Diet can bring out the best of microbial behaviours: microbiome symbiosis

Microorganisms within the human intestine ferment carbohydrate sources, which reach the colon into the SCFA acetate, propionate and butyrate⁽ Reference Conterno, Fava and Viola ^{15 )}. These small organic acids have been shown in animal studies to regulate incretin or gut hormone production, thereby controlling satiety and food intake⁽ Reference Conterno, Fava and Viola ^{15 ,} Reference Lin, Frassetto and Kowalik ^{72 ,} Reference Cani, Possemiers and Van de Wiele ^{74 )}. They also stimulate the gut hormone glucagon-like peptide-2, which is involved in maintaining gut barrier function, a defense mechanism, which can impede the uptake of inflammatory compounds such as lipopolysaccharide (LPS) from the gut lumen that trigger the low-grade chronic inflammation and subsequent insulin resistance associated with obesity and CVD⁽ Reference Tappenden, Albin and Bartholome ^{73 ,} Reference Cani, Possemiers and Van de Wiele ^{74 )}. Similarly, SCFA have been shown to regulate adipocyte hormone production, not least of leptin⁽ Reference Xiong, Miyamoto and Shibata ^{75 )}, the obesity hormone, and to control inflammatory processes in adipose tissue⁽ Reference Al-Lahham, Roelofsen and Rezaee ^{76 )}, processes intimately involved in CVD risk, and possibly also impact on the way energy is stored or burnt in the body controlling adiposity and thermogenesis⁽ Reference Conterno, Fava and Viola ^{15 ,} Reference Gao, Yin and Zhang ^{77 )}.

Fig. 1. (colour online) A schematic representation of how diet shapes the human gut microbiota and impacts on chronic disease risk.

Microbial deconjugation of BA and the enterohepatic circulation of BA is a core activity of the human gut microbiota and is thought to directly regulate cholesterol levels in the blood⁽ Reference Jones, Begley and Hill ^{78 ,} Reference Jones, Tomaro-Duchesneau and Martoni ^{79 )}. Conjugated BA are secreted into the small intestine to aid micelle formation and fat absorption. About 5 % of BA may pass to the distal ileum and colon where they undergo deconjugation by the gut microbiota reducing their absorbability and increasing their loss in faeces. Since BA synthesis from cholesterol is under tight control in the liver, BA lost in faeces must be replaced driving hepatic uptake and metabolism of circulating cholesterol⁽ Reference Charach, Grosskopf and Rabinovich ^{80 ,} Reference Matsubara, Li and Gonzalez ^{81 )}. Indeed, increased loss of BA or cholesterol in faeces has long been suggested as a mode of action of dietary fibres and whole-plant foods in lowering blood cholesterol⁽ Reference Kay and Truswell ^{82 –} Reference Sembries, Dongowski and Mehrländer ^{84 )}. However, more recent studies are suggesting that this may not be the whole picture. Intriguingly, deconjugated BA may also be absorbed⁽ Reference Jones, Martoni and Prakash ^{30 )}. BA and deconjugated BA are emerging as important cell-signalling molecules, interacting with nuclear receptors such as Farnesoid X receptor (FXR), Pregnane X receptor (PXR) and Peroxisome proliferator-activated receptor alpha (PPARα) and G-protein-coupled receptors such as TGR-5, which regulate inflammation, xenobiotic detoxification, lipid metabolism, cholesterol biosynthesis and uptake, BA metabolism, glucose homoeostasis and thermogenesis⁽ Reference Matsubara, Li and Gonzalez ^{81 ,} Reference Cyphert, Ge and Kohan ^{85 –} Reference Sayin, Wahlström and Felin ^{88 )}. However, the specific mechanistic studies in animals and human dietary interventions including analysis of the gut microbiota and their metabolic activities with foods capable of modulating the enterohepatic circulation of BA (e.g. probiotics, prebiotics, polyphenols and whole-plant foods) are needed to confirm which gut bacteria are involved in BA metabolism and how their modulation through dietary means alters BA profiles entering the enterohepatic circulation and any subsequent impact on CVD risk.

Other metabolites derived from microbial metabolism in the gut have also been shown to reduce the risk of metabolic disease, at least in animal studies. The neurotransmitter γ-aminobutyric acid is also produced by gut bacteria including certain lactic acid bacteria⁽ Reference Barrett, Ross and O'Toole ^{89 )}. Gamma-Aminobutyric acid given orally is also a strong regulator of inflammation and immune function, and has been shown to reduce the risk of metabolic disease by improving insulin sensitivity through reduced inflammation in laboratory animals on a high-fat, obesogenic diet⁽ Reference Tian, Dang and Yong ^{90 )}. Certain gut bacteria, most notably the bifidobacteria, have been shown to produce folate, a key metabolite C₁ metabolism which lowers circulating levels of homocysteine, an independent risk factor of CVD⁽ Reference D'Aimmo, Mattarelli and Biavati ^{91 )}. Animal studies have shown that feeding folate-producing bifidobacteria can increase plasma folate concentrations, and that co-administration of the Bifidobacterium strain with a substrate for its growth within the intestine, the prebiotic inulin, can increase plasma folate concentrations further in a synergistic manner⁽ Reference Pompei, Cordisco and Amaretti ^{92 )}. Gut bacteria may also interact with dietary fats. We have shown that both the quantity and type of dietary fat can reshape the microbial community structure within the gut⁽ Reference Cani, Amar and Iglesias ^{16 ,} Reference Fava, Gitau and Griffin ^{18 )}. Conversely, it has been shown in a study at Teagasc in Ireland that bifidobacteria capable of biohydrogenation of fatty acids, when administered to pigs or mice along with α-linoleic acid can increase concentrations of beneficial fats, EPA, DHA and conjugated linoleic acid concentrations in extra-intestinal organs including adipose tissue, liver and brain⁽ Reference Wall, Ross and Shanahan ^{93 ,} Reference Wall, Ross and Shanahan ^{94 )}, indicating that at least certain bifidobacterial strains can alter dietary fats in vivo, and increase body stores of beneficial fats. Conjugated linoleic acid, EPA and DHA have long been studied for anti-inflammatory properties and association with reduced risk of CVD and other metabolic diseases⁽ Reference Tricon, Burdge and Williams ^{95 ,} Reference Pfeuffer, Fielitz and Laue ^{96 )} (Fig. 1).

Diet can bring out the worst of microbial behaviours: microbiome dysbiosis

Choline-deficient diets have long been used to induce insulin resistance and metabolic disease in animal models and the gut microbiota is recognised to play an important role in the co-metabolic processing of choline and phosphatidylcholine in man⁽ Reference al-Waiz, Mikov and Mitchell ^{97 ,} Reference Koteish and Mae Diehl ^{98 )}. Dumas et al.⁽ Reference Dumas, Barton and Toye ^{99 )} investigated the impact of choline metabolism disruption on the development of metabolic disease and non-alcoholic fatty liver disease in mice prone to disease (129S6) and in mice resistant to disease (BALBc) using NMR-based metabonomics. These investigators found that low plasma levels of phosphatidylcholine and increased urinary excretion of methylamines (dimethylamine, trimethylamine (TMA) and trimethylamine-N-oxide, (TMAO)) was associated with insulin resistance and fatty liver phenotype. Importantly these metabolites result from host:microbiota co-metabolic processing of choline and phosphatidylcholine⁽ Reference al-Waiz, Mikov and Mitchell ^{97 )}. Dumas et al.⁽ Reference Dumas, Barton and Toye ^{99 )} demonstrated that microbial conversion of choline into methylamines is induced by high-fat feeding and mimics the disease effects of choline-deficient diets. Other studies have shown that high-fat, low fermentable fibre diets, typically used to induce metabolic disease in laboratory animals decimate the gut microbiota. Cani et al.⁽ Reference Cani, Amar and Iglesias ^{16 )} showed that high-fat, low fermentable fibre feeding leads to significant die off in dominant and putatively beneficial gut bacteria including bifidobacteria, butyrate-producing Firmicutes and polysaccharide degrading Bacteroidetes. Interestingly, high-fat feeding did not change numbers of Gram-negative sulphate-reducing bacteria (SRB) in these experiments. Decimation of beneficial gut bacteria was accompanied by translocation of bacterial cell wall LPS across the gut wall, either through increased permeability due to reduced tight junction control or carried with fat absorbed from the gut. LPS is an inflammatory cell wall constituent of Gram-negative bacteria and triggers inflammatory cascade through Toll-like receptor-4 or CD14, and NF-κB, and in the high-fat-fed animals, plasma LPS triggers low-grade chronic inflammation and subsequent insulin resistance and body weight gain⁽ Reference Cani, Amar and Iglesias ^{16 )}. Interestingly, Zhang et al.⁽ Reference Zhang, Zhang and Wang ^{17 )} more recently showed that in another murine model of metabolic disease (ApoA-I knockout mice on high-fat diet), the SRB, Desulfovibrionaceae, showed higher relative abundance in animals with insulin resistance, especially insulin-resistant animals fed high-fat diets. In this study too, numbers of bifidobacteria were decimated by high-fat feeding. SRB, which convert mainly dietary sulphate to H₂S, are considered to play an important role in human H₂S metabolism⁽ Reference Deplancke, Finster and Graham ^{100 ,} Reference Carbonero, Benefiel and Gaskins ^{101 )}, have been implicated in inflammatory bowel disease and their LPS is highly inflammatory. More recently, in common with certain other gut bacteria, SRB have also been shown to be capable of converting choline to TMA under anaerobic conditions, using a glycyl radical enzyme⁽ Reference Craciun and Balskus ^{102 )}. TMA in human subjects is further converted into TMAO by the hepatic enzyme flavin-dependent monooxygenase 3. Interestingly, TMA, the microbial precursor of TMAO, also acts as a substrate for methanogenesis, being metabolised to inoxious CH₄, raising the intriguing possibility that modulation of the relative abundance of methanogens:SRB within the human gut might regulate the production of cardiotoxic TMAO or inoxious CH₄ gas. The relative proportions and activity of methanogens:SRB have long been of interest but mainly in relation to explaining H₂ disposal⁽ Reference Gibson, Cummings and Macfarlane ^{103 )}. Interestingly, Fernandes et al.⁽ Reference Fernandes, Wang and Su ^{104 )} have recently confirmed that methane production and carriage of detectable Archaea (mainly methanogens in human subjects) are inversely related to BMI and that methanogenesis appears to allow more efficient production and absorption of SCFA from dietary fibre fermentation. While the ecological processes regulating the relative abundance of gut SRB and methanogens are not fully understood, ingestion of fermentable fibre increases methanogenesis in CH₄-producing subjects, while sulphate or dietary protein appear to stimulate the numbers of SRB and presumably their metabolic activities within the gut microbiota⁽ Reference Christl, Gibson and Cummings ^{105 –} Reference Khalil, Walton and Gibson ^{109 )}.

In a large clinical cohort, Wang et al.⁽ Reference Wang, Klipfell and Bennett ^{13 )} showed in 2011 that plasma concentrations of choline, TMAO and betaine, all metabolites of phosphatidylcholine metabolism, are predictive of CVD. While all three up-regulated multiple macrophage scavenger receptors, choline and TMAO feeding induced arteriosclerosis in mice. Moreover, the authors confirmed the essential role of the gut microbiota in TMAO production from dietary choline in germ-free animals, while broad spectrum antibiotics lowered macrophage cholesterol accumulation, foam cell formation and aortic lesions in high-fat-fed conventional animals. Koeth et al.⁽ Reference Koeth, Wang and Levison ^{14 )} more recently showed that another compound, l-carnitine with a TMA structure similar to that of choline and commonly found in meat, may also be converted to TMA by the gut microbiota and contribute to the increased CVD risk associated with high red meat consumption⁽ Reference Bernstein, Sun and Hu ^{110 ,} Reference Micha, Michas and Mozaffarian ^{111 )}. They confirmed microbial involvement in TMA(O) formation from dietary l-carnitine using a stable isotope dilution method and broad spectrum antibiotic microbiota suppression in five healthy omnivorous subjects and in germ-free and antibiotic-treated animals. Conversion of dietary l-carnitine to TMAO did not appear to occur in long-term (>1 year) vegans or vegetarians, suggesting an adoptive response of the gut microbiota in omnivores to TMA production from l-carnitine derived from red meat. Compared with the vegans and vegetarians, omnivores had elevated plasma TMAO concentrations and significantly higher relative abundance of faecal clostridia and peptostreptococci, groups of bacteria commonly associated with protein fermentation. Furthermore, the authors confirmed the direct and dose-dependent association between fasting plasma l-carnitine levels and prevalent coronary artery disease in a large cohort of elective cardiac evaluation subjects (n 2595) and elevated l-carnitine was significantly associated with risk of major adverse cardiac events. When corrected for plasma TMAO these associations disappeared suggesting that TMAO mediated disease risk rather than l-carnitine itself. TMAO also impacted on BA metabolism in the liver at multiple levels including cholesterol transporters and suppression of BA synthetic enzymes Cyp7a1 and Cyp27a1 and animals fed TMAO had a significantly lower BA pool compared with those fed standard rodent chow.

These studies have provided a clear pathological mechanism involving the gut microbiota: mammalian co-metabolic processing of choline, phosphatidylcholine and l-carnitine which appears responsive to red meat consumption but which is much reduced or absent in vegetarians on high-fibre diets. This novel mechanism also fits neatly into existing data from epideamiological studies implicating red meat consumption in CVD risk, and whole-plant food or fibre with low risk of CVD. It also fits with the important role of the enterohepatic circulation of BA in regulating cholesterol metabolism and CVD risk via the activities of nuclear receptor FXRα and G-protein-coupled receptor TGR-5 (Fig. 1).

Reducing CVD risk with probiotics

Probiotics are defined as ‘live microorganisms which when administered in adequate amount confer a health benefit on the host’ and represent a very direct means of modulating the composition of the gut microbiota by adding exogenous microorganisms^{( 112 )}. The most common probiotics are lactobacilli and bifidobacteria⁽ Reference Tuohy, Probert and Smejkal ^{113 )}. The notion that probiotics may impact on CVD risk stems from early suggestions that yoghurt consumption may reduce blood cholesterol levels in nomadic dairying peoples⁽ Reference Hepner, Fried and St Jeor ^{114 ,} Reference Mann, Spoerry and Gray ^{115 )}. Some studies from the 1970s and 1980s found that cholesterol levels could be reduced following consumption of large volumes of yoghurt or fermented milk⁽ Reference Hepner, Fried and St Jeor ^{114 –} Reference Rossouw, Burger and Van der Vyver ^{116 )}. For example, Mann et al.⁽ Reference Mann, Spoerry and Gray ^{115 )} found that massive (4 l/d) consumption of milk fermented with a ‘wild’ strain of Lactobacillus, resulted in a significant reduction in serum cholesterol in twenty-four Maasai warriors. However, many of these early studies used milk or pasteurised yoghurt as control, confounding interpretation and also, did not characterise the microorganisms used to ferment the milk to any great extent. Similarly, studies were often conducted in healthy subjects within normal cholesterol ranges who do not alter the cholesterol levels readily in response to dietary change.

Later studies in the 1990s began to recognise that milks fermented with different lactic acid bacteria may elicit different effects (Table 1). For example, Andersson and Gilliland⁽ Reference Anderson and Gilliland ^{117 )} found a significant reduction in total cholesterol in fourteen hypercholesterolaemic subjects when consuming Lactobacillus acidophilus L1, a human-derived strain, compared with L. acidophilus ATCC 43121, isolated from the porcine gut. Agerholm-Larsen et al.⁽ Reference Agerholm-Larsen, Bell and Grunwald ^{124 )} reported a metanalysis of studies using the probiotic fermented milk Gaio^®, containing Enterococcus faecium (with the potential probiotic activity) and two strains of Streptococcus thermophilus for milk acidification. From six studies using this product, the authors concluded that short-term intake (4–8 weeks) of this fermented milk produced significant lowering of total and LDL-cholesterol (by 4 and 5%, respectively) but that further studies were required to confirm any long-term effects. Hlivak et al.⁽ Reference Hlivak, Odraska and Ferencik ^{125 )} more recently, using another strain of Enterococcus faecium, M-74 in combination with selenium, found a 12% reduction in serum cholesterol after a 1-year intervention in forty-three volunteers who consumed the probotic or placebo. Interestingly, not only is this one of the few long-term studies to confirm the cholesterol-lowering effects of probiotics, but it was also conducted with the probiotic in lyophilised powder form delivered in a capsule containing 2×10⁹ colony-forming units of E. faecium M-74. The test product however, also contained 50 mg/dose of organically bound selenium, which may independently lower serum cholesterol⁽ Reference Rees, Hartley and Day ^{126 )}. However, many other early studies have also reported no effect of probiotic intervention in lowering serum cholesterol levels. In general, early probiotic studies have rarely been conducted in large cohorts presumably because of costs associated with culture-based faecal microbiology, and seldom reached the statistical power necessary to adequately test the cholesterol-lowering potential of even the most promising strains⁽ Reference Jackson and Lovegrove ^{127 )}. Moreover, it has become apparent that probiotics or probiotic effects are strain specific with particular probiotic health effects, e.g. immune modulation, production of antimicrobial compounds or the ability to lower cholesterol being present in one strain and absent in another strain belonging even to the same species. A number of mechanisms have been proposed including binding of cholesterol within the gut lumen or incorporation of cholesterol into bacterial cell walls, production of SCFA which might modulate cholesterol synthesis in the liver or increased deconjugation of BA impacting on cholesterol absorption or increasing faecal excretion of bile driving hepatic BA synthesis from circulating cholesterol⁽ Reference Pereira and Gibson ^{128 )}. Although a few of these putative mechanisms have been shown in human subjects, they have been used in vitro as screening tools in an attempt to improve probiotic efficacy. Jones et al.⁽ Reference Jones, Martoni and Parent ^{29 )} investigated the ability of a Lactobacilus reuteri strain NCIMB 30242 microencapsulated in a yoghurt formulation to reduce the cholesterol levels in hypercholesterolaemic individuals (n 114). Importantly, this strain had been selected as a putative cholesterol-lowering probiotic because of its bile salt hydrolase activity. These authors found that consuming the yoghurt containing the microencapsulated probiotic L. reuteri NCIMB 30242 twice daily for 6 weeks with a combined daily dose of about 10¹¹ viable bacteria, decreased serum total cholesterol and LDL-cholesterol by 4·81 and 8·92%, respectively, compared with the placebo. Non-HDL cholesterol and apoB-100 were also significantly reduced compared with the placebo group. In a second study, this time using the same strain in the lyophylised form, the authors confirmed the cholesterol-lowering ability in 127 hypercholesterolaemic individuals⁽ Reference Jones, Martoni and Prakash ^{30 )}. Upon probiotic supplementation for 9 weeks, LDL-cholesterol, total cholesterol, non-HDL cholesterol and apoB-100 were significantly reduced by 11·64, 9·14, 11·30 and 8·41%, respectively. Moreover, the subjects receiving the probiotic had lower serum levels of plant sterols, a surrogate marker for cholesterol absorption and higher plasma levels of deconjugated BA, leading the authors to suggest a novel cholesterol-lowering mechanism for this strain through reduced cholesterol absorption via the action of deconjugated BA on hepatic FXRα, a nuclear receptor responsible for controlling lipid absorption from the gut by regulating the expression of lipid transporters on the gut wall. Thus it appears that by right choice of probiotic strain it may be possible to lower CVD risk through improved profiles of disease biomarkers especially plasma cholesterol levels.

Table 1. Selected studies where recognised probiotic strains, delivered in fermented milk/yoghurt or pure form, have been investigated for cholesterol-lowering effects in hypercholesterolaemic individuals (after⁽ Reference Jackson and Lovegrove ^{127 )})

CFU, colony forming units; FM, fermented milk; TC, total cholesterol; T2D, type 2 diabetes.

Reducing CVD risk with prebiotics

A certain class of fermentable carbohydrates has been identified as of particular interest for their ability to modulate the relative abundance of gut bacteria considered beneficial for human health. Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health⁽ Reference Petschow, Doré and Hibberd ^{49 ,} Reference Gibson and Roberfroid ^{129 )}. Common prebiotics include the fructans, inulin and oligofructose, lactulose, galatooligosaccharides, arabinogalactan, arabinoxylan, pectic-oligosaccharides, β-glucan and resistant starches, all of which have been shown to increase the relative abundance of bifidobacteria within the human gut microbiota⁽ Reference Tuohy, Brown and Klinder ^{22 )}. Bifidobacteria are a group of bacteria particularly equated with gut health because of their saccharolytic nature, history of use as probiotics and because they dominate the gut microbiota of breastfed infants⁽ Reference Gibson and Roberfroid ^{129 )}. In parallel, many of these compounds have long been under investigation for their ability to modify blood lipid profiles mainly because of their ability to bind cholesterol or BA in the upper gut and increase sterol excretion, or for some, through their gel-forming nature causing a bulking effect in the intestine and triggering satiety⁽ Reference Delzenne, Daubioul and Neyrinck ^{130 ,} Reference Slavin ^{131 )}. Animal studies have consistently shown that dietary intervention with prebiotics, especially the fructans inulin and oligofructose, can reduce serum TAG⁽ Reference Delzenne, Daubioul and Neyrinck ^{130 )}. Elevated TAG, together with elevated total and LDL-cholesterol, low HDL-cholesterol and elevated total cholesterol:HDL-cholesterol characterise dyslipidaemia, and represent an important modifiable risk factor for CVD. Brigenti⁽ Reference Brighenti ^{132 )} conducted a metanalysis of human studies investigating the ability of fructans to impact on serum TAG and concluded that 81% of fifteen trials with inulin or oligofructose reduced serum TAG. Although this effect was small (about 7·5% reduction in serum TAG) and only statistically significant in five of the interventions, the effect appeared to be independent of gender, type of prebiotic, background diet, overweight, dyslipidaemia or diabetes. However, the low number of studies considered in the metanalysis limited the power to adequately assess the impact of these confounders. More recently, Jackson and Lovegrove⁽ Reference Jackson and Lovegrove ^{127 )} reviewed the current evidence on the ability of prebiotics (as well as probiotics and synbiotics) to alter blood lipids including cholesterol levels. They found that the majority of prebiotic studies were conducted with either inulin or oligofructose (a hydrolytic product of inulin) and that despite ample evidence for TAG lowering and often cholesterol-lowering effects of prebiotic dietary supplementation in animals, prebiotic trials in human subjects gave favourable if inconsistent modulation in lipid levels⁽ Reference Jackson and Lovegrove ^{127 )}. Table 2 summarises dietary intervention studies in human subjects with recognised prebiotics⁽ Reference Jackson and Lovegrove ^{127 )}. Although eight out of the twenty-one studies showed that prebiotic intervention did not change the blood lipids levels, the remainder showed statistically significant lowering of lipid biomarkers of CVD risk, usually TAG and/or total cholesterol. Although, prebiotics appeared to be more efficacious in the hyperlipidaemic, more studies are needed in the specific target groups, especially those fulfilling the criteria for the metabolic syndrome and in the obese. Fermentation of prebiotics, as with other fermentable fibres, leads to the production of SCFA. Demigné et al.⁽ Reference Demigné, Morand and Levrat ^{153 )} showed that in vitro, propionate was an efficient inhibitor of cholesterol biosynthesis in rat hepatocytes when acetate was the main substrate available. This in vitro observation was confirmed later in vivo. However, acetate only becomes the main substrate for cholesterol biosynthesis in the liver when animals are fed high-fibre diets. This indicates that SCFA produced during high fibre diets are unlikely to increase circulating cholesterol levels despite increasing availability of the substrate for cholesterol⁽ Reference Jackson and Lovegrove ^{127 )}. This explains also why prebiotic interventions do not always lead to lower cholesterol in human studies, as hepatic cholesterol biosynthesis from acetate in human subjects, especially those on a low fibre, high-fat Western-style diet, is likely to contribute very little to circulating cholesterol levels⁽ Reference Jackson and Lovegrove ^{127 )}. Kok et al.⁽ Reference Kok, Morgan and Williams ^{154 )} showed that oligofructose induces the gut hormones glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 of rats fed normal chow and that this contributed to improved glucose homoeostasis and lipid metabolism. Glucose-dependent insulinotropic polypeptide induces insulin and stimulates lipoprotein lipase in adipocytes, while glucagon-like peptide-1 stimulates insulin secretion, improves insulin sensitivity and contributes to satiety by reducing gastric emptying. More recently, Cani et al.⁽ Reference Cani, Neyrinck and Fava ^{19 )} have shown that the prebiotic oligofructose can reverse metabolic endotoxemia by reducing gut permeability and LPS leakage into circulation. They also proposed the term ‘metabolic endotoxemia’ to describe the chronic low-grade systemic inflammation characteristic of obesity and type 2 diabetes⁽ Reference Cani, Amar and Iglesias ^{16 )}. Improved gut barrier activity resulted from prebiotic induction of the gut hormone glucagon-like peptide-2, a result confirmed in a later animal experiment⁽ Reference Jones, Begley and Hill ^{78 )}. More recently, Everard et al.⁽ Reference Teixeira, Grześkowiak and Franceschini ^{155 )} confirmed Cani's earlier work that the prebiotic oligofructose could reduce gut permeability and associated metabolic endotoxemia and obesity in high-fat-fed animals through induction of intestinal endocannabinoids. They also found that prebiotic oligofructose-induced microbiota modulation also resulted in increased relative abundance of Akkermansia muciniphila, a mucus dwelling and degrading intestinal commensal. The authors found that the same physiological effects could be brought about by ingestion of Ak. muciniphila alone as a ‘probiotic’, but that the effect depended on microbial viability.

Table 2. Dietary interventions investigating the ability of common prebiotics (inulin, oligofructose/fructooligosaccharides and galactooligosaccharides) to improve blood lipid profiles in human subjects (after⁽ Reference Jackson and Lovegrove ^{127 ,} Reference Brighenti ^{132 )})

TC, total cholesterol; FOS, fructooligosaccharides; scFOS, short-chain FOS; OFS, oligofructose; GOS, galactooligosaccharides; B-GOS, Bi²muno GOS; Gluc, glucose; LPa, lipoprotein(a).

However, it must be emphasised that it is often difficult to extrapolate animal data to the human situation, where the genetic background is much more diverse, diets are open and vary greatly between individuals and where other life-style factors are sometimes not reported or underreported (e.g. alcohol intake, drugs and smoking, and high fat, high sugar convenience foods). Moreover, the quantities of prebiotic used in animal studies targeting metabolic disease and cholesterol are much higher than those routinely used in human dietary interventions. 10% w/w is the typical dose used in mouse studies and equates to more than 50 g/d prebiotic for human subjects, using mean European adult male body weight⁽ Reference Reagan-Shaw, Nihal and Ahmad ^{156 )}. Interestingly, this level of fermentable fibre intake in human subjects correlates well with estimates of dietary fibre intake for ancient diets and traditional high-fibre diets in the Mediterranean, Africa and China⁽ Reference Tuohy, Gougoulias and Shen ^{31 )}.

Reducing CVD risk with whole-grain cereals: oats as a case study

Whole-grain cereals, and in particular oats and fibre fractions derived from oats, have long been considered as foods capable of reducing CVD risk by lowering blood cholesterol levels. Whole-grain oats comprise a number of different classes of biologically active molecule capable of modulating cholesterol metabolism in mammals, including mono- and di-unsaturated fatty acids, fibres such as β-glucan, arabinoxylans and resistant starch, phenolic compounds such as dehydroferulates and avenathramides, and lignans which are converted into the phytoeostrogens enterolactone and enterodiol by gut bacteria⁽ Reference Ryan, Kendall and Robards ^{157 ,} Reference Borneo and León ^{158 )}. However, early studies attributed the cholesterol-lowering effect of oats to their gel-forming nature within the gut and/or cholesterol/BA binding. β-Glucan is the major structural polysaccharides accounting for about 85% of oat cell wall and is a soluble fibre, which, depending on molecular length, forms viscous gels in aqueous solutions. Tiwari and Cummins⁽ Reference Tiwari and Cummins ^{159 )} performed a recent metanalysis on the ability of β-glucans to reduce blood cholesterol and glucose levels. Considering 126 clinical studies with different doses of β-glucans from either oats or barley, the authors concluded that there was a significant inverse relationship between β-glucan intake and blood total cholesterol, LDL-cholesterol and TAG levels. Moreover, there was a significant dose–response between β-glucan ingestion and cholesterol-lowering activity, with 3 g/d of either oat or barley β-glucan being sufficient to lower blood total cholesterol (by −0·30 mm). β-Glucan from either oats or barley are one of the few fibres to have attained health claims status with the European Food Safety Authority^{( 160 )}. While the ability of oats and β-glucans to increase excretion of BA and cholesterol in faeces appears to be well established in the literature, the underlying mechanism still remains to be determined. β-Glucans have been proposed to alter the viscosity of the unstirred layer lining the intestinal mucosa thus reducing the flow of BA towards the epithelial cell surface and reducing BA/cholesterol absorption. The gel-forming nature of β-glucans has been suggested to trap BA/cholesterol micelles thereby reducing the efficiency of cholesterol absorption, and finally, β-glucans, especially those of small molecular weight, have been suggested to bind BA, directly inhibiting their reabsorption in the small intestine and driving them distally into the colon where they are deconjugated/hydrolised by the resident microbiota⁽ Reference Gunness and Gidley ^{161 )}. Whether one or all of these mechanisms holds is still a matter of debate. However, other possibilities also exist, not least the ability of oats or β-glucan to impact on the gut microbiota and their metabolic activities. Andersson et al.⁽ Reference Andersson, Axling and Xu ^{162 )} investigated the ability of oat bran (27%) to lower blood cholesterol in two substrains of C57BL/6 mice on artherogenic diet (low choline diet), the C57BL/6 NCrl and the C57BL/6JBomTac strains, compared with control fibres. In the C57BL/6 NCrl mice, dietary intervention with oat bran reduced blood cholesterol with a concomitant increase in faecal BA excretion and increased hepatic BA synthesis. No change in cholesterol, BA excretion or synthesis was observed in the C57BL/6JBomTac. However, the C57BL/6JBomTac mice carried significantly lower intestinal populations of Enteorbacteriaceae, Akkermansia and Bacteroides fragilis than the C57BL/6 NCrl mice, and changes within the relative abundance of the gut microbiota appeared to predict cholesterol response to oat bran. This in vivo experiment suggests that the gut microbiota play a critical role in the cholesterol-lowering ability of oat bran. In vitro we have shown that different oat preparations can mediate a prebiotic modulation of the human gut microbiota, giving significant increases in bifidobacteria, lactobacilli and SCFA⁽ Reference Hughes, Shewry and Gibson ^{163 –} Reference Connolly, Tuohy and Lovegrove ^{167 )}. We also showed that β-glucan increases the relative abundance of bifidobacteria in C57BL/6 mice fed high-fat diet, and regluates food intake by suppressing neuronal signals in appetite centres of the brain⁽ Reference Arora, Loo and Anastasovska ^{20 )}. The BA sequestrant, colestyramine has been shown to trigger satiety via reduced gastric emptying in healthy human subjects⁽ Reference Psichas, Little and Lal ^{168 )}, suggesting that factors controlling the enterohepatic circulation of BA may contribute to satiety as well as regulating blood cholesterol levels. Interestingly, recent studies showing that probiotic microorganisms can reduce cholesterol uptake from the intestine via modulation of BA signalling, discussed earlier⁽ Reference Jones, Martoni and Parent ^{29 ,} Reference Jones, Martoni and Prakash ^{30 )} raise another possibility that oats and prebiotics in general (including certain polyphenols) may selectively stimulate gut bacteria capable of hydrolysing bile salts modifying the enterohepatic circulation of BA both in terms of quantity and chemical profile⁽ Reference Nokoff and Rewers ^{39 ,} Reference Jones, Tomaro-Duchesneau and Martoni ^{79 ,} Reference Ooi, Ahmad and Yuen ^{169 –} Reference Kim, Yi and Lee ^{171 )}. Intestinal lactobacilli and bifidobacteria commonly encode bile salt hydrolysing enzymes and changes in their relative abundance wrought by probiotics, prebiotics and polyphenols could increase also their relative contribution to modifying the pool of BA returning from the gut and acting as signalling molecules in the liver, the gut wall and systemically through receptors FXRα and TGR-5 to impact on lipid absorption from the intestine, lipid and glucose homoeostasis, thermogenesis and inflammation⁽ Reference Jones, Martoni and Prakash ^{30 ,} Reference Charach, Grosskopf and Rabinovich ^{80 ,} Reference Davidson, Maki and Synecki ^{146 ,} Reference Yamashita, Kawai and Itakura ^{148 ,} Reference Alles, de Roos and Bakx ^{149 )}.