Gastrointestinal microbiology - part 8 ppsx

simultaneous administration of other competing organisms. In all these future experiments, it might be wise to keep in mind that it is a constant “trialogue” of interactions between intestinal microbes, epithelium, and GALT. As pointed out elsewhere (6) these interactions are probably dynamic, reciprocal, and combinatorial, making it difficult to separate out a single tune in this cacophony of noise. Utilization of gnotobiotic animals might represent a suitable reductionistic “noise filter,” allowing us to study host-microbe cross-talks in greater details. For more information on the role of the intestinal microbiota on the immune system, see the chapter by Moreau elsewhere in this book. CONCLUSION For more than a century, germ-free and gnotobiotic animals have been used to investigate the influence of the intestinal microbiota and specific members of the intestinal microbiota on the functioning and health of the host. This has provided much insight into the intricate relation between the host and its microbes. However, as outlined above, much still remains to be studied, and germ-free animals will remain an important tool in the study of the interactions between the intestinal microbiota and the host. REFERENCES 1. Nuttal GHF, Thierfelder H. Tierisches leben ohne Bakterien im Verdauungskanal. Hoppe Seyler’s Zeitsch Physiol Chemie 1896–1897; 33:62–73. 2. Gustafsson BE. Lightweight stainless steel systems for rearing germfree animals. Ann N Y Acad Sci 1959; 78:17–28. 3. Midtvedt T. Influence of antibiotics on biochemical intestinal microflora-associated characteristics in man and animals. In: Gillessen G, Opferkuch W, Peters G, Pulverer G, eds. The Influence of Antibiotics on the Host-Parasite Relationship III. Berlin, Heidelberg: Springer Verlag, 1989:209–215. 4. Midtvedt T, Bjo ¨ rneklett A, Carlstedt-Duke B, et al. 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Born Germ-Free—Microbial Dependent 283 16 Modifying the Human Intestinal Microbiota with Prebiotics Ross Crittenden The Preventative Health Flagship, Food Science Australia, Werribee, Victoria, Australia Martin J. Playne Melbourne Biotechnology, Hampton, Victoria, Australia INTRODUCTION The aim of both prebiotic and probiotic functional food ingredients is to improve the health of consumers by selectively altering the composition and/or activity of microbial populations within the gastrointestinal tract. While the probiotic approach endeavors to directly deliver supplemental beneficial bacteria to the gut, prebiotics offer an alternative strategy. Rather than supplying an exogenous source of live bacteria, prebiotics aim to selectively stimulate the proliferation and/or activity of desirable bacterial populations already resident in the consumer’s intestinal tract. The prebiotic strategy offers a number of practical and theoretical advantages over modifying the intestinal microbiota using probiotics or antibiotics. This chapter aims to provide an overview of the prebiotic approach, modes of action, and an evaluation of their effectiveness in modulating intestinal microbial populations and providing health benefits to consumers. The production, properties and applications of prebiotics are outlined and likely future developments in prebiotics are discussed. However, before exploring the concept of modifying the intestinal microbiota using prebiotics, it is perhaps pertinent to first reflect briefly on why we might want to alter the composition and activity of the intestinal microbiota in the first place. WHY MODIFY THE INTESTINAL MICROBIOTA? Far from being inconsequential to our lives, the bacteria residing within our gastrointestinal tracts are highly important to our health and well-being. They provide us with a barrier to infection by intestinal pathogens (1), much of the metabolic fuel for our colonic epithelial cells (2), and contribute to normal immune development and func- tion (3,4). Intestinal bacteria have also been implicated in the etiology of some chronic diseases of the gut such as inflammatory bowel disease (IBD) (5,6). As we age, changes 285 occur in the composition of the intestinal microbiota that may contribute to an increased level of undesirable microbial metabolic activity and subsequent degenerative diseases of the intestinal tract (7,8). Modifying the composition of the intestinal microbiota to restore or maintain a beneficial population of micro-organisms would appear to be a reasonable approach in cases where a deleterious or sub-optimal population of micro-organisms has colonized the gut. The difficulty facing intestinal microbiologists is trying to determine what constitutes a “normal,” healthy intestinal microbiota. A switch in recent years from culture-based, phenotypic examination of microbial ecosystems to the application of culture- independent, molecular techniques has helped speed progress. It has also provided new insights into the great diversity of bacteria within the human intestinal tract. Historical estimates based on culture methods did recognize the complexity of the ecosystem, placing the number of bacterial species within the gastrointestinal microbiota at around 400, dominated by perhaps 30–40 (9). However, it is now believed to be far richer, with the number of identified taxa expected to eventually exceed 1000 (10). It is clear that we are only at the very beginning of understanding the role of individual bacterial populations in health and disease and their interactions with each other, the host, and the diet. Addressing these fundamental questions is an essential prerequisite to targeted disease intervention strategies involving modification of the intestinal microbiota. While acknowledging that the science of manipulating the intestinal microbiota to achieve improved health is still very much in its infancy, progress is being made, and strategies that may lead to tangible health benefits in specific populations are emerging. THE PREBIOTIC STRATEGY TO MODIFYING THE INTESTINAL MICROBIOTA For a variety of reasons, the two bacterial genera most often advocated as beneficial organisms with which to augment the intestinal microbiota are lactobacilli and bifidobacteria, both of which are common members of the human intestinal microbiota (11,12). These bacteria are numerically common, non-pathogenic, non-putrefactive, non- toxigenic, saccharolytic organisms that appear from available knowledge to provide little opportunity for deleterious activity in the intestinal tract. As such, they are reasonable candidates to target in terms of restoring a favorable balance of intestinal species. While the probiotic strategy aims to supplement the intestinal microbiota via the ingestion of live bacteria, the prebiotic strategy aims to stimulate the proliferation and/or activity of beneficial microbial populations already resident in the intestine. The characteristics shared by all successful prebiotics is that they remain largely undigested during passage through the stomach and small intestine and selectively stimulate only beneficial populations of bacteria in the colon. That is not to say that prebiotics cannot be theoretically designed to target bacteria within the stomach and small intestine, but rather those currently developed tend to target bifidobacteria, which predominantly reside in the colon. Importantly, prebiotics should not stimulate the proliferation or pathogenicity of potentially deleterious micro-organisms within the intestinal microbiota. To date, most prebiotics have been non-digestible carbohydrates, particularly oligosaccharides. Since the prebiotics identified to date promote the proliferation of bifidobacteria in particular, they are often referred to as bifidogenic factors or bifidus factors. Historically, lactobacilli and bifidobacteria have been targeted as beneficial organisms with which to augment the intestinal tract. However, as discussed later in this chapter, the manipulation more broadly Crittenden and Playne286 of the metabolic activity of the microbiota is of increasing interest for improving intestinal health (13). A number of largely prophylactic health targets have been proposed for prebiotics that, as might be expected, overlap considerably with the targets of probiotic interventions. The mechanisms of action remain largely theoretical, but rational hypotheses have been developed as our understanding of the intestinal microbiota has advanced. Proposed benefits in the gut include protection against enteric infections, increased mineral absorption, immunomodulation, trophic and anti-neoplastic effects of short chain fatty acids (SCFA), fecal bulking, and reduced toxigenic microbial metabolism (Figs. 1–4). A BRIEF HISTORY OF THE DEVELOPMENT OF BIFIDUS FACTORS AND PREBIOTICS Bifidogenic or bifidus factors were recognized as early as 1954 with Gyorgy et al. (14,15) describing such components in milk and colostrum, including a range of amino sugars and non-glycosylated casein peptides. Glycoproteins from whey were also shown to have bifidogenic potential (16) along with lactoferrin (17,18). Bifidogenic effects have been reported for pantethine from carrot extracts (19,20) and for 2-amino- 3-carboxy-1,4-naphthoquinone (ACNQ), a compound isolated from Propionibacterium freudenreichii (21,22). Interest in bifidogenic compounds accelerated with the identification of non- digestible oligosaccharides (NDOs) in human milk as major factors responsible for maintaining an intestinal microbiota numerically dominated by bifidobacteria in breast- feeding infants. In contrast, infants fed cow’s milk-based formula developed a mixed microbiota, including higher levels of potentially deleterious organisms (23,24). Human DC IL-10 TGF-β IFN-γ M cell prebiotics of bifidobacteria allergy prevention immunomodulation colonocytes lamina propria IL-12 suppression IL-4 IgE-mediated allergy stimulate growth intestinal lumen TrTr Th1 Th2 Figure 1 Proposed mechanisms of immunomodulation by prebiotics for the prevention of IgE- mediated food allergies that are mediated by a skewing of the immune response at the T helper (Th) cell level towards a Th2 response. Prebiotics stimulate the growth of bifidobacteria that are sampled by the gut-associated lymphoid tissue via M-cells or dendritic cells (DC). The commensal bacteria drive a counterbalancing Th1 response producing interferon-g (IFN-g), and/or a tolerogenic response by regulatory T-cells (Tr) producing the anti-inflammatory cytokines interleukin-10 (IL-10) and transorming growth factor-b (TGF-b) that quell the allergenic Th2 response. Modifying the Human Intestinal Microbiota with Prebiotics 287 milk oligosaccharides (HMOs) (discussed later in this chapter) were then, and remain today, too complex to be synthesized commercially. However, other NDOs were shown to replicate the bifidogenic effect of milk oligosaccharides. The Japanese research community in particular studied the ability to modify the intestinal microbiota using lactulose and fructo- and galacto-oligosaccharides. Although often lacking rigorous design, early studies (25–30) at least provided the impetus for later, randomized controlled studies that have demonstrated the notion that some NDOs selectively promote the proliferation of bifidobacteria in the intestinal tract. Concurrently in the late 1980s and early 1990s, interest was rising in the use of probiotics to modify the intestinal microbial balance. The term “prebiotic” was coined by Tr DC M cell Tr prebiotics colonization resistance immunomodulation anti-inflammatory intestinal lumen lamina propria SCFA trophic effects on mucosa eliminate pro-IBD microbial antigens colonocytes suppress inflammation IBD TGF-β IL-10 stimulate growth and fermentation Figure 2 Proposed mechanisms by which prebiotics may ameliorate inflammatory bowel disease (IBD). Abbreviations: DC, dendritic cell; IL, interleukin; SCFA, short chain fatty acids; Tr, regulatory T cell; TGF, transforming growth factor. prebiotics antimicrobials antagonism SCFA lower intestinal pH nutrient and niché competition selective growth and fermentation block ligand adhesion to mucosa Figure 3 Proposed mechanisms by which prebiotics may enhance colonization resistance against bacterial pathogens in the gastrointestinal tract. Abbreviation: SCFA, short chain fatty acids. Crittenden and Playne288 Gibson and Roberfroid in 1995 (31) and effectively linked these two concepts for promoting beneficial populations of intestinal bacteria. Gibson and Roberfroid (31) broadened the narrow bifidogenic target to include the specific stimulation of any potentially beneficial microbial genera. There is an obvious potential for synergy between prebiotic and probiotic ingredients, and hence, foods containing both prebiotic and probiotics ingredients were termed “synbiotics.” CURRENTLY AVAILABLE PREBIOTIC CARBOHYDRATES The prebiotics most commonly used as functional food ingredients are non-digestible oligosaccharides (NDOs), of which a variety of types are commercially available (32). Most of these NDOs are natural components of many common foods including honey, milk, and various fruits and vegetables (32–34). Commercially, they are produced as food ingredients by four main processes: 1. Extraction and purification from plants, e.g., soybean oligosaccharides and inulin from chicory 2. Controlled enzymatic degradation of polysaccharides, e.g., xylo-oligosacchar- ides, isomalto-oligosaccharides, and some fructo-oligosaccharides 3. Enzymatic synthesis from disaccharides, e.g., some fructo-oligosaccharides, galacto-oligosaccharides and lactosucrose (32,33) 4. Chemical isomerization, e.g., lactulose. Ca ++ Mg ++ prebiotics selective fermentation alleviate constipation increased mineral absorption antagonism of putrefactive bacteria fewer toxic microbial metabolites SCFA lower intestinal pH reduced cancer ris k reduced cancer risk trophic and anti-neoplastic effects induce peristalsis de novo lipo genesis controlled serum lipids and cholesterol increased fecal bulk Figure 4 Proposed mechanisms by which the selective fermentation of prebiotics and subsequent production of short chain fatty acids (SCFA) improve bowel habit, increase dietary mineral absorption, and may reduce the risk of colon cancer. Modifying the Human Intestinal Microbiota with Prebiotics 289 In nearly all cases, the commercial oligosaccharide products contain a range of oligosaccharide structures of differing molecular weights and often with a variety of glycosidic linkages between sugar moieties. To date, the largest number of reported studies and the most consistent evidence accumulated for prebiotic effects have been for fructo-oligosaccharides and the polyfructan inulin (34–39). Good evidence from human studies also exists for the prebiotic activities of galacto-oligosaccharides (40–43) and lactulose (44–47). Boehm and Stahl (48) have summarized 28 of the human studies conducted on the physiological effects of galacto-oligosaccharides and fructans (fructo- oligosaccharides and inulin). Most of these studies were between one and three weeks in duration. Commercial food-grade oligosaccharide was fed at between 8 and 15 g/day in most experiments. Higher levels (40 g/day) were fed when inulin was used. They list 14 trials on galacto-oligosaccharides involving 298 adults and 27 infants, and another 14 with fructans involving 238 adults and 34 infants. In nearly all cases, only healthy volunteers were tested. A number of other NDOs, to which less rigorous study has been so far applied, have at least indications of prebiotic potential. These include lactosucrose (49–52), gluco- (53), xylo- (54,55), isomalto- (56–59), and soybean oligosaccharides (60–63). Additionally, bifidogenic effects have been reported for lactitol (45), polydextrose (64) and glucono-d- lactone (65) in small human feeding studies. Evidence that some dietary fibers, such as resistant starches (66–72), arabinoxylan (73,74) and plant gums (75) have prebiotic potential is accumulating, but to date remains limited largely to in vitro and animal studies. These large carbohydrates may have some advantages in the intestinal tract over rapidly fermented oligosaccharides. They minimize rapid gas formation and osmotic effects in the gut, which can lead to intestinal discomfort, flatulence and diarrhea at high doses of NDOs (typically above 15–20 g per day). Additionally, they persist as substrates for saccharolytic fermentation more distally in the colon where carbohydrate limitation is believed to promote toxigenic microbial reactions leading to an increased risk of colorectal cancer (76–79). The molecular structure of the prebiotic can be expected to determine its physiological effects as well as which microbial species are able to utilize it as a carbon and energy source in the bowel. However, it appears that despite the diversity in molecular sizes, sugar compositions, and structural linkages within the range of prebiotic carbo- hydrates, it is the bifidobacteria that are almost universally observed to respond. Some established and emerging prebiotics, including lactulose (46), galacto-oligosaccharides (40,80,81) and resistant starches (69,71) have been sporadically reported to stimulate intestinal Lactobacillus populations. Indeed, some lactobacilli have been shown to possess the metabolic machinery to use fructo-oligosaccharides (82,83). Despite this, bifidobacteria remain the major beneficiaries of these substrates in the gut. Given the benefits attributed to probiotic lactobacilli, the development of novel prebiotics directly targeting Lactobacillus species remains an opportunity. The rise in these beneficial bacterial populations during prebiotic feeding has often been shown to be accompanied by concomitant reductions in the numbers of putrefactive organisms such as clostridia and Bacteroides spp. and Enterobacteriaceae (31,44–46,60,84), possibly due to antagonism by SCFA production, acidification of the colonic environment, or direct antagonism (Figs. 3–4). 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