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    Neurobiological impacts of kiwifruit consumption in a pig model and its effects on sleep and mood in young adults : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2024) Kanon, Alexander Putra
    Kiwifruit (KF) positively impacts gut health, specifically in alleviating gastrointestinal symptoms and improving laxation. Emerging evidence also suggests that consuming KF influences sleep and mood, with most studies indicating improvements in subjective measures of these attributes. Previous research has explored the mechanisms behind these effects using in vitro and rodent models, which have considerable differences to human physiology. This study explores the impact of New Zealand KF on various brain physiological aspects in animal models and humans. It explores the antioxidant neuroprotective potential of KF, examines alterations in the gut microbiome composition and bioamine concentrations, analyses temporal bioamine concentration effects in plasma and brain regions, and assesses the acute effects on human sleep quality and mood. Findings reveal that in one week, consumption of both green and gold KF reduced oxidative potential in plasma, increased concentrations of 5-Hydroxyindoleacetic Acid (5HIAA, a serotonin metabolite), and induced changes in the abundance of specific microbial genera along the colon of adult pigs, a more representative model of human physiology. Furthermore, green KF enhances antioxidant protective potential in plasma and various brain regions, while gold KF elevates plasma vitamin C levels and tends to reduce acetylcholinesterase activity across the entire brain. Temporal effects highlight distinct patterns in metabolite concentrations between green and gold KF, with γ-Aminobutyric Acid (GABA) and serotonin exhibiting notable interactions in different brain regions. Good and poor sleepers consuming KF before sleep had improved sleep quality and mood. Fresh KF facilitates easier sleep onset for good sleepers, while freeze-dried KF leads to increased ease of awakening in the morning for poor sleepers. Notably, both forms of KF increase the urinary excretion of 5HIAA and reduce feelings of sleepiness while increasing alertness. The inclusion of the fruit skin appears to increase improvements in sleep quality, suggesting a more noticeable effect. These studies provide valuable insights into the neurobiological effects of KF and support its potential as a functional food to improve sleep in humans.
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    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 Jamil
    The 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.
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    The effect of milk processing on protein digestion and amino acid absorption in the gastrointestinal tract of pigs as a model human : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Manawatū, New Zealand
    (Massey University, 2024-06-19) Ahlborn, Natalie Gisela Marlis
    Globally, milk is processed using heat and homogenisation to improve food safety and extend shelf life. These common processing techniques can alter the native structures present in milk, including protein structures. However, the impact of these processing-induced changes on the digestion of milk protein and subsequent absorption of amino acids in the human body is not yet fully understood. The overall objective of this research was to understand how heat treatment and homogenisation affect milk protein coagulation and digestion in the stomach, and to investigate how changes to gastric coagulation (curd formation) influence amino acid (AA) absorption in the small intestine and AA concentrations in blood circulation. Due to the limited accessibility of the human gastrointestinal tract, pigs were used as a model of the human. An initial study using raw bovine (cow), caprine (goat), and ovine (sheep) milk established the role of gastric curd formation in small intestinal AA absorption in piglets at a single postprandial time point. Specifically, differences in the retention of AA in the gastric curd were responsible for differences in the small intestinal AA absorption across milk of different species. A separate study using bovine milk as a milk model was then conducted to determine the effect of heat treatment and homogenisation on the kinetics of milk protein digestion and small intestinal AA absorption. The selected processing treatments were pasteurisation, ultra-high temperature treatment (UHT), and homogenisation. Raw milk was included as a comparator. In the stomach, heat treatment and homogenisation altered the strength and structure of the curd formed during gastric digestion, which in turn affected both milk protein hydrolysis and the rate of AA entering the small intestine. Differences in the release of digested protein and AA into the small intestine were reflected in the kinetics of AA absorption of the processed milk types. For example, UHT milk had both a faster rate of AA entering the small intestine and a faster rate of AA absorption. Processing also altered the appearance of some AA in blood circulation; however, these differences were not directly reflective of the differences observed in their small intestinal absorption kinetics. In conclusion, this PhD research demonstrated that the rate of small intestinal AA absorption was modulated by gastric curd formation, indicating that milk processing could be used as a strategy to modulate protein digestion and AA absorption in the gastrointestinal tract.
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    Ruminants' milk in early postnatal brain development in a pig model of the human infant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Jena, Ankita
    Given the rapid brain development in the early postnatal period and its sensitivity towards changes in the external environment like nutrition, this period is of utmost importance for determining later life health and well-being. Emerging evidence suggests a link between the triad of early life nutrition, the gut and brain axis and its potential for optimising or retrograding early postnatal brain development. In this context, human breast milk has been most studied. However, whether the development of the brain is responsive towards milk from ruminant species used to make milk formula via modulating gut-derived molecules has not been well understood. The aim, therefore, of the thesis was to evaluate the effects of milk from bovine, caprine and ovine species on circulatory blood plasma metabolites, brain tissue metabolites and brain tissue gene expression in piglets and establish associations between changes in plasma metabolite profile with neurochemical and molecular features of the brain. The hypothesis was that metabolites in the peripheral circulation would differ between different ruminant milk consumption, influencing brain metabolite and gene expression. Liquid chromatography-mass spectrometry-based metabolomics was used to profile the plasma, hippocampal, prefrontal cortex, and striatal tissue metabolite relative abundances. NanoString technology was used to evaluate the expression of genes associated with neuro- and cognitive development in the hippocampus, prefrontal cortex, and striatum tissue samples. Multi-omics data integration was used to explore the correlation between plasma and brain lipid profiles. The results showed that the relative intensity of plasma metabolites differed between bovine, caprine and ovine milk treatments, and lipid metabolites were the predominant features. The bovine group had a higher relative intensity of plasma lipids (e.g., saturated triglycerides, phosphatidylcholine, sphingomyelin) than the ovine and caprine milk groups, except for unsaturated triglycerides, which had a higher intensity in the ovine milk group. Metabolite profiling of brain regions indicated that the relative intensity of lipid metabolites, mainly phospholipids, changed in response to different milk treatments. Further analysis showed that in the striatum and hippocampus, the relative intensity of phospholipids in the bovine milk group was higher than in the ovine and caprine milk groups. In contrast, the relative intensity of phospholipids in the prefrontal cortex was higher in the ovine milk group than in the other milk groups. Gene expression profiling showed that the expression of genes in the striatum and hippocampus associated with neurotransmission differed between milk treatments. Both increased and decreased gene expressions were observed in response to ovine milk treatment, whereas a similar gene expression pattern was observed between the caprine and ovine milk treatments. No effect of milk treatments was observed on the prefrontal cortex gene expressions. Striatal and hippocampal lipid relative intensities showed a positive association with that of plasma lipids and the prefrontal cortex showed negative associations. Thus, this PhD research findings suggest that consuming different ruminant milk can impact early postnatal brain development by influencing the peripheral circulatory metabolites in piglets as a model of human infants
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    Fibre fermentation in the ileum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Hoogeveen, Anna Maria Elisabeth Hoogeveen
    Recently, several studies have suggested that the microbes present in the ileum (i.e., the end of the small intestine) can ferment dietary fibre resulting in organic acid production and contribute to the overall gastrointestinal tract (GIT) fermentation. However, studying human ileal fermentation is challenging due to inaccessibility of the small intestine. The aim was to validate a newly developed and optimised in vivo/in vitro ileal fermentation assay based on the growing pig as an animal model for human adults. After the assay was validated, this method was used to quantify ileal fermentation and compare this with large intestinal fermentation. In addition, the effect of diet on ileal fermentation and which factor was a greater contributor to in vitro ileal fermentation (inoculum or substrate) were studied. Firstly, in vitro ileal organic matter (OM) fermentability was similar to in vivo fermentability in the conventional grown pig. Artificially rearing and inoculating young pigs with an infant faecal inoculum did not improve the model. Secondly, the ileal microbiota from pigs and human ileostomates was found to have similar in vitro OM fermentability and organic acid production for arabinogalactan, fructooligosaccharides and pectin, even though some differences were found in the ileal microbial community. Therefore, the in vivo/in vitro ileal fermentation assay using conventional pigs is a preferred and valid model for studying ileal fermentation in the adult human. It was found that ileal fermentation was quantitatively significant and similar in magnitude to hindgut fermentation when using this validated assay. However, the microbial community and organic acid production (mainly acetic acid) in the ileum differed. It was also found that partly replacing cellulose with more fermentable fibres in the diet affected the ileal microbial community and its fermentative capacity in growing pigs. Lastly, the substrate (i.e., different fibre sources) was found to have a greater effect on ileal fermentation than the inoculum (i.e., different ileal microbiota obtained by feeding pigs different diets). In conclusion, this work has demonstrated the quantitatively significant contribution of ileal fermentation to overall GIT fermentation, and that the in vivo/in vitro ileal fermentation assay using the growing pig is a valid assay for studying ileal fermentation in the adult human. Dietary intervention can be used to shape ileal microbiota and fermentation.
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    Effect of Kiwifruit actinidin on the digestion of gluten proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Jayawardana, Isuri Achintha
    Gluten proteins are resistant to complete proteolysis by the human gastrointestinal tract (GIT) enzymes, due to their high proline- and glutamine-rich peptide sequences. Proline confers resistance to proteolysis by digestive enzymes, producing indigestible proline-rich peptides, some of which can trigger immunogenic reactions that are responsible for gluten-related health disorders such as coeliac disease, wheat allergy and gluten sensitivity. At present, gluten-free diets (GFD) are the only promising therapy for gluten-related health disorders. However, maintaining a lifelong GFD is challenging. As an alternative therapy, gluten-specific enzymes to hydrolyse immunogenic peptides have shown promising results. Most of these are of microbial origin. Identification of natural alternative enzymes is desirable, with fruit-borne enzymes a possible solution. Actinidin, a cysteine protease found in most green kiwifruit (Actinidia deliciosa), is suggested as an effective exogenous enzyme, to be utilized in this category. The objective of this PhD study was to evaluate the effect of actinidin on the digestion of gluten and gluten-derived immunogenic peptides in the GIT. The effectiveness of actinidin was tested using different in vitro GIT models and an animal (pig) preclinical model with purified gluten or whole wheat bread as sources of gluten, and purified actinidin or and fresh green kiwifruit as sources of actinidin. Analytical techniques such as free amino nitrogen determination, enzyme-linked immunosorbent assay and both targeted and untargeted mass spectrometry were used to determine the degree of hydrolysis (DH), R5 gluten epitopes and immunogenic peptides respectively. Actinidin hydrolysed peptide bonds adjacent to proline residues in the 33-mer peptide, one of the most immunogenic gluten peptides. The gastric DH of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05). Actinidin at a concentration of > 2.7 U/mL and pH > 2 during hydrolysis was considered ideal for gluten hydrolysis. Actinidin increased (P < 0.05) the rate of acceleration of DH of gluten and reduced the amount of R5 epitopes present in the small intestine using a semi-dynamic in vitro GIT digestion model. Actinidin also accelerated the gastric hydrolysis of wheat proteins in whole wheat soda bread, which was reflected in a faster reduction of R5 epitopes in the gastric conditions and the rate of reduction (P < 0.05) of most of the immunogenic marker peptides present in the small intestine. In vivo, the presence of dietary actinidin in the form of green kiwifruit significantly (P < 0.01) enhanced the gastric digestion of wheat proteins in whole wheat soda bread fed to pigs as a model of human GIT digestion. The amount of R5 epitopes was lower (P < 0.01) in the stomach, proximal and distal small intestine and terminal ileum of pigs fed diets containing green kiwifruit (P <0 .05). The number of immunogenic peptides in the proximal small intestine was low in the pigs fed green kiwifruit diet compared to that of the pigs fed yellow kiwifruit diet (control). In addition, a diet containing green kiwifruit markedly reduced (P < 0.05) the amount of seven gluten immunogenic marker peptides including the 33-mer peptide in the stomach chyme of pigs. Actinidin was able to survive peptic proteolysis and gastric pH conditions until 300 min postprandial in pigs. Taken together, these results suggest that actinidin enhanced the rate of proteolysis of both purified gluten and gluten in a food matrix and reduced the amount of immunogenic gluten epitopes reaching the small intestine during GIT digestion in vitro and in vivo. Actinidin was able to reduce both the amount of and the time of exposure to immunogenic peptides in the small intestinal lumen, therefore it is a promising candidate to be considered in oral enzyme therapy for gluten-related health disorders.
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    Structural changes in milk of different species during digestion : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Food Technology), Massey University, Manawatū, New Zealand
    (Massey University, 2021) Roy, Debashree
    Cow milk is the most abundant type of mammalian milk produced in the world. It has been widely explored industrially as well as academically. However, non-cow milk (e.g. water buffalo, goat, and sheep milk) consumption is significant and forms an important nutritional source for people in many countries. The interest in non-cow milks has increased because of several anecdotal experiences reported about the nutritional and digestive benefits of these milks. However, there is very little scientifically validated information available. The overall objective of this PhD study was to investigate how some of the non-cow milks (such as goat and sheep milk) are structurally different (or similar) to cow milk, especially in their coagulation behaviour under the gastric environment. The potential implications of structural changes on the delivery of nutrients under dynamic gastric digestion conditions were also explored. Dynamic in vitro and in vivo gastric digestion models were employed for this study. It was found that milk from different species vary in their natural macronutrient composition, structure, and acid-gelation behaviour. The fundamental mechanism of coagulation of proteins under the dynamic in vitro gastric digestion conditions was found to be similar for different species milk. The in vivo gastric digestion studies revealed comparable results, although goat and sheep milk curds had relatively lower rates of strengthening and relatively more open microstructure. Both the dynamic in vitro and in vivo studies revealed that the release of fat globules from the coagulated curd was directly proportional to the breakdown (or hydrolysis) of the protein in the curd during gastric digestion. The studies clearly showed that the curd formation and its disintegration in the stomach is a key factor influencing the rate of delivery of macronutrients to the small intestine. The results from this thesis contribute to the knowledge of how composition along with structure impact the release of nutrients at various stages of gastric digestion of different mammalian milks. The information gained from this study might have important consequences for developing dairy products with improved structures for controlled delivery or release of nutrients to meet the special dietary needs of consumers.
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    Functional characterisation of coq8 in Drosophila : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Genetics, Massey University
    (Massey University, 2018) Hura, Angelia Josephine
    With the increasing number of novel mutations being discovered by whole genome and whole exome sequencing, functional studies are increasingly required to determine whether specific mutations are responsible for the disease phenotypes. Drosophila, with its vast set of genetic and molecular tools as well as robust behavioural assays, is an ideal model for functional characterisation. Coenzyme Q biosynthesis is highly conserved from yeast to humans and involves a number of genes in the enzymatic pathway including COQ8A. The role of COQ8A in CoQ biosynthesis is not clear. However, mutations in COQ8A have been associated with autosomal recessive cerebellar ataxia, which is characterised by gait ataxia, cerebellar degeneration and CoQ10 deficiency. This project aimed to characterise the phenotypes resulting from the reduction of coq8 expression (the Drosophila homologue of COQ8A) to develop a model of coq8 deficiency that could be used to characterise COQ8A mutations functionally. RNAi knockdown of coq8 resulted in severe developmental delay, larval lethality, locomotor impairment, a decrease in ATP production, as well as developmental deficits and neurodegeneration in the Drosophila eye. Reintroduction of wild-type Drosophila coq8 partially rescued the larval lethality, restored locomotor function and also primarily rescued the necrotic phenotype in the eye. This model could, therefore, be used to determine whether a specific mutation impaired function, such that it would not rescue the deficiency. As a proof-of-principle, two mutant variants of coq8, I295P and L520*, which were modelled on the human COQ8A mutations L277P and c.1506+1G>A (which results in a truncated protein) did not rescue the coq8 deficiency, indicating that they disrupted normal coq8 function. However, the reintroduction of human COQ8A did not restore function but instead exacerbated the necrotic and neurodegenerative phenotype in the eye suggesting that it may be impairing the mitochondrial function of wild-type coq8. Drosophila provides the means to characterise disease-causing genetic mutations functionally. Here we have developed a model that can be used to study the role of coq8 in Drosophila and have found that Drosophila coq8 and human COQ8A differ in function.