APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1990, p. 3112-3116 Vol. 56, No.

10
0099-2240/90/103112-05$02.00/0
Copyright X3 1990, American Society for Microbiology

Enhancing the Viability of Lactobacillus plantarum Inoculum by

Immobilizing the Cells in Calcium-Alginate Beads
Incorporating Cryoprotectants
LOUISE KEARNEY,* MARY UPTON, AND AIDEN Mc LOUGHLIN
Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, Ireland
Received 25 March 1990/Accepted 18 July 1990

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in
determining viability. The rate of rehydration and the volume of fluid used have been identified as two
important factors. One possible means of controlling these is by immobilizing the cells before lyophilization
within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the
properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate
beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The
properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The
beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size
decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the
microenvironment within the two bead types was probably different. The protective effect of the bead
microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the
alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during
rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the
immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.

Lyophilization is frequently used to preserve lactic acid
bacterial starter cultures involved in dairy and food fermen-
tations. Microbial cell survival during the lyophilization
procedure is dependent on many factors, including the strain
of organism, the age of the culture, growth conditions, and
the lyophilization conditions. The subsequent rehydration of
lyophilized cultures can also have effects on microbial
viability. Research by Leach and Scott (12) illustrated how
the rehydration fluid, its water activity, and the rate of its
addition determined cell survival during rehydration. De
Valdez et al. (7) also reported the importance of rehydration
fluid volume on viability. Thus, control of the rehydration
environment is crucial in insuring adequate survival rates.
One possible method of achieving this could be by lyophi-
lizing the cells in Ca-alginate beads, in which subsequent
rehydration would be in the controlled protective microen-
vironment of the bead. The importance of the bead micro-
environment in determining the properties of the immobi-
lized cells was studied by Mattiasson and Hahn-Hagerdal
(13). They proposed a model to suggest that the low water
activity and/or oxygen deficiency of the bead microenviron-
ment was responsible for the altered properties of the
immobilized cells.
Fages (8) found that Azotospirillum lipoferum survived
well during lyophilization and subsequent rehydration in
immobilized beads of Ca-alginate when certain cyroprotec-
tive agents were added. De Valdez et al. (5) reported 0.75 M
adonitol with skim milk and 1 M glycerol with skim milk to
be useful suspending media for insuring the survival of
Lactobacillus plantarum in free cell culture during lyo-
philization. When L. plantarum is lyophilized in Ca-alginate
beads with these cryoprotectants, the cells may also benefit
from the protective bead microenvironment.
*
Corresponding author.
3112
Work by de Valdez et al. (6) showed the rate of moisture
loss during lyophilization and the resultant residual moisture
content to be dependent on the nature of the cryoprotectant.
It has already been established that lyophilization and rehy-
dration of Ca-alginate beads alters their physical properties.
Cheetham (3) reported on the incomplete rehydration of
alginate beads after lyophilization. Drying of beads was
found by Klein et al. (10) and Klein and Wagner (11) to
increase their mechanical strength. Since the composition of
the cryoprotectant influences the residual moisture content,
their incorporation into Ca-alginate beads to protect the cells
during lyophilization could also modify the physical proper-
ties of these beads and hence the microenvironment to which
the immobilized cells are exposed. Thus, the bead size and
mechanical strength were determined in beads incorporating
0.75 M adonitol with skim milk and 1 M glycerol with skim
milk.
To evaluate the protective effect of the alginate bead with
the cryoprotectants on cell survival during rehydration, free
and immobilized cultures lyophilized with both cryopro-
tectants were rehydrated with fluids at different pHs and
with fluids incorporating salts, and the viabilities were com-
pared. A range of pH values and salt solutions were exam-
ined, since starter culture rehydration often occurs in such
unfavorable conditions.
MATERIALS AND METHODS
Immobilization procedure. The immobilization procedure
used was based on that of Bashan (1). L. plantarum ATCC
8014 was grown in MRS broth (Oxoid Ltd.) (4) at 30°C for 18
h and then harvested by centrifugation for 20 min at 10,000
rpm in an RC2-B Sorvall refrigerated centrifuge. Cells from
50 ml of the broth were suspended in 50 ml of the cryopro-
tective agents formulated to have a final concentration of 1
M glycerol (M&B Chemicals) with 10% skim milk (Oxoid) or
0.75 M adonitol (BDH Chemicals) with 10% skim milk and
VOL. 56, 1990 IMMOBILIZED L. PLANTARUM WITH ENHANCED VIABILITY 3113

TABLE 1. Physical properties of Ca-alginate beads incorporating 1 M glycerol and skim milk or 0.75 M adonitol and skim milk
before and after lyophilization and rehydration
Before lyophilization After lyophilization and rehydration
Cryoprotectant Diameter Strength Diameter Strength
Shape (mm) (N/MM2) Shape (mm) (N/mm2)

1 M glycerol + skim milk Spherical 2.09 (± 0.31)" 0.38 (± 0.05) Spherical 1.19 (± 0.19) 2.04 (± 0.04)
0.75 M adonitol + skim milk Spherical 2.43 (± 0.27) 0.24 (± 0.04) Irregular 1.75 (± 0.71) 0.63 (± 0.02)
a Numerical results are given ± standard deviations.

then mixed for 30 min with 50 ml of a sterile alginate solution
(BDH) at a final concentration of 2%. Beads were then
formed by the dropwise addition of this solution to a gently
stirred 0.1 M calcium chloride solution (BDH). Equimolar
concentrations of the corresponding polyol were included in
the calcium chloride solution to eliminate its diffusion from
the Ca-alginate beads. After curing for 2 h in the salt and
polyol solution, the beads were lyophilized on an Edwards
freeze-drier (model EF03). The primary drying was carried
out at -60°C for 3 h, during which the pressure was reduced
to 10-1 torr. The ampules were then attached to a secondary
drying manifold, dried overnight, and then sealed under
vacuum.
The various physical properties of the beads and cell
viability were examined within hours of completing lyoph-
ilization. Any storage of beads was at 4°C.
Preparation of free cell cultures. L. plantarum cells were
grown, centrifuged, and suspended in the cryoprotectants as
above. The suspended cells were dispensed into tubes and
lyophilized in the same manner as the immobilized cells.
100
Bead size determination. Before and after lyophilization
and rehydration in distilled water, alginate beads incorporat-
ing the cryoprotectants were measured by direct micro-
scopic observation with a x4 lens. For each set, 40 beads
were examined.
Mechanical strength of alginate beads. The mechanical
strength before and after lyophilization and rehydration in
distilled water was determined by using a JJ Tensile Testing
Machine, type T500. The load cell had a force of 4 N, the
speed was 5 mm/min, and the paper/cross head ratio was
25:1. Results were plotted on a JJ XY Plotter, type PL200.
For each set 12 beads were tested.
Immobilized and free cell survival rates. Survival after
lyophilization of the free and immobilized cells with 1 M
glycerol and skim milk or 0.75 M adonitol and skim milk as
cryoprotectants was evaluated by using the standard plate
count on MRS agar. After incubation for 48 h at 300C, all
colonies were of uniform size and were counted. The free
cells were suspended in 0.5 ml of Ringer solution (Oxoid),
110

100

80

90

60 80

a ZI
*.
U)*. 70
40

60

20
50
Fl Immobilized cells
+ Free cells

I I I I
l I I
Il l
I I I I I
I I f
6 7 8 2 3 4 5 6 7
2 3 4 5

pH
pH
FIG. 1. Rehydration of free and immobilized L. plantarum cul- FIG. 2. Rehydration of free and immobilized L. plantarum cul-
tures with water at different pH values after lyophilization with 1 M tures with water at different pH values after lyophilization with 0.75
glycerol and skim milk as a cryoprotectant. M adonitol and skim milk as a cryoprotectant.
3114 KEARNEY ET AL.
and the immobilized cells were suspended in 1 ml of Ringer
solution per bead. Before counting, the alginate beads were
dissolved in 0.2 M potassium phosphate buffer (pH 6.8).
Rehydration fluids. Free and immobilized L. plantarum
cells were rehydrated in various fluids after lyophilization
with either cryoprotectant. Tolerance to pH was evaluated
by rehydration in distilled water adjusted to pH 7.0, 6.5, 6.0,
5.5, 5.0, 4.5, 4.0, 3.5, and 3.0 with lactic acid. The salts
examined were 3% NaCl, 0.0156% NaNO2, and 3% NaCl-
0.0156% NaNO2; the pH values of these solutions were 6.55,
6.8, and 6.55, respectively. Free cells were rehydrated in 0.5
ml of fluid, and the immobilized cells were rehydrated in a
volume of 1 ml per bead. Rehydration was carried out at
30°C for 10 min as recommended by de Valdez et al. (7) for
lactic acid bacteria. Viability was determined by standard
plate counting on MRS agar. Before counting, the alginate
beads were dissolved in 0.2 M potassium phosphate buffer
(pH 6.8).
RESULTS
Ca-alginate beads incorporating 1 M glycerol with skim
milk and 0.75 M adonitol with skim milk had different shapes
and sizes (Table 1). Before lyophilization, beads with both
cryoprotectants were spherical, but those with adonitol were
slightly larger. After lyophilization and rehydration, beads
incorporating glycerol retained their spherical shape, but
their diameter was reduced by 43%. In comparison, the
beads containing adonitol had a less uniform shape, with the
average diameter reduced by 30%. These decreases in bead
diameter resulted in a more compact structure with a higher
concentration of alginate per unit area and hence an altered
microenvironment.
The effect of the increased concentration of alginate per
unit area after rehydrating the lyophilized beads is reflected
in the increase in the mechanical strength of the beads (Table
1). Beads that incorporated glycerol while decreasing in size
by 43% after lyophilization required a fivefold increase in the
mechanical force required to disrupt them. In comparison,
beads containing adonitol exhibiting a decrease in size of
30% had their mechanical strength increased only 2.6 times.
Hence, the nature of the cryoprotectant also affects bead
structure, as reflected by its influence on the resistance to
mechanical force.
The survival rates of free and immobilized cells after
lyophilization are shown in Table 2. The data presented are
the results of triplicate experiments. Lyophilizing L. plan-
tarum in alginate beads in the absence of polyols resulted in
survival rates lower than those of the free cell culture.
However, when the polyols were incorporated, the survival
rates of the immobilized cultures surpassed that of the free
cells. The highest survival rates were obtained when 0.75 M
adonitol with skim milk was used as the cryoprotectant.
To establish whether an immobilized environment confers
additional protection to cells during rehydration with subop-
timal regeneration fluids, lyophilized free and immobilized
TABLE 2. Survival of free and immobilized L. plantarum
cultures during lyophilization
t% Survival (± SD)
Cryoprotectant
Free cells Immobilized cells
10% skim milk 8.4 (± 3.2) 1.1 (± 0.5)
1 M glycerol + skim milk 36.1 (± 4.3) 44.7 (± 3.6)
0.75 M adonitol + skim milk 64.6 (± 3.6) 85.9 (± 4.3)
APPL. ENVIRON. MICROBIOL.
50
s=2.50
40 _
30
a) s=1.26
20
10
o
H20 NaCI NaNO2 NaCI +
NaNO2
(pH 7.0) (pH 6.6) (pH 6.8) (pH 6.6)
salt
* Immobilized cells
* Free cells
a = standard deviation
FIG. 3. Survival after rehydration of L. plantarum cultures in
salt solutions for 10 min at 30°C after lyophilization with 1 M
glycerol and skim milk as a cryoprotectant.
cultures were rehydrated with fluids at various pH values
and salt contents. The data presented for these studies are
the result of triplicate experiments.
Figure 1 shows the effect of pH on the viability during
rehydration of cultures with 1 M glycerol with skim milk as
the cryoprotectant. At all pH values examined the immobi-
lized cultures had higher survival rates. The optimum pH for
rehydrating L. plantarum was 4.5. At this pH the immobi-
lized cells exhibited total viability retention. This increase of
over 50% in viability from that at neutral pH reflects the
acidophilic nature of this organism. In comparison, decreas-
ing the pH from 7.0 to 4.5 increased the free cell survival by
only 15%.
Rehydration of lyophilized cultures with 0.75 M adonitol
and skim milk as the cryoprotectant in fluids with adjusted
pH values also revealed immobilized cells to have higher
survival rates (Fig. 2). Again the pH optimum was found to
be 4.5. At this point the immobilized cells exhibited no
significant cell loss, whereas the free cells incurred a mor-
tality rate in excess of 20%. At pH values higher than the
optimum, the survival rates of both free and immobilized
cells surpassed those of comparable cultures with glycerol.
Rehydrating lyophilized L. plantarum cultures incorporat-
ing 1 M glycerol with skim milk in salt solutions reduced the
survival rates of both free and immobilized cultures (Fig. 3).
Although the immobilized cultures showed similar sensitiv-
ity to 3% NaCl and 0.0156% NaNO2 individually, the free
cells were more inhibited by the NaCl. Combined, the salts
had a more severe effect on both culture types, reducing the
viability of the immobilized cells by 21 to 23% and that of the
free cells by 17 to 18%.
VOL. 56, 1990
100
ss3.44
80
s-4.26
60
8)
82
40
20
0
HF N NaCi NaN02
(piH 7.0) (pH 6.6) (pH 6.8)
sal
* Immobilized cells
* Free cells
= standard deviation
a
FIG. 4. Survival after rehydration of L. plantarum cultures in
salt solutions for 10 min at 30°C after lyophilization with 0.75 M
adonitol and skim milk as a cryoprotectant.
When lyophilized cultures with 0.75 M adonitol and skim
milk were rehydrated with salt solutions (Fig. 4), both free
and immobilized cells showed greater sensitivity to 3% NaCl
than to 0.0156% NaNO2. Combining the salts further re-
duced viability in both cultures. On a comparative basis the
salts had a more deleterious effect on the free cells than on
the immobilized ones, reducing the viability of the former by
41% and of the latter by 15%. However, both free and
immobilized cultures with adonitol were more resistant to
salts than were the comparable cultures with glycerol.
DISCUSSION
The physical properties of the Ca-alginate beads were
dependent on the nature of the incorporated cryoprotectant.
After lyophilization, beads with both cryoprotectants exhib-
ited decreases in size, but to a greater extent in those
incorporating glycerol. The resultant higher alginate concen-
tration per unit volume in the beads with glycerol led to the
expected higher mechanical strength. The irregular shape of
the beads incorporating 0.75 M adonitol and skim milk after
lyophilization could also be indicative of inherent differences
in the Ca-alginate structure when this cryoprotectant was
used in comparison with glycerol and skim milk. Casas et al.
(2) showed the physical properties of carageenan carob-bean
gum beads to be dependent on the nature of the incorporated
polyol. The results presented here show that this also
appears to be true for Ca-alginate beads. The skim milk
component could also have an effect on the alginate struc-
ture due to its high protein content. Research by Tanaka et
al. (14) led them to suggest that the Ca-alginate structure
may be altered by the incorporation of various substances
IMMOBILIZED L. PLANTARUM WITH ENHANCED VIABILITY 3115
such as proteins, whereas Imeson et al. (9) have shown
direct modifications resulting from protein-alginate interac-
tions at various pH values. These differences in the physical
properties reflect differences in the microenvironment of the
two bead types, which could have an influence on the
immobilized cells.
The importance of the rehydration environment on cell
it-2 21 survival is reflected by the higher survival rates of immobi-
lized cells as compared with those of free cell cultures when
polyols were in the lyophilization medium. Therefore algi-
nate beads combined with the polyols do confer additional
protection during lyophilization and rehydration.
Research by Leach and Scott (12) and de Valdez et al. (7)
showed that the rate and volume of rehydration fluid used
can have a large effect on cell viability of lyophilized
cultures, possibly by protecting the cells from osmotic
shock. In cells immobilized in alginate beads, rehydration is
not instantaneous; the rate and volume of fluid involved are
controlled by the diffusion properties and limited volume of
the alginate bead, and thus osmotic shock is avoided.
The nature of the cryoprotectants also influences the
NaCI+ survival of the immobilized cells during rehydration with the
NaN02 various fluids. Incorporating adonitol rendered the cells
(pH 6.6) more resistant to neutral pH values than if glycerol was
used. However, at the pH optimum of 4.5, immobilized
cultures with both cryoprotectants exhibited no significant
losses, thus confirming that the rehydration process plays a
key role in determining cell viability of cultures that have
been lyophilized. The influence of the cryoprotectants on
tolerance to salts also shows that adonitol exerts a greater
protective effect. Thus during rehydration with suboptimal
fluids the modifications conferred by adonitol on the alginate
bead microenvironment appear to be more conducive to cell
survival.
It can thus be concluded that Ca-alginate beads incorpo-
rating different polyols have different physical properties,
and that the microenvironment created by the polyols with
skim milk within the bead increases the cell survival rate.
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