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Applied and Environmental Microbiology, February 2000, p. 606-613, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bacteriocin Production with Lactobacillus
amylovorus DCE 471 Is Improved and Stabilized by Fed-Batch
Fermentation
Raf
Callewaert and
Luc
De Vuyst*
Research Group of Industrial Microbiology,
Fermentation Technology and Downstream Processing, Department of
Applied Biological Sciences, Vrije Universiteit Brussel, Brussels,
Belgium
Received 16 August 1999/Accepted 24 November 1999
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ABSTRACT |
Amylovorin L471 is a small, heat-stable, and hydrophobic
bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were
studied with fed-batch fermentations. A twofold increase in bacteriocin
titer was obtained when substrate addition was controlled by the
acidification rate of the culture, compared with the titers reached
with constant substrate addition or pH-controlled batch cultures
carried out under the same conditions. An interesting feature of
fed-batch cultures observed under certain culture conditions (constant
feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up
to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The
model showed that bacterial growth was dependent on both the energy and
the complex nitrogen source. Bacteriocin production was growth
associated, with a simultaneous bacteriocin adsorption on the producer
cells dependent on the lactic acid accumulated and hence the viability
of the cells. Both bacteriocin production and adsorption were inhibited
by high concentrations of the complex nitrogen source.
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INTRODUCTION |
Lactic acid bacteria are known to
have an antagonistic activity toward a variety of microorganisms.
Bacteriocin production is one of the properties responsible for the
antibacterial activity against closely related species and possibly
gram-positive food spoilers and pathogens (10, 18).
Bacteriocins produced by lactic acid bacteria are either small
thermostable peptides or large thermolabile proteins. Large numbers
of bacteriocin producers have been found among different genera
of the lactic acid bacteria (10, 11, 13, 14, 29).
Bacteriocins are of interest for potential application in the food
industry because of their antimicrobial activity and their
technologically favorable properties (10, 30).
Optimal bacteriocin production in batch fermentation usually requires
complex media and well-controlled physical conditions, such as
temperature and pH (3, 4, 9, 17, 20, 23, 27, 28, 34).
Bacteriocin production is correlated with bacterial growth, implying
that the volumetric bacteriocin production is dependent on the total
biomass formation (6, 9, 23). After reaching a maximal
bacteriocin activity in the fermentation medium during the active
growth phase, often a drastic decrease in soluble bacteriocin activity
occurs. This disappearance of bacteriocin activity was ascribed to
proteolytic inactivation (9, 16), protein aggregation
(6, 7), and adsorption of the bacteriocin molecules to the
cell surface of the producer cells (6, 26, 27, 33).
Mørtvedt-Abildgaard et al. (23) proposed adding ethanol to
the fermentation medium to prevent aggregation. Matsusaki et al.
(21) found that the use of calcium in the fermentation medium prevented the adsorption of nisin Z to the producer cells. Fed-batch fermentation technology allows the obtaining of high cell
densities through the continuous supply of fresh medium. The growth
rate can be controlled by the application of growth-limiting feeding
strategies. Bacteriocin production in fed-batch fermentation to prevent
substrate inhibition has already been described for nisin
(8) and epidermin and gallidermin (32).
Bacteriocin production was modeled in a few cases only (19, 20,
26-28). The models were set up with a term for growth-associated
bacteriocin production and a term for bacteriocin degradation or adsorption.
Lactobacillus amylovorus DCE 471 produces a small,
heat-stable, and strongly hydrophobic bacteriocin, named amylovorin
L471, with a narrow inhibitory spectrum (7). The effects of
different physical and chemical factors on the production of amylovorin L471 in batch fermentation have already been studied (6,
19). A decrease in the activity of soluble amylovorin L471 after
the active growth phase was due to adsorption of the bacteriocin
molecules to the producer cells. In this paper, fed-batch fermentations were studied to examine the nutritional requirements of bacteriocin production. In addition, it is shown that amylovorin L471 production could be improved and stabilized by adequate nutrient feeding. A
mathematical model was set up to simulate L. amylovorus DCE 471 cell growth and bacteriocin production
during fed-batch fermentation.
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MATERIALS AND METHODS |
Bacterial strains, maintenance, inoculum preparation, and media.
L. amylovorus DCE 471 was used as the producer strain of the
bacteriocin amylovorin L471. Lactobacillus delbrueckii
subsp. bulgaricus LMG 6901T was used as a
sensitive indicator organism for detection of bacteriocin activity. The
strains were stored at 
80°C in MRS medium (Oxoid, Basingstoke,
United Kingdom) containing 25% (vol/vol) glycerol. Cultures were
propagated twice in MRS medium (Oxoid; initial pH 6.5, 12 h,
37°C) prior to use as the inoculum (1% [vol/vol]) for the
fermentation experiments. The transfer inoculum was 1% (vol/vol). Fermentations were carried out in modified MRS medium adjusted to pH
5.0 (i.e., MRS medium containing different concentrations of glucose
and complex nitrogen source [CNS; see below]). The CNS consisted of
the following (in grams per gram of CNS): Lab Lemco powder (Oxoid),
0.36; yeast extract (E. Merck, Darmstadt, Germany), 0.18; and
bacteriological peptone (Oxoid), 0.46. All media were sterilized by
being heated at 121°C for 20 min. Glucose was sterilized separately
and added aseptically to the medium. Bottom and overlay agar media were
prepared by addition of, respectively, 15 and 7 g of granulated
agar (Oxoid) to 1 liter of MRS medium (Oxoid).
Fermentation experiments.
A 15-liter stainless steel Biostat
C fermentor (B. Braun Biotech International, Melsungen, Germany) that
was in situ sterilizable was used throughout this study. The software
program Micro-MFCS for Windows NT (B. Braun Biotech International) was
used to control the fermentation process. Fermentations were carried
out, without aeration, at a controlled temperature of 37°C and at a
controlled constant pH of 5.0 through the automatic addition of 10 N
NaOH. Slow agitation (50 rpm) was maintained to keep the medium
homogeneous. Fermentations were carried out in duplicate.
For constant fed-batch fermentations (Table
1), the fermentor was filled with 8 liters of modified MRS medium containing 2 g (each) of glucose and
CNS per liter (fermentations 1, 4, and 5). From the start of the
fermentation, 4 liters of concentrated glucose (120 g
liter
1) and CNS (264 g liter1) was added to
the fermentation medium at constant feed rates of 0.13, 0.36, and 0.06 liters per h for fermentations 1, 4, and 5, respectively (Table 1).
This would result in a final added concentration of 40 g of
glucose per liter and 88 g of CNS per liter in the end volume of
12 liters. This proportion of the energy source to the CNS was chosen
because it was optimal for cell growth and bacteriocin production in
batch fermentation (6). In fermentations 2 and 3, the
initial CNS and glucose concentration of the modified MRS medium were
112 and 57 g liter1, respectively (Table 1). During
fermentation, concentrated glucose and CNS were added, respectively,
which would again result in a final added concentration of 40 g of
glucose per liter and 88 g of CNS per liter in the end volume of
12 liters.
For acidification-controlled fed-batch fermentations (Table
1), the
starting volume consisted of 8 liters of modified MRS
medium with
11 g of glucose per liter and 25 g of CNS per liter
(fermentation 6). The fermentor was fed with concentrated glucose
and
the CNS (see above) at an automatically controlled feeding
rate
according to the amount of alkali (10 N NaOH) consumed at
each time.
Because of the homofermentative character of
L. amylovorus DCE 471, glucose was completely converted to lactic acid. The
amount of
alkali consumed to neutralize the lactic acid formed
was hence
proportional to the amount of glucose converted to lactic
acid and thus
was a measure of glucose
consumption.
Samples were withdrawn aseptically from the fermentation medium at
regular time intervals and analyzed for cell growth and
bacteriocin
production.
Analysis of cell growth.
Cell dry mass (CDM) determinations
were performed by filtrating 50 ml of fermentation medium through
0.45-µm-pore-size filters (type HA; Millipore Corporation, Bedford,
Mass.) and subsequent drying at 105°C for 24 h. Standard
deviations were 0.11 g of CDM per liter. Viable cell numbers were
enumerated by plating on MRS agar and expressed as CFU per milliliter.
Quantitative determination of bacteriocin titers.
Amylovorin
L471 activity was measured by an adaptation of the critical dilution
method used for the assay of bacteriocins (7). Serial
twofold dilutions of cell-free culture supernatant containing
bacteriocin were spotted (10 µl) onto fresh indicator lawns of
L. delbrueckii subsp. bulgaricus LMG
6901T. These lawns were prepared by propagating fresh
cultures to an optical density at 600 nm of 0.45 and adding 100 µl of
the cell suspension to 3.5 ml of the overlaid agar. Overlaid agar
plates were incubated at 37°C for at least 24 h. Bacteriocin
activity is expressed in millions of arbitrary units (MAU). One
arbitrary unit is defined as the reciprocal of the highest dilution
displaying a clear zone of inhibition. When a turbid zone followed a
clear zone, the critical dilution was taken as the average of the two dilutions.
Glucose and lactate concentration determination.
Glucose
consumption and lactate production were determined by high-performance
liquid chromatography (Waters Corporation, Milford, Mass.). Samples
were pretreated with 20% (wt/vol) trichloroacetic acid to precipitate
proteins. A prepacked column, RT 300-7,8 Polyspher OA KC (Merck), and a
differential refraction detector (Waters) were used. As the mobile
phase, a 0.005 N H2SO4 solution was used at a
fixed flow rate of 0.4 ml min1. Standard deviations were
0.04 g of glucose per liter and 0.03 g of lactic acid per liter.
Model development.
Bacterial growth and bacteriocin
production with L. amylovorus DCE 471 in batch fermentation
have been modeled by Lejeune et al. (19). However, the
logistic equation used for cell growth was not suitable for the
simulation of fed-batch fermentation processes (unpublished data). The
dynamic model developed in this work is based on Monod growth kinetics.
Changes in volume as a result of nutrient feeding
(
Fin) and sampling (
Fout)
are given by the following equation:
|
(1)
|
where
V is the volume of the fermentation medium (in
liters) and
Fin and
Fout
are the inflow and outflow, respectively (in
liters per
hour).
Medium compounds are divided into two parts: an energy source (glucose)
and a CNS that provides the necessary building blocks
(e.g., amino
acids, peptides, vitamins) for cell synthesis. Both
compounds are
necessary for bacterial growth and bacteriocin production
(see below).
On the other hand, high glucose concentrations and
lactic acid inhibit
cell growth. Biomass formation is hence given
by the following
equation:
|
(2)
|
where
X is the biomass (in grams of CDM per liter); µ and µ
max are the specific growth rate and
the maximal specific
growth rate, respectively (per hour);
S
is the concentration of
the energy source (in grams of glucose per
liter);
N is the concentration
of the CNS (in grams of CNS
per liter); and
KS and
KN
are the
corresponding Monod constants for glucose (in grams of glucose
per liter) and CNS (in grams of CNS per liter), respectively;
L is the lactic acid concentration (in grams of lactic acid
per
liter); and
KiS and
KiL are the inhibition constants for glucose
(in
grams of glucose per liter) and lactic acid (in grams of lactic
acid
per liter),
respectively.
Substrate (glucose and CNS) consumption is described by the linear
equations
|
(3)
|
|
(4)
|
where
YX/S and
YX/N are the yield coefficients of biomass for
glucose (in grams of CDM per gram of glucose) and CNS (in grams
of CDM
per gram of CNS), respectively;
mS and
mN are the maintenance
coefficients for glucose
(in grams of glucose per gram of CDM
per hour) and CNS (in grams of CNS
per gram of CDM per hour),
respectively; and
SF
and
NF are the concentrations of glucose
(in
grams of glucose per liter) and CNS (in grams of CNS per liter)
in the
feed, respectively. Because the maintenance metabolism
of lactic acid
bacteria seems to be strongly dependent on growth
rate, a similar
limitation by glucose and CNS was introduced (
19,
25).
Since glucose is homofermentatively converted to lactic acid by
L. amylovorus, formation of lactic acid is described by the
equation
|
(5)
|
where
YL/S is the yield coefficient of
lactic acid for glucose (in grams of lactic acid per gram of
glucose).
Bacteriocin production is described as growth associated and is
inhibited by high concentrations of the CNS. Bacteriocin adsorption
is
proportional to the bacteriocin titer, is dependent on the
physiological state of the cells and in particular on the lactic
acid
concentration, and is inhibited by the CNS. These observations
were
also made by Meghrous et al. (
22) and Parente et al.
(
26).
Consequently, soluble bacteriocin activity in the
fermentation
medium is given by the equation
|
(6)
|
where
B is the bacteriocin titer (in MAU per liter),
kB is the specific bacteriocin production (in
MAU per gram of CDM),
KiBN is the CNS inhibition
constant for bacteriocin production
(in grams of CNS per liter),
k'B is the specific bacteriocin adsorption
rate
(per hour),
K'iBN is the CNS inhibition constant
for bacteriocin
adsorption (in grams of CNS per liter), and
K'BL is the lactic
acid constant for bacteriocin
adsorption (in grams of lactic acid
per
liter).
Determination of biokinetic parameters.
The Euler
integration method was used to simulate fermentation runs according to
the model equations. Model parameters were determined by nonlinear
regression. The sum of the squared differences between simulated and
experimental values was minimized by varying the values of the model
parameters. The growth parameters µmax, YL/S, and YX/S were
obtained from simulation of batch fermentations run at 37°C and
controlled pH of 5.0 in modified MRS medium with initial concentrations
of 40 g of glucose per liter and 88 g of CNS per liter
(6). The parameters KS,
KiL, and mS were obtained from simulations of fed-batch fermentations with a high initial CNS
concentration and feeding of concentrated glucose. The parameters KN, YX/N,
mN, and KiS were obtained
from simulations of fed-batch fermentations with a high initial glucose
concentration and feeding of the concentrated CNS.
Calculation of Fin and
Fout.
The inflow (Fin)
was calculated as the sum of alkali, glucose, and CNS supplies. These
experimental values were monitored online. For the simulation of the
fermentation profiles, the supply of alkali was calculated from the
simulated amount of lactic acid produced (equation 5). One mole of
lactic acid was neutralized with 1 mol of NaOH. For the simulation of
acidification-controlled fed-batch fermentations, the supplies of
glucose and CNS were calculated from the simulated supply of alkali.
Simulated values were compared to the experimental values (see
Results). The outflow (Fout) was calculated from
the experimental sample volumes during discrete time intervals (see Results).
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RESULTS |
Influence of a constant addition of glucose and/or CNS.
Three
fed-batch fermentations were carried out with constant feeding of
glucose and/or CNS during approximately 30 h from the start of the
fermentation (Fig. 1, 2, and
3).
Fermentations started with a low initial concentration of glucose
and/or CNS. Feeding of concentrated glucose and/or CNS would result in
a medium with a final concentration of 40 g of glucose per liter
and 88 g of CNS per liter in the final volume of 12 liters (Table
1). This experimental design enabled us to find out the effect of glucose and CNS limitation on cell growth and bacteriocin production.
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FIG. 1.
Fed-batch fermentation of L. amylovorus DCE
471 with constant addition of glucose and CNS from the start of the
fermentation during 30.5 h.  , biomass (X; grams of
CDM per liter);  , residual glucose (S; grams of glucose
per liter);  , lactic acid (L; grams of lactic acid per
liter);  , bacteriocin activity (B; MAU per liter);  ,
CFU (log CFU per milliliter). The corresponding lines represent
simulated values. The line without experimental points represents the
residual CNS (N; grams of CNS per liter).
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FIG. 2.
Fed-batch fermentation of L. amylovorus DCE
471 with constant addition of glucose from the start of the
fermentation during 27.0 h. Symbols and lines are similar to those
in Fig. 1, except that for clarity, the residual CNS is expressed in
grams of CNS per liter ×21. The values of the CNS on the
left y axis have to be multiplied by 2.
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FIG. 3.
Fed-batch fermentation of L. amylovorus DCE
471 with constant addition of CNS from the start of the fermentation
during 28.5 h. Symbols and lines are similar to those in Fig. 1.
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In the first fed-batch fermentation experiment, glucose and the CNS
were added to the fermentation medium at a constant rate
of 0.13 liters
per h (Fig.
1). An exponential growth phase of
8 h was followed by
a decrease in the growth rate and finally
a linear growth phase after
12 h of fermentation, when the glucose
concentration became growth
limiting. Bacteriocin production started
at the beginning of the
exponential growth phase and reached a
maximum of 6.4 MAU per liter in
this phase. In the beginning of
the linear growth phase, when glucose
was growth limiting, the
amylovorin titer increased to 12.8 MAU per
liter and remained
constant afterwards. A final maximal biomass of
5.0 g of CDM per
liter was reached. The maximal viable cell number
was 1.5 × 10
9 CFU per ml and remained constant upon
prolonged
fermentation.
In a second fed-batch experiment, only glucose was fed to the
fermentation medium at a constant feeding rate of 0.15 liters
per h
(Fig.
2). A profile similar to that of
the first fermentation
was obtained. Due to limitation of glucose, a
linear growth phase
of about 20 h followed the exponential growth
phase of 9 h. A
bacteriocin activity of 6.4 MAU per liter was
reached at the end
of exponential growth. The bacteriocin titer varied
between 6.4
and 12.8 MAU per liter during linear growth. After the
linear
growth phase, at 30 h of fermentation, the bacteriocin
activity
decreased. The maximal biomass obtained was 5.0 g of CDM
per liter.
The maximal viable cell number was 1.9 × 10
9 CFU per ml and decreased slowly upon prolonged
fermentation.
In a third fed-batch experiment, only CNS was fed to the fermentation
medium at a constant feeding rate of 0.14 liters per
hour (Fig.
3). Again, a linear growth phase followed
an exponential
growth phase of approximately 9 h. Glucose was
still sufficiently
available, indicating that the CNS was growth
limiting. The activity
obtained in the exponential growth phase was 6.4 MAU per liter
and increased further to a maximal activity of 12.8 MAU
per liter.
Thereafter, the soluble bacteriocin activity decreased
sharply.
The maximal viable cell number was 1.5 × 10
9
CFU per ml and decreased along with the bacteriocin activity.
A maximal
biomass of 4.6 g of CDM per liter was reached when glucose
was
completely
consumed.
Influence of time of addition of glucose and/or CNS.
In the
fermentations described above, bacteriocin production could be improved
by increasing the availability of nutrients. In the fourth and fifth
constant fed-batch fermentation experiments, glucose and CNS were added
much more slowly and much more quickly, respectively (Table 1).
Slow, constant addition of glucose and CNS (i.e., addition within
72 h of fermentation) resulted in low growth rates. Amylovorin
L471 was again mainly produced during the exponential growth phase
of
approximately 9 h. At the end of this phase, the medium reached
a
maximal activity of 4.8 MAU per liter. From 9 h to the end of
the
fermentation, growth was restricted by the slow addition of
glucose.
The bacteriocin titer decreased slowly to a value of
0.4 MAU per liter
after 60 h of fermentation. The presence of
small measurable
amounts of glucose in the fermentation medium
at the end of the
fermentation (0.02 and 0.05 g of glucose per
liter after,
respectively, 30 and 60 h of fermentation) indicated
that another
factor (e.g., a compound supplied with the CNS) became
growth limiting
or that growth was severely inhibited by lactic
acid or bacteriocin
molecules adsorbed to the cells. The maximal
biomass production (4.4 g
of CDM per liter) was lower than those
of the other fed-batch
fermentation runs due to the stronger nutrient
limitation. The viable
cell numbers reached a maximum of 9.7 ×
10
8 CFU per
ml after 9 h of fermentation and decreased slowly upon
further
fermentation.
Fast addition of glucose and CNS (i.e., addition within 11 h of
fermentation) resulted again in an exponential growth phase
of 8 h. A bacteriocin titer of 8.0 MAU per liter was obtained
in this phase,
and it increased further to a maximal value of
12.8 MAU per liter. A
high biomass production was obtained with
a maximum of 5.9 g of
CDM per liter. The maximal viable cell number
was 2.1 × 10
9 CFU per ml. After 14 h of fermentation, glucose
was completely
consumed, resulting in a drop in viable cell number and
a corresponding
decrease in bacteriocin
activity.
Acidification-controlled addition of the substrates.
In order
to prolong the exponential growth phase and hence the concomitant
bacteriocin production phase, substrate was added to the fermentation
medium in an acidification-controlled manner (Fig.
4). The biomass increase was exponential
during feeding, since glucose and CNS were present in excess. The
glucose concentration increased slowly until 12 h, when feeding
stopped; apparently, the ratio of glucose to alkali in the feed was not
well adjusted to keep the glucose concentration constant in the medium.
At 15 h, the growth phase stopped due to a depletion of glucose.
Biomass production was similar, as compared to a fast, constant
fed-batch fermentation (addition within 11 h of fermentation). A
maximal biomass of 5.8 g of CDM per liter was reached. The maximal
bacteriocin titer averaged 25.6 MAU per liter, which is twice the
amount obtained during constant fed-batch fermentation. This titer
decreased strongly when glucose was completely consumed. At this time,
the amount of viable cells, which had reached a maximum of 2.1 × 109 CFU per ml, decreased drastically too.
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FIG. 4.
(a) Fed-batch fermentation of L. amylovorus
DCE 471 with an acidification-controlled addition of glucose and CNS
from the start of the fermentation during 12 h. Symbols and lines
are similar to those in Fig. 1. (b) Addition of glucose, CNS, and
alkali. ×, cumulative volume of glucose and CNS (liters); +,
cumulative volume of alkali (liters [10 N NaOH]). (c) Volume and flow
rate in and out of the fermentor. Solid line, inflow
(Fin; liters per hour); broken line, outflow
(Fout; liters per hour);
atyp0250;, volume (liters).
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Determination of cell growth and bacteriocin production model
parameters.
Biokinetic cell growth parameters were determined by
simulating the profiles of both batch and constant fed-batch
fermentation runs according to the model described above (Table
2). The maximal specific growth rate
(µmax), the yield coefficient of biomass for
glucose (YX/S), the cell maintenance coefficient
for glucose (mS), and the yield coefficient of
lactic acid for glucose (YL/S) were comparable
to those obtained for other batch fermentations (19).
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TABLE 2.
Biokinetic cell growth parameters for L. amylovorus DCE 471 determined by simulation of batch and
fed-batch fermentation profiles
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Bacteriocin production was clearly growth associated. The specific
bacteriocin production obtained from simulation of the
different
fermentation runs was approximately 4.7 MAU per g of
CDM (Table
3). When the fermentation was started
with a high
initial concentration of the CNS, similar to that in batch
fermentations,
a bacteriocin activity lower than expected was obtained
(Fig.
2). Consequently, the specific bacteriocin production had to be
adjusted to 2.4 MAU per g of CDM, similar to that in a comparable
batch
fermentation in modified MRS medium (40 g of glucose per
liter, 88 g of CNS per liter, pH 5.0, 37°C) (results not shown).
The maximal
volumetric bacteriocin titer in the latter fermentation
was 11.2 MAU
per liter and corresponded to a maximal biomass of
5.3 g of CDM
per liter (
6). The introduction of a term of inhibition
of
the bacteriocin production by the CNS was able to demonstrate
the
resulting reduction in specific bacteriocin production (equation
6).
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TABLE 3.
Biokinetic parameters for bacteriocin production
determined by simulation of fed-batch fermentation profiles
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The decrease in bacteriocin activity accompanied a decrease in viable
cell numbers (Fig.
3 and
4). In contrast, bacteriocin
activity remained
stable when no decrease in viable cells was
observed (Fig.
1).
Bacteriocin adsorption seemed to be dependent
on the viability of the
producer cells, since a higher lactic
acid concentration coincided with
a lower number of viable cells
and hence a larger amount of bacteriocin
molecules adsorbed to
the cells (see equation 6). A value of 90 g
of lactic acid per
liter was found for the lactic acid constant for
bacteriocin adsorption
(Table
3). In addition, the CNS concentration
seemed to have
an inhibitory effect on bacteriocin adsorption (see
equation
6).
Finally, the model was validated by simulation of both a slow and a
fast constant fed-batch fermentation (results not shown)
and an
acidification-controlled fed-batch fermentation (Fig.
4).
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DISCUSSION |
Amylovorin L471 is produced during the active growth phase,
implying that the volumetric bacteriocin production is dependent on the
total biomass formation. Hence, conditions that provide high cell
densities favor a high bacteriocin production. Indeed, an increased
nutrient supply in batch fermentation results in an increased biomass
and volumetric bacteriocin production. However, the specific
bacteriocin production decreases under these conditions (6,
19). Furthermore, during batch fermentation of L. amylovorus DCE 471, a rapid decline in bacteriocin activity is
observed after the growth-associated bacteriocin production phase
(6, 7, 19). This decline is concomitant with the depletion
of the energy source, glucose, at the end of the fermentation. It is
due to adsorption of the bacteriocin molecules to the producer cells (6).
In this paper, bacteriocin production was improved by fed-batch
fermentation and could be stabilized, provided an appropriate feeding
strategy was applied. This cultivation technique allowed a controlled
growth through limitation of glucose or the CNS. In addition, the high
nutrient supply did not inhibit growth or bacteriocin production. Slow
growth due to limitation of the energy or CNS was not beneficial for
amylovorin L471 production. Limitation of the CNS resulted in a
stagnation of amylovorin L471 production and a decrease in soluble
bacteriocin activity in the fermentation medium. Under glucose-limiting
conditions in a constant fed-batch fermentation, no amylovorin L471
production could be observed. Stronger limitation of the energy source
resulted in a decrease in the viable cell number and a concomitant
decrease in the soluble amylovorin L471 titer. Hence, for optimal
bacteriocin production, the energy source and other nutrients should be
sufficiently available.
When substrates were sufficiently available during a fast, constant
fed-batch fermentation or an acidification-controlled fed-batch
fermentation of L. amylovorus DCE 471, bacterial growth was
optimal. A maximal specific bacteriocin production and volumetric bacteriocin titer seemed to have been achieved. Growth limitation by
lactic acid is probably the major reason for the restricted bacteriocin production.
Slow growth upon prolonged fermentation resulted in weak or no
amylovorin L471 production. A minimal growth rate seemed to be
necessary for bacteriocin production. Although some bacteriocins are mainly produced in the stationary growth phase (2,
3, 15), generally bacteriocin production occurs only in the
active growth phase (9, 23, 28). In contrast, Ten Brink et
al. (31) reported on acidocin B production by
nongrowing L. acidophilus M46 cells.
Usually, during batch fermentation experiments, the amylovorin L471
titer decreases drastically after reaching a peak of activity in the
active growth phase. A drop in CFU is also observed, apparently indicating cell death. This phenomenon was shown to be due to adsorption of amylovorin L471 to the producer cells
(6). The apparent loss of bacteriocin could be
avoided during fed-batch fermentation. Compared to
batch fermentations, a less drastic decrease or no decrease in the
amylovorin L471 titer was observed after reaching maximal activity.
Constant addition of glucose and CNS at an appropriate rate could
stabilize the bacteriocin titer. Similarly, acidophilicin LA-1 activity
decreased sharply in batch fermentation of L. acidophilus
LA-1 when the death phase started. However, bacteriocin activity was
stabilized by adding concentrated medium (5). Ferreira and
Gilliland (12) explained the drastic drop in CFU by
demonstrating the presence of a mixed culture of L. acidophilus NCFM cells containing a major fraction of sensitive
nonproducing strains and a minor fraction of resistant bacteriocin-producing strains. Resistant bacteriocin-producing cells formed large and small colonies on MRS agar. Two
morphologically different colonies were also observed for L. amylovorus DCE 471 plated on MRS agar (7). A limited
immunity of the producing cells to their own bacteriocin may explain
this phenomenon. For several bacteriocin producers, it is already well
documented that genes for bacteriocin production and immunity are
regulated and transcribed simultaneously (1, 24). This
implies that bacteriocin nonproducing cells are sensitive to their own
bacteriocin. When bacteriocin production stops during fermentation,
cells become sensitive because they do not further produce immunity
proteins. During fed-batch fermentation, certain nutrient compounds
necessary for bacteriocin production may become limited or exhausted,
resulting in more sensitive bacteria and causing a decrease in viable
cell numbers. Constant feeding of nutrients enables continuous growth of at least part of the biomass. As a consequence, amylovorin L471 was still produced by this part of the L. amylovorus population due to the growth-associated
bacteriocin production. The other part of the culture, nonproducers and
sensitive cells, mainly adsorbed the bacteriocin molecules to their
cell surfaces. While the first fraction, the growing cells, were able
to form colonies on MRS agar, the second fraction, the nongrowing
cells, were not. Hence, stabilization of the bacteriocin titer could be
explained by assuming that bacteriocin production compensated for the
loss of bacteriocin in the medium by adsorption. Once the energy or CNS
was insufficient, the production/adsorption equilibrium was lost,
resulting in a decrease in bacteriocin activity. In addition, the CNS
possibly interfered with bacteriocin adsorption. The latter was also
shown for nisin adsorption on Lactococcus lactis cells (22). However, these hypotheses require additional
experimental data.
In this work, an unstructured mathematical model was set up that was
able to describe the effect of nutrient limitations on growth and
bacteriocin production or adsorption of L. amylovorus DCE
471 in fed-batch fermentation. The model was validated under different fed-batch fermentation conditions. From this model, the
following conclusions can be extrapolated. Bacteriocin production and
adsorption were considered to occur simultaneously. Whereas at a low
growth rate, bacteriocin adsorption dominated, at a high growth rate,
bacteriocin production dominated. In the exponential growth phase,
bacteriocin adsorption was low, resulting in a high specific
bacteriocin production. Subsequent reduction of the growth rate upon prolonged fermentation resulted in increased bacteriocin adsorption. Adequate nutrient feeding stabilized the bacteriocin titer
by equilibrating the bacteriocin production and adsorption. However, too strong nutrient limitation resulted in a
decrease in bacteriocin activity; furthermore, the CNS inhibited
both bacteriocin production and adsorption. Finally, the
specific amylovorin L471 production seemed to have attained a
natural maximum during acidification-controlled fed-batch fermentation.
The presented results obtained by fed-batch fermentation are
interesting regarding the development of continuous fermentation processes. During continuous processes, the concentrations of nutrients
can be controlled in order to improve and stabilize the bacteriocin
production. In addition, the continuous removal of inhibitory
metabolites such as lactic acid or the bacteriocin itself should
maximize volumetric bacteriocin production, especially at dilution
rates that are high enough to provide the necessary nutrients.
| |
ACKNOWLEDGMENTS |
The research presented in this paper was financially supported by
the Research Council of the Vrije Universiteit Brussel, the Fund for
Scientific Research
Flanders, and the Biotechnology Programme of the
Commission of the European Community (grants BIO2-CT943055 and
ERB-CIPACT-940160).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Research Group
of Industrial Microbiology, Fermentation Technology and Downstream
Processing (IMDO), Department of Applied Biological Sciences, Vrije
Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium.
Phone: 32-2-6293245. Fax: 32-2-6292720. E-mail:
ldvuyst{at}vub.ac.be.
| |
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Applied and Environmental Microbiology, February 2000, p. 606-613, Vol. 66, No. 2
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Top
Abstract
Introduction
