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Identification of Bis(methylsulfanyl)methane and Furan-2(5H)-one as Volatile Marker Compounds for the Differentiation of the White Truffle Species Tuber magnatum and Tuber borchii
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Food and Beverage Chemistry/Biochemistry

Identification of Bis(methylsulfanyl)methane and Furan-2(5H)-one as Volatile Marker Compounds for the Differentiation of the White Truffle Species Tuber magnatum and Tuber borchii
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  • Philipp Schlumpberger
    Philipp Schlumpberger
    TUM School of Natural Sciences, Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, Germany
    Leibniz Institute for Food Systems Biology at the Technical University of Munich (Leibniz-LSB@TUM), Lise-Meitner-Straße 34, 85354 Freising, Germany
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  • Martin Steinhaus*
    Martin Steinhaus
    Leibniz Institute for Food Systems Biology at the Technical University of Munich (Leibniz-LSB@TUM), Lise-Meitner-Straße 34, 85354 Freising, Germany
    TUM School of Natural Sciences, Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, Germany
    *Email: [email protected]. Phone: +49 8161 71 2991.
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    Orcidhttps://orcid.org/0000-0002-9879-1474
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Journal of Agricultural and Food Chemistry

Cite this: J. Agric. Food Chem. 2024, 72, 17, 10023–10030
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https://doi.org/10.1021/acs.jafc.4c00714
Published April 17, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Some truffles are expensive and, therefore, are prone to food fraud. A particular problem is the differentiation of high-priced Tuber magnatum truffles from cheaper Tuber borchii truffles, both of which are white truffles with similar morphological characteristics. Using an untargeted approach, the volatiles isolated from samples of both species were screened for potential marker compounds by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOFMS) and statistical analysis of the obtained semiquantitative data. Results suggested bis(methylsulfanyl)methane and furan-2(5H)-one as compounds characterizing T. magnatum and T. borchii, respectively. Exact quantitation of both volatiles by conventional one-dimensional gas chromatography–mass spectrometry in combination with stable isotopologues of the target compounds as internal standards confirmed both as marker compounds. The method is suitable to be used in the routine analysis for the objective species differentiation of T. magnatum and T. borchii.

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Introduction

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Truffles are the hypogeous fruiting bodies of some ascomycete fungi, in particular those of the genus Tuber in the Tuberaceae family. (1,2) Their hyphae form an ectomycorrhiza with the roots of various trees and shrubs such as oaks, elms, poplars, hazels, willows, pines, and cedars. (2−5) The symbiotic relationship serves to exchange nutrients between the fungus and the plant. (3)
The fruiting bodies of all of the Tuber species are edible. According to their color, these are typically classified as white or black truffles. Both are highly appreciated, particularly in French, Spanish, Italian, and Greek cuisines. Whereas black truffles are used in the kitchen mainly as funds or essences, white truffles are typically not cooked but added in thin slices to the finished dish. Risotto, pasta, pizza, omelet, salad, and polenta are just some popular dishes that are commonly served with a topping of white truffle slices. (3,6)
Only two species predominantly share the white truffle market: Tuber magnatum Picco, yielding the Alba truffle or Piedmont truffle, and Tuber borchii Vittad., previously named Tuber albidum, yielding the Bianchetto truffle. (1,3,5,7) Both species are native to Europe. The occurrence of T. magnatum is not limited to the Piedmont region of Italy, but this species is also found in other Italian regions, in Croatia, Hungary, Romania, and Slovenia. (1,2,5) Italy is also the center of occurrence of T. borchii; however, its growing region extends over an even larger geographical area than that of T. magnatum and includes England, Finland, Germany, Hungary, Poland, and Switzerland. (5,8)
Truffles are among the most expensive foods. This particularly applies to T. magnatum. (5) An increased demand in combination with a declining crop has caused the prices to rise substantially in recent years. (3,4) In January 2024, the price of a T. magnatum truffle ranged from 2000 to 3000 $. T. borchii truffles were significantly cheaper and cost ∼250 to 700 $. (9) The higher price of T. magnatum truffles is associated with their stronger and richer aroma–a major factor for their great popularity–and with difficulties in their cultivation. (1,6) Whereas other species, including T. borchii, are successfully cultivated in truffle plantations, cultivation of T. magnatum has rarely been effective to date. (3,4,10)
In contrast to the substantial differences in price and availability, the appearances of T. magnatum and T. borchii truffles are often very similar. T. magnatum truffles are typically 2–6 cm in size but can reach up to 15 cm. (11) The surface is smooth and usually pale ochre in color. (11,12) Reddish spots occasionally occur. (12) A cross-section of a T. magnatum truffle shows light brown flesh with white veins. (5) T. borchii truffles, on average, are smaller than T. magnatum truffles and typically show sizes of 2–3 cm. (11) The surface is just as smooth and pale ochre-colored as the surface of T. magnatum truffles; reddish spots are also often present. (5,11) The flesh may be darker in color and the veins may be wider compared to T. magnatum truffles. (5,11)
Given the morphological similarities between T. magnatum and T. borchii truffles in combination with a high morphological variability within each species, it is often impossible to unequivocally assign an individual truffle to one or the other species on the basis of the morphology alone. Consequently, the risk of cheap and readily available T. borchii truffles being marketed as T. magnatum truffles is high. The vast price difference makes selling T. borchii truffles fraudulently labeled as T. magnatum truffles a lucrative business. Chemical marker compounds characterizing each species would be beneficial to counteracting this type of food fraud.
Studies on the differentiation of truffle species based on chemical analyses have already been published. (13−19) A widely applied approach is based on untargeted metabolomics using liquid chromatography (LC) in combination with mass spectrometry (MS). (20,21) For example, Li et al. (20) were able to differentiate the European truffle species T. melanosporum and the four Chinese truffle species T. indicum, T. panzhihuanense, T. sinoaestivum, and T. pseudoexcavatum by a comprehensive LC–MS profiling including the analysis of amino acids, saccharides and nucleosides, alkaloids, flavonoids, carnitines, organic acids, phenols, alcohols, esters, and sulfur compounds. Individual marker compounds, however, were not determined. Creydt and Fischer (21) concentrated on the analysis of the lipidome. In the two white truffle species included in the study, namely, T. magnatum and T. borchii, they identified 33 metabolites. The analysis of 26 of these metabolites proved to be suitable for distinguishing between the two white truffle species.
Another approach that has been evaluated for the differentiation of truffle species is the analysis of volatiles by gas chromatography (GC) in combination with MS. Pelusio et al. (22) compared the white truffle species T. magnatum and the black truffle species T. melanosporum on the basis of volatile sulfur compounds analyzed by headspace solid phase microextraction (HS–SPME) and GC–MS. (Methylsulfanyl)methane, (methyldisulfanyl)methane, dimethyltrisulfane, and bis(methylsulfanyl)methane were identified as important organosulfur volatiles; however, these were not suitable to differentiate between the two species when samples of different harvest years were considered. Kiss et al. (23) used a modified Likens-Nickerson apparatus to isolate the volatiles from Hungarian black truffles of the species T. aestivum and T. brumale. GC–MS analysis identified 102 and 104 volatiles in T. aestivum and T. brumale, respectively. Semiquantitative data revealed apparent differences in the composition of the volatile fraction between the two species. Whereas in T. aestivum, 2-methylbutan-1-ol was the predominating volatile followed by hexadecanoic acid, 2-phenylethan-1-ol, butan-2-ol, 1-octen-3-ol, and 2-methylpropan-1-ol, the most abundant volatiles in T. brumale were methoxybenzenes such as 1,4-dimethoxybenzene, 1-methoxy-3-methylbenzene, and foremost 1,2,4-trimethoxybenzene. D’Auria et al. (24) compared the volatiles in three samples of the common white truffle species T. borchii with the volatiles in four samples of the rare white truffle species T. asa-foetida by HS–SPME–GC–MS. Among the 12 volatiles identified, six compounds including 2-methylpropan-1-ol, 3-methylbutanal, and 3-methylbutan-1-ol were common to both species, whereas 3-methylthiophene, xylene, α-pinene, 3,7-dimethylocta-1,3,6-triene, 3-acetyl-1-propyl-5,6-dihydro-2-naphthol, and 9-(diphenylmethylidene)-9H-fluorene were only identified in T. borchii and butan-2-one, tetrahydrofuran, benzene, butan-2-yl formate, 2-methylbutan-1-ol, and toluene were only found in T. asa-foetida. Among them, however, only 3-methylthiophene and toluene were suitable as marker compounds for T. borchii and T. asa-foetida, respectively, as only these two compounds were identified in all samples of the particular species. Zhang et al. (25) compared a sample of a black Chinese truffle with a sample of a white Chinese truffle. Volatiles were isolated by solvent extraction and solvent-assisted flavor evaporation (SAFE), and analyzed by comprehensive two-dimensional gas chromatography in combination with time-of-flight MS (GC×GC–TOFMS). Fifty-eight and 47 volatiles were identified in the black and white truffles, respectively. The authors suggested that the approach might be suitable to discriminate between the two species.
Only two studies were available that compared the volatiles in the two white truffle species, T. magnatum and T. borchii. Mauriello et al. (26) analyzed the volatilome of 11 different truffle species, including five samples each of T. magnatum and T. borchii by HS–SPME–GC–MS. The T. magnatum samples were characterized by the presence of bis(methylsulfanyl)methane, (methylsulfanyl)methane, (methyldisulfanyl)methane, and (methyltrisulfanyl)methane, whereas numerous volatiles were identified in the T. borchii samples that were absent in the T. magnatum samples, including 1,3-xylene, 2-benzothiophene, 2-methylbutan-1-ol, 2-methylbuta-1,3-diene, 2-methylfuran, 2-methylpropan-1-ol, 3-methylbutan-1-ol, 3-methylthiophene, (3Z)-3,7-dimethylocta-1,3,6-triene, butan-2-ol, butan-2-yl formate, decane, ethanol, ethenylbenzene, octan-3-one, penta-1,2-diene, pentanal, tetradecanal, tetradecane, and toluene. Gioacchini et al. (27) analyzed the volatiles of six different truffle species, including T. magnatum and T. borchii, by HS–SPME–GC–MS. The mass spectral data of the individual runs were processed into an average mass spectrum. Specific mass-to-charge ratio (m/z) values were assigned to different compound classes, such as alcohols, aldehydes, etc., and their intensities were successfully used for species differentiation.
In summary of the literature overview, differentiation of T. magnatum and T. borchii truffles on the basis of the analysis of volatile marker compounds seemed feasible. However, previous studies suffered from some deficiencies. For example, structure assignments were only based on mass spectral libraries and were not confirmed by analysis of authentic reference compounds. Semiquantitative data obtained in the untargeted approaches were not confirmed by exact quantitations, e.g., using GC–MS in combination with isotopically substituted internal standards. Furthermore, in the HS–SPME–GC–MS approaches, it remained unclear whether the suggested marker volatiles were genuine truffle constituents or thermal artifacts formed in the hot injector during desorption from the fiber. Accordingly, our study aimed to use the recently developed automated solvent-assisted flavor evaporation (aSAFE) approach (28) to reproducibly prepare artifact-free volatile isolates from numerous T. magnatum and T. borchii truffle samples, screen the volatiles for potential marker compounds by GC×GC–TOFMS analysis in combination with statistical analysis of the semiquantitative data, unequivocally assign the structures of the potential marker compounds, and finally verify the marker compounds by exact quantitation using GC–MS analysis in combination with isotopologues of the target compounds as internal standards.

Materials and Methods

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Truffle Samples

Truffle samples with confirmed authenticity (17 × T. magnatum, samples M 01 C – M 17 C; 6 × T. borchii, samples B 01 C – B 06 C) were provided by a specialized retailer (La Bilancia, Munich, Germany). Their authenticity was assessed based on unequivocal morphological characteristics. Truffles with an equivocal morphology and truffles with damages were excluded. Truffle samples with unconfirmed authenticity (2 × T. magnatum, samples M 18 U and M 19 U; 1 × T. borchii, sample B 07 U) were obtained from Internet shops. All truffle samples were collected between 2018 and 2020. The fresh material was shock-frosted with liquid nitrogen and stored at–24 °C before analysis.

Reference Compounds and Stable Isotopically Substituted Volatiles

Isotopically unmodified reference compounds bis(methylsulfanyl)methane and furan-2(5H)-one were purchased from Merck (Darmstadt, Germany). (2H8)Naphthalene was also from Merck. (2H8)Bis(methylsulfanyl)methane was synthesized as detailed in the literature. (29) (2H2)Furan-2(5H)-one was synthesized according to a procedure published previously (30) but with some modifications. In brief, 3a,4,7,7a-tetrahydro-4,7-epoxy-2-benzofuran-1,3-dione (abcr, Karlsruhe, Germany) was deuterated with sodium borodeuteride (Cambridge Isotope Laboratories, Tewksbury, MA, USA) under an argon atmosphere. Acidic workup led to the intermediate (3,3-2H2)-3a,4,7,7a-tetrahydro-4,7-epoxy-2-benzofuran-1(3H)-one, which was extracted by dichloromethane instead of chloroform and then purified by chromatography (3 cm column diameter) on silica gel 60 (0.040–0.063 mm; VWR, Darmstadt, Germany; 60 g). After being washed with n-hexane/ethyl acetate (50/50, v/v; 150 mL), the compound was eluted with n-hexane/ethyl acetate (25/75, v/v; 150 mL) and ethyl acetate (200 mL). The solvents were removed in vacuo, and the purified (3,3-2H2)-3a,4,7,7a-tetrahydro-4,7-epoxy-2-benzofuran-1(3H)-one was heated to 150 °C at 15 mbar to distill off the target product (5,5-2H2)furan-2(5H)-one (96% purity by GC–flame ionization detector) obtained in a retro-Diels–Alder reaction.

Organic Solvents

Dichloromethane was obtained from CLN (Freising, Germany) and freshly distilled through a column (120 cm × 5 cm) packed with Raschig rings. Ethyl acetate was obtained from J. T. Baker (Phillipsburg, NJ, USA) and n-hexane from Merck.

GC×GC–TOFMS Analysis

Truffles were cooled with liquid nitrogen and ground in the frozen state with a laboratory mill GrindoMix GM200 (Retsch, Haan, Germany) at 10,000 rpm (2 × 3 s). Dichloromethane (50 mL) and (2H8)naphthalene (1 μg) were added to the powder (2 g). Under ice-cooling, anhydrous sodium sulfate (6 g) was added, and the mixture was homogenized with an Ultra-Turrax T25 (IKA, Staufen, Germany) at 13,500 rpm for 20 s. After continuous stirring overnight at room temperature and under light exclusion, the mixture was filtered through a folded filter, and the residue was washed with dichloromethane (10 mL). The combined extracts were subjected to aSAFE at 40 °C using a valve open/closed time combination of 0.2 s/10 s. (28) The obtained volatile fraction was concentrated to a final volume of 1 mL using a Vigreux column (50 × 1 cm) and a Bemelmans microdistillation device. (31) For each truffle sample, a duplicate or triplicate workup was performed.
The truffle volatile isolates were stored in amber glass vials at −24 °C prior to analysis with a GC×GC–TOFMS system. This was equipped with a 6890 gas chromatograph (Agilent, Waldbronn, Germany), a Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland), a Cooled Injection System (CIS) 4 (Gerstel, Mülheim/Ruhr, Germany), and a DB-FFAP GC column, 30 m × 0.25 mm i.d., 0.25 μm film thickness (Agilent) in the first dimension (1D). The end of this column was connected to a dual-stage quad-jet thermal modulator (Leco, Mönchengladbach, Germany) which cryofocused the volatiles in the eluate with the help of liquid nitrogen and transferred them in portions to the column in the second dimension (2D), which was a DB-1701 column, 3 m × 0.18 mm i.d., 0.18 μm film (Agilent) and installed in the secondary oven located inside the primary oven. The end of the second column was connected to the inlet (250 °C) of a Pegasus III TOFMS (Leco). Helium at a constant flow of 2.0 mL/min served as the carrier gas. The injection volume was 1 μL. The injection mode was splitless. The initial temperature of the primary oven was 40 °C (2 min), followed by a gradient of 6 °C/min to 95 °C (5 min), a gradient of 3 °C/min to 155 °C, and a final gradient of 4 °C/min to 230 °C (5 min). The initial temperature of the secondary oven was 70 °C (2 min), followed by a gradient of 6 °C/min to 125 °C (5 min), a gradient of 3 °C/min to 185 °C, and a final gradient of 6 °C/min to 250 °C (5 min), resulting in a total run time of 58 min. The modulator was operated with a temperature offset of +50 °C relative to the secondary oven temperature. The modulation period was 4 s, with a hot pulse time of 1 s. Mass spectra were generated in electron ionization (EI) mode at 70 eV with a scan rate of 100 spectra/s and a scan range of m/z 35–300. The temperature of the transfer line was 250 °C and the temperature of the ion source was 230 °C. Data files were recorded by ChromaTOF software (Leco). Each truffle volatile isolate was analyzed in triplicate.
Data preprocessing and statistical analysis was accomplished by using ChromSpace and ChromCompare+ software (Markes International, Llantrisant, United Kingdom). At first, GC×GC–TOFMS raw data were imported into ChromSpace. Preprocessing started with retention time alignment to compensate for retention time shifts. One chromatogram with medial retention times (tR) was manually selected as a reference, and the algorithm adjusted the tR of all other chromatograms in both the first dimension (1tR) and the second dimension (2tR). The aligned chromatograms were subjected to integration by means of a tile-sum algorithm. In brief, the entire chromatogram was divided into overlapping tiles in size of 120 s × 1 s (1D × 2D). The overlap was 50% in all directions, i.e., the tile borders were located in the center of the neighboring tiles. For each tile and each m/z value, a summed intensity was calculated. To retain the entire information, no data filtering was applied at this point, and the software parameters “area”, “height”, and “width” were thus set to zero.
The output was a set of 121,296 “features”. Each feature was defined by three parameters (1tR of the tile center, 2tR of the tile center, m/z value) and the corresponding intensities in the individual GC×GC–TOFMS runs. These data were imported into ChromCompare+, and for each GC×GC–TOFMS run data set, the truffle species was manually added. The m/z intensities in each GC×GC–TOFMS run data set were normalized using feature F41817, which originated from the internal standard (2H8)naphthalene as a reference (cf. Supporting Information, Table S2). The normalized m/z intensities were subjected to a log10 transformation followed by a principal component analysis (PCA). In the PCA, both biological replicates (2 or 3 workups) and technical replicates (3 GC×GC–TOFMS runs) were not averaged but treated independently. Using the feature discovery tool in ChromCompare+, the number of features included in the PCA was stepwise reduced.

GC–MS Quantitation of Bis(methylsulfanyl)methane and Furan-2(5H)-one

Dichloromethane (20 mL), the internal standards (2H8)bis(methylsulfanyl)methane (0.0743–14.9 μg) and (2H2)furan-2(5H)-one (0.138–2.76 μg), and anhydrous sodium sulfate (1.5 g) were added to cryomilled truffles (0.5 g) under ice-cooling, and the mixture was homogenized with an Ultra-Turrax T25 (IKA) at 13500 rpm for 20 s. Extraction, filtration, aSAFE, and concentration were performed as detailed before. At a concentrate volume of ∼1 mL, 100 μL was sampled for the GC–MS analysis of the higher concentrated marker compound. The remaining ∼900 μL was further concentrated to a final volume of ∼100 μL by using a Bemelmans microdistillation device, (31) and this portion was used for the analysis of the lower concentrated marker compound. For each truffle sample, a triplicate workup was performed.
The truffle volatile isolates were stored in amber glass vials at −24 °C prior to analysis with a GC–MS system consisting of a 7890B gas chromatograph, a Combi PAL autosampler, a multimode inlet used in splitless mode, a DB-FFAP column, 30 m × 0.25 mm i.d., 0.25 μm film thickness, and a Saturn 220 ion trap mass spectrometer (Agilent). Helium at a constant flow of 1.2 mL/min served as the carrier gas. The injection volume was 2 μL. For the analysis of bis(methylsulfanyl)methane, the initial inlet temperature was 40 °C (2 min), followed by a gradient of 6 °C/min to 118 °C, a gradient of 40 °C/min to 230 °C, and a final gradient of 900 °C/min to 250 °C. The initial oven temperature was 40 °C (2 min), followed by a gradient of 6 °C/min to 118 °C and a gradient of 40 °C/min to a final temperature of 230 °C (5 min). A rather mild temperature program in the injector that paralleled the temperature program in the oven was found to be crucial to avoid thermal degradation of the target compound during injection. The analysis of furan-2(5H)-one was performed with an initial inlet temperature of 40 °C, followed by a gradient of 900 °C/min to 250 °C (5 min), and a final gradient of 900 °C/min to 280 °C. The initial oven temperature was 40 °C (2 min), followed by a gradient of 6 °C/min to 166 °C and a gradient of 40 °C/min to a final temperature of 230 °C (5 min). Mass spectra were generated in EI mode at 70 eV with a scan range of m/z 35–250. Data analysis was performed using MS Workstation 7.0.2 software (Agilent).
Peak areas of the analyte and the respective internal standard were obtained from extracted-ion chromatograms (EICs) of the characteristic quantifier ions. The concentration of the marker compound was calculated from the acquired peak areas of the analyte and the internal standard, the amount of truffle sample used for the workup, and the amount of internal standard added by applying a calibration line equation. The calibration line equation was obtained by linear regression applied to the data obtained from the GC–MS analysis of analyte/standard mixtures in different concentration ratios. The quantifier ions and the calibration line equations are summarized in the Supporting Information file, Table S3. The individual concentrations obtained from the triplicate workups and the standard deviations are available in the Supporting Information file, Tables S4 and S5.

Results and Discussion

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Screening for Marker Compounds

The untargeted volatilomics approach selected for the marker compound screening combined aSAFE for volatile isolation and comprehensive two-dimensional gas chromatography–mass spectrometry with a GC×GC–TOFMS instrument for volatile analysis. SAFE is a markedly gentle isolation technique that preserves the composition of the volatile fraction during isolation. (28,32) aSAFE, the automated variant of SAFE, additionally provides high reproducibility due to the electronically controlled switching technique, which operates with fixed settings independently of the user. (28) Finally, GC×GC–TOFMS combines an extraordinarily high separation efficiency on the chromatographic level with a high linear range on the mass spectrometric level, reported to allow for the semiquantitation of over 1000 compounds in a single run. (33)
The approach was applied to 17 T. magnatum and 6 T. borchii samples of confirmed authenticity. Each sample was subjected to duplicate or triplicate workup (depending on sample size), and each truffle volatile isolate was analyzed in triplicate by GC×GC–TOFMS analysis. The GC×GC–TOFMS raw data were subjected to retention time alignment and feature detection. The result was >100,000 features, which reflected the complexity of the volatile fraction of the truffle samples. Intensity data were normalized and subjected to log10 transformation followed by PCA with stepwise reduction of the number of features. Finally, differentiation of the two white truffle species, T. magnatum and T. borchii, was achieved based on only five features. For each of these, application of a Welch’s t test to the intensity values showed mean values that significantly differed between T. magnatum and T. borchii (p-values < 0.001; cf. Supporting Information, Table S1).
The biplot of the PCA based on the five features is depicted in Figure 1. The two species were clearly separated into two distinct clusters. The features characterizing the T. magnatum samples were F11518 and F12848, whereas the features characterizing the T. borchii samples were F41304, F42634, and F43432. The two clusters associated with the T. magnatum and T. borchii samples were clearly separated along principal component 1 (PC1). PC1 alone accounted for 93.3% of the total variance. In combination with PC2 (6.5%), 99.8% of the total variance was covered. Three T. magnatum data points were not included in the 95% confidence ellipse of the cluster. However, we did not consider them as outliers as all three were derived from the same biological sample and thus obviously reflected the biological variability within T. magnatum.

Figure 1

Figure 1. Biplot of the principal component analysis based on the five most relevant features obtained in the untargeted marker screening approach.

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An alternative visualization of the differentiation between T. magnatum and T. borchii on the basis of the five previously identified features is depicted in Figure 2. For each feature, the normalized intensities associated with T. magnatum and T. borchii were displayed as box plots. Features F11518 and F12848 showed higher intensities in T. magnatum, and features F41304, F43432, and F42634 showed higher intensities in T. borchii. Most importantly, in all five features, the T. magnatum and T. borchii data were well separated with no overlap.

Figure 2

Figure 2. Box plots showing the semiquantitative intensity values of the five most relevant features (A–E) obtained in the untargeted marker screening approach.

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As the next step, the compounds behind the five crucial features were identified. The feature characteristics (1tR, 2tR, and m/z values) are summarized in Table S2. Features F11518 and F12848, characterizing T. magnatum, showed the same m/z value (61) and were derived from neighboring tiles. This suggested that both features originated from a single compound. Likewise, features F41304, F43432, and F42634, characterizing T. borchii, all showed an m/z value of 55 and their tiles were also adjacent. Thus, there was, most probably, also only one underlying compound. The exact positions of the two crucial compounds in the GC×GC chromatograms were determined using EICs based on the features’ m/z values, 61 and 55, respectively, and the associated mass spectra were compared to database spectra. (34) In both cases, the database search returned hits with match and reverse match factors >900 and probabilities >97%, suggesting that the compound characterizing T. magnatum was bis(methylsulfanyl)methane and the compound characterizing T. borchii was furan-2(5H)-one. GC×GC–TOFMS analysis of authentic reference compounds confirmed the structure assignments: when analyzed under the same conditions, the reference compounds returned the same values for 1tR, 2tR, and identical EI mass spectra as obtained from the truffle volatile isolates (Supporting Information, Figures S1 and S2). In summary, the untargeted screening suggested bis(methylsulfanyl)methane as a marker compound for T. magnatum and furan-2(5H)-one as a marker compound for T. borchii.

Verification of the Marker Compounds by Exact Quantitation

Although the marker compound screening was successful, it must be considered that the differentiation between T. magnatum and T. borchii depicted in Figures 1 and 2 was based on semiquantitative data only. Furthermore, GC×GC–TOFMS analysis is expensive and unsuitable for routine analysis in truffle companies or commercial laboratories. Consequently, we aimed to verify the marker compound properties of bis(methylsulfanyl)methane and furan-2(5H)-one by a targeted approach that combines the accuracy of the results with a more straightforward GC–MS system. Thus, stable isotopologues of the target compounds were used as internal standards. This fully compensates for losses during workup and analysis and allows for the acquisition of accurate quantitative data independent of workup details and instrumental platforms. Furthermore, an economic one-dimensional system readily available in quality control laboratories was used for the GC–MS measurements.
The targeted quantitation approach was applied to 13 T. magnatum samples and six T. borchii samples of confirmed authenticity, all of which had previously been included in the untargeted marker compound screening. Three additional samples whose authenticity was not confirmed were analyzed, among which two were labeled as T. magnatum and one as T. borchii. Each sample was subjected to a triplicate workup. The results (Figure 3) confirmed the outcome of the semiquantitative analyses (cf. Figure 2). The concentration of bis(methylsulfanyl)methane (Figure 3A) in the 13 confirmed T. magnatum samples ranged from 237 ± 27 to 4360 ± 260 μg/kg (Supporting Information, Table S4). By contrast, bis(methylsulfanyl)methane was undetectable in the six confirmed T. borchii samples. Integration of the background noise in these samples indicated theoretical maximum bis(methylsulfanyl)methane concentrations below 56 μg/kg. Thus, the bis(methylsulfanyl)methane concentration in the T. magnatum samples was consistently higher than in the T. borchii samples, with an empty window of 154 μg/kg between the two data sets when the error bars were considered (Figure 3A, range between the red lines). This corresponded to a factor of 3.75. Likewise, the concentration of furan-2(5H)-one in the six confirmed T. borchii samples was consistently higher than that in the T. magnatum samples (Figure 3B). Whereas the values in the T. borchii samples ranged from 1490 ± 80 to 5010 ± 160 μg/kg, the values in the T. magnatum samples ranged only from 137 ± 23 to 487 ± 25 μg/kg (Supporting Information, Table S5). Considering the error bars, this corresponded to an empty window between the two concentration intervals of 898 μg/kg (Figure 3B, range between the red lines), resulting in a factor of 2.75.

Figure 3

Figure 3. Concentrations of bis(methylsulfanyl)methane (A) and furan-2(5H)-one (B) in samples of Tuber magnatum and Tuber borchii with confirmed and unconfirmed authenticity.

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In summary, the data showed that the quantitation of the two volatile marker compounds bis(methylsulfanyl)methane and furan-2(5H)-one (Figure 4) is a suitable analytical approach to distinguish between T. magnatum and T. borchii. This conclusion was supported by the results obtained from the samples without confirmed authenticity, which were purchased on the Internet. Both the two samples sold as T. magnatum and the sample sold as T. borchii showed concentrations of bis(methylsulfanyl)methane and furan-2(5H)-one in the expected ranges, indicating that they were correctly labeled (Figure 3, gray bars). In view of the low requirements regarding instrumentation, the method is directly available to be used in routine analysis for the objective species differentiation of T. magnatum and T. borchii.

Figure 4

Figure 4. Marker compounds bis(methylsulfanyl)methane and furan-2(5H)-one characterizing the white truffle species Tuber magnatum and Tuber borchii, respectively.

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Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c00714.

  • t- and p-Values associated with the difference in the intensity values of the five crucial features between T. magnatum and T. borchii; characteristics of the five crucial features and the internal standard as obtained from the GC×GC–TOFMS screening; stable isotopically substituted internal standards, quantifier ions, and calibration lines used in the targeted quantitation of the marker compounds; individual concentration values used for calculating the mean values and standard deviations; signals and mass spectra obtained for bis(methylsulfanyl)methane and furan-2(5H)-one in the truffle volatile isolates and from the respective reference compounds by GC×GC–TOFMS analysis (PDF)

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Author Information

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  • Corresponding Author
    • Martin Steinhaus - Leibniz Institute for Food Systems Biology at the Technical University of Munich (Leibniz-LSB@TUM), Lise-Meitner-Straße 34, 85354 Freising, Germany;  TUM School of Natural Sciences, Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, Germany;  Orcidhttps://orcid.org/0000-0002-9879-1474;  Email: [email protected]
  • Author
    • Philipp Schlumpberger - TUM School of Natural Sciences, Department of Chemistry, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, Germany;  Leibniz Institute for Food Systems Biology at the Technical University of Munich (Leibniz-LSB@TUM), Lise-Meitner-Straße 34, 85354 Freising, Germany
  • Funding

    The project was supported by funds of the Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support program (grant no. 2816504314).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank Julia Bock and Inge Kirchmann for their skillful technical assistance in sample preparation and quantitation. Eva Bauersachs and Jörg Stein provided helpful support with the synthesis and purification of (2H2)furan-2(5H)-one.

Abbreviations

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1D

first dimension

2D

second dimension

1tR

retention time in the first dimension

2tR

retention time in the second dimension

aSAFE

automated solvent-assisted flavor evaporation

CIS

cooled injection system

EI

electron ionization

EIC

extracted-ion chromatogram

GC

gas chromatography

GC×GC–TOFMS

comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry

GC–MS

gas chromatography–mass spectrometry

HS–SPME

headspace solid-phase microextraction

LC

liquid chromatography

MS

mass spectrometry

m/z

mass-to-charge ratio

PC

principal component

PCA

principal component analysis

SAFE

solvent-assisted flavor evaporation

tR

retention time

References

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This article references 34 other publications.

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    Application of the aroma ext. diln. anal. (AEDA) on distillates prepd. by solvent extn./SAFE-distn. from white Alba truffle (WAT; Tuber magnatum pico) and Burgundy truffle (BT; Tuber uncinatum), revealed twenty odor-active regions in the FD-factor range of 16 to 4096 in WAT and twenty-five in BT. The identification expts. in combination with the FD-factors showed clear differences in the overall set of key odorants of both fungi. While 3-(methylthio)propanal (potato-like) followed by 2- and 3-methylbutanal (malty), 2,3-butanedione (buttery) and bis(methylthio)methane (garlic-like) showed the highest FD-factors in WAT, 2,3-butanedione, phenylacetic acid (honey-like) and vanillin (vanilla-like) appeared with the highest FD-factors in BT. Odor activity values (ratio of concn. to odor thresholds), which were calcd. on the basis of quant. data obtained by stable isotope diln. assays, revealed bis(methylthio)methane, 3-methylbutanal and 3,4-dihydro-2-(H)pyrrol (1-pyrroline) with OAVs above 1000 as key contributors the aroma of WAT. In BT, 1-pyrroline and 2,3-butanedione showed the highest OAVs of 1530 and 1130, resp. Aroma recombination expts. successfully mimicked the overall aroma profiles of both fungi when all odorants showing OAVs above one were combined. Omission expts. confirmed the amine-, sperm-like smelling 1-pyrroline, identified for the first time as key odorant in both truffle species, as very important odorant in both fungi.
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  1. Eva Bauersachs, Andreas Dunkel, Veronika Mall, Klaas Reglitz, Martin Steinhaus. Using a combined volatilomics and sensomics approach to identify candidate markers for the differentiation of variously preserved not from concentrate (NFC) orange juices. Food Chemistry 2025, 480 , 143966. https://doi.org/10.1016/j.foodchem.2025.143966
  2. Na Li, Guanyu Li, Xuan Guan, Aihua Li, Yongsheng Tao. Volatile aroma compound-based decoding and prediction of sweet berry aromas in dry red wine. Food Chemistry 2025, 463 , 141248. https://doi.org/10.1016/j.foodchem.2024.141248
  3. P. Schlumpberger, M. Steinhaus. Entwicklung einer GC‐MS‐Methode zur eindeutigen Differenzierung der weißen Trüffelsorten Tuber magnatum und Tuber borchii. Lebensmittelchemie 2024, 78 (S3) https://doi.org/10.1002/lemi.202459053
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  • Abstract

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    Figure 1

    Figure 1. Biplot of the principal component analysis based on the five most relevant features obtained in the untargeted marker screening approach.

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    Figure 2

    Figure 2. Box plots showing the semiquantitative intensity values of the five most relevant features (A–E) obtained in the untargeted marker screening approach.

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    Figure 3

    Figure 3. Concentrations of bis(methylsulfanyl)methane (A) and furan-2(5H)-one (B) in samples of Tuber magnatum and Tuber borchii with confirmed and unconfirmed authenticity.

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    Figure 4

    Figure 4. Marker compounds bis(methylsulfanyl)methane and furan-2(5H)-one characterizing the white truffle species Tuber magnatum and Tuber borchii, respectively.

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  • References

    This article references 34 other publications.

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      Truffles are hypogeous fungi which live in symbiosis with plant host roots in order to accomplish their life cycle. Some species, such as Tuber magnatum Pico, the 'white truffle', and Tuber melanosporum Vittad., the 'black truffle', are highly appreciated in many countries because of their special taste and smell. The great demand for the black and white truffles, the increasing attention towards other species of local interest for the rural economy (such as T. aestivum) together with a drop in productivity, have stimulated researchers to develop projects for a better understanding of the ecol. of truffles by exploiting the new approaches of environmental microbiol. and mol. ecol. Specific primers have been developed to identify many morphol. similar species, the distribution of T. magnatum has been followed in a selected truffle-ground, the phylogeog. of T. melanosporum and T. magnatum has been traced, and the microorganisms assocd. with the truffles and their habitats have been identified.
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      The biochemistry and biological properties of the world's most expensive underground edible mushroom: Truffles
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      Food Research International (2011), 44 (9), 2567-2581CODEN: FORIEU; ISSN:0963-9969. (Elsevier B.V.)
      This review is to provide an update in the recent truffle research with particular emphasis on the chem. properties (nutritional and arom. profile) and their potential biol. activities such as antioxidant, antiviral, anti-microbial, hepatoprotective, anti-mutagenic, anti-inflammatory, anti-carcinogenic, and anti-tuberculoid. In addn., some of the diversification patterns (e.g., biogeog., cultivar, and morphol.) and preservation of truffles are briefly introduced. A few snapshot summary tables are also incorporated to give further detailed guidance for each section, spanning in particular the findings in the last ten years (2000-2010). It is quite clear that further scientific studies need to pay greater attention on how to incorporate these biochem. and biol. properties into the value-added truffles and truffle related products.
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      Belfiori, B.; Riccioni, C.; Paolocci, F.; Rubini, A. Characterization of the reproductive mode and life cycle of the whitish truffle T. borchii. Mycorrhiza 2016, 26, 515– 527,  DOI: 10.1007/s00572-016-0689-0
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      Leo, A. Tartufo.com. https://www.tartufo.com/en/truffle-prices/ (accessed January 12, 2024).
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      Mustafa, A. M.; Angeloni, S.; Nzekoue, F. K.; Abouelenein, D.; Sagratini, G.; Caprioli, G.; Torregiani, E. An Overview on truffle aroma and main volatile compounds. Molecules 2020, 25, 5948,  DOI: 10.3390/molecules25245948
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      An overview on truffle aroma and main volatile compounds
      Mustafa, Ahmed M.; Angeloni, Simone; Nzekoue, Franks Kamgang; Abouelenein, Doaa; Sagratini, Gianni; Caprioli, Giovanni; Torregiani, Elisabetta
      Molecules (2020), 25 (24), 5948CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Truffles are underground edible fungi that grow symbiotically with plant roots. They have been globally considered as one of the most expensive foods because of their rarity, unique aroma, and high nutritional value as antioxidant, anti-inflammatory, antiviral, hepatoprotective, anti-mutagenic, antituberculoid immunomodulatory, antitumor, antimicrobial, and aphrodisiac. The unique flavor and fragrance of truffles is one of the main reasons to get worldwide attraction as a food product. So, the aim of this review was to summarize the relevant literature with particular attention to the active aroma components as well as the various sample prepn. and anal. techniques used to identify them. The major anal. methods used for the detn. of volatile org. compds. (VOC) in truffles are gas chromatog. (GC), proton-transfer-reaction mass spectrometry (PTR-MS), and electronic nose sensing (EN). In addn., factors influencing truffle aroma are also highlighted. For this reason, this review can be considered a good ref. for research concerning aroma profiles of different species of truffles to deepen the knowledge about a complex odor of various truffles.
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      Buendía, E.; Rodríguez, A. Trufamania. https://www.trufamania.com/truffles-home.htm (accessed January 12, 2024).
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      Graziosi, S.; Hall, I. R.; Zambonelli, A. The mysteries of the white truffle: its biology, ecology and cultivation. Encyclopedia 2022, 2, 1959– 1971,  DOI: 10.3390/encyclopedia2040135
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      Culleré, L.; Ferreira, V.; Venturini, M. E.; Marco, P.; Blanco, D. Potential aromatic compounds as markers to differentiate between Tuber melanosporum and Tuber indicum truffles. Food Chem. 2013, 141, 105– 110,  DOI: 10.1016/j.foodchem.2013.03.027
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      Potential aromatic compounds as markers to differentiate between Tuber melanosporum and Tuber indicum truffles
      Cullere, Laura; Ferreira, Vicente; Venturini, Maria E.; Marco, Pedro; Blanco, Domingo
      Food Chemistry (2013), 141 (1), 105-110CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
      The Tuber indicum (Chinese truffle) and Tuber melanosporum (Black truffle) mushroom species are morphol. very similar, but their aroma is very different. The black truffle aroma is much more intense and complex and is appreciated more gastronomically. Differences in the aroma compds. compn. in the 2 species could help in fraud detection. The compds. were analyzed by GC with olfactometric evaluation (GC-O). Eight important odorants were identified. In the order of arom. significance, these were 1-octen-3-one, 1-octen-3-ol, Et isobutyrate, Et 2-methylbutyrate, 3-methyl-1-butanol, iso-Pr acetate, dimethyldisulfide (DMDS), and dimethylsulfide (DMS). Comparison of the aroma profiles revealed that T. indicum had significant arom. contribution of 1-octen-3-one and 1-octen-3-ol (with modified frequencies MF% of 82 and 69%, resp.), while T. melanosporum these values were <30%. Et isobutyrate, Et 2-methylbutyrate, and iso-Pr acetate MF% values were also higher, while DMS and DMDS values were lower (30-40%) compared to T. melanosporum (>70%). The volatile profiles of both species were also studied by means of headspace solid-phase microextn. GC-MS (HS-SPME-GC-MS). The family of C8 compds. (3-octanone, octanal, 1-octen-3-one, 3-octanol, 1-octen-3-ol) was present in T. indicum at much higher levels. The presence of 1-octen-3-ol was higher ∼100-fold, while 1-octen-3-one was detected in T. indicum only (no chromatog. peak in T. melanosporum). As well as showing the greatest chromatog. differences, these 2 compds. were also the most powerful from the arom. viewpoint in the T. indicum olfactometry. The GC-O and HS-SPME-GC-MS together or sep. could be used as screening techniques to distinguish between T. indicum and T. melanosporum and thus avoid possible fraud.
    14. 14
      El Karkouri, K.; Couderc, C.; Decloquement, P.; Abeille, A.; Raoult, D. Rapid MALDI-TOF MS identification of commercial truffles. Sci. Rep. 2019, 9, 17686,  DOI: 10.1038/s41598-019-54214-x
      14
      Rapid MALDI-TOF MS identification of commercial truffles
      El Karkouri Khalid; Couderc Carine; Decloquement Philippe; Abeille Annick; Raoult Didier; El Karkouri Khalid; Couderc Carine; Decloquement Philippe; Abeille Annick; Raoult Didier
      Scientific reports (2019), 9 (1), 17686 ISSN:.
      Truffles are edible mushrooms with similar morphological characteristics, that make it difficult to distinguish between highly prized truffles (such as the Perigord black T. melanosporum) and inexpensive truffles (such as the Asian Black T. indicum). These biological and economic features have led to several misidentifications and/or fraudulent profit in the truffle markets. In this paper, we investigate Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) biotyping to identify 34 commercial fresh truffles from Europe and Asia. The MALDI-TOF MS clustering rapidly distinguished seven Tuber species identified by ITS phylogenetic analysis. The tasty T. melanosporum was clearly differentiated from the Chinese and less expensive truffles. These cheaper mushrooms were marketed as T. indicum but corresponded to a mix of three species. In total, the method confirmed misidentifications in 26% of commercial specimens. Several unknown blind-coded truffles were rapidly identified, with scores >= 2, using the Bruker Biotyper algorithm against MS databases. This study demonstrates that MALDI-TOF MS is a reliable, rapid and cheaper new tool compared with molecular methods for the identification of truffle species and could be used to control frauds in the truffle markets. It could also be useful for the certification of truffle-inoculated seedlings and/or diversity in forest ecosystems.
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      Schelm, S.; Siemt, M.; Pfeiffer, J.; Lang, C.; Tichy, H. V.; Fischer, M. Food authentication: identification and quantitation of different Tuber species via capillary gel electrophoresis and real-time PCR. Foods 2020, 9, 501,  DOI: 10.3390/foods9040501
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      Krauss, S.; Vetter, W. Geographical and species differentiation of truffles (Tuber spp.) by means of stable isotope ratio analysis of light elements (H, C, and N). J. Agric. Food Chem. 2020, 68, 14386– 14392,  DOI: 10.1021/acs.jafc.0c01051
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      Segelke, T.; von Wuthenau, K.; Neitzke, G.; Muller, M. S.; Fischer, M. Food authentication: species and origin determination of truffles (Tuber spp.) by inductively coupled plasma mass spectrometry and chemometrics. J. Agric. Food Chem. 2020, 68, 14374– 14385,  DOI: 10.1021/acs.jafc.0c02334
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    18. 18
      Sommer, K.; Krauss, S.; Vetter, W. Differentiation of European and Chinese truffle (Tuber sp.) species by means of sterol fingerprints. J. Agric. Food Chem. 2020, 68, 14393– 14401,  DOI: 10.1021/acs.jafc.0c06011
      18
      Differentiation of European and Chinese Truffle (Tuber sp.) Species by Means of Sterol Fingerprints
      Sommer, Katrin; Krauss, Stephanie; Vetter, Walter
      Journal of Agricultural and Food Chemistry (2020), 68 (49), 14393-14401CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      The increasing demand of valuable truffles (Tuber sp.) has prompted new areas of naturally growing truffles entering the market. Hence, the identification of valueless Tuber species is an important task to prevent food fraud. Here, we show that sterol patterns are suited to differentiate five Tuber species (Tuber magnatum, Tuber melanosporum, Tuber aestivum, Tuber albidum, and Tuber indicum varieties) from each other. Next to the known main sterols of Tuber, ergosterol and brassicasterol, occurrence of minor sterols in differing shares resulted in characteristic fingerprints in the five Tuber species, irresp. of the country of origin. A total of 27 sterols were evaluated, and we proposed assignment criteria of main sterol relations as well as eight distinct biomarkers within the minor compds. for the differentiation of European and Chinese truffles.
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      Mix, T.; Janneschutz, J.; Ludwig, R.; Eichbaum, J.; Fischer, M.; Hackl, T. From nontargeted to targeted analysis: feature selection in the differentiation of truffle species (Tuber spp.) using 1H NMR spectroscopy and support vector machine. J. Agric. Food Chem. 2023, 71, 18074– 18084,  DOI: 10.1021/acs.jafc.3c05786
      There is no corresponding record for this reference.
    20. 20
      Li, X.; Zhang, X.; Ye, L.; Kang, Z.; Jia, D.; Yang, L.; Zhang, B. LC-MS-based metabolomic approach revealed the significantly different metabolic profiles of five commercial truffle species. Front. Microbiol. 2019, 10, 2227,  DOI: 10.3389/fmicb.2019.02227
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      Creydt, M.; Fischer, M. Food authentication: truffle species classification by non-targeted lipidomics analyses using mass spectrometry assisted by ion mobility separation. Mol. Omics 2022, 18, 616– 626,  DOI: 10.1039/D2MO00088A
      21
      Food authentication: truffle species classification by non-targeted lipidomics analyses using mass spectrometry assisted by ion mobility separation
      Creydt, Marina; Fischer, Markus
      Molecular Omics (2022), 18 (7), 616-626CODEN: MOOMAW ISSN:. (Royal Society of Chemistry)
      Truffles are appreciated as food all over the world because of their extraordinary aroma. However, quantities are limited and successful cultivation in plantations is very labor-intensive and expensive, or even impossible for some species. These factors make truffles a very valuable food, which is why it is particularly rewarding and tempting to declare inferior species of truffles as more expensive species and thereby counterfeit them. The various species differ in their aroma and thus in their culinary value, but the adulterations cannot be detected on the basis of pure morphol. For this reason, the objective of the present study was to develop a non-targeted lipidomics approach using ion mobility spectrometry-mass spectrometry to distinguish between the white truffle species Tuber magnatum and T. borchii as well as the black truffle species T. melanosporum, T. aestivum and T. indicum. Several hundred features were detected, which were present in significantly different concns. in the studied truffle species. The most important of them were identified using MS/MS spectra and collision cross section (CCS) values. Some compds. were detected whose CCS values have not yet been published and may facilitate identification by other researchers in the future. Just a few marker substances are sufficient to be able to distinguish both black and white truffle species with 100% accuracy. These results can be used for the development of rapid tests, which in the best case will allow truffle anal. directly on-site.
    22. 22
      Pelusio, F.; Nilsson, T.; Montanarella, L.; Tilio, R.; Larsen, B.; Facchetti, S.; Madsen, J. Headspace solid-phase microextraction analysis of volatile organic sulfur compounds in black and white truffle aroma. J. Agric. Food Chem. 1995, 43, 2138– 2143,  DOI: 10.1021/jf00056a034
      22
      Headspace Solid-Phase Microextraction Analysis of Volatile Organic Sulfur Compounds in Black and White Truffle Aroma
      Pelusio, Fabio; Nilsson, Torben; Montanarella, Luca; Tilio, Roberto; Larsen, Bo; Facchetti, Sergio; Madsen, Jorgen
      Journal of Agricultural and Food Chemistry (1995), 43 (8), 2138-43CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Headspace solid-phase microextn. (HS-SPME) combined with gas chromatog.-ion trap mass spectrometry (GC-ITMS) is shown to be a powerful technique for detection of volatile org. sulfur compds. (sulfur VOCs) in aromas of white truffles (Tuber magnatum Pico) and black Perigord truffles (Tuber melanosporum). For both species all of the compds. previously identified during several studies were detected in single analyses, and in the case of white truffles three new sulfur compds. were identified: di-Me di- and trisulfide and 1,2,4-trithiolane. Comparison with traditional headspace Tenax adsorption/desorption GC-MS analyses of the aromas showed that the HS-SPME technique is less suited for quant. analyses, esp. because the polydimethylsiloxane fiber coating used in the SPME device strongly discriminates more polar and very volatile compds. With the Tenax adsorption anal. two new sulfur compds. were identified in black truffle aroma: 1-(methylthio)propane and 1-(methylthio)-1-propene. The predominant sulfur compds. are di-Me sulfide and bis(methylthio)methane in white truffle aroma and di-Me sulfide in black truffle aroma. On evapn. of the sulfur compds. from cuttings of black truffle a distinct mushroom odor appeared that is ascribed to the considerable contents of 1-octen-3-ol and other C8 compds., characteristic for mushroom aroma, that are present in the black truffle aroma.
    23. 23
      Kiss, M.; Csóka, M.; Győrfi, J.; Korány, K. Comparison of the fragrance constituents of Tuber aestivum and Tuber brumale gathered in Hungary. J. Appl. Bot. Food Qual. 2011, 84, 102– 110
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    24. 24
      D’Auria, M.; Rana, G. L.; Racioppi, R.; Laurita, A. Studies on volatile organic compounds of Tuber borchii and T. asa-foetida. J. Chromatogr. Sci. 2012, 50, 775– 778,  DOI: 10.1093/chromsci/bms060
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    25. 25
      Zhang, N.; Chen, H.; Sun, B.; Mao, X.; Zhang, Y.; Zhou, Y. Comparative analysis of volatile composition in Chinese truffles via GC × GC/HR-TOF/MS and electronic nose. Int. J. Mol. Sci. 2016, 17, 412,  DOI: 10.3390/ijms17040412
      25
      Comparative analysis of volatile composition in Chinese truffles via GC × GC/HR-TOF/MS and electronic nose
      Zhang, Ning; Chen, Haitao; Sun, Baoguo; Mao, Xueying; Zhang, Yuyu; Zhou, Ying
      International Journal of Molecular Sciences (2016), 17 (4), 412/1-412/16CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      To compare the volatile compds. of Chinese black truffle and white truffle from Yunnan province, this study presents the application of a direct solvent extn./solvent-assisted flavor evapn. (DSE-SAFE) coupled with a comprehensive two-dimensional gas chromatog. (GC × GC) high resoln. time-of-flight mass spectrometry (HR-TOF/MS) and an electronic nose. Both of the anal. methods could distinguish the aroma profile of the two samples. In terms of the overall profile of truffle samples in this research, more kinds of acids were detected via the method of DSE-SAFE. Besides, compds. identified in black truffle (BT), but not in white truffle (WT), or vice versa, and those detected in both samples at different levels were considered to play an important role in differentiating the two samples. According to the anal. of electronic nose, the two samples could be sepd., as well.
    26. 26
      Mauriello, G.; Marino, R.; D’Auria, M.; Cerone, G.; Rana, G. L. Determination of volatile organic compounds from truffles via SPME-GC-MS. J. Chromatogr. Sci. 2004, 42, 299– 305,  DOI: 10.1093/chromsci/42.6.299
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      Gioacchini, A. M.; Menotta, M.; Bertini, L.; Rossi, I.; Zeppa, S.; Zambonelli, A.; Piccoli, G.; Stocchi, V. Solid-phase microextraction gas chromatography/mass spectrometry: a new method for species identification of truffles. Rapid Commun. Mass Spectrom. 2005, 19, 2365– 2370,  DOI: 10.1002/rcm.2031
      27
      Solid-phase microextraction, gas chromatography/mass spectrometry: A new method for species identification of truffles
      Gioacchini, Anna Maria; Menotta, Michele; Bertini, Luana; Rossi, Ismaela; Zeppa, Sabrina; Zambonelli, Alessandra; Piccoli, Giovanni; Stocchi, Vilberto
      Rapid Communications in Mass Spectrometry (2005), 19 (17), 2365-2370CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      This study describes a rapid method to identify different truffle species by anal. of their volatile compd. fraction using static headspace solid-phase microextn. gas chromatog./mass spectrometry. The volatile org. compds. (VOCs) were extd. using a new 2-cm 50/30 μm DVB/CAR/PDMS fiber placed for 10 min in the headspace of the truffle sample with the vial maintained at 20°C (in a thermostatically controlled anal. room). The mass spectra of the VOC chromatograms were represented as 'fingerprints' of the analyzed samples. Next, stepwise factorial discriminant anal. afforded a limited no. of characteristic fragment ions that allowed a classification of the truffle species studied. This new method provides an effective approach to rapid quality control and identification of truffle species by anal. of their volatile fraction. Moreover, this method offers the advantage of minimizing thermal, mech., and chem. modifications of the truffles, thereby reducing the risk of anal. artifacts.
    28. 28
      Schlumpberger, P.; Stübner, C. A.; Steinhaus, M. Development and evaluation of an automated solvent-assisted flavour evaporation (aSAFE). Eur. Food Res. Technol. 2022, 248, 2591– 2602,  DOI: 10.1007/s00217-022-04072-1
      28
      Development and evaluation of an automated solvent-assisted flavor evaporation (aSAFE)
      Schlumpberger, Philipp; Stuebner, Christine A.; Steinhaus, Martin
      European Food Research and Technology (2022), 248 (10), 2591-2602CODEN: EFRTFO; ISSN:1438-2377. (Springer)
      Artifact-avoiding isolation of the volatiles from foods is a crucial step before anal. of odor-active compds. by gas chromatog. (GC). In the past 20 years, solvent extn. followed by solvent-assisted flavor evapn. (SAFE) has become the std. approach, particularly prior to GC-olfactometry. The manual valve of the SAFE equipment, however, leads to suboptimal yields and the risk of a contamination of the volatile isolate with non-volatiles. We thus developed an automated SAFE (aSAFE) approach by replacing the manual valve with an electronically controlled pneumatic valve. The aSAFE provides clearly higher yields than the manual SAFE (mSAFE), notably from exts. high in lipids and for odorants with comparably high b.ps. Addnl., aSAFE substantially reduces the risk of non-volatiles being transferred to the volatile isolate. Full automatisation is possible by combining the aSAFE approach with an automated liq. nitrogen refill system as well as an endpoint recognition and shut-off system.
    29. 29
      Schmidberger, P. C.; Schieberle, P. Characterization of the key aroma compounds in white Alba truffle (Tuber magnatum Pico) and Burgundy truffle (Tuber uncinatum) by means of the sensomics approach. J. Agric. Food Chem. 2017, 65, 9287– 9296,  DOI: 10.1021/acs.jafc.7b04073
      29
      Characterization of the Key Aroma Compounds in White Alba Truffle (Tuber magnatum pico) and Burgundy Truffle (Tuber uncinatum) by Means of the Sensomics Approach
      Schmidberger, Philipp C.; Schieberle, Peter
      Journal of Agricultural and Food Chemistry (2017), 65 (42), 9287-9296CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Application of the aroma ext. diln. anal. (AEDA) on distillates prepd. by solvent extn./SAFE-distn. from white Alba truffle (WAT; Tuber magnatum pico) and Burgundy truffle (BT; Tuber uncinatum), revealed twenty odor-active regions in the FD-factor range of 16 to 4096 in WAT and twenty-five in BT. The identification expts. in combination with the FD-factors showed clear differences in the overall set of key odorants of both fungi. While 3-(methylthio)propanal (potato-like) followed by 2- and 3-methylbutanal (malty), 2,3-butanedione (buttery) and bis(methylthio)methane (garlic-like) showed the highest FD-factors in WAT, 2,3-butanedione, phenylacetic acid (honey-like) and vanillin (vanilla-like) appeared with the highest FD-factors in BT. Odor activity values (ratio of concn. to odor thresholds), which were calcd. on the basis of quant. data obtained by stable isotope diln. assays, revealed bis(methylthio)methane, 3-methylbutanal and 3,4-dihydro-2-(H)pyrrol (1-pyrroline) with OAVs above 1000 as key contributors the aroma of WAT. In BT, 1-pyrroline and 2,3-butanedione showed the highest OAVs of 1530 and 1130, resp. Aroma recombination expts. successfully mimicked the overall aroma profiles of both fungi when all odorants showing OAVs above one were combined. Omission expts. confirmed the amine-, sperm-like smelling 1-pyrroline, identified for the first time as key odorant in both truffle species, as very important odorant in both fungi.
    30. 30
      Kirk, D. N.; McLaughlin, L. M.; Lawson, A. M.; Setchell, K. D. R.; Patel, S. K. Synthesis of the [2H]-labelled urinary lignans enterolactone and enterodiol. J. Chem. Soc., Perkin Trans. 1985, 1, 35– 37,  DOI: 10.1039/p19850000035
      There is no corresponding record for this reference.
    31. 31
      Bemelmans, J. M. H. Review of isolation and concentration techniques. In Progress in Flavour Research; Land, G. G., Nursten, H. E., Eds.; Applied Science Publishers: London, UK, 1979; pp 79– 88.
      There is no corresponding record for this reference.
    32. 32
      Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation – a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237– 241,  DOI: 10.1007/s002170050486
      32
      Solvent assisted flavor evaporation. A new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrixes
      Engel, Wolfgang; Bahr, Wolfgang; Schieberle, Peter
      European Food Research and Technology (1999), 209 (3-4), 237-241CODEN: EFRTFO; ISSN:1438-2377. (Springer)
      A compact and versatile distn. unit was developed for the fast and careful isolation of volatiles from complex food matrixes. In connection with a high vacuum pump (5 × 10-3 Pa), the new technique, designated solvent assisted flavor evapn. (SAFE), allows the isolation of volatiles from either solvent exts., aq. foods, such as milk or beer, aq. food suspensions, such as fruit pulps, or even matrixes with a high oil content. Application of SAFE to model solns. of selected aroma compds. resulted in higher yields from both solvent exts. or fatty matrixes (50% fat) compared to previously used techniques, such as high vacuum transfer. Direct distn. of aq. fruit pulps in combination with a stable isotope diln. anal. enabled the fast quantification (60 min including MS anal.) of compds. such as the very polar and unstable 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberries (3.2 mg/kg) and tomatoes (340 μg/kg). Furthermore, the direct distn. of aq. foods, such as beer or orange juice, gave flavorful aq. distillates free from non-volatile matrix compds.
    33. 33
      Tranchida, P. Q.; Purcaro, G.; Maimone, M.; Mondello, L. Impact of comprehensive two-dimensional gas chromatography with mass spectrometry on food analysis. J. Sep. Sci. 2016, 39, 149– 161,  DOI: 10.1002/jssc.201500379
      There is no corresponding record for this reference.
    34. 34
      NIST/EPA/NIH Mass Spectral Library (NIST 17) and NIST Mass Spectral Search Program (Version 2.3); National Institute of Standards and Technology: Gaithersburg, 2017.
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c00714.

    • t- and p-Values associated with the difference in the intensity values of the five crucial features between T. magnatum and T. borchii; characteristics of the five crucial features and the internal standard as obtained from the GC×GC–TOFMS screening; stable isotopically substituted internal standards, quantifier ions, and calibration lines used in the targeted quantitation of the marker compounds; individual concentration values used for calculating the mean values and standard deviations; signals and mass spectra obtained for bis(methylsulfanyl)methane and furan-2(5H)-one in the truffle volatile isolates and from the respective reference compounds by GC×GC–TOFMS analysis (PDF)


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