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Iron (Fe )-catalyzed glucosamine browning at 50°C: identification and quantification of major flavor compounds for antibacterial activity Yuliya Hrynets, Abhishek Bhattacherjee, Maurice Ndagijimana, Daylin Johana Hincapie Martinez, and Mirko Betti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00761 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 10, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Iron (Fe2+)-catalyzed glucosamine browning at 50°C: identification and quantification of major flavor compounds for antibacterial activity

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Yuliya Hrynetsa, Abhishek Bhattacherjeea, Maurice Ndagijimanaa, Daylin Johana Hincapie Martinez a and Mirko Bettia*

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Affiliations:

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a

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410 Agriculture/Forestry Centre

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Edmonton, AB T6G 2P5 Canada

Department of Agricultural, Food and Nutritional Science, University of Alberta

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*Corresponding Author: Dr. M. Betti;

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E-mail: [email protected]

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Tel: (780) 248-1598;

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ABSTRACT

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Glucosamine browning at 50°C with (GlcN/Fe2+) or without iron (GlcN) was studied over time

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from 0 to 48 h. Generation of reactive oxygen species (ROS), H2O2 and 1O2, along with α-

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dicarbonyls, fructosazine and deoxyfructosazine were evaluated. Singlet oxygen generation

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increased over time and was greater in GlcN/Fe2+ caramel solution. The presence of iron

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significantly increased the concentration of α-dicarbonyls at an early incubation time (3 h).

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Fructosazine and deoxyfructosazine were the major degradation products at 48 h comprising

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together up to 37 and 49% in GlcN and GlcN/Fe2+, respectively. GlcN/Fe2+ (48 h) exhibited a

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MIC50 against highly heat-resistant Escherichia coli AW 1.7, at pH 5, but not at pH 7. Despite

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several antimicrobial compounds being produced during browning, GlcN/Fe2+ created a

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synergistic environment for the fructosazine-organic acids to confer their antimicrobial activity.

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GlcN caramel solutions have the potential to serve as both flavoring compounds and

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antimicrobial agents in formulated food systems.

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KEYWORDS: Glucosamine, Iron, α-Dicarbonyl compounds, Fructosazines, Organic acids,

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Escherichia coli AW 1.7.

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2 ACS Paragon Plus Environment

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INTRODUCTION

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Glucosamine (GlcN, 2-amino-2-deoxy-d-glucose; chitosamine) is a popular natural health

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product used by North American consumers to prevent and treat osteoarthritis.1 Recently, GlcN

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self-condensation products (Figure 1), such as fructosazine (FR; [2,5-bis(D-arabino-

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tetrahydroxybutyl)pyrazine]) and deoxyfructosazine (DOFR; [2-(D-arabino-tetrahydroxybutyl)-

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5-(D-erythro-2,3,4-trihydroxybutyl)pyrazine])), are gaining increased attention as flavoring

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agents in both food and tobacco industries.2 FR and DOFR have been identified in roasted

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peanuts,3 caramel4 and soy sauce.5 More than just a flavor compound, DOFR confers an anti-

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diabetic6 function and activity against immunological and anti-inflammatory diseases.7 Research

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has been conducted to economically produce FR and DOFR using chitin-derived GlcN as a

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reactant.8,9

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Hrynets et al.10 studied the non-enzymatic browning of GlcN in a liquid system at 37°C.

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The authors showed that GlcN, being a Heyns compound, degrades rapidly producing highly

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reactive α-dicarbonyl compounds (α-DCs), including glucosone (G), 3-deoxyglucosone (3-DG),

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methylglyoxal (MGO), glyoxal (GO) and diacetyl (DA). These compounds are known to be

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important intermediates in flavor generation and some of them also possess a proven

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antimicrobial activity (i.e. MGO, GO and DA). MGO, in particular, is very well-studied and it is

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responsible for the antibacterial activity of Manuka honey.11 In fact, 1 kg of GlcN produces up to

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5.7 g of total α-DCs when heated at 37°C for 12 d.10 This shows that GlcN, under certain

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conditions, could be used to produce caramel solution with both flavoring and antimicrobial

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capacities. Moreover, heme-iron can play a major role in degrading GlcN. In our latest study,12

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heme-iron induced large levels of α-DCs in the GlcN-myoglobin reaction system in a catalytic

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manner.



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Although participation and activity of transition metals in the Maillard reaction (MR) is

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still a matter of debate, the majority of the studies support the theory that metals can cause a

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catalytic effect on MR progress. In this scenario, MR products likely complex with metal ions13

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and many of these complexes catalyze the formation of α-DCs.14,15 The use of iron ions (Fe2+ or

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Fe3+) can speed up MR.16 Iron acts as a pro-oxidant,16,17 promoting the decomposition of the

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Amadori adduct into α-DCs,18 3-DG and GO in particular.18,19 At the same time,

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monosaccharides can autoxidize by a transition metal-catalyzed reaction yielding α-DCs, H2O2

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and free radical intermediates.20 Since GlcN is very unstable and reactive molecule, even at

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37°C, it was hypothesized that GlcN could produce a caramel solution rich in flavor and

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antimicrobial compounds at mild conditions. This was intended then to minimize problems

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associated with unwanted toxic compounds with greater non-enzymatic browning temperatures.

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The objective of this study was to investigate the effect of Fe2+ on GlcN browning at a moderate

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temperature (50°C) acceptable for food production systems, and at different incubation times (0,

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3, 12, 24 and 48 h). Kinetics of the browning reaction was monitored by measuring ROS, H2O2

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and 1O2, pH, α-DCs, and also FR and DOFR formation over time. Furthermore, the antimicrobial

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activity of GlcN caramel solutions produced at different incubation times was tested against

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highly heat-resistant Escherichia coli (E. coli) AW 1.7, a gram-negative bacteria commonly

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found in beef carcass.21 MIC50 for each of the identified compounds was determined in its pure

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form against E. coli AW 1.7.

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MATERIALS AND METHODS

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Chemicals

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D-Glucosamine hydrochloride, ammonium iron (II) sulfate hexahydrate; HPLC-grade methanol

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and formic acid, G (2-keto-d-glucose), GO (ethanedial; 40% in H2O), MGO (2-oxopropanal;


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40% in H2O), DA (butane-2,3-dione), o-OPD (1,2-diaminobenzene), diethylene triamine

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pentaacetic acid (DTPA), (2-(N-morpholino)ethanesulfonic acid)) (MES) were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). 3-DG (3-Deoxy-D-erythro-hexosulose) was obtained

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from Cayman Chemical (Ann Arbor, MI, USA). SPE tC-18 Sep-Pak Vac 6 cc columns were

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from Waters (Milford, MA, USA). Hydrogen peroxide and Amplex Red hydrogen

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peroxide/peroxidase assay kit were from Fisher Scientific (Burlington, ON, Canada). Singlet

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oxygen sensor green (SOSG) reagent was from Thermo Fisher (Waltham, MA, USA). FR and

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DOFR were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of

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analytical grade and buffers were prepared with Milli-Q purified water (Millipore, Bedford, MA,

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USA).

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Design of the Experiment

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The non-enzymatic degradation of GlcN and GlcN in presence of iron (GlcN/Fe2+) was

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evaluated over time at 50°C. Generation of ROS, hydrogen peroxide and singlet oxygen, was

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evaluated first. For this purpose, 3 independent trials were conducted. For each trial a total of 30

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tubes (3 tubes × treatment × incubation time) were prepared at 150 g/L GlcN, from which 6

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tubes were left untreated as controls at time zero, and 24 tubes were randomly assembled within

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the incubator and collected at 3, 12, 24 and 48 h. Next, α-DCs and pH were determined using the

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same design as described above. To determine the concentration of FR and DOFR, 3

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independent trials were conducted followed the same protocol as described for ROS and

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subjected to UHPLC-PDA analyses. Selected samples for each treatment were also subjected to

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HPLC-ESI-MS/MS to unambiguously identify FR and DOFR. For the antimicrobial assay, 2

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independent trials were performed, where for each trial a total of 20 tubes (2 tubes × treatment ×

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incubation time) were prepared at 150 g/L. After incubation, antimicrobial activity (MIC50) of



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GlcN caramel solutions was evaluated against E. coli AW 1.7. Finally, MIC50 of identified flavor

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and antimicrobial compounds were determined using pure standards. Each compound was tested

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against E. coli AW 1.7 in three independent experiments.

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Preparation of GlcN and GlcN/Fe+2 Aliquots

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Filtered (0.2 µm sterile filters) 150 g/L GlcN and GlcN/Fe2+ solutions in water were incubated at

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50°C for 3, 12, 24 or 48 h in sterile tubes. The effect of added iron was evaluated at 0.5 mM. The

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pH of the original solutions at time zero was adjusted to 7.40 with 1 M KOH and left unadjusted

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during incubation over time.

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Singlet Oxygen (1O2) Generation

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In vitro production of 1O2 in GlcN and GlcN/Fe2+ reaction mixtures was determined using a

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SOSG reagent. A SOSG stock solution (5 mM) was freshly prepared each time by adding 33 µL

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of methanol to a 100 µg vial, and kept in darkness at 4°C. SOSG with the final concentration of

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10 µM was added to all samples and incubated in darkness for 15 min at 50°C. Then the

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fluorescence was measured at λexc = 504 nm and λem = 525 nm. Thermally- treated sensor reagent

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solutions were kept under identical conditions as control experiment. The emission intensity of

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the control sample was subtracted from the emission intensities of the incubation time sample

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values and fitted with a simple linear regression using GraphPad Prism software.

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Production of Hydrogen Peroxide in Vitro

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H2O2 production in GlcN and GlcN/Fe2+ control and incubated solutions was determined using a

135

kit. Hydrogen peroxide, in the presence of horseradish peroxidase, reacts with Amplex Red

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reagent (10-acetyl-3,7-dihydroxuphenoxazine) in a 1:1 stoichiometry to generate resorufin, a red-

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fluorescent oxidation product. These reagents were included in the Amplex Red hydrogen

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peroxide/peroxidase kit and the assay was conducted in flat-bottom 96-well microplates


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according to the manufacturer’s instructions. For determination of free H2O2, 100 µl of samples

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or standards were mixed with Amplex Red working solution (prepared per the manufacturer’s

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directions, containing 0.2 U/mL horseradish peroxidase) and incubated at room temperature for

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30 min protected from light. Fluorescence emission of the formed product, resorufin, was

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measured at λexc = 540 nm and a λem = 590 nm using a Spectra Max M3 multi-mode microplate

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reader (Molecular Devices, Sunnyvale, CA, USA). To monitor the presence of bound H2O2,

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more stable form of H2O2, samples were vortexed extensively for 1 min to remove free H2O2 and

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the 25 mM H2SO4 was added to release bound H2O2.22 Once liberated, H2O2 was determined in

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the same way as described above for free H2O2. To calculate free and bound H2O2

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concentrations, a standard curve was prepared from a 200 µM H2O2 stock solution. Following the

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subtraction of zero time values, the mean values were fitted on a curve with a non-linear fitting

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model using GraphPad Prism software.

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Measurement of pH

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A pH meter (Orion 2-star, Thermo Scientific, Beverly, MA, USA) was used for the

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determination of systems’ pH values at each incubation time.

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Analyses of Free α-Dicarbonyl Compounds

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Solid-Phase Extraction (SPE) and Pre-Column Derivatization

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Extraction of α-DCs was performed following the method described in Hrynets et al.10: 6 mL

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sample was applied to a pre-conditioned SPE tC-18 Sep-Pak cartridge and washed with 2 mL of

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water at a flow rate of ≤ 2 mL/min. This polar fraction was spiked with 6 mg of o-OPD and the

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pH adjusted to 3.00 ± 0.02 using 4 N HCl. Obtained aliquots were incubated at 37°C for 1 h in

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the presence of 11 mM DTPA. The quinoxaline derivatives were eluted from another SPE

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cartridge with 4 mL of a MeOH/H2O mixture (90/10, v/v).10



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Reverse-Phase Ultra-high Performance Liquid Chromatography with Photodiode Array

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Detection (RP-UHPLC–PDA) and MS Identification of Quinoxaline Derivatives

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The experiments were performed using UHPLC apparatus (Shimadzu, Columbia, MD, USA) as

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reported in Hrynets et al.10 As a stationary phase, an Ascentis Express ES-C18 column (Sigma-

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Aldrich, MO, USA) was applied with the gradient mixture of (eluent A) 0.1% formic acid in

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water and 100% methanol (eluent B). The flow rate was 0.3 mL/min and injection volume of 5

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µL. Identification of extracted α-DCs was based on comparison with the retention time and

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absorption spectra of known reference compounds, exact mass and MS/MS fragmentation of the

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respective quinoxalines.10 MS analyses were based on direct injection of collected sample`s

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peaks into an Ion Max electron spray ionization (ESI) source (Thermo Fisher Scientific)

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mounted on a LTQ Orbitrap XL (Thermo Scientific, San Jose, CA, USA) with the acquisition

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conditions similar to those reported previously.10

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Quantification

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Peaks resolved in UHPLC were quantified using standard curves (coefficient determinations of

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R2 ≥ 0.99) obtained by treating a reference compounds with o-OPD by the same procedure used

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for the reaction mixtures. To construct standard curves each quinoxaline derivative was diluted

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to final concentrations in the following ranges: 500 - 16000 (G), 120 - 2000 (3-DG), 2 - 200

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(GO), 1 - 50 (MGO) and 5 - 50 µM (DA). Each concentration was analyzed in triplicate. The

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average limit of detection (LOD) was calculated as 2.15 ± 0.07 (G), 0.27 ± 0.00 (3-DG), 0.13 ±

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0.00 (GO), 0.09 ± 0.00 (MGO) and 0.18 ± 0.00 µM (DA). The average limit of quantification

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(LOQ) was determined as 6.52 ± 0.21 (G), 0.81 ± 0.01 (3-DG), 0.39 ± 0.01 (GO), 0.27 ± 0.01

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(MGO), and 0.55 ± 0.02 µM (DA) based on a signal-to-noise ratio (S/N) of 3:1 and 10:1 for

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LOD and LOQ, respectively.


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Analyses of Fructosazine (FR) and Deoxyfructosazine (DOFR)

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UHPLC-PDA Conditions and Quantification

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LC separations were performed on the similar UHPLC apparatus as described above. Spectral

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data from all peaks were accumulated in range 190 - 600 nm, and chromatograms recorded at

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275 nm. The revered-phase separation was performed on an Ascentis Express ES-C18 column at

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a flow rate of 0.9 mL/min and injection volume of 10 µL. Gradient elution was performed

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according to the modified method of Shimamura et al.23 with a binary system consisting of (A)

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0.1% aqueous formic acid and (B) 100 % methanol. The gradient program was: 0 - 5 % B (5 - 15

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min), 5 - 50 % B (15 - 25 min), 50 - 5 % B (25 - 35 min), and re-equilibration for 10 min. Area

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integration of eluting peaks was obtained at 275 nm. Quantification was performed by LC-PDA

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using an external calibration curve with a set of seven standard dilutions. The LOD and LOQ

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were 1.30 ± 0.01 and 4.29 ± 0.03 µg/mL, respectively for FR, and were 0.07 ± 0.11 and 0.21 ±

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0.36 µg/mL, respectively for DOFR. The calibration curves for FR and DOFR were linear with a

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correlation coefficient of 0.999. Each data point was analyzed at least in triplicate.

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HPLC-ESI-MS/MS

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Peak identification was performed using a 1200 series HPLC unit (Agilent, Palo Alto, CA, USA)

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connected to a 4000 Q TRAP LC-MS/MS System (Applied Biosystems, Concord, ON, Canada).

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The HPLC separation was performed under exactly the same separation conditions as described

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above for UHPLC. Ionization was achieved using ESI in the positive mode at a spray voltage of

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4 kV and source temperature 500°C. N2 was used as a nebulizing and heating gas at 40 psi. Full

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scan data were acquired by scanning from m/z 50 to 1000. In product ion scan experiments,

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MS/MS product ions were produced by collision-induced dissociation (CID) of selected

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precursor ions using nitrogen as a collision gas under collision energy of 20 eV. The curtain gas



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pressure was set to 20 psi and the declustering and entrance potentials at 40 and 20 V,

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respectively.

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Bacterial Strain and Culture Conditions

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Heat-resistant E. coli AW 1.7 isolated from beef carcass21 was routinely grown on Difco Luria-

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Bertani (LB) medium. For cell plate counting, 15.0 g of agar was added to 1 L of the LB

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medium. Broth and agar media were prepared with distilled water and final pH of 7.0 ± 0.2,

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before autoclaving (121°C for 20 min). Cells were incubated at 37°C under aerobic conditions

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for 24 h for further utilization in growth inhibition analyses. The LB medium was adjusted to pH

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5.0 ± 0.2 using MES.

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Antimicrobial Activity Testing

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The antimicrobial activity of GlcN and GlcN/Fe2+ samples was determined by critical dilution

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assay.24 Prior to the antimicrobial test, E. coli was aerobically subcultured twice in LB liquid

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medium at 37°C. Filtered (Millipore sterile 0.22 µM filters) GlcN and GlcN/Fe2+ solutions were

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divided into two parts: pH 5 and pH 7. 100 µL of sample and 100 µL of LB broth medium were

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mixed, and two-fold serial dilutions of the mixtures were prepared on a sterile flat-bottom 96-

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well polystyrene microtiter plates (Corning, Inc., Corning, NY, USA). Then, 50 µL of overnight

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E. coli bacterial culture were added into the microtiter wells. The final concentration of GlcN

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and GlcN/Fe2+ samples in microtiter plates ranged from 50.0 to 0.098 g/L whereas the initial cell

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count was 1 × 106 CFUs/mL. Microtiter plates were incubated for 16 h at 37°C and at the end of

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the incubation, the antimicrobial activity of samples was evaluated by optical density at 630 nm.

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GlcN and GlcN/Fe+2 samples were analyzed under the same conditions but without addition of

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the bacterial cells. The percentage of inhibition was calculated as: [1-(ODsample−ODnegative

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control)/(ODpositive control–ODnegative control)]×100,

where negative and positive controls were LB


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medium and LB medium inoculated with E. coli AW 1.7 in the absence of antimicrobial

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compounds, respectively. GraphPad Prism software was used to extrapolate the minimum

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inhibitory concentration (MIC) values. MIC50 was defined as the compound concentration

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required to inhibit replication by 50%.

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Determination of GlcN-derived Compounds within E. coli AW 1.7

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500 µl of GlcN/Fe2+ (48 h) solution was incubated with E. coli AW 1.7 (grown at pH 5 and 7)

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cell suspension (500 µl containing 1 × 106 CFUs) for 4 h at 37°C. After incubation the reaction

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mixture was pelleted by centrifugation at 1100 rpm for 10 min. The recovered cell pellet was

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collected while the supernatant was discarded. Bacterial cells were further washed three times

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with PBS and resuspended in PBS (pH 7.40). Bacterial solution was treated with ice-cold RIPA

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buffer (0.15 mM NaCl/0.05 mM Tris⋅HCl, pH 7.2/1% Triton X-100/1% sodium

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deoxycholate/0.1% SDS) and agitated for 20 min at 4°C. The suspension was further sonicated

243

for 30 min and then was centrifuged at 12000 rpm at 4°C for 20 min. After centrifugation,

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supernatant containing the bacterial intracellular content was collected and analyzed by direct

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injection mass spectrometry.

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ESI-Ion-Trap MS

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Direct infusion analyses were conducted using 4000 Q TRAP MS/MS instrument (without trap

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column) equipped with an ESI source. A 1 mL Hamilton syringe was attached to a syringe pump

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and used to infuse samples directly into the ESI chamber at a rate of 100 µL/min. The parameters

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were adjusted as described in HPLC-ESI-MS/MS section. The declustering potentials and

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collision energies were tuned to maximize the intensity of the fragments. Full scan mass spectra

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were acquired over the range m/z 50-1000. For MSn experiments, ions of interest were

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fragmented with compounds-dependent collision energy.



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Statistical Analysis

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All of the data were analyzed by analyses of variance (ANOVA) using the PROC MIXED

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procedure according to SAS methods (v. 9.3, SAS Institute, Cary, NC, USA). Post hoc multiple

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comparison test was Tukey's test (p < 0.05). Results of free and bound H2O2, pH and DOFR

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formation were fitted in GraphPad Prism software for Windows (v. 6, San Diego, CA, USA)

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using a non-linear curve-fitting model with the following equation: Y=Y0 + (Ymax-Y0)×(1-exp[-

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K*x]). 1O2 generation was fitted to a simple linear regression model: Y = mx + c. All results

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were expressed as a mean ± standard deviation.

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RESULTS AND DISCUSSION

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Generation of ROS and pH Changes

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Formation of Singlet Oxygen (1O2) Detected by SOSG

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ROS like 1O2 and H2O2 are normally produced during non-enzymatic browning reactions25 and

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may be important antimicrobial compounds in caramels due to their reactivity toward organic

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molecules. Furthermore, the formation of 1O2 also can be indicative of photosensitizer molecules

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produced during GlcN browning. Browning compounds like melanoidins are involved in type II

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photoreactions producing 1O2 from triplet ground oxygen (3O2).26 SOSG, which emits green

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fluorescence around 525 nm in the presence of 1O2, is a highly selective indicator for monitoring

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1

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detection in GlcN and GlcN/Fe2+ solutions was monitored by recording the emission of SOSG.

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Figure 2 A shows that SOSG fluorescence (FL) emission signal increased linearly in both GlcN

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and GlcN/Fe2+ upon heating, indicating increase in 1O2. In solutions where GlcN degradation

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was Fe2+-induced, significantly greater 1O2 generation was found at 12, 24 and 48 h. Browning

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compounds (pre- and melanoidins) are responsible for 1O2 generation. The absorbance at both

O2 generation. As the concentration of 1O2 is proportional to the emission of SOSG, 1O2


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320 and 420 nm is normally used for pre- and melanoidins evaluation, respectively, increases

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over time.10 Markham and Sammes27 also reported that pyrazines can act as photosensitizers.

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Hence, the large concentration of FR and DOFR (hydroxyalkyl pyrazines), particularly in

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GlcN/Fe2+ generated in the current study (reported below), may be involved in the increased

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formation of 1O2 over time.

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Hydrogen Peroxide Production

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Figure 2 C, D shows the concentrations of bound and free H2O2. The generation of bound H2O2

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was significantly greater at 12, 24 and 48 h in GlcN/Fe2+ as compared to GlcN. Addition of Fe2+

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in the reaction significantly increased free H2O2 at each time tested. Previous studies highlighted

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a few mechanisms of H2O2 generation, like superoxide anion which may be formed by reduction

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of oxygen with alkoxy radicals, which are then reduced to give H2O2. Alkoxy radicals can be

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produced by autoxidation of sugar degradation products.28 Involvement of 1O2 has been also

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proposed due to its ability to rapidly oxidize molecules containing carbon-carbon double bonds

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to form hydroperoxides.29 Thornalley30 showed the reduction of oxygen by the enediol tautomer

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of simple monosaccharides via superoxide intermediacy produces H2O2 and α-DCs (i.e. G, MGO

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and GO). A greater formation in the presence of Fe2+ could occur with metal-catalyzed reduction

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of oxygen via the formation of a complex between oxygen and a metal ion and an

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aminoreductone or enediol;30 so the formation of aminoreductone and enediol structures have

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been proposed during GlcN degradation.10 Prolonged heating (above 24 h) was not substantiated

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by an increase in H2O2 production, where a plateau was reached at 2.6 and 5.9 µM for bound

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H2O2 in GlcN and GlcN/Fe2+, respectively. For free H2O2 the plateau was reached at 9.1 and 20.4

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µM in GlcN and GlcN/Fe2+, respectively. This suggests that the most of the H2O2 produced

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within half time of the reaction (24 h). This could be caused by acidification (Figure 2 B) of the



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reaction system, since enolization and further autoxidation of monosaccharides are largely

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dictated by pH.

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pH Kinetics During the Reaction Progress

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Throughout the incubation of GlcN and GlcN/Fe2+ solutions, a significant drop in the pH was

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observed (Figure 2 B). After 2 days of incubation the final pH of both experimental treatments

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without and with iron reached a plateau of 4.46 and 4.43, respectively. No significant differences

306

in pH at each incubation point was observed in the presence of iron, and the rate constant was

307

slightly greater for GlcN/Fe2+ (0.121) as compared to GlcN (0.117). These results can be

308

partially attributed to the formation of carboxylic and hydroxycarboxylic acids occurring during

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monosaccharide (i.e. d-glucose, d-fructose, etc.) decomposition.31 For example, Brands and van

310

Boekel32 demonstrated the formation of acetic and formic acids by the splitting of 1-

311

deoxyglucosone (1-DG) and 3-DG, respectively. A preliminary study by our research group

312

confirmed the production of formic and acetic acids (Figure S1, Table S1).

313

Identification and Quantification of Free α-Dicarbonyls in GlcN and GlcN/Fe2+

314

The representative UHPLC chromatograms and MS identification analyses of the major α-DCs

315

in GlcN and GlcN/Fe2+ are reported in Figure S2 and Table S2, respectively. The concentration

316

of α-DCs produced by GlcN and GlcN/Fe2+ was determined over time and results are presented

317

in Figure 3. The major α-DC identified in GlcN and GlcN/Fe2+ mixtures was 3-DG (Figure 3 A).

318

However, with the GlcN/Fe2+ treatment significantly greater concentrations were found at 3 (925

319

mg/L) and 24 h (725 mg/L) as compared to GlcN alone (656 mg/L; 580 mg/L), suggesting a

320

catalyzing effect of Fe2+ on 3-DG generation. After 3-DG in GlcN/Fe2+ reached its maximum

321

concentration it significantly declined with prolonged heating, whereas with GlcN, heating from

322

24 to 48 h significantly increased the production of 3-DG. Fallico and Ames19 also reported the


Page 15 of 37


323

accelerated production of 3-DG when iron was added to a glucose-Phe reaction mixture. Wolff

324

and Dean33 found that a Fenton reaction-mediated oxidation of glucose produced α-DCs by

325

autoxidation of glucose enediol. The decrease in 3-DG upon heating could be due to its

326

degradation to MGO34,35 or by its transformation into more advanced reaction products. As a

327

matter of fact, the concentration of MGO increased upon incubation (Figure 3 D). In addition,

328

based on the reaction pathways originally proposed by Klinger et al.,36 Hrynets et al.10 reported a

329

mechanism by which 3-DG can condense to form pyrazine compounds in the presence of

330

ammonia. The predominance of 3-DG can be also explained by reaction’s low pH (Figure 2 B)

331

since acidic conditions favor 1,2-enolization of Heyns compounds (i.e. GlcN).10,37 Addition of

332

iron also increased (p < 0.05) the formation of G at earlier incubation times (3 h), where 524 and

333

433 mg/L were found in GlcN/Fe2+ and GlcN, respectively. After reaching a maximum

334

concentration, a significant drop at 12 h was found in both GlcN and GlcN/Fe2+ with no further

335

significant increase upon heating. G is formed via autoxidation of glucose or oxidation of the

336

Amadori/Heyns compound,10,38 so the addition of iron could stimulate pro-oxidant capacity of

337

the incubation system generating G. Kawakishi et al.39 found that G was a main product formed

338

from Cu2+-catalyzed autoxidation of the Amadori compound. GO concentration significantly

339

increased in presence of iron at 3 h, however the greatest concentration was reached at 12 h

340

where no statistical difference was found between GlcN and GlcN/Fe2+. Prolonged heating

341

caused the drop in GO production in both systems tested. Few pathways were reported for GO

342

formation, including C-2/C-3 scission of G,40 enolization/oxidation pathways of 3-DG,41 or C-

343

2/C-3 retroaldolization cleavage of GlcN with consequential rearrangements.10

344

The amount of MGO increased steadily in GlcN and GlcN/Fe2+. At 12 and 48 h of incubation the

345

effect of iron was to create significantly less MGO (2.6; 4.4 mg/L) as compared to GlcN (3.2; 6.2



Page 16 of 37

346

mg/L). The production of DA was promoted in the presence of iron, reaching a maximum at 12 h

347

(12.4 mg/L) compared to GlcN (9.3 mg/L), while in GlcN solution greatest concentration was

348

reached at 24 h. The concentration of DA observed in this study is comparable to that found in

349

diacetyl-rich foods, like popcorn and margarine.42 Overall, the concentrations of G, 3-DG, GO

350

and DA (expressed as total α-DCs) were the greatest in GlcN/Fe2+ at 3 and 24 h of incubation

351

while at the end of the reaction (48 h) no significant difference was observed. This confirms that

352

a more oxidative environment induces a fast degradation of α-hexosamines like the Heyns`s

353

compound.43

354

Identification and Quantification of Fructosazine and Deoxyfructosazine

355

The goal of this study was to determine the major products of GlcN degradation with flavor and

356

antimicrobial capacity. FR and DOFR were identified and quantified in GlcN and GlcN/Fe2+

357

treatments. Identification of these polyhydroxyalkyl pyrazines from GlcN had been described

358

before,10,44 however quantification based on authentic standards has not been reported. In the

359

previous study10 resolved peaks of fructosazines were noticed during LC separation of

360

quinoxaline derivatives of α-DCs. Since FR and DOFR share similar structural elements

361

(pyrazine ring), it was presumed that GlcN caramel solution could be resolved using the same

362

methodological approach as for α-DCs. Figure 4 A shows a representative chromatogram for

363

GlcN incubated for 48 h. MS spectra as well as MS/MS product ion spectra were recorded for all

364

of the peaks resolved in UHPLC. Based on the above-mentioned characteristics, two of the

365

products were assigned to FR and DOFR (as marked in Figure 4 B). For unequivocal peak

366

identification, the chromatographic and spectroscopic behaviors (retention times, UV, MS and

367

MS/MS) of the peaks in GlcN were compared to those of authentic standards of FR and DOFR.

368

Therefore, the presence of FR and DOFR eluted at 3.5 and 4.3 min, respectively could be


Page 17 of 37


369

confirmed. The MS profile of FR (Figure 4 C) showed the base peak at m/z 321.2 (100%), while

370

the last peak eluted, DOFR (Figure 4 A), showed the base peak at m/z 305.2 (100%). The

371

product ion scan of FR showed a loss of two water molecules (Figure 4 E). Fragments

372

corresponding to the loss of one ([M-18]+; fragment at m/z 287.1) and two ([M-36]+; fragment at

373

m/z 268.4) water molecules from molecular ion were detected during MS/MS fragmentation of

374

DOFR (Figure 4 F). The fragmentation patterns of FR and DOFR corresponded to the

375

fragmentation of authentic standards reported in Figure S3, another time confirming correct

376

identification. For the best of our knowledge this is the first time the MS/MS fragmentation

377

pattern of GlcN-derived FR and DOFR have been reported. The quantification results are shown

378

in Figure 5 A, B. The production of FR was significantly greater in the presence of Fe2+ at all

379

tested times except 3 h. At the end of the reaction, 48 h, the concentration of FR produced from

380

150 g/L of GlcN/Fe2+ was 36.4 g/L, which is 1.7 times greater (p < 0.05) than that from GlcN.

381

On percentage basis, 14% of GlcN was transformed in FR at 48 h, while 24% of FR was

382

produced at the same incubation time from GlcN/Fe2+. These results once again demonstrate the

383

catalyzing effect of iron on GlcN degradation, in particular on FR generation. Interestingly, a

384

different pattern was observed with the production of DOFR, where a catalyzing effect of iron

385

was also observed at 48 h (p < 0.05), but no other incubation times (Figure 5 B). By 48 h, the

386

addition of Fe2+ resulted in 3.3 g/L more DOFR generated compared to GlcN. A different

387

degradation pattern was also observed, where DOFR reached a plateau by 24 h, while prolonged

388

heating in the presence of iron increased FR. Overall, by 48 h, 23 and 25% of the original GlcN

389

was transformed to DOFR in GlcN and GlcN/Fe2+, respectively. FR and DOFR are likely then

390

the major degradation products of GlcN, comprising together up to 37 and 49% of degradation in

391

GlcN and GlcN/Fe2+, respectively.



392

Page 18 of 37

Antimicrobial Action of GlcN Caramel Solution and its Reaction Products

393

Action of GlcN caramel solutions on growth inhibition of E. coli. The inhibitory activity

394

of GlcN and GlcN/Fe2+ caramel solutions produced at different times was tested against the heat-

395

resistant E. coli AW 1.7, a major contaminant in the meat industry, beef in particular. Figure 6 A

396

shows the activity of GlcN and GlcN/Fe2+ at pH 5. The MIC50 was only achieved in the presence

397

of iron and 48 h heating (p < 0.05), while GlcN incubated alone for 48 h could inhibit only 31%

398

of E. coli growth. Even though the presence of iron helped to inhibit on average 11% more of

399

bacterial growth as compared to GlcN, no MIC50 was achieved at pH 7 (Figure 6 B). Firstly,

400

these findings highlight the catalytic effect of iron in determining the antimicrobial activity of

401

GlcN caramel solutions, and secondly, the effect of pH, where GlcN caramel solution was more

402

active against the bacteria in moderate acidic media. Since we were able to identify and quantify

403

the major chemical products from GlcN/Fe+2 caramel solution, it was important to analyze the

404

antimicrobial activity (MIC50) of the pure standards of these products against E. coli AW 1.7. By

405

comparing the concentration of identified antimicrobial compounds in GlcN/Fe+2 caramel with

406

the MIC50 of the pure components, it is possible to understand which specific components confer

407

antimicrobial power to GlcN/Fe+2 caramel solution.

408

Organic acids. As mentioned in the section describing pH kinetics, a 3 log pH decrease

409

was observed in both GlcN and GlcN/Fe+2, and was associated with the production of acetic and

410

formic acids. The MIC50 at two different pH values (5 and 7) was 0.6, 1.0 g/L for formic acid

411

and 0.6, 1.1 g/L for acetic acid (Figure 6 C). Although acid quantification is not reported here,

412

considering ∆pH and the respective acids Ka, their concentration in both GlcN and GlcN/Fe+2

413

caramel solutions was in the range of MIC50. However, the pH decrease in the reaction system in

414

the absence or presence of iron was the same, implying that the antimicrobial effect achieved


Page 19 of 37


415

was not just due to a pH effect. However, the production of organic acids may potentiate the

416

action of other antimicrobial compounds; for instance, Przybylski and Witter45 reported that

417

acetic acid was able to cause sublethal injury to E. coli, implying the disruption of the

418

lipopolysaccharide layer on the membrane surface.

419

Reactive oxygen species: H2O2. As reported in Figure 2 (C, D), H2O2 produced during

420

GlcN/Fe2+ (48 h) browning reached its maximum total concentration of 27.3 µM. The MIC50 of

421

H2O2 standard was 1.7 and 3.4 g/L at pH 5 and 7, respectively. Even though H2O2 is among the

422

best known antimicrobial compounds, its concentration found in GlcN/Fe2+ caramel solution was

423

much less than the concentration required to inhibit E. coli AW 1.7 at pH 5. This means that

424

H2O2 alone is not the major determinant of antimicrobial activity of GlcN/Fe2+. Moreover,

425

addition of catalase (data not shown) to GlcN/Fe2+ (48 h) did not reduce its MIC50, reinforcing

426

this conclusion.

427

α-DCs. Five commercial α-DCs quantified in GlcN/Fe+2 were tested against E. coli AW

428

1.7. All these α-DCs showed a MIC50 at both pH values tested. Among these, MGO was the most

429

effective followed by DA, GO, 3-DG and G. The MIC50 at pH 5 was 0.05 (MGO), 0.1 (DA), 0.4

430

(GO), 1.0 (3-DG), and 23.5 g/L (G) (Figure 6 C). Increasing the pH caused an increase in MIC50

431

by 4.3, 3.0, 2.0, 1.7 and 1.3 times for MGO, DA, GO, 3-DG and G, respectively. The reaction of

432

α-DCs with cysteine residues causes an interference in both DNA and protein synthesis; this has

433

been ascribed to α-DC’s ability to inhibit microbial growth.46,47 The concentration of α-DCs

434

determined in 50 g/L GlcN/Fe2+ (48 h) caramel solution was 0.015 (MGO), 0.003 (DA), 0.001

435

(GO), 0.23 (3-DG) and 0.12 g/L (G), which is significantly less compared to the concentration

436

required for inhibiting 50% of E. coli AW 1.7 growth. Furthermore, the concentration of the

437

major α-DCs was not significantly different among the treatments at 48 h, reducing the role of



Page 20 of 37

438

α-DCs in determining the antimicrobial activity of GlcN/Fe+2 caramel solution. Supporting these

439

conclusions on the observations is also the effect of "GlcN matrix" on reducing the antimicrobial

440

activity of some α-DCs. For instance, when fresh GlcN was added at different ratios to 3-DG,

441

the antimicrobial activity of the latter was reduced (Figure S4). This is likely due the instability

442

and reactivity of 3-DG that could be "quenched" by the component of GlcN matrix before

443

exerting its effect on E. coli AW 1.7.

444

Fructosazines. The MIC50 of FR was 3.6 g/L at pH 5 while at pH 7 MIC50 was not

445

reached (Figure 6 D); only 40% of the E. coli AW 1.7 growth was inhibited at a plateau

446

concentration of 6.6 g/L. DOFR did not achieve an MIC50 at any pH value tested (Figure S5).

447

Since the FR concentration found in GlcN/Fe2+ was greater than in GlcN caramel solutions

448

without Fe2+ and well above the MIC50 of pure FR standard, it is likely that the antimicrobial

449

activity of GlcN/Fe2+ is mainly due to FR and its interaction with pH. To the best of our

450

knowledge, this is the first time that the antimicrobial activity of FR has been reported. This is an

451

important contribution to the literature because FR is also a flavoring agent, and possessing an

452

additional antimicrobial activity may increase its utility in food products. However, FR-pH

453

synergy seems to be the key focus for achieving a microbial growth reduction by 50% (Figure 6

454

D). It is possible that the acidic conditions induced by organic acids induced a permeabilization

455

of the outer membrane of E. coli AW 1.7 to promote the uptake of FR. FR would then be able to

456

induce DNA fragmentation and inhibit bacterial growth. Sumoto et al.48 demonstrated DNA

457

strand breaking activity of FR in plasmid pBR322, which in addition was catalyzed by Cu2+.

458

Preliminary direct evidence of such mechanism is reported in Figure S6 where peak at m/z 359.4,

459

a potassium adduct of FR (refer to MS/MS fragmentation in Figure S7), was presented within the

460

collected bacterial intracellular content.


Page 21 of 37


461

In conclusion, this study was able to confirm the role of iron in catalyzing GlcN browning, and

462

to identify and quantify the major GlcN-derived flavor and antimicrobial compounds. FR and

463

DOFR are the major products of GlcN caramel solution, and between these two, FR showed a

464

more important role as an antimicrobial agent against the heat-resistant E. coli AW 1.7.

465

Production of acid, along with flavoring and antimicrobial compounds, makes GlcN caramel

466

solution a promising food additive indeed.

467

ABBREVIATIONS USED

468

ANOVA, analysis of variance; CID, collision-induced dissociation; DA, diacetyl; α-DC, alpha-

469

dicarbonyl compounds; 3-DG, 3-deoxyglucosone; DOFR, deoxyfructosazine; ESI, electrospray

470

ionization; FL, fluorescence; FR, fructosazine; G, glucosone; GlcN, glucosamine; GO, glyoxal;

471

LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MGO,

472

methylglyoxal; MIC, minimum inhibitory concentration; MR, Maillard reaction; MS, mass

473

spectrometry; MS/MS, tandem mass spectrometry; OPD, o-phenylenediamine; PDA, photo

474

diode array detector; ROS, reactive oxygen species; SOSG, singlet oxygen sensor green; SPE,

475

solid phase extraction; UHPLC, ultra-high performance liquid chromatography.

476

ACKNOWLEDGMENTS

477

The authors would like to thank Dr. Michael Gänzle (Department of AFNS, University of

478

Alberta, Edmonton, Canada) for providing E. coli Aw 1.7. The authors thank Dr. Yuan Yuan

479

Zhao (Department of AFNS University of Alberta, Edmonton, Canada) for technical assistance

480

with mass spectrometry experiments. We also thank Dr. James Harynuk`s research group from

481

the Department of Chemistry (University of Alberta, Edmonton, Canada) for helping with

482

preliminary analyses of organic acids production.

483

FUNDING SOURCES



Page 22 of 37

484

This research was funded by grant from Alberta Livestock Meat Agency (ALMA), Alberta

485

Innovates – Bio Solutions (Al Bio), and Natural Sciences and Engineering Research Council of

486

Canada (NSERC).

487

Supporting information. Figure S1. Sample GC×GC-TOF MS chromatograms of control, GlcN

488

and GlcN/Fe2+ incubated for 48 h; Figure S2. UHPLC chromatograms of quinoxaline derivatives

489

of free α-dicarbonyl compounds; Figure S3. MS/MS fragmentation pattern of (A) fructosazine

490

and (B) deoxyfructosazine authentic standards; Figure S4. Effect of GlcN addition at different

491

weight ratios on the ability of 3-deoxyglucosone (3-DG) to inhibit the growth of E. coli AW 1.7;

492

Figure S5. Concentration-response analyses of E. coli AW 1.7 growth inhibition by

493

deoxyfructosazine (DOFR) at pHs 5 and 7; Figure S6. Mass spectrometry profile of intercellular

494

uptake of GlcN/Fe2+ (48 h): (A) control (non –treated E. coli AW 1.7), Inset: enlargement of the

495

respective spectra in the range of m/z 290-390; (B) pH 5 and (C) pH 7. Figure S7. MS/MS

496

fragmentation of the peak at m/z 359.4 obtained from the sample representing the intercellular

497

uptake of GlcN/Fe2+ (48 h) by E. coli AW 1.7. The peak at m/z 359.4 is potassium adduct of

498

fructosazine (m/z 321.4); Table S1. First (1tR) and second (2tR ) dimension times, area, quant

499

masses, similarity and chemical abstract system (CAS) number for formic and acetic acids

500

identified in GlcN and GlcN/Fe2+ solutions after 48 h incubation; Table S2. Retention time,

501

structure, MS and MS/MS data of the α-dicarbonyl compounds identified in GlcN and GlcN/Fe2+

502

treatments. This material is available free of charge via the Internet at http://pubs.acs.org.

503

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metabolic weapons. Comp. Biochem. Physiol. C 2007, 146, 88−110.

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(44) Candiano, G.; Ghiggeri, G.M.; Gusmano, R.; Zetta, L.; Benfenati, E.; Icardi, G. Reaction of

615

2-amino-2-deoxyglucose

616

bis(tetrahydroxybutyl)pyrazine. Carbohydr. Res. 1988, 184, 67-75.

617 618

and

lysine:

Isolation

and

characterization

of

2,5

(45) Przybylski, K.S.; Witter, L.D. Injury and recovery of Escherichia coli after sublethal acidification. Appl. Environ. Microbiol. 1979, 37, 261-265.



619 620

Page 28 of 37

(46) Leoncini, G.; Maresca, M.; Bonsignore, A. The effect of methylglyoxal on the glycolytic enzymes. FEBS Lett. 1980, 117, 17-18.

621

(47) Fraval, H.N.; McBrien, D.H. The effect of methylglyoxal on cell division and the synthesis

622

of protein and DNA in synchronous and asynchronous cultures of Escherichia coli B/r. J.

623

Gen. Microbiol. 1980, 117, 127-134.

624

(48) Sumoto, K.; Irie M.; Mibu, N.; Miyano, S.; Nakashima, Y.; Watanabe, K. et al. Formation

625

of pyrazine derivatives from D-glucosamine and their deoxyribonucleic acid (DNA) strand

626

breaking activity. Chem. Pharm. Bull. 1991, 39, 792-794.

627 628 629 630 631 632 633 634 635 636 637 638 639


Page 29 of 37


640 641 642

FIGURE CAPTIONS

643

Figure 1. The mechanism of glucosamine self-condensation to dihydrofructosazine, fructosazine

644

(FR) and deoxyfructosazine (DOFR).

645 646

Figure 2. Development of SOSG fluorescence (FL) as an indicator of 1O2 formation (A);

647

Changes in pH of GlcN and GlcN/Fe2+ solutions incubated over time at 50°C (B); Concentration

648

(µM) of (C) free and (D) bound H2O2 produced from GlcN and GlcN/Fe2+ solutions over time at

649

50°C incubation. The results are presented as mean ± SD (n = 9). * Indicates the significant

650

differences (p < 0.05) between GlcN and GlcN/Fe2+ at specific time of incubation.

651 652

Figure 3. Concentration (mg/L) of free α-dicarbonyl compounds: (A) 3-deoxyglucosone; (B)

653

glucosone, (C) glyoxal, (D) methylglyoxal, (E) diacetyl and (F) total α-DCs produced from 150

654

g/L of GlcN and GlcN/Fe2+ experimental solutions incubated from 3 to 48 h at 50°C. The results

655

are presented as mean ± SD (n = 9). * Indicates the significant differences (p < 0.05) between

656

GlcN and GlcN/Fe2+ at specific time of incubation.

657 658

Figure 4. Identification of fructosazine (FR) and deoxyfructosazine (DOFR) as degradation

659

products of GlcN and GlcN/Fe2+ solutions incubated over time at 50°C. (A) Representative

660

UHPLC chromatograms of (A) GlcN incubated for 48 h; (B) reference mixture of FR (0.3

661

mg/mL) and DOFR (0.037 mg/mL); MS chromatogram of (C) peak FR and (D) peak DOFR



Page 30 of 37

662

obtained from GlcN/Fe2+ incubated for 3 h; Product ion scans (MS/MS spectra) and structures of

663

(E) FR and (F) DOFR obtained from GlcN/Fe2+ incubated for 3 h.

664 665

Figure 5. Concentration (g/L) of (A) FR and (B) DOFR produced from 150 g/L of GlcN and

666

GlcN/Fe2+ solutions incubated from 3 to 48 h at 50°C. The results are presented as mean ± SD (n

667

= 9). * Indicates the significant differences (p < 0.05) between GlcN and GlcN/Fe2+ at specific

668

time of incubation.

669 670

Figure 6. Inhibitory effect on the growth of E. coli AW 1.7 by (A) GlcN and (B) GlcN/Fe2+

671

treatments at a concentration of 50 g/L; (C) Antimicrobial activity of different standards

672

quantified in GlcN caramel; (D) Inhibition of E. coli AW 1.7 by FR at pH 5 and 7. The

673

highlighted area in the graph and a broken line crossing y-axis at 10 g/L (1%) refers to a region

674

where antimicrobial activity against studied strain was less than 1% v/v concentration. Results in

675

(A) and (B) are reported as mean ± SD (n = 4), while in (C) and (D) n = 3.* Indicates the

676

significant differences (p < 0.05) between GlcN and GlcN/Fe2+ at specific time of incubation.

677 678 679 680 681 682 683 684 30 ACS Paragon Plus Environment

Page 31 of 37


685 686

FIGURES Figure 1.

Fructosazine (FR) - 2H

Glucosamine

-2 H2O

Dihydrofructosazine -HO 2

Glucosamine

De oxyfructosazine (DO FR)



Page 32 of 37

Figure 2.

A

B

* ∆=0.8

* ∆=1.5 ∆=0.4

*

D

C

*

* *

∆=0.2

*

* * *


Page 33 of 37


Figure 3. A

B *

* *

C C

D *

*

*

*

E

F * *



Page 34 of 37

Figure 4. A

DOFR

B FR

DOFR

FR DOFR

D

C

DOFR

FR

E

F


Page 35 of 37


Figure 5. GlcN/Fe2+

GlcN 40

A

* *

30

GlcN

GlcN/Fe2+

40

B

*

* 30

20

20

10

10

0

0

0

12 24 36 Incubation time (h)

48

0

12 24 36 Incubation time (h)

48



Page 36 of 37

Figure 6.

B

A

* * * *

C

*

D


Page 37 of 37


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