Purification and Partial Characterization of Melanoidins Fractions from Toasted Oak Heartwood , Comparison with Melanoidins from Roasted Coffee

During the cooking, processing, and storage of food products, a whole range of browning reactions occurs, initiated by the reaction of a carbohydrate with a compound possessing a free amino group. Melanoidins formed, influence food quality, mainly their colour, their flavour, and their antioxidant activities. Melanoidins are complex Maillard reaction products. We developed a method to isolate coffee melanoidins and melanoidins from toasted oak wood. We noted that coffee is richer in melanoidin compounds than oak wood. We presented a partial characterization of melanoidins fractions from toasted oak heartwood, and a comparison with melanoidins from roasted coffee. Mass spectra of the fractions isolated from toasted oak wood indicate the presence of pentose and hexose-based oligosaccharides with different degrees of polymerisation. The presence of the oligosaccharide moieties, as well as their degradation products found in the oak wood melanoidins, supports the postulated carbohydrate-based origin of melanoidins.


Introduction
During the cooking, processing, and storage of food products, a whole range of browning reactions occurs, initiated by the reaction of a carbohydrate with a compound possessing a free amino group.Melanoidins occur in many stored and processed foods.They influence food quality, mainly owing to their colour, their flavour, their antioxidant activities (Manzocco et al., 2001;Morales et al., 2005;Lindenmeier et al., 2002;Charles-Bernard et al., 2005;Ames, 1998;Delgado-Andrade & Morales, 2005;Delgado-Andrade et al., 2005;Wang et al., 2011;Borrelli et al., 2002;Andriot et al., 2004).Melanoidins are present in several food products, such as coffee, roasted malt, breakfast cereals, bread… Melanoidins are complex Maillard reaction products.Melanoidins are formed from cyclizations, dehydrations, retroaldolizations, rearrangements from nitrogen compounds and sugars, isomerizations, and condensations of low molecular weight Maillard reaction products.Due to the high reactivity of the intermediates, a complex polymerization takes place, resulting in brown-colored high molecular weight melanoidins.The composition of melanoidin chemical structures is relatively unknown, however, due to the complexity of the products that are generated in the reaction (Bekedam et al., 2008;Kim & Lee, 2009).Based on model systems, different suggestions have been made for melanoidins structures (Tressl et al., 1998;Hofmann, 1988;Cä mmerer et al., 2002;Nunes & Coimbra, 2007).However, their complexity and structures depend on the nature and number of possible reactants and the reaction conditions.Therefore the Maillard reaction in foods and the structures of the resulting melanoidins are presumed to be much more complex than in model systems, and different melanoidin structures may coexist (Adams et al., 2005).Different hypotheses have been formulated on the structural backbone of melanoidins.A first hypothesis states that a melanoidin skeleton is constituted mainly from sugar degradation products, polymerized through aldol-type condensations and probably branched via amino compounds (Kato & Tsuchida, 1981;Yaylayan & Kaminsky;1998;Cä mmerer & Kroh;1995).Tressl et al. proposed a complex macromolecular structure consisting of repeating units of furans and pyrroles, linked by polycondensation reactions.Hofmann et al. identified low-molecular weight chromophores and postulated the generation of melanoidin-type colorants by a cross-linking reaction between these low molecular weight substances and noncolored high molecular weight biopolymers, such as proteins.
One of the strategies in melanoidin characterization is the chemical or thermal degradation of melanoidins, 18-19  followed by the identification of the decomposition products formed, giving information on structural domains of the melanoidin network.In addition, thermal destruction of melanoidins leads to the formation of volatiles that contribute to the development of aroma in roasted food systems (Kuntcheva et al., 1998;Adams et al., 2005;Adams et al., 2003).
In coffee matrix the amount of melanoidins is often represented 25% of the dry matter.During toasting of oak wood used in making barrels, melanoidins are formed.The aim of this study was to develop a method to isolate coffee melanoidins and melanoidins from toasted oak wood.We compared melanoidins from coffee and from toasted oak wood by different analytical techniques: absorption spectra, IR, viscosity, headspace/GC/MS, elemental analysis, LC/ESI/MS, NMR.

Materials
Different toasted oak wood were tested: the first one was a no toasted oak wood, and the others, oak wood with different degree of toasting.

Isolation of Coffee Melanoidins
According to the technique described by Hofmann et al., coffee (10 g) was extracted with hot pure water (80-90°C) until no coloured material could be extracted.The aqueous phase was extracted twice with dichloromethane (200 ml) to eliminate lipids and then freeze-dried.The obtained fraction was the Total Fraction Melanoidins coffee (TFMC).
We used HPLC/DAD technique in order to separate purified compounds.The compounds were collected and the different purified fractions were dried and frozen.

Isolation of Oak Wood Melanoidins
According to Hofmann et al. (2001) in the case of isolation of coffee melanoidins, oak wood (10 g) was extracted with hot pure water (80-90°C) until no coloured material could be extracted.The aqueous phase was extracted twice with dichloromethane (200 ml) to eliminate lipids and then freeze-dried.The obtained fraction was the Total Fraction Melanoidins Wood (TFMW).
We used different analytical techniques in order to separate purified compounds.First technique consisted to optimize the HPLC/DAD conditions.The compounds were collected and the different purified fractions were dried and frozen.The second one consisted to optimize low pressure gel chromatography: choice of gel, solvents, flow in order to separe and collected purify fractions; we used UV at 280 nm for detection.Fractions were obtained in quantity more important than in the case of liquid chromatography.

HPLC/DAD Analysis
HPLC/UV-Visible analyses were performed with a Waters separation module system, a Waters UV-Visible detector, and Millenium32 chromatography manager software.UV-visible spectra were recorded at 280 nm and 260 nm.The column was a reverse-phase Sunfire C18 (5 m packing, 250 x 5 mm i.d.) protected with a guard column of the same material; solvent A, water/formic acid (98:2, v/v); solvent B, acetonitrile/water/formic acid (80:18:2, v/v).The column was placed at ambient temperature (T=21°C).The elution program was performed at a constant flow of 1 ml/min, passing from 5 to 30% of B in 40 min., and then rising to 40% of B in 10 min., and finally to 100% of B in 5 min., followed by washing and re-equilibrating the column during 15 min.The injection volume was 20 l.

Semi-preparative HPLC
The fractions were collected and purified by semi preparative HPLC.Analyses were performed with a Waters separation module system, a Waters UV-Visible detector, and Millenium 32 chromatography manager software.UV-visible spectra were recorded at 280 nm and 260 nm.The column was a reverse-phase Sunfire Prep C18 (5 m packing, 250 x 10 mm i.d.) protected with a guard column of the same material; solvent A, water/formic acid (98:2, v/v); solvent B, acetonitrile/water/formic acid (80:18:2, v/v).The column was placed at ambient temperature (T=21°C).The elution program was performed at a constant flow of 3 ml/min, passing from 5 to 30% of B in 40 min., and then rising to 40% of B in 10 min., and finally to 100% of B in 5 min., followed by washing and re-equilibrating the column during 15 min.The injection volume was 200l.

LC/ESI/MS Analysis
LC/ESI/MS, LC/ESI/MS/MS and ESI/HR/MS were performed on a Q-Star TM instrument using an electrospray ionization source in negative-ion mode.Ion spray voltage was selected at 4.5 KV, the capillary temperature was selected at 275°C, and the source temperature was selected at 400°C.The values of sheath gas flow rate were: nebulisation gas=2.85l/min, turbo gas=4.8l/min, curtain gas=1.48l/min.For MS/MS measurements, the energy collision and the gas collision were respectively -30 eV and 5. Isolation window was 1 uma.For HR/MS analysis, we used an internal calibration using PPG.The resolution obtained was 12 000.
The column was a reverse-phase Interchim C18 (10 m packing, 250 x 4.7 mm i.d.) protected with a guard column of the same material; solvent A, water/acetic acid (98:2, v/v); solvent B, acetonitrile/water/acetic acid (80:18:2, v/v).The column was placed at ambient temperature (T=21°C).The elution program was performed at a constant flow of 1 ml/min using the LC inlet and splitter outlet connection permitting to reduce the flow at 0.5 ml/min, passing from 5 to 30% of B in 40 min., and then rising to 40% of B in 10 min., and finally to 100% of B in 5 min., followed by washing and re-equilibrating the column during 15 min.The injection volume was 20 l.

Absorption Spectra
Spectrophotometric measurements were performed using an Anthelie Secomam TM spectrophotometer and UV-Visible spectra were recorded with a spectrophotometer fitted with a quartz cell (1 cm).The wine-like samples were diluted (1/20) and then their spectra measurements were taken.

IR
The Spectrum was recorded with a Nicolet iS50 FT-IR Fourier Transform Infrared Spectrometer (Thermo Scientific, Madison, WI) equipped with a DTGS/KBr detector, KBr beamsplitter and an iS50-ATR diamond accessory.Each spectrum is obtained from the accumulation of 500 scans using an aperture of 150 at 45° of incidence from 4000 to 400 cm-1 with a 4 cm-1 resolution, and an Happ-Genzel apodisation.Then spectrum was treated for ATR correction, thereafter a small offset was applied for baseline.Attributions were done taking in account the formula C 24 H 49 O 23 N, references, tables and softwares (Omnic, KnowItAll).

Headspace/GC/MS
The total fraction of melanoidins (TFMC and TFMW) obtained from toasted oak wood and coffee were heated at 250°C (10 min), and the produced volatiles were analyzed by SPME-GC-MS.For the analysis of the SPME extracts a Thermo coupled with, and a HP5-MS column (30m x 0.25 mm i.d., 0.25 m) was used.Working conditions were as follows: injector, 250°C; transfer line, 250°C; oven temperature, start 40°C and hold 2 min, programmed from 40 to 120°C at 4°C/min and from 120 to 240°C at 30°C/min, hold 2 min; carrier gas (He), 1.2 ml/min; splitless; ionization EI, 70 eV; acquisition parameters, scanned m/z 40-600.Compounds were identified by comparison of their mass spectra and retention times with those of reference compounds and by comparison with the NIST Mass Spectral Library.When only MS data were available, identities were considered to be tentative.

NMR Experiments
NMR experiments were performed at 298 K using a Bruker Avance 800 MHz spectrometer equipped with a 5 mm TCI ( 1 H / 13 C / 15 N/ 2 H) cryoprobe with Z-gradients and ATM accessory.All 1 H NMR and 13 C chemical shifts are given with respect to tretramethylsilane in D 2 O as an external reference. 1H NMR spectra was acquired with water suppression using excitation sculpting (pulsed field gradients applied for 3 ms).1D 1 H spectra was acquired with 8 transients were typically collected at frequency of 800.23 MHz with a flip angle of 90° (7.5 µs), a spectral width of 10000 Hz, acquisition time of 1.64 s and 32 K data points.Data were processed with multiplication prior to Fourier transformation by an exponential function (line broadening was 0.3 Hz) and with zero filling. 13C NMR spectra.1D 13 C spectra was acquired with power gated decoupling using 30° flip angle, spectral widths of 48 kHz consisting of 64 K data points, acquisition time of 0.68 s.Data were processed with multiplication prior to Fourier transformation by an exponential function (line broadening was 2 Hz) and with zero filling.

Elemental Analysis
The analyzes were performed on an elemental analyzer Thermo Finnigan Flash EA 2000 equipped with an autosampler of 32 samples and a chromatographic column.The system is managed by the Eager 300 software Metered elements C, H, N, S and O.The results are provided with an absolute accuracy of ± 0.2% and are valid for a minimum of two tests.
The combustion of the sample takes place at high temperature (940°C) in the presence of tungstic anhydride (WO 3) in a stream of oxygen for a very short (15s).This decomposition gives CO 2 , H 2 O, SO 2 , NO 2 , nitrogen oxides are reduced to N2 (nitrogen) with copper.The entire system is swept by a stream of helium.
For oxygen, pyrolysis of samples were performed under a stream of helium and gas chromatography with a gas-solid stationary phase compound formed CO.Because of the filling system of the reaction tube, the dosage of oxygen can be achieved with the samples containing fluorine.

Isolation and Purification of Melanoidins
Isolation of melanoidin fraction in the case of coffee: yield = 2, 5 g from 10 g of powder (25% of dry matter) and in the case of toasted oak wood : yield = 0,5 g from 10 g of powder (5% of dry matter).
We noted first the difference of yield in the two cases.Coffee matrix was richer in melanoidins than toasted oak wood, it is the most abundant substrate, the percentage of melanoidins was more important because coffee is more roasted than toasted oak wood.
We developed an analytical HPLC method in order to analyze the total fraction composition from coffee and toasted oak wood.HPLC/DAD technique (Figures 1 and 2) permits us to well separate several peaks which present the same spectrum UV-Visible with a maximum of absorbance at 260 nm, specific of melanoidin spectrum.Each peak was collected by semi-preparative HPLC.In the case of roasted coffee we obtained seven fractions: G1, G2, G3, G4, G5, G6, G7, see Figure 1.At the end of the HPLC chromatogram, the peak of polymers and apolar substances is low.In the case of toasted oak wood, we obtained four fractions: F1, F2, F3, F4, see Figure 2. The peak of polymers and apolar substances is more important.HPLC chromatograms show the excellent purity of each product collected nearly 100% (Figures 1 and 2, Table 1).In the case of toasted oak wood, we developed a second method using low pressure gel chromatography to obtain higher quantitatively product and to separate the polymer form.The different fractions, obtained and detected by UV detector at 280 nm, were collected manually: P1, P2, P3, P4, P5, P6, P7 were the obtained fractions (Figure 3, Table 1).HPLC chromatograms show the excellent purity of each product collected nearly 100%.We retained the second method in order to obtain pure fractions from TFMW, we separated thanks to this technique the polymer form no fractionated by HPLC column.
The fractions P were analyzed by different analytical techniques: LC/ESI-MS, IR and NMR.

Thermal Degradation Studies
The total fraction of melanodins (TFMC and TFMW) obtained respectively from coffee and toasted oak wood were heated at 250°C (10 min) and the produced volatiles were analyzed by SPME-GC-MS (Figures 4 and 5).To this purpose, firstly we subjected the isolated melanoidins from oak wood to thermal destruction at 250°C in an inert atmosphere.The especially selected high temperature was to ensure a sufficient degree of conversion of the melanoidin molecules and to provide us with better information relating to their ability to generate volatile products.In the case of coffee, the data are given in Table 2; as can be seen the mixture contains 20 components.The furan group is represented in eight compounds, the total amount being 60%.The highest content is that of 2(3H)-furanone, 5-acetyldihydro-, and 5-hydroxymethyldihydrofuran-2-one.Alicyclic derivatives were found (12%) as well as maltol, see Table 2.The headspace profil of the oak wood melanoidins (Figure 5) consisted mainly of furans (74% of the total GC peak area), a small amount of pyrroles and pyrazines, see Table 3. Furfural, maltol, and isomaltol, which are important compounds in the headspace of heated oak wood melanoidins, are typical caramelization products of sugars.In Figures 6 and 7, some representative structures of compounds identified in the headspace of heated melanoidins respectively from TFMC and TFMW are depicted.
From the results presented it can be seen that during manufacturing process destruction of melanoidins is likely to take place, producing volatile components which take part in the formation of the flavour complex in the finished product.

Elemental Analysis (C, H, O, N) and Viscosimetry
The chemical investigations in our study revealed that coffee and toasted oak wood melanodins have different elemental (CHON) compositions and similar viscosity values, see Table 4. Coffee melanoidin fraction (TFMC) contain 2,65% of nitrogen, 42,15% of carbon, 5,10% hydrogen, and 50,10% of oxygen.Toasted oak wood melanoidin fraction (TFMW) contain 1,95% of nitrogen, 39,95% of carbon, 6,86% hydrogen, and 50,96% of oxygen, and the formula for their ratio is C 24 H 49 O 23 N.These data are similar to those obtained for the quantitative element composition of known model and food melanodins (Cä mmerer & Kroh, 1995;Margarita et al., 1996).The percentage of nitrogen is more important in the case of coffee fraction; it was easily explain by the fact that coffee contained a large majority of melanoidins.Viscosity of each fraction is around 1,466 mPa.s in the case of oak wood and 1,545 mPa.s in the case of coffee.The viscosity in each case varied according to the temperature (Table 5).More temperature increased and more viscosity decreased and inversely.n (cm -1 ) Figure 10.
The IR spectra (Figure 10) of the different fractions : P1, P2, P3, P4, P5, P6, P7 are similar in the case of toasted oak wood, and they are typical of polymeric compounds with spreading bands.In the same way the IR spectra (Figure 9) of the different fractions: G1, G2, G3, G4, G5, G6, G7 in the case of coffee are similar and present the same characteristic bands.
Nevertheless some characteristic absorptions are observed which allow assignment to group frequencies.Characteristic frequencies in the case of oak wood (Table 6) are observed for C-OH groups at 1035 cm -1 , C=O (-COOH) groups at 1700 cm -1 , C=N and C=C groups at 1644 cm -1 .The band at 2946 cm -1 can be attributed to the hydrogen stretching vibrations in CH 2 and CH 3 groups, the broad band at 3382 cm -1 to the -O-H and -N-H stretching.The same bands are observed in the case of coffee, see Table 7.

Structure of Melanoidin Polymer
Melanoidins are high molecular weight amino-carbonyl compounds.In general, the separation of melanoidins from food and other biological samples is very difficult and therefore, most of the chemical and biological studies about melanoidins have done on model melanoidins.Although the chemical structure of melanoidins is not understood clearly, some part of the chemical structure of model melanodins have been elucidated by NMR techniques (Allard et al., 1997;Fang & Schmidt-Rohr, 2009;Gniechwitz et al., 2008).
Figure 16 presented 1 H NMR spectra of each P purified fractions from toasted oak wood.
Figures 17 and 18 presented respectively 1 H NMR and 13 C spectra of P4 fraction with experimental data. 1 H -13 C correlations were obtained with normal 2D techniques HSQC and HMBC (Table 8).
These correlations permitted to observe and confirm some groups and functions: aldehyde >C=O, heteroaromatic >C=N-, alkene, aromatic >C=C<, anomeric proton and carbon, and sugar rings.These results were in correlation with those obtained in IR and ESI/MS.
The determination of the exact chemical structure of these compounds will be achieved by using other classical battery of 1D and 2D NMR structural experiments.

Discussion
We studied the extractible forms of melanoidins produced by toasting oak heartwood and coffee beans.For these two different sources of melanoidins, maillard reactions represent an important transformation of the raw material, improving the quality of products and develop a large part of these aromatic interest and diversity.
Because coffee melanoidins are well documented, we used it as a model to develop our specific method on the toasted oak heartwood melanoidins.
We noted that the quantities of melanoidins in coffee and oak wood are very different.Coffee is richer in melanoidin compounds than oak wood.This is probably due to the roasting step that is different in the two cases.
The roasting process, in the case of coffee, is more favorable to the production of melanoidins than the toasting process in the case of oak wood.The roasting temperatures and the toasting temperatures are comparable but the times in each case is different.
Specialists situate "roasting zone" between 185 and 240°C, the optimum being between 210 and 230°C.Above of these temperatures starts over-roasting.The time of the roasting is usually 12 to 15 minutes.The temperature roasting and its conduct have considerable influence on the qualities of coffee.
In the case of oak wood, we noted the presence of a larger amount of melanoidin polymers probably in relation with the heating time (± 50-55 min).The time of roasting coffee is around 15 to 20 min in the case of traditional roasting.This may explain the lower extraction because more the degree of polymerization increase and more the extractability decrease.So in oak wood, the extractible melanoidins are less present than in the case of coffee; this may also explain the difference in content melanoidins in both matrices.
In addition of the total amount of melanoidins, the polymeric status of both melanoidins are different.
After the maillard reaction, inducing the formation of melanoidins, the thermic treatment permitted the degradation of melanoidins, source of various aroma : vanilla for vanillin, almond for furfural, toasted almond for 5-methyl furfural, toasted for maltol.
The Pyrolysis GC of coffee melanoidins product less of various compounds, about 20, against 28 for Pyrolysis/GC of oak wood melanoidins.It exist probably a relation between degree of polymerization and number of molecules product and identify by pyrolysis technique.
Pyrolysis is the decomposition of an organic compound by a significant increase in its temperature (450°C) to obtain other products.At this high temperature, polymerized melanoidins, more present in oak wood melanoidins, are decomposed and produce more simple organic compounds.
From measurements of the viscosity of the solution for different concentrations of dissolved polymer, one can calculate the viscosity-average molecular weight (ref).
Oak wood melanoidins contain less nitrogen (1,9%) than coffee melanoidins (2,6%).Green coffee contain from 1 to 3% total nitrogen, engaged in various combinations, the main ones are proteins and alkaloids (ref).Amino acids in general and certain amino acids containing sulfur (cystine and methionine especially), contained in the coffee proteins play an important role in the formation of the roasted coffee aroma.Among the amino acids that make up green coffee proteins, some of them, such as arginine, cysteine, lysine and serine, showed a high decrease in their amount during roasting (ref review).Oak wood contain less than 1% total nitrogen.And toasting process have also an impact on the degradation compounds.
We noted that the absorption maximum is about 260 nm, characteristic of nucleic acid absorbance.Nucleic acids are macromolecules with nitrogen groups, that is to say relatively large complex molecules.They enter in the family biomolecules as they are of great importance in the kingdom of life.
The nucleic acids consist of a chain of nucleotides linked by phosphodiester bonds.Nucleotides always consist of two basic components: a sugar (ose with 5 carbons or pentose) and nucleic base or nitrogenous base.This corroborates the observations made by LC/ESI/MS technique.Mass spectra of the different fractions in the case of oak wood, show the presence of pentoses linked with another element.We proposed a hypothetic structure with a degree of pentose polymerization, DP=5.
The IR spectra present some characteristic frequencies and bands similar in the case of oak wood melanoidins and coffee melanoidins : C-OH groups, C=O (-COOH) groups, C=N and C=C groups, band corresponding to the hydrogen stretching in CH 2 and CH 3 groups, band corresponding to the -O-H and -N-H stretching.
3.3.3LC/ESI/MS AnalysisCarbohydrates are the most important substrates in the Maillard reaction and melanoidin formation.Both, monosaccharide degradation products and intact sugars play a role in the formation of the melanoidin backone(ref).Therefore, to obtain further insight into the carbohydrate composition of the melanoidins, the isolated melanoidins fractions (P) from TFMW low pressure gel chromatography were analysed using LC/ESI/MS.Mass spectrum of Fraction P1, obtained by electrospray ionisation in the negative ion mode, was obtained (Figure11).

Table 1 .
Proportions of each purified fraction from coffee and toasted oak wood melanoidin total fractions

Table 2 .
Data for the thermal degradation products of melanoidins from coffee

Table 3 .
Data for the thermal degradation products of melanoidins from toasted oak wood

Table 4 .
Elemental compositions of coffee melanodin fraction and toasted oak wood melanoidin fraction

Table 5 .
Viscosty measurements from coffee and toasted oak wood fractions

oak wood melanoidin fraction (TFMW) Fractions Viscosity at 15°C (mPa.s) Viscosity at 20°C (mPa.s) Viscosity at 25°C (mPa.s)
3.3.2UV-Visibleand IR SpectraThe UV and IR spectra are characteristics of melanoidins (Figures 8, 9 and 10).The absorption maximum at about 260 nm is broad; this is characteristic of polymer compounds containing a large number of chromophores which a different nature.

Table 6 .
Assigments of the bands in infrared spectra of toasted oak wood melanoidin fraction

Table 7 .
Assigments of the bands in infrared spectra of coffee melanoidin fraction