Tmax, the material will undergo viscous flow, resulting in loss of the
pore structure, which is defined as the collapse phenomenon by
Pikal and Shah [8]. Collapsed dried products generally have high
residual water content and a lengthy reconstitution time and may
also display a loss of functional properties. For amorphous material,
Tmax is usually associated to the glass transition temperature of the
maximally freeze-concentrated phase (Tg
0 ). For simple binary
aqueous solutions, the collapse was shown to take place 1–3
C
above Tg
0 [8–11]. However, when adding bacterial pellets to these
solutions, the collapse of the dried porous structure occurred at a
temperature about 10
C higher than Tg
0 [9, 12]. The presence of
LAB cells confers significant robustness to the freeze-dried product, thus allowing the use of higher product temperatures during
primary drying than expected from the protective solution.
Differential scanning calorimetry (DSC) and freeze-drying
microscopy (FDM) are two complementary tools used to characterize the freeze-drying behavior of a solution [13]. DSC determines the phase changes occurring in a sample following cooling
and heating, and it is the method of choice for measuring glass
transition temperature in liquid (Tg
0 ) and solid (Tg) samples. By
making it possible, the direct microscopic observation of the
porous dried structure following sublimation FDM gives an estimation of the collapse temperature (Tcoll).
The composition of the protective solution added to bacteria
before the freeze-drying process affects Tg
0 , Tcoll, and Tg, thus
constituting a key element for optimizing both the product quality
and the process. Table 1 summarizes the main protective molecules
used for preserving LAB and their associated mechanisms of action.
An appropriate choice of molecules is needed to prevent freezingand drying-induced damage and to preserve biological activity
during processing, storage, and rehydration. The development of
a protective solution must integrate the maximization of Tmax.
Consequently, the concentration of molecules lowering the Tmax
value should be reduced or still better if they can be replaced by
molecules increasing the Tmax value. Table 2 summarizes the
physical properties (Tg
0 and Tcoll) of the most commonly used
protective molecules for preserving lactic acid bacteria.
Many protective solutions and freeze-drying cycles have been
separately developed on an empirical basis. However, it is today
widely accepted that formulation and cycle development have to be
performed concomitantly, on a more rational and scientific basis
involving a deep characterization of the formulation’s physical
behavior. In the current chapter, a logical stepwise approach for
developing a protective formulation for lactic acid bacteria that is
compatible with an efficient freeze-drying process is described. In
addition, methods are presented to determine physical properties
of the freeze-dried product, glass transition temperature, water
activity, and water content.
Physical Properties and Freeze-Drying of Bacteria
705
pore structure, which is defined as the collapse phenomenon by
Pikal and Shah [8]. Collapsed dried products generally have high
residual water content and a lengthy reconstitution time and may
also display a loss of functional properties. For amorphous material,
Tmax is usually associated to the glass transition temperature of the
maximally freeze-concentrated phase (Tg
0 ). For simple binary
aqueous solutions, the collapse was shown to take place 1–3
C
above Tg
0 [8–11]. However, when adding bacterial pellets to these
solutions, the collapse of the dried porous structure occurred at a
temperature about 10
C higher than Tg
0 [9, 12]. The presence of
LAB cells confers significant robustness to the freeze-dried product, thus allowing the use of higher product temperatures during
primary drying than expected from the protective solution.
Differential scanning calorimetry (DSC) and freeze-drying
microscopy (FDM) are two complementary tools used to characterize the freeze-drying behavior of a solution [13]. DSC determines the phase changes occurring in a sample following cooling
and heating, and it is the method of choice for measuring glass
transition temperature in liquid (Tg
0 ) and solid (Tg) samples. By
making it possible, the direct microscopic observation of the
porous dried structure following sublimation FDM gives an estimation of the collapse temperature (Tcoll).
The composition of the protective solution added to bacteria
before the freeze-drying process affects Tg
0 , Tcoll, and Tg, thus
constituting a key element for optimizing both the product quality
and the process. Table 1 summarizes the main protective molecules
used for preserving LAB and their associated mechanisms of action.
An appropriate choice of molecules is needed to prevent freezingand drying-induced damage and to preserve biological activity
during processing, storage, and rehydration. The development of
a protective solution must integrate the maximization of Tmax.
Consequently, the concentration of molecules lowering the Tmax
value should be reduced or still better if they can be replaced by
molecules increasing the Tmax value. Table 2 summarizes the
physical properties (Tg
0 and Tcoll) of the most commonly used
protective molecules for preserving lactic acid bacteria.
Many protective solutions and freeze-drying cycles have been
separately developed on an empirical basis. However, it is today
widely accepted that formulation and cycle development have to be
performed concomitantly, on a more rational and scientific basis
involving a deep characterization of the formulation’s physical
behavior. In the current chapter, a logical stepwise approach for
developing a protective formulation for lactic acid bacteria that is
compatible with an efficient freeze-drying process is described. In
addition, methods are presented to determine physical properties
of the freeze-dried product, glass transition temperature, water
activity, and water content.
Physical Properties and Freeze-Drying of Bacteria
705