Potentials of truffles in nutritional and medicinal applications: a review

Truffles, the symbiotic hypogeous edible fungi, have been worldwide regarded as a great delicacy because of their unique flavor and high nutritional value. By identifying their bioactive components such as phenolics, terpenoids, polysaccharides, anandamide, fatty acids, and ergosterols, researchers have paid attention to their biological activities including antitumor, antioxidant, antibacterial, anti-inflammatory, and hepatoprotective activities. In addition, numerous factors have been investigating that can affect the quality and productivity of truffles to overcome their difficulty in culturing and preserving. To provide the information for their potential applications in medicine as well as in functional food, this review summarizes the relevant literature about the biochemical composition, aromatic and nutritional benefits, and biological properties of truffles. Besides, various factors affecting their productivity and quality as well as the preservation methods are also highlighted.


Background
Truffles are hypogeous ascomycetes fungi growing underground in depth between 5 and 10 cm. More than a hundred different kinds of truffle species were known worldwide, and new species are being discovered consistently. Taxonomically, edible truffles belong to family Tuberaceae and Pezizaceae, and to order Pezizales [1]. Tuberaceae is showing the diverse lineages of exclusively truffle forming fungi, and truffles belonging to this family have been widely consumed for centuries [2]. For example, the genus Tuber from the Tuberaceae family comprised of approximately more than 180 species globally [3]. Most culinary-grade truffles discovered in northern temperate forests of Europe, Australia, New Zealand, Asia, and North America generally belong to genus Tuber [4,5]. On the other hand, desert truffles growing in the arid and semi-arid regions such as Syria, Iraq, Kuwait, Saudi Arabia, Morocco, Egypt, South Africa, and Tunisia, generally belong to the genus Terfezia and Tirmania [1,6]. More details in the phylogeny of truffles are presented in Fig. 1.
In general, truffles tend to be firm, dense, and woody in comparison with mushrooms that is soft and fragile. White truffles include Tuber magnatum, T. borchii, T. maculatum, T. oregonense, T. latisporum, T. japonicum, and Tirmania nivea [7]. Among them, T. magnatum generally found in Italy and East Europe is the high cost truffle species [8]. In 2000 and 2001, it reached €8000 per kilogram on several Italian markets [9]. Black truffles include Tuber melanosporum, T. aestivum, T. brumale, T. uncinatum, T. indicum, T. himalayense, and Terfezia claveryi [7]. Truffles exhibit a large genome size. For example, T. melanosporum showed a genome size of 125 Mb with a high transposable element and repetitive DNA content which comprised more than 58% of the genome [10].
Truffles are ectomycorrhizal having symbiotic root association. In a complex life cycle, the mycelia establish a symbiotic interaction with host organisms predominantly with the roots of various trees, both gymnosperms, and angiosperms, such as hazel, poplar, pine, eucalyptus, and

Open Access
Fungal Biology and Biotechnology contain more than 60 volatiles, of which 2,4-dithiapentane is the most common [22]. The mixture of DMS and 2-methylbutanal has been also used for a long time by the food industry to reproduce T. melanosporum aroma [15]. Apart from the aromatic feature, their biochemical composition encompasses multiple nutritional and medicinal benefits. Truffles are rich in various types of essential nutritional products including carbohydrates, proteins, fats, minerals, lipids, and amino acids [7]. In addition, they are rich in phenolics, terpenoids, polysaccharides, and phytosterols which are related to their antitumor, antioxidant, antibacterial, hepatoprotective, anti-inflammatory, and immunomodulatory properties [11]. In recent decades, researchers have paid great attention to the bioactive compounds derived from diverse truffle species and their potential in nutritional and medicinal applications [7,23]. This review summarizes and updates the current status of knowledge on the chemical composition of truffles, their biological activities as well as nutritional and medicinal potential, and various factors contribute to their productivity and quality.

Aroma profiles of truffles
The unique flavor of truffles is one of the main reasons to get worldwide attraction as a food product. Previous studies have focused on screening and identifying the volatile organic compounds (VOCs), namely the aroma active compounds, and characterized more than 200 VOCs in various truffle species. The major compounds responsible for the aroma in diverse truffle species were well documented in 2011 reviews [7,15]. Our review aimed to provide an update on research conducted since 2011 about the analysis of active aroma components as well as the analytical techniques to identify them (Table 1).
Truffles possess significant variability in their aroma profiles from species to species. In general, sulfur compounds such as dimethyl sulfide (DMS) and dimethyl disulfide (DMDS), 1-octen-3-ol, and 2-methyl-1-propanol have been identified in most truffle species including Tuber melanosporum, Tuber aestivum, and Tuber borchii [15]. In Tuber magnatum (white truffle), bis (methylthio) methane (BMTM) was reported as the most critical aroma active compound [24]. To deepen the knowledge about a complex odor of various truffle species, researchers have developed multiple methods to analyze truffle's aroma. Traditionally, VOCs have been comprehensively profiled by solid-phase microextraction (SPME), which is generally followed by gas chromatography-mass spectrometry (GC-MS) [25][26][27]. Torregiani et al. analyzed 20 raw truffles, truffle sauces, and truffle-flavored oils using headspace SPME with GC-MS (HS-SPME-GC-MS). Consequently, BMTM was predominantly detected both in raw white truffles and oils flavored with white truffle but not detected or detected only in low amounts in the T. melanosporum (black truffle) samples; instead, 1-octen-3-ol was detected in high amounts in the raw black truffle and oil samples flavored with black truffle [28]. The HS-SPME-GC-MS technique enabled that Liu and Li profiled for the first time a total of 57 volatiles in T. liyuanum; a novel truffle species that have been identified in recent years. In this new truffle species, aldehydes and aromatics were the main chemical constituents, and 3-octanone, phenylethyl alcohol, isopentane, and methylbutane contributed to the significant aromaticity [29].
The GC-MS based analysis expressed limitations to determine the correlation of quantified volatiles to the olfactory stimulus as this technique could not give information about human perception. Moreover, the perceived odor presented frequently at lower concentrations than the instrumental detection limit [30]. To overcome these limitations and to identify the key aroma contributors amongst detected volatiles, the flavor dilution (FD) factor by aroma extract dilution analysis (AEDA) or/ and the odor activity value (OAV) could be determined, which are based on the gas chromatography-olfactometry (GC-O) technology [31]. Splivallo and Ebeler performed the GC-O based analysis to investigate whether thiophene derivatives contributed to the human-sensed aroma in T. borchii. Consequently, they identified a total of 13 main aroma active compounds that showed FD factor higher than 5 and determined that thiophene derivatives; particularly in 3-methyl-4,5-dihydrothiophene, were the important contributors to the human-sensed aroma in T. borchii [32]. Feng et al. performed the SPME extraction combined with GC-O technology. They determined 24 compounds as key contributors to the overall aroma in three Chinese truffle varieties which present floral, mushroom, and sweet odor [24]. In addition to these studies, numerous researchers performed GC-O based analysis together with SPME-GC-MS to find the critical aroma contributor among the comprehensively profiled truffle volatiles [19,[32][33][34].
The olfactometry based analysis considering the entire aroma mixture has been regarded as the most useful method for estimating the contribution of key aromaactive compounds. Although it represents the standardized method for determining the aroma concentration, it does not determine the single compounds and their contribution to the aroma, and the large number of dilution of AEDA is a time-consuming process [30]. Liu  HS-SPME-GC-MS Hexanal and 1-octen-3-ol were key volatiles in T. boudieri and T. nivea fruiting bodies. P. lefebveri with weak aroma exhibits low levels of both volatiles. T. nivea contains dimethyl disulfide, methional, benzaldehyde and benzenacetaldehyde, 3-methylbutanal and 2-methylbutanal. Total volatile levels in T. nivea are about twofold higher than in T. boudieri. T. boudieri has around tenfold higher volatile levels than in P. lefebveri [25] T. formosanum n.g.

Truel, Spain
HS-SPME-GC-MS The effect of freezing on the aroma of T. melanosporum ascocarps was evaluated.

T. liyuanum
China HS-SPME-GC-MS A total of 57 volatiles in T. liyuanum were detected for the first time. Among them, 3-octanone, phenylethyl alcohol, isopentana, and methylbutana were the key contributor to the aroma. This study also suggested the optimal extraction condition for the screening of aroma active compounds: sample amount, 2 g; extraction temperature, 80 °C; extraction time, 40 min; SPME fibre of carboxen/PDMS [29] Page 5 of 17 Lee et al. Fungal Biol Biotechnol (2020)

GC-MS, GC-IRMS
The differences among home-made and commercial truffle oils were evaluated.
The δ13C value of 2,4-dithiapentane in most flavored oil samples was obtained by GC-IRMS and GC-MS. GC-IRMS could not distinguish between synthetic and natural flavors, while the metabolic profiling using GC-MS revealed that two sulfur containing volatiles, dimethyl sulfone and dimethyl sulfoxide, were exclusively detected in commercial oils [22] T. magnatum Pico Italy HS-SPME MDGC-C-IRMS Bis(methylthio)methane was analyzed to discriminate natural and synthetic aroma.
The Stable isotope ratio (δ13C) values of this volatile compound in genuine truffles attained between − 43 and − 34‰, while those from synthetic aroma exhibited more negative values [42] n.g not given samples; because the fermentation condition significantly affects aroma attributes, the changing condition oneat-a-time and the repeating AEDA and OAV are laborious. The olfactometry-based method required a total of 5278 dilution samples from 26 fermentation conditions to determine the FD, while the aroma evaluation method scored 504 fermentation samples from various fermentation conditions to identify potential aroma compounds, among them the key volatiles was determined by PCA. Consequently, seven key volatiles identified by GC-O based analysis were also characterized by the new approach along with an additional two significant aroma contributors, 2,3-butanediol, and trimethylpyrazine. The results showed the potential of an aroma evaluation method combined with PCA to study key aroma compounds of fermentation samples [31]. Vita  They revealed for the first time that the Chinese white truffle exhibited more sulfur-containing volatiles (2.894 µg/g in total) than the Chinese black truffle (0.040 µg/g in total) in terms of both amounts and contents based on fresh weight of samples used in extraction [40]. In recent times, researchers have increasingly recognized the authenticity and traceability of flavor compounds in truffles. The authenticity and traceability can be determined by GC coupled with combustion-isotope ratio mass spectrometry (GC-C-IRMS), which exploits 13 C/ 12 C ratio abundance of the main aroma contributor in foods [41,42]. Wernig et al. used GC-IRMS and GC-MS to discriminate the home-made and commercial truffle oils. The GC-IRMS could not differentiate between natural and synthetic flavors, while the metabolic profiling using GC-MS revealed that two sulfurcontaining volatiles, dimethyl sulfone, and dimethyl sulfoxide were exclusively detected in commercial oils [22]. To discriminate the natural aroma from the synthetic one, Sciarrone et al. evaluated bis-(methylthio) methane known as one of the key aroma contributors in T. magnatum pico, using the improved multidimensional GC-C-IRMS with simultaneous quadrupole MS, by minimizing the effect of column bleeding on stable isotope ratio (δ 13 C) measurement. Consequently, the δ 13 C values of bis-(methylthio) methane from genuine truffle samples attained between − 42.6 and − 33.9‰, while those from synthetic aroma exhibited more negative values [42]. More details about the recent aroma related studies are listed in Table 1 (cited on page # 35).
Various truffle species including T. aestivum, T. borchii, T. magnatum, T. melanosporum, desert truffles, and Chinese truffles exhibited a significant amount of unsaturated fatty acid (UFA) such as oleic acid and linoleic acid, accounting for more than 60% of their total fatty acid content [48,49]. For example, the fatty acid in the fruiting bodies of T. pinoyi was mainly composed of oleic and linoleic acid, which possessed 32.29% and 29.72%, respectively [45]. Yan et al. demonstrated that linoleic and oleic acid is the most abundant fatty acid in T. latisporum, T. subglobosum, and T. pseudohimalayense. The dried powder of these three Chinese truffles contains 246.81-365.24 mg/100 g linoleic acid and 94.04-250.97 mg/100 g oleic acid, respectively [48]. Linoleic acid is an essential fatty acid and acts as a precursor of 1-octen-3-ol, which is one of the vital aroma active compounds in most truffle species [15,50]. Oleic acid exhibited potentials in reducing cholesterol levels, preventing cardiovascular, and inhibiting cancer progression [51,52]. In terms of their high UFA content, truffles might be considered as a potential nutritional and medicinal food. Minerals are the essential dietary constituents that are required for numerous metabolic reactions, rigid bone formation, the transmission of nerve impulses, and regulation of water and salt balance [53]. Truffles provide a significant amount and variety of minerals. In particular, the major minerals such as potassium, magnesium, calcium, and phosphorus were abundant in European and desert truffles [44,46,54,55]. Truffles are also known as a rich source of amino acids, especially the sulfurcontaining amino acids (cysteine and methionine), while they generally remain limited in other plant-derived foods [56].
Extracellular enzymes play a role in nutrient acquisition and symbiotic fungus-host interaction helping truffles to penetrate and colonize into roots of the host plants [57]. T. maculatum produced seven extracellular enzymes, including amylase, catalase, cellulase, laccase, lipase, peroxidase, and xylanase; however, T. aestivum produced amylase, catalase, and peroxidase [58,59].
Truffles can be considered as a source of protein but not appropriate for human's daily diet because of their high price [7]. However, it is not suitable to regard them only as a costly source of protein because truffles possess great capability in both the diversity and amount of nutritionally valuable compounds such as fiber and minerals. Moreover, Bouatia et al. demonstrated that T. pinoyi either exhibited no or very low levels of antinutrient compounds including cyanide, oxalate, mercury, arsenic, and cadmium [47]. From this perspective, truffles can be consumed as a potential nutritional source with safety.

Bioactive compounds in truffles
Truffles are rich in bioactive compounds such as ascorbic acid, ergosterol, phenolics, flavonoids, terpenoids, phytosterol, and polysaccharides. In particular, truffles contain abundant flavonoids that are appreciated as an important secondary metabolite of natural products due to their diverse biological properties involving antioxidative, anti-inflammatory, anti-mutagenic, and anticancer properties, while edible mushrooms cannot synthesize, therefore, do not contain these valuable metabolites [60,61]. From this perspective, researchers have paid attention to the biological activities of truffles and their application in medicinal domains. For example, chemical analysis of three Chinese truffle species revealed that the methanol extracts of them contained abundant antioxidant sources, including 4.59-4.63 g/100 g ascorbic acid, 144-271 mg of β-carotene/100 g carotenoid, 450-735 mg gallic acid equivalents (GAE)/100 g phenolics, and 611-1355 mg of rutin/100 g flavonoid, which could be used to prevent diseases related to oxidative damage [48]. Furthermore, the desert truffle T. nivea exhibited relatively higher total phenolic content (1.39 g GAE/100 g fresh mass (FM)) as compared to other phenolic-rich fruits such as cherries (44-88 mg GAE/100 g FM), strawberries (54-94 mg GAE/100 g FM), or onions (142-428 mg GAE/100 g FM). In particular, their methanolic extract comprised a high number of flavonoids (74.52 mg GAE/g extract) and tannins (19.78 mg catechin equivalent (CE)/g extract). These abundant bioactive compounds in truffle extract were correlated to the 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scavenging and lipid peroxidation inhibitory properties together with the antimicrobial properties against seven species of bacteria [62][63][64][65]. Ergosterol, the biological precursor of vitamin D 2 , is also one of the abundant bioactive compounds in diverse truffle species [66]. Villares et al. reported that T. melanosporum, T. aestivum, and T. indicum presented total ergosterol amount of 1.90 mg/g DM, 1.86 mg/g DM, and 1.37 mg/g DM, respectively [67]. This bioactive compound and its derivatives have exhibited various health-promoting activities including antioxidant, antiinflammatory, and antihyperlipidemic activities [68][69][70].

Biological activities of truffles Antitumor activity
Recently, many research studies have been conducted to find out the anticancer properties of truffles. According to Beara et al., methanol extracts from T. aestivum and T. magnatum possessed significant in vitro cytotoxic effects against different cancer cell lines (HeLa, MCF-7, and HT-29), besides the prominent activity of the water extract towards breast adenocarcinoma (MCF-7) [71]. In another study conducted by Khadri et al., silver nanoparticles were synthesized from aqueous extract of T. claveryi, which is particularly rich in amino acids and flavonoids. The synthesized nanoparticles exhibited significant in vitro cytotoxic effects (IC 50 = 10 mg/mL) against MCF-7, which was evaluated by the colorimetric sulforhodamine B assay [72]. Using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay, Dahham et al. determined the significant in vitro cytotoxic effects of hexane and ethyl acetate extract obtained from T. claveryi on different cancer cell lines, including the human brain carcinoma cell line (U-87 MG) with IC 50 values of 50.3 µg/mL DW of extract. Furthermore, this study showed the anti-angiogenic efficacy of hexane extract using rat aorta ring assay, the hexane extract inducing the reduction of mitochondrial membrane potential as well as the condensation of nuclei in U-87 MG cells. Their anticancer properties would be Page 10 of 17 Lee et al. Fungal Biol Biotechnol (2020) 7:9 correlated to their bioactive compounds including stigmasterol, β-sitosterol, squalene, and lupeol [73]. Phytosterols played a role in the inhibition of cancer cell growth mechanism, angiogenesis, and stimulation of cancer cell apoptosis [74]. Stigmasterol, one of the major phytosterols, downregulated lipid peroxidation and increased the levels of glutathione, superoxide dismutase, and catalase in the liver, and consequently decreased tumor volume, viable cell count, and increased mean survival time of Ehrlich ascites carcinoma tumor-bearing mice [75]. Squalene involved in natural triterpene potentially inhibited cancer cell proliferation without disturbing the normal biochemical pathways [76]. Truffles comprised significant amounts of polysaccharides possessing anticancer activity. Most polysaccharides in fungi are β-glucan polymers having the main chain which contains β-(1 → 3) linkage with some β-(1 → 6) branches [77]. Polysaccharides and their derivates play important roles in various biological processes, including pathogenesis prevention, signal recognition, and cell-cell communication [78]. It has been reported from previous studies that these macromolecules have oncogenesis-and metastasis-preventing abilities, including immunopotentiation activity against tumors. The polysaccharides in truffles could stimulate lymphocyte and macrophage division and synthesis of cytokines such as interferons, interleukins, and immunoglobulins directed against cancer antigens [79][80][81]. According to Attia et al., polysaccharides extract from T. claveryi possessed anticancer effect on Ehrlich's ascites carcinoma cells, arresting G2 phase and reducing the percentage of G0/G1 phase in the cell cycle [82]. Zhao et al. revealed that 52 polysaccharides isolated from T. aestivum, T. indicum, T. melanosporum, and T. sinense fermentation systems as well as from their fruiting bodies showed antitumor activities against A549, HCT-116, HepG2, HL-60, and SK-BR-3 cells lines. This study found that the fraction from the fermentation system, which contained β-d-glucan having triple helix conformation (high-chain stiffness), showed significantly higher cancer cell inhibition rate than those from fruiting bodies. Moreover, the heteropolysaccharides with having lower molecular weight exhibited higher antitumor activity than those with higher molecular weight [83].
The oleic acid content in truffles also contributed to the anticancer activities. Oleic acid could suppress the overexpression of HER2, an oncogene correlated to the invasion and metastasis of cancer cells. Moreover, they could increase the intracellular ROS production and caspase 3 activity and consequently induce the cancer cell apoptosis [84,85].
Pacioni et al. found that the fruiting bodies of T. melanosporum contained the major endocannabinoid metabolic enzymes such as N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL), and anandamide which is a fatty acid neurotransmitter derived from arachidonic acid and showed anticancer activity [86]. According to Picardi et al. [87], anandamide inhibited the angiogenesis of highly invasive and metastatic breast cancer cells. Patsos et al. [88] revealed that PG-EAs, the cyclooxygenase 2 (COX-2) dependent metabolites of anandamide, induced apoptosis of colorectal cancer cells, while anandamide stimulated non-apoptotic cell death in COX-2 overexpressed colorectal cancer cells. In EGFR-overexpressed prostate cancer cells, anandamide decreased EGFR levels by interacting with cannabinoid CB1 receptor, and consequently, inhibited the cancer cell proliferation [89].

Antioxidant and anti-inflammatory activities
Truffles comprise of secondary antioxidant metabolites such as phenolics, flavonoids, tocopherol, carotenoids, and phytosterols. As a free radical scavenger, phenolics can be used as an efficient antioxidant due to their reducing abilities as hydrogen-or electron-donating agents [90]. According to Beara et al. [71], water and methanolic extracts of T. aestivum and T. magnatum possessed moderate antioxidant properties related to their phenolic contents of 12-18 and 13-19 mg GAE/g DW, respectively. According to Villares et al., the methanolic extract of T. aestivum, T. indicum, and T. melanosporum comprised phenolics and ergosteryl ester of 1.88 mg/g DW and 0.36 mg/g DW, 1.52 mg/g DW and 0.09 mg/g DW, and 1.20 mg/g DW and 1.10 mg/g DW, respectively. Consequently, the highest phenolics and ergosteryl ester levels of T. aestivum were closely correlated to its greatest antioxidant power amongst these three species [67].
Desert truffle species also comprised of diverse antioxidant secondary metabolites. T. nivea showed much higher total phenolic content than that of T. pinoyi, and possessed greater antioxidant potential than T. pinoyi [19,46]. According to Hamza and Jdir, ascorbic acid content (10.63 mg/100 g), and the combined content of carotenoids (1.17 mg/100 g), and anthocyanins (29.1 mg/100 g) in T. nivea were responsible to their antioxidant potential [46]. Doğan and Aydın revealed that the high scavenging effect of T. boudieri would correlate with catechin, the predominant phenolics present in this truffle [91].
Apart from desert truffles, Chinese truffle species also possessed high antioxidant properties. Chen et al. [92] evaluated the great DPPH radical scavenging ability of purified water-soluble polysaccharides from T. huidongense. Yan  activities, in particular, T. latisporum and T. pseudohimalayense, which were more abundant in phenolic compounds, possessed much stronger antioxidant potential than T. subglobosum [48]. The antioxidant compounds including phenolics, terpenoids, and polysaccharides also possess anti-inflammatory properties [77,93]. Oxidative stress is often correlated to inflammatory disorders, particularly diabetes mellitus (DM). It is due to the activity of hyperglycemia, the typical indicator of DM, causes over-production of free radical [94]. Zhang et al. evaluated the effects of aqueous extract from T. melanosporum on the streptozotocin induced hyperglycemic rat. Consequently, the rat treated with truffle extract showed the reduced levels of glucose like the rat treated with the standard antidiabetic drug, glibenclamide. The hypoglycemic effect of truffle extract was significantly correlated with Nrf2 and NF-kB pathways, and the regulation of enzymatic and nonenzymatic antioxidant including superoxide dismutase, catalase, vitamin E, and vitamin C [95]. In addition to hypoglycemic effect, Janakat and Nassar revealed the hepatoprotective effect of aqueous extract from T. claveryi on liver damage caused by oxidative stress [96]. Moreover, Beara et al. [71] demonstrated that T. magnatum extract inhibited the COX-1 and 12-LOX pathway products, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (12-HHT), thromboxane B 2 (TXB 2 ), and 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), which overexpressed in various inflammatory diseases.

Antimicrobial activity
The antimicrobial activity of desert truffles has been well reported for a few decades. Janakat et al. [97] demonstrated that the aqueous extract obtained from T. claveryi inhibited the growth of Staphylococcus aureus by 66.4%. According to Owaid et al., silver nanoparticles synthesized by Tirmania sp. revealed the antibacterial activity towards gram-negative and gram-positive bacteria, predominantly the infectious bacteria of the eyes, Pseudomonas aeruginosa [98]. In another study conducted by Casarica et al., the aqueous extract from T. claveryi possessed the antimicrobial properties against Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus, and consequently, their usage was suggested to treat eye infections caused by those three bacterial species [99]. Another desert truffle, T. nivea, from Tunisia arid zone was also exhibited by the remarkable inhibitory activity against three species of gram-positive and four species of gram-negative bacteria including Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, and Bacillus subtilis [46]. Dib-Bellahouel and Fortas found that the growth of Bacillus subtilis and Staphylococcus aureus was inhibited by ethyl acetate extract of T. pinoyi which contained pyrazines and its derivatives as the antimicrobial active compounds [100]. Stojković et al. [45] revealed that the aqueous and methanolic extract from T. pinoyi efficiently inhibited the growth of Staphylococcus aureus in chicken soup kept in the refrigerator and at room temperature. Table 2 summarizes more details about studies investigating the antimicrobial activity of truffles.

Aphrodisiac activity
Traditionally, truffles have been used as an effective sexual enhancer due to their constituent of androstenol as a steroidal pheromone [101,102]. While truffle hunting, animals might recognize the odor of this chemical marker. Androstenol was also found in the underarm perspiration of men and urine of women and increased sexual attractiveness higher [11,102]. The alcoholic extract of T. boudieri increased the levels of luteinizing hormone and testosterone significantly in rats and consequently possessed the aphrodisiac activity owing to its androgen enhancing properties [103]. However, Al-Damegh demonstrated that the androgenic property of truffles was more related to psychological effect because the flavonoids in truffles were present in the form of glycosides, which act as antagonists for male sex hormones [102].

Other potential applications
The bioactive compounds in truffles potentiate the use of truffles in other medicinal usages such as anti-depressants, cholesterol reducer, and immunostimulant. For example, the blood cholesterol-reducing effect of linoleic acid has been widely reported [104,105]. In the meanwhile, truffles comprise abundant amino acids involving l-tyrosine which is a precursor of neurotransmitters catecholamines deeply involved in neural circuits [44,106,107]. Pattanayak et al. studied the cytotoxic effect of water-soluble heteroglycan obtained from T. rufum on human blood lymphocytes. Consequently, they discovered the potential of this polysaccharide as an immunostimulant, in terms of its ameliorative activities by maintaining the redox balance [108].

Various factors affecting productivity and quality of truffles
Like most edible fungi, the growth pattern, nutritional quality, and biological activities of truffles could be changed by various factors such as climate, soil, or the presence of contaminating microorganisms [8]. For example, dehydrative low-temperature stress induces accumulation of dehydrins in truffles proposed to play a protective role [109] and upregulation of stress-responsive genes including heat shock proteins and cell wall remodeling genes in T. melanosporum [110]. High temperatures and hydric stress in summer, together with decreased summer precipitation, could also lead to the genetic or morphological change and reduction of subsequent winter harvest [111]. Leonardi et al. evaluated the effect of heat stress on eleven T. borchii strains from different geographical and ecological provenance. Ten from eleven tested strains showed the significant growth reduction accompanying by increased hyphal septation at 34 °C, while the best performances of mycelial growth and biomass production were at 22 °C. In terms of global climate change, a suitable selection of strain which is tolerant in temperature change could be one of the successful strategies for truffle cultivation [112]. Edaphic factors such as pH, moisture, and nutrient also influence the richness and composition of truffles [113]. Hall et al. [114] demonstrated that a soil pH of 8.0-8.5 was an appropriate condition for the T. melanosporum cultivation. Ge et al. revealed that soil pH is strongly correlated to the levels of minerals in truffles such as K + , Ca 2+ , and Mg 2+ , and host plants generally produced more T. lyonii fruiting bodies at soil pH of 6.6-7.3 [115].
Meanwhile, truffle productivity and quality could be improved by regulating preharvest factors. As truffles are hypogeous fungi, they interact with soil micro-fauna and microorganisms, which can enhance the truffle formation and their antagonistic, competitive, or synergistic activities [8]. Frey-Klett et al. demonstrated that co-inoculating, adding a small amount of helper bacterium to the ectomycorrhizal fungus, could lead to much easier and faster-growing than ectomycorrhizal fungi. Thus, this method can be one of the efficient methods for optimizing the mycorrhization techniques as well as minimizing the inoculation cost [116]. Tang's research team has developed and improved the submerged fermentation process for enhancing not only truffle biomass but also the production of polysaccharides, VOCs, and sterols in truffles [117]. For example, they demonstrated that the addition of Mg 2+ and K + could enhance the production of extracellular polysaccharides during the submerged fermentation of T. sinense. According to Vahdatzadeh and Splivallo, genetic features deeply influenced the truffle aroma formation, and the human nose could distinguish the flavor among the strains. From this regard, strain selection could be effectively used for improving the truffle flavor [118].
Truffles are highly perishable and very sensitive to pathogen contamination and water loss [119]. During storage processes such as frozen or sterilization, the aroma quality and texture of truffles had been changed. Therefore, preservation methods play a critical role in the maintenance or improving their quality. Saltarelli et al. [55] recommended a storage temperature at 4 °C to preserve the biochemical and microbiological characteristics of fresh T. aestivum, T. magnatum, and T. melanosporum. Al-Ruqaie demonstrated that freezing after blanching in 4% boiling NaCl solution is the most effective preservation method for maintaining the quality of T. claveryi and T. nivea [120]. Rivera et al. recommended lyophilization as an adequate method to better preserve the aroma of T. melanosporum, while Palacios et al. suggested that rehydration after lyophilization recovered the aromatic profile changed through lyophilization [121,122]. According to Tirillini et al. [123], the addition of 10% linoleic acid (w/w) significantly reduced total bacterial load and enhance the nutritional value of truffle sauce.
Irradiation is one of the ways affecting microbial populations, shelf life, chemical composition, and sensory characteristics of truffles. Adamo et al. [124] demonstrated that the irradiated truffles showed a microbial load of five orders of magnitude less than the non-irradiated. Reale et al. [125] revealed that irradiation at 1.5 kilo gray (kGy) was the most effective way for extending the shelf life of truffles as well as minimizing the alteration of sensorial features of fresh truffles. According to Culleré et al. [126], the ionizing radiation at 1.5 and 2.5 kGy increased the levels of practically all the primary odorant of T. melanosporum and T. aestivum, and therefore, these type of irradiation were considered as an effective way for maintaining the truffle aroma. Tejedor-Calvo et al. recommended that electron beam or gamma irradiation (both at 1.5 kGy and 2.5 kGy) followed by storage up to 21 days were the efficient methods to enhance truffles quality and shelf life. In the study, the irradiation exhibited no influence on the total carbohydrates, β-glucans, and overall sterols levels. Although this method significantly reduced protein and chitin levels, storage after the irradiation maintained the levels of all determined compounds unchanged except for the phenolics and ergosterol levels that increased after 21 days storage [127]. Storing fresh truffles under high carbon dioxide conditions is also associated with their quality. Mencarelli et al. [128] revealed that CO 2 treatment on T. aestivum at 5 °C could keep well their fragrance and robust defense system. According to Hajjar et al., storing T. uncinatum in high CO 2 and low O 2 atmospheres played a decisive role in maintaining their antioxidative properties by inhibiting polyphenol metabolism, anaerobic pathways, and polyamine biosynthesis. The antioxidant enzymes in truffles were active when they are kept in the soil with lower O 2 conditions; therefore, removing truffles from lower to higher O 2 conditions could weaken their antioxidant defense system [119].

Conclusions
The unique flavor of truffles has made them the high demanded foodstuffs; however, they are not enough to be regarded only as high-priced foodstuffs because of their nutritional and medicinal potentials as well as no known side effects. Diverse truffle species are rich in nutrients such as carbohydrates, protein, fiber, minerals, fatty acids, and amino acids. Apart from their high value in the nutritional domain, their potential in therapeutic agents is ample as well. Recent studies have identified diverse bioactive compounds such as phenolics, flavonoids, and polysaccharides in truffles and revealed their potentials as anticancer, antioxidant, antimicrobial, anti-inflammatory, antidiabetic agents. Nevertheless, most studies have focused on in vitro studies and limited researches on the application of truffles in practical therapy has been investigated. Future studies need to optimize the extraction process to obtain a high yield of the bioactive compounds. Moreover, it is needed to focus on their bioassay against different diseases and pathogens to incorporate these biological values effectively into the truffles derived natural products as well as potential medicines. In this regard, more innovative methods are also expected to overcome the difficulty in preserving truffles to enhance their shelf life whilst maintaining their bioactive compounds and causing no harmful effects. These attempts would enable the practical usage of truffles in nutritional and medicinal fields.