Summary
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.
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References
M. Alander I. De Smet L. Nollet W. Verstraete A. Wright Particlevon T. Mattila-Sandholm (1999) ArticleTitleThe effect of probiotic strains on the microbiota of the simulator of the human intestinal microbial ecosystem (SHIME) International Journal of Food Microbiology 46 71–79 Occurrence Handle10.1016/S0168-1605(98)00182-2 Occurrence Handle10050686
P. Audet C. Paquin C. Lacroix (1988) ArticleTitleImmobilized growing lactic acid bacteria with k-carrageenan-locust bean gum gel Applied Microbiology and Biotechnology 29 11–18 Occurrence Handle10.1007/BF00258344
H. Buyukgungor (1992) ArticleTitleStability of Lactobacillus bulgaricus immobilized in kappa-carrageenan gels Journal of Chemical Technology and Biotechnology 53 173–175
E. Caplice G.F. Fitzgerald (1999) ArticleTitleFood fermentations: role of microorganisms in food production and preservation International Journal of Food Microbiolology 50 131–149 Occurrence Handle10.1016/S0168-1605(99)00082-3
R. Carcoba A. Rodriguez (2000) ArticleTitleInfluence of cryoprotectants on the viability and acidifying activity of frozen and freeze-dried cells of the novel starter strain Lactococcus lactis ssp. lactis CECT 5180 European Food Research Technology 211 433–437 Occurrence Handle10.1007/s002170000194
C.P. Champagne N. Gardner (2001) ArticleTitleThe effect of protective ingredients on the survival of immobilized cells of Streptococcus thermophilus to air and freeze-drying Electronic Journal of Biotechnology 4 1–8
C.P. Champagne N.J. Gardner L. Soulignac J.P. Innocent (2000) ArticleTitleThe production of freeze-dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milk Journal of Applied Microbiology 88 124–131 Occurrence Handle10.1046/j.1365-2672.2000.00927.x Occurrence Handle10735251
C.P. Champagne C. Lacroix I. Sodini-Gallot (1994) ArticleTitleImmobilized cell technologies for the dairy industry CRC Critical Reviews in Biotechnology 14 109–134 Occurrence Handle8069933
W.P. Charteris P.M. Kelly L. Morelli J.K. Collins (1998) ArticleTitleDevelopment and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract Journal of Applied Microbiology 84 759–768 Occurrence Handle9674129
F.J. Chavarri M. De Paz M.M. Nueez (1988) ArticleTitleCryoprotective agents for frozen concentrated starters from non-bitter Streptococcus lactis strains Biotechnology Letters 10 11–16
C.A.L.S. Colaco C.J.S. Smith S. Sen D.H. Roser Y. Newman S. Ring D.H. Roser (1994) Chemistry of protein stabilization by trehalose J.L. Cleland R. Langer (Eds) Formulation and Delivery of Proteins and Peptides American Chemical Society Washington DC USA 216–240
R. Coppola B. Giagnacovo M. Iorizzo L. Grazia (1998) ArticleTitleCharacterization of lactobacilli involved in the ripening of Soppressata molisana, a typical southern Italy fermented sausage Food Microbiology 15 347–353
E. Corton M. Piuri F. Battaglini S.M. Ruzal (2000) ArticleTitleCharacterization of Lactobacillus carbohydrate fermentation activity using immobilized cell technique Biotechnology Progress 16 59–66 Occurrence Handle10662490
J.H. Crowe L.M. Crowe D. Chapman (1984) ArticleTitlePreservation of membranes in anhydrobiotic organisms: the role of trehalose Science 223 701–703
J.H. Crowe L.M. Crowe A.S. Carpenter A.S. Rudolph C. Aurell-Winstrom B.J. Spargo Y.I. Anchordoguy (1988) ArticleTitleInteraction of sugars with membranes Biochimica et Biophysica Acta 947 367–384 Occurrence Handle3285894
L.M. Crowe J.H. Crowe A. Rudolph C. Womersley L. Appel (1985) ArticleTitlePreservation of freeze-dried liposomes by trehalose Archives of Biochemistry and Biophysics 242 240–247 Occurrence Handle4051504
R. Diefenbach H. Keweloh H.J. Rehm (1992) ArticleTitleFatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coli Applied Microbiology and Biotechnology 36 530–534 Occurrence Handle1368205
K.I. Draget G. Skjak-Brek O. Smidrod (1997) ArticleTitleAlginate based new materials International Journal of Biological Macromolecules 21 47–55 Occurrence Handle9283015
H. Ertesvag S. Valla (1998) ArticleTitleBiosynthesis and applications of alginates Polymer Degradation and Stability 59 85–91
M.L. Fernandez-Murga A.P. De Ruiz Holgado G.F. De Valdez (1998) ArticleTitleSurvival rate and enzyme activities of Lactobacillus acidophilus following frozen storage Cryobiology 36 315–319 Occurrence Handle9679081
A. Groboillot D.K. Boadi D. Poncelet R.J. Neufeld (1994) ArticleTitleImmobilization of cells for application in the food industry CRC Critical Reviews in Biotechnology 14 75–107 Occurrence Handle8069936
Z. Hubalek (2003) ArticleTitleProtectants used in the cryopreservation of microorganisms Cryobiology 46 205–229 Occurrence Handle12818211
G. Jan P. Leverrier V. Pichereau P. Boyaval (2001) ArticleTitleChanges in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii Applied and Environmental Microbiology 67 2029–2036 Occurrence Handle11319077
C. Lacroix C. Paquin J.P. Arnaud (1990) ArticleTitleBatch fermentation with entrapped growing cells of Lactobacillus casei. Optimization of the rheological properties of the entrapment gel matrix Applied Microbiology and Biotechnology 32 403–408
S.B. Leslie E. Israeli B. Lighthart J.H. Crowe L.M. Crowe (1995) ArticleTitleTrehalose and sucrose protect both membranes and proteins during drying Applied and Environmental Microbiology 61 3592–3597 Occurrence Handle7486995
P. Leverrier Y. Fremont A. Roualt P. Boyaval G. Jan (2005) ArticleTitleIn vitro tolerance to digestive stresses of propionibacteria: influence of food matrices Food Microbiology 22 11–18
P. Louis H.G. Truper E.A. Galinski (1994) ArticleTitleSurvival of Escherichia coli during drying and storage in the presence of compatible solutes Applied Microbiology and Biotechnology 41 684–688
M.C. Machado C.S. Lopez H. Heras E.A. Riva (2004) ArticleTitleOsmotic response in Lactobacillus casei ATCC393: biochemical and biophysical characteristics of membrane Archives of Biochemistry and Biophysics 422 61–70 Occurrence Handle14725858
S. Maicas (2001) ArticleTitleThe use of alternative technologies to develop malolactic fermentation in wine Applied Microbiology & Biotechnology 56 35–39
A.J. McLoughlin C.P. Champagne (1994) ArticleTitleImmobilized cells in meat fermentation CRC Critical Reviews in Biotechnology 14 179–192 Occurrence Handle8069934
F. Nazzaro R. Coppola M. Marotta L. Maurelli M. De Rosa F. Addeo (1999) ArticleTitleEffect of added carbohydrates on some dried lactic acid bacteria Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent 64 253–258
F. Nazzaro A. Sorrentino M. Malinconico P. Orlando (2001) ArticleTitleIn vitro studies on some probiotic properties of immobilized lactic acid bacteria after freezing and freeze-drying Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent Sect 2: Industrial Biotechnology and Biocatalysis 66/3a 307–312
J.C. Piard M. Desmazeaud (1992) ArticleTitleInhibiting factors produced by lactic acid bacteria. 2-Bacteriocins and other antimicrobial-substances Lait 72 113–130
H. Prevost C. Divies (1987) ArticleTitleFresh fermented cheese production with continuous prefermented milk by a mixed culture of mesophilic lactic streptococci entrapped in Ca-alginate Biotechnology Letters 9 789–794
H. Prevost C. Divies (1992) ArticleTitleCream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beads Biotechnology Letters 14 583–588
E. Selmer-Olsen S.E. Birkeland T. Sorgaug (1999) ArticleTitleEffect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydration Journal of Applied Microbiology 87 429–437 Occurrence Handle10540246
O. Smidsrod G. Skjak-Braek (1990) ArticleTitleAlginate as immobilization matrix for cells Trends in Biotechnology 8 71–78 Occurrence Handle1366500
I. Sodini C.Y. Boquien G. Corrieu C. Lacroix (1997) ArticleTitleMicrobial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous pre-fermentation of milk Enzyme and Microbial Technology 20 381–388 Occurrence Handle9084207
J. Visick S. Clark (1995) ArticleTitleRepair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins Molecular Microbiology 16 835–845 Occurrence Handle7476182
R.F. Vogel G. Bocker P. Stolz M. Ehrmann D. Fanta W. Ludwig B. Pot K. Kersters K.H. Schleifer W.P. Hammes (1994) ArticleTitleIdentification of lactobacilli from sourdough and description of Lactobacillus pontis sp. novis International Journal of Systematic Bacteriology 44 223–229 Occurrence Handle8186088
M.V. Yabannavar D.I.C. Wang (1991) ArticleTitleAnalysis of mass transfer for immobilized cells in an extractive lactic acid fermentation Biotechnology and Bioengineering 37 544–550
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Giulio, B.D., Orlando, P., Barba, G. et al. Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying. World J Microbiol Biotechnol 21, 739–746 (2005). https://doi.org/10.1007/s11274-004-4735-2
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DOI: https://doi.org/10.1007/s11274-004-4735-2