Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies

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Highlights

  • Functionality from human gut microbiota can be preserved for fermentation studies.

  • Diversity declines in Bacteroidetes phylum after handling human feces.

  • Preservation techniques can protect phylogenetic groups in a fecal inoculum.

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.

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).

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