Formation of Phenolic Compounds from d -Galacturonic Acid

Article abstract of DOI:10.1021/acs.jafc.8b04158

Aqueous d-galacturonic acid (d-GalA) model systems treated at 130 °C at different pH values show an intense color formation, whereas other reducing sugars, such as d-galactose (d-Gal), scarcely react. GC-MS measurements revealed the presence of several ph

Full text of DOI:10.1021/acs.jafc.8b04158

Subscriber access provided by UNIV OF LOUISIANA
Food and Beverage Chemistry/Biochemistry
Formation of Phenolic Compounds from D-Galacturonic Acid
Alexandra Urbisch, U. Einhorn-Stoll, Hanna Kastner, Stephan Drusch, and Lothar W. Kroh
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04158 • Publication Date (Web): 08 Oct 2018
Downloaded from http://pubs.acs.org on October 11, 2018
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 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 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.
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 31
Journal of Agricultural and Food Chemistry
Formation of Phenolic Compounds from D-Galacturonic Acid
Alexandra Urbisch a*, Ulrike Einhorn-Stoll b, Hanna Kastner b, Stephan Drusch b and
Lothar W. Kroh a
a Technische Universität Berlin, Food Chemistry and Analytics, Germany
b Technische Universität Berlin, Food Technology and Food Material Sciences, Germany
Gustav-Meyer-Allee 25, 13355 Berlin, Germany
*Corresponding author: a.urbisch@tu-berlin.de, phone: +49 30 314 72782,
fax: +49 30 314 72 585
RECEIVED DATE (to be automatically inserted after your manuscript is accepted
if required according to the journal that you are submitting your paper to)
TITLE RUNNING HEAD
Formation of Phenolic Compounds from D-Galacturonic Acid
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 31
1
ABSTRACT
2
3
Aqueous D-galacturonic acid (D-GalA) model systems treated at 130 °C at different pH
values show an intense color formation, whereas other reducing sugars, like D-galactose
(D-Gal), scarcely react. GC-MS measurements revealed the presence of several phenolic
compounds e.g. 3,8-dihydroxy-2-methyl-4H-chromen-4-one (chromone) and 2,3-
dihydroxybenzaldehyde (2,3-DHBA). These phenolic compounds, especially 2,3-DHBA,
possess an intense browning potential and cannot be found within heated model solutions
of reducing sugars. Investigations regarding the formation of these substances show that
α-ketoglutaraldehyde plays an important role as an intermediate product. In addition, MS
analysis of model systems of norfuraneol in combination with 2,3-DHBA showed the
formation of oligomers that could also be detected in D-GalA model systems, leading to
the assumption that, besides reductic acid, these compounds are jointly responsible for
the strong color formation during the heat treatment of D-GalA.
4
5
6
7
8
9
10
11
12
13
14
15
KEYWORDS
16
17
18
19
D-galacturonic acid, 2,3-dihydroxybenzaldehyde, 3,8 dihydroxy-2-methyl-4H-chromen-4-
one, D-galactose furfural, polygalacturonic acid, pectin, polyphenol, catechol,
methylcatechol, α-ketoglutaraldehyde
2
ACS Paragon Plus Environment

Page 3 of 31
Journal of Agricultural and Food Chemistry
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
INTRODUCTION
Since the beginning of food processing, the thermal formation of reaction products formed
via non-enzymatic browning has increasingly become of interest. Depending on the
application, these mechanisms are either desirable or not. Uronic acids are abundant in
nature and consequently play a very important role in the formation of browning and
volatile products in groceries. For D-galacturonic acid (D-GalA), which is present in the
backbone of the pectin molecule, a drastic influence on the color formation was already
reported in several studies, e.g. Hodge et al.1-3 Uronic acids in general show a stronger
browning potential than reducing sugars, such as D-galactose (D-Gal).2,4-6 It was observed
that one of the major chromophore developing substances in model systems of these
sugar acids is reductic acid. It is formed after decarboxylation of D-GalA and further
dehydration reactions that lead to the reactive α-ketoglutaraldehyde. Bornik succeeded
in verifying this important intermediate through a derivatization reaction with
dinitrophenylhydrazine and subsequent MS analysis.7 This intermediate then cyclizes into
carbocyclic pentenones, such as reductic acid.6 In model reaction systems, the pure
substance showed the development of reddish-brown polymers.8 This color drift into red
could also be observed in D-galacturonic acid model systems. Still, the intense browning
formation of heat-treated D-GalA model systems without amine catalysis cannot solely be
explained through the formation of reductic acid.
Another interesting pathway for the formation of browning precursors could be observed
by Popoff and Theander. They were able to prove that several polyphenolic reaction
products were formed during the heat treatment of reducing sugars and uronic acid.5 They
assumed that the polyphenolic compounds may have an influence on the formation of
color within the model systems but did not explore the matter further. Later, the same
group, postulated that the phenolic compounds were intermediates of the temperature
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 31
45
46
47
48
49
50
51
52
53
54
55
56
57
induced degradation of uronic acids and that unsaturated aldehydes take part in their
formation mechanism.9
The aim of the present paper is to attempt a more in-depth analysis of the reaction variety
of uronic acids. GC-MS measurements were conducted to reveal the product spectrum
and kinetics during the heat treatment of D-GalA and oligo- or polymer representatives,
such as pectin or polygalacturonic acid, at different reaction conditions. The analysis of
the degradation products focuses primarily on polyphenolic compounds in combination
with the formation of color. Aqueous model systems of D-GalA were therefore heated at
temperatures between 130 °C to 160 °C and pH values between pH 3 and 8. Single
compounds that appeared to possess a high browning potential were individually heated
and characterized using various analytical methods. Furthermore, the formation
mechanism of these compounds was reconstructed through the reaction of known
intermediates.
4
ACS Paragon Plus Environment

Page 5 of 31
Journal of Agricultural and Food Chemistry
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
MATERIALS AND METHODS
Chemicals. The following compounds were obtained commercially: D-galacturonic acid
monohydrate, furfural, norfuraneol, 2,3-dihydroxybenzaldehyde, D-glucuronic acid,
scopoletin, L-arabinose and the solvents (HPLC- or GC ultra-grade) were purchased from
Sigma-Aldrich (Steinheim, Germany); L-alanine, D-galactose, D-xylose, and sodium
acetate were purchased from Merck (Darmstadt, Germany), MN Polyamid SC 6 was
purchased from Macherey-Nagel (Düren, Germany)
Syntheses.
3,8-dihydroxy-2-methyl-4H-chromen-4-one. 3,8-dihydroxy-2-methyl-4H-chromen-4-one
(chromone) was synthesized using a slightly modified method of Lindgren and
Pernemalm 1980.10 5.0 g of D-xylose were dissolved in 200 mL of an acetate buffer
(0.5 mol/L) solution and the pH was adjusted to 4.5. The solution was heated for 48 hours
under reflux and, after cooling down, extracted with ethyl acetate (3x100 mL). The
combined extract was dried with anhydrous sodium sulfate and the solvent completely
removed under a nitrogen stream. Clean-up was done using a SPE phase. The residue
was then resolved in water and added onto the polyamide cartridge (MN Polyamid SC 6,
particle size 0.05-0,16 mm, Macherey-Nagel, Düren, Germany). The cartridge was rinsed
with 10 mL of distilled water, after which the product was eluted with a mixture of
methanol/water/acetic acid (90:5:5, v:v:v). The organic solvent was removed by vacuum
distillation and the residual was freeze-dried. Purity was determined via HPLC-RI and
GC-MS detection. The product was yielded in 92 % purity. GC-MS tR = 32.11 min. m/z
192 [M+, 100 %], 163 (15 %), 146 (10 %), 137 (42 %), 121 (38 %), 108 (7 %), 79 (9 %),
65 (11 %)
Model Reaction. D-galacturonic acid. To investigate the formation of volatile and
potential chromophore-building substances, D-galacturonic acid (0.25 mol/L), pectin
(0.25 mol/L D-GalA equivalent) and polygalacturonic acid (PGA) (0.25 mol/L D-GalA
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 31
84
85
equivalent) were dissolved in water and the pH was adjusted with NaOH and HCl to 3.0,
6.0 and 8.0 (to prevent the formation of lumps the polymer compounds were added to a
stirred solution). The use of buffering solutions was avoided, as they are known to change
the reaction process. The thusly prepared solutions were filled into headspace vials and
heated in heating blocks at 130 °C for varying lengths of time, up to a maximum of
4 hours. The solutions were cooled down and spiked with an internal standard and
subsequently extracted three times with 10 mL dichloromethane. The solvent was
removed under a nitrogen stream and 250 µL of the residue dissolved in dichloromethane
was analyzed by GC-MS (Shimadzu, QP 2010, Duisburg, Germany). The browning
intensity of the heated solutions was measured with a UV/VIS spectrometer at 420 nm
and converted into CIELab colors (Specord® 40, Analytik Jena, Jena, Germany). The
change in the pH value was measured after cooling down to room temperature with a pH-
electrode (pH-Meter 761 Calimatic, Knick). Furthermore, the formation of potential
browning polymers and the degradation of the pectin molecule were measured with
HPLC-SEC-UV-VIS/RI (size exclusion chromatography) at 420 nm and a refractometric
index detector, as described previously by Wegener et al.11
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
2,3-dihydroxybenzaldehyde. The concentration of model reaction systems containing
2,3-dihydroxybenzaldehyde (6) was 0.025 mol/L. Spiked model systems contained the
doped substance in concentrations that were also found in D-galacturonic acid or pectin
systems.
Qualification and Quantitation.
To ensure a correct assignment of the peaks in the GC chromatogram, standard
substances were measured and compared with the sample. Quantitation was done using
an internal standard (scopoletin) and response factors were determined with reference
substances. A three-fold determination was performed.
6
ACS Paragon Plus Environment

Page 7 of 31
Journal of Agricultural and Food Chemistry
110
Gas Chromatography−Mass Spectrometry
111
112
113
114
115
116
117
118
119
120
121
122
123
GC-MS investigations were performed on a Shimadzu GC-QP 2010, using a capillary
DP-5 column (60 m, 0.25 mm, 0.25 µm, Supelco SLB-5MS, Bellefonte, PA, USA). The
measurements of the samples were performed via split injection (1:5), using helium as
carrier gas, with a flow rate of 2 mL/min. The injection temperature was set to 270 °C and
the initial temperature, for the temperature gradient, to 30 °C. After 3 minutes, the
temperature was raised to 120 °C at a rate of 5 °C/min. Subsequently, the temperature
was increased to 200 °C, at a rate of 20 °C/min, and held for 5 minutes. Again, the
temperature was raised to 250 °C, at a rate of 20 °C/min. This temperature was held for
5 minutes, before it was ultimately increased to 320 °C, at a rate of 20 °C/min, and held
for another 15 minutes. To ensure that the all molecules remain in the gas phase the
interface temperature was adjusted to 270 °C and the ion source to 200 °C. An ionization
energy of 70 eV was used and a mass range of 35-400 m/z was scanned. The solvent
cut time was set to 5 minutes.
124
Electrospray Ionization-Mass Spectrometry
125
126
127
128
129
130
ESI-MS investigations were performed on a LTQ Orbitrap XL (Thermo Fischer Scientific,
Bremen, Germany), between a mass range of 150-1000 m/z. The source voltage was set
to 3.54 kV and a current of 1.01 uA. The capillary temperature was 300 °C. The samples
were diluted with methanol/water to 1 mg/mL and directly injected to the spectrometer
with a syringe pump at a flow rate of 20 µL/min. Measurements were performed using a
negative electrospray ionization mode.
131
132
133
134
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Parameters used for the matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS) were as follows. All experiments were performed on a
Bruker Daltonics Autoflex III smartbeam MALDI TOF mass spectrometer (Bruker
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 31
135
136
137
138
139
140
141
142
Daltonics, Bremen) equipped with a 50 Hz pulsed N2 laser (wavelength 337 nm). The ion
source was set to a voltage of 19.00 kV, lens and reflector had a voltage of 8.50 and
20.00 kV. Laser intensity was set to 60 %. The MALDI matrix was prepared by dissolving
25 mg/mL 2,5-dihydroxybenzoic acid in acetonitrile/water (30/70, v/v) and 0.1 %
trifluoroacetic acid. Samples were dissolved in methanol and diluted to 1 mg/mL. 0.5 µL
matrix were spread on a MALDI plate and mixed with 0.5 µL of sample. The solution could
dry at room temperature and build a layer of crystals. The scanning range was set from
400-2000 m/z.
8
ACS Paragon Plus Environment

Page 9 of 31
Journal of Agricultural and Food Chemistry
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
RESULTS and DISCUSSION
Polyphenol formation during the degradation of D-galacturonic acid
Uronic acids, in this case D-galacturonic acid, as well as reducing sugars, such as
D-galactose, are structurally very similar. Both substances have of a pyranoid backbone
and a carbonyl group at C-1 that is next to the C-4 OH function included in the glycosidic
bond of oligo- and polymers. Sugar acids consist furthermore of a carboxylic group
located at C-6 that influences the reactivity of the uronic acids. Earlier works have already
shown that D-GalA reacts far more drastically than D-Gal although they are structurally
related. The heat treatment of aqueous model systems of D-GalA, for example, showed
a ten times higher browning compared to that of D-Gal and L-Ara at temperatures of
130 °C and a heating time of 240 minutes (figure 1).8 In addition to products, such as
furfural and norfuraneol that are also generated during the degradation of D-Gal and
D-Ara, 2-furoic acid, 5-formyl-2-furoic acid and reductic acid are specific degradation
products for uronic acids that may influence the color development. Alongside these
already known degradation products (e.g. Bornik (2013)7, Madson (1979)2, Isbell
(1944))6, Popoff and Theander were able to determine the formation of phenolic reaction
products during the reaction of sugars and uronic acids in aqueous solutions under reflux
at a temperature of 96 °C and a heating time of 48 hours. Based on these experiments,
one can see that the use alone of D-glucuronic acid under basic conditions forms phenolic
compounds. Reducing sugars react to form phenolic compounds under Maillard-Reaction
(glycine) conditions. Without amino acid catalysis, only traces of phenolic products can
be detected. The reaction products found include catechol (1), methylcatechol (2), 3,8-
dihydroxy-2-methyl-4H-chromen-4-one (chromone, 3), 5,6,7,8-tetrahydro-3,5-dihydroxy-
2-methyl-8-oxobenzo-pyrene (4), trihydroxybenzaldehyde, 2,3-dihydroxybenzoic acid
and 2,3-dihydroxyacetophenone.5
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 31
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
In the present study, it was examined what kind of reaction products are formed
throughout the degradation of D-galacturonic acid (0.25 mol/L) during heat treatment at
130 °C, in acidic and basic conditions, in comparison to D-Gal model reaction systems.
Buffering solutions were not used to prevent a possible influence of these substances on
the reaction. In accordance with other publications, furfural (3.5 mmol/L, pH 3, 130°C, 2
h) was found to be one of the main products produced in model systems in an acidic
milieu, followed by norfuraneol (0.02 mmol/L, pH 3, 130°C, 2 h)8, 12. In addition to these
already very well-known oxygen heterocycles, degradation products, such as short chain
aldehydes, e.g. glycolaldehyde amounting to 1 % or acetaldehyde, amounting to 25 % of
the analyzed aldehydes, could be detected. These aldehydes may contribute to the
formation of phenolic compounds. Along with these well-known sugar degradation
products, several phenolic compounds were also able to be identified 5,13-16. Besides the
already described phenolic compounds, such as 1, 2, 3 and 4, we could detect
2,3-dihydroxybenzaldehyde (6) and 3,4-dihydroxyacetophenone (5) (figure 2). These
additional phenolic substances could be identified via comparison with reference
substances and interpretation of EI-MS and ESI-MS/MS spectra. The present objective
is to examine whether, and to what extent, these phenolic compounds can form
chromophoric polymers that influence the color of the D-GalA model system. It is known
that phenolic compounds show a strong tendency to polymerize due to oxidative coupling
reactions, resulting in colored compounds. For instance, an aqueous solution of 6 gains
up to 100 times more color than the D-GalA solution.
Given the fact that 6 and high concentrations of 3 are detected in aqueous D-GalA model
systems (table 1), they are of special interest and deserving of further investigations
concerning their reactivity.
Alongside D-GalA model-solutions, 6 could also be detected in di-, tri- and
polygalacturonic acid systems, as well as in pectin. The calculated concentrations
10
ACS Paragon Plus Environment

Page 11 of 31
Journal of Agricultural and Food Chemistry
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
detected in these model-solutions are listed in table 1. One can see that the concentration
of 6 found in pectin is more than twice as high as in the PGA system. This is mainly
because pectin consists of neutral sugars, primarily pentoses (L-arabinose, D-xylose) that
may also degrade into furfural and smaller amounts of α-ketoglutaraldehyde and thus
enhance the possibility for the formation of 6. In heated model systems of D-Gal, no
polyphenols could be detected. The assumption that neutral sugars positively influence
the formation of polyphenols could be confirmed through the introduction of L-Ara or D-Gal
to polygalaturonic acid model systems. As figure 3 shows, this addition leads to a
significant increase in the concentration of 6. Especially the use of pentoses, shown here
for L-Ara, lead to a drastic enhancement of 6 after only 120 minutes. Along with an
increasing of polyphenol concentration, an increase of the furfural concentration, a
precursor molecule of 6, could also be registered. This is because furfural is the main
degradation product of pentoses. The comparison of color formed through D-GalA
polymers, such as polygalacturonic acid, shows that the introduction of neutral sugars to
the pure substance not only leads to an increase in the concentration of 6, but it also
causes an increase of the color formation. This rise of a factor of 2.3 with the addition of
L-arabinose goes hand in hand with an increase in the concentration of 6 and furfural.
211
212
Analyses regarding the involvement of degradation products in the formation of
polyphenols and their color-characteristic
213
214
215
216
217
218
a) 2,3-DHBA
To gain further insights into the browning potential of 6, model systems of D-GalA were
conducted in combination with 6, in concentrations found in the sugar acid systems.
Although 6 was only added in µM concentrations, the increase in color intensity compared
to D-GalA on its own is clearly visible (figure 1). In addition, model reaction systems
containing only 6 in higher concentrations were performed at 130 °C for up to 4 hours.
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 31
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
After 60 minutes in a slightly acidic milieu, the solution started to develop a brownish color
that drastically intensified after 2 hours and turned into a nearly black solution after
4 hours of heating. This effect even increases in a mild alkaline milieu. Investigations of
the model reaction systems show that these reactions may, as well, take place during the
processing of food (storage, drying) and may lead to an undesired color formation. If one
compares the temperature induced browning of the polyphenols, e.g. 6, with other
browning precursor molecules, such as furfural (0.025 M, 130 °C, 4 h, pH 3, absorption
at 420 nm: 0.10) or norfuraneol (0.025 M, 130 °C, 4 h, pH 3, absorption at 420 nm: 0.54),
during caramelization, they are unable to reach the browning values at 420 nm of 6.
Similar results occur during the investigation of D-GalA and L-Ara model systems, where
ten times higher browning values (420 nm) are found in D-GalA samples. Furfural, the
main degradation product of L-Ara, only exhibits a very low browning potential.
In literature, several methods of coupling phenolic compounds have already been
described, e.g. oxidative coupling reactions.17,18 These could lead to the formation of high
molecular weight products that potentially have an influence on the browning of the
reaction solution. To examine the products, samples of pure heated solutions of 6 were
investigated with LC-MS. The results reveal the presence of polymerization products,
such as the dimer. High molecular weight products could only be detected with SEC-UV-
VIS/RI (figure 4) (7 to 20 min, >400 to 10 kDa). This is probably due to the diversity of
reaction pathways that may take place and that lead to a multitude of different polymers
in individual low concentrations. Nevertheless, not only phenolic compounds may react
together. Within heat treated sugar acid model systems, the reaction of phenolic
compounds with degradation products, such as heterocyclic or short chained molecules
formed through D-GalA, is very likely as well and may even enhance the diversity. To
prove that polymerization reactions also take place within mixtures of polyphenoles and
O-heterocyles, norfuraneol was mixed with 6 and heated for 4 hours at 130 °C and pH 3.
12
ACS Paragon Plus Environment

Page 13 of 31
Journal of Agricultural and Food Chemistry
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
HR-ESI-MS measurements of these model systems revealed that a condensation
reaction between these two substances takes place, leading to the condensation product
with a mass of 233.05 m/z (figure 6, right corner). This mass could also be found within
ESI-MS investigations of D-GalA model systems. In addition, further products of
polymerization could be observed.
To compare the different molecular weight domains of polymers formed in the polyphenol
and uronic acid model system, size exclusion chromatography was performed in
combination with a refractometric index and UV-detector. The D-GalA model system
shows a broad peak at 20 min that splits into three molecular weight domains. High
molecular weight fractions were not observed. Model systems of 6 show, in addition to
the 2,3-DHBA peak, oligomeric (20 to 25 min,10 to 5 kDa) and, in minor concentrations,
polymeric polyphenolic compounds (around 12 min, 7 to 20 min, >400 to 10 kDa) (figure
4, left). The investigations of D-GalA and 2,3-DHBA model reaction systems after heat
treatment showed similar molecular weight domains in the low (25 to 35 min, 5 to <1 kDa)
to intermediate molecular weight fractions (figure 4, left). Analyzed with an UV-detector
(figure 4, right) at 420 nm, the same samples show a peak at 22.3 minutes. The intense
signal leads to the prediction that the substance responsible for this peak is formed in
D-GalA, as well as in model systems of 6, and has a high browning potential. The
polymeric substances of 6 also lead to a signal at a detection wavelength of 420 nm,
indicating that these compounds also contribute to the color of the polyphenolic system.
They could not be detected within the sugar acid solution, most likely due to their low
concentration.
b) 3,8-dihydroxy-2-methyl-4H-chomen-4-one
For its investigations, 3 was synthesized using a slightly modified method of Lindgren and
Pernemalm 1980.10 GC/MS measurements were used to confirm its identity. This
provided a reliable identification and enabled subsequent quantitation in model systems
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 31
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
to be performed. As already described above, 3 belongs to the major polyphenolic
degradation products in heated aqueous D-GalA model systems (130 °C, pH 3, figure 5,
left). Model reactions systems performed at 160 °C, at pH 3, show a great increase up
to 60 minutes, in comparison to 130 °C, where 3 could not be determined up to this point.
After this point in time, the concentrations start to decline, due to degradation and
polymerization reactions. A similar course could also be registered for furfural.
Furthermore, the formation of 5,6,7,8-Tetrahydro-3,5-dihydroxy-2-methyl-8-oxobenzo-
pyren as a precursor molecule of 3 was measured.
To have a closer look at the characteristics of 3, tests concerning its browning potential
and possible formation mechanisms were conducted. Model systems of pure 3 (aqueous
solutions of 3 are slightly yellow) under different conditions displayed the formation of
brown polymers of medium molecular weight (figure 5, right). Nevertheless, the browning
intensity of model systems of 6, conducted under the same conditions, could not be
reached. One explanation could be that 3 does not have the same polymerization
potential as 6 and partly accumulates within the system.
286
287
288
289
290
291
292
293
294
295
Formation pathways of the investigated polyphenols
a) 2,3-DHBA
Popoff and Theander (1972)5 already described the formation of 1 and 2 in uronic acid
model systems, albeit using different temperatures, time intervals and pH values. The
formation of 6 in D-GalA systems has not yet been described. To elucidate possible
formation mechanisms, the formation out of smaller degradation products was considered
in a first step. The results demonstrate that furfural is a key component. One pathway
already described for the formation of polyphenolic products is the ring opening of
O-heterocyclic compounds (e.g 5-hydroxymethylfurfural) under hydrothermal conditions.
This ring-opening is followed by an addition reaction.19 Since HMF is not a degradation
14
ACS Paragon Plus Environment

Page 15 of 31
Journal of Agricultural and Food Chemistry
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
product of D-GalA, the main product furfural was used in combination with short-chained
aldehydes (glycolaldehyde, acetaldehyde, glyoxal and formaldehyde) that may be
possible reaction partners in the D-GalA model reaction system.
Furthermore, the reactions were carried out under more moderate temperatures, since
the formation of phenolic compounds in D-GalA model systems could already be
observed at 130 °C and small concentrations could be measured even in systems heated
at 100 °C. During the investigations of furfural with glycolaldehyde and acetaldehyde,
small amounts of 6 could be measured. Since only small concentrations of 6 are formed
within the heterocyclic systems, it was concluded that pathway A cannot be the favored
course for the formation of 2,3-DHBA, since an acid catalyzed ring opening must take
place. If D-GalA itself is used, pathway B can occur, which leads through the formation of
-ketoglutaraldehyde. The occurrence of -ketoglutaraldehyde was postulated in 1944
by Isbell6 and proven by Bornik7,8. Accordingly, higher concentrations of 6 can be
measured in uronic acid model systems. This assumption is supported by considerations
of Ahmad et al., (1993), who postulated a possible formation mechanism for
2,3-dihydroxyacetophenone in which an unsaturated aldehyde reacts with acetol9. Both
products occur during the degradation of D-GalA.
To exclude that 6 degrades itself from a higher molecular weight product, examinations
with chromone-like structures were conducted. One of the major products found in the
D-GalA model systems is 3 and could be an intermediate product for the formation of 6.
To demonstrate this reaction mechanism, 3 was firstly synthesized10 and then heated in
a model system at 130 °C. GC-MS investigations that have been used to identify the
phenolic compounds within the model systems did not provide any trace of 6. Thus, the
respective formation mechanism was no longer considered and the reaction pathway
through furfural and -ketoglutaraldehyde under acidic conditions should be the preferred
one (pathway A and B, figure 6).
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 31
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
b) 3,8-dihydroxy-2-methyl-4H-chromen-4-one
M/z of 702, 815, 928 and 1024 were detected within MALDI-TOF-MS experiments, which
could belong to the medium molecular weight substances found within the chromone
system during SEC measurements. The same m/z were registered in model systems of
norfuraneol in combination with furfural, which leads to the assumption that a possible
formation of 3 originates from these two O-heterocycles. GC-MS investigations of the
model systems of norfuraneol and furfural confirmed this hypothesis, since 3 could be
detected. According to model reaction systems of furfural and glycolaldehyde used for
the formation mechanism of 6, only small concentrations of 3 were registered, leading
again to the conclusion that precursor molecules of furfural and norfuraneol are
responsible for the formation of chromone and further chromone-like structures (figure 6,
pathway A). Again, this suggests that precursor molecules of these two compounds are
responsible for the formation of 3. α-ketoglutaraldehyde, in particular, moves into focus,
since it is one of the compounds that is not found during the degradation of hexoses and
only in very small concentrations in pentoses.7 Ahmad et al. (1993), postulated a
formation pathway that uses an unsaturated aldehyde and an enediol as educts9, similar
to those of 6. These degradation intermediates of D-GalA can also be found in the
postulated formation mechanisms of norfuraneol and furfural, which underscores the
above-mentioned assumption.
To investigate temperature and pH dependency of the formation of 3 from norfuraneol
and furfural, the reaction partners were heated to 150 °C, at pH-values of 1, 2, 3, 5. The
lowering of the pH-value to 1 should enhance the ring opening and raise the yield.
Consequently, the oxygen atom located within the ring should become protonated and
the attack of an oxonium ion at the adjacent carbon atom should be facilitated. However,
the conducted experiments show a rapid and strong degradation of norfuraneol, only
allowing a formation of 3 to occur in trace amounts. During the reaction at pH 2, the
16
ACS Paragon Plus Environment

Page 17 of 31
Journal of Agricultural and Food Chemistry
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
concentration of 3 doubles in relation to pH 3, which is a sign that the catalytic ring
opening occurs (without norfuraneol reacting with itself). The enhancement to a pH value
of 5 does not lead to any further changes in the concentration of 3. Along with the pH
value, the temperature of 150 °C also has an impact on the formation of 3. Furthermore,
model systems heated at this temperature and a pH value of 2, show more than a twofold
increase in the yield of the product in comparison to 130 °C. The increase to a
temperature of 160 °C does not further enhance the formation. Under these conditions,
an enforced degradation can be registered, most likely attributable to polymerization.
Moreover, norfuraneol could not be detected anymore after 2 hours of heating, so that
further formation of 3 was no longer possible.
In addition to the variation of reaction conditions for the norfuraneol, furfural model
systems, the addition of different compound classes such as alcohols, aldehydes and
carboxylic acids, were also investigated. At a pH value of 3 and a temperature of 130 °C
(standard conditions), the addition of acetic acid (pH was adjusted to 3 again after the
addition) leads to an increase in concentration of 3 of 50 %, whereas no influence can be
registered during the addition of the other compound classes.
In summary, the temperature and pH dependent degradation of D-GalA leads first of all
to the formation of typical carbohydrate reaction products, such as furfural, and
norfuraneol or short chain aldehydes to a broad substance spectrum, from which different
phenolic compounds can be formed. 2,3-DHBA was detected for the first time and plays
an important role. Although only found in small amounts within the heated D-GalA model
systems, it possesses a high browning potential. 3,8-dihydroxy-2-methyl-4H-chromen-4-
one, on the other hand, possesses a comparatively low browning potential and partly
accumulates within the system, thus becoming one of the main components found within
the D-GalA model system. Conducted experiments with the pure substances show the
formation of medium to high molecular weight compounds that proportionally contribute
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 31
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
to the total browning of the D-GalA system. According to current knowledge, the formation
of both substances passes through the intermediate stage of α-ketoglutaraldehyde. Two
different reaction pathways derive from this intermediate, leading, on the one hand, to
furfural and, on the other, to reductic acid through the elimination of water. Through
addition reactions, a chain elongation is possible. Depending on the reaction partner,
acetaldehyde or norfuraneol, the compounds 2,3-dihydroxybenzaldehyde or rather
3,8-dihydroxy-2-methyl-4H-chromen-4-one
are
formed.
In
both
cases,
α-ketoglutaraldehyde reacts as the key component of the D-GalA degradation. Given that
reducing sugars, such as D-Gal or D-Glc, do not degrade into α-ketoglutaraldehyde, the
participation of phenolic compounds in color formation remains limited to the reaction of
uronic acids.
ABBREVIATIONS USED
2,3-DHBA, 2,3-dihydroxybenzaldehyde; L-Ara, L-arabinose; chromone, 3,8-dihydroxy-2-
methyl-4H-chromen-4-one; DAD, diode array detector; ESI, electrospray-ionization; Fur,
furfural; D-Gal, D-galactose; D-Glc, D-glucose D-GalA, D-galacturonic acid; GC, gas
chromatography; HPLC, high performance liquid chromatography; MALDI-TOF-MS,
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS, mass
spectrometry; RI, refractometric index detector SEC, size-exclusion chromatography,
ACKNOWLEDGEMENT
The authors are grateful for the financial support of the Forschungsgemeinschaft (DFG-
project “Structure-depending degradation reactions of pectins and their impact on non-
enzymatic browning and technological functionality”) (DR 806/4-1).
Thanks are also extended to Luzia Hammer and Isabell Buggisch for the support of the
sample analyses.
18
ACS Paragon Plus Environment

Page 19 of 31
Journal of Agricultural and Food Chemistry
400
401
402
403
404
405
406
407
408
409
SUPPORTING INFORMATION
Figure 1: ESI mass spectrum of a heated 2,3-DHBA model system.
Figure 2: ESI mass spectrum of a heated model system of a mixture of 2,3-DHBA and
norfuraneol
Figure 3: MALDI-TOF spectrum of 2,5-dihydroxybenzoic acid (matrix)
Figure 4: MALDI-TOF spectrum of a heated model system of chromone
Figure 5: GC-MS spectrum of 3,8-dihydroxy-2-methyl-4H-chomen-4-on
Table 1: chromone concentration in various model systems of furfural and norfuraneol
performed at different temperature and pH values.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 31
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
REFERENCES
(1) Hodge J. E; Chemistry of Browning Reactions in Model Systems. J. Agric. Food
Chem. 1953, 15, 928–943
(2) Madson, M. A.; Feather M. S. The acid-catalyzed decarboxylation of D-xyluronic,
D-galacturonic, and D-glycera-D-gulo-hepturonic acid. Carbohydr. Res., 1979, 70,
307-311.
(3) Scott, R. W.; Moore, W. E.; Effland Marylin J.; Millett, M. A. Ultraviolet
Spectrophotometric determination of hexoses, pentoses, and uronic acids after their
reactions with concentrated sulfuric acid. Analytical Biochemistry, 1967, 21, 68–80.
(4) Ahmad, T.; Andersson, R.; Olsson, K.; Westerlund, E. On the formation of reductic
acid from pentoses or hexuronic acids. Carbohydr. Res. 1993, 247, 217–222.
(5) Popoff, T.; Theander, O. Formation of aromatic compounds from carbohydrates: Part
1. Reation of D-glucuronic acid, D-galacturonic acid, D-xylose, D- arabinose in slightly
acidic, aqueous solution. Carbohydr. Res. 1972, 22, 135–149.
(6) Isbell, H. S. Synthesis of vitamin C from pectic substances. J. of the Franklin Institute
1944, 238, 140–141.
(7) Bornik, M.-A.; Kroh, L. W. D-Galacturonic acid as a highly reactive compound in
nonenzymatic browning. 1. Formation of browning active degradation products. J.
Agric. Food Chem. 2013, 61, 3494–3500.
(8) Bornik, M.-A. D-Galacturonsäure in der nicht-enzymatischen Bräunungsreaktion:
Qualität und Sicherheit: Tagungsband. Dissertation, TU Berlin, Berlin, 2012.
(9) Ahmad, T.; Andersson, R.; Olsson, K.; Theander, O. On the formation of 2,3-
dihydroxyacetophenone from pentoses or hexuronic acids. Carbohydr. Res. 1993,
247, 211–215.
20
ACS Paragon Plus Environment

Page 21 of 31
Journal of Agricultural and Food Chemistry
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
(10) Lindgren, G.; Pernemalm, P.-Å. A High Performance Preparative Procedure for
the Isolation of Degradation Products from D-Xylose. J. of Liquid Chromatography
1980, 3, 1737–1742.
(11) Wegener, S.; Kaufmann, M.; Kroh, L. W. Influence of L-pyroglutamic acid on the
color formation process of non-enzymatic browning reactions. Food Chemistry 2017,
232, 450–454.
(12) Conrad, C. M. The decarboxylation of D-galacturonic acid with special reference to
the hypothetical formation of L-arabinose, J. American Chemical Society 1931, 53,
2282-2287.
(13) Popoff, T.; Theander, O. Formation of aromatic compounds from Carbohydrates.
III: Reaction of D-Glucose and D-Fructose in slightly acidic, aqueous solution. Acta
Chemica Scandinavica 1976, 30, 397–402.
(14) Popoff, T.; Theander, O. Reactions of D-Xylose and D-Glucose in alkaline, aqueous
solutions. Carbohydr. Res. 1976, 48, 13–21.
(15) Olsson, K.; Pernemalm, P.-A.; Theander, O. Formation of aromatic compounds
from Carbohydrates. VII: Reaction of D-Glucose and Glycine in slighty acidic,
aqueous conditions. Acta Chemica Scandinavica 1978, 32, 249–256.
(16) Olsson, K.; Pernemalm, P.-A.; Popoff, T.; Theander, O. Formation of aromatic
compounds from Carbohydrates. V: Reaction of D-Glucose and methylamine in
slightly acidic, aqueous conditions. Acta Chemica Scandinavica 1977, 31, 469–474.
(17) Kobayashi, S.; Higashimura, H. Oxidative polymerization of phenols revisited.
Progress in Polymer Science 2003, 28, 1015–1048.
(18) Smejkalova, D.; Conte, P.; Piccolo, A. Structural characterization of isomeric
dimers from the oxidative oligomerization of catechol with a biomimetic catalyst.
Biomacromolecules 2007, 8, 737–743.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 31
459
460
461
(19) Luijkx, G. C.; van Rantwijk, F.; van Bekkum, H. Hydrothermal formation of 1,2,4-
benzenetriol from 5-hydroxymethyl-2-furaldehyde and D-fructose. Carbohydr. Res.
1993, 28, 131–139
22
ACS Paragon Plus Environment

Page 23 of 31
Journal of Agricultural and Food Chemistry
462
463
464
465
466
FIGURE CAPTIONS
Figure 1: Color (absorption at 420 nm) of aqueous model reaction systems
(0.015 mol/L) consisting of D-GalA, 2,3-DHBA, L-Ara, D-Gal and D-GalA doped with
2,3-DHBA heated at 130 °C and a pH of 6. Samples were taken after 0, 120 and 240
minutes.
467
468
469
470
Figure 2: Polyphenolic compounds that are formed during the heat treatment of D-Gal.
catechol (1); methylcatechol (2); 3,8-dihydroxy-2-methyl-4H-chromen-4-one (chromone)
(3); 5,6,7,8-tetrahydro-3,5-dihydroxy-2-methyl-8-oxobenzo-pyrene (4); 3,4-dihydroxy-
acetophenone (5); 2,3-dihydroxybenzaldehyde (2,3-DHBA) (6)
471
472
Figure 3: Formation of furfural and 2,3-DHBA in pure polygalacturonic acid and with
addition of the sugars D-Gal and L-Ara at 130 °C and pH 3.
473
474
475
476
477
478
479
Figure 4: Molecular weight distribution of formed degradation products of D-GalA and
2,3-DHBA solutions heated at 130°C and a pH of 8. The measurements were performed
using the size exclusion chromatography in combination with a RI and a parallel detection
at a wavelength of 420 nm.
Figure 5: a) Concentrations of chromone and furfural, found in D-GalA model systems at
130 °C and a pH of 3 (GC-MS). b) SEC investigation of a heated chromone model system,
showing an intense signal at 420 nm at around 18 min.
480
481
482
483
484
485
Figure 6: Formation scheme of 2,3-DHBA and chromone, originating, on the one hand,
from furfural (pathway A) and, on the other hand, from D-GalA (pathway B).
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 31
Table 1: Concentrations of 2,3-DHBA, catechol, methylcatechol and chromone found in different model systems heated
for 120 minutes at 130 °C and a pH of 3. NQ: not quantitated
concentration
[µmol/L]
concentration
[µmol/L]
D-GalA
2,3-DHBA
196.2 ± 4.3
6.60 ± 0.1
4.40 ± 0.2
1.32 ± 0.1
47.40 ± 1.8
196.2 ±
4.3
2,3-DHBA (6)
catechol (1)
D-GalA
DiGalA
TriGalA
PGA
1.8*
NQ
methyl-
catechol (2)
3,4-dihydroxy-
acetophenone (5)
0.3 ± 0.01
1573.4 ±
0.14
chromone (3)
Pektin
* Only detected in one sample
24
ACS Paragon Plus Environment

Page 25 of 31
Journal of Agricultural and Food Chemistry
Figure captions
Figure 1
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 31
Figure 2
26
ACS Paragon Plus Environment

Page 27 of 31
Journal of Agricultural and Food Chemistry
Figure 3
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 31
Figure 4
28
ACS Paragon Plus Environment

Page 29 of 31
Journal of Agricultural and Food Chemistry
Figure 5
29
ACS Paragon Plus Environment