Glycerol Induces Reuterin Production and Decreases Escherichia Coli Population in an in Vitro Model of Colonic Fermentation With
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library RESEARCH ARTICLE Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces Valentine Cleusix, Christophe Lacroix, Sabine Vollenweider & Gwenae¨ lle Le Blay Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Zurich, Switzerland Correspondence: Gwenaelle Le Blay, Abstract Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Lactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the Institute of Technology, ETH Zentrum, LFV C presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This 25.2, CH-8092 Zurich, ¨ Switzerland. strain has been shown to prevent intestinal infections in vivo; however, its Tel.: 141 44 632 3293; fax: 141 44 632 mechanisms of action, and more specifically whether reuterin production occurs 1403; e-mail: gwenaelle.leblay@ilw. within the intestinal tract, are not known. In this study, the effects of L. reuteri agrl.ethz.ch ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors Received 14 June 2007; revised 5 October were inoculated with adult immobilized fecal microbiota and the effects of daily 2007; accepted 9 October 2007. 8 À1 First published online 20 November 2007. addition of L. reuteri into one of the reactors (c.10 CFU mL ) without or with glycerol were tested on major bacterial populations and compared with addition of DOI:10.1111/j.1574-6941.2007.00412.x glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus–Enterococcus group and decreased Escher- Editor: Julian Marchesi ichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli Keywords concentration during the addition of glycerol (in presence or absence of L. reuteri) in vitro fermentation model; immobilized could be due to in situ reuterin production because 1,3-propanediol, a typical intestinal microbiota; Lactobacillus reuteri product of glycerol fermentation, was detected during the addition of glycerol. ATCC 55730; reuterin; glycerol. L. reuteri ATCC 55730 was associated with therapeutic Introduction effects. It reduces the duration of watery diarrhea caused by A healthy gastrointestinal tract is colonized by a highly rotavirus or bacteria (Salmonella, Shigella and Campylobacter complex bacterial community, which plays a major role in species) in children (Weizman et al., 2005), and the occur- human health and nutrition. This bacterial community is rence of respiratory and gastrointestinal diseases in healthy notably involved in the protection of the gastrointestinal adults (Tubelius et al., 2005). Other L. reuteri strains have tract by acting as a barrier against colonization by patho- also been shown to protect against enteropathogens in mice, genic bacteria. Among commensal bacteria, lactobacilli and monkeys, chickens and turkeys (Casas & Dobrogosz, 2000). bifidobacteria, including those ingested as probiotics have Different mechanisms have been proposed to account for attracted much attention for their potential to treat or the protective action of probiotic bacteria, and more espe- prevent intestinal infections. cially L. reuteri toward enteropathogens, such as stimulation Lactobacillus reuteri ATCC 55730 (also designated as of the immune system, competition-exclusion, or produc- SD2112) is a well-known obligate heterofermentative pro- tion of antimicrobial compounds such as organic acids or biotic, isolated from human breast milk, and widely used as reuterin. Lactobacillus reuteri is known to produce reuterin a food additive to improve human gastrointestinal health (3-hydroxypropionaldehyde, 3-HPA), a broad-spectrum (Casas & Dobrogosz, 2000). Lactobacillus reuteri ATCC antimicrobial substance active in a wide range of pH values 55730 can survive low pH as it passes through the stomach against Gram-positive and Gram-negative bacteria (espe- to reach the human intestine and colonize it transiently. cially Escherichia coli), yeasts, fungi, protozoa and viruses Clinical trials have shown that the administration of (Vollenweider & Lacroix, 2004). Reuterin production and c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol 63 (2008) 56–64 Published by Blackwell Publishing Ltd. All rights reserved Effect of glycerol on human intestinal microbiota 57 accumulation occur during the anaerobic growth of Feces collection and preparation of the L. reuteri in the presence of glycerol and low concentrations fecal inoculum of glucose, under conditions of pH and reduction potential A fecal sample (46.5 g) was collected from a healthy, found in the small and large intestines (Chung et al., 1989). nonmethanogenic human adult who had not received anti- Indeed, reuterin is believed to provide an ecological compe- biotic therapy for the last 3 months. Promptly after defeca- titive advantage to L. reuteri and to be involved in coloniza- tion, the sample was diluted with prewarmed (37 1C) tion resistance against potential pathogens (Edens et al., prereduced (0.05% L-cystein) peptone water (0.1%, pH 7) 1997). Reuterin synthesis in the colon would occur when to obtain a final fecal concentration of 20% (w/v). The sufficient amounts of glycerol become available as a product s suspension was then homogenized in a Stomacher (Sew- of luminal microbial fermentations, digestion of luminal ard, Norfolk, UK) for 2 min and filtered through a 150 mm fats, sloughed mucus and desquamated epithelial cells, and nylon membrane (Sefar AG, Ruschlikon,¨ Switzerland) to intestinal clearing of endogenous plasma glycerol (Casas & remove large particles, under continuous CO bubbling. Dobrogosz, 2000). Very little information is available on the 2 When about 10 mL of the slurry were filtered, the filtrate production and/or availability of glycerol for reuterin synth- was immediately transferred to an anaerobic chamber (Coy esis in the colon. Moreover, reuterin is very difficult to Laboratories, Ann Arbor, MI) with a nitrogen atmosphere quantify in vivo due to its aldehyde function that reacts with containing 5% (v/v) hydrogen, for immobilization in amino and sulfhydryl groups readily available in the intes- polysaccharide beads. tine (Casas & Dobrogosz, 2000). In this study, the capacity of L. reuteri ATCC 55730 to Immobilization technique produce reuterin from glycerol in a complex bacterial ecosystem similar to that of the human intestine was tested. The immobilization procedure in 1–2 mm polysaccharide To estimate reuterin production, two specific markers were gel beads made of gellan (2.5% w/v) and xanthan gum analysed: 1,3-propanediol, a typical product of glycerol (0.25% w/v) was based on a dispersion process in a two- fermentation formed from reuterin reduction (Biebl et al., phase system, as already described (Cinquin et al., 2006). 1999), and Escherichia coli highly sensitive to reuterin The entire process was completed in anaerobic conditions activity (Rasch, 2002). With this objective, pure reuterin within 1 h. and L. reuteri ATCC 55730 (in absence or presence of glycerol) were added in an in vitro model of colonic Culture medium fermentation with immobilized fecal microbiota recently The culture medium used to simulate adult human ileal developed and validated (Cinquin et al., 2004, 2006). chyme was the same as previously validated by Macfarlane Immobilization was shown to preserve bacterial diversity et al. (1998) with one modification. The reducing agent and induce high cell density in beads and fermentation (L-cystein) was replaced by titanium (III) citrate (0.6 mM) effluents as a result of immobilized fecal microbiota growth, to avoid binding of L-cystein sulfhydryl groups with reuterin. cell release from beads and possibly growth of free cells in A vitamin solution described by Gibson & Wang (1994) and the bulk medium. Moreover, a very high stability renders sterilized by filtration (Minisart 0.2 mm, Sartorius, this model particularly suitable for long-term fermentation Gottingen,¨ Germany) was added (0.5 mL LÀ1) separately to studies and comparative testing of different conditions with the autoclaved (15 min, 121 1C) culture medium. Depending the same microbiota in the same experiment (Cinquin et al., on the test period, this medium was supplemented with 2006). different glycerol concentrations (10 or 100 mM) or with 1.3 mM of pure reuterin. Materials and methods Fermentation procedures Duplicate single-stage reactors based on the model de- Bacterial strain and reuterin production scribed by Cinquin et al. (2004) were used to mimic the Lactobacillus reuteri ATCC 55730 was used for reuterin microbial ecosystem of the adult proximal colon. Two production and fermentation experiments. It was routinely stirred glass reactors with a volume of 300 mL (Sixfors, cultivated in Mann, Rogosa and Sharpe (MRS) aerobically at Infors, Bottmingen, Switzerland) were filled with 210 mL 37 1C for 14 h. Reuterin was purified as described by culture medium and 90 mL freshly inoculated beads. Batch Vollenweider et al. (2003). The molarity of the reuterin fermentations were first carried out for bead colonization solution was measured by the assay described