Chemical Screening of Metabolites Profile from Romanian Tuber spp.

Truffles are the rarest species and appreciated species of edible fungi and are well-known for their distinctive aroma and high nutrient content. However, their chemical composition largely depends on the particularities of their grown environment. Recently, various studies investigate the phytoconstituents content of different species of truffles. However, this research is still very limited for Romanian truffles. This study reports the first complete metabolites profiles identification based on gas chromatography-mass spectrometry (GC-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) of two different types of Romania truffles: Tuber magnatum pico and Tuber brumale. In mass spectra (MS) in positive mode, over 100 metabolites were identified from 14 secondary metabolites categories: amino acids, terpenes, alkaloids, flavonoids, organic acids, fatty acids, phenolic acids, sulfur compounds, sterols, hydrocarbons, etc. Additionally, the biological activity of these secondary metabolite classes was discussed.


Introduction
At present, truffles (Tuberaceae family, Tuber genus) are considered an emblem of culinary refinement. Because of their nutritive and very particular organoleptic properties, they are considered as one of the most precious foodstuffs. Truffles were assigned mythical qualities in antiquity and then later in the Middle Age because they grow in the ground and are rarely found [1][2][3][4][5][6][7].
From ancient times, truffles have been considered aphrodisiacs. This property is attributed to the outstanding chemical constituents able to mime the male reproductive hormones (androsterone). There are reports about the truffle flavor is associated with perspiration, clay, garlic, mildew, and a faint onion smell [1,5]. There have been several studies on the volatile organic compounds (VOC) and the components involved in flavor. However, the chemical composition of truffles largely depends on the soil characteristics, environmental conditions, and especially the host trees [1][2][3][4][5][7][8][9][10][11][12].
The truffle's growth in natural conditions depends on continuously changing climate conditions causing a restriction of their natural area, which directly influences their prices. Preserving truffles and their complex flavor still represents a challenge for the

Results and Discussion
The truffles chemical composition is highly complex and it is not yet fully described, especially since it is directly dependent on several factors, of which the most important are: host tree and soil parameters. Two solvents were selected with low polarity to achieve the extraction of truffles metabolites.
Thus, in dichloromethane, a polar aprotic solvent is expected to extract lipophilic compounds, such as fatty acids, terpenes, steroids, etc. Moreover, high polarity fractions (amino acids, alkaloids, carbohydrates, etc.) were extracted in methanol. The bioactive compounds screening from the truffles sample were tentatively identified by gas-chromatography coupled with mass spectroscopy (GC-MS) and electrospray ionization-quadrupole time-offlight mass spectrometry (ESI-QTOF-MS) analysis.
Even though gas-chromatography coupled with mass spectroscopy (GC-MS) is one of the most common analytic techniques and is essential in the investigation of natural products due to their features, robustness and high sensitivity allow affordable and highly accurate separation and identification of metabolites [36].
Usually, gas-chromatography (GC) is used mainly for the separation of relatively low molecular weight metabolites such as amino acids, carbohydrates, organic acids, fatty acids, sterols, etc. [36].
A comparison of the total ion chromatographs of both truffle extracts presents the similarities and the differences regarding the metabolite types separated from the analyzed samples. The results are summarized in Table 1, which presents the GC-MS tentative compounds identification corresponding to Tuber magnatum pico and Tuber brumale samples. Truffle samples were diluted in methanol and characterized by ESI-TOF mass spectroscopy (ESI-QTOF-MS). The spectra revealed a complex mixture of molecules from which a few molecules were detected. Thus, mass spectra analysis showed the presence of 103 compounds in Tuber magnatum pico and 105 compounds from the Tuber brumale. Major of these phytochemicals are fatty acids, fatty esters, and sterols. The truffles samples were carried out in positive mode.
The spectra disclose a very complex mixture of molecules from which only some molecules were detected. A total of 109 identified metabolites were attributed to different chemical classes such as amino acids, saccharides, flavonoids, aldehyde, ketone, esters, sulfur compounds, terpenoids, phenolic acids, steroids, hydrocarbons, and other data confirming results already published in the literature [7,10,15,17,[19][20][21][22][23][24][25][26][27][28][29]. The results of the GC-MS were confirmed by ESI-QTOF-MS analysis.     The proportion of each metabolite categories distributed in two species truffles investigated was presented in the figures below. There is a distinction regarding the metabolite numbers accumulated in T. brumale (105), which was slightly larger than in T. magnatum pico (103). It was found that for T. brumale, the number of steroids and sulfur compounds was significantly higher than in T. magnatum pico. More amino acids were present in T. magnatum pico than T. brumale. In both truffle samples investigated, different amino acids were identified, and most of them are essential amino acids (valine, threonine, leucine, lysine, methionine) with few non-essential amino acids (ornithine, asparagine, cysteine) [7,25]. Previous studies revealed that each of these categories of metabolites identified in truffle samples exhibit biological activity [7,[22][23][24]52]. For instance, sinapine, an alkaloid from T. brumale, possesses antioxidant and anti-inflammatory properties [7]. Aldehydes, alcohols, esters, and sulfur compounds are considered as responsible for the special truffle flavor [7,22,53,59]. Despite numerous studies, there is no complete description of the truffles' very complex VOC assemble. Moreover, it is even more difficult to distinguish between each flavor component [7,10,38,40,45,53]. Some of them have been identified and presented in Table 3 [1,7,10,39,40,45]. In black truffles, such as T. brumale, the presence of sulfur compounds in large numbers is considered to be decisive for their specific aroma [1,7,10,39,40,45]. The environmental conditions lead to differences in the VOC profile between the same type of truffles harvested in different seasons.  Winter truffles have to develop more VOC molecules than white truffles, since the growing conditions are quite different between them [1,7,10,38,40,45]. Our results support this hypothesis. Among the winter truffles investigated, T. brumale contains more VOC molecules than white truffle, T. magnatum pico. Dipropyl trisulfide and bis (2-methyl-3 furyl) disulfide are the two sulfur compounds that have been identified only in our black truffle sample (T. brumale). More recently, truffles' ergosteroid have been integrated into the VOC category with a characteristic sulfurous aroma [54]. Ergosta-5,7,22-trien-ß-ol, ergosterol, and brassicasterol were tentatively identified by ESI-QTOF-MS in T. brumale.
It should be mentioned that in both truffles, androstenone was identified, a steroidal pheromone with a distinct scent with various and completely different descriptions (floral, vanilla, sandalwood, sweaty, urine, or even without any odor [1,57]). It is estimated that due to the presence of this pheromone it is possible to train pigs or dogs to detect truffles [1,57], The predominant sulfur compounds in white truffle aroma are dimethyl sulfide and bis(methylthio)methane and dimethyl sulfide in black truffle aroma [40]. Disulfides derivates has bacteriostatic and antifungal properties [43]. The phenolic compound 4-aminophenol has shown to have an anti-inflammatory role [7].
The glycosylceramide identified in both truffles investigated is a sphingolipid type containing glucose residue [20,54]. This compound is highly bioactive with multiple roles in the organism: cell growth apoptosis, antitumor activity, and lowering cholesterol [20,54].
The flavor of the VOC metabolites identified in the investigated truffles is displayed in Table 3 and Figure 1. The key aroma of the investigated Romanian truffles is influenced by environmental conditions (soil parameters, tree host, etc.). Their fragrances are unique: medium sulfuric with sweet fruity, nutty, and floral notes [40].
The glycosylceramide identified in both truffles investigated is a sphingolipid type containing glucose residue [20,54]. This compound is highly bioactive with multiple roles in the organism: cell growth apoptosis, antitumor activity, and lowering cholesterol [20,54].
The flavor of the VOC metabolites identified in the investigated truffles is displayed in Table 3 and Figure 1. The key aroma of the investigated Romanian truffles is influenced by environmental conditions (soil parameters, tree host, etc.). Their fragrances are unique: medium sulfuric with sweet fruity, nutty, and floral notes [40].

Screening and Classification of Metabolites
A total of 109 metabolites were assigned to different chemical categories: amino acids, saccharides, nucleoside, flavonoids, organic acids, phenols and alcohol, esters, sulfur compounds, terpenoids and sesquiterpenes, aldehyde and ketones, phenolic acids, fatty acids, hydrocarbons, vitamins, alkaloids, and other ( Table 4).
The data analysis reported in Table 4 allowed obtaining charts for T. magnatum pico

Screening and Classification of Metabolites
A total of 109 metabolites were assigned to different chemical categories: amino acids, saccharides, nucleoside, flavonoids, organic acids, phenols and alcohol, esters, sulfur compounds, terpenoids and sesquiterpenes, aldehyde and ketones, phenolic acids, fatty acids, hydrocarbons, vitamins, alkaloids, and other (Table 4). Table 4. Classification of metabolites identified in truffles samples on chemical categories.

Sample Fraction Chemical Class Metabolite Name
Tuber magnatum pico

Sample Fraction Chemical Class Metabolite Name
Tuber magnatum pico

Sample Fraction Chemical Class Metabolite Name
Tuber magnatum pico

Sample Fraction Chemical Class Metabolite Name
Tuber brumale

Sample Fraction Chemical Class Metabolite Name
Tuber brumale  The data analysis reported in Table 4 allowed obtaining charts for T. magnatum pico and T. brumale, which are presented in Figures 2 and 3

Materials and methods
Fresh fruiting bodies of Tuber magnatum pico (50 g) and Tuber brumale (50 g) were collected in late November 2019 from the area of the Eastern Carpathians and offered by Cromatec Plus after prior taxonomically and authentication. The truffles samples were rapid frozen in liquid nitrogen (−196 °C), ground and sieved to obtain a particle size lower than 0.5 mm, and kept at −80 °C to avoid enzymatic conversion or metabolites degradation.
For each analysis, 2 g of dried sample was subject to sonication extraction in 25 mL solvent (methanol/ dichloromethane = 1:1) for 20 min at 45 °C, with a frequency of 50 kHz. The solution was concentrated using a rotavapor and the residue was dissolved in MeOH. The extract was centrifuged and the supernatant was filtered through a 0.2-μm syringe filter and stored at −18 °C until analysis.

Reagents
All used reagents were GC grade. Methanol and dichloromethane were purchased from VWR (Wien, Austria).

GC-MS Separation Conditions
The oven temperature program was 80 °C for 9 min, then raised to 220 °C (5 °C/min), to 280 °C (10 °C/ min.), and finally held at this temperature for 20 min. The temperature of the injector was 260 °C and the temperature at the interface was 200 °C.

Materials and Methods
Fresh fruiting bodies of Tuber magnatum pico (50 g) and Tuber brumale (50 g) were collected in late November 2019 from the area of the Eastern Carpathians and offered by Cromatec Plus after prior taxonomically and authentication. The truffles samples were rapid frozen in liquid nitrogen (−196 • C), ground and sieved to obtain a particle size lower than 0.5 mm, and kept at −80 • C to avoid enzymatic conversion or metabolites degradation.
For each analysis, 2 g of dried sample was subject to sonication extraction in 25 mL solvent (methanol/dichloromethane = 1:1) for 20 min at 45 • C, with a frequency of 50 kHz. The solution was concentrated using a rotavapor and the residue was dissolved in MeOH. The extract was centrifuged and the supernatant was filtered through a 0.2-µm syringe filter and stored at −18 • C until analysis.

Reagents
All used reagents were GC grade. Methanol and dichloromethane were purchased from VWR (Wien, Austria).

GC-MS Separation Conditions
The oven temperature program was 80 • C for 9 min, then raised to 220 • C (5 • C/min), to 280 • C (10 • C/min.), and finally held at this temperature for 20 min. The temperature of the injector was 260 • C and the temperature at the interface was 200 • C.

Mass Spectrometry
MS experiments were conducted on an EIS-QTOF-MS analysis from Bruker Daltonics, Billerica, MA, USA. All mass spectra were acquired in the positive ion mode within a mass range of (100-2500) m/z, with a scan speed of 2.1 scans/second. The source block temperature was kept at 80 • C. The reference provided in positive ion mode a spectrum with fair ionic coverage of the m/z range scanned in full-scan MS. The resulting spectrum is a sum of scans over the total ion current (TIC) acquired at 25-85 eV collision energy to provide the full set of diagnostic fragment ions.
Peak assignment to specific ion was based on the standard library, the NIST/NBS-3 (National Institute of Standards and Technology/National Bureau of Standards) spectral database. According to the peak, the resolution area was determined from the total ion current (TIC) or from the estimated selected ions integration. The results are presented in Table 1. The mass spectra of the compounds were compared with those from NIST/EPA/NIH Mass Spectral Library, and the identified compounds are presented in Table 2.

Conclusions
The proposed analytical methodology for the chemical screening of these Romanian truffles type allowed obtaining their metabolite profile. The number of metabolites (amino acids, steroids, and sulfur compounds) was different in both truffle species.
The different proportion of total metabolites identified between T. brumale and T. magnatum pico can be considered as evidence of the influence exerted by genetic and environmental conditions. Each of the chemical categories were detailed, including their biological activity. Moreover, we evaluated the profile of the key aroma compounds. However, studies on Romanian truffles are in the early stages considering that these fungi are still an unvalued source of compounds with high economic value. Further investigations are necessary to disclose the influence of the external factors (environmental condition, host tree, etc.) on the metabolic mechanism of truffles.