Food Chemistry 305 (2020) 125504

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Full length article

Cold non-enzymatic browning of glucosamine in the presence of T

metmyoglobin induces glucosone and deoxymyoglobin formation
⁎
Xue Zhaoa, Yuliya Hrynetsb, Mirko Bettib,
a
Key Laboratory of Meat Products Processing, Ministry of Agriculture, Jiangsu Collaborative Innovation Centre of Meat Production and Processing, Quality and Safety
Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
b
Department of Agricultural, Food and Nutritional Science, University of Alberta, 410 Agriculture/Forestry Centre, Edmonton, AB T6G 2P5, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: Glucosamine (GlcN) and GlcN-myoglobin reaction systems were incubated at 4 °C to verify that GlcN can go
Glucosamine through non-enzymatic browning at this low temperature, and to test the hypothesis that certain reductones
Myoglobin from GlcN non-enzymatic browning can promote the formation of deoxy- and oxymyoglobin from metmyoglobin
Glucosone reduction. Remarkably, alpha-dicarbonyls and self-condensation products, fructosazine and deoxyfructosazine,
Dihydrofructosazine
were produced at this relatively low temperature. The presence of myoglobin shifted GlcN non-enzymatic
2-Amino-2,4-dideoxy-3-keto-D-glucose
browning toward the formation of glucosone and fructosazine. When glucosone (250–2000 mg/L) was incubated
Iron reduction
with myoglobin it contributed to the formation of deoxymyoglobin, indicating its capacity to reduce metmyo-
globin. This study opens the possibility of using GlcN in meat products to increase oxy- and deoxymyoglobin and
enhance the color of meat.

1. Introduction reaction can be regarded as a complex system in which a network of

In food science, a reductone is a general term used to indicate a class
of compounds produced during the Maillard or caramelization reactions
(non-enzymatic browning) that possess a reducing capacity. The me-
chanism of reduction is similar to that of ascorbic acid, a common re-
ducing agent and antioxidant found in fruits and vegetables. From a
chemical point of view, the term reductone describes a structure with
an enediol or eneminol next to a carbonyl. However, in the latter case
the term amino-reductone is more appropriate. However, any com-
pound produced from the Maillard reaction that possesses a reducing
ability can also be regarded as a reductone. Simple examples of re-
ductones are glucosone and 1-deoxyglucosone, both α-dicarbonyl
compounds (α-DCs) produced from the Maillard reaction as a con-
sequence of the direct oxidation of the Amadori/Heyns compounds or
an enolization reaction, respectively. Some reductones possess a more
complex structure like the heterocyclic ones, such as 3(2H)-furanone
and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (Kanzler,
Haase, Schestkowa, & Kroh, 2016). Kanzler, Haase, and Kroh (2014)
demonstrated that some of these reductones also possess radical
scavenging activity. For instance, 1-deoxyglucosone and its stable de-
rivative enediol (maltol, 3-hydroxy-2-methyl-4H-pyran-4-one) have the
ability to significantly quench radicals. Glucosone has also shown ra-
dical scavenging activity to a lesser degree. Hence, the Maillard
multiple reactions generates ROS and radicals and at the same time
scavenging capacity capable of mitigating the dangerous effects. On the
other hand, the reducing ability of these compounds can become useful
in food products like meat, where having a reduced form of iron in the
prosthetic group of myoglobin can have a positive effect on the ap-
pearance of the product.
In this regard, Hrynets, Ndagijimana, and Betti (2015a) reported
that the Heyns compound glucosamine (GlcN) can go through an oxi-
dative reaction forming glucosone at 37 °C. In another study (Hrynets,
Bhattacherjee, Ndagijimana, Hincapie Martinez, & Betti, 2016) a cata-
lytic effect of ferrous iron (Fe++) was demonstrated on the formation
of glucosone at a relatively short incubation period of 3 h. Furthermore,
the same research group discovered that when GlcN was incubated with
metmyoglobin at 37 °C, the chemical state of metmyoglobin changed to
deoxy- and oxymyoglobin forms, indicating the role of reductones in
reducing Fe+++ iron to Fe++ (Hrynets, Ndagijimana, & Betti, 2015b).
It was postulated that glucosone, dihydrofructosazine (DHFR) and other
compounds produced during the glycation reaction could be re-
sponsible for increasing the amount of deoxy- and oxymyoglobin.
Furthermore, it may be possible that the direct dehydration of GlcN
induces the formation of an amino-reductone, a compound known for
its strong reducing capacity (Shimamura, Takamori, Ukeda, &
Sawamura, 2003). For instance, ascorbinic acid (an eneminol form of

⁎
Corresponding author.
E-mail addresses: 2017208024@njau.edu.cn (X. Zhao), hrynets@ualberta.ca (Y. Hrynets), mirko.betti@ualberta.ca (M. Betti).

https://doi.org/10.1016/j.foodchem.2019.125504
Received 10 May 2019; Received in revised form 4 September 2019; Accepted 8 September 2019
Available online 10 September 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
X. Zhao, et al.
ascorbic acid) possesses greater reducing activity than ascorbic acid
itself. Therefore, if these hypotheses are confirmed, it would have
practical consequences, since production of these reductones could
occur at refrigerated conditions (i.e. 0–4 °C), and thus GlcN could be
possibly used to enhance the color of meat.
Therefore, this study aims to evaluate the non-enzymatic browning
of GlcN in the presence or absence of myoglobin (Mb), to understand if
heme iron has a catalytic effect at this low temperature. Glucosone and
other major α-dicarbonyl compounds as well as heterocyclic com-
pounds, like fructosazine and deoxyfructosazine were determined.
2. Materials and methods
2.1. Chemicals
Horse skeletal muscle myoglobin (Mb, ≥98%), fructose (Fru,
≥99%), D-glucosamine hydrochloride (GlcN, ≥99%), ascorbic acid
(AA, ≥99%), potassium phosphate monobasic and dibasic, HPLC-grade
solvents (acetonitrile, formic acid, methanol), glucosone (2-keto-D-glu-
cose; ≥98%), glyoxal (ethanedial; 40% in H2O), methylglyoxal (2-ox-
opropanal; 40% in H2O), diacetyl (butane-2,3-dione; ≥95.0%), 1,2-
diaminobenzene (o-OPD), dansyl chloride (5-(dimethylamino)-1-naph-
thalenesulfonyl chloride, 99%), ammonium acetate and ammonium
hydroxide solutions were from Sigma-Aldrich (St. Louis, MO).
Fructosazine (FR; 2,5-bis(D-arabino-1,2,3,4-tetrahydroxybutyl)pyr-
azine, > 98%), deoxyfructosazine (DOFR, 2-(D-arabino-tetra-
hydroxybutyl)-5-(D-erythro-2,3,4-trihydroxybutyl))pyrazine, ≥98%)
and 3-deoxyglucosone (3-deoxy-D-erythro-hexosulose; ≥ 95%) were
from Cayman Chemical (Ann Arbor, MI). SPE tC-18 Sep-Pak Vac 6 mL
columns were obtained from Waters (Milford, MA). Filtration mem-
branes (0.22 μm) were from Millipore (Billerica, MA). All buffers and
reaction mixtures were prepared with Milli-Q purified distilled water
(Millipore, Bedford, MA).
2.2. Experimental setup
At the initial stage of this study, to verify the formation of reductone
compounds, GlcN-, Fru- and Fru-NH3-Mb model systems were produced
and color changes as well as chemical state of Mb were determined. Fru
and Fru-NH3 model systems were chosen being the precursors of GlcN
in non-enzymatic browning reaction. In this case, a total of 66 vials (2
vials × GlcN/Fru/Fru-NH3 × incubation time) were randomly located
within the fridge and incubated over time at 0.5, 1, 2, 3, 6, 9, 12, 15 and
18 days at 4 °C. Three independent trials were performed, resulting in
six replicates per treatment. In the second part of the study, identifi-
cation and quantitation of the major reductones were conducted.
Alpha-DCs (i.e. glucosone) and fructosazines were identified and
quantitated using liquid chromatography and mass spectrometry ana-
lyses. The reduction of GlcN due to conversion into its reaction products
was also monitored over time. After quantitation of potential re-
ductones produced during GlcN caramelization, verification experi-
ments with known quantities of glucosone were conducted in the third
part of this study.
2.3. Preparation of experimental aliquots
Mb was dissolved in 50 mM sodium phosphate buffer (pH 7.4) to
reach a concentration of 0.2% and was then mixed with different
concentrations (0.75, 1.5 or 3.0%) of GlcN, Fru or Fru in the presence of
ammonia (Fru-NH3). The pH of the resulted aliquots was adjusted to
7.4, if needed. Afterwards, the sample aliquots were incubated at 4 °C
for up to 18 days. Carbohydrate-free Mb solution was a negative con-
trol, while the positive control contained Mb (0.2%) mixed with
200 ppm of ascorbic acid.
2
Food Chemistry 305 (2020) 125504
2.4. Detection of color changes during incubation
2.4.1. Determination of chemical state of myoglobin
The samples were scanned spectrophotometrically using
Spectramax M5 multimode microplate reader (Molecular Devices,
Sunnyvale, CA). Oxy-, deoxy- and metMb were calculated using the
modified Krzywicki equations (Tang, Faustman, & Hoagland, 2004).
2.4.2. Colorimetric measurements
Color was evaluated with regard to the Hunter coordinates using a
Konica Minolta CR-400 chroma meter (Ramsey, NJ). The instrument
was calibrated against a white tile. The parameters chroma (C*), and
hue angle (H°) were calculated using the CIE L*(lightness), a* (redness),
and b* (yellowness) values with the following equations:
C* = [(a*2 + b*2)1/2] and H° = [arctan (b*/a*)] (Hong & Betti, 2016).
Each color space value reported was the mean of three measurements.
2.5. Analyses of free α-dicarbonyl compounds
The extraction of α-DCs by solid-phase extraction was conducted as
described in Hrynets et al. (2015a). Briefly, 6 mL of the sample were
passed through a conditioned SPE tC-18 SepPak cartridge (SPE1) at a
flow rate < 2 mL/min. Then 2 mL water were used to elute the polar
substance and total 8 mL were collected as SPE-F1. The SPE-F1 was
spiked with 6 mg of o-OPD and incubated at 37 °C for 1 h in the pre-
sence of 11 mM DTPA after pH adjustment to 3.00 ± 0.02 using 4 N
HCl. The quinoxaline derivatives were eluted with 4 mL of a methanol/
H2O mixture (90:10, v/v). Obtained samples were separated by using
HPLC (Agilent 1100 system; Agilent Technologies, Inc., Santa Clara,
CA) consisting of a G-1312 binary pump, a G-1328A injector, a G-
1322A degasser, and a G-1315A photodiode array detector, equipped
with an Ascentis Express ES- C18 column (150 × 4.6 mm, 2.7 μm par-
ticle size; Sigma-Aldrich). The separation conditions were as described
previously (Hrynets et al., 2015a).
The major eluted α-DCs were identified by comparison of their re-
tention times to the reference compounds and by mass spectrometry
analyses. Mass spectra were recorded on a linear ion trap mass spec-
trometer 4000 QTRAP (AB Sciex, ON, Canada) coupled to the HPLC
system (Thermo Finnigan, Dreieich, Germany) equipped with a ther-
mostated autosampler and an Ascentis Express ES-C18 column. The
HPLC separation conditions were the same as described before (Hrynets
et al., 2015a). The mass spectrometer was operated in positive elec-
trospray ionization mode (ESI). The spray needle voltage was 4.0 kV,
source temperature was 450 °C, the capillary voltage was 35 V and the
sheath gas was 40 AU. The derivatives were first characterized by their
molecular masses obtained in the full scan mode set in the range of m/z
50 to 1000. In product ion scan experiments, MS/MS product ions were
produced by collision-induced dissociation (CID) of selected precursor
ions using nitrogen as a collision gas under collision energy of 20 eV.
The curtain gas pressure was set to 20 psi, and the declustering and
entrance potentials were set at 40 and 20 V, respectively. The mass and
MS/MS fragmentation patterns were compared to authentic standards
and to the reference data. Mass spectrometry analyses were performed
in duplicates. Followed identification, the quinoxaline derivatives were
quantitated using standard curves (R2 > 0.99) obtained by treating
standards with o-OPD. The average limits of detection (LODs) were
calculated as 5.8 ± 0.6 (glucosone), 1.6 ± 0.4 (3-deoxyglucosone),
1.2 ± 0.1 (glyoxal) and 0.8 ± 0.0 μg/mL (diacetyl). The average
limits of quantitation (LOQs) were 17.4 ± 1.8 (glucosone), 4.8 ± 1.2
(3-deoxyglucosone), 3.6 ± 0.3 (glyoxal), and 2.4 ± 0.1 μg/mL (dia-
cetyl). LODs and LOQs were calculated as the concentration of analyte
that produces a signal-to-noise (S/N) ratio of 3:1 and 10:1, respectively.
2.6. Assay of fructosazine (FR) and deoxyfructosazine (DOFR)
FR and DOFR were quantitated using the same HPLC apparatus and
X. Zhao, et al. Food Chemistry 305 (2020) 125504

Fig. 1. Time-course changes of (A, D) oxy-, (B, E) deoxy- and (C, F) metMb contents in Mb solutions (negative control) during reaction with GlcN (A, B, C) and Fru/
Fru-NH3 (D, E, F). Mb solutions (negative control) were mixed with ascorbic acid (positive control), 0.75, 1.5 or 3.0% GlcN, Fru or Fru-NH3 and incubated from 0.25
to 6 d at 4 °C. All graphs show the mean ± SD (n = 6). All plots were constructed using a cubic spline curve function (GraphPad 8 software). Fru: fructose; GlcN:
glucosamine; Mb: myoglobin.

C18 reversed-phase column as described above for α-DCs. The mobile
phases were 0.1% formic acid in water (A) and methanol (B) flowing
under gradient elution of 0–5% B from 5 to 15 min, 5–50% B from 15 to
25 min and 50–5% B from 25 to 35 min at a flow rate of 0.4 mL/min.
The injection volume was 10 μL, and the UV detector recorded a signal
at 275 nm. Identification was performed by comparison with the re-
tention time of standard solutions and by mass spectrometric analyses
(Hrynets et al., 2016). FR and DOFR were quantitated using standard
curves (R2 > 0.99). The LODs and LOQs were 1.26 ± 0.02 and
3.78 ± 0.06 μg/mL, respectively, for FR, and were 0.07 ± 0.12 and
0.21 ± 0.36 μg/mL, respectively, for DOFR.
2.8. Statistical analysis
The data were subjected to ANOVA using the PROC MIXED proce-
dure of SAS (v. 9.3; SAS Institute Inc., Cary, NC). Differences among
means at the 5% level were determined by the least significance dif-
ference test. Each reaction condition was performed at least in duplicate
on three different days. The day of trial replication was used as a
random variable. Cubic spline curves showing the changes in chemical
state of Mb were generated by GraphPad Prism 8 to smoothly connect
the points in the figure.

3. Results and discussion

2.7. Derivatization and identification of dansylated glucosamine
3.1. Chemical state of myoglobin and color changes
A volume of 400 μL of a sample solution was treated with 80 μL of
Reductones produced during glycation of Mb, a primary meat pig-
2 M NaOH and 120 μL of saturated sodium carbonate (Lu, Hrynets, &
ment, at 37 °C in the presence of GlcN, are likely involved in the for-
Betti, 2017). To each mixture, 800 μL of freshly prepared dansyl
mation of deoxy- and oxyMb, indicating that ferric iron in Mb is re-
chloride solution (10 mg/mL in acetone) were added followed by
duced to its ferrous form (Hrynets et al., 2015b). The chemical state of
vortex-mixing for 30 s. The solution was protected from light by alu-
Mb influences the color of meat and meat products, where its oxyge-
minum foil and kept at 40 °C for 45 min. The residual dansyl chloride
nated form, oxyMb, is the main analog in muscle foods that contributes
was removed by adding 32 μL of ammonium hydroxide, vortexed for
to the red color associated with fresh meat (Bou et al., 2008). Therefore,
1 min, and left to react at room temperature in darkness for 30 min.
in this study, it was necessary to understand if the same effect would
Subsequently, the volume of the mixture was made up to 2 mL with
occur in cold refrigerated conditions, so that GlcN could eventually be
acetonitrile and filtered through 0.22-μm filters prior to HPLC injec-
used as a novel food ingredient to preserve the color of meat during
tions. The chromatographic system and a column used for elution of
refrigerated storage. Hence, GlcN-Mb and Fru-Mb model systems with
dansyl chloride derivatives were the same as described above for α-DCs
different GlcN and Fru concentrations (0.75, 1.5 or 3.0%) as well as
and pyrazines. The gradient elution system consisted of 0.1 M ammo-
Fru-NH3 were produced and the chemical state of Mb was evaluated. As
nium acetate (phase A) and acetonitrile (phase B) started at 20% B,
mentioned in the Materials and Methods section, Fru and Fru-NH3 were
increased via linear gradient to 75% B at 80 min, then returned to in-
chosen being the precursors of GlcN during non-enzymatic browning.
itial composition within 10 min with a flow rate of 0.4 mL/min. The
Ascorbic acid was used as a positive control to produce reducing con-
injection volume was 10 μL and detection was at 254 nm. The identi-
ditions, which facilitated the reduction of oxidized metMb and there-
fication was performed using the same mass spectrometer as described
fore color changes. Indeed, ascorbic acid is the main reducing agent
above for FR and DOFR.
responsible for changes in the Mb oxidation states (Gianelli, Flores, &
Toldrá, 2005).

3
X. Zhao, et al. Food Chemistry 305 (2020) 125504

Fig. 2. Color mapping showing the changes in (A) lightness (B) hue angle and (C) chroma values of Mb solutions (negative control) incubated in the presence of
ascorbic acid (positive control), 0.75, 1.5 and 3.0% GlcN, Fru or Fru-NH3 from 0.5 to 18 d at 4 °C. The graphs show the mean value calculated from six replicates
(n = 6). Part (D) of the figure shows representative photographs of Mb incubated in the presence of different concentrations of GlcN or Fru over time at 4 °C. AA
refers to the positive control prepared by incubating Mb with ascorbic acid. NC refers to the negative control corresponding to incubated Mb only.

As demonstrated in Fig. 1C, untreated Mb was mainly in the Fe+++
form (86%). The presence of Fru or Fru-NH3 did not change the che-
mical state of metMb within 9 days (Fig. 1F). Prolonged incubation with
Fru resulted in decrease in metMb and increase in deoxyMb by up to
8%. No color changes for Fru-treated samples were visible to the naked
eye (Fig. 2D). Incubation of Mb in the presence of GlcN resulted in a
slight decrease of metMb with a concomitant increase in oxyMb at the
earlier times of incubation (Fig. 1A, B, C). The addition of 3.0% GlcN
was almost comparable to the addition of ascorbic acid (200 ppm)
within 1 d of incubation showing oxyMb contents of 29 ± 1 and
27 ± 1%, respectively. Prolonged incubation resulted in a decrease in
metMb and an increase in deoxyMb, most likely due to the release of
oxygen from oxyMb. After 18 d incubation with different concentra-
tions of GlcN, 25–35% of Mb was in the deoxygenated form (Fig. 1B).
The changes from brown to different intensities of red color during
incubation from 1 to 9 d were clearly evident to the naked eye (Fig. 2D).
Color values of L* (lightness), chroma and hue are shown in Fig. 2.
For better visualization the data were processed using a color map,
where darker colors indicate larger values for particular treatment
within measured color characteristics. In general, the L*, denoting
black (0) to white (100) color space scale, varied from 32.5 to 41.0
(Fig. 2A). Lighter color samples were found for treatment of Mb with
ascorbic acid within 9–18 d, while samples of Mb incubated with 3.0%
GlcN were very light at the beginning of incubation (0.5–3 d) and
progressively became darker with longer incubation times. Since the
changes in L* values do not necessarily correlate with the visually ob-
served browning (Rufian-Henares, Garcia-Villanova, & Guerra-
Hernandez, 2004), the color was expressed by means of the hue angle
and chroma describing color tonality and its vividness (or purity), re-
spectively. The hue angle values, which is the actual color (i.e. red,
yellow, blue, etc.), ranged from 26.4 to 56.7, signifying high color
variability of Mb incubated with GlcN (Fig. 2B). In general, positive
control and treatments with GlcN were of red or orange color, while
negative control and treatments with Fru were of yellowish tonality. It
is important to note that treatments with GlcN were red at early in-
cubation times. As for the chroma, the treatments with Fru or Fru-NH3
had lesser values (Fig. 2C) indicating dullness of the color, while greater
chroma values of Mb-ascorbic acid and Mb-GlcN treatments signified

4
X. Zhao, et al. Food Chemistry 305 (2020) 125504

Fig. 3. Time-course formation of quinoxaline derivatives of (A) glucosone, (B) 3-deoxyglucosone, (C) glyoxal, (D) diacetyl and heterocyclic pyrazines (E) fructosazine
and (F) deoxyfructosazine in GlcN or GlcN-Mb solutions incubated from 1 to 6 d at 4 °C. All graphs show the mean ± SD (n = 6).

more color purity (Fig. 2C). Based on the results of Mb chemical state
and subsequent changes in color, the samples of Mb treated with 3.0%
GlcN were chosen for further analyses. We also propose that observed
changes in color are consistent with the production of reducing com-
pounds originating during the non-enzymatic browning of GlcN at this
relatively low temperature. The next paragraphs focus on the identifi-
cation and possible quantitation of a reductone compound that can be
possibly responsible for the production of the reduced form of Mb.
3.2. The role of glucosone in promoting reduced form of Mb
3.2.1. Generation of glucosone and its reduction potential in model systems
To verify the presence and correct identification of α-DCs, HPLC
chromatograms and MS/MS fragmentation patterns are presented in
Supplementary Figs. S1 and S2, respectively. As previously mentioned,
glucosone is a reductone that might be involved in the promotion of
deoxy- and oxyMb forms in Mb-GlcN model systems. Fig. 3A shows the
glucosone concentration over time (up to 6 d), along with other non-
reducing α-DCs, originated in GlcN incubated alone (control) and GlcN-
Mb model systems. Interestingly, GlcN incubated alone can produce α-
DCs, even at 4 °C, but even more remarkably, the presence of Mb dra-
matically increases the concentration of glucosone at each incubation
time. After 2 d of incubation, the GlcN-Mb produced 246 ± 10 mg/L of
glucosone which was 12.6 times the amount of glucosone found in GlcN
incubated alone. At 3 d the concentration of glucosone in Mb-GlcN did
not significantly changed as compared to 2 d, while in GlcN alone a 3.7-
times increase in concentration was found. At 6 d the glucosone con-
centration was 258 ± 36 and 679 ± 8 mg/L in GlcN and GlcN-Mb,
respectively. Considering the relatively low temperature used in this
study, the concentration of glucosone, especially in the presence of Mb,
is rather impressive. Interestingly, 3-deoxyglucosone, a non-reductone
α-dicarbonyl, was significantly greater at all incubation times in GlcN
alone as compared to GlcN-Mb (Fig. 3B). Furthermore, while glucosone
is the major α-DC in GlcN-Mb model system, 3-deoxyglucosone is the
major one in GlcN control. Glucosone is proposed to be formed by the
oxidation of the Heyns compound (Hrynets et al., 2015a), while 3-
deoxyglucosone is formed through 1,2-enolization. Hence, it is ap-
parent the Mb shifts the reaction toward a direct oxidation of GlcN
rather than the enolization pathway. To verify the reducing capacity of
glucosone toward the Fe+++ in Mb, glucosone-Mb model systems were
produced with different concentrations of glucosone (Fig. 4). Glucosone
at 2000 mg/L significantly increased the production of deoxyMb at each
incubation time with the maximum increase of 10% observed at 6 d. Its
presence at 500 and 1000 mg/L was also able to increase the deoxyMb
content by 4.5 and 6%, respectively. The total increase in deoxyMb in
GlcN-Mb model systems was up to 40% (Fig. 1B); this indicates that
glucosone concentration of 679 ± 8 mg/L found in this study con-
tributed a 10–12% increase. Therefore, glucosone, at least in part, is
responsible for the production of deoxyMb in this study. To the best of
our knowledge, this is one of the first studies to show the potential of a

5
X. Zhao, et al.
Fig. 4. Time-course changes of deoxyMb content in Mb solutions (negative
control) incubated in presence of glucosone. Mb solutions were mixed with 250,
500, 1000 and 2000 mg/L of glucosone and incubated from 0.5 to 6 d at 4 °C.
The graph shows the mean ± SD (n = 6).
Maillard-generated reductone (glucosone) to reduce a transition metal
like Fe+++ in Mb. Other molecules beside glucosone are likely re-
sponsible for this increase of deoxyMb.
3.3. Other α-dicarbonyls and a proposed new pathway for glyoxal and
diacetyl formation
In relation to the production of short-chain α-DCs, GlcN-Mb pro-
duced significantly less glyoxal compared to the GlcN control (Fig. 3C),
while diacetyl was not detected at all in GlcN-Mb (Fig. 3D). Glucosone
is the precursor of glyoxal through retroaldolization reaction (Hrynets
et al., 2016). However, in this study this seems to be in contradiction;
the greatest amount of glucosone found in GlcN-Mb did not result in
greater glyoxal production. On the contrary, the concentration of
glyoxal was significantly greater in the GlcN control at all tested times
as compared to GlcN-Mb. This indicates that glucosone may not be the
major precursor of glyoxal in this study. Here we postulate that both
glyoxal and diacetyl are formed through the double dehydration of
GlcN followed by a retroaldol reaction, as reported in the proposed
mechanism in Fig. 5. Therefore, differently to what was reported in
Hrynets et al. (2016), diacetyl can be generated not only through the
interconversion reaction of Heyns to Amadori compound, but also from
the direct dehydration and subsequent retroaldol degradation of GlcN.
As is evident in the mass spectrum of untreated GlcN shown in Fig. S3,
the molecular ion peaks were detected at m/z 162.0 and 144.0, corre-
sponding to single and double dehydrated base peak [M + H]+ at m/z
180.0. Furthermore, detection of dehydrated forms of GlcN discussed
provides evidence for a possible new pathway for diacetyl production
from GlcN.
Fig. 5. Proposed mechanism of glyoxal, diacetyl and amino-reductone formation from glucosamine. RA: retroaldolization.
6
Food Chemistry 305 (2020) 125504
3.4. Generation of fructosazine and deoxyfructosazines
As reported in several studies, GlcN can easily condense at a rela-
tively low temperature (37–50 °C) to form DHFR (Hrynets et al., 2015b,
2016). This intermediate product is rather unstable and upon oxidation
or dehydration can form FR and DOFR, respectively. Thus, it was of
interest to understand if such a condensation reaction would occur at
4 °C, and if Mb could direct the condensation reaction products toward
the oxidation of DHFR. Figs. S4 and S5 show HPLC chromatograms and
MS/MS identification of FR and DOFR in GlcN and GlcN-Mb treatments.
Fig. 3 (panels E and F) clearly indicates the production of FR and DOFR
occur at 4 °C and that Mb shifts the condensation of GlcN toward FR
(i.e. 200 mg/L at 6 d), since DOFR concentration in GlcN-Mb was rather
low (10 mg/L at 6 d). Hence in GlcN-Mb the production of FR is at least
twenty times greater than the DOFR concentration. Since FR is mainly
produced through oxidation of DHFR, it indicates that Mb promotes the
oxidation of DHFR rather than its dehydration to DOFR. Since DHFR is
a rather unstable molecule and is difficult to be separated by liquid
chromatography, it is somewhat challenging to test its reducing capa-
city in the model system. However, a study conducted by Shimamura
et al. (2003) demonstrated the capacity of DHFR to reduce tetrazolium.
3.5. Dehydration of GlcN and possible formation of an amino-reductone
structure
The GlcN molecule, as other common hexoses, can be dehydrated
forming intermediate compounds like amino-reductone and α-DCs. At
least theoretically, upon GlcN dehydration the amino-reductone (2-
amino-2,4-dideoxy-3-keto-D-glucose) reported in Fig. 5 could possibly
be produced. Derivatization with dansyl chloride was used to determine
possible forms of dehydrated GlcN molecules and chromatograms are
presented in Fig. 6A. The peak of dansylated GlcN was found at a re-
tention time of 13.5 min, while the peak of dansylated derivative of
dehydrated GlcN (−1 H20) eluted at 18.7 min. Mass spectrometry
analyses, reported in Fig. 6B, showed protonated molecular ion masses
of the respective peaks at m/z 412.7 and 394.7, verifying the presence
of dansylated GlcN and single dehydrated dansylated GlcN. The iden-
tifications of the peaks at m/z 162.0755 and 144.0649 in Hrynets et al.
(2015a) also confirm this hypothesis. As proposed in the reaction
scheme shown in Fig. 5, the dehydration at C3–C4 can result in the
formation of two possible forms, one of which could be an amino-re-
ductone (2-amino-2,4-dideoxy-3-keto-D-glucose). This amino-re-
ductone, at least theoretically, could promote formation of deoxyMb.
4. Conclusions
This research demonstrated that reductone compounds can be
X. Zhao, et al. Food Chemistry 305 (2020) 125504

Fig. 6. (A) HPLC chromatograms of dansyl derivatives of GlcN and dehydrated GlcN detected at λ = 254 nm in GlcN and GlcN-Mb solutions incubated from 0 to 3 d
at 4 °C. Top right panel shows GlcN dansylation reaction. (B) Mass spectrometry analyses of peaks eluted at (B1) 13.5 and (B2) 18.7 min, corresponding to protonated
molecular ions of dansyl GlcN and dansyl dehydrated GlcN, respectively; Arrows show MS/MS fragmentation patterns of the respective peaks at m/z 412.7 and m/z
394.7.

generated in GlcN-Mb model system, promoting formation of deoxyMb
at 4 °C. Among the reductones identified in this study, we report that
glucosone formation is promoted through the oxidation of the Heyns
compound GlcN, probably due to a peroxidase-like activity of Mb.
DHFR and 2-amino-2,4-dideoxy-3-keto-D-glucose are other possible re-
ductones that could be responsible for the reduction of Fe+++ in
MetMb. From a food science perspective, the ability of GlcN to produce
reductones could eventually be exploited in meat products to enhance
the color. However, as reported in another study (Hrynets et al., 2016),
singlet oxygen can be generated during GlcN non-enzymatic browning,
possibly inducing lipid oxidation. Studies in real meat samples are
going to verify the applicability of GlcN as a color enhancer. This

7
X. Zhao, et al.
research also proposes a new pathway for the formation of glyoxal and
diacetyl through double dehydration and C2–C3 retroaldolization re-
action.
Authors' contributions
XZ performed the majority of the experiments. YH wrote the
manuscript. MB conceived the idea, designed the study, supervised the
study and edited the manuscript.
Funding
This work was supported by the grants from the Alberta Livestock
Meat Agency (ALMA), Alberta Innovates Bio Solutions (Al Bio), and the
Natural Sciences and Engineering Research Council of Canada (NSERC).
Xue Zhao acknowledges China Scholarship Council (CSC) scholarship
for six months' student exchange programme.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.125504.
Food Chemistry 305 (2020) 125504
References
Bou, R., Guardiola, F., Codony, R., Faustman, C., Elias, R. J., & Decker, E. A. (2008). Effect
of heating oxymyoglobin and metmyoglobin on the oxidation of muscle microsomes.
Journal of Agricultural and Food Chemistry, 56, 9612–9620.
Gianelli, M. P., Flores, M., & Toldrá, F. (2005). Interaction of soluble peptides and pro-
teins from skeletal muscle with volatile compounds in model systems as affected by
curing agents. Journal of Agricultural and Food Chemistry, 53, 1670–1677.
Hong, P. K., & Betti, M. (2016). Non-enzymatic browning reaction of glucosamine at mild
conditions: Relationship between colour formation, radical scavenging activity and
α-dicarbonyl compounds production. Food Chemistry, 212, 234–243.
Hrynets, Y., Bhattacherjee, A., Ndagijimana, M., Hincapie Martinez, D. J., & Betti, M.
(2016). Iron (Fe2+)-catalyzed glucosamine browning at 50°C: Identification and
quantification of major flavor compounds for antibacterial activity. Journal of
Agricultural and Food Chemistry, 64, 3266–3275.
Hrynets, Y., Ndagijimana, M., & Betti, M. (2015a). Studies on the formation of Maillard
and caramelization products from glucosamine incubated at 37°C. Journal of
Agricultural and Food Chemistry, 63, 6249–6261.
Hrynets, Y., Ndagijimana, M., & Betti, M. (2015b). Rapid myoglobin aggregation through
glucosamine-induced α-dicarbonyl formation. PLoS One, 10, e0139022.
Kanzler, C., Haase, P. T., & Kroh, L. W. (2014). Antioxidant capacity of 1-deoxy-D-erythro-
hexo-2,3-diulose and D-arabino-hexo-2-ulose. Journal of Agricultural and Food
Chemistry, 62, 2837–2844.
Kanzler, C., Haase, P. T., Schestkowa, H., & Kroh, L. W. (2016). Antioxidant properties of
heterocyclic intermediates of the Maillard reaction and structurally related com-
pounds. Journal of Agricultural and Food Chemistry, 64, 7829–7837.
Lu, X., Hrynets, Y., & Betti, M. (2017). Transglutaminase-catalyzed amination of pea
protein peptides using the biogenic amines histamine and tyramine. Journal of the
Science of Food and Agriculture, 97, 2436–2442.
Rufian-Henares, J. A., Garcia-Villanova, B., & Guerra-Hernandez, E. (2004). Generation of
furosine and color in infant/enteral formula-resembling systems. Journal of
Agricultural and Food Chemistry, 52, 5354–5358.
Shimamura, T., Takamori, A., Ukeda, H., & Sawamura, M. (2003). Reduction mechanism
of tetrazolium salt XTT by a glucosamine derivative. Bioscience, Biotechnology, and
Biochemistry, 67, 295–299.
Tang, J., Faustman, C., & Hoagland, T. A. (2004). Krzywicki revisited: Equations for
spectrophotometric determination of myoglobin redox forms in aqueous meat ex-
tracts. Journal of Food Science, 69, C717–C720.