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White Acqualagna truffle (Tuber magnatum Pico): Evaluation of volatile and non-volatile profiles by GC-MS, sensory analyses and elemental composition by ICP-MS

Tu, 26.11.2024
| Original article from: Food Chemistry, Volume 439, 2024, 138089
In the study published in the Food Chemistry journal, the researchers provided the first comprehensive characterization of the Acqualagna white truffle.
<ul><li><strong>Photo:</strong> <cite>Food Chemistry, Volume 439, </cite>2024, 138089: Graphical abstract</li></ul>
  • Photo: Food Chemistry, Volume 439, 2024, 138089: Graphical abstract

In the study published in the Food Chemistry journal, the researchers from the University of Camerino, Italy provided the first comprehensive characterization of the Acqualagna white truffle, the world's most expensive edible underground mushroom.

The volatile profile, dominated by bis(methylthio)methane (78.72%), was analyzed by GC–MS and correlated with sensory descriptors such as garlic-like, nutty, floral, and pungent notes. Non-volatile analysis, including ICP-MS, revealed a high content of essential elements like potassium, phosphorus, sulfur, calcium, and magnesium, as well as the assimilation of rare earth elements (REE) from the soil. These findings highlight the unique properties of Acqualagna white truffles, emphasizing their connection to local soil conditions and potential for regional food valorization.

The original article

White Acqualagna truffle (Tuber magnatum Pico): Evaluation of volatile and non-volatile profiles by GC-MS, sensory analyses and elemental composition by ICP-MS

Diletta Piatti, Riccardo Marconi, Giovanni Caprioli, Marco Zannotti, Rita Giovannetti, Gianni Sagratin

Food Chemistry, Volume 439, 2024, 138089

https://doi.org/10.1016/j.foodchem.2023.138089

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Highlights

  • First chemical characterization of Acqualagna white truffle (Tuber magnatum Pico).
  • Valorization of local precious food with global impact.
  • Presented the correspondence between GC–MS molecules and sensory analysis odorants.
  • The truffle was linked to the soil through elemental composition by ICP-MS analysis.

Abstract

The White Truffle is the most expensive edible underground mushroom. In this study the first characterization of the Acqualagna white truffle was delivered, taking into consideration the soil of origin and the human perception. The volatile profile was identified by GC–MS and compared with the descriptors obtained by sensory analysis. The non-volatile characterization was done using elemental composition by ICP-MS analysis, elemental analysis, and spectrophotometric assays. The volatile profile consists mainly of bis(methylthio)methane (78.72%) and other minor constituents, linked to seven odorant descriptors: garlic-like, nutty-like, geosmine-like, floral, mushroom-like, pungent and green/herbal. ICP-MS revealed that truffle has a higher content of K, P, S, Ca and Mg (97% of the elements investigated) and that it assimilates the Rare Earth Elements (REE) from the soil without discriminating them. In conclusion, this project is the first step for the enhancement of local food, linked to the territory conditions in which it is produced.

2.3. Aroma characterization by GC–MS

2.3.1. Sample preparation

White truffles (1 g) from Acqualagna were finely ground using a truffle slicer (approximately 0.2 mm in thickness). These small slices of the truffle were put in the vial (20 mL) and closed with a screw cap equipped with a PTFE- silicon septum. The headspace solid phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC–MS) was used to characterize the volatile profile of fresh white truffles using a previous publish method (Torregiani et al., 2017) with some modification.

2.3.2. Headspace solid-phase microextraction (HS-SPME)

Each vial was heated under stirring at around 250 rpm, the temperature was 60 °C, for 15 min in a heating platform. An AgilentChem workstation was used for the GC–MS system. After this time, the fiber DVB/PDMS/CWR/PDMS, (Agilent Technologies, Santa Clara, California, USA), 80 µm (50//30 µm) thickness was exposed to the headspace of the sample for 15 min. Once the extraction time had ended, the fiber was removed from the vial and placed in the injection port of the gas chromatograph for the rapid desorption of the analytes. A desorption time of 15 min, with an injection temperature of 250 °C, was sufficient to desorb most of the analytes from the fiber. After desorption from the fiber the headspace of the sample went in contact with the stationary phase in the column. The fiber was cleaned before each microextraction to prevent contamination using a blank GC–MS run with the same conditions mentioned above.

2.3.3. GC–MS analysis

A gas chromatograph and mass selective detector were used in combination to study the volatile profile of Acqualagna truffles. In particular, it was used an Agilent 8890 gas chromatograph (GC) coupled with a 5977B mass spectrometer (MSD) from Agilent (Santa Clara, California, USA). The system was also constituted of a PAL RTC 120 autosampler (Switzerland). The separation was performed by HP-5 MS capillary column (30 m l. × 0.25 mm i.d., 0.1 μm f.t., Agilent), supplied by Agilent (Santa Clara, California, USA) and coated with 5% phenylmethylpolysiloxane. The carrier gas was He (99.999%) flowing at 3 mL min−1 in splitless mode. The oven was thermostatted at 35 °C (5 min) then ramp went to 60 °C, 3 °C min−1, up to 70 °C, 1 °C min−1 and 200 °C at 5 °C min−1, hold 2 min, finally the temperature arrives 300 °C at 15 °C min−1 and remain 5 min. The mass spectra were acquired in full scan in the range 40/400 uma and the mass spectrometer used the electron impact (EI) mode with an ionization voltage of 70 eV to produce the spectra of the separated compounds. Peak assignment of the chromatograph was based on the computer matching of the mass spectra with the WILEY275 and NIST 08 using a matching quality of over 60%, published literature, and Kovats retention indices (RI) which were calculated based on n-alkane (C7–C30) series (Sigma Aldrich) under the same chromatographic condition. The relative percentage content of each compound was determined using a peak area normalization procedure based on the total ion flow chromatogram and consequentially expressed as a relative percentage (%) by calculating the ratio of each individual peak area to the sum of the peak areas of all target compounds. Only peaks with area percentage > 0.01 were considered and analyzed by comparing relative peak areas.

2.4. Sensory analysis

To establish how the olfactory characteristics of white truffle from Acqualagna are perceived by the consumer a study of sensory analysis was realized. Identification and selection of descriptors for establishing a sensory profile by quantitative descriptive analysis of white Acqualagna truffle were estimated as the protocols defined in ISO 11035:1994 (Sensory analysis – identification and selection of descriptors for establishing a sensory profile by a multidimensional approach). The room where the sensory analyses were carried out must conform to ISO 8589:2007, in particular for: lighting, temperature, noise and odours. Individual booths should be set up, so that the assessors can work on their own without distraction (modular mobile units can be used). The samples were prepared as follows: about 10 g of each truffle sample was put into the glass goblet (one for each assessor), which is then covered with a plastic lid to preserve the characteristic volatile profile of the truffle. The panel was constructed following ISO 8586:2012 (Sensory analysis – General guidelines for the selection, training and monitoring of selected assessors and expert sensory assessors) and consisted of 16 assessors. The panel leader was responsible for coordinating the trials, collecting and processing the results and overseeing the selection, training and monitoring of the performance of all the members of the panel. The panel participated in three sensory sessions of 45 min to train their ability to recognize and describe different aroma attributes in according to ISO 8586:2012. After the training sessions the panelists ultimately came to a final agreement regarding truffle's aroma description. (sulfury or garlic-like, cabbage-like, malty, cooked potato-like, fatty or green and mushroom-like). The following reference compounds were used to prepare the judges: bis(methylthio)methane for sulfury or garlic-like note, dimethyl sulfide for cabbage-like note, 3-methylbutanal for malty note, 3-(methylthio)propanal for cooked potato-like note, (E,E)-2,4-nonadienal for fatty or green note, and finally 1-octen-3-one for mushroom-like note (Feng et al., 2019, Schmidberger and Schieberle, 2017). The single judgment of each panelist was evaluated. Judges were required to generate the maximum number of descriptors defining all the olfactory sensations produced by truffles and to write them down in the appropriate form provided for all the tests. In this phase, no aromatic aspect of the product was overlooked and any distortion due to the influence of an individual in the group was avoided. Secondly, the judges discussed as a group and compare their perceptions under the guidance of the panel leader. This encouraged them to analyze the different components of product perception and to link it to a precise descriptor. All descriptive terms were then collected, including synonyms. In according to ISO 11035:1994, during the group discussions and in the presence of the samples, the panel leader proceeded with the selection of the descriptors. Hedonistic, quantitative, irrelevant words, that can describe the product in their own terms, were eliminated from the discussion. Finally, were identified several descriptors that can be used in the sensory analysis of the product (ISO 11035:1994).

2.5. Sample preparation for non-volatile profile

The samples of truffle used for spectrophotometric tests, elemental analysis and ICP-MS, were obtained in the same way. As described in section 2.1, we took 9 samples every 15 days. Each of these groups was divided into 3 distinct groups of 3 samples. The truffles of each group were carefully sliced (with a thickness of approximately 2 mm) and grounded by using a mortar with liquid nitrogen. For elemental analysis and ICP-MS a part of each blend of truffles obtained was freeze-dried at − 54 °C and 0.05 mbar, through a BUCHI Lyovapor™ L-200 freeze-dryer (Büchi Labortechnik AG, Flawil, Switzerland). The lyophilized samples were ground in a mortar and the obtained powders were maintained at 4 °C until further analyses.

2.6. Phenolic profile and antioxidant activity

2.6.1. Preparation of extracts

Extraction of flavonoids and phenolic compounds were carried out as follows: 1 g of the ground fresh truffles was extracted with 10 mL of ethanol 70% (drug-solvent ratio 1:10), as described by Sezer et al., 2017. Extractions were performed in screw-capped tubes, at room temperature in an ultrasound water bath at 40 kHz (FALC, Treviglio, Italy) for 20 min. The samples were centrifuged at 5000 rpm for 10 min using an IEC CL10 Centrifuge (Thermo Fisher Scientific, Waltham, USA). The supernatant was used for spectrophotometric assays and each trial was performed in triplicate.

2.6.2. Determination of the total phenolic content

The TPC of the extract was determined by the Folin-Ciocalteu method reported by Giusti et al., 2018 with slight variations. Briefly, 0.5 mL of the extract or gallic acid was mixed with 2.5 mL of 0.1 M Folin-Ciocalteu reagent in a polypropylene conical tube and after 5 min of incubation in the dark at room temperature was added 7 mL sodium carbonate (Na2CO3) solution 7.5%. The mixture was allowed to stand for 2 h in the dark, and absorbance was measured at 765 nm with an Agilent Technologies (Cary 8454 UV–Vis, Woburn, Massachusetts, USA) spectrophotometer, using the respective solvent as blank. Results were calculated by comparing the absorbance of samples with the standard calibration curve of gallic acid. TPCs were expressed as milligrams of gallic acid equivalents (GAE)/g dry weight (DW).

2.6.3. Determination of the total flavonoid content

Total Flavonoid Content (TFC) was determined following a method described by Laurita et al., 2021. Briefly, 0.5 mL of extract solution, 0.15 mL of NaNO2 (0.5 M), 3.2 mL of methanol (30% v/v) were mixed. After 5 min 0.15 mL of AlCl36H2O (0.3 M) and after other 5 min, 1 mL of NaOH (1 M) were added. The solution was mixed well and was incubated for 30 min in the dark at room temperature. The absorbance was measured, against the blank reagent, at 506 nm. The standard calibration curve for TFC was made using rutin standard solution under the same procedure as described above. TFC was expressed as mg of rutin equivalents (RT)/g dry weight (DW).

2.6.4. Radical scavenging activity assay

The antioxidant activity was determined using the DPPH method. Free radical scavenging activity of the extracts against radical 2,2-diphenyl-1-picrydrazyl (DPPH) was estimated spectrophotometrically as described by Giusti et al., 2017 with some modifications. According to the procedure, 0.5 mL of extract solution or standard (ascorbic acid) or blank (ethanol) was mixed with 4.5 mL of ethanolic solution of DPPH (0.1 mM). DPPH stock solution was prepared by dissolving 3.95 mg DPPH in 100 mL ethanol and kept at 4 °C, protected from light. After 30 min of incubation in the dark at room temperature, the DPPH disappearance was measured spectrophotometrically at 517 nm using (Agilent Technologies, Cary 8454 UV–Vis, Woburn, Massachusetts, USA) spectrophotometer. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the reference antioxidant for the calibration curve and the results were expressed as mg Trolox equivalent (TE)/kg dry weight (DW).

2.7. Elemental analysis

A ThermoFisher Scientific™ FLASH 2000 CHNS/O Analyzers (Thermo Fisher Scientific, Waltham, USA) was used to detect and quantify basic elements, such as C, H, N and S in Acqualagna truffles. The freeze-dried sample was ground, weighed and packed carefully in a tin capsule with an oxidizer (vanadium pentoxide) to be introduced into the instrument in solid form. Calibration samples were prepared by using BBTO (2,5-Bis(5-tert-butyl-benzoxazol-2-yl)thiophene). The truffle is heated and combusted in a furnace at 950 °C with a constant flow of helium stream 140 mL/min in a temporarily enriched oxygen atmosphere (flow oxigen 250 mL/min) occurred the combustion and were generated four reduced components: N2, CO2, H2O and SO2. These was separated in a chromatographic column and detected by a detector. Data were processed with specific software.

2.8. Quantitative elemental analysis by ICP-MS

The freeze-dried truffle was previously mineralized, by acid digestion, using a Berghof speedwave 4 microwave mineralizer (Berghof, Eningen, Germany). The acid digestion was performed in Teflon vessels using 4 mL 30%–32% H2O2, 1 mL 65% −69% HNO3, 0.05 g of freeze-dried truffles powder and 50 µL of a solution containing 2 mg/l of Au, Be and Ru added as recovery standard. The latter solution was appropriately prepared from the single-element standard solutions (1 g/L, ICP-MS grade, Fluka Analytical, Merck, Darmstadt, Germania). The digestion program follows three steps indicated in the Table 1. The mineralized sample was then transferred to a plastic tube and then diluted 1:10 with ultrapure water (resistivity 18.2 MΩ cm) produced by Millipore Milli-Q system (Millipore, Molsheim, France). In addition, the soil, where the truffles have been collected, was dried in oven at 100 °C for 24 h, diluted in a 1:5 ratios with ultrapure water, stirred for 24 h, and finally allowed to settle for 2 h. The obtained supernatant (release water) was filtered by PTFE-0.2 µm filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany). Both, the mineralized truffle solutions and the release water samples were characterized by an ICP-MS 7500cx series (Agilent Technologies, Santa Clara, CA, USA). The ICP-MS instrumentation operated under the following conditions: power 1550 W, carrier gas 0.9 L/min, make-up gas 0.00 L/min, sample depth 7 mm, nebulizer pump 0.1 r.p.s. and spray chamber temperature 2 °C. The 7500cx series can operate in NoGas/He mode, in order to overcome most of the polyatomic interference by the collision cell. A solution containing 45Sc, 115In, 140Ce and 209Bi (10 mg/l) was properly prepared from the single-element standard solutions (1 g/L, ICP-MS grade, Fluka Analytical, Merck, Darmstadt, Germania) and used as the internal standard for ICP-MS measurements. Standard solutions of the investigated elements were prepared by dilution with 1.0% HNO3, conveniently prepared from stock solution (Fluka Analytical, Merck, Darmstadt, Germania). The calibration line for the micro-elements (Li, Be, B, Al, Ti, V, Cr, Mn, Fe, Co, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ru, Pd, Ag, Cd, Sn, Sb, Cs, Ba, Pb, U) was performed using the following solutions: 0.01 ppb; 0.10 ppb; 1.00 ppb; 5.00 ppb; 10.0 ppb; 50.0 ppb; 100.0 ppb and 500.0 ppb. For the macro-elements (Na, Mg, P, S, K, Ca), the calibrations line was performed by using the following standard solutions: 0.50 ppm; 1.00 ppm; 2.50 ppm; 5.00 ppm; 10.0 ppm; 25.0 ppm and 50.0 ppm. The calibration of Hg element was operated as follow: 0.1 ppb; 0.5 ppb; 1.0 ppb; 5.0 ppb; 10.0 ppb. For the rare earth elements (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Th), the calibration line was performed by using the following standard solutions: 0.001 ppb, 0.01 ppb; 0.10 ppb; 1.00 ppb; 5.00 ppb; 10.0 ppb; 50.0 ppb; 100.0 ppb and 500.0 ppb, in this case as internal standard was used only 115In and 209Bi. All the calibration standards solutions for the ICP-MS analysis were prepared using ICP-MS calibration standards (10 mg/L, Agilent Technologies, Santa Clara, CA, USA).

4. Conclusions

Today there are no other studies in the literature on Acqualagna truffle, so this is the first study to obtain its characterization with different approaches: GC–MS, panelists analyses, ICP-MS, elemental analysis and spectrophotometric assays. Sensory panelists' analysis reported a good correlation with our instrumental data obtained with GC–MS. The key aroma compounds of our truffle was sulfury or garlic-like which corresponds to sulphur compounds such as bis(methylthio)methane and dimethyl sulfide. The Acqualagna T. magnatum was studied by ICP-MS and 48 elements were detected and quantified. Our white truffle was characterized by five main elements K, P, S, Ca and Mg, which give 97% of the total mass while the other elements investigated represent only 3%. CHNS elemental analysis showed in the tested sample that carbon to nitrogen ratio was higher than 6:1, despite sulfur and hydrogen being present in smaller amounts they still play important roles in the chemistry of truffles. Furthermore, this truffle exhibited good antioxidant activity so it could be valued by consumers for its positive impact on human health. The strong association between the elements identified in soil and in T. magnatum testifies to the fundamental role of the soil environment for the elementary composition of truffles.

In conclusion, this pioneering study on Acqualagna white truffles and it is could be a starting point considering that these fungi are still an unvalued source of compounds with high economic value.

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