Addresses Gut Group, University of Dundee, Ninewells Hospital Medical School, Dundee DD1 9SY, United Kingdom

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1 Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut George T Macfarlane and Sandra Macfarlane There is increasing interest in the human colonic microbiota and in the way its metabolic activities impact on host health and well-being. For most practical purposes, however, the large bowel is inaccessible for routine investigation, and a variety of animal and in vitro model systems have been developed to study the microbiota. In vitro models range from simple closed systems using pure or defined mixed populations of bacteria, or faecal material, to more sophisticated complex multistage continuous cultures that are able to simulate many of the spatial, temporal and environmental attributes that characterize microbiological events in different regions of the large gut. Recent developments using these systems have enabled modelling of surface colonisation and biofilm development, a hitherto neglected area of study. Addresses Gut Group, University of Dundee, Ninewells Hospital Medical School, Dundee DD1 9SY, United Kingdom Corresponding author: Macfarlane, George T (g.t.macfarlane@dundee.ac.uk) This review comes from a themed issue on Food biotechnology Edited by Christophe Lacroix and Beat Mollet Available online 2nd February /$ see front matter # 2006 Elsevier Ltd. All rights reserved. DOI /j.copbio Introduction The human large intestine is a specialized digestive organ. It harbours large numbers of microorganisms that exist in dynamic but stable communities. Several hundred different species and strains of bacteria have been identified in faecal material [1], as well as on mucosal surfaces in the gut [2,3]. The metabolic activities of the microbiota play a key role in host physiology in health and disease. For example, through fermentation, colonic microorganisms are responsible for the final stages of the digestive process, and through the formation of short chain fatty acids (SCFA), contribute to energy reclamation from the gut contributing up to 10% of the body s metabolic requirements [4]. Butyrate is the principal fuel for the colonic epithelium and also has anticancer properties, while acetate is utilized by the brain, heart and in peripheral tissues [4]. Although exfoliated epithelial cells and other host-produced substances such as mucus, pancreatic enzymes, and other secretions are broken down and recycled by the microbiota, its species composition and biochemical activities are principally determined by diet, particularly carbohydrate availability [4]. The large intestine is an open system in the sense that food residues from the small bowel enter at one end, and faecal material is periodically excreted at the other end. Because of its anatomy, and the mechanics of movement of digestive materials through the large intestine, bacteria that are able to colonise food residues in the proximal bowel, and maintain significant populations, serve as inocula for new substrates entering the colon. There are several distinct anatomical regions in the large gut: the proximal bowel comprises the caecum and ascending colon, which is followed by the transverse and descending colons, and the sigmoid/rectum. Measurements made with human sudden death victims have shown that substrate availability, bacterial populations, and the metabolism of the microbiota vary along the length of the gut [5,6], and this is strongly affected by intestinal transit time [5,7]. The nutritional and environmental determinants, together with the anatomical, physiological and microbiological factors that affect metabolic processes in the large gut, are complex and interdependent. In particular, the chemical composition of the fermentable substrate, the amount of substrate available, its physical form (e.g. particle size, solubility, association with undigestible complexes such as tannins, lignin and silica etc.), the bacterial species composition of the microbiota, ecological factors (e.g. competitive and cooperative interactions between different groups of bacteria), and the availability of inorganic electron acceptors such as NO 3 and SO 4 2 all affect the way substrates are digested and fermentation products are formed. The colonic microbiota can be studied in vivo using healthy human volunteers, hospital patients, ileostomists, sudden death victims and animal models. Apart from investigations with healthy volunteers and ileostomists, access to other human materials, such as gut contents and tissues, is restricted for practical and ethical reasons. Moreover, there are limitations on the types of foods or drugs that can be administered to human volunteers.

2 Models for intestinal fermentation Macfarlane and Macfarlane 157 Despite this, investigations involving the use of humans potentially provide the best models for studying the microbiota. However, they often require specialist facilities and are expensive and time-consuming, which usually limits study numbers. Furthermore, many of these investigations suffer from low compliance and high drop-out rates. The availability of animal models to investigate metabolic processes mediated by intestinal microorganisms is of great value in that the animals can be given controlled diets, while researchers have direct access to intestinal contents as well as tissues and organs at autopsy. They can be used to compare the metabolic effects of drugs and toxins in germfree and conventional states, while various knockouts are available for immunological studies. However, animal models are relatively expensive compared with in vitro models, and special handling facilities are needed in some circumstances. Moreover, animal digestive physiologies differ from humans; for example, rodents are coprophages, whereas in pigs, which are generally considered to be similar to humans, the upper digestive tract is much more heavily colonised by microorganisms, while their lower bowel is proportionally much larger in size. In healthy people, the inaccessibility of the digestive tract has meant that most studies on the gut microbiota have been made in vitro. In vitro modelling of the digestive tract is particularly useful for investigating microbial processes such as carbohydrate and protein fermentation, steroid and bile acid metabolism, hydrogen formation and disposal, mutagen formation and degradation, transformations of xenobiotic substances, and the metabolism of lignans and phytooestrogens. In vitro models involve the use of pure cultures, defined mixed cultures and faecal material, and can range from simple batch style fermentations performed in serum bottles, to sophisticated ph controlled multistage continuous culture systems designed to simulate environmental conditions that occur in different parts of the colon (see Box 1). The effectiveness of such model systems depends on the problem being studied, and all have advantages and disadvantages. Thus, fermentation experiments involving the use of serum bottles are inexpensive and allow large numbers of substrates and/or faecal samples to be tested. However, these fermentations are uncontrolled and need to be of short duration to avoid selection of non-representative microbial populations, which could lead to distortions in fermentation profiles. These problems can be circumvented, to some extent, through the use of continuous cultures. Chemostats work effectively at high microbial cell population densities, characteristic of those found in the large bowel. By controlling the rate and composition of nutrient feed, these models allow tight control of bacterial metabolism and environmental conditions. The human large intestine is often likened to a continuous culture system, however, Box 1 General characteristics and limitations of in vitro models. General characteristics Range from simple batch culture vessels to single-stage continuous or semi-continuous fermenters, to complex multistage continuous culture systems Generally inexpensive to operate Easy to set-up Rapid turnaround and throughput of samples Continuous culture systems allow precise manipulation of environmental variables Useful for mechanistic studies Facilitate use of radioactive, toxic and genotoxic substances No ethical considerations Potential limitations of in vitro models No host immune or neuroendocrine system functionality Other biotic factors usually not incorporated into the models (e.g. gut absorptive processes, digestive tract secretions or defensive systems) Epithelial and other colonic biofilms not usually reproduced Difficult to control changes in microbiota community structure in the models following inoculation, particularly in closed systems this is an oversimplification. Only the caecum and ascending colon exhibit characteristics of a continuous culture. This part of the gut receives digesta from the small bowel, and acts as a mixing chamber. Colonic material then passes to the distal bowel for storage, and further mixing does not occur. Owing to restrictions in substrate supply, the bacteria are effectively growing under batch or fedbatch conditions. It is therefore difficult to reproduce the fermentation dynamics of the large bowel in its entirety in vitro. Multistage continuous cultures probably come closest in this regard, they facilitate long-term studies and allow perturbations to the microbiota to be investigated under steady-state conditions. Models based on the system shown in Figure 1, which was validated using measurements made on colonic contents taken from sudden death victims [5,6], are being increasingly used in fermentation studies on the gut. The breakdown of complex carbohydrates is one of the most important functions carried out by the colonic microbiota and is an important factor in maintaining gut health [8,9]. This review focuses on recent advances in the application of in vitro models to investigate the metabolism and ecology of human intestinal microorganisms, with particular emphasis on carbohydrate utilization and fermentation product formation. Carbohydrate utilization Considerable work has been carried out in recent years to develop new non-digestible carbohydrates aimed at stimulating the growth of specific groups of beneficial

3 158 Food biotechnology Figure 1 Schematic diagram of a typical three-stage continuous culture system used to simulate nutritional and environmental conditions in the human large intestine. Sterile culture medium is fed to vessel 1, which represents the proximal bowel and sequentially feeds into vessel 2 and vessel 3, which simulate the transverse and distal colons, respectively. Owing to progressive substrate utilization, nutritional and ph gradients are established across the fermentation system that are analogous to conditions observed in vivo.

4 Models for intestinal fermentation Macfarlane and Macfarlane 159 bacteria (predominately bifidobacteria and lactobacilli) in the large bowel [10]. Other desirable aims would be to increase levels of carbohydrate fermentation in the distal colon and thus reduce levels of proteolytic metabolites, which would require the development of slowly digestible carbohydrates, and the discovery of preferential substrates for the new groups of butyrate-producing anaerobes that have recently been identified in the gut, such as eubacteria and roseburias [11,12]. Many investigations on the fermentation characteristics of these so-called prebiotics [13] have been made in vitro using multistage continuous culture models of the gut [14,15,16,17]. For example, when the prebiotic effects of chicory inulin were investigated in the SHIME (Simulator of the Human Intestinal Microbial Ecosystem) model [15], results showed that its metabolism by faecal bacteria resulted in proportionately increased levels of propionate and butyrate formation. Culture-based analysis of the microbiota and denaturing gradient gel electrophoresis suggested that there was only a weak bifidogenic effect, but molecular analysis using real-time PCR demonstrated major increases in bifidobacterial numbers throughout the fermentation system. Unquestionably, the majority of studies on prebiotics have been performed with various fructo-oligosaccharides (FOS) and inulins [18]; however, there is potentially a wide variety of carbohydrates available to act in the prebiotic role. Thus, galacto-oligosaccharides (GOS) were shown to be bifidogenic in the first two fermentation vessels of the three-stage gut model [16 ]; the lack of bifidogenicity in vessel 3 was ascribed to complete utilization of the substrate by faecal bacteria in vessels 1 and 2. When GOS produced from maltodextrin by Gluconobacter oxydans were tested in a three-stage colon simulator, it was found (on the basis of fluorescence in situ hybridisation measurements) that lactic acid bacteria (lactobacilli/enterococci) and bifidobacteria were stimulated in all three vessels of the model [17]. Unfortunately, direct comparisons of fermentability and relative prebiotic potentials cannot be made from the above studies for two reasons: firstly, the systems received inocula from different individuals, each comprising unique microbiotas, and secondly, the models were operated at different system retention times (R), which would influence patterns of substrate utilization and the colonisation abilities of individual bacterial populations. The significance of retention time in relation to microbial community structure and fermentation product formation was demonstrated in the study by Child et al. [19 ], where a threestage system was operated at either R=60 or R=20 h, which corresponded to normal and fast colonic retention times. This study also showed that substrate availability in the individual culture vessels was a major determinant affecting bacterial colonisation and cellular morphologies. Recent work on the metabolism of intestinal bacteria using batch cultures [20 ] and single-stage fermenter systems [8] has shown how different polymerised carbohydrates affect bacterial community structure, and shows the way in which metabolic cross-feeding is important in butyrate production in the gut. Interestingly, the data indicate that many low G+C Gram-positive species can be selected against during in vitro incubations, which could distort the results of comparative substrate fermentation experiments. Metabolism of xenobiotics Intestinal bacteria are able to transform ingested xenobiotic compounds into either less active or more bioactive metabolites [21 23]. In vitro modelling systems are particularly useful for studying these substances, because many are highly toxic. Polycyclic aromatic hydrocarbons (PAH) can be transformed by human enzymes into toxic metabolites. Because soil is an important route of entry for PAHs into the body, a five-stage model gastrointestinal simulator representing the stomach, small intestine, proximal, transverse and distal colon was used to assess the release of these compounds from contaminated soil [24]. In a subsequent study, it was demonstrated for the first time that PAHs could also be bioactivated by intestinal bacteria, with increased formation of oestrogenic metabolites [25 ]. This is of importance because it demonstrates that risk levels for ingested xenobiotics must take into account microbial biotransformations that occur as they pass through the digestive tract. The same model system was also used to show that production of the potent phytooestrogen 8-prenylnaringenin, which is found in hops, occurs only in the distal colon [26]. The isoflavone genistein, found mainly in soyabeans, has anticancer properties that might be lost on transit through the gastrointestinal tract, owing to degradation by the gut microbiota; however, a recent study showed that this degradation can be reduced by FOS [27 ]. This observation suggests that modulation of the intestinal microbiota with prebiotics could be beneficial in maintaining the anticancer activity of genistein as it passes through the gut. Arabinoxylans (AX) are increasingly being used as additives in the food industry as they can decrease the moisture content of baked products and reduce staleness, however, there have been few studies on the outcome of AX fermentation in the gut and its effects on immature gut microbiotas. Hopkins et al. [28] compared the fermentation of AX and ferulic acid cross-linked AX in phcontrolled single-stage anaerobic batch culture fermenters inoculated with faeces from children age 16 months to 7 years. It was shown that the microbial ecology and metabolism of the children s microbiotas closely resembled that of adults, and that cross-linked AX were safe for use in young children. Studies on antimicrobial activities Several recent investigations involving the use of singleand multistage fermentation systems have focussed on stress factors that affect colonisation of the upper digestive tract and the effects of antimicrobial substances on colonic bacterial community composition and pathogen

5 160 Food biotechnology survival. Modelling studies using aspirates taken from bacterial overgrowth in patients receiving nutritional support during enteral feeding, where a percutaneous endoscopic gastrostomy (PEG) tube is placed directly into the stomach, demonstrated that a simple chemostat incorporating a PEG tube could accurately reproduce duodenal and gastric microbiotas found in these individuals [29,30]. Low ph was found to be considerably less inhibitory to bacteria and yeasts than was previously thought, especially if the organisms were growing in biofilms on the surfaces of feeding tubes; however, growth under acid conditions did induce morphological changes and pseudohyphae formation in yeasts. Work on the effects of antimicrobial substances using multistage continuous culture models of the colon showed that sphingosine, which usually protects the skin [31], manifested strong broad-spectrum antibacterial effects when incubated with pure cultures of bacteria, whereas under environmental conditions simulating the colon no changes in bacterial populations were observed. These observations were attributed to neutralisation of the sphingosine by proteins, long-chain fatty acids and surfactants. A three-stage fermentation system was used to investigate the effects of piperacillin and tazobactam, an antibiotic/b-lactamase inhibitor combination with a broad spectrum of activity, on the ability of the pathogen Clostridium difficile to proliferate [32]. Previous work had shown that C. difficile, after being inoculated as spores, had been unable to establish in this model in the absence of antibiotics [33]. However, the more recent study demonstrated that, despite the fact that the antibiotic had devastating effects on microbial community structure in the model system, the pathogen was still unable to become established. It was concluded that factors other than colonisation resistance mediated by the normal gut microbiota, which is normally thought to be one of the most important factors protecting against infection by this pathogen, were preventing C. difficile growth, but these were not identified. Studies on bacterial biofilms Extensive biofilm formation occurs in the colon. Until recently, however, few studies have investigated the composition and metabolic activities of bacteria growing in these structures, and little is known of their ecology or physiological significance. Biofilms occur on the gut mucosa and in the lumen attached to food particles [34]. Because of the difficulty in accessing and studying mucosal populations in vivo, several model systems have been used to simulate these structures in vitro. These range from simple single-stage fermenters to more complex two- and three-stage systems. In one model, mucin beads were encased in a dialysis membrane filled with a faecal inoculum [35]. An interesting addition was the use of a polyethylene glycol solution to remove excess water and metabolites, to simulate absorption by the colonic epithelium. A novel two-stage fermentation system that involved suspending mucin gels in modified chemostats has been used to study the ability of human intestinal bacteria to colonise mucus and establish biofilm communities [36 ]. The mucin gels were shown to be rapidly colonised by a heterogeneous mixture of intestinal bacteria in a sequential manner. Cinquin et al. [37] have developed and validated a new three-stage continuous model with immobilised faecal bacteria to simulate the infant colonic ecosystem. With the same model, they were able to study modulation of the infant microbiota by purified exopolysaccharides from Lactobacillus rhamnosus. Unlike FOS, the exopolysaccharide was shown to have no prebiotic effect on the infant gut microbiota [38 ]. Conclusions The human colonic microbiota is an organ of great metabolic complexity, the robustness of which allows it to be studied in its entirety in vitro. Many models based on well-established fermentation technologies are now available for investigating the biochemical potential of the microflora, and its interactions with drugs and nutrients. The effectiveness of these model systems will be greatly enhanced by recent developments in molecular analytical methods, particularly those involved in the tracking of microbial population shifts and the nutritional regulation of gene expression. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Macfarlane S, Macfarlane GT: Bacterial diversity in the large intestine. Adv Appl Microbiol 2004, 54: Macfarlane S, Furrie E, Cummings JH, Macfarlane GT: Chemotaxonomic analysis of bacterial populations colonizing the rectal mucosa in patients with ulcerative colitis. Clin Infect Dis 2004, 38: Hold GL, Pryde SE, Russell VJ, Furrie E, Flint HJ: Assesment of microbial diversity in human colonic samples by 16S rdna sequence analysis. FEMS Microbiol Ecol 2002, 39: Macfarlane GT, Cummings JH: The colonic flora, fermentation and large bowel digestive function. In The Large Intestine: Physiology, Pathophysiology and Disease. Edited by Phillips SF, Pemberton JH, Shorter RG. Raven Press; 1991: Macfarlane GT, Gibson GR, Cummings JH: Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol 1992, 72: Macfarlane GT, Macfarlane S, Gibson GR: Use of a three-stage compound continuous culture system to investigate bacterial growth and metabolism in the human colonic microbiota. Microbiol Ecol 1998, 35: Cummings JH, Hill MJ, Bone ES, Branch WJ, Jenkins DJA: The effect of meat protein and dietary fiber on colonic function and metabolism. Part II. Bacterial metabolites in feces and urine. Am J Clin Nutr 1987, 32: Duncan SH, Scott KP, Ramsey AG, Harmsen HJM, Welling GW, Stewart CS, Flint HJ: Effects of alternative dietary substrates on competition between Human colonic bacteria in an anaerobic fermentor system. Appl Environ Microbiol 2003, 69:

6 Models for intestinal fermentation Macfarlane and Macfarlane Cummings JH, Pomare EW, Branch WJ, Naylor CPE, Macfarlane GT: Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 1987, 28: Rastall RA, Gibson GR, Gill HS, Guarner F, Klaenhammer TR, Pot B, Reid G, Rowland IR, Sanders ME: Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbial Ecol 2005, 52: Hold GL, Schwiertz A, Aminov RI, Blaut M, Flint HJ: Oligonucleotide probes detect quantitatively significant groups of butyrate producing bacteria in human feces. Appl Environ Microbiol 2003, 69: Aminov RI, Walker AW, Duncan SH, Harmsen HJM, Welling GW, Flint HJ: Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of Human gut bacteria related to Roseburia spp. or Eubacterium rectale. Appl Environ Microbiol 2006, 72: Van Loo J: The specificity of the interaction with intestinal bacterial fermentation by prebiotics determines their physiological efficacy. Nutr Res Rev 2004, 17: Probert HM, Apajalahti JHA, Rautonen N, Stowell J, Gibson GR: Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system. Appl Environ Microbiol 2004, 70: A comparative study on carbohydrate fermentation involving the use of a three-stage continuous culture system to investigate the effects of FOS, polydextrose and lactitol on fermentation product formation and selected faecal bacterial populations. SCFAs were measured in the gut simulator, and bacterial communities were studied using fluorescent in situ hybridisation in association with 16S rrna oligonucleotide probes, together with species-specific PCR for bifidobacterial species determinations, and percent GC profiling. The results showed that FOS and polydextrose were bifidogenic in all three culture vessels of the gut model system. Conversely, lactitol suppressed bifidobacteria and bacteroides and had general inhibitory effects on the microbiota, however, the sugar alcohol stimulated SCFA formation and was shown to be a butyrigenic substrate. 15. Van de Wiele TV, Boon N, Possemiers S, Jacobs H, Verstraete W: Prebiotic effects of chicory inulin in the simulator of the human intestinal ecosystem. FEMS Microbiol Ecol 2004, 51: Tzortzis G, Goulas AK, Gee JM, Gibson GR: A novel galactooligosacccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo. J Nutr 2005, 135: In vitro and in vivo trials were performed on the microbiological effects of a novel mixture of GOS, manufactured enzymically using Bifidobacterium bifidum, using a three-stage gut simulator and pigs, respectively. Selected bacterial communities were studied using FISH technology. Bifidobacterial numbers were stimulated by 1% GOS in the in vitro model, in vessels corresponding to the proximal and transverse colons, while no effects were seen with lactobacilli. In the pig feeding study, inclusion of 1.6% GOS in the diet had no significant effects on bifidobacterial or lactobacillus numbers in colonic contents compared with controls. However, in animals fed 4% GOS, bifidobacterial numbers were significantly higher than in the control animals and those fed 1.6% GOS, showing that, in vivo, higher substrate concentrations were needed to exert bifidogenic effects than in the in vitro model. Fermentation product measurements were only made in the pig studies, so comparisons could not be made with the in vitro fermentation of GOS. 17. Wichienchot S, Prasertsan P, Hongpattarakere T, Gibson GR, Rastall RA: In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora. Curr Iss Intest Microbiol 2006, 7: Macfarlane S, Macfarlane GT, Cummings JH: Prebiotics in the gastrointestinal tract. Aliment Pharmacol Therap 2006, 24: Child MW, Kennedy A, Walker AW, Bahrami B, Macfarlane S, Macfarlane GT: Studies on the effect of system retention time on bacterial populations colonising a three-stage continuous culture model of the human large gut using FISH techniques. FEMS Microbiol Ecol 2006, 55: One of the most detailed in vitro studies on microbial community structure involving the use of 23 different FISH probes belonging to the major groups of bacteria found in the large intestine. The results showed that while the gut model provided a good representation of the original faecal inoculum, some minor groups of bacteria were unable to establish in the fermentation system. Numbers of butyrate-producing Roseburia spp. were greatly lowered when the system retention time was changed from 60 to 20 h, which correlated with marked reductions in butyrate formation and increased acetate production 20. Belenguer A, Duncan SH, Calder A, Holtrop G, Louis P, Lobley GE, Flint HJ: Two routes of metabolic cross-feeding between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human gut. Appl Environ Microbiol 2006, 72: A very important study based on a series of pure and co-culture experiments that show how metabolic cross-feeding can occur between nonbutyrate-forming bifidobacteria and butyrate-producing Roseburia sp., Anaerostipes caccae and Eubacterium hallii. Two distinct mechanisms of cross-feeding were identified involving the utilization of bifidobacterium fermentation products, such as lactate and acetate by A. caccae and E. hallii, and the sequestration of hydrolysis products formed by bifidobacteria growing on FOS by the roseburia and E. hallii. These data could explain how high levels can be produced from FOS and inulins in the large gut. 21. Decroos K, Eeckhaut E, Possemiers S, Verstraete W: Administration of equol- producing bacteria alters the equol production status in the simulator of the gastrointestinal tract microbial ecosystem (SHIME). J Nutr 2006, 136: Vanhaecke L, Van Hoof N, Van Brabandt W, Soenen B, Heverick A, De Kimpe N, De Keukeleire D, Verstraete W, Van de Wiele T: Metabolism of the food-associated carcinogen 2-amino-1- methy-6-phenylimidazo[4,5-b]pyridine by human intestinal microbiota. J Agric Food Chem 2006, 54: Humblot C, Combourieu B, Vaisanen ML, Furet JP, Delort AM, Rabot S: 1 H nuclear magnetic resonance spectroscopy-based studies of the metabolism of food-borne carcinogen 2-amino- 3-methyimidazo[4,5- f]quinoline by human intestinal microbiota. Appl Environ Microbiol 2005, 71: Van de Wiele TR, Verstraete W, Siciliano SD: Polycyclic aromatic hydrocarbon release from a soil matrix in the in vitro gastrointestinal tract. J Environ Qual 2004, 33: Van de Wiele T, Vanhaecke L, Boeckaert C, Peru K, Headley J, Verstraete W, Siciliano S: Human colonic microbiota transform polycyclic aromatic hydrocarbons to estrogenic metabolites. Environ Health Perspect 2005, 113:6-10. A novel study that followed the microbial biotransformation of polycyclic aromatic hydrocarbons (PAH) in a five-stage gut model, using a newly optimised liquid chromatography-mass spectrometry method to detect the production of PAH metabolites. Pure PAH and PAH-contaminated soils were used in the study, and the oestrogenicity of the PAHs and whether or not they were able to induce signal transduction were measured. The colonic microbiota was shown to directly bioactivate PAHs, and this activation was not reduced in the presence of soil. 26. Possemiers S, Bolca S, Grootaert C, Heverick A, Decroos K, Dhooge W, De Keukeleire D, Rabot S, Verstraete W, Van De Wiele T: The prenylflavenoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine. J Nutr 2006, 136: Steer TE, Johnson ET, Gee JM, Gibson GR: Metabolism of the soyabean isoflavone glycoside genistein in vitro by human gut bacteria and the effect of prebiotics. BJN 2003, 90: This paper demonstrates the benefits of regulating the metabolism of the gut microbiota using prebiotics. FOS significantly reduced breakdown of the isoflavone genistein in batch culture experiments and in a three-stage model system. Genistein degradation decreased sequentially in each of the multistage fermentation vessels. Levels of degradation with FOS were 22% (vessel 1), 24% (vessel 2) and 26% (vessel 3), compared with 87%, 95% and 93% with no additions. Increased numbers of lactobacilli and bifidobacteria were also found with FOS, concomitant with reduced counts of bacteroides and clostridia. 28. Hopkins MJ, Englyst H, Macfarlane S, Furrie E, Macfarlane GT, McBain AJ: Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children. Appl Environ Microbiol 2003, 6:

7 162 Food biotechnology 29. O May GA, Reynolds N, Smith AR, Kennedy A, Macfarlane GT: Effect of ph and antibiotics on microbial overgrowth in the stomach and duodenum of patients undergoing percutaneous endoscopic gastrostomy feeding. J Clin Microbiol 2005, 43: O May G, Reynolds N, Macfarlane GT: Effect of ph on an in vitro model of the gastric microbiota in enteral nutrition patients. Appl Environ Microbiol 2005, 71: Possemiers S, Van Camp J, Bolca S, Verstraete W: Characterization of the bactericidal effect of dietary sphingosine and its activity under intestinal conditions. Int J Food Microbiol 2005, 105: Baines SD, Freeman J, Wilcox MH: Effect of piperacillin/ tazobactam on Clostridium difficile growth and toxin production in a human gut model. J Antimicrob Chemother 2005, 55: Freeman J, O Neill FJ, Wilcox MH: The effects of cefotaxime and desacetylcefotaxime upon Clostridium difficile proliferation and toxin production in a triple-stage model of the human gut. J Antimicrob Chemother 2003, 52: Macfarlane S, Macfarlane GT: Composition and metabolic activities of bacterial biofilms colonizing food residues in the human gut. Appl Environ Microbiol 2006, 72: Probert HM, Gibson GR: Development of a fermentation system to model sessile bacterial populations in the human colon. Biofilms 2004, 1: Macfarlane S, Woodmansey EJ, Macfarlane GT: Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system. Appl Environ Microbiol 2005, 71: A combination of culture and scanning electron confocal microscopy were used to study colonisation of artificial mucin gels in a two-stage fermentation system. Mucin gels immersed in the model system were found to be rapidly colonised by bacteria and phylogenetically and metabolically distinct from their non-adherent counterparts. FISH demonstrated the presence of bacteroides growing in microcolonies, while bifidobacteria were more dispersed on the gels. Mucin was found to be extensively degraded by the luminal bacteria, and the majority of glycoprotein oligosaccharides in the mucin gels were utilized by the adherent bacteria. 37. Cinquin C, Le Blay G, Fliss I, Lacroix C: New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota. FEMS Microbiol Ecol 2006, 57: Cinquin C, Le Blay G, Fliss I, Lacroix C: Comparative effects of exopolysaccharides from lactic acid bacteria and fructooligosaccharides on infant gut microbiota tested in an in vitro colonic model with immobilized cells. FEMS Microbiol Ecol 2006, 57: The authors demonstrated that their recently validated three-stage model of the infant gut could be used to study modulation of the microbiota by prebiotics. FOS was compared with purified exopolysaccharide from L. rhamnosus. The prebiotic, at a concentration of 9.8 g l 1, was shown to increase numbers of lactobacilli and decrease coliforms in both gel beads and in the non-adherent fraction in all three vessels. Reduced ammonia production and increased organic acid formation were also found with FOS. The exopolysaccharide from L. rhamnosus was shown to have no prebiotic effect, and was not metabolised by the infant microbiota.