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Item The links between human breath methane, dietary fibre digestion, and the gut microbiota : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutrition Science at Massey University, Manawatu, New Zealand(Massey University, 2022) Payling, Laura MarieThe concentration of methane that is exhaled in human breath has been associated with the composition and fibre-fermenting function of the human colonic microbiota. The current research aimed to investigate whether composition and fibre fermentation function of the colonic microbiota differ in individuals who are low breath methane emitters (LE) or high breath methane emitters (HE). Healthy adult individuals (18) were recruited and breath tested. Unexpectedly, they showed positive correlations between breath hydrogen and methane. Then, the highest and lowest breath methane emitters provided faecal samples used for shotgun metagenomic sequencing and as faecal inocula for in vitro colonic fermentations with dietary fibres (β-glucan and lignocellulose). Individuals who were LE reported higher dietary vitamin E, fibre, and fat intakes and a Bacteroides-driven microbiota composition compared to HE individuals who reported a greater starch intake and a Prevotella-driven microbiota composition. The faecal microbiota from individuals who were HE had a greater abundance of taxa from the Methanobrevibacter genus and more methane gas production during in vitro colonic fibre fermentation compared to the microbiota of individuals who were LE; however, the results were variable within the HE group. There was a greater rate and extent of dietary fibre fermentation in LE compared to HE individuals during in vitro colonic fermentation, which aligned with the greater fibre intakes of LE individuals. Furthermore, the faecal microbiota of LE individuals showed increased beneficial organic acid production and a greater abundance of functional pathways related to amino acid metabolism compared to HE during in vitro colonic fermentation. These results did not align with published research on human breath methane and the gut microbiota. However, there was consensus with emerging hypotheses suggesting that there are important pathways involved in hydrogen sulphide production and hydrogen utilisation that are largely unexplored. Further investigations in these areas could help redefine our understanding of fibre fermentation by the human colonic microbiota.Item 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 HoogeveenRecently, 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.Item Process for recovery of smooth fibre ingredient from pomace : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Food Technology at Massey University, Manawatu, New Zealand(Massey University, 2020) Eblaghi, MarziehThis research aimed to develop a process to convert apple pomace into a food ingredient which can provide functional properties such as water-binding in baked goods, stability in aqueous suspension and smooth mouthfeel. This was achieved by modifying the apple pomace through three main steps: heating, shearing and enzymatic hydrolysis. Firstly, the effect of sample preparation (addition of water to fresh pomace, temperature and shearing apple pomace) on the solubility of pectin was investigated. Secondly, kinetics of main reactions involved in pomace while heating at temperatures between 90-140 °C (10 °C intervals) and incubation times between 0-360 min, were studied at bench scale. These reactions were: solubilisation and depolymerisation reactions of pectin, degradation of sugars and production of secondary products such as organic acids and 5-hydroxymethylfurfural (5-HMF). After that, the kinetics of these reactions were modelled for determining the rate constants and activation energies. The kinetic models of pectin solubilisation and 5-HMF formation were then used for scaling up the hydrothermal process in a more complex heat transfer situation, using a pilot scale retort. Finally, the effect of particle size distribution and molecular weight of solubilised components (mainly pectin) on physicochemical and sensorial properties of pomace material was investigated. Solubilisation of pectin at room temperature was independent of addition of water and shearing treatment of pomace. However, heating at temperatures > 100 °C, combined with increasing the amounts of water added to pomace (from 0 to 8 mL water/ g pomace) resulted in increasing the pectin solubility up to a pomace-water ratio of 1:2. The maximum amount of solubilised pectin (~ 605 μmol galacturonic acid/ g dry pomace) was determined when heating pomace at 130 and 140 °C for 15 and 7 min, respectively. Hydrothermal depolymerisation of pectin through acid hydrolysis and β-elimination reactions also showed temperature-dependent behaviour. Depolymerisation reactions resulted in degradation of pectin polymers into ethanol-soluble forms (galacturonic acid). Depolymerisation seemed more likely to happen from non-esterified sites of pectin polymers, as suggested by the high degree of esterification of the remaining insoluble pectin. Increasing amounts of glucose and fructose were observed in the serum phase of pomace to about five times their initial values when pomace was heated at temperatures >120 °C. This was accompanied by a reduction in sucrose content, suggesting hydrothermal hydrolysis of sucrose to its subunits. A complete conversion of sucrose was recorded at temperatures > 120 °C and times > ~30 min. Glucose, fructose and galacturonic acid underwent further transformation at temperatures > 100 °C forming secondary products of organic acids, (such as acetic acid, formic acid and lactic acid), furfural and 5-HMF. Modelling the kinetics of pectin solubilisation and 5-HMF production resulted in activation energies of 81 and 105 kJ/mol, respectively. The effects of hydrothermal treatment were modelled using COMSOL for heating a slab of pomace in a pilot scale retort with a maximum steam temperature of 125 °C. In this model, heat transfer through the pomace and chemical reactions of pectin solubilisation and 5-HMF production were predicted. The aim for this model was to identify conditions permitting solubilisation enough to double the amount of pectin from the amount initially present at room temperature while limiting the production of 5-HMF to the range permitted in food standards (bulk averaged). The validity of the model was confirmed in the pilot plant condition. Another objective of this research study was to investigate the effects of particle size distributions and molecular weight of pectin on sensory properties and physical stability of pomace samples. A controlled modification of particle size and molecular weight of solubilised pectin was achieved by fractionation of heat-treated pomace into insoluble solid and serum parts. Shearing insoluble particles for 5 min showed significant particle size reduction from 496 μm (initial shearing for 2 min) to 165 μm. Further shearing did not affect the average size of the particles. Microscopy revealed the effect of shearing in separating cell aggregations, resulting in individual cells. Shearing also fractured some individual cells, the oval shape of most cells was still visible. The pectin in the serum phase of pomace before and after heat treatment was analysed for molecular weight distribution. Results confirmed the effectiveness of heat treatment on solubilising high molecular weight pectins into the serum phase. Two ranges of high (between 4000-73 kDa) and low (< 73 kDa) pectin molecular weights were analysed in heat-treated pomace. These two ranges were separated from each other by ultrafiltration. High molecular weight of fraction (with the average size of 380 kDa) was enzymatically hydrolysed into two pectin components with average molecular weights of 30 and 150 kDa. Finally, six pomace ingredients were produced from the pomace fractions with two insoluble particle size distributions (with the average size ranges of 500 and 160 μm) and three ranges of molecular weights (380, 150 and 30 kDa) being blended. Particle size reduction had a significant effect on the physical stability of pomace suspensions. Samples containing particles >496 μm showed phase separation during storage (5 days at 4 °C), while samples with smaller particle size did not show any phase separation.Item Dietary fibre intake : validity of a short-term food frequency questionnaire : association with gastrointestinal health and public perceptions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutrition and Dietetics, Massey University, Albany, New Zealand(Massey University, 2018) Micah, JohnIntroduction: Dietary fibre is an important constituent of the diet as it plays a key role in reducing the risk of certain diseases. There are several validated dietary assessment tools that measure current fibre intakes; however these are lengthy, cannot be self-administered or classify dietary fibre intakes. The beneficial effects of fibre consumption have led to dietary recommendations that encourage adequate intake, yet there are a limited number of studies that have investigated the effect fibre has on gut symptoms or examined the perceived benefits versus barriers to eating fibre containing foods. Objectives: This study aimed to validate a tool that measures short term dietary fibre intake against a 4-day food record, and compare dietary fibre intake to gastrointestinal symptoms. The study also aimed to survey perceived benefits and barriers to dietary fibre intake. Methods: One hundred and five healthy male and female participants aged 19-65 years completed the study. All eligible participants completed a 4-day diet record, a food frequency questionnaire based 9-item dietary fibre intake tool (DFiT), a daily gastrointestinal symptom diary and a 15-item dietary fibre perceptions survey. Agreement between the 4-day diet record and DFiT was analysed using a paired t-test, correlation coefficient, cross-classification, weighted k statistic and Bland Altman analysis. Results: The DFiT was accurate in classifying but not estimating total dietary fibre intakes. When different levels of dietary fibre intakes were compared to markers of gastrointestinal health, there were no associations found for occurrence or severity for gastrointestinal symptoms. However, high fibre consumers pass one additional bowel motion per day and had softer stool than low fibre consumers. The survey of perceptions showed that the majority of participants agreed with the health benefits, however just over half of participants identified with barriers. There were some differences in responses between genders, levels of dietary fibre intake and socioeconomic status. Conclusion: The DFiT is a valid, simple, short and easy to use questionnaire for classifying but not estimating total short term dietary fibre intakes. In the context of sustainability and shift towards a higher consumption of dietary fibre, it is important to further investigate the effect of dietary fibre on gastrointestinal symptoms and perceptions of barriers.Item Characterisation of food fibres and their effect on starch digestion in an in-vitro system at physiological shear rates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Anatomy and Physiology at Massey University, New Zealand(Massey University, 2017) Yap, Sia-YenThe fast pace of life promotes the excessive consumption of processed starchy food containing high levels of sugar, salt and oil; which can increase the prevalence of type II diabetes, colon and cardiovascular diseases. The addition of dietary fibres in the diet increases the viscosity of digesta, delays mixing in the gut, and promotes laxation. However, few studies attempt to quantify the possible physical and chemical effects of either soluble (food gums) and insoluble (largely cellulose) fibre in the diet. These effects may encompass the retention of water inside the fibre particles, between particles in the fibre mass and direct effects of the chemical nature of the fibre on the digestion process. In this study, the fractions of water held in the various partitions of insoluble particulate dietary fibres are quantified. The relationship between the volume fraction of soluble and insoluble dietary fibres in simulated digesta at physiological concentrations and the rheological properties of the suspension at physiological shear rates is determined. Furthermore, the impact of fibre and shear rates on the digestion of starch in-vitro at physiological shear rates was measured. This work provides the first quantitative assessment of the effects of the physical attributes of dietary fibre on the digestion of starch in-vitro, at physiological shear rates. In this work, four insoluble fibre types were used to construct aqueous suspensions containing solid volume fractions similar to those of pig digesta from the small intestine; these suspensions also were shown to have similar rheological properties to those of pig digesta at physiological shear rates. In addition, a soluble fibre (Guar gum) was used to construct solutions with viscosities comparable to those of the particulate suspensions. Gelatinised and partially gelatinised starch was added to these suspensions and its rate of digestion at 37°C under simulated small intestinal conditions was measured at shear rates covering the reported physiological range. Important results from this work include: - The proportion of water retained by a given volume of hydrated mass of large fibre particles (AllBran®) was double that of smaller particles (wheat fibre). For all of the solid particles used, the proportion of water sequestered by the intra-particulate voids was less than 4% of the volume of the particles, similar proportions were determined for indigestible particles recovered from the colon of pigs and from human faeces. - Food fibre systems containing less than 20% by volume (solid volume fraction, φ = 0.20) of insoluble dietary fibres showed Newtonian rheological properties and the viscosity of these suspensions could be predicted from φ by the Maron-Pierce model. Starch/fibre suspensions prepared with φ below 20% (φ = 0.68-0.98) had a similar viscosity to that of starch/guar suspension comprising 10% (w/v) starch and 0.4% (w/v) guar. During in-vitro digestion, the viscosity of the starch/fibre suspensions decreased logarithmically over the first 20 minutes during which about 30% of the starch was hydrolysed, this was followed by a prolonged period of slow digestion as the slowly digested starch (SDS) and resistant starch (RS) were hydrolysed. The rate of starch digestion was independent of the type of insoluble fibre and was not affected by suspension viscosities used providing shear rates could be maintained within physiological levels. For guar, rates of digestion were slowed probably due to non-competitive inhibition of the amylase by the guar. - When shear rates were below the physiological range (0.1 s-1) or gelatinisation was incomplete, the rate of digestion became linear over the first 20 minutes of digestion suggesting that the rate of digestion was limited by transport processes at low shear in viscous suspensions. - This study provides useful information regarding the limiting concentration of particles and hence viscosity of digesta in the gut if rates of digestion are to be maximised. Additionally, it is suggested that guar, even at low concentration may reduce glycemia by reducing rates of amylolysis.Item Studies on methodology in dietary fibre analysis : a neutral detergent fibre method using glucoamylase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University(Massey University, 1979) Sills, Victor ErnestThe dietary fibre content of foods is conveniently and rapidly determined by the neutral and acid detergent methods devised originally by Van Soest and associates. A serious disadvantage of the neutral detergent method relates to the interference caused by starch during filtration when the method is applied to cereals and cereal products. In these circumstances the results of neutral detergent fibre (NDF) measurements are variable and often over-estimated . A study of the starch-lipid reaction which takes place when cereal products are heated in Van Soest's neutral detergent solution showed that although the precipitate derived from pure wheat starch and lipid is soluble in hot water this action is often far from complete when much fibrous cereal matter is present. Much of the starch appears to be occluded in the NDF residue which then takes on a gummy-like character and tends to clog the filter. Southgate recently recommended purified amyloglucosidase from Aspergillus niger (Boehringer) for the purpose of hydrolysing starch in cereal samples before starting the neutral detergent extraction. Present studies have been concerned with the development of this enzymatic procedure with the aim of devising improved methodology and enhancing existing knowledge of the behavioural characteristics of amyloglucosidases from A. niger and from an alternative source, Rhizopus spp. Preliminary investigations showed that amyloglucosidase from A. niger (Boehringer) was completely effective as a starch hydrolysing agent in the pretreatment of a cereal substrate but that in order to use the enzyme economically it was necessary to use a semi micro version of Van Soest's neutral detergent extraction procedure. The main features of the new method are as follows: preparation of a subsample of lipid-free food sample of fine particle size; gelatinization of starch before enzyme treatment; treatment with the minimum quantity of enzyme (2 mg); extraction with neutral detergent at half the normal rate; separation of detergent solution from the residue by means of centrifugation; dehydration of the residue with acetone before filtration; special techniques for filtration, drying and weighing procedures. A table of NDF values for various cereal products determined by the semi micro procedure is presented. The results agree, for the most part, with the results of other workers in this field, the exceptions being for cornflakes, rolled oats and puffed wheat. The coefficients of variation for the NDF values compare favourably with those of other workers. A semi micro version of Van Soest's acid detergent method of evaluating dietary fibre was devised and is described with supporting analytical data. Tests performed with a low cost preparation of amyloglucosidase from Rhizopus spp (Sigma) showed that the crude enzyme was capable of fully hydrolysing the starch component of cereal products before commencing the neutral detergent extraction procedure but that it also seriously reduced the NDF values. In order to establish the cause of the discrepancies two approaches were made: an attempt was made to analyse the products of enzymatic hydrolysis; and a study of the effect of enzyme concentration on the yield of neutral detergent fibre was undertaken. The former approach proved impracticable, the latter suggested that either impurities in the crude enzyme preparation were responsible or the amyloglucosidase itself was active towards one or more components of dietary fibre. In order to determine which of the alternative explanations was correct small amounts of the crude enzyme preparation were purified by means of anion exchange chromatography using DEAE cellulose and one of two buffer systems, one based on citrate-phosphate, the other on tris-HCl. The citrate-phosphate conditions reported by Pazur and Lineback et al for the column separation of amyloglucosidase of A. niger were found to be quite unsuitable for the enzyme from Rhizopus spp. and a new set of conditions had to be determined for this enzyme. The activity of small amounts of the purified enzyme (< 1mg) was estimated by an improvised visual method using buffered 1% wheat starch, and the effect of the enzyme on cereal fibre was determined by means of the semi micro neutral detergent procedure using 0.08-0.2 g wholemeal flour as a substrate. It was found that both crude and purified forms of the enzyme caused a loss of ca 30% NDF from wholemeal flour, from which it was concluded that amyloglucosidase from Rhizopus spp was not a suitable enzyme for use in the neutral detergent method of measuring fibre. A literature review of the known chemistry of the amyloglucosidases of A. niger and R. delemar showed that differences in molecular structure reported by Pazur and others could account for their different electrophoretic properties. In the light of the present work it appears that another important biochemical difference between these enzymes relates to the activity of the Rhizopus enzyme towards the dietary fibre component of cereals.Item The influence of habitual dietary intake on the responsiveness of the gut microbiota to a dietary intervention : 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, 2017) Healey, Genelle RosePreliminary evidence suggests that inter-individual variability in gut microbiota response to a dietary intervention is influenced by baseline gut microbiota composition. Differing habitual dietary intakes lead to distinctions in baseline gut microbiota composition making it plausible that habitual dietary intake may also influence gut microbiota response. Prior to conducting this research no studies had been undertaken to determine whether habitual dietary intake has an impact on gut microbiota responsiveness. Therefore, the aim of this research was to investigate the influence habitual dietary intake as on gut microbiota response to a dietary intervention. Initially, secondary data analysis was conducted to determine whether there was any support for the hypothesis that individuals with differing habitual dietary intakes would have gut microbiota that respond in a distinctive manner to a dietary intervention. The secondary data analysis results demonstrated that dietary groups rich in dietary fibre had the greatest impact on gut microbiota responsiveness. An in vitro three-stage colonic model system study was conducted to determine whether media with differing fermentable carbohydrate (i.e. dietary fibre) contents influenced gut microbiota response to an inulin-type fructan prebiotic. It was demonstrated that differing prebiotic driven changes in organic acids and bacterial taxa occurred between the low (LFC) and high fermentable carbohydrate (HFC) content media. The results of the secondary data analysis and in vitro study provided evidence to suggest that a human intervention study was warranted. A randomised, double-blind, placebo-controlled, cross-over, human intervention study in 34 healthy participants was undertaken to determine whether habitual dietary fibre intake influenced gut microbiota response to an inulin-type fructan prebiotic. The results of the human intervention study demonstrated that the low habitual dietary fibre (LDF) group harboured gut microbiota that were less responsive to the inulin-type fructan prebiotic than the high habitual dietary fibre (HDF) group. Future studies which aim to modulate the gut microbiota via dietary change or to determine the prebiotic potential of a novel fermentable substrate should take habitual dietary fibre intakes into consideration when recruiting participants or analysing the data. This will help reduce the confounding influence of inter-individual variability in gut microbiota responsiveness and ensure the true efficacy of a dietary intervention is demonstrated.