title: Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies.
abstract: This study investigated the optimal preservation approach to prepare human feces as inoculum for in vitro fermentations as an alternative to the use of fresh feces. The four treatments studied were: Treatment 1) fresh feces resuspended in dialysate solution+glycerol; Treatment 2) fresh feces resuspended in dialysate solution+glycerol and then stored at -80°C; Treatment 3) fecal sample frozen with 1.5 g glycerol; and Treatment 4) fecal sample frozen. All the treatments contained 8.75 g of feces, 3.5 ml dialysate and 4.9 ml glycerol when inoculated in TIM-2 in vitro system. Treatment 1 (fresh fecal preparation) was used as a reference. The effects were evaluated in terms of i) metabolic activity and ii) composition of the microbiota using fermentation experiments in the TIM-2 in vitro system. In all treatments, high levels of acetate were produced followed by n-butyrate and propionate. However, the metabolic activity of the bacteria, in terms of short-chain fatty acid production, was affected by the different treatments. Microbiota composition was analyzed using the IS-pro profiling technique. Diversity in Actinobacteria, Firmicutes, Fusobacteria and Verrucomicrobia and Proteobacteria groups seemed to be preserved in all treatments whereas it was observed to decline in the Bacteroidetes group. Preparing a human fecal inoculum resuspended in dialysate solution with glycerol and then stored at -80°C showed high similarities to the results obtained with fresh feces, and is proposed as the optimal way to freeze fecal material as an alternative to fresh feces for in vitro fermentation studies.
authors.affiliation: Top Institute of Food & Nutrition, PO Box 557, 6700 AA Wageningen, The Netherlands; Maastricht University, School of Nutrition, Toxicology and Metabolism (NUTRIM), Faculty of Health, Medicine and Life Sciences, Department of Human Biology, PO Box 616, 6200 MD, Maastricht, The Netherlands; The Netherlands Organization for Applied Scientific research, TNO, PO Box 360, 3700 AJ, Zeist, The Netherlands. Electronic address: marisol.aguirremorales@tno.nl.
authors.forename: Marisol
authors.initials: M
authors.lastname: Aguirre
authors.affiliation: VU University Medical Center, Department of Medical Microbiology and Infection Control, 1081 BT Amsterdam, The Netherlands. Electronic address: a.eckhauer@vumc.nl.
authors.forename: Anat
authors.initials: A
authors.lastname: Eck
authors.affiliation: The Netherlands Organization for Applied Scientific research, TNO, PO Box 360, 3700 AJ, Zeist, The Netherlands. Electronic address: marjorie_koenen@hotmail.com.
authors.forename: Marjorie E
authors.initials: ME
authors.lastname: Koenen
authors.affiliation: VU University Medical Center, Department of Medical Microbiology and Infection Control, 1081 BT Amsterdam, The Netherlands; Maastricht University Medical Center, Department of Medical Microbiology, PO Box 5800, 6202 AZ, Maastricht, The Netherlands. Electronic address: p.savelkoul@vumc.nl.
authors.forename: Paul H M
authors.initials: PH
authors.lastname: Savelkoul
authors.affiliation: VU University Medical Center, Department of Medical Microbiology and Infection Control, 1081 BT Amsterdam, The Netherlands. Electronic address: D.Budding@vumc.nl.
authors.forename: Andries E
authors.initials: AE
authors.lastname: Budding
authors.affiliation: Top Institute of Food & Nutrition, PO Box 557, 6700 AA Wageningen, The Netherlands; The Netherlands Organization for Applied Scientific research, TNO, PO Box 360, 3700 AJ, Zeist, The Netherlands. Electronic address: koen.venema@outlook.com.
authors.forename: Koen
authors.initials: K
authors.lastname: Venema
mesh_terms: D000328:Adult; D001419:Bacteria / Q000737:chemistry / Q000378:metabolism*; D015925:Cryopreservation / Q000379:methods*; D005227:Fatty Acids / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D005260:Female; D005285:Fermentation; D006801:Humans; D008297:Male; D064307:Microbiota; D008875:Middle Aged; D011309:Preservation, Biological / Q000379:methods*
chemical_list: D005227:Fatty Acids
keywords: Cryopreservation; Gut microbiota; In vitro model; Inoculum preparation