D-galacturonic acid as a highly reactive compound in nonenzymatic browning. 1. Formation of browning active degradation products

Article abstract of DOI:10.1021/jf303855s

Thermal treatment of an aqueous solution of d-galacturonic acid at pH 3, 5, and 8 led to rapid browning of the solution and to the formation of carbocyclic compounds such as reductic acid (2,3-dihydroxy-2-cyclopenten-1-one), DHCP (4,5-dihydroxy-2-cyclopenten-1-one), and furan-2-carbaldehyde, as degradation products in weak acidic solution. Studies on their formation revealed 2-ketoglutaraldehyde as their common key intermediate. Norfuraneol (4-hydroxy-5-methyl-3-(2H)-furanone) is a typical alkaline degradation product and formed after isomerization. Further model studies revealed reductic acid as an important and more browning active compound than furan-2-carbaldehyde, which led to a red color of the model solution. This red-brown color is also characteristic of thermally treated uronic acid solutions.

Full text of DOI:10.1021/jf303855s

Article
pubs.acs.org/JAFC
D‑Galacturonic Acid as a Highly Reactive Compound in
Nonenzymatic Browning. 1. Formation of Browning Active
Degradation Products
Maria-Anna Bornik and Lothar W. Kroh*
Institute of Food Technology and Food Chemistry, Technical University of Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
ABSTRACT: Thermal treatment of an aqueous solution of D-galacturonic acid at pH 3, 5, and 8 led to rapid browning of the
solution and to the formation of carbocyclic compounds such as reductic acid (2,3-dihydroxy-2-cyclopenten-1-one), DHCP (4,5-
dihydroxy-2-cyclopenten-1-one), and furan-2-carbaldehyde, as degradation products in weak acidic solution. Studies on their
formation revealed 2-ketoglutaraldehyde as their common key intermediate. Norfuraneol (4-hydroxy-5-methyl-3-(2H)-furanone)
is a typical alkaline degradation product and formed after isomerization. Further model studies revealed reductic acid as an
important and more browning active compound than furan-2-carbaldehyde, which led to a red color of the model solution. This
red-brown color is also characteristic of thermally treated uronic acid solutions.
KEYWORDS: D-galacturonic acid, colored compounds, nonenzymatic browning, furan-2-carbaldehyde, reductic acid
The objectives of the present investigations were therefore to
characterize key intermediates and quantitate degradation
products formed in aqueous unbuffered solutions of D-
galacturonic acid at pH 3.0, 5.0, and 8.0, respectively, and to
evaluate the influence of certain reaction intermediates as
precursors in colorant formation to obtain more detailed
information on the chemical reactions involved in the
degradation. The so-formed caramel-like structures and the
resulting color were further investigated by gel permeation
chromatography (GPC) with refractive index and diode array
detection.
INTRODUCTION
■
Thermal treatment of food and food products causes changes
mostly by nonenzymatic browning of reducing carbohydrates,
amino acids, and proteins. Those complex and multiple
reaction pathways are well known, and a lot of intermediates
and reaction products have been postulated, identified, and
characterized in the last decades.1,2 Uronic acids have the same
structure as reducing sugars with an additional carboxylic group
on the C-6. They can also take part in nonenzymatic browning
reactions, but reaction pathways have not been studied in a
systematical way so far.3−7
It is known that in model studies uronic acids especially D-
galacturonic acid, the main monomer of pectin, produces more
brown color than reducing sugars, but surprisingly little is
known about the structures of the compounds responsible for
this typical reddish-brown color. Model studies already revealed
that the kinetics of the formation of browning is different in
reducing sugars and uronic acids.8 In comparison to pentoses
and hexoses, less information is yet available regarding
degradation products and intermediates of uronic acids.
Furanoic compounds such as furan-2-carbaldehyde, 2-furanoic
acid, and 5-formyl-2-furanoic acid as well as reductic acid (2,3-
dihydroxycyclopent-2-en-1-on), an aci-reductone and structure
analogue of L-ascorbic acid, are postulated as degradation
products in acidic aqueous solutions. Decarboxylation of D-
galacturonic acid to L-arabinose as the first step of reaction
followed by dehydration and cyclization yielding typical
degradation products of pentoses has been assumed as the
main degradation pathway.4 Stutz and Deuel found out that
only small amounts of L-arabinose are formed during thermal
treatment of D-galacturonic acid and that there have to be other
and more important degradation pathways.9 Our studies also
revealed that decarboxylation is not the only degradation
pathway leading to the known degradation products because of
their imbalanced concentrations in model studies of L-arabinose
and D-galacturonic acid under the same reaction conditions.
MATERIALS AND METHODS
■
Chemicals. The following compounds were obtained commer-
cially: D-galacturonic acid monohydrate (Chemodex), barium
carbonate, furan-2-carbaldehyde, norfuraneol, Amberlite 4400 OH−,
2-ketoglutaric acid, 5-formyl-2-furanoic acid, 2-furanoic acid (Sigma),
tetrabutylammonium hydrogen sulfate (Acros), sulfuric acid, succinic
acid, malic acid (Fluka). Furan-2-carbaldehyde was freshly distilled at
300 °C in high vacuum prior to use. Solvents were of HPLC grade
(Merck).
Synthesis of Reductic Acid [2,3-Dihydroxycyclopent-2-en-1-
one]. Following a procedure of Reichstein and Oppenauer with
modifications of Goldstein,7,10 D-galacturonic acid (3 g) was dissolved
in 15 mL of 1 N sulfuric acid in a sealed Pyrex reaction tube and
heated for 90 min at 150 °C. After cooling the solution to room
temperature and filtration of the residue the sulfuric acid was
precipitated with barium carbonate. After filtration of the precipitate
the solution was filtrated and percolated through a strong cation
exchange column (Dowex 50x8, H+ form, 50−100 mesh) and after
that percolated on a strong anion exchange column (Amberlite 4400
OH−). Nonionic compounds are not adsorbed on the anionic
material; anions such as reductic acid are adsorbed and can be
displaced from the resin with 0.1 N formic acid. Control of the
Received: October 6, 2012
Revised: March 11, 2013
Accepted: March 15, 2013
Published: March 15, 2013
© 2013 American Chemical Society
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displaced fractions was done on silica plates (silica gel 60 F254) with
n-butanol/glacial acid/water (4:2:1) as an eluent. Detection of reductic
acid was done under UV light, and detection of partly co-eluating,
unreacted D-galacturonic acid was done by dipping the plate into a
methanolic 10% sulfuric acid solution and heating it at 120 °C for 10
min. The effluents from the resin containing only reductic acid were
pooled and concentrated to dryness under reduced pressure at 35 °C.
The light brown syrup was washed with cold acetone to obtain crude
crystals, which were recrystallized from a mixture of ethyl acetate and
ethyl alcohol or sublimed.
min) was detected. Quantitation was performed by external calibration
with pure reference compounds.
α-Dicarbonyl Compounds as Quinoxalines. The derivatized
samples were analyzed by HPLC-DAD using a flow rate of 0.5 mL/
min, an oven temperature of 40 °C, and a solvent gradient starting
with a mixture (95/5; v/v) of water and acetonitrile and increasing the
acetonitrile content after 5 min to 10% within 3 min. After another 5
min the content was increased to 20% within 7 min and after 5 min
increased to 50% within 10 min. Quantitation was performed by
comparing the peak areas with those of a standard solution at 317 nm
containing known amounts of each pure reference or pure authentic
quinoxaline isolated and elucidated from reaction mixtures in our
working group.
Aldehydes as 2,4-Dinitrophenylhydrazones. Identification of
reaction products was achieved by LC-MS measurements in the
total ion mode. HPLC separation (Thermo Fischer Scientific Accela
pump, CTC PAL autosampler) was carried out by injecting the
derivatized samples using a solvent gradient starting with a mixture
(40/60; v/v) of water and acetonitrile and increasing the acetonitrile
content after 7 min to 100% within 13 min. Ionization (TSQ Vantage
System with Ion Max Source, H-ESI II probe) in the ESI negative
mode followed, using a spray voltage of 3 kV, a vaporizer temperature
of 450 °C, a sheath gas pressure of 60 psi, and a capillary temperature
of 270 °C. Spectra were obtained by setting the scan at 50 to 1000 m/
z. Thermo Excalibur 2.1.0.1139 software was used for the identification
of the substances.
Gas Chromatography−Mass Spectrometry (GC-MS). GC was
performed on a Shimadzu GC-2010 by using a capillary DP-5 column
(30 m × 0.25 mm, 0.25 mm, Supelco SLB-5MS, Bellefonte, PA, USA).
The samples were applied by split injection (1:10) at an injection
temperature of 250 °C and an oven temperature of 100 °C. After 4
min, the temperature of the oven was raised at a rate of 8 °C/min to
200 °C and held for 10 min, then raised again at a rate of 40 to 275 °C
and held for 2 min. The flow of the carrier gas, helium, was 2 mL/min.
MS analysis was performed with a GCMS-QP2010plus (Shimadzu) in
tandem with the GC.
Model Reactions. D-Galacturonic Acid. To investigate the
degradation behavior and the formation of key intermediates and
degradation products, D-galacturonic acid monohydrate (0.5 M) was
dissolved in water, and the pH adjusted with sodium hydroxide and
hydrochloric acid solutions to 3.0, 5.0, or 8.0. Aliquots of the solution
were filled into glass ampules and incubated at 100 °C for 2 h in a
heating block. The reaction was stopped at various time points and
worked up as follows. Browning was measured as the absorption at
420 nm with a spectrophotometer (UV-1650 PC, Shimadzu, Duisburg,
Germany), and the CIELab color with a reflectance attachment with a
Spectralon integrating sphere (Specord 40, Analytik Jena, Jena,
Germany). pH values were measured with a pH electrode (SenTix
Mic, WTW, Weilheim, Germany; pH meter CG 820, Schott, Mainz,
Germany) after cooling the sample to room temperature. For organic
acids and aci-reductones, samples were diluted (1:10) with 1% m-
phosphoric acid and 10 mM dithiothreitol and measured by HPLC-
DAD. For furans and furanones, samples were diluted (1:100; v/v)
with 10 mM phosphate buffer (pH 6.0) and subjected to HPLC-DAD.
For quinoxalines, o-phenylenediamine hydrochloride (OPD; 20 mM)
was added (1:1; v/v) and the samples were reincubated for at least 3 h
at room temperature and subsequently analyzed by HPLC-DAD. For
aldehydes, 2,4-dinitrophenylhydrazine (DNPH) and toluene were
added to the sample (1:2:1; v/v/v) and reincubated for 12 h at room
temperature and in the dark. The samples were vigorously shaken, and
the organic layer was taken and dried under a stream of nitrogen. The
residue was dissolved in acetonitrile and subjected to LC-MS. For
unknown carboxylic acids, dicarboxylic acids, and sugar derivatives, an
aliquot of the solution was dried under a stream of nitrogen and
derivatized with 200 mL of N,O-bis(trimethylsilyl)acetamide with 5%
trimethylchlorosilane in anhydrous pyridine (1:1; v/v) and subjected
to GC-MS.
Reductic Acid and Furan-2-carbaldehyde. To investigate the
browning behavior of degradation products of D-galacturonic acid, 0.05
M reductic acid or furan-2-carbaldehyde with and without L-alanine
(0.005 M) was dissolved in water and the pH adjusted to 5.0 or 8.0.
The solutions were further treated and analyzed like the model
solutions of D-galacturonic acid. All experiments were conducted in
three independent replications, and the arithmetical mean of all
quantified values was calculated.
Characterization and Quantitation. Organic Acids and aci-
Reductones. The diluted samples were analyzed by HPLC-DAD using
a phosphate buffer (20 mM, pH 2.8) as an isocratic eluent and
monitoring the effluent at wavelengths of 214 nm for organic acids
(formic acid (3.9 min), acetic acid (6.3 min), succinic acid (11.4 min),
2-ketoglutaric acid (6.9 min), malic acid (4.2 min)) and 254 nm for
reductic acid (7.5 min) detection. Identification of the organic acids
was accomplished by comparing their retention time with authentic
known acids and by comparing with the results of GC-MS analysis
after silylation of the sample. Standard curves were prepared for each
compound, and the peak areas of the compounds in the samples were
measured and the concentrations calculated from the standard curves.
Furans and Furanones. The diluted samples were analyzed by
HPLC-DAD using a solvent gradient starting with a mixture (99/1; v/
v) of phosphate buffer (5 mM, pH 6.0) and methanol and increasing
the methanol content after 10 min to 20% within 5 min and after
another 5 min to 99% within 5 min. By monitoring the effluent at a
wavelength of 280 nm, furan-2-carbaldehyde (6.7 min), 5-formyl-2-
furanoic acid (16.5 min), and norfuraneol (3.6 min) were detected,
and by monitoring at a wavelength of 254 nm 2-furanoic acid (8.5
High-Performance Liquid Chromatography (HPLC)-DAD.
The HPLC apparatus consisted of one pump (LC-9A, Shimadzu), a
gradient mixer (FCV-9 AL, Shimadzu), a column oven (VDS Optilab,
Berlin, Germany), and an autosampler (Gina50, Dionex, Germering,
Germany). The effluent was monitored by a diode array detector
(DAD, Gynkotek UVD 340S, Gynkotek, Germering, Germany)
operating in a wavelength range between 200 and 500 nm. Separations
of organic acids and aci-reductones were performed on a stainless steel
column (150 × 4.6 mm), packed with ODS-AQ material (3 mm,
YMC, Kyoto, Japan), with a flow rate of 0.6 mL/min at a column
temperature of 25 °C. Separations of furans and furanones were
performed on a stainless steel column (125 × 4.6 mm, Macherey-
̃
Nagel, DA1/4 ren, Germany), packed with an RP-18 material
(Nucleosil, 3 mm), with a flow rate of 0.5 mL/min at a column
temperature of 40 °C. For the separation of α-dicarbonyl compounds
as quinoxalines the same column, but a column temperature of 35 °C,
was used.
Gel Permeation Chromatography−Refractive Index Detec-
tion (GPC-RI). For the characterization of the molecular weight
distribution of the thermally treated samples at 100 °C for 2 h, the
samples were diluted with water and analyzed by GPC-RI. The
molecular distribution of the reacted D-galacturonic acid was compared
to standard solutions of D-glucose, a pullulan standard of 40 kDa, and a
reaction mixture consisting of D-glucose and L-alanine.
RESULTS AND DISCUSSION
■
Thermal treatment of unbuffered solutions of D-galacturonic
acid started at pH 3.0, 5.0, or 8.0 at 100 °C led to 10 times
higher browning after 2 h compared to L-arabinose and D-
glucose or D-galactose.
The browning was measured as the absorption at 420 nm. It
was highest at pH 5.0 and a little lower at pH 3.0 and 8.0 at the
beginning of the reaction. The pH was not controlled during
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further reaction. The color formed was the same for all three
starting pH values and more reddish compared to model
solutions of reducing sugars treated in the same way. CIELab
measurements revealed that the lightness is much lower and the
+a value (red) is much higher in the thermally treated D-
galacturonic acid solutions (L = 60, +a = 30) compared to
reducing sugars (L = 90, +a = 0).
Unlike the decreasing pH value of the reducing sugars, the
pH value of D-galacturonic acid solutions increased until it
remained steady at about 6.5. Only at pH 3.0 did the value
remain stable during the whole reaction time. During the
degradation of reducing sugars higher amounts of short-chain
organic acids were formed due to oxidative cleavage reactions
of the α-dicarbonyl compounds, leading to the decrease in pH
and a deceleration in reaction speed.11 Thermal treatment of D-
galacturonic acid tended to result in less acidic heterocyclic or
carbocyclic compounds, which are responsible for the increase
in the pH value and an acceleration of the reaction.
Feather postulated the existence of a unique mechanism for
the formation of reductic acid from uronic acid based on their
labeling experiments,15 but they could not verify this
mechanism any further. The other known mechanism
responsible for the formation of reductic acid is the same as
in pentoses assuming furan-2-carbaldehyde as precursor.13,16,17
Our experiments indicated different isomers of 2-ketoglutar-
aldehyde (4) as the common intermediate in the formation of
reductic acid (5), DHCP (6), and furan-2-carbaldehyde (7)
from D-galacturonic acid. One of these isomers, 4c, can also be
formed by dehydration of pentoses 2. Decarboxylation of D-
galacturonic acid (1) to L-arabinose (2) takes place only in
concentrated mineral acids.18 Thus, decarboxylation of 1 is not
a major degradation pathway of D-galacturonic acid under the
slightly acidic conditions investigated in this work. Eliminative
decarboxylation of D-galacturonic acid led to the formation of a
very reactive 4,5-unsaturated 4-deoxy-L-arabinose (3), which
after ring-opening and dehydration yielded the enolic form of
2-ketoglutaraldehyde (4a) and further cyclized to reductic acid
(5). Figure 1 shows the postulated degradation pathway of D-
galacturonic acid leading to the postulated precursor and key
intermediates of the known degradation products under slightly
acidic to alkaline reaction conditions.
So far, 2-ketoglutaraldehyde was only supposed as the key
intermediate,13,16,19 but could not be identified and charac-
terized in heat-treated solutions of D-galacturonic acid. It is
susceptible to secondary reactions and therefore has a decisive
influence on the product spectrum. Such a reactive intermediate
may be transformed into a stable derivative with trapping
reagents and thus be protected from further reactions. While
our postderivatization with OPD was not successful, post-
derivatization with DNPH led to a stable tris(2,4-dinitrophe-
nylhydrazone) derivative, which we could then analyze by LC-
MS. We identified 2-ketoglutaraldehyde as a direct intermediate
of the D-galacturonic acid degradation for the first time. The
total ion chromatogram of the model solution derivatized with
DNPH is shown in Figure 3. The peak at 11.07 min represents
the tris(2,4-dinitrophenylhydrazone) derivative of 2-ketoglutar-
aldehyde, as the mass spectrum in Figure 4 shows.
The confirmation of 2-ketoglutaraldehyde as a key
intermediate of D-galacturonic acid also gives further indication
for eliminative decarboxylation as the main degradation
pathway under slightly acidic to neutral conditions. Due to
the trans position of the substituents on C-4 and C-5 of D-
galacturonic acid in its lowest energy 1C4 chair conformation, a
hydrogen bond can form a six-center intermediate stage. In a
concerted mechanism dehydratization of C-4 and decarbox-
ylation at C-5 proceed simultaneously and a 4,5-unsaturated 4-
deoxypentose (3a), a labile precursor in the formation of 2-
ketoglutaraldehyde, is formed.
We suppose that after further dehydratization the postulated
4,5-unsaturated 4-deoxypentose 3b can yield the enolic form of
2-ketoglutaraldehyde (4a), which leads directly to the
formation of reductic acid (5) and after further isomerization
also to DHCP (6) and furan-2-carbaldehyde (7). The
formation of the enolic form of 2-ketoglutaraldehyde (4a)
instead of 4c also gives a reasonable explanation for the high
concentration of reductic acid (5) and the relatively low
concentration of furan-2-carboxyldehyde (7) compared to
similar model reactions with L-arabinose (2). L-Arabinose was
relatively stable under the same reaction conditions, and we
recovered 64% of the intact pentose and only 60 mM furan-2-
Table 1 gives an overview of the concentrations of the
formed products of thermally treated D-galacturonic acid
Table 1. Concentration of the Main Degradation Products
Reductic Acid (RA), DHCP, Furan-2-carbaldehyde (FF),
and Norfuraneol (Norf) of D-Galacturonic Acid after
Thermal Treatment at 100 °C for 2 h with Different pH
Values at the Start of the Reaction and the Resulting
Absorption at 420 nm (Abs420
)
a
GalA
[mM]
RA
[mM]
DHCP
FF
[mM]
Norf
[mM]
pH Abs420
[mM]
GalA
3
5
8
2.3
4.0
3.3
182
153
160
1.9
0.2
0.4
61.5
39.8
12.5
1.8
n.d.
0.3
0.2
n.d.
n.d.
a
Quantified as malic acid equivalent.
solutions after 2 h. In model solutions started at pH 5.0
about 31% of the unreacted uronic acid could be detected, but
the sum of the main degradation products is only around 8%.
Therefore we suppose that the other 61% of the degraded D-
galacturonic acid is mostly incorporated into colored polymer
structures.
Teleman et al. postulated that at very low pH values the
hemiacetal ring of the D-galacturonic acid opens and
dehydration on the C-3 proceeds. This leads to the postulated
but unstable 3-deoxyhexosulosuronic acid, which readily forms
a furanoic intermediate that is transformed to the stable
degradation products 5-formyl-2-furanoic acid and 2-furanoic
acid.12 These stable furanoic acids could not be detected in any
of the investigated model reactions of D-galacturonic acid in this
work. A pH of 3.0 and a temperature of 100 °C seemed to be
inadequate for this reaction pathway. Instead the highest
concentrations of reductic acid and furan-2-carbaldehyde were
formed. 4,5-Dihydroxy-2-cyclopenten-1-one (DHCP), an iso-
mer of reductic acid, was also formed during thermal
degradation of D-galacturonic acid. Isbell proposed DHCP as
the main precursor in the formation of reductic acid,13 but
Ahmad et al. determined that DHCP is only an isomer but not
a tautomer of reductic acid, and therefore there have to be
different formation pathways.14 We observed that DHCP is
preferably formed at pH 3, but according to Ahmad et al.5 we
also suppose that it is not the precursor or a tautomer of
reductic acid but an alternative degradation product of D-
galacturonic acid.
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Figure 1. Degradation pathways of D-galacturonic acid in slightly acidic to alkaline media. Decarboxylation to L-arabinose (2) or eliminative
decarboxylation to 4,5-unsaturated 4-deoxy-L-arabinose (3) to 2-ketoglutaraldehyde (4) leading to reductic acid (5), DHCP (6), and furan-2-
carbaldehyde (7). Isomerization to D-tagaturonic acid (8) and formation of norfuraneol (9) or oxidation to galactaric acid (10).
Figure 2. Polymerization of reductic acid due to aldol condensation. Because of the highly conjugated structure, the color of the formed polymer
appears red.
carbaldehyde as well as 0.2 mM 3-deoxypentosulose as the
main degradation products.
Investigations of the molecular size distribution by GPC
showed that compared to reducing sugars not only is the
browning more intense and the color different but also the
polymer structure seemed to be different.21 Yet, due to the
strong interaction of the carboxylic groups with the column
material of the GPC and the totally different behavior
compared to pullulans, an estimation of the molecular size
was not possible.
To gain more insight into precursors of colored products, it
might be promising to identify the chromophoric structures
formed by reacting certain intermediates known as uronic acid
degradation products. For example, furan-2-carbaldehyde is also
known as one of the main reaction products formed from
pentoses during thermal treatment. It was reported that this
We also observed that at higher pH values at the beginning
of the reaction isomerization of the D-galacturonic to D-
tagaturonic acid (8) was favored over other reaction pathways.
The highly reactive norfuraneol (9), a methylene active aroma
impact compound, was formed to a higher degree at pH 5 and
8 compared to pH 3. The formation pathway of norfureneol
was postulated by Kraehenbuehl et al.20 Besides isomerization,
also oxidation of D-galacturonic acid to galactaric acid (10)
occurred to a higher extent compared to model reactions
started at lower pH values. Compared to pH 3 at pH 8 reductic
acid and DHCP were formed only in small amounts, but no
furan-2-carboxyaldehyde could be detected at all (see Table 1).
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Figure 3. Confirmation of 2-ketoglutaraldehyde as a key intermediate in the degradation of D-galacturonic acid by LC-MS after postderivatization
with DNPH. Total ion chromatogram of the tris(2,4-dinitrophenylhydrazine) derivative (tR 11.07 min).
Figure 4. Mass spectrum of 2-ketoglutaraldehyde as the tris(2,4-dinitrophenylhydrazine) derivative at m/z 653 [M − 1].
aldehyde easily reacts with other carbohydrate degradation
products containing an activated methylene group, such as
norfuraneol, giving rise to yellow reaction products.
primary amines or carboxylic acids to form more intense and
more highly cross-linked structures. Our observations provided
more evidence that reductic acid is the most reactive
degradation product of D-galacturonic acid and that DHCP is
more stable and accumulates in the solution, as shown in Table
1. In contrast to the literature, the cyclopentenone ring of the
reductic acid in our experiments was still intact.22 The furan
ring of the furan-2-carbaldehyde can be reopened, for example,
in an amine-catalyzed reaction.23 After this ring-opening several
condensation reactions between methylene active intermediates
The browning activity of neither reductic acid nor DHCP has
been investigated so far. We suppose that both can form
colored polymeric resins, and this may be one of the reasons for
the higher browning activity of D-galacturonic acid compared to
reducing sugars. We suggest a Michael addition as the
polymerization reaction for DHCP and an aldol condensation
for the polymerization of reductic acid (see Figure 2). The
colored condensation products in turn could further react with
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Figure 5. GPC chromatogram with refractive index and UV detection at 420 nm of the aqueous solution of reductic acid after thermal treatment for
120 min at 120 °C. A pullulan standard represents the high molecular weight fraction of 40 kDa (domain B), and a D-glucose solution represents the
low molecular weight fraction, <1 kDa (domain C). Domain A is the newly formed polymer with a molecular weight of >40 kDa and a relatively
strong absorption at 420 nm.
and carbonyl compounds can take place, which lead to the
formation of several chromophoric compounds.23
the formation of reductic acid, DHCP, and furan-2-
carbaldehyde.
Our investigations showed that at pH 5 typical degradation
products of acidic D-galacturonic acid solution such as reductic
acid and DHCP are formed as well as products of alkaline
solution such as norfuraneol and galactaric acid. All these
compounds possess reactive groups that would allow their
further involvement in nonenzymatic browning, possibly
including the formation of colored materials. Because of the
large diversity of degradation products, which can contribute to
browning due to synergistic effects on the formation of
chromophoric and/or polymer structures, browning activity is
highest under slightly acidic conditions. Reductic acid is
unequivocally established as a precursor of chromophoric
structures, and therefore we suppose that it contributes to color
formation in the course of nonenzymatic browning of D-
galacturonic acid as well. Furan-2-carbaldehyde is also a color
precursor but only in association with other reactive
degradation compounds of D-galacturonic acid such as
norfuraneol and reductic acid or other nitrogen-containing
compounds, for example, amines or pyrrols, present in the heat-
treated solution.
We therefore reacted reductic acid, as well as furan-2-
carbaldehyde, with L-alanine, respectively, to obtain colored
condensation products. The solution of reductic acid already
changed its appearance after 5 min and led to an intense red-
colored solution after a reaction time of 2 h. Amino acids
additionally increased the red color formation, but there was no
acceleration of the reaction. Reductic acid was degraded rapidly,
but no Dulkin and Friedemann degradation products such as 2-
ketoglutaric acid or succinic acid could be detected.22 GPC
measurements revealed that high molecular weight compounds
were formed (domain A), which accounted for the absorption
at 420 nm and the red color (see Figure 5). We suppose that
these polymer structures are formed in an autocatalytic reaction
and directly from reductic acid or its dehydro form due to an
aldol condensation. We suppose that a highly conjungated, high
molecular weight structure is the reason for the red color of the
reductic acid model solutions.
Freshly distilled furan-2-carbaldehyde treated in the same
way as reductic acid did not give rise to color formation or
polymeric structures. Only in combination with L-alanine,
pyrrol-2-carbaldehyde, or norfuraneol did the solution turn
slightly yellow after 2 h. Thus, reductic acid surely contributes
to color formation in the course of thermally treating D-
galacturonic acid, whereas furan-2-carbaldehyde contributes to
browning only in the presence of amines and/or methylene
active compounds and polymer formation is much slower.
In summary, the browning activity of D-galacturonic acid is
up to 10 times higher compared to pentoses and hexoses. The
reason for the higher browning activity and the different color is
a divergence in the reaction pathways because of the carboxylic
group on C-6 of the uronic acid leading to different key
intermediates and degradation products. We suggest the main
key intermediate of D-galacturonic acid to be 2-ketoglutar-
aldehyde and its isomers, which are formed after eliminative
decarboxylation at elevated temperatures in slightly acidic to
neutral solutions. 2-Ketoglutaraldehyde is the main precursor in
We also investigated the influence of amino acids on the
browning behavior of D-galacturonic and identified some new
and specific degradation products, which we will present in our
next paper.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: lothar.kroh@tu-berlin.de. Phone: +49 30 314 72 583.
Fax: +49 30 314 72 585.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
Ara, L-arabinose; DAD, diode array detector; DHCP, 4,5-
dihydroxy-2-cyclopenten-1-one; DNPH, 2,4-dinitrophenylhy-
drazine; FF, furan-2-carbaldehyde; GalA, D-galacturonic acid;
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Journal of Agricultural and Food Chemistry
Article
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GC, gas chromatography; GPC, gel permeation chromatog-
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mass spectrometry; OPD, o-phenylenediamine; Norf, norfur-
aneol; RA, reductic acid
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