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: Stimulation of TNF-α release in monocytes by honey

authors: Tonks, A.

subjects: honey

abstract: Although evidence exists for the antibacterial effects of honey there is limited objective evidence for direct promotion of healing. We investigated the effect of manuka, pasture and an artificial honey on macrophage function. Reactive oxygen intermediate (ROI) production was assessed by luminol enhanced chemoluminescence and tumour necrosis factor-alpha (TNF-α) release was determined by immunoassay. ROI production was significantly (P<0.001) decreased by pasture honey and manuka honey. TNF-α release was significantly enhanced (P<0.001) in unprimed MM6 cells by manuka and pasture honey but was not altered in primed cells. These results could explain the suggested therapeutic properties of honey in promoting wound healing.

text: Stimulation of TNF-α release in monocytes by honey Although evidence exists for the antibacterial effects of honey there is limited objective evidence for direct promotion of healing. We investigated the effect of manuka, pasture and an artificial honey on macrophage function. Reactive oxygen intermediate (ROI) production was assessed by luminol enhanced chemoluminescence and tumour necrosis factor-alpha (TNF-α) release was determined by immunoassay. ROI production was significantly (P<0.001) decreased by pasture honey and manuka honey. TNF-α release was significantly enhanced (P<0.001) in unprimed MM6 cells by manuka and pasture honey but was not altered in primed cells. These results could explain the suggested therapeutic properties of honey in promoting wound healing.

year: 2001

title: Te kaumoana Poronīhia: Ngā terenga onamata

authors: Tuaupiki, Jackie Whetumarama

subjects: Kōrero nehe

abstract: Tā tēnei tuhinga e whai ake nei, he wānanga i ngā pūtake o te whanaketanga mai o ngā iwi, nāna ngā motu o Poronīhia i whakakāinga, ka utaina te karangatanga ki runga ki a rātou, he iwi Poronīhia. Ko te pātai matua, i ahu mai ngā iwi Poronīhia i whea? Tēnei, ka hahaina ngā whakaaro Māori me ngā whakaaro Tauiwi e pā ana ki te pūtakenga mai o te iwi tāngata ki te ao. I konei, ka kitea te rerekē o te titiro ā-ao a ēnei iwi tāngata.

Ka ū te titiro ki Hawaiki, e tau ana ngā kōrero tuku iho a te ao Māori, ki te ao Poronīhia hoki. E mea ana te Māori, he tūrangawaewae taurikura a Hawaiki, he pā whakawairua hoki. Ka riariakina e au ngā whakapae mō tēnei karangatanga kāinga i mau roa ai i ōna iwi kōrero.

Ka huri rā ki te āta whakatewhatewha i ngā rangahau mō te horanga haeretanga o te iwi tāngata mai i whirika, ki  hia ki te tonga mā rāwhiti, ā, tau atu rā ki roto i ngā whaitua moana o Poronīhia. I konei, kua āta wānangahia e au ngā whakapae huhua mō ngā take i horapa haere ai ēnei iwi ki roto o Poronīhia, ka tahi. Ka rua, ka tirohia ngā tohe e pā ana ki tō rātou āheinga ki te whakatere i ō rātou waka. Ko ēnei kōrero, he whakatuwhera i te pūtea mātauranga mō runga i ngā mahi whakatere waka a ngā tūpuna Poronīhia.

text: Te kaumoana Poronīhia: Ngā terenga onamata Tā tēnei tuhinga e whai ake nei, he wānanga i ngā pūtake o te whanaketanga mai o ngā iwi, nāna ngā motu o Poronīhia i whakakāinga, ka utaina te karangatanga ki runga ki a rātou, he iwi Poronīhia. Ko te pātai matua, i ahu mai ngā iwi Poronīhia i whea? Tēnei, ka hahaina ngā whakaaro Māori me ngā whakaaro Tauiwi e pā ana ki te pūtakenga mai o te iwi tāngata ki te ao. I konei, ka kitea te rerekē o te titiro ā-ao a ēnei iwi tāngata.

Ka ū te titiro ki Hawaiki, e tau ana ngā kōrero tuku iho a te ao Māori, ki te ao Poronīhia hoki. E mea ana te Māori, he tūrangawaewae taurikura a Hawaiki, he pā whakawairua hoki. Ka riariakina e au ngā whakapae mō tēnei karangatanga kāinga i mau roa ai i ōna iwi kōrero.

Ka huri rā ki te āta whakatewhatewha i ngā rangahau mō te horanga haeretanga o te iwi tāngata mai i whirika, ki  hia ki te tonga mā rāwhiti, ā, tau atu rā ki roto i ngā whaitua moana o Poronīhia. I konei, kua āta wānangahia e au ngā whakapae huhua mō ngā take i horapa haere ai ēnei iwi ki roto o Poronīhia, ka tahi. Ka rua, ka tirohia ngā tohe e pā ana ki tō rātou āheinga ki te whakatere i ō rātou waka. Ko ēnei kōrero, he whakatuwhera i te pūtea mātauranga mō runga i ngā mahi whakatere waka a ngā tūpuna Poronīhia.

year: 2017