An Insight by Molecular Sensory Science Approaches to Contributions and Variations of the Key Odorants in Shiitake Mushrooms

An insight using molecular sensory science approaches to the contributions and variations of the key odorants in shiitake mushrooms is revealed in this study. Odorants were extracted by headspace solid phase microextraction (HS-SPME) and direct solvent extraction combined with solvent-assisted flavor evaporation (DSE-SAFE) in fresh and hot-air-dried shiitake mushrooms. Among them, 18 and 22 predominant odorants were determined by detection frequency analysis (DFA) and aroma extract dilution analysis (AEDA) combined with gas chromatography-olfactometry (GC-O) in the fresh and dried samples, respectively. The contributions of these predominant odorants in the food matrix were determined by quantification and odor activity values (OAVs) with aroma recombination verification. There were 13 and 14 odorants identified as key contributing odorants to overall aroma, respectively. 1-Octen-3-ol and 1-octen-3-one were the most key contributing odorants in the fresh samples in contributing mushroom-like odor. After hot-air-drying, the OAV and concentrations on dry basis of the key contributing odorants changed, due to oxidation, degradation, caramelization and Maillard reactions of fatty acids, polysaccharides and amino acids. 1-Octen-3-ol was reduced most significantly and degraded to 1-hydroxy-3-octanone, while phenylethyl alcohol increased the most and was formed by phenylalanine. In hot-air-dried samples, lenthionine became the most important contributor and samples were characterized by a sulfury odor. Overall contributions and variations of odorants to the aroma of shiitake mushrooms were revealed at the molecular level.


Introduction
Shiitake mushrooms (Lentinus edodes), belonging to fungi phylum tricholoma, are one of most important traditional delicacies and medicinal fungi in Asia on account of their nutritional characteristics, medicinal properties and distinctive flavor [1,2]. They hold the second highest market share in the global market consumption of edible fungi [3]. Generally, shiitake mushrooms are rich in proteins, vitamins, minerals, and low in fat and cholesterol [3,4]. In addition, they have the medicinal properties of being anti-tumoral, antimicrobial, lowering cholesterol activity, lowering blood pressure, improving liver function and strengthening the immune system [5,6]. What is more, shiitake mushrooms are mainly popular for their unique flavor properties and are used as a basis for various dishes [7,8].
Due to their short shelf-life, shiitake mushrooms are usually dehydrated for preservation. Hot-air-drying is the most widely used method for production of dehydrated fruits and vegetables, not only because it is low cost, with easy control and short process size and without mechanical damage were picked out for analysis. The pilei of shiitake mushrooms were further divided into four equal parts.

Hot-Air-Drying Processing
The drying process was carried in a constant-temperature drying oven (DHG-9053A, Jinghong Laboratory Equipment Co., Shanghai, China). The samples were hot air dried at 60 • C in the oven with an air flow speed of 1 m/s and an air humidity of 10%. The samples were spread in a single layer on the tray. The drying process lasted for 10 h until the samples reached constant weight. The water content determination was replicated three times and the average was reported. The water content of pilei was 3.94 ± 0.33% (on wet basis) at 10 h, meeting the national food safety standards for edible fungi and its products (GB7096-2014). After the drying was completed, the dried products were kept in sealed aluminum foil bags and stored at −80°C until analysis.

HS-SPME Analysis
Chopped samples (0.5 g) with saturated NaCl (4 mL) solution were hermetically sealed in a 20 mL headspace glass having a PTFE/silicone septum (Supelco, Bellefonte, PA, USA) and a magnetic screw cap. Then the samples were incubated at 40 • C for 40 min. A stainless steel needle containing divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 µm) SPME fiber (Supelco, Bellefonte, PA, USA) was inserted through the septum of the sample vial to extract the volatile compounds at 40 • C for 40 min. The sample vials were shaken in the agitator at 250 rpm/min during incubating and extracting.

DSE-SAFE Analysis
The samples were extracted following Erten and Cadwallader [21] with some modifications. The fresh (150 g) and dried (18.05 g) shiitake mushrooms with equal contents on a dry basis were broken, and extracted in a PTFE centrifuge bottle with diethyl ether. Then the ether layer was collected by centrifugation. Extraction and centrifugation were repeated twice more with fresh diethyl ether. Subsequently, the extracts were subjected to solvent assisted flavor evaporation (SAFE), and concentrated using the Vigreux column (45°C).

Gas Chromatography-Olfactometry (GC-O)
GC-O analysis was performed using an Agilent 7890B GC (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a flame ionization detector (FID), a sniffing port (ODP3; Gerstel, Mülheim an der Ruhr, Germany), a cool on-column injector and a split/splitless (S/SL) injector. Separations were performed on a fused silica capillary column (either Rtx®-Wax, 30 m length × 0.32 mm i.d. × 0.25 µm film, Restek; or HP-5, 30 m length × 0.32 mm i.d. × 0.25 µm film; Agilent Technologies Inc., USA). The sample (1.0 µL) extracted by DSE-SAFE was injected cold on-column at 35 • C in track oven mode, and that extracted by HS-SPME was injected S/SL injector at 250 • C in splitless mode. Nitrogen was used as carrier gas at a fixed flow rate of 1.53 mL/min. The oven temperature was programmed from 35 • C, increased by 2 • C/min to 130 • C, then increased to 250 • C at 8 • C/min and held for 10 min. The flow of the carrier gas was split in a 1:1 ratio at the end of the capillary column. One part was directed to FID (250 • C) and the other to the sniffing port (200 • C). A series of n-alkanes C7-C30 for the DB-wax and the HP-5 was used to determine linear retention indices (RI).

Detection Frequency Analysis (DFA)
DFA using a panel of four judges (two males and two females) was applied to obtain the odor pattern of shiitake mushrooms following the methodology described by Pang et al. [32]. In total, eight GC-O runs were performed (two runs for each assessor). The detection frequency (DF) for an odor with the same RI and a similar description was summed. At the sniffing port any odorant that had total DF ≥ 2 (reported by at least two assessors) was arbitrarily considered to have aroma potential activity.

Aroma Extract Dilution Analysis (AEDA)
AEDA was used to detect the potential odorants of the DSE-SAFE isolates. The concentrated extract was diluted stepwise in a series of 1:3n with diethyl ether. Each dilution was performed using the GC-O conditions described above until no odor was detected. Flavor dilution (FD) factor of the odorants at the sniffing port was used as a measure for the intensity of a compound, and the higher FD factors were concluded to have a greater relative importance [33].

Gas Chromatography-Mass Spectrometry (GC-MS)
The odorants were determined by the Agilent 7890B/5977B GC-MS instrument (Agilent Technologies Inc., USA) with VF-WAXms and HP-5MS capillary columns (both 30 m × 0.25 mm × 0.25 µm, Agilent Technologies Inc., USA). The sample (0.4 µL) extracted by DSE-SAFE was injected in split ratio (10:1), and that extracted by HS-SPME was injected in splitless mode, with the inlet at 250 • C. The carrier gas was 99.999% pure helium at a fixed flow rate of 1.68 mL/min. The initial oven temperature was 35 • C, ramped at 2 • C/min to 130 • C, then increased to 250 • C at 8 • C/min and held for 10min. Mass detector was conducted in an electron impact mode at 70 eV and the ion source temperature was 230 • C. Mass spectra (MS) were scanned from 50 to 550 amu.

Identification of Odorants
Odorants were positively identified by matching several criteria of unknown odorants to authentic reference standards, including odor characteristics, retention index (RI, on polar and nonpolar GC columns) and electron-impact mass spectra (MS). If one or more of the above criteria could not be met, the compound was considered tentatively identified. RI was calculated by injection of series of n-alkanes as described by Van Den Dool and Kratz [34].

Quantitation of the Predominant Odorants
Quantitation was performed following Zhang et al. [20] with some modifications. Positively identified odorants exhibiting high FD factors (FD ≥ 3) or DF factors (DF = 8) were quantitated by constructing standard curves, which were the linear regressions of mass ratio (target/internal standard) of standard substance versus selected ion area ratio (target/internal standard) [21]. Odorants extraction was performed as above described except that shiitake mushroom samples were first spiked with 800 µL mixture of internal standards (0.01 mg/mL). Mixed internal standards with five levels of concentration were prepared for the calibration and standards were analyzed in triplicate. These solutions were detected by GC-MS as described in Section 2.9, except that mass spectrometry was conducted in the single ion monitoring (SIM) mode. The concentration of a target compound was determined based on the ratio of its selected ion peak area to that of selected ion peak area of its corresponding internal standard (IS) ( Table 3), and the formula as follows: Rf was the response factor (1/slope).

Calculation of Odor Activity Values (OAV)
The OAV of an odorant was determined by dividing its concentration by its published odor detection threshold in water [21].

Aroma Analysis
Aroma was determined by 20 experienced panelists. The judgements of the panelists were averaged. Broken fresh shiitake mushrooms (20 g) and dry shiitake mushrooms powder (2 g) were presented in covered and odorless plastic vessels at room temperature. The assessors were asked to evaluate the intensities of selected characteristic aromas (mushroom, grass, metallic, sulfury, caramel, fatty, and cabbage for fresh shiitake mushrooms; mushroom, chocolate, sulfury, caramel, sweaty, seasoning-like, and cooked potato-like for dry shiitake mushrooms). The intensities were ranged on a six-point scale from 0 (not perceivable) over 1, 2, ..., to 5 (strongly perceivable). The descriptors were compared with aqueous solutions of the reference odorants, and the concentrations of the reference odorants in different odor intensities are shown in (Table 1).

Aroma Recombination Experiments.
All odorants with OAVs ≥ 1 were prepared by mixing them in water solution at their actual concentrations determined in the fresh and hot-air-dried shiitake mushrooms. Then the solution was added to the deodorized matrix, which was prepared using the method of Zhang et al. [20]. Subsequently, the recombination samples, the fresh and hot-air-dried shiitake mushrooms, were each placed in closed, odorless plastic vessels and evaluated by the panelists as explained in Section 2.13.

Statistical Analysis
All statistical analyses were performed in triplicate, with the experimental results expressed as means ± SD. The data were analyzed using the Statistical Program for Social Sciences (SPSS 20.0, Chicago, IL, USA) software for analysis of variance and Duncan's test. The significance was established at p < 0.05. The aroma recombination experiments data were collected and analyzed by Excel (Microsoft Office 2018, Redmond, WA, USA).

Odorants Identified by HS-SPME
A total of 23 odorants were detected by HS-SPME in the fresh shiitake mushrooms ( Table 2). Among them, 17 compounds were positively identified, six compounds were tentatively identified and three compounds were unknown. Most odorants were related to mushroom, herbaceous tinge and sulfury descriptors.
Thirteen compounds were detected by DFA in the fresh samples by all assessors (DF = 8), indicating that they contributed more actively to the aroma of fresh samples and were considered as the predominant odorants. These consisted of four aldehydes, four sulfur compounds, two alcohols, two ketones, and one nitrogen-containing compound. Aldehydes, sulfur compounds, alcohols and ketones have been reported as the main volatile compounds in fresh shiitake mushrooms [35,36]. In addition, 3-octanone, 1-octen-3-one and 1-octen-3-ol exhibited a mushroom-like odor, which was the main odor attribute of shiitake mushrooms. The sulfur compounds, such as 3-(methyl-thio)-1-propanal, 3-(methylthio)-1-propanol, 1,2,4,5-tetrathiane and lenthionine, were also found as predominant odorants in fresh samples and offered cooked potato-like, cabbage-like, sulfury and burnt aroma properties.

Odorants Identified by DSE-SAFE
As shown in (Table 2), 21 odorants were positively and seven compounds tentatively identified in fresh shiitake mushrooms by DSE-SAFE. Three compounds were not identified.
Twenty-three odorants exhibited high FD factors (FD ≥3) by AEDA, indicating that these 23 compounds made major contributions to the overall aroma and were the predominant odorants of fresh shiitake mushrooms. Among these, phenylacetaldehyde (floral), trans-4,5-epoxy-(E)-2-decenal (metallic), 1,2,4,5-tetrathiane (sulfury, burnt), 1,2,4,6tetrathiepane (sulfury) and lenthionine (sulfury, burnt) had the highest FD factors of 729, followed by 3-methyl-butanal (chocolate), 3-(methyl-thio)-1-propanal (cooked potato-like), dimethyl tetra-sulfide (mushroom) with FD factors of 243. The relatively high FD factors of sulfur compounds, like 1,2,4,5-tetrathiane, 1,2,4,6-tetrathiepane, lenthionine, 3-(methylthio)-1-propanal and dimethyl tetra-sulfide, could demonstrate that the sulfur compounds also provided major aroma properties to fresh samples. However, Tian et al. [12] reported that dimethyl tetra-sulfide and 1,2,4,6-tetrathiepane were not detected in fresh shiitake mushrooms. This might be due to the different types of GC column and detector. Only the polar column was used in the study of Tian et al. [12], while dimethyl tetra-sulfide and 1,2,4,6-tetrathiepane were identified as the predominant odorants on the nonpolar column in this study. Therefore, the use of columns with different polarities could more comprehensively identify odorants, since there were many types of aroma compounds with different polarities in the food matrix. In addition, 1-octen-3-one and 1-octen-3-ol were also more important odorants in fresh samples with FD factors of 81, which was consistent with the result identified by DFA. Furthermore, only 13 compounds were consistent with the odor-active compounds detected by Schmidberger and Schieberle [3] in raw shiitake mushrooms. This difference might be due to the different varieties, cultural practices and collection time of shiitake mushrooms, and the distinct extraction and identification methods applied [37].

Odorants Identified by HS-SPME
Among the 25 odorants detected by HS-SPME in the dried shiitake mushrooms, 19 compounds were positively identified, six compounds were tentatively identified, and only one compound was not identified ( Table 2). Most odorants were related to mushroom-like, sulfury, sweet and sweaty descriptors. Based on DFA, 15 compounds had DF of 8, illustrating that these components were detected in the dried shiitake mushrooms by all assessors. Although Tian et al. [12] reported that sulfur compounds and acids were the main volatile compounds in dried shiitake mushrooms, the sulfur compounds and acids were further demonstrated in this study as the predominant odorants in the dried samples, and not only the main volatiles. The sulfur compounds, including dimethyl trisulfide, 1,2,4,5-tetrathiane and lenthionine, performed higher DF factors and contributed to a stronger sulfury odor to dried samples. The acids, like isovaleric acid and phenylacetic acid, contributed sweaty and floral odors, respectively.
In addition, there was some inconsistency between DF determined by DFA, FD determined by AEDA and OAV. In the fresh shiitake mushrooms, benzaldehyde and (E)-2octen-1-ol had the highest DF factors (DF = 8), and isovaleric acid and octanoic acid had relatively high FD factors (FD = 27), but they all had lower OAVs (OAVs < 1). Similarly, 2-ethyl-1-hexanol, benzaldehyde and (E)-2-octen-1-ol had the highest DF factors (DF = 8), and 1-hexanol and 3-(methyl-thio)-1-propanol had relatively high FD factors (FD = 27), but they all had lower OAVs (OAVs < 1) in the dried shiitake mushrooms. This might be due to DFA and AEDA were carried out on the extracts of shiitake mushrooms, which eliminated the influence of food matrix. However, OAV took into account the effect of the food matrix [31]. This inconsistency indicated that the food matrix affected the contribution of volatile components to the aroma. Moreover, ethyl acetate, acetic acid, phenylethyl alcohol and 3-hydroxy-4,5-dimethyl-2(5H)-furanone were identified as predominant odorants in dried shiitake mushrooms by AEDA and their OAVs were greater than or equal to 1, but DFA showed they were non-essential. 1-Octen-3-ol had relatively high OAV and DF factor, but it was not identified as predominant odorants by AEDA in the fresh samples. This might be due to the fact that DFA and AEDA were based on different extraction methods, HS-SPME and DSE-SAFE. HS-SPME specializes in extracting highly volatile compounds and DSE-SAFE extracted semi-volatile and volatile compounds [21]. Thus, the combination of DFA and AEDA could more comprehensively identify the odorants of shiitake mushrooms.

Aroma Recombination Verification
It was well accepted that the predominant odorants obtained by the OAV concept could be confirmed by aroma reconstitution experiments [48]. For this purpose, reconstitution models were prepared containing odorants with high OAVs (OAV ≥ 1) in their actual concentrations in the fresh and dried shiitake mushrooms, respectively. The similarities between the recombination and original samples were judged by the included angle cosine analysis [20]. The results for aroma are summarized in Figure 1, and show that the average similarity degrees were 0.9987 for the fresh samples, and 0.9977 for the dried samples. Thus, it was corroborated that the identification and quantitation of predominant odorants of the fresh and dried shiitake mushrooms could be considered successful, due to the fact that the reconstitution odorants exhibited high similarities to the original samples. Besides, it is shown in (Figure 1) that the mushroom-like odor was reduced after drying. This was due to the decreased OAVs of 3-octanone, 1-octen-3-one and 1-octen-3-ol after drying, which contribute mushroom-like odor. Moreover, the sulfury odor was more intense after drying. This was related to the fact that dimethyl trisulfide and lenthionine had higher OAVs in dried shiitake mushrooms. Similarly, the enhancement of sweaty and caramel-like odors after drying might be due to the increased OAVs of isovaleric acid and 4-hydroxy-2,5-dimethyl-3(2H)-furanone, respectively.  Besides, it is shown in (Figure 1) that the mushroom-like odor was reduced after drying. This was due to the decreased OAVs of 3-octanone, 1-octen-3-one and 1-octen-3-ol after drying, which contribute mushroom-like odor. Moreover, the sulfury odor was more intense after drying. This was related to the fact that dimethyl trisulfide and lenthionine had higher OAVs in dried shiitake mushrooms. Similarly, the enhancement of sweaty and caramel-like odors after drying might be due to the increased OAVs of isovaleric acid and 4-hydroxy-2,5-dimethyl-3(2H)-furanone, respectively.

Variations and Aroma Chemistry of the Key Contributing Odorants in Shiitake Mushrooms
after Hot-Air-Drying Figure 2 shows the key contributing odorants in shiitake mushrooms before and after dying and (Figure 3) illustrates the aroma chemistry of the changes.

Conclusions
A comprehensive molecular sensory science approach, including SPME, DSE-SAFE, GC-O, DFA, AEDA, OAV and aroma recombination verification, was explored to identify and quantify the predominant odorants and their contributions to the aroma of fresh and hot-air-dried shiitake mushrooms and their variations induced by hot-air-drying. 1-Octen-3-ol, lenthionine, 1-octen-3-one and trans-4,5-epoxy-(E)-2-decenal were the key contributing odorants in fresh shiitake mushrooms. This accounted for the fact that the shiitake mushrooms were characterized by mushroom-like, sulfury and metallic odor before hot-air-drying. After hot-air-drying, mushroom-like odor was diminished, following the decrease of 1-octen-3-ol, 1-octen-3-one and 3-octanone due to thermal decomposition and oxidation. However, sulfury, caramel, and seasoning-like odor stood out in the aroma due to the increase or formation of lenthionine, dimethyl trisulfide, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 3-hydroxy-4,5-dimethyl-2(5H)-furanone by the reactions of fatty acids, amino acids and polysaccharides. Ultimately, lenthionine became the most important contributor to aroma with the highest OAV, and the hot-air-dried shiitake mushrooms featured a sulfury odor. Overall, the predominant odorants contributing to the aroma of shiitake mushrooms before and after hot-air-drying were revealed at the molecular level. The contributions and variations in the odorants of shiitake mushrooms and their mechanism were preliminarily studied.