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Encapsulation of Lactobacillus Casei
Encapsulation of Lactobacillus Casei
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 relatively 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 studied. Among various cell immobilization
methods, entrapment 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 Caalginate capsule based on Nigam et al. l1 was developed;
however, Ca-alginate gels are chemically unstable on con-
Engineering Research Center, Korea Advanced Inst. of Science and Technology, 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
0141-0229/96/$15.00
PII SOl41-0229(96)00016-6
Immobilization of cells
Encapsulation.
Fe~e~tation
conditions
Analytical rnet~~ds
10
15
20
Time (h)
Figure 1 Comparison
of the lactic acid production by Caalginate entrapped and encapsulated L. casei cells during the
second and third batch of fermentations: second batch with capsules, 0; third batch with capsules, 0; second batch with beads,
q; and third batch with beads, W. Bars indicate standard deviation from the mean on the basis of four replicate experiments
429
Papers
the production rate by the beads (0.625 gl- h-l) significantly decreased (P < 0.05) compared with that by the capsules (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 comparison of the cell densities within both immobilized matrices 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 fermentation, while the number of viable cells remarkably decreased 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 concentration 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 capsule membrane. In the gel-core beads formed by the conventional 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 interphasic 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 fermentations were almost the same even though the rate of
3
%
M
zB
r^
g
0.6
00
F
u
f
=
8
0.4
28
0.2
Time (h)
Figure 3
10
100
80
430
Enzyme Microb.
Technol.,
Encapsulation
of Lactobacillus
0.0
0
Time (h)
Figure 4
431
Papers
100
80
0.6
ti)
i?
1s
60
.L(
Y
*a
8
ha
s
L
0.4
40
c7
20
20
0
80;
4
OIOO
0
t
10
15
20
25
30
LO
15
20
25
Time(h)
Figure 5 Time courses of lactic acid production by chitosanand 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, respectively. 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 immobilized 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 maintained 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
432
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 Caalginate 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 instead of Ca*+ and a mixture of chitosan and BaCl, as the
hardening solution for the preformed Ba-alginate capsules.
References
I.
Bibal, B., Vayssier, Y., Goma, G., and Pareiileux. A. Highconcentration cultivation of L.4acrococcus cremons in a ceil-recycle
reactor. Biotechnol. Bioeng. 1991, 37,14&754
Norton, S., Lacroix, C., and Vuillemard, J.-C. Kinetic study of continuous whey permeate fermentation by immobilized Lmrobacillus
helveticus for lactic acid production.
Enzyme Microb. Technol.
1994, 16,457-m
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