Formation of Reactive Intermediates, Color, and Antioxidant Activity in the Maillard Reaction of Maltose in Comparison to d -Glucose

Article abstract of DOI:10.1021/acs.jafc.7b04105

In this study, the Maillard reaction of maltose and d-glucose in the presence of l-alanine was investigated in aqueous solution at 130 °C and pH 5. The reactivity of both carbohydrates was compared in regards of their degradation, browning, and antioxidant activity. In order to identify relevant differences in the reaction pathways, the concentrations of selected intermediates such as 1,2-dicarbonyl compounds, furans, furanones, and pyranones were determined. It was found, that the degradation of maltose predominantly yields 1,2-dicarbonyls that still carry a glucosyl moiety and thus subsequent reactions to HMF, furfural, and 2-acetylfuran are favored due to the elimination of d-glucose, which is an excellent leaving group in aqueous solution. Consequently, higher amounts of these heterocycles are formed from maltose. 3-deoxyglucosone and 3-deoxygalactosone represent the only relevant C₆-1,2-dicarbonyls in maltose incubations and are produced in nearly equimolar amounts during the first 60 min of heating as byproducts of the HMF formation.

Full text of DOI:10.1021/acs.jafc.7b04105

Subscriber access provided by GRIFFITH UNIVERSITY
Article
Formation of Reactive Intermediates, Color, and Antioxidant Activity
in the Maillard Reaction of Maltose in Comparison to D-Glucose
Clemens Kanzler, Helena Schestkowa, Paul T. Haase, and Lothar W. Kroh
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04105 • Publication Date (Web): 07 Sep 2017
Downloaded from http://pubs.acs.org on September 8, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 35
Journal of Agricultural and Food Chemistry
Formation of Reactive Intermediates, Color, and Antioxidant Activity
in the Maillard Reaction of Maltose in Comparison to -Glucose
D
Clemens Kanzlera,*, Helena Schestkowaa, Paul T. Haasea, Lothar W. Kroha
a Institut für Lebensmitteltechnologie und Lebensmittelchemie, Lebensmittelchemie und
Analytik, Technische Universität Berlin, GustavꢀMeyerꢀAllee 25, TIB 4/3ꢀ1, Dꢀ13355 Berlin,
Germany
* Address of correspondence: Clemens Kanzler, Institut für Lebensmitteltechnologie und
Lebensmittelchemie, Lebensmittelchemie und Analytik, Technische Universität Berlin,
GustavꢀMeyerꢀAllee 25, Dꢀ13355 Berlin, Germany. Telephone: +49ꢀ30ꢀ31472404; Fax: +49ꢀ
30ꢀ31472585; eꢀMail: clemens.kanzler@tuꢀberlin.de
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 35
1
ABSTRACT
2
3
In this study the Maillard reaction of maltose and Dꢀglucose in presence of Lꢀalanine was
investigated in aqueous solution at 130 °C and pH 5. The reactivity of both carbohydrates was
compared in regards of their degradation, browning and antioxidant activity. In order to
identify relevant differences in the reaction pathways, the concentrations of selected
intermediates such as 1,2ꢀdicarbonyl compounds, furans, furanones, and pyranones were
determined. It was found, that the degradation of maltose preꢀdominantly yields 1,2ꢀ
dicarbonyls that still carry a glucosyl moiety and thus subsequent reactions to HMF, furfural,
4
5
6
7
8
9
and 2ꢀacetylfuran are favored due to the elimination of Dꢀglucose, which is an excellent
10
11
12
13
14
leaving group in aqueous solution. Consequently, higher amounts of these heterocycles are
formed from maltose. The only relevant C6ꢀ1,2ꢀdicarbonyls 3ꢀdeoxyglucosone and 3ꢀ
deoxygalactosone in maltose incubations are produced in nearly equimolar amounts during
the first 60 min of heating as byꢀproducts of the HMF formation.
15 KEYWORDS
16
Maillard reaction; maltose; antioxidant activity; reductones; color formation.
2
ACS Paragon Plus Environment

Page 3 of 35
Journal of Agricultural and Food Chemistry
17 INTRODUCTION
18
The majority of food items is processed today, and thus heatꢀtreated for preservation
reasons and/or to develop certain organoleptic characteristics that meet the expectations of the
consumer.1 A main reason for the changes in qualities such as taste, flavor, texture, and –
most obviously – color are nonꢀenzymatic browning reactions, caused by the thermal
degradation of reducing carbohydrates. In presence of amino components the reactions taking
place are summarized under the term of Maillard reaction.2–4 Besides colorants and aromaꢀ
active compounds reducing substances are formed in the course of the Maillard reaction, that
are able to influence the oxidative stability of foods.5 In our previous publications, we could
identify 1,2ꢀdicarbonyls,6; 7 such as 1ꢀdeoxyglucosone and glucosone, as well as heterocyclic
intermediates,8 such as Furaneol, 3,5ꢀdihydroxyꢀ6ꢀmethylꢀ2,3ꢀdihydroꢀ4Hꢀpyranꢀ4ꢀone
(DHHM), maltol, and isomaltol as reductones. The heterocyclic reductone ethers show
considerably higher antioxidant capacities than the 1,2ꢀdicarbonyls and in analogy to ascorbic
acid these compounds are prooxidants in presence of redoxꢀactive metal ions.
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
But before the impact of these intermediates on complex matrices, such as food or living
cells, can be discussed, the general reaction pathways and possible key compounds have to be
identified. Although the Maillard reaction of monoꢀ and disaccharides might be well
described in principle, most studies are based on analysis of either 1,2ꢀdicarbonyls or their
subsequent degradation products in form of furans, furanones, and pyranones. In our
approach, we analyzed both groups of early intermediates starting from maltose (Mal) in
presence of
Lꢀalanine (Ala) in a closed, aqueous system at 130 °C and a pH value of 5 in
direct comparison to
Dꢀglucose (Glc). The focus on Mal was chosen, because of the
disaccharide’s high relevance in food systems. Browning, antioxidant activity, degradation of
the respective carbohydrate component, formation of 1,2ꢀdicarbonyl compounds, and
heterocycles were investigated in systems of both carbohydrates. Additionally, incubations of
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 35
42
43
44
45
46
47
48
49
50
51
52
53
54
Mal in combination with Lꢀproline (Pro) and Lꢀlysine (Lys) as well as without addition of an
amino component were analyzed. As in previous investigations,6; 7 Ala was chosen as
reference amino acid because of its simple, chemically inert sideꢀchain. Lys with its two
amino functions (in αꢀ and εꢀposition) and the secondary amine Pro were used to investigate
the influence of amines with higher or lower nucleophilicity than Ala. Because of its
additional amino function in the side chain, Lys is able to form crossꢀlinks between different
intermediates and reacts even if it is bound in peptides or proteins, for which reason Lys has a
special role in the Maillard reaction.
To gain further knowledge about their stability and their contribution to color and
antioxidant properties the heterocyclic intermediates HMF, maltol, isomaltol, DHHM, and
Furaneol were incubated with Ala under identical conditions as carbohydrate and amino acid
mixtures and analyzed in regards of reactant degradation, browning, and antioxidant activity.
55 MATERIALS AND METHODS
56
57
58
59
60
61
62
63
64
65
66
Chemicals. 2ꢀacetylfuran, 5ꢀhydroxymethylfurfural, ethylmaltol, Furaneol, piperidine,
ꢀalanine, ꢀlysine, and ꢀproline were purchased from Acros organics (Geel, Belgium);
L
L
L
aqueous hydrogen chloride solution (1 M) was purchased from BerndꢀKraftꢀGmbH
(Duisburg, Germany); orthoꢀphenylenediamine was purchased from Fluka (Steinheim,
Germany); aqueous sodium hydroxid solution (1 M), iron(III) nitrate nonahydrate, potassium
dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, and paraꢀtoluidine
were purchased from Merck (Darmstadt, Germany); acetic acid, and
Dꢀglucose were
purchased from Roth (Karlsruhe, Germany); 1,10ꢀphenanthroline, 2ꢀacetylpyrrole, 2,2'ꢀazinoꢀ
bis(3ꢀethylbenzothiazolineꢀ6ꢀsulphonic acid), disodium edetate dehydrate, Furaneol, furfural,
maltol, potassium persulfate, sodium bathocuproinsulfonate, Trolox, and maltose
monohydrate were purchased from SigmaꢀAldrich (Steinheim, Germany); methanol was
4
ACS Paragon Plus Environment

Page 5 of 35
Journal of Agricultural and Food Chemistry
67
purchased from VWR chemicals (Darmstadt, Germany); tetrabutylammonium
hydrogensulfate was purchased from TCI (Eschborn, Germany).
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
Synthesis. The syntheses of DHHM and isomaltol were carried out as described in our
previous publication.8 Maltose phenylosazone was synthesized according to Oikawa et al.,9
and used for the synthesis of the maltosone quinoxaline derivative (maltosoneꢀQ) as described
by Smuda et al.10 3ꢀDeoxymaltose bis(benzoylhydrazone) was synthesized as described below
and used for the synthesis of 3ꢀdeoxymaltosoneꢀQ according to Smuda et al.10 The isolation of
1ꢀdeoxymaltosoneꢀQ from a reaction mixture of Mal, Lys, and orthoꢀphenylenediamine
(OPD) was performed as described by Smuda et al.10 Spectroscopic data of all obtained
quinoxaline derivatives with maltose backbone are in line with ref. 10. 3ꢀDeoxygalactose
bis(benzoylhydrazone) and 3ꢀdeoxygalactosoneꢀQ were synthesized according to Hellwig et
al.11 The spectroscopic data of 3ꢀdeoxygalactosoneꢀQ are in line with ref. 10.
3ꢀDeoxymaltose bis(benzoylhydrazone). The synthesis was carried out as described by El
Khadem et al.12 and Madsen et al.13 with some modifications. 50.0 g of maltose monohydrate
(139 mmol), 13.75 g of paraꢀtoluidine (128 mmol), and 25.0 g of sodium chloride were
suspended in 475 mL of water and 25 mL of acetic acid. The mixture was stirred for 12 h at
60 °C under argon atmosphere. Subsequently, 13.75 g of benzhydrazide (303 mmol) were
added, the reaction mixture was stirred for additional 48 h at 60 °C under argon atmosphere,
cooled down, and stored at –21 °C overnight. The residue was filtered off and washed with
iceꢀcold water.
HPLC-DAD Analysis of 1,2-Dicarboyl Compounds. The following system and settings
were used: pump, Shimadzu LC20AD; auto sampler, Shimadzu SILꢀ10AF; column oven,
Shimadzu CTOꢀ20A; detector, Shimadzu SPDꢀM20A; communication module, Shimadzu
CBMꢀ20A; software, Shimadzu LCsolution v1.22 SP1; column, MacheryꢀNagel EC250/4.6
Nucleodur C18 100ꢀ5; injection volume, 40 µL; flow, 0.5 mL/min; column temperature,
35 °C; eluent A, water; eluent B, methanol; gradient: 0 min, 17.5 % B; 15 min, 17.5 % B;
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 35
93
94
35 min, 28 % B; 55 min, 49 % B; 60 min, 95 % B; 65 min, 95 % B; 70 min, 17.5 % B;
detector range, 190–600 nm; quantitation wavelength, 318 nm. The 1,2ꢀdicarbonyl
compounds in the samples were trapped with OPD and analyzed as quinoxaline derivatives.
For derivatization 400 µL freshly prepared sample were incubated with 400 µL OPD solution
(50 mM in methanol/water, 1:1, v/v) for 24 h at room temperature. The samples were stored at
–20 °C prior to analysis.
95
96
97
98
99
GlucosoneꢀQ (tR = 28.3 min), 1ꢀdeoxyglucosoneꢀQ (tR = 36.0 min), 3ꢀdeoxyglucosoneꢀQ
(tR = 40.6 min), 3ꢀdeoxypentosoneꢀQ (tR = 46.0 min), and 1.4ꢀdideoxyglucosone (tR = 53.8)
are part of the mix standard used in our working group.14; 6; 7 MaltosoneꢀQ (tR = 26.2 min) and
3ꢀdeoxymaltosoneꢀQ (tR = 34.7 min) were synthesized as standards for quantitation.
1ꢀDeoxymaltosoneꢀQ (tR = 25.4 min) and 3ꢀdeoxygalactosoneꢀQ (tR = 41.2 min) were
synthesized as authentic references, but quantified as maltosoneꢀQ and 3ꢀdeoxyglucosonꢀQ,
respectively.
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
HPLC-DAD Analysis of Heterocyclic Compounds. The following system and settings
were used: pump, Shimadzu LC9A; auto sampler, Shimadzu SCLꢀ6B; column oven,
Shimadzu CTOꢀ6B; detector, Shimadzu SPDꢀM10A; communication module, Shimadzu
CBMꢀ10A; software, Shimadzu ClassꢀLC10 v1.64A; column, MacheryꢀNagel EC250/4.6
Nucleosil C18 120ꢀ5; injection volume, 10 µL; flow, 0.5 mL/min; column temperature, 40 °C;
eluent A, phosphate buffer (5 mM, pH 6.0) with tetrabutylammonium hydrogensulfate (2.5
mM) and disodium edetate (1 mM); eluent B, methanol; gradient: 0 min, 5 % B; 5 min, 5 %
B; 15 min, 20 % B; 20 min, 20 % B; 25 min, 95 % B; 35 min, 95 % B; 40 min, 5 % B;
detector range, 190–500 nm; quantitation wavelength, 285 nm. The samples were diluted and
filtered (syringe filter, nylon, 0.45 µm) prior to analysis.
The formation of HMF (tR = 21.0 min) and furfural (tR = 24.2 min) in carbohydrate
incubations and the degradation of maltol (tR = 26.3 min), Furaneol (tR = 26.3 min), DHHM
6
ACS Paragon Plus Environment

Page 7 of 35
Journal of Agricultural and Food Chemistry
118
(tR = 16.2 min), HMF, and isomaltol (tR = 30.0 min) in incubations of heterocyclic
intermediates was analyzed by HPLCꢀDAD.
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
HPLC-PAD Analysis of Carbohydrates. The system and method described in a
publication of Wegener et al.15 were used.
GC-MS Analysis of Heterocyclic Compounds. The system and settings described in our
previous publication8 were used. 800 µL sample were mixed with 100 µL internal standard
(aqueous solution of ethylmaltol with a concentration of 10 mM) and extracted three times
with 800 µL ethyl acetate. The solvent was reduced to around 200 µL under nitrogen stream
and the samples were subjected to GCꢀMS analysis.
The determination of DHHM (tR = 24.3 min), maltol (tR = 23.4 min), isomaltol
(tR = 18.5 min), and 2ꢀacetylfuran (tR = 16.1 min) in carbohydrate incubations was performed
with GCꢀMS.
UV/Vis Measurements. For the browning measurements, a BioꢀTek Instruments
UVIKON XL photospectrometer was used. The absorbance at 420 nm was measured in a
quartz cuvette against water. The samples were diluted with water when necessary
(absorbance > 1.4) and turbid samples were syringe filtered (nylon, 0.2 µm) before analysis.
Microplate Assays. For the TEAC and phenanthroline assay, a Tecan Infinite M200
microplate reader was used. Micro plates with 96 wells were purchased from TPP
(Trasadingen, Switzerland).
TEAC Assay. The aqueous 2,2'ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ6ꢀsulphonic acid)
(ABTS) radical cation solution was prepared by mixing 25 mL of ABTS solution (10 mM)
and 25 mL of potassium persulfate solution (3.5 mM). Before use, the mixture was incubated
overnight at room temperature in the dark and diluted 12:100 in PBS buffer (5 mM phosphate,
pH 7.2–7.4) to prepare the radical working solution. The calibration was performed with
seven trolox standards (0.010−0.100 mM; diluted in PBS buffer). 100 µL of sample were
filled in each well and the initial absorbance was measured at 734 nm. After addition of
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 35
144
145
146
147
148
149
150
151
152
153
154
100 µL radical working solution, the samples were incubated for 120 min and the final
absorbance was measured at 734 nm.
Phenanthroline Assay. The phenanthroline assay was carried out as described in our
previous publication.8
Incubation of Carbohydrates and Heterocyclic Intermediates. An overview of the
prepared reaction mixtures is given in table 1. Prior to incubation the pH value of the mixtures
was adjusted to (5.0 ± 0.1) with aqueous solutions of hydrochloric acid or sodium hydroxide.
The pH adjusted mixtures were sealed in ampules (each 2.5 mL) and heated at (130 ± 1) °C in
a heating block for 0, 30, 60, 120, 180, and 300 min. Every sample was prepared in triplicate
(all results are given as means ± standard deviation).
155 RESULTS & DISCUSSION
156
157
158
159
160
161
162
163
164
165
166
167
168
Degradation, Formation and Transformation of Carbohydrates. The samples of the
carbohydrate and amino acid mixtures obtained after 300 min of heating were analyzed with
HPLCꢀPAD for degradation of the used carbohydrate component as indication of the
reactivity of the respective system, for the hydrolysis of maltose, and for transformation of
Glc to Dꢀfructose (Fru) (table 2). Without amino component, 6 % of the initial amount of Mal
degraded, wherein around 4 % was cleaved to Glc. The conversion of Mal was accelerated
under amino catalysis leading to a degradation of 10 % (Pro), 26 % (Ala), and 32 % (Lys),
respectively. In all systems considerable amounts of Glc (between 3 to 9 % of the initial Mal
concentration) could be found, originating from hydrolysis of Mal or Mal specific Maillard
reaction intermediates. Of course, these concentrations do not represent the accumulated
amount of Glc formed in these systems, because Glc undergoes Maillard reaction as well.
Under identical conditions Glc is even more reactive than Mal, showing a slightly higher
conversion of 29 % in combination with Ala.
8
ACS Paragon Plus Environment

Page 9 of 35
Journal of Agricultural and Food Chemistry
169
In samples obtained from Glc/Ala, around 5 % of the initial Glc amount is converted to Fru
via isomerization.16 Only trace amounts of Fru can be found in Mal systems, because of the
much lower concentrations of Glc present in these samples.
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
Color Formation and Antioxidant Activity of Carbohydrate Incubations. Maillard
reaction mixtures are typically associated with the formation of color, mostly measured as
absorbance at 420 nm, and an increasing antioxidant activity, determined by different
photometric assays, depending on the chosen reaction conditions.5 In general, both properties
tend to increase over the course of the heating time, resulting in a direct, linear correlation.
Such a trend was observed with two different antioxidant assays for all samples prepared for
this study. The color formation and the antioxidant activities did correspond to the
degradation of the used carbohydrate component in each system. For example, Mal/Lys
showed the strongest degradation of Mal out of all Mal systems and the highest
browning/antioxidant activity, followed by Mal/Ala, Mal/Pro, and Mal. The same trends in
color for the used combinations of carbohydrates and amino components could be observed
under different conditions by Kwak et al.17
The results of the correlation between antioxidant activity (measured with the TEAC assay
and expressed as mmol trolox equivalents (TE) per L) and color are shown in Figure 1. The
same plot for the phenanthroline assay is to be found in the supporting information in Fig. Sꢀ1
(all values for color and the antioxidant activity are given in Table Sꢀ1).
The benefit of using both methods is, that it is possible to distinguish between the total
antioxidant activity of the sample, based on the radical scavenging ability determined with the
TEAC assay, and the metal reducing properties, measured with the phenantroline assay.
Whereas the first method detects all reductones formed in the Maillard reaction, the latter
excludes reductones with complexing abilities, such as maltol, isomaltol, or various pyridinꢀ4ꢀ
ones.8 Both properties correlate linear with the browning of the samples, but the metal
reducing substances in the samples caused antioxidant activities that are around 75 % lower
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 35
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
than the total reducing abilities. This suggests that complexing substances in Maillard reaction
mixtures have a huge impact on the overall antioxidant abilities of the corresponding samples.
Considering, only metal reducing reductones initiate redox cycling and consequently cause
radical generation, these results indicate that antioxidants formed during the Maillard reaction
are mostly beneficial to the oxidative stability of foods and only a fraction bears the risk of
prooxidative effects. In addition, the amount of radical generation caused by metal reducing
reductones is most likely compensated by the antioxidant activity of the sample anyways.
Formation of 1,2-Dicarbonyl Compounds in Carbohydrate Incubations. The general
reaction pathways concerning the formation of 1,2ꢀdicarbonyl compounds from Mal were
thoroughly investigated by Smuda et al.10 These experiments were carried out in phosphate
buffer (pH 7.4) at moderate temperatures (50 °C) and with long incubation times (up to 7
days) with Lys. The focus of the present work was to compare the reactivity of Mal and Glc in
presence of Ala in an unbuffered aqueous model system (initial pH value of 5.0) at elevated
temperatures (130 °C) and short reaction times (up to 300 min) to investigate the reaction
pathways without influence of a complex matrix and to induce strong color formation.
Without buffer the pH value dropped depending on the reactants to pH 4.7 ± 0.0 (Glc/Ala),
4.4 ± 0.0 (Mal/Ala), 3.9 ± 0.1 (Mal), 4.5 ± 0.1 (Mal/Pro), and 3.7 ± 0.0 (Mal/Lys) after
300 min. There were significant differences in the formed amount of reaction intermediates to
expect in comparison to the results of Smuda et al.,10 because pH value,18 temperature,19 and
phosphate20; 21 are known to drastically affect the Maillard reaction.
The concentrations of the main intermediates obtained from Mal and Glc after 300 min
heating time are summarized in Table 3. As already described by Smuda et al.10 degradation
of Mal under Maillard conditions predominantly yields 1,2ꢀdicarbonyl compounds with an
intact backbone of Mal, such as 3ꢀdeoxymaltosone (3ꢀDM), 1ꢀdeoxymaltosone (1ꢀDM), and
maltosone. 3ꢀdeoxyglucosone (3ꢀDG), 3ꢀdeoxygalactosone (3ꢀDGal), and 3ꢀdeoxypentosone
(3ꢀDP) were the only 1,2ꢀdicarbonyls with C6ꢀ or C5ꢀbody that could be found to a relevant
10
ACS Paragon Plus Environment

Page 11 of 35
Journal of Agricultural and Food Chemistry
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
extend. In addition, small amounts of glucosone and 1,4ꢀdideoxyglucosone were detected in
individual samples. Whereas Smuda et al.10 could only find trace amounts of 3ꢀDM and
identified 1ꢀDM as the quantitative most important 1,2ꢀdicarbonyl compound in Mal/Lys
incubations under mild conditions, at 130 °C both compounds were equally relevant in
systems of Mal containing Ala, Pro, or Lys. The differences in the formation of 1ꢀDM and 3ꢀ
DM are most likely attributed to the pH dependent degradation of their precursor in form of
the Amadori rearrangement product (ARP).22; 23 Under acidic conditions the ARP is mainly
degraded to 3ꢀdeoxyosones via 1,2ꢀenolization. On the other hand, the 2,3ꢀenolization and the
formation of 1ꢀdeoxyosones is favored at pH values higher than 7. In addition, phosphate is
known to abstract protons of ARPs24 and could possibly mediate the 2,3ꢀenolization.
In analogy to the color formation Mal systems with Lys showed the highest 1ꢀDM
concentration (272 µM) followed by incubations with Ala (162 µM), Pro (99 µM), and
without amino acid addition (2 µM). However, the 3ꢀDM concentrations did not correspond
to the browning at 420 nm. The reaction mixture with Lys had the highest concentration of 3ꢀ
DM (459 µM), but the caramelization model produced higher amounts of 3ꢀDM (310 µM)
than the Maillard systems with Ala (139 µM) and Pro (59 µM). Maltosone could only be
quantified in the Mal incubations without amino acid (30 µM) after 300 min, but was found in
Mal/Ala and Mal/Pro samples between 30 and 180 min and in Lys samples between 30 and
60 min (data not shown).
When the formation of the respective osones, 1ꢀdeoxyosones, and 3ꢀdeoxyosones in the
Mal/Ala system is compared to Glc/Ala, it is evident that Glc produces higher amounts of 3ꢀ
deoxyosones absolutely and in relation to the osones and 1ꢀdeoxyosones. Furthermore, 3ꢀDP
was only found in trace amounts in the Glc/Ala incubations, but belonged to the main
intermediates in Mal/Ala samples. The importance of 3ꢀDP in the Maillard reaction of Mal
was already recognized by Hollnagel et al.25 and later by Smuda et al.10 The authors explain
the preferred formation from Mal in comparison to Glc with two different mechanisms, but
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 35
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
starting from the corresponding ARP both pathways involve an oxidation step, hydrolytic 1,3ꢀ
dicarbonyl cleavage, and elimination of the Glc moiety (supporting information Fig. Sꢀ2). But
the investigations of Smuda et al.10 strongly suggest the formation via maltosone. However, in
both cases the elimination of Glc is the driving force of the reaction, because of its good
leaving group ability in aqueous solution. This explains the higher amounts of 3ꢀDP formed in
Mal/Ala samples in comparison to Glc/Ala. The substantial differences in the 3ꢀDP
concentrations obtained from incubation of Mal with different amino compounds are likely
caused by the reactivity of the 3ꢀDP degradation product furfural and are discussed later on.
In contrast to Hollnagel et al.26 1,4ꢀdideoxyglucosone (1,4ꢀDDG) could not be identified as
dominating 1,2ꢀdicarbonyl compound in the Maillard reaction of maltose in our study. But
these investigations were carried out in an open, dry system with
Lꢀglycine and most
importantly in presence of OPD, because the authors chose to use a preꢀderivatization method
for the quantification of 1,2ꢀdicarbonyls. As amino compound OPD takes influence on the
Maillard reaction and the trapping of the 1,2ꢀdicarbonyls withdrawals them from the reaction
equilibrium. Therefore, the results are hard to compare to the present study. But there are
indications, that the formation of 1,4ꢀDDG might be favored from Mal over Glc that will be
discussed in the section regarding the formation of Oꢀheterocycles.
Formation of HMF from 3-Deoxyosones in Carbohydrate Incubations. Generally, the
5ꢀ(hydroxymethyl)ꢀ2ꢀfuraldehyde (HMF) concentration was 2 to 10ꢀfold higher than the
summarized concentrations of its precursors. HMF is formed from 3ꢀDM, 3ꢀDG, and 3ꢀDGal
after acetalyzation and aromatization through elimination.27; 28 The crucial intermediate is the
unsaturated 3,4ꢀdideoxyglucosonꢀ3ꢀene (3,4ꢀDGE), which exists as E and Z isomer, but only
the Z form will yield HMF.29 Starting from 3ꢀDM instead of 3ꢀDG or 3ꢀDGal, Glc is
eliminated instead of water to form 3,4ꢀDGE (Figure 2). Due to the fact, that Glc is a much
better leaving group in an aqueous system, higher amounts of HMF could be found in the
incubations of Mal/Ala in comparison to Glc/Ala, even though the concentrations of the
12
ACS Paragon Plus Environment

Page 13 of 35
Journal of Agricultural and Food Chemistry
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
respective precursors in form of the different 3ꢀdeoxyosones are higher in the Glc system
(Table 3). The favored formation of HMF from oligosaccharides in consequence of the better
leaving group ability of the respective carbohydrate moiety was described earlier by Kroh,30
but under different conditions (caramelization in a dry system).
As sideꢀreaction 3,4ꢀDGE is hydrolyzed producing the C6ꢀ3ꢀdeoxyosones 3ꢀDG and 3ꢀ
31
DGal.29;
Both compounds might undergo cleavage reactions or form HMF after
dehydration. Mal/Ala systems should theoretically yield nearly equal amounts of both C6ꢀ
bodies in consequence of the degradation of 3ꢀDM, whereas Glc/Ala should produce an
excess of 3ꢀDG and significant smaller amounts of 3ꢀDGal as the only byꢀproduct. The latter
is clearly shown by the data presented in Table 3, but equal concentrations of 3ꢀDG and 3ꢀ
DGal were only to be found at the beginning of the reaction (30–60 min) in Mal/Ala
incubations (Fig. 3). With increasing heating time the relative quantity of 3ꢀDG rose,
indicating that the degradation of Glc is getting more important due to the elimination of the
monosaccharide from 3ꢀDM in course of the formation of 3,4ꢀDGE. On the other hand,
starting from Glc (Glc/Ala incubation) always an excess of 3ꢀDG was produced.
Investigations in dry systems did show, that Mal/Ala only forms 3ꢀDG.32 The reason for
this observation is the lack of water in these systems and following the reaction scheme in
Fig. 2 liberated Glc is the only source of 3ꢀDG and there is no alternative way to form 3ꢀ
DGal.
Formation of O-Heterocycles in Carbohydrate Incubations. In contrast to HMF, the
heterocyclic intermediates furfural, 2ꢀacetylfuran, isomaltol, DHHM, and maltol were formed
in concentrations comparable to the 1,2ꢀdicarbonyl compounds. The favored formation of 3ꢀ
DP from Mal entails higher concentrations of the subsequently formed furfural and in
consequence the system Mal/Ala contained the 3ꢀfold amount of furfural (143 µM) than
Glc/Ala (43 µM). The lower concentrations of furfural in incubations with Pro (27 µM) and
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 35
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
Lys (90 µM) might be attributed to the formation of unique reaction products of the respective
amino acids with furfural as reported by Hofmann33 or Murata et al.34
DHHM – the main degradation product of 1ꢀdeoxyglucosone (1ꢀDG)35; 36 – was the
quantitative most important heterocycle past HMF. Because 1ꢀDG is mainly produced from
Glc degradation, the highest DHHM concentration can be found in the Glc/Ala system
(487 µM). In Mal incubations without amino acid addition neither 1ꢀDG nor DHHM could be
detected. Even though, in Mal/Pro and Mal/Ala the 1,2ꢀdicarbonyl precursor is not to be
found, DHHM concentrations of 113 µM and 138 µM, respectively, indicate the intermediate
occurrence of 1ꢀDG in these systems. The highest DHHM content of all Mal incubations was
detected in combination with Lys (237 µM) and at the same time Mal/Lys was the only Mal
system in which 1ꢀDG could be quantified (34 µM).
As described in literature35 and as observed in our experiments, maltol is exclusively
formed in incubations containing Mal. Although, the concentration of the respective precursor
1ꢀDM increased in the order Mal (2 µM), Mal/Pro (99 µM), Mal/Ala (162 µM), and Mal/Lys
(272 µM), there were lower concentrations of the pyranꢀ4ꢀone found in Mal/Lys (112 µM)
than in Mal/Ala (171 µM). This suggests that in combination with Lys different degradation
pathways of 1ꢀDM are important, for instance the formation of maltosine.37
Kim et al.38 postulated the formation of 2ꢀacetylfuran starting from DHHM with 1ꢀDG as
intermediate stage. The mechanism can be transferred to Mal or Glc reaction mixtures starting
directly from the respective 1ꢀdeoxyosone (for 1ꢀDM see Fig. 4). The first steps, including the
reduction of 1ꢀDM and the elimination of Glc, resulting in 1,4ꢀDDG are equal to the “peeling
off” mechanism described by Hollnagel et al.26 and Pfeifer et al.14 But the favored formation
of 1,4ꢀDDG from Mal in comparison to Glc, as reported by the named authors, was not
observed, as discussed earlier. In fact, its concentration was similar in the incubations of both
carbohydrates. However, the amount of the subsequently formed 2ꢀacetylfuran differed
strongly and incubations with Mal showed substantially higher amounts of the furan
14
ACS Paragon Plus Environment

Page 15 of 35
Journal of Agricultural and Food Chemistry
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
compound, indicating that the “peeling off” mechanism occurs indeed. The formation of 2ꢀ
acetylfuran from DHHM, as described by Kim et al.,38 seems not to be of importance in
carbohydrate reaction mixtures. Although the Glc/Ala system contained the highest amount of
the pyranꢀ4ꢀone, it did not produce any 2ꢀacetylfuran. On the contrary, the Mal systems
formed 2ꢀacetylfuran, despite the fact that they showed lower DHHM concentrations in
comparison to Glc/Ala.
Degradation, Color Formation, and Antioxidant Activity in Incubations of O-
Heterocycles. Since Furaneol,39 DHHM,38 and isomaltol40 are known as highly reactive
intermediates, their final concentrations in Maillard reaction mixtures of Mal are of limited
value for the estimation of their contribution to relevant properties of the respective systems.
To investigate the stability and their influence on color and antioxidant activity, selected
heterocycles were incubated with Ala at 130 °C. In consideration of our previous
investigations,8 isomaltol, Furaneol, DHHM, and maltol were chosen to represent different
classes of reductone ethers and in addition, HMF was used, because of the high quantities
found in Mal reaction mixtures. The starting concentration of the Oꢀheterocycles was chosen
a decimal power lower (20 mM instead of 200 mM) to reflect the real concentrations and on
account of their limited solubility in water in comparison to the carbohydrates.
The most stable Oꢀheterocycles under these conditions were the aromatic compounds
maltol and HMF, which concentrations were not measurably reduced. Furaneol, DHHM, and
isomaltol on the other hand showed a strong degradation. The Furaneol content was reduced
by around 78 % after 300 min, the DHHM content by 98 %, and isomaltol was not detected in
the respective samples (supporting information Fig. Sꢀ3). But in contrast to carbohydrate
incubations, there is no general connection between the degradation of the respective reactants
and color formation. For instance, both DHHM and isomaltol degraded quickly, but DHHM
samples did not produce colorants, whereas isomaltol samples were strongly colored
(supporting information Fig. Sꢀ4). The browning of isomaltol is most likely caused by
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 35
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
condensation of isomaltol and one of its main degradation products – a C4ꢀfuranone – to a red
colored aromatic compound as described in literature.40 Even though, the concentration of
HMF stayed nearly constant in course of the heating time, incubations of HMF/Ala yielded
yellow solutions and showed the second strongest browning of all investigated Oꢀ
heterocycles. This might be attributed to the formation of small amounts of high colored
polymers through vinylogous aldol addition.41 Furaneol and DHHM are known to form
mostly colorless degradation products, as result of oxidation, reduction, or cleavage reactions.
Consequently, both compounds did not show a measurable browning after incubation with
Ala.
The reaction mixtures resulting from Oꢀheterocycles and Ala were analyzed in regards of
their antioxidant activities with means of the TEAC and phenanthroline assay. In contrast to
carbohydrate systems, which form various reductones in course of their degradation and
consequently exhibited increasing antioxidant activities with increasing heating time and
color formation, the samples derived from Oꢀheterocycles showed a more complex picture
(Fig. 5). Because of their reducing abilities maltol, Furaneol, DHHM and isomaltol showed
antioxidant activities even in samples that are not thermally treated (0 min). The presence of
Ala and the adjusted pH value did influence the antioxidant properties of the reductones
considerably. Whereas isomaltol showed lower antioxidant capacities as the other reductones
when tested isolated,8 the antioxidant activity of the isomaltol/Ala system was much higher
than the activities of maltol, Furaneol, and DHHM in combination with Ala.
Maltol/Ala incubations did not show any changes in their antioxidant activity in the course
of 300 min heating time confirming the thermal stability of maltol. The decreasing antioxidant
activities of Furaneol, DHHM, and isomaltol incubations indicate that the reductone function
is destroyed in most degradation pathways of reductone ethers. But the antioxidant activities
measured after 300 min do not correspond to the remaining concentrations of the used
reductones. This is most obvious for isomaltol which degraded completely in course of the
16
ACS Paragon Plus Environment

Page 17 of 35
Journal of Agricultural and Food Chemistry
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
heating, but still exhibited around 36 % of the initial antioxidant activity. Therefore, certain
degradation products seem to maintain the reducing properties of isomaltol.
HMF is the only compound that showed a behavior similar to the carbohydrates. Lacking a
reductone structure the untreated HMF/Ala samples did not show an antioxidant activity, but
with increasing heating time the activity of the samples rose. However, these antioxidant
activities are considerably lower than the activities of the samples containing reductone ethers
(small diagram in Fig. 5).
In general, the phenanthroline assay showed the same results, except that incubations of
metal chelating reductones, such as maltol and isomaltol, did not exhibit measurable
antioxidant activities, due to the mechanism of this method, which is based on the reduction
of metal ions (supporting information Fig. Sꢀ5).
Differences between the Maillard Reaction of Mal and Glc. Looking at the
intermediates analyzed in this investigation, the product range of the Ala catalyzed
degradation of Mal and Glc bears many similarities. Both systems produced predominantly
1,2ꢀdicarbonyl compounds with an intact carbohydrate backbone. The only relevant C6ꢀ1,2ꢀ
dicarbonyls in Mal/Ala incubations were 3ꢀDG and 3ꢀDGal, which are most probably byꢀ
products of the HMF formation and are not primarily formed from Glc after hydrolysis of
Mal. The relevance of Glc degradation in Mal systems increased during the time of
incubation, because several reaction pathways liberate Glc, for instance the formation of
HMF, furfural, or 2ꢀacetylfuran, and because hydrolysis of Mal gains in importance. The
Maillard reactions of both carbohydrates differ mainly in regard of relative and absolute
quantities of the various intermediates. The summarized concentration of all analyzed 1,2ꢀ
dicarbonyl compounds were slightly higher starting from Glc/Ala. 3ꢀDG was found in a 6ꢀ
fold higher concentration than 1ꢀDG after 300 min (Table 3) in Glc/Ala, whereas in Mal/Ala
the ratio of 3ꢀDM to 1ꢀDM was around 0.86:1. The ratios of 3ꢀDG/3ꢀDGal and the respective
dependencies were discussed in detail in the section about HMF formation.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 35
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
The only 1,2ꢀdicarbonyl compounds that could be found in higher concentrations in Mal
incubations than in Glc incubations besides the Mal specific 1,2ꢀdicarbonyls was 3ꢀDP. The
driving force of this reaction is the elimination of Glc in case of the formation from the
disaccharide.42 Starting from the monosaccharide water has to be eliminated, which is the less
favorable leaving group in an aqueous setup. The higher concentration of 3ꢀDP was directly
translated into a higher concentration of the subsequently formed furfural.
Besides furfural also HMF and 2ꢀacetylfuran were formed in higher quantities from Mal,
as consequence of the elimination of a glucosyl moiety from the respective intermediates. An
exception was the degradation of 1ꢀdeoxyosones to DHHM or maltol. Maltol was exclusively
formed in Mal systems, whereas DHHM was found in both systems, but in significantly
higher amounts in Glc incubations. However, even the summarized concentration of both
pyranones in Mal/Ala was lower than the DHHM concentration in Glc/Ala. Considering that
only low concentrations of 1ꢀDG were found in comparison to subsequently formed DHHM,
the reaction seems to be fast. Maltol on the other hand was detected in almost identical
concentrations as its precursor 1ꢀDM, indicating a rather slow conversion. In addition, the
formation of 2ꢀacetylfuran is a competitive pathway to the maltol formation in the degradation
of 1ꢀDM, but seems to be irrelevant starting from 1ꢀDG.
But, even though the concentrations of most Oꢀheterocycles, especially of HMF as typical
indicator substance for heat treatment, were considerably higher in Mal incubations than in
Glc incubations, Glc showed a faster degradation and a stronger color formation under
identical conditions. These contradictory observations might be explained by results obtained
from the incubations of Oꢀheterocycles with Ala. Of course, the degradation of the respective
reactants and the color yields in these experiments have to be interpreted carefully, because
the behavior of these compounds could drastically change in complex Maillard reaction
mixtures. Nevertheless, these results indicate that furans and completely conjugated pyranons
preferably formed by Mal, namely HMF, furfural, and maltol, are rather stable compounds,
18
ACS Paragon Plus Environment

Page 19 of 35
Journal of Agricultural and Food Chemistry
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
whereas Glc predominantly produced the highly reactive DHHM. The stability of HMF can
also be seen in the carbohydrate incubations, because it does accumulate in amounts roughly a
decimal power higher than all other intermediates. Despite its low thermal stability in
presence of Ala, DHHM is detected in relatively high concentrations in carbohydrate
incubations, indicating that it has to be formed in even higher intermediate concentrations and
might have a crucial role in the reaction cascade. And although, the trends in color formation
of the incubations of DHHM/Ala and HMF/Ala suggest the contrary, it is likely that DHHM
and/or its degradation products contribute to browning reactions. But this hypothesis has to be
verified in further investigation through the utilization of more complex model systems.
The absence of other highly reactive heterocycles in the carbohydrate systems, such as
furanone derivatives or isomaltol, might indicate that these compounds react too fast under
the used conditions to be tracked as intermediates. This could be investigated in following
studies by the analysis of their characteristic degradation products. Hence, in order to fully
understand the role of heterocyclic intermediates in the Maillard reaction on browning
additional work is needed. Especially the isolation and structure elucidation of the formed
colorants by means of MS and NMR is of great interest.
The antioxidant properties of the carbohydrate incubations cannot be explained by the
concentrations of the analyzed reductones, indicating that several of the subsequently formed
intermediates and end products also contribute to reducing and complexing abilities of the
respective samples. However, our data suggest a connection between the formation of colored
and antioxidant substances. A possible explanation might be condensation reactions that
integrate intact reductones in the skeleton of melanoidin polymers, for instance the aldol
condensation of furanones and furanꢀ2ꢀaldehyd derivatives.43 Furthermore, the incubations of
HMF/Ala did show an increase of antioxidant properties indicating that the polymerization of
nonꢀreducing Maillard intermediates might lead to the formation of reducing functional
groups.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 35
454 ABBREVATIONS USED
455
456
457
458
459
460
461
462
463
464
465
466
1,4ꢀDDG, 1,4ꢀdideoxyglucosone; 1ꢀDG, 1ꢀdeoxyglucosone; 1ꢀDM, 1ꢀdeoxymaltosone;
3,4ꢀDGE, 3,4ꢀdideoxyglucosonꢀ3ꢀene; 3ꢀDG, 3ꢀdeoxyglucosone; 3ꢀDGal, 3ꢀ
deoxygalactosone; 3ꢀDM, 3ꢀdeoxymaltosone; 3ꢀDP, 3ꢀdeoxypentosone; Abs[420], absorbance
at 420 nm; ABTS, 2,2'ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ6ꢀsulphonic acid); Ala, ꢀalanine;
ARP, Amadori rearrangement product; DAD, diode array detector; DHHM, 3,5ꢀdihydroxyꢀ6ꢀ
methylꢀ2,3ꢀdihydroꢀ4Hꢀpyranꢀ4ꢀone; F, dilution factor; Fru, ꢀfructose; GC, gas
chromatography; Glc, ꢀglucose; Lys, ꢀlysine; Mal, maltose; MS, mass spectrometry; nd, not
detected; NMR, nuclear magnetic resonance (spectroscopy); Pro, ꢀproline; OPD, orthoꢀ
L
D
D
L
L
phenylendiamin; PAD, pulsed amperometric detector; PBS, phosphateꢀbuffered saline; Q,
quinoxaline; TE, trolox equivalent(s); TEAC, trolox equivalent antioxidant capacity; tR,
retention time.
467 ACKNOWLEDGEMENT
468
This publication uses data collected for the PhD thesis of Clemens Kanzler.32
20
ACS Paragon Plus Environment

Page 21 of 35
Journal of Agricultural and Food Chemistry
469 REFERENCES
470
471
472
[1] van Boekel, M. A. J. S.; Fogliano, V.; Pellegrini, N.; Stanton, C.; Scholz, G.; Lalljie, S.
et al. A review on the beneficial aspects of food processing. Mol. Nutr. Food Res. 2010,
54, 1215–1247.
473
474
[2] Hodge, J. E. Dehydrated Foods, Chemistry of Browning Reactions in Model Systems. J.
Agric. Food Chem. 1953, 1, 928–943.
475
476
477
[3] Ledl, F.; Schleicher, E. Die MaillardꢀReaktion in Lebensmitteln und im menschlichen
Körper – neue Ergebnisse zu Chemie, Biochemie und Medizin. Angew. Chem. 1990,
102, 597–626.
478
479
[4] Hellwig, M.; Henle, T. Baking, ageing, diabetes: a short history of the Maillard reaction.
Angew. Chem. Int. Ed. Engl. 2014, 53, 10316–10329.
480
481
482
[5] Manzocco, L.; Calligaris, S.; Mastrocola, D.; Nicoli, M. C.; Lerici, C. R. Review of
nonꢀenzymatic browning and antioxidant capacity in processed foods. Trends Food Sci.
Tech. 2000, 11, 340–346.
483
484
[6] Kanzler, C.; Haase, P. T.; Kroh, L. W. Antioxidant capacity of 1ꢀdeoxyꢀ
Dꢀerythroꢀhexoꢀ
2,3ꢀdiulose and ꢀarabinoꢀhexoꢀ2ꢀulose. J. Agric. Food Chem. 2014, 62, 2837–2844.
D
485
486
487
[7] Haase, P. T.; Kanzler, C.; Hildebrandt, J.; Kroh, L. W. Browning Potential of C6ꢀalphaꢀ
Dicarbonyl Compounds under Maillard Conditions. J. Agric. Food Chem. 2017, 65,
1924–1931.
488
489
490
[8] Kanzler, C.; Haase, P. T.; Schestkowa, H.; Kroh, L. W. Antioxidant Properties of
Heterocyclic Intermediates of the Maillard Reaction and Structurally Related
Compounds. J. Agric. Food Chem. 2016, 64, 7829–7837.
491
492
[9] Oikawa, N.; Müller, C.; Kunz, M.; Lichtenthaler, F. W. Hydrophilically functionalized
pyrazoles from sugars. Carbohydr. Res. 1998, 309, 269–279.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 35
493
494
[10] Smuda, M.; Glomb, M. A. Novel insights into the maillard catalyzed degradation of
maltose. J. Agric. Food Chem. 2011, 59, 13254–13264.
495
496
[11] Hellwig, M.; Degen, J.; Henle, T. 3ꢀdeoxygalactosone, a "new" 1,2ꢀdicarbonyl
compound in milk products. J. Agric. Food Chem. 2010, 58, 10752–10760.
497
498
[12] El Khadem, H.; Horton, D.; Meshreki, M. H.; Nashed, M. A. New route for the
synthesis of 3ꢀdeoxyaldosꢀ2ꢀuloses. Carbohydr. Res. 1971, 17, 183–192.
499
500
501
[13] Madson, M. A.; Feather, M. S. An improved preparation of 3ꢀdeoxyꢀDꢀerythroꢀhexosꢀ2ꢀ
ulose via the bis(benzoylhydrazone) and some related constitutional studies. Carbohydr.
Res. 1981, 94, 183–191.
502
503
504
[14] Pfeifer, Y. V.; Kroh, L. W. Investigation of reactive alphaꢀdicarbonyl compounds
generated from the Maillard reactions of Lꢀmethionine with reducing sugars via their
stable quinoxaline derivatives. J. Agric. Food Chem. 2010, 58, 8293–8299.
505
506
507
[15] Wegener, S.; Kaufmann, M.; Kroh, L. W. Influence of lꢀ pyroglutamic acid on the color
formation process of nonꢀenzymatic browning reactions. Food Chem. 2017, 232, 450–
454.
508
509
510
[16] Harris, D. W.; Feather, M. S. Evidence for a Cꢀ2→Cꢀ1 intramolecular hydrogenꢀ
transfer during the acidꢀcatalyzed isomerization of Dꢀglucose to Dꢀfructose. Carbohydr.
Res. 1973, 30, 359–365.
511
512
[17] Kwak, E.ꢀJ.; Lim, S.ꢀI. The effect of sugar, amino acid, metal ion, and NaCl on model
Maillard reaction under pH control. Amino Acids 2004, 27, 85–90.
513
514
[18] Ajandouz, E. H.; Puigserver, A. Nonenzymatic Browning Reaction of Essential Amino
Acids. J. Agric. Food Chem. 1999, 47, 1786–1793.
515
516
[19] Martins, S. I.; van Boekel, M. A. A kinetic model for the glucose/glycine Maillard
reaction pathways. Food Chem. 2005, 90, 257–269.
22
ACS Paragon Plus Environment

Page 23 of 35
Journal of Agricultural and Food Chemistry
517
518
[20] Rizzi, G. P. Role of phosphate and carboxylate ions in maillard browning. J. Agric.
Food Chem. 2004, 52, 953–957.
519
520
[21] Bell, L. N. Maillard reaction as influenced by buffer type and concentration. Food
Chem. 1997, 59, 143–147.
521
522
[22] Martins, S. I.; Jongen, W. M.; van Boekel, M. A. A review of Maillard reaction in food
and implications to kinetic modelling. Trends Food Sci. Technol. 2000, 11, 364–373.
523
524
[23] Martins, S. I.; van Boekel, M. A. Kinetics of the glucose/glycine Maillard reaction
pathways. Food Chem. 2005, 92, 437–448.
525
526
[24] Gil, H.; Salcedo, D.; Romero, R. Effect of phosphate buffer on the kinetics of glycation
of proteins. J. Phys. Org. Chem. 2005, 18, 183–186.
527
528
529
[25] Hollnagel, A.; Kroh, L. W. 3ꢀdeoxypentosulose: an αꢀdicarbonyl compound
predominating in nonenzymatic browning of oligosaccharides in aqueous solution. J.
Agric. Food Chem. 2002, 50, 1659–1664.
530
531
532
[26] Hollnagel, A.; Kroh, L. W. Degradation of oligosaccharides in nonenzymatic browning
by formation of α‑dicarbonyl compounds via a “peeling off” mechanism. J. Agric. Food
Chem. 2000, 48, 6219–6226.
533
534
535
[27] Antal, M. J., JR; Mok, W. S.; Richards, G. N. Mechanism of formation of 5ꢀ
(hydroxymethyl)ꢀ2ꢀfuraldehyde from
Dꢀfructose and sucrose. Carbohydr. Res. 1990,
199, 91–109.
536
537
538
[28] Usui, T.; Yanagisawa, S.; Ohguchi, M.; Yoshino, M.; Kawabata, R.; Kishimoto, J. et al.
Identification and determination of αꢀdicarbonyl compounds formed in the degradation
of sugars. Biosci. Biotechnol. Biochem. 2007, 71, 2465–2472.
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 35
539
540
541
[29] Hellwig, M.; Nobis, A.; Witte, S.; Henle, T. Occurrence of (Z)ꢀ3,4ꢀDideoxyglucosonꢀ3ꢀ
ene in Different Types of Beer and Malt Beer as a Result of 3ꢀDeoxyhexosone
Interconversion. J. Agric. Food Chem. 2016, 64, 2746–2753.
542
[30] Kroh, L. W. Caramelisation in food and beverages. Food Chem. 1994, 51, 373–379.
543
544
545
546
[31] Mittelmaier, S.; Fünfrocken, M.; Fenn, D.; Pischetsrieder, M. 3ꢀDeoxygalactosone, a
new glucose degradation product in peritoneal dialysis fluids: identification,
quantification by HPLC/DAD/MSMS and its pathway of formation. Anal. Bioanal.
Chem. 2011, 399, 1689–1697.
547
548
549
[32] Kanzler, C. Furane, Pyrrole und Furanone – ihr Beitrag zur Farbe und den
antioxidativen Eigenschaften in der MaillardꢀReaktion der Maltose. Dissertation,
Technische Universität Berlin, 2017.
550
551
552
[33] Hofmann, T. Characterization of the Chemical Structure of Novel Colored Maillard
Reaction Products from Furanꢀ2ꢀcarboxaldehyde and Amino Acids. J. Agric. Food
Chem. 1998, 46, 932–940.
553
554
555
[34] Murata, M.; Totsuka, H.; Ono, H. Browning of furfural and amino acids, and a novel
yellow compound, furpipate, formed from lysine and furfural. Biosci. Biotechnol.
Biochem. 2007, 71, 1717–1723.
556
557
[35] Yaylayan, V. A.; Mandeville, S. Stereochemical control of maltol formation in Maillard
reaction. J. Agric. Food Chem. 1994, 42, 771–775.
558
559
560
[36] Voigt, M.; Smuda, M.; Pfahler, C.; Glomb, M. A. Oxygenꢀdependent fragmentation
reactions during the degradation of 1ꢀdeoxyꢀDꢀerythroꢀhexoꢀ2,3ꢀdiulose. J. Agric. Food
Chem. 2010, 58, 5685–5691.
561
562
[37] Kramhöller, B.; Pischetsrieder, M.; Severin, T. Maillard reactions of lactose and
maltose. J. Agric. Food Chem. 1993, 41, 347–351.
24
ACS Paragon Plus Environment

Page 25 of 35
Journal of Agricultural and Food Chemistry
563
564
[38] Kim, M.ꢀO.; Baltes, W. On the role of 2,3ꢀdihydroꢀ3,5ꢀdihydroxyꢀ6ꢀmethylꢀ4(H)ꢀpyranꢀ
4ꢀone in the Maillard reaction. J. Agric. Food Chem. 1996, 44, 282–289.
565
566
[39] KunertꢀKirchhoff, J.; Baltes, W. Model reactions on roast aroma formation. Z. Lebensm.
Unters. For. 1990, 190, 14–16.
567
568
569
[40] Goodwin, J. C.; Hodge, J. E.; Weisleder, D. Preparation and structure of an unusual
dimeric furan from the acid decomposition of isomaltol. Carbohydr. Res. 1986, 146,
107–112.
570
571
572
[41] Nikolov, P. Y.; Yaylayan, V. A. Thermal decomposition of 5ꢀ(hydroxymethyl)ꢀ2ꢀ
furaldehyde (HMF) and its further transformations in the presence of glycine. J. Agric.
Food Chem. 2011, 59, 10104–10113.
573
574
[42] Rakete, S.; Klaus, A.; Glomb, M. A. Investigations on the Maillard reaction of dextrins
during aging of Pilsner type beer. J. Agric. Food Chem. 2014, 62, 9876–9884.
575
576
[43] Ledl, F.; Severin, T. Bräunungsreaktionen von Pentosen mit Aminen. Z. Lebensm.
Unters. For. 1978, 167, 410–413.
577
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 35
TABLES
Table 1: Overview of the prepared incubations of carbohydrates and heterocyclic
intermediates.
carbohydrate/
heterocycle
concentration (mM)
amino acid
concentration (mM)
Glc
200
200
200
200
200
20
Ala
–
200
200
200
200
200
20
Mal
Mal
Ala
Pro
Lys
Ala
Ala
Ala
Ala
Ala
Mal
Mal
HMF
maltol
Furaneol
DHHM
isomaltol
20
20
20
20
20
20
20
20
26
ACS Paragon Plus Environment

Page 27 of 35
Journal of Agricultural and Food Chemistry
Table 2: Carbohydrate concentrations in reaction mixtures of Glc/Ala, Mal, Mal/Pro,
Mal/Ala, and Mal/Lys after 300 min of heating at 130 °C and pH 5.
concentration (mM)
Glc/Ala
nd
Mal
188 ± 19
16 ± 1
nd
Mal/Pro
179 ± 12
10 ± 1
nd
Mal/Ala
148 ± 2
18 ± 0
1 ± 0
Mal/Lys
135 ± 2
35 ± 1
nd
Mal
Glc
Fru
141 ± 2
9 ± 1
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 35
Table 3: Concentrations of 1,2ꢀdicarbonyl compounds and heterocyclic intermediates formed
from the degradation of Glc/Ala, Mal, Mal/Pro, Mal/Ala, and Mal/Lys after 300 min of
heating at 130 °C and pH 5.
concentration (µM)
Glc/Ala
23 ± 3
93 ± 8
548 ± 48
76 ± 9
4 ± 6
14 ± 2
nd
Mal
nd
Mal/Pro
nd
Mal/Ala
4 ± 0
Mal/Lys
nd
glucosone
1ꢀdeoxyglucosone
3ꢀdeoxyglucosone
3ꢀdeoxygalactosone
3ꢀdeoxypentosone
1,4ꢀdideoxyglucosone
maltosone
nd
nd
nd
34 ± 3
172 ± 3
55 ± 6
21 ± 1
13 ± 5
nd
79 ± 9
48 ± 7
3 ± 1
nd
23 ± 2
12 ± 1
35 ± 4
nd
137 ± 12
71 ± 3
92 ± 3
16 ± 0
nd
30 ± 9
2 ± 2
310 ± 35
nd
1ꢀdeoxymaltosone
3ꢀdeoxymaltosone
HMF
nd
99 ± 11
59 ± 8
487 ± 57
27 ± 2
113 ± 1
87 ± 7
nd
162 ± 3
139 ± 5
272 ± 16
459 ± 13
nd
1210 ± 113 1694 ± 137
2064 ± 91 6518 ± 107
furfural
45 ± 13
487 ± 13
nd
43 ± 9
nd
143 ± 10
138 ± 3
171 ± 2
nd
90 ± 9
237 ± 10
112 ± 6
34 ± 5
DHHM
maltol
nd
isomaltol
nd
nd
2ꢀacetylfuran
nd
nd
43 ± 3
54 ± 2
85 ± 2
28
ACS Paragon Plus Environment

Page 29 of 35
Journal of Agricultural and Food Chemistry
578 FIGURE CAPTIONS
579
580
581
582
583
584
585
586
587
Figure 1: Correlation between antioxidant activity (TEAC) and color in all carbohydrate
incubations (n = 30, m = 3).
Figure 2: Formation of HMF from 3ꢀDM with 3,4ꢀDGE as relevant intermediate and the
C6ꢀ3ꢀdeoxyosones 3ꢀDG and 3ꢀDGal as byꢀproducts.
Figure 3: Ratio of 3ꢀDG to 3ꢀDGal in incubation of Glc/Ala and Mal/Ala in the course of
300 min heating time.
588
589
Figure 4: Formation of 2ꢀacetylfuran from 1ꢀDM through reduction, βꢀelimination of Glc,
cyclization, and elimination of two moles of water adopted from Kim et al.38 and
Hollnagel et al.26 R’ represents a glucosyl moiety.
590
591
592
593
594
Figure 5: Changes in the antioxidant activity of maltol/Ala, Furaneol/Ala, DHHM/Ala,
HMF/Ala, and isomaltol/Ala at pH 5 and 130 °C measured with the TEAC assay.
29
ACS Paragon Plus Environment