Freeze drying (lyophilization) is a dehydration process which allows water to sublimate directly from solid phase to vapour phase at and below the freezing temperature of the material. Sub-atmospheric pressure (< 40 Pa) is maintained in most freeze-drying operations and the condensed water is immediately removed (Pikal, 2007). Freeze drying has been for decades one of the most preferred preservation methods for culture collection maintenance (Morgan et al., 2006). Due to high viability losses, an initial bacterial load of greater than 107 viable cells/mL has been recommended to ensure sufficient cells survive the freeze-drying process, thereby giving better success in storage, reconstitution and propagation (Bozoglu et al., 1987).
At commercial scale, operational and capital costs of freeze drying are very high. The freeze-drying process operates in batch mode and requires long drying times and large drying units to achieve mass production. Even so, freeze drying is currently the only drying method used at commercial scale for production of starter cultures intended for use as primary acid producers in dairy fermentations.
It is reported that the majority of bacterial death occurring during freeze drying happens during the freezing stage before the drying (sublimation) process commences. A slow freezing rate leads to higher bacterial death in the subsequent sublimation stage (Uzunova-Doneva and Donev, 2002). Rapid freezing, with formation of smaller ice crystals, favours better bacterial survival. On the other hand, formation of large ice crystals during slow freezing causes structural and physiological injury to the bacterial cells and causes damage to cell membranes that cannot be repaired upon subsequent drying or rehydration (Gardiner et al., 2000).
Many studies have exploited the addition of ‘protectant’ substances to enhance survival, and have investigated the use of low-cost food ingredients as protectants rather than substances such as glycine betaine (Cleland et al., 2004). Recent examples include work by Jagannath et al. (2010), who studied the survival of various probiotic bacteria after freeze drying. The survival obtained ranged from 67% to 70% depending on bacterial species. Zamora et al. (2006) compared the survival of twelve strains of lactic acid bacteria after freeze drying and reported a range from 3.3% to 100% depending on the bacterial type and protectant type used. For example, the survival of four strains of Lactococcus garviae was reported to be 100% when non-fat skim milk was used as the protectant (Zamora et al., 2006). Reddy et al. (2009) studied survival of three probiotic lactic acid bacteria with eleven different protectants (at various solids concentrations), and suggested that these protected not only the viability of the probiotic lactic acid bacteria but also their functional properties.