Symposium: Probiotic Bacteria: Implications for Human Health

Aspects of In Vitro and In Vivo Research Approaches Directed Toward Identifying Probiotics and Prebiotics for Human Use1
Glenn R. Gibson2 and Roy Fuller*
Department of Food Science and Technology, The University of Reading, Reading, UK and *Russett House, Ryeish Green, Reading, UK
ABSTRACT The microbiota of the human gastrointestinal tract plays a key role in nutrition and health. Through the process of fermentation, gut bacteria metabolize various substrates (principally dietary components) to end products such as short-chain fatty acids and gases. This anaerobic metabolism is thought to contribute positively toward host daily energy requirements. However, under certain circumstances, the fermentative process may produce undesirable metabolites. This may cause the onset of gut disorders that can be manifest through both acute and chronic conditions. Moreover, the gut ora may become contaminated by transient pathogens that serve further to upset the normal community structure. There has been a recent increase in the use of dietary components that help to maintain, or even improve, the gut microora balance. Probiotics are live microbial feed supplements added to appropriate food vehicles (usually fermented milks), whereas prebiotics are dietary carbohydrates that have a selective metabolism in the colon and serve to increase numbers of bacteria seen as desirable. Because of their purported health-promoting properties, lactic acidproducing bacteria, including bidobacteria, are the usual target organisms. The market value and biological potential of both approaches are enormous. This article will summarize how efcacious types can be identied. J. Nutr. 130: 391S395S, 2000. KEY WORDS:

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gut microbiology

probiotic

prebiotic

A delicate balance exists between the human intestinal microora and its host. Upset of this community structure may lead toward the symptoms of acute gastroenteritis, and there is also the possibility of more chronic disorders (inammatory bowel disease, colonic cancer). It is therefore important that gut microora interactions be controlled and sustained in an optimal manner. Many different environmental factors may affect the gut microbial ecology; these include diet, medication, stress, age and general living conditions. Knowledge of the gut microora and its interactions has led to the development of dietary strategies that serve to sustain, or even improve normal gastrointestinal microbiology. Both probiotics and prebiotics are popular concepts that have been developed to target the gastrointestinal microora. Denitions and development The Greek meaning of the word probiotic is for life. One early formal denition was that of Parker (1974), i.e., Organisms and substances which contribute to intestinal microbial
1 Presented at the symposium entitled Probiotic Bacteria: Implications for Human Health as part of the Experimental Biology 99 meeting held April 1721 in Washington, DC. This symposium was sponsored by the American Society for Nutritional Sciences and was supported in part by an educational grant from the National Dairy Council. The proceedings of this symposium are published as a supplement to The Journal of Nutrition. Guest editor for this supplement was Douglas B. DiRenzo, National Dairy Council, Rosemont, IL. 2 To whom correspondence should be addressed.

balance. However, this was subsequently rened by Fuller (1989) whose revised denition is A live microbial feed supplement which benecially affects the host animal by improving its intestinal microbial balance. This version emphasizes the need for the supplement to be composed of viable microorganisms and is the most widely used and accepted denition. Probiotics, therefore, aim to produce a benecial effect on the host by administration of viable microorganisms such as those found in traditional yogurts and other fermented foods, as well as powders, tablets, liquid suspensions and lyophilized forms in capsules. Monocultures as well as mixed species (up to nine) can be used in individual products. Primarily, these are lactic acid excreting bacteria such as lactobacilli, streptococci, lactococci or bidobacteria, although some yeasts and other fungi are also used. Metchnikoff (1907) rst developed the concept of what we now know as probiotics at the beginning of this century. His hypothesis was that the complex microbiota of the colon was having an adverse effect on the host through what he termed the autointoxication effect. As such, he believed that modication of the activity of the colonic microora could occur through the ingestion of soured milks. The theory was developed after he observed that Bulgarian peasants consumed large quantities of such milks and exhibited longevity. Metchnikoff isolated the bacteria responsible and used them in human feeding trials. After Metchnikoffs death, Rettger and colleagues became interested in the mechanism of the probiotic

0022-3166/00 $3.00 2000 American Society for Nutritional Sciences.

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effects and researched the use of intestine-derived species (Rettger et al. 1935). The eld then took a number of scientic progressions to reach todays situation in which live microbial feed additions are ubiquitous. Three landmark observations were as follows: 1) germ-free animals are more susceptible to infection than are their conventional counterparts (Collins and Carter 1978); 2) oral antibiotics increase susceptibility of animals to infection (Freter 1955); and 3) administration of fecal enemas may control antibiotic-associated diarrhea (Schwass et al. 1984). For a prebiotic, the view is that the human large intestine contains indigenous bacteria that are benecial, benign and detrimental for host health; it has been dened as a nondigestible food ingredient that benecially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Gibson and Roberfroid 1995). In this context, a prebiotic is a dietary ingredient that reaches the large intestine in an intact form and has a specic metabolism therein, one directed toward benecial rather than harmful bacteria. This would ultimately lead to a marked change in the gut microora composition. Preferred target organisms for prebiotics are species belonging to the Lactobacillus and Bidobacterium genera. The most efcient prebiotics may also reduce or suppress numbers and activities of organisms seen as pathogenic. For the substrate to be classied as a prebiotic, the following three criteria should be met: 1) the substrate must not be hydrolyzed or absorbed in the stomach or small intestine; 2) it must be selective for benecial commensal bacteria in the colon by encouraging the growth/metabolism of the organisms; and 3) it will alter the microora to a healthy composition by inducing benecial luminal/systemic effects within the host. Any food substrate that enters the colon is a potential prebiotic; however, selectivity of the fermentation is a required determinant. Nondigestible oligosaccharides (NDO) are dietary substrates that seem to possess good prebiotic potential. Much of the early and ongoing work on prebiotics has been carried out in Japan. The search for bidobacteria-promoting substances began by screening a range of carbon sources for their ability to increase these organisms in pure culture. For example, Yazawa et al. (1978) screened a range of dietary carbohydrates for their ability to promote bidobacteria in comparison to other intestinal isolates. Further studies used mixed culture, animal models and human trials to determine the efcacy of oligosaccharides to modulate the gut ora composition (for reviews, see Crittenden 1999, Gibson et al. 1999). The term prebiotic was rst coined in the mid-1990s (Gibson and Roberfroid 1995). Methods for testing probiotics and prebiotics Various different in vitro and in vivo approaches have been used to measure the efcacy of probiotics and prebiotics. For probiotics, challenge tests may be undertaken to determine survivability. Here, the test strain would be added to a fermenter and/or administered to a laboratory animal or humans. Subsequent effects, including those on immune function, lipids and pathogens, would then be determined. One important requirement is that the probiotic strain be detected in the study. Here, specic metabolic traits may be of some value, as are selected antibiotic resistance markers. However, neither is likely to be as efcacious as the use of certain molecular markers, which allow high discrimination and are genetically conserved. There have been some studies in which the green uorescent protein is expressed by target strains and thus

allows the probiotic to be tracked (Collins and Gibson 1999). For prebiotics, it is important that their nondigestibility as well as selective fermentation be determined. Substrate integrity in the upper gastrointestinal tract is a requirement and can be measured using either in vitro conditions that simulate this environment or an ileostomy model (Gibson et al. 1999). A popular approach for determining bacterial fermentability is to use agars thought to be selective for gut microorganisms and measuring the response of predominant colonic genera during prebiotic fermentation. However, such methods are not wholly reliable; they do not recover the full gut diversity, and the techniques are both laborious and susceptible to operator subjectivity. There has been a large shift toward the use of molecular procedures in microbiology generally and gut bacteriology specically. For a review of applicable techniques see OSullivan (1999). The simplest in vitro fermenters are static batch cultures; the substrate is added at a known concentration to a vessel containing a fecal suspension, or dened cultures, incubated anaerobically at 37C for a short period (usually 24 48 h) and sampled at regular intervals (Wang and Gibson 1993). This method takes a relatively short time; thus, it can act as a quick screening procedure when substrates are being compared. Moreover, only small volumes are required, which is important if the test material is in short supply. However, batch fermenters are closed systems in which the substrate is limited and the culture follows a typical bacterial growth curve; therefore, they may be used only for short time course experiments. Continuous culture systems (chemostats) can be used to simulate the intestinal conditions more closely. By varying dilution rates and other parameters, optimum conditions for growth can be determined under steady-state conditions (Gibson and Wang 1994a). Semicontinuous cultures in which the medium is added and spent culture removed at specic intervals have also been used. There are other variations on the single-stage chemostat approach. For example, Gibson and Wang (1994b) used a two-vessel system, with a membrane between the two chambers, called a diffusion chemostat. This allowed diffusion of growth factors and metabolites between the vessels, but not of the cells themselves. Multistage chemostats have also been developed and are used as efcient gut models in that each vessel represents a different physicochemical region of the intestine. The model validated (against gut contents from human sudden death victims) by Macfarlane et al. (1998) consists of three vessels aligned in series. The rst has nutrient-rich, fast transit and acidic conditions; the third has much less substrate, slow and neutral conditions (see Fig. 1 for a diagrammatic representation of the system). The rst vessel is set to resemble the proximal colon, the second the transverse colon and the third the distal colon. The ultimate test for probiotic or prebiotic functionality is the in vivo situation, in particular, well-controlled human studies. Animals, usually rats or mice, have been used to determine the effect of substrate on the fecal microora. Animals associated with their normal ora may be used for certain effects such as gas production, weight changes and toxicology. However, differences exist between animal and human microora; thus comparative results are likely to be compromised. Gnotobiotic rats may be used to investigate interactions between the host and the organisms, but these situations do not resemble the usual situation in the gut. Human oraassociated rats, or mice, give one representation of the situation in the human colon, although the intestinal physiology is not the same. Obviously, the best test of efciency is a human volunteer trial with placebo control and blind coded samples. How-

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FIGURE 1 Diagrammatic representation of the three-stage multiple continuous culture chemostat as used to mimic the human colonic microbial environment. Vessel 1 (V1) has a low operating volume, reduced pH, rapid transit and high substrate availability. This approximates the physicochemical conditions in the proximal colon. Vessel 3 (V3) has the highest operating volume, neutral pH, slow transit and low substrate availability. This approximates to the distal colon. Vessel 2 has imposed physicochemical parameters intermediate between V1 and V3 and resembles the transverse colon. Vessel 1 is fed with a substrate input that is typical of the Western diet (i.e., a mixture of carbohydrates, proteins, vitamins); each vessel is inoculated with feces and operated anaerobically at 37C. The system operates as a cascade in that V1 sequentially feeds V2, which then feeds V3. After a period of equilibration, such that the imposed conditions prevail on the microora inoculum, the model can be inoculated with a probiotic(s) or prebiotic(s). Subsequently, strain recoveries and fermentation characteristics can be monitored. Through comparisons on the bacteriology and enzymology of gut contents from sudden death victims, the system has been validated as efcacious (Macfarlane et al. 1998).

ing to the gut, some degree of longevity was clearly evident in situ. Such strains would presumably be very effective probiotics; however, should attachment be a key facet, it may be that the appropriate receptors are present only in certain individuals. Nevertheless, to reduce the necessity for continuous administration of cultures, it may be advantageous to use an adhering strain. A number of other selection criteria may be important for the success of probiotics. Some examples are shown in Table 1. Not every probiotic will have all of these characteristics, but it is desirable that as many as possible are present. Nondigestible oligosaccharides (NDO) seem to be preferred prebiotics. These are oligomeric carbohydrates, whose osidic bond allows resistance to intestinal digestive enzymes but can be metabolized by bacteria. As such, they do fulll certain criteria for prebiotic functionality in that they are resistant to hydrolysis in the upper gastrointestinal tract of humans, but may be fermented by the colonic microbiota (Roberfroid 1997, Van Loo et al. 1995). NDO for which in vitro and in vivo data have been published to support a prebiotic effect include lactulose, inulin-type fructans, galactooligosaccharides, soybean oligosaccharides (rafnose and stachyose), isomaltooligosaccharides, glucooligosaccharides and xylooligosaccharides (Table 2). Currently, few comparative data exist on the relative efciencies of these molecules, i.e., carried out in the same study. It is often the case that a prebiotic effect is suggested without a full and careful investigation of the fermentation prole. It is critical that as many components of the gut microbiota as possible be measured during fermentation studies. These should include at least bacteroides, bidobacteria, clostridia, gram-positive cocci, coliforms, lactobacilli, total aerobes and total anaerobes. Pure bacterial studies should also be supported by mixed culture work because it is the competitive ecological effect that is critical. As mentioned earlier, developments in

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TABLE 1
Examples of criteria that aid the selection of efcacious probiotics

ever, drawbacks still exist in that such trials may be difcult and expensive to set up. Moreover, unlike trials using animals, different regions of the gut are inaccessible, because only fecal material is readily available. Mechanisms Little is known about the way in which probiotics can inuence the gut microora; therefore, mechanisms for benecial effects on the host are difcult to determine. However, it may be assumed that resistance to low (stomach) pH, bile acids and ability to compete effectively in the gut ecosystem are likely to enhance probiotic effects. For prebiotics, strain survival is not required, but the nonviable food component should enter the large gut intact and have a selective fermentation therein. One facet that would improve survival of a probiotic agent would be an ability to attach successfully to the gut epithelium. McCartney et al. (1996) carried out a study in which ribotyping was used to identify population dynamics of lactic acid bacteria in two individuals over a 1-y period. In one volunteer, the ora existed in a relatively transient state with varying ribopatterns frequently being detected. However, in the other volunteer, some indigenous strains of lactobacilli and bidobacteria were detectable over the entire 12-mo period. Although it is unclear whether these strains were attach-

Strain originthose isolated from the same species as the intended use ought to have an enhanced chance of survival Safetyprobiotics should be generally recognized as safe (GRAS) with minimal possibilities for the transfer of antibiotic resistance Survivabilityboth in the product and after ingestion. Strains that have improved resistance to acid, bile secretions and able to attach to the gut epithelium would have better survival characteristics Production characteristicsable to be grown in bulk culture, without genetic variation Processingrobust enough to withstand the rigors associated with incorporation into oral delivery systems Sensory propertieswhen added to foods, the quality should not be diminished Microbiological propertiesrequired to survive in the gastrointestinal microbial ecosystem Effects on the consumerno adverse side effects such as bloating and effects on gut transit should occur Adherenceto enhance survival in the gut Effects on pathogensmany probiotics are able to inhibit adverse microorganisms by the production of acid, bacteriocins or competitive exclusion Modulation of metabolic activitiessuch as the inactivation of procarcinogens Immunomodulationprobiotics may affect the immune system such that improved pathogen resistance occurs, as well as positive aspects with respect to food allergy

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TABLE 2
Examples of in vitro and in vivo studies designed to determine the efcacy of candidate prebiotics1
Candidate prebiotic Lactulose Fructooligosaccharides In vitro study Fadden and Owen (1992) Wang and Gibson (1993) Gibson and Wang (1994a and b) McBain and Macfarlane (1997) In vivo study Suzuki et al. (1985) Hidaka et al. (1986) Williams et al. (1994) Bouhnik et al. (1994) Gibson et al. (1995) Buddington et al. (1996) Kleesen et al. (1997) Andrieux and Szylit (1992) Ito et al. (1990 and 1993) Rowland and Tanaka (1993) Bouhnik et al. (1997) Benno et al. (1987) Hayakawa et al. (1990) Kohomoto et al. (1991) Kaneko et al. (1994) Djouzi et al. (1995) Okazaki et al. (1990) Downloaded from jn.nutrition.org by guest on September 5, 2012

Galactooligosaccharides

Tanaka et al. (1983) Durand et al. (1992) Bouhnik et al. (1997) Tamura (1983) Hayakawa et al. (1990) Saito et al. (1992) Kohmoto et al. (1988) Djouzi et al. (1995) Hopkins et al. (1998)

Soybean oligosaccharides Isomaltooligosaccharides Glucooligosaccharides Xylooligosaccharides

1 The original articles should be referred to for details of the studies carried out. Note that the in vitro trials contained both pure and mixed culture fermentation data. The in vivo studies cited were both animal and human work, with various degrees of control exerted.

the application of molecular biological techniques to human gut bacteriology are of enormous value in this respect in that they offer the degree of delity, automation and discrimination that is required. Fructooligosaccharides are the most extensively studied NDO in terms of their prebiotic properties. These carbohydrates contain both GpyFn (-D-glucopyramosyl-[-D-fructofuranosyl]n-1-D-fructofuranoside) and FpyFn (-D-fructopyranosyl-[-D-fructofuranosyl]n-1-D-fructofuranoside) molecules, with the number of fructose units varying from 2 to 70. They are available either as inulin, which is the storage carbohydrate in many thousands of plants, or can be synthesized enzymatically from sucrose (Van Loo et al. 1995). Bidobacteria possess a cell-bound -fructofuranosidase enzyme that allows preferred utilization of fructooligosaccharides over sucrose (Muramatsu et al. 1994) and clearly offers this genus a competitive advantage in the human gut. The fructose moiety is then metabolized in the specic bidus pathway. Similarly, bidobacterial -galactosidase activity likely allows a prebiotic effect for soybean oligosaccharides (Desjardins et al. 1990). Galactooligosaccharides are manufactured from lactose by transglycosylation reactions and consist of galactosyl derivatives of lactose with 13 and 1 6 linkages. The purported prebiotic nature of galactooligosaccharides may be due to the linkage-specicity of the Bidobacterium -galactosidase (Dumortier et al. 1994). Isomaltooligosaccharides (1 6 linked) and glucooligosaccharides (1 6 linked) are candidate prebiotics, as are xylooligosaccharides. However, specic enzymes for the degradation of these molecules have not yet been evaluated; thus the explanatory mechanism for any purported prebiotic effect is not yet evident. SUMMARY This article has introduced the rationale for probiotic and prebiotic use in humans. Both approaches serve to improve the gut microora composition because the importance of intestinal microbiology in health and disease is now clear. Other reviews in this series will identify the possible health outcomes. However, it is clear that microora management

through diet is achievable. Importantly, the scientic tools for determining probiotic and prebiotic effects now exist and should be exploited. In particular, this involves a molecular approach to the fermentation process in conjunction with well-controlled human trials. LITERATURE CITED

Andrieux, C. & Szylit, O. (1992) Effects of galacto-oligosaccharides (TOS) on bacterial enzyme activities and metabolite production in rats associated with a human faecal ora. Proc. Nutr. Soc. 51: 7A (abs.). Benno, Y., Endo, K., Shiragami, N., Sayama, K. & Mitsuoka, T. (1987) Effects of rafnose intake on human fecal microora. Bid. Microora 6: 59 63. Bouhnik, Y., Flourie, B., DAgay-Abensour, L., Pochart, P., Gramet, G., Durand, M. & Rambaud, J. C. (1997) Administration of transgalacto-oligosaccharides increases fecal bidobacteria and modies colonic fermentation metabolism in healthy humans. J. Nutr. 127: 444 448. Bouhnik, Y., Flourie, B., Ouarne, F., Riottot, M., Bisetti, N., Bornet, F. & Rambaud, J. C. (1994) Effects of prolonged ingestion of fructo-oligosaccharides (FOS) on colonic bidobacteria, fecal enzymes and bile acids in humans. Gastroenterology 106: A598 (abs.). Buddington, R. K., Williams, C. H., Chen, S. & Witherly, S. A. (1996) Dietary supplement of neosugar alters the fecal ora and decreases activities of some reductive enzymes in human subjects. Am. J. Clin. Nutr. 63: 709 716. Collins, F. M. & Carter, P. B. (1978) Growth of salmonellae in orally infected germfree mice. Infect. Immunol. 21: 41 47. Collins, M. D. & Gibson, G. R. (1999) Probiotics, prebiotics and synbiotics: dietary approaches for the modulation of microbial ecology. Am. J. Clin. Nutr. 69: 10521057. Crittenden, R. G. (1999) Prebiotics. In: Probiotics: A Critical Review (Tannock, G., ed.). Horizon Scientic Press, Wymondham, UK. Desjardins, M. L., Roy, D. & Goulet, J. (1990) Growth of bidobacteria and their enzyme proles. J. Dairy Sci. 73: 299 307. Djouzi, Z., Andrieux, C., Pelenc, V., Somarriba, S., Popot, F., Paul, F., Monsan, P. & Szylit, O. (1995) Degradation and fermentation of -gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats. J. Appl. Bacteriol. 79: 117127. Dumortier, V., Brassart, C. & Bouquelet, S. (1994) Purication and properties of a -galactosidase from Bidobacterium bidum exhibiting a transgalactosylation reaction. Biotechnol. Appl. Biochem. 19: 341354. Durand, M., Cordelet, C., Hannequart, G. & Beaumatin, P. (1992) In vitro fermentation of a galacto-oligosaccharide by human bacteria in continuous culture. Proc. Nutr. Soc. 51: 6A (abs.). Fadden, K. & Owen, R. W. (1992) Faecal steroids and colorectal cancer: the effect of lactulose on faecal bacterial metabolism in a continuous culture model of the large intestine. Eur. J. Cancer Prev. 1: 113127. Freter, R. (1955) The fatal enteric cholera infection in the guinea pig achieved by inhibition of normal enteric ora. J. Infect. Dis. 97: 57 65. Fuller, R. (1989) Probiotics in man and animals. J. Appl. Bacteriol. 66: 365 378.

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Gibson, G. R. & Wang, X. (1994a) Enrichment of bidobacteria from human gut contents by oligofructose using continuous culture. FEMS Microbiol. Lett. 118: 121128. Gibson, G. R. & Wang, X. (1994b) Regulatory effects of bidobacteria on the growth of other colonic bacteria. J. Appl. Bacteriol. 77: 412 420. Gibson, G. R., Beatty, E. R., Wang, X. & Cummings, J. H. (1995) Selective stimulation of bidobacteria in the human colon by oligofructose and inulin. Gastroenterology 108: 975982. Gibson, G. R. & Roberfroid, M. B. (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125: 1401 1412. Gibson, G. R., Rastall R. A. & Roberfroid, M. B. (1999) Prebiotics. In: Colonic Microbiota, Nutrition and Health (Gibson, G. R. & Roberfroid., M. B., eds.) Kluwer Academic, Dordrecht, The Netherlands. Hayakawa, K., Mizutani, J., Wada, K., Masai, T., Yoshihara, I. & Mitsuoka, T. (1990) Effects of soybean oligosaccharides on human faecal ora. Microb. Ecol. Health Dis. 3: 293303. Hidaka, H., Eida, T., Takizawa, T., Tokunaga, T. & Tashiro, Y. (1986) Effects of fructooligosaccharides on intestinal ora and human health. Bid. Microora 5: 3750. Hopkins, M. J., Cummings, J. H. & Macfarlane, G. T. (1998) Inter-species differences in maximum specic growth rates and cell yields of bidobacteria cultured on oligosaccharides and other simple carbohydrate sources. J. Appl. Microbiol. 85: 381386. Ito, M., Deguchi, Y., Miyamori, A., Matsumoto, K., Kikuchi, H., Matsumoto, K., Kobayashi, Y., Yajima, T. & Kan, T. (1990) Effects of administration of galactooligosaccharides on the human fecal microora, stool weight and abdominal sensation. Microb. Ecol. Health Dis. 3: 285292. Ito, M., Kimura, M., Deguchi, Y., Miyamori-Watabe, A., Yajima, T. & Kan, T. (1993) Effects of transgalactosylated disaccharides on the human intestinal microora and their metabolism. J. Nutr. Sci. Vitaminol. 39: 279 288. Kaneko, T., Kohmoto, T., Kikuchi, H., Shiota, M., Iino, H. & Mitsuoka, T. (1994) Effects of isomaltooligosaccharides with different degrees of polymerisation on human fecal bidobacteria. Biosci. Biotechnol. Biochem. 58: 2288 2290. Kleessen, B., Sykura, B., Zunft, H.-J. & Blaut, M. (1997) Effects of inulin and lactose on fecal microora, microbial activity and bowel habit in elderly constipated persons. Am. J. Clin. Nutr. 65: 13971402. Kohmoto, T., Fukui, F., Takaku, H., Machida, Y., Arai, M. & Mitsuoka, T. (1988) Effect of isomalto-oligosaccharides on human fecal ora. Bid. Microora 7: 61 69. Kohmoto, T., Fukui, F., Takaku, H. & Mitsuoka, T. (1991) Dose-response test of isomaltooligosaccharides for increasing fecal bidobacteria. Agric. Biol. Chem. 55: 21572159. Macfarlane, G. T., Macfarlane, S. & Gibson, G. R. (1998) Validation of a three-stage compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colonic microbiota. Microb. Ecol. 35: 180 187. McBain, A. J. & Macfarlane, G. T. (1997) Investigations of bidobacterial ecology and oligosaccharide metabolism in a three-stage compound continuous culture system. Scand. J. Gastoenterol. 32: 32 40.

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McCartney, A. L., Wenzhi, W. & Tannock, G. W. (1996) Molecular analysis of the composition of the bidobacterial and lactobacillus microora of humans. Appl. Environ. Microbiol. 62: 4608 4613. Metchnikoff, E. (1907) The Prolongation of Life. William Heinemann, London, UK. Muramatsu, K., Onodera, S., Kikuchi, M. & Shiomi, N. (1994) Substrate specicity and subsite afnities of -fructofuranosidase from Bidobacterium adolescentis G1. Biosci. Biotechnol. Biochem. 58: 16421645. Okazaki, M., Fujikawa, S. & Matsumoto, N. (1990) Effects of xylooligosaccharide on growth of bidobacteria. J. Jpn. Soc. Nutr. Food Sci. 43: 395 401. OSullivan, D. J. (1999) Methods for analysis of the intestinal microora. In: Probiotics: A Critical Review (Tannock, G., ed.). Horizon Scientic Press, Wymondham, UK. Parker, R. B. (1974) Probiotics, the other half of the antibiotic story. Anim. Nutr. Health 29: 4 8. Rettger, F., Levy, M. N., Weinstein, K. & Weiss, J. E. (1935) Lactobacillus acidophilus and Its Therapeutic Application. Yale University Press, New Haven, CT. Roberfroid, M. B. (1997) Health benets of non-digestible oligosaccharides, In: Dietary Fiber in Health and Disease (Kritchevsky, D. & Boneld, C., eds.), pp. 211219. Plenum Press, New York, NY. Rowland, I. R. & Tanaka, R. (1993) The effects of transgalactosylated oligosaccharides on gut ora metabolism in rats associated with a human faecal microora, J. Appl. Bacteriol. 74: 667 674. Saito, Y., Takano, T. & Rowland, I. (1992) Effects of soybean oligosaccharides on the human gut microora in in vitro culture. Microb. Ecol. Health Dis. 5: 105110. Schwass, A., Sjolin, S. Trottestam, V. & Aransson, B. (1984) Relapsing Clostridium difcile enterocolitis cured by rectal infusion of normal faeces. Scand. J. Infect. Dis. 16: 211215. Suzuki, K., Endo, Y., Uehara, M., Yamada, H., Goto, S., Imamura, M. & Shioza, S. (1985) Effect of lactose, lactulose and sorbital on mineral utilisation and intestinal ora. J. Jpn. Soc. Nutr. Food Sci. 38: 39 42. Tamura, Z. (1983) Nutriology of bidobacteria. Bid. Microora 2: 316. Tanaka, R., Takayama, H., Morotomi, M., Kuroshima, T., Ueyama, S., Matsumoto, K., Kuroda, A. & Mutai, M. (1983) Effects of administration of TOS and Bidobacterium breve 4006 on the human fecal ora. Bid. Microora 2: 1724. Van Loo, J.A.E., Coussement, P., De Leenheer, L., Hoebregs, H. & Smits, G. (1995) On the presence of inulin and oligofructose as natural ingredients in the western diet. CRC Crit. Rev. Food Sci. Nutr. 35: 525552. Wang, X. & Gibson, G. R. (1993) Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J. Appl. Bacteriol. 75: 373380. Williams, C. H., Witherly, S. A. & Buddington, R. K. (1994) Inuence of dietary neosugar on selected bacterial groups of the human faecal microbiota. Microb. Ecol. Health Dis. 7: 9197. Yazawa, K., Imai, K. & Tamura, Z. (1978) Oligosaccharides and polysaccharides specically utilisable by bidobacteria, Chem. Pharm. Bull. 26: 3306 3311.

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