Grainger, Megan N.C.Prentice, Erica J.Kaur, Harmanpreet2025-12-082025-12-082025https://hdl.handle.net/10289/17811New Zealand’s multi-million dollar apiculture industry exports over 10,000 tonnes of honey annually. Each country has import guidelines to ensure honey quality. The diastase activity assay is used to determine whether honey has been exposed to harsh conditions (excess storage time or high temperatures will denature diastase) and reports the activity as diastase number (DN), where DN must be >8 for exports. Unfortunately, mānuka honey (NZ’s high value honey with unique properties) fails the diastase assay more often than other NZ floral honeys. Bioactive compounds [methylglyoxal (MGO), dihydroxyacetone (DHA) and 3-phenyllactic acid (3PLA)] in mānuka honey were predicted to decrease diastase activity by forming modifications on diastase. Hence, the extent, diversity, location and functional impacts of these modifications on diastase were investigated. Two fresh mānuka honeys, a clover control and clover spiked (MGO, DHA, 3PLA, 3PLA+MGO and 3PLA+DHA) honey samples were stored at 27 °C for ~200 days and periodically tested to observe chemical compositional changes in relation to the loss of diastase activity. The results corroborated previous data; DN decreased in honey over time with increased loss rates observed in mānuka. In the spiked clover samples both DHA and MGO increased rates of diastase activity loss and these rates were enhanced by 3PLA (a proton donor). Enzyme kinetics (enzyme-substrate binding and enzyme rate constants) suggest that denaturation is not the sole mechanism for reduced DN values in honey over time, rather, surface level modifications from bioactive compounds are affecting the affinity of diastase for the substrate. Diastase was isolated from the honeys (days 0 and ~200) using SDS-PAGE gels and analysed with LC-MS/MS to identify surface modifications on diastase. Results showed evidence of diastase modification over the time trial for all honeys. This occurred in unique locations for both mānuka honeys and MGO and 3PLA+DHA spiked clover honeys. Some of these locations were in proximity to the active site of diastase. The most abundant honey protein, MRJP1, was used to accurately identify trends in modification changes on proteins over time as a model for the effect occurring on diastase (since similar reactive amino acids are present on both MRJP1 and diastase). All honeys (except the clover control) had an increased number of modifications. The largest increases were observed for the MGO, DHA and 3PLA+MGO spiked honeys. Fewer modifications were detected for the 3PLA+DHA spiked honey despite this having the most pronounced changes in the enzyme kinetic parameters. This is likely because the modification library focused on MGO-induced modifications and did not account for DHA modifications. In summary, MGO modifications on proteins in honey were observed. DHA modifications and the role of 3PLA as a proton donor were deduced from the enzyme kinetic parameters. While time and temperature denature diastase, surface modifications on diastase may be contributing to the accelerated loss of diastase activity observed for mānuka honey. This necessitates the reassessment of diastase activity as a test for honey quality, especially in the context of complex honey matrices (such as mānuka honey).enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis