Collapse Temperature of Freeze-Dried Lactobacillus bulgaricus Suspensions and Protective Media Fernanda Fonseca,* Ste ´ phanie Passot, Olivier Cunin, and Miche ` le Marin UMR Ge ´nie et Microbiologie des Proce ´de ´s Alimentaires, Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the “microcollapse” temperature (T μc , beginning of a local loss of structure) and the “collapse” temperature (T c , beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 °C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of “robustness” to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 °C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 °C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temper- ature T c was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells. 1. Introduction Freeze-drying (or lyophilization) is widely used to stabilize pharmaceuticals, microorganisms, and many food and biological products. This process makes it possible to minimize the degradation reactions and to maintain an adequate physical, chemical, and biological stability of the product during long-term storage, even at ambient temperatures. However, it is an expensive drying process (low productivity), which remains advan- tageous for high-value foods or labile biomaterials. As a general rule, primary drying is the most time-consuming stage of the process, and it is well known that raising the temperature significantly increases productivity. An optimized freeze-drying process must be carried out at the maximum allowable product temperature, which depends on the physical state of the frozen and dried products. As a general rule, solutes crystallize or remain amor- phous after freezing depending on several factors: chemi- cal nature, initial concentration, and interaction with other solutes and the freezing procedure applied. In biological materials, the usual freezing behavior corre- sponds to noncrystalline solutes (proteins, sugars, etc.) that remain amorphous, and in this case, the maximum allowable temperature is the collapse temperature (1- 3). Nevertheless, in a binary (one-solute) aqueous solu- tion, if the solute crystallizes during freezing, the eutectic temperature corresponds to the highest allowable tem- perature. During primary drying, if the product temper- ature is higher than the collapse temperature, the amorphous material will undergo viscous flow, resulting in loss of the pore structure obtained by freezing, which is defined as the collapse phenomenon by Pikal and Shah (3). Collapsed dried products generally have a high residual water content and lengthy reconstitution times and may also present a loss of functional properties. Moreover, in the pharmaceutical industry, collapse is normally cause for rejection of the vials due to the lack of material elegance. Since a small variation of temper- ature can greatly modify the primary drying time as well as the dried product structure, an accurate determination of the collapse temperature is critical for the process * To whom correspondence should be addressed. Tel: (33) (0) 1 30 81 59 40. Fax: (33) (0) 1 30 81 55 97. E-mail: fonseca@ grignon.inra.fr. 229 Biotechnol. Prog. 2004, 20, 229-238 10.1021/bp034136n CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/09/2003