Isolation of Deoxyfructosazine and Its 6-Isomer from the Nondialyzable Melanoidin Hydrolyzate

The nondialyzable melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was hydrolyzed. The hydrolyzate was fractionated by DEAE-cellulose column and Dowex 50 W column. Deoxyfructosazine and its 6-isomer were respectively isolated from main two fractions, and identified. Even on boiling the melanoidin in aqueous solution, these pyrazines as well as imidazoles and β-hydroxy pyridines in the melanoidin were liberated.

Furthermore, amounts of these heterocyclic compounds liberated from the nondialyzable melanoidin and the fractionated melanoidins (fractionated into five fractions on DEAE-cellulose column according to the method described previously) were examined.

The results obtained seem to suggest that these heterocyclic compounds are not present probably as a molecular skelton or an inclusion compound in the melanoidin, but as a small moiety of the melanoidin molecule with loose chemical bond.     

Melanoidins as major colourant in sugarcane molasses based distillery effluent and its degradation

Melanoidins are natural condensation products of sugar and amino acids produced by non-enzymatic Maillard amino-carbonyl reaction taking place between the amino and carbonyl groups in organic substances. Melanoidins extensively exist in food products, drinks and wastewaters released from distilleries and fermentation industries. Melanoidins are very important from the nutritional, physiological and environmental aspects and due to their structural complexity, dark colour and offensive odor, these pose serious threat to soil and aquatic ecosystem that release of melanoidins cause increased load of recalcitrant organic material to natural water bodies. This then causes the problems, like reduction of sunlight penetration, decreased photosynthetic activity and dissolved oxygen concentration whereas on land, it causes reduction in soil alkalinity and inhibition of seed germination. Further, due to the possibility of complexation reactions of introduced melanoidins with metal ions, they could influence the biogeochemical cycle of many constituents in natural waters. This review presents an overview to dramatic progress to understand the synthesis, chemical structure and degradation pathway of melanoidins as well as microbial strategies for the degradation and decolourisation of melanoidins.     

Exudates from Rice Callus Tissues Resulting from the Lack of Cuticle and Wax Layers

Nondialyzable melanoidins prepared from a glucose-glycine system were investigated as to their decolorization and degradation products on ozone treatment. Melanoidins were decolorized to degrees of 84 and 97% after ozonolysis at — 1°C for lOmin and 90min, respectively, and the mean molecular weight of melanoidins decreased from 7000 to 3000 after ozonolysis for 40 min. The major components of electrofocused melanoidins before and after ozone treatment had pis of 3.00 and 2.86, respectively, the pI 3.00 band being significantly affected.

IR measurement showed that the absorption at 1290 cm^?1 disappeared and that at 1720 cm^?1 newly appeared on ozonolysis, respectively, and the absorption at 1620 cm^?1 disappeared on acid hydrolysis after ozonolysis.

Furthermore, the major degradation products in the ether-soluble fractions obtained from ozone-treated melanoidins were identified as butanedioic acid, glycolic acid, 2-hydroxybutanoic acid and so on.

In the aqueous fraction, one of the major products was glycine, which was produced to the level of 1.05% on ozonolysis which increased to 5.75% per melanoidin on acid hydrolysis after ozonolysis. From these findings and the IR results, it is postulated that glycine was considerably incorporated into melanoidin molecules as the amide form.     

Physiological relevance of dietary melanoidins

Melanoidins are the final products of the Maillard reaction. The main dietary sources of melanoidins are coffee, bread crust, bakery products, black beer and cocoa. Although the chemical structures of melanoidins are widely unknown, data from gravimetric techniques allow to roughly estimate a daily intake in the order of 10 g with a Western diet. Melanoidins contribute to the sensorial properties, modulating texture and flavour of foods. Growing evidence also suggests that melanoidins have health beneficial properties, such as chemopreventive, antioxidant and antimicrobial activities, and the ability to chelate different minerals. In the gastrointestinal tract, melanoidins behave not only as antioxidants, but also as dietary fibre by promoting the growth of bifidobacteria. This array of biological activities suggests the need for analytical techniques to identify the melanoidin structures and to control their formation during thermal food processing.     

Role of hydroxycinnamates in coffee melanoidin formation

Melanoidins are the high molecular weight brown end products of the Maillard reaction. They are formed during heat processing of foods like coffee, bread, malt, and beef. A chemical definition of these food polymers is still impossible, despite several efforts to determine their structure. In the last years, the interest in research on melanoidins has increased due to their biological activities. Coffee brew is one of the main sources of melanoidins in human diet. Various melanoidin fractions were obtained by applying chromatographic separation techniques or specific isolation procedures, allowing, a partial view on structural features and diversity of coffee brew melanoidins. Different melanoidin populations can be found with respect to total carbohydrate contents and their structural features. In this paper, the recent advances in research on coffee melanoidin structures and formation mechanisms are reviewed. The participation of hydroxycinnamates in melanoidin formation, especially true for coffee melanoidins, is a hypothesis older than three decades, but only recently more consistent data have been obtained for their presence. Although the role of hydroxycinnamates in melanoidin formation is not yet completely understood, it was demonstrated that the interaction between phenolic compounds and melanoidins can be of non-covalent or of covalent nature. The most likely linkage point is through the protein fragments incorporated in the coffee melanoidin during the roasting process, although carbohydrates, such as arabinose, seem to be possible binding sites for the chlorogenic acid derivatives on these brown structures, too.     

Relationship between the Molecular Weight and the Color Intensity of Color Components of Melanoidin from the Glycine-Xylose System

The molecular weights of color components (designated as P1, P2, P3, P4, P5, P6, P7 and P8 in order of elution from a DEAE-cellulose column) isolated by the conversion of color components of melanoidin produced from the glycine-xylose system in an oxidative browning were studied in relation to the color intensity. The molecular weights of P5, P6, P7 and P8 estimated by gel filtration on Sephadex G–50, G–75 and G–150 using dextran as a standard were approximately 2,140, 3,550, 5,600 and 14,200, respectively. The molecular weights of P1, P2, P3 and P4 could not be estimated by the gel filtration method because of their low values.

On the other hand, a linear relationship between Kd, the distribution coefficient in dextran gel, and E₄₅₀ (E) of the color components was observed. Thus, there is considered to be a linear relationship between log E and log molecular weight (M). The correlation coefficient between log E and log M was calculated to be from 0.96 to 0.98 in the visible wavelength region. Therefore, the equation, E=k × M^α was adopted. The value, E=2.15 × M^0.29 was obtained from melanoidin prepared from the glycine-xylose system. The molecular weights of P1, P2, P3 and P4 were calculated from the equation to be 290, 360, 700 and 1,200, respectively. The equation, E=k × M^α, was demonstrated to be reasonably applicable to the melanoidin from Glu-, Lys-, Gly₂-, Gly-Leu-, and Gly₃-xylose systems. It is concluded that the polymerization of the structural unit in melanoidin occurs in an oxidative browning and that their color tone is darkened and the color in melanoidin is increased by polymerization according to the equation, E=k × M^α.     

Enolization and racemization reactions of glucose and fructose on heating with amino-acid enantiomers and the formation of melanoidins as a result of the Maillard reaction

This study investigated the enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and the formation of melanoidins as a result of the Maillard reaction. The study measured reducing sugars and L- and D- amino acids using HPLC as an index for the amount of enolization of the sugars and isomerization of the amino acids. Additionally, the absorption of melanoidins was measured at different wavelengths (420, 450, 470, 490 nm); the UV–Vis spectra and the extinction coefficient were determined for the formation of melanoidins. Melanoidins were, rather arbitrarily, defined by a high-molecular-weight (HMW) if it was above a lower limit of 12.4 kDa, which was the nominal cut-off value in the dialysis system used. A remarkable enolization reaction of the sugars was observed in the course of the Maillard reaction. Especially, in the Fru/D-Asn model system, the degree of sugar enolization was more than in the other model systems. All of the FDAA (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide) amino acids were separated by TLC. The racemization of the amino acids was higher in the fructose-amino acids systems. Isomer formation was the highest in the Fru/D-Asn system. The L- and D- isomers showed different absorptions in the UV–Vis spectra, although these had similar shapes. The absorption of the melanoidins formed from glucose was higher than that formed from fructose. In particular, the sugar–asparagine system showed different characteristics according to the L- and D-isomers. The differences in the extinction coefficients of the melanoidins was significant (P < 0.05), except for the sugar–lysine system.     

Isolation and characterization of aerobic bacteria capable of the degradation of synthetic and natural melanoidins from distillery effluent

Melanoidins, complex biopolymer of amino-carbonyl compounds are the major coloring and polluting constituents of distillery wastewaters. In this study, three aerobic melanoidin-degrading bacteria (RNBS1, RNBS3 and RNBS4) were isolated from soil contaminated with distillery effluent and characterized as Bacillus licheniformis (RNBS1), Bacillus sp. (RNBS3) and Alcaligenes sp. (RNBS4) by biochemical tests and 16S rRNA gene sequence analysis. The degradation of synthetic and natural melanoidins was studied by using the axenic and mixed bacterial consortium. Results have revealed that the mixed consortium was more effective compared to axenic culture decolorizing 73.79 and 69.83% synthetic and natural melanoidins whereas axenic cultures RNBS1, RNBS3 and RNBS4 decolorized 65.88, 62.56 and 66.10% synthetic and 52.69, 48.92 and 59.64% natural melanoidins, respectively. The HPLC analysis of degraded samples has shown reduction in peak areas compared to controls, suggesting that decrease in color intensity might be largely attributed to the degradation of melanoidins by isolated bacteria.     

Purification and Some Properties of Melanoidin Decolorizing Enzymes,P-III and P-IV,from Mycelia of Coriolus versicolor Ps4a

Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48,400 ~ 50,000, an optimum pH of 5.5 and an optimum temperature of 30~35°C. P-III required glucose and 02 for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.

On the other hand, P-IV had a molecular weight of 43,800 ~ 45,000, an optimum pH of 4.0~4.5 and an optimum temperature of 30~35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CMB and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.

Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.     

Decolourisation of synthetic and spentwash melanoidins using the white-rot fungus Phanerochaete chrysosporium JAG-40

Phanerochaete chrysosporium JAG-40 was isolated from the soil samples saturated with spilled molasses collected from a sugar mill. This isolate decolourised synthetic and natural melanoidins present in spentwash in liquid fermentation; up to 80% in 6 days at 30 degrees C under aerobic conditions. A large inoculum size stimulated fungal biomass production, but this gave less decolourisation of pigment; 5% w/v (dry weight) mycelial suspension was found optimum for maximum decolourisation in melanoidin medium supplemented with glucose and peptone. Gel-filtration chromatography showed that larger molecular weight fractions of melanoidin were decolourised more rapidly than small molecular weight fractions.     

Proteinoids as complexes of polyamino acids with melanoidins

Proteinoids have been demonstrated as complexes of amino acid polymers with melanoidin pigments. Some physico-chemical properties of proteinoid pigments were studied in comparison with the standard melanoidins. Proteinoid pigments were able to enhance oxidoreduction and hydrolysis reactions, and their activity was comparable with the activity of the corresponding polyamino acid components or even of the entire proteinoids. The pigmented proteinoids had relatively strong ESR signal indicating the presence of free radicals into melanoidin components. Hypothetical participation of proteinoid melanoidin pigments in prebiotic evolution is discussed.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

The Bacteriophage-Inactivating Effect of Basic Amino Acids; Arginine,Histidine, and Lysine

Some physicochemical properties and substrate specificity of acid protease B isolated from Scytalidium lignicolum were investigated.

The molecular weight determined by the sedimentation equilibrium and sedimentation velocity method was 21,000 and 19,000~20,000, respectively. The isoelectric point was determined as 3.0 using the Tiselius electrophoresis apparatus, 3.2 by isoelectric focusing, respectively.

The enzyme did not contain histidine and was composed of 188 amino acid residues. Substrate specificity against various synthetic peptides was different from those of the acid proteases which were inactivated by S–PI and DAN.     

Involvement of Amino Acid 36 in TM1 in Voltage Sensitivity in Mouse Na<Superscript>+</Superscript>/Glucose Cotransporter SGLT1

Human SGLT1 protein is an established sodium-glucose cotransporter. Despite widespread use of the mouse as a model organism, the mouse SGLT1 homologue has yet to be functionally characterized. Additionally, the crystal structure of a sugar transporter homologue, Vibrio SGLT, has recently been described, however, it offers limited information about the role of transmembrane segments outside of the core ligand binding domains. In particular, the amino acids in TM1 were not assigned in the structure. To examine the contribution of TM1 to the function of SGLT1, we have cloned and characterized the biophysical properties of SGLT1 from mouse, mSGLT1, and compared it to a clone containing an amino acid substitution in TM1, F36S. As predicted, both proteins formed functional Na⁺/sugar cotransporters, but F36S-mSGLT1 showed decreased rates of sugar uptake and decreased apparent affinities for both Na⁺ and sugar compared to mSGLT1. Analysis of pre-steady-state currents and comparison with the crystal structure of Vibrio SGLT provide plausible mechanisms to explain the differences in function of these two proteins. Our data suggest that amino acids in TM1, which are not involved in ligand binding and translocation pathways, significantly influence the functional properties of sodium-glucose carrier proteins.     

Isolation and Identification of Two β-Hydroxy-pyridine Derivatives from the Nondialyzable Melanoidin-hydrolyzate

The hydrolyzate of the melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was fractionated into the two fractions of non-adsorbate and adsorbate on Amberlite IR-120 (H⁺-form). In the present paper, the adsorbed fraction (Fraction B) was examined.

Paper chromatographic examination of the Fraction B indicated the presence of at least eight compounds positive to diazotized sulphanilic acid reagent. The two compounds of them (indicated orange and orange-yellow color) were isolated and identified as 2-methyl-5-hydroxy-pyridine and 2-hydroxymethyl-5-hydroxy-pyridine, respectively.

It is probable that these compounds would loosely be bound as a small moiety in the melanoidin molecule.     

Separation of l-Leucine-pyruvate and l-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.

The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.     

Physiological Effects of Nondialyzable Melanoidin in Rats

The absorption and excretion of melanoidins, a mixture of the low- and high-molecular weight components (LMM and HMM), and the LMM component prepared from a l-lysine^d-glucose system, at pH 7.4 and 9.0, respectively, and the effects of these melanoidins on cholesterol metabolism were examined in rats. Weanling male rats of the Wistar strain weighing about 50 g were fed diets containing 10% casein (10C) with 3% of a melanoidin or 25% casein (25C) with 4% of the melanoidin for 12 weeks, and the 25C diet including 3% of the melanoidin or LMM for 8 weeks. The growth and organ weights of the melanoidin-supplied groups were not different from those of the control ones. In rats given the melanoidin diets with both protein levels, the kidneys became dark brownish due to the accumulation of the melanoidin, though the accumulated amount was extremely small (nearly 0.5 mg/g wet kidney), and gel filtration chromatography of a water extract of the kidneys on Sephadex G-75 showed that the deposited melanoidin was the LMM component. Most of the ingested melanoidin, however, was excreted in the feces, and on comparison of the gel chromatographic patterns, the melanoidin groups were found to have more fecal LMM than the control ones. When rats were provided with LMM, HMM increased in their feces. The addition of melanoidin suppressed the level of plasma total cholesterol and elevated the fecal excretion of total lipids and total cholesterol. The urinary contents of protein and total creatinine did not differ from those in the control groups.     

A Red,Fluorescent Protein from Silkworm

Nondiffusable melanoidin obtained from a glycine-xylose system was heated in aqueous media, and the resulting chemical changes, as affected by the presence of oxygen, pH value, temperature and the addition of transitional metals, were investigated.

Melanoidin, when heated at 90°C in an aqueous solution, caused remarkable discolorization accompanied by the development of fluorescence, oxygen consumption and a noticeable variation of reductone content. Heated melanoidin became polydispersive in molecular weight on gel filtration chromatograms. There appeared reductones, ninhydrin-positive substances, fluorescent substances, aromatic amines, aliphatic carbonyls, and aliphatic primary amines and/or methylene groups in diffusâtes of melanoidin heated in various media.

An increase in pH value favored oxidative discolorization, while an increase in the concentration of transitional metals except Mn²⁺ restrained the discolorization. In the absence of oxygen, heated melanoidin brought about a slight strengthening of color and the accumulation of reductones ca. fifteen times more than the initial level, while in the presence of oxygen the increase of reductone content at the early period was followed by a rapid decrease.

According to the results obtained, the ambivalent reactivity of melanoidin, i.e. polymerization (colorization) and depolymerization (discolorization), was discussed in relation to influencing factors. A mechanism for the production of reductones in heated melanoidin was also proposed.     

Decolourisation and detoxification of synthetic molasses melanoidins by individual and mixed cultures of Bacillus spp

The decolourisation of synthetic melanoidins (i.e., GGA, GAA, SGA, and SAA) by three Bacillus isolates (Bacillus thuringiensis (MTCC 4714), Bacillus brevis (MTCC 4716) and Bacillus sp. (MTCC 6506)) was studied. Significant reduction in the values of physicochemical parameters was noticed alongwith the decolourisation of all four melanoidins (10% v/v). B. thuringiensis (MTCC 4714) caused maximum decolourisation followed by B. brevis (MTCC 4716) and Bacillus sp. (MTCC 6506). A mixed culture comprised of these three strains was capable of decolourising all four melanoidins. The medium that contained glucose as a sole carbon source showed 15% more decolourisation than that containing both carbon and nitrogen sources. Melanoidin SGA was maximally decolourised (50%) while melanoidin GAA was decolourised least ( approximately 06%) in the presence of glucose as a sole energy source. The addition of 1% glucose as a supplementary carbon source was essential for co-metabolism of melanoidin complex. The decolourisation of synthetic melanoidin by three Bacillus spp. significantly reduced the toxicity to the tubificid worm (Tubifex tubifex, Müller).     

Heterogenous Degradation of Myofibrillar Proteins

Pectic substances were extracted from the vegetables with oxalate buffer of pH 4.25 and, after saponification, fractionated into two components, weakly acidic pectic polysaccharide (WAP) and pectic acid, by DEAE-cellulose and Sephadex G-100 chromatographies. The galacturonic acid content (17.3~25.8%) of WAPs was much lower than that of pectic acids, though the neutral sugar compositions of both pectic substances were almost the same. The arabinose-galactose side chains were found to be very long or highly branched in WAPs compared with those in pectic acids.

All the WAPs were appreciably hydrolyzed by exo- and endopolygalacturonases. The limited-degradation products (the residual polysaccharides; i.e., the rhamnogalacturonan segments) obtained by endopolygalacturonase from both WAPs and pectic acids showed a similar behavior on Sephadex G-100 and Sepharose CL-4B gel filtrations; each of the rhamnogalacturonan segments was eluted in the void volume of the Sephadex G-100 column. From these results, we concluded that WAPs are probably an inherent pectic component of the cell walls of the vegetables.