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

SummaryIn 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.

[1]  H. Ertesvåg,et al.  Biosynthesis and applications of alginates , 1998 .

[2]  R. Coppola,et al.  Characterization of lactobacilli involved in the ripening of soppressata molisana, a typical southern Italy fermented sausage , 1998 .

[3]  C. Diviès,et al.  Cream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beads , 1992, Biotechnology Letters.

[4]  Ana Rodríguez,et al.  Influence 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 , 2000 .

[5]  L. Morelli,et al.  Development 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 , 1998, Journal of applied microbiology.

[6]  C. Diviès,et al.  Fresh fermented cheese production with continuous pre-fermented milk by a mixed culture of mesophilic lactic streptococci entrapped in Ca-Al ginate , 1987, Biotechnology Letters.

[7]  C. Champagne,et al.  The production of freeze‐dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milk , 2000, Journal of applied microbiology.

[8]  J. Piard,et al.  Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances , 1992 .

[9]  D. Juang,et al.  The applicability of the API 20E and API Rapid NFT systems for the identification of bacteria from activated sludge , 2001 .

[10]  J. Crowe,et al.  Preservation of freeze-dried liposomes by trehalose. , 1985, Archives of biochemistry and biophysics.

[11]  E. Cortón,et al.  Characterization of Lactobacillus Carbohydrate Fermentation Activity Using Immobilized Cell Technique , 2000, Biotechnology progress.

[12]  G. Jan,et al.  In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices , 2005 .

[13]  Z. Hubálek,et al.  Protectants used in the cryopreservation of microorganisms. , 2003, Cryobiology.

[14]  J. Crowe,et al.  Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose , 1984, Science.

[15]  G. Fitzgerald,et al.  Food fermentations: role of microorganisms in food production and preservation. , 1999, International journal of food microbiology.

[16]  K. Draget,et al.  Alginate based new materials. , 1997, International journal of biological macromolecules.

[17]  C. Champagne,et al.  Immobilized cells in meat fermentation. , 1994, Critical reviews in biotechnology.

[18]  C. Lacroix,et al.  Batch fermentation with entrapped growing cells of Lactobacillus casei. Optimization of the rheological properties of the entrapment gel matrix. , 1990 .

[19]  D. I. Wang,et al.  Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentation , 1991, Biotechnology and bioengineering.

[20]  J H Crowe,et al.  Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying , 1995, Applied and environmental microbiology.

[21]  K. Schleifer,et al.  Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov. , 1994, International journal of systematic bacteriology.

[22]  D. Poncelet,et al.  Immobilization of cells for application in the food industry. , 1994, Critical reviews in biotechnology.

[23]  W. Verstraete,et al.  The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). , 1999, International journal of food microbiology.

[24]  Koide K. Iwamoto.  Alginate as immobilization matrix for cells , 1990 .

[25]  G. Valdez,et al.  Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage , 1998 .

[26]  C. Colaco,et al.  Chemistry of Protein Stabilization by Trehalose. , 1995 .

[27]  B. Spargo,et al.  Interactions of sugars with membranes. , 1988, Biochimica et biophysica acta.

[28]  C. Lacroix,et al.  Immobilized cell technologies for the dairy industry. , 1994, Critical reviews in biotechnology.

[29]  G. Jan,et al.  Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii , 2001, Applied and Environmental Microbiology.

[30]  J E Visick,et al.  Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins , 1995, Molecular microbiology.

[31]  G. Corrieu,et al.  Microbial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous prefermentation of milk. , 1997, Enzyme and microbial technology.

[32]  C. Champagne,et al.  The effect of protective ingredients on the survival of immobilized cells of Streptococcus thermophilus to air and freeze-drying , 2001 .

[33]  Christophe Lacroix,et al.  Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel , 1988, Applied Microbiology and Biotechnology.

[34]  S. Birkeland,et al.  Effect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydration , 1999, Journal of applied microbiology.

[35]  S. Maicas.  The use of alternative technologies to develop malolactic fermentation in wine , 2001, Applied Microbiology and Biotechnology.

[36]  H. Rehm,et al.  Fatty acid impurities in alginate influence the phenol tolerance of immobilized Escherichia coli , 2004, Applied Microbiology and Biotechnology.

[37]  H. G. Trüper,et al.  Survival of Escherichia coli during drying and storage in the presence of compatible solutes , 1994, Applied Microbiology and Biotechnology.

[38]  E. Rivas,et al.  Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane. , 2004, Archives of biochemistry and biophysics.