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Item The dynamics of drug resistance evolution and diagnosis in Mycobacterium tuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics/Genomics, Massey University, Manawatu, New Zealand(Massey University, 2024-07-30) Fong, Yang (Richard)Tuberculosis (TB) remains a critical global health challenge with over 10.4 million new cases annually, complicated by rising antimicrobial resistance (AMR) threatening to surpass cancer mortality by 2050. This PhD thesis establishes a systematic diagnostic framework addressing AMR challenges through progressive research from fundamental microbiome characterization to innovative diagnostic applications in resource-limited settings like Myanmar. The "Microbiome Dataset from the Upper Respiratory Tract of Patients Living with HIV, HIV/TB and TB from Myanmar" establishes the foundational understanding of microbial community structures in complex clinical presentations (n=309 isolates). This microbiome characterization reveals critical signatures that directly inform direct sequencing strategies for enhanced MTBC detection in polymicrobial environments, addressing a fundamental challenge in AMR detection. Next, the "Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar" validates and expands these microbiome-informed approaches through comprehensive whole genome sequencing surveillance, establishing genotype-phenotype correlations that achieve 97.8% concordance with phenotypic testing. This genomic profiling directly addresses AMR surveillance gaps by enabling rapid resistance prediction. Subsequently by "Unveiling Hr-TB in Myanmar: Comprehensive Genotypic and Phenotypic Insights for Improved TB Management" demonstrates targeted application of microbiome-informed diagnostic approaches to isoniazid mono-resistant TB, a clinically critical AMR variant frequently missed by conventional methods. The integrated microbiome-genomic approach enhances MTBC detection accuracy by 23% compared to standard methods, reducing diagnostic time from weeks to under one week. Future perspectives translate these discoveries into field-deployable MDA primer systems for point-of-care AMR detection using portable MinION sequencing technology. This systematic progression from microbiome foundation to diagnostic innovation establishes a replicable technological blueprint for next-generation TB AMR diagnostics, supporting Myanmar's National TB Control Program while providing a framework for global TB elimination efforts Keywords: Mycobacterium tuberculosis (MTB), Tuberculosis (TB), Antimicrobial Resistance (AMR), Isoniazid Mono-Resistant (Hr-TB), Drug-Resistant Tuberculosis (DR-TB), Whole-genome sequencing (WGS), Resistance associated Mutations, Epidemiology, Surveillance, Rapid Diagnosis, Microbiome, Yangon, Myanmar.Item Control of citrinin and pigments produced by Monascus purpureus during the fermentation of red fermented rice : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu Campus, New Zealand(Massey University, 2024) Abdul Halim, FarawahidaMonascus spp. is a fungal starter used to produce red fermented rice (RFR). This product has some health benefits and contains pigments that can act as natural colour and flavouring agents. However, Monascus spp. can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. In this current research, Coconut Cream Agar (CCA) was developed as a simple and rapid semiquantitative method to screen CIT–producing Monascus spp. isolates from RFR. Two Monascus purpureus isolates, MF1 and MS1, were isolated. These isolates were identified based on the macroscopic and microscopic observations, and deoxyribonucleic acid (DNA) sequencing [internal transcribed spacer (ITS) and beta–tubulin (β–tubulin) genes]. Further analysis showed that these isolates contained polyketide synthase (pksCT) and ctnA genes, which are CIT biosynthesis genes. These isolates exhibited fluorescence on CCA and were confirmed as CIT producers by Ultra–high performance liquid chromatography with a fluorescence detector (UHPLC–FLD). A toxicity test was conducted using Artemia salina to determine the toxicity of CIT. The results showed that LC50 was 66 µg/mL. The fungal growth, CIT, pigments production, and pH of M. purpureus isolates were characterized on CCA for 30 days. Decreasing CIT levels were observed after incubation of MF1 and MS1 on CCA for 8 and 7 days, respectively. The pigments increased during this incubation time. There were similar trends for CIT and pigments observed during the production of RFR. CIT increased to a maximum level after 5 days of incubation and pigments increased from 5–9 days. There appears to be a relationship between pigments production and CIT levels during the growth of M. purpureus. Mixing CIT and pigments extracted from MF1 and MS1 resulted in a reduction of CIT by 26–68% and 16–45%, respectively. The results on specific pigments and their effect on CIT showed that ankaflavin, monascin, monascorubrin, rubropunctatin, and monascorubramine significantly reduced CIT, with the highest reduction produced by ankaflavin. The findings of this study suggest pigments production could be optimized to control CIT levels in Monascus–fermented products.Item Modulation of enteric neural activity and its influence on brain function and behaviour : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatu, New Zealand(Massey University, 2024) Parkar, Nabil JamilThe gut-brain axis (GBA) facilitates bidirectional communication between the enteric nervous system (ENS) of the gastrointestinal (GI) tract and the central nervous system (CNS). The location of the ENS along the GI tract enables it to serve as a relay station along the GBA. A key regulator of the GBA is the diverse population of microbial communities inhabiting the GI tract, known as the gut microbiota. Due to its proximity to the ENS, gut microbes significantly influence ENS functions, such as gut motility, and also impact brain function and behavior. A diverse and healthy gut microbiota is crucial for normal GI physiology and mental health. Understanding the physiological host factors that influence and control the gut microbiota is essential for grasping its variability in health and states of dysbiosis. Movement of luminal content along the GI tract, primarily driven by rhythmic contractions of GI smooth muscles, affects gut microbiota growth and population dynamics. This research involved a series of ex vivo and behavioral experiments in rodents to better understand ENS control of gut motility and its impact on anxiety-related behavior. Initially, the effect of a specific pharmacological agent on colonic motility patterns was evaluated using ex vivo techniques. Observations from this study provided fundamental insights into ENS function and its regulation of colonic motility, laying the foundation for further research on how altered colonic motility via ENS manipulation affects gut microbiota composition and anxiety-related behavior. The second study investigated whether pharmacological modulation of the ENS, resulting in reduced colonic motility, affected the gut microbiota. Results revealed significant changes in gut microbiota composition, including decreased abundance of certain bacterial species and alterations in community structure. The final study aimed to understand the relationship between ENS manipulation, brain function, and behavior by inducing changes in gut motility. Anxiety-related behavior was assessed in rats using open field and elevated plus maze tests, focusing on those exposed to a pharmacological agent that slowed colonic motility via specific ENS receptors. To determine if behavior changes involved specific neural pathways, brain gene expression in key regions was studied. Additionally, the potential relationship between gut microbiota and brain function was explored, assessing if ENS modulation and behavioral effects correlated with changes in gene expression and microbiota profiles in the large intestine. Findings indicated that ENS modulation altered anxiety-related behavior in a sex-specific manner, with female rats showing increased anxiety and corresponding changes in brain and proximal colon gene expression compared to males. This study highlighted sexually dimorphic gut-brain communication and suggested multiple genes/pathways may influence anxiety-related behavior in females. This comprehensive exploration through three interrelated studies has provided new insights into the regional specificity of ENS receptors in regulating colonic motility, the impact of slowed gut transit on microbiota composition, and the physiological consequences of ENS modulation on brain function and anxiety, with associated sex differences. A combined analysis of these findings discusses their implications for understanding the ENS as a key player in regulating the gut-brain axis.Item An observational study of farmer-reported clinical mastitis in New Zealand dairy ewes.(Taylor and Francis Group, 2024-07-01) Chambers G; Laven R; Grinberg A; Ridler A; Velathanthiri NAIMS: To describe the incidence, aetiology, treatment, and outcomes of farmer-reported clinical mastitis on New Zealand dairy sheep farms. METHODS: A prospective cohort study was conducted on 20 spring-lambing New Zealand sheep milking farms over the 2022-2023 season. Clinical mastitis was defined as a change in the appearance of milk and/or signs of inflammation in the gland. Farmers were required to report all cases of clinical mastitis and collect information on affected ewes' demographics, clinical features, treatments (where applicable), and outcomes. Milk samples from mastitic glands were submitted for microbiological culture and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF). RESULTS: Partial or complete clinical mastitis data were available for 236 cases from 221 ewes on 18/20 study farms. Clinical mastitis was diagnosed in 0-6% of ewes at the farm level, with an overall incidence of 1.8 (95% CI = 1.0-3.2)% using the study data, or 2.3 (95% CI = 1.6-3.3)% using the study data and farmer estimates that included unreported cases. Cases occurred mostly in early lactation, with 59% detected during the lambing period (August-October), at a median of 7 (IQR 3, 40) days in milk. The majority of cases featured clots in the milk (59%), swelling (55%), and unevenness (71%) of the glands. Pyrexia (rectal temperature ≥ 40.0°C) was diagnosed in 25% of cases and depression (lethargy, inappetence, or inability to stand) in 26% of cases. Treatment was given to 46% of cases, with tylosin being the most commonly used treatment (50% of treated cases). The most common outcome was immediate drying off to be culled without treatment (32%), followed by still milking and recovered but with lasting problems (25%). Nearly half of all the milk samples submitted were culture negative. Streptococcus uberis (14%), non-aureus staphylococci (12%), and Staphylococcus aureus (11%) were the most common isolates, found on 12, 8 and 8 of the 16 farms with microbiological data, respectively. CONCLUSIONS: Clinical mastitis affected up to 6% of ewes at the farm level. Systemic signs were observed in one quarter of affected ewes, suggesting a role for supportive treatment. Clinical mastitis can be severe and challenging to fully resolve in New Zealand dairy sheep. CLINICAL RELEVANCE: This is the first systematic study of clinical mastitis in New Zealand dairy ewes. It provides baseline information specific to New Zealand conditions for farmers, veterinarians, and other advisors to guide the management of mastitis for the relatively new dairy sheep industry in New Zealand.Item Genomics and eDNA provide a holistic understanding of microbial communities and zoonoses in Aotearoa's waters : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Palmerston North, New Zealand(Massey University, 2024-03-18) Davis, Meredith TaylorIn Aotearoa, water quality and freshwater ecosystem health is declining. Much of that decline has been blamed on livestock farming and is quantified by using invertebrate communities or Escherichia coli levels. However, the wider microbial community, particularly archaea and eukaryotes, have been overlooked in the current ecosystem health measures. Some of the more well-known impacts of microorganisms on waterways are sporadically measured (e.g., algae blooms, drinking water contamination by faeces, and shellfish toxins) but the drivers of their community structure, both individually and as a whole, in Aotearoa is poorly understood. This thesis is a start at rectifying this knowledge gap. The studies in this thesis were approached with a One Health perspective and provide a quantitative, holistic analysis of microbial communities by investigating three significant challenges facing Aotearoa’s freshwater ecosystems. Those challenges - the spread of waterborne disease, eutrophication, and microbial biogeochemical cycling - were investigated using microbiological cultures and environmental DNA, analysed with metagenomic and other molecular methods. I was able to determine that targeted testing for genetic loci associated with antimicrobial resistance and Shiga toxin-producing E. coli virulence was a useful in monitoring three Canterbury waterways for human health. Furthermore, enteropathogenic human and bovine strains of E. coli appeared unresponsive to in-stream nitrate-nitrogen concentrations of 0, 1, and 3 mg/L and native in-stream biota in microcosms. However, environmentally sourced E.coli imported as part of the in-stream biota survived longer in NO3-N concentrations of 1 and 3 mg /L than at 0 mg/L. Microorganism groups (e.g., archaea, bacteria, and microbial eukaryotes) responded to different environmental, spatio-temporal, and physico-chemical drivers depending on taxonomic level. As a group, lotic pressures and dispersal outweighed other drivers in community structuring. Archaeal communities were highly correlated with Austral season and the most abundant functional groups reflected a likely response to common agricultural pollutants found the Waiotahe catchment and in many rural rivers across Aotearoa (e.g., nitrogen pollution and livestock waste/effluents). The drivers commonly associated with bacterial survival (e.g., conductivity and temperature) were less important than dispersal and lotic pressures, particularly at lower taxonomic levels. Cultured E. coli concentrations from sediment and/or the water column were poorly indicators of Campylobacter, Enterobacter, and Enterococcus relative abundances. Additionally, neither Enterobacter nor Enterococcus relative abundances were correlated with E. coli/E. cloacae group concentrations or Campylobacter relative abundances. These findings have important implications for water quality monitoring and recreational human health risk assessments in Aotearoa. Currently, microbiological water testing is limited to bathing season (i.e., late spring to early autumn) and to culturing either Enterococcus in saline/brackish water or E. coli in freshwater. Effective water quality monitoring must include both water and benthic substrates to accurately portray the entire riverscape. Genetic loci associated with zoonotic human pathogens are present in some of Aotearoa’s waterways and they are likely a result of catchment land use, livestock farming, and effluent contamination. Additionally, genetic loci can be detected with collection methods similar to those employed for current water quality monitoring using Escherichia coli and some molecular methods are more specific (i.e., not proxies). Metagenomic methods allowed for the discernment of microbial communities and core biomes from genetic information extracted from environmental samples. Microbial communities were affected by many different in-stream conditions; however, dispersal and the pressures associated with lotic systems proved to be more important than adjacent land use, precipitation levels, or season. In contrast, archaeal communities were better explained by season. It is clear that water monitoring in Aotearoa needs an overhaul and to incorporate new technology in a thoughtful and ecologically informed manner. A review of the current methods and new technologies should be undertaken by a multi-disciplinary group of experts in the fields of microbiology, epidemiology, and freshwater and microbial ecology. Community buy in and the inclusion of Māori values and indigenous rights should be at the forefront of any proposed changes to freshwater restoration and conservation.Item Spoilage bacteria in ewe milk and the sheep dairy farm environment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science/Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2023-03) Risson, AlexisMilk spoilage bacteria are capable of impacting the stability, processability, and overall quality of dairy products. These contaminants are highly diverse and can be found at various stages of the dairy production, but the majority originate from the dairy farm environment where they contaminate raw milk. On bovine dairy farms, research has identified several routes of transmission of spoilage microorganisms into raw milk, as well as some of the factors that modulate this process. This knowledge has aided the design, development and implementation of interventions aiming to limit the on-farm contamination of raw milk and thereby improve the microbiological quality of cow dairy products. In contrast, the microbial communities associated with the ovine dairy production have been largely overlooked. This lack of understanding of the ecology and transmission of spoilage bacteria on sheep dairy farms and in dairy products hinders attempts at reducing the farm-borne contamination of ewe milk by spoilage organisms. In this thesis, I set out to identify and characterise the microbial communities associated with the ovine dairy production, with a focus on bacteria capable of impacting ewe milk quality. In the first part, I compiled, developed, and subsequently used an extensive set of culture methods targeting the main types of bacteria implicated in dairy spoilage, namely spore-forming bacteria, and Pseudomonas species. These methods were applied to survey the microbial communities present in the farm environment of dairy ewes that were grazed or housed. For the first time, this approach provides a detailed overview of the ecology of spoilage bacteria found on sheep dairy farms and enables the identification of numerous bacterial species with spoilage potential. The diversity and principal reservoirs of spoilage bacteria were found to vary between the two farms, possibly as a result of their farming practices. In the second part, I performed source-tracking of the contaminants isolated from milking cups using phylogenetic tools such as DNA fingerprinting and whole-genome sequencing. According to this approach, sources of milking cup, and thus possibly raw milk contamination were found to vary between the two farms. In the barn environment, the silage-faeces axis was identified as the main source of spoilage bacteria in the wider farm environment and in milking cups. There was also some contribution, albeit minimal, from bedding materials and animal drinking water as potential intermediaries. In the pasture environment, the transmission of bacteria in the environment was low, but the origin of the contaminants found in milking cups was multiple, dependent on the bacteria considered, and included soil, pasture, animal faeces and drinking water. In addition, the phylogeny of Thermoactinomyces spp., which were found to be particularly abundant in both housing and grazing environments, was studied in more depth using whole-genome sequencing. Finally, I surveyed the microbiological quality of ewe raw milk samples and sheep dairy products produced in New Zealand using the culture methodology developed for the farm studies. This work provided a glimpse of the diversity and abundance of the spoilage microbiota of sheep milk and dairy products produced in New Zealand. It was found that the microbiological quality of sheep dairy products was high, however, the presence of a highly diverse range of spoilage bacteria highlights their potential to impact the quality of a variety of dairy products.Item Host-microbiota interactions underlying functional gastrointestinal disorders and the impact of gold kiwifruit consumption : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Albany, Manawatū, Palmerston North, New Zealand(Massey University, 2023-05-18) Carco, CaterinaThe microbial ecosystem exists in a mutualistic relationship with its host, contributing to a healthy gastrointestinal tract. Growing evidence supports the role of microbial-immune interactions in functional gastrointestinal disorders (FGIDs), including irritable bowel syndrome (IBS). However, these mechanisms are poorly understood. It has been hypothesised that taxonomic and gene abundance in the faecal microbiota and gene expression of peripheral blood mononuclear cells (PBMCs) could discriminate FGID subtypes (functional constipation (FC), IBS constipation, functional diarrhoea (FD), IBS diarrhoea) from each other and healthy subjects (controls). A second hypothesis was that consuming two gold kiwifruit daily for four weeks had a different effect on the microbial composition and gene abundance than psyllium in constipation predominant FGID subjects or controls. A systems biology approach was used to address these hypotheses. Different microbial compositional and gene abundance profiles were associated with constipation and/or diarrhoea, particularly facultative anaerobes and obligate fermenters, and genes related to tyrosine metabolism, secretion systems and micronutrient utilisation. Differentially PBMC expressed immunoglobulin variable domain genes were shared among FGIDs, except for IBS constipation. Increased expression levels of interferon-induced genes and those linked to the complement system and platelet functions characterised the immune signature of functional constipation. Increased expression levels of immunoglobulin variable domain associated with immunoglobulin E/G receptor-mediated pathways characterised the immune signature of IBS diarrhoea and FD. Further analyses showed that computationally selected microbial, immune gene and symptomatic variables were associated with constipation or diarrhoea predominant FGIDs, and that symptoms remain the best way to discriminate among FGIDs or controls than PBMC genes or microbial taxa except for FC, which was best discriminated from other FGIDs or controls by selected PBMC genes. Eggerthella and Bacteroides were the only genera that differed between subjects consuming gold kiwifruit or psyllium or between each intervention compared to pre-intervention levels, regardless of the digestive health status of the subjects. This PhD thesis presents novel insights into the host-microbiota interactions underlying FGIDs and the microbiota responses to daily consumption of two gold kiwifruit over four weeks in constipation predominant FGID subjects. The knowledge generated can be used for future research on food-based treatments supporting gastrointestinal health and comfort.Item Further development of a cell culture based approach to model the diet-derived impacts on the faecal microbiome and potential host health in the domestic dog : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2023) Phimister, FrancisGlobally, diets that promote and optimise health and a ‘healthy microbiome’ are becoming increasingly popular for pets. However, the impacts of novel diet ingredients and formulations on the health of the host and their microbiome require testing to ensure there are no unforseen detrimental effects. However, there are currently a very limited number of canine-specific intestinal cell lines and the capacity to utilise these cells to model host-food interactions is limited. Thus, this doctoral project aimed to develop an in vitro model of the canine intestine, using a previously established canine intestinal epithelial cell (cIEC) line. This could then be used to characterise the canine response to dietary challenges to the gut microbiota. As there is limited research that has assessed the interactions of the gut microbiota with the canine intestine, the initial step of this project was to evaluate the current knowledge of the intestinal inflammatory response to bacterial ligands and diet-derived metabolites (Chapter Two). This literature review indicated that prior to investigating the interactions between bacteria and the canine intestine an evaluation of the canine intestinal response to these challenge compounds was required. Building on the knowledge base established in Chapter Two, key microbes associated with health in the dog required identification. Thus, this thesis provided the first meta-analysis of the available literature on the relationship of dietary nutrients and their impact on the gut microbiota in the dog (Chapter Three). The hypothesis of this meta-analysis was that dietary protein and dietary fats would have singificant impacts on the faecal microbiota of the dog and additionally, that this analysis would reveal bacterial genera associated with these dietary macronutrients.In the meta-analysis the novel discovery was made that despite its low relative abundance,Sharpea was the genera most associated with causing the shifts in microbial profiles in response to changes in both crude protein, and crude fats, thus confirming the hypotheses. Early results indicated that the methods required to further refine the existing cIEC line as an in vitro model of the canine intestine were sub-optimal and required further development. These method developments are detailed in Chapter Four. Experiments in this chapter assessed methods to promote cell growth and differentiation in a cellZscope system, which automatically performed barrier integrity assessments, whilst inside a temperature-controlled incubator. The inclusion of 150 nM hydrocortisonein cell culture media during the initial 48 hours of cellular differentiation, and subsequent removal of hydrocortisone after this period successfully enabled the cIEC to differentiate and form confluent monolayers in the cellZscope system. These methods were intended to be used going forwards in an apically anaerobic system that would more closely resemble conditions seen in vivo and would have allowed the simultaneous culture of the oxygen-requiring cIEC and anaerobic bacteria. Work utilising this model was stopped due to complications that arose from the Covid-19 pandemic, but work conducted and experiments that were planned are explored in Chapter Six. Utilising the refined methods from Chapter Four, the inflammatory response of the cIEC to butyrate and bacterial lipopolysaccharides (LPS) were characterised (Chapter Five). This was performed to address gaps in the literature highlighted in Chapter Two.It was hypothesised that the stimulation with bacterial LPS would cause a pro-inflammatory response, whilst the stimulation with butyrate would cause an anti-inflammatory response. Furthermore, it was also hypothesised that the stimulation of the cIEC with both LPS and butyrate would cause the butyrate to reduce the pro-inflammatory response, and the LPS to reduce the anti-inflammatory response. It was observed that LPS induced a pro-inflammatory response in the cIEC, which butyrate was able to mitigate in most instances. Overall, the methods developed and refined in this project will be able to be utilised in future experiments utilising these cells, such as evaluating new pet food ingredients for beneficial effects and exploring how changes in the gut microbiome impact gut health in the dog. It can take the knowledge established in Chapter Three to further investigate the impacts of the bacterial genera on the health of the dog. Futhermore, it can utilise the immune responses observed in Chapter Five to better understand the relationship between inflammation and diet in the dog. Future work can build on the knowledge discovered and presented in this thesis to fully understand the impact of diet changes on the health of the dog, and further define the microbial profile of the ‘healthy microbiome’ for the dogItem Characterization of mānuka and rosemary oils as antimicrobial and antioxidant agents for meat applications : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2023) Kaur, RamandeepThe usage of chemical preservatives in meat products has been associated with adverse health effects, which is driving consumers' preferences towards natural preservatives. Raw and processed meats have been linked to cancers due to the presence of nitrate/nitrite as a chemical preservative. In recent years, extensive innovative and promising approaches have been exploited to entirely or partially replace synthetic preservatives. Plant-based natural preservatives possessing antioxidant and antimicrobial characteristics can be ideal for food applications. Essential oils are aromatic liquids extracted from different plant parts, such as leaves, bark, roots, and seeds, and are rich sources of bioactive compounds like monoterpenes, sesquiterpenes, and oxygenated sesquiterpenes and phenolic compounds. As per the literature, essential oils possessing antioxidant and antimicrobial characteristics have been reported to decline the rate of oxidative reactions and microbial growth. This study aims to harness the potential of essential oil obtained from the indigenous plant of New Zealand, i.e., mānuka, as a natural antioxidant and antimicrobial agent for meat preservation than chemical preservatives like nitrates/nitrites. As per the available literature, β-triketones are responsible for the antimicrobial characteristics of mānuka oil. We hypothesise that using antioxidants and antimicrobial bioactive compounds of Leptospermum scoparium (mānuka) will improve the shelf life and stability of the meat products. The first research objective characterised and compared the antioxidant and antimicrobial potential of mānuka oil with different triketone contents (5, 25, and 40 %) and kānuka oil with a commonly used natural preservative, i.e., rosemary oil. In chemical composition, kānuka oil possessed higher levels of α-pinene, while rosemary oil exhibited higher amounts of 1,8 cineole and α-pinene as primary compounds. In mānuka oils, the concentration of other compounds decreased as triketone content increased from 5 to 40 %. A comparison of the antioxidant characteristics of these oils was also made with chemical antioxidants, i.e., butylated hydroxytoluene (BHT). It was observed that mānuka oils possess higher antioxidant properties than rosemary and BHT (at both the lowest tested concentrations of 0.1 % and 1 %). In the antimicrobial efficacies assay results, all mānuka oils showed more effectiveness against Listeria monocytogenes and Staphylococcus aureus than Salmonella and Escherichia coli. However, the inhibition effect of rosemary oil was greater against Salmonella and Escherichia coli than mānuka oil (Chapter 3). The minimum inhibitory concentration of all mānuka oils required to inhibit Listeria monocytogenes and Staphylococcus aureus was below 0.04 %, while kānuka and rosemary oil inhibited these microbes at 0.63 and 2.5 %, respectively. On the other hand, a minimum 2.5 % concentration of all oils was needed to inhibit Salmonella and Escherichia coli. These results indicated that mānuka oil can be used as an antimicrobial agent, particularly against tested Gram-positive microbes (at a very concentration of 0.04 %) in meat products, while rosemary oil can be used against all tested microbes at 2.5 %. However, meat constituents such as fats have a significant effect on the efficacies of added bioactive compounds, therefore, it is essential to have insights into the lipophilicity of added essential oils and their bioactive compounds. In the next research experiment, to confirm the lipophilic behaviour of chemical compounds present in mānuka oil, the octanol-water partition coefficient of beta-triketones (leptospermone, isoleptospermone, and flavesone), α-pinene and γ-terpinene were elucidated using shake flask method (Gas chromatography and mass spectrometry) and predicted using EPI software (Chapter 4). High values of the octanol-water partition coefficient of these compounds indicate their more affinity towards the fat than the water. Further, when the concentration of the compounds separated in 3 and 12 % beef-fat and water systems was determined, all compounds showed higher concentrations in water of the low-fat system than in the high-fat. The findings pointed out that essential oils may exert an antioxidant effect in the high-fat system to prevent lipid oxidation; however, their antimicrobial effect may be reduced due to the presence of fat, and higher concentrations of these oils may be needed to achieve an antimicrobial effect against selected microbes. In the third research experiment, selected mānuka and rosemary oils were used as natural antioxidants and antimicrobial agents in low and high-fat meat pastes prepared from commercial-breed and wagyu beef tenderloins, respectively (Chapter 5). These effects were compared against the chemical preservative sodium nitrate and butylated hydroxytoluene during refrigerated storage of meat pastes at 4 °C. In commercial and wagyu beef pastes, a lower number of Listeria monocytogenes and Staphylococcus aureus were observed in mānuka and rosemary oil treatments than in the sodium nitrate and control samples (without added preservative). Rosemary oil also delayed the growth of Salmonella and Escherichia coli more than mānuka oil added and control samples. In terms of oxidative stability, mānuka oil added wagyu beef pastes were more stable and showed the lowest lipid oxidation values than all treatments. In commercial beef samples, no significant difference between essential oils added samples, either mānuka or rosemary oil and control samples was observed. There was a significant change in pH values of all wagyu and commercial beef samples, whilst these changes were greater in untreated samples (controls) than in the essential oils-treated samples. Despite the promising antioxidant and antimicrobial characteristics of essential oils, these are rarely utilised in food products owing to their easy degradation, low water solubility, low stability, and unwanted odour and flavour. The application of essential oils in encapsulated form is an effective and innovative approach to overcome these limitations by covering the core materials (oil droplets) in carrier materials. In addition, it improves stability and provides controlled release and targeted delivery of essential oils in foods. In the next research objective, mānuka and rosemary oils-containing nonentities (nanoemulsions and nanocapsules) made of sodium alginate and whey protein were fabricated and compared for their thermal stability and release characteristics (Chapter 6). The particle size and zeta potential of prepared nanoentities were between 100 -600 nm and -10 to -40 mV, confirming that the obtained nanoemulsions and nanocapsules were stable and in the nano range. The obtained nanoentities were observed to be more thermostable, sustained release profile than the free form of oils while showing a lower in vitro antioxidant effect. The release mechanism of the essential oil from nanoemulsions and nanocapsules was also studied using different mathematical models. The release mechanism of essential oil from nanoemulsions and nanocapsules followed Higuchi’s law, which indicates that the solvent first penetrates the encapsulated matrix and then dissolves the embedded oil droplets through the diffusion process. The delayed or sustained release from encapsulated oil might influence the antioxidant and antimicrobial activity of essential oils in meat pastes. However, a food matrix made up of different constituents can affect the partitioning and release of essential oils from the carrier material, and consequently, their preservative effect may vary according to the meat paste. An improvement in the antioxidant activity of oils after emulsification was observed as nanoemulsions of both oils had the lowest TABRS values in crossbred and wagyu pastes (Chapter 7). Mānuka oil and its nanoentities had more antioxidant effects than rosemary oil. In wagyu pastes, there was a significant difference in nanoemulsions added pastes than the other treatments, while in crossbred pastes, no significant differences were noted between free oils and nanoentities containing beef pastes. Despite the antioxidant efficacies, the antimicrobial activity of free, nanoemuslfisied and nanoencapsulated oils was also determined in the wagyu and crossbred beef pastes during refrigerated storage (4 °C) of two weeks. These antimicrobial effects were compared against controls (without added preservatives) and sodium nitrite-added paste samples. There was a significant increase in microbial counts of all inoculated-paste samples, whilst this increase was lower in preservatives added samples than in the controls. In wagyu and crossbred beef pastes, mānuka oil and its nanoentities delayed the growth of Listeria monocytogenes and Staphylococcus aureus, and mānuka-nanoemulsions exhibited the lowest number of these microbes than all other treatments. However, rosemary oil and its nanoforms effectively inhibited Salmonella and Escherichia coli during refrigerated storage at 4 °C. To better understand the mechanism for the antimicrobial activity of essential oils against selected pathogens, cell viability membrane integrity and the release of intracellular compounds and proteins through fluorescence-based assays were determined. In all these assay results, mānuka and rosemary oils treatment of Listeria monocytogenes, Staphylococcus aureus, Salmonella and Escherichia coli exhibited a decline in cell viability, disrupted cell-wall permeability and enhanced release of intracellular compounds and proteins from cells than the untreated cells. Scanning electron micrographs also confirmed that these mechanisms were responsible for the antibacterial efficacy of mānuka and rosemary oil. To correlate the effect of fat content on varied antimicrobial characteristics of essential oils in meat pastes, the partitioning of essential oils in different phases, such as octanol, beef and water, was determined. Overall, the work showed that mānuka oil has the potential to be used in meat pastes as an antimicrobial agent, especially against tested Gram-positive (Listeria monocytogenes and Staphylococcus aureus). In addition, this oil can be used to completely replace synthetic antioxidants like butylated hydroxytoluene to inhibit lipid oxidation in high-fat meat systems. Due to the lipophilic nature of oils, the fat content of meat systems significantly affects the partitioning of these oils in water and fat phases, which in turn affect their antimicrobial efficacies.Item Drivers of obesity : associations of physical activity, sedentary behaviour and diet on metabolic health and the gut microbiota : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Tāmaki Makaurau, Aotearoa New Zealand(Massey University, 2023) Slater, JoanneBackground: Regular physical activity (PA) and limited time spent sedentary are important for almost all aspects of health, including prevention and treatment of obesity. Aim: To describe the PA and sedentary behaviour (SB) of healthy, lean and obese, Pacific and NZE women, aged 18-45 years; and to explore the associations of PA and SB with diet, BF%, biomarkers of metabolic health, and gut microbiota composition. Methods: Pacific (n = 142) or NZE (n = 162) women aged 18–45 years with a self- reported body mass index of either 18.5–25.0 kg/m2 or ≥30.0 kg/m2 were recruited. Whole body dual-energy X-ray absorptiometry was used to subsequently stratify participants as either low (<35%) or high (≥35%) BF%. Eight-day accelerometery assessed PA and SB levels. Meeting the PA guidelines was defined as accumulation of ≥ 30 minutes of moderate or greater intensity activity on ≥ 5 days per week OR 150 minutes of moderate to vigorous PA (MVPA) per week. Dietary intake was assessed using a 5-day food record. Fasting blood was analysed for biomarkers of metabolic health, and whole body dual-energy X-ray absorptiometry was used to estimate body composition. Bulk DNA was extracted from faecal samples and the metagenomic sequences associated with the microbiota were analysed using MetaPhlAN and QIIME2 software. Adjusted multivariate regression models were conducted to explore the associations between PA, SB and diet, body composition and biomarkers of metabolic health, and between PA, SB and gut microbiota composition. Results: Less than half Pacific women were meeting the PA guidelines (high-BF%; 39% and low-BF%; 47%) versus 81% of low-BF% and 65% of high-BF% NZE women. Low-BF% Pacific women were more sedentary than all other women (p<0.05): Pacific low- 10.4 and high-BF% 9.93 and NZE low- 9.69 and high-BF% 9.96 hours/day. Every additional 10-minutes spent in MVPA was associated with 0.9% lower total and trunk fat and 0.7% lower gynoid fat in all women (p<0.05). Among Pacific women; every 100 cpm increase in total PA was associated with 6% lower fasting plasma insulin. Every 10-minute increase in MVPA was associated with 8% lower fasting plasma insulin in both ethnic groups (p<0.05). Among NZE women, every one-hour increase in sedentary time was associated with 0.8% higher gynoid fat (p<0.05), and longer weighted median sedentary bout length was associated with higher BF% (gynoid fat 0.3%, total body 0.4%, trunk 0.4%, android 0.4% and visceral fat 0.4% (p<0.05)) and 14% higher C-reactive protein (CRP) (p<0.05). No associations between SB and body composition or metabolic markers were found among Pacific women. There was no significant difference in average total energy intake between Pacific and NZE women or BF% groups. No women were consuming more than the carbohydrate AMDR (>65% total energy). Pacific women’s mean daily starch intake was significantly higher than NZE women (g/day, and % total energy intake). Only the NZE low-BF% groups mean fibre intake was above the recommended daily intake of ≥25g/day. All the women that were in the lowest quartile of fibre intake, and particularly the NZE women, had a lower odds of meeting the PA guidelines (OR 0.72 (p=0.008) and OR 0.66 (p=0.021) respectively) compared to women in the top three quartiles of fibre intake. All the women that were in the lowest quartile of polyunsaturated fat intake, especially Pacific women had a lower odds of meeting the PA guidelines compared women in the top three quartiles (0.76, p=0.027 and OR 0.67, p=0.030 respectively). Among NZE women, every one SD increase in total PA (197 cpm/day) was associated with 36.3% higher relative abundance of Erysipelotrichaceae (p=0.031) and 37.9% lower relative abundance of Verrucomicrobiaceae (p=0.029). Every one SD increase in SB (1.45 hours/day) was associated with a 28% lower relative abundance of Erysipelotrichaceae (p=0.030). Every one SD increase in NZE women’s total PA was associated with 23.1% higher Firmicutes:Bacteroidetes ratio (p=0.031), whereas among Pacific women, every 1 SD increase in MVPA was associated with 22.8% lower (p=0.034) Firmicutes:Bacteroidetes ratio. Conclusion: Increased time spent in PA of all intensities and breaking-up prolonged SB was associated with healthier body composition and lower metabolic disease risk in Pacific and NZE women. Compared to NZE, the impact of increased total PA on fasting insulin may be greater in Pacific women and inflammation may be a pathway through which SB impacts cardiovascular risk, especially for NZE women. Although higher total PA and lower SB was associated with some aspects of the gut microbiota composition, more needs to be known about the mechanisms driving associations between PA SB and the gut microbiota to enable these findings to be interpreted.