Effect of Freezing on Gut Microbiota Composition and Functionality for In Vitro Fermentation Experiments

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.

The impact of freezing on the post-mortem human microbiome

“…Observing similar microbiome signatures in the donors before and after freezing is unsurprising as it is generally accepted that for a variety of human-related samples -including fecal matter (Cardona et al, 2012;Pérez-Burillo et al, 2021), skin and saliva (Marotz et al, 2021;Furuhashi et al, 2022), meconium (Xin et al, 2021), and milk (Lyons et al, 2021) -freezing is sufficient to preserve microbial composition. This is supported by the universal knowledge that low temperatures can slow or stop the proliferation of bacteria already present within samples and hinder the establishment of potential contaminants.…”

“…However, although classified as statistically non-significant, our results still show that there were changes to the microbiome after freezing. Pérez-Burillo et al (2021) have speculated that certain taxa may become impaired due to freezing, allowing competing taxa to thrive in their absence after thawingClick or tap here to enter text.. This change in ecological relations may explain the presence or absence of taxa, such as Deinococcota and Myxococcota phyla, despite the overall stability of the microbiome after freezing.…”

Gut microbiota and faecal cytokine profiles of rural Cambodian infants and links to early life nutrition and diarrhoeal episodes

“…Previous research has investigated the effects of storage conditions on microbiota analysis and reported alterations in Bacteroides abundance, although this has focused on storage conditions while frozen or number of freeze thaw cycles (Pérez-Burillo et al, 2021; Poulsen et al, 2021). Room temperature storage of faecal samples for up to 72 hours has been reported to reduce genus including Bacteroides in adult faecal samples over time (Roesch et al, 2009).…”

Impact of Freeze Storage on the Estimation of Phenotypic Antimicrobial Resistance Prevalence in Escherichia coli Collected from Faecal Samples from Healthy Humans and Chickens

“…We hypothesise that the observed increases in E. coli counts in samples stored at −80 °C may be linked to the differential mortality of other bacterial species in the sample, thus providing E. coli with a competitive advantage. It has been shown that freezing faecal samples may change the bacterial community structure [ 16 ], although some studies have only found marginal changes based on 16S rRNA analyses [ 11 ]. Escherichia coli have been generally described as relatively resilient organisms under different environmental conditions [ 17 ].…”