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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.