Download The kinetics of the conversion of DHA to MGO

Kinetics of formation
of methylglyoxal in
maturing New Zealand
mānuka honey
Merilyn Manley-Harris
Chemistry, School of Science University of Waikato
Acknowledgements:
Megan N.C. Grainger,
Adrian Owens,
Joseph R. Lane,
Richard J. Field
Steens Honey
Kevin Gibbs
Graham Platt
The University of Waikato
Doctoral Scholarship, New
Zealand Federation of
Graduate Women Charitable
Trust Waikato Branch Merit
Award for Doctoral Study and
Shirtcliffe Fellowship. The
Claude McCarthy Travel
Scholarship and Fulbright New
Zealand Travel Award.
Dihydroxyacetone (DHA) is a triose (3C) sugar in the same
sugar series as fructose; it occurs naturally as a phosphate
derivative in various biochemical cycles including the
respiratory cycle in plant and animal cells.
This is often represented as a dehydration reaction (loss
of water, H2O) but in fact is multiple steps involving
catalytic species such as H+ (acid) or OH- (base).
Stoichiometry
The balance of reactants and products
1 molecule of DHA→1 molecule of MGO
Mechanism
The sequence of individual simple steps by which this occurs
Rate-determining step
The slowest step in the reaction mechanism – this controls the
overall rate of the reaction.
Chemical kinetics
The rate at which a reaction occurs
Catalyst
A substance that accelerates the rate of a chemical reaction
Federoňko, M.; Königstein, J.(1969) Coll. Czech. Chem.
Commun. 34, 3881-3894.
Both acid (H+) and base (OH-) catalysis
Note that the reaction that forms MGO is irreversible
The Honey Matrix
Highly dehydrating with low water activity
Large amounts of sugar present with lots of capacity for
H-bonding – binding other molecules that might be potential
catalysts for example water
pH (measure of capacity to donate H+) is moot in this environment
– only measured in diluted honey
Lots of other compounds present arising from the flower itself but
also from the bee – proteins (enzymes), amino acids, phenolics
Care must be taken in drawing analogies with published data
based around aqueous solutions of pure compounds.
Aim of the research
Establish a chemical model for the conversion of DHA to
MGO in honey that can be used to simulate the
transformation in computer modelling.
• Other chemical species (perturbants) that participate
in the mechanism of conversion as catalysts, thus
changing the kinetics.
• Competing reactions that consume DHA, MGO or
catalysts thus changing both stoichiometry and/or
kinetics.
Methodology 1
Loss of DHA and appearance of MGO were monitored over
periods up to two years at different temperatures in the
following systems:
• Mānuka honeys unmodified
• Mānuka honeys doped to a specific level of DHA
• Clover honeys doped to a specific level of DHA
• Artificial honey: Glucose, Fructose, pH adjusted with
gluconic acid (3.8-4) doped with specific levels of DHA or
MGO and with perturbants added singly or in groups.
This allowed determination of overall rates of both
reactions, stoichiometry (efficiency of conversion) and the
effect of model perturbants.
Results I
In clover honey doped with DHA loss of DHA and
appearance of MGO both showed exponential (first
order) behaviour until the DHA:MGO ratio was 0.4:1
and with an efficiency of conversion of ~0.9 at 27oC.
In contrast, in mānuka honey exponential growth only
occurred until the DHA:MGO ratio reached
approximately 2:1 at which point the MGO
concentration remained stable until eventually it
began to decline until the DHA:MGO ratio reached
~0.6:1 with loss of MGO. Efficiency of conversion in the
exponential growth region at 27oC was ~0.5.
Results I
At 4 oC no loss of DHA or gain of MGO was observed
over one year.
Rates of DHA loss and MGO gain at 20, 27 and 37oC in a
doped clover honey were compared
700
9
600
8
7
500
6
400
5
4
300
3
200
2
100
1
0
0
0
100
200
300
Day
400
500
600
MGO (mg/kg)
MGO (mmol/kg)
Variable
37 deg C
27 deg C
20 deg C
4 deg C
Results I
MGO gain in mānuka honey during the exponential
growth period showed similar behaviour but in
mānuka honey the rate of loss of DHA was much
higher and increased much more with increased
temperature than in the clover honey.
This indicates that the conversion of DHA to MGO in
mānuka honey is less efficient than in clover honey and
also that the efficiency declines with increasing
temperature.
Conclusions I
• Mānuka honey contains substances (perturbants)
that can remove both DHA and MGO either by
reacting with them directly or by catalysing their
reaction with another compound.
• Either these substances are not present in clover
honey or mānuka honey contains more of these
substances than clover honey.
• The side reactions require more energy to occur
than the main reaction and therefore receive a
greater rate acceleration as temperature increases.
Conclusions I
• As the rate of DHA conversion slows because the amount
of DHA is falling, the side reactions become more
prominent in their effect
• The “plateau” represents a period of time in which the
rate of formation of MGO is just balanced by the loss of
MGO to side reactions.
• After the “plateau” the side reactions become dominant
and there is nett loss of MGO.
Results II
Perturbants studied in artificial honeys included:
Primary and secondary aliphatic amines and an aliphatic
amide
Proline – a secondary amino acid
Alanine – a primary amino acid
Lysine – a primary amino acid
Iron II
Results II
Results II
• The primary amine accelerated DHA loss and MGO gain
with ~88% efficiency of conversion.
• The secondary amine accelerated the loss of DHA but not
the gain of MGO and the efficiency of conversion was
~2%
• The amide had no effect upon the reaction.
Results II
In systems in which the artificial honey was doped with
MGO
• The secondary amine consumed all the MGO in 2-3 days.
• The primary amine consumed MGO but less rapidly
(76.6% in two days).
• The amide had no effect upon the MGO.
Results II
Alanine increased the rate of formation of MGO more than
proline or a mixture of alanine and proline; the latter system
showed behaviour analogous to a real honey.
12
500
400
8
300
6
200
4
100
2
0
0
0
100
200
300
Time (days)
400
500
MGO (mg/kg)
MGO (mmol/kg)
10
Variable
Control
Proline
Alanine
Proline + Alanine
Possible side reactions that are occurring
• DHA and MGO can react with amines in Maillard-type reactions
• Products of these reactions may be reversible or irreversible
• Products of reactions of secondary and primary amines with
DHA and MGO are different
• Both DHA and MGO may react with each other or themselves
• Amine catalysts may be consumed by Maillard-type reaction
with DHA, MGO or sugars
Dimer →2 DHA
2 DHA → Dimer
DHA → Enediol
Enediol →DHA
Enediol →Enolic
Enolic → MGO
2 DHA → Aldol
DHA + MGO → DHA-MGO product
DHA-MGO product → DHA + MGO
DHA-MGO product → Dead end DHA-MGO product
DHA + Alanine → Imine
Imine → DHA + Alanine
Imine → Maillard-like product
Aldol+ Alanine → 2 MGO + Alanine
Alanine + Compound A → Alanine-A product
DHA + Proline → DHA-Proline product
DHA-Proline product → DHA + Proline
DHA + Proline → MGO + Proline
MGO + Proline → MGO-proline product
MGO-Proline product - MGO + Proline
MGO + Proline → MGO side product + Proline
DHA + Iron → DHA-Iron product
DHA-Iron product → DHA + Iron
DHA + Iron → MGO + Iron
Results II
Correlations between possible perturbants and loss of both
DHA and MGO
Compound
R2 (%) for correlation between
compound and DHA loss
R2 (%) for correlation between compound
and MGO loss
Proline
Glycine
Total primary
amino acids
Total amino acids
Tyrosine
Phenyllactic acid
Syringic acid
Methyl syringate
Sum of phenolic
acids
Total acidity
82
85
86
94
77
56
92
92
98
86
70
91
74
80
58
73
99
71
87
89
Conclusions II
50
Variable
953 A
953 B
Simulation
4000
40
30
2000
20
DHA (mg/kg)
DHA (mmol/kg)
3000
1000
10
0
0
0
100
200
300
Time (days)
400
500
Experimental (circles and squares) and simulated (triangles) data for loss
of DHA in mānuka honey 953.
Conclusions II
900
Variable
953 A
953 B
Simulation
12
800
700
600
8
500
6
400
MGO (mg/kg)
MGO (mmol/kg)
10
300
4
200
2
100
0
100
200
300
Time (days)
400
500
Experimental (circles and squares) and simulated (triangles) data for gain of MGO
in mānuka honey 953.
Results III
What is the rate-determining step?
As a solid DHA exists as an exceptionally stable dimer.
In honey it is likely also to exist as the dimer.
Only the monomer can form MGO.
Upon dissolving in water the dimer is in equilibrium with
the monomer.
This equilibrium is several steps which require catalysis by
H+ and OH-
Effect of incremental addition of water on the rate of
conversion of dimeric DHA to monomer in DMSO a model
for the honey matrix.
0.9
0.8
Rate constant k (min-1)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
10
20
30
Concentration of D2O (m%)
40
50
Conclusions III
• Rate of change of DHA dimer to monomer and hence
formation of MGO is controlled by the lack of available
donors of catalytic H+ and OH-.
• These potential donors, especially water are likely to be
bound up with the sugar by H-bonding.
• The presence of additional H+ donors such as phenolic acids
will accelerate the reaction of DHA but can potentially also
accelerate side reactions thereby reducing the overall yield
of MGO.
The Real World Take-Home Message
Do not store mānuka honeys at elevated temperatures for
prolonged periods.
Quite apart from the effect on DHA/MGO there is HMF to
consider.
The Real World Take-Home Message
The reaction continues during transport and after packaging
for the lifetime of the honey.
You should consider the conditions of transport and
corresponding shelf-life claims as well as advice to purveyors
and purchasers of packaged honey.
The Real World Take-Home Message
Each mānuka honey has a subtly different individual profile.
Accurate prediction of maturation outcomes will therefore be
difficult to ascertain without consideration of the
concentrations of perturbants.
Bibliography
Adams, C. J.; Manley-Harris, M.; Molan, P. C. The origin of methylglyoxal in New
Zealand mānuka (Leptospermum scoparium) honey. Carbohydrate Research, 2009,
344, 1050-1053.
Grainger, M.N.C.; Manley-Harris, M.; Lane, J.R.; Field, R.J. (2016) Kinetics of
conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey:
Part I - Honey systems. Food Chemistry, 202, 484-491.
Grainger, M.N.C.; Manley-Harris, M.; Lane, J.R.; Field, R.J. (2016) Kinetics of
conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey:
Part II - Model systems. Food Chemistry, 202. 492-499.
Grainger, M.N.C.; Manley-Harris, M.; Lane, J.R.; Field, R.J. (2016) Kinetics of
conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey:
Part III - a model to simulate the conversion. Food Chemistry, 202, 500-506.
Owens, A. (2016) The kinetics of the dissociation of the dihydroxyacetone dimer in
aprotic media. MSc thesis, University of Waikato.