Effect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydration

Authors


E. Selmer-Olsen, Department of Food Science, Agricultural University of Norway, PO Box 5036, N-1432 Ås, Norway (e-mail: eirik.selmer-olsen@inf.nlh.no).

Abstract

When lactic acid bacteria are used industrially as fermentation starters it is important to obtain stable and highly viable bacterial cultures. Six strains of Lactobacillus encapsulated in Ca-alginate gel beads were investigated to determine whether dehydration, storage and rehydration may inflict injury. A negative relationship between leakage of lactate dehydrogenase and survival rates was found. Mesophilic lactobacilli showed only negligible leakage compared with thermophilic strains when dehydrated at 30 °C to a level of 0·11 g H20 (g dry wt)−1. The choice of an appropriate suspending medium to be introduced before drying was therefore very important for thermophilic lactobacilli in order to increase the survival rates during dehydration, storage and rehydration. The osmoregulatory solutes tested were adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS). Less injury was inflected during dehydration for Lactobacillus helveticus with adonitol, glycerol and NFMS. Survival rates for the strains subjected to immobilization, dehydration, storage and rehydration varied with the strain and the protective solute when fluidized-bed drying was used at 5 °C to a level as high as 0·34 g H20 (g dry wt)−1. Non-fat milk solids gave the best protection for thermophilic lactobacilli, while adonitol and NFMS were best for mesophilic lactobacilli.

Introduction

The lactobacilli are a diverse group of micro-organisms of widespread use in food and feed fermentations. Lyophilization, involving both freezing and drying, or drying is frequently used to preserve lactic acid bacterial (LAB) starter cultures. Maximum survival of LAB starters during the drying process and subsequent storage is of vital importance technologically and economically.

When LAB are dried in Ca-alginate gel beads together with protective agents, the cells may benefit both from the agent and from the protective bead micro-environment. The ionotropic gelation of alginate with divalent cations, such as calcium, is a procedure that is compatible with most bioactive materials (Thu et al. 1996). The procedure used in the present work for immobilization with drying and rehydration of Ca-alginate gel beads is very good for retaining the quality and properties of the beads.

Defence mechanisms to cope with different stresses appear to be a prerequisite in all organisms. The study of stress response in LAB has started only recently (Rallu et al. 1996). The tolerance of bacteria to stresses varies with growth conditions and further treatment. Injury may be produced by a variety of stressful conditions; thus immobilization, dehydration and rehydration are stress conditions involving several factors (Sørhaug 1992). Information on the sites of interference and nature of these phenomena together with knowledge of the induction of stress proteins, particularly those which provide resistance against drying, can be important to produce encapsulated, dried starter cultures with high survival rates even after prolonged storage.

Freezing of bacterial cells in the presence of suitable cryoprotectants has been shown to result in a minimum loss of viability which contrasts with freeze-drying involving the removal of water which reduces the viability of cells (Castro et al. 1997). The major factors of injury from drying of bacterial cells are probably osmotic shock with membrane damage and the removal of bound water which affects the properties of many hydrophilic macromolecules in cells. Thus, retaining critical levels of bound water, perhaps by adding a suitable hydrophilic additive(s), should be practised when drying LAB starters (Ray 1993).

The nature of the membrane changes during drying may involve adjustments in the unsaturated:saturated fatty acid ratio, an increase in membrane permeability to intracellular enzymes, a decrease in the activity of the membrane-bound enzyme ATPase, changes in the structure of sensitive proteins and a reduced capability to maintain ΔpH across the cell membrane (Castro et al. 1997; Lievense and van’t Riet 1994). Cell membranes are in a gel phase at room temperature and undergo a transition from gel to liquid crystal phase upon dehydration. As the membranes pass through this phase transition there may be regions with packing defects, making the membranes leaky. Adding a protective solution before drying lowers the transition temperature (Tm) of the dry membranes by replacing the water between the lipid headgroups, preventing the phase transition and its accompanying leakage upon rehydration (Leslie et al. 1995; Lievense and van’t Riet 1994; Lou et al. 1994).

Among several others, adonitol, betaine, glycerol and skim milk have been reported as efficient suspending agents for freeze-drying or convective drying of bacterial cells (Sinha et al. 1974; Valdéz et al. 1983a, 1983b, 1985a; Barbour & Priest 1986; Kearney et al. 1990; Selmer-Olsen et al. 1996).

When the external osmolarity becomes high, bacteria can compensate for the increased inward pressure by transporting solutes into the cell (Archer 1996). Work with LAB has shown that survival of cultures subjected to drying was considerably enhanced when betaine, for example, was accumulated by the cells. Betaine was also found in these bacteria when subjected to salt stress (Kets et al. 1996).

The present work aimed to study the expression of leakage and cell injury and the efficiency of some agents in protecting immobilized LAB during dehydration, storage and rehydration.

Materials and methods

Organisms, media and cultivation

The homofermentative or facultatively heterofermentative LAB used were Lactobacillus helveticus CNRZ 303 (Centre National de Recherches Zootechniques, Jouy-en-Josas, France), Lact. delbrückii ssp. bulgaricus-12 (CHR HANSEN, Hørsholm, Denmark), Lact. helveticus INF-II, Lact. plantarum INF-9a, Lact. casei ssp. casei INF-15d and Lact. casei ssp. pseudocasei INF-13i (Department of Food Science, Agricultural University of Norway, Ås, Norway). Stock cultures in MRS broth (Difco, Detroit, MI, USA) supplemented with 15% (v/v) glycerol were kept at −80 °C. Growth of Lact. helveticus and Lact. delbrückii ssp. bulgaricus was at 39 °C while the other lactobacilli were grown at 30 °C; the inoculation rate was 1% (v/v) of a culture in exponential phase transferred three times every 24 h before use. The lactobacilli were all grown in MRS in a 2-l batch fermenter at 30 and 39 °C, respectively. pH was maintained above 6·0 by a Radiometer Titrator system (Radiometer, ETS822 system, Copenhagen, Denmark) with 1·5 n NaOH and stirring at 250 rev min−1.

All cultures were harvested in early stationary phase by centrifugation at 10 000 g for 5 min at <5 °C. Cells were washed twice with Ringer solution (quarter-strength; Oxoid, Basingstoke, UK) and resuspended in Ringer solution (quarter-strength).

Cell immobilization

The cells in suspension were mixed with an equal amount of previously heat-treated (80 °C, 1 min) and cooled 4% (w/v) Na-alginate solution (Protanal, LF 10/60; Pronova Biopolymer A/S, Drammen, Norway) to a final concentration of 20 g dry cell mass l−1 2% (w/v) alginate solution. Ca-alginate gel beads were formed by the drop-wise addition of this mixture to a gently stirred 0·05 mol l−1 Ca-lactate solution, pH 6·9 and <5 °C, based on the procedure of Smidsrød & Skjåk-Brrk (1990). After stirring for 30 min, the beads were washed with sterile Ringer solution to remove excess calcium ions and free cells, transferred to Ringer solution and kept at 2–3 °C. The bead diameter was 3·0 ± 0·1 mm, determined according to Øyaas et al. (1995).

Expressions of cell injury

Lactate dehydrogenase (LDH) of the lactobacilli was used as an intracellular marker to evaluate the leakage through the cell envelope. The specific amount of LDH activity was determined by the method of McKay & Baldwin (1974). Total LDH activity was determined in the supernatant fluid after lysozyme treatment and sonication of whole cells according to Tobiassen et al. (1997).

Alginate beads were dehydrated in petri dishes by exposure to air (20% relative humidity) at 30 °C for 5 h to a level of 0·11 g H2O (g dry wt)−1 ± 0·01 (s.d.). The alginate beads in microtubes were subsequently stored at 2–3 °C above a saturated solution of LiCl (aw 0·12) in a sealed box for 7 or 28 d. Equal amounts of alginate beads were rehydrated in cheese whey (CW) permeate, pH 6·9, at 20 °C for 2 h prior to suspending beads in 0·2 mol l−1 NaH2PO4, pH 6·9, at 20 °C. Both the rehydration and suspending supernatant fluids were mixed and centrifuged in centrifugal concentrators (MICROSEPTM, 10K; Pall Filtron AB, Lund, Sweden) at 3000 g for 90 min at <5 °C (fixed angle rotor) to obtain detectable levels of enzyme activity.

Survival rates, expressed as colony-forming units (cfu), were determined with plate counts on both MRS agar (MRSA) and MRSOA containing 0·15% (w/v) oxgall (Difco) to assess sublethal cellular injury (King and Su 1993). Duplicate plates were prepared for each dilution and counted after 72 h incubation at, respectively, 30 and 39 °C for mesophilic and thermophilic lactobacilli. The difference in cfu on MRSA before and after drying, storage and rehydration represented cells which had died. The difference in cfu on MRSA and MRSOA was due to sublethally injured cells among the survivors.

Protective solutes

Ca-alginate gel beads containing cells were suspended in protective solutes, twice the volume of the beads, and kept on ice with mixing 10 times during 1 h prior to drying to allow for equilibration between cells and protective solutes. Protective solutes were added after gel formation to prevent leakage during the following residence time in the gelling bath. The following additives were incorporated and tested for their protective effect: 0·5 mol l−1 adonitol (Sigma, St. Louis, MO, USA), 0·5 mol l−1 betaine (glycine betaine; Sigma), 0·5 mol l−1 glycerol (BDH Laboratory Supplies, Poole, UK) and 10% (w/v) reconstituted non-fat milk solids (NFMS; Merck, Darmstadt, Germany). The pH was adjusted to 6·9 for all additives and the osmolality of the protective solutes was 534, 567, 531 and 267 mOsm for adonitol, betaine, glycerol and NFMS, respectively. The osmolality of the control Ringer solution (quarter-strength) was 107 mOsm. Osmolality was measured using an osmometer (The AdvancedTM Osmometer, Model 3D3; Advanced Instruments, Norwood, MA, USA). The solutions were heat treated at 80 °C for 1 min and cooled (2–3 °C) prior to use.

Dehydration, rehydration and storage

Ca-alginate beads containing cells were dried in a home-made laboratory fluidized-bed dryer. The simultaneous drying of four (five) separate samples was conducted under controlled air flow, temperature (5 °C) and relative humidity (55 ± 2·5%) of the entering air. The drying time was either 6 h, for Lact. helveticus CNRZ 303 to find survival as a function of overall moisture content (Fig. 1), or 3 h, for studying the influence of protective solutes during storage of all strains (Fig. 2). The storage of dehydrated beads was in ampoules of polypropylene in a glass flask at 2–3 °C.

Figure 1.

Residual survival of Ca-alginate-entrapped Lactobacillus helveticus CNRZ 303 with drying solutes after fluidized-bed drying (5 °C to a level of 0·15–0·25 g H2O (g dry wt)−1) as a function of overall moisture content. Drying solutes: ▪, Ringers; □, adonitol; ○, betaine; ▵, glycerol; ●, reconstituted non-fat milk solids

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Figure 2.

Figure 2.

The influence of Ringer solution (control for Lactobacillus helveticus CNRZ 303 (a)), adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS) on residual specific lactic acid production rate (qp/qp0) and residual survival (N/N0) of dried, stored and rehydrated Lactobacillus strains in Ca-alginate gel beads compared with qp0 and N0, activity and colony counts, respectively, for immobilized cells prior to drying. Entrapped cells in Ca-alginate gel beads were dehydrated in a fluidized-bed dryer (5 °C to a level of 0·34 g H2O (g dry wt)−1). (b) Lact. delbrückii subsp. bulgaricus-12; (c) Lact. helveticus INF-II; (d) Lact. plantarum INF-9a; (e) Lact. casei subsp. casei INF-15d; (f) Lact. casei subsp. pseudocasei INF-13i. □, Ringer (qp) (CNRZ 303); ▪, adonitol (qp); bsl00020, betaine (qp);bsl00021, glycerol (qp); bsl00024, NFMS (qp); –+–, Ringer (cfu) (CNRZ 303); —○—, adonitol (cfu); —▵—, glycerol (cfu); —□—, betaine (cfu);—×—, NFMS (cfu)

Rehydration was conducted at room temperature to allow cell repair. Ultrafiltered CW permeate, previously heat-treated at 80 °C for 1 min, was used and the pH was maintained at 6·9 by titration with 0·25 n NaOH. The rehydration was stopped when the gel bead volume was identical to freshly made beads. This was achieved within 2–3 h; however, this was somewhat dependent on the protective additive used. The gel bead volume was verified by determining the length of 30 beads giving the actual mean diameter of beads. The mechanical strength of the beads was measured by ‘back extrusion’ according to Steffe & Osario (1987).

Activity measurements and plate counts were made before and after drying and rehydration. Dehydration of each Lactobacillus strain was made in duplicate and representative results from experiments with nearly equal water content (±0·05 (s.d.)) are shown. The variation due to protectants is reflected in the residual water content (Fig. 1).

Activity and survival determination

The specific lactic acid-producing activity was determined by assessing the lactic acid production in CW permeate for a known amount of Ca-alginate gel beads (cell mass). The experiments were conducted in a stirred (500 rev min−1) batch reactor of 300 ml working volume, at a controlled temperature of 30 and 39 °C, respectively. The pH was maintained at 5·5 for all strains by titration with 0·25 n NaOH and activity was determined from the slope of the respective plots within the linear range of 2 h.

Survival rates, expressed as cfu, were determined with the plate count agar method by plating serial dilutions in duplicate on MRSA after dissolving the beads in 0·2 mol l−1 NaH2PO4, pH 6·9. Plates were incubated at 30 and 39 °C, respectively for 48 h.

To verify whether any of the strains grow in CW permeate and thus interfere with the results during the time of activity measurements, we compared incubations with and without chloramphenicol (5 μg ml−1) which blocks protein synthesis (Roy 1991).

Statistical analysis

The effect of protective solute and strain of Lactobacillus on activity expressed as specific lactic acid production rate was tested by analysis of variance (anova) based on the model:

image

where α and β are effects of the protective solute and strain of Lactobacillus and ɛ is the error term which is assumed to have a normal distribution, mean zero and variance σ2 (Montgomery 1991). Statistical analysis was performed by the SAS statistical program (SAS, Cary, NC, USA).

Results

Expression of leakage and cell injury

The intracellular marker, LDH, of the lactobacilli was assayed to evaluate the leakage through the cell envelope. The specific LDH activity of supernatant fluids relates, inversely, well with the viability of the cell population (Table 1, Fig. 3). The total LDH activities after lysozyme and ultrasound treatment of undried cells were determined as: 5790, 11 330, 3170, 5060, 5040 and 9290 dA (g dw cells·min)−1 for the respective lactobacilli as shown in Table 1.

Table 1. Lactobacillus strains were immobilized in Ca-alginate beads, dried, stored and rehydrated; the table shows the influence of these steps on survivors, injured cells among survivors and release of lactate dehydrogenase (LDH)
OrganismSurvivors
Treatment*
Survivors injured
(%)
Specific LDH activity
(%)
(dA (g dw cells min)−1)
  • *

    All cell samples were immobilized, dried and stored for respective times.

  • Ratio of number of viable counts after drying, storage and rehydration relative to number of viable counts of recently made Ca-alginate beads before drying. Table values are means of duplicate determinations.

  • Injured cells among survivors, i.e. per cent survivors injured by plate counts on MRSA with (MRSOA) and without oxgall (MRSA). Table values are means of duplicate determinations.

  • ND, Not determined.

Lact. helveticus CNRZ-303Prior to drying10077700
0 d storage 0·1681043
7 d storage<0·190939
30 d storage<0·187909
100 d storage<0·1NDND
Lact. delbrückii subsp. bulgaricus-12Prior to drying10094405
0 d storage<0·150959
7 d storage<0·1ND959
30 d storage<0·1501170
100 d storage<0·1NDND
Lact. helveticus INF-IIPrior to drying10090102
0 d storage 0·387329
7 d storage 0·179395
30 d storage 0·180417
100 d storage<0·1NDND
Lact. plantarum INF-9aPrior to drying100≈010
0 d storage315321
7 d storage92021
30 d storage5740
100 d storage8NDND
Lact. casei subsp. casei INF-15dPrior to drying100411
0 d storage14929
7 d storage191022
30 d storage10524
100 d storage12NDND
Lact. casei subsp. pseudocasei INF-13iPrior to drying100≈03
0 d storage4236
7 d storage4415
30 d storage4769
100 d storage45NDND
Figure 3.

Specific lactate dehydrogenase (LDH) activity in supernatant fluids of Ca-alginate-entrapped cells prior to drying and dried, stored and rehydrated cells with respective strains of lactobacilli presented in Table 1, as a function of their residual logarithmic colony count. ●, Lactobacillus helveticus CNRZ 303; □, Lact. delbrückii ssp. bulgaricus-12; ▵, Lact. helveticus INF-II; ×, Lact. plantarum INF-9a; ○, Lact. casei ssp. casei-15d; ▪, Lact. casei ssp. pseudocasei INF-13i. Mesophilic lactobacilli are supplemented in a minor figure. Entrapped cells in Ca-alginate gel beads were dehydrated in petri dishes by exposure to air (30 °C to a level of 0·10–0·12 g H2O (g dry wt)−1)

The leakage of total intracellular LDH during de- and rehydration was related, but in different ways, to the mesophilic and thermophilic lactobacilli (Fig. 3). Lysozyme treatment and sonication of the respective lactobacilli (Table 1) after dehydration, storage for 100 d and rehydration, gave LDH values (as a percentage of the total LDH activities of undried cells) as follows: 61, 101, 81, 108, 80 and 85%. This indicates either leakage and final cell death during rehydration or retained intracellular LDH activity during low temperature storage, even though the cells are injured or even dead. An interesting observation was the relationship between LDH activities observed in supernatant fluids after lysozyme treatment and LDH activities observed after sonication. For the thermophilic group stored for 100 d, 76–89% of activity was detected in the spheroplast buffer compared with 1–14% for undried cells. The corresponding change for the mesophilic group was 4–15% and 0·2–2%. The survival rate for the thermophilic group was < 0·1% while the mesophilic group was at the same level as observed for 28 d (Table 1). These results indicate marked differences between the two groups of lactobacilli for the chosen drying conditions (30 °C, 0·11 g H2O (g dry wt)−1). Experiments to express cell injury were performed twice and representative results showed that the fraction of injured cells among survivors was significantly higher for the thermophilic than for the mesophilic lactobacilli.

Effect of protective solutes

The detailed study of viability and water losses during dehydration of Lact. helveticus CNRZ 303 was expressed by plotting the residual colony counts as survivor cells (N/N0) vs water content (X g H2O (g dry weight)−1; Fig. 1). The viability varied with the protective solute used. Improved survival during dehydration was obtained with adonitol, glycerol and NFMS. The fall in viability with all solutes, was not uniform either regarding time (data not shown) or water loss. The results for the solute betaine showed less protection in Fig. 1 than in Fig. 2 at the same moisture content.

For the fluidized-bed drying of both thermophilic and mesophilic lactobacilli we chose a final water concentration just below the level for dehydration inactivation. This was done to study the effects of protective solutes during storage and at unfavourable conditions. The mean value for dried samples was 0·34 g H2O (g dry wt)−1 ± 0·05 (s.d.). Drying without protective solutes under these conditions gave negligible survival as shown with Lact. helveticus CNRZ 303 dried with Ringer solution (Fig. 2).

Activity measurements in the present study (Fig. 2) showed a significant effect between strains of Lactobacillus (P < 0·005) and between protective agents (P < 0·05). Considering the mesophilic lactobacilli and thermophilic lactobacilli as separate groups after storage for 28 d, adonitol and NFMS gave the best protection for mesophilic lactobacilli, while NFMS was optimal for thermophilic lactobacilli. Glycerol had less protective effect for thermophilic lactobacilli (Lact. helveticus CNRZ 303) during storage (Fig. 2) than during dehydration (Fig. 1).

Only CW permeate obtained after fermentative coagulation of milk, i.e. containing the chelating compound lactic acid, gave appropriate rehydration of Ca-alginate gel beads. The optimal recovery conditions for dried Lact. helveticus were rehydration at 20–30 °C and pH 6·5–7·0 (data not shown). The mechanical strength of the beads was measured by ‘back extrusion’ and was found to be slightly weaker than for the starting material (η1; 0·46 Pa·s vs 0·67 Pa·s).

A small increase in activity was observed within 2 h for rehydrated lactobacilli without addition of chloramphenicol, which suggests that cell repair has started. A comparison has been performed to verify whether there was a linear relation between survival and activity. The correlation coefficients (r) for the studied protective solutes adonitol, betaine, glycerol and NFMS were, respectively, 0·87, 0·66, 0·76 and 0·68 (n ≥ 16 for each protective solute).

Discussion

Expression of leakage and cell injury

Lou et al. (1994) measured the rate of DNA hydrolysis after dehydration of Lact. plantarum. By using a method where DNase diffuses into cells with injured cell envelopes and hydrolyses the intracellular DNA they showed that dehydration inactivation was associated with evidence of membrane damage.

We assumed that varying degrees of membrane damage might be associated with differences in survival between the strains. Different growth optima for the two strains of Lact. helveticus and Lact. delbrückii ssp. bulgaricus compared with the other strains may relate to different melting points of the phospholipids in the membranes. Based on this assumption, the drying of lactobacilli can have the potential to be more successfully conducted at temperatures as high as that for optimum growth. This point was substantiated by Valdéz et al. (1985b) who showed that rehydration at 0 °C gave less survival for freeze-dried Lact. helveticus than rehydration at higher temperatures for other lactobacilli. However, when protective agents are used at such temperatures the effect of the metabolic activity of the cell and the subsequent pH reduction are factors to consider.

In Figs 1 and 2, it can be seen that the survival of thermophilic Lact. helveticus CNRZ 303 was nearly zero at low temperatures without protective agents. Therefore, we chose 30 °C, in an attempt to reduce membrane changes during dehydration.

Significantly more LDH leaked out during lysozyme treatment of whole dried cells compared with undried thermophilic lactobacilli. The fraction of sublethally injured cells among the survivors was higher for thermophilic than for mesophilic lactobacilli which was confirmed by results on leakage and cell injury. Cytoplasmic membrane damage and release of LDH from injured cells could occur during dehydration, rehydration or through autolysis of cells. The protective effect of agents tested (particularly for strains Lact. helveticus and Lact. delbrückii ssp. bulgaricus) appears to be accompanied by less membrane injury as indicated also by the low values of the LDH analyses.

Effect of protective solutes

Adonitol, which cannot be metabolized by most lactobacilli, has shown a protective effect during freeze-drying by controlling the water content (Valdéz et al. 1985a; Kearney et al. 1990). This may be due to the steric conformations of its hydroxyl groups which may replace water molecules in protein structures (Valdéz et al. 1983b).

Lactic acid bacteria confronted with a lowered aw (osmotic stress) over a long period respond by accumulation of compatible solutes such as betaine and carnitine (Hutkins et al. 1987; Kets et al. 1996). A strain of Lactococcus lactis ssp. lactis contained large pools of proline or glycine betaine as a result of specific transport, when grown under conditions of high osmotic strength (Molenaar et al. 1993). The quaternary amine N,N,N-trimethylglycine (betaine) has been reported to be an important osmoprotective molecule in several groups of Gram-negative bacteria (Cairney et al. 1985; Perroud & Le Rudulier 1985; Hutkins et al. 1987). Betaine is a metabolically inert compound, has no net charge, does not inhibit cytosolic enzymes and, because of its dipolar nature, no counterion accumulation is necessary to maintain electroneutrality (Hutkins et al. 1987; Kashket 1987).

Glycerol has the ability to maintain the available water at the optimal level for the cell during dehydration (Lievense and van’t Riet 1994).

Skim milk is expected to prevent cellular injury by stabilizing the cell membrane constituents (Valdéz et al. 1983b; Kearney et al. 1990; Castro et al. 1995). Calcium in milk increases survival after either freezing or freeze-drying (King & Su 1993). Linders (1996) found that some carbohydrates had the ability to minimize the dehydration inactivation of Lact. plantarum during fluidized-bed drying.

Summing up, the different protectants have high water-binding potentials together with other mechanisms for stabilizing proteins as well as mechanisms to stabilize membrane structures by hydrogen bonding with the phosphate of the phospholipid when water is removed and replaced by sugar molecules, for example.

Low air temperatures should help to avoid thermal inactivation and keep the metabolic activity at a low level. This was the reason for choosing 0–5 °C during preparation for drying and storage. The Ca-alginate beads were quite shrunk after dehydration and the diffusion of oxygen was thought to be limited. Further, storage stability increases with decreasing temperature (Lievense and van’t Riet 1994).

The osmolarity of the rehydration solution, the pH, rehydration temperature and rehydration volume are all factors expected to have some influence on survival rates (Mäyrä-Mäkinen & Bigret 1993). An appropriate nutritional energy source in the rehydration medium may also be important for cell recovery.

Concerning the survival rate, the time interval between rehydration with repair (recovery) in a rich medium and measuring can influence the result. Some divergence was observed between survival, as determined by cfu, and rate of lactic acid production (Fig. 2). Injured cells can repair during plate incubation (MRSA) (Lievense and van’t Riet 1994; Sørhaug 1992). However, only a limited time of 2 h was available for repair during activity measurements. The plate count method can also be misleading, since disaggregation of the chains of bacteria during the dissolving of beads can differ and influence the apparent numbers of survivors. In contrast to the drying conditions chosen for expression of cell injury, both fluidized-bed drying temperature and level of water content (Fig. 2) were not optimal, as indicated by cell leakage and survival during storage. The effect of protective solutes was shown under the unfavourable conditions chosen. In fact, suspension of Lactobacillus strains encapsulated in alginate beads in the presence of respective protective solutes appeared to be a possible strategy for a short period of storage with potential for further survival improvements.

It is important to be aware that the best protective agents during dehydration are not necessarily optimal for protection during storage of the cells (Crowe et al. 1987). Nevertheless, this was the situation for the solutes used in this study (Figs 1 and 2).

The choice of optimal water content for storage of cells is dependent on whether the goal is high survival rates immediately after drying or a low inactivation rate during storage.

The lactobacilli are probably not able to accumulate compatible solutes during a short drying process and therefore such compounds should be accumulated prior to drying, either by simple uptake of added agents or by activating the accumulation mechanisms.

Acknowledgements

The financial support from the Norwegian Research Council and TINE Norwegian Dairies BA is gratefully acknowledged.

Ancillary