P U B L I C A T I O N S CODON Italian Journal of Food Science, 2022; 34 (2): 67–73 ISSN 1120-1770 online, DOI 10.15586/ijfs.v34i2.2202 67 P U B L I C A T I O N S CODON Proximate analysis of lipid composition in Moroccan truffles and desert truffles Fatima Henkrar1*, Lahsen Khabar2* 1Plant Biotechnology and Physiology Laboratory, Faculty of Sciences, University Mohammed V-Rabat, Morocco; 2Botanical, Mycology and Environment Laboratory, Faculty of Sciences, University Mohammed V-Rabat, Morocco *Corresponding Authors: Fatima Henkrar, Plant Biotechnology and Physiology Laboratory, Faculty of Sciences, University Mohammed V-Rabat, Morocco. Email: f.henkrar@um5r.ac.ma; Lahsen Khabar, Botanical, Mycology and Environment Laboratory, Faculty of Sciences, University Mohammed V-Rabat, Morocco. Email: l.khabar@um5r.ac.ma Received: 18 March 2022; Accepted: 17 May 2022; Published: 10 June 2022 © 2022 Codon Publications OPEN ACCESS PAPER Abstract Lipid composition in truffle is essential for nutraceutical and medicinal purposes. Currently, there is no data regarding the lipid content in Moroccan truffles. Therefore, we determined the fatty acid and sterol composition of six Moroccan truffles and desert truffles. The gas chromatography analysis revealed the predominance of fatty palmitic, oleic and linoleic acids. The prominent sterol components were brassicasterol and ergosterol. Besides, the sterol analysis discriminated between the Tuber and Terfezia truffles. These differences seem to be exploitable at a taxonomic level. This is a preliminary report disclosing the fatty acid and sterol composition of Moroccan truf- fles, indicating the potential use of lipids analysis, especially sterol analysis, as biomarker for truffles distinction. Keywords: desert truffles, discrimination, fatty acid, gas chromatography, Moroccan truffle, sterol Introduction The truffles are edible ascocarp of hypogenous ascomyce- tes fungi that grow underground (Khabar et al., 2001; Lee et al., 2020). The term ‘desert truffles’ is used to describe truffles growing particularly in arid and semi-arid areas (Khabar et al., 2001; Morte et al., 2021), such as Morocco, Algeria, Tunisia, Egypt, South Africa, Saudi Arabia, Iraq, Syria and Kuwait (Khabar, 2014; Lee et al., 2020). The genera found abundantly in those areas are Terfezia and Tirmania. Besides, other genera exist as well, namely Delastria and Picoa (Khabar, 2014). In Mediterranean countries, especially in North Africa, the truffles are harvested in abundance and known as ‘Terfass,’ also called ‘Kame,’ ‘Kholassi,’ ‘Zoubaïdi,’ ‘Truffles of the deserts’ and ‘Truffles of the sands’ because of their development in sandy soil (Khabar, 2014). The determination of lipid composition in truffles is essen- tial both for lipid analysis as well as for nutraceutical and medicinal purposes. The truffles contain only 4–9% by dry weight of total lipids (Tang et al., 2011). Fatty acids and phytosterols are the main lipid compounds in truffle fruiting bodies, which are well known for their potential human benefits. Several studies through global chromato- graphic analysis demonstrated that desert truffles are rich in fatty acids, both saturated and unsaturated that have many positive effects on health (Akyüz, 2013; Al-Shabibi et al., 1982; Bokhary et al., 1989; Doğan and Aydın, 2013; Veeraraghavan et al., 2021). For example, in Terfezia boud- ieri from Tunisia, Hamza et al. (2016) reported that essen- tial fatty acids, like linoleic and oleic acids, account for 76% of the total fat content. Linoleic acid or omega-6 is an essential fatty acid and one of the most aromatic com- pounds in most truffle species (Lee et al., 2020), which has protective and antioxidative effects beneficial for human health (Sokoła-Wysoczańska et al., 2018). While, oleic acid, a bioactive compound, has the aptitude in reduc- ing cholesterol levels (Lee et al., 2020). Another example of Terfezia claveryi from Saudi Arabia, which is closely related to T. boudieri (Dahham et al., 2018), was found to have arachidic, myristic, palmitic, behenic, pentadecanoic, stearic, heneicosanoic, nonadecanoic and margaric acids 68 Italian Journal of Food Science, 2022; 34 (2) Henkrar F and Khabar L collected directly from their natural environments and transported to the laboratory. Under the fume hood, the samples were surface sterilised with ethanol, peeled and then fragmented by hand. Several pieces were taken from the glebe and stored in pillboxes at −64°C. Alternatively, other samples were sun-dried for 2 months before being stored at −64°C. The different steps of extraction, separation and analy- sis of lipids were released at the Laboratory of Myco- logy, Phytopathology and Environment of the Littoral France University, following the method of Fontaine et al. (2001). Extraction of total lipids The extraction was performed with approximately 20–40 mg of freeze-dried material (pieces of truffle glebe). The solvent used for extraction was a mixture of dichloromethane and methanol (2:1 v/v) with 0.05% BHT (Butylated hydroxytoluene; Sigma) as antioxidant. The freeze-dried fungal material was first ground in 40 ml of the solvent using ultra-turrax homogenizer. The first extractions were performed in the dark to preserve the ergosterol, a photosensitive sterol. The extraction of total lipids was carried out under reflux (1 h at 70°C) with some pieces of pumice stone. After filtration, the lipid extract was recovered under nitrogen blowdown and rotary evaporator at 60°C. This step was repeated thrice. Separation of fatty acids and total sterols by saponification The crude lipid extract was used to separate fatty acids and total sterols by saponification. The crude extract was saponified under reflux (1 h at 90°C) in 2 ml of 6% (w/v) methanolic potash and some pieces of pumice. After cooling, two successive cold extractions with hex- ane were performed. The first extraction permitted the as saturated fatty acids along with unsaturated fatty acids (palmitoleic, oleic, erucic and linoleic) (Bokhary et al., 1989). The lipid composition of desert truffles depends highly on the species as well as growing environments. For instance, T. boudieri from Saudi Arabia was rich in penta- decanoic, margaric, stearic and arachidic acids (Bokhary et al., 1989). Whereas, the same species from Turkey con- tained mainly oleic, linoleic, linolenic, palmitic, palmi- toleic, stearic and behenic acids (Akyüz, 2013). The most identified phytosterols in truffle reports were brassicast- erol and ergosterol. Harki et al. (1996) reported that the prominent components identified in Tuber melanosporum were ergosterol and brassicasterol, accounting for about 90% of total sterols. As well, the major sterol components in the Tuber ascocarps were brassicasterol and ergosterol, accounting for about 17–64% and 25–67% of total sterols, respectively (Tang et al., 2012). In Terfezia truffles, brassi- casterol levels were 98% of the total sterols, while ergos- terol was present in lower amounts (Tejedor-Calvo et al., 2021). Other phytosterols such as beta-sitosterols, stig- masterol and campestanol were also present in low con- tents (Dahham et al., 2018). The six species included in this study were natives of Morocco. Terfezia arenaria, commonly called ‘Pink Terfess of Mamora,’ was harvested from acidic soil, in semi-arid climate under Helianthemum guttatum. It is an appreci- ated edible fungus, detected by the ‘mark’ method (Khabar, 2014) unlike Delastria rosea, known as ‘Bitter Terfess of Taida’ due to its bitter flavour. It was collected under Pinus pinaster var. atlantica and Pinus halepensis in Mamora forest between November and December (Khabar, 2014). Similarly, Tuber oligospermum was also collected under P. pinaster var. atlantica in Mamora forest, starting from December until April. The ascocarp of T. boudieri origi- nated from Bouaarfa region, collected from limestone soil, under arid and sub-Saharan climate, and Terfezia lepto- derma was obtained from Mamora forest from the acidic soil under H. guttatum at the beginn ing of February until May. Tuber asa, commonly called ‘Terfass male of Terfass’ because of its hard consistency, collected as well under H. guttatum on acidic soil of Mamora forest, towards the end of February at the same time as T. leptoderma (Khabar, 2014). The aims of this work were 1) to determine the lipid profile of the six Moroccan truffle species, 2) to determine the relation between the genus, species and lipid composi- tion of truffles, and 3) to determine whether the lipid profile can be used as a tool for species or genus distinction. Materials and methods Fungus material Six Moroccan truffle species were used in this experi- ment. The ascocarps of different species (Table 1) were Table 1. The name and location of six Moroccan truffle species used in this study. Species name Location Terfezia leptoderma (1) Mamora Forest under Helianthemum T. leptoderma (2) Mamora Forest under Pinus pinaster Terfezia arenaria Mamora Forest Terfezia boudieri Bouaarfa Tuber asa Mamora Forest Tuber oligospermum Mamora Forest under P. pinaster Delastria rosea Mamora Forest under P. pinaster Italian Journal of Food Science, 2022; 34 (2) 69 Lipid composition of Moroccan truffles Statistical analysis The data were analysed using R studio software. The means and standard deviations were calculated. The pairwise comparisons among means were performed using two-way ANOVA and Tukey HSD test. To indicate significant differences, we used the multcompLetters4() function from the multcompView package. Results and discussions The objective of this work was to define the nature and proportion of fatty acids and sterols in six species of Moroccan truffles and to determine if this lipid pro- file could be used as a classification tool to discriminate between species or genus. This study was conducted for the first time on Moroccan truffles, disclosing distinctly the fatty acid and sterol components of truffles and des- ert truffles grown in Morocco. Fatty acid composition The fatty acid composition of Moroccan truffles has not been reported previously, and studies on fatty acid content of other truffles are scarce. The first and most reported studies on fatty acid composition were focused on Tirmania pinoyi, Tirmania nivea, T. boudieri, T. clav- eryi and Picoa lefebvrei from Saudi Arabia (Bokhary et al., 1989; Bokhary and Parvez, 1995) and T. claveryi from Iraq (Al-Shabibi et al., 1982). Recently, other stud- ies appeared on fatty acid composition of T. boudieri from Iraq (Dahham et al., 2018), T. boudieri from Turkey (Hamza et al., 2016), T. claveryi and Picoa juniperi from Spain (Murcia et al., 2003) as well as T. nivea from Libya (Shah et al., 2020). The chromatographic analysis results for the identifica- tion of fatty acids compositions are presented in (Table 2 and Figure 1). Seven fatty acids were detected in the six truffles species used in this experiment; four saturated fatty acids [palmitic (C16:0), stearic (C18:0), arachidic (C20:0) and behenic (C22:0)], and three unsaturated fatty acids [palmitoleic (C16:1), oleic (C18:1) and lin- oleic (C18:2)]. Bokhary et al. (1989) reported that pal- mitic, stearic, oleic and linoleic acids were predominant in T. nivea and T. boudieri which originated from Saudi Arabia. As well, the Turkish T. boudieri was also rich in behenic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids (Akyüz, 2013), which agree with our results and particularly with T. boudieri. Furthermore, a recent study on fatty acid composition in Tuber maculatum, Tuber aestivum/uncinatum, Tuber borchii, T. melanosporum and T. nivea revealed the dominance of palmitic, stearic, oleic and linoleic acids followed recovery of unsaponifiable fraction (sterols), while the second one enabled the retrieval of saponifiable fraction (fatty acid). To perform the first cold extraction, one vol- ume of distilled water was added to the cooled saponified extract, followed by six volumes of hexane. This mixture was then vigorously stirred for 1 min with a vortex. After decantation, the organic phase (upper layer), which con- tains the unsaponifiable elements, was taken out and dehydrated with anhydrous sodium sulfate. This step was repeated three times, and the recovered extract was concentrated in a rotary evaporator at 50°C. For the fatty acid recuperation, the aqueous phase was acidified to pH 1 with 1 M HCl to liberate them from their saline com- bination. Afterwards, the acidified phase was extracted by performing three extractions with hexane. The concentra- tion of these extracts was done under nitrogen. Fatty acid analysis The fatty acids were solubilise in 1 ml boron trifluo- ride–methanol (14% w/v). The methylation reaction was carried out for 2 min at 90°C in a water bath and then stopped by immersing the tubes in ice. After addition of 1 ml of distilled water and 2 ml of hexane, the tubes were vortexed for 30 s. The organic phase (upper phase) was taken out and dehydrated by adding anhydrous sodium sulfate. This step was repeated thrice. The methylated fatty acids were purified on silica gel of TLC (20 × 20 cm, type Silicagel F 254, Merck) with a solvent system com- posed of diethyl ether/hexane (10/90; v/v). The fatty acid spots were detected by primuline and eluted in about 1 ml of dichloromethane. Thereafter, the fatty acids were immediately taken up in 25–100 µl of hexane and injected into the gas chromatograph. Fatty acids were identified by comparing their relative retention times with internal standard such as methyl C 17:0 (methyl margarate) as well as other known standards (Alltech). Sterol analysis To obtain a better separation in gas chromatography, ste- rols were acetylated either for 12 h at room temperature or 2 h at 60°C by the mixture of toluene/acetic anhydride/ pyridine (1/2/1; v/v/v). Sterol acetates were purified on silica gel thin layer with dichloromethane as migration solvent. Cholesterol acetate (1 mg/ml) was used as a con- trol to localise acetylated products after spraying with 0.01% (w/v) primuline solution. The acetylated sterols were taken up in 25–100 µl of hexane and injected into the chromatograph. The sterol acetates were identified by comparing their relative retention times against an inter- nal standard, cholesterol in alcohol form (non-acetylated) along with brassicasterol and other known acetylated standards. 70 Italian Journal of Food Science, 2022; 34 (2) Henkrar F and Khabar L for D. rosea where the opposite was true; the level of oleic acid was higher than linoleic acid. The same results were also reported by Hamza et al. (2016). The T. boudieri was characterised by its higher content of linoleic acid (54.10%) compared to oleic and palmitic acids that repre- sented 22 and 20.40%, respectively (Hamza et al., 2016). Linoleic acid level was considerably high in Tuber com- pared to Terfezia species. The rate of oleic acid was, on the other hand, slightly lower in Tuber genus compared to Terfezia genus. This remark goes with Tejedor-Calvo et al. (2021), who reported that linoleic acid content in Tuber brumal and T. melanosporum reached 78.3 and by traces of polyunsaturated fatty acids (Shah et al., 2020). The main fatty acids detected were palmitic, oleic and linoleic acids. The other fatty acids were also present but at lower levels. Similar findings were also reported in var- ious species of Terfezia and Tuber (Hamza et al., 2016; Tejedor-Calvo et al., 2021). Our results demonstrated that the rate of palmitic acid (C16:0) is appreciably equal in all the species studied. We could also notice that the linoleic acid level was generally higher compared to the oleic acid (C18:1) level in all the species studied except Figure 1. Fatty acid composition of the six Moroccan truffle species used in this study. Table 2. Fatty acid composition of the six Moroccan truffle species through gas–liquid chromatography analysis (percentage of dry weight of the lipid fraction). Species C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 C 20:0 C 22:0 Terfezia leptoderma (1) 16.080 ± 0.936a 0.913 ± 0.372a 1.600 ± 0.185a 27.530 ± 0.598a 48.513 ± 1.179a 4.570 ± 0.589a 0.790 ± 0.147a T. leptoderma (2) 17.456 ± 2.167a 1.733 ± 0.221a 1.026 ± 0.047a 26.106 ± 0.740a 48.633 ± 1.075a 5.753 ± 0.247a 0.803 ± 0.100a Terfezia arenaria 21.470 ± 1.822b 1.066 ± 0.133a 3.253 ± 0.206a 27.020 ± 0.727a 38.803 ± 0.525b 8.563 ± 0.577b 0.900 ± 0.095a Terfezia boudieri 20.823 ± 1.019b 0.830 ± 0.303a 2.433 ± 0.508a 28.333 ± 1.028a 42.770 ± 1.127c 4.580 ± 0.545a 0.710 ± 0.156a Tuber asa 16.470 ± 1.483a 0.940 ± 0.096a 1.810 ± 0.259a 15.983 ± 0.879b 61.186 ± 0.315d 4.773 ± 0.396a 0.740 ± 0.070a Tuber oligospermum 16.670 ± 0.138a 1.090 ± 0.389a 2.023 ± 0.200a 20.593 ± 0.450c 54.926 ± 1.601e 4.720 ± 0.500a 0.780 ± 0.105a Delastria rosea 12.420 ± 1.574c 1.230 ± 0.166a 1.263 ± 0.112a 60.160 ± 0.916d 19.836 ± 1.237f 4.503 ± 0.678a 0.830 ± 0.052a Data shown as mean ± standard deviation (n = 3). Different superscript letters in the same column indicate a statistically significant difference (P < 0.05). Italian Journal of Food Science, 2022; 34 (2) 71 Lipid composition of Moroccan truffles Sterol composition The sterol composition of Moroccan truffles has never been reported before. The first report on sterol was that by Weete et al. (1985), which mentioned about both Terfezia and Tuber genera. Further, other studies princi- pally on Tuber species including T. melanosporum (Harki et al., 1996; Sancholle et al., 1988), Tuber magnatum, T. melanosporum, T. aestivum, Tuber albidum and Tuber indicum were released (Sommer et al., 2020). Four sterols (brassicasterol, ergosterol and lanosterol) were identified in the ascocarps of the examined truffle species (Table 3 and Figure 2). Furthermore, the main 61.12%, respectively, compared to T. leptoderma and T. arenaria which noticed only 51.3 and 30.9%, respec- tively. The fatty acid results of Moroccan truffles and desert truffles proved their richness in unsaturated and healthy fatty acids such as linoleic acid, suggesting their equivalent culinary value compared to European truffles. Regarding fatty acid discrimination, it seems that these criteria could not differentiate clearly between the spe- cies of the two genera of Tuber and Terfezia. Indeed, the ratio of linoleic acid or oleic acid was approximately equal between the different species of the two genera. Nevertheless, the fatty acid composition could distin- guish between Delastria and other two genera. Figure 2. Sterol composition of the six Moroccan truffle species. Table 3. Sterol composition of the six Moroccan truffle species through gel permeation chromatography analysis. Sterols Brassicasterols Ergosterols Lanosterol (1.31) n.i. (1.42) Terfezia leptoderma (1) 96.870 ± 1.260a 3.016 ± 0.621a 0 ± 0a n.d. T. leptoderma (2) 92.410 ± 2.606a 8.153 ± 0.143a 0 ± 0a n.d. Terfezia arenaria 96.833 ± 1.045a 0 ± 0b 2.003 ± 0.532a n.d. Terfezia boudieri 97.190 ± 2.416a 0 ± 0b 3.110 ± 0.298a n.d. Tuber asa 40.526 ± 2.377b 23.196 ± 0.718c 17.470 ± 0.856b 12.220 ± 2.186b Tuber oligospermum 46.006 ± 2.001c 21.470 ± 1.990c 16.223 ± 4.441b 17.286 ± 1.997b Delastria rosea 21.343 ± 1.770d 42.9633 ± 0.621d 22.826 ± 3.471c 12.886 ± 1.806b Data shown as mean ± standard deviation (n = 3). Lanosterol and n.i. compounds are reported with their retention time (in minutes) between brackets. Different superscript letters in the same column indicate a statistically significant difference (P < 0.05). n.i., not identified; n.d., not detected. 72 Italian Journal of Food Science, 2022; 34 (2) Henkrar F and Khabar L demonstrating the richness of Moroccan truffles in essential unsaturated fatty acid, such as linoleic acid. There was a slight difference between Tuber and Terfezia species in fatty acid component, which is not sufficient to differentiate between them. However, sterol analysis distinguished between these two genera. Hence, a com- parison of their sterol composition with reported data seems to be plausible for Tuber and Terfezia distinction. Finally, a deeper study on other nutrient compounds and bioactive molecules of Moroccan truffles as well as their antioxidant evaluation is predetermined to improve their edible and culinary interest through their health benefits. References Akyüz, M., 2013. Nutritive value, flavonoid content and radical scavenging activity of the truffle (Terfezia boudieri Chatin). Journal of Soil Science and Plant Nutrition. 13:143–151. Al-Shabibi, M.M.A., Toma, S.J. and Haddad, B.A., 1982. Studies on Iraqi truffles. I. Proximate analysis and characterization of lipids. Canadian Institute of Food Technology Journal. 15:200–202. https://doi.org/10.4067/S0718-95162013005000013 Bokhary, H.A. and Parvez, S., 1995. Studies on the chemical compo- sition of the Ascomycete fungus Phaeangium lefebvrei Pat. 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Similar results were disclosed in black truffle, where the sterol compo- sition of T. melanosporum was analysed and ergosterol along with brassicasterol were identified as the major components (90%) (Harki et al., 1996). As well, the exam- ination of Tuber sinense, T. aestivum, T. indicum, Tuber himalayense and T. borchii revealed the predominance of brassicasterol and ergosterol, with 17–64% and 25–67% of total sterols, respectively (Tang et al., 2012). Besides, the ratio of ergosterol to brassicasterol changes according to the genera studied. In Terfezia species, bras- sicasterol was the main sterol identified, accounting for 92–97% of the total sterols, affirming the results of Weete et al. (1985), who reported that brassicasterol levels were 98% of total sterols in Terfezia truffles, while ergosterol was registered at very low amounts (0–8%). On the other hand, in Tuber species (Tuber asa and Tuber oligosper- mum) and D. rosea, the ergosterol represented a consid- erable amount compared to Terfezia species, accounting for 23, 21 and 43% of the sterols, respectively. These species also contained 40, 46 and 21% of brassicast- erol, respectively. A recent study by Tejedor-Calvo et al. (2022) demonstrated that ergosterol and brassicasterol were the two main sterols in T. claveryi and T. aestivum ascocarps, with differences in ergosterol to brassicasterol ratio depending on the ascocarp genus. Lanosterol was also detected in Tuber species, as well as in D. rosea, in considerable quantities, accounting for 16 and 23% of the sterols, respectively. While in Terfezia, they were either totally absent or present in very small quantity (approx- imately 2–3%). The high amount of brassicasterol in Terfezia will increase the quality interest of the Moroccan genera, knowing that brassicasterol has several health benefits, such as antioxidative activity and anti-infective properties. Finally, Terfezia genus was distinguished by the high bras- sicasterol content, while the Tuber genera and Delastria were characterised by the equivalent amount of ergos- terol and brassicasterol. Hence, sterol analysis proved their importance to highlight differences between species and to separate the Tuber from Terfezia truffles collected in Morocco. These differences seem to be exploitable at the taxonomic level. Conclusion The lipid composition and concentration were highly influenced by truffle speciation and their growing area. 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