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CQ-VR, Chemistry Research Centre, Department of Chemistry, Universidade de Tras-os-Montes e Alto Douro, 5000-801 Vila Real,
Portugal
ABSTRACT: Phenolic compounds are incorporated into coee melanoidins during roasting mainly in condensed form (4262
mmol/100 g) and also in ester-linked form (1.11.6 mmol/100 g), with incorporation levels depending on the green coee
chlorogenic acid content. The phenolic compounds are incorporated in dierent coee melanoidin populations, but mainly in
those soluble in 75% ethanol (82%), a signicant correlation between the amount of phenolic compounds and the amount of
protein and color characteristics of the dierent melanoidin populations being observed. The incorporation of phenolic
compounds into coee melanoidins is a signicant pathway of chlorogenic acid degradation during roasting, representing 23% of
the chlorogenic acids lost. These account for the nearly 26% of the material not accounted for by polysaccharides and proteins
present in coee melanodins. The cleavage mechanism and the eciency of alkaline fusion used to release condensed phenolics
from coee melanoidins suggest that the phenolic compounds can be linked to the polymeric material by aryl-ether, stilbene type,
and/or biphenyl linkages.
KEYWORDS: coee, melanoidins, chlorogenic acids, phenolic, roasting, infusions
INTRODUCTION
Received:
Revised:
Accepted:
Published:
7843
March
July 3,
July 5,
July 5,
31, 2014
2014
2014
2014
Article
b*
h* = tan1
a*
C* = (a*2 + b*2 )1/2
E* = [(L*)2 + (a*)2 + (b*)2 ]1/2
Total Phenolic Groups Content. The total phenolic groups
content of the coee samples was determined with the Folin
Ciocalteu reagent.4 For all coee samples, a 0.33 or 1 mg/mL solution
was prepared. To 1 mL of the coee sample solution was added 0.5
mL of FolinCiocalteu reagent. After mixing of the sample, 1 mL of a
saturated Na2CO3 solution was added, and ltered demineralized
water was added to reach a total volume of 10 mL. After 1 h of
reaction, the absorbance of the sample was measured at 725 nm. 5CQA was used as reference.
Adsorbed Phenolic Compounds. For determination of coee
melanoidins adsorbed phenolic compounds, a 5 mg/mL solution of
coee melanoidins was analyzed by direct injection (100 L) on a RPHPLC according to the method described previously for CGA
determination. For determination of the eect of melanoidins in the
determination of free phenolic compounds, melanoidin solutions (5
mg/mL) were spiked with dierent concentrations of 5-CQA and
incubated overnight at room temperature. Each solution was analyzed
by RP-HPLC as described previously, and the areas were compared
with that of aqueous solutions of 5-CQA with the same
concentrations.
Alkaline Saponication. For determination of ester-linked
phenolic compounds, melanoidins were subjected to alkaline
saponication by using a procedure that prevented oxidation of
phenolic compounds.24 To 750 L of the melanoidin solution (12
mg/mL) was added 750 L of a 2 M NaOH solution containing 2%
(w/w) ascorbic acid and 20 mM ethylenediaminetetraacetic acid. After
incubation for 1 h at 30 C, the mixture was quenched to pH 1 with
330 L of 5 M HCl to precipitate most of the coee material,
preventing precipitation during further analysis. The mixture was
stored for 2 h at 4 C, the precipitate was removed by centrifugation,
and the supernatant was analyzed by reversed-phase HPLC.
Experiments were performed at least in duplicate. The identication
of caeic, ferulic, and 4-hydroxycinnamic acids was made by
comparison of their retention times and UVvis spectra with that of
pure standards analyzed under the same conditions. Caeic, ferulic,
and 4-hydroxycinnamic acids were quantied by external calibration.
Alkaline Fusion. In a nickel crucible were weighed 1 g of solid
NaOH and 100 mg of zinc dust, and, after fusion of the mixture at 350
C,6,25 5 mg of HMWM was added (or 5 mg of melanoidin fractions).
After 10 s, the nickel crucible was removed and rapidly cooled on ice.
The fusion cake was solubilized by adding 6 M HCl, and 200 L of
internal standard solution was added (3,4-dimethoxybenzoic acid, 1
mg/mL) and acidied to pH 12 with 6 M HCl. The acidic mixture
was extracted four times with 30 mL of diethyl ether. After evaporation
of the organic solvent, carefully and with protection, the residue was
derivatized and analyzed by GC-EI/MS. Samples were derivatized with
0.5 mL of pyridine and 0.5 mL of N,O-bis(trimethylsilyl)triuoroacetamide and heated at 70 C for 30 min. After cooling to
room temperature, the silyl derivatives of phenolic compounds were
7844
Article
Table 1. Soluble Sugar, Caeine, and Protein Contents (Grams per 100 g, Dry Basis) of Original, Control, and in Bean Green
and Roasteda Coee Models
coee
green
original
control
CGAcoee
roasted
original
control
CGAcoee
a
glucose
fructose
sucrose
total
caeine
proteinb
0.018 0.001
0.018 0.001
0.017 0.001
0.60 0.00
0.59 0.00
0.58 0.00
7.45 1.32
5.78 1.10
5.08 0.98
8.07 1.32
6.39 1.10
5.69 0.99
1.37 0.01
1.12 0.02
1.07 0.05
14.0 0.9
14.1 0.7
14.0 1.1
0.018 0.001
0.018 0.001
0.017 0.001
0.008 0.002
0.019 0.001
0.032 0.025
0.64 0.06
0.99 0.02
3.07 0.07
0.66 0.07
1.07 0.05
3.16 0.02
1.42 0.15
1.23 0.12
1.11 0.07
9.5 0.7
12.3 0.9
11.8 0.5
Not corrected for weight loss during roasting. b(Ntotal Ncaffeine) 6.25.
Table 2. Chlorogenic Acid (CGA) Composition of Original, Control, and in Bean Green and Roasteda Coee Model (Grams
per 100 g, Dry Basis)
green
CGA
control
roasted
CGAcoee
original
3-CQA
5-CQA
4-CQA
total CQA
0.53
3.36
0.007
3.90
0.02
0.02
0.001
0.03
0.44
2.92
0.001
3.37
0.03
0.17
0.001
0.20
0.44
3.34
0.006
3.79
0.01
0.20
0.002
0.20
0.48
1.04
0.17
1.70
1-FQA
3-FQA
5-FQA
4-FQA
total FQA
0.01
0.70
0.05
0.003
0.77
0.00
0.05
0.01
0.005
0.06
0.01
0.60
0.04
0.003
0.65
0.00
0.07
0.00
0.004
0.07
0.01
0.63
0.04
0.003
0.68
0.00
0.05
0.00
0.004
0.06
0.01 0.00
0.76 0.02
nd
nd
0.77 0.02
3-CoQA
5-CoQA
4-CoQA
total CoCQA
nd
0.007 0.001
0.04 0.02
0.05 0.02
3-CQL
4-CQL
3-FQL
4-FQL
1-FQL
total FQL
original
nd
nd
nd
nd
nd
nd
nd
0.008 0.003
0.03 0.02
0.04 0.02
nd
nd
nd
nd
nd
nd
nd
0.006 0.002
0.03 0.02
0.03 0.02
nd
nd
nd
nd
nd
nd
0.04
0.10
0.03
0.16
nd
nd
nd
nd
0.04
0.09
0.01
0.02
0.04
0.08
control
0.43
1.12
0.20
1.75
0.02
0.07
0.02
0.11
0.01 0.00
0.69 0.04
nd
nd
0.69 0.04
nd
nd
nd
nd
0.02
0.03
0.00
0.01
0.01
0.02
0.04
0.09
0.01
0.02
0.06
0.09
CGAcoee
0.53
1.07
0.11
1.72
0.00
0.02
0.00
0.02
0.01 0.00
0.88 0.01
nd
nd
0.89 0.01
nd
nd
nd
nd
0.00
0.01
0.00
0.00
0.00
0.01
0.04
0.09
0.04
Nd
0.03
0.07
0.00
0.00
0.05
0.03
0.02
3,4-diCQA
3,5-diCQA
total diCQA
0.18 0.00
0.30 0.01
0.48 0.01
0.16 0.02
0.26 0.05
0.42 0.07
0.31 0.01
0.48 0.01
0.79 0.02
0.01 0.00
0.07 0.01
0.08 0.01
0.01 0.00
0.07 0.01
0.08 0.01
nd
0.06 0.02
0.06 0.02
total (CGA)
5.19 0.13a
4.48 0.09ab
5.43 0.20bc
2.53 0.19
2.52 0.16
2.67 0.00
Not corrected for weight loss during roasting. Identical letters in the same row are signicantly dierent.
Article
Table 3. Total Sugara Composition (Grams per 100 g, Dry Basis) of Original, Control, and in Bean Green and Roasted Coee
Modelsb
green
original
control
CGAcoee
ANOVA
roasted
original
control
CGAcoee
ANOVA
a
Ara
Man
1.71 0.04
1.86 0.81
2.12 0.44
0.758
22.7 0.2
26.3 7.9
30.8 2.2
0.359
1.62 0.20
1.99 0.27
1.92 0.05
0.276
18.0 2.5
20.3 2.2
20.6 0.4
0.444
Gal
12.7 0.2
13.2 2.2
14.7 0.7
0.412
7.61 1.01
8.86 1.18
9.37 0.07
0.276
Glc
Glcpolymeric
total
totalpolymeric
11.2 1.0
9.81 3.58
10.3 1.3
0.835
7.61
6.60
7.44
48.3 1.1
51.2 4.5
57.9 4.6
0.169
44.7
48.0
55.0
8.41 1.23
9.30 0.80
10.2 0.2
0.259
8.07
8.75
8.60
35.7 5.0
40.4 4.4
42.0 1.1
0.370
35.4
39.9
40.4
Values expressed in anhydrosugar residues. bNot corrected for weight loss during roasting.
7847
18.4
3.86
1.44
0.26
2.16
14.9
3.73
22.5
2.39
37.3
6.7
56.0
32.7
16.8
50.5
34.8
12.5
52.7
g/100 g HMWM
1.3b
0.1
3.8
1.8
0.3a
0.9
0.6
1.2
0.0121
5.94 0.6ab
1.70 0.4
11.3 1.7
13.2 0.9
11.5
2.7
19.2
16.7
11.1
3.9
14.2
16.8
Ara
0.234
20.1
38.7
11.1
3.5
17.1
34.9
7.9
4.0
20.2
30.1
11.6
5.5
Man
2.0
9.2
1.9
0.2
1.9
1.1
1.7
0.4
0.3
3.0
0.2
0.4
0.7a
2.0
2.6
1.2
1.3ab
0.8
3.7
0.3
0.0296
12.5
3.5
25.3
14.8
17.2 1.9b
4.85 0.22
30.4 5.5
20.4 2.2
19.8
9.4
32.8
23.8
Gal
0.15ab
1.27
0.08
0.05
0.09a
0.09
0.07
1.48
0.0277
ab
1.02 0.28
1.13 0.31
0.87 0.48
0.45
1.54
0.49
0.44
0.50
0.53
0.50
1.63
Glc
4.0
10.1
7.6
1.5
5.2
0.2
11.0
4.4
1.3
0.3
3.4
4.4
0.0940
38.6
44.9
48.7
32.3
46.2
44.0
57.9
41.6
51.6
44.0
59.1
47.7
total
1.0ab
0.4
0.5
0.7
0.8ab
0.2
0.5
0.3
0.3a
0.1
0.5
0.4
0.0030
18.9
4.8
14.7
27.9
14.1
5.2
12.3
14.6
9.9
2.3
12.1
14.3
protein
Phenolics measured by the FolinCiocalteu method and expressed as 5-CQA equivalents. Identical letters in the same column are signicantly dierent.
ANOVA
original
soluble solids
HMWM
Et50
Et75
EtSn
control
soluble solids
HMWM
Et50
Et75
EtSn
CGAcoee
soluble solids
HMWM
Et50
Et75
EtSn
g/100 g coee
0.03
0.05a
0.01
0.02
0.11
0.08
0.00ab
0.03
0.02
0.19
0.00
0.15b
0.09
0.13
0.07
0.0234
0.70
1.27
0.56
0.88
1.27
0.74
0.89
0.89
0.56
1.07
0.75
1.40
0.85
0.82
1.37
Kmix405 nm
2.99
1.11
2.40
3.19
2.24
1.75
1.88
2.44
3.63
1.58
2.85
3.61
MBI
0.049
6.19
1.55
3.13
8.20
4.50
1.57
1.93
7.28
4.59
1.16
1.69
5.36
0.36ab
0.16
0.27
0.45
0.61b
0.04
0.13
0.18
0.24a
0.10
0.20
0.33
phenolicsa
Table 4. Yield and Chemical Composition (Grams per 100 g, Dry Basis) of the High Molecular Weight and Resulting Ethanol-Precipitated Fractions of the Original, Control,
and in Bean Coee Models
Article
Ester-Linked Phenolic Compounds. The presence of esterlinked phenolic compounds in the HMWM from original,
control, and CGAcoee was determined after saponication.
This procedure, known to release ester-linked phenolic
compounds, 33 allowed the release of simple phenolic
compounds from the HMWM, including caeic, ferulic, and
coumaric acids (Figure 2). Caeic acid was the main cinnamic
acid detected in all analyzed HMWMs. These simple phenolic
compounds released by alkaline saponication have their origin
in the adsorbed CGA present but also, probably, in the CGA
incorporated into melanoidins, with structures where the
cinnamic acids are esteried and maintain unchanged their
structure. The amount of phenolic compounds released by
saponication ranged from 1.1 to 1.6 mmol/100 g, which was
signicantly higher than that found adsorbed in the HMWM.
For calculating the amount of ester-linked phenolic compounds
in the HMWM, the amount of simple phenolics released by
saponication was subtracted from the amount of adsorbed
phenolics (Figure 1a). As can be observed, the amount of esterlinked phenolic compounds present in the HMWM ranged
from 0.5 to 1.4 mmol/100 g. The values are in the range of
those found in a previous study,15 although lower values have
been reported14 or even not detected after alkaline hydrolysis,6,9 probably due to their oxidation during extraction.24 The
amount of ester-linked phenolic compounds present in the
CGAcoee model was signicantly higher than that present in
the HMWM of control, showing that the incorporation of
phenolic compounds in the CGAcoee resulted in a
signicantly higher amount of ester-linked phenolic compounds
in the HMWM.
7848
Article
Figure 3. Nature and relative abundance of phenolic compounds released by alkaline fusion of the high molecular weight material recovered from
the in bean CGAcoee model.
Article
sition, including melanogalactomannans, melanoarabinogalactans, and melanoidins containing low amounts of carbohydrates.6,7 These macromolecules can be roughly separated by
ethanol precipitation. Galactomannans and melanogalactomannans can be obtained by precipitation in 50% ethanol
solutions (Et50), arabinogalactans and melanoarabinogalactans
can be obtained by precipitation in 75% ethanol solutions
(Et75), and a second melanoarabinogalactan fraction and the
melanoidins with low amounts of carbohydrates remain soluble
in 75% ethanol solutions (EtSn). The yield and chemical
composition of the fractions obtained by ethanol fractionation
are shown in Table 4. For all coees, the amount of material
recovered was highest on the EtSn fraction (53%, on average)
followed by the Et50 fraction (35%), and the Et75 fraction
accounted for the lowest amount (13%). This distribution of
the dierent melanoidin populations is in accordance with
previous ethanol fractionation procedures in Arabica and
Robusta coees.6,16,20,34,35 The amount of protein recovered
in the fractions isolated from the dierent coees increased
signicantly with the increase of the ethanol concentration, in
accordance with Bekedam et al.4 The amount of protein
recovered in the EtSn and Et75 fractions from CGAcoee was
signicantly higher than that of control. The phenolic
compounds present in the each fraction also increased with
the increase in ethanol solubility, being also observed a
signicantly higher amount of total phenolics in the Et75 and
EtSn fractions recovered from CGAcoee when compared to
control. A signicant correlation between the amount of
protein and phenolic compounds present in the ethanol
fractions (r = 0.830, p < 0.05) was also observed. When the
percentage of phenolic compounds present in each fraction is
calculated in relation to the amount of phenolic compounds
present in the HMWM, it can be observed that the phenolic
compounds present in the EtSn fraction represent nearly 83%
of the HMWM phenolic compounds, followed by the Et50
fraction (15%) and the Et75 (12%). These results show that the
melanoidin population soluble in ethanol contained the
majority of the phenolic compounds incorporated into the
HMWM during roasting, which is in accordance with Nunes et
al.16 and Bekedam et al.4 The MBI increased for all coee
fractions with the increase of their solubility in ethanol. For the
Et75 and EtSn fractions recovered from the CGAcoee model,
an average of 30% increase in the MBI was observed. In these
fractions, the amounts of protein and phenolic compounds are
signicantly higher for CGAcoee model when compared to
the control. Also, a signicant correlation between the phenolic
compounds and MBI (r = 0.674, p < 0.05) is observed. The
amount of phenolic compounds was also signicantly
correlated with the color of the dierent melanoidin
populations (r = 0.782, p < 0.05), a signicant correlation
between the amount of protein and MBI (r = 0.749, p < 0.05)
being also observed.
As the melanoidin populations soluble in 75% ethanol
contained the majority of the phenolic compounds present in
the HMWM, the nature of the phenolic compounds present in
these melanoidin populations was further studied (Figure 1b).
The EtSn fraction did not contain adsorbed phenolic
compounds, which can be explained by the experimental
procedure used, including dialysis and urea solubilization,
probably resulting in the loss of the adsorbed phenolics. The
amount of ester-linked phenolic compounds was in the range of
that found for the HMWM, and there was no signicant
dierence between the amounts of ester-linked phenolics for
Article
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