(A) Decrease in Soret band absorbance; (B) images and the percentages of metmyoglobin (MetMb), deoxymyoglobin (DeoMb) and oxymyoglobin (OxyMb) in native Mb and Mb-GlcN conjugates over glycation period from 0.5 to 12 days.

<p>(A) Decrease in Soret band absorbance; (B) images and the percentages of metmyoglobin (MetMb), deoxymyoglobin (DeoMb) and oxymyoglobin (OxyMb) in native Mb and Mb-GlcN conjugates over glycation period from 0.5 to 12 days.</p

Protein oxidation (carbonyl content) in Mb and Mb conjugated with GlcNAc, Glc and GlcN from 0 to 12 days.

<p>The results are mean ± standard deviation of three independent experiments. Data were fitted (except Mb-GlcN) with the non-linear fitting by GraphPad Prism software using following exponential equation: <i>y</i> = A(1-e<i>^-kt</i>), where <i>y</i> is the product concentration, A is the initial value at <i>t</i>₀, <i>k</i> is the reaction rate, and <i>t</i> is time.</p

Studies on the Formation of Maillard and Caramelization Products from Glucosamine Incubated at 37 °C

This experiment compared the in vitro degradation of glucosamine (GlcN), <i>N</i>-acetylglucosamine, and glucose in the presence of NH₃ incubated at 37 °C in phosphate buffer from 0.5 to 12 days. The reactions were monitored with UV–vis absorption and fluorescence emission spectroscopies, and the main products of degradation, quinoxaline derivatives of α-dicarbonyl compounds and condensation products, were determined using UHPLC-UV and Orbitrap mass spectrometry. GlcN produced two major dicarbonyl compounds, glucosone and 3-deoxyglucosone, ranging from 709 to 3245 mg/kg GlcN and from 272 to 4535 mg/kg GlcN, respectively. 3,4-Dideoxyglucosone-3-ene, glyoxal, hydroxypyruvaldehyde, methylglyoxal, and diacetyl were also detected in lower amounts compared to glucosone and 3-deoxyglucosone. Several pyrazine condensation products resulting from the reaction between dicarbonyls and GlcN were also identified. This study determined that GlcN is a significantly unstable molecule producing a high level of degradation products at 37 °C

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation

<div><p>The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with <i>N</i>-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.</p></div

Concentration of the major α-dicarbonyl compound produced during incubation of Mb in the presence of GlcN from 0 to 12 days.

<p>The values are represented as mean ± standard deviation (calculated from three independent trials). G, glucosone; 3-DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal; DA, diacetyl. Different letters within each α-dicarbonyl compound indicate statistical significant difference (<i>p</i> < 0.05).</p

Retention time, MS and MS/MS data of the α-dicarbonyl compounds detected Mb-GlcN conjugates.

<p>Retention time, MS and MS/MS data of the α-dicarbonyl compounds detected Mb-GlcN conjugates.</p

A deconvoluted ESI-MS spectra of Mb incubated at 37°C for various times in the presence of GlcNAc, Glc and GlcN.

<p>The experimental conditions were the same as those used to obtain the spectra in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139022#pone.0139022.g001" target="_blank">Fig 1</a>. Inset spectrum (A) shows the spectrum of GlcN incubated for 12 days in the region of 7000–18000 Da.</p

Fructosazine, a Polyhydroxyalkylpyrazine with Antimicrobial Activity: Mechanism of Inhibition against Extremely Heat Resistant <i>Escherichia coli</i>

Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity against heat-resistant <i>Escherichia coli</i> AW 1.7. This study investigated fructosazine’s antimicrobial mechanism of action and compared it to that of riboflavin. Fructosazine–acetic acid was effective in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity using 1-<i>N-</i>phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by <i>E. coli</i> was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h, except 2, 3, and 10 h. Fructosazine generates ¹O₂, which is partially why it damages <i>E. coli</i>. DNA fragmentation was confirmed by fluorescence microscopy, and the fructosazine–acetic acid was the second most intense treatment after riboflavin–acetic acid. Electron microscopy revealed membrane structural damage by fructosazine at pH 5 and 7. This study provides evidence that fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging membrane integrity, and fragmenting DNA

UHPLC analyses of quinoxaline derivatives of α-dicarbonyl compounds produced from Mb-GlcN conjugates over time.

<p>(A) Chromatograms of (I) a reference quinoxaline mixture of glucosone (G), 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA). (II) Representative chromatogram of Mb-GlcN conjugate incubated for 1 d, derivatized with <i>o</i>-OPD and acquired by UHPLC with UV detection at 314 nm. Numbers indicate the peaks of the quinoxalines of (1) G, (2) unidentified, (3) 3-DG, (4) GO, (5) HPA, (6) 3,4- DGE, (7) MGO, (8) DA and a, b, c peaks corresponding to non-OPD derived GlcN condensation products.</p

Sous-Vide Nonenzymatic Browning of Glucosamine at Different Temperatures

Sous-vide is an increasingly popular method of cooking under controlled conditions of temperature and time inside vacuumed pouches to preserve the nutritional and sensory qualities of food. Sous-vide nonenzymatic browning of glucosamine (GlcN) was investigated at 50, 60, and 70 °C for 12 h. Changes investigated were pH, color, level of browning, and the concentrations of the key Maillard and caramelization reaction products, including α-dicarbonyls and pyrazines. The concentrations of undesired 4-methylimidazole (4-MEI), 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), and 5-hydroxymethylfurfural (5-HMF) were also determined. Six types of caramels were produced of unique composition with no detectable levels of 4-MEI. GlcN caramels produced under vacuum were more acidic and lighter in color, containing significantly less flavorful diacetyl, but more fructosazine (FR) as compared to nonvacuum caramels. THI concentration was well below the toxicity levels for all studied caramels. Principal component analyses showed that the incubation temperature played a key role in determining the composition of caramels