In Vitro Models to Study the Fermentation of Carbohydrates by the Colonic Microflora

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The most commonly used in vitro models to study anaerobic fermentation of carbohydrates by mixed bacteria population, particularly fecal bacteria, are:47

• Batch culture fermenters (50-350 ml working volume) that are inoculated either with pure culture of selected genera or species of bacteria or with fecal slurry (5-10% w/v fresh feces homogenized in anaerobic buffer pH 7.0 and subsequently sieved) and the carbohydrate (±1% w/v) to be studied. Feces are collected anaerobically from volunteers with no preceding history of gastrointestinal disorder and who have not been prescribed antibiotics for at least 3 months. Before incubation, the slurry is gazed out with argon leaving a slight positive pressure of gas. The fermenters are then incubated for up to 48 h and samples of gas and liquid are taken for analyses at regular time intervals. The gas samples are analyzed for H2, CH4, and CO2 concentrations by gas liquid chromatog-raphy. In the liquid samples, the concentration of SCFAs is determined by gas liquid (GLC) or high-pressure liquid (HPLC) chromatography. Lactate and eventually succinate are also quantified by GLC after meth-ylation. The concentration of the residual carbohydrates is measured by using the adequate specific methodology, being either enzymatic and/or physical (GLC or HPLC).

• Multichamber continuous culture systems that have been developed in an attempt to reproduce some of the different physical and nutritional characteristics of the different segments (ascending, transverse, and descending) in the colon.15 48 Such a system is composed of three vessels aligned in series so that adequate culture medium pumped in vessel 1 (320 ml, pH 5.5) sequentially feeds vessel 2 (320 ml, pH 6.2) and then vessel 3 (500 ml, pH 6.8) at dilution rates of 0.15/h, 0.15/h, and 0.08/h, respectively. Vessel 1, rich in nutrients with a fast transit and acidic pH, is similar to proximal colon, whereas vessel 3, poor in fermentable substrate and with a neutral pH, resembles the distal colon where bacteria grow slowly. All of the system is maintained in oxygen-free conditions at 37°C, and the pH is strictly controlled in each vessel.24 After inoculation with a fresh fecal sample (16% w/v), the system is allowed to stabilize for 2 weeks after which time additional carbohydrate (15 g/d) is pumped in for 2 weeks. The concentration of SCFAs and carbohydrate substrate is deter mined (as indicated above for batch fermenters) in samples periodically removed from the vessels.

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