Abstract
The gut microbiota has a profound effect on human health and is modulated by food and bioactive compounds. To study such interaction, in vitro batch fermentations are performed with fecal material, and some experimental designs may require that such fermentations be performed with previously frozen stools. Although it is known that freezing fecal material does not alter the composition of the microbial community in 16S rRNA gene amplicon and metagenomic sequencing studies, it is not known whether the microbial community in frozen samples could still be used for in vitro fermentations. To explore this, we undertook a pilot study in which in vitro fermentations were performed with fecal material from celiac, cow’s milk allergic, obese, or lean children that was frozen (or not) with 20% glycerol. Before fermentation, the fecal material was incubated in a nutritious medium for 6 days, with the aim of giving the microbial community time to recover from the effects of freezing. An aliquot was taken daily from the stabilization vessel and used for the in vitro batch fermentation of lentils. The microbial community structure was significantly different between fresh and frozen samples, but the variation introduced by freezing a sample was always smaller than the variation among individuals, both before and after fermentation. Moreover, the potential functionality (as determined in silico by a genome-scaled metabolic reconstruction) did not differ significantly, possibly due to functional redundancy. The most affected genus was Bacteroides, a fiber degrader. In conclusion, if frozen fecal material is to be used for in vitro fermentation purposes, our preliminary analyses indicate that the functionality of microbial communities can be preserved after stabilization.
Highlights
The gut microbiota is known to affect host health, playing a critical role in the modulation of the physiological processes of the related to chronic conditions such as diabetes type II [1], colorectal cancer [2], obesity [3], neurological disorders [4], inflammatory bowel disease [5], celiac disease [6], or food allergies [7]
This paper describes the effect of freezing fecal material as a means of preservation before in vitro fermentation experiments
The microbial community in the fecal samples was incubated over 6 days in a complex medium as in [23], with the aim of giving the cells time to recover from the effects of freezing
Summary
The gut microbiota is known to affect host health, playing a critical role in the modulation of the physiological processes of the related to chronic conditions such as diabetes type II [1], colorectal cancer [2], obesity [3], neurological disorders [4], inflammatory bowel disease [5], celiac disease [6], or food allergies [7]. The possibility of optimizing the gut microbiota via diet and bioactive compounds has received much attention This task has a number of challenges due to the complexity of diets, the microbiome, and human physiology, and requires different approaches ranging from in vivo to in vitro or in silico studies [8]. The effect that storage time and temperature can have on fecal microbial communities has been extensively studied in healthy subjects [10,11,12,13,14], subjects with type II diabetes [15], or with inflammatory bowel disease [16,17]. For the fecal material transplant (FMT) consensus [21], the “gold standard” is to store the feces with glycerol at a 50:50 proportion at −80 ◦ C, as glycerol will allow for the preservation of the cell structure, which is essential for FMT
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