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Aims: To investigate the effects of whey retentate from Bifidobacteria fermented milk. Methods and Results: The simulator of the human intestinal microbial ecosystem (SHIME) was used. The composition of the microbiota and its metabolic activities were analysed. Changes in the microbial composition became apparent within 15 days of the treatment in the vessels representing the ileum and the large intestine. The whey retentate favoured the growth of endogenous bifidobacteria and induced a decrease in Bacteroides fragilis and in sulfipho-reducing clostridia, especially Clostridium perfringens. After the administration was stopped, these populations tended to revert to their original levels, except for the streptococci and the staphylococci populations. The treatment also led to an increase in acetic acid, CH4 and CO2 production, suggesting overgrowth of some anaerobic bacteria. Ammonium, generally considered as undesirable, declined. Conclusions: The whey retentate clearly altered the microbial community in the SHIME. Significance and Impact of the Study: Whey retentate appears to exert a beneficial effect on the in vitro gastrointestinal system; these findings warrant confirmation by in vivo studies.