Developing an index of dietary estimation of fermentable carbohydrate to allow dietary analysis for epidemiological studies

It has been proposed that long-term consumption of diets rich in non-digestible carbohydrates (NDCs), such as cereals, fruit and vegetables might protect against several chronic diseases, however, it has been difficult to fully establish their impact on health in epidemiology studies. The wide range properties of the different NDCs may dilution their impact when they are combined in one category for statistical comparisons in correlations or multivariate analysis. Several mechanisms have been suggested to explain the protective effects of NDCs, including increased stool bulk, dilution of carcinogens in the colonic lumen, reduced transit time, lowering pH, and bacterial fermentation to short chain fatty acids (SCFA) in the colon. However, it is very difficult to measure SCFA in humans in vivo with any accuracy, so epidemiological studies on the impact of SCFA are not feasible. Most studies use dietary fibre (DF) or Non-Starch Polysaccharides (NSP) intake to estimate the levels, but not all fibres or NSP are equally fermentable. It has been proposed that long-term consumption of diets rich in non-digestible carbohydrates (NDCs), such as cereals, fruit and vegetables might protect against several chronic diseases, however, it has been difficult to fully establish their impact on health in epidemiology studies. The wide range properties of the different NDCs may dilution their impact when they are combined in one category for statistical comparisons in correlations or multivariate analysis. Several mechanisms have been suggested to explain the protective effects of NDCs, including increased stool bulk, dilution of carcinogens in the colonic lumen, reduced transit time, lowering pH, and bacterial fermentation to short chain fatty acids (SCFA) in the colon. However, it is very difficult to measure SCFA in humans in vivo with any accuracy, so epidemiological studies on the impact of SCFA are not feasible. Most studies use dietary fibre (DF) or Non-Starch Polysaccharides (NSP) intake to estimate the levels, but not all fibres or NSP are equally fermentable. The first aim of this thesis was the development of the equations used to estimate the amount of FC that reaches the human colon and is fermented fully to SCFA by the colonic bacteria. Therefore, several studies were examined for evidence to determine the different percentages of each type of NDCs that should be included in the final model, based on how much NDCs entered the colon intact and also to what extent they were fermented to SCFA in vivo. Our model equations are FC-DF or NSP$ 1: 100 % Soluble + 10 % insoluble + 100 % NDOs¥ + 5 % TS** FC-DF or NSP 2: 100 % Soluble + 50 % insoluble + 100 % NDOs + 5 % TS FC-DF* or NSP 3: 100 % Soluble + 10 % insoluble + 100 % NDOs + 10 % TS FC-DF or NSP 4: 100 % Soluble + 50 % insoluble + 100 % NDOs + 10 % TS *DF: Dietary fibre; **TS: Total starch; $NSP: non-starch polysaccharide; ¥NDOs: non-digestible oligosaccharide The second study of this thesis aimed to examine all four predicted FC-DF and FC-NSP equations developed, to estimate FC from dietary records against urinary colonic NDCs fermentation biomarkers. The main finding of a cross-sectional comparison of habitual diet with urinary excretion of SCFA products, showed weak but significant correlation between the 24 h urinary excretion of SCFA and acetate with the estimated FC-DF 4 and FC-NSP 4 when considering all of the study participants (n = 122). Similar correlations were observed with the data for valid participants (n = 78). It was also observed that FC-DF and FC-NSP had positive correlations with 24 h urinary acetate and SCFA compared with DF and NSP alone. Hence, it could be hypothesised that using the developed index to estimate FC in the diet form dietary records, might predict SCFA production in the colon in vivo in humans. The next study in this thesis aimed to validate the FC equations developed using in vitro models of small intestinal digestion and human colon fermentation. The main findings in these in vitro studies were that there were several strong agreements between the amounts of SCFA produced after actual in vitro fermentation of single fibre and different mixtures of NDCs, and those predicted by the estimated FC from our developed equation FC-DF 4. These results which demonstrated a strong relationship between SCFA production in vitro from a range of fermentations of single fibres and mixtures of NDCs and that from the predicted FC equation, support the use of the FC equation for estimation of FC from dietary records. Therefore, we can conclude that the newly developed predicted equations have been deemed a valid and practical tool to assess SCFA productions for in vitro fermentation.

The structural and mechanical integrity of historic wood

Little is known about historic wood as it ages naturally. Instead, most studies focus on biological decay, as it is often assumed that wood remains otherwise stable with age. This PhD project was organised by Historic Scotland and the University of Glasgow to investigate the natural chemical and physical aging of wood. The natural aging of wood was a concern for Historic Scotland as traditional timber replacement is the standard form of repair used in wooden cultural heritage; replacing rotten timber with new timber of the same species. The project was set up to look at what differences could exist both chemically and physically between old and new wood, which could put unforeseen stress on the joint between them. Through Historic Scotland it was possible to work with genuine historic wood from two species, Oak and Scots pine, both from the 1500’s, rather than relying on artificial aging. Artificial aging of wood is still a debated topic, with consideration given to whether it is truly mimicking the aging process or just damaging the wood cells. The chemical stability of wood was investigated using Fourier-transform infrared (FTIR) microscopy, as well as wet chemistry methods including a test for soluble sugars from the possible breakdown of the wood polymers. The physical properties assessed included using a tensile testing machine to uncover possible differences in mechanical properties. An environmental chamber was used to test the reaction to moisture of wood of different ages, as moisture is the most damaging aspect of the environment to wooden cultural objects. The project uncovered several differences, both physical and chemical, between the modern and historic wood which could affect the success of traditional ‘like for like’ repairs. Both oak and pine lost acetyl groups, over historic time, from their hemicellulose polymers. This chemical reaction releases acetic acid, which had no effect on the historic oak but was associated with reduced stiffness in historic pine, probably due to degradation of the hemicellulose polymers by acid hydrolysis. The stiffness of historic oak and pine was also reduced by decay. Visible pest decay led to loss of wood density but there was evidence that fungal decay, extending beyond what was visible, degraded the S2 layer of the pine cell walls, reducing the stiffness of the wood by depleting the cellulose microfibrils most aligned with the grain. Fungal decay of polysaccharides in pine wood left behind sugars that attracted increased levels of moisture. The degradation of essential polymers in the wood structure due to age had different impacts on the two species of wood, and raised questions concerning both the mechanism of aging of wood and the ways in which traditional repairs are implemented, especially in Scots pine. These repairs need to be done with more care and precision, especially in choosing new timber to match the old. Within this project a quantitative method of measuring the microfibril angle (MFA) of wood using polarised Fourier transform infrared (FTIR) microscopy has been developed, allowing the MFA of both new and historic pine to be measured. This provides some of the information needed for a more specific match when selecting replacement timbers for historic buildings.

An investigation into the properties of non-digestible carbohydrates that selectively promote colonic propionate production

Short chain fatty acids (SCFA), including propionate, are produced by the bacterial fermentation of carbohydrates in the colon. Propionate has many potential roles in health, including inhibiting cholesterol synthesis, de novo lipogenesis and increasing satiety. The profile of SCFA produced is determined by both the substrate available and the bacteria present and may be influenced by environmental conditions within the lumen of the colon. Whilst it may be beneficial to increase colonic propionate production, dietary strategies to achieve this are unproven. Adding propionate to food leads to poorer organoleptic properties, and oral propionate is absorbed in the small intestine. The optimum way to selectively increase colonic propionate would be to select fermentable carbohydrates that selectively promote propionate production. To date, few studies have undertaken a systematic assessment of the factors leading to increased colonic propionate production making the selection of propiogenic carbohydrates challenging. The aim of this thesis was to identify the best carbohydrates for selectively increasing propionate production, and to explore the factors which control propionate production. This work started with a systematic review of the literature for evidence of candidate carbohydrates, which led to a screen of ‘propiogenic’ substrates using in vitro batch fermentations and mechanistic analysis of the impact of pH, bond linkage and orientation using a range of sugars, polysaccharides and fibre sources. A new unit for SCFA production was developed to allow comparison of results from in vitro studies encompassing a range different methodologies found in the literature. The systematic review found that rhamnose yielded the highest rate and proportion of propionate production whereas, for polysaccharides, β-glucan ranked highest for rate and guar gum ranked highest for molar production, but this was not replicated across all studies. Thus, no single NDC was established as highly propiogenic. Some substrates appeared more propiogenic than others and when these were screened in vitro. Laminarin, and other β-glucans ranked highest for propionate production. Legume fibre and mycoprotein fibre were also propiogenic. A full complement of glucose disaccharides were tested to examine the role glycosidic bond orientation and position on propionate production. Of the glucose disaccharides tested, β(1-4) bonding was associated with increased proportion of propionate and α(1-1) and β(1-4) increased the rate and proportion of butyrate production. In conclusion, it appears that for fibre to affect satiety, high intakes of fibre are needed, and which a major mechanism is thought to occur via propionate. Within this thesis it was identified that rather than selecting specific fibres, increasing overall intakes of highly fermentable carbohydrates is as effective at increasing propionate production. Selecting carbohydrates with beta-bonding, particularly laminarin and other β(1-4) fermentable carbohydrates leads to marginal increases in propionate production. Compared with targeted delivery of propionate to the colon, fermentable carbohydrates examined in this thesis have lesser and variable effects on propionate production. A more complete understanding of the impact of bond configurations in polysaccharides, rather than disaccharides, may help selection or design of dietary carbohydrates which selectively promote colonic propionate production substrates for inclusion in functional foods. Overall this study has concluded that few substrates are selectively propiogenic and the evidence suggests that similar changes in propionate production may be achieved by modest changes in dietary fibre intake