Characterization of Odor-Active Compounds, Polyphenols, and Fatty Acids in Coffee Silverskin

For the first time the volatile fraction of coffee silverskin has been studied focusing on odor-active compounds detected by gas chromatography-olfactometry/flame ionization detector (GC-O/FID) system. Two approaches, namely headspace (HS) analysis by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and odor-active compounds analysis by gas chromatography-olfactometry/flame ionization detector (GC-O/FID), have been employed to fully characterize the aroma profile of this by-product. This work also provided an entire characterization of the bioactive compounds present in coffee silverskin, including alkaloids, chlorogenic acids, phenolic acids, flavonoids, and secoiridoids, by using different extraction procedures and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system. Coffee silverskin was shown to be a good source of caffeine and chlorogenic acids but also of phenolic acids and flavonoids. In addition, the fatty acid composition of the coffee silverskin was established by GC-FID system. The results from this research could contribute to the development of innovative applications and reuses of coffee silverskin, an interesting resource with a high potential to be tapped by the food and nutraceutical sector, and possibly also in the cosmetics and perfumery.


Introduction
Coffee is one of the most consumed beverages in the world and an important agricultural product in the international trade. Coffee companies generate a significant amount of liquid and solid wastes (by-products); around 90% of the weight of coffee cherries (mostly pulp) is discarded during processing as agricultural waste or by-product [1]. Several authors have previously proposed different approaches to reuse the coffee by-products in order to reduce their disposal [2][3][4][5][6][7]. Among these by-products is coffee silverskin (CS), which is the major residue generated during the roasting process. It is a thin tegument that covers the coffee seeds, also known as coffee beans (CB). During roasting, CB expand and this thin layer is detached [8]. Although CS accounts for only a minimal fraction of the whole

Odor-Active Compound Identification by GC-O/FID and GC×GC-TOF
The first identification step of odor-active compounds in CS and CB was performed by comparing the experimental linear retention indices (LRI) and the odor descriptions of the odorants present in different chromatogram regions, recorded during the AEDA, to outcomes obtained in previous works of coffee odorants [13,14], and to data compiled in the Leibniz-LSB@TUM Odorant Database [15]. In case of matching, authentic reference compounds were injected into GC-O/FID to confirm the proposed structures. The second step of identification was the comparison of the GC-O analysis of the concentrated volatile extracts and reference compounds using a second column with different polarity . Finally, to confirm the proposed structures, samples were analyzed by GC×GC-TOF. Before injection, volatile extracts of CS and CB were separated into different fractions, as detailed below in Materials and Methods section. Each fraction was then analyzed by GC-O and by comprehensive two-dimensional GC-MS, together with reference compounds, to identify odor-active compounds. As an example, Figure 1 reports the TIC of 2D-GC-MS plots of a mixture of thirteen reference compounds (a) and a sample of acidic volatiles (AV) fraction of coffee silverskin (b).
Molecules 2020, 25, x FOR PEER REVIEW 3 of 22 different polarity . Finally, to confirm the proposed structures, samples were analyzed by GC×GC-TOF. Before injection, volatile extracts of CS and CB were separated into different fractions, as detailed below in Materials and Methods section. Each fraction was then analyzed by GC-O and by comprehensive two-dimensional GC-MS, together with reference compounds, to identify odor-active compounds. As an example, Figure 1 reports the TIC of 2D-GC-MS plots of a mixture of thirteen reference compounds (a) and a sample of acidic volatiles (AV) fraction of coffee silverskin (b).   Table 1 shows all odors detected by GC-O and the assigned odorant structures with their LRI calculated on DB-FFAP and DB-5 columns, the odor descriptions and the flavor dilution (FD) factors for CS and CB. A case of unseparated odorants was observed for 3-methylbutanoic acid and 2-methybutanoic acid, which were characterized by a cheesy aroma. These two compounds were not separated on DB-FFAP column as well as on DB-5; the MS studies demonstrated the presence of both isomers in CS volatile fraction. Additionally, it was only possible to assert the presence of 2-methylbutanal or 3-methylbutanal.

Compound
Several classes of compounds such as organic acids (especially short chain fatty acids), furans, furfurals, ketones, aldehydes, alcohols, pyridines, phenols, and lactones were detected in CS. The volatile substances qualitative and quantitative profile in roasted CB and their silverskin depends on the chemical composition of the raw seeds, their origin and maturation degree, and also on the roasting conditions [36]. About 1000 volatile organic compounds (VOCs) have been previously identified in different types of roasted CB with different analytical methods [13]. The classes of VOCs typically found are furans, pyrazines, ketones, phenols, alcohols, aldehydes, organic acids, esters, lactones, pyridines and sulfur compounds [21]. Their formation is usually due to the chemical processes involved during the roasting process and their presence and quantity is highly related to the roasting intensities. For example, some flavor compounds, such as furfural derivatives and furanones deriving from reactions involving sugars and lipids of green CB, seem to be in relatively high concentrations under mild roasting conditions (light roasting degree) than under higher roasting intensities (dark roasting degree). Pyridines and pyrroles, which can derive from the Maillard reaction, are mainly formed at high roasting intensities. Also, other VOCs formed from the degradation of chlorogenic acids (phenols and lactones) are found in greater amounts at high roasting temperatures [37].
The classes of VOCs detected in the present study were in accordance with the ones found in literature [36,37]. In fact, roasted coffee contains mainly furans, pyrazines, pyridines, alcohols, ketones, phenols, some aldehydes, and short chain fatty acids (SCFAs). In particular, acetic acid was the most abundant volatile compound detected by HS-SPME-GC-MS technique in terms of peak area percentage in the analyzed CS. Somporn et al. [36] found acetic acid as the most abundant VOC in roasted coffee. Then, other SCFAs were present in considerable amount in the sample under investigation, such as formic, propanoic, butanoic, 3-methylbutanoic, 3-methyl-2-butenoic acid, pentanoic,4-methylpentanoic acid, hexanoic, heptanoic, octanoic and nonanoic acids. In fact, during roasting process, carbohydrates like sucrose, begin to breakdown, leading to the formation of SCFAs such as acetic and formic. Depending on roasting conditions, acetic acid concentration can become 25 times higher than its initial green bean concentration. Overall acetic acid reaches its maximum level at light or medium roasts, then quickly dissipates as roasting progresses due to its high volatility [38]. At low concentrations SCFAs show pleasant and sweet-like sensory characteristics, but at higher amounts can impart ferment-like flavors. The presence of SCFAs, especially acetic, propionic and butanoic acids, makes CS a potential functional food. In fact, SCFAs have several beneficial effects: they are able to enhance the growth of beneficial intestinal bacteria, decrease blood pressure, and reduce fat absorption and the presence of pathogenic bacteria in the intestinal tract [39].
Regarding the other classes of VOCs detected in the sample, furans are generally associated with the aromas of nuts and caramel. For instance, the detected 2-furanmethanol is known to give bitter and toasted flavor, and was also found by Colzi et al. [40] as the most abundant compound in the lipid extract from the spent coffee in capsules. Then, furfural derivatives can be formed from monosaccharides and from the reaction between a sugar and an amino acid at high temperatures, suggesting that they are formed during the roasting step. The presence of furfural in the sample contributed to sweet, bread-like and caramel flavor [37]. Other classes of compounds important for CS aroma included aldehydes, such as hexanal, which is associated to grassy and green oily aroma and 2-methylbutanal and 3-methylbutanal, which are associated with malty aroma. Several pyrazines and pyridines, molecules responsible for toasted, nut and chocolate flavor notes [40], were identified by HS-SPME-GC-MS. Important for coffee aroma are also some phenolic compounds such as guaiacol (2-methoxyphenol), 4-vinylguaiacol, 4-ethylguaiacol and 4-hydroxy-3-methoxybenzaldehyde. These phenols arise from thermal degradation of chlorogenic acids, and these volatiles could have a role in flavor differentiation between arabica and robusta, as the two species contain significantly different amounts of chlorogenic acids [21]. In particular, guaiacol, 4-methylphenol, phenol, and 2-methylphenol were detected by HS-SPME-GC-MS system. Butyrolactone was also detected in the sample. Its presence in coffee volatiles has been reported in many studies since the 1960s. It possesses butter and coconut-like flavors and it may play an important role in the flavor of coffee and other food and beverages [37].

Fatty Acid Profile
The lipid content of CS investigated in this study was 7.49 ± 0.01 g/100 g. Table 3 presents the % fatty acid composition found in CS lipids.

Bioactive Compounds in Coffee Silverskin (CS)
Thirty bioactive compounds, including alkaloids, chlorogenic acids, phenolic acids, flavonoids, and secoiridoids were quantified in CS by using HPLC-MS/MS triple quadrupole. The analytical method was validated by investigating the linearity, reproducibility and the sensitivity as reported in a previous work [42]. Several extraction methods such as liquid-solid extraction assisted and not by sonication, and various solvents were evaluated for their ability to extract analytes from CS matrix. Table 4 reports the contents (in mg kg −1 ) of 30 analytes resulting from the eight different extraction methods. The highest content of total bioactive compounds was obtained with Method 4 (2005.613 ± 42.118 mg kg −1 ) and Method 2 (1910.549 ± 55.406 mg kg −1 ). In both cases ultrasound assisted extractions (UAE) were carried out but employing EtOH/H 2 O and H 2 O, respectively. Excluding the concentration of caffeine and chlorogenic acids to the total content of bioactive compounds, the highest concentrations were found for Method 4 (4.391 ± 0.228 mg kg −1 ) and 5 (4.226 ± 0.093 mg kg −1 ). Therefore, Method 4, i.e., an ethanol/water (70/30) extraction assisted by sonication, was the best procedure in term of extraction efficiency not only for caffeine and chlorogenic acids but also for polyphenols. This solvent was the best for the extraction of sixteen polyphenols from pulse samples as well [43]. Interesting levels of polyphenols were also obtained when the extraction was performed under acid condition (Method 5). This could be due to the prevention of polyphenols oxidation at low pH [43].
A total of 17 bioactive compounds were found in CS; caffeine (731.5−845.5 mg kg −1 , RSD 2.5−5.5%) and chlorogenic acids (total contents: 974.6−1155.7 mg kg −1 , RSD 1.8−6.3%) were the analytes present in the highest concentrations. Therefore, CS can be considered a good source to recover caffeine and chlorogenic acid and an interesting starting material for nutraceutical formulations. In fact, it has been reported that chlorogenic acids are an important class of biologically active dietary polyphenols, which are associated with several beneficial effects such as antioxidant activity, antibacterial, hepatoprotective, cardioprotective, anti-inflammatory, antipyretic, neuroprotective, anti-obesity, free radicals scavenger, and a central nervous system (CNS) stimulator [44]. The concentrations of caffeine and chlorogenic acid in this study were slightly lower than those reported previously in [44] and [40]. This could be due to various factors affecting the coffee sample such as coffee variety, processing method and roasting degree. In fact, the contents of chlorogenic acids and other polyphenols can be influenced by roasting degree and processing method [36,45] and the levels of caffeine can fluctuate depending on the used roasted beans from 0.1 to 2.0% (dry weight) [46]. The present work is one of the first on the quantification of unconjugated phenolic acids in CS; all 7 monitored phenolic acids were found in CS, as it was shown in Table 4. Vanillic (0.880−1.472 mg kg −1 , RSD 2.1−3.6%) and caffeic acid (0.858−1.420 mg kg −1 , RSD 3.2−6.2%) were the most abundant followed by syringic acid (0.094−0.356 mg kg −1 , RSD 1.8−4.2%). Shikimic acid ranged from 0.198 to 0.520 mg kg −1 (RSD 3.1−4.6%); it is an important intermediate in the biosynthesis of lignin, aromatic amino acids and most alkaloids in plants and microorganisms [47]. This study is the first on the quantification of flavonoids including flavonols, flavan-3-ols, flavanone and anthocyanins, alkaloid (quinine), xanthone, iridoid and secoiridoids. Among these polyphenols, four molecules of flavonols such as rutin, hyperoside, kaempferol 3-glucoside, and quercitrin, were detected above their limit of detection (from 0.002 to 0.069 mg kg −1 , RSD 3.2−5.8%). A flavanone, i.e., naringin (0.002−0.034 mg kg −1 , RSD 4.6−6.3%), was found in the CS extracts of four extraction methods. Coffee silverskin can be considered not only a good source of caffeine and chlorogenic acids but also a resource of polyphenols such as phenolic acids and flavonoids.

Coffee Silverskin and Coffee Beans Preparation and Odor-Active Compounds Extraction
Coffee silverskin (CS) and coffee bean (CB) samples, from 100% Coffea arabica L. var. Catuai Rosso coming from Naranjo, Santa Cruz region (Guatemala), were provided by Perfero Caffè (Altidona, Italy) roasting company. The coffee berries were submitted to natural method which consisted of sun-drying the berries on raised bed with wire mesh (African bed) for 24 days. About 200 g of coffee silverskin were collected after the roasting process from 20 kg of green coffee. The roasting process was performed during 16 min and it reached the maximum temperature of 220 • C. Samples were kept in vacuum sealed bags at −20 • C.
Just before the extraction process, CS was immersed in liquid nitrogen and milled by GM 200 Retsch GrindoMix (time: 10 s; speed: 4000 rpm; in both rotation direction). CB was processed into a powder through 6875 Freezer/Mill High Capacity Cryogenic Grinder (SPEX SamplePrep, Stanmore, UK) using the following program: pre-cool, 2 min; run time, 1 min; cool time, 1 min; cycle, 3; rate, 14 cps. The volatile compounds in 20 g of CS or CB powder were extracted with 250 mL of dichloromethane under stirring at room temperature for 2 h. After filtration with filter paper, the volatile compounds were removed from the extract by Solvent Assisted Flavour Evaporation (SAFE) at 40 • C. The SAFE distillate was dried by adding anhydrous sodium sulfate and concentrated to 1 mL by using a Vigreux column (50 × 1 cm) and then a Bemelmans microdistillation device [50]. The concentrated volatile extracts were kept at −20 • C and the odor evaluation of a small amount of CS and CB extracts using fragrance test strips demonstrated the aroma equivalence to the starting materials.

Odorants Analysis: GC-O/FID and AEDA
A Trace GC Ultra gas chromatograph (Thermo Scientific, Dreieich, Germany) was equipped with a cold-on-column injector, a flame ionization detector (FID) and a tailor-made sniffing port [51]. Two types of fused silica columns were used for volatile separation: (a) DB-FFAP (30 m × 0.32 mm i.d., 0.25 µm film thickness); (b) DB-5 (30 m × 0.32 mm i.d., 0.25 µm film thickness) (both Agilent J&W, United States). The carrier gas was helium (He) at 60 KPa (DB-FFAP) and 65 KPa (DB-5) and the injection volume was 1 µL. The initial temperature of the oven was 40 • C (2 min), the gradients were at 6 • C/min to 230 • C for DB-FFAP and to 240 • C for DB-5, and held at 230 • C (DB-FFAP) and 240 • C (DB-5) for 5 min. The end of the analytical column was connected to a deactivated Y-shaped glass splitter which divided the column effluent in two equal parts that were directed via deactivated fused silica capillaries (50 cm × 0.25 mm i.d.) to the FID (250 • C) and the sniffing port (230 • C), respectively.
The concentrated volatile extracts of CS and CB were injected into the GC-O/FID system. The GC-O/FID analyses were carried out by three trained and experienced sniffers (two males, one female: aged 26−40) using the DB-FFAP column as well as the DB-5 column. The training consisted in weekly sensory evaluation sessions of reference odorants dissolved in water and the evaluation of reference mixtures by GC-O analysis. Each sniffer during the GC-O analysis, placed the nose in the region above the top of the sniffing port and evaluated the odor of the effluent. The positions and the descriptions of the odors were marked on the FID chromatogram registered by a recorder. On both columns, an experimental linear retention index (LRI) of each odor was calculated from their retention times and the retention times of adjacent n-alkanes by linear interpolation according to van Den Dool and Kratz [16]. Each sniffer repeated the analysis until data was reproducible.
Aroma Extract Dilution Analysis (AEDA) was performed by stepwise diluting, the concentrated coffee volatile extracts with dichloromethane (1:2, 1:4, 1:8, 1:16, 1:32, etc.). Each diluted sample was then injected into the GC-O/FID system using the DB-FFAP column. A flavor dilution (FD) factor was assigned to each odor-active compound, representing the dilution factor of the highest diluted sample in which the odorant was detected during GC-O/FID analysis by any of the three sniffers.

Fractionation of Coffee Silverskin and Coffee Beans Volatiles
The fractionation of volatile extracts was performed to simplify the CS and CB SAFE distillate and, consequently, to have less coelution during GC separation, aimed to facilitate the MS identification. Seven different fractions, i.e., acidic volatiles (AV), 5 neutral and basic volatiles (NBVA-E) and volatile thiols (VT), were prepared according to odor-active compounds commonly reported in coffee [19,21,52]. A SAFE distillate was prepared as described above and was extracted with aqueous sodium carbonate solution (0.5 mol L −1 ) in three portions (300 mL total). The organic phase (dichloromethane), containing the neutral and basic volatiles, was dried with anhydrous sodium sulfate and concentrated to 0.5 mL by using a Vigreux column and then a Bemelmans microdistillation device (NBV). The aqueous phase, containing the acidic volatiles, was washed with dichloromethane (50 mL) and then acidified with hydrochloric acid (32%) to pH 2. Acidic volatiles were re-extracted in three portions with dichloromethane (300 mL total) and the remaining water was removed by drying over anhydrous sodium sulfate. Finally, the organic phase was concentrated to 0.5 mL (AV). The fraction of NBV was further separated on a slurry of purified silica gel (9 g) in pentane using a water-cooled (12 • C) glass column (1 cm i.d.). The elution was carried out with five different mixtures of pentane and diethyl ether: A, 100:0; B, 90:10; C, 70:30; D, 50:50; E, 0:100 (v:v; 50 mL each). The eluate was collected in five portions of 50 mL and eluate portions were concentrated to 0.5 mL (NBVA-E). Another SAFE distillate was used to prepare a volatile thiol fraction by following a published procedure [48]. Briefly, the concentrated volatile extracts of CS and CB were applied onto mercurated agarose gel (1 g) in a glass column (0.5 cm i.d.). Then, the column was rinsed with dichloromethane (50 mL) and the volatile thiols were eluted with dithiothreitol (10 mmol/L) in dichloromethane (50 mL). The excess of dithiothreitol was removed by SAFE distillation, and the distillate was concentrated to 0.5 mL (VT).

GC×GC-TOF
The system consisted of a 6890 Plus gas chromatograph (Agilent Technologies, Waldbronn, Germany) and a Pegasus III TOFMS (Leco, Mönchengladbach, Germany). The GC was equipped with a KAS4 injector (Gerstel, Mühlheim/Ruhr, Germany). The injector was connected to a fused silica column, DB-FFAP, 30 m × 0.25 mm i.d., 0.25 µm film (Agilent). The end of this column was connected to a second fused silica column, DB-5, 2 m × 0.15 mm i.d., 0.30 µm film (Agilent). The front part of this column was passed through a liquid nitrogen-cooled dual-stage quad-jet thermal modulator (Leco), the major part was installed in a secondary oven mounted inside the primary GC oven, and the column end was connected via a heated (250 • C) transfer line to the MS inlet. Helium at 2 mL/min constant flow served as the carrier gas. The temperature of the first oven was 40 • C for 2 min, ramped up at 6 • /min to 230 • C, and held for 5 min at 230 • C. The modulation time was 4 s. The temperature of the secondary oven was 70 • C for 2 min, ramped up at 6 • /min to 250 • C, and held for 5 min at 250 • C. The mass spectrometer was operated in the EI mode at 70 eV with a scan range of m/z 35−350 and a scan rate of 100 spectra/s. Data evaluation was performed by means of GC Image (GC Image, Lincoln, NE, USA).

Volatile Substance Composition Analysis by HS-SPME-GC-MS
An aliquot of 0.5 g of triturated CS was weighed in a 10 mL screw cap vial with pierceable septum with 2 mL of water and 0.4 g of NaCl. Then the sample was conditioned at 40 • C for 20 min under agitation. A solid-phase microextraction fiber coated with 50/30 µm divinylbenzene/Carboxen/ polydimethylsiloxane (DVB/CAR/PDMS), 1 cm long, was then exposed to the headspace of the sample for 1 h and then the fiber was retracted and exposed for 10 min into the hot injector (260 • C) of a 6850 gas chromatograph (Agilent, Santa Clara, CA, USA). The splitless injection (splitless time, 4 min) was used. The GC was coupled with a 5973 N mass spectrometer detector (Agilent). The GC was equipped with a capillary column coated with polyethylene glycol, DB-WAX, 60 m × 0.25 mm i.d., 0.25 µm film thickness (Agilent J&W). The end of the column was connected via a heated (260 • C) transfer line to the MS inlet. The carrier gas was helium at 1.2 mL/min. The initial oven temperature was 35 • C (min), the gradients were at 2.5 • C/min to 120 • C and 15 • C/min to 250 • C and held for 3.33 min. The mass spectrometer was operated in the EI mode at 70 eV with a scan range of m/z 29−400. Identification of eluted molecules was performed by comparison of the experimental linear retention indices, calculated with reference to linear alkanes, according to van Den Dool and Kratz [16], with those reported in literature, and with comparison of the experimental mass spectra with those of the NIST 08 library. Blank analysis was performed in order to identify contaminants.

Lipid Extraction from Coffee Silverskin
Silverskin lipids were obtained by Folch method extraction [53]. An aliquot of triturated sample (10 g) was dissolved in 160 mL of a solvent mixture of chloroform/methanol 2:1. The sample was homogenized for 3 min by Ultraturrax (Yellow Line DI 25s basic immersion-type homogenizer). The solution was filtered and the solvent was collected in a graduated 200 mL cylinder. The filter was washed with 40 mL of fresh solvent mixture, reaching a final volume of 200 mL. The solution was put in a separating funnel and washed with 40 mL of aqueous potassium chloride solution (0.88%). The organic phase was recovered in a flask and dried over anhydrous sodium sulfate. Subsequently, the solvent was removed with the use of a rotavapor until constant weight. Lastly, the lipid extract was recovered with 4 mL of chloroform and stored in a refrigerator at −20 • C.

Fatty Acid Composition Analysis by GC-FID
The fatty acids were derivatized to form the corresponding fatty acid methyl esters (FAMEs). A proper aliquot of the dried lipid extract (10 mg) of CS was dissolved in 1 mL of n-hexane and 100 µL of KOH 2N in methanol were added to the solution and shaken for 2 min with the help of a vortex device. Thereafter, the reaction was quenched by the addition of 1.5 mL of a saturated brine. The mixture was vortexed for 2 min and centrifuged for 5 min (5000 rpm). The upper layer was transferred to a 4 mL vial and anhydrous sodium sulfate was used to eliminate any remaining water. This mixture was further vortexed and centrifuged.
The supernatant was analyzed in a 6850 gas chromatograph (Agilent) equipped with a split/splitless injector (260 • C) and a flame ionization detector (250 • C). Compounds were separated using a fused-silica capillary column coated with 50% of cyanopropylphenyl-dimethylpolysiloxane, DB-225MS™, 30 m × 0.25 mm i.d., 0.25 µm film thickness (Agilent). The carrier gas used was hydrogen at a flow rate of 3.7 mL/min. The injection was performed in split mode (split ratio 30:1) and the injection volume was 1 µL. The initial oven temperature was 40 • C (3 min), ramped at 20 • C/min to 220 • C (5 min) and at 20 • C/min to 240 • C (1 min). FAMEs were identified by comparing retention times of analytes with reference solutions obtained from the FAMEs Mix. The results were expressed as relative percentage of each fatty acid, after correcting FAMEs peak areas using the theoretical response factors [54].

Coffee Silverskin Preparation and Bioactive Compounds Extraction
Just before the extraction process, CS was immersed in liquid nitrogen and milled by Ariete Blendy 570 grinder (Florence, Italy) through five cycles of 5 s. The extraction of the bioactive compounds was based on extraction methods optimized by Kamgang Nzekoue et al. [42] with some modifications. The following two sections describe the tested procedures: Ultrasound-assisted extraction (UAE) and liquid-solid extraction (LSE) without sonication. At the end eight extraction processes have been tested.  7), have been tested. Only for Method 5, after the extraction the pH of the sample was adjusted to 6 by adding 1 M KOH. After sonication, the sample was filtrated with filter paper and an aliquot of supernatant was collected, centrifuged at 13,000× g rpm for 10 min and filtrated with 0.2 µm syringeless filter. Eventually, it was injected into HPLC-MS/MS system.

Liquid-Solid Extraction (LSE)
The extraction of 10 g of CS powder was performed with 100 mL of H 2 O (Method 8) keeping the sample in a water bath under magnetic stirring for 30 min at 80 • C. After extraction, the sample was cooled at room temperature and filtrated with filter paper. Then, an aliquot was centrifuged at 13,000× g rpm for 10 min, filtrated with 0.2 µm syringeless filter and injected into HPLC-MS/MS system.

HPLC-MS/MS Analyses
The HPLC-MS/MS studies were performed following a previous work of Kamgang Nzekoue et al. [42]. Briefly, the analysis was carried out by using a 1290 Infinity series liquid chromatograph (Agilent) and a 6420 Triple Quadrupole (Agilent) equipped with an electrospray ionization (ESI) source operating in negative and positive ionization mode. In fact, the instrument allowed to perform a one run with polarity switching without any problems. The separation of target compounds was achieved on a Kinetex PFP analytical column, 100 mm × 2.1 mm i.d., particle size 2.6 µm (Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of water (A) and methanol (B) both with formic acid 0.1% at a flow rate of 0.2 mL min −1 in gradient elution mode. The composition of the mobile phase varied as follows: 0-2 min, isocratic condition, 20% B; 2-15 min, 80% B; 15-18 min, isocratic condition, 80% B; 18-23 min, 100% B; 23-35 min, 20% B. The injection volume was 2 µL. The temperature of the column was 30 • C and the temperature of the drying gas in the ionization source was 350 • C. The gas flow was 10 L/min, the nebulizer pressure was 172.369 kPa and the capillary voltage was 4000 V. Detection was performed in the Dynamic "multiple reaction monitoring" (Dynamic-MRM) mode and the Dynamic-MRM peak areas were integrated for quantification. The most abundant product ion was used for quantitation, and the other for qualification. The selected ion transitions and the mass spectrometer parameters including the specific time window for each compound (delta retention time) are reported in Table 5. Table 5. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) acquisition parameters, working as Dynamic "Multiple Reaction Monitoring" mode, including retention time (Rt) and delta retention time (∆ Rt) for each transition.

Conclusions
For the first time the volatile fraction of coffee silverskin has been studied using two approaches, i.e., HS analysis by SPME-GC-MS and odor-active compounds analysis by GC-O/FID system. Our studies demonstrated that coffee silverskin contains an interesting odor-active compound fraction with high similarity to coffee beans. Although beans are characterized by more complex and intense aroma, coffee silverskin remains an important co-product to be exploited in food industry, for instance in novel food production. In this context, it will assume an important role for further research on odorant quantification and sensory tests in order to determine the key aroma compounds. This work also provided an entire characterization of bioactive compounds together with the fatty acid composition. This research increased knowledge on coffee silverskin and it is hoped that the results can contribute to the development an its original application in food and nutraceutical sector. In addition, in the optical of more sustainable economy, this work could encourage the use of coffee silverskin in certain industrial fields and therefore, it can contribute to the decrease in coffee waste and the disposal costs. This may lead to an eco-friendlier coffee production and consumption.