title: Analysis of Amino Acids in Mānuka Honey

authors: Lloyd, Alicia Marie

subjects: Amino acid

abstract: Honey, a natural product produced by honeybees, has a complex matrix of sugars proteins, minerals, vitamins, enzymes and free amino acids. Amino acids, obtained from the nectar of plants, account for 50-30 mg/kg of honey, the most abundant of which is proline, a secondary amino acid originating mainly from the haemolymph of bees.
Three methods were investigated for the analysis of amino acids in honey. The first used HPLC-UV with pre-column derivatisation, the second HPLC-MS with hydrophobic interaction chromatography, and finally HPLC-MS with aTRAQ™ derivatisation.
The HPLC-UV method involves derivatisation of amino acids by OPA-MPA and FMOC. A fully automated injection program analysed seventeen primary amino acids in 19 minutes. Ultimately, the detection by UV had inadequate sensitivity, and the secondary amino acid proline could not be detected. The method was rejected for these main reasons.
ZIC-pHILIC chromatography paired with LC-MS-MS gave high-quality separation of twenty one amino acids, detected using scheduled MRM, in 10 minutes. Amino acid recovery out of vial was low for the majority, this variation originating from sample preparation. In vial loss of amino acids could not be recovered and so investigation into the last method was initiated.
The final method used an aTRAQ™ kit which labels amino acids with a Δ8 reagent for analysis and also provides Δ0 labelled internal standards for comparison. Forty eight amino acids and internal standards can be accurately detected by MRM’s in 18 minutes. Sample preparation was optimised for honey and the method was validated.
The amino acid content of ten honeys were compared to values obtained from Massey University. Small differences in the majority of amino acids were observed. Mānuka and clover honeys from this data set were also compared, it was found that phenylalanine and tyrosine were at much higher concentrations in clover honey.
Seven mānuka honeys stored in different conditions, warm and cold, were analysed. Applied statistical analysis with the hypothesis that the warm honeys would have lower amino acid concentrations than the cold, found this to be true for seventeen amino acids. Glutamine and then lysine were at decreased concentrations after warm storage in the most honey samples.
The amino acid content of honey, analysed by this method can be used to investigate: botanical origin of honey, speed of honey production/harvest, effect on DHA conversion, inaccurate labelling, and sugar syrup addition.

text: Analysis of Amino Acids in Mānuka Honey Honey, a natural product produced by honeybees, has a complex matrix of sugars proteins, minerals, vitamins, enzymes and free amino acids. Amino acids, obtained from the nectar of plants, account for 50-30 mg/kg of honey, the most abundant of which is proline, a secondary amino acid originating mainly from the haemolymph of bees.
Three methods were investigated for the analysis of amino acids in honey. The first used HPLC-UV with pre-column derivatisation, the second HPLC-MS with hydrophobic interaction chromatography, and finally HPLC-MS with aTRAQ™ derivatisation.
The HPLC-UV method involves derivatisation of amino acids by OPA-MPA and FMOC. A fully automated injection program analysed seventeen primary amino acids in 19 minutes. Ultimately, the detection by UV had inadequate sensitivity, and the secondary amino acid proline could not be detected. The method was rejected for these main reasons.
ZIC-pHILIC chromatography paired with LC-MS-MS gave high-quality separation of twenty one amino acids, detected using scheduled MRM, in 10 minutes. Amino acid recovery out of vial was low for the majority, this variation originating from sample preparation. In vial loss of amino acids could not be recovered and so investigation into the last method was initiated.
The final method used an aTRAQ™ kit which labels amino acids with a Δ8 reagent for analysis and also provides Δ0 labelled internal standards for comparison. Forty eight amino acids and internal standards can be accurately detected by MRM’s in 18 minutes. Sample preparation was optimised for honey and the method was validated.
The amino acid content of ten honeys were compared to values obtained from Massey University. Small differences in the majority of amino acids were observed. Mānuka and clover honeys from this data set were also compared, it was found that phenylalanine and tyrosine were at much higher concentrations in clover honey.
Seven mānuka honeys stored in different conditions, warm and cold, were analysed. Applied statistical analysis with the hypothesis that the warm honeys would have lower amino acid concentrations than the cold, found this to be true for seventeen amino acids. Glutamine and then lysine were at decreased concentrations after warm storage in the most honey samples.
The amino acid content of honey, analysed by this method can be used to investigate: botanical origin of honey, speed of honey production/harvest, effect on DHA conversion, inaccurate labelling, and sugar syrup addition.

year: 2015

title: Enzyme activity down to −100°C

authors: Bragger, J.M.

subjects: Alkaline phosphatise

abstract: The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to −97°C and −100°C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below −100°C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.

text: Enzyme activity down to −100°C The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to −97°C and −100°C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below −100°C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.

year: 2000

title: 2016 Subtidal Ecological Survey of Tauranga Harbour and development of benthic health models

authors: Clark, Dana

abstract: This report summarises the results of biological and physical data collected from a broad-scale subtidal survey of Tauranga Harbour conducted between March and May 2016. The survey was designed to understand more fully the role of various anthropogenic stressors on ecosystem health and feed into management of the harbour. Community-based models of ecosystem health called Benthic Health Models (BHMs) were developed to assess ecosystem health in response to mud and metal loading. This is the first comprehensive quantitative survey of Tauranga Harbour’s subtidal environment since 1990/91. 
The research was conducted as part of the Oranga Taiao Oranga Tangata (OTOT) programme. The wider research project aims to provide knowledge and toolsets to support co-management of estuaries. The three phases of this programme include 1) gathering Mātauranga Māori (a body of knowledge of Māori experience in the area) from local iwi/hapū, 2) consolidating the ecological knowledge of the Tauranga Harbour and providing modelling and indicators of estuarine ecosystem health, resilience and functioning, and 3) creating an Integrative Spatial Planning Tool (ISPT) that can help inform decision making in the harbour. The subtidal survey fits into the latter two research objectives, as the information collected will be used to develop indicators of ecosystem health, which will be used as components of the ISPT. 
Water, sediment and benthic macrofauna samples were collected from 45 subtidal sites across the harbour. Subtidal sediments were predominantly sandy with low levels of nutrients and metal loading. Upper reaches of channels tended to have higher mud, organic content and nutrient concentrations than sites closer to the main channels. Maximum subtidal sediment metal concentrations were well below guideline values and all metals, except lead, were less than median national values for intertidal estuarine sites. Highest metal concentrations were in the urbanised southern harbour or in areas of high mud deposition. 
Many of the numerically dominant macrofaunal taxa were present in both the 1990/91 and 2016 subtidal surveys, with pipi the most abundant bivalve in both surveys. Although the 2016 survey was not designed to quantitatively measure large bivalves, compared with the 1990/91 survey, fewer scallops and horse mussels were observed. The apparent decline in these species is concerning as large bivalves stabilise the sediment and provide complex physical structure to soft sediment habitats, providing predation refuges and settlement substrate for epifauna. The invasive Asian date mussel, on the other hand, has also become common in the harbour. 
Mud and metals were identified as key variables affecting the ecology of the harbour. To determine how environmental gradients in these variables affected ecosystem health, two Benthic Health Models (BHMs) were developed based on variability in community structure, one for each environmental gradient. The BHM approach is a useful management tool that can be used to determine the relative health of benthic communities at a single point in time, or track sites over time to assess whether communities are moving towards a more healthy or unhealthy state. It has been found to be more sensitive to changing ecosystem health than simple univariate community measures because it preserves more information about the community. 
Most sites were ranked in the lower BHM groups, suggesting Tauranga Harbour had fairly healthy subtidal communities with regard to mud and metal impacts. This was supported by values from other biotic indices, which indicated that all subtidal sites in Tauranga would be classified as having ‘good’ ecological status. Sites identified as most impacted by elevated mud and metals were generally located in the upper reaches of estuaries or near the urbanised southern portion of the harbour. In terms of mud, this to some extent reflects the natural progression of an estuary from land to sea; however, the rates of accumulation of sediments have been accelerated because of anthropogenic land-based activities. Although currents and mud were highly coupled in Tauranga’s subtidal environment, the Mud BHM could draw out community responses that were only associated with mud.

text: 2016 Subtidal Ecological Survey of Tauranga Harbour and development of benthic health models This report summarises the results of biological and physical data collected from a broad-scale subtidal survey of Tauranga Harbour conducted between March and May 2016. The survey was designed to understand more fully the role of various anthropogenic stressors on ecosystem health and feed into management of the harbour. Community-based models of ecosystem health called Benthic Health Models (BHMs) were developed to assess ecosystem health in response to mud and metal loading. This is the first comprehensive quantitative survey of Tauranga Harbour’s subtidal environment since 1990/91. 
The research was conducted as part of the Oranga Taiao Oranga Tangata (OTOT) programme. The wider research project aims to provide knowledge and toolsets to support co-management of estuaries. The three phases of this programme include 1) gathering Mātauranga Māori (a body of knowledge of Māori experience in the area) from local iwi/hapū, 2) consolidating the ecological knowledge of the Tauranga Harbour and providing modelling and indicators of estuarine ecosystem health, resilience and functioning, and 3) creating an Integrative Spatial Planning Tool (ISPT) that can help inform decision making in the harbour. The subtidal survey fits into the latter two research objectives, as the information collected will be used to develop indicators of ecosystem health, which will be used as components of the ISPT. 
Water, sediment and benthic macrofauna samples were collected from 45 subtidal sites across the harbour. Subtidal sediments were predominantly sandy with low levels of nutrients and metal loading. Upper reaches of channels tended to have higher mud, organic content and nutrient concentrations than sites closer to the main channels. Maximum subtidal sediment metal concentrations were well below guideline values and all metals, except lead, were less than median national values for intertidal estuarine sites. Highest metal concentrations were in the urbanised southern harbour or in areas of high mud deposition. 
Many of the numerically dominant macrofaunal taxa were present in both the 1990/91 and 2016 subtidal surveys, with pipi the most abundant bivalve in both surveys. Although the 2016 survey was not designed to quantitatively measure large bivalves, compared with the 1990/91 survey, fewer scallops and horse mussels were observed. The apparent decline in these species is concerning as large bivalves stabilise the sediment and provide complex physical structure to soft sediment habitats, providing predation refuges and settlement substrate for epifauna. The invasive Asian date mussel, on the other hand, has also become common in the harbour. 
Mud and metals were identified as key variables affecting the ecology of the harbour. To determine how environmental gradients in these variables affected ecosystem health, two Benthic Health Models (BHMs) were developed based on variability in community structure, one for each environmental gradient. The BHM approach is a useful management tool that can be used to determine the relative health of benthic communities at a single point in time, or track sites over time to assess whether communities are moving towards a more healthy or unhealthy state. It has been found to be more sensitive to changing ecosystem health than simple univariate community measures because it preserves more information about the community. 
Most sites were ranked in the lower BHM groups, suggesting Tauranga Harbour had fairly healthy subtidal communities with regard to mud and metal impacts. This was supported by values from other biotic indices, which indicated that all subtidal sites in Tauranga would be classified as having ‘good’ ecological status. Sites identified as most impacted by elevated mud and metals were generally located in the upper reaches of estuaries or near the urbanised southern portion of the harbour. In terms of mud, this to some extent reflects the natural progression of an estuary from land to sea; however, the rates of accumulation of sediments have been accelerated because of anthropogenic land-based activities. Although currents and mud were highly coupled in Tauranga’s subtidal environment, the Mud BHM could draw out community responses that were only associated with mud.

year: 2018