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

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

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

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

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

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

(A) Fluorescence emission spectra; (B) far-UV spectra analyses; (C) secondary structure composition; (D) maximum fluorescence intensity of Thioflavin T (λ = 482 nm).

<p>The data points all have SD bars, but some are illegible and lie within the symbols; (E) transmission electron micrographs of native Mb and Mb incubated in the presence of GlcN at specific time points.</p

MALDI-TOF/TOF mass spectra of Mb incubated at 37°C for various times in the presence of GlcNAc, Glc and GlcN.

<p>Glycation adducts are marked with a star, with the number of stars corresponding to the number of adducts. Inset spectrum (A) shows the spectrum of GlcN incubated for 12 days in the region of m/z 6000–18000.</p

Iron (Fe²⁺)‑Catalyzed Glucosamine Browning at 50 °C: Identification and Quantification of Major Flavor Compounds for Antibacterial Activity

Glucosamine browning at 50 °C with (GlcN/Fe²⁺) or without iron (GlcN) was studied over time from 0 to 48 h. Generation of reactive oxygen species (ROS), H₂O₂, and ¹O₂, along with α-dicarbonyls, fructosazine, and deoxyfructosazine, was evaluated. Singlet oxygen generation increased over time and was greater in GlcN/Fe²⁺ caramel solution. The presence of iron significantly increased the concentration of α-dicarbonyls at an early incubation time (3 h). Fructosazine and deoxyfructosazine were the major degradation products at 48 h comprising together up to 37 and 49% in GlcN and GlcN/Fe²⁺, respectively. GlcN/Fe²⁺ (48 h) exhibited a MIC₅₀ against highly heat-resistant Escherichia coli AW 1.7 at pH 5, but not at pH 7. Despite several antimicrobial compounds being produced during browning, GlcN/Fe²⁺ created a synergistic environment for the fructosazine-organic acids to confer their antimicrobial activity. GlcN caramel solutions have the potential to serve as both flavoring compounds and antimicrobial agents in formulated food systems