Quality in Relation to Other Models with the Same Applicability
When microbial metabolism of rutin (quercetin-rhamnoglucoside) and chlorogenic acid was studied in a one compartment fermentation model using four individual donors (Rechner et al. 2004), the patterns of the metabolite formations varied according to the subject. The formation of 3,4-dihydroxyphenylacetic or -propionic acid from rutin and chlorogenic acid, respectively, showed maxima at 8–10 h using 10 % (w/v) suspension (Rechner et al. 2004). In contrast, using the diluted suspension (5 %, w/v) pooled and homogenized under strict anaerobic conditions, the corresponding value was shown in 2 h and complete dehydroxylation was observed in 8 h (Aura et al. 2002). The inoculum in VTT one compartment system has to be diluted 1 % in order to observe the deglycosylation of quercetin derivatives or anthocyanins in different experiments (Aura 2005). It is likely that strict application of anaerobic conditions enables faster conversions, when vulnerable anaerobic microbial suspension is used as a source of activity.
When in vitro colon models using monogastric pig and human faeces as inocula were compared, human inoculum showed more efficient fermentation for several sources of DF, whereas pig inoculum was more efficient in cellulose digestion. The pig inoculum produced less SCFA and more gas compared with the human inoculum. These differences were attributed to an adaptation of human microbiota to a more diverse diet compared to the pig feed and an adaptation of pig microbiota to a cellulose-rich diet (Jonathan et al. 2012).
The latest description of the model is from Nordlund et al. (2012). In vitro colon model for measurement of SCFA and phenolic acids was started by weighing 100 mg per 10 ml of incubation suspension (dry w/v) of plant foods or their fractions to the bottles, and hydrating with 20 % of the volume of medium 1 day before inoculation. Human faeces were collected from at least 3 (usually from 4–6) healthy volunteers, who had not received antibiotics for at least 6 months and had given a written consent. Freshly passed faeces were immediately taken in an anaerobic chamber or closed in a container with an oxygen consuming pillow (Anaerocult Mini; Merck, Darmstadt, Germany) and a strip testing the anaerobiosis (Anaerotest; Merck, Darmstadt, Germany). Faecal suspension was prepared under strictly anaerobic conditions. Equal amounts of faecal samples were pooled and diluted to a 12.5 % (w/v) or 20.8 % (w/v) suspension, depending on the application and 80 % of the volume of the incubation suspension was dosed to the fermentation bottles to obtain a 10 % or 16.7 % (w/v) final faecal concentrations as described previously (Aura 2005). Lower concentrations can be used. For example 5 % suspension is suitable for isolated components, because the metabolite responses from faecal control are high in more dense inoculum and the substrate concentration should be below the saturation point of the substance to keep the substrate in the solution and not to suppress the activity of the microbiota.
The fermentation experiments were performed in triplicate and a time course of 0, 2, 4, 6, 8 and 24 h is followed using the same inoculum for all the substrates. Incubation is performed at 37 °C in tightly closed bottles and in magnetic stirring (250 rpm). Faecal background is incubated without addition of the supplements (Aura 2005). Headspace is sampled for the measurement of gas pressure (Nordlund et al. 2012) and the liquid space is sampled for SCFA and phenolic acid metabolite analyses (Nordlund et al. 2012; Aura et al. 2013).