Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids

To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.     

Assessment of initial stages of Maillard reaction in dehydrated onion and garlic samples

The initial steps of the Maillard reaction in freshly laboratory-freeze-dried and commercial dehydrated onion and garlic samples have been assessed by quantitative determination of 2-furoylmethylamino acids, obtained after acid hydrolysis of the corresponding Amadori compound. In freshly prepared samples, hardly any presence of 2-furoylmethylamino acids was detected, whereas in commercial samples, onion contained much more important levels of 2-furoylmethylamino acids as compared to garlic species. 2-Furoylmethyl-gamma-aminobutyric acid (1), 2-furoylmethyl-lysine (furosine; 2), and 2-furoylmethylarginine (3) were identified in all commercial dehydrated onion samples, with compound 3 being the most abundant. All garlic samples presented slightly higher levels of 2 than 3 with no presence of 1. The observed differences between onion and garlic commercial samples may be due to their very different content of reducing sugars. Moreover, some variations found in 2-furoylmethyl derivatives within both onion and garlic species could be also attributed to different processing and storage conditions during the manufacture of these products. The findings of this study show the first evidence of important levels of Amadori compounds in dehydrated garlic and onion samples, as well as the usefulness of 2-furoylmethyl derivatives as quality indicators for the early detection of the Maillard reaction in onion and garlic products.     

Formation of early and advanced Maillard reaction products correlates to the ripening of cheese

The present study deals with the characterization of the ripening of cheese. A traditional German acid curd cheese was ripened under defined conditions at elevated temperature, and protein and amino acid modifications were investigated. Degree of proteolysis and analysis of early [Amadori compound furosine (6)] and advanced [N(ε)-carboxymethyllysine (4), N(ε)-carboxyethyllysine (5)] Maillard reaction products confirmed the maturation to proceed from the rind to the core of the cheese. Whereas 6 was decreased, 4 and 5 increased over time. Deeper insight into the Maillard reaction during the ripening of cheese was achieved by the determination of selected α-dicarbonyl compounds. Especially methylglyoxal (2) showed a characteristic behavior during storage of the acid curd cheese. Decrease of this reactive structure was directly correlated to the formation of 5. To extend the results of experimental ripening to commercial cheeses, different aged Gouda types were investigated. Maturation times of the samples ranged from 6 to 8 weeks (young) to more than 1 year (aged). Again, increase of 5 and decrease of 2 were able to describe the ripening of this rennet coagulated cheese. Therefore, both chemical parameters are potent markers to characterize the degree of maturation, independent of coagulation.     

Relative quantification of N(epsilon)-(Carboxymethyl)lysine,imidazolone A,and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry

The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, has received increasing recognition from nutritional science and medical research. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform relative and simultaneous quantification of the Amadori product, which is an early glycation product, and of N(epsilon)-(carboxymethyl)lysine and imidazolone A, two important advanced glycation end products. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1, 4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 degrees C. After enzymatic digestion with endoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE) and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were used for relative quantification of the three Maillard products. Amadori product, N(epsilon)-(carboxymethyl)lysine, and imidazolone A were the main glycation products formed under these conditions. Their formation was dependent on glucose concentration and reaction time. The kinetics were similar to those obtained by competitive ELISA, an established method for quantification of N(epsilon)-(carboxymethyl)lysine and imidazolone A. Inhibition experiments showed that coincubation with N(alpha)-acetylargine suppressed formation of imidazolone A but not of the Amadori product or N(epsilon)-(carboxymethyl)lysine. The presence of N(alpha)-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations of imidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completely inhibited the formation of N(epsilon)-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved to be a new analytical tool for the simultaneous, relative quantification of specific products of the Maillard reaction. For the first time, kinetic data of defined products on specific sites of glycated protein could be measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillard reaction in the course of food processing.     

Synthesis and characterization of gamma-N-(2-furoylmethyl)aminobutyric acid.

The product of acid hydrolysis of the Amadori compound gamma-N-(1-deoxy-D-fructosyl)aminobutyric acid was isolated and identified by (1)H NMR and (13)C NMR as gamma-N-(2-furoylmethyl)aminobutyric acid. This compound is an analogue to furosine, formed during acid hydrolysis of the corresponding Amadori compound. The retention time of the isolated compound was the same as that of the main peak observed in acid hydrolysates of stored orange juice powder. gamma-N-(2-Furoylmethyl)aminobutyric acid can be a useful indicator of the early stages of Maillard reaction in foods containing free gamma-aminobutyric acid.     

Identification and quantification of aminophospholipid-linked Maillard compounds in model systems and egg yolk products

While the Maillard reaction of free amino acids and proteins is a well-established process, no defined structures from the nonenzymatic browning of aminophospholipids in foodstuffs have been described so far. Phosphatidylethanolamine (PE)-linked glucosylamines (Schiff-PE), Amadori products (Amadori-PE), 5-hydroxymethylpyrrole-2-carbaldehydes (Pyrrole-PE), and carboxymethyl (CM-PE) as well as carboxyethyl (CE-PE) derivatives were detected and quantified by liquid chromatography- electrospray mass spectrometry (LC-(ESI)MS). Model incubations of soy-PE and D-glucose were employed to firmly establish the LC-(ESI)MS procedure. Analyses of spray-dried egg yolk powders and lecithin products derived therefrom show one-fourth of the native D-glucose content of egg yolk to be transformed to Amadori-PE, corresponding to a PE derivatization quota of 11-15.5 mol %. Schiff-PE and Pyrrole-PE were present only in low amounts, no CM-PE and CE-PE could be identified in any of the investigated samples. The high glycation rate of egg yolk PE will influence the emulsifying properties and perhaps even the oxidation resistance of the respective products.     

Reactivities of D-glucose and D-fructose during glycation of bovine serum albumin.

Glycation of bovine serum albumin by D-glucose and D-fructose under dry-heating conditions was studied. The reactivities of D-glucose and D-fructose, with respect to their ability to utilize primary amino groups of proteins, to cross-link proteins, to develop Maillard fluorescence, and to reduce protein solubility in the presence and absence of air (molecular oxygen) were investigated. D-Glucose showed a higher initial rate of utilization of primary amino groups than D-fructose, both in the presence and in the absence of oxygen. Subsequent reactions of the Amadori and Heyns rearrangement products, cross-linking, development of Maillard fluorescence, oxidation, and fragmentation, indicated that the alpha-hydroxy carbonyl group of Amadori products is more reactive than the aldehydo group of Heyns products. D-Fructose showed a greater sensitivity than D-glucose toward the presence of oxygen at the initial stages of the Maillard reaction. The presence or absence of oxygen in the glycation mixture did not seem to have an influence on the nature of products generated in the glycation mixtures during the advanced stages of the Maillard reaction.     

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.     

Mass spectrometric detection and formation of D-amino acids in processed plant saps, syrups, and fruit juice concentrates

Liquid and syrupy dietary saps and juices of plant origin, characterized by the presence of large quantities of saccharides (glucose, fructose, or sucrose) and containing amino acids, were analyzed for the presence of D-amino acids using enantioselective gas chromatography-mass spectrometry. D-amino acids were detected in processed saps and juices of trees (maple, palm, birch), fruits (grape, apple, pear, pomegranate, date), and various other plants (agave, beetroot, sugar cane, carob). D-Ala was detected in all plant products and amounted to approximately 34% D-Ala (relative to L-Ala + D-Ala) in Canadian maple syrups, to approximately 13% in palm saps, and to 48 and 13% D-Ala, respectively, in concentrated grape juices (Spanish Arrope and Turkish Pekmez). Varying amounts and kinds of other D-amino acids were also detected. To test the hypothesis that racemization, that is, partial conversion of L-amino acids into their corresponding D-enantiomers, occurs at reversible stages of the Maillard reaction, the Amadori compound fructose-L-phenylalanine was synthesized. On heating at 200 degrees C for 5 (20) min, release of 10.8% (24.2%) D-Phe was detected. From the data it is concluded that the Amadori compounds formed in the course of the Maillard reaction are pecursors of D-amino acids in foodstuffs.     

Simultaneous quantitative analysis of maillard reaction precursors and products by high-performance anion exchange chromatography

A new analytical setup allowing the simultaneous analysis of precursors and products of the Maillard reaction is described. It is based on high-performance anion exchange chromatography with electrochemical (ECD) and diode array detectors (DAD) coupled in series. Chromatography and detection were optimized to permit simultaneous monitoring of compounds relevant to the Maillard reaction, such as the sugar, the amino acid, and the corresponding Amadori compound as well as the cyclic intermediates 5-(hydroxymethyl)-2-furaldehyde, maltol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one. Separation was achieved on a CarboPac PA-1 column using a gradient of sodium acetate in aqueous sodium hydroxide. The Amadori compound, glucose, and glycine were monitored by an ECD operating in the integrated amperometry mode. The number of analyzed compounds was further increased by coupling the ECD with a DAD for the analysis of ultraviolet-active constituents. This method was successfully applied to model Maillard reaction mixtures based on glucose and glycine.     

Storage stability assessment of freeze-dried royal jelly by furosine determination

The effect of freeze-drying and the assessment of the storage stability of freeze-dried royal jelly (RJ) were investigated by the determination of furosine and blocked lysine. The level of furosine in the RJ samples collected from cells at different times (1, 2, and 3 days after grafting) showed that the Maillard reaction had already occurred in the hive as indicated by the increase in furosine: from 9.6 to 20.8 mg/100 g of protein. Freeze-dried RJ was more prone to the early stage of the Maillard reaction than fresh RJ, as confirmed by the significantly higher furosine values found after 12 months, both at 4 degrees C (253.4 versus 54.9 mg/100 g of protein) and at room temperature (884.3 versus 332.5 mg/100 g of protein). After 18 months at room temperature, the lyophilized samples reached a furosine level of 1440.4 mg/100 g of protein, which corresponded to the blocked lysine levels, amounting to 24% of total lysine.     

The fluorescence of advanced Maillard products is a good indicator of lysine damage during the Maillard reaction

The objective of this study was to determine whether in heat-treated milk-resembling models or milk there is a lag phase, before lactulosyllysine (LL) is converted into advanced Maillard products (AMP), and if there is a step during the heat treatment where LL is actively degraded into AMP. For that purpose, a low temperature (60-85 degrees C) and a long heat treatment (15-90 h) were chosen. We observe that the heat treatment first induces a parallel increase in furosine and AMP fluorescence, confirming that AMP are produced very early during the heat treatment. At this step, both indicators are correlated with each other and precisely reflect the lysine damage. After a time, however, furosine reaches a steady-state concentration, whereas AMP fluorescence still increases, remaining correlated with the lysine blockage. Nevertheless, heat treatment applied to milk does not reach this step so that AMP fluorescence appears as a rapid alternative to furosine quantification.     

Presence of 2-furoylmethyl derivatives in hydrolysates of processed tomato products

Acid hydrolysis of Amadori compounds yields the corresponding 2-furoylmethylamino acids (2-FM-AA) that can be analyzed by ion-pair HPLC. The relative proportions of the different 2-FM-AA present in the hydrolysates of tomato products were determined to assess their usefulness as indicators of quality. In the lyophilized tomato samples stored at 50 degrees C and a(w) = 0.44 the formation of 2-FM derivatives of alanine, gamma-aminobutyric acid (GABA), asparagine, aspartic acid, glutamic acid, lysine, serine, and threonine was detected. In commercial tomato products the most abundant 2-FM-AA was 2-FM-GABA (from traces to 26.4 mg/100 g of dry matter) followed by 2-FM-lysine (furosine). Differences in 2-FM-AA contents among samples may be related to processing and storage conditions.     

Kinetics of formation of three indicators of the maillard reaction in model cookies: influence of baking temperature and type of sugar

The Maillard reaction (MR), despite its impact on flavor, color, and texture of cereal products, must be controlled for possible deleterious effects on protein nutritional quality. The present study aims to simultaneously monitor three indicators of the MR reaction (acid-released lysine, furosine, and carboxymethyllysine (CML)) by GC/MS in model cookies and evaluate the effect of formulation and baking temperature. Whereas furosine followed a bell-shape kinetic, indicative of an intermediary compound, CML linearly accumulated, proving to be a good indicator of the advanced MR. Acid-released lysine continuously decreased during baking. A reference baking level was defined to compare differently processed cookies using fluorescence synchronous spectra, highly sensitive to the dough physicochemical properties. Furosine was maximal in glucose-containing cookies, but only accounted for 5-50% lysine blockage, depending on the sugar and baking temperature. High oven temperatures and the use of fructose as the sugar source were associated with lowest the lysine damage and CML formation.     

Qualitative determination of specific protein glycation products by matrix-assisted laser desorption/ionization mass spectrometry Peptide mapping

The nonenzymatic reaction between reducing sugars and proteins, known as the Maillard reaction, has received increased recognition from nutritional science and medical research. The development of new analytical techniques for the detection of protein-bound Maillard products is therefore crucial. In this study, we applied peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to investigate the formation of structurally specific Maillard products on glycated lysozyme (AGE-lysozyme), produced upon incubation with D-glucose. In parallel, we synthesized N(epsilon)-(carboxymethyl)lysine-modified lysozyme (CML-lysozyme) and N(epsilon)-(carboxyethyl)lysine-modified lysozyme, two well-described glycation products, as model substances. 3-Deoxyglucosone-modified lysozyme and methylglyoxal-modified lysozyme were prepared as examples of glycation products incubated with dicarbonyl compounds. We were able to detect specific modifications on AGE-lysozyme, which were assigned to CML, imidazolone A, and the Amadori product.     

Nonenzymatic browning of lactose and caseinate during dry heating at different relative humidities

Dry mixtures of lactose and caseinate were heated at 60 degrees C for up to 96 h at different relative humidities (RHs) ranging from 29 to 95%. The resulting nonenzymatic browning was studied by determining lactulosyl lysine formation in the caseinate (as measured by the conversion to furosine), amount of reacted lactose, loss of lysine, color formation, and fluorescent intensity. For each measurement, the maximum reaction occurred at intermediate RHs. While there is general agreement between the results obtained by different methods, discrepancies are understandable given the complex nature of nonenzymatic browning. It was shown that the degradation of the Amadori product, lactulosyl lysine, increased with RH. Moreover, the Maillard reaction, as opposed to caramelization of lactose, was the major pathway at all RHs. Visible browning occurred when the destruction of Amadori product became dominant, and interactions between sugar fragments and caseinate were not the rate-limiting steps in the nonenzymatic browning.     

Reactions of monosaccharides during heating of sugar-casein systems: building of a reaction network model

The Maillard reaction is important during the heating and processing of foods for its contribution to food quality. To control a reaction as complex as the Maillard reaction, it is necessary to study the reactions of interest quantitatively. In this paper the main reaction products in monosaccharide-casein systems, which were heated at 120 degrees C and pH 6.7, were identified and quantified, and the reaction pathways were established. The main reaction routes were (i) sugar isomerization, (ii) degradation of the sugar into carboxylic acids, and (iii) the Maillard reaction itself, in which not only the sugar itself but also its reaction products react with the epsilon-amino group of lysine residues of the protein. Significant differences in reaction mechanism between aldose and ketose sugars were observed. Ketoses seemed to be more reactive in the sugar degradation reactions than their aldose isomers, and whereas the Amadori product was detected as a Maillard reaction intermediate in the aldose-casein system, no such intermediate could be found in the ketose-casein system. The reaction pathways found were put together into a model, which will be evaluated by kinetic modeling in a subsequent paper.     

Characterization and functional properties of lactosyl caseinomacropeptide conjugates

Ovine caseinomacropeptide (CMP) was modified with lactose through Maillard reaction under 44% relative humidity and 40 degrees C for various periods (0-11 days). Different lactosylated CMP forms were separated by capillary electrophoresis and reversed phase high-performance liquid chromatography (RP-HPLC) and identified by RP-HPLC coupled with electrospray ionization mass spectrometry (ESI-MS). Around 55-60% of CMP was lactosylated under the conditions assayed, with the monolactosylated form being the most abundant one, followed by the di-, tri-, and tetralactosylated species. During the first days of incubation amino acid analyses showed a decrease in lysine and NH(2)-terminal methionine, which coincided with an increase in the furosine content. However, from the ninth day of incubation, further degradation of Amadori compounds prevailed over their formation. Solubility, heat stability, and emulsifying capacity of the native and modified CMP were investigated. Lactosylation improved the emulsifying activity, but it did not modify the great solubility and heat stability of native CMP.     

High-pressure effects on Maillard reaction between glucose and lysine

Glucose-lysine model systems prepared over a range of pH values (5-10) in unbuffered and buffered media were incubated at 60 degrees C either under atmospheric pressure or at 400 MPa. The results obtained showed that high pressure affected in different ways the different stages of the Maillard reaction and that such effects were strongly influenced by pressure-induced changes in the pH of the systems. In unbuffered media, at an initial pH < or =8.0, the formation of Amadori rearrangement products (ARP) was not considerably affected by pressure, whereas the intermediate and advanced stages of the Maillard reaction were suppressed, suggesting a retardation of the degradation of the ARP. In buffered media, at pH values < or =8.0, pressure slowed the Maillard reaction from the initial stages. These effects are attributed to the pH drop caused by the pressure-induced dissociation of the acid groups. In unbuffered and buffered media at initial pH = 10.2, high pressure accelerated the formation and subsequent degradation of ARP, leading to increased levels of intermediate and advanced reaction products.     

Monitoring carbonyl-amine reaction between pyruvic acid and alpha-amino alcohols by FTIR spectroscopy--a possible route to Amadori products

The carbonyl-amine reaction between pyruvic acid and alpha-amino alcohols was monitored by Fourier transform infrared spectroscopy at a temperature range between 20 and 100 degrees C and under acidic and basic conditions. To avoid interference, the reactions were conducted in the absence of solvent using liquid reactants such as methyl pyruvate, pyruvic acid, ethanolamine, and 1-amino-2,3-propanediol. Analysis of the time- and temperature-dependent spectra indicated that under basic conditions and at room temperature, the initial imine formation and its subsequent isomerization through a 1,3-prototropic shift occur very rapidly and the reaction goes to completion within 12 min. Interestingly, the isomerization product of the initial imine is the so-called Schiff base intermediate formed when the corresponding amino acid and the reducing sugar react during a typical Maillard reaction. Furthermore, the detailed studies also indicated that during the first 30 s, the rate of formation of the initial imine was faster than the rate of its isomerization; however, after 60 s, its rate of isomerization becomes faster than the rate of its formation. The data also indicated that under acidic conditions, this isomerization was prevented from occurring and the reaction was terminated at the initial imine formation stage. In addition, temperature-dependent spectra indicated that the isomerization of the Schiff's base into eneaminol can be achieved at or above 60 degrees C and its subsequent rearrangement into Amadori product can be attained at temperatures above 80 degrees C even under basic conditions, thus providing a novel route to Maillard reaction products starting from a keto acid and an amino alcohol. This observation was also confirmed through identification of the common Amadori product in both keto acid/amino alcohol and sugar/amino acid mixtures, by the application of tandem mass spectrometry and chemical ionization techniques.