title: An investigation of the accelerated loss of diastase (α-amylase) activity in mānuka honey

authors: Kaur, Harmanpreet

abstract: New 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).

text: An investigation of the accelerated loss of diastase (α-amylase) activity in mānuka honey New 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).

year: 2025

title: Sustainable composite fused deposition modelling filament using post-consumer recycled polypropylene

authors: Pickering, Kim L.

abstract: Post-consumer recycled polypropylene (PP) with differing harakeke and hemp fibre contents was used to fabricate a range of 3D printing feedstock filaments. The most successful filaments in terms of tensile properties contained 30 wt % harakeke and had a tensile strength and Young’s modulus of 41 MPa and 3.8 GPa respectively. Comparing these results to those of post-consumer recycled PP filament, showed improvements in tensile strength and Young’s modulus of 77% and 275%. The composite that showed the least shrinkage consisted of 30 wt % harakeke with a shrinkage value of 0.34% corresponding to a net reduction of 84% relative to post-consumer PP.

text: Sustainable composite fused deposition modelling filament using post-consumer recycled polypropylene Post-consumer recycled polypropylene (PP) with differing harakeke and hemp fibre contents was used to fabricate a range of 3D printing feedstock filaments. The most successful filaments in terms of tensile properties contained 30 wt % harakeke and had a tensile strength and Young’s modulus of 41 MPa and 3.8 GPa respectively. Comparing these results to those of post-consumer recycled PP filament, showed improvements in tensile strength and Young’s modulus of 77% and 275%. The composite that showed the least shrinkage consisted of 30 wt % harakeke with a shrinkage value of 0.34% corresponding to a net reduction of 84% relative to post-consumer PP.

year: 2017

title: He pakiriha

authors: Temara, Pou

subjects: Tikanga

abstract: Kua tae ki te wā hai kōrero i ētahi tikanga marae, ētahi tikanga kawa e kawa ana ki tā te mātanga titiro. Tērā ētahi tikanga e whakamahia ana e ngā kaumātua o nāianei e uru atu ana ki te pae o rorirori, ka mutu, e ai ki tā ngā kaumātua nei, ko te mutunga mai o te tika ā rātau i whakatau ai. Nā hei whakarāpopoto i te whakaaro whānui, i ētahi wā kāore ko te mea e tika ana ki ō tātau whakaaro te huarahi pai. Ko tā te tikanga he kimi i te huarahi e tika ana, kia kore ai e whara ngā whakaaro o te tangata. Ko te tikanga ehara i te mea i whakairotia ki te kōhatu, engari e taea ana te neke i ngā pae o te tikanga kia pai ai te whakahaere a te tangata i āna mahi, kia tau ai te rangimārie, kia mau tonu ai te mana o te tangata. E whakapae ana ahau e kūmea ana e ētahi kaumātua ētahi tikanga kia hāngai ki tā rātau i whakaaro ai kai te tika, kai te hē rānei. Mā ētahi tauira e mārama ai te kaipānui ki te kaupapa me te whakaaro o te kaituhi.

text: He pakiriha Kua tae ki te wā hai kōrero i ētahi tikanga marae, ētahi tikanga kawa e kawa ana ki tā te mātanga titiro. Tērā ētahi tikanga e whakamahia ana e ngā kaumātua o nāianei e uru atu ana ki te pae o rorirori, ka mutu, e ai ki tā ngā kaumātua nei, ko te mutunga mai o te tika ā rātau i whakatau ai. Nā hei whakarāpopoto i te whakaaro whānui, i ētahi wā kāore ko te mea e tika ana ki ō tātau whakaaro te huarahi pai. Ko tā te tikanga he kimi i te huarahi e tika ana, kia kore ai e whara ngā whakaaro o te tangata. Ko te tikanga ehara i te mea i whakairotia ki te kōhatu, engari e taea ana te neke i ngā pae o te tikanga kia pai ai te whakahaere a te tangata i āna mahi, kia tau ai te rangimārie, kia mau tonu ai te mana o te tangata. E whakapae ana ahau e kūmea ana e ētahi kaumātua ētahi tikanga kia hāngai ki tā rātau i whakaaro ai kai te tika, kai te hē rānei. Mā ētahi tauira e mārama ai te kaipānui ki te kaupapa me te whakaaro o te kaituhi.

year: 2017