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

Abstract

Strict 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.

Figure 1

Set‐up of preservation experiments. Early stationary phase cultures were incubated for 30 min in buffers containing sucrose and inulin (SI), sucrose, inulin and glycerol (GSI) and in control buffer lacking protectants (control) before processing for preservation. Viability and growth were assessed at three different time points indicated by red dots. The first assessment was performed with fresh cultures after incubation in the protective and control buffers (t ₀). The second assessment was conducted with the processed cultures immediately after freezing, respectively, lyophilization (t ₁) and the third assessment with cryopreserved and lyophilized samples stored for 3 months at −80°C or 4°C, respectively (t ₂).

Figure 2

Impact of cryoprotectants on membrane integrity of cryopreserved and lyophilized strict anaerobes. Percentage of intact cells in fresh (t ₀), and cryopreserved (Cryo) and lyophilized (Lyo) B. thetaiotaomicron (A), E. hallii (B), B. obeum (C), R. intestinalis (D), A. caccae (E) and F. prausnitzii (F) after 3 months storage in control (no protectant) and treated cultures (t₂) (SI and GSI).