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1.
A method using high-performance liquid chromatography with UV and electrospray ionization mass spectrometry detection was developed for monitoring the alpha-dicarbonyl compound profiles generated from nonenzymatic browning using o-phenylenediamine (OPD) as a trapping agent. The alpha-dicarbonyl compounds were generated by the "dry" reaction of sodium caseinate and lactose heated at various relative humidities (RHs). The proportions of alpha-dicarbonyls formed were different for samples heated at low, intermediate, and high RHs. This study shows that relatively large amounts of 3-deoxypentosulose and galactosyl 2-pentosulose are produced under high RHs, while galactosyl hexosulose and 1,4-dideoxyhexosulose are elevated under low RH conditions. Both caramelization and Maillard reaction pathways contributed to the generation of alpha-dicarbonyls.  相似文献   

2.
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.  相似文献   

3.
Color-generating reactions of protein-bound lysine with carbohydrates were studied under thermal as well as under physiological conditions to gain insights into the role of protein/carbohydrate reactions in the formation of food melanoidins as well as nonenzymatic browning products in vivo. EPR spectroscopy of orange-brown melanoidins, which were isolated from heated aqueous solutions of bovine serum albumin and glycolaldehyde, revealed the protein-bound 1,4-bis(5-amino-5-carboxy-1-pentyl)pyrazinium radical cation (CROSSPY) as a previously unknown type of cross-linking amino acid leading to protein dimerization. To verify their formation in foods, wheat bread crust and roasted cocoa as well as coffee beans, showing elevated nonenzymatic browning, were investigated by EPR spectroscopy. An intense radical was detected, which, by comparison with the radical formed upon reaction bovine serum albumin with glycolaldehyde, was identified as the protein-bound CROSSPY. The radical-assisted protein oligomerization as well as the browning of bovine serum albumin in the presence of glycolaldehyde occurred also rapidly under physiological conditions, thereby suggesting CROSSPY formation to be probably involved also in nonenzymatic glycation reactions in vivo.  相似文献   

4.
The formation of alpha-dicarbonyl-containing substances and Amadori rearrangement products was studied in the glycine-catalyzed (Maillard reaction) and uncatalyzed thermal degradation of glucose, maltose, and maltotriose using o-phenylenediamine as trapping agent. Various degradation products, especially alpha-dicarbonyl compounds, are formed from carbohydrates with differing degrees of polymerization during nonenzymatic browning. The different Amadori rearrangement products, isomerization products, and alpha-dicarbonyls produced by the used carbohydrates were quantified throughout the observed reaction time, and the relevance of the different degradation pathways is discussed. In the Maillard reaction (MR) the amino-catalyzed rearrangement with subsequent elimination of water predominated, giving rise to hexosuloses with alpha-dicarbonyl structure, whereas under caramelization conditions more sugar fragments with an alpha-dicarbonyl moiety were formed. For the MR of oligosaccharides a mechanism is proposed in which 1,4-dideoxyosone is formed as the predominating alpha-dicarbonyl in the quasi-water-free thermolysis of di- and trisaccharides in the presence of glycine.  相似文献   

5.
Recent studies have hypothesized that pyrrole formation and polymerization may be contribute to the nonenzymatic browning produced in both oxidized lipid/protein reactions and the Maillard reaction. To develop a methodology that would allow investigation of the contribution of this browning mechanism, the kinetics of formation of color, fluorescence, and pyrrolization in 4, 5(E)-epoxy-2(E)-heptenal/lysine and linolenic acid/lysine model systems were studied. In both cases similar kinetics for the three measurements were observed at the two temperatures assayed (37 and 60 degrees C), and there was a high correlation among color, fluorescence, and pyrrolization measurements obtained as a function of incubation time. Because the color and fluorescence production in the 4,5(E)-epoxy-2(E)-heptenal/lysine system is a consequence of pyrrole formation and polymerization, the high correlations observed with the unsaturated fatty acid also suggest a contribution of the pyrrole formation and polymerization to the development of color and fluorescence observed in the fatty acid/lysine system. Although the contribution of other mechanisms cannot be discarded, all of these results suggest that when the pyrrole formation and polymerization mechanism contributes to the nonenzymatic browning of foods, a high correlation among color, fluorescence, and pyrrolization measurements should be expected.  相似文献   

6.
The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods.  相似文献   

7.
Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.  相似文献   

8.
The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic browning reactions is changing at the same time that the browning (by the pyrrole polymerization mechanism) is being produced. The antioxidative activities of eight model pyrroles (pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole, pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde) as well as the browning reaction of 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and the dimer (DIM) produced during HMP browning were determined. The results obtained suggest that the AAs observed in nonenzymatic browning reactions are the result of the AAs of the different oxidized lipid/amino acid reaction products formed. Thus, the different pyrrole derivatives produced in these reactions had different AAs, and the highest AAs were observed for alkyl-substituted pyrroles without free alpha-positions. Because some of these pyrrole derivatives are implicated in nonenzymatic browning production and this browning production implies the loss of hydroxyl groups and the transformation of some pyrroles with one type of substitution into others, changes in AA during browning production were observed, and the resulting DIM derivative was more antioxidant than HMP or higher polymers. These results explain the AA observed in fatty acid/protein mixtures after slight oxidation and suggest that, when the pyrrole polymerization mechanism is predominant, slightly browned samples may be more antioxidant than samples in which nonenzymatic browning has been highly developed.  相似文献   

9.
The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin.  相似文献   

10.
Reaction of folic acid with reducing sugars and sugar degradation products   总被引：2，自引：0，他引：2  
The reaction of folic acid with reducing sugars (nonenzymatic glycation) under conditions that can occur during food processing and preparation was studied by high-performance liquid chromatography with diode array detection. N-(p-Aminobenzoyl)-L-glutamic acid, a well-established oxidation product, was detected in the reaction mixtures. Furthermore, a new product was isolated and identified as N2-[1-(carboxyethyl)]folic acid (CEF). CEF was the main product that was formed by the nonenzymatic glycation of folic acid. For preparation, N2-[1-(carboxyethyl)]folic acid was obtained in high yields when folic acid and dihydroxyacetone (DHA), a sugar degradation product, were heated at 100 degrees C in phosphate buffer. Mixtures of folic acid and different sugars or DHA were heated under variation of reaction time and temperature, and CEF was quantified. Up to 50% of the vitamin was converted to CEF, with highest yields formed from maltose (49%) and lactose (43%).  相似文献   

11.
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.  相似文献   

12.
Whole milk powder with an initial water content of 4.4% (w/w) and a water activity of 0.23 stored in hermetically sealed vials for up to 147 days below (37 and 45 degrees C) and above (55 degrees C) the glass transition temperature (T(g) determined to have the value 48 degrees C) showed a strong temperature dependence for quality deterioration corresponding to energies of activation close to 200 kJ/mol for most deteriorative processes. The glass transition was found not to cause any deviation from Arrhenius temperature dependence. Lactose crystallization, which occurred as a gradual process as monitored by isothermal calorimetry, is concluded to liberate bound water (a(w) increase to 0.46) with a modest time delay (approximately 2 days at 55 degrees C) and with concomitant surface browning as evidenced by an increasing Hunter b-value. Browning and formation of bound hydroxymethyl-furfural determined by HPLC seem to be coupled, while formation of another Maillard reaction product, furosine, occurred gradually and was initiated prior to crystallization. Initiation of lipid oxidation, as detected by lipid-derived radicals (high g-value ESR spectra), and progression of lipid oxidation, as detected by headspace GC, seem not to be affected by lactose crystallization and browning, and no indication of browning products acting as antioxidants could be determined.  相似文献   

13.
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.  相似文献   

14.
Whole milk powder was stored in closed vials at 60 degrees C to induce crystallization of lactose within a short time scale. After an induction period of 3-4 days simultaneous crystallization of lactose, increase of water activity, formation of browning products, and increase of radical content took place. Radicals detected before lactose crystallization were characterized by a narrow ESR spectrum (g = 2.006) and could be depleted by removal of oxygen and therefore were assigned to oxidation processes. Late-stage radicals present after crystallization of lactose gave much wider spectra (g = 2.0048) and were independent of oxygen availability and assigned to late-stage Maillard reaction products. The study indicates that the processes of lactose crystallization, browning, and formation of radical species (g = 2.0048) are strongly coupled, while lipid oxidation is less dependent on the other processes.  相似文献   

15.
The extent of the Maillard reaction was studied by measuring furosine and color formation in infant and enteral formula-resembling model systems prepared by mixing calcium caseinate, laboratory-obtained or commercial whey protein with lactose or dextrinomaltose (ingredients similar to those used in infant and enteral formula manufacture) and heating the mixture at 100, 120, or 140 degrees C for 0-30 min. The furosine determination was performed by HPLC and the color determination by measuring colorimetric parameters L, a, and b in a reflection photometer. The first steps of the Maillard reaction could be followed by furosine determination when initial ingredients had low thermal damage. Hence, furosine may be an indicator of low thermal damage in ingredients with <100 mg/100 g of protein. At the concentrations used in these model systems, similar to those in infant and enteral formulas, furosine values (indirect measure of lysine losses) were higher in lactose than in dextrinomaltose systems, in which only glucose, maltose, maltotriose, and maltotetraose among all of the sugars present showed reactivity with casein. Finally, the advanced steps could be followed by color determination when the initial ingredients had high thermal damage or the model systems were heated at high temperature or for a long time. Among the parameters assayed, b was the most sensitive.  相似文献   

16.
The solubility and chemical changes due to the Maillard reaction were investigated in milk protein concentrate powder containing 80% protein (MPC80) during storage at temperatures and relative humidities in the ranges of 25-40 °C and 44-84%, respectively. The Maillard reaction was studied by measuring furosine (a product of lactosylated protein after digestion with acid) and free hydroxymethylfurfural (HMF) contents by HPLC and L*, a*, b* values with a color-meter. Furosine, free HMF, and browning in MPC80 increased during storage, whereas the solubility decreased. The correlation between the Maillard reaction and solubility loss was explored in modified MPC80 to which glucose was added to enhance the rate of the Maillard reaction. More furosine and brown pigments were observed in the glucose-containing MPC80 than in MPC80 with added lactose. The opposite trend occurred for solubility, suggesting that the Maillard reaction may be a cause of solubility loss in MPC powder.  相似文献   

17.
Role of phosphate and carboxylate ions in maillard browning   总被引：4，自引：0，他引：4  
The Maillard reaction of carbohydrates and amino acids is the underlying chemical basis for flavor and color formation in many processed foods. Phosphate and other polyatomic anions will accelerate the rate of Maillard browning, and this effect has been explained by invoking enhanced proton abstraction from intermediate Amadori compounds. In this work, the effect of phosphate and carboxylate ions on browning was measured for a series of reducing sugars with and without the presence of beta-alanine. Significant browning was observed for sugars alone suggesting that polyatomic anions contribute to Maillard browning by providing reactive intermediates directly from sugars. A mechanism is proposed for decomposition of sugars by polyatomic anions and efforts to trap reactive species using o-phenylenediamine (OPD) are described. The results of this study suggest how complications may arise from the popular usage of phosphate buffers in the study of Maillard reaction kinetics. In addition, the results imply how phosphates may be useful for enhancing browning during food processing.  相似文献   

18.
The reaction of malondialdehyde with casein was studied in aqueous solution to evaluate the impact of this lipid oxidation product on food protein modification. By using multiresponse modeling, a kinetic model was developed for this reaction. The influence of temperature and pH on protein browning and malondialdehyde degradation was evaluated. The hypothesis that one malondialdehyde unit leads to the cross-linking of two casein-bound lysine residues was in accordance with the data. At higher malondialdehyde concentrations, a different reaction mechanism was operative, probably involving a dihydropyridine cross-link. The results obtained were compared with the reaction of casein with 2-oxopropanal, a well-studied alpha-dicarbonyl compound. The reaction of casein with 2-oxopropanal followed a different reaction pathway. Comparison of the degree of browning of casein by reaction with malondialdehyde and 2-oxopropanal showed a considerably higher degree of browning induced by malondialdehyde. This research has shown that kinetic modeling is a useful tool to unravel reaction mechanisms. Clearly, the contribution of lipid oxidation products, such as malondialdehyde, to protein modification (both in food and in vivo) can be substantial and needs to be taken into account in future studies.  相似文献   

19.
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.  相似文献   

20.
Experimental data comparing the alkali extraction/ion exclusion chromatographic method with the Monier-Williams method for determination of total sulfite are presented in (a) enzymatic and nonenzymatic browning systems, (b) vegetables containing naturally occurring sulfite, and (c) a carbohydrate-type food additive, erythorbic acid. Excellent agreement, with a linear correlation coefficient of 0.99, was observed in fresh potato samples homogenized with sulfite and allowed to react for different time intervals (enzymatic browning system). A good overall correlation was observed in dehydrated, sulfited apple samples heated for different times (nonenzymatic browning system); however, as heating time increased, higher results were obtained by the Monier-Williams method than by the alkali extraction/ion exclusion chromatographic method. The results of determining sulfite in the alkali trapping solution following acid distillation or acid treatment without heat suggested that this deviation was due to a fraction of sulfite bound to the browning reaction products in such a way that it was released by acid distillation but not by alkali extraction or acid treatment without heat. Similar behavior was demonstrated in cabbage with naturally occurring sulfite, which was released by acid distillation but not by alkali extraction or acid treatment without heat. The ion exclusion chromatographic method could overcome interference by the volatile caramelization reaction products in the Monier-Williams determination of erythorbic acid.  相似文献   

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