Prebiotic inulin-type fructans and galacto-oligosaccharides: definition, specificity, function, and application in gastrointestinal disorders

bs_bs_banner doi:10.1111/jgh.13700 REVIEW ARTICLE Prebiotic inulin-type fructans and galacto-oligosaccharides: definition, specificity, function, and application in gastrointestinal disorders Bridgette Wilson and Kevin Whelan * Faculty of Life Sciences and Medicine, Diabetes and Nutritional Sciences Division, King s College London, London, UK Key words galacto-oligosaccharides, inulin, irritable bowel syndrome, low FODMAP, prebiotics. Accepted for publication 21 December 2016. Correspondence Professor Kevin Whelan, Diabetes and Nutritional Sciences Division, King s College London, 150 Stamford Street, London, SE1 9NH, UK. Email: kevin.whelan@kcl.ac.uk Disclosures: B. W. is undertaking a doctoral fellowship funded by Clasado Biosciences Ltd. K. W. is currently or has recently been in receipt of research funding from a range of research and charitable bodies, including the Broad Medical Research Program, British Dietetic Association, Core, Crohn s and Colitis UK, Kenneth Rainin Foundation, Medical Research Council and National Institute of Health Research, as well as industry bodies including the Californian Dried Prune Board, Clasado Biosciences Ltd., Danone, Dr Schar, and Nestle. None of these funding bodies contributed to the content or writing of this manuscript. Abstract Prebiotics are non-digestible selectively fermented dietary fibers that specifically promote the growth of one or more bacterial genera in the gastrointestinal tract and thus provide health benefit to the host. The two most investigated prebiotics being the inulin-type fructans and galacto-oligosaccharides. Prebiotic specificity is mediated through speciesspecific gene clusters within saccharolytic bacteria controlled by signaling sensors for various substrates. Prebiotic health benefits are attributed to immune regulation and bacterial metabolite production. In humans, prebiotic supplementation leads to increased growth of specific gut microbiota (e.g., bifidobacteria), immune modulation, and depending on the bacterial augmentation, short-chain fatty acid production. Irritable bowel syndrome and Crohn s disease are gastrointestinal disorders associated with reductions in some gut bacteria and greater mucosal inflammation. Prebiotic supplementation studies have shown some promise at low doses for modulation of the gut bacteria and reduction of symptoms in IBS; however, larger doses may have neutral or negative impact on symptoms. Studies in Crohn s disease have not shown benefit to bacterial modulation or inflammatory response with prebiotic supplementation. Dietary restriction of fermentable carbohydrates (low FODMAP diet), which restricts some naturally occurring prebiotics from the diet, has shown efficacy in improving symptoms in irritable bowel syndrome, but it lowers the numbers of some key gut microbiota. Further research is required on the effect of prebiotics in gastrointestinal disorders and, in particular, on their use in conjunction with the low FODMAP diet. Introduction The prebiotic effect is defined as the selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus (era)/species in the gut microbiota that confer(s) health benefits to the host. 1 While much attention is now focussed on dietary restriction of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (low FODMAP diet) for the management of functional gastrointestinal symptoms, the removal of these naturally occurring prebiotic fibers has a marked impact on the gut microbiome with unknown long-term health effects. The aim of this review is to discuss the definition, mechanisms, function, and application of prebiotics in relation to gastrointestinal disorders, with a specific focus on irritable bowel syndrome (IBS) and Crohn s disease. Prebiotics. The key characteristics of a prebiotic are that they are as follows: (1) non-digestible by endogenous enzymes in the human gut; (2) selectively fermented by specific genera/species of resident gut microbiota; and (3) that this results in a targeted increase in specific bacteria that confers health benefits to the host. 1 These health benefits are diverse and can include immune modulation through increased intestinal-specific immunoglobulins and immuno-regulatory interleukins, and a reduction in proinflammatory interleukins. Health benefits also include the production of short-chain fatty acids acetate, propionate, butyrate and lactate that reduce luminal ph, which in turn, may be important in preventing colonization with acid-sensitive enteropathogens. Acetate production also contributes, through cross-feeding, to the production of butyrate, which is a primary substrate for colonocytes thus contributing to epithelial integrity. 1 Prebiotics are usually dietary carbohydrates. The two major carbohydrates that fulfill the criteria for prebiotics are inulin-type fructans and the galacto-oligosaccharides (GOS), although many other classes are under investigation. Inulin-type fructans are polymers of fructose linked by β-2,1 bonds with a terminal α-linked glucose. Longer chains are inulin (degree of polymerization, 2 60), and shorter chains are oligofructose/fructo-oligosaccharides (FOS) (degree of 64 Journal of Gastroenterology and Hepatology 2017; 32 (Suppl. 1): 64 68

B Wilson and K Whelan Prebiotic fructans and GOS polymerization, 2 8). 1 Inulin-type fructans can be extracted from plants (e.g., inulin, oligofructose), produced from partial hydrolysis of inulin (oligofructose), or synthesized enzymatically from sucrose (FOS). They have been shown in various studies to promote the growth of bifidobacteria, bacteroides, and lactobacilli. 1 Prebiotic GOS are polymers of galactose with a terminal glucose monomer. The prebiotic GOS, termed β-gos, have terminal β-linked glucose and are commercially produced using enzymatic transferase activity of β-galactosidases on lactose (degree of polymerization 2 8). 2 These differ from the type of GOS commonly found in the generative part of plants such as beans and pulses that consist of α-linked galactose, α-linked glucose, and a terminal β-linked fructose (e.g., raffinose, stachyose, and verbascose). 3,4 Because of the three types of bonds in plant GOS, they can be fermented by any bacteria possessing any of the three enzymes able to digest them, explaining why it has been demonstrated in vitro that plant GOS is less selective for specific bacterial growth than β-gos. 5 Prebiotic research has therefore largely focused on β-gos, which specifically promotes the growth of bifidobacteria. Prebiotic bacterial specificity. Bacteria have differing specificity for the prebiotics that they can utilize. Specific gene clusters within the bacterial genome dictate the saccharolytic enzymes that the bacteria can produce and, therefore, whether they can metabolize the prebiotic substrate. Table 1 outlines the basis of bacterial specificity for the most common prebiotics. The genomic determinants of prebiotic fermentation are only recently being investigated and mostly for the bacteroides genus. For example, Bacteroides thetaiotaomicron contains 88 polysaccharide utilization loci genes that dictate the type and variety of substrate it can metabolize. 6 These loci are controlled by 33 genes encoding hybrid two-component (HTC) signaling sensors. One of these gene clusters, the fructan utilization locus, is conserved to varying degrees across the Bacteroides genus and is controlled by a fructose-binding HTC signaling sensor. Deletion mutation of the fructan HTC signaling sensor gene prevents B. thetaiotaomicron from growing on fructan-containing media in vitro, indicating the key role of this sensor in fructan utilization. To further demonstrate genetic specificity for inulin utilization, in vivo studies used gnotobiotic mice co-inoculated with Bacteroides caccae (rapid inulin fermenter) and B. thetaoitaomicron (poor-inulin fermenter) fed an inulin-only diet. 6 B. caccae outcompeted the B. thetaiotaomicron; however, transfer of the gene locus coding for inulin utilization from the B. caccae to the B. thetaiotaomicron nullified this effect, demonstrating that this gene cluster contains the required genomic machinery for efficient and preferential inulin utilization. Numerous studies have demonstrated that supplementation with inulin-type fructans increases the growth of bifidobacteria in healthy humans. 1 A double-blind placebo-controlled crossover study of 30 healthy volunteers supplemented with 5 or 8 g/d inulin for 2 weeks increased bifidobacteria with both doses. However, there were more responders with 8 g/d. A higher number of bifidobacteria at baseline led to a reduced response to supplementation indicating that inulin supplementation has a more marked effect on those with low initial concentrations of bifidobacteria. 7 Genetic mechanisms for β-gos specificity have been identified within bifidobacteria. Bifidobacteria express higher activity of β-galactosidase, the saccharolytic enzyme for digestion of the β-linked galactose, than many other members of the gut microbiota. In addition, most strains of bifidobacteria produce more than one isotype of these enzymes, which would indicate that bifidobacteria have a rapid and broad enzyme response to the presence of this type of prebiotic. 2 In vitro studies suggest that bifidobacteria internalize some oligosaccharides prior to digestion. Oligosaccharides and disaccharides have been shown to be preferentially utilized by some bifidobacteria before glucose in mixed media. 2 In addition, a Bifidobacterium adolescentis enzyme, β-galactosidase II, which digests longer chain oligosaccharides, contains no transmembrane sequence indicating that the enzyme is not externalized, and therefore, metabolizes only internalized oligosaccharides. 8 The internalization of prebiotic oligosaccharides prior to bacterial digestion may prevent scavenging by other opportunistic bacteria. To confirm the growth specificity of β-gos, a faecal homogenate study used variable prebiotic substrates at a concentration of 1% (w/v) and found that β-gos specifically increases the maximum specific growth rate for Bifidobacterium spp without increasing growth of other bacteria. 5 β-gos specifically increases the growth of bifidobacteria in vivo. A study compared two types of β-gos at doses of 7 g/d in a randomized cross-over control study in 59 healthy volunteers and showed that both had a significant bifidogenic effect. 9 Supplementation with 5.5 g/d β-gos in a double blinded 10-week crossover trial in 40 elderly human volunteers showed that there were significant increases in bifidobacteria. 10 Prebiotics and immune modulation. The impact of prebiotics on specific bacterial growth and metabolism appears to have an impact on immune signaling as shown by in vivo Table 1 Prebiotics, the major bacterial groups that degrade them and the fermentation products Prebiotics Sources Bacterial enzyme required to hydrolyse prebiotic Major bacterial groups with specificity for this prebiotic and their fermentation products Inulin type fructans β-fructofuranosidase (fructanase) Bifidobacteria (acetate, lactate); Inulin Chicory (inulin) Lactobacilli (lactate); Oligofructose Enzymatic hydrolysis of inulin (oligofructose) Bacteroides (acetate, propionate) Fructo-oligosaccharides Enzymatic synthesis from sucrose (FOS) β-galacto-oligosaccharides Enzymatic trans-galactosylation of lactose β-galactosidase Bifidobacteria (acetate, lactate) Journal of Gastroenterology and Hepatology 2017; 32 (Suppl. 1): 64 68 65

Prebiotic fructans and GOS B Wilson and K Whelan studies in mice and in humans. Faecal IgA is increased in Balb/c mice fed FOS at 3% and 5% of dietary intake. 11 Other shorter chain inulin-type fructans have been shown to increase faecal IgA in murine studies, but inulin does not. 11 Gnotobiotic mice have twofold fewer IgA-positive cells than wild-type mice indicating the importance of the gut microbiota in the production of this immunoglobulin. In addition to immunoglobulin stimulation, interleukin-1β production by peritoneal macrophages was suppressed in the FOS-fed mice. IL-1β is one of the first cytokines released following macrophage stimulation; it may be that this blunted response indicates an immuno-regulatory effect of FOS consumption. 11 The immune-modulating effects of inulin-type fructans and GOS prebiotics have been investigated in humans. In healthy humans, inulin-type fructans modulate numerous gut immune markers in multiple studies. 12 Faecal IgA is increased, Peyer s patches express greater IL-10 and IFN-γ, and activity of immune cells in the spleen is elevated. β-gos has also been shown to impact on immune function. In the previously described β-gos supplementation trial in elderly subjects, the immune-regulatory cytokine IL-10 was increased, and IL-1β stimulation was decreased compared with placebo. 10 Microbial effects of dietary prebiotics. Human milk oligosaccharides, found naturally in human breast milk, have some structural similarities to β-gos and lead to greater number of bifidobacteria in breastfed babies. 1 However, whilst inulin-type fructans occur naturally within many plant-based foods, no studies have investigated whether naturally occurring prebiotics in foods specifically impact the gut microbiota. However, there is evidence that diets high in plant-based products and dietary fiber, which would, therefore, be high in naturally occurring non-digestible oligosaccharides, do impact the gut microbiota. Two studies comparing a western diet to rural African diet in both adults and children found a high-fiber diet in the African populations was associated with a greater bacteroides to firmicutes ratio (increased by twofold in children and 4.7-fold in the adults) and greater butyrate content of stool. 13,14 In the adult study, this was associated with lower biomarkers for inflammation and cancer-associated proliferation markers. The effect of a high fiber diet on the gut microbiota is not specificand, therefore, does not fit the definition of being prebiotic as such. However, the potential benefit to health should not be discounted. Prebiotics in gastrointestinal disorders. A shift from a stable intestinal environment occurs when the gut microbiota community is temporarily or permanently altered and is termed dysbiosis. Factors that may lead to dysbiosis include antibiotics, diet, host immune system, inflammation, and infectious gastroenteritis. 15 Dysbiosis is seen in numerous gastrointestinal disorders including IBS, and Crohn s disease, and may play a key role in their pathogenesis and possibly in management. Some of the common features of dysbiosis in IBS and Crohn s disease are reduced microbial diversity, lower bifidobacteria, lower bacteroides to firmicutes ratio in IBS and in Crohn s disease, and decreased Faecalibacterium prausnitzii. Prebiotics in irritable bowel syndrome. Irritable bowel syndrome is a chronic functional bowel disorder whose etiology is not entirely understood, although a role for the gut microbiota exists, including previous gastroenteritis, dysbiosis, alterations in colonic gas production, and low-grade inflammation have all been identified in some people with IBS. Luminal bifidobacteria are negatively associated with pain in both healthy controls and IBS, 16,17 and bifidobacteria have been found to be lower in IBS compared with healthy controls. 1 Therefore, as a therapeutic target, specific prebiotic-stimulated growth of bifidobacteria is promising. However, to date, there have been few randomized control trials (RCTs) investigating the effect of prebiotics on IBS. Two studies in adults with IBS at doses of 6 g/d of oligofructose and 20 g/d of inulin showed no improvement in symptom or stool output measures. Another trial showed an improvement in composite symptom score with 5 g/d of short-chain FOS in the per-protocol population, but this was not analyzed intention to treat, with a high non-compliance rate and only 50/105 being included in the per protocol analysis. 18 One single-center 12-week parallel crossover trial, which used a β-gos, showed a dose-dependent stimulation of bifidobacteria at 3.5 and 7.0 g/d. Symptom relief measured as a global assessment was significantly improved compared with placebo in both groups receiving the prebiotic, but the lower dose resulted in lower symptoms scores flatulence, bloating, and stool consistency when individual symptoms were assessed and clustered. 19 Therefore, the type and dose of prebiotics are likely to be important when considering prebiotic use in IBS. Prebiotics in Crohn s disease. Crohn s disease is an inflammatory bowel disease caused by an inappropriate mucosal inflammatory response against commensal gut microbiota in genetically susceptibility individuals. 18 Animal studies demonstrate the effectiveness of inulin-type fructan prebiotics in treating Crohn s disease via beneficial modulation of the gut microbiota, 18 but the evidence in humans is less convincing. A pilot study of 10 patients showed promise using 15 g/d oligofructose/inulin for 3 weeks in active Crohn s disease leading to increased bifidobacteria and reduction in disease activity (Harvey Bradshaw Index) compared with baseline. 20 Two large RCTs using 15 g/d oligofructose/inulin in 103 patients with active Crohn s disease 21 and 20 g/d oligofructose/inulin in 67 patients with mild/moderately active or inactive disease 22 did not confirm this finding. Furthermore, the prebiotic group in the first study had greater severity of symptoms and, in the second study, had a trend toward greater withdrawal rates due to symptoms. 21,22 Therefore, high-dose inulin-type fructans prebiotics may actually worsen symptoms in active Crohn s disease, perhaps explaining why they also consume lower amounts of naturally occurring fructans and rarely use prebiotic supplements. 23,24 Contrasting approaches of prebiotics and the low FODMAP diet. While using prebiotics to enhance the growth of bifidobacteria seems logical to reduce symptoms of IBS, studies are limited by the type and dose of prebiotic used. On the other hand, there is growing evidence for the mechanisms and efficacy of the low FODMAP diet for treating functional gastrointestinal symptoms in IBS 25 and possibly in inflammatory bowel disease. 26,27 Inulin, oligofructose, and GOS are all restricted in this diet. Two RCTs provide clinical evidence of the efficacy of the low FODMAP diet in the management of symptoms in 66 Journal of Gastroenterology and Hepatology 2017; 32 (Suppl. 1): 64 68

B Wilson and K Whelan Prebiotic fructans and GOS IBS. 28,29 However, bifidobacteria were reduced in both studies (significantly so in the first), and stool ph was higher after the low FODMAP diet in the second study. These alterations in the gut microbiota may be of concern if raised colonic ph enables enteropathogenic colonization. Use of probiotics or prebiotics with narrow specificity for non-gas forming bifidobacteria concurrently with the low FODMAP diet may be able to modulate gut microbiota so long as this does not significantly reduce the efficacy of symptom improvement in IBS. There are limited studies of the use of the low FODMAP diet in managing functional gastrointestinal symptoms in Crohn s disease, 26,27,30 and only one has investigated the effect on the gut microbiota. 30 Conclusion Prebiotics have potential benefits in maintaining diverse gut microbiota, providing substrate and promoting the growth of specific saccharolytic bacteria that produce metabolites (e.g., short-chain fatty acids) that are beneficial for gut health. Most prebiotic studies in humans are in healthy populations and not in gastrointestinal disease. In IBS, there are a limited number of clinical trials of prebiotics. Although the trials have numerous limitations, beneficial effects, if they exist, may relate to the type and dose of prebiotics used. Two large RCTs in active Crohn s disease showed no impact of prebiotics on the microbiota and indeed worsened symptoms, albeit at high daily doses (15 and 20 g/d) and only using inulintype fructans. 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