Use of the Extended Fujita method for representing the molecular weight and molecular weight distributions of native and processed oat beta-glucans

Beta 1–3, 1–4 glucans (“beta-glucans”) are one of the key components of the cell wall of cereals, complementing the main structural component cellulose. Beta-glucans are also an important source of soluble fbre in foods containing oats with claims of other benefcial nutritional properties such as plasma cholesterol lowering in humans. Key to the function of beta-glucans is their molecular weight and because of their high polydispersity - molecular weight distribution. Analytical ultracentrifugation provides a matrix-free approach (not requiring separation columns or media) to polymer molecular weight distribution determination. The sedimentation coefficient distribution is converted to a molecular weight distribution via a power law relation using an established procedure known as the Extended Fujita approach. We establish and apply the power law relation and Extended Fujita method for the frst time to a series of native and processed oat beta-glucans. The application of this approach to beta-glucans from other sources is considered

Increasing dietary oat fibre decreases the permeability of intestinal mucus

This study investigates the influence of the dietary fibre β-glucan on nutrient composition and mucus permeability. Pigs were fed a standard diet or a diet containing twice the β-glucan content for 3 days (n = 5 per group), followed by the collection of small intestinal mucus and tissue samples. Samples of the consumed diets were subjected to in vitro digestion to determine β-glucan release, nutrient profile and assessment of mucus permeability. In vitro digestion of the diets indicated that 90% of the β-glucan was released in the proximal small intestine. Measurements of intestinal mucus showed a reduction in permeability to 100 nm latex beads and also lipid from the digested enhanced β-glucan diet. The data from this study show for the first time that reducing mass transfer of bile and lipid through the intestinal mucus layer may be one way in which this decrease in bile reabsorption by soluble fibre is enabled

Sodium alginate decreases the permeability of intestinal mucus

In the small intestine the nature of the environment leads to a highly heterogeneous mucus layer primarily composed of the MUC2 mucin. We set out to investigate whether the soluble dietary fibre sodium alginate could alter the permeability of the mucus layer. The alginate was shown to freely diffuse into the mucus and to have minimal effect on the bulk rheology when added at concentrations below 0.1%. Despite this lack of interaction between the mucin and alginate, the addition of alginate had a marked effect on the diffusion of 500 nm probe particles, which decreased as a function of increasing alginate concentration. Finally, we passed a protein stabilised emulsion through a simulation of oral, gastric and small intestinal digestion. We subsequently showed that the addition of 0.1% alginate to porcine intestinal mucus decreased the diffusion of fluorescently labelled lipid present in the emulsion digesta. This reduction may be sufficient to reduce problems associated with high rates of lipid absorption such as hyperlipidaemia

Technical tip: high-resolution isolation of nanoparticle–protein corona complexes from physiological fluids

Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP–protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches

The impact of oat structure and β-glucan on in vitro lipid digestion

Oat β-glucan has been shown to play a positive role in influencing lipid and cholesterol metabolism. However, the mechanisms behind these beneficial effects are not fully understood. The purpose of the current work was to investigate some of the possible mechanisms behind the cholesterol lowering effect of oat β-glucan, and how processing of oat modulates lipolysis. β-Glucan release, and the rate and extent of lipolysis measured in the presence of different sources of oat β-glucan, were investigated during gastrointestinal digestion. Only a fraction of the original β-glucan content was released during digestion. Oat flakes and flour appeared to have a more significant effect on lipolysis than purified β-glucan. These findings show that the positive action of β-glucan is likely to involve complex processes and interactions with the food matrix. This work also highlights the importance of considering the structure and physicochemical properties of foods, and not just the nutrient content

Effects of grain source and processing methods on the nutritional profile and digestibility of grain amaranth

Amaranth grain is reputed to have a high nutritional value, and as a plant, be tolerant to adverse weather conditions. This suggests that grain amaranth could be useful in tackling malnutrition and the growing burden of cardiometabolic diseases. However, there is insufficient knowledge at present about how the nutrient composition and digestibility of amaranth grain varies with growing environment, crop genotype, and post-harvest processing. We investigated the effect of the source and processing of amaranth grains on the digestibility of protein and lipid present in the grains. There was variation in the composition and digestibility of raw grains from different sources, indicating a role of genotype and/or growing environment which warrants further investigation. The greatest differences in digestibility were measured between the different processing techniques. This indicates that efforts to increase the cultivation and consumption of grain amaranth need to be supported by education about effective processing and preparation techniques

Transport of Particles in Intestinal Mucus under Simulated Infant and Adult Physiological Conditions: Impact of Mucus Structure and Extracellular DNA

The final boundary between digested food and the cells that take up nutrients in the small intestine is a protective layer of mucus. In this work, the microstructural organization and permeability of the intestinal mucus have been determined under conditions simulating those of infant and adult human small intestines. As a model, we used the mucus from the proximal (jejunal) small intestines of piglets and adult pigs. Confocal microscopy of both unfixed and fixed mucosal tissue showed mucus lining the entire jejunal epithelium. The mucus contained DNA from shed epithelial cells at different stages of degradation, with higher amounts of DNA found in the adult pig. The pig mucus comprised a coherent network of mucin and DNA with higher viscosity than the more heterogeneous piglet mucus, which resulted in increased permeability of the latter to 500-nm and 1-µm latex beads. Multiple-particle tracking experiments revealed that diffusion of the probe particles was considerably enhanced after treating mucus with DNase. The fraction of diffusive 500-nm probe particles increased in the pig mucus from 0.6% to 64% and in the piglet mucus from ca. 30% to 77% after the treatment. This suggests that extracellular DNA can significantly contribute to the microrheology and barrier properties of the intestinal mucus layer. To our knowledge, this is the first time that the structure and permeability of the small intestinal mucus have been compared between different age groups and the contribution of extracellular DNA highlighted. The results help to define rules governing colloidal transport in the developing small intestine. These are required for engineering orally administered pharmaceutical preparations with improved delivery, as well as for fabricating novel foods with enhanced nutritional quality or for controlled calorie uptake

Interactions of bile salts with a dietary fibre, methylcellulose, and impact on lipolysis

Methylcellulose (MC) has a demonstrated capacity to reduce fat absorption, hypothetically through bile salt (BS) activity inhibition. We investigated MC cholesterol-lowering mechanism, and compared the influence of two BS, sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), which differ slightly by their architecture and exhibit contrasting functions during lipolysis. BS/MC bulk interactions were investigated by rheology, and BS behaviour at the MC/water interface studied with surface pressure and ellipsometry measurements. In vitro lipolysis studies were performed to evaluate the effect of BS on MC-stabilised emulsion droplets microstructure, with confocal microscopy, and free fatty acids release, with the pH-stat method. Our results demonstrate that BS structure dictates their interactions with MC, which, in turn, impact lipolysis. Compared to NaTC, NaTDC alters MC viscoelasticity more significantly, which may correlate with its weaker ability to promote lipolysis, and desorbs from the interface at lower concentrations, which may explain its higher propensity to destabilise emulsions

The effects of increased butyrate delivered as butyrylated starch on large bowel physiology in the rat.

Introduction: Short chain fatty acids (SCFA) are produced by large bowel fermentation of dietary carbohydrates including resistant starch (RS) and non-starch polysaccharides (NSP). SCFA (particularly butyrate) play a major role in maintaining large bowel function and may reduce the incidence of colonic disease. Butyrate is the preferred metabolic substrate of colonocytes and is believed to play a key role in modulating epithelial cell cycle, mucosal immune response and gut motility. Increasing large bowel butyrate supply requires intakes of NSP or (RS) much higher than those currently consumed in western diets. Recent studies have shown large bowel butyrate is increased by the ingestion of butyrylated starch but the characteristics and physiological effects of its ingestion in animal models of colonic disease have not yet been investigated. Aims and Methods: The experiments in in vitro and in rats described in this thesis examined: the effects of production techniques and cooking on the capacity of butyrylated starch to deliver butyrate to the large bowel. They investigated the effects of increased butyrate levels on large bowel function in: (i) normal rats, (ii) the dextran sulphate sodium (DSS) rat model of ulcerative colitis (UC) and (iii) the high dietary protein rat model of colonocyte genetic damage. Results: Starch type, pre-treatment and the degree of butyrylation influenced the in vitro digestion and fermentation characteristics of butyrylated starch before and after cooking. Butyrylated starch was less susceptible to small intestinal digestion RS as than high amylose maize starch (HAMS) in vitro. Feeding diets containing 10% cooked butyrylated starch delivered significantly greater amounts of butyrate to the large bowel of rats than 10% raw or cooked HAMS. Butyrate did not influence colonocyte proliferation throughout the large bowel of the rat but increased distal colonic IL-18 concentrations and decreased longitudinal smooth muscle contractility. Feeding HAMS or butyrylated HAMS (HAMSB) to rats during DSS induced UC and during 7 days of recovery resulted in increased mucosal damage compared to low amylose maize starch (LAMS) fed rats. When rats were fed HAMS or HAMSB during the 7 days of recovery only, there was no significant difference in mucosal damage. Genetic damage, as measured by the comet assay, was 2 fold higher in rats fed high protein diet compared with those fed a low protein diet. Concurrent feeding of high protein and either HAMS or HAMSB resulted in significantly less genetic damage. Genetic damage in rats fed 20% HAMSB was half the levels of the 20% HAMS group, and was the same as the low protein diet. Conclusions: Butyrylated starch delivered butyrate to the large bowel in rats effectively, was less susceptible to small intestinal digestion and had greater stability following cooking than the unmodified base starch. Increased digesta butyrate did not affect large bowel function or colonocyte proliferation in the normal rat; the effects on mucosal damage in the DSS rat model of ulcerative colitis were inconclusive. Increased luminal butyrate prevented high-protein induced colonocyte genetic damage. Butyrylated starches have potential to assist with the maintenance of bowel health and to contribute to reduced risk of colonic disease in the community.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200