7. Mix cell pellets (CP) with the protective solution (PS) at a ratio
of 1:2 (1 g of cells and 2 g of protective solution). Suspend cells
thoroughly by using a spatula and a vortex mix until complete
cell resuspension. Maintain the cell sample at 4
C.
8. Aseptically aliquot the final protected bacterial suspensions into
appropriate containers for freeze-drying (about 1 cm product
fill depth).
9. Place the containers onto the precooled (at 4
C) shelves of a
freeze-dryer (see Note 9).
10. Place calibrated thermocouples at the bottom of some containers, and fix them with specific holders (rubber band for
instance) (see Note 10).
11. Decrease shelf temperature to À50
C at 1–0.5
C/min, and
hold this temperature until the sample temperature stabilizes
to ensure complete solidification of the product (about 2 h).
12. After freezing the samples, cool the condenser to <À65
C,
switch on the vacuum pump, and decrease the chamber pressure to 10 Pa. Then increase the shelf temperature to À10
C at
1
C/min to initiate the ice sublimation phase (see Note 11).
13. Monitor the process variables during the freeze-drying process
(shelf, condenser, product temperatures, and pressure) in order
to detect any dysfunction, and also to identify the end of
sublimation. The end of sublimation is detected by following
the decrease of the pressure measured by the Pirani gauge.
When the pressure value given by the Pirani gauge reaches
the pressure value given by the capacitance manometer, the
sublimation step is finished (see Fig. 5).
14. Increase the shelf temperature to 25
C at 0.25
C/min to
initiate the secondary drying phase, and reduce the chamber
pressure to minimal value (see Note 12).
15. After 10 h of desorption, break the vacuum by injecting dry air,
vacuum-pack the freeze-dried product in aluminum bags, and
store at temperatures 20
C below the sample’s Tg value (or at
À20
C while waiting for Tg results).
3.4 Characterization
of the Freeze-Dried
Product
1. Manipulate the freeze-dried sample inside a dry chamber, grind
the freeze-dried cake into powder (see Note 13).
2. Measure the water activity: fill the plastic cup with powder until
three-quarters full, place the cup in the a w -meter previously
conditioned at 25
C, close the equipment, and start the measure (stability criteria for a w measurement of 5 min).
3. Measure the glass transition temperature (Tg) of the freezedried sample by DSC: briefly place 10 mg of freeze-dried
powder in 50-μL Perkin Elmer DSC sealed aluminum pan.
Use an empty sealed aluminum pan as the reference. Apply
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Fernanda Fonseca et al.
of 1:2 (1 g of cells and 2 g of protective solution). Suspend cells
thoroughly by using a spatula and a vortex mix until complete
cell resuspension. Maintain the cell sample at 4
C.
8. Aseptically aliquot the final protected bacterial suspensions into
appropriate containers for freeze-drying (about 1 cm product
fill depth).
9. Place the containers onto the precooled (at 4
C) shelves of a
freeze-dryer (see Note 9).
10. Place calibrated thermocouples at the bottom of some containers, and fix them with specific holders (rubber band for
instance) (see Note 10).
11. Decrease shelf temperature to À50
C at 1–0.5
C/min, and
hold this temperature until the sample temperature stabilizes
to ensure complete solidification of the product (about 2 h).
12. After freezing the samples, cool the condenser to <À65
C,
switch on the vacuum pump, and decrease the chamber pressure to 10 Pa. Then increase the shelf temperature to À10
C at
1
C/min to initiate the ice sublimation phase (see Note 11).
13. Monitor the process variables during the freeze-drying process
(shelf, condenser, product temperatures, and pressure) in order
to detect any dysfunction, and also to identify the end of
sublimation. The end of sublimation is detected by following
the decrease of the pressure measured by the Pirani gauge.
When the pressure value given by the Pirani gauge reaches
the pressure value given by the capacitance manometer, the
sublimation step is finished (see Fig. 5).
14. Increase the shelf temperature to 25
C at 0.25
C/min to
initiate the secondary drying phase, and reduce the chamber
pressure to minimal value (see Note 12).
15. After 10 h of desorption, break the vacuum by injecting dry air,
vacuum-pack the freeze-dried product in aluminum bags, and
store at temperatures 20
C below the sample’s Tg value (or at
À20
C while waiting for Tg results).
3.4 Characterization
of the Freeze-Dried
Product
1. Manipulate the freeze-dried sample inside a dry chamber, grind
the freeze-dried cake into powder (see Note 13).
2. Measure the water activity: fill the plastic cup with powder until
three-quarters full, place the cup in the a w -meter previously
conditioned at 25
C, close the equipment, and start the measure (stability criteria for a w measurement of 5 min).
3. Measure the glass transition temperature (Tg) of the freezedried sample by DSC: briefly place 10 mg of freeze-dried
powder in 50-μL Perkin Elmer DSC sealed aluminum pan.
Use an empty sealed aluminum pan as the reference. Apply
714
Fernanda Fonseca et al.