2. A freeze-drying cryo-stage (FDCS 196, Linkam Scientific
Instruments, Surrey, UK) equipped with a programmable temperature controller (TMS94, Linkam), a liquid nitrogen cooling system, a vacuum pump (Edwards E2M1.5, Linkam), and a
Pirani gauge.
3. A Retiga 2000R digital monochrome camera (QImaging, Surrey, Canada).
4. 16-mm quartz coverslips, 13-mm glass coverslips, and metallic
spacers.
2.3 Freeze-Dryer
1. A pilot-scale freeze-dryer equipped with temperaturecontrolled shelves (À50 to 50
C), capacitive manometer for
pressure control and sensors for monitoring the product temperature (thermocouples) and the end of sublimation (Pirani
gauge or moisture sensor or pressure rise test device) [14] (see
Note 1).
2. A computer and software associated with the freeze-dryer
allowing control of the equipment, data acquisition, and
visualization.
3. Suitable containers for freeze-drying the product (vials, trays,
aluminum cups, etc.).
4. Aluminum bags for conditioning the freeze-dried samples.
5. Equipment for sealing aluminum bags under vacuum.
2.4 Formulations
for Freeze-Drying
Bacteria
1. Bacterial pellet obtained after fermentation and concentration
steps. The protocol described by Fonseca et al. [15] can be
followed to produce the bacterial pellet.
2. Protective molecules (see Table 1). A protective solution for
preserving lactic acid bacteria usually involves three components: (a) a stabilizer that can form hydrogen bonds with
proteins and polar head groups of membrane phospholipids
and act as water substitute when water is removed during
desorption of the unfrozen water (disaccharides for instance);
(b) a polysaccharide or polymer of high molecular weight that
increase the glass transition temperature of the solution (Tg
0
higher than Tg
0 of disaccharides); (c) an antioxidant molecule
to limit oxidative reactions following storage of the freezedried product; and (d) in some cases additional molecules
such as specific cryoprotective agents (amino acid, etc.) or
surfactants. Table 2 summarizes the physical properties (Tg
0
and Tcoll) of the most commonly used protective molecules for
preserving lactic acid bacteria.
708
Fernanda Fonseca et al.
Instruments, Surrey, UK) equipped with a programmable temperature controller (TMS94, Linkam), a liquid nitrogen cooling system, a vacuum pump (Edwards E2M1.5, Linkam), and a
Pirani gauge.
3. A Retiga 2000R digital monochrome camera (QImaging, Surrey, Canada).
4. 16-mm quartz coverslips, 13-mm glass coverslips, and metallic
spacers.
2.3 Freeze-Dryer
1. A pilot-scale freeze-dryer equipped with temperaturecontrolled shelves (À50 to 50
C), capacitive manometer for
pressure control and sensors for monitoring the product temperature (thermocouples) and the end of sublimation (Pirani
gauge or moisture sensor or pressure rise test device) [14] (see
Note 1).
2. A computer and software associated with the freeze-dryer
allowing control of the equipment, data acquisition, and
visualization.
3. Suitable containers for freeze-drying the product (vials, trays,
aluminum cups, etc.).
4. Aluminum bags for conditioning the freeze-dried samples.
5. Equipment for sealing aluminum bags under vacuum.
2.4 Formulations
for Freeze-Drying
Bacteria
1. Bacterial pellet obtained after fermentation and concentration
steps. The protocol described by Fonseca et al. [15] can be
followed to produce the bacterial pellet.
2. Protective molecules (see Table 1). A protective solution for
preserving lactic acid bacteria usually involves three components: (a) a stabilizer that can form hydrogen bonds with
proteins and polar head groups of membrane phospholipids
and act as water substitute when water is removed during
desorption of the unfrozen water (disaccharides for instance);
(b) a polysaccharide or polymer of high molecular weight that
increase the glass transition temperature of the solution (Tg
0
higher than Tg
0 of disaccharides); (c) an antioxidant molecule
to limit oxidative reactions following storage of the freezedried product; and (d) in some cases additional molecules
such as specific cryoprotective agents (amino acid, etc.) or
surfactants. Table 2 summarizes the physical properties (Tg
0
and Tcoll) of the most commonly used protective molecules for
preserving lactic acid bacteria.
708
Fernanda Fonseca et al.