerged culture. After treatment with an Ultra-Turrax (5 s, 10,000 rpm), the material was transferred into vials and immediately frozen (−20 °C) until GC analysis (“disrupted mycelium”). For analysis of the substrate, IM was autoclaved prior to sampling. Headspace Solid Phase Microextraction. For HS-SPME analysis, standard solution (2 mL), PSA-IM samples (2 g), or autoclaved molasses (2 g) were added to a 20 mL headspace vial and sealed with a silicone/PTFE screw cap. The samples were incubated for 15 min, followed by a headspace extraction for 45 min by means of an MPS 2XL multipurpose sampler (GERSTEL). The SPME fibers (length 1 cm) (Supelco, Steinheim, Germany) were coated with the following sorbent materials: PDMS/DVB (polydimethylsiloxane/ divinylbenzene, 65 μm) and DVB/CAR/PDMS (divinylbenzene/ carboxen/polydimethylsiloxane, 50/30 μm). Incubation and extraction were performed at 40 °C with an agitation rate of 250 rpm. The analytes were desorbed in an SPME liner (0.75 mm i.d.) (Supelco) within the inlet of the GC system at 250 °C for 90 s. Afterward, the fibers were baked out at the recommended conditioning temperature in the needle heater station of the MPS 2 XL. Stir Bar Sorptive Extraction. For SBSE analysis, 10 mL vials were completely filled with the standard solution or the PSA-IM samples. Ten millimeter stir bars with 0.5 mm PDMS coatings (Twister, GERSTEL) were added, and the vials were sealed with silicone/PTFE caps. The samples were magnetically stirred at 1000 rpm in a water bath adjusted to 22 °C for 3 h. For method development, extraction temperatures of 30 and 40 °C were tested as well. After extraction, the stir bars were removed with forceps, rinsed with deionized water, carefully dried with lint-free tissues, and placed in a conditioned thermal desorption unit (TDU) liner (GERSTEL). The TDU temperature was held at 40 °C for 0.5 min, then raised at 120 °C/min to 250 °C, and held for 10 min to desorb the stir bars in splitless mode. The analytes were cryo-focused in a Cold Injection System 4 (CIS) (GERSTEL) equipped with a Tenax liner (GERSTEL) in solvent−vent mode (40 mL/min). The CIS was heated at 12 °C/s from −100 to 250 °C and held for 5 min to release the analytes to the GC column with the selected split vent flow rate. Aroma Dilution Analysis. Aroma dilution analyses with HS-SPME and SBSE were performed by varying the split ratios in the GC inlet.15,16 On the basis of the definition of the split ratio (SR) used by Kim et al., the split vent flow rate V̇ split vent was adjusted according to

of our knowledge, the use of SBSE-GC-O for this purpose has been validated for the first time. The aroma-active volatiles of the substrate, the submerged culture, and the mycelium were compared to monitor the effects of the involved processing steps.

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MATERIALS AND METHODS

Materials and Chemicals. P. sapidus (DSM No. 8266) was obtained from the German Collection of Microorganisms and Cell Cultures (Brunswick, Germany). For the preculture and main culture media, chemicals were purchased from Carl Roth (Karlsruhe, Germany), Sigma-Aldrich (Taufkirchen, Germany), and Th. Geyer (Hamburg, Germany). Isomaltulose molasses was obtained from Suedzucker (Offstein, Germany). Authentic aroma standards of analytical grade were purchased from Alfa Aesar (Karlsruhe, Germany), Biozol (Eching, Germany), Carl Roth, Chempur (Karlsruhe, Germany), Fisher Scientific (Schwerte, Germany), Sigma-Aldrich, TCI Deutschland (Eschborn, Germany), Th. Geyer, and VWR (Darmstadt, Germany). Stereoisomers of 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one and 4,5epoxy-(E)-2-decenal were provided by Nils H. Schebb, University of Wuppertal. Dried dill leaves were bought from a local supermarket. Prior to analysis, calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth) was heated for 3 h at 550 °C to remove impurities. Ethanol (Carl Roth) was used for the preparation of stock solutions of aroma compounds. Citric acid (C6H8O7·H2O) (Carl Roth) and disodium hydrogen phosphate (Na2HPO4·2H2O) (Fisher Scientific) were used for the citrate−phosphate buffer. Helium 5.0 (Praxair, Duesseldorf, Germany) and nitrogen 5.0 (Praxair) were used for gas chromatography. Preparation of Standard Solutions. For method optimization and validation, the following representative authentic aroma standards were chosen (with respective qualifier ions used for analysis): pentanal (m/z 58), ethyl 2-methyl butanoate (m/z 102), ethyl 3-methylbutanoate (m/z 88), hexanal (m/z 56), 3-octanone (m/z 99), octanal (m/z 84), 1-octen-3-one (m/z 70), nonanal (m/z 57), (E)-2-octenal (m/z 57), (E)-2-nonenal (m/z 70), (S)-(+)-carvone (m/z 82), (E,E)-2,4-decadienal (m/z 81), 3-methoxybenzaldehyde (m/z 135), 2-phenylethanol (m/z 91), nerolidol (mixture of (E)- and (Z)-isomers) (m/z 69), (E)-methyl cinnamate (m/z 131), ethyl p-anisate (m/z 135), and 3,4-dimethoxybenzaldehyde (m/z 166). Stock solutions of each standard in ethanol were prepared in equimolar concentrations (20 μmol/mL) and stored at −20 °C. A standard solution was prepared with 100 μL of each stock solution in distilled water (ad 500 mL). One milliliter of the standard solution was transferred to 10 mL vials. The vials were filled completely with distilled water and sealed with a silicone/PTFE screw cap. For variation of the salt concentration of the standard solutions, CaCl2 was weighed directly into the vials. Synthesis and Extraction of References. A mixture of 3,9-epoxyp-menth-1-ene stereoisomers was synthesized according to a modified method described by Strauss et al. and Bonnländer et al.:13,14 1 μL of 10-hydroxygeraniol ((E,E)-2,6-dimethyl-2,6-octadiene-1,8-diol) stock solution (50 mg in 500 μL ethanol) was added to 5 mL of citrate− phosphate buffer (pH 2.5) in a 20 mL headspace vial. The vial was incubated at 95 °C and 250 rpm for 20 min in an agitator module of a Multipurpose Sampler (MPS; GERSTEL, Muelheim an der Ruhr, Germany). After being cooled to room temperature, the vial was extracted by means of HS-SPME and SBSE. Fifty milligrams of dried dill leaves was dispersed in 1 mL of H2O in a 20 mL vial and extracted by means of HS-SPME and SBSE. Preculture and Fermentation. P. sapidus was cultivated with industrial side-streams according to the revised method of Bosse et al.7 A cubic piece of overgrown agar stock culture was transferred to an Erlenmeyer flask (250 mL) containing malt extract medium (2%) and homogenized by means of an Ultra-Turrax T25 homogenizer (IKA, Staufen, Germany) for 30 s at 10,000 rpm. The preculture was grown on a rotary shaker (150 rpm, 25 mm shaking diameter) (Orbitron, Infors, Einsbach, Germany) for 6 days in darkness at 24 °C. The main culture medium contained yeast extract (3.0 g/L), MgSO4·H2O (1.5 g/L), KH2PO4 (0.5 g/L), and 1 mL/L trace ele-

̇ vent = Vcolumn ̇ Vsplit (SR − 1)

(1)

where V̇ column is the column flow rate and SR is the split ratio. 2394

DOI: 10.1021/acs.jafc.6b05292 J. Agric. Food Chem. 2018, 66, 2393−2402

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Journal of Agricultural and Food Chemistry The following split ratios were applied: splitless, 2:1, 4:1, 8:1, 16:1, 32:1, 64:1, 128:1, and 256:1 (splitless and 2:1 only for HS-SPME). The method was validated prior to sample analysis. GC-MS/MS-O Analysis. Gas chromatography was performed with an Agilent 7890A gas chromatograph (Agilent Technologies, Waldbronn, Germany) connected to an Agilent 7000B triplequadrupole mass spectrometry (MS/MS) detector (Agilent Technologies). A polar Agilent J&W VF-WAXms column (30 m × 0.25 mm i.d., 0.25 μm) was used. Helium was used as the carrier gas with a constant column flow rate of 1.56 mL/min. The gas flow was split 1:1 into the MS/MS detector and into an Olfactory Detection Port 3 (ODP) (GERSTEL). The GC oven temperature was held at 40 °C for 3 min, increased at 5 °C/min to 240 °C, and held for 5 min. For the purposes of identification, each sample was additionally analyzed with a modified temperature program (40 °C for 5 min, heating at 3 °C/min, 240 °C for 12 min). Further applied conditions were as follows: septum purge flow rate, 3 mL/min; scan mode, total ion current (TIC) in Q1; scan range, m/z 33−300; electron ionization energy, 70 eV; source temperature, 230 °C; quadrupole temperatures, 150 °C; MS/MS transfer line temperature, 250 °C; He quench gas, 2.25 mL/min; N2 collision gas, 1.5 mL/min; ODP 3 transfer line temperature, 250 °C; ODP 3 mixing chamber temperature, 150 °C; ODP 3 makeup gas, N2. GC-MS analyses were carried out with an Agilent 7890B gas chromatograph (Agilent Technologies) equipped with a 5977B mass spectrometry detector (Agilent Technologies). The following conditions were applied: carrier gas, helium; constant flow rate, 1.2 mL/min; inlet temperature, 250 °C; split ratio, 10:1; septum purge flow rate, 3 mL/min; 30 m × 0.25 mm i.d., 0.25 μm Agilent J&W DB-5ms column; temperature program, 40 °C for 3 min, heating at 5 °C/min, 290 °C for 12 min; scan mode, TIC; scan range, m/z 33−300; electron ionization energy, 70 eV; source temperature, 230 °C; quadrupole temperature, 150 °C; transfer line temperature, 250 °C. Compound Identification. The odor-active compounds were detected by means of gas chromatography−olfactometry (GC-O). For their identification, the odor impressions (at comparable peak intensities), retention indices (RI), and mass spectra were compared to authentic standards on two columns of different polarities, published data, and the NIST 2011 MS library. Sensory Analysis. Sensory analysis of the PSA-IM mycelium was conducted by a trained panel (n = 12). The panelists were trained and tested prior to analysis. The sensory samples were prepared by dispersing the freeze-dried mycelium 1:10 (w/w) in water or in a 1:5:5 (w/w/w) oil-in-water (o/w) emulsion. The samples were analyzed by retronasal tasting. Eight previously defined flavor attributes were evaluated on a unipolar line scale (0−9). Statistics. Generally, samples were analyzed in a randomized order, and the peak areas were obtained in selected ion monitoring (SIM). For optimization of SBSE extraction, analyses were performed in triplicate and are reported as means with standard deviations. For validation of aroma dilution analysis by means of SBSE, the standard solution was analyzed for each split ratio in duplicate. Linear regression analysis of the binary logarithmic plots of the means of peak areas in SIM versus the split ratios was performed. For identification of the odor-active compounds, the samples were analyzed at least in triplicate in split mode with the lowest applicable split ratio.

tions, which were previously identified in submerged cultures of basidiomycetes. The compounds were selected to cover a broad range of logarithmic octanol−water partition coefficients (log Ko/w) and vapor pressure values as indicators for the level of polarity and volatility, respectively. For HS-SPME, two adsorptive fiber types with mixed coatings (PDMS/DVB and DVB/CAR/PDMS) were compared. The DVB/CAR/PDMS fiber enabled a slightly better extractability of highly volatile compounds, whereas the PDMS/DVB fiber extracted marginally more of the less volatile compounds. However, a key observation was that the applied desorption and baking-out conditions were not sufficient to completely remove the analytes from the DVB/CAR/PDMS coating. When air blanks were measured with the DVB/CAR/ PDMS fiber after previous sample analysis, certain compounds were still detectable via GC-MS and olfactometry. These carryover effects occurred primarily with aromatic compounds, such as benzaldehyde and p-anisaldehyde, which presumably had a high affinity for the carboxen phase. As this was not the case for PDMS/DVB fibers, this fiber type was used for further analyses. For SBSE applied in direct immersion, the nonpolar PDMS coating was used. For SBSE, increasing the temperature from 22 to 40 °C led to a decline of extractability of most volatile compounds. Only a few less volatile compounds (e.g., methyl (E)-cinnamate and ethyl p-anisate) were extracted in significantly higher amounts at 30 °C compared to 22 °C. In contrast, the extractability by HS-SPME was improved for most tested compounds by increasing the temperature from 30 to 50 °C. Exceptions were the highly volatile compounds pentanal, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and hexanal, which were extracted less with increasing temperature. It was thus decided to perform HS-SPME extraction at 40 °C to favor more volatile compounds and to limit potential thermal artifact formation during the extraction of the samples. CaCl2 was used to adjust the ionic strength. This type of salt was selected for the purpose of enzyme inhibition and to increase the cellular rigidity during sample preparation and extraction. Increasing the CaCl2 concentration from 0 to 20% led to a significantly improved extraction of compounds with log Ko/w < 3 by SBSE. This observed “salting-out” effect of hydrophilic compounds was in good agreement with publications, in which the effect of the salt concentration on the extractability by HS-SPME and SBSE was analyzed.19−21 Validation of ADA by HS-SPME and SBSE. Aroma extract dilution analysis (AEDA), in which a solvent extract or distillate of a sample is diluted stepwise and sniffed by means of GC-O, is a well-established procedure for the identification of odor-active compounds. The highest dilution factor, at which a compound is still perceivable, is defined as the flavor dilution (FD) factor.22 However, this approach of sample dilution is not applicable for nonexhaustive methods such as HS-SPME and SBSE, as these methods strongly depend on the composition of the sample matrix, which is altered by dilution.23 An alternative approach was described for HS-SPME first by Kim et al.15 This research group did not modify the sample itself, but the split ratios in the GC inlet, in order to specifically dilute the carrier gas flow. They were successful in demonstrating the applicability of this approach for a range of split ratios of 8:1 to 128:1, which corresponded to the FD factors. More recently, Zhang et al. were able to extend the linear range with an optimized method.16 In the present study, this procedure was applied for investigating the potency of odor-active

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RESULTS AND DISCUSSION Method Optimization. SBSE and HS-SPME are nonexhaustive extraction methods.17 The recovery of analytes thus strongly depends on the selected extraction parameters. Especially the sorbent material, extraction time and temperature, ionic strength, and agitation rate were found to strongly influence the recovery.18,19 Prior to analysis of the samples, these parameters were optimized to enhance the extractability of the volatile analytes. This was performed with an aqueous standard solution containing 18 authentic aroma compounds in equimolar concentra2395

DOI: 10.1021/acs.jafc.6b05292 J. Agric. Food Chem. 2018, 66, 2393−2402

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Journal of Agricultural and Food Chemistry

Table 1. Results of Aroma Dilution Analysis of IM and PSA-IM (Homogenized Nonseparated Submerged Culture and Disrupted Mycelium) by Means of HS-SPME and SBSEa FD factorsd RIb

IM

no. VF-WAXms DB-5ms 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

128h 16

ndo ndo 8 128 nd 8 8 ndo nd nd 8 ndo 32h ndo

nd nd nd nd 4 nd nd 4 nd nd

128 4 2 4 nd 128 nd nd 4 256

>128 nd 128 32 nd ndo >128

128 nd ndo 16 nd 128 32 8 64 >128 >128 ndo 8 nd >128 >128 32

>128 >128 ndo ndo 16 >128 ndo nd 128 8 >128

MS, RI, O, STD MS, RI, O, STD

nd nd nd nd nd

8 32j nd 4 32

32 >128j 16 32 >128

ndo 16j nd ndo 16

MS, RI, O, STD

nd

1

16

ndo

nd nd nd nd

nd nd 2 nd

16 8 64 0.989 for the split ratios from 8:1 to 128:1. The calculated slopes of the linear regression lines were in the range from −0.96 to −1.27 and close to the ideal value of −1.15 It was thus possible to demonstrate that the concept of ADA was also applicable and reliable for SBSE. However, if the range was extended to higher or lower split ratios, the regression coefficients significantly declined. This effect was especially observable for less volatile compounds. These findings were thus well in accordance with the findings of Kim et al., who stated that an unstable carrier gas flow at too high or too low split ratios technically limited this procedure.15 For this reason, reliable determination of FD factors with the applied SBSE method was performed in the 2397

DOI: 10.1021/acs.jafc.6b05292 J. Agric. Food Chem. 2018, 66, 2393−2402

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Journal of Agricultural and Food Chemistry

Figure 1. Comparison of GC-MS chromatograms (VF-WAXms): (A) homogenized submerged culture (SBSE); (B) homogenized submerged culture (HS-SPME); (C) culture medium (SBSE); (D) culture medium (HS-SPME); (E) disrupted mycelium (SBSE); (F) disrupted mycelium (HS-SPME) (HS-SPME in splitless mode, SBSE in split mode with split ratio 10:1).

The ADA resulted in the detection of overall 14 reproducible odor impressions with FD ≥ 4 at the ODP (Table 1). Typical perceived notes were “caramel-like”, “cotton candy”, “roasty”, or “buttery”. Among the most potent aroma compounds were furan derivatives, such as 4-hydroxy-2,5-dimethyl-3(2H)furanone (65, FDSPME 64), 2-acetylfuran (35, FDSPME 16), and α-dicarbonyls, like 2,3-butanedione (3, FDSPME 32) and 2,3pentanedione (6, FDSPME 16). Strecker aldehydes, such as 3-methylbutanal (2, FDSPME 16), 2-methylbutanal (FDSPME < 4), phenylethanal (FDSPME < 4), and methyl propanal (FDSPME < 4) were identified as further typical Maillard reaction products (MRP). Identification of Key Aroma Compounds of P. sapidus Cultivated with Isomaltulose Molasses. The submerged culture of PSA-IM exhibited a pleasant anisic, fruity odor. Overall, 62 odor impressions with FD ≥ 4 were perceived in

reproducible manner by ODP sniffing after either HS-SPME or SBSE (Table 1). A number of aliphatic compounds, which are typically generated by enzymatic peroxidation of unsaturated fatty acids, were formed by the fungus.24,25 1-Octen-3-one (17, FDSPME 256, FDSBSE > 128) with a typical “mushroom” odor was identified as a key aroma compound. By way of comparison, the corresponding alcohol 1-octen-3-ol (30, FDSPME 4, FDSBSE 16) marginally contributed to the overall aroma, even though its relative peak area was larger. Various aliphatic aldehydes were also perceivable at the highest split ratios by both HS-SPME and SBSE. The identified saturated aldehydes, such as hexanal (9, FDSPME 256), typically imposed “green”, “fruity” notes. In contrast, the unsaturated aldehydes, such as (E)-2-octenal (27, FDSPME 256), (E,E)-2,4-nonadienal (54, FDSPME 256), and 2398

DOI: 10.1021/acs.jafc.6b05292 J. Agric. Food Chem. 2018, 66, 2393−2402

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Journal of Agricultural and Food Chemistry (E,E)-2,4-decadienal (59, FDSPME 256), exhibited “fatty”, “fried”, or “waxy” notes. Many of the corresponding (Z)- and (E,Z)-isomers of the alkenals were identified as well, but with lower FD values. Another key aroma compound, which was also presumably generated by oxidation of unsaturated fatty acids, was trans-4,5-epoxy-(E)-2-decenal (63, FDSPME 256, FDSBSE > 128).25,26 The second main group of identified key aroma compounds comprised arylic compounds. Within this group, p-anisaldehyde (64, FDSPME 256, FDSBSE > 128) with its typical “anisic, bitter almond” note was the most potent odor compound. p-Anisaldehyde can be synthesized de novo and has been identified previously in Pleurotus spp. and other white rot fungi.27−30 Additionally, the corresponding esters methyl p-anisate (67) and ethyl p-anisate (69) were identified in the submerged culture. Both esters had been identified in other basidiomycota.29,31 (E)-Methyl cinnamate (66, FDSBSE 32) was identified in the submerged PSA-IM cultures as well. The corresponding (E)-cinnamic acid was reported as an intermediate metabolite in the biosynthesis of p-anisaldehyde from L-phenylalanine in Bjerkandera adusta.28 The third major class of identified aroma compounds comprised terpenoids. Within this group, the odors 37 (FDSPME 8, FDSBSE 16) and 47 (FDSPME 256, FDSBSE > 128) possessed characteristic “anisic, tarragon” impressions. The latter odor was related to the highest peaks in the HS-SPME and SBSE chromatograms of the submerged PSA-IM culture (Figure 1). Together with p-anisaldehyde (64), 47 was likely to be the key contributor to the overall “anisic” aroma of the submerged PSA-IM culture. For peaks 37 and 47 almost identical MS spectra were recorded. By comparison of the MS spectra with the NIST database, the compounds were tentatively identified as stereoisomers of the bicyclic monoterpene 3,9-epoxy-p-menth1-ene (3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran). These compounds were detected not only in the PSA-IM culture but also in the PSA precultures (grown in malt extract medium). As 3,9-epoxy-p-menth-1-ene was not identified in the substrate IM or in any of the other used components of the preculture and main culture, they were most likely formed due to metabolic activities of PSA. To the best of our knowledge, 3,9-epoxy-pmenth-1-ene has not yet been reported in Pleurotus spp. or other basidiomycetes but was identified after biotransformation of nerol by Botrytis cinerea.32 In total, eight stereoisomers of 3,9-epoxy-p-menth-1-ene exist. It was reported that the transisomers of 3,9-epoxy-p-menth-1-ene are sterically less favorable and thus less likely to be formed.33 Among the cis-isomers, only the (3S,3aS,7aR)-isomer (“dill ether”) possesses a characteristic dill-like odor, whereas the other stereoisomers are assigned “fruity, tarragon” odors.33,34 To support the identification, a modified approach for the synthesis of isomeric 3,9-epoxy-pmenth-1-ene by thermal acid-catalyzed conversion of 10-hydroxygeraniol was applied.13,14 The obtained mixture was extracted and analyzed under the same conditions as the PSA-IM samples. Among the formed reaction products, two peaks were identified as 3,9-epoxy-p-menth-1-ene isomers with identical RI and MS spectra as 37 and 47 in the PSA-IM samples on polar and nonpolar GC columns. Furthermore, dried dill leaves were analyzed by HS-SPME-GC-MS-O, in which only enantiopure (3S,3aS,7aR)-dill ether occurs as its key aroma compound.33−37 This enantiomer was identified in the dill sample by sniffing and MS at the same retention indices on both columns as 37 from PSA-IM. In conclusion, 3,9-epoxy-p-menth-1-ene (37) identified at RIWAX 1512 and RIDB‑5 1187 in PSA-IM may

presumably be attributed to the dill ether (3S,3aS,7aR) and/or its enantiomer (3R,3aR,7aS). The latter enantiomer was considered to be more likely, because an “anisic, fruity” odor was perceived for 37, not the characteristic dill ether odor. Under the premise that only cis-isomers were present, 3,9-epoxy-pmenth-1-ene (47) identified at RIWAX 1598 and RIDB‑5 1233 in PSA-IM most likely represented its (3R,3aS,7aR)- and/or (3S,3aR,7aS)-enantiomers. The odors 70 (“coconut, peach, flowery”, FDSBSE > 128) and 71 (“flowery, coconut”, FDSBSE 16) were assigned to two separated peaks with highly similar mass spectra. Their spectra matched well with that of 3,6-dimethyl-3a,4,5,7a-tetrahydro-1benzofuran-2(3H)-one. The lactone has a similar stereochemical structure as 3,9-epoxy-p-menth-1-ene. Guth et al. investigated the properties of the eight possible 3,6-dimethyl-3a,4,5,7a-tetrahydro1-benzofuran-2(3H)-one stereoisomers and described their odors as “coconut”-like.38 The (3S,3aS,7aR)-isomer (“wine lactone”) is naturally present as a key aroma compound in white wine varieties.38 For identification, the RI values and mass spectra of 70 and 71 were compared to those of synthesized cis-3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran2(3H)-one stereoisomers. The obtained data on both columns for 70 were in agreement with those of the (3S,3aS,7aR)/ (3R,3aR,7aS)-enantiomers, whereas 71 matched those of the (3R,3aS,7aR)/(3S,3aR,7aS)-enantiomers. Even though the absolute configurations still have to be elucidated, these results demonstrated the ability of PSA to produce cis-3,6-dimethyl3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-ones. To the best of our knowledge, these compounds have not been described in basidiomycetes before. The perceived odor 43 (FDSBSE > 128) was comparable to those of the 3,9-epoxy-p-menth-1-ene isomers (“anisic, tarragon”). The comparison of the measured MS spectrum with the NIST database suggested 3,9-epoxy-p-mentha-1,8(10)-diene as the most probable compound. This compound was identified in Ledum groenlandicum. The reported RI values (RIWAX 1547 and RIDB‑5 1185) matched those of the present study quite well (RIWAX 1561 and RIDB‑5 1189).39 Further identified highly potent terpenoidic compounds were verbenone (55, FDSBSE 32) and 4-acetyl-1-methylcyclohexene (41, FDSBSE 128). Verbenone has been reported in PSA as a biotransformation product of α-pinene previously.11 4-Acetyl-1methylcyclohexene can be formed by chemical conversion of limonene but was not reported as a fungal metabolite to the best of our knowledge.40 Furthermore, the comparative ADA of IM and the submerged PSA-IM culture was a helpful tool to investigate whether a compound originated mainly from the substrate or from fungal metabolism. From this, it was found that some of the potent aroma compounds from IM were still detectable after the fermentation, even though they played only a marginal role in the overall aroma of PSA-IM. Among them were the highly odoractive MRP 2,3-butanedione (3) and 4-hydroxy-2,5-dimethyl3(2H)-furanone (65). Comparison of ADA via HS-SPME and SBSE. One of the intentions of the present study was to compare the applicability of the developed HS-SPME and SBSE procedures for aroma dilution analyses. For that purpose, the homogenized submerged PSA-IM culture was analyzed via both approaches after identical sample preparation prior to extraction. Overall 33 and 54 reproducible odors with FD ≥ 4 were perceived with the applied extraction procedures of HS-SPME and SBSE, respectively. The chromatograms obtained by SBSE 2399

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Journal of Agricultural and Food Chemistry and HS-SPME were comparable for RIWAX < 2000 (retention times roughly 128). It was thus possible to efficiently extract highly volatile compounds from the submerged culture not only by HS-SPME but also by an optimized SBSE procedure. For RIWAX > 2000 (Figure 1), the peak intensities were substantially lower in the HS-SPME chromatograms. This was thus reflected by significantly lower FDSPME factors compared to the respective FDSBSE factors for compounds such as (E)-methyl cinnamate (66) and methyl p-anisate (67). Within this interval, several odors were still perceivable even in the highest applicable split ratios for SBSE. Overall, a good extractability for a broad range of volatile compounds in PSA-IM was found for SBSE. A drawback of both extraction approaches was the technical limitation, which set an upper limit for the split flow rate and thus for the determination of the FD factors. This became obvious for aroma compounds that were still clearly perceived via sniffing even with the highest split ratio. Presumably, some of these odors would have also been detected in higher split ratios. This was especially true for SBSE, for which a narrower linear range of applicable split ratios of five dilution steps was possible. Nevertheless, the applied methodology was appropriate to identify the key aroma compounds. The comparable results of the two ADA indicated the reliability of the applied approach. Because fewer compounds were detected by HS-SPME, ADA of the disrupted mycelium was performed only by means of SBSE. Separation of the Mycelium from the Culture Medium. As only the mycelium is intended to be further used as a protein source in food applications, the key changes in the aroma profile related to the separation of the mycelium from the culture medium were investigated. For better accessibility of the intracellular compounds, the mycelium was disrupted prior to extraction. The aroma profiles of the homogenized submerged culture and of the culture medium were highly similar. In contrast, the peak intensities were substantially lower for the disrupted mycelium. This observation was in line with the results of ADA. Overall, significantly fewer odors were perceived from the disrupted mycelium at the ODP. The decrease of the FD factors due to the removal of the liquid culture medium was especially observable for arylic compounds such as p-anisaldehyde (64) and the anisates (67, 69). These findings were in good agreement with the data of Gutierrez et al.27 Mainly lipid peroxidation products, such as 1-octen-3-one (17, FDSBSE > 128), (E)-2-octenal (27, FDSBSE 128), (E)-2-nonenal (38, FDSBSE 128), (E,E)-2,4-nonadienal (54, FDSBSE > 128), (E,E)-2,4-decadienal (59, FDSBSE > 128), and (E)-4,5-epoxy-(E)-2-decenal (63, FDSBSE > 128), were identified as the most potent aroma compounds from the mycelium (see Figure 2). The terpenoid compound 3,9-epoxy-p-menth-1-ene (47) was perceived with the highest possible FD factor (>128) in the disrupted mycelium, too.

Figure 2. Selection of identified aroma compounds. Compounds 37, 43, and 47 were identified tentatively.

Sensory Analysis of the Mycelium. To additionally support the data from ADA, sensory analysis of the PSA-IM mycelium was carried out by a trained panel (Figure 3). The

Figure 3. Sensory analysis (retronasal) of mycelium (submerged PSA culture on IM) by a trained panel (n = 12). Unipolar line scale (0−9) was used. Sensory samples were prepared by dispersion of mycelium 1:10 (w/v) in water and 1:5:5 (w/v/v) in water−oil mixture.

samples for the retronasal tasting were prepared by dispersing the mycelium in water or in o/w emulsion. The results were comparable for the two matrices. Among the tested flavor attributes, the highest intensities were measured for “sweet”, “mushroom”, “apple”, and “anisic”. The “sweet” flavor was likely caused by the residual nonmetabolized sugars from the molasses. The identified MRP 4-hydroxy-2,5-dimethyl-3(2H)furanone (65, “cotton candy, caramel”) might have synergistically enhanced the “sweet” flavor.41 The “mushroom-like” note was most likely mainly caused by 1-octen-3-one (17). On the basis of the observation that arylic compounds such as p-anisaldehyde (64) were predominantly enriched in the culture medium, the tentatively identified benzofuran compounds (43, 47) were likely to affect the “anisic” note to a large extent. In conclusion, ADAs by means of HS-SPME and SBSE were efficient tools to identify the key aroma compounds of the substrate, the submerged PSA-IM culture, and the mycelium. 2400

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However, the limited range of measurable FD factors of ADA compared to AEDA was the major drawback. The comparative ADA was helpful to investigate whether the identified compounds of the submerged culture originated from the substrate or from the fungal metabolism. It was furthermore demonstrated that the aroma compounds mainly partitioned in the liquid culture medium. The instrumentally identified key aroma compounds related well with the results of the sensory analysis of the mycelium. The results of the present study will be used to optimize the flavor of the mycelium for potential food applications.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05292. Method optimization: variation of extraction temperature (SBSE) (Figure S1), extraction temperature (HS-SPME) (Figure S2), and CaCl2 content (SBSE) (Figure S3). Validation of aroma dilution analysis (SBSE) (Table S1; Figure S4). Experimental data (MS, RI, odors) obtained for the identification of 3,9-epoxy-p-menth-1-ene (Table S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(M.A.F.) Phone: +49 641 99-34902. Fax: +49 641 99-34909. E-mail: [email protected]. ORCID

Holger Zorn: 0000-0002-8383-8196 Marco Alexander Fraatz: 0000-0002-5028-9653 Funding

This project (HA project 478/15-20) has been funded within the framework of HessenModellProjekte, financed with funds of LOEWE − Landes-Offensive zur Entwicklung Wissenschaftlich-oekonomischer Exzellenz, Foerderlinie 3: KMU-Verbundvorhaben (State Offensive for the Development of Scientific and Economic Excellence). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Suedzucker AG − Central Department Research, Development and Services (Offstein, Germany) for providing the isomaltulose molasses for the experiments and to Nils Helge Schebb for providing authentic aroma standards from the former working group of Helmut Guth.

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ABBREVIATIONS USED ADA, aroma dilution analysis; AEDA, aroma extract dilution analysis; CIS, cooled injection system; DI, direct immersion; FD, flavor dilution; HS, headspace; IM, isomaltulose molasses; log Ko/w, logarithmic partition coefficient of analyte between octanol and water; MRP, Maillard reaction product; ODP, olfactory detection port; o/w, oil-in-water; PSA, Pleurotus sapidus; PSA-IM, Pleurotus sapidus cultivated with isomaltulose molasses; RI, retention index; SBSE, stir bar sorptive extraction; SR, split ratio; TDU, thermal desorption unit 2401

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