Identification of 2,4-Dihydroxy-2,5-dimethyl-3(2H)-thiophenone as a Low-Molecular-Weight Yellow Pigment in Soy Sauce

The color of soy sauce is considered to be mainly attributable to melanoidins formed by the Maillard reaction. However, the chemical structure of melanoidins cannot be clarified, because melanoidins are high-molecular-weight heterogeneous polymers. We isolated a low-molecular-weight pigment from soy sauce and identified 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone as this pigment formed by the Maillard reaction, although its contribution to the total color of soy sauce was very low.     

Identification of 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone as a low-molecular-weight yellow pigment in soy sauce

The color of soy sauce is considered to be mainly attributable to melanoidins formed by the Maillard reaction. However, the chemical structure of melanoidins cannot be clarified, because melanoidins are high-molecular-weight heterogeneous polymers. We isolated a low-molecular-weight pigment from soy sauce and identified 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone as this pigment formed by the Maillard reaction, although its contribution to the total color of soy sauce was very low.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.     

Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine

Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300–360 nm under acidic and neutral conditions and at 320–390 nm under alkaline conditions.     

Identification of the Blue Pigment Formed in a D-Glucose-Glycine Reaction System

D-Glucose (0.7 M), glycine (0.3 M), and sodium hydrogencarbonate (0.1 M) were dissolved in aqueous 30% ethanol at pH 8.0 and left at 50 °C for 4 d in a dark room under nitrogen displacement. The resulting blue pigment was isolated and purified from the blue solution by anionic exchange and gel filtration chromatography. This blue pigment, which is designated Blue-G1, was identified as 5-[1,4-bis-carboxymethyl-5-(2,3,4-trihydroxybutyl)-1,4-dihydropyrrolo[3,2-b]pyrrol-2-ylmethylene]-1,4-bis-carboxymethyl-2-(2,3,4-trihydroxybutyl)-4,5-dihydropyrrolo[3,2-b]pyrrol-1-ium. Blue-G1 had two symmetrical pyrrolopyrrole structures with a trihydroxybutyl group. Blue-G1 had a polymerizing activity, suggesting it to be an important Maillard reaction intermediate through the formation of melanoidins.     

Identification of a novel blue pigment as a melanoidin intermediate in the D-xylose-glycine reaction system

Some blue pigments were formed in the D-xylose (1 M)-glycine (0.1 M) reaction system. A novel blue pigment, designated as Blue-M2 (blue Maillard intermediate-2), was identified as 5-[1,4-dicarboxymethyl-5-(2,3-dihydroxypropyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2-ylmethylene]-1,4-dicarboxymethyl-2-{5-[N-carboxymethyl(2,3,4-trihydroxytetrahydrofuran-2-yl)methylamino]-2-hydroxymethyl-4-(1,2,3-trihydroxypropyl)tetrahydrofuran-3-yl}-4,5-dihydropyrrolo-[3,2-b]pyrrole-1-ium. Blue-M2 is presumed to have been generated by the reaction between Blue-M1, which was identified as the major blue pigment in a previous paper (Hayase et al., Biosci. Biotechnol. Biochem., 63, 1512-1514 (1999)), and di-D-xyluloseglycine. Blue pigments are important Maillard reaction intermediates through the formation of melanoidins.     

Mechanism of formation of the Maillard protein cross-link pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors

Pentosidine is a recently discovered fluorescent protein cross-link from human extracellular matrix that involves lysyl and arginyl residues in an imidazo (4, 5b) pyridinium ring. Pentosidine could be synthesized in vitro by the reaction of ribose, lysine, and arginine. The potential biological significance of the molecule prompted us to investigate its mechanism of formation from D-ribose and key Maillard intermediates, as well as from other potential precursor sugars. The yield of pentosidine from N alpha-t-Boc-lysine, N alpha-t-Boc-arginine, and D-ribose was highest at pH 9.0 and 65 degrees C, but was unaffected by reactant ratios at alkaline pH suggesting an important role for base catalysis. Ribated Boc-lysine on incubation with N alpha-t-Boc-arginine afforded a fluorescent compound with UV, fluorescence, 1H NMR, and MS properties identical with those from native or synthetic pentosidine. 3-Deoxypentosone, however, was not a major pentosidine precursor. Pentosidine became slowly detectable in bovine serum albumin incubated with 0.25 M and 1.0 M glucose and reached, at 30 days, 13.2 and 17 pmol/mg bovine serum albumin, respectively. Spectroscopical properties of glucose-derived pentosidine were identical with those from ribose-derived pentosidine. Pentosidine formed from glucated Boc-lysine with N alpha-t-Boc-arginine in higher yields than from glucose under standard conditions. Fructose, and unexpectedly ascorbate, also formed pentosidine in similar yields as glucose. The discovery that pentosidine can form not only from pentoses but also from hexoses and ascorbate raises major new questions concerning biochemical pathways of the Maillard reaction in vivo.     

Formation of a thiamine artifact during chromatography: A single column procedure for the separation of thiamine and the thiamine mono-, di-, and triphosphate esters

Chromatography of a preparation of [¹⁴C]thiamine pyrophosphate (thiamine-PP) on Dowex 1X8 in the formate form produced an unexpected peak, X-1, which was eluted just prior to the thiamine-PP peak. An ammonium formate, pH 4.5, gradient was the eluant. Rechromatography of either peak X-1 or thiamine-PP produced the same two peaks. The radioactive specific activity per micromole labile phosphate was the same for the two peaks. Peak X-1 appears to be a thiamine compound formed in the presence of formate solutions. This procedural artifact was circumvented by the substitution of acetate for formate. By varying the pH as well as the ionic strength a single column procedure has been developed that separates thiamine and the three phosphate esters quantitatively in micromolar amounts.     

A crystalline pigment produced from 2-hydroxypyridine by arthrobacter crystallopoietes n.sp.

Summary A bacterium was isolated from soil which utilizes 2-hydroxypyridine as sole source of carbon and nitrogen. When grown on solid medium with this substrate massive amounts of green rectangular crystals are deposited extracellularly in the colony mass. The pigment producing organism proved to be a hitherto undescribed species to which the name Arthrobacter crystallopoietes was applied. The pigment formed is characterized qualitatively by the following properties: it is an oxidation product of 2-hydroxypyridine probably still containing a six-membered heterocyclic ring; it exists as an anion with an intense blue color in neutral or slightly alkaline solution and as a metal salt in the deposited crystals; it precipitates from acid solution as a red water-insoluble free acid; it can be reversible oxidized and reduced, being colorless in the reduced form; and in solution it is spontaneously oxidized by air, the reaction being very rapid at alkalineph. The ultraviolet, visible and infrared spectra of the blue and red forms are presented. The properties of the pigment show that it is a member of a chemically poorly defined group of compounds termed azaquinones and that it is related to but not identical with pigments produced by the bacterial oxidation of nicotine, nicotinic acid and isonicotinic acid.This investigation was supported in part by grants G9882 and GB736 from the National Science Foundation.     

Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogenSclerotium hydrophilum

Sclerotium hydrophilum was shown to be auxoheterotrophic for thiamine, with the addition of this vitamin being required for the induction of sclerotia on defined media, but riboflavin and pyridoxine also have a positive effect. In the absence of thiamine, an increase in glucose concentration lead to a decrease in the yield of sclerotia; however, the addition of thiamine negate this inhibition and, instead, as the glucose concentration increased a higher proportion of sclerotial initials matured. Overall it was found that thiamine, specifically the pyrimidine component of thiamine, is crucial for initiating sclerotium production, while glucose stimulates maturation. The effect of light on sclerotium production was found to be complex and dependent on the growth medium. Light is not required for either the induction or maturation of sclerotia, but continuous irradiation of developing cultures with either white light or black light induces an endogenous rhythm whereby sclerotia are formed every 48h. When exposed to alternating light/dark regimes mycelium that formed in the light does not mature sclerotia, but dark-formed mycelium does, even if it is subsequently exposed to light.     

Identification of N epsilon-(carboxyethyl)lysine,one of the methylglyoxal-derived AGE structures,in glucose-modified protein: mechanism for protein modification by reactive aldehydes

We have developed a separation system for N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) by HPLC equipped with a styrene-divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N(alpha)-acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.     

2,3-Dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) generated by the Maillard reaction in foods affect autonomic nervous activity and central nervous activity in human

ABSTRACT

This study investigated the effect of the odors generated by the glycine/glucose Maillard reaction and the potent odorants 2,3-dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) on the human mood and integrative physiological activity. The score of certain subjective moods, especially anger-hostility, and tension-anxiety were decreased significantly after inhalation of the Maillard reaction sample and DMHF, and fatigue-inertia mood was also significantly decreased by DMHF, suggesting a sedative effect of these odors on mood, while 3DP had no effect. Miosis rate and fingertip temperature increased significantly following inhalation of the odor from the Maillard reaction sample and both potent odorants, suggesting that the parasympathetic nervous system dominates through suppression of the sympathetic activity. The physiological relaxing effect of these odors was also confirmed by decreased flicker frequency value and decreased oxyhemoglobin in the prefrontal cortex.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

Heliothrix oregonensis,gen. nov., sp. nov., a phototrophic filamentous gliding bacterium containing bacteriochlorophyll a

An unusual filamentous, gliding bacterium was found in a few hot springs in Oregon where it formed a nearly unispecific top layer of microbial mats. It contained a bacteriochlorophyll a-like pigment and an abundance of carotenoids. There were no chlorosomes or additional chlorophylls. The organism was aerotolerant and appeared to be photoheterotrophic. It was successfully co-cultured with an aerobic chemoheterotroph in a medium containing glucose and casamino acids. Although it has many characteristics in common with the genus Chloroflexus, the lack of chlorosomes and bacteriochlorophyll c and the aerobic nature of this organism indicate that it should be placed in a new genus. This conclusion is supported by 5S rRNA nucleotide sequence data.     

Identification of the blue pigment formed in a D-glucose-glycine reaction system

D-Glucose (0.7 M), glycine (0.3 M), and sodium hydrogencarbonate (0.1 M) were dissolved in aqueous 30% ethanol at pH 8.0 and left at 50 °C for 4 d in a dark room under nitrogen displacement. The resulting blue pigment was isolated and purified from the blue solution by anionic exchange and gel filtration chromatography. This blue pigment, which is designated Blue-G1, was identified as 5-[1,4-bis-carboxymethyl-5-(2,3,4-trihydroxybutyl)-1,4-dihydropyrrolo[3,2-b]pyrrol-2-ylmethylene]-1,4-bis-carboxymethyl-2-(2,3,4-trihydroxybutyl)-4,5-dihydropyrrolo[3,2-b]pyrrol-1-ium. Blue-G1 had two symmetrical pyrrolopyrrole structures with a trihydroxybutyl group. Blue-G1 had a polymerizing activity, suggesting it to be an important Maillard reaction intermediate through the formation of melanoidins.     

Formation mechanism of cross‐linking Maillard compounds in peptide–xylose systems

The formation mechanism of Maillard peptides was explored in Maillard reaction through diglycine/glutathione(GSH)/(Cys‐Glu‐Lys‐His‐Ile‐Met)–xlyose systems by heating at 120 °C for 30–120 min. Maximum fluorescence intensity of Maillard reaction products (MRPs) with an emission wavelength of 420~430 nm in all systems was observed, and the intensity values were proportional to the heating time. Taken diglycine/GSH–[¹³C₅]xylose systems as a control, it was proposed that the compounds with high m/z values of 379 and 616 have the high molecular weight (HMW) products formed by cross‐linking of peptides and sugar. In (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the m/z value of HMW MRPs was not observed, which might be due to the weak signals of these products. According to the results of gel permeation chromatography, HMW MRPs were formed by Maillard reaction, especially in (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the percentage of Maillard peptides reached 52.90%. It was concluded that Maillard peptides can be prepared through the cross‐linking of sugar and small peptides with a certain MW range. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical‐mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the γ‐linkage, H‐Glu(Cys‐Gly‐OH)‐OH (GSH). Screening for products formed by GSH with D ‐glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N‐(1‐deoxy‐D ‐fructos‐1‐yl)‐pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N‐terminal glutamic acid and the formation of pyroglutamic acid‐related Amadori product were accompanied by generation of Cys‐Gly‐derived Amadori and thiazolidine compounds. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Flow injection fluorometry of protein using hypochlorite-thiamine reagent

This paper describes a flow injection protein assay based on the formation of N-chlorides. Thiamine, which gives fluorescent thiochrome on reaction with N-chlorides, is used as a reagent. The protein sample is first mixed with the carrier solution containing sodium hypochlorite to chlorinate peptide bonds. The fluorescence reagent, containing thiamine and sodium nitrite, is then delivered to the mixture; the sodium nitrite decomposes active chlorine. The assay is sensitive, reproducible, and linear over a range from 20 ng to 2 micrograms of bovine serum albumin. The fluorescence intensity reflects the correct amount of protein because the thiochrome formed is proportional to the number of peptide bonds.     

Heterooligosaccharide synthesis catalyzed by α-glucosidase from Bacillus stearothermophilus

α-Glucosidase from Bacillus stearothermophilus was used as a catalyst for oligosaccharide synthesis by reversed hydrolysis. The yield of disaccharides and trisaccharides depended strongly on the units of enzyme activity added, and on the stability of the enzyme under reaction conditions. When glucose was the only saccharide present in the reaction mixture with α-glucosidase, isomaltose (51%), nigerose (25%), maltose (14%) and kojibiose (10%) were formed. In 50% glucose solution, disaccharide concentrations reached up to 400 mmol/l and trisaccharides were also produced. When other saccharides (mannose or xylose), in addition to glucose, were present in the reaction mixture, both homodisaccharides and heterodisaccharides were formed, their quantity being dependent on the glucose/saccharide acceptor ratios. The highest yields of oligosaccharides were observed with glucose alone, consistent with the observation that the enzyme stability was highest with glucose as the sole saccharide.