Nutraceutical potential, antioxidant and antibacterial activities of Terfezia boudieri Chatin, a wild edible desert truffle from Tunisia arid zone

2.1 Chemicals and spectrophotometric measurements

DPPH and chemical standards were purchased from Sigma Aldrich Co. (St. Louis, USA). All other chemicals and reagents used were of analytical grade and were purchased from Carlo Erba Reagenti SpA (Starda Rivoltana, Italy), Merck (Darmstadt, Germany) and Kemika (Zagreb, Croatia). Spectrophotometric measurements were performed by UV–VIS spectrophotometer (UV mini-1240) model Shimadzu.

2.2 Truffles

Truffles of T. boudieri were collected during the months of March and April 2009 from Southern area of Tunisia (Medenine, Benguerdenne: latitude 32°57′09″N, longitude 11°38′26″ E, with an arid climate characterized by a mean rainfall of 150 mm/year). After harvest, the fresh truffles were dried in the shadow, until constancy of the mass (20 days). Then, they were ground into fine powder and stored at ambient temperature in a dry place and in the dark until use.

2.3 Volatile compounds’ extraction

The truffle dry matter was submitted to hydrodistillation for 4 h using a Clevenger-type apparatus and the collector solvent used was n-hexane (2 ml). After evaporation of the solvent under nitrogen flow, the volatile compounds were stored in sealed vials protected from the light at −20 °C until analysis by GC and GC/MS.

2.4 Lipids’ extraction

The truffle lipids were extracted using chloroform/methanol as previously described by Zouari et al. (2010). After lipids’ extraction, methyl esters of the fatty acids were prepared as follows. A sample containing 50 mg lipids was dissolved in 500 μl n-hexane. Then, 200 μl of potassium hydroxide 2 M in methanol was added and the solution was mixed for 2 min in a vortex mixer. After phase separation, the upper layer of n-hexane containing the fatty acid methyl esters was analyzed by GC and GC/MS.

2.5 Volatiles’ and fatty acids’ analyses

2.6 Physicochemical composition and mineral concentration

Dry matter was determined by oven-drying at 105 °C to constant mass (AOAC, 1997). Crude proteins were analyzed according to the Kjeldhal method. A factor of 6.25 was used for conversion from total nitrogen into crude protein (AOAC, 1997). The fat content was determined by Soxhlet extraction with hexane for 8 h at the boiling point of the solvent. The ash content was determined by combustion of the samples at 550 °C for 8 h. Then, different mineral constituents such as (potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), manganese (Mn) and copper (Cu)) were analyzed separately using an atomic absorption spectrophotometer (Hitachi Z6100, Tokyo, Japan). Total phosphorus (P) content was determined using a molybdenum-blue colorimetric method (Murcia et al., 2003). The carbohydrate content was estimated by difference of mean values: $$\text{Carbohydrate}=[\text{total solids}-(\text{protein}+\text{lipids}+\text{minerals})]\text{.}$$

2.7 Preparation of T. boudieri extracts

The dried truffle powder (25 g) was soxhlet-extracted successively using solvents of increasing polarity as follows: petroleum ether, followed by dichloromethane, chloroform, ethyl acetate and finally with methanol during 6 h for each solvent. The volume of each solvent used is 300 ml which was then evaporated under vacuum and the residual solvent was removed by flushing with nitrogen. Finally, the obtained extracts were kept in the dark at +4 °C until further analysis. For water extraction, the fungi powder (50 g) was macerated during 24 h in 200 ml distilled water, with continuous stirring at room temperature. After that, the macerate was filtered through Whatman No.1 filter paper. The same procedure was repeated twice with the obtained residue, and then the total filtrate (macerate) was lyophilized. Moreover, a hot water extract was prepared by mixing 50 g of powdered truffles with 200 ml of distilled water at 50 °C. The mixture was stirred for 3 h and then, the extract was filtered and lyophilized.

2.8 Chemical analysis

The aqueous and the organic fractions of the truffles were selected for preliminary chemical analysis to explore the major classes of phytoconstituents. Phenolic content of T. boudieri extracts was determined by the Folin–Ciocalteu method (Dewanto et al. 2002). Gallic acid monohydrate was used as standard for the calibration curve. Phenolic content was expressed as mg gallic acid equivalent (GAE)/g extract. Flavonoid content of the samples was determined as previously described (Dewanto et al., 2002) and catechin was used as standard. The results were expressed as mg catechin equivalent (CE)/g extract. Tannin content in different extracts was also measured using the modified vanillin assay described by Sun et al. (1998). Tannin content was expressed as mg catechin equivalent (CE)/g extract. The dried truffles of T. boudieri were also subjected for carotenoid and anthocyanin contents which were estimated as previously described by Lichtenthaler and Wellburn (1983) and by Rodriguez-Saona and Wrolstad (2001), respectively. Furthermore, ascorbic acid content was determined according to Al-Laith (2010). All tests were carried out in triplicate and the results were averaged.

2.9 DPPH radical-scavenging and antioxidant activities

The DPPH radical-scavenging activity and the antioxidant activity using β-carotene/linoleic acid assay of T. boudieri extracts were measured as previously described (Yildirim et al., 2001; Koleva et al., 2002; Zouari et al., 2011). The DPPH radical-scavenging activity and the antioxidant activity of T. boudieri extracts were presented by IC₅₀ values, defined as the concentration of the extract needed to provide 50% inhibition. Therefore, IC₅₀ values were calculated from the graph plotting DPPH radical-scavenging activity or antioxidant activity percentages against extract concentration. Lower IC₅₀ values reflected better scavenging or antioxidant activities.

2.10 Antimicrobial activity

2.11 Statistical analysis

Excel (Microsoft Corporation, USA) and SPSS (version 12.1, SPSS, Chicago, IL, USA) were used for statistical analysis. Data are expressed as means ± SD. To assess the variation of the variables among samples, a one-way ANalysis Of VAriance (ANOVA) was performed. Statistical significance between means was determined using Duncan’s multiple range tests and set at p < 0.05.

3.1 Physicochemical composition of T. boudieri truffle

The results of the nutrient composition expressed on a dry mass basis were presented in Table 1. The average moisture content of the fresh truffle was 78.23 g/100 g which in close agreement with those reported for fresh desert truffles (Tirmania nivea) from various Middle Eastern origins (Al-Laith, 2010). However, moisture content as high as 93% was reported for some American edible mushrooms (Dubost et al. 2007). Carbohydrates were the most abundant macronutrients (62.03 g/100 g) in T. boudieri truffle as was found previously for other desert truffles which contain approximately 60% carbohydrates (Kagan-Zur and Roth-Bejerano, 2008). The protein content of T. boudieri truffle (26.12 g/100 g) was found to be higher than the value reported for Terfezia claveryi ascocarps (16 g/100 g) (Bokhary and Parvez, 1993). The ash content in T. boudieri truffle was 4.49 g/100 g which is close to those (from 4.9 to 5.9%) previously reported for different desert truffles such as T. claveryi, T. nivea, and Tirmania pinoyi (Hussain and Al-Ruqaie, 1999). Table 2 presents the concentrations of different minerals (K, Ca, Mg, Fe, Na, P, Mn and Cu) in T. boudieri truffle. Potassium, phosphorus, iron, and calcium were found to be particularly abundant in T. boudieri truffle as was previously reported for European truffles (Wang and Marcone, 2011). The intake of T. boudieri could be expected to contribute a large proportion of the essential mineral requirement in the body. Table 1 shows that T. boudieri truffle contains a high fat content (8 g/100 g) as compared to those (from 2.81% to 7.42%) reported for different desert truffles (Wang and Marcone, 2011). Composition of the fatty acids of T. boudieri truffle was investigated using both GC and GC/MS techniques. Percentages and retention indices of the identified fatty acids are listed in Table 3 in the order of their elution on the HP-5MS column. The global chromatographic analysis resulted in the identification of four fatty acids, accounting for 99.9% of the total fat content. Interestingly, the T. boudieri truffle was characterized by its higher content of the essential linoleic acid (n-6) (54.10%). Furthermore, oleic acid (n-9) and palmitic acid represent 22% and 20.40%, respectively (Table 3).

3.2 Volatile compounds of T. boudieri truffle

In addition to their nutritional value, truffles have gained popularity as they have a unique aroma profile which was highly desirable in various food. The volatile compounds of T. boudieri truffle were extracted by hydrodistillation and analyzed by GC and GC/MS techniques. The identified compounds, their percentages and their experimental retention indices are listed in Table 4. Eight compounds were identified, accounting for 98.29% of the total volatiles. The volatile profile identified in the T. boudieri truffle seems to be original since, volatiles were dominated by fatty acids. In fact, palmitic acid (65.21%) and linoleic acid (15.7%) were the major compounds. Aldehydes such as hexanal (1.18%) and phenols such as thymol (2.65%) were found to be minor compounds. More than 200 volatile compounds have been reported from various truffle species. However, the volatile compounds identified so far, by GC/MS analysis, are hydrocarbons, alcohols, aldehydes, esters, ketones, benzene derivatives and sulfur compounds (Culleré et al. 2010). Bellesia et al. (1998) found that the major contributors to the aroma profile of Tuber melanosporum from central Italy were four volatile compounds including two aldehydes (2- and 3-methylbutanal) and two alcohols (2- and 3-methylbutanol). Moreover, Bellesia et al. (2001) found that octen-3-ol (mushroom alcohol) was the main volatile product of the Tuber borchii truffle.

3.3 Antioxidant properties of T. boudieri truffle

3.4 Antimicrobial activity of T. boudieri truffle

The antimicrobial activity of T. boudieri truffle extracts against seven species of bacteria was assessed by evaluating the determination of minimum inhibitory concentration (MIC) values (mg of extract/ml of medium). As can be seen in Table 6, truffle extracts showed varying degrees of antibacterial activity against all tested strains. The MIC values of microbial strains were in the range of 0.5–8 mg/ml depending on the nature of the extract and the bacterial strain. Table 6 shows that methanolic extract seems to be the most effective on the tested strains. Furthermore, B. subtilis, S. aureus, E. faecalis, and E. coli were the most susceptible bacteria for this truffle extract. Hussain and Al-Ruqaie (1999) reported that methanolic extract of Terfezia truffles had antimicrobial activity against a wide range of both Gram-positive and Gram-negative bacteria. Furthermore, some researchers have also investigated the impact of extract solvents on the antimicrobial activity of truffles (Hussain and Al-Ruqaie, 1999; Janakat et al. 2004, 2005). The methanol extract of Tirmania truffles has been considered to provide the most antimicrobial inhibitory activity as compared with water and ethyl acetate extracts of Tirmania truffles.