Chapter 38
Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach
for Developing a Freeze-Drying Protocol Based on Physical
Properties
Fernanda Fonseca, Ame ´ lie Girardeau, and Ste ´ phanie Passot
Abstract
Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The
development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of
the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process.
Physical properties of protective solutions, such as glass transition and collapse temperatures, are key
elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter
provides a stepwise approach for developing a protective formulation for the long-term preservation of LAB
and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures
of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried
products, are described.
Key words Glass transition, Collapse, Lactic acid bacteria, Lyoprotectants, Freeze-drying, Lyophilization, Formulation, Stabilizers
1 Introduction
From ancient times, lactic acid bacteria (LAB) have been used for
preserving agricultural raw materials. Today LAB are of great
importance for the food, health products (probiotics), and biotechnology industries. The market of concentrated cultures ready to use
(starters) is continuously growing. The probiotic market is indeed
expected to increase by 7% per year for the next 5 years, reaching
$69 billion USD by 2023 [1].
Freeze-drying is an essential and valuable preservation method
to ensure the long-term stability of bioactive products. This
method makes it possible to remove the majority of water at temperatures far below 0
C (usually between À40 and À20
C) by
sublimation, the phase transition from ice to water vapor. However,
during this low-temperature desiccation process, bacteria are
exposed to several environmental stresses (thermal, osmotic,
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_38, © Springer Science+Business Media, LLC, part of Springer Nature 2021
703
Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach
for Developing a Freeze-Drying Protocol Based on Physical
Properties
Fernanda Fonseca, Ame ´ lie Girardeau, and Ste ´ phanie Passot
Abstract
Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The
development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of
the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process.
Physical properties of protective solutions, such as glass transition and collapse temperatures, are key
elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter
provides a stepwise approach for developing a protective formulation for the long-term preservation of LAB
and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures
of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried
products, are described.
Key words Glass transition, Collapse, Lactic acid bacteria, Lyoprotectants, Freeze-drying, Lyophilization, Formulation, Stabilizers
1 Introduction
From ancient times, lactic acid bacteria (LAB) have been used for
preserving agricultural raw materials. Today LAB are of great
importance for the food, health products (probiotics), and biotechnology industries. The market of concentrated cultures ready to use
(starters) is continuously growing. The probiotic market is indeed
expected to increase by 7% per year for the next 5 years, reaching
$69 billion USD by 2023 [1].
Freeze-drying is an essential and valuable preservation method
to ensure the long-term stability of bioactive products. This
method makes it possible to remove the majority of water at temperatures far below 0
C (usually between À40 and À20
C) by
sublimation, the phase transition from ice to water vapor. However,
during this low-temperature desiccation process, bacteria are
exposed to several environmental stresses (thermal, osmotic,
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_38, © Springer Science+Business Media, LLC, part of Springer Nature 2021
703