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. 2018 Jan;11(1):163-175.
doi: 10.1111/1751-7915.12844. Epub 2017 Oct 4.

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Lea Bircher  1 , Clarissa Schwab  1 , Annelies Geirnaert  1 , Christophe Lacroix  1
Affiliations

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Lea Bircher et al. Microb Biotechnol. 2018 Jan.

Abstract

Interest in faecal microbiota transplantation (FMT) has increased as therapy for intestinal diseases, but safety issues limit its widespread use. Intestinal fermentation technology (IFT) can produce controlled, diverse and metabolically active 'artificial' colonic microbiota as potential alternative to common FMT. However, suitable processing technology to store this artificial microbiota is lacking. In this study, we evaluated the impact of the two cryoprotectives, glycerol (15% v/v) and inulin (5% w/v) alone and in combination, in preserving short-chain fatty acid formation and recovery of major butyrate-producing bacteria in three artificial microbiota during cryopreservation for 3 months at -80°C. After 24 h anaerobic fermentation of the preserved microbiota, butyrate and propionate production were maintained when glycerol was used as cryoprotectant, while acetate and butyrate were formed more rapidly with glycerol in combination with inulin. Glycerol supported cryopreservation of the Roseburia spp./Eubacterium rectale group, while inulin improved the recovery of Faecalibacterium prausnitzii. Eubacterium hallii growth was affected minimally by cryopreservation. Our data indicate that butyrate producers, which are key organisms for gut health, can be well preserved with glycerol and inulin during frozen storage. This is of high importance if artificially produced colonic microbiota is considered for therapeutic purposes.

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Figures

Figure 1
Figure 1
Microbial composition of donor faeces and fermentation effluents. Relative abundance of microbial phyla (A) and families (B) in the fermentation effluents and corresponding donors was analysed by V4 region 16S rRNA gene sequencing.
Figure 2
Figure 2
Percentage of main metabolites in fermentation effluent samples. Ratios of major metabolites acetate, propionate and butyrate were calculated from the concentrations measured by HPLC‐RI giving an acetate:propionate:butyrate ratio of 1:0.2:0.8 for effluent 1.1, 1:0.4:1.3 for effluent 1.2 and 1:0.5:0.4 for effluent 2.
Figure 3
Figure 3
Kinetics of main metabolites production after reactivation of effluent microbiota stored for 3 months. Main metabolites acetate, propionate and butyrate were analysed by HPLC‐RI after reactivation in batch fermentations. Each point represents the average of three replicates with standard deviation.
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