2.5 Water Activity
and Water Content
Measurement
of the Freeze-Dried
Product
1. A chamber continuously purged with dried air for manipulating hygroscopic dried samples.
2. a w -meter LabMaster-aw (Novasina, Precisa, Poissy, France).
3. Plastic cups for measuring water activity (Novasina, Precisa,
Poissy, France).
4. Karl Fisher titration method: Metrohm KF 756 apparatus
(Herisau, Switzerland) with reagent Hydranal
® Coulomat AG
(Honeywell Fluka, Seelze, Germany).
5. Hydranal™ methanol dry (Honeywell Fluka, Seelze,
Germany).
6. 1 mL Microliter™ syringe (Hamilton, Bonaduz, Switzerland).
7. 4 mL glass vials, septum, and caps.
8. Mortar and pestle.
3 Methods
Figure 2 depicts a stepwise approach for developing a protective
formulation for lactic acid bacteria that is compatible with an
efficient freeze-drying process. This approach lies in the measurement of the formulation’s physical properties (Tg
0 and Tcoll).
3.1 Determination
of the Effect of Cells
and Supernatant
on the Physical
Properties (Tcoll
and Tg
0 )
1. Perform bacterial growth according to your own production
protocol or to the protocol described by Fonseca et al. [15].
2. Prepare a protective solution (PS) composed of 200 g/L of
sucrose and 9 g/L of NaCl (see Note 2).
3. Transfer the cell suspension to centrifugation flasks.
4. Centrifuge (17,000 Â g, 4
C, 30 min) to concentrate cell
suspension, and remove the culture supernatant (see Note 3).
5. 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.
6. Determine the glass transition temperature of the maximally
freeze-concentrated phase (Tg
0 ) of the protected bacterial suspension (mixture CP/PS) by using DSC. Briefly, place 15 μL of
the suspension in 50-μL Perkin Elmer DSC sealed aluminum
pan, and note the sample weight. Use an empty sealed aluminum pan as the reference. Apply the following temperature
program to the sample and the reference: cooling from 20 to
À100
C at 10
C/min, holding at À100
C for 1 min, and
heating from À100 to 20
C at 10
C/min. Plot the first
derivative of the heat flow recorded during the heating scan,
and identify the Tg
0 value associated to the higher heat capacity
Physical Properties and Freeze-Drying of Bacteria
709
and Water Content
Measurement
of the Freeze-Dried
Product
1. A chamber continuously purged with dried air for manipulating hygroscopic dried samples.
2. a w -meter LabMaster-aw (Novasina, Precisa, Poissy, France).
3. Plastic cups for measuring water activity (Novasina, Precisa,
Poissy, France).
4. Karl Fisher titration method: Metrohm KF 756 apparatus
(Herisau, Switzerland) with reagent Hydranal
® Coulomat AG
(Honeywell Fluka, Seelze, Germany).
5. Hydranal™ methanol dry (Honeywell Fluka, Seelze,
Germany).
6. 1 mL Microliter™ syringe (Hamilton, Bonaduz, Switzerland).
7. 4 mL glass vials, septum, and caps.
8. Mortar and pestle.
3 Methods
Figure 2 depicts a stepwise approach for developing a protective
formulation for lactic acid bacteria that is compatible with an
efficient freeze-drying process. This approach lies in the measurement of the formulation’s physical properties (Tg
0 and Tcoll).
3.1 Determination
of the Effect of Cells
and Supernatant
on the Physical
Properties (Tcoll
and Tg
0 )
1. Perform bacterial growth according to your own production
protocol or to the protocol described by Fonseca et al. [15].
2. Prepare a protective solution (PS) composed of 200 g/L of
sucrose and 9 g/L of NaCl (see Note 2).
3. Transfer the cell suspension to centrifugation flasks.
4. Centrifuge (17,000 Â g, 4
C, 30 min) to concentrate cell
suspension, and remove the culture supernatant (see Note 3).
5. 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.
6. Determine the glass transition temperature of the maximally
freeze-concentrated phase (Tg
0 ) of the protected bacterial suspension (mixture CP/PS) by using DSC. Briefly, place 15 μL of
the suspension in 50-μL Perkin Elmer DSC sealed aluminum
pan, and note the sample weight. Use an empty sealed aluminum pan as the reference. Apply the following temperature
program to the sample and the reference: cooling from 20 to
À100
C at 10
C/min, holding at À100
C for 1 min, and
heating from À100 to 20
C at 10
C/min. Plot the first
derivative of the heat flow recorded during the heating scan,
and identify the Tg
0 value associated to the higher heat capacity
Physical Properties and Freeze-Drying of Bacteria
709