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The glassy state of the freeze-concentrated medium formed during freezing: effects on cryopreservation of Lb. bulgaricus cells

Abstract : Improvement of the cryopreservation conditions for lactic acid bacteria (LAB) need to identify and control the factors determining cellular injury. Previous study has shown that fast cooling rates obtained by direct immersion of straws into liquid nitrogen resulted in the minimum loss of acidification activity and viability on thawing, provided that samples were stored at low temperatures (<80 C) [Fonseca, F., Marin, M., Morris G.J. (2006) ‘‘Stabilization of frozen Lactobacillus bulgaricus in glycerol suspensions: freezing kinetics and storage temperature effects’’. Applied and Environmental Microbiologie, 72: 6474–6482.]. It has been demonstrated that the cell damage which occurs in rapidly cooled cells during storage at high sub zero temperatures (20 C) is caused by an osmotic imbalance during warming rather than intracellular ice formation.The aim of this study was to determine whether the manipulation of the physical state of the frozen lactic acid concentrates (e.g immobilizing the cells in a glassy matrix) would allow long-term storage at samples at temperatures higher than 80 C. Studies were carried out on Lactobacillus delbrueckii subsp. bulgaricus CFL1 with different cryoprotectants (glycerol, polyvinylpirrolidone, sucrose). Samples were frozen in 0.5 mL capacity straws by immersion in liquid nitrogen and then stored at different subzero temperatures (from 20 C to 196 C) for 1 month. The LAB acidification activity and viability were determined before and after freezing and during storage by using the CINAC system and plate counts, respectively. Differential scanning calorimetry was used to determine glass transition temperatures (Tg0 1 and Tg0 2) of the residual freeze concentrated medium. Straws were examined following each storage condition using the cryostage of a scanning electron microscope (CryoSEM). Dynamic viscosity of residual unfrozen protective solutions that cells are exposed to during 358 Abstracts / Cryobiology 55 (2007) 324–378 freezing and storage was measured at different subzero temperatures [Kerr, W. L. and D. S. Reid (1994). ‘‘Temperature dependence of the viscosity of sugar and maltodextrin solutions in coexistence with ice.’’ Lebensmittel Wissenschaft und Technologie 27: 225–231]. The acidification activity and the viability of Lb. bulgaricus CFL1 decreased during storage when the bacterial suspensions were stored in a rubbery state. The significant retardation effects on the loss of acidification activity observed in the range of temperature corresponding to the glassy states suggested a diffusion controlled degradation process. The threshold temperature for cell stabilisation varied according to the cryoprotectant used. In the presence of PVP or sucrose, storage temperatures below Tg0 1 seemed necessary to stabilize cells, while in the presence of glycerol Tg0 2 appeared low enough to avoid cell degradation. The LAB acidification activity and viability was related to the ice crystal size and distribution in the frozen samples, as well as to the increased viscosity during freezing of protective aqueous solutions.
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Fernanda Fonseca, Stéphanie Passot, Stéphanie Cenard, Michèle Marin, G. J. Morris. The glassy state of the freeze-concentrated medium formed during freezing: effects on cryopreservation of Lb. bulgaricus cells. 44. Anual Meeting of the Society for Cryobiology, Jul 2007, Lake Louise, Alberta, Canada. ⟨10.1016/j.cryobiol.2007.10.106⟩. ⟨hal-02755460⟩

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