5. Further adjustment of the composition of the protective solution can be required according to the biological activity recovery after freeze-drying and storage. In any case, the
concentration of solutes in the protective solution should not
be higher than 250 g/L.
6. After the centrifugation step and the removal of culture supernatant, add an equivalent volume of saline solution, suspend
cells thoroughly by using a spatula and a vortex mixer until
complete cell resuspension, centrifuge, and remove the supernatant. This operation can be repeated once.
7. Increasing the antioxidant concentration up to 10 g/L may be
necessary, particularly for strains that are sensitive to oxidation
reactions.
8. Use a laminar hood for transferring and manipulating cell
samples. If a laminar hood is not available, work close to a gas
burner.
9. Samples can be frozen outside the freeze-dryer (for instance, in
a À80
C freezer or by immersion in liquid nitrogen). In this
case, the samples must be transferred to the freeze-drier after
precooling the shelves to À50
C to avoid thawing the samples.
10. Place thermocouples at different locations in the freeze-dryer.
If only one shelf is used, place them in the center of
the container (and center of the shelf) and on the borders of
the recipient (and the borders of the shelf) to take into account
the heterogeneity of the product temperature within the sample batch. The samples located at the border of the shelf receive
an additional heat flow rate and display product temperatures
up to 4
C higher than central samples [19, 20].
11. The values of shelf temperature, chamber pressure, and heating
rate may require some adjustment dependent on the type of
freeze-dryer and the type of container. The selection of an
optimal process condition for primary (sublimation) and secondary (desorption) steps can be guided by specific interactive
software ([21], LyOptM software [22]).
12. The values of shelf temperature, heating rate, and desorption
time can be modified according to the properties of the formulation and bacterial sensitivity.
13. Place all the materials (plastic cups, vials, septum, stoppers,
spatula, mortar and pestle, etc.) needed for manipulating the
freeze-dried product and preparing sample for water activity,
water content, and glass transition temperature measurement
in the chamber continuously purged with dried air, the day
before the analysis.
Physical Properties and Freeze-Drying of Bacteria
717
concentration of solutes in the protective solution should not
be higher than 250 g/L.
6. After the centrifugation step and the removal of culture supernatant, add an equivalent volume of saline solution, suspend
cells thoroughly by using a spatula and a vortex mixer until
complete cell resuspension, centrifuge, and remove the supernatant. This operation can be repeated once.
7. Increasing the antioxidant concentration up to 10 g/L may be
necessary, particularly for strains that are sensitive to oxidation
reactions.
8. Use a laminar hood for transferring and manipulating cell
samples. If a laminar hood is not available, work close to a gas
burner.
9. Samples can be frozen outside the freeze-dryer (for instance, in
a À80
C freezer or by immersion in liquid nitrogen). In this
case, the samples must be transferred to the freeze-drier after
precooling the shelves to À50
C to avoid thawing the samples.
10. Place thermocouples at different locations in the freeze-dryer.
If only one shelf is used, place them in the center of
the container (and center of the shelf) and on the borders of
the recipient (and the borders of the shelf) to take into account
the heterogeneity of the product temperature within the sample batch. The samples located at the border of the shelf receive
an additional heat flow rate and display product temperatures
up to 4
C higher than central samples [19, 20].
11. The values of shelf temperature, chamber pressure, and heating
rate may require some adjustment dependent on the type of
freeze-dryer and the type of container. The selection of an
optimal process condition for primary (sublimation) and secondary (desorption) steps can be guided by specific interactive
software ([21], LyOptM software [22]).
12. The values of shelf temperature, heating rate, and desorption
time can be modified according to the properties of the formulation and bacterial sensitivity.
13. Place all the materials (plastic cups, vials, septum, stoppers,
spatula, mortar and pestle, etc.) needed for manipulating the
freeze-dried product and preparing sample for water activity,
water content, and glass transition temperature measurement
in the chamber continuously purged with dried air, the day
before the analysis.
Physical Properties and Freeze-Drying of Bacteria
717