113 research outputs found
Investigation of extractable materials from biochar
No full textBiochar has been used to improve soil productivity and has been a subject of discussion since 1804. However, research and development of biochar for environmental purposes on a global scale are a recent development. Due to the increase of its uses and interest in biochar as soil amendment, there is a need to understand the intrinsic chemistry of biochar to understand how this might affect its action in the soil.
In this work two principal topics were addressed:
1) Investigation of volatile organic compounds in biochar that has been derived from various biomasses and the effect of different temperatures of pyrolysis
2) Identification of some chemical structures of biochar.
GC-MS analysis identified 60 extractable organic compounds. With respect to pyrolysis temperature, GC-MS results of Green Waste chars and Sucrose chars shows that extractable organic compounds changed their proportions with differing pyrolysis temperatures. MALDI-TOF and high resolution mass spectrometry results suggested that the characteristic ions for biochar that appear in MALDI-TOF spectra with m/z values of 301,317, 413,429 and 453 are plasticizers whereas 685/ 701 are ions, [M+Na] ⁺/ [M+K] ⁺ respectively that are intrinsic to biochar
Characterisation of watersoluble polysaccharides produced during prehydrolysis of pinus radiata
No full textAn aqueous prehydrolysate (or prehydrolysis liquor) was produced during a mild
hot-water prehydrolysis (90 minute ramp to 175 C) of commercial radiata pine
wood chips. Oligosaccharide and polysaccharide material was separated from the
concentrated prehydrolysate using solvent precipitation after most of the noncarbohydrate
material was removed.
These polymeric carbohydrates were fractionated based on charge and molecular
weight by size-exclusion chromatography (SEC). The fractions were each
analysed by a number of methods including MALDI-ToF mass spectrometry, and
NMR. A number of different types of carbohydrate polymer structures were
found that were produced due to the partial de-polymerisation of the wood
hemicelluloses during the prehydrolysis process.
The O-acetylated (galacto)glucomannans were the most extensively characterised.
These partially-acetylated hexose-based polymers were the main type found and
accounted for approximately 54% by mass of the polymeric carbohydrates. Most
appeared to contained between 5 and 79 hexose units with differing degrees of
acetylation. The average mol ratio of components in these polymers was
calculated to be approximately 3.7 : 1.3 : 1 : 0.2 (D-mannosyl : acetyl : D-glucosyl
: D-galactosyl). They had a structure consistent with a linear backbone of β-1,4-
linked D-mannopyranosyl and β-1,4-linked D-glucopyranosyl units with acetyl
groups attached at C-2 and C-3 positions of some D-mannopyranosyl units. The
terminal D-galactopyranosyl units were likely to be attached at 1,4,6-linked Dmannopyranosyl
branch points. Of the neutral (non-anionic) polysaccharides, this
type was most prevalent in the higher molecular weight fractions.
Anionic pentose-based polymers with a backbone of β-1,4-linked D-xylopyranosyl
units were also characterised. Identified as (arabino)glucuronoxylans, they
featured uronic acid groups consistent with 4-O-methyl-α-D-glucopyranosyluronic
acids attached to the C-2 position of some D-xylopyranosyl units. Smaller
amounts of terminal α-L-arabinofuranosyl units likely to be attached at β-1,3,4-
linked D-xylopyranosyl branch points were also detected. These polymers
appeared to mostly contain between 5 and 40 pentose units with between 1 and 4
uronic acid groups attached.
The anionic fractions (approximately 30% by mass) also contained large amounts
of D-galactopyranosyl and L-arabinosyl units along with some D-glucuronic and
D-galacturonic acid residues. This suggested the presence of carbohydrates
produced from the partial hydrolysis of arabinogalactans and pectins.
The smaller molecular weight fractions of non-anionic polysaccharides were
enriched in both 1,4-linked D-galactopyranosyl units and non-acetylated hexosebased
polymers that contained between 5 and 30 hexose units; this suggested that
significant amounts 1,4-galactan derived carbohydrates were present. Small
amounts of oligomers containing only pentose units were detected in these smaller
molecular weight fractions along with what appeared to be other uncharged
fragments of the polysaccharide-types that were present in the anionic fractions
Isolation, Characterization and Synthesis of a Phenyl-substituted Pyrrole Isolated from the Flavonoid Fraction of Mānuka Honey
No full textCompound 1, which occurs in the flavonoid fraction of mānuka honey and showed a statistical correlation with the non-peroxide antibacterial activity of the honeys, was extracted from fifteen kilograms of mānuka honey using Amberlite XAD-2 resin and liquid-liquid extraction, and isolated by a combination of Sephadex-LH20 column chromatography and HPLC. Characterization of 1 was achieved by one and 2D- 1H and 13C NMR spectroscopy and GC-MS and 1 was identified as 2-formyl-5-(2-methoxyphenyl)-pyrrole. In addition to 1, two other non-flavonoids were isolated from the flavonoid fraction and their identities confirmed as caffeic acid and p-coumaric acid. Synthesis of 9 (3-hydroxy-1-(2-methoxyphenyl)-3-(oxazol-4-yl) propan-1-one), an intermediate in the route to 1, gave a yield of 67.5% as a pale yellow crystals after crystallization from CH2Cl2/hexane. Synthesis of 1 from 9 only resulted in barely traceable amount of 1. The dominant product after recrystallization from CH2Cl2/hexane was 10 ((E)-1-(2-methoxyphenyl)-3-(oxazol-4-yl)prop-2-en-1-one) which was the dehydrated analogue of 9. The synthesis of 1 was repeated. The product mixture was fractionated on a silica gel column, followed by two cycles of preparative layer chromatography applied to the fractions which contained 1 and yield 0.36 mg of 1 (0.00179 mmol, 0.2%)
Analysis of Nucleosides and Nucleotides in Milk and Infant Formula
No full textNucleotides have been routinely supplemented to infant formulas due to the important roles they play in metabolism and to replicate the higher concentrations typically found in human milk. A method utilising anion exchange solid-phase extraction clean-up and liquid chromatography was developed for the rapid, routine determination of supplemented cytidine 5′ monophosphate, uridine 5′ monophosphate, inosine 5′ monophosphate, guanosine 5′ monophosphate, and adenosine 5′ monophosphate in bovine milk-based infant formula. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92–101% and repeatability of 1.0–2.3%. An extension study demonstrated the expansion in scope to a wider range of different infant formula products including milk protein and hydrolysate-based products, low and high fat products, soy protein-based and elemental products, adult nutritional and infant formulations, in both ready-to-feed and powder forms.
The development of a method to measure the total potentially available nucleosides (TPAN) in human milk has made an important contribution to further understanding the distribution of nucleosides and nucleotides. This method was applied in a lactation study of bovine milk with colostrum and milk samples collected from two herds over the course of the first month post-partum, pooled within each herd by stage of lactation and the TPAN concentrations were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate affinity gel extraction, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the nutritionally available nucleoside pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations.
The TPAN method was subsequently applied to the analysis of mature bovine, caprine, and ovine milk. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN (374.1 µmol dL-1), with lower concentrations in caprine milk (97.4 µmol dL-1) and bovine milk (7.9 µmol dL-1). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest.
A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry was developed. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution, with mass spectrometric detection, and quantitation by stable isotope labelled internal standard technique. A single laboratory validation study was performed with recoveries of 80.1–112.9% and repeatability relative standard deviations of 1.9–7.2%. The method was validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysate-based infant formula
Identification of the floral source of New Zealand honeys
No full textDepending on the nectar source, honey is either unifloral (derived mostly from one plant type), or polyfloral (derived from multiple plant types). Unifloral honey has characteristic sensory properties, and is therefore of greater commercial value. Currently, identification of floral source involves pollen counting, a specialised and labour intensive process. The current research was aimed at developing an alternative, rapid, chemistry-based method of floral identification. The aroma of honey depends on volatile compounds present; these may be derived from the plant from which nectar was taken. Therefore by identifying volatiles in honey it could be possible to identify floral source. Solid-phase microextraction (SPME) is a technique that is useful for the headspace analysis of volatile compounds; when coupled with GC-MS it provides a powerful tool for fingerprinting volatiles in honey. GC-MS chromatograms of ten New Zealand unifloral honey types were obtained after headspace SPME extraction. Statistical analysis of the GC-MS chromatographic data was used to discriminate between floral types. Probability plots were used to identify compounds indicative of floral source; this method discriminated between honey types with 90% success. Hierarchical cluster analysis and principal component analysis were used to study the structure of the data. Learning algorithms in Weka (machine-learning software) were used to build models of data to classify honey types. The logistic model tree algorithm classified 89.8% of samples correctly. Such a model has the potential to be used to classify future honey samples, once further samples have been tested to validate the model
A Survey of Dihydroxyacetone in Nectar of Leptospermum Scoparium from Several Regions of New Zealand
No full textMānuka honey has been identified as having non-peroxide antimicrobial activity (NPA). This medical benefit has led to mānuka honey becoming a major export for New Zealand. Hives are currently located in preserved or regenerating mānuka bush. With the growth in the mānuka honey industry, interest has arisen to determine why mānuka trees produce the non-peroxide active honey. Identification of this reason will allow mānuka plantations to be planted that are expected to provide honey with a high level of non-peroxide activity. These plantations will also allow marginal land to become more productive due to mānuka being a resilient plant able to grow under harsh conditions.
It has been shown that the majority of the non-peroxide activity arises from the presence of methylglyoxal (MGO). In fresh honey a minimal amount of MGO was present, but a large amount of dihydroxyacetone (DHA) was found. Previous work has shown that DHA can undergo chemical conversion into MGO under the correct conditions. A similar conversion is postulated to take place in honey, testing suggests that this conversion is a non-enzymatic reaction. Preliminary surveys have been carried out which have identified DHA in the nectar of mānuka owers.
This thesis describes a wider survey of mānuka trees around New Zealand.The trees were sampled in the flowering seasons of 2009 and 2010 between October and January. Flowers were picked and frozen for processing and an aqueous soaking method was developed to extract the DHA and sugar from a pooling of 20 owers. Analyses of the samples were carried out by gas chromatography with flame ionisation detection. This method was further improved to include the nectar extraction and measurement of DHA levels within a single flower.
To allow the DHA to be related back to honey, it was measured in ratio to the total sugar (Tsugar) in the nectar to give the ratio DHA/Tsugar. It was confirmed that DHA/Tsugar measured in the nectar of the mānuka flower does vary within and between the regions surveyed. Suggested causes of within region variation are the age of the tree, micro-environments and possibly genetics. Variation between regions is strongly suggested to be genetically linked. Using the work by Adam set al.(2008, 2009), it was possible to predict honey NPA values based upon the DHA/Tsugar found in the nectar and these values were comparable with the measured NPA of the honey as supplied by beekeepers. Only a poor correlation of DHA/Tsugar was found with the soil components measured by Kiefer(2010); with the leaf oil components measured by Janusch(2010) some correlation was found, when these were correlated across all the sampled regions. When each region was correlated individually, the correlation proved much stronger, suggesting a link, though most likely indirect, to the mānuka oil chemotype. Using these survey results, mānuka trees have been identified for the purpose of breeding and on-going study
Development of a Method for the Quantitative Detection of Honey in Imported Products.
No full textThe carbohydrate composition of Asian honeys was determined using analysis of per-O-trimethylsilylated sugar alditols by GC-FID. This method was established to detect the presence and quantify honey in imported products scheduled for investigation by the Ministry of Agriculture and Fisheries (MAF) Biosecurity, because the import of honey products is regulated. The Asian honeys analysed had a carbohydrate composition within the limits set for honey by the Codex Alimentarius Commission, and had a disaccharide profile similar to honeys from elsewhere in the world. Kojibiose, and peaks corresponding to turanose/nigerose and turanose/maltulose, which are carbohydrates not common in nature, were present in all the honey samples analysed. A reference database of the sugar content of these honeys was created; and the presence of these disaccharides together in imported products under investigation would indicate that the product contains honey. Several samples were found to be adulterated, mostly with sucrose syrup and also with glucose syrup through improper bee-feeding. This method is suitable for detection of the presence of honey in a product being investigated but might encounter problems when quantitation of the honey at low levels of honey addition is required, due to the poor precision of the method. This low precision resulted from the difficulty in getting a homogeneous honey sample and quantifying the small or poorly resolved peaks in the chromatograms. A report on the analysis of actual samples supplied by MAF is presented in Appendix A; quantitation of the monosaccharides, the ratio of glucose:fructose and ratio of disaccharides to monosaccharides could be used to quantitate the amount of honey present and this method is recommended for future use
In vivo utilisation of fructooligosaccharides by sheep faecal bifidobacteria and in vitro antagonistic effects against intestinal pathogens
No full textThe application of pro-, pre, and synbiotics has been studied primarily in humans and some other monogastric animals. Very few studies have been made to determine their effects on the ruminant intestinal microflora. This project tested whether or not four commercial food-grade oligo- and polysaccharides (three FOS products and one polysaccharide control, Arabinogalactan) could modify the hindgut microflora of sheep towards a more salutary community in which the health-promoting bacterial groups bifidobacteria and lactobacilli predominate, whereas the potential intestinal pathogens and putrefactive bacteria E. coli and sulphite reducing clostridia are suppressed.
A fructo-6-phosphate phosphokatolase (F6PPK) enzyme-based identification protocol was developed and optimised for identifying and large-scale screening of presumptive bifidobacteria isolates from gut contents or faecal samples.
An in vivo experiment was then carried out to determine the bifidogenic effect (promotion of bifidobacteria by prebiotics) and the associated antimicrobial effect (suppression of potential pathogens due to the increase in the populations of bifidobacteria) of the four oligo- and polysaccharides on sheep hindgut microflora. Twelve fistulated sheep were managed in a balanced, two Latin square, cross-overdesign experiment, which was run in 5 consecutive periods, with each of 5 treatments (Arabinogalactan, Fibruline, Raftilose, Yacon, and an "acidified saline" carbohydrate-free control) administered to two sheep in each period. Each period consisted of a 1 week of stabilisation to the pelleted diet (no oligo- and polysaccharides), followed by 14 days of daily abomasal supplementation of oligo and polysaccharide/acidified saline, followed by about 12 days of normal pelleted diet. In each period, sheep faecal bifidobacteria, lactobacilli, E. coli/ Enterobacteriaceae, sulphite reducing clostridia, and total anaerobes were enumerated on the day -4 of the "stabilisation" period; days 3 & 9 of the "treatment supplementation" period, and days 15, 16, 19 & 26 after cessation of infusions. Raftilose, Yacon, and Fibruline all exerted significant bifidogenic effects on sheep faecal bifidobacteria after 9 days of daily dosing. Raftilose produced the greatest stimulation of bifidobacteria, reaching counts of approximately 107 CFU/g of faeces. No significant changes in the populations of bifidobacteria were observed in Arabinogalactan-treated sheep. Raftilose and Yacon significantly increased the number of lactobacilli, reaching approximately 107 CFU/g of faeces, after 9 days of daily dosing. With Fibruline, the lactobacilli increase after 9 days of administration was not significant. Arabinogalactan did not elevate the populations of lactobacilli. All four carbohydrate treatments significantly increased the number of total anaerobes to approximately 107 to 108 CFU/g of faeces after 9 days of daily dosing. Supplementation of the test oligo- and polysaccharides had no significant effect on the other determined groups of gut microflora: sulphite reducing clostridia and E. coli/Enterobacteriaceae. There were no significant changes in sheep faecal pH and dry matter content with the four treatments.
Further in vitro antagonistic experiments were carried out to determine whether or not the isolated sheep faecal bifidobacteria inhibited the growth of potential intestinal pathogens in fermentation broth containing the bifidogenic FOS. One hundred and seventeen bifidobacterial isolates from sheep faeces, were screened for their capacity to utilise different oligo- and polysaccharides. Eighteen of these, with strong fermentation patterns, were selected for further study. In the first preliminary experiment, the 18 isolates plus 2 reference cultures of bifidobacteria were divided into 5 groups of 4 strains each. The 5 groups of bifidobacteria were compared for their in vitro antagonistic activities against E. coli in Peptone Yeast Extract broth containing Yacon, Raftilose, or Fibruline as primary carbon sources. Two groups, exerting 100% antagonistic effects against E. coli after 48-hour anaerobic co-culture at 37°C, were selected for further examination. The eight individual strains in these two groups were tested individually for their antagonistic activities against E. coli in PY broth containing Yacon or Raftilose. Fibruline was eliminated due to its low antagonistic activity by bifidobacteria. Six isolates, showed 100% inhibitory effects against E. coli, which made them particularly promising for use as probiotics. The pH of the fermented broths showed a clear negative correlation with the rank transformed or angular transformed inhibition rate of E. coli. One bifidobacterial strain, P5-Po4-37, was subsequently investigated for its in vitro antagonistic activity against E. coli by determination of bacterial growth kinetics over 60 hours anaerobic incubation at 37°C in PY broth containing Raftilose or Yacon. In this experiment, two different concentrations of bifidobacteria, 107 to 108 CFU/mL and 103 to 104 CFU/mL, were incubated with 104 to 105 CFU/mL of E. coli. After 30 and 48-60 hours of incubation, the growth of E. coli was completely inhibited by both the higher and lower concentrations of bifidobacteria, respectively. The presence of E. coli did not affect the growth of bifidobacteria. With the higher inoculum level, the populations of bifidobacteria increased by only approximately 1.30 log10 cycles; whereas with the lower inoculum level, the populations of bifidobacteria increased by approximately 5.62 log10 cycles, to attain the same maximum viable counts at approximately 109 CFU/mL. Fermentation products were analysed in Raftilose containing PY fermented broth. The inhibitory activity of strain P5-Po4-37 was associated with the production of acetic and lactic acids. Strain P5-Po4-37 also exerted strong antagonistic activities against Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella Dublin, and Salmonella Menston. These findings indicated that Yacon and Raftilose potentiate an organic acid mediated inhibitory action of bifidobacterial strain P5-Po4-37 against the test potential intestinal pathogens. To date, this demonstration of inhibitory activities has been convincingly made only in in vitro studies. The combination of Yacon or Raftilose and bifidobacterial strain P5-Po4-37 may exert a promising synbiotic effect on sheep hindgut microflora, which will be investigated in vivo in the near future
Development of a Method for Trace Analysis of Dioctyl Sodium Sulfosuccinate by Liquid Chromatography Mass Spectrometry and its Application to Samples from the MV Rena Incident
No full textThe grounding of the MV Rena off the coast of Tauranga, New Zealand in 2011 prompted the application of Corexit® oil spill dispersants in an attempt to mitigate the impact of the spilled oil to coastal ecosystems. A quantitative method was developed employing sonication assisted extraction from beach sand followed by sample clean up by solid phase extraction and analysis by liquid chromatography tandem mass spectrometry for the analysis of Corexit® component dioctyl sodium sulfosuccinate (DOSS) at trace levels. The chromatographic method included the use of a C8 stationary phase with a gradient elution and quantitation by an internal standard approach based on mass spectrometer instrument response. Validation studies gave recoveries of 72 ± 1.7% with an accuracy of 94%. Calibration curves were shown to have a linear range of 0 – 200 μg.L⁻¹. Application of the method to the available environmental samples showed no presence of DOSS.
An attempt was made to apply the instrumental method to heavy fuel oil (HFO) tar-balls and oiled sand samples. Multiple extraction techniques were trialled including liquid-liquid extraction, sonication assisted extraction and anion exchange chromatography with recovery experiments for each method being carried out. Analysis of the extracts showed that quantitative recovery of DOSS had not been achieved for any of the methods investigated.
The difficulties associated with extracting DOSS from HFO tar-balls and oil sands suggest that the application of DOSS to surface oil slicks results in the preferential partitioning of DOSS into HFO. This has implications with respect to the distribution of DOSS in the environment following application, subsequent environmental monitoring and the degradation of HFO components by microbial communities
Quantification of Nitro-toxins in Karaka (Corynocarpus laevigatus) Drupes
No full textThe karaka (Corynocarpus laevigatus) is a tree native to New Zealand (NZ), found throughout the North and South Islands. Traditionally, many parts of the karaka were used by Māori, however the parts of the karaka of most interest are the berry and nut as they were valuable food sources, in particular the nut. Karaka was known to be a toxin-bearing food, however implementation of a traditional baking and soaking process meant that the nuts were left in states fit for consumption, more often than not. The nuts also had the benefit of being able to be stored for future use when other food sources were short in supply. Karaka was readily consumed up until the 1950s, after this consumption rates decreased rapidly, after the toxic effects were associated with the incorrect preparation of the nuts.
Studies on karaka have revealed that the toxicity of the nuts primarily arises from various nitropropanoyl glucopyranoses (NPGs), twelve of which have been detected in karaka. The quantity of these NPGs has been infrequently studied and it is often only one of the NPGs-karakin which is quantified, although it is believed the toxicity in nuts arises collectively from all of the NPGs. As a result, there is the need to be able to quantify the NPGs in karaka and look at potential toxin removal techniques. If toxin removal can be accomplished, there is the potential for karaka to be a marketable ‘traditional’ or ‘Kiwi’ food item. Additionally, the toxins removed as a by-product may prove to have their own potential commercial applications, for example as insecticides or repellents.
Since all the NPGs are assumed to be toxic, a method was developed to quantify the total toxicity in the nuts. The method involves the release of NPA via acid hydrolysis of the nitropropanoyl ester groups of the NPGs. The NPA is then able to be measured and subsequently quantified using HPLC. The method incorporates a correction factor (CF) that was determined to give the original NPA content at a time of zero (NPA0), accounting for the unavoidable loss of NPA that arises from the hydrolysis method.
The average quantity of NPA in karaka nuts was found to range from 50.25 to 138.62 g kg-1 (dry weight) which is equivalent to 5.0 to 13.9%. These are much higher percentages than any previously reported because the method used in this study measured total NPA. Earlier methods measured a limited range of NPGs (often a single NPG) unhydrolysed and could have missed NPA arising from other compounds that contribute to total toxicity. Additionally, the concentrations of NPA in karaka nuts were influenced by a number of factors including intra and inter tree variation, storage conditions and ripeness. Although quantification was focused on determining NPA content in nuts, a test conducted also showed NPA to be present in increasingly lower levels in the berry flesh, shell and pellicle respectively.
Nuts were subjected to various potential industrial processing techniques, treatment times and conditions, in order to determine their efficacy for toxin removal. Treatments included boiling, microwave cooking, soxhlet extraction, oven roasting, autoclaving and cold-water treatments. The efficiency of each treatment varied considerably, with both heat and water proving to be beneficial in toxin removal. However, treatments involving water were found to be more effective than heat treatments alone. Out of all treatment types, times and conditions tested, none were found to leave nuts with a NPA concentration lower than the estimated safe level of daily consumption for a 70 kg adult (< 1.75 mg). Additionally, only some of the treatments resulted in nuts being left in visually appealing states. At present, toxin removal by a single process in order to render karaka nuts safe to consume appears to be an impractical activity. However, if treatment protocols were combined and modified, there is the potential for the development of an effective detoxification process. Furthermore, tests on the treatment solutions revealed that NPA can be obtained in solution as a by-product of nut treatment; with cold-water proving to be more effective than treatments that also used heat. Obtaining NPA as a by-product of toxin removal in nuts appears to be a feasible option for a natural source of NPA
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