Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying

  • B. De Giulio
  • P. Orlando
  • G. Barba
  • R. Coppola
  • M. De Rosa
  • A. Sada
  • P. P. De Prisco
  • F. NazzaroEmail author


In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subspbulgaricus, Streptococcus salivarius subsp.thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 °C in 32% final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared to isotonic solution, especially after freeze-drying procedures (log c.f.u./ml ranging between 1.16 and 2.08, P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated. Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freeze-drying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and 94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp. bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain probiotic formulations more resistant to a variety of stressful conditions.


Alginate cryo-preservation freeze-drying lactic acid bacteria trehalose 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alander, M., De Smet, I., Nollet, L., Verstraete, W., Wright, A., Mattila-Sandholm, T. 1999The effect of probiotic strains on the microbiota of the simulator of the human intestinal microbial ecosystem (SHIME)International Journal of Food Microbiology467179CrossRefPubMedGoogle Scholar
  2. Audet, P., Paquin, C., Lacroix, C. 1988Immobilized growing lactic acid bacteria with k-carrageenan-locust bean gum gelApplied Microbiology and Biotechnology291118CrossRefGoogle Scholar
  3. Buyukgungor, H. 1992Stability of Lactobacillus bulgaricus immobilized in kappa-carrageenan gelsJournal of Chemical Technology and Biotechnology53173175Google Scholar
  4. Caplice, E., Fitzgerald, G.F. 1999Food fermentations: role of microorganisms in food production and preservationInternational Journal of Food Microbiolology50131149CrossRefGoogle Scholar
  5. Carcoba, R., Rodriguez, A. 2000Influence of cryoprotectants on the viability and acidifying activity of frozen and freeze-dried cells of the novel starter strain Lactococcus lactis ssp. lactis CECT 5180European Food Research Technology211433437CrossRefGoogle Scholar
  6. Champagne, C.P., Gardner, N. 2001The effect of protective ingredients on the survival of immobilized cells of Streptococcus thermophilus to air and freeze-dryingElectronic Journal of Biotechnology418Google Scholar
  7. Champagne, C.P., Gardner, N.J., Soulignac, L., Innocent, J.P. 2000The production of freeze-dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milkJournal of Applied Microbiology88124131CrossRefPubMedGoogle Scholar
  8. Champagne, C.P., Lacroix, C., Sodini-Gallot, I. 1994Immobilized cell technologies for the dairy industryCRC Critical Reviews in Biotechnology14109134PubMedGoogle Scholar
  9. Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K. 1998Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tractJournal of Applied Microbiology84759768PubMedGoogle Scholar
  10. Chavarri, F.J., De Paz, M., Nueez, M.M. 1988Cryoprotective agents for frozen concentrated starters from non-bitter Streptococcus lactis strainsBiotechnology Letters101116Google Scholar
  11. Colaco, C.A.L.S., Smith, C.J.S., Sen, S., Roser, D.H., Newman, Y., Ring, S., Roser, D.H. 1994Chemistry of protein stabilization by trehaloseCleland, J.L.Langer, R. eds. Formulation and Delivery of Proteins and PeptidesAmerican Chemical SocietyWashington DC USA216240ISBN 0841229597Google Scholar
  12. Coppola, R., Giagnacovo, B., Iorizzo, M., Grazia, L. 1998Characterization of lactobacilli involved in the ripening of Soppressata molisana, a typical southern Italy fermented sausageFood Microbiology15347353Google Scholar
  13. Corton, E., Piuri, M., Battaglini, F., Ruzal, S.M. 2000Characterization of Lactobacillus carbohydrate fermentation activity using immobilized cell techniqueBiotechnology Progress165966PubMedGoogle Scholar
  14. Crowe, J.H., Crowe, L.M., Chapman, D. 1984Preservation of membranes in anhydrobiotic organisms: the role of trehaloseScience223701703Google Scholar
  15. Crowe, J.H., Crowe, L.M., Carpenter, A.S., Rudolph, A.S., Aurell-Winstrom, C., Spargo, B.J., Anchordoguy, Y.I. 1988Interaction of sugars with membranesBiochimica et Biophysica Acta947367384PubMedGoogle Scholar
  16. Crowe, L.M., Crowe, J.H., Rudolph, A., Womersley, C., Appel, L. 1985Preservation of freeze-dried liposomes by trehaloseArchives of Biochemistry and Biophysics242240247PubMedGoogle Scholar
  17. Diefenbach, R., Keweloh, H., Rehm, H.J. 1992Fatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coliApplied Microbiology and Biotechnology36530534PubMedGoogle Scholar
  18. Draget, K.I., Skjak-Brek, G., Smidrod, O. 1997Alginate based new materialsInternational Journal of Biological Macromolecules214755PubMedGoogle Scholar
  19. Ertesvag, H., Valla, S. 1998Biosynthesis and applications of alginatesPolymer Degradation and Stability598591Google Scholar
  20. Fernandez-Murga, M.L., De Ruiz Holgado, A.P., De Valdez, G.F. 1998Survival rate and enzyme activities of Lactobacillus acidophilus following frozen storageCryobiology36315319PubMedGoogle Scholar
  21. Groboillot, A., Boadi, D.K., Poncelet, D., Neufeld, R.J. 1994Immobilization of cells for application in the food industryCRC Critical Reviews in Biotechnology1475107PubMedGoogle Scholar
  22. Hubalek, Z. 2003Protectants used in the cryopreservation of microorganismsCryobiology46205229PubMedGoogle Scholar
  23. Jan, G., Leverrier, P., Pichereau, V., Boyaval, P. 2001Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichiiApplied and Environmental Microbiology6720292036PubMedGoogle Scholar
  24. Lacroix, C., Paquin, C., Arnaud, J.P. 1990Batch fermentation with entrapped growing cells of Lactobacillus casei. Optimization of the rheological properties of the entrapment gel matrixApplied Microbiology and Biotechnology32403408Google Scholar
  25. Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H., Crowe, L.M. 1995Trehalose and sucrose protect both membranes and proteins during dryingApplied and Environmental Microbiology6135923597PubMedGoogle Scholar
  26. Leverrier, P., Fremont, Y., Roualt, A., Boyaval, P., Jan, G. 2005In vitro tolerance to digestive stresses of propionibacteria: influence of food matricesFood Microbiology221118Google Scholar
  27. Louis, P., Truper, H.G., Galinski, E.A. 1994Survival of Escherichia coli during drying and storage in the presence of compatible solutesApplied Microbiology and Biotechnology41684688Google Scholar
  28. Machado, M.C., Lopez, C.S., Heras, H., Riva, E.A. 2004Osmotic response in Lactobacillus casei ATCC393: biochemical and biophysical characteristics of membraneArchives of Biochemistry and Biophysics4226170PubMedGoogle Scholar
  29. Maicas, S. 2001The use of alternative technologies to develop malolactic fermentation in wineApplied Microbiology & Biotechnology563539Google Scholar
  30. McLoughlin, A.J., Champagne, C.P. 1994Immobilized cells in meat fermentationCRC Critical Reviews in Biotechnology14179192PubMedGoogle Scholar
  31. Nazzaro, F., Coppola, R., Marotta, M., Maurelli, L., De Rosa, M., Addeo, F. 1999Effect of added carbohydrates on some dried lactic acid bacteriaMededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent64253258Google Scholar
  32. Nazzaro, F., Sorrentino, A., Malinconico, M., Orlando, P. 2001In vitro studies on some probiotic properties of immobilized lactic acid bacteria after freezing and freeze-drying Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit GentSect 2: Industrial Biotechnology and Biocatalysis66/3a307312Google Scholar
  33. Piard, J.C., Desmazeaud, M. 1992Inhibiting factors produced by lactic acid bacteria. 2-Bacteriocins and other antimicrobial-substancesLait72113130Google Scholar
  34. Prevost, H., Divies, C. 1987Fresh fermented cheese production with continuous prefermented milk by a mixed culture of mesophilic lactic streptococci entrapped in Ca-alginateBiotechnology Letters9789794Google Scholar
  35. Prevost, H., Divies, C. 1992Cream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beadsBiotechnology Letters14583588Google Scholar
  36. Selmer-Olsen, E., Birkeland, S.E., Sorgaug, T. 1999Effect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydrationJournal of Applied Microbiology87429437PubMedGoogle Scholar
  37. Smidsrod, O., Skjak-Braek, G. 1990Alginate as immobilization matrix for cellsTrends in Biotechnology87178PubMedGoogle Scholar
  38. Sodini, I., Boquien, C.Y., Corrieu, G., Lacroix, C. 1997Microbial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous pre-fermentation of milkEnzyme and Microbial Technology20381388PubMedGoogle Scholar
  39. Visick, J., Clark, S. 1995Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteinsMolecular Microbiology16835845PubMedGoogle Scholar
  40. Vogel, R.F., Bocker, G., Stolz, P., Ehrmann, M., Fanta, D., Ludwig, W., Pot, B., Kersters, K., Schleifer, K.H., Hammes, W.P. 1994Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. novisInternational Journal of Systematic Bacteriology44223229PubMedGoogle Scholar
  41. Yabannavar, M.V., Wang, D.I.C. 1991Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentationBiotechnology and Bioengineering37544550Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • B. De Giulio
    • 1
  • P. Orlando
    • 2
  • G. Barba
    • 1
  • R. Coppola
    • 1
    • 3
  • M. De Rosa
    • 4
  • A. Sada
    • 1
  • P. P. De Prisco
    • 1
  • F. Nazzaro
    • 1
    Email author
  1. 1.Institute of Food Science and Technology – CNRAvellinoItaly
  2. 2.Institute of Protein Biochemistry – CNRNapoliItaly
  3. 3.Di.S.T.A.A.M. – University of MoliseCampobassoItaly
  4. 4.Department of Experimental Medicine, Biotechnology and Molecular Biology SectionMedical School – Second University of NaplesNapoliItaly

Personalised recommendations