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Applied and Environmental Microbiology, February 2000, p. 869-873, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Survival of Bifidobacterium longum
Immobilized in Calcium Alginate Beads in Simulated Gastric
Juices and Bile Salt Solution
Ki-Yong
Lee and
Tae-Ryeon
Heo*
Department of Biological Engineering, Inha
University, Inchon, Korea
Received 3 May 1999/Accepted 10 November 1999
 |
ABSTRACT |
Bifidobacterium longum KCTC 3128 and HLC 3742 were
independently immobilized (entrapped) in calcium alginate beads
containing 2, 3, and 4% sodium alginate. When the bifidobacteria
entrapped in calcium alginate beads were exposed to simulated gastric
juices and a bile salt solution, the death rate of the cells in the
beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads
affected the numbers of survivors after exposure to these solutions;
however, the death rates of the viable cells were not affected.
Accordingly, a mathematical model was formulated which expressed the
influences of several parameters (gel concentration, bead size, and
initial cell numbers) on the survival of entrapped bifidobacteria after
sequential exposure to simulated gastric juices followed by a bile salt
solution. The model proposed in this paper may be useful for estimating
the survival of bifidobacteria in beads and establishing optimal
entrapment conditions.
| |
TEXT |
It has been reported that the
microencapsulation of bifidobacteria can ensure greater survival in
gastric and intestinal environments (18). Immobilized cells
exhibit many advantages over free cells, including the maintenance of
stable and active biocatalysts, high volumetric productivity, improved
process control, the protection of cells against damage, and reduced
susceptibility to contamination (12, 19). Recently, yogurt
products containing encapsulated lactic acid bacteria have been
distributed under the brand name Doctor-Capsule (Bingrae Co.,
Kyunggi-do, Korea) in Korea. Among the available techniques for
immobilizing living cells, entrapment in Ca alginate beads has been
frequently used for the immobilization of lactic acid bacteria
(22). Alginate has the benefits of being nontoxic to the
cells being immobilized, and it is an accepted food additive
(17). This study used mathematical modelling to represent
the influences of alginate concentration, bead size, and initial cell
numbers on the survival rate of entrapped bifidobacteria against
simulated gastric juices and bile salt solution.
Entrapment of bifidobacteria.
Bifidobacterium
longum KCTC 3128 (ATCC 15707) was purchased in lyophilized
form from the Korea Collection for Type Cultures (Genetic Resource
Center, Taejon, Korea). B. longum HLC 3742 was screened from
feces from a healthy Korean (14). These two types of
bacteria were transferred twice in Trypticase-protease peptone-yeast extract (TPY) broth at 37°C. Cultivation was carried out in a 2.5-liter fermentor containing 1,000 ml of TPY broth. After cultivation for 20 h, cultures were collected by centrifugation
(3,000 × g, 10 min), washed, and resuspended in a
0.85% NaCl saline solution to approximately 109 cells/ml.
Sodium alginate (medium viscosity) obtained from Sigma Chemical Co. was
used in this study. Various amounts of sodium alginate (5, 7.5, and
10 g) were autoclaved at 121°C for 15 min in powder and then
individually dissolved in 250-ml resuspended cell solutions in aseptic
vinyl bags. The solutions were thoroughly mixed with a Stomacher 400 (Seward Co., London, United Kingdom) laboratory blender. By using
aseptic and compressed air, filtered by autoclaved 5-, 1-, and
0.22-µm-pore-size filters sequentially set in the compressor, the
cell-alginate mixtures were ejected dropwise through a 20-gauge nozzle
into a 0.1 M CaCl2 solution in a clean bench. Large,
medium, and small beads (mean diameters, about 1.03, 1.75, and 2.62 mm,
respectively) containing 2, 3, and 4% sodium alginate were obtained by
controlling the compressed air pressure through a 20-gauge nozzle. The
beads were made to be spherical and were prepared to be relatively
uniform in size. The total viable numbers of untrapped bifidobacteria
expressed as CFU were determined by the plate count method using TPY
agar. To count the viable cell numbers in beads, 100 particles were washed with a sterile saline solution and dissolved for 10 min in 30 ml
of a sterile 0.1 M sodium citrate solution with the aid of a Stomacher
400 (Seward Co.). The cultivation of bifidobacteria on a TPY agar plate
was done anaerobically under N2 (75%), H2 (10%), and CO2 (5%) at 37°C for 48 h.
Survival of bifidobacteria in simulated gastric juices.
Simulated gastric juices without pepsin (0.08 M HCl containing 0.2%
NaCl [pH 1.55]) were prepared by Rao et al. (18). One hundred single beads in each of six cap tubes containing 10 ml of
simulated gastric juices were incubated anaerobically at 37°C for
3 h. At an interval of 30 min during incubation, all the beads from one cap tube sample were harvested, washed with physiological saline, and immediately assayed for cell enumeration. To prepare samples for untrapped bifidobacteria, 10-ml volumes of cultures were
centrifuged (10 min, 3,000 × g). The pellets were
resuspended in a 0.85% saline solution and collected by
centrifugation. The supernatants were discarded, and 10 ml of simulated
gastric juices was added to each cap tube containing the recovered
cells. The cells were then lightly agitated, incubated at 37°C for
the same time as described for the entrapped cells, and finally assayed for cell enumeration. Figure 1 shows that
the log of the surviving cells decreased proportionally with the time
that the entrapped bifidobacteria were exposed to simulated gastric
juices. The death rate (slope of line) of B. longum KCTC
3128 and HLC 3742 entrapped in the beads decreased proportionally with
increased bead size and alginate concentration (Fig. 1A and B). The
viable cell numbers of untrapped B. longum KCTC 3128 and HLC
3742 rapidly decreased from 1.28 × 109 and 1.18 × 109 CFU/ml, respectively, to below 1 × 103 CFU/ml within 30 min. Therefore, the survival of
entrapped bifidobacteria was higher than that of untrapped cells. In
the presence of simulated gastric juices, the change of viable cell
numbers in a bead (death rate) is given by
dNg/dtg, where
Ng is the log viable cell numbers and
tg is the exposure time. Assuming that the beads
have a constant cell density, the log viable cell numbers of
bifidobacteria in a bead can be given by the following equation:
|
(1)
|
where Ngo is the log viable cell numbers in
a bead before exposure to simulated gastric juices. As shown in Fig.
1C, the absolute value of the cell death rate in beads decreased
proportionally with increased alginate concentration in the beads.
Therefore, the change in cell death rate in a bead after exposure to
simulated gastric juices related to the change in alginate
concentration can be expressed by:
|
(2)
|
where C is the alginate concentration of a bead and
g is the change in the cell death rate in a
bead in relation to a change in alginate concentration of beads. In
Fig. 1D, the value of g increased
proportionally with increased bead size. Therefore, the change in
g related to the change in bead size can be
written as:
|
(3)
|
where S is the bead size and
g is the change in
g in relation to a change in bead size.
Accordingly, by using equations 1, 2, and 3, the viable cell numbers in
a bead with a certain alginate concentration and bead size in the
presence of simulated gastric juices can be expressed as follows:
|
(4)
|
where gso is the slope of the cell death
rate when S is zero, and
(dNg/dtg)co
is the change in viable cell numbers in beads when C is
zero. These three values, including g, were
calculated from the experimental data. From the results shown in Fig.
1C, we know that
(dNg/dtg)co was not a constant and increased proportionally with the increase in
bead size (data not shown). This change is expressed by:
|
(5)
|
where 
g is the change in
(dNg/dtg)co
in relation to change in bead size. Equation 5 can be integrated as
follows:
|
(6)
|
where
(dNg/dtg)coso
is
(dNg/dtg)co
when S is zero. Therefore, equation 4 can be expressed as
follows:
|
(7)
|
To examine the effect of the initial cell loading in the beads on
the survival of bifidobacteria, three 2.6-mm-diameter beads containing
3% alginate and different initial cell loads (approximately 108, 109, and 1010 cells/ml) were
also tested by the same methods as described above. As expected, the
death rates of the viable cells in all three different kinds of beads
had the same values (data not shown). Accordingly, the initial cell
numbers did not affect the death rates of the viable cells.
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FIG. 1.
Survival of entrapped bifidobacteria after exposure to
simulated gastric juices at 37°C for 3 h. (A) Survival of
B. longum KCTC 3128; (B) Survival of B. longum
HLC 3742; (C) death rate of bifidobacteria in beads; (D) change in
death rate of bifidobacteria in beads in relation to the change in bead
size. Symbols:  and  , 2% alginate beads;  and  , 3%
alginate beads;  and  , 4% alginate beads;
···, small beads;  · , medium
beads; , large beads;  , death rate of B. longum KCTC
3128;  , death rate of B. longum HLC 3742;
··· ···,
change in death rate of B. longum KCTC 3128;
- -,
change in death rate of B. longum HLC 3742;
- -, untrapped B. longum
KCTC 3128; - -, untrapped B. longum HLC 3742.
|
|
Survival of bifidobacteria in a bile salt solution.
To
determine the effect of bile salts on bifidobacteria survival, 100 single beads were separately put into each of three cap tubes
containing 10 ml of a bile salt solution dissolved in 0.6% oxgall
(Difco Co.) which had been sterilized by autoclaving at 121°C for 15 min and then incubated anaerobically at 37°C for 6 h. The
influences of the entrapment parameters on the survival of the
bifidobacteria exposed to bile salt solution were tested by the same
methods as described for the simulated gastric juices. As expected, the
log of the surviving cells in beads decreased proportionally with the
exposed time as shown in Fig. 2. The
alginate concentration had a mild effect on cell viability (Fig. 2A and B). However, the absolute value of the cell death rate in the beads
decreased proportionally with increased alginate gel concentration and
bead size (Fig. 2C). As with the pattern for survival of simulated gastric juices, the change in cell death rate in a bead after exposure
to bile salt solution in relation to the change in alginate concentration (b) increased proportionally
with increased bead size (data not shown). Therefore, the survival of
entrapped bifidobacteria in bile salt solution can be also expressed in the same manner with equation 7 and can be defined as follows:
|
(8)
|
where
Nb is the log viable cell number in a
bead with a certain alginate concentration and bead size in the
presence of a
bile salt solution and
tb is the
exposure time to a bile salt
solution. Also,
b is the change in cell death rate in
a bead
after exposure to a bile salt solution in relation to a
change in
alginate concentration, and
b is the change
in
b in relation to change in bead size.
b is the slope of
(
dNb/dtb)
co
in relation to change in
bead size.
b,
bso,
b,
Co, and
Nbo were all
calculated by using the experimental data.
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FIG. 2.
Survival of entrapped bifidobacteria after exposure to
bile salt solution at 37°C for 6 h. (A) Survival of B. longum KCTC 3128; (B) Survival of B. longum HLC 3742;
(C) death rate of bifidobacteria in beads. Symbols: and , 2%
alginate beads; and , 3% alginate beads; and , 4%
alginate beads; ···, small beads; · , medium
beads; , large beads; , death rate of B. longum KCTC
3128; , death rate of B. longum HLC 3742;
--, untrapped B. longum
KCTC 3128; --, untrapped B. longum HLC 3742.
|
|
However, in the case where some entrapped bifidobacteria are
sequentially exposed to simulated gastric juices and a bile salt
solution, the log viable cell numbers in a bead before exposure
to a
bile salt solution in equation 8 (
Nbo) are the
same as the
log viable cell numbers in a bead after exposure to
simulated
gastric juices in equation 7 (
Ng).
Therefore,
S, the bead size
of equation 8, can be mismatched
with the intrinsic bead size.
The reduction in actual bead volume
occupied by survivors with
the exposure time to simulated gastric
juices, given by
dV/dtg,
is proportional to
the death rate of log viable cell numbers in
a bead, where
V
is the bead volume occupied by viable cells,
tg is the exposure time to simulated gastric juices, and
is the
cell
density of a bead:
|
(9)
|
Assuming that a bead has a complete spherical shape, it can be
represented by:
|
(10)
|
where
R is the radius of a bead. Accordingly, the
substitution of equation 10 into equation 9 estimates the change in
viable
cell numbers in the beads. That is:
|
(11)
|
Thus, equation 11 can be integrated to give a bead radius as a
function of time, and it can be rearranged by:
|
(12)
|
where
Ro is the initial radius of a bead
before exposure to simulated gastric juices. Thus, the viable cell
numbers in a
calcium alginate bead after sequential exposure to
simulated gastric
juices followed by a bile salt solution can be
expressed by using
equations 7, 8, and 12. That is:
|
(13)
|
where
S' is the actual bead size occupied by viable
cell numbers after exposure to simulated gastric juices, that is,
2R.
One hundred single beads, 3% alginate beads with
2.62-mm diameters,
were put separately into each of six cap tubes
containing 10 ml
of simulated gastric juice, and the cap tubes were
incubated anaerobically
at 37°C for 3 h. Every 1 h during
incubation, all the beads from
one cap tube sample were harvested,
washed with physiological
saline, and immediately assayed for cell
enumeration. After 3
h, the solutions in the remaining three cap
tubes were changed
from simulated gastric juice to 10 ml of a bile salt
solution
and then incubation was continued for 6 h. Figure
3 shows that
the number of survivors
after sequential exposure to simulated
gastric juices and a bile salt
solution estimated from equation
13 (
Ngb) is not
in disagreement with the measured data; therefore,
this equation has
credibility. However, the experimental data
were slightly lower than
the calculated data, and there was some
scatter in the measured data.
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FIG. 3.
Theoretical and experimental survival of bifidobacteria
entrapped in 3% alginate beads containing three different initial cell
numbers after sequential exposure to simulated gastric juices for
3 h followed by bile salt solution for 6 h. The lines were
calculated by using equation 13. The points marked by symbols ( and
) are experimental data. Symbols: , the viable cell numbers of
B. longum KCTC 3128; , the viable cell numbers of
B. longum HLC 3742.
|
|
The influences of the entrapment parameters (alginate concentration,
bead size, and initial cell number) on the survival of
the
bifidobacteria were quantitatively and systematically determined.
Although some authors have studied the survival of bifidobacteria
and
lactic acid bacteria in the presence of gastric and/or intestinal
juices (
2,
5,
15,
16), this is the first report, as
far as
is known, to present a mathematical quantification of the
survival of
entrapped bifidobacteria when sequentially exposed
to simulated gastric
juices and a bile salt solution. The mathematical
quantification
described above was made possible by introducing
a new entrapping
procedure whereby alginate gel, containing bifidobacteria,
was prepared
by the addition of sterilized sodium alginate powder
to a suspended
cell solution. Prior to the experiment described
above, another
experiment had been conducted by the conventional
entrapping procedures
(
4,
13,
17), with a cell suspension
added to a sterile
sodium alginate solution. However, the experimental
results obtained
from the conventional entrapping procedures did
not provide sufficient
information to study the survival characteristics
of bifidobacteria
entrapped in calcium alginate beads in simulated
gastric juices and
bile salt solution. It is thought that the
nonuniform cell distribution
in the gel beads resulted in the
insufficient information. This
phenomenon is related to mixing
problems that result when the inoculum
is added to the polymer
solution during bead preparation, since this
polymer solution
has a very high viscosity and produces a nonuniform
cell distribution
in the gel beads (
23). Sodium alginate
concentrations from 2
to 4% were tested in this study. As expected,
the higher the concentration
of alginate in the beads, the lower the
death rate of the cells
in the beads (Fig.
1 and
2). It has been
reported that the lower
diffusion rate of glucose and ethanol in
more-concentrated alginate
gels is due to a decrease in the number and
length of the pores
rather than a decrease in the pore diameter
(
7). It was found
that gel concentrations below 2% did not
form spherically shaped
beads. This result was in agreement with other
investigations
(
9,
20). It has also been shown that
low-viscosity droplets
are less able to retain their spherical shape
against drag forces
upon collision with a solution. Yet, a high
concentration of sodium
alginate (5% or more) cannot form small
droplets because of its
physiological characteristics as a dough.
Accordingly, cell entrapment
is limited to the range of gel
concentrations that form spherical
beads.
In relation to bead size, the survival of cells in beads is higher with
larger beads as shown in Fig.
1 and
2. Sheu et al.
(
21)
indicated that larger bead diameters provided more protection
for
Lactobacillus bulgaricus in frozen desserts. Very large
beads,
however, can cause a coarseness of texture in live microbial
feed
supplements, and small beads cannot provide sufficient protection
for the bacteria. Therefore, bifidobacteria should be entrapped
within
a limited range of bead
sizes.
Two strains of
B. longum (KCTC 3128 and HLC 3743), as used
in this study, have a distinct value for each parameter in equation
13. Other bifidobacteria may have their distinct values. Species
of
Bifidobacterium have been reported to differ in their
susceptibility
to gastric acidity (
2). In addition, they
have a different
tolerance of bile salts (
6,
8). This
suggests that the values
of each parameter in equation 13 will be
affected by the kind
of
bifidobacteria.
In order to survive and reach the colon in quantities large enough to
facilitate colonization, a large number of initial cells
must be
entrapped in the beads. Hannoun and Stephanopoulos (
7)
reported that a larger cell load weakened the gel. Moreover, there
is
difficulty with high-concentration cultivation of bifidobacteria.
Berrada et al. (
2) demonstrated that not all
commercial bifidobacterium-fermented
milks can bring enough
living bifidobacteria to the human intestine
to ensure a health
benefit. Accordingly, most researchers have
chosen to use
Bifidobacterium strains that are more resistant
to gastric
acid and bile salts (
2,
6,
8,
10,
15,
16). As another
effective way, Rao et al. (
18) developed a
preliminary
procedure for the microencapsulation of
Bifidobacterium pseudolongum with cellulose acetate phthalate by using phase
separation-coacervation.
Their results showed that microencapsulated
bacteria were more
resistant than unencapsulated bacteria against
sequential stress
in simulated gastric and intestinal juices. However,
among the
techniques for immobilizing living cells, gel entrapment
using
natural biopolymers is favored by most researchers for various
reasons (
1,
11), including nontoxicity of the matrix
(crucial
for food-related application), simplicity of immobilization
technique,
high viability, and productivity of the immobilized cells.
Until
now, most immobilization techniques for bifidobacteria or
probiotic
bacteria have been developed to test the hypothesis that
immobilized
cells survive better than nonimmobilized cells (
3,
4,
18,
21,
22).
From this study, it was found that the survival of entrapped
bifidobacteria was strongly dependent on various parameters,
including
alginate concentration, bead size, initial cell numbers,
and bacterial
species. The mathematical model outlined in this
article should be
useful in evaluating the influence of various
parameters on the
survival of entrapped bifidobacteria under sequential
stress in the
gastrointestinal tract and for establishing the
optimal conditions for
the entrapment of bifidobacteria. This
model may have an important role
in food processing and pharmaceutical
preparations.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant (BK21 project) from the Ministry
of Education, Korea.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Postal Code
402-751, Department of Biological Engineering, Inha University,
Yonghyun-dong 253, Nam-gu, Inchon, Korea. Phone: 82-032-860-7511. Fax:
82-032-873-4429. E-mail: heotary{at}dragon.inha.ac.kr.
| |
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Applied and Environmental Microbiology, February 2000, p. 869-873, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Crittenden, R., Weerakkody, R., Sanguansri, L., Augustin, M.
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