- Email: [email protected]
Prebiotics
Best Practice & Research Clinical Gastroenterology Vol. 18, No. 2, pp. 287 –298, 2004 doi:10.1053/ybega.2004.445, available online at http://www.sciencedirect.com
5 Prebiotics Thea Scantlebury Manning*
BSc, PhD
Research fellow
Glenn R. Gibson
BSc, PhD
Professor and Head of Food Microbial Science Unit Food Microbial Science Unit, School of Food Biosciences, Reading University, Science and Technology Center, Earley Gate, Whiteknights Road, Reading RG6 6BZ, UK
In nutritional sciences there is much interest in dietary modulation of the human gut. The gastrointestinal tract, particularly the colon, is very heavily populated with bacteria. Most bacteria are benign; however, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders. Bifidobacteria and lactobacilli are thought to be beneficial and are common targets for dietary intervention. Prebiotic is a non-viable food ingredient selectively metabolized by beneficial intestinal bacteria. Dietary modulation of the gut microflora by prebiotics is designed to improve health by stimulating numbers and/or activities of the bifidobacteria and lactobacilli. Having an ‘optimal’ gut microflora can increase resistance to pathogenic bacteria, lower blood ammonia, increase stimulation of the immune response and reduce the risk of cancer. This chapter examines how prebiotics are being applied to the improvement of human health and reviews the scientific evidence behind their use. Key words: prebiotic; oligosaccharides; gut microflora; bifidobacteria; lactobacilli functional food.
Biological functions of the human large intestine include waste storage (and its excretion) and the absorption of water as well as essential minerals. However, because of a slow transit time, near-neutral pH and high substrate availability, the colon harbours a very complex and diverse bacterial microflora.1 The microflora in the human large intestine is thought to compromise about 95% of total cells in the body, representing 1012 cells/g dry weight contents, making the organ a highly specialized and active area of the body. Through the activities of the resident microflora, the colon plays a major role in host nutrition and welfare.2 Dietary modulation of the human gut flora can be of some benefit to health. In recent years, the functional food concept has moved towards the situation whereby improved gut (microbial) functionality is the main current driving force. The colon is by far the most intensely populated region of the gastrointestinal tract and is therefore a major target for dietary intervention. * Corresponding author. Tel.: þ44-118-935-7220; Fax: þ44-118-935-7222. E-mail address: [email protected] (T. S. Manning). 1521-6918/$ - see front matter Q 2003 Elsevier Ltd. All rights reserved.
288 T. S. Manning and G. R. Gibson
The gut microflora ferments a range of substances, mainly provided by the diet, that cannot be digested by the host in the small intestine and are available for fermentation by the colonic microflora. These include resistant starch, non-starch polysaccharides (dietary fibre), oligosaccharides, proteins, amino acids, etc. In a typical adult, about 100 g of food ingested each day reaches the large intestine and is therefore susceptible to fermentation by the gut flora. The two main types of anaerobic fermentation that are carried out in the gut are saccharolytic and proteolytic. The main end-products of carbohydrate metabolism are the short-chain fatty acids (SCFAs), principally acetate, propionate and butyrate. These may be further metabolized systemically or locally to provide energy generation for the host. The end-products of proteolytic fermentation include phenolic compounds, amines and ammonia, all of which are toxic. The proximal colon (right side) is essentially a site of saccharolytic fermentation, whereas the more distal (left side) sees a depletion of available carbohydrate and more protein metabolism. Dietary modulation of the human gut microflora is a popular area of the nutritional sciences. This is driven by the fact that the gastrointestinal tract, particularly the colon, is very heavily colonized and that the composition of the flora can be modulated. Undoubtedly, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders. However, bifidobacteria and lactobacilli are considered to be examples of health-promoting constituents of the microflora. Lactobacilli may aid digestion of lactose in lactose-intolerant individuals, reduce constipation and infantile diarrhoea, help resist infections such as salmonellae, prevent traveller’s diarrhoea and help to relieve irritable bowel syndrome (IBS).3 Bifidobacteria are thought to stimulate the immune system, produce B vitamins, inhibit pathogen growth, reduce blood ammonia and blood cholesterol levels, and help to restore the normal flora after antibiotic therapy.4 Health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens, cholesterol reduction and lowering of gas distension.4 Harmful effects are carcinogen production, intestinal putrefaction, toxin formation, diarrhoea/constipation, liver damage and intestinal infection. Bifidobacteria and lactobacilli are therefore common targets for dietary intervention that improves health. GENERAL ASPECTS OF PREBIOTICS Dietary modulation of the human gut flora has been carried out for many years. In humans, there are positive aspects to the gut fermentation. For instance, bifidobacteria and lactobacilli may help to reduce the risk of disease for the reasons given above.2,5 The definitive health outcomes, and their mechanisms of effect, are being gradually uncovered and there is currently much interest in increasing numbers and activities of these bacteria in the large gut, preferably at the expense of more harmful species. One approach whereby commensal bifidobacteria and/or lactobacilli are selectively promoted by the intake of certain non-viable substrates, is known as prebiotics. Gibson and Roberfroid4 first described a prebiotic as a ‘non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health’. As diet is the main factor controlling the intestinal microflora it is possible to modulate the composition of the microflora through foods. A prebiotic substrate is selectively utilized by beneficial components of the indigenous gut flora but does not promote potential pathogens such as toxin-producing clostridia, proteolytic bacteroides and toxigenic Escherichia coli. In this manner, a ‘healthier’ microflora composition is obtained whereby the bifidobacteria and/or lactobacilli become predominant in the intestine
Prebiotics 289
and exert possible health-promoting effects. For a dietary substrate to be classed as a prebiotic, at least three criteria are required: (1) the substrate must not be hydrolysed or absorbed in the stomach or small intestine, (2) it must be selective for beneficial commensal bacteria in the colon such as the bifidobacteria, (3) fermentation of the substrate should induce beneficial luminal/systemic effects within the host. A range of substrates of dietary origin, or produced by the host, are available for fermentation by the colonic microflora. Through diet, resistant starch (RS) is the most quantitatively important.6 Non-starch polysaccharides (NSP) form the next largest contribution and include plant-derived substrates such as pectin, cellulose, hemicellulose, guar and xylan. Sugars and oligosaccharides such as lactose, lactulose, raffinose, stachyose and fructo-oligosaccharides (FOS) also escape absorption in the small intestine and are metabolized by species of colonic bacteria. Mucin glycoproteins, which are produced by goblet cells in the colonic epithelium, are predominant endogenous substances fermented in the colon. Related mucopolysaccharides such as chondroitin sulphate and heparin, and pancreatic and bacterial secretions, are also available for the intestinal microflora.7 Finally, proteins and peptides originating in the diet, in pancreatic secretions or produced by bacteria are also available8, although to a lesser extent than the carbohydrates. The premise behind prebiotics is therefore to stimulate certain indigenous bacteria resident in the gut rather than introducing exogenous species as is the case with probiotics. Ingesting a diet containing non-digestible carbohydrates that are selectively fermented by indigenous beneficial bacteria is the prebiotic principle. Any dietary component that reaches the colon intact is a potential prebiotic; however, most of the interest in the development of prebiotics is aimed at non-digestible oligosaccharides. It may be possible to take in prebiotics more naturally through the diet. Many fruit and vegetables contain prebiotic oligosaccharides such as FOS. Examples are onion, garlic, banana, asparagus, leek, Jerusalem artichoke, chicory. However, the probable situation is that levels in these foods are too low to exert any significant effect. Our (unpublished) data indicate that at least 4 g/days but more preferably 8 g/days of FOS would be needed to significantly (ca. one log10 value) elevate bifidobacteria in the human gut. Hence, there exists much value in the approach of fortification of commonly ingested foodstuffs with prebiotics. As the majority of bacteria resident in the gut microflora are present in the colon, prebiotics are usually directed towards lower gut bacteriology. As mentioned, current prebiotics seem to be mainly confined to oligosaccharides that are non-digestible in the upper gut, and confer the degree of fermentation selectivity that is required, for example, specifically towards bifidobacteria. Oligosaccharides are sugars consisting of between approximately two and 20 saccharide units, i.e. they are short-chain polysaccharides. Apart from those, which occur naturally in fruits and vegetables, and are extractable, others can be commercially produced through the hydrolysis of polysaccharides (e.g. dietary fibres, starch) or through enzymatic generation. The following oligomers have been suggested as having prebiotic potential9: † † † † † †
Lactulose Fructo-oligosaccharides Galacto-oligosaccharides Soybean oligosaccharides Lactosucrose Isomalto-oligosaccharides
290 T. S. Manning and G. R. Gibson
† Gluco-oligosaccharides † Xylo-oligosaccharides † Palatinose Structure–function relationships The prebiotic properties of carbohydrates are likely to be influenced by the following factors: Monosaccharide composition Recognized prebiotics are built primarily from glucose, galactose, xylose and fructose. The prebiotic potential of oligosaccharides composed of other monosaccharides is not known at the present time. Glycosidic linkage The linkage between the monosaccharide residues is a crucial factor in determining both selectivity of fermentation and digestibility in the small intestine. Fermentation of FOS prebiotics is selective because of a cell-associated b-fructofuranosidase in the bifidobacteria. Molecular weight Polysaccharides are generally not prebiotic in their metabolism but oligosaccharides are.10 Inulin has the highest molecular weight, but most of the carbohydrate in inulin has a degree of polymerization less than 25, with an average of about DP 14.11 The effect of molecular weight on prebiotic properties can be seen from the fact that xylan is not selective whereas xylo-oligosaccharides are thought to be.12,13 Similar effects occur with pectin.14,15 Increased molecular weight Most current prebiotics are of relatively small DP, the exception being inulin. It is thought that the oligosaccharides must be hydrolysed by cell-associated bacterial glycosidases prior to uptake of the resultant monosaccharides. It is, therefore, reasonable to assume that the longer the oligosaccharide the slower the fermentation—and hence the further the prebiotic effect will penetrate more effectively throughout the colon. For example, long-chain inulin may exert a prebiotic effect in more distal colonic regions compared with the lower-molecular-weight FOS, which may be more quickly fermented in the saccharolytic proximal bowel. Potential food applications The current concept of a prebiotic is an oligosaccharide that is selectively fermented by bifidobacteria and lactobacilli.4 Due to the difficulties of characterizing the colonic microflora at the species level, virtually all of the data on prebiotic properties of oligosaccharides are on microflora changes at the genus level. It may, however, be desirable to develop prebiotics which are targeted at particular species of
Prebiotics 291
Bifidobacterium and Lactobacillus. Such targeted prebiotics might be considered for several applications, as follows. Synbiotics with defined health benefits Many probiotic strains have been developed to have particular health benefits, such as immune stimulation or anti-pathogen activity. In addition, commercial probiotic strains are selected for their survival characteristics such as resistance to acid and bile, and their ability to be freeze-dried.16,17 Availability of prebiotics specifically targeted at these strains (not just genus-level changes) would enable the development of synbiotic versions with enhanced survivability in the gut. Infant formulae It has long been thought that the gut flora of the breast-fed infant is dominated by bifidobacteria and that this is not the case for formula-fed infants.18,19 This is seen to be one reason for the improved resistance to infection that the latter group experiences. If prebiotics could be developed with particular selectivity towards those bifidobacteria that are present in the guts of breast-fed infants, a new range of synbiotic formula foods could be envisaged. Functional foods for the elderly Above the age of about 55 –60 years, faecal bifidobacterial counts have been shown to markedly decrease compared to those of younger people.20,21 This decrease in bifidobacteria is a cause for concern as the natural elderly gut flora may have become compromised through reduced bifidobacterial numbers, resulting in a diminished ability to resist colonization with invading pathogens. Prebiotics may be potentially utilized as a dietary intervention in the attempt to restore the microflora (i.e. bifidobacteria) balance of the gut in the elderly population concurrently with indirectly providing antipathogenic protection. As prebiotics exploit the use non-viable dietary components to improve gut health, the range of foods into which they can be added is much wider than that for probiotics, where culture viability needs to be maintained. This has the advantage that heat stability, or exposure to oxygen is not an issue. As such, virtually any carbohydrate containing food is susceptible to supplementation. Potential applications for prebiotics as food ingredients to improve the gastrointestinal health of the consumer are listed below: † † † † † † † † † † †
Beverages and fermented milks Health drinks Bakery products Table spreads Sauces Infant formulae and weaning foods Cereals Biscuits Confectionery, cakes, desserts Snack bars Soups
292 T. S. Manning and G. R. Gibson
† Salad dressings † Dairy products HEALTH-RELATED ASPECTS AND APPLICATIONS At present, most prebiotics are selected on the basis of their ability to promote the growth of lactic-acid-producing microorganisms. Fructo-oligosaccharides, lactulose and glucooligosaccharides (GOS) are all popular prebiotics. In Europe, most success has been gained with FOS. In human studies, after a short feeding period, FOS stimulate bifidobacteria in the lower gut.22 Similarly, lactulose is an efficient prebiotic—as demonstrated through the use of molecular probes in a human volunteer trial.23 In Europe, FOS, GOS and lactulose have been shown to be prebiotics, as evidenced by their ability to change the gut flora composition after a short feeding period.9 The Japanese market is more widespread. A recent volunteer trial was carried out at the University of Reading.23 Here, shortbread containing 7 g/day FOS was fed to human subjects and the effects upon faecal bacteria determined as compared to a placebo (FOS not added). The nature of the trial was a crossover approach in that volunteers took active and placebo shortbread but neither they nor the investigators were aware of which was ingested. Moreover, the bacteriology was carried out using a (culture-independent) probing approach that relied upon differences in 16SrRNA profiles for the confirmation of identity. The data clearly showed that the use of FOS exerted a profound effect upon bifidobacteria. A number of benefits can be ascribed to prebiotic intake.9 However, some areas of interest are described below. Protection against colon cancer Many common diseases of the human large bowel arise in the distal colon, particularly colonic cancer.24 Prebiotics have been postulated to be protective against the development of colon cancer.25 – 29 The second most prevalent cancer in humans is colon cancer30; in addition, it is thought that tumours arise 100 times more often in the large intestine compared to the small intestine.31 For this reason, many researchers believe that the colonic microflora has an important role to play in the development of bowel cancer.32 It is known that several species of bacteria commonly found in the colon produce carcinogens and tumour promoters from the metabolism of food components. Interest in a diet-mediated intervention towards colon cancer arises due to the slow, progressive nature of the disease and the fact that we can influence colonic microbiology by diet. There have been several studies on the use of prebiotics in cancer prevention, mainly focusing on animal models. It is thought that prebiotics may protect against development of colon cancer through at least two mechanisms: (i)
Production of protective metabolites. Butyrate is a common fermentation end product and is known to stimulate apoptosis in colonic cancer cell lines and it is also the preferred fuel for healthy colonocytes.33,34 For these reasons, it is generally believed that it is desirable to increase the level of butyrate formed in the large gut. Some prebiotics are known to have this effect14,35, although it must be borne in mind that lactobacilli and bifidobacteria do not produce butyrate. Known butyrate producers in the gut are clostridia and eubacteria.6 Development of prebiotics which stimulate (benign) eubacteria but not (toxic) clostridia would be a desirable enhancement.
Prebiotics 293
(ii) Subversion of colonic metabolism away from protein and lipid metabolism. It is possible that prebiotics would induce a shift in bacterial metabolism in the colon towards more benign end products. An obvious target would be to shift the metabolism of clostridia and bacteroides away from proteolysis to a saccharolysis. Lactic acid bacteria are believed to have inhibitory effects on several bacteria that produce carcinogenic enzymes and are themselves non-producers. Moreover, prebiotics may indirectly modify the activities of enzymes produced by the lactic acid bacteria that are involved in carcinogenesis, such as azoreductases, nitroreductases, bglucuronidase, etc.36 To date, few prebiotics have been evaluated in animal and human trials. Inulin, for instance has been shown to inhibit the formation of aberrant crypt foci in rats.26 Human studies are low in number and tend to focus on faecal markers of carcinogenesis rather than being epidemiological in nature. FOS, GOS and resistant starch have all been investigated in this regard. FOS has been found to reduce genotoxic enzymes concomitant with increasing bifidobacteria27, and resistant starch has been found to reduce sterols, secondary bile acids and genotoxic enzymes, although no microbiological studies were performed.29 However, a recent study on GOS found no significant changes in bifidobacteria or in markers of carcingenesis37. At first sight these results mightseem curious, as GOS are known prebiotics.38 However, the starting populations of bifidobacteria in the volunteers were rather high (9.2 –9.4 log). It has been noted previously39 that the magnitude of the response to prebiotics by bifidobacteria depends on the starting levels. It is apparent that we currently have an inadequate knowledge of the effects of various prebiotics upon the risk of colon cancer; more studies are needed to address this. Development of prebiotics with the goal of reducing biomarkers of cancer would, however, be very desirable.
Effects on pathogens Good evidence for the success of prebiotics lies in their ability to improve resistance to pathogens by increasing bifidobacteria and lactobacilli. Lactic-acid-excreting microorganisms are known for their inhibitory properties.40 In humans, viruses, protozoa, fungi and bacteria can all cause acute gastroenteritis. Metabolic end-products, such as acids excreted by these microorganisms, may lower the gut pH to levels below those at which pathogens are able effectively to compete. Also, many lactobacilli and bifidobacterial species are able to excrete natural antibiotics, which can have a broad spectrum of activity. For the bifidobacteria, some species are able to exert antimicrobial effects on various Gram-positive and Gram-negative intestinal pathogens.41 A recent study in mice has shown that FOS and inulin protected against enteric and systemic pathogens and tumour inducers.42 This includes the verocytotoxin strain of Escherichia coli O157:H7 and campylobacters. A rational way to reduce the food-poisoning burden may be fortify certain components of the intestinal flora such that it becomes much more resistant to invasion. This is achievable through the use of prebiotics that target bifidobacteria and/or lactobacilli. Taking this further, some other gut-related conditions more chronic than acute gastroenteritis, additionally labeled microbiological pathogens, may also be susceptible to prevention or treatment by altering the gut flora. Examples would include ulcerative colitis, bowel cancer, peptic ulcers, pseudomembranous colitis and Candida-induced conditions.
294 T. S. Manning and G. R. Gibson
Improved calcium absorption There has been increasing interest in recent years in the possibility of increasing mineral (particularly calcium) absorption through the consumption of prebiotics. Although the small intestine is the principal site of calcium absorption in humans, it is thought that significant amounts are absorbed throughout the length of the gut, consequently, maximizing of colonic effects is desirable. Several mechanisms have been postulated for increased calcium absorption induced by prebiotics.43 They include the following. (i) Fermentation of prebiotics such as inulin results in a significant production of SCFA, leading to a reduction in lumenal colonic pH. This is likely to increase calcium solubility and overall levels in the gut. (ii) Phytate (myoinositol hexaphosphate) is a component of plants that reaches the colon largely intact.44 It also forms stable, insoluble complexes with divalent cations, such as calcium, rendering them unavailable for transport. Fermentation results in bacterial metabolism of phytate, thereby liberating calcium. (iii) It is postulated that a calcium exchange mechanism operates in the colon. In this system, SCFAs enter the colon in a protonated form and then dissociate in the intracellular environment. The liberated proton is then secreted into the lumen in exchange for a calcium ion. Numerous animal studies have indicated that prebiotics increase absorption of calcium from the colon and decreased losses from bone tissue.45 However, very few human studies have been carried out. In one study, the feeding of 40 g inulin/day for 28 days to nine healthy subjects resulted in a significant increase in calcium absorption.46 A lower dose, 15 g inulin, FOS or GOS/day, when fed to 12 healthy subjects for 21 days, resulted in no significant effect on absorption of calcium or iron.47 In a later study, 12 adolescent boys (aged 14 – 16) were fed 15 g FOS/day for 9 days in a placebo-controlled trial against sucrose.48 The data showed a 10.8% increase in calcium balance with no significant effect on urinary excretion. Effects on blood lipids There is interest in the food industry in developing functional foods to modulate blood lipids such as cholesterol and triglycerides. It is widely believed that elevated cholesterol levels in the blood represent a risk factor for coronary heart disease, with low-density lipoproteins (LDL) being of most concern.49 There is also evidence that lactic acid bacteria may be able to reduce total and LDL cholesterol levels. The mechanisms by which lactic acid bacteria, and hence, indirectly, prebiotics, influence blood lipids are not clearly understood at the present time. It is possible that some lactic acid bacteria may be able to assimilate cholesterol directly.49,50 There is evidence that FOS decrease the de novo synthesis of triglycerides by the liver. The means by which this occurs is not fully understood but the effect appears be exerted at the transcriptional level. It is also possible that prebiotics (such as inulin) can modulate insulin-induced inhibition of triglyceride synthesis.50 Human studies on the lipidlowering properties of prebiotics when consumed at a realistic (tolerable) dose are, however, contradictory and the role in lipid reduction remains to be confirmed.51 Immunological effects Lactic acid bacteria are thought to stimulate both non-specific host defence mechanisms and certain types of cell involved in the specific immune response.
Prebiotics 295
The result is often increased phagocytic activity and/or elevated immunological molecules such as secretory IgA, which may affect pathogens such as salmonellae and rotavirus. Most attention in this respect has been diverted towards the intake of probiotics (lactic acid bacteria)52,53 and interactions between cell wall components and immune cells. As prebiotics serve a similar end point to lactic acid bacteria (i.e. improved composition of the gut microflora) similar effects may occur through their intake. A recent animal study have shown that prebiotics had an effect on immune functions.54
Practice points † health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens, cholesterol reduction and lowering of gas distension † harmful effects of microflora are carcinogen production, intestinal putrefaction, toxin formation, diarrhoea/constipation, liver damage and intestinal infection † a prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (i.e. bifidobacteria and lactobacilli), and thus improves the host’s health † prebiotics have been postulated to be protective against the development of colon cancer † evidence supports the ability of prebiotics to improve resistance to pathogens by increasing bifidobacteria and lactobacilli † numerous animal studies have indicated that prebiotics increase absorption of calcium from the colon and decreased losses from bone tissue † evidence of the ability of prebiotics to lower lipids remains controversial † prebiotics may have an effect on immune functions
Research agenda If progress in the use of dietary intervention directed towards particular gut bacteria is to be exploited, a sound research base is required. Some areas of interest may include: † the application of advanced molecular procedures that help to identify the gut microbial diversity as well as allow effective tracking of changes in microflora in response to diet (it is likely that a large number of gut bacteria have not hitherto been characterized, and culture-independent methodologies may help to overcome this) † the prebiotic potential of dietary ingredients, an identification of those foodstuffs that can be fortified and the optimal dose required † a definition of prebiotics which act at the species level and have a high degree of selectivity and contain multiple biological activities † whether certain target groups are more susceptible to the approach (elderly, weaning stage, formula-fed infants, hospitalized patients) † a determination of the health consequences that are associated with modulation of the gut flora
296 T. S. Manning and G. R. Gibson
SUMMARY The microflora of the gastrointestinal tract is key for nutrition and health of the host. Modulation of the microflora can occur through diets that contain prebiotics. The approach of using diet to induce microbial change offers a very straightforward approach towards improved health. In terms of new developments, it is important that the definitive health bonuses associated with prebiotic intake be determined. This is especially relevant given the broad applicability of their use. It is likely that prevention of acute gastroenteritis through fortification of certain components of the gut microflora is an important aspect. Moreover, improved protection from more chronic gut disorders that have been associated with bacteria (inflammatory bowel disease, colon cancer, irritable bowel syndrome) may also be possible. It may also be the case that certain target populations, such as infants, the elderly and hospitalized persons, are more susceptible to the approach. The health benefits that have been suggested are varied but also very important. In addition to good human volunteer studies we also need to enhance our mechanistic understanding of the health effects of prebiotics.
REFERENCES 1. Simon GL & Gorbach SL. The human intestinal microflora. Digestive Diseases and Sciences 1986; 31: 147S– 162S. *2. Gibson GR & Roberfroid MB. Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 3. Salminen S, Ramos P & Fonden R. Substrates and lactic acid bacteria. In Salminen S & von Wright A (eds) Lactic Acid Bacteria. New York: Marcel Dekker, 1993. *4. Gibson GR & Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 1995; 125: 1401–1412. 5. Sanders MA. Overview of functional foods: emphasis on probiotic bacteria. International Dairy Journal 1998; 8: 341–347. 6. Cummings JH & Macfarlane GT. The control and consequences of bacterial fermentation in the human colon. Journal of Applied Bacteriology 1991; 70: 443–459. *7. Salminen S, Bouley C, Boutron-Ruault M-C et al. Functional food science and gastrointestinal physiology and function. British Journal of Nutrition 1998; 80: S147– S171. 8. Macfarlane GT, Cummings JH & Allison C. Protein degradation by human intestinal bacteria. Journal of General Microbiology 1986; 132: 1647–1656. *9. Gibson GR, Berry Ottaway P & Rastall RA. Prebiotics: New Developments in Functional Foods. Oxford: Chandos Publishing Limited, 2000. * 10. Wang X & Gibson GR. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. Journal of Applied Bacteriology 1993; 75: 373–380. 11. De Leenheer L. Production and use if inulin: industrial reality with a promising future. In van Bekkum H, Roper H & Voragen AGJ (eds) Carbohydrates as Organic Raw Materials III. Weinheim: VCH, 1994. 12. Okazaki M, Fujikawa S & Matsumoto N. Effects of xylooligosaccharide on growth of bifidobacteria. Journal of the Japanese Society of Nutrition and Food Sciences 1990; 43: 395– 401. 13. Jaskari J, Kontula P, Siitonen A et al. Oat b-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains. Applied Microbiology and Biotechnology 1998; 49: 175 –181. * 14. Olano-Martin E, Mountzouris KC, Gibson GR & Rastall RA. In vitro fermentability of dextran, oligodextran and maltodextrin by human gut bacteria. British Journal of Nutrition 2000; 83: 247 –255. 15. Olano-Martin E, Mountzouris KC, Gibson GR & Rastall RA. Continuous production of oligosaccharides from pectin in an enzyme membrane reactor. Journal of Food Sciences 2001; 66: 966–971. 16. Svensson U. Industrial perspectives. In Tannock G (ed.) Probiotics: a Critical Review. Wymondham: Horizon Scientific Press, 1999, pp 57–64. 17. Lee Y-K, Nomoto K, Salminen S & Gorbach SL. Handbook of Probiotics. New York: Wiley, 1999. 18. Cooperstock MS & Zedd AJ. Intestinal flora of infants. In Hentges DJ (ed.) Human Intestinal microflora in Health and Disease. London: Academic Press, 1983, pp 79–99.
Prebiotics 297 19. Benno Y, Sawada K & Mitsuoka T. The intestinal microflora of infants: composition of fecal flora in breastfed and bottle-fed infants. Microbiology and Immunology 1984; 28: 975–986. 20. Mitsuoka T. Bifidobacteria and their role in human health. Journal of Industrial Microbiology 1990; 6: 263–268. 21. Kleessen B, Sykura B, Zunft H-J & Blaut M. Effects of inulin and lactose on fecal microflora, microbial activity and bowel habit in elderly constipated persons. American Journal of Clinical Nutrition 1997; 65: 1397– 1402. * 22. Gibson GR, Beatty ER, Wang X & Cummings JH. Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 1995; 108: 975 –982. * 23. Tuohy KM, Kolida S, Lustenberger A & Gibson GR. The prebiotic effects of biscuits containing partially hydrolyzed guar gum and fructooligosaccharides—a human volunteer study. British Journal of Nutrition 2001; 86: 341 –348. 24. Rowland IR. Metabolic interactions in the gut. In Fuller R (ed.) Probiotics: the Scientific Basis. Andover, UK: Chapman & Hall, 1992. 25. Rowland IR & Tanaka R. The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora. Journal of Applied Bacteriology 1993; 74: 667 –674. 26. Reddy BS, Hamid R & Rao CV. Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition. Carcinogenesis 1997; 18: 1371– 1374. 27. Bouhnik Y, Flourie B, Riottot M et al. Effects of fructo-oligosaccharides ingestion on faecal bifidobacteria and selected metabolic indexes of colon carcinogenesis in healthy humans. Nutrition and Cancer 1996; 26: 21–29. 28. Buddington RK, Williams CH, Chen S-C & Witherly SA. Dietary supplementation of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. American Journal of Clinical Nutrition 1996; 63: 709–716. 29. Hylla S, Gostner A, Dusel G et al. Effects of resistant starch on the colon in healthy volunteers: possible implications for cancer prevention. American Journal of Clinical Nutrition 1998; 67: 136 –142. * 30. Gibson GR & Macfarlane GT. Intestinal bacteria and disease. In Gibson SAW (ed.) Human Health—the Contribution of Microorganisms. London: Springer-Verlag, 1994. 31. Morotomi M, Guillem JG, LoGerfo P & Weinsten IB. Production of diacylglycerol, an activator of protein kinase C by human intestinal microflora. Cancer Research 1990; 50: 3595–3599. 32. Rowland IR (ed.) Role of the Gut Flora in Toxicity and Cancer. London: Academic Press, 1998. 33. Prasad KN. Butyric acid: a small fatty acid with diverse biological functions. Life Sciences 1980; 27: 1351– 1358. 34. Kim YS, Tsao D, Morita A & Bella A. Effect of sodium butyrate and three human colorectal adenocarcinoma cell lines in culture. Falk Symposium 1982; 31: 317–323. 35. Videla S, Vilaseca J, Antolin M et al. Dietary inulin improves distal colitis induced by dextran sodium sulfate in the rat. American Journal of Gastroenterology 2001; 96: 1468–1493. 36. Reddy BS. Prevention of colon cancer by pre- and probiotics: evidence from laboratory studies. British Journal of Nutrition 1998; 80: S219–S223. 37. Alles MS, Hartemink R, Meyboom S et al. Effect of transgalactooligosaccharides on the composition of the human intestinal microflora and on putative risk markers for colon cancer. American Journal of Clinical Nutrition 1999; 69: 980–991. 38. Schoterman HC & Timmermans HJAR. Galacto-oligosaccharides. In Gibson GR & Angus F (eds) Prebiotics and Probiotics. LFRA Ingredients Handbook. Leatherhead: Food RA Publishing, 2000. 39. Rycroft CE, Jones MR, Gibson GR & Rastall RA. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. Journal of Applied Bacteriology 2001; 91: 878– 887. 40. Fuller R (ed.) Probiotics 2: Application and Practical Aspects. Andover, UK: Chapman & Hall, 1997. 41. Mackey BM & Gibson GR. Escherichia coli 0157-from farm to fork and beyond. Society of General Microbiology Quarterly 1997; 24: 55– 57. 42. Buddington KK, Danohoo JB & Buddington RK. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumour inducers. Journal of Nutrition 2002; 132: 472– 477. 43. Fairweather-Tait SJ & Johnson IT. Bioavailability of minerals. In Gibson GR & Roberfroid MB (eds) Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 44. Cummings JH, Hill MJ, Houston H et al. The effect of meat protein and dietary fibre on colonic function and metabolism. 1. Changes in bowel habit, bile acid excretion and calcium absorption. American Journal of Clinical Nutrition 1979; 32: 2086–2093. 45. Greger JL. Nondigestible carbohydrates and mineral bioavailability. Journal of Nutrition 1999; 129: 1434S–1435S. 46. Coudray C, Bellanger J, Castiglia-Delavaud C et al. Effect of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. European Journal of Clinical Nutrition 1997; 51: 375 –380.
298 T. S. Manning and G. R. Gibson 47. van den Heuvel E, Schaafsma G, Muys T & van Dokkum W. Non-digestible oligosaccharides do not interfere with calcium and non-haeme iron absorption in young, healthy men. American Journal of Clinical Nutrition 1998; 67: 445 –451. 48. van den Heuvel E, Muys T, van Dokkum W & Schaafsma G. Oligofructose stimulates calcium absorption in adolescents. American Journal of Clinical Nutrition 1999; 69: 544 –548. 49. Delzenne NM & Williams CM. Actions of non-digestible carbohydrates on blood lipids in humans and animals. In Gibson GR & Roberfroid MB (eds) Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 50. Delzenne NM & Kok NN. Biochemical basis of oligofructose-induced hypolipidaemia in animal models. Journal of Nutrition 1999; 129: 1467S–1470S. 51. Williams CM. Effects of inulin on lipid parameters in humans. Journal of Nutrition 1999; 129: 1471S–1473S. 52. Schiffrin EJ, Rochat F, Link-Amster HA et al. Immune system stimulation by probiotics. Journal of Dairy Science 1995; 78: 1597–1606. 53. Perdigon G & Alvarez S. Probiotics and the immune state. In Fuller R (ed.) Probiotics: the Scientific Basis. London: Chapman & Hall, 1992, pp 146–180. * 54. Swanson KS. Prebiotics and probiotics: impact on gut microbial populations, nutrient digestibilities, fecal protein catabolite concentrations and immune functions of humans and dogs. Dissertation and Abstracts International 2002; 63: 746.
5 Prebiotics Thea Scantlebury Manning*
BSc, PhD
Research fellow
Glenn R. Gibson
BSc, PhD
Professor and Head of Food Microbial Science Unit Food Microbial Science Unit, School of Food Biosciences, Reading University, Science and Technology Center, Earley Gate, Whiteknights Road, Reading RG6 6BZ, UK
In nutritional sciences there is much interest in dietary modulation of the human gut. The gastrointestinal tract, particularly the colon, is very heavily populated with bacteria. Most bacteria are benign; however, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders. Bifidobacteria and lactobacilli are thought to be beneficial and are common targets for dietary intervention. Prebiotic is a non-viable food ingredient selectively metabolized by beneficial intestinal bacteria. Dietary modulation of the gut microflora by prebiotics is designed to improve health by stimulating numbers and/or activities of the bifidobacteria and lactobacilli. Having an ‘optimal’ gut microflora can increase resistance to pathogenic bacteria, lower blood ammonia, increase stimulation of the immune response and reduce the risk of cancer. This chapter examines how prebiotics are being applied to the improvement of human health and reviews the scientific evidence behind their use. Key words: prebiotic; oligosaccharides; gut microflora; bifidobacteria; lactobacilli functional food.
Biological functions of the human large intestine include waste storage (and its excretion) and the absorption of water as well as essential minerals. However, because of a slow transit time, near-neutral pH and high substrate availability, the colon harbours a very complex and diverse bacterial microflora.1 The microflora in the human large intestine is thought to compromise about 95% of total cells in the body, representing 1012 cells/g dry weight contents, making the organ a highly specialized and active area of the body. Through the activities of the resident microflora, the colon plays a major role in host nutrition and welfare.2 Dietary modulation of the human gut flora can be of some benefit to health. In recent years, the functional food concept has moved towards the situation whereby improved gut (microbial) functionality is the main current driving force. The colon is by far the most intensely populated region of the gastrointestinal tract and is therefore a major target for dietary intervention. * Corresponding author. Tel.: þ44-118-935-7220; Fax: þ44-118-935-7222. E-mail address: [email protected] (T. S. Manning). 1521-6918/$ - see front matter Q 2003 Elsevier Ltd. All rights reserved.
288 T. S. Manning and G. R. Gibson
The gut microflora ferments a range of substances, mainly provided by the diet, that cannot be digested by the host in the small intestine and are available for fermentation by the colonic microflora. These include resistant starch, non-starch polysaccharides (dietary fibre), oligosaccharides, proteins, amino acids, etc. In a typical adult, about 100 g of food ingested each day reaches the large intestine and is therefore susceptible to fermentation by the gut flora. The two main types of anaerobic fermentation that are carried out in the gut are saccharolytic and proteolytic. The main end-products of carbohydrate metabolism are the short-chain fatty acids (SCFAs), principally acetate, propionate and butyrate. These may be further metabolized systemically or locally to provide energy generation for the host. The end-products of proteolytic fermentation include phenolic compounds, amines and ammonia, all of which are toxic. The proximal colon (right side) is essentially a site of saccharolytic fermentation, whereas the more distal (left side) sees a depletion of available carbohydrate and more protein metabolism. Dietary modulation of the human gut microflora is a popular area of the nutritional sciences. This is driven by the fact that the gastrointestinal tract, particularly the colon, is very heavily colonized and that the composition of the flora can be modulated. Undoubtedly, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders. However, bifidobacteria and lactobacilli are considered to be examples of health-promoting constituents of the microflora. Lactobacilli may aid digestion of lactose in lactose-intolerant individuals, reduce constipation and infantile diarrhoea, help resist infections such as salmonellae, prevent traveller’s diarrhoea and help to relieve irritable bowel syndrome (IBS).3 Bifidobacteria are thought to stimulate the immune system, produce B vitamins, inhibit pathogen growth, reduce blood ammonia and blood cholesterol levels, and help to restore the normal flora after antibiotic therapy.4 Health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens, cholesterol reduction and lowering of gas distension.4 Harmful effects are carcinogen production, intestinal putrefaction, toxin formation, diarrhoea/constipation, liver damage and intestinal infection. Bifidobacteria and lactobacilli are therefore common targets for dietary intervention that improves health. GENERAL ASPECTS OF PREBIOTICS Dietary modulation of the human gut flora has been carried out for many years. In humans, there are positive aspects to the gut fermentation. For instance, bifidobacteria and lactobacilli may help to reduce the risk of disease for the reasons given above.2,5 The definitive health outcomes, and their mechanisms of effect, are being gradually uncovered and there is currently much interest in increasing numbers and activities of these bacteria in the large gut, preferably at the expense of more harmful species. One approach whereby commensal bifidobacteria and/or lactobacilli are selectively promoted by the intake of certain non-viable substrates, is known as prebiotics. Gibson and Roberfroid4 first described a prebiotic as a ‘non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health’. As diet is the main factor controlling the intestinal microflora it is possible to modulate the composition of the microflora through foods. A prebiotic substrate is selectively utilized by beneficial components of the indigenous gut flora but does not promote potential pathogens such as toxin-producing clostridia, proteolytic bacteroides and toxigenic Escherichia coli. In this manner, a ‘healthier’ microflora composition is obtained whereby the bifidobacteria and/or lactobacilli become predominant in the intestine
Prebiotics 289
and exert possible health-promoting effects. For a dietary substrate to be classed as a prebiotic, at least three criteria are required: (1) the substrate must not be hydrolysed or absorbed in the stomach or small intestine, (2) it must be selective for beneficial commensal bacteria in the colon such as the bifidobacteria, (3) fermentation of the substrate should induce beneficial luminal/systemic effects within the host. A range of substrates of dietary origin, or produced by the host, are available for fermentation by the colonic microflora. Through diet, resistant starch (RS) is the most quantitatively important.6 Non-starch polysaccharides (NSP) form the next largest contribution and include plant-derived substrates such as pectin, cellulose, hemicellulose, guar and xylan. Sugars and oligosaccharides such as lactose, lactulose, raffinose, stachyose and fructo-oligosaccharides (FOS) also escape absorption in the small intestine and are metabolized by species of colonic bacteria. Mucin glycoproteins, which are produced by goblet cells in the colonic epithelium, are predominant endogenous substances fermented in the colon. Related mucopolysaccharides such as chondroitin sulphate and heparin, and pancreatic and bacterial secretions, are also available for the intestinal microflora.7 Finally, proteins and peptides originating in the diet, in pancreatic secretions or produced by bacteria are also available8, although to a lesser extent than the carbohydrates. The premise behind prebiotics is therefore to stimulate certain indigenous bacteria resident in the gut rather than introducing exogenous species as is the case with probiotics. Ingesting a diet containing non-digestible carbohydrates that are selectively fermented by indigenous beneficial bacteria is the prebiotic principle. Any dietary component that reaches the colon intact is a potential prebiotic; however, most of the interest in the development of prebiotics is aimed at non-digestible oligosaccharides. It may be possible to take in prebiotics more naturally through the diet. Many fruit and vegetables contain prebiotic oligosaccharides such as FOS. Examples are onion, garlic, banana, asparagus, leek, Jerusalem artichoke, chicory. However, the probable situation is that levels in these foods are too low to exert any significant effect. Our (unpublished) data indicate that at least 4 g/days but more preferably 8 g/days of FOS would be needed to significantly (ca. one log10 value) elevate bifidobacteria in the human gut. Hence, there exists much value in the approach of fortification of commonly ingested foodstuffs with prebiotics. As the majority of bacteria resident in the gut microflora are present in the colon, prebiotics are usually directed towards lower gut bacteriology. As mentioned, current prebiotics seem to be mainly confined to oligosaccharides that are non-digestible in the upper gut, and confer the degree of fermentation selectivity that is required, for example, specifically towards bifidobacteria. Oligosaccharides are sugars consisting of between approximately two and 20 saccharide units, i.e. they are short-chain polysaccharides. Apart from those, which occur naturally in fruits and vegetables, and are extractable, others can be commercially produced through the hydrolysis of polysaccharides (e.g. dietary fibres, starch) or through enzymatic generation. The following oligomers have been suggested as having prebiotic potential9: † † † † † †
Lactulose Fructo-oligosaccharides Galacto-oligosaccharides Soybean oligosaccharides Lactosucrose Isomalto-oligosaccharides
290 T. S. Manning and G. R. Gibson
† Gluco-oligosaccharides † Xylo-oligosaccharides † Palatinose Structure–function relationships The prebiotic properties of carbohydrates are likely to be influenced by the following factors: Monosaccharide composition Recognized prebiotics are built primarily from glucose, galactose, xylose and fructose. The prebiotic potential of oligosaccharides composed of other monosaccharides is not known at the present time. Glycosidic linkage The linkage between the monosaccharide residues is a crucial factor in determining both selectivity of fermentation and digestibility in the small intestine. Fermentation of FOS prebiotics is selective because of a cell-associated b-fructofuranosidase in the bifidobacteria. Molecular weight Polysaccharides are generally not prebiotic in their metabolism but oligosaccharides are.10 Inulin has the highest molecular weight, but most of the carbohydrate in inulin has a degree of polymerization less than 25, with an average of about DP 14.11 The effect of molecular weight on prebiotic properties can be seen from the fact that xylan is not selective whereas xylo-oligosaccharides are thought to be.12,13 Similar effects occur with pectin.14,15 Increased molecular weight Most current prebiotics are of relatively small DP, the exception being inulin. It is thought that the oligosaccharides must be hydrolysed by cell-associated bacterial glycosidases prior to uptake of the resultant monosaccharides. It is, therefore, reasonable to assume that the longer the oligosaccharide the slower the fermentation—and hence the further the prebiotic effect will penetrate more effectively throughout the colon. For example, long-chain inulin may exert a prebiotic effect in more distal colonic regions compared with the lower-molecular-weight FOS, which may be more quickly fermented in the saccharolytic proximal bowel. Potential food applications The current concept of a prebiotic is an oligosaccharide that is selectively fermented by bifidobacteria and lactobacilli.4 Due to the difficulties of characterizing the colonic microflora at the species level, virtually all of the data on prebiotic properties of oligosaccharides are on microflora changes at the genus level. It may, however, be desirable to develop prebiotics which are targeted at particular species of
Prebiotics 291
Bifidobacterium and Lactobacillus. Such targeted prebiotics might be considered for several applications, as follows. Synbiotics with defined health benefits Many probiotic strains have been developed to have particular health benefits, such as immune stimulation or anti-pathogen activity. In addition, commercial probiotic strains are selected for their survival characteristics such as resistance to acid and bile, and their ability to be freeze-dried.16,17 Availability of prebiotics specifically targeted at these strains (not just genus-level changes) would enable the development of synbiotic versions with enhanced survivability in the gut. Infant formulae It has long been thought that the gut flora of the breast-fed infant is dominated by bifidobacteria and that this is not the case for formula-fed infants.18,19 This is seen to be one reason for the improved resistance to infection that the latter group experiences. If prebiotics could be developed with particular selectivity towards those bifidobacteria that are present in the guts of breast-fed infants, a new range of synbiotic formula foods could be envisaged. Functional foods for the elderly Above the age of about 55 –60 years, faecal bifidobacterial counts have been shown to markedly decrease compared to those of younger people.20,21 This decrease in bifidobacteria is a cause for concern as the natural elderly gut flora may have become compromised through reduced bifidobacterial numbers, resulting in a diminished ability to resist colonization with invading pathogens. Prebiotics may be potentially utilized as a dietary intervention in the attempt to restore the microflora (i.e. bifidobacteria) balance of the gut in the elderly population concurrently with indirectly providing antipathogenic protection. As prebiotics exploit the use non-viable dietary components to improve gut health, the range of foods into which they can be added is much wider than that for probiotics, where culture viability needs to be maintained. This has the advantage that heat stability, or exposure to oxygen is not an issue. As such, virtually any carbohydrate containing food is susceptible to supplementation. Potential applications for prebiotics as food ingredients to improve the gastrointestinal health of the consumer are listed below: † † † † † † † † † † †
Beverages and fermented milks Health drinks Bakery products Table spreads Sauces Infant formulae and weaning foods Cereals Biscuits Confectionery, cakes, desserts Snack bars Soups
292 T. S. Manning and G. R. Gibson
† Salad dressings † Dairy products HEALTH-RELATED ASPECTS AND APPLICATIONS At present, most prebiotics are selected on the basis of their ability to promote the growth of lactic-acid-producing microorganisms. Fructo-oligosaccharides, lactulose and glucooligosaccharides (GOS) are all popular prebiotics. In Europe, most success has been gained with FOS. In human studies, after a short feeding period, FOS stimulate bifidobacteria in the lower gut.22 Similarly, lactulose is an efficient prebiotic—as demonstrated through the use of molecular probes in a human volunteer trial.23 In Europe, FOS, GOS and lactulose have been shown to be prebiotics, as evidenced by their ability to change the gut flora composition after a short feeding period.9 The Japanese market is more widespread. A recent volunteer trial was carried out at the University of Reading.23 Here, shortbread containing 7 g/day FOS was fed to human subjects and the effects upon faecal bacteria determined as compared to a placebo (FOS not added). The nature of the trial was a crossover approach in that volunteers took active and placebo shortbread but neither they nor the investigators were aware of which was ingested. Moreover, the bacteriology was carried out using a (culture-independent) probing approach that relied upon differences in 16SrRNA profiles for the confirmation of identity. The data clearly showed that the use of FOS exerted a profound effect upon bifidobacteria. A number of benefits can be ascribed to prebiotic intake.9 However, some areas of interest are described below. Protection against colon cancer Many common diseases of the human large bowel arise in the distal colon, particularly colonic cancer.24 Prebiotics have been postulated to be protective against the development of colon cancer.25 – 29 The second most prevalent cancer in humans is colon cancer30; in addition, it is thought that tumours arise 100 times more often in the large intestine compared to the small intestine.31 For this reason, many researchers believe that the colonic microflora has an important role to play in the development of bowel cancer.32 It is known that several species of bacteria commonly found in the colon produce carcinogens and tumour promoters from the metabolism of food components. Interest in a diet-mediated intervention towards colon cancer arises due to the slow, progressive nature of the disease and the fact that we can influence colonic microbiology by diet. There have been several studies on the use of prebiotics in cancer prevention, mainly focusing on animal models. It is thought that prebiotics may protect against development of colon cancer through at least two mechanisms: (i)
Production of protective metabolites. Butyrate is a common fermentation end product and is known to stimulate apoptosis in colonic cancer cell lines and it is also the preferred fuel for healthy colonocytes.33,34 For these reasons, it is generally believed that it is desirable to increase the level of butyrate formed in the large gut. Some prebiotics are known to have this effect14,35, although it must be borne in mind that lactobacilli and bifidobacteria do not produce butyrate. Known butyrate producers in the gut are clostridia and eubacteria.6 Development of prebiotics which stimulate (benign) eubacteria but not (toxic) clostridia would be a desirable enhancement.
Prebiotics 293
(ii) Subversion of colonic metabolism away from protein and lipid metabolism. It is possible that prebiotics would induce a shift in bacterial metabolism in the colon towards more benign end products. An obvious target would be to shift the metabolism of clostridia and bacteroides away from proteolysis to a saccharolysis. Lactic acid bacteria are believed to have inhibitory effects on several bacteria that produce carcinogenic enzymes and are themselves non-producers. Moreover, prebiotics may indirectly modify the activities of enzymes produced by the lactic acid bacteria that are involved in carcinogenesis, such as azoreductases, nitroreductases, bglucuronidase, etc.36 To date, few prebiotics have been evaluated in animal and human trials. Inulin, for instance has been shown to inhibit the formation of aberrant crypt foci in rats.26 Human studies are low in number and tend to focus on faecal markers of carcinogenesis rather than being epidemiological in nature. FOS, GOS and resistant starch have all been investigated in this regard. FOS has been found to reduce genotoxic enzymes concomitant with increasing bifidobacteria27, and resistant starch has been found to reduce sterols, secondary bile acids and genotoxic enzymes, although no microbiological studies were performed.29 However, a recent study on GOS found no significant changes in bifidobacteria or in markers of carcingenesis37. At first sight these results mightseem curious, as GOS are known prebiotics.38 However, the starting populations of bifidobacteria in the volunteers were rather high (9.2 –9.4 log). It has been noted previously39 that the magnitude of the response to prebiotics by bifidobacteria depends on the starting levels. It is apparent that we currently have an inadequate knowledge of the effects of various prebiotics upon the risk of colon cancer; more studies are needed to address this. Development of prebiotics with the goal of reducing biomarkers of cancer would, however, be very desirable.
Effects on pathogens Good evidence for the success of prebiotics lies in their ability to improve resistance to pathogens by increasing bifidobacteria and lactobacilli. Lactic-acid-excreting microorganisms are known for their inhibitory properties.40 In humans, viruses, protozoa, fungi and bacteria can all cause acute gastroenteritis. Metabolic end-products, such as acids excreted by these microorganisms, may lower the gut pH to levels below those at which pathogens are able effectively to compete. Also, many lactobacilli and bifidobacterial species are able to excrete natural antibiotics, which can have a broad spectrum of activity. For the bifidobacteria, some species are able to exert antimicrobial effects on various Gram-positive and Gram-negative intestinal pathogens.41 A recent study in mice has shown that FOS and inulin protected against enteric and systemic pathogens and tumour inducers.42 This includes the verocytotoxin strain of Escherichia coli O157:H7 and campylobacters. A rational way to reduce the food-poisoning burden may be fortify certain components of the intestinal flora such that it becomes much more resistant to invasion. This is achievable through the use of prebiotics that target bifidobacteria and/or lactobacilli. Taking this further, some other gut-related conditions more chronic than acute gastroenteritis, additionally labeled microbiological pathogens, may also be susceptible to prevention or treatment by altering the gut flora. Examples would include ulcerative colitis, bowel cancer, peptic ulcers, pseudomembranous colitis and Candida-induced conditions.
294 T. S. Manning and G. R. Gibson
Improved calcium absorption There has been increasing interest in recent years in the possibility of increasing mineral (particularly calcium) absorption through the consumption of prebiotics. Although the small intestine is the principal site of calcium absorption in humans, it is thought that significant amounts are absorbed throughout the length of the gut, consequently, maximizing of colonic effects is desirable. Several mechanisms have been postulated for increased calcium absorption induced by prebiotics.43 They include the following. (i) Fermentation of prebiotics such as inulin results in a significant production of SCFA, leading to a reduction in lumenal colonic pH. This is likely to increase calcium solubility and overall levels in the gut. (ii) Phytate (myoinositol hexaphosphate) is a component of plants that reaches the colon largely intact.44 It also forms stable, insoluble complexes with divalent cations, such as calcium, rendering them unavailable for transport. Fermentation results in bacterial metabolism of phytate, thereby liberating calcium. (iii) It is postulated that a calcium exchange mechanism operates in the colon. In this system, SCFAs enter the colon in a protonated form and then dissociate in the intracellular environment. The liberated proton is then secreted into the lumen in exchange for a calcium ion. Numerous animal studies have indicated that prebiotics increase absorption of calcium from the colon and decreased losses from bone tissue.45 However, very few human studies have been carried out. In one study, the feeding of 40 g inulin/day for 28 days to nine healthy subjects resulted in a significant increase in calcium absorption.46 A lower dose, 15 g inulin, FOS or GOS/day, when fed to 12 healthy subjects for 21 days, resulted in no significant effect on absorption of calcium or iron.47 In a later study, 12 adolescent boys (aged 14 – 16) were fed 15 g FOS/day for 9 days in a placebo-controlled trial against sucrose.48 The data showed a 10.8% increase in calcium balance with no significant effect on urinary excretion. Effects on blood lipids There is interest in the food industry in developing functional foods to modulate blood lipids such as cholesterol and triglycerides. It is widely believed that elevated cholesterol levels in the blood represent a risk factor for coronary heart disease, with low-density lipoproteins (LDL) being of most concern.49 There is also evidence that lactic acid bacteria may be able to reduce total and LDL cholesterol levels. The mechanisms by which lactic acid bacteria, and hence, indirectly, prebiotics, influence blood lipids are not clearly understood at the present time. It is possible that some lactic acid bacteria may be able to assimilate cholesterol directly.49,50 There is evidence that FOS decrease the de novo synthesis of triglycerides by the liver. The means by which this occurs is not fully understood but the effect appears be exerted at the transcriptional level. It is also possible that prebiotics (such as inulin) can modulate insulin-induced inhibition of triglyceride synthesis.50 Human studies on the lipidlowering properties of prebiotics when consumed at a realistic (tolerable) dose are, however, contradictory and the role in lipid reduction remains to be confirmed.51 Immunological effects Lactic acid bacteria are thought to stimulate both non-specific host defence mechanisms and certain types of cell involved in the specific immune response.
Prebiotics 295
The result is often increased phagocytic activity and/or elevated immunological molecules such as secretory IgA, which may affect pathogens such as salmonellae and rotavirus. Most attention in this respect has been diverted towards the intake of probiotics (lactic acid bacteria)52,53 and interactions between cell wall components and immune cells. As prebiotics serve a similar end point to lactic acid bacteria (i.e. improved composition of the gut microflora) similar effects may occur through their intake. A recent animal study have shown that prebiotics had an effect on immune functions.54
Practice points † health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens, cholesterol reduction and lowering of gas distension † harmful effects of microflora are carcinogen production, intestinal putrefaction, toxin formation, diarrhoea/constipation, liver damage and intestinal infection † a prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (i.e. bifidobacteria and lactobacilli), and thus improves the host’s health † prebiotics have been postulated to be protective against the development of colon cancer † evidence supports the ability of prebiotics to improve resistance to pathogens by increasing bifidobacteria and lactobacilli † numerous animal studies have indicated that prebiotics increase absorption of calcium from the colon and decreased losses from bone tissue † evidence of the ability of prebiotics to lower lipids remains controversial † prebiotics may have an effect on immune functions
Research agenda If progress in the use of dietary intervention directed towards particular gut bacteria is to be exploited, a sound research base is required. Some areas of interest may include: † the application of advanced molecular procedures that help to identify the gut microbial diversity as well as allow effective tracking of changes in microflora in response to diet (it is likely that a large number of gut bacteria have not hitherto been characterized, and culture-independent methodologies may help to overcome this) † the prebiotic potential of dietary ingredients, an identification of those foodstuffs that can be fortified and the optimal dose required † a definition of prebiotics which act at the species level and have a high degree of selectivity and contain multiple biological activities † whether certain target groups are more susceptible to the approach (elderly, weaning stage, formula-fed infants, hospitalized patients) † a determination of the health consequences that are associated with modulation of the gut flora
296 T. S. Manning and G. R. Gibson
SUMMARY The microflora of the gastrointestinal tract is key for nutrition and health of the host. Modulation of the microflora can occur through diets that contain prebiotics. The approach of using diet to induce microbial change offers a very straightforward approach towards improved health. In terms of new developments, it is important that the definitive health bonuses associated with prebiotic intake be determined. This is especially relevant given the broad applicability of their use. It is likely that prevention of acute gastroenteritis through fortification of certain components of the gut microflora is an important aspect. Moreover, improved protection from more chronic gut disorders that have been associated with bacteria (inflammatory bowel disease, colon cancer, irritable bowel syndrome) may also be possible. It may also be the case that certain target populations, such as infants, the elderly and hospitalized persons, are more susceptible to the approach. The health benefits that have been suggested are varied but also very important. In addition to good human volunteer studies we also need to enhance our mechanistic understanding of the health effects of prebiotics.
REFERENCES 1. Simon GL & Gorbach SL. The human intestinal microflora. Digestive Diseases and Sciences 1986; 31: 147S– 162S. *2. Gibson GR & Roberfroid MB. Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 3. Salminen S, Ramos P & Fonden R. Substrates and lactic acid bacteria. In Salminen S & von Wright A (eds) Lactic Acid Bacteria. New York: Marcel Dekker, 1993. *4. Gibson GR & Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 1995; 125: 1401–1412. 5. Sanders MA. Overview of functional foods: emphasis on probiotic bacteria. International Dairy Journal 1998; 8: 341–347. 6. Cummings JH & Macfarlane GT. The control and consequences of bacterial fermentation in the human colon. Journal of Applied Bacteriology 1991; 70: 443–459. *7. Salminen S, Bouley C, Boutron-Ruault M-C et al. Functional food science and gastrointestinal physiology and function. British Journal of Nutrition 1998; 80: S147– S171. 8. Macfarlane GT, Cummings JH & Allison C. Protein degradation by human intestinal bacteria. Journal of General Microbiology 1986; 132: 1647–1656. *9. Gibson GR, Berry Ottaway P & Rastall RA. Prebiotics: New Developments in Functional Foods. Oxford: Chandos Publishing Limited, 2000. * 10. Wang X & Gibson GR. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. Journal of Applied Bacteriology 1993; 75: 373–380. 11. De Leenheer L. Production and use if inulin: industrial reality with a promising future. In van Bekkum H, Roper H & Voragen AGJ (eds) Carbohydrates as Organic Raw Materials III. Weinheim: VCH, 1994. 12. Okazaki M, Fujikawa S & Matsumoto N. Effects of xylooligosaccharide on growth of bifidobacteria. Journal of the Japanese Society of Nutrition and Food Sciences 1990; 43: 395– 401. 13. Jaskari J, Kontula P, Siitonen A et al. Oat b-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains. Applied Microbiology and Biotechnology 1998; 49: 175 –181. * 14. Olano-Martin E, Mountzouris KC, Gibson GR & Rastall RA. In vitro fermentability of dextran, oligodextran and maltodextrin by human gut bacteria. British Journal of Nutrition 2000; 83: 247 –255. 15. Olano-Martin E, Mountzouris KC, Gibson GR & Rastall RA. Continuous production of oligosaccharides from pectin in an enzyme membrane reactor. Journal of Food Sciences 2001; 66: 966–971. 16. Svensson U. Industrial perspectives. In Tannock G (ed.) Probiotics: a Critical Review. Wymondham: Horizon Scientific Press, 1999, pp 57–64. 17. Lee Y-K, Nomoto K, Salminen S & Gorbach SL. Handbook of Probiotics. New York: Wiley, 1999. 18. Cooperstock MS & Zedd AJ. Intestinal flora of infants. In Hentges DJ (ed.) Human Intestinal microflora in Health and Disease. London: Academic Press, 1983, pp 79–99.
Prebiotics 297 19. Benno Y, Sawada K & Mitsuoka T. The intestinal microflora of infants: composition of fecal flora in breastfed and bottle-fed infants. Microbiology and Immunology 1984; 28: 975–986. 20. Mitsuoka T. Bifidobacteria and their role in human health. Journal of Industrial Microbiology 1990; 6: 263–268. 21. Kleessen B, Sykura B, Zunft H-J & Blaut M. Effects of inulin and lactose on fecal microflora, microbial activity and bowel habit in elderly constipated persons. American Journal of Clinical Nutrition 1997; 65: 1397– 1402. * 22. Gibson GR, Beatty ER, Wang X & Cummings JH. Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 1995; 108: 975 –982. * 23. Tuohy KM, Kolida S, Lustenberger A & Gibson GR. The prebiotic effects of biscuits containing partially hydrolyzed guar gum and fructooligosaccharides—a human volunteer study. British Journal of Nutrition 2001; 86: 341 –348. 24. Rowland IR. Metabolic interactions in the gut. In Fuller R (ed.) Probiotics: the Scientific Basis. Andover, UK: Chapman & Hall, 1992. 25. Rowland IR & Tanaka R. The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human faecal microflora. Journal of Applied Bacteriology 1993; 74: 667 –674. 26. Reddy BS, Hamid R & Rao CV. Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition. Carcinogenesis 1997; 18: 1371– 1374. 27. Bouhnik Y, Flourie B, Riottot M et al. Effects of fructo-oligosaccharides ingestion on faecal bifidobacteria and selected metabolic indexes of colon carcinogenesis in healthy humans. Nutrition and Cancer 1996; 26: 21–29. 28. Buddington RK, Williams CH, Chen S-C & Witherly SA. Dietary supplementation of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. American Journal of Clinical Nutrition 1996; 63: 709–716. 29. Hylla S, Gostner A, Dusel G et al. Effects of resistant starch on the colon in healthy volunteers: possible implications for cancer prevention. American Journal of Clinical Nutrition 1998; 67: 136 –142. * 30. Gibson GR & Macfarlane GT. Intestinal bacteria and disease. In Gibson SAW (ed.) Human Health—the Contribution of Microorganisms. London: Springer-Verlag, 1994. 31. Morotomi M, Guillem JG, LoGerfo P & Weinsten IB. Production of diacylglycerol, an activator of protein kinase C by human intestinal microflora. Cancer Research 1990; 50: 3595–3599. 32. Rowland IR (ed.) Role of the Gut Flora in Toxicity and Cancer. London: Academic Press, 1998. 33. Prasad KN. Butyric acid: a small fatty acid with diverse biological functions. Life Sciences 1980; 27: 1351– 1358. 34. Kim YS, Tsao D, Morita A & Bella A. Effect of sodium butyrate and three human colorectal adenocarcinoma cell lines in culture. Falk Symposium 1982; 31: 317–323. 35. Videla S, Vilaseca J, Antolin M et al. Dietary inulin improves distal colitis induced by dextran sodium sulfate in the rat. American Journal of Gastroenterology 2001; 96: 1468–1493. 36. Reddy BS. Prevention of colon cancer by pre- and probiotics: evidence from laboratory studies. British Journal of Nutrition 1998; 80: S219–S223. 37. Alles MS, Hartemink R, Meyboom S et al. Effect of transgalactooligosaccharides on the composition of the human intestinal microflora and on putative risk markers for colon cancer. American Journal of Clinical Nutrition 1999; 69: 980–991. 38. Schoterman HC & Timmermans HJAR. Galacto-oligosaccharides. In Gibson GR & Angus F (eds) Prebiotics and Probiotics. LFRA Ingredients Handbook. Leatherhead: Food RA Publishing, 2000. 39. Rycroft CE, Jones MR, Gibson GR & Rastall RA. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. Journal of Applied Bacteriology 2001; 91: 878– 887. 40. Fuller R (ed.) Probiotics 2: Application and Practical Aspects. Andover, UK: Chapman & Hall, 1997. 41. Mackey BM & Gibson GR. Escherichia coli 0157-from farm to fork and beyond. Society of General Microbiology Quarterly 1997; 24: 55– 57. 42. Buddington KK, Danohoo JB & Buddington RK. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumour inducers. Journal of Nutrition 2002; 132: 472– 477. 43. Fairweather-Tait SJ & Johnson IT. Bioavailability of minerals. In Gibson GR & Roberfroid MB (eds) Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 44. Cummings JH, Hill MJ, Houston H et al. The effect of meat protein and dietary fibre on colonic function and metabolism. 1. Changes in bowel habit, bile acid excretion and calcium absorption. American Journal of Clinical Nutrition 1979; 32: 2086–2093. 45. Greger JL. Nondigestible carbohydrates and mineral bioavailability. Journal of Nutrition 1999; 129: 1434S–1435S. 46. Coudray C, Bellanger J, Castiglia-Delavaud C et al. Effect of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. European Journal of Clinical Nutrition 1997; 51: 375 –380.
298 T. S. Manning and G. R. Gibson 47. van den Heuvel E, Schaafsma G, Muys T & van Dokkum W. Non-digestible oligosaccharides do not interfere with calcium and non-haeme iron absorption in young, healthy men. American Journal of Clinical Nutrition 1998; 67: 445 –451. 48. van den Heuvel E, Muys T, van Dokkum W & Schaafsma G. Oligofructose stimulates calcium absorption in adolescents. American Journal of Clinical Nutrition 1999; 69: 544 –548. 49. Delzenne NM & Williams CM. Actions of non-digestible carbohydrates on blood lipids in humans and animals. In Gibson GR & Roberfroid MB (eds) Colonic Microbiota, Nutrition and Health. Dordrecht: Kluwer Academic Press, 1999. 50. Delzenne NM & Kok NN. Biochemical basis of oligofructose-induced hypolipidaemia in animal models. Journal of Nutrition 1999; 129: 1467S–1470S. 51. Williams CM. Effects of inulin on lipid parameters in humans. Journal of Nutrition 1999; 129: 1471S–1473S. 52. Schiffrin EJ, Rochat F, Link-Amster HA et al. Immune system stimulation by probiotics. Journal of Dairy Science 1995; 78: 1597–1606. 53. Perdigon G & Alvarez S. Probiotics and the immune state. In Fuller R (ed.) Probiotics: the Scientific Basis. London: Chapman & Hall, 1992, pp 146–180. * 54. Swanson KS. Prebiotics and probiotics: impact on gut microbial populations, nutrient digestibilities, fecal protein catabolite concentrations and immune functions of humans and dogs. Dissertation and Abstracts International 2002; 63: 746.