Anthony R. Bird

Diets that differ in their FODMAP content alter the colonic luminal microenvironment

Objective A low FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols) diet reduces symptoms of IBS, but reduction of potential prebiotic and fermentative effects might adversely affect the colonic microenvironment. The effects of a low FODMAP diet with a typical Australian diet on biomarkers of colonic health were compared in a single-blinded, randomised, cross-over trial. Design Twenty-seven IBS and six healthy subjects were randomly allocated one of two 21-day provided diets, differing only in FODMAP content (mean (95% CI) low 3.05 (1.86 to 4.25) g/day vs Australian 23.7 (16.9 to 30.6) g/day), and then crossed over to the other diet with ≥21-day washout period. Faeces passed over a 5-day run-in on their habitual diet and from day 17 to day 21 of the interventional diets were pooled, and pH, short-chain fatty acid concentrations and bacterial abundance and diversity were assessed. Results Faecal indices were similar in IBS and healthy subjects during habitual diets. The low FODMAP diet was associated with higher faecal pH (7.37 (7.23 to 7.51) vs 7.16 (7.02 to 7.30); p=0.001), similar short-chain fatty acid concentrations, greater microbial diversity and reduced total bacterial abundance (9.63 (9.53 to 9.73) vs 9.83 (9.72 to 9.93) log10 copies/g; p<0.001) compared with the Australian diet. To indicate direction of change, in comparison with the habitual diet the low FODMAP diet reduced total bacterial abundance and the typical Australian diet increased relative abundance for butyrate-producing Clostridium cluster XIVa (median ratio 6.62; p<0.001) and mucus-associated Akkermansia muciniphila (19.3; p<0.001), and reduced Ruminococcus torques. Conclusions Diets differing in FODMAP content have marked effects on gut microbiota composition. The implications of long-term reduction of intake of FODMAPs require elucidation. Trial registration number ACTRN12612001185853.

The impact of diet and lifestyle on gut microbiota and human health.

There is growing recognition of the role of diet and other environmental factors in modulating the composition and metabolic activity of the human gut microbiota, which in turn can impact health. This narrative review explores the relevant contemporary scientific literature to provide a general perspective of this broad area. Molecular technologies have greatly advanced our understanding of the complexity and diversity of the gut microbial communities within and between individuals. Diet, particularly macronutrients, has a major role in shaping the composition and activity of these complex populations. Despite the body of knowledge that exists on the effects of carbohydrates there are still many unanswered questions. The impacts of dietary fats and protein on the gut microbiota are less well defined. Both short- and long-term dietary change can influence the microbial profiles, and infant nutrition may have life-long consequences through microbial modulation of the immune system. The impact of environmental factors, including aspects of lifestyle, on the microbiota is particularly poorly understood but some of these factors are described. We also discuss the use and potential benefits of prebiotics and probiotics to modify microbial populations. A description of some areas that should be addressed in future research is also presented.

A comparison of five methods for extraction of bacterial DNA from human faecal samples.

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.

Resistant starch as a prebiotic and synbiotic: state of the art

Non-infectious diseases such as CHD and certain cancers have become major causes of death and disability in affluent countries. Probiotics (principally lactic acid bacteria; LAB) may assist in lowering the risk of these diseases. Experimental studies with probiotics have given generally inconclusive outcomes for infectious disease and for biomarkers for non-infectious disease. In part this situation may reflect their inability to colonise the adult human gut effectively. Prebiotics can assist in promoting colonisation, and resistant starch (RS), as a high-amylose starch, is a prebiotic and synbiotic. This starch exerts its synbiotic action through adhesion of the bacteria to the granule surface. Consumption of RS assists in recovery from infectious diarrhoea in man and animals. A rice porridge, high in RS, appears to modify the autochthonous porcine large-bowel microflora favourably through lowering Escherichia coli and coliform numbers. Many of the beneficial effects of RS on large-bowel function appear to be exerted through short-chain fatty acids (SCFA)formed by bacterial fermentation. In man LAB are found in relatively highest numbers in milk-fed infants where the profile of fermentation products differs quite markedly from that in adults. It appears unlikely that ingestion of current probiotics will alter either total SCFA or the proportions of the major acids. More emphasis needs to be given to the investigation of the effects of complex carbohydrates, including RS, on the autochthonous microflora of the human large bowel.

Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations

Very low-carbohydrate diets are often used to promote weight loss, but their effects on bowel health and function are largely unknown. We compared the effects of a very low-carbohydrate, high-fat (LC) diet with a high-carbohydrate, high-fibre, low-fat (HC) diet on indices of bowel health and function. In a parallel study design, ninety-one overweight and obese participants (age 50.6 (sd 7.5) years; BMI 33.7 (sd 4.2) kg/m(2)) were randomly assigned to either an energy-restricted (about 6-7 MJ, 30 % deficit) planned isoenergetic LC or HC diet for 8 weeks. At baseline and week 8, 24 h urine and faecal collections were obtained and a bowel function questionnaire was completed. Compared with the HC group, there were significant reductions in the LC group for faecal output (21 (sd 145) v. - 61 (sd 147) g), defecation frequency, faecal excretion and concentrations of butyrate ( - 0.5 (sd 10.4) v. - 3.9 (sd 9.7) mmol/l) and total SCFA (1.4 (sd 40.5) v. - 15.8 (sd 43.6) mmol/l) and counts of bifidobacteria (P < 0.05 time x diet interaction, for all). Urinary phenols and p-cresol excretion decreased (P < or = 0.003 for time) with no difference between diets (P > or = 0.25). Faecal form, pH, ammonia concentration and numbers of coliforms and Escherichia coli did not change with either diet. No differences between the diets were evident for incidences of adverse gastrointestinal symptoms, which suggests that both diets were well tolerated. Under energy-restricted conditions, a short-term LC diet lowered stool weight and had detrimental effects on the concentration and excretion of faecal SCFA compared with an HC diet. This suggests that the long-term consumption of an LC diet may increase the risk of development of gastrointestinal disorders.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barley increases the levels of cell wall (1,3;1,4)-β-D-glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.

Resistant starch prevents colonic DNA damage induced by high dietary cooked red meat or casein in rats

In previous studies we have shown that high levels of dietary protein (as casein) result in increased levels of colonic DNA damage, measured by the comet assay, and thinning of the colonic mucus layer in rats when dietary resistant starch (RS) is negligible. Feeding RS abolishes these effects. This study aimed to establish whether a diet high in protein as cooked red meat would have similar effects and whether RS was protective. Rats were fed a diet containing 15% or 25% casein, or 25% cooked lean red beef, each with or without the addition of 48% high amylose maize starch (a rich source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic DNA damage compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% greater DNA damage than the high casein diet but reduced mucus thickness to a similar degree to casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. Caecal and faecal short chain fatty acid pools were also increased by inclusion of RS in the diet. Because DNA damage is an early step in the initiation of cancer, these findings suggest that increased DNA damage due to high dietary protein as cooked red meat or casein could increase colorectal cancer risk but inclusion of resistant starch in the diet could significantly reduce that risk.

Fecal Butyrate Levels Vary Widely among Individuals but Are Usually Increased by a Diet High in Resistant Starch

Butyrate and other SCFA produced by bacterial fermentation of resistant starch (RS) or nonstarch polysaccharides (NSP) promote human colonic health. To examine variation in fecal variables, especially butyrate, among individuals and the response to these fibers, a randomized cross-over study was conducted that compared the effects of foods supplying 25 g of NSP or 25 g of NSP plus 22 g of RS/d over 4 wk in 46 healthy adults (16 males, 30 females; age 31-66 y). Fecal SCFA levels varied widely among participants at entry (butyrate concentrations: 3.5-32.6 mmol/kg; butyrate excretions: 0.3-18.2 mmol/48 h). BMI explained 27% of inter-individual butyrate variation, whereas protein, starch, carbohydrate, fiber, and fat intake explained up to 16, 6, 2, 4, and 2% of butyrate variation, respectively. Overall, acetate, butyrate, and total SCFA concentrations were higher when participants consumed RS compared with entry and NSP diets, but individual responses varied. Individual and total fecal SCFA excretion, weight, and moisture were higher than those for habitual diets when either fiber diet was consumed. SCFA concentrations (except butyrate) and excretions were higher for males than for females. Butyrate levels increased in response to RS in most individuals but often decreased when entry levels were high. Fecal butyrate and ammonia excretions were positively associated ((2) = 0.76; P < 0.001). In conclusion, fecal butyrate levels vary widely among individuals but consuming a diet high in RS usually increases levels and may help maintain colorectal health.

Two high-amylose maize starches with different amounts of resistant starch vary in their effects on fermentation, tissue and digesta mass accretion, and bacterial populations in the large bowel of pigs.

Four groups of young pigs (n 6) were fed a diet containing 50% maize starch as either a highly digestible waxy starch (control; 0% amylose) or one of three resistant starch (RS) diets, namely a high-amylose maize starch (HAMS; 85% amylose), this starch subjected to hydrothermal treatment (HTHAMS; 85% amylose), or a blend of HAMS and HTHAMS included in equal amounts, for 21 d. Food intake and live weight at the end of the study were similar among the four groups. Ileal starch digestibility was lower in pigs fed the three RS diets but was greater for HAMS(88%) than for HTHAMS (70%; P<0.05). Faecal output and large bowel digesta mass, and concentrations and pools of individual and total SCFA were higher (by about two- to threefold; all P<0.05) and digesta pH lower (by about 1 unit, all P<0.001) in pigs fed either HAMS or HTHAMS compared to the controls. These differences in biomarkers were seen along the length of the large bowel. Colon length was 0.5-0.9 m longer (19-35%) in pigs fed the high-RS diets relative to those fed the highly digestible starch diet (P<0.05). Faecal and proximal colonic lactobacilli and bifidobacteria numbers were higher (by 1 and 3 log units; P< 0.05) in pigs fed the HAMS or HTHAMS diets. Although both high-amylose starches promoted fermentation throughout the large bowel, the data suggest that the effects of HTHAMS may be more pronounced in the distal region compared to those of HAMS.