Article pubs.acs.org/JAFC
D‑Galacturonic
Acid: A Highly Reactive Compound in Nonenzymatic Browning. 2. Formation of Amino-Specific Degradation Products Steffen Wegener, 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 aqueous solutions of D-galacturonic acid and L-alanine at pH 3, 5, and 8 led to rapid and more intensive nonenzymatic browning reactions compared to similar solutions of other uronic acids and to Maillard reactions of reducing sugars. The hemiacetal ring structures of uronic acids had a high impact on browning behavior and reaction pathways. Besides reductic acid (1,2-dihydroxy-2-cyclopenten-1-one), 4,5-dihydroxy-2-cyclopenten-1-one (DHCP), furan-2-carboxaldehyde, and norfuraneol (4-hydroxy-5-methyl-3-(2H)-furanone) could be detected as typical products of nonenzymatic uronic acid browning reactions. 2-(2-Formyl-1H-pyrrole-1-yl)propanoic acid (FPA) and 1-(1-carboxyethyl)-3-hydroxypyridin-1-ium (HPA) were identified as specific reaction products of uronic acids with amine participation like L-alanine. In contrast, the structurally related D-galacturonic acid methyl ester showed less browning activity and degradation under equal reaction conditions. Pectinspecific degradation products such as 5-formyl-2-furanoic acid and 2-furanoic acid were found but could not be verified for Dgalacturonic acid monomers alone. KEYWORDS: Maillard reaction, D-galacturonic acid, D-glucuronic acid, D-galacturonic acid methyl ester, colored compounds, nonenzymatic browning, pyrrole-2-carboxaldehyde, 3-hydroxypyridine, L-alanine
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of uronic acid browning.5 However, until now this suggested compound could not be confirmed by analytical methods, and nitrogen-containing degradation products of D-galacturonic acid and amino acids have not been postulated so far. Therefore, the objectives of the present investigation were to characterize and quantify amino compound-specific degradation products formed in unbuffered aqueous solutions of D-galacturonic acid and L-alanine at different starting pH values. The role of αketoglutaraldehyde as the key intermediate in the formation of browning products was investigated in reactions of Dgalacturonic acid with different amino acids. Moreover, browning behaviors of D-galacturonic acid, D-galacturonic acid methyl ester, and D-glucuronic acid were compared to get more information about the influence of uronic acid ring structures on degradation pathways.
INTRODUCTION Besides scientific aspects, there is a special significance in uronic acids’ Maillard reaction behavior for technological applications. Foods rich in uronic acids such as juices often suffer from undesired browning reactions during thermal processing that effect product quality in a negative way.1 On the contrary, there are certain fields of application in which products could benefit from the use of uronic acids. Different preliminary tests by our working group have shown a faster browning and a better texture of bun crumbs when the dough contained a low monadic D-galacturonic acid portion. A better understanding of uronic acids’ Maillard reaction behavior allows a wide range of options for controlling beneficial and adverse effects on certain foods to emerge, and thus the overall product quality could be improved. Model studies already revealed that kinetics of nonenzymatic browning reactions are different in reducing sugars and uronic acids2 and that uronic acids, especially D-galacturonic acid, produce a more intense and reddish brown color.3 2Ketoglutaraldehyde has already been confirmed as a key intermediate in the formation of reductic acid (1,2-dihydroxy2-cyclopenten-1-one), 4,5-dihydroxy-2-cyclopenten-1-one (DHCP), and furan-2-carboxaldehyde, which is formed due to eliminative decarboxylation of D-galacturonic acid in slightly acidic to neutral media with 4,5-unsaturated 4-deoxy-Larabinose as the expected precursor.3 Little is known about the reaction of D-galacturonic acid with amino acids. Heyns and Schulz as well as Eichner and Ciner-Doruk investigated the Maillard reaction and discovered that the Amadori compounds formed from uronic acids are not very stable and that preferably reverse reactions are taking place.4,5 They postulated that an Amadori rearrangement is not essential for the dehydration of uronic acids because it can also take place on the N-glycoside, leading to 3-deoxyhexosulosuronic acid as the key intermediate © 2015 American Chemical Society
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MATERIALS AND METHODS
Chemicals. The following compounds were obtained commercially: D-galacturonic acid monohydrate (Chemodex, Switzerland); furan-2-carboxaldehyde, norfuraneol, γ-aminobutyric acid (GABA), 2furanoic acid, 5-formyl-2-furanoic acid, D -glucuronic acid, (trimethylsilyl)diazomethane (Sigma, Germany); N-methylpyrrole-2carboxylic acid (Maybridge, USA); tetrabutylammonium hydrogensulfate (Acros, Germany); L-alanine, L-aspartic acid, D-galactose, L-arabinose (Fluka, Germany); and phosphorus pentoxide (Fisher Scientific, Germany). Furan-2-carboxaldehyde was freshly distilled at 300 °C at reduced pressure (100 mbar) prior to use. Solvents were of HPLC grade (Merck, Germany). Reductic acid was synthesized as previously described.3 Received: Revised: Accepted: Published: 6457
March 3, 2015 May 27, 2015 June 25, 2015 June 25, 2015 DOI: 10.1021/acs.jafc.5b01121 J. Agric. Food Chem. 2015, 63, 6457−6465
Article
Journal of Agricultural and Food Chemistry
α-dicarbonyl compounds, o-phenylenediamine hydrochloride (OPD; 20 mM) was added (1:1; v/v). Because advance tests had shown no further chemical change after 3 h of reaction time, the samples were reincubated for at least 3 h at room temperature to obtain UV-active quinoxalines and subsequently analyzed by HPLC-DAD. For identification and quantification of FPA the carboxyl group was methylated with (trimethylsilyl)diazomethane and subjected to GCMS. Model solutions (0.5 M) of D-glucuronic acid, D-galactose, and Darabinose with 0.05 M L-alanine were treated and analyzed as the model reactions of D-galacturonic acid. D-Galacturonic Acid versus D-Galacturonic Acid Methyl Ester. The concentrations of the model reactions were 0.05 M of the carbonyl compound and 0.005 M of the amino acid L-alanine. The procedure and the measurements were the same as in the higher concentrated model systems of D-galacturonic acid. All experiments were conducted in three independent replications, and the arithmetical mean of all quantified values was calculated. For quantification five-point calibration curves were determined from parent solutions of mixes of different pure reference compound groups. Qualification and Quantification. Organic Acids and Acireductones. The diluted samples were analyzed by HPLC-DAD (degasser, Gastorr GT-103; pump, Shimadzu LC-9A; autosampler, Dionex GINA50; column oven, VDS optilab; guard column, YMC ODS-AQ 4.0 × 20 mm, 3 μm; column, YMC-Pack ODS-AQ 150 × 4.6 mm, 3 μm; detector, Gynkotek UVD340S; software, Chromeleon v6.00 build 435) using a phosphate buffer (20 mM, pH 2.8) as an isocratic eluent with a flow rate of 0.6 mL/min, an oven temperature of 25 °C, and an injection volume of 40 μL. The effluent was monitored 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 compounds was tentatively accomplished by comparing their retention times with those of authentic known commercially available references or pure authentic compounds isolated and elucidated from reaction mixtures in our working group.3 For quantification pure reference compounds were used. Furans and Furanones. The diluted samples were analyzed by HPLC-DAD (setup as described above; guard column, MachereyNagel CC 8/4 Nucleosil 120/3 C18; column, Macherey-Nagel EC 125/3 Nucleosil 120-3 C18) 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 with a flow rate of 0.5 mL/min, an oven temperature of 40 °C, and an injection volume of 40 μL. By monitoring the effluent at a wavelength of 280 nm for furan-2carboxaldehyde (6.7 min) and norfuraneol (3.6 min) and at a wavelength of 254 nm for 2-furanoic acid (8.5 min) the compounds were detected. Quantification was performed by external calibration with standard solutions containing known amounts of either commercially available references or pure authentic compounds isolated and elucidated from reaction mixtures in our working group.3 α-Dicarbonyl Compounds as Quinoxalines. The derivatized samples were analyzed by HPLC-DAD (setup as described above; guard column, Macherey-Nagel CC 8/4 Nucleosil 120/3 C18; column, Macherey-Nagel EC 250/4.6 Nucleosil 120-5 C18) using 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 with a flow rate of 1.0 mL/min, an oven temperature of 35 °C, and an injection volume of 40 μL. Quantification 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.11 Identification of unknown quinoxalines was accomplished by LC-MS measurements in the total ion mode of the pre- and postderivatized and diluted samples. The gradient used for the separation was the same (Thermo Fischer Scientific pump Accela, CTC PAL autosampler) as for the HPLC-DAD measurements.
Syntheses. D-Galacturonic Acid Methyl Ester. (Trimethylsilyl)diazomethane (TMSDM) was used for the esterification of Dgalacturonic acid instead of explosive and toxic diazomethane6 or methanolic hydrochloric acid7,8 used in most other publications. Methylation of carboxylic acids with TMSDM is more specific and can take place under relatively mild conditions.9 One hundred milligrams of D-galacturonic acid (0.5 mmol) was dried over phosphorus pentoxide and dissolved in 5 mL of absolute methanol. A solution of 0.2 M TMSDM diluted with diethyl ether was added dropwise until the yellow color of the reagent persisted. The reaction proceeded instantaneously and nearly quantitatively without any influence of other reactive groups. The reaction was easily monitored by the disappearance of the yellow color of TMSDM. The reaction mixture was stirred for 30 min at room temperature and concentrated under reduced pressure to give the corresponding methyl ester. The yellow syrup was purified by recrystallization from 1,4-dioxane and yielded after freeze-drying 83.1 mg (0.39 mmol) of a white powder, which was instantaneously used for model reactions. Purity was confirmed by GC-MS after derivatization with trifluoroacetic acid anhydride (TFAA). For this purpose aliquots of the freeze-dried substance were resolved in 325 μL of dichloromethane. One hundred and fifty microliters of TFAA and 25 μL of pyridine were added, and the sample was shaken for 30 s and afterward derivatized for 3 h at room temperature. After that, 500 μL of dichloromethane was added and the diluted derivatized sample subjected to GC-MS. For the GC-MS analysis of the D-galacturonic acid methyl ester the same setup was used as for pyrrole-2-carboxaldehyde (see below). FPA. D-Galacturonic acid (10.345 g; 50 mmol) and L-alanine (4.595 g; 52 mmol) were dissolved in 100 mL of distilled water and stirred for 2 h under reflux. The resulting brown/black reaction mixture was extracted with 40 mL of ethyl acetate/diethyl ether (1:1) three times. The combined extracts were concentrated in a nitrogen stream and purified by silica gel flash chromatography (Merck silica gel 60, 0.040− 0.063 mm, 230−400 mesh) with ethyl acetate/diethyl ether (1:1) as solvent. Removing the solvent at 30 °C in vacuo yielded 16.6 mg (0.1 mmol) of yellow crystals. Tentative identification and purity control were carried out by GC-MS (setup as described below) after derivatization with TMSDM. For this purpose 10 μL of Nmethylpyrrole-2-carboxylic acid as internal standard (9 mM in methanol) was added to an aliquot of 10 μL of FPA in methanol (5.5 mM). After that, 150 μL of a TMSDM solution (20 mM) in methanol/diethyl ether was slowly added for derivatization. After 30 min at 25 °C in a reaction tube, the sample was subjected to GC-MS (see Figure 3). A purity of 98% was achieved. Model Reaction. D-Galacturonic Acid versus D-Glucuronic Acid and Reducing Sugars. To investigate the degradation behavior and the formation of key intermediates and degradation products, Dgalacturonic acid monohydrate (0.5 M) and L-alanine, L-aspartic acid, or GABA (0.05 M) were dissolved in water. The above-mentioned concentrations were chosen to adapt the model systems as close as possible to foods rich in uronic acids such as juices or jams in which the uronic acid compounds occur in a surplus. The pH value was adjusted to 3.0, 5.0, or 8.0 by adding thinned hydrochloric acid and sodium hydroxide solution. In this case, the application of buffer substances was avoided by choice, because buffers are known to cause unintended side reactions.10 Aliquots of the model solution were filled into glass ampules and incubated in a heating block at 100 °C for 2 h. The reaction was stopped at various points in time 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 Spectralon integrating sphere (Specord 40, Analytik Jena, Jena, Germany). pH values were measured after the sample had been cooled to room temperature with a pH-electrode (SenTix Mic, WTW, Weilheim, Germany; pH-meter CG 820, Schott, Mainz, Germany). For the analysis of the organic acids, aci-reductones, and pyridines, samples were diluted (1:10) with 1% metaphosphoric acid and 10 mM dithiothreitol and measured by HPLC-DAD. For the quantification of furans, furanones, and pyrroles, samples were diluted (1:100; v/v) with 10 mM phosphate buffer (pH 6.0) and subjected to HPLC-DAD. For 6458
DOI: 10.1021/acs.jafc.5b01121 J. Agric. Food Chem. 2015, 63, 6457−6465
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Journal of Agricultural and Food Chemistry Ionization (TSQ Vantage System with Ion Max Source, H-ESI II Probe) in the ESI positive 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 m/z 50−1000. Software Thermo Excalibur 2.1.0.1139 was used for the identification of the substances. Pyrroles and Hydroxypyridines. The diluted samples were analyzed by HPLC-DAD (see above) in the case of 3-hydroxypyridines using the same method as for organic acids, but monitoring the effluent at a wavelength of 280 nm and in the case of pyrrole-2-carboxaldehyde derivatives with the same method mentioned for furans but monitoring at a wavelength of 293 nm. Quantification was performed by external calibration with pure authentic compounds isolated and elucidated from reaction mixtures in our working group. For the identification and quantification of FPA a GC-MS method was established. Therefore, the carboxyl group had to be methylated with (trimethylsilyl)diazomethane. For that, 100 μL of a model solution was spiked with 10 μL of N-methylpyrrole-2-carboxylic acid in methanol (2 mM) as internal standard in a 1 mL reaction tube. The sample mixture was dried under nitrogen stream and resolved in 100 μL of distilled water. After that, the sample was extracted five times with 100 μL of diethyl ether/ethyl acetate (1:1). The combined extracts were dried under nitrogen stream and resolved in 100 μL of methanol. For derivatization 300 μL of TMSDM (20 mM) was added, and after 30 min at 25 °C, the sample was subjected to GC-MS analysis (Shimadzu GC-2010, AOC-20i autosampler, GCMSQP2010plus mass spectrometer, Duisburg, Germany) using a fused silica capillary column (Supelco SLB-5 ms, 30 m × 0.25 mm, 0.25 μm). The derivatized samples were applied by a split injection technique at an injection temperature of 250 °C and an oven temperature of 80 °C. The temperature was increased at a rate of 6 °C/min to 104 °C, held for 1 min, increased again at a rate of 10 °C/ min to 164 °C, and then increased at a rate of 12 °C/min to 280 °C. Helium 5.0 was used as a carrier gas with a flow rate of 2 mL/min. Software Lab Solutions version 2.71 was used for the identification of the substances.
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browning trend. More precisely, after a reaction time of 120 min at 100 °C, a D-galacturonic acid model solution exceeded twice the value of the absorption at 420 nm of an equimolar Dglucuronic acid reaction model (results not shown). Figure 2 shows the influence of several amino acids on browning intensities of the D-galacturonic acid solutions
Figure 2. Nonenzymatic browning of D-galacturonic acid (GalA) solutions with L-alanine (Ala), L-aspartic acid (Asp), or γ-aminobutyric acid (GABA) after thermal treatment at 100 °C and a starting pH value of 5.0, measured as absorption at 420 nm.
(starting pH 5.0), which were heated at 100 °C for different time intervals. As expected, GABA promotes browning the most followed by Ala and Asp. Both might degrade more rapidly in the presence of dicarbonyl compounds (Strecker degradation), resulting in a loss of browning-fostering molecules. In comparison to a starting pH of 5.0, an initial pH value of 3.0 caused less absorption at 420 nm, whereas starting at pH 8.0 induced a higher absorption. Despite different browning intensities all starting pH values led to the same order of priority concerning the amino acid influence on color development (results not shown). Not only the addition of amino acids but also the structure of the carbonyl compound can affect color formation, especially the presence of the carboxyl group on C-6 and the stereochemistry of a 1C4-conformation in the pyranoic form. Eliminative decarboxylation gives a plausible explanation for the carboxyl group’s strong influence. Eliminative decarboxylation preferably occurred in the presence of D-galacturonic acid with the substituents at C-4 and C-5 in trans position and at elevated temperatures under slightly acidic to neutral conditions. Table 1 shows the browning values and the corresponding CIELab values for the thermally treated model solutions (2 h) and the calculated yellowness (YI), chroma (C), and saturation (S). Compared to the slightly yellow model solutions of the previously heated reducing sugars (Ara, Glc) the color of uronic acids Maillard reactions was reddish and CIELab measurements revealed that not only the lightness was considerably lower, but the +a (red) as well as the +b (yellow) values were higher in the thermally treated D-galacturonic acid solutions compared to reducing sugars. In the presence of amino acids (e.g., L-alanine) the lightness of the model solutions was lower than without an amino acid, the +a value increased from +19 to +36, whereas the +b value decreased from +54 to +44. Also, the structure of the amino acid influences color and browning intensity. The browning value was highest with GABA (13.1) and lowest with L-aspartic acid (4.5); the average value of L-alanine was (9.9).
RESULTS AND DISCUSSION
Formation of Colored Compounds. Thermal treatment of unbuffered solutions of D-galacturonic acid and L-alanine at pH 5.0 and a temperature of 100 °C for 2 h led to a 6 times higher browning compared to L-arabinose, a 7 times higher browning compared to D-galactose, and an almost 3 times higher browning compared to D-galacturonic acid alone (see Figure 1). Its epimer D-glucuronic acid offers a decreased
Figure 1. Nonenzymatic browning of D-galacturonic acid (GalA) solutions with and without L-alanine (Ala) compared to solutions of Larabinose (Ara), D-glucose (Glc), and D-galactose (Gal) with L-alanine thermally treated at 100 °C and a starting pH value of 5.0, measured as absorption at 420 nm. 6459
DOI: 10.1021/acs.jafc.5b01121 J. Agric. Food Chem. 2015, 63, 6457−6465
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Table 1. CIELab Values of Maillard Reaction Models of D-Galacturonic Acid (GalA) Alone or with L-Alanine (Ala), L-Aspartic Acid (Asp), or γ-Aminobutyric Acid (GABA) Compared to Model Solutions of L-Arabinose (Ara) and D-Glucose (Glc) with LAlanine at a Reaction Temperature of 100 °C after 2 h and a pH Value of 5.0 at the Beginning of the Reactiona Ara + Ala Glc + Ala GalA GalA + Ala GalA + Asp GalA + GABA
L
a (red)
b (yellow)
YI
C
S (%)
Abs420
90 98 67 56 56 21
−1 −2 19 36 28 30
22 11 54 44 43 −13
35 16 114 113 111 90
22 11 57 57 57 33
24 11 65 71 71 84
2.3 1.5 4.0 9.9 4.5 13.1
a
Yellowness (YI), chroma (C), and saturation (S) were calculated according to the method of Hunter and Harold.22 Browning intensity (Abs420) was measured as the absorption at 420 nm.
Figure 3. Total ion chromatogram of the FPA as a degradation product of D-galacturonic acid (GalA) and L-alanine by GC-MS after postderivatization with TMSDM and total ion chromatogram of the methylated FPA derivative (tR 14.73 min) with its embedded fragmentation pattern and the internal standard N-methylpyrrole-2-carboxylic acid (tR 10.42 min).
which are involved in the formation of polymeric dyes.14 Higher pH values as well as the addition of amino compounds accelerated the degradation reaction of D-galacturonic acid and led to a higher absorption at 420 nm and an increase in the +a value. Reactive Intermediates of D-Galacturonic Acid. Our earlier experiments confirmed that 2-ketoglutaraldehyde is a key intermediate in the formation of reductic acid, DHCP, and furan-2-carboxaldehyde.3 This would support the eliminative decarboxylation mechanism being the main degradation pathway of D-galacturonic acid under slightly acidic to neutral conditions. A concerted mechanism involving a simultaneous dehydration at C-4 and decarboxylation at C-5 of GalA resulting in the formation of 4,5-unsaturated 4-deoxy-Larabinose, as labile precursor for 2-ketoglutaraldehyde formation, can be assumed. After dehydration, the postulated 4,5unsaturated 4-deoxypentose can yield the enol form of 2ketoglutaraldehyde, which leads directly to the formation of reductic acid or, after isomerization, to DHCP and furan-2carboxaldehyde. However, because Amadori compounds of Duronic acids are unstable, the 4-deoxypentose can also react with the amino group to form imine intermediates, which then may cyclize to N-heterocyclic compounds such as pyrroles and pyridines. 3-Hydroxypyridinium compounds have already been found as degradation products of D-glucuronic acid and peptides by Horvat et al., who postulated a formation pathway that includes the intermediate formation of an Amadori compound.12 So far, pyrroles have not been identified as degradation products of thermally treated uronic acids. Figure 3 shows the total ion chromatogram of the GC-MS analysis of an unknown degradation product, which has been isolated from the model
Furthermore, differences in yellowness, saturation, and chroma could be detected. These three parameters are much higher in thermally treated D-galacturonic acid solutions than in model solutions of reducing sugars as well. These findings emphasize the differences in browning behavior and in the formation of polymeric dyes between uronic acids and common reducing sugars. The browning of D-glucuronic acid was slower than that of D-galacturonic acid, but the characteristic red color of the model was equal (data not shown). Color formation by Dgalacturonic acid methyl ester was also slower than that of the unesterified D-galacturonic acid and the browning index detectable after 2 h of heating at 100 °C without amino acid catalysis was only around half of the value of unesterified Dgalacturonic acid (data not shown). As described for the D-galacturonic acid model solutions without amino acid, the pH value of D-galacturonic acid solutions with L-alanine increased steadily from starting pH 5.0 until it reached 5.8 after 120 min (data not shown). Only in model solutions starting at pH 3.0 did the value not change with reaction progress. The pH increase may be caused by heterocyclic or carbocyclic compounds formed during thermally induced decomposition of D-galacturonic acid. These heterocyclic compounds are supposed to be less acidic than short-chained dicarbonyl cleavage products as formic or acetic acid. In addition, a loss of the carboxyl group of the uronic acid itself due to eliminative decarboxylation leads to a deacidification of the model system. In contrast to the almost colorless to slightly yellow color of the common reducing sugar solutions, the color of uronic acid model solutions treated under the same conditions was red-brown. This typical red-brown color of Dgalacturonic acid solutions is caused by specific degradation products, such as reductic acid and the pyrroles discussed later, 6460
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via the formation of the Amadori compound 2c shown in Figure 5 alone. Amadori compounds are not easily formed under these conditions, because the intermediate N-glucosides 2a−2c are not stable and preferably cleaved back to the free uronic and amino acid 1. A direct dehydration of the Nglycoside should lead to the formation of 3-deoxyhexosulosuronic acid 6, which then preferably degrades to furan-2carboxaldehyde and reductic acid.3,5 Compared to the relatively high concentrations of FPA and HPA no furan-2-carboxaldehyde was found in the solutions and only traces of 3deoxyhexosulosuronic acid could be detected by LC-MS after postderivatization of the solution with OPD (determined as quinoxaline). Thus, a formation pathway of pyrrole-2carboxaldehyde derivatives and 3-hydroxypyridines distinct from the formation of furan-2-carboxaldehyde and 3-deoxyhexosulosuronic acid is postulated (see Figure 6). Due to eliminative decarboxylation of D-galacturonic acid, the 4,5unsaturated 4-deoxy-L-arabinose is formed. In the presence of amino acids a nucleophilic addition reaction at C-5 followed by dehydration and cyclization reactions leads to the sixmembered N-heterocyclic compound HPA. As a competitive reaction the nucleophilic addition at C-1 yields a Schiff base. A concerted cyclization dehydration reaction via a carbenium ion 10, the (3Z)-configuration of which is mandatory for cyclization,13 results in the five-membered N-heterocyclic compound FPA. Besides pyrroles such as FPA that are formed via a breakdown of the carbohydrate backbone and the incorporation of amino compounds, another reaction pathway was postulated by De Kimpe et al. This pathway suggests pyrroles being built from smaller fragmentation products. Methylglyoxal was identified as an important precursor leading via condensation reactions to highly substituted pyrroles.14 3-Hydroxypyridine derivatives are known to be stable compounds and therefore not relevant as color precursors,15 which was experimentally confirmed (data not shown). However, Tressl et al. identified certain pyrroles as important precursors of polymerization and color and postulated an acidcatalyzed formation of so-called type II polymers. Furan-2carboxaldehyde could also take part in this polymerization, but decelerated the polymerization speed.16 Type II polymers possess a red color, but after longer exposure to air, they turn black because of oxidation reactions. The higher the reaction temperature and the higher the amount of amino acids present in the model solutions of uronic acids, the higher was the
solution of GalA and L-alanine by diethyl ether/ethyl acetate extraction. After methylation with TMSDM, this compound could be identified by GC-MS as FPA and thus also verified pyrroles in general as typical reaction products of D-galacturonic acid and amino acids for the first time. In addition, the formation of pyrrole-2-carboxaldehyde derivatives could also be confirmed in the reaction of D-galacturonic acid with other amino compounds such as L-glycine and GABA, but not with Laspartic acid (data not shown). These pyrrole-2-carboxaldehydes are also formed during thermal treatment of Dgalacturonic acid methyl ester (see Table 3). The concentration of pyrrole-2-carboxaldehydes formed depends not only on the ratio of carbonyl to amino compound but also on the structure of the carbonyl used (see Figure 4), as
Figure 4. Comparison of FPA formation in model reactions between L-alanine (Ala) and D-galacturonic acid (GalA), D-glucuronic acid (GlcA), and L-arabinose (Ara) at 100 °C and a starting pH value of 8.0.
well as on the reaction temperature, initial pH value, and reaction time of the model solution. The highest amounts were formed with ratios of uronic acid to amino acid between 2:1 and 1:1, pH values 100 °C (data not shown). Table 2 gives an overview of the concentration of the most important products formed during thermal treatment of Dgalacturonic acid compared to D-glucuronic acid solutions with and without the presence of L-alanine at 100 °C after 2 h. Relatively high concentrations of FPA 4 and HPA 5 in the reaction of both uronic acids with L-alanine cannot be explained
Table 2. Concentrations (Micromolar) of the Main Degradation Products Reductic Acid (RA), DHCP, Furan-2-carboxaldehyde (FF), Norfuraneol (Norf), FPA, HPA, and Glyoxal and the Sum of Short-Chain α-Dicarbonyl Compounds (DC) in Model Solutions of D-Glucuronic Acid (GlcA) with and without the Participation of L-Alanine (Ala) Compared to Model Reactions of D-Galacturonic Acid (GalA) after Thermal Treatment for 2 h at 100 °C and with Different Initial pH Values GalA GalA + Ala
GlcA GlcA + Ala
a
pH
RA
DHCPa
FF
Norf
5 8 5 8
150 370 610 700
40000 nd 12000 nd
nd nd nd nd
260 2200 290 4400
5 8 5 8
630 330 420 860
22000 5200 41000 nd
nd nd nd nd
850 200 3900 1900
FPA
840 180
360 59
HPA
glyoxal
DCb
230 130
69 110 40 100
110 170 71 160
180 nd
190 280 110 160
320 460 430 430
quantified as malic acid equivalent. bSum of glyoxal, 2-oxopropanal, diacetyl, and 3-deoxypentosulose. 6461
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Figure 5. Formation of FPA 4 and HPA 5 via the Amadori reaction pathway. 2a−2c formed of D-galacturonic acid 1 and L-alanine12 by decarboxylation, cyclization. and dehydrations reactions. Direct dehydrations of the N-glycoside 2b without the formation of an Amadori compounds led to the formation of 3-deoxyhexosulosuronic acid 6.
Figure 6. Alternative formation pathway of N-heterocyclic compounds due to imine formation on C-5 or C-1 of the 4,5-unsaturated 4-deoxy-Larabinose 8 with L-alanine and further cyclization and dehydration to FPA 4 or HPA 5.
concentration of pyrrole-2-carboxaldehyde derivatives and the darker was the color of the model solution (data not shown). The polymerization speed along with the polymer’s solubility depended on the structure of the amino acid. In equimolar solutions of D-galacturonic acid with L-alanine or GABA at a reaction temperature of 160 °C, the associated pyrrole-2carboxaldehyde derivative (FPA, FPG) was the main degradation product. Figure 7 illustrates the pyrrole-2carboxaldehyde derivative formation in relation to the browning of the respective model solutions measured at 420 nm. In the above-mentioned reaction with L-alanine the FPA formation was very fast and the maximum concentration was reached after 5 min. Afterward, it slightly decreased because D-galacturonic acid was probably degraded in large part, but because of the accumulated FPA, polymerization could still take place; only the polymerization speed slowed. In the case of L-alanine the polymers were still soluble in the aqueous solution after 2 h of continuous heating. With GABA, the polymerization was even faster than with L-alanine; insoluble black particles were formed
Figure 7. Nonenzymatic browning progress as the absorption at 420 nm and FPA and FPG concentrations of D-galacturonic acid (GalA) solutions with L-alanine (Ala) or γ-aminobutyric acid (GABA) at a reaction temperature of 160 °C, respectively.
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Figure 8. β-Elimination of the pyranoic and the furanoic forms of D-galacturonic acid methyl ester 1a, 1b and further degradation to 5-formyl-2furanoic acid 16 and β-elimination of the furanoic form of the methyl ester 1b leading to furan-2-carboxaldehyde 18.
nal, diacetyl, and 3-deoxpentosulose, respectively, were 3−4 times higher in D-glucuronic acid models. On the other hand, cyclic nitrogen-containing products such as FPA and HPA are formed on a larger scale in D-galacturonic acid browning reactions. The most obvious reason for these differences in reaction behavior is their special cyclic conformation, as mentioned above. However, more structural elucidation is necessary to clarify this issue. A comparison of degradation products detected after thermal treatment of D-glucuronic and D-galacturonic acid model reactions with and without L-alanine, respectively, after 2 h of reaction at 100 °C and starting pH values of 5.0 and 8.0 can be found in Table 2. Reactive Intermediates of D-Galacturonic Methyl Ester. The monomeric methyl ester of D-galacturonic acid prefers the furanoic form 1b (shown in Figure 8) as well, and therefore β-elimination at C-3 is preferred.20 Subsequent dehydration and elimination of the labile formic acid methyl ester group is supposed to lead to the formation of furan-2carboxaldehyde (FF) 18 as the main degradation product. This pathway would give an explanation for the comparatively high amounts of FF detected for GalA methyl ester models compared to free D-galacturonic acid model reactions as mentioned above. When the reaction was started at pH 3.0, the pH value remained stable because neither alkaline hydrolysis leading to higher pH values nor acid ester cleavage can take place at these conditions (see Table 3). Starting GalA methyl ester model reactions at pH 5.0, deesterification and β-elimination proceeded simultaneously and caused a decrease in the pH value from 5.0 to about 4.0. Except for DHCP, all other described degradation products of D-galacturonic acid were formed, but mostly in a lesser extent, with the exception of FF (Table 3). Concerning reductic acid, the thermal D-galacturonic methyl ester degradation yields only a small amount of it compared to its unesterified derivative. Methylation seems to prevent a β-ketoacid-like decarboxylation leading to reductic acid via 2-ketoglutaraldehyde. Starting thermal degradation at pH 3.0, the methyl ester was partially hydrolyzed to the free acid, which could be further converted into its typical and highly browning active degradation products such as reductic acid. In contrast, less acidic starting conditions (pH 5.0) caused a decrease in reductic acid. Under these conditions β-elimination of the relatively stable methyl ester
after about 20 min. After reaching an absorption plateau at 420 nm at about 5 min of reaction time, the absorption of the solution with GABA started to decrease again because of the formation of insoluble black particles. Experiments with several uronic acid derivatives gave indication that the reason for the differences in browning behavior lies within the preferred ring structure of these compounds in aqueous solution. D-Galacturonic acid prefers the lowest energy 1C4 chair conformation with the substituents at C-4 and C-5 in trans position.17 This can be the reason for a relieved eliminative decarboxylation under neutral to slightly acidic conditions resulting in the formation of 2-ketoglutaraldehyde and its isomers and their secondary degradation products discussed recently.3 D-Glucuronic acid mainly exists in the more labile furanoic hemiacetal form, which is stabilized by a γ-lactone ring under acidic conditions.18 Due to this lactone ring the proton of the C-4 is acidified and can easily be withdrawn by a base in a β-elimination step at C-3. Dax et al. postulated a degradation pathway that leads to the formation of glyoxal and 3-formyl-2-oxopropanoic acid in acidic solutions of D-glucuronic acid and the formation of 2-ketoglutaraldehyde under more alkaline conditions.19 This postulated pathway could be confirmed by our investigations, which proved glyoxal as the main α-dicarbonyl compound also formed by the degradation of D-galacturonic acid under slightly acidic conditions. 2-Ketoglutaraldehyde emerged as a key intermediate in browning and the formation of other typical degradation products such as reductic acid and DHCP. In addition, the furanoic compound norfuraneol could be verified as a main degradation product (see Table 2). In reactions with Dgalacturonic acid it can especially be found under alkaline conditions, and in participation of L-alanine the amount formed is doubled. In contrast, D-glucuronic acid model reactions yield only small portions of norfuraneol. Adding L-alanine increases the norfuraneol amount to the level found in alkaline D-galacturonic browning reactions. As mentioned above, this can be explained by D-glucuronic acid mainly existing as γ-lactone in aqueous solutions, which hinders a direct degradation. However, in the presence of amino compounds the degradation reaction toward norfuraneol is catalyzed to a high degree. Compared to model reactions with D-galacturonic acid, the total concentration of the short-chain α-dicarbonyl compounds glyoxal, 2-oxopropa6463
DOI: 10.1021/acs.jafc.5b01121 J. Agric. Food Chem. 2015, 63, 6457−6465
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to eliminative decarboxylation of D-galacturonic acid and an associated carbonyl−amine condensation, Schiff bases of the 4,5-unsaturated 4-deoxy-L-arabinose are formed, which after cyclization and dehydrations lead to the corresponding Nheterocyclic compounds. By comparison of results obtained for D-galacturonic acid model systems with those of the corresponding methyl ester, the ester compound shows less browning activity and degradation under equal reaction conditions. 5-Formyl-2furanoic acid and 2-furanoic acid could not be identified as specific reaction products of monomeric esters even though thermal pectin decomposition is known to yield these acids. Further short-chain acidic cleavage products such as formic or acetic acid could not be verified as degradation products for both D-galacturonic acid and its methyl ester. In conclusion, uronic acids are assumed to be responsible for color alteration processes in thermally treated pectin-containing foods. Especially some kinds of juices such as apple juice are susceptible to color change because amino−carbonyl reactions between the endogenous uronic and free amino acids can easily take place during industrial applied pasteurization and storage processes. In this case not only does the undesired color diminish quality but also a loss of free amino acids connected with a decline in nutritional quality is to be expected. For industrial use our findings may implicate changes in cultivation procedures resulting in a reduced free uronic acid content of respective fruits and vegetables and the consideration of negative effects caused by uronic acids during special engineering technologies. However, besides color effects uronic acids affect food quality in many other ways such as texture, taste, and scent, and thus more studies concerning their roles in foods and food technology and their influence on the end product are strongly recommended before a final evaluation.
Table 3. Concentrations (Micromolar) of the Main Degradation Products Reductic Acid (RA), Furan-2carboxaldehyde (FF), Norfuraneol (Norf), FPA, and HPA in Model Solutions of D-Galacturonic Acid Methyl Ester (GalAMe) with and without L-Alanine (Ala) Compared with Model Reactions of D-Galacturonic Acid (GalA) with and without Ala after Thermal Treatment for 2 h at 100 °C and with Different Initial pH Values GalA GalA + Ala
GalA-Me GalA-Me + Ala
pH
RA
FF
Norf
FPA
HPA
3 5 3 5
7 39 38 29
9 2 2 38
nd 15 29 368
50 14
203 84
3 5 3 5
12 6 2 2
17 30 43 58
nd nd nd nd
35 41
157 84
seemed to be preferred, leading via a 2,3-unsaturated intermediate to FF as already described. In the presence of Ala a decrease of the browning active RA could be seen at all pH values, probably caused by amino− carbonyl reactions leading to colored polymers. However, the formation of other less reactive compounds such as FF, FPA, or HPA was strongly promoted. Concerning the pH influence, FPA formation seemed analogous to FF, whereas HPA was formed only after acidic ester hydrolysis to a greater extent. Pectins are important biopolymers that naturally occur in the more ligneous parts of fruits and vegetables. Their backbone mainly consists of D-galacturonic acid methyl ester and Dgalacturonic acid units, which are both present in 4C1 chair conformation. When pectins are exposed to alkaline conditions, the methyl ester 1a is particularly degraded by β-elimination at C-4. Therefrom unsaturated oligogalacturonides occur, which decompose to shortened saturated oligogalacturonides and further degrade via furan-typical dehydration rearomatization reactions to 5-formyl-2-furanoic acid 16.21 2-Furanoic acid, which was also experimentally verified, is supposed to be formed in the same reaction pathway. Neither furanoic acid could be detected during the degradation of monomeric D-galacturonic acid methyl ester alone, thus confirming the different degradation pathways preferred by the furanoic or pyranoic ring structure according to the constitution of the uronic acid derivative (see Figure 8). In summary, it can be stated that D-galacturonic acid has emerged as a very potent browning active carbonyl compound within the Maillard reaction compared to other related carbonyl compounds such as D-glucuronic acid or L-arabinose. Model reactions of D-galacturonic acid and L-alanine provoke a >6 times higher browning intensity compared to similar reactions of Ara, Gal, and GalA, respectively. CIELab measurements revealed thereby a uronic acid specific red cast formed during the Maillard reaction. Besides reductic acid, DHCP, furan-2-carboxaldehyde, and norfuraneol, pyrrole-2-carboxaldehyde and 3-hydroxypyridine1-ium amino acid derivatives were identified as specific Maillard reaction products of uronic acids. The so-formed relatively high concentrations cannot likely be explained by the Amadori rearrangement pathway alone becaue uronic acid N-glucosides are not stable and easily decompose to their starting products. Thus, an alternative reaction pathway has been suggested. Due
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AUTHOR INFORMATION
Corresponding Author
*(L.W.K.) E-mail:
[email protected]. Phone: +49 30 314 72 583. Fax: +49 30 314 72 585. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED Ara, L-arabinose; Ala, L-alanine; Asp, L-aspartic acid; DAD, diode array detector; DHCP, 4,5-dihydroxy-2-cyclopenten-1one; DNPH, 2,4-dinitrophenylhydrazine; FF, furan-2-carboxaldehyde; FPA, (2-(2-formyl-1H-pyrrol-1-yl)propanoic acid; FPG, (4-(2-formyl-1H-pyrrol-1-yl)butanoic acid); GABA, γaminobutyric acid; Gal, D-galactose; GalA, D-galacturonic acid; GalA-Me, D-galacturonic acid methyl ester; GC, gas chromatography; GlcA, D-glucuronic acid; GPC, gel permeation chromatography; HPA, 1-(1-carboxyethyl)-3-hyroxypyridin-1ium; HPLC, high-performance liquid chromatography; MS, mass spectrometry; OPD, o-phenylenediamine; Norf, norfuraneol; RA, reductic acid; TMSDM, (trimethylsilyl)diazomethane
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REFERENCES
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