Article

Cite This: J. Agric. Food Chem. 2019, 67, 5197−5203 pubs.acs.org/JAFC

Basic Structure of Melanoidins Formed in the Maillard Reaction of

3‑Deoxyglucosone and γ‑Aminobutyric Acid
Philipp Bruhns,*,† Clemens Kanzler,† Andreas G. Degenhardt,‡ Timo J. Koch,‡ and Lothar W. Kroh†
†
Institut für Lebensmitteltechnologie und Lebensmittelchemie, Fachgebiet Lebensmittelchemie und Analytik, Technische
Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
‡
Pfeifer & Langen GmbH & Co. KG, Aachener Straße 1042a, 50858 Köln, Germany
*
S Supporting Information
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ABSTRACT: Melanoidins are formed in foods during processing through the Maillard reaction between carbohydrates and
amino compounds. The aim of this study was to draw conclusions about the formation mechanism and the structure of
Downloaded via ADAMA MAKHTESHIM LTD on March 8, 2021 at 10:24:25 (UTC).

melanoidins formed at low water contents and low temperatures. In the Maillard reaction of D-glucose and γ-aminobutyric acid
at low water contents 3-deoxyglucosone is the most important intermediate. Therefore, we used the reaction of 3-
deoxyglucosone with γ-aminobutyric acid or β-alanine as a simplified model system. The degradation of 3-deoxyglucosone and
the color formation of the formed melanoidins were determined. In addition, the reaction mixture was analyzed with high-
resolution mass spectrometry and a Kendrick analysis was applied. Oligomers consisting of up to four molecules of 3-
deoxyglucosone and three amino acids and their respective dehydration products with furanoidic structure were detected. The
melanoidin structure of C−C linked monomeric units postulated by Kroh et al. could be confirmed.
KEYWORDS: Maillard reaction, melanoidins, α-dicarbonyl compounds, 3-deoxyglucosone, high-resolution mass spectrometry,
Kendrick analysis

■ INTRODUCTION
During the Maillard reaction between carbohydrates and
amino compounds a wide range of low molecular mass
compounds, such as organic acids, aldehydes, 1,2-dicarbonyl,
and heterocyclic compounds, are formed.1 The formation of
these compounds leads to changes in flavor, taste, and texture
of food during processing and has been the object of a
multitude of studies.2−5 Several reaction pathways in the early
stages of the Maillard reaction have already been described
well.1−4 In addition to intermediates of low molecular mass,
high molecular mass melanoidins are formed as end products
of the reaction. These products can be responsible for the color
development in food. They are thought to be formed through
polymerization of sugar degradation products to high
molecular mass polymers. The mass of the molecules depends
on the reaction conditions and the involved amino
compound.6 So far, the structures and the reaction pathways
are unknown. In Maillard reaction systems only several low
molecular mass compounds have been identified and several
proposals for the polymerization mechanisms of melanoidins
exist.7−13 The postulated mechanisms and melanoidin
structures can be divided into polymers with a backbone
consisting of heterocyclic compounds and polymers formed
through polymerization of carbohydrates. For example, Heyns
and Hauber,14 and later Tressl et al.,11,12 found oligomers
consisting of furanes and pyrroles. In model reactions starting
from heterocyclic compounds Tressl et al. could identify
oligomers consisting of up to 12 monomeric units.11 In
contrast, Kroh et al.13,7 and Huyghues-Despointes and
Yaylayan10 postulated the polymerization of earlier intermedi-
ates, such as α-dicarbonyl compounds or Amadori products via
aldol and nucleophilic addition reactions. Doubtless the
melanoidin formation in the Maillard reaction, in a complex
matrix like food, will be a mix of several mechanisms, resulting
in a variety of different structures.
The analysis of Maillard reaction products is difficult
because of the wide range of formed intermediates and end
products and can be overcome only by separation and analysis
of single compounds. As a powerful analysis technique for the
analysis of complex mixtures, high-resolution mass spectrom-
etry was used in this study. This technique can provide
information on hundreds of compounds simultaneously and be
selective when needed. Kuhnert et al.15 applied this technique
for the analysis of caramelization products of several
monosaccharides and showed the possibilities of modern
interpretation tools as Kendrick and van Krevelen analysis.
They could identify a series of homologous oligomers of up to
six monomeric units of carbohydrates bound through
glycosidic bonds and their dehydration products.16 In Maillard
model systems with several different carbohydrates and amino
acids they could show the formation of oligomers of similar
structure and in addition the formation of products consisting
of carbohydrates and amino acids.17
The aim of the present study was to bring new insights into
the structure of melanoidins formed during the Maillard
reaction. Therefore, selected model systems at mild reaction
conditions were used to reduce the complexity of the reaction.
The model reaction products were analyzed by high-resolution
mass spectrometry. In addition, modern data interpretation
Received: January 9, 2019
Revised: April 3, 2019
Accepted: April 3, 2019
Published: April 24, 2019

© 2019 American Chemical Society 5197 DOI: 10.1021/acs.jafc.9b00202

J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry
tools were applied with the aim to identify oligomers. On the
basis of the structure of these oligomers, the formation
mechanism of these high molecular mass molecules was
elucidated.
Preliminary experiments showed that in the Maillard
reaction of D-glucose with γ-aminobutyric acid high color
development already occurs under mild reaction conditions
(50 °C and water contents <30%). Under these conditions
almost 100% of the D-glucose degrades via the Amadori
product to 3-deoxy-D-erythro-hexo-2-ulose (3-deoxyglucosone)
(see Supporting Information). For our current approach, 3-
deoxyglucosone was synthesized and heated with γ-amino-
butyric acid without addition of water at low temperatures. In
addition to the determination of 3-deoxyglucosone degrada-
tion and color formation the formed products were analyzed
by mass spectrometry. This simplified Maillard system starting
from 3-deoxyglucosone leads to a more directed pathway of
melanoidin formation, which allows one to draw conclusions
about the melanoidin structure through mass spectrometric
analysis.
■ MATERIALS AND METHODS
The following chemicals were obtained commercially: acetic acid
(Roth, Karlsruhe, Germany), acetonitrile (VWR, Darmstadt,
Germany), γ-aminobutyric acid (Alfa Aesar, Karlsruhe, Germany),
β-alanine (Sigma-Aldrich, Munich, Germany), benzaldehyde (Merck,
Hohenbrunn, Germany), benzhydrazide (Sigma-Aldrich), diethyl
ether (Roth), ethanol (VWR), D-glucose (Roth), methanol (VWR),
o-phenylenediamine (Fluka, Steinheim, Germany), pullulan standard
(PSS Polymer Standard Service GmbH, Valkenburg, Netherland), and
p-toluidine (Merck, Hohenbrunn, Germany).
Preparation of Maillard Mixtures. 3-Deoxyglucosone or D-
glucose was mixed in an equimolar ratio with γ-aminobutyric acid or
β-alanine and homogenized in a mortar. Ten milligrams of the 3-
deoxyglucosone/amino acid mixture or 100 mg of the D-glucose/
amino acid mixture was transferred into a vial (1.5 mL), and the vial
was sealed and heated in an aluminum block in a drying cabinet
(Heraeus, Function Line, Typ T6) at 50 °C. After defined reaction
times, the reaction was stopped by cooling in a freezer. The samples
were diluted with 1 mL of purified water for further analysis.
Synthesis of 3-Deoxyglucosone. 3-Deoxyglucosone was
synthesized according to Hellwig et al.18 with modifications according
to Haase et al.19
High-Performance Liquid Chromatography with Diode
Array Detector (HPLC-DAD) Analysis of α-Dicarbonyl Com-
pounds. The determination of α-dicarbonyl compounds was carried
out with HPLC-DAD as published before.20
UV−Vis Spectrophotometry. The color was determined after
dilution with purified water by absorption measurement at 420 nm
with a spectrophotometer (Specord 200 Plus, Analytik Jena, Jena,
Germany; software: WinAspect Plus Version 4.1). For the
comparability of all model systems the extinction was multiplied
with the dilution factor and calculated as extinction per mole of 3-
deoxyglucosone. All measurements were carried out with a quartz
cuvette against water.
Electrospray Ionization Mass Spectrometry (ESI-HRMS). The
mass spectrometry analyses were carried out on a Thermo Fisher
Scientific LTQ Orbitrap XL in positive and negative electrospray
ionization mode. Samples were direct infused via a syringe pump. For
data interpretation the 300 most intense signals in a m/z range
between 200 and 1000 were used.
Elemental Analysis. The elemental analysis was carried out on a
FlashEA 1112 Organic Elemental analyzer (Thermo Fisher Scientific).
The amount of oxygen was calculated.
Size Exclusion Chromatography (SEC) UV−Vis. The molec-
ular size distribution was analyzed by size exclusion chromatography
with an UV−vis and refractive index (RI) detector (pump, Shimadzu
5198
Article
LC-20ADSP; degasser, Uniflows LC-10AT; autosampler, Merck
Hitachi AS 4000; two columns, Agilent PL Aquagel-OH MIXED-H
8 μm; detector (UV−vis), Shimadzu SPD-10A; detector (IR),
Shimadzu RID-6A; communication module, Schimadzu CBM-20A;
software, Shimadzu LabSolutions v5.71 SP1). The samples were
diluted 1:10 with purified water. The analysis was carried out at room
temperature. The flow rate was 1 mL/min and the mobile phase was
purified water. The detection of colored reaction intermediates was
monitored at a wavelength of 420 nm. As reference for the molecular
mass a mixture of pullulans between 10 and 400 kDa was used.
Dialysis of Melanoidin. The reaction mixture was dissolved in
purified water (1.5 g/100 mL) and transferred into dialysis tubes
[molecular weight cut-off (MWCO), 12.000−14.000 Da; ZelluTrans,
Roth]. The tubes were stored in purified water for 72 h. The water
was exchanged every 10 h. After dialysis, the samples were freeze-
dried.
■
RESULTS AND DISCUSSION
Degradation and Color Formation of 3-Deoxygluco-
sone under Mild Reaction Conditions. 3-Deoxyglucosone
was heated in the absence of an amino compound and water at
50 °C. Under these conditions the α-dicarbonyl compound is
relatively stable and only 4% degrade in 48 h and no color
formation can be observed. With addition of γ-aminobutyric
acid in an equimolar ratio, the reactivity increases considerably.
Ninety percent of 3-deoxyglucosone degrades in only 3 h and
the color formation correlates with the high degradation rate
(Figure 1A). After these first 3 h the initially fast color
Figure 1. Model reaction of 3-deoxyglucosone (3-DG) with γ-
aminobutyric acid (GABA) at 50 °C. (A) 3-Deoxglucosone
degradation and color formation. Three repetitions of the model
reaction were performed. (B) Size exclusion chromatogram of the
reaction mixture after 8 h.
development slows down and seems to be limited by the low
remaining concentration of 3-deoxyglucosone. In contrast, the
insignificant degradation of the reactive α-dicarbonyl com-
pound in absence of an amino acid shows that caramelization
reactions are not important under mild conditions and the
degradation occurs only in the presence of an amino acid. A
typical degradation reaction for the caramelization of 3-
deoxyglucosone at a higher temperature would be the
formation of hydroxymethylfurfural (HMF).21 Neither in the
Maillard reaction of 3-deoxyglucosone with γ-aminobutyric
acid nor in other experiments with D-glucose and γ-amino-
butyric acid, the formation of HMF was observed in low
moisture systems. Also, the formation of other low molecular
mass cleavage products as glyoxal or methylglyoxal was not
detected.
On the other hand, size-exclusion chromatography revealed
that mainly high molecular mass compounds were formed and
the signal of a UV−vis detector at 420 nm showed that only
high molecular mass compounds are responsible for the color
DOI: 10.1021/acs.jafc.9b00202
J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry
of the reaction mixture (Figure 1B). The retention time of the
high molecular mass fraction is lower than the retention time
of the 400 kDa pullulan standard, indicating a higher
hydrodynamic volume than the pullulan. Apart from these
polymers and the residual colorless 3-deoxyglucosone, no other
compounds could be detected. All observations suggest that
the α-dicarbonyl compound reacts through a polymerization
with the amino acid.
Formation of a Schiff Base from 3-Deoxyglucosone.
In the same way as D-glucose reacts with an amino acid in the
initial stage of the Maillard reaction, 3-deoxyglucosone is most
probably attacked by a nucleophile, that is, the amino function
of γ-aminobutyric acid (Figure 2). The attack at the carbonyl
Figure 2. Two possible reaction mechanisms of a nucelophilic attack
of the amino acid at the C1 (a) or C2 (b) of the 3-deoxyglucosone.
group at C1 is presumably preferred over the attack at C2
because the aldehyde at C1 should be more reactive compared
to the ketone structure at the C2. The reactivity of the C2 is
lower because of the higher electron density through the
electron-donating effect of the second bound carbon atom.
The reaction product, the Schiff base from 3-deoxyglucosone,
could not be isolated until now, which indicates high reactivity
and fast degradation.
Looking at the formation mechanism of 3-deoxyglucosone in
a Maillard system starting from D-glucose, 3-deoxyglucosone is
formed via the Amadori product followed by a vinylogous β-
elimination of water resulting in a Schiff base of 3-
deoxyglucosone. A hydrolysis of the amino acid leads
ultimately to 3-deoxyglucosone.2 This last reaction step is
the reverse reaction of the nucleophilic attack of the amino
acid at 3-deoxyglucosone as described above for the reaction of
an amino acid with 3-deoxyglucosone.
This means that there is an equilibrium between the free α-
dicarbonyl compound and the Schiff base, which is influenced
by the presence of free water. Therefore, in addition to the
formation of the free α-dicarbonyl compound, another
degradation pathway of the Schiff base from 3-deoxyglucosone
must exist. Under water-free conditions, the equilibrium is
shifted toward the Schiff base and thereby the reaction rate
increases, resulting in a higher color formation rate. The
addition of water reduces the stability of the Schiff base and
shifts the equilibrium to the free 3-deoxyglucosone and
decreases the reaction rate. Consequently, a determination of
the actual 3-deoxyglucosone concentration of a dry reaction
mixture is only possible with a completely water-free workup.
Probably the determined amounts of 3-deoxyglucosone are
only formed after addition of water during the analysis.
5199
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Analysis of Melanoidins by High-Resolution Mass
Spectrometry. After addition of water to the reaction
mixtures, these were analyzed by high-resolution mass
spectrometry (ESI-orbitrap). The mass spectrum of the
Maillard model reaction of 3-deoxyglucosone with γ-amino-
butyric acid is shown in Figure 3A. It consists of several
hundred signals and every single signal can originate from a
number of different isomers.
Figure 3. (A) Mass spectrum and (B) Kendrick plot with water as
mass increment of the Maillard reaction of 3-deoxyglucosone with γ-
aminobutyric acid at 50 °C in the positive ion mode using a direct
infusion into an ESI-Orbitrap-MS instrument. The size of the data
points correlates with the signal intensity.
A list of the 50 most intense signals is given in the
Supporting Information. To simplify the interpretation of the
complex spectrum, a Kendrick analysis was carried out.22 As
Kendrick mass increment the mass of water was used and
normalized from mIUPAC = 18.0106 to the Kendrick mass of
mKM = 18.0000. The Kendrick mass defect is the difference
between the exact Kendrick mass and the nominal Kendrick
mass. For visualization the Kendrick mass defect was plotted as
a function of the Kendrick mass (Figure 3B).
Molecules that differ only in the number of water molecules
show the same Kendrick mass defect and can be identified by a
series of data points on the same height (y-axis) in the plot.
With this tool it was possible to visualize the existence of a
number of homologous series of compounds whose structures
differ only in the number of eliminated water molecules. With
use of the exact mass and calculation of the molecular formula,
it was possible to identify oligomers consisting of 3-
deoxyglucosone and γ-aminobutyric acid as monomeric units.
Oligomers containing up to four 3-deoxyglucosone and three
γ-aminobutyric acid units could be identified (Table 1).
For every oligomer, with a different number of incorporated
3-deoxyglucosone and amino acid (AA) molecules, the
Kendrick plot revealed a homologous series of hydrated and
dehydrated oligomers. These series are colored red (1× AA),
maroon (2× AA), and rose (3× AA) in Figure 3B. The
homologous series with the same color but different Kendrick
mass defect differ in the number of incorporated 3-
deoxyglucosone molecules. The number of incorporated
amino acids was evaluated by comparison of the mass spectra
of the reaction of 3-deoxyglucosone with γ-aminobutyric acid
and of the reaction with β-alanine. For example, the spectrum
from γ-aminobutyric acid contains a signal at m/z 248.1124
(C10H18O6N) and the spectrum from β-alanine at m/z
234.0967 (C9H16O6N). Both signals result from the product
of one molecule of 3-deoxyglucosone and one molecule of the
corresponding amino acid. The difference between both is
exactly 14.0157. This corresponds to the mass of a methylene
group, which is the difference between γ-aminobutyric acid and
β-alanine. It was possible to identify the correct molecular
DOI: 10.1021/acs.jafc.9b00202
J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry Article

Table 1. High-Resolution ESI-MS (Orbitrap) Data of the Reaction Mixture of 3-Deoxyglucosone (3-DG) with γ-Aminobutyric
Acid (GABA) at 50 °C in Positive Ion Mode and Negative Ion Mode (Red Signals in Figure 3)
m/z [M + H]+
structure assignment molecular formula exp theor rel. error [ppm] rel. intens. m/z [M − H]−, exp
−3× H2O C10H12O3N 194.0811 194.0817 3.1
−2× H2O C10H14O4N 212.0913 212.0923 4.7 12.9 210.0769
−H2O C10H16O5N 230.1019 230.1028 3.9 2.4 228.0868
3-DG GABA C10H18O6N 248.1124 248.1134 4.0 6.3 246.0976
+H2O C10H20O7N 266.1230 266.1240 3.8 4.9 264.1082
−2× H2O C14H21O5N2 297.1440 297.1450 3.4 32.2 295.1291
−H2O C14H23O6N2 315.1546 315.1556 3.2 0.8
3-DG 2× GABA C14H25O7N2 333.1651 333.1662 3.3 0.6
+H2O C14H27O8N2 351.1755 351.1767 3.4 0.5
−3× H2O C16H22O8N 356.1333 356.1345 3.4 0.7 354.1185
−2× H2O C16H24O9N 374.1439 374.1451 3.2 4.1 372.1292
−H2O C16H26O10N 392.1545 392.1557 3.1 5.3 390.1397
2× 3-DG GABA C16H28O11N 410.1649 410.1662 3.2 19.6 408.1501
+H2O C16H30O12N 428.1756 428.1768 2.8 3.6
−4× H2O C20H27O8N2 423.1755 423.1767 2.8 4.1 421.1608
−3× H2O C20H29O9N2 441.1859 441.1873 3.2 20.8 439.1711
−2× H2O C20H31O10N2 459.1965 459.1979 3.0 16.7 457.1817
−H2O C20H33O11 N2 477.2070 477.2084 2.9 13.8 475.1923
2× 3-DG 2× GABA C20H35O12N2 495.2175 495.2190 3.0 26.6 493.2030
−4× H2O C24H34O9N3 508.2280 508.2295 2.9 1.2
−3× H2O C24H36O10N3 526.2386 526.2401 2.8 3.4
−2× H2O C24H38O11N3 544.2494 544.2506 2.2 1.1
−H2O C24H40O12N3 562.2599 562.2612 2.3 1.1 560.2448
2× 3-DG 3× GABA C24H42O13N3 580.2703 580.2718 2.6 0.3
−4× H2O C22H30O12N 498.1606
−3× H2O C22H32O13N 518.1861 518.1874 2.5 0.9 516.1712
−2× H2O C22H34O14N 536.1966 536.1979 2.4 1.1 534.1818
−H2O C22H36O15N 554.2072 554.2085 2.3 1.9 552.1921
3× 3-DG GABA C22H38O16N 572.2177 572.2191 2.4 1.6 570.2028
+H2O C22H40O17N 590.2281 590.2296 2.5 0.5 588.2132
−5× H2O C26H35O12N2 567.2176 567.2190 2.5 2.1 565.2025
−4× H2O C26H37O13N2 585.2281 585.2296 2.6 1.6 583.2131
−3× H2O C26H39O14N2 603.2387 603.2401 2.3 3.8 601.2237
−2× H2O C26H41O15N2 621.2493 621.2507 2.2 4.7 619.2342
−H2O C26H43O16N2 639.2598 639.2613 2.4 4.3 637.2447
3× 3-DG 2× GABA C26H45O17N2 657.2705 657.2718 1.9 0.4
−5× H2O C30H42O13N3 652.2702 652.2718 2.5 0.9 650.2551
−4× H2O C30H44O14N3 670.2809 670.2823 2.1 4.3 668.2656
−3× H2O C30H46O15N3 688.2916 688.2929 1.9 1.7 686.2761
−2× H2O C30H48O16N3 706.3021 706.3035 1.9 2.6 704.2866
−H2O C30H50O17N3 724.3123 724.3140 2.3 0.4
3× 3-DG 3× GABA
−3× H2O C28H40O18N 678.2235
−2× H2O C28H44O19N 696.2340
−H2O C28H46O20N 714.2445
4× 3-DG 1× GABA C28H48O21N 732.2548
+H2O
−5× H2O C32H45O17N2 727.2549
−4× H2O C32H47O18N2 747.2805 747.2824 2.5 0.3 745.2655
−3× H2O C32H49O19N2 765.2915 765.2930 1.9 0.5 763.2760
−2× H2O C32H51O20N2 783.3022 783.3035 1.6 0.7 781.2866
−H2O C32H53O21N2 801.3125 801.3141 2.0 0.3 799.2970
4× 3-DG 2× GABA C32H55O22N2 817.3076
−5× H2O C36H52O18N3 814.3232 814.3240 0.9 0.5
−4× H2O C36H54O19N3 832.3337 832.3346 1.1 0.6 830.3182
−3× H2O C36H56O20N3 850.3445 850.3457 1.4 0.3 848.3286
−2× H2O C36H58O21N3 868.3551 868.3563 1.4 0.4
−H2O

5200 DOI: 10.1021/acs.jafc.9b00202

J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry
Table 1. continued
structure assignment molecular formula
4× 3-DG 3× GABA
formula and composition, even of high molecular mass
oligomers through comparison of the high-resolution mass
spectra from reaction mixtures of both amino acids. In the
reaction of 3-deoxyglucosone with β-alanine and with γ-
aminobutyric acid, analogous signals, considering the different
molecular masses of the reagents, were found.
In addition, dehydrated isomers of all oligomers with a loss
of up to five molecules of water were detected. Here, the
number of lost water molecules increases with increasing
number of 3-deoxyglucosone molecules. Furthermore, hy-
drated oligomers were found, but almost exclusively in
oligomers containing a higher number of 3-deoxyglucosone
molecules as amino acid molecules. They result from the
hydration of one of the carbonyl groups of a 3-deoxyglucosone
unit.
The peak at m/z 248 (C10H18O6N) is the product of the
nucleophilic substitution reaction of 3-deoxyglucosone and γ-
aminobutyric acid as postulated in Figure 2. The addition of a
second γ-aminobutyric acid leads to m/z 333 (C14H25O7N2).
Also, the reaction with a second molecule of 3-deoxyglucosone
is possible and leads to m/z 410 (C16H28O11N) or the reaction
with a second molecule of γ-aminobutyric acid and 3-
deoxyglucosone which results in the peak at m/z 495
(C20H35O12N2). Most of the main peaks can be explained by
oligomers consisting of 3-deoxyglucosone and γ-aminobutyric
acid units and their respective dehydration products.
From the above explained data it is not possible to predict
the structure and reaction mechanism because of the high
amount of possible isomers, but conceivable is a polymer-
ization through aldol reactions as postulated by Kroh et al.13,7
Through a keto−enol tautomerism, 3-deoxglucosone or its
Schiff base can form a 2,3-enediol, which can nucleophilic
attack a carbonyl group of another 3-deoxglucosone or Schiff
base, forming a C−C bond as shown in Figure 4. The attack
can occur at C1 of the imine or at C2 of the carbonyl. In
general, carbonyl groups have a stronger electron-withdrawing
Figure 4. Formation of an 2,3-enediol through a tautomerism and
formation of oligomers through an aldol reaction. (a) Nucleophilic
attack at C1; (b) nucleophilic attack at C2.
Article
m/z [M + H]+
exp theor rel. error [ppm] rel. intens. m/z [M − H]−, exp
effect and should be preferred to an imine group. The observed
increase in reactivity by addition of the amino acid and
formation of the Schiff base in comparison to that of the free 3-
deoxyglucosone might result from the electron-withdrawing
effect of the partially protonated imine group. Schiff bases are
stronger bases than aldehydes and are in equilibrium with their
protonated form. The positive charge of the protonated Schiff
base is delocalized between two resonance structures, with the
positive charge either at the carbon or at the nitrogen.
Therefore, the electron density decreases and the nucleophilic
attack of another 3-deoxyglucosone or its Schiff base is favored
and occurs at the C1.
As shown in Figure 5, it is possible to stabilize the postulated
structure of oligomers by formation of a pyranose or furanose
Figure 5. Postulated melanoidin structure of an oligomer consisting
of three 3-deoxyglucosone units and two amino acids and conceivable
dehydration products.
ring in every monomeric unit through a nucleophilic attack of
the hydroxyl group at C5 or C6 and the carbonyl group at C2
in the same manner as free carbohydrate derivatives. In these
oligomers, unsaturated structures, possibly aromatic rings with
structures similar to those of HMF, can be formed through loss
of water molecules. For the oligomer with three 3-
deoxyglucosone units, it was possible to detect the loss of
five molecules of water (Figure 5); theoretically, the loss of up
to six molecules of water is possible. Elimination of water with
increasing reaction time would lead to the formation of
multiple C−C double bonds. The resulting melanoidin
structure consisting of carbohydrate units with a high number
of hydroxyl groups and repetitive unsaturated units can explain
the good water solubility. Even after a reaction time of 8 h the
melanoidins from 3-deoxyglucosone and γ-aminobutyric acid
as well as from β-alanine are water-soluble as no precipitation
after dissolving in water was observed. In comparison, a
structure consisting mainly of heterocyclic compounds would
be insoluble in water already at low molecular mass.
In addition, an aldol reaction with other byproducts is
possible, for example the reaction of the proposed oligomers
with heterocyclic compounds. The combination of different
reaction pathways and the resulting structures generates the
variety of the formed products.
MS/MS Experiments. Besides the elucidation of the
molecular mass and calculation of the molecular formula, it
is of interest to study the molecular structure to obtain insights
into the linkage of the monomeric units. From these structures
the formation mechanism of melanoidins can be elucidated. As
discussed before, a linkage of the monomeric units through an
5201 DOI: 10.1021/acs.jafc.9b00202
J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry
aldol reaction is possible. This would lead to a stable C−C
bond between the 3-deoxyglucosone units in contrast to a less
stable C−O−C bond resulting from a glycosidic linkage. In a
MS/MS experiment two fragments with the difference of m/z
18 would be typical for a glycosidic linkage because the bond
breakage could occur on both sides of the glycosidic oxygen
atom. In MS2 spectra of polysaccharides such peaks show often
the highest intensities.23,24
For this study tandem-MS experiments were applied in
negative and positive ion mode. Figure 6 shows the MS2
Figure 6. MS2 spectra of (A) m/z 493 and (B) m/z 475 in negative
ion mode using a direct infusion into an ESI-Orbitrap-MS instrument.
spectra of two of the most intense peaks (m/z 493 and 475) of
the reaction of 3-deoxyglucosone and γ-aminobutyric acid. The
oligomer of m/z 493 consists of two 3-deoxyglucosone and
two γ-aminobutyric acid units and m/z 475 is the respective
dehydration product after the loss of one water molecule.
The peak at m/z 493 shows a minimal fragmentation with a
neutral loss of 18 (H2O) and of 90 (C3H6O3), which is a
typical fragmentation product of saccharides.23 The fragmen-
tation of the peak m/z 475 can be explained by fragmentation
mechanisms, which are also typical for oligosaccharides (Figure
7).23 This includes the neutral loss of 18 (H2O), of 90
Figure 7. Observed fragmentation pathways in the negative ion mode.
(C3H6O3), and of 86 (C4H6O2) due to the loss of the amino
acid and of 44 (CO2). Typical fragments of a glycosidic bond
could not be observed.
Also, the fragmentation peaks of all other studied peaks in
negative ion mode did not show these fragments. All
fragmentations can be explained by the six mechanisms from
a structure consisting of monomeric units bond via aldol
reaction shown in Figure 7.
In contrast, the fragmentations observed in the 3-
deoxglucosone/γ-aminobutyric acid system correspond to the
typical fragmentation mechanisms of C−C bond glyco-
sides.23,25−27 This validates the postulated C−C linkage of 3-
deoxyglucosone units.
The MS2 spectra in positive ion mode differ from the spectra
in negative ion mode by additional peaks of the breakage of the
postulated C−C bond, occurring in low intensities. For
example, the MS2 spectrum of the peak m/z 477 (dehydration
product of the oligomer consisting of two 3-deoxyglucosone
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Article
and two amino acid units) shows two peaks at m/z 230 and
345. They might result from the two different methods of C−
C bond breakage as illustrated in Figure 8.
Figure 8. Fragmentation pathway of the peak m/z 477 in the positive
ion mode.
The peak at m/z 230 may originate from a single 3-
deoxyglucosone unit with one molecule of γ-aminobutyric acid.
The peak at m/z 345 may originate also from a 3-
deoxyglucosone unit with one molecule of γ-aminobutyric
acid plus a C1 body with a second molecule of γ-aminobutyric
acid. The detection of both fragments proves the postulated
C−C linkage via an aldol reaction as the main mechanism of
the polymerization.
Elementary Analysis. Further indication for the postu-
lated structure provides the elemental analysis of a dialyzed
melanoidin from the reaction of D-glucose with γ-aminobutyric
acid. The partly dehydrated oligomer M2 in Figure 5 has a
calculated elemental composition of 51.8% C, 6.4% H, 4.7% N,
and 37.2% O. The measured elemental composition of the
dialyzed melanoidin from the reaction of D-glucose and γ-
aminobutyric acid is similar to this with 51.2% C, 5.9% H, 4.8%
N, and 38.2% O. Therefore, the structure of oligomer M2 can
be considered as a typical fragment of melanoidins formed
under these reaction conditions.
Furthermore, it is possible to specify the ratio of
incorporated carbohydrates to amino acids from the ratio
between the amount of carbon and nitrogen. For each amino
acid one and a half carbohydrate units are incorporated, which
corresponds to the postulated structure. This shows that the
amino acid not only increases the reactivity but is also
incorporated into the melanoidins.
The found elemental composition is comparable to the
results obtained by Cämmerer and Kroh28 of melanoidins from
Maillard reaction model systems with D-glucose and glycine.
The elemental composition as well as the postulated structure
is comparable to our current results. In contrast, a melanoidin
structure consisting only of heterocyclic compounds, as
suggested by Tressl et al.,11 would have a different C/O
ratio and therefore cannot be the basic structure of
melanoidins formed under mild conditions. However, our
study shows that especially under dry reaction conditions and
at low temperatures a targeted reaction occurs via the
degradation to 3-deoxyglucosone followed by a direct
polymerization to the proposed basic melanoidin structure.
The proven elimination of water molecules leads to
fragments with unsaturated parts, which could lead after
DOI: 10.1021/acs.jafc.9b00202
J. Agric. Food Chem. 2019, 67, 5197−5203
Journal of Agricultural and Food Chemistry
further rearrangements to the formation of chromophoric
systems.
The found results broaden the knowledge about the
formation mechanism of high molecular mass melanoidins
under mild reaction conditions. This reaction can occur in all
foods containing carbohydrates and amino acids during
manufacturing and processing under low water content, for
example, during drying or baking processes.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.9b00202.
Additional tables of high-resolution mass spectrometry
data for the reaction mixture of 3-deoxyglucosone with
γ-aminobutyric acid or β-alanine and tables with MS2
data (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: p.bruhns@tu-berlin.de (P.B.). Tel.: +49 (0)30 314-
72404.
ORCID
Philipp Bruhns: 0000-0001-8859-8243
Clemens Kanzler: 0000-0002-1461-5386
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS USED
3-DG,3-deoxy-D-erythro-hexo-2-ulose; AA,amino acid; β-Ala,β-
alanine; GABA,γ-aminobutyric acid; HMF,hydroxymethylfur-
fural; HRMS,high-resolution mass spectroscopy
■
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DOI: 10.1021/acs.jafc.9b00202
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