Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections
- 961 Downloads
The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project (www.embarc.eu). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894T, best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414T should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96T using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587T expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406T did not survive the freeze-drying process under all tested conditions.
KeywordsFreeze-drying Bacteria Lyoprotectant Viability Biological resource centers Residual moisture content
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7, 2007–2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project).
- Hoefman S, Van Hoorde K, Boon N, Vandamme P, De Vos P, Heylen K (2012) Survival or revival: Long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. Plos One 7(4). doi:10.1371/journal.pone.0034196Google Scholar
- Saarela M, Virkajarvi I, Alakomi HL, Mattila-Sandholm T, Vaari A, Suomalainen T, Matto J (2005) Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients. J Appl Microbiol 99(6):1330–1339CrossRefPubMedGoogle Scholar
- Sakane T (1997) Viabilities of dried cultures of various bacteria after preservation for over 20 years and their prediction by the accelerated storage test. Microbiol Cult Coll 13(1):1–7Google Scholar
- Schoug A, Olsson J, Carlfors J, Schnurer J, Hakansson S (2006) Freeze-drying of Lactobacillus coryniformis Si3—effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties. Cryobiology 53(1):119–127Google Scholar
- Tymczyszyn EE, Gomez-Zavaglia A, Disalvo EA (2007) Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus. J Appl Microbiol 102(3):845–851Google Scholar