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Annals of Microbiology

, Volume 57, Issue 4, pp 537–544 | Cite as

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

  • Mariantonietta Succi
  • Patrizio Tremonte
  • Anna Reale
  • Elena Sorrentino
  • Raffaele Coppola
Food Microbiology Original Articles

Abstract

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (−20 and −80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at −80 °C.

Key word

cryotolerance freezing protective media probiotic lactobacilli 

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References

  1. Accolas J.P., Auclair J. (1967). Storage of highly concentrated suspensions of lactic acid bacteria in the frozen state. Lait, 47: 253–260.CrossRefGoogle Scholar
  2. Brashears M.M., Gilliland S.E. (1995). Survival during frozen and subsequent refrigerated storage ofLactobacillus acidophilus cells as influenced by the growth phase. J. Dairy Sci., 78, 2326–2335.PubMedGoogle Scholar
  3. Chavarri F.J., De Paz M., Nuñez M. (1988). Cryoprotective agents for frozen concentrated starters from non-bitterStreptococcus lactis strains. Biotechnol. Lett., 10: 11–16.CrossRefGoogle Scholar
  4. Coppola R., Iorizzo M., Sorrentino A., Sorrentino E., Grazia L. (1996). Resistenza al congelamento di lattobacilli mesofili isolati da insaccati e paste acide. Industrie Alimentari, 35: 349–356.Google Scholar
  5. Coppola R., Nanni M., Iorizzo M., Sorrentino A., Sorrentino E., Grazia L. (1997). Survey of lactic acid bacteria isolated during the advanced stages of the ripening of Parmigiano Reggiano cheese. J. Dairy Res., 64: 305–310.CrossRefGoogle Scholar
  6. Coppola R., Nanni M., Iorizzo M., Sorrentino A., Sorrentino E., Chiavari C., Grazia L. (2000). Microbiological characteristics of Parmigiano Reggiano cheese during the cheesemaking and the first months of the ripening. Lait, 80: 479–480.CrossRefGoogle Scholar
  7. Coppola R., Succi M., Tremonte P., Reale A., Salzano G., Sorrentino E. (2005). Antibiotic susceptibility ofLactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. Lait, 85: 1–12.CrossRefGoogle Scholar
  8. de Antoni G.L., Pérez P., Abraham A., Anon M.C. (1989). Trehalose, a cryoprotectant forLactobacillus bulgaricus. Cryobiology, 26: 149–153.CrossRefGoogle Scholar
  9. Fernández Murga M.L., Cabrera G.M., Font de Valdéz G., Disalvo A., Seldes A.M. (2000). Influence of growth temperature on cryotolerance and lipid composition ofLactobacillus acidophilus. J. Appl. Microbiol., 88: 342–348.CrossRefGoogle Scholar
  10. 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.CrossRefPubMedGoogle Scholar
  11. Fonseca F., Béal C., Corrieu G. (2001). Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage. Cryobiology, 43: 189–198.CrossRefPubMedGoogle Scholar
  12. Fonseca F., Marin M., Morris G.J. (2006). Stabilization of frozenLactobacillus delbrueckii subsp.bulgaricus in glycerol suspensions: freezing kinetics and storage temperature effects. Appl. Environ. Microbiol., 72: 6474–6482.CrossRefPubMedGoogle Scholar
  13. Font de Valdéz G., de Giori G., de Ruiz Holgado A., Oliver G. (1983). Protective effect of adonitol on lactic acid bacteria subjected to freeze-drying. Appl. Environ. Microbiol., 45: 302–304.Google Scholar
  14. Foschino R., Fiori E., Galli A. (1996). Survival and residual activity ofLactobacillus acidophilus frozen cultures under different conditions. J. Dairy Res., 63: 295–303.CrossRefGoogle Scholar
  15. Gibson C.A., Landerkin G.B., Morse P.M. (1965). Survival of strains of lactic streptococci during freezing storage. J. Dairy Res., 32: 151–156.CrossRefGoogle Scholar
  16. Gilliland S.E., Lara R.C. (1988). Influence of storage at freezing and subsequent refrigeration temperatures on b-galactosidase activity ofLactobacillus acidophilus. Appl. Environ. Microbiol., 54: 898–902.PubMedGoogle Scholar
  17. Gilliland S.E., Speck L.M. (1974). Frozen concentrated cultures of lactic acid bacteria — A review. J. Milk Food Technol., 37: 107–111.Google Scholar
  18. Golberg I., Eschar L. (1977). Stability of lactic acid bacteria to freezing as related to their fatty acid composition. Appl. Environ. Microbiol., 33: 489–496.Google Scholar
  19. Goldin B.R., Gorbach S.L., Saxelin M., Barakat S., Gualtieri L., Salminen S. (1992). Survival ofLactobacillus species (strain GG) in human gastrointestinal tract. Dig. Dis. Sci., 37: 121–128.CrossRefPubMedGoogle Scholar
  20. Johnson M., Ray B., Speck M.L. (1984). Freeze injury in cell wall and its repair inLactobacillus acidophilus. Cryoletters, 5: 171–176.Google Scholar
  21. Kim W.S., Dunn N.W. (1997). Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance. Curr. Microbiol., 35: 59–63.CrossRefPubMedGoogle Scholar
  22. Klaenhammer T.R., Kleeman E.G. (1981). Growth characteristics, bile sensitivity and freeze damage in colonial variants ofLactobacillus acidophilus. Appl. Environ. Microbiol., 41: 1461–1467.PubMedGoogle Scholar
  23. Korpela R., Peuhkuri K., Lahteenmaki T., Sievi E., Saxelin M., Vapaatalo H. (1997).Lactobacillus rhamnosus GG shows antioxidative properties in vascular endothelial cell culture. Milchwissenschaft, 52: 503–505.Google Scholar
  24. Leach R.D., Sandine E. (1976). Numerical relationship between strains in frozen concentrates of lactic streptococcal starter cultures. J. Dairy Sci., 59: 1392–1397.Google Scholar
  25. Majamaa H., Isolauri, E. (1997). Probiotics: a novel approach in the management of food allergy. J. Allergy Clin. Immunol., 99: 179–185.CrossRefPubMedGoogle Scholar
  26. Mazur P. (1977). The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology, 14: 251–272.CrossRefPubMedGoogle Scholar
  27. Millar M.R., Bacon C., Smith S.L., Walker V., Hall M.A. (1993). Enteral feeding of premature infants withLactobacillus GG. Arch. Dis. Child., 69: 483–487.CrossRefPubMedGoogle Scholar
  28. Monnet C., Béal C., Corrieu G. (2003). Improvement of the resistance ofLactobacillus delbrueckii ssp.bulgaricus to freezing by natural selection. J. Dairy Sci., 86: 3048–3053.PubMedCrossRefGoogle Scholar
  29. Panoff J.M., Thammavongs B., Guéguen M. (2000). Cryoprotectants lead to phenotypic adaptation to freezethaw stress inLactobacillus delbrueckii ssp.bulgaricus CIP 10102T. Cryobiology, 40: 264–269.CrossRefPubMedGoogle Scholar
  30. Salminen S., Isolauri E., Salminen E. (1996). Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie Van Leeuwenhoek, 70: 347–358.CrossRefPubMedGoogle Scholar
  31. Saxelin M. (1997).Lactobacillus GG — a human probiotic strain with thorough clinical documentation. Food Rev. Int., 13: 293–313.CrossRefGoogle Scholar
  32. Saxelin M., Ahokas M., Salminen S. (1993). Dose response on the faecal colonisation ofLactobacillus strain GG administered in two different formulations. Microbiol. Ecol. Health Dis., 6: 119–122.CrossRefGoogle Scholar
  33. Smittle R.B., Gilliland S.E., Speck M. (1972). Death ofLactobacillus bulgaricus resulting from liquid nitrogen freezing. Appl. Microbiol., 24: 551–554.PubMedGoogle Scholar
  34. Smittle R.B., Gilliland S.E., Speck M., Walter W.M.J. (1974). Relationship of cellular fatty acid composition to survival ofLactobacillus bulgaricus in liquid nitrogen. Appl. Microbiol., 27: 738–743.PubMedGoogle Scholar
  35. Stadhouders J., Hup G., Jansen L.A. (1971). A study of the optimum conditions of freezing and storing concentrated mesophilic starters. Neth. Milk. Dairy J., 25: 229–239.Google Scholar
  36. Succi M., Tremonte P., Reale A., Sorrentino E., Grazia L., Pacifico S., Coppola R. (2005). Bile salt and acid tolerance ofLactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol. Letters, 244: 129–137.CrossRefGoogle Scholar
  37. Thunnell R.K., Sandine W.E., Bodyfelt F.W. (1984). Frozen starters from internal-pH-control-grown cultures. J. Dairy Sci., 67: 24–36.CrossRefGoogle Scholar
  38. Tomás M.S.J., Ocaña M.E., Nader-Macías M.E. (2004). Viability of vaginal probiotic lactobacilli during refrigerated and frozen storage. Anaerobe, 10: 1–5.CrossRefGoogle Scholar
  39. Van De Guchte M., Serror P., Chervaux C., Smokvina T., Ehrlich S.D., Maguin E. (2002). Stress response in lactic acid bacteria. Antonie van Leeuwenhock, 82: 187–216.CrossRefGoogle Scholar
  40. Wright C.T., Klaenhammer T.R. (1983). Survival ofLactobacillus bulgaricus during freezing and freeze-drying after growth in the presence of calcium. J. Food Sci., 48: 773–777.CrossRefGoogle Scholar
  41. Zago M., Fornasari M.E., Rossetti L., Bonvini B., Scano L., Carminati D., Giraffa G. (2007). Population dynamics of lactobacilli in Grana cheese. Ann. Microbiol., 57 (3): 349–353.CrossRefGoogle Scholar

Copyright information

© University of Milan and Springer 2007

Authors and Affiliations

  • Mariantonietta Succi
    • 1
  • Patrizio Tremonte
    • 1
  • Anna Reale
    • 1
  • Elena Sorrentino
    • 1
  • Raffaele Coppola
    • 1
  1. 1.Dipartimento di Scienze e Tecnologie Agro-alimentari, Ambientali e MicrobiologicheUniversità degli Studi del MoliseCampobassoItaly

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