ELSEVIER

Encapsulation of Lactobacillus casei

cells in liquid-core alginate capsules
for lactic acid production
Ik-Keun Yoo,* Gi Hun Seong ,* Ho Nam Chang,* and Joong Kon Park
*Department of Chemical Engineering and BioProcess Engineering Research Center, Korea
Advanced institute of Science and Technology (EXI ST), Taejon, Korea Department of Chemical
Engineering, Kyungpook National University, Taegu, Korea
The immobilization method of Lactobacillus casei cells was investigated using alginate capsules that possess an
interphosic membrane and a liquid core. The capsules were found to offer more space for cellular growth than
gel-core beads, which resulted in I.5-fold higher cell concentration than in the latter; however, the Ca-alginate
structure was unstable during repeated batch fermentations for lactic acid production. Ba-alginate capsules were
chemically and physically more stable than the Ca-alginate capsules in phosphate and lactate solutions. Attempts
were also made to use various hardening agents to stabilize the structure of the Ba-alginate capsules. It was
found that the treatment with a mixture of chitosan and BaCl, solution gave the best results for hardening.
Finally, stable lactic acid production was possible with a productivity of more than 2.7 g l-h- by L. casei cells
immobilized in chitosan-coated Ba-alginate capsules. The cell leakage from the capsules was maintained rela-
tively low during repeated batch fermentations.
Keywords: Lactic acid; barium-alginate capsules; encapsulation; alginate gel stability; Lactobucillus casei
Introduction
Given the low productivity of batch processes for lactic acid
production, recent research has focused on increasing the
cell concentration in the reactor.14 Cell immobilization is
one of the most attractive methods in maintaining high cell
concentration in the reactor and has been extensively stud-
ied. Among various cell immobilization methods, entrap-
ment in Ca-alginate beads has commonly been used for
immobilization of lactic acid bacteria.4-9 On the other hand,
encapsulation of cells in a liquid-core capsule which offers
more space for cellular growth than entrapment will be a
good method for a high density culture. In the previous
work of our group, encapsulation of yeast cells in a Ca-
alginate capsule based on Nigam et al. l1 was developed;
however, Ca-alginate gels are chemically unstable on con-
Address reprint requests to Dr. Ho Nam Chang, Director, Bioprocess En-
gineering Research Center, Korea Advanced Inst. of Science and Technol-
ogy, Daeduk Science Town, Taejon 305-701, South Korea
Received 29 June 1995; revised 27 November 1995; accepted 11 January
1996
Enzyme and Microbial Technology 18: 0 1996 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
tact with various cation-chelating agents such as phosphate,
citrate, and lactate which can cause bead disruption or dis-
solution.* Many hardening techniques have been investi-
gated to improve the stability of Ca-alginate gels9,* but
most procedures are complicated, time consuming, and/or
costly. Recently, it was reported that barium alginate beads
were chemically and physically more stable in electrolyte
solutions than conventional calcium alginate beads.13,14
The aim of this investigation was to compare the method
of entrapment and encapsulation of Lactobacillus casei cells
in Ca-alginate gels. Also, different metal ions other than
calcium and various coating agents were tested to improve
the stability of alginate gel in a lactate solution. Finally, the
production of lactic acid with chemically stabilized alginate
capsules containing L. casei cells was studied.
Materials and methods
Materials
Sodium alginate (Na-alginate) was obtained from Junsei (Tokyo,
Japan, Cat. No. 13035-1201). Xanthan gum (Cat. No. G1253),
chitosan (Cat. No. C3646), and polyethyleneimine (PEI, Cat. No.
P3143) were supplied by Sigma (St. Louis, MO, U.S.). Yeast
extract and MRS medium were from Difco (Detroit, MI, U.S.). All
other chemicals were of reagent grade quality.
Enzyme and Microbial Technology 19:426-433, 1996
0 1996 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
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PII SOl41-0229(96)00016-6
Encapsulation of Lactabacillus casei cells in liquid-core: I.-K. Yoo et al.
~~crooorga~ism and substrate
L. cusei ssp. r~~nosus (ATCC 10863) was used throughout this
investigation, The strain was maintained at 4C on MRS-agar. The
production medium consisted of 40 ml MRS broth or GY medium
containing per liter: 1 g Na-acetate * 3H,O; 0.5 g K,HPO,; 0.5 g
K&PO,; 0.2 g MgSO, - 7H,O; 0.03 g MnSO, - H,O; and 0.03 g
FeSO, * 7H,O. Glucose, yeast extract, and CaCo, concentrations
were variable.
Immobilization of cells
Encapsulation. Cells of L. casei for encapsulation were obtained
from culture grown in 50 ml of MRS broth at 42C for 24 h. The
cells were harvested from the fe~entation broth by cen~~gation
at 10,000 g for 10 min and washed thoroughly with 0.85% saline.
The cells were resuspended in 10 ml of a 1.0% (w/v) sterile CaCl,
or BaCl, solution containing 0.15% (w/v) xanthan gum. This cell
suspension with a density of about 0.4 g dry wt. 1-l was dropped
through a 22G bluntly ended needle into 130 ml of sterile 0.6%
(w/v) Na-alginate and 0.1% (v/v) Tween 20 solution stirred by a
magnetic bar. Xanthan gum and Tween 20 were used for making
clean spherical capsules. The schematic diagram of the experimen-
tal apparatus was the same as that described in our previous
work. The capsules formed were washed with sterile saline to
remove excess sodium alginate. The capsules were resuspended in
various coating and hardening solutions stirred with a magnetic bar
for 30 min. A 1.0% CaCl, solution was used for hardening of
Ca-alginate capsules. The chitosan solution for the Ba-alginate
capsule coating was prepared as described by Yoshioka et a1.l5
The coating solutions were discarded and the resulting alginate
capsules (3-3.5 mm diameter) were washed with sterile saline.
Entrapment. Harvested cells (0.04 g dry weight) were mixed with
100 ml of a 1 .O% sterile sodium alginate solution. The mixture was
added dropwise into a sterile 1.0% CaCl, solution at room tem-
perature while stirring it continuously. The beads were hardened in
this solution for 1 h. The beads (2-2.5 mm diameter) were then
washed with sterile saline prior to use to remove excess calcium
ions and untrapped cells.
Fe~e~tation conditions
A 50 ml sample of immobilized cells prepared above was first
grown in 150 ml MRS broth at 42C for 24 h. Batch fermentations
were then performed in 250-ml Erlenmeyer flasks containing 40
ml of medium and 15-40 ml of alginate beads or capsules with
immobilized cells. For the cultures grown in GY medium, 2.5-5 g
of CaCO, was added to the flasks to prevent significant decrease
in pH by produced lactic acid. The flasks were incubated at 42C
in a shaking incubator (Vision Scientific Co., Seoul, Korea) ad-
justed to 100 pm. After each batch fermentation, beads or cap-
sules were washed two times with fresh medium for the next batch
culture.
Analytical rnet~~ds
Free cell concentrations were determined by measuring the optical
density (OD) of the fermentation broth at 620 mn using a standard
curve to correlate OD and dry cell weight. For the cultures grown
in GY medium, the optical density was measured after removing
CaCO, particles with 1 M HCl. The cell density within Ca-alginate
beads or capsules was determined after dissolving the beads or
capsules in McIkaine buffer (0.2 M Na$POd, 0.1 M citric acid).
The number of viable cells liberated from the Ca-alginate gels was
obtained by plating appropriate dilutions on MRS agar and incu-
bating them at 42C for 36 h. In the case of Ba-alginate capsules,
capsules were placed in a 0.5 M EDTA solution with continuous
stirring and then broken by pinching, since the McIlvaine buffer
was not effective for liquefying Ba-alginate capsules. After cells
were completely released from the ruptured capsules, cell density
inside the capsules was determined by measuring the optical den-
sity at 620 nm. The lactic acid concentration was measured by
HPLC using an Aminex HPX-87H column (Bio-Rad Co., Rich-
mond, VA, U.S.) and a refractive index detector (Hitachi L-6000.
Tokyo, Japan). Glucose concentration was determined by enzy-
matic assay kits of Glucose-E kits (Youngdong Pharm. Co.. Seoul.
Korea).
Results are the average of at least three independent trials.
Statistical analyses on the data were carried out by using Duncans
new multiple range test.
Results and discussion
Comparison of encapsulation and entrapment for
lactic acid production
Lactic acid production by encapsulated cells was compared
with that by entrapped cells using Ca-alginate gels. Figure
1 shows the lactic acid production during the second and
third batch of fermentations in 40 ml of MRS broth con-
taining 0.5 g 1-l CaCI, and 15 ml of alginate beads or
capsules with immobilized cells. The addition of CaCl, was
done to keep the capsules from swelling as reported in our
previous work. As shown in Figure I , the Ca-alginate
beads showed a little higher but not statistically significant
(P > 0.05) rate of lactic acid Production (1.9 1 g 1-l h-) than
that by the capsules (1.75 gl- h-) during the first 7 h of the
second batch; however, during the 20 h of the third batch,
0
0 5 10 15 20
Time (h)
Figure 1 Comparison of the lactic acid production by Ca-
alginate entrapped and encapsulated L. casei cells during the
second and third batch of fermentations: second batch with cap-
sules, 0; third batch with capsules, 0; second batch with beads,
q ; and third batch with beads, W. Bars indicate standard devia-
tion from the mean on the basis of four replicate experiments
Enzyme Microb. Technot., 1996, vol. 19, November 1 429
Papers
the production rate by the beads (0.625 gl- h-l) signifi-
cantly decreased (P < 0.05) compared with that by the cap-
sules (0.81 g l-h-l). As reported earlier,1037 this could be
due to the limitation in increasing biomass per unit volume
of the conventional gel-core beads. Figure 2 shows the com-
parison of the cell densities within both immobilized ma-
trices during repeated batch fermentations in terms of dry
cell weight per liter of gels and the number of viable cells
per milliliter of gels. As can be seen, there was no increase
in dry cell weight within beads during the third batch fer-
mentation, while the number of viable cells remarkably de-
creased compared with that after the second batch. The cell
density inside the Ca-alginate capsules after the third batch
was about 65 g dry wt 1-l gels, which was 1.5-fold higher
than that of the beads. Until the third batch, the free cell
concentrations were less than 0.15 g dry wt 1-l in both
immobilized matrices. In the fourth batch, serious leakage
of cells was observed in the beads and the free cell concen-
tration was more than 0.8 g dry wt 1-l while significant cell
leakage was not observed in the capsules yet (0.2 g dry wt
1-l). The latter may be due to the barrier effect of the cap-
sule membrane. In the gel-core beads formed by the con-
ventional method, cells are homogeneously distributed both
near the surface of and inside the beads at the stage of bead
formation. During fermentation, the cells inside the beads
grow poorer than near the surface of beads due to mass
transfer limitation. The cells may then be released into the
surrounding medium once the matrix space near the surface
of beads has been occupied. On the other hand, the inter-
phasic membrane of the capsule can act as a barrier to retain
microbial cells inside the capsule.
The fermentation using a GY medium containing 50 g 1-l
glucose and 15 g 1-l yeast extract was carried out with 15 ml
of Ca-alginate capsules. As shown in Figure 3a, the final
concentrations of lactic acid produced in three repeated fer-
mentations were almost the same even though the rate of
100
3
%
M
z 80
B
10
0 1 2 3
Repeated batch No.
Figure 2 Comparison of the cell density within Ca-alginate
capsules and beads during repeated batch fermentations: im-
mobilized cells within capsules, 0; viable cells within capsules,
0; immobilized cells within beads, Cl; and viable cells within
beads, H
r^
g 0.6
F
u
f 0.4
=
8
8 2 0.2
t
00
Time (h)
Figure 3 Time profiles of lactic acid production (a) and cell
leakage (b) during repeated batch fermentations by Ca-alginate-
encapsulated cells: (symbols are for graph a, graph b). First
batch (0, 0); second batch (Cl, n ); and third batch (A, A). The
standard deviation from the mean is based on three replicate
experiments
lactic acid production in the third batch was slightly slower
than the previous ones. During fermentations with MRS
medium, the addition of 0.5 g 1-l CaCl, could prevent the
capsules from swelling and also cell leakage from the cap-
sules to some extent. On the other hand, the free cell density
in GY medium containing 0.5 g 1-i CaCl, even after the
third batch significantly increased compared to that after the
second batch (P < 0.05) as shown in Figure 3b; therefore, it
could be concluded that a higher concentration of lactic acid
brought about a decrease in the stability of Ca-alginate
structure.
Enhancement of capsule stability in lactate solution
Although there have been numerous reports on the $J;%;
tion of lactic acid with Ca-alginate entrapped cells,
the problem of cell leakage was not solved yet due to the
decalcification of Ca-alginate beads by lactic acid.6V8 We
first investigated the stability of alginate capsules gelled
with various metal ions besides Ca2+ in a high concentration
of phosphate and lactate solution. Among the tested metal
ions such as Ba*+, Fe3+, Mn2+, Zn+, and Ca*+, only the
430 Enzyme Microb. Technol., 1996, vol. 19, November 1
Encapsulation of Lactobacillus casei ceils in liquid-core: I.-K. Yoo et al.
b~um-gelled aiginate capsules remained stable for more
than 100 h at a concentration of 0.1 M phosphate and 0.5 M
Na-lactate solution (pH 6 at 42C). The capsules gelled
with other ions swelled with time and eventually disinte-
grated in less than 3 h. When making Ca-alginate beads, it
has been a common procedure to harden the preformed
beads in a CaCl, solution for a while. In this study, various
coating agents were investigated for the hardening solution
of the preformed barium alginate capsules in the hopes that
the gel stability would be more enhanced. Based on previ-
ous reports, 2y20,2 the following solutions were prepared for
a hardening solution of the preformed Ba-alginate capsules:
1.0% (w/v) BaCl, solution; 0.5% BaCl, and 0.5% gelatin
solution; 0.5% BaCl, and 0.5% PEI solution; 0.5% BaCI,
and 0.5% chitosan solution; and 0.1 M Al(NO,), solution.
After treatment with the above hardening solutions for 30
min, the Ba-alginate capsules were placed in the solution of
0.1 M phosphate and 0.5 M Na-lactate for 100 h. The gela-
tin-or Al(NO,),-hardened capsules were easily crushed be-
tween two fingers compared to the other capsules; therefore,
the Ba-alginate capsules treated with the other hardening
solutions such as BaCl,, PEI, and chitosan were selected for
encapsulation of L. casei cells.
As shown in Figure 4, the lactic acid production and the
leakage of cells using 25 ml of Ba-alginate capsules were
compared with the Ca-alginate ones. In these fermentations,
CaCl, was not added to the MRS medium, so we observed
a rem~kable increase in free cell density as the Ca-alginate
capsules with immobilized cells were used for four repeated
batches. Treatment of Ba-alginate capsules with PEI as pro-
posed by Veliky and WilliamszO was ineffective to avoid
cell leakage and caused severe shrinkage of the capsules. As
can be seen. the free cell ~oncen~ations in the Ba-alginate
capsules treated with 1% BaCl, solution were not signifi-
cantly different (P > 0.05) from those in the capsules treated
with a mixture of 0.5% chitosan and 0.5% BaCl,; however,
the capsules treated with a mixture of chitosan and BaCl,
were a little stronger between two fingers than the capsules
treated with only BaCl, solution. Consequently, a mixture
of 0.5% chitosan and 0.5% BaCl, was used for hardening
solution in the following fermentations. The cell densities
inside the capsules after the fourth batch were in the range
of 70-80 g dry wt I- gels.
Lactic acid production by immobilized L. casei cells
in alginate capsule
Batch fe~entations were performed using a GY medium
containing 30 g 1-l yeast extract and supplemented to con-
tain 40, 70, and 100 g I- of glucose. Figure 5a shows the
time courses of lactic acid production by 25 ml of Ba-
alginate capsules with immobilized cells treated with a mix-
ture of 0.5% chitosan and 0.5% BaCl,. There was little
difference in the rates of lactic acid production between the
fermentations with different initial glucose concentrations.
During the first 12.5 h of fermentation, the productivities of
lactic acid were 2.9, 3.3, and 3.1 g I-h-l in the medium
with 40, 70, and 100 g 1-l of glucose, respectively. The
highest concentration of lactic acid (98.1 g 1-l) was obtained
in the medium with 100 g 1-l glucose; no residual glucose
was found after 32 h of fermentation. Nomura et al. re-
0.0
0 4 8
Time (h)
Figure 4 Time profiles of lactic acid production (a) and ceils
leakage (b) during the fourth batch of fermentation by alginate
capsules treated with various hardening agents (symbols are for
graph a, graph b). Ca-alginate capsules (0, W; Ba-alginate cap-
sules hardened by a BaCI, solution FY, VI; Ba-alginate capsules
hardened by a mixture of PEI and BaCI, (A, A); and Ba-alginate
capsules hardened by a mixture of chitosan and BaCI, (0, m).
The standard deviation from the mean is based on three repli-
cate experiments
ported that the m~imum condensation of lactic acid (80 g
I-) was obtained by L. delbrueckii entrapped in Ca-alginate
beads after 120 h of fermentation while Roukas and
Kotzekidou reported that the maximum concentration of
lactic acid (41.3 g 1-l) was observed with coimmobilized L.
casei and L. luctis cells in Ca-alginate beads after 48 h of
fermentation.
A yeast extract concentration varied from 5-30 g 1-l in a
GY medium containing 70 g 1-l of glucose. As shown in
Figure 5b, the rate of lactic acid production by encapsulated
cells increased concomi~~y with the increase of yeast ex-
tract concentration. For instance, the productivities of lactic
acid during the first 9.5 h of fermentation were 2.6, 3.3, and
3.5 g I-h- in the medium with 5, 15, and 30 g 1-i of yeast
extract, respectively. This difference seems to be due to a
higher concen~ation of yeast extract resulting in a higher
growth rate of cells inside the capsules. At the end of fer-
mentations, cell mass concentrations inside the capsules
reached approximately 70, 100, and 110 g dry wt I- gels in
Enzyme Microb. Technol., 1996, vol. 19, November 1 431
Papers
100
80
ti) 60
.L(
Y
*a
8
40
ha
20
0
80; 5 10 15 20 25 30
;
0 5 LO 15 20 25
Time(h)
Figure 5 Time courses of lactic acid production by chitosan-
and BaCI,-coated Ba-alginate encapsulated ceils in media with
different initial glucose (a) and yeast extract (bi concentrations
(g I-): G 40 and YE 30,O; G 70 and YE 30, Cl; GIOO and YE 30,
A; G 70 and YE 15, g; and G 70 and YE 5, 0 where glucose is G
and yeast extract is YE
the medium with 5, 15, and 30 g 1-l of yeast extract, re-
spectively. Guoqiang et ~1.~ reported that the productivity of
lactic acid by L. casei immobilized in Ca-alginate beads
was 1.6 g l-h-l in the medium with 10 g 1-l yeast extract.
Repeated batch fermentations were carried out five times
successively using 40 ml of Ba-alginate capsules with im-
mobilized cells. As shown in Figure 6, the conversions of
glucose to lactic acid were in the similar range besides the
fifth batch when the fermentation medium was replaced
with a fresh one every 24 h. Until the fourth batch, the
productivity of lactic acid during 24 h of fermentation was
more than 2.7 g l-h-i and no residual glucose was found;
however, in the fifth batch, the pr~uctivity was less than
2.6 g I-h- and the concentration of residual glucose was
about 2 g I-. Cell leakage from the capsules was main-
tained relatively low during repeated fermentations; the
concentration of free cells after five batches was less than
0.28 g dry wt 1-l. By contrast, the concentration of free cells
in the fermentation broth using Ca-alginate capsules with
0
3
Y
c7
20
~
t 4
i
0.6 s
L
1
i?
u
0.4 s
OIOO
0 1 2 3 4 5
Repeated batch No.
Figure 6 Lactic acid production (0) and cells leakage (0) during
repeated batch fermentations by ~hitosan-coated Ba-alginate
encapsulated cells in GY media containing 70 g I- glucose and
15 g I- yeast extract. Recycling took place every 24 h
immobilized cells was more than 0.55 g dry wt 1-i even
after three batches as shown in Figure 3b. Boyaval and
Goulet6 reported that serious cell leakage due to the decal-
cification of Ca-alginate beads was observed during con-
tinuous lactic acid fe~en~tion by L. ~e~vetic~~ entrapped
in Ca-alginate beads. Champagne et al9 found that a double
coating of poly-r,-lysine and alginate reduced cell leakage
by a factor of approximately 50 during milk fermentation by
Luctococcus la& immobilized in Ca-alginate beads.
Conclusion
The entrapment in conventions Ca-alginate beads has been
a popular method for immobilization of lactic acid bacteria;
however, it seems that there are two disadvantages for lactic
acid production using immobilized cells in Ca-alginate
beads. One is the limitation of space for cellular growth due
to gel-core structure and the other is an instability of Ca-
alginate structure due to the decalcification of Ca-alginate
beads by lactic acid. In this study, the encapsulation method
in alginate capsules was used for immobilization of L,. casei
cells. It was found that the encapsulation method gave
higher cell density and lactic acid productivity compared to
the entrapment. The stability of alginate capsules was also
improved using Ba2+ as the gelling agent for alginate in-
stead of Ca*+ and a mixture of chitosan and BaCl, as the
hardening solution for the preformed Ba-alginate capsules.
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