Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes

Abstract

SummaryStrict anaerobic gut microbes have been suggested as ‘next‐generation probiotics’ for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (−80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant‐free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant‐free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process‐ and species‐specific.

Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions

“…A previous study reported that following 6 months of frozen storage of stools in 10% glycerol, CFU counts of six bacterial groups (bifidobacteria, Escherichia coli, total coliforms, lactobacilli, total anaerobic bacteria, or total aerobes) were similar to baseline values (Costello et al, 2015). Similarly, Bircher et al (2018) showed that the gut commensal Bacteroides thetaiotaomicron (phylum Bacteroidetes) was resistant to cryopreservation for 3 months in terms of CFU counts and membrane integrity. Another study, based on a PMA treatment combined with qPCR, showed a reduced overall viability but no significant changes in composition of viable microbiota frozen fecal samples (Papanicolas et al, 2019).…”

Planktonic and Sessile Artificial Colonic Microbiota Harbor Distinct Composition and Reestablish Differently upon Frozen and Freeze-Dried Long-Term Storage

“…Lyophilization and cryopreservation can impair growth and can lead to lethal damage for a certain fraction of preserved bacterial cells, clearing niches for more-resistant taxa that are less competitive in fresh microbiota (51). The levels of tolerance of freezing and drying differ greatly between different anaerobic gut microbes (34) and might explain the observed shifts. Previous preservation studies demonstrated that Gram-positive bacteria have an advantage over Gram-negative bacteria in surviving lyophilization (52,53).…”

“…A storage period of 9 months was selected to represent the conditions suggested for storing fecal material for fecal microbiota transplantation (FMT) (33). To improve preservation survival, the protectants sucrose and inulin (both 5% [wt/vol]) and combinations of sucrose and inulin (both 5% [wt/vol]) and glycerol (15% [vol/vol]), previously developed for stabilizing strict anaerobic gut microbes, were added for lyophilization and cryopreservation, respectively (34). Composition and metabolic activities of preserved plankM and sessM were evaluated in small-scale, strictly anaerobic batch cultures over 24 h, and the results were compared to those seen with batch cultures inoculated with fresh microbiota.…”

Effect of osmoprotectants on the survival of bacterial endophytes in lyophilized beet roots

“…Significantly higher cell density of culturable bacteria observed in sugar beet lyophilized roots can be attributed to high content of sucrose (Haankuku et al, 2015). This sugar acts as a natural osmoprotectant, allowing a better viability of microorganisms during lyophilization (Bircher et al, 2018). Another explanation of obtained results can be associated with higher ability of sugar beet endophytes to grow on solid medium.…”