Skip to main content

Freeze-Drying of Lactic Acid Bacteria

  • Protocol
  • First Online:
Cryopreservation and Freeze-Drying Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1257))

Abstract

Lactic acid bacteria are of great importance for the food and biotechnology industry. They are widely used as starters for manufacturing food (e.g., yogurt, cheese, fermented meats, and vegetables) and probiotic products, as well as for green chemistry applications. Freeze-drying or lyophilization is a convenient method for preservation of bacteria. By reducing water activity to values below 0.2, it allows long-term storage and low-cost distribution at suprazero temperatures, while minimizing losses in viability and functionality. Stabilization of bacteria via freeze-drying starts with the addition of a protectant solution to the bacterial suspension. Freeze-drying includes three steps, namely, (1) freezing of the concentrated and protected cell suspension, (2) primary drying to remove ice by sublimation, and (3) secondary drying to remove unfrozen water by desorption. In this chapter we describe a method for freeze-drying of lactic acid bacteria at a pilot scale, thus allowing control of the process parameters for maximal survival and functionality recovery.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jennings T (2002) Lyophilization: introduction and basic principles. CRC, Washington, DC

    Google Scholar 

  2. Roy ML, Pikal MJ (1989) Process control in freeze drying: determination of the end point of sublimation drying by an electronic moisture sensor. J Parenter Sci Technol 43:60–66

    CAS  Google Scholar 

  3. Searles JA, Carpenter JF, Randolph TW (2001) The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf. J Pharm Sci 90:860–871

    Article  CAS  Google Scholar 

  4. Fonseca F, Passot S, Cunin O, Marin M (2004) Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media. Biotechnol Prog 20:229–238

    Article  CAS  Google Scholar 

  5. Fonseca F, Passot S, Lieben P, Marin M (2004) Collapse temperature of bacterial suspensions: the effect of cell type and concentration. Cryo Letters 25:425–434

    Google Scholar 

  6. Passot S, Cenard S, Douania I, Tréléa IC, Fonseca F (2012) Critical water activity and amorphous state for optimal preservation of lyophilised lactic acid bacteria. Food Chem 132:1699–1705

    Article  CAS  Google Scholar 

  7. Font de Valdez G, Savoy de Giori G, Pesce de Ruiz Holgado A, Oliver G (1983) Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying. Cryobiology 20:560–566

    Article  CAS  Google Scholar 

  8. Miyamoto-Shinohara Y, Sukenobe J, Imaizumi T, Nakahara T (2006) Survival curves for microbial species stored by freeze-drying. Cryobiology 52:27–32

    Article  Google Scholar 

  9. Crowe JH, Crowe LM, Carpenter JF, Rudolph AS, Wistrom CA, Spargo BJ, Anchordoguy TJ (1988) Interactions of sugars with membranes. Biochim Biophys Acta 947:367–384

    Article  CAS  Google Scholar 

  10. Crowe JH, Crowe LM, Carpenter JF (1993) Preserving dry biomaterials: the water replacement hypothesis, part 1. Biopharmacology 6:28–33

    CAS  Google Scholar 

  11. Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 61:3592–3597

    CAS  Google Scholar 

  12. Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P (2004) Relevant factors for the preparation of freeze-dried lactic acid bacteria. Int Dairy J 14:835–847

    Article  CAS  Google Scholar 

  13. Kurtmann L, Carlsen CU, Skibsted LH, Risbo J (2009) Water activity-temperature state diagrams of freeze-dried Lactobacillus acidophilus (La-5): influence of physical state on bacterial survival during storage. Biotechnol Prog 25:265–270

    Article  CAS  Google Scholar 

  14. Castro HP, Teixeira PM, Kirby R (1995) Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres. Appl Microbiol Biotechnol 44:172–176

    Article  CAS  Google Scholar 

  15. Champagne CP, Gardner N, Brochu E, Beaulieu Y (1991) The freeze-drying of lactic acid bacteria. A review. Can Inst Food Sci Technol 24:118–128

    Article  Google Scholar 

  16. Meng XC, Stanton C, Fitzgerald GF, Daly C, Ross RP (2008) Anhydrobiotics: the challenges of drying probiotic cultures. Food Chem 106:1406–1416

    Article  CAS  Google Scholar 

  17. Tymczyszyn EE, Sosa N, Gerbino E, Hugo A, Gomez-Zavaglia A, Schebor C (2012) Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix. Int J Food Microbiol 155:217–221

    Article  CAS  Google Scholar 

  18. Santivarangkna C, Aschenbrenner M, Kulozik U, Foerst P (2011) Role of glassy state on stabilities of freeze-dried probiotics. J Food Sci 76:R152–R156

    Article  CAS  Google Scholar 

  19. Santivarangkna C, Kulozik U, Foerst P (2006) Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying. Lett Appl Microbiol 42:271–276

    Article  CAS  Google Scholar 

  20. Castro HP, Teixeira PM, Kirby R (1996) Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying. Biotechnol Lett 18:99–104

    Article  CAS  Google Scholar 

  21. Teixeira P, Castro H, Kirby R (1996) Evidence of membrane lipid oxidation of spray-dried Lactobacillus bulgaricus during storage. Lett Appl Microbiol 22:34–38

    Article  CAS  Google Scholar 

  22. Schoug A, Olsson J, Carlfors J, Schnurer J, Hakansson S (2006) Freeze-drying of Lactobacillus coryniformis Si3: effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties. Cryobiology 53:119–127

    Article  CAS  Google Scholar 

  23. Zhang J, Du GC, Zhang YP, Liao XY, Wang M, Li Y, Chen J (2010) Glutathione protects Lactobacillus sanfranciscensis against freeze-thawing, freeze-drying, and cold treatment. Appl Environ Microbiol 76:2989–2996

    Article  CAS  Google Scholar 

  24. Passot S, Tréléa IC, Marin M, Galan M, Morris GJ, Fonseca F (2009) Effect of controlled ice nucleation on primary drying stage and protein recovery in vials cooled in a modified freeze-dryer. J Biomech Eng 131:0745111–0745115

    Article  Google Scholar 

  25. Fonseca F, Béal C, Corrieu G (2000) Method of quantifying the loss of acidification activity of lactic acid starters during freezing and frozen storage. J Dairy Res 67:83–90

    Article  CAS  Google Scholar 

  26. Fonseca F, Marin M, Morris GJ (2006) Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: freezing kinetics and storage temperature effects. Appl Environ Microbiol 72:6474–6482

    Article  CAS  Google Scholar 

  27. Streit F, Corrieu G, Béal C (2010) Effect of centrifugation conditions on the cryotolerance of Lactobacillus bulgaricus CFL1. Food Bioprocess Technol 3:36–42

    Article  Google Scholar 

  28. Fonseca F, Béal C, Mihoub F, Marin M, Corrieu G (2003) Improvement of cryopreservation of Lactobacillus delbrueckii subsp. bulgaricus CFL1 with additives displaying different protective effects. Int Dairy J 67:83–90

    Article  Google Scholar 

  29. Tréléa IC, Passot S, Marin M, Fonseca F (2009) Model for heat and mass transfer in freeze-drying pellets. J Biomech Eng 131:0745011–0745017

    Article  Google Scholar 

  30. Chavez BE, Ledeboer AM (2007) Drying of probiotics: optimization of formulation and process to enhance storage survival. Drying Technol 25:1193–1201

    Article  CAS  Google Scholar 

  31. Font de Valdez G, Savoy de Giori G, Pesce de Ruiz Holgado A, Oliver G (1985) Rehydration conditions and viability of freeze-dried lactic acid bacteria. Cryobiology 22:574–577

    Article  Google Scholar 

Download references

Acknowledgment

This work was supported by the National Institute of Agronomic Research (Paris, France) and by the Paris Institute of Technology for Life, Food and Environmental Sciences (AgroParisTech, Paris, France).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Fernanda Fonseca Ph.D. .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Fonseca, F., Cenard, S., Passot, S. (2015). Freeze-Drying of Lactic Acid Bacteria. In: Wolkers, W., Oldenhof, H. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, vol 1257. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-2193-5_24

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2193-5_24

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-2192-8

  • Online ISBN: 978-1-4939-2193-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics