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Molecules 2020, 25, 2993; doi:10.3390/molecules25132993 www.mdpi.com/journal/molecules
Article
Characterization of Odor-Active Compounds,
Polyphenols, and Fatty Acids in Coffee Silverskin
Simone Angeloni 1,2, Serena Scortichini 3, Dennis Fiorini 3, Gianni Sagratini 1, Sauro Vittori 1,
Silva D. Neiens 4, Martin Steinhaus 4, Valtcho D. Zheljazkov 5, Filippo Maggi 1,*
and Giovanni Caprioli 1
1 School of Pharmacy, University of Camerino, via Sant’ Agostino 1, I-62032 Camerino (MC), Italy;
simone.angeloni@unicam.it (S.A.); gianni.sagratini@unicam.it (G.S.); sauro.vittori@unicam.it (S.V.);
giovanni.caprioli@unicam.it (G.C.)
2 International Hub for Coffee Research and Innovation, 62020 Belforte del Chienti (MC), Italy
3 School of Science and Technology, Chemistry Division, University of Camerino, V. S. Agostino 1,
I-62032 Camerino (MC), Italy; serena.scortichini@unicam.it (S.S.); dennis.fiorini@unicam.it (D.F.)
4 Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Lise-Meitner-Straße 34,
85354 Freising, Germany; silva.neiens@gmail.com (S.D.N.); martin.steinhaus@tum.de (M.S.)
5 Department of Crop and Soil Science, 431A Crop Science Building, 3050 SW Campus Way,
Oregon State University, Corvallis, OR 97331, USA; Valtcho.jeliazkov@oregonstate.edu
* Correspondence: filippo.maggi@unicam.it; Tel.: +39-073-740-4506
Academic Editor: Chiara Emilia Cordero
Received: 12 June 2020; Accepted: 29 June 2020; Published: 30 June 2020
Abstract: 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.
Keywords: coffee silverskin; GC-O; aroma; volatile compounds; bioactive compounds; SPME
1. 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 [2–7]. Among
these by-products is coffee silverskin (CS), which is the major residue generated during the roasting
Molecules 2020, 25, 2993 2 of 22
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 coffee cherry (1–2%), it contains high concentrations of dietary fiber (6880%)
and polysaccharides (6070%). The total sugars content in CS varies greatly (212%) and it contains
also fat (23%), protein (1619%) and ash (57%) [1]. In addition, some bioactive compounds, e.g.,
caffeine, chlorogenic acids and melanoidins, responsible for different biological activities including
antioxidant [5,9], occur in CS. Therefore, some authors proposed the use of CS as raw material for
the recovery of functional compounds of potential interest. Indeed, CS is a rich source of soluble and
insoluble dietary fibers (4 and 64%, respectively), which can be used for food enrichment [1]. Recent
studies demonstrated that CS could be a valuable source of bioactive compounds such as
melanoidins, caffeine and polyphenols, with potential applications of CS extracts as functional
ingredients in cosmetic and nutraceutical formulations [8,10]. Other authors have suggested a
potential use of this coffee residue as adsorbent material for removal of toxic metals from
contaminated water [11], as a source of cellulose for paper production [5], and as a prospective
ingredient in the food industry. Indeed, Martinez-Saez et al. [6] suggested the use of CS for the
production of a novel beverage to be used in body weight control.
Several studies have reported the nutritional composition of CS and the content of bioactive
compounds such as caffeine, chlorogenic acids, melanoidins and polyphenols [2,9,12]. Regarding
polyphenols, the majority of the previously conducted research estimated their content in CS by
measuring total phenolic and/or flavonoid content and none has investigated the individual
concentrations of different polyphenolic and other bioactive compounds. Moreover, different
authors have proposed innovative CS reuses and applications, but to the best of our knowledge, the
aroma of this coffee by-product has not been reported. Therefore, there is a dearth of investigation
focused on CS odorants which could be fascinating for food and food flavor industries. Furthermore,
the characterization of odor-active compounds may foster role in the development of novel foods.
Hence, the first objective of this research was to characterize the volatile fraction of CS employing
two different techniques, i.e., analysis of odor-active compounds by gas
chromatography-olfactometry/flame ionization detector (GC-O/FID) and analysis of headspace (HS)
volatiles by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). In
the first case, after fractionation of the volatile extracts, the proposed chemical structures of odorants
were confirmed by comparative analysis with reference compounds using comprehensive
two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) and the intensity of each
odor was studied by Aroma Extract Dilution Analysis (AEDA). The second objective of this study
was to provide an extensive characterization of the bioactive compounds present in CS. For this
purpose, different extraction procedures, (i.e., liquid–solid extraction assisted and not by sonication,
testing various solvents), were applied for the quantification of 30 bioactive compounds including
alkaloids, chlorogenic acids, phenolic acids, flavonoids and secoiridoids, by using high performance
liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Finally, the fatty acid
composition was also investigated by GC-FID analysis. The present research will contribute to
increase knowledge on volatile fraction, bioactive compound characterization and fatty acid
composition of CS and, therefore, may facilitate the development of innovative applications of this
product while reducing the amount of agricultural wastes that end up in landfills.
2. Results and Discussion
2.1. 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
Molecules 2020, 25, 2993 3 of 22
analysis of the concentrated volatile extracts and reference compounds using a second column with
different polarity (DB-5). 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).
(a)
(b)
Figure 1. The two plots report the 2D-GC-MS total ion current chromatograms (TICs) of a mixture of
thirteen reference compounds (a) and a sample of acidic volatiles (AV) fraction of coffee silverskin
(b). The regions highlighted in sky blue represented the identified compounds.
Molecules 2020, 25, 2993 4 of 22
2.2. Odor-Active Compounds in Coffee Silverskin (CS) by GC-O/FID and GC×GC-TOF Analysis
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.
Table 1. Odors and odorants described and identified in coffee silverskin (CS) and coffee beans (CB)
with their linear retention indices (LRI) and flavor dilution (FD) factors measured in DB-FFAP.
No.
Odorant
Odor
LRI
b
FD
c
FFAP
DB-5
CS
CB
1 2-/3-Methylbutanal a malty 953 659 4 64
2 2,3-Butanedione a butter-like 980 606 16 512
3 2,3-Pentanedione butter-like 1055 700 2 256
4 3-Methyl-2-buten-1-thiol a garlic-like, thiol 1097 819 8 1024
5 1-Octen-3-one a mushroom-like 1293 983 16 <1
6 unknown roasty, fatty 1298 - <1 256
7 2-Acetyl-1-pyrroline a roasty 1330 922 16 32
8 Dimethyl trisulfide spicy, cabbage, sulfurous 1364 967 16 128
9 2-Ethyl-5-Methylpyrazine roasty, nutty 1380 989 <1 128
10 2,3,5-Trimethylpyrazine fatty, roasty, earthy 1398 1007 8 128
11 unknown roasty, fatty 1402 - <1 256
12 2-Isopropyl-3-methoxypyrazine a green, earthy 1420 1093 32 2048
13 2-Furfurylthiol a pungent, coffee-like 1427 907 64 2048
14 3-Ethyl-2,5-dimethylpyrazine roasty, popcorn, earthy 1439 1084 8 1024
15 Acetic acid pungent, vinegar 1443 624 16 256
16 3-(Methylthio)propionaldehyde a cooked potato 1452 904 128 4096
17 2,3-Diethyl-5-methylpyrazine roasty, fatty 1480 1157 64 2048
18 2-Isobutyl-3-methoxypyrazine a green pea-like, green bell
pepper 1512 1177 128 4096
19 (E)-2-Nonenal greasy, green, roasty 1524 1157 64 256
20 3,7-Dimethylocta-1,6-dien-3-ol sweet, fruity, citrus 1536 1102 1 64
21 6-Acetyl-2,3,4,5-tetrahydropyridine a roasty, popcorn 1556 1054 <1 2048
22 unknown roasty, green 1572 - <1 2048
23 unknown fatty, roasty 1581 - 32 <1
24 Butanoic acid sweaty, cheese 1618 819 4 1024
25 2-Acetylpyrazine roasty 1625 1020 8 16
26 2-Acetylthiazole roasty 1638 1020 16 256
27 2-/3-Methylbutanoic acid cheese-like 1658 860 128 1024
28 unknown meaty, greasy 1693 1212 32 32
29 unknown silverskin-like 1718 - 512 512
30 unknown spicy 1724 - 32 256
31 2-Acetyl-2-thiazoline fatty, roasty 1750 1100 8 256
32 unknown caramel-like 1787 - <1 128
33 (E,E)-2,4-Decadienal meaty, gravy-like 1800 1315 64 1024
34 2-Hydroxy-3-methyl-2-cyclopenten-1-one spicy, burnt paper, smoky 1827 1029 8 512
35 unknown pungent, spicy, clove-like 1832 - 128 256
36 2-Methoxyphenol clove-like, phenolic 1854 1087 1024 16,384
37 2-Phenylethanol sweet, honey 1906 1111 4 <1
38 2-Phenyl-2-butenal green, phenolic 1926 1278 8 64
38 5-Methyl-2-Methoxyphenol a phenolic, clove-like 1937 1185 <1 256
40 unknown smoky 1957 1123 64 <1
41 3-Hydroxy-2-methyl-4-pyrone caramel-like 1969 1115 64 512
42 trans-4,5-Epoxy-(E)-2-decenal a metallic 1997 1377 256 512
Molecules 2020, 25, 2993 5 of 22
43 γ-Nonalactone fruity, coconut 2026 1364 64 1024
44 4-Hydroxy-2,5-dimethylfuran-3(2H)-one caramel-like 2032 1064 8192 16,384
45 unknown burnt paper, seasoning 2059 - 128 256
46 4-Methylphenol fecal, horse stable-like,
pee-like 2079 1080 16 256
47 4-Methyloctanoic acid a goaty, sheepy 2091 - 512 2048
48 γ-Decalactone a peach-like, lemon-like 2147 1471 32 <1
49 unknown rubber-like 2178 - 32 <1
50 3-Ethylphenol phenolic, leather-like 2181 1169 <1 512
51 2-Methoxy-4-vinylphenol clove-like 2197 1315 4096 8192
52 unknown seasoning, phenolic 2269 1357 128 <1
53 Indole fecal 2448 1298 8 64
54 3-Methylindole fecal 2493 1387 32 256
55 Phenylacetic acid honey 2555 1259 128 512
56 4-Hydroxy-3-methoxybenzaldehyde vanilla, chocolate-like 2572 1402 256 512
57 unknown saliva-like 2619 - 64 128
a GC×GC-MS analysis did not result in a clear mass spectrum, but comparison of linear retention
index and odor quality with respective data of an authentic reference compound allowed for
unequivocal structure assignment. b Experimental linear retention index (LRI), calculated according
to van Den Dool and Kratz [16]. c Only more powerful odors (unknown chemical structure) with, for
CS, FD > 16 and for CB, FD > 128 are reported.
A total of 40 odorants were identified in CS. The odorants with the highest FD factors were
4-hydroxy-2,5-dimethylfuran-3(2H)-one (furaneol) with 8192, 2-methoxy-4-vinylphenol
(4-vinylguaiacol), 4096, and 2-methoxyphenol (guaiacol), 1024. The furanone possessed caramel-like
notes, while the others were described as clove-like and phenolic. These volatiles were reported in
several studies as important odor-active compounds, which contribute to coffee flavor [17–21]. High
FD factors, from 512 to 128, were also found for 4-methyloctanoic acid, 512,
trans-4,5-epoxy-(E)-2-decenal, 256, 4-hydroxy-3-methoxybenzaldehyde (vanillin), 256,
3-(methylthio)propionaldehyde (methional), 128, 2-isobutyl-3-methoxypyrazine, 128,
2-/3-methylbutanoic acid, 128, and phenylacetic acid, 128. 4-Methyloctanoic acid is a
4-alkyl-branched-chain fatty acid (vBCFA). Such compounds are responsible for the goaty-sheepy
flavor of sheep and goat milk [22] and for the first time, we identified this fatty acid in coffee and
coffee products. trans-4,5-Epoxy-(E)-2-decenal is an important volatile compound associated with
metallic and blood-like odor; some behavioral studies reported that mammalian predators are as
attracted by this single volatile compound as they are by the odor of real blood [23,24]. It has been
identified in a coffee surrogate, namely chicory coffee [25] but, to the best of our knowledge, never in
coffee or coffee beverages. 4-Hydroxy-3-methoxybenzaldehyde, 3-(methylthio)propionaldehyde,
2-isobutyl-3-methoxypyrazine, 2-/3-methylbutanoic acid and phenylacetic acid are common
odorants reported in coffee beans and brews [17,19,26]. With FD factors from 64 to 16, fifteen
volatiles were identified in CS: 2-furfurylthiol, 2,3-diethyl-5-methylpyrazine, (E)-2-nonenal,
(E,E)-2,4-decadienal, 3-hydroxy-2-methyl-4-pyrone (maltol), γ-nonalactone,
2-isopropyl-3-methoxypyrazine, γ-decalactone, 3-methylindole (skatole), 2,3-butanedione,
1-octen-3-one, 2-acetyl-1-pyrroline, dimethyl trisulfide, acetic acid, 2-acetylthiazole and
4-methylphenol. 2-Furfurylthiol and 2-isopropyl-3-methoxypyrazine, are important volatiles in
coffee, which possess (1) coffee-like, roasty and pungent odor, and (2) green, earthy odor. [13,27].
Moreover, for 2-furfurylthiol a high Odor Activity Value (OAV) was reported in arabica and robusta
coffee. [18,27]. The other odor-active compounds, with FD factors 64-16, have already been described
in roasted beans and coffee beverages, except for γ-nonalactone. This lactone has never been
detected in those matrices but some studies reported its presence in green beans [28,29]. Volatiles
identified in CS with low FD factors (8-1) were 3-methyl-2-buten-1-thiol, 2,3,5-trimethylpyrazine,
3-ethyl-2,5-dimethylpyrazine, 2-acetylpyrazine, 2-acetyl-2-thiazoline,
2-hydroxy-3-methyl-2-cyclopenten-1-one, 2-phenyl-2-butenal, indole, 2-/3-methylbutanal, butanoic
Molecules 2020, 25, 2993 6 of 22
acid, 2-phenylethanol, 2,3-pentanedione, and 3,7-dimethylocta-1,6-dien-3-ol (linalool). All of these
volatiles have been identified in previous works on coffee [30].
2.3. Odor-Active Compounds in Coffee Beans (CB) by GC-O/FID and GC×GC-TOF Analysis and Comparison
with Coffee Silverskin (CS)
The GC-O/FID analysis of a concentrated volatile extract of coffee beans resulted in a large
number of odors (about 150, 1 ≤ FD ≤ 16,384) and almost 2.5 times more than those of CS (63 odors, 1
≤ FD ≤ 8192). For the direct comparison, it was necessary to keep the amount of the two matrices and
the solvent/sample ratio consistent during the extraction process. Therefore, the stepwise diluted
(1:2, 1:4, 1:8, 1:16) volatile extracts of CB were analyzed by GC-O/FID. A reasonable number of odors
was found in the sixteen times diluted sample (1:16) hence the same was chosen as starting point of
our studies. Our results showed that almost all odorants in CB occurred with higher FD factors than
in CS (Table 1). Some volatiles were identified only in CB, such as 2-ethyl-5-methylpyrazine,
6-acetyl-2,3,4,5-tetrahydropyridine, 5-methyl-2-methoxyphenol and 3-ethylphenol. To the best of
our knowledge, this is the first report on 5-methyl-2-methoxyphenol, a phenol derivate, and
6-acetyl-2,3,4,5-tetrahydropyridine, a pyridine derivate in CB. It has been reported that phenol
compounds can be formed during roasting from quinic and caffeic acid and maybe also
5-methyl-2-methoxyphenol was formed from these molecules. [31,32].
6-Acetyl-2,3,4,5-tetrahydropyridine is commonly present in the volatile fraction of baked products,
e.g., bread and pretzels, and possesses a roasty and popcorn odor [33,34]. 3-Ethylphenol was
described, after evaluation on the sniffing port, as phenolic, clove-like and has been already found in
coffee [30,35]. On the other hand, three odorants, such as 1-octen-3-one, 2-phenylethanol and
γ-decalactone were identified only in CS. The first odorant was described as mushroom-like, the
second possessed sweet, honey-like notes and the third was described as peach-like and lemon-like.
The most intensive odors in CB, in terms of FD factor were: 2-methoxyphenol, 16,384,
4-hydroxy-2,5-dimethylfuran-3(2H)-one, 16,384, and 2-methoxy-4-vinylphenol, 8192. These
molecules were the most intense in CS as well. Other thirteen identified compounds occurred with
high FD factors (from 1024 to 4096): 3-methyl-2-buten-1-thiol, 3-ethyl-2,5-dimethylpyrazine,
butanoic acid, 2-/3-methylbutanoic acid, (E,E)-2,4-decadienal, γ-nonalactone,
2-isopropyl-3-methoxypyrazine, 2-furfurylthiol, 2,3-diethyl-5-methylpyrazine,
6-acetyl-2,3,4,5-tetrahydropyridine, 4-methyloctanoic acid, 3-(methylthio)propionaldehyde and
2-isobutyl-3-methoxypyrazine. Among these, 3-methyl-2-buten-1-thiol,
3-ethyl-2,5-dimethylpyrazine, 6-acetyl-2,3,4,5-tetrahydropyridine and butanoic acid may account for
more intense roasty and popcorn aromas and also notes of thiol-like, and cheese-like in CB, since the
ratio of CB and CS flavor dilution factors for the above-mentioned molecules were 128, 128, >2048,
and 256, respectively. Other identified odorants with high FD factors (from 256 to 512) were
2,3-pentanedione, acetic acid, (E)-2-nonenal, 2-acetylthiazole, 2-acetyl-2-thiazoline,
5-methyl-2-methoxyphenol, 4-methylphenol, 3-methylindole, 2,3-butanedione,
2-hydroxy-3-methyl-2-cyclopenten-1-one, 3-hydroxy-2-methyl-4-pyrone,
trans-4,5-epoxy-(E)-2-decenal, 3-ethylphenol, phenylacetic acid, and
4-hydroxy-3-methoxybenzaldehyde. The FD factor ratio of CB and CS demonstrated that butter-like
2,3-pentanedione, (ratio: 128), spicy and smoky 2-hydroxy-3-methyl-2-cyclopenten-1-one, (ratio: 64),
phenolic and clove-like 5-methyl-2-methoxyphenol, (ratio >256), and phenolic and leather-like
3-ethylphenol (>512) were more intense in CB than in CS. In contrast, some odorants including two
important actors of coffee flavor, were found with similar FD factors in both matrices:
4-hydroxy-2,5-dimethylfuran-3(2H)-one, 2-methoxy-4-vinylphenol,
4-hydroxy-3-methoxybenzaldehyde, trans-4,5-epoxy-(E)-2-decenal, 2-acetyl-1-pyrroline and
2-acetylpyrazine. In conclusion, our study revealed a potent odorant fraction in CS and therefore,
this co-product can be considered as source of interesting and pleasant aroma and could be
exploited in the food and other industries.
Molecules 2020, 25, 2993 7 of 22
2.4. Volatile Substances Composition by HS-SPME-GC-MS Analysis
This study aimed to determine for the first time the volatile profile of CS. The main volatile
substances detected by HS-SPME-GC-MS are presented in Table 2.
Table 2. Volatile compounds detected in coffee silverskin (CS) by HS-SPME-GC-MS; their
experimental linear retention indices (LRI) on a polyethyleneglycol coated column, their abundances
in terms of peak areas percentages, and their relative standard deviations (RSD %, n = 3).
Compound Detected a
LRI
b
(exptl)
LRI
c
(lit) Area % RSD %
2-Methylbutanal 903 910 1.44 14.77
3-Methylbutanal 907 913 2.86 12.76
1-Chloropentane 927 941 0.74 10.22
2-Ethylfuran 938 951 0.22 13.26
Pentanal 966 971 1.56 5.18
Hexanal 1069 1077 8.47 2.35
1-(1-Cyclohexen-1-yl)-ethanone 1109 / 0.25 6.61
3-Methyl-1-pentene 1116 / 0.22 22.82
Heptanal 1173 1179 1.68 0.45
D-Limonene d 1180 1190 0.87 12.27
2-Methyl-2-butenal 1189 1129 0.25 10.43
2-Hexenal 1208 1209 0.65 18.39
2-Pentylfuran 1218 1230 1.80 10.33
Styrene d 1240 1247 0.79 16.55
1-Pentanol 1252 1255 0.73 11.35
2,6-Dimethylpyridine 1255 1248 0.59 19.12
Methylpyrazine 1259 1262 0.41 8.88
1-Octen-3-one 1292 1298 0.46 13.78
(E)-2-Heptenal 1314 1318 1.73 2.66
4,6-Dimethylpyrimidine 1318 1363 0.12 11.91
3-Methyl-2-buten-1-ol 1321 1317 0.25 7.30
2-Heptanol 1323 1318 0.42 7.86
2,6-Dimethylpyrazine 1324 1330 0.55 0.37
Ethylpyrazine 1327 1338 0.26 11.96
6-Methyl-5-hepten-2-one 1329 1333 1.01 9.79
2,3-Dimethylpyrazine 1342 1345 0.31 14.72
1-Hexanol 1355 1352 0.33 7.73
2-Ethyl-5-methylpyrazine 1381 1382 0.72 1.48
2-Ethyl-6-methylpyrazine 1388 1389 0.87 12.52
2-Ethyl-3-methylpyrazine 1401 1406 0.32 9.68
Trimethylpyrazine 1403 1404 0.22 5.32
(Z)-3-Ethyl-2-methyl-1,3-hexadiene 1405 / 0.22 3.04
(E)-2-Octenal 1422 1425 1.78 2.99
Acetic acid 1441 1431 3.68 12.88
trans-Linalool oxide d 1443 1460 7.15 4.29
Furfural 1450 1452 5.56 3.57
4-Ethenyl-1,4-dimethylcyclohexene 1456 / 0.46 15.96
cis-Linaloloxide d 1471 1475 2.17 6.24
Formic acid 1492 1492 1.11 16.27
Benzaldehyde 1509 1520 4.86 2.33
Furfuryl acetate 1530 1539 0.24 2.79
Propanoic acid 1536 1535 1.15 1.03
3,7-Dimethylocta-1,6-dien-3-ol 1555 1554 0.41 1.34
5-Methyl-2-furfural 1570 1579 4.45 0.95
3-Furoic acid 1572 / 0.34 5.78
3-Methoxypyridine 1584 1581 0.33 11.85
6-Methyl-3,5-heptadiene-2-one 1591 1590 1.08 4.74
3-Methyl-2-cyclohexen-1-one 1593 1592 0.76 5.61
Molecules 2020, 25, 2993 8 of 22
1,5-Dimethyl-2-pyridone 1600 / 0.65 3.61
2-Acetyl-5-methylfuran 1607 1608 0.59 10.28
Pyrrole-2-carboxaldehyde 1611 1610 1.88 5.62
Butyrolactone 1620 1618 0.54 7.11
Butanoic acid 1625 1628 0.38 6.54
Acetophenone 1645 1640 1.89 2.59
2-Furanmethanol 1659 1662 1.86 2.46
3-Methylbutanoic acid 1668 1665 3.55 13.46
1-Adamantol d 1682 1661 0.61 2.15
2,6,6-Trimethyl-2-cyclohexene-1,4-dione 1690 1680 0.51 7.05
2-Methoxypyrimidine 1709 / 0.35 14.04
5-Methyl-2-furanmethanol 1719 1720 0.24 18.74
3,4-Dimethyl-2,5-furandione 1725 / 0.52 18.53
Pentanoic acid 1734 1733 1.32 3.97
3,5,5-Trimethylcyclohexene 1744 / 0.30 15.36
Methyl salycilate d 1770 1771 0.52 8.45
1-(4-Methylphenyl)-ethanone 1773 1771 1.16 8.63
3-Methyl-2-butenoic acid 1789 1802 0.71 0.57
4-Methyl-pentanoic acid 1797 1795 0.22 13.01
Phenylethyl acetate 1813 1807 0.26 10.02
1-Furfurylpyrrole 1822 1817 0.26 7.75
Hexanoic acid 1841 1846 4.06 8.01
2-Methoxyphenol 1858 1853 0.73 9.51
Benzyl alcohol 1877 1879 1.13 8.87
Phenylethyl Alcohol 1916 1913 3.64 5.84
Heptanoic acid 1946 1942 0.72 4.78
1-(1H-pyrrol-2-yl)-ethanone 1974 1972 1.09 5.76
Phenol 1996 1999 0.48 8.31
1H-Pyrrole-2-carboxaldehyde 2028 2032 1.26 11.98
Octanoic acid 2051 2060 0.75 15.38
2-Methylphenol 2077 2060 0.26 3.61
4-Methylphenol 2085 2079 0.25 10.72
Nonanoic acid 2158 2168 0.45 9.43
a Compounds reported are those which had peak area values higher than 500,00. b Experimental
linear retention index calculated according to van Den Dool and Kratz [16]. c Linear retention indices
reported in literature (NIST 2017). d D-Limonene, (R)-4-isopropenyl-1-methyl-1-cyclohexene;
Styrene, ethenylbenzene; trans-Linalool oxide,
trans-2-methyl-2-vinyl-5-(1-hydroxy-1-methylethyl)tetrahydrofuran; cis-Linaloloxide,
5-(3,3-dimethyloxiran-2-yl)-3-methylpent-1-en-3-ol; 1-Adamantol, tricyclo[3.3.1.1(3,7)]decan-1-ol;
Methyl salycilate, methyl 2-hydroxybenzoate.
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
Molecules 2020, 25, 2993 9 of 22
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].
2.5. 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.
Table 3. Average fatty acid % composition ± standard deviation (n = 3) of coffee silverskin (CS)
lipids.
Fatty Acid Composition (%)
C14:0 1.28 ±0.03
C15:0 0.21 ± 0.01
C16:0 35.64 ± 0.19
C17:0 0.14 ± 0.01
C18:0 6.38 ± 0.09
Molecules 2020, 25, 2993 10 of 22
C18:1 n-9 5.77 ± 0.10
C18:1 c-11 0.64 ± 0.01
C18:2 n-6 27.62 ± 0.10
C18:3 n-6 0.75±0.01
C18:3 n-3 0.88 ± 0.04
C20:0 8.62 ± 0.04
CLA a 0.33 ± 0.01
C22:0 10.70 ± 0.06
C24:0 1.03 ± 0.03
SFA 64.01 ± 0.12
MUFA 6.41 ± 0.08
PUFA 29.58 ± 0.04
a Conjugated Linoleic Acid (C18:2, c-9, t-11).
Palmitic acid (C16:0) was the main fatty acid found (36%), followed by linoleic acid (C18:2 n-6,
28%), behenic acid (C22:0, 11%), and arachidic acid (C20:0, 9%). This profile is in agreement with the
results obtained by Costa et al. [41]. In general, CS contained mainly saturated fatty acids (64%),
followed by polyunsaturated (30%) and monounsaturated (6%) ones. A high concentration of SFAs
together with a significant amount of phenolic compounds could limit the CS degradation due to
lipid oxidation. The high content of linoleic acid (27.6 ± 0.10%), which is the second most abundant
FA, could have positive effects on HDL cholesterol concentrations, reducing the risk of
cardiovascular diseases [12].
2.6. 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/H2O and H2O, 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).
Table 4. Contents (mg kg−1) of bioactive compounds in CS extracted with eight processes.
Extraction Solvents a
No Analytes b MeOH H2O MeOH/
H2O
EtOH/
H2O MeOH pH 2 EtOH MeOH/
EtOH H2O c
1 Shikimic acid 0.328 0.520 0.390 0.502 0.323 0.198 0.273 0.252
2 Gallic acid 0.209 0.310 0.223 0.250 0.182 0.190 0.174 0.161
3 Loganic acid n.d. d n.d. n.d. n.d. n.d. n.d. n.d. n.d.
4
5-CQA
940.420
940.400
961.800
985.705
854.909
903.109
910.350
900.785
5 Swertiamarin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
6 Gentiopicroside n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
7 (+)-Catechin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
8 Del-3,5-diglu n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
9 Sweroside n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
10 3-CQA 112.530 100.800 111.960 128.512 100.636 105.027 102.275 101.869
11 Caffeine 761.800 845.280 785.280 845.516 768.436 800.727 731.500 817.385
12
Cya-3-glu
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
13 Vanillic acid 1.410 0.880 1.090 1.472 1.320 1.364 1.342 1.138
14 Caffeic acid 1.250 0.858 1.030 1.361 1.420 1.127 1.125 1.038
15 (−)-Epicatechin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
16 Syringic acid 0.203 0.094 0.113 0.295 0.356 0.184 0.169 0.156
Molecules 2020, 25, 2993 11 of 22
17 p-Coumaric acid 0.128 0.100 0.120 0.150 0.221 0.116 0.107 0.098
18 Ferulic acid 0.183 0.152 0.183 0.204 0.139 0.166 0.152 0.141
19 3,5-diCQA 27.480 21.007 25.208 41.489 19.097 22.982 22.900 20.138
20 Quinine n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
21 Naringin n.d. 0.002 0.003 0.009 0.034 n.d. n.d. n.d.
22 Rutin 0.021 0.042 0.051 0.024 0.039 0.019 0.017 0.016
23
Hyperoside
0.004
0.010
0.012
0.003
0.009
0.004
0.003
0.003
24 Trans-cin acid 0.066 0.055 0.066 0.066 0.082 0.060 0.055 0.050
25 Resveratrol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
26 Amarogentin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
27 Kae-3-glu 0.044 0.037 0.045 0.052 0.069 0.040 0.037 0.034
28 Quercitrin 0.002 0.002 0.002 0.003 0.032 0.002 0.002 0.002
29 Quercetin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
30 Isogentisin n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Tot bio compounds
1846.079
1910.549
1887.576
2005.613
1747.304
1835.315
1770.481
1843.266
a Each sample was analyzed in triplicate (n = 3) and RSD values were from 2.3 to 9.8%. b Del-3,5-diglu,
Delphinidin-3,3-diglucosiede; Cya-3-glu, Cyanidin-3-glucoside; Trans-cin acid, Trans-cinnamic acid;
Kae-3-glu, Kaempferol-3-glucoside; Tot bio compounds, Total bioactive compounds. c Liquid–solid
extraction without sonication. d n.d., not detectable.
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.
Molecules 2020, 25, 2993 12 of 22
3. Materials and Methods
3.1. Reagents and Standards
Cyanidin-3-glucoside chloride, delphinidin-3,5-diglucoside chloride, and
kaempferol-3-glucoside were purchased from PhytoLab (Vestenbergsgreuth, Germany). The other
27 analytical standards of bioactive compounds were supplied by Sigma-Aldrich (Milan, Italy).
Individual stock solutions of each analyte, at a concentration of 1000 mg L−1, were prepared by
dissolving pure standard compounds in HPLC-grade methanol and storing them in glass-stoppered
bottles at 4 °C. Afterwards, standard working solutions at various concentrations were prepared
daily by appropriate dilution of the stock solution with methanol. HPLC-grade formic acid 99−100%
was purchased from Merck (Darmstadt, Germany) and hydrochloric acid (HCl) 37% from Carlo
Erba Reagents (Milan, Italy). HPLC-grade acetonitrile and methanol were supplied by
Sigma-Aldrich (Milano, Italy). Deionized water was obtained from a Milli-Q Reagent Water System
(Bedford, MA, USA). All other solvents and chemicals were analytical grade. All solvents and
solutions for HPLC-MS/MS were filtered through a 0.2 µm polyamide filter from Sartorius Stedim
(Göttingen, Germany). Before HPLC analysis, all samples were filtered with Phenex™ RC 4 mm 0.2
µm syringeless filter, Phenomenex (Castel Maggiore, Italy). Chloroform (CAS: 67-66-3) and sodium
chloride (CAS: 7647-14-6) were purchased from Carlo Erba (Milan, Italy); while methanol (CAS:
67-56-1) was purchased from Fisher Scientific (Leicestershire, UK). Pure potassium chloride (CAS:
7447-40-7) was obtained from PanReac Quimica (Barcelona, Spain). Pure sodium chloride (CAS:
7647-14-5) was purchased by Carlo Erba (Milan, Italy). Anhydrous sodium sulphate (CAS:
7757-82-6) was purchased from Sigma-Aldrich (Milan, Italy). The potassium hydroxide
(CAS:1310-58-3) was obtained from ProLabo (Fontenay-sous-Bois, France) and the Supelco 37
components, FAME Mix was purchased from Sigma-Aldrich (Milan, Italy).
4-Hydroxy-3-methoxybenzaldehyde was purchased from Merck (Darmstadt, Germany),
2-methoxy-4-vinylphenol from Alfa Aesar (Karlsruhe, Germany), while other reference compounds
of odor-active molecules were purchased from Sigma Aldrich (Taufkirchen, Germany).
Dichloromethane, diethyl ether, and pentane were freshly distilled before use. Silica gel 60
(0.040−0.063 mm) was purchased from VWR (Darmstadt, Germany) and purified as described
previously [48]. Mercurated agarose gel was prepared from Affi-Gel 10 (Bio-Rad, Munich, Germany)
[49]. All other chemicals were analytical grade.
3.2. 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.
Molecules 2020, 25, 2993 13 of 22
3.3. 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.
3.4. 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).
Molecules 2020, 25, 2993 14 of 22
3.5. 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).
3.6. 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 minutes into
the hot injector (260 °C) of a 6850 gas chromatograph (Agilent, Santa Clara, United States). 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.
3.7. 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.
3.8. 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
Molecules 2020, 25, 2993 15 of 22
mixture was vortexed for 2 minutes and centrifuged for 5 minutes (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].
3.9. 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.
3.9.1. Ultrasound Assisted Extractions (UAE)
10 g of CS powder were extracted with 100 mL of solvent using FALC ultrasonic bath (FALC,
Treviglio, Italy) at a frequency of 40 KHz for 120 minutes at 20 °C. Seven solvents such as MeOH
(Method 1), H2O (Method 2), MeOH/H2O (50/50, v/v) (Method 3), EtOH/H2O (70/30, v/v) (Method 4),
MeOH pH 2 (Method 5), EtOH (Method 6), EtOH/MeOH (50/50, v/v) (Method 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.
3.9.2. Liquid–Solid Extraction (LSE)
The extraction of 10 g of CS powder was performed with 100 mL of H2O (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.
3.10. 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
Molecules 2020, 25, 2993 16 of 22
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.
Molecules 2020, 25, 2993 17 of 22
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 (Δ Rt) for each transition.
No. Compounds Precursor ion
(m/z)
Product Ion
(m/z)
Fragmentor
(V)
Collision
Energy
(V)
Polarity Retention Time
(Rt) (min)
Delta
Retention
Time (Δ Rt)
1 Shikimic acid 173 173 87 0 Negative 1.40 3
- - - -
2 Gallic acid 169 125 a 92 12 Negative 2.37 3
51 36
3 Loganic acid 375 213 a 126 8 Negative 3.13 3
113 16
4 5-Caffeoylquinic acid 353 191 a 102 12 Negative 3.58 3
179 12
5 Swertiamarin 419 179 a 100 4 Negative 4.89 3
89 16
6 Gentiopicroside 357 177 a 50 10 Positive 5.33 3
73 28
7 (+)-Catechin 289 245 a 121 8 Negative 5.48 3
109 24
8 Delphinidin-3,5-diglucoside 463 300 a 165 24 Negative 5.64 3
271 48
9 Sweroside 403 125 a 102 12 Negative 5.95 3
179 4
10 3-Caffeoylquinic acid 353 191 a 92 12 Negative 6.22 3
85 48
11 Caffeine 195 138 a 107 20 Positive 6.50 3
110 24
12 Cyanidin-3-glucoside 449 287 a 121 20 Positive 6.50 3
403 16
13 Vanillic acid 167 108 a 78 16 Negative 6.70 3
152 8
14 Caffeic acid 179 135 a 87 12 Negative 6.87 3
134 24
15 (−)-Epicatechin 289 245 a 126 8 Negative 7.03 3
109 20
16 Syringic acid 197 182 a 92 8 Negative 7.48 3
Molecules 2020, 25, 2993 18 of 22
123 20
17 p-Coumaric acid 163 119 a 83 12 Negative 8.47 3
93 32
18 Ferulic acid 193 134 a 88 12 Negative 9.16 3
178 8
19 3,5-Dicaffeoylquinic acid 515 353 a 117 8 Negative 9.82 3
191 28
20 Quinine 325 79 a 135 44 Positive 10.1 5
81 32
21 Naringin 579 271 a 210 32 Negative 10.17 3
151 48
22 Rutin 609 300 a 195 40 Negative 10.34 3
271 50
23 Hyperoside 463 300 a 160 24 Negative 10.43 3
271 44
24 Trans-cinnamic acid 149 131 a 44 8 Positive 10.79 3
77 36
25 Resveratrol 227 185 a 131 12 Negative 10.92 3
143 20
26 Amarogentin 585 227 a 145 16 Negative 11.05 3
245 16
27 Kaempferol-3-glucoside 447 284 a 163 24 Negative 11.24 3
227 50
28 Quercitrin 447 300 a 155 24 Negative 11.24 3
301 16
29 Quercetin 301 151 a 126 16 Negative 13.03 3
179 12
30 Isogentisin 257 242 a 116 16 Negative 16.31 3
214 24
a These product ions were used for quantification, the others to confirm the analytes.
Molecules 2020, 25, 2993 19 of 22
4. 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.
Author Contributions: Conceptualization, F.M. and G.C.; methodology, S.A. and S.S.; validation, S.A.; D.F. and
S.S.; investigation, S.A. and G.C.; resources, G.C.; data curation, G.S., S.V., S.D.N. and M.S.; writing—original
draft preparation, S.A., G.S. and D.F.; writing—review and editing, F.M., S.V., M.S., G.C., and V.D.Z.;
supervision, M.S., S.D.N. and G.C.; funding acquisition, G.C. and S.V. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the University of Camerino (Fondo di Ateneo per la Ricerca – Year 2018,
Grant no. FPI000051) assigned to Dr. Giovanni Caprioli.
Acknowledgments: The authors are grateful to Simonelli Group S.p.A. (Belforte del Chienti, Macerata, Italy)
for partial economic support of fellowship for S A and to Perfero Caffè (Altidona, Italy) roasting company for
kindly provided samples.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open
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