Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies
Introduction
The human gut harbors a community of microorganisms commonly known as the microbiota. This community is dominated by anaerobic bacteria and consists of at least 1014 members with a wide variety of species (± 500–1000) (Bäckhed et al., 2004).
The intestinal microbiota in humans has been demonstrated to be highly active and able to ferment indigestible compounds from the host's diet (Flint et al., 2007). The type of diet determines whether the fermentation process occurring in the gut is dominantly saccharolytic or proteolytic (Scott et al., 2013). The metabolites from these two types of fermentation include mainly short-chain fatty acids (SCFA), specifically acetate, propionate and butyrate (Flint et al., 2007), and branched-chain fatty acids (BCFA) including principally iso-butyric, iso-valeric and 2-methylbutyric acids (Bergman, 1990) Metabolites such as acetate, propionate and butyrate are of particular interest since they have been found to be involved in lipid metabolism, reduction of food intake, improvement of tissue insulin sensitivity and intestinal barrier, and energy balance (Al-Lahham et al., 2010, Al-Lahham et al., 2011, Ferchaud-Roucher et al., 2005, Peng et al., 2009, Roediger, 1982, Scheppach, 1994). As a consequence, increasing evidence shows that the composition and activity of the intestinal microbiota is associated with the overall health state of humans, including obesity. Food components affect the composition and activity of the gut microbiota. Therefore, the fermentation characteristics of an ample number of substrates have been studied (Cardelle-Cobas et al., 2012, Fassler et al., 2006, Hernot et al., 2009).
Part of these studies include experiments performed in in vitro systems which offer a high flexibility in their design since there are less limitations in regard to costs and ethical constraint when compared to human trials. For such in vitro studies, the use of a well-preserved fecal sample or inoculum is crucial to perform reproducible experiments and to guarantee the robustness and reliability of these experiments. The use of a standardized inoculum provides the opportunity to perform a large number of studies with the same microbiota for different substrates. This contributes to more reproducible assays that can be performed over a long period of time, which is impossible with a single fresh fecal sample.
There is a lack of literature addressing the possible variations in the microbial activity and composition induced by storage and preparation of a human fecal inoculum for in vitro studies. In experiments performed in rumen fluid, canine, and equine feces, freezing has been found to damage and disrupt the bacterial cell membrane, which causes the release of intracellular contents that subsequently led to loss of (members of the) communities (Bosch et al., 2013, Murray et al., 2012, Prates et al., 2010, Rose et al., 2010). Moreover, some groups of bacteria have been observed to be seriously damaged after freezing and thawing such as certain Gram-negative bacteria (Murray et al., 2012). Alterations in the kinetics of fermentation as well as production of gases have also been found to occur after manipulation (Murray et al., 2012, Pastorelli et al., 2014). However, these negative effects were not observed during the preparation of the human fecal inoculum by Rose et al. (2010) which, to our knowledge, is one of the few studies that have validated the use of fresh and frozen human microbiota. These authors observed that the viable cells in the microbiota stored for 44 weeks at − 80 °C were not affected and the microbial diversity of this inoculum did not substantially differ from the fresh one, although in that previous study the direct comparisons between a frozen and fresh inoculum were not performed as the main goal. As explained before, there is a lack of information about an appropriate treatment to preserve human feces for in vitro fermentation experiments. Furthermore, these previous findings need to be expanded. Thus, the purpose of this study was to determine the optimal conditions to prepare a human fecal inoculum to be used in the TNO dynamic in vitro proximal colon model (TIM-2) (Venema et al., 2000). Four different treatments to prepare human fecal inocula were studied and their efficacy was evaluated by monitoring the composition and activity (in terms of SCFA and BCFA production) of the microbiota under standard fermentation experiments. A potential alternative to fresh feces was successfully found.
Section snippets
Fecal samples
Participants involved in this study were non-smokers and had not used antibiotics, prebiotics, probiotics or laxatives 3 weeks prior to the donation. Fresh fecal samples were directly collected in a closed box containing an anaerobic strip (AnaeroGen™, Oxoid, Cambridge, UK). Donations and treatment preparations were handled under strict anaerobic conditions in an anaerobic chamber (Bactron IV, Sheldon manufacturing, Cornelius, OR USA) containing 5% H2, 5% CO2, and 90% N2. A pool of feces was
Results
The use of a freshly collected fecal sample is the optimal way to guarantee minimal perturbation of a microbial inoculum for in vitro fermentation experiments. Therefore, Treatment 1 (fresh fecal preparation) was considered as a reference in the study.
Discussion
Freshly collected fecal samples may not always be available. In some circumstances, the use of fresh human microbiota is not possible because donors live far away from the laboratory or because they are not continuously available to repeatedly participate at various times during the study. In addition, the composition and activity of repeated donations are likely to be different from day to day. Therefore, these samples are not always identical.
To guarantee a constant inoculum over time for
Conclusion
We conclude that preparing a human fecal inoculum resuspended in dialysate solution with glycerol and then stored at − 80 °C after snap-freezing in liquid nitrogen (Treatment 2) is a viable alternative to fresh feces (resuspended in the same buffer) for in vitro fermentation studies.
Further experiments are recommended to i) test the optimal time and alternatives for thawing as e.g., discussed by Hamilton et al. (2012) who tested thawing using an ice-bath, ii) study the extent of the effects of
Acknowledgments
The authors thank Malieka van der Lugt-Degen and Linda Poort for assisting during the ABI runs. This study was partly funded by the Top Institute Food & Nutrition (GH004) (TIFN, Wageningen, The Netherlands).
References (52)
- et al.
To pool or not to pool? Impact of the use of individual and pooled fecal samples for in vitro fermentation studies
J. Microbiol. Methods
(2014) Protectants used in the cryopreservation of microorganisms
Cryobiology
(2003)- et al.
Galacto-oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a C-labeling technique
J. Nutr.
(2012) - et al.
The effect of freezing on the fermentative activity of equine faecal inocula for use in an in vitro gas production technique
Anim. Feed Sci. Technol.
(2012) - et al.
Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers
J. Nutr.
(2009) - et al.
Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation
Anim. Feed Sci. Technol.
(2010) - et al.
In vitro and in vivo fermentation of glycerol by rumen microbes
Anim. Feed Sci. Technol.
(1993) - et al.
Microbial hydrolysis of steviol glycosides
Food Chem. Toxicol.
(2008) - et al.
Bile salt biotransformations by human intestinal bacteria
J. Lipid Res.
(2006) Utilization of nutrients by isolated epithelial cells of the rat colon
Gastroenterology
(1982)