Amadori Compounds Research Articles

Analysis of Umami Taste Compounds in a Fermented Corn Sauce by Means of Sensory-Guided Fractionation.

Corn sauce, an ingredient obtained from the fermentation of enzymatically hydrolyzed corn starch and used in culinary applications to provide savory taste, was investigated in this study. The links between its sensory properties and taste compounds were assessed using a combination of analytical and sensory approaches. The analyses revealed that glutamic acid, sodium chloride, and acetic acid were the most abundant compounds, but they could not explain entirely the savory taste. The addition of other compounds, found at subthreshold concentrations (alanine, glutamyl peptides, and one Amadori compound), contributed partly to close the sensory gap between the re-engineered sample and the original product. Further chemical breakdown, by a sensory-guided fractionation approach, led to the isolation of two fractions with taste-modulating effects. Analyses by mass spectrometry and nuclear magnetic resonance showed that the fractions contained glutamyl peptides, pyroglutamic acid, glutamic acid, valine, N-formyl-glutamic acid, and N-acetyl-glutamine.

Analytical strategies to depict the fate of the Maillard reaction in foods

Thousands of molecules are formed as result of the reaction between reducing carbonyls and amino groups, making the Maillard reaction one of the functional orchestrators of food quality. Upon the formation of the Amadori and Heyns compounds, a bewildering network of reactions takes place and a parallelism between the optimization of analytical tools and the significance of the molecules formed can be traced. This review dives into the chemical aspects of the MR highlighting the interrelation among analytical techniques, dietary advanced glycation end-products, food quality and human health.

The quantification of free Amadori compounds and amino acids allows to model the bound Maillard reaction products formation in soybean products

The quantification of protein bound Maillard reaction products (MRPs) is still a challenge in food chemistry. Protein hydrolysis is the bottleneck step: it is time consuming and the protein degradation is not always complete. In this study, the quantitation of free amino acids and Amadori products (APs) was compared to the percentage of blocked lysine by using chemometric tools. Eighty thermally treated soybean samples were analyzed by mass spectrometry to measure the concentration of free amino acids, free APs and the protein-bound markers of the Maillard reaction (furosine, Nε-(carboxymethyl)-l-lysine, Nε-(carboxyethyl)-l-lysine, total lysine). Results demonstrated that Discriminant Analysis (DA) and Correlated Component Regression (CCR) correctly estimated the percent of blocked lysine in a validation and prediction set. These findings indicate that the measure of free markers reflects the extent of protein damage in soybean samples and it suggests the possibility to obtain rapid information on the quality of the industrial processes.

Global proteomic analysis of advanced glycation end products in the Arabidopsis proteome provides evidence for age-related glycation hot spots

Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.

Simultaneous quantification of ten Amadori compounds in tobacco using liquid chromatography with tandem mass spectrometry.

Amadori compounds are aroma precursors formed in the initial phase of the Maillard reaction. Based on their similar structures, simultaneous quantification of more than six Amadori compounds in tobacco has not been reported yet. In this study, a simple and rapid method was developed to simultaneously quantify ten Amadori compounds including the isomers of Fructose-isoleucine and Fructose-leucine in tobacco. The separation was performed on an Atlantis T3 column (2.1 × 250 mm, 5 μm) by gradient elution using acetonitrile and water as the mobile phases. The quantification method was systematically evaluated and proven to be sensitive and accurate. The linearity was good, with correlation coefficients of 0.9977-0.9999. The limits of detection and quantitation were 1.354-2.532 and 4.516-8.444 ng/mL, respectively. The recoveries were 84.0-119.6%, and the relative standard deviations were 1.33-5.40%. The method was used to analyze the changes in the amounts of ten Amadori compounds in tobacco before and after tobacco primary processing. The analysis shows that the Maillard reaction occurs during the short processing period.

Evaluation of the Availability and Antioxidant Capacity of Maillard Compounds Present in Bread Crust: Studies in Caco-2 Cells.

Bread crust is one of the major contributors to the intake of Maillard reaction products (MRP). MRP improve the organoleptic properties of foods and can provide biological actions such as antioxidant properties. The transport and availability of Amadori compounds (measured as furosine) and hydroxymethylfurfural (HMF)—early and intermediary MRP—from enzymatically digested bread crust (BC) and from its soluble low-molecular weight (LMW) and high-molecular weight (HMW) fractions were investigated in the Caco-2 cell line. The absorption of the early and final MRP pool was tested by measuring the absorbance recovery (280 and 420 nm). The ability of soluble BC or its fractions to lessen the production of reactive oxygen species (ROS) was examined. Amadori compounds (furosine) were transported across Caco-2 cell monolayers from the soluble BC in percentages ranging between 40% and 56%; the lower amount of the compound supplied, the higher transport rate. However, HMF transport rate (35%) was unaffected by the initial amount of the compound. Amadori compounds and HMF contained in the LMW fraction were more efficiently transported than those present in the HMW fraction, suggesting improved absorption when supplied as free forms or linked to LMW compounds. Absorbance recovery at 280 nm was higher from the LMW fraction, whereas higher recovery was detected for the HMW fraction at 420 nm. The digested BC—but not its isolated fractions—was able to significantly reduce ROS production at basal conditions and after subjecting cells to an oxidant. A clear positive action of BC on the antioxidant defence is manifested, seemingly attributable to the combined presence of soluble LMW and HMW products.

Synthesis and biological evaluation of a novel Amadori compound.

Here, we report the synthesis, purification, ESI MS and NMR characterization, as well as the SEM analysis of a fructosyl thiophenyl-substituted triazolyl-thione L-alanine (denominated Fru-L-TTA). This novel fructosyl derivative was obtained by solution synthesis using the Amadori reaction, in analogy to other natural fructosyl-amino acids, and fully characterized. In particular, we report an accurate NMR/MS/SEM characterization of Fru-L-TTA alongside some biological properties, and investigated to compare the properties of the artificial derivative of this work with the natural counterparts. In particular, Fru-L-TTA shares with natural fructosyl-amino acids the possibility to inhibit the colony formation of prostate cancer cells and additionally decreases their migration.

Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana.

Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought.

Amadori compounds as potent inhibitors of angiotensin-converting enzyme (ACE) and their effects on anti-ACE activity of bell peppers

The angiotensin-converting enzyme (ACE) inhibitor activity of Amadori compounds (ACs) was evaluated in vitro. The results revealed that the N-(1-deoxy-d-fructose-1-yl) glutamic acid was the most active with the lowest IC50 value (0.2590 mM), followed by N-(1-deoxy-d-fructose-1-yl) histidine (0.5676 mM); others showed weak ACE inhibition. Besides, we further studied the change of AC content and corresponding anti-ACE activity during heating of the bell pepper. The AC content in fresh bell pepper was low and drying of the bell pepper led to a significant increase, especially at the beginning five days, in N-(1-deoxy-d-fructose-1-yl) glutamic acid, N-(1-deoxy-d-fructose-1-yl) histidine and the total of eight ACs respectively from nd (not detected) −5.53, 0.76–6.47 and 11.17–100.01 mg/g; simultaneously, the IC50 values for bell pepper extracts decreased from 0.2161 to 0.04627 mM during thermal processing, showing anti-ACE activity increase, which illustrated that the ACs formed during heating of the bell pepper can contribute to the increase of ACE inhibitory activities.

The Simultaneous Analysis of Amadori and Heyns Compounds in Dried Fruits by High Performance Liquid Chromatography Tandem Mass Spectrometry

Amadori and Heyns compounds, which play important roles in food flavor, color, and nutritional value, are generated during the Maillard reaction. It is difficult to analyze the compounds simultaneously because the structures of Amadori and Heyns compounds are very similar. To develop a method to distinguish and analyze Amadori and Heyns compounds simultaneously by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), studies of mass spectrometry fragments of the Heyns compounds obtained by electrospray ionization were performed and two characteristic fragment ion pairs of Amadori ([AA-H + CH2]+/[M + H]+) and Heyns ([M + H-2H2O–CH3OH-CO]+/[M + H]+) compounds were selected by comparing the cleavage mechanism between them. The calibration curves established with ion pairs of Fru-Pro (fructose-proline), Fru-Leu (fructose-leucine), and Fru-Val (fructose-valine) were used to calculate the content of the corresponding Amadori and Heyns compounds. The method displayed good linearity with correlation coefficients no less than 0.9989. The limit of detection was no more than 0.125 ng/mL. The relative standard deviation was less than 4.31 % and the spiked recoveries ranged from 90.55 % to 110.91 %. The method is accurate and reliable for simultaneously determining Amadori and Heyns compounds in dried raisins and jujubes and was helpful for evaluating and controlling the quality of dried fruits.

Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.

A study of different indicators of Maillard reaction with whey proteins and different carbohydrates under adverse storage conditions

This study examined different indicators of each stage of Maillard reaction under adverse storage conditions in a system with whey proteins and lactose or glucose. The analysis of lysine loss by the o-phthaldialdehyde method can be considered a good indicator of the early stage, showing considerable differences in reactivity when systems with mono and disaccharides were analyzed. Capillary electrophoresis proved to be a sensitive method for evaluating the extent of glycosylation of the native proteins, providing valuable information when the loss of lysine was not significant. The estimation of the Amadori compound from the determination of total 5-hydroxymethyl-2-furfuraldehyde would have correlate well with reactive lysine content if the advanced stages of the reaction had not been reached. For assessing the occurrence of the intermediate and final stages, the measurement of free 5-hydroxymethyl-2-furfuraldehyde and color, proved not to be suitable for storage conditions.

Root‐specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N.tomentosiformis and in most N.tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N.benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG.

Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I

Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37°C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods.

Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds

We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid.

Structure–reactivity relationship of Amadori rearrangement products compared to related ketoses

Structure-reactivity relationships of Amadori rearrangement products compared to their related ketoses were derived from multiple NMR spectroscopic techniques. Besides structure elucidation of six Amadori rearrangement products derived from d-glucose and d-galactose with l-alanine, l-phenylalanine and l-proline, especially quantitative 13C selective saturation transfer NMR spectroscopy was applied to deduce information on isomeric systems. It could be shown exemplarily that the Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-proline exhibits much higher isomerisation rates than d-fructose, which can be explained by C-1 substituent mediated intramolecular catalysis. In combination with a reduced carbonyl activity of Amadori compounds compared to their related ketoses which results in an increased acyclic keto isomer concentration, the results on isomerisation dynamics lead to a highly significant increased reactivity of Amadori compounds. This can be clearly seen, comparing approximated carbohydrate milieu stability time constants (ACuSTiC) which is 1 s for N-(1-deoxy-d-fructos-1-yl)-l-proline and 10 s for d-fructose at pD 4.20 ± 0.05 at 350 K. In addition, first NMR spectroscopic data are provided, which prove that α-pyranose of (amino acid substituted) d-fructose adopts both, 2C5 and 5C2 conformation.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation.

Kinetics of combined thermal pretreatment and anaerobic digestion of waste activated sludge from sugar and pulp industry

The purpose of this work was to evaluate the effects of thermal pretreatment temperature (from 100°C to 200°C) and time (15–60min) on the characteristics and the anaerobic biodegradability of wasted activated sludge from sugar and pulp mills. Reaction Curve model and First-order kinetics were used to obtain a better understanding of the data from the anaerobic biodegradability tests. Sludge characteristics and methane production prior and after thermal pretreatment confirmed that temperature exerted the major influence on sludge solubilization and biodegradability. The optimal methane yield (182mlCH4/gVSFeed) and ultimate methane production rate (72.1mlCH4/gVSFeedd) was obtained at 165°C for 30min. However, the negative effects of melanoidins and Amadori compounds appeared and the rising trend of biodegradability reversed at 200°C. The growth rate of methane yield topped out at 476% at 100°C, and the maximum methane yield (range from 28.9 to 72.1mlCH4/gVSFeedd) were also observed at the first day, showing the radical kinetics improvement after pretreatment. This remarkable initial methane yield was attributed to the raising share of the rapidly biodegradable organics generated in thermal pretreatment, which were estimated with methane yield percentage of day 3. Both Reaction Curve model and First-order kinetics fitted with the experimental data well, and a Simplified Reaction Curve model without lag time provides a feasible tool to estimate methane yield of the rapidly biodegradable substrates.

Direct UV determination of Amadori compounds using ligand-exchange and sweeping capillary electrophoresis.

The detection of Amadori compounds poorly absorbing in the UV region is challenging. To cope with this problem, a simple and effective method using ligand-exchange and sweeping capillary electrophoresis was first developed. With this method, Amadori compounds can be detected by UV at 236 nm without derivatization steps and separated based on the stable levels of Amadori compound-Cu(2+) complexes. Under optimal conditions with background electrolyte of 50 mmol/L copper sulfate, pH 3.94, and separation voltage 25 kV, baseline separations of the following five Amadori compounds, N-(1-deoxy-D-fructos-1-yl) valine (Fru-Val), N-(1-deoxy-D-fructos-1-yl) leucine (Fru-Leu), N-(1-deoxy-D-fructos-1-yl) alanine (Fru-Ala), N-(1-deoxy-D-fructos-1-yl) methionine (Fru-Met), N-(1-deoxy-D-fructos-1-yl) glutamic acid (Fru-Glu), were achieved within a 20 min run. The method was applied to analyze these Amadori compounds in two tomato products. Fru-Glu could be quantitated sensitively down to 0.706 mg/L in MS powder (a kind of tomato powder), 0.726 mg/L in MS liquid (a kind of concentrated tomato juice), and the limit of quantification for the others ranged from 0.792 to 1.084 mg/L. The most abundant Amadori compound was Fru-Glu (1057.5 mg/100 g) in MS powder; however, Fru-Met was rarely detected in MS liquid. The accuracy of the method was tested by recovery measurement of the spiked samples, which yielded good results (94.2-106.0%) with relative standard deviation lower than 5%. Simultaneously, the method was also applied in the analysis of Fru-Glu content during a tomato puree evaporating concentration process. The first significant increase in Fru-Glu content coincided with the increment rate of soluble solids, which indicated that fast evaporation of water could be a critical step for the formation of Amadori compounds. These results show that this method is convenient and reliable for rapid analysis of Amadori compounds in tomato products.

Purification, characterization and antiglycation activity of a novel polysaccharide from black currant

A novel polysaccharide fraction (BCP-1) was extracted from the black currant fruit by ultrasound-assisted compound enzyme and purified by chromatography on macroporous resin D4006, anion-exchange Q-Sepharose FF and Sephadex G-100 columns. BCP-1 consisted of galacturonic acid, xylose, mannose, glucose and galactose in a ratio of 1.00:3.14:1.83:17.90:1.98 and its molecular weight was 14,050Da. The preliminary structure features of BCP-1 were investigated by FT-IR and NMR. SEM and Congo red test showed that BCP-1 had honeycomb-like structure, but no triple helix structure. BCP-1 exhibited significant inhibitory abilities on protein glycation. Especially, BCP-1 showed obvious inhibitory effects on the formation of dicarbonyl compounds and AGEs (% inhibition of 66.95±0.33% and 67.15±0.40% respectively), but weaker inhibition Amadori compound production (max. 37.15±0.36% inhibition). This suggested that the inhibitory action of BCP-1 on protein glycation was more effective on the later phases of dicarbonyl compounds and AGEs formation.