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Applied and Environmental Microbiology, March 1999, p. 974-981, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Temperature and pH Conditions That Prevail during
Fermentation of Sausages Are Optimal for Production of the
Antilisterial Bacteriocin Sakacin K
Frédéric
Leroy and
Luc
de Vuyst*
Division of Industrial Microbiology,
Fermentation Technology and Downstream Processing (IMDO),
Department of Applied Biological Sciences, Vrije Universiteit Brussel,
B-1050 Brussels, Belgium
Received 11 September 1998/Accepted 15 December 1998
 |
ABSTRACT |
Sakacin K is an antilisterial bacteriocin produced by
Lactobacillus sake CTC 494, a strain isolated from Spanish
dry fermented sausages. The biokinetics of cell growth and bacteriocin
production of L. sake CTC 494 in vitro during laboratory
fermentations were investigated by making use of MRS broth. The data
obtained from the fermentations was used to set up a predictive model
to describe the influence of the physical factors temperature and pH on
microbial behavior. The model was validated successfully for all
components. However, the specific bacteriocin production rate seemed to
have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the
inactivation of sakacin K; the optimal pH for cell growth did not
correspond to the pH for maximal sakacin K activity. Furthermore, cells
grew well at 35°C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for
implementation as a novel bacteriocin-producing sausage starter culture
with antilisterial properties, considering the fact that the
temperature and acidity conditions that prevail during the fermentation
process of dry fermented sausages are optimal for the production
of sakacin K.
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INTRODUCTION |
Fermentation is a worldwide and
ancient preservation technique, probably one of the oldest
methods known (51). It is commonly employed to preserve or
enhance the organoleptic attributes and microbiological safety of
foods. Indigenous microorganisms have been responsible for fermentation
traditionally, but starter cultures can now be added to induce
fermentation and favorable processing conditions can be selected to
ensure desired quality (6, 23). These processes encourage
the development of a desirable safe microflora, which is important for
preventing the outgrowth of spoilage bacteria and food-borne pathogens.
With the increasing demand for biological preservation techniques, the
application of lactic acid bacteria (LAB) as starter or protective
cultures is gaining interest (20). Some LAB show special
promise as they do not pose any health risk to man and are able to
prevent the outgrowth of undesirable bacteria and opportunistic
pathogens such as Staphylococcus aureus and Listeria monocytogenes. Microbial antagonism is due to the production of metabolites such as lactic acid, acetic acid, diacetyl, hydrogen peroxide, and bacteriocins. Bacteriocins are, in general, small cationic peptides (30 to 60 amino acid residues) with high isoelectric points and amphiphilic characteristics that inhibit at micromolar concentrations the growth of bacterial species closely related to the
producing organism and thus provide this organism with a selective
advantage over its natural competitors (13).
Many of the antimicrobial activities associated with
Lactobacillus meat isolates were proven to be bacteriocin
mediated (17, 30, 40). In most cases activity against
Listeria monocytogenes was detected. Examples of such
bacteriocins are sakacins A, M, and P (19, 39, 41, 45),
curvacin A (45), curvaticins 13 and FS47 (18,
43), plantaricin BN (25), lactocin 705 (47), acidocin B (44), salivaricin B
(44), and bavaricin MN (25, 49). Antilisterial
activities by LAB have been demonstrated for fermented meat systems,
such as with American-style fermented meat products (2, 14,
36), Italian salami (5), and Spanish-style dry
fermented sausages (22). Although no Listeria
outbreak due to the consumption of fermented meat products has yet been
reported, several health authorities have expressed their concern
(22). A high rate of patient fatality (circa 30%)
(1) and the resistance of Listeria to low
temperature, pH, and water activity and to high concentrations of NaCl
have indeed made the bacterium a major concern for the modern
food industry (35). Gahan et al. even warn us about
acid-adapted Listeria mutants that have an increased ability
to survive in low-pH foods (15).
The application of bacteriocin-producing lactic acid starter cultures
may be a potential solution for preserving fermented meat products from
the outgrowth of Listeria monocytogenes (22). However, although the results of active inhibition of
Listeria outgrowth in meat are encouraging, bacteriocin
activity in meat was shown to be less effective than in broth
(42), probably due to partial inactivation by proteases,
limited diffusion in the food matrix, and unspecific binding to food
ingredients such as fat particles (8, 20). Therefore, the
production of bioavailable, active bacteriocins must be increased
(4). Careful selection of strains adapted to certain food
environments and food processing conditions such as temperature and pH
is absolutely necessary.
In this paper, the kinetics of in vitro cell growth and bacteriocin
production of a Lactobacillus sake starter strain during laboratory fermentations were investigated by making use of MRS broth.
The data obtained from the fermentations was used to set up a
predictive model to describe the influence of the physical factors
temperature and pH on microbial behavior. The bacterium investigated
was the bacteriocinogenic strain L. sake CTC 494, which
has previously been isolated from dry fermented sausages and has been
characterized by Hugas et al. (21). The bacteriocin produced
was designated sakacin K; it has a bacteriolytic effect on
Listeria monocytogenes. L. sake CTC 494 has excellent
starter culture capacities. Besides producing bacteriocin, it is highly competitive in the meat environment and imparts good sensorial and
organoleptic qualities to the final product (22).
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MATERIALS AND METHODS |
Microorganisms and media.
L. sake CTC 494, a
producer of sakacin K (22), and Listeria innocua
LMG 13568, which was used as an indicator organism to determine
bacteriocin activity levels, were stored at 
80°C in MRS broth
(9) (Oxoid, Basingstoke, United Kingdom) and brain heart
infusion (Oxoid), respectively, both of which contained 25% (vol/vol)
glycerol as a cryoprotectant. L. sake CTC 494 was kindly provided by M. Hugas (Institut de Recerca i Technologia Agroalimentàries, Meat Technology Center, Monells, Spain). The strains were propagated twice at 30°C for 12 h before
experimental use. Solid medium was prepared by adding 1.5% agar
(Oxoid) to the broth. The overlays needed for the estimation of the
bacteriocin titer were prepared with 0.7% agar.
Fermentation experiments.
In order to investigate the
influence of temperature and pH on both the growth and the bacteriocin
production of L. sake CTC 494, a series of
fermentations was performed with MRS broth (9).
Fermentations were carried out in a 15-liter laboratory fermentor
(Biostat C; B. Braun Biotech International, Melsungen, Germany) containing 10 liters of MRS broth. The vessel was sterilized in situ at
121°C for 20 min. Glucose was sterilized separately and aseptically
added to the fermentor. For the preparation of the inoculum, 10 ml of
MRS broth was inoculated with 0.5 ml of a freshly prepared
L. sake CTC 494 culture and incubated for 12 h at
30°C. This preculture was added to 90 ml of MRS broth. After 11 h of growth at 30°C this culture was used to inoculate the fermentor (1%, vol/vol). Temperature and pH control was performed on-line (Micro-MFCS for Windows NT; B. Braun Biotech International). The pH was
controlled to within pH 0.05 of the set point by automatic addition of
10 M NaOH. Temperature stayed within 0.1°C of the set point. Moderate
agitation (50 rpm) was performed to ensure homogeneity of the broth.
During the first experiments the pH was maintained at 6.5 while the
fermentations were carried out at 20, 25, 30, and 35°C.
In a second
series of experiments the temperature was held at
30°C while the
fermentations were carried out with pHs of 4.5,
5.0, 5.5, and 6.5. For
the validation of the model, two additional
fermentations were
performed, one at a temperature of 25°C and
a pH of 5.5 and the other
at a temperature of 23°C and a pH of
5.0.
Assays.
Samples were withdrawn aseptically from the
fermentor in order to determine cell dry mass (CDM), bacteriocin
activity, lactic acid concentration, and residual glucose
concentration. CDM was determined after membrane filtration
(0.45-µm-pore-size filters, type HA; Millipore, Bedford, Mass.) of a
known volume of fermentation liquor, followed by washing the
filter with demineralized water and drying it overnight at 105°C.
Microcentrifugation (13,000 × g for 10 min) was
performed in order to obtain cell-free samples, needed for the
measurement of lactic acid and residual glucose concentration with a
high-performance liquid chromatograph (Waters Corporation, Milford,
Mass.) and also for the estimation of bacteriocin activity levels.
High-performance liquid chromatography analysis was performed as
described previously (11). The soluble bacteriocin activity
was determined by a modified critical dilution method (10).
Briefly, serial twofold dilutions of cell-free culture supernatant with
sodium phosphate buffer (50 mmol liter
1, pH 6.5) were
spotted (10 µl) onto indicator lawns. The lawns were prepared by
adding fresh cultures of Listeria innocua LMG 13568 with an
optical density at 600 nm of 0.45 to 3.5 ml of brain heart infusion
overlay agar. Overlaid agar plates were incubated at 30°C. Activity
was expressed in arbitrary units (AU), corresponding to 10 µl of the
highest dilution causing a definite zone of inhibition on the lawn of
the indicator organism.
Model development.
Cell growth was modeled with the equation
for logistic growth (46); this equation has been frequently
used to describe the growth of LAB (24, 27, 31):
|
(1)
|
where
X is the CDM concentration (in grams of CDM per
liter),
t is time (in hours), µ
max is the
maximum specific growth rate
(per hour), and
Xmax is the maximum attainable CDM concentration
(in grams of CDM per liter) under a given set of conditions. By
this
model, the specific growth rate [µ = µ
max (1
X/
Xmax)] decreases
linearly as cell
concentration
increases.
Glucose consumption can be described by using the maintenance
energy model of Pirt (
32):
|
(2)
|
where
S is the residual glucose concentration (in
grams of glucose per liter),
YX/S is the cell
yield coefficient (in grams
of CDM per gram of glucose), and
mS is the maintenance coefficient
(in grams of
glucose per gram of CDM per
hour).
Lactic acid production can be calculated from the consumption of
glucose, with
YL/S (in grams of lactic acid per
gram of glucose)
being a yield coefficient for the conversion of
glucose into lactic
acid:
|
(3)
|
where
L is lactic acid production (in grams of lactic
acid per
liter).
Sakacin K is produced as a primary metabolite, and its titer increases
with CDM (this paper). When CDM production stagnates
as the growth
curve reaches the stationary phase, bacteriocin
production ceases and
the activity decreases due to proteolytic
degradation, aggregation, or
adsorption to the cells (
10,
11,
31). The soluble
bacteriocin activity (
B), which was measured
in the
cell-free supernatant, can be expressed in arbitrary units
per liter as
follows (
31):
|
(4)
|
where
kb is the specific bacteriocin
production (in arbitrary units per gram of CDM) and
kinact is the specific rate of bacteriocin
degradation (in liters per gram of CDM per hour). However, this
equation has never been validated
experimentally.
The relation between maximum specific growth rate and temperature can
be obtained with the equation of Ratkowsky et al. (
33):
|
(5)
|
where
a (per hour per degree Celsius squared) and
Tmin (in degrees Celsius) are regression
coefficients, with
Tmin being
the theoretical
minimum temperature for cell growth. Wijtzes et
al. have demonstrated
that for
Lactobacillus curvatus Tmin
is
independent of the pH (
48).
Growth behavior can also be expressed as a function of pH with a
parabolic equation (
48):
|
(6)
|
where
b (per hour) is a regression coefficient and
pH
min and pH
max are the theoretical minimum and
maximum pHs for cell growth,
respectively. Neither pH
min
nor pH
max showed a trend as a function
of temperature for
the growth of an
L. curvatus strain (
48).
Combining equations 5 and 6 as
|
(7)
|
yields a general equation for the combined effect of temperature
and pH on the maximum specific rate of growth, with
c (per
hour per degree Celsius squared) being a regression coefficient
(
48).
Equations
1,
2,
3, and
4 were integrated with the Euler integration
technique with Microsoft Excel version 7.0. All parameters
needed for
the modeling were estimated by manual adjustment until
a good
visual fit was
obtained.
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RESULTS |
Modeling of the fermentation profiles.
The influence of
temperature and pH on both the growth and the bacteriocin production of
L. sake CTC 494 was assessed. The data concerning the
consumption of glucose and the production of biomass, lactic acid, and
bacteriocin in fermentations maintained at constant pHs and
temperatures were fitted with equations 1 to 4. Biokinetic parameters
were varied manually until a good visual fit of the curves was
obtained. Figure 1 is included as an
illustrative example, representing a fermentation run at a controlled
temperature and pH of 20°C and 6.5, respectively. The experimental as
well as the modeled evolutions of CDM concentration and bacteriocin
titer (Fig. 1a) and of residual glucose concentration and lactic acid
production (Fig. 1b) are shown. To check the repeatability of the
results, one of the fermentations (30°C, pH 6.5; see below), which
was chosen randomly, was carried out in fourfold. Standard deviations
of 0.03 g of CDM g of glucose1, 0.04 g of
glucose g of CDM1 h1, 0.05 h1, 0.11 g of CDM liter1, 48 AU
ml1, and 0.02 liter g of CDM1
h1 for YX/S, mS,
µmax, Xmax,
kb, and kinact,
respectively, were obtained. Validation of the model further
contributes to the reliability of the experiments (see below).
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FIG. 1.
Modeling of the biomass (in grams of CDM per liter;  )
and bacteriocin production (in arbitrary units [in millions] per
liter; ×) (a) and the lactic acid production (in grams per liter; )
and glucose consumption (in grams per liter;  ) (b) of L. sake CTC 494 in MRS broth at a controlled temperature and pH of
20°C and pH 6.5.
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Bacteriocin activity increased rapidly while cells were growing
exponentially. This finding confirmed equation 4, which supposes
that
the production rate of bacteriocin is related to the production
rate of
biomass, and indicated clearly that the production of
bacteriocin by
L. sake CTC 494 follows primary metabolite kinetics
(
11,
12,
24). Once cell mass production began to level off,
bacteriocin activity decreased rather quickly. This decrease was
more
pronounced at the high temperatures and pH values, indicating
greater
proteolytic degradation or adsorption (Tables
1 and
2).
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TABLE 1.
Influence of temperature on µmax,
YX/S, mS, Xmax,
Bmax, kb, and
kinact of L. sake CTC 494 in
MRS broth at a constant pH of 6.5
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TABLE 2.
Influence of pH on µmax,
YX/S, mS, Xmax,
Bmax, kb, and
kinact of L. sake CTC 494 in
MRS broth at a constant temperature of 30°C
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For all experiments the yield coefficient,
YL/S, for lactate production was equal to
1 g g
1. This means that all glucose was converted
into lactic acid,
confirming the homofermentative character of
L. sake CTC 494.
The other parameters
(µ
max,
YX/S,
mS,
Xmax,
kb, and
kinact) were
dependent on pH and
temperature (see
below).
Influence of temperature.
Table 1 and Fig.
2 demonstrate the influence of
temperature at a constant pH of 6.5 on the different parameters used in
the model. They indicate that at a pH of 6.5 both bacteriocin and cell
mass production were optimal at a temperature between 20 and 25°C. At
35°C, no bacteriocin was produced and only 61% of the biomass
obtained at 20°C was achieved. Hence, the bacteriocin-producing isolate L. sake CTC 494 clearly showed mesophilic
growth behavior.
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FIG. 2.
Influence of temperature on the biomass production
(YX/S [] in grams of CDM per gram of
glucose, mS [ ] in grams of glucose per gram
of CDM per hour, and Xmax [] in grams of
CDM per liter) (a) and the bacteriocin production
(Bmax [] in arbitrary units per milliliter,
kb [] in arbitrary units [in thousands]
per gram of CDM, and kinact [] in liters
per gram of CDM per hour) (b) of L. sake CTC 494 in MRS
broth at a constant pH of 6.5. Lines are drawn according to the
model.
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Above 25°C the specific bacteriocin production decreased
rather rapidly as a function of temperature to become 0 at
34°C.
Furthermore, the degradation of bacteriocin activity
(
kinact)
became more important if temperature
increased, probably due to
a higher protease activity or
cell-bacteriocin or bacteriocin-bacteriocin
interaction. This
degradation of bacteriocin activity resulted
in low bacteriocin titers
when a fermentation temperature of more
than 30°C was
applied.
Figure
3 shows the relation between
temperature and maximum specific growth rate as calculated with the
model of Ratkowsky
et al. (equation 6). Cells grew faster with higher
temperature.
The cell yield, however, decreased because the energy
needed for
maintenance is higher when temperature increases.
Apparently,
the maintenance coefficient is correlated
(
r2 = 0.98) with the inverse of the cell yield
coefficient, as is
represented in Fig.
4.
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FIG. 3.
Influence of temperature on the maximum specific growth
rate (per hour) of L. sake CTC 494 in MRS broth at pH
4.5 (), pH 5.5 (), and pH 6.5 (). Lines drawn are according to
the model.
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FIG. 4.
Correlation between the cell yield coefficient
(YX/S [in grams of CDM per gram of glucose])
and the inverse of the maintenance coefficient
(mS [in grams of glucose per gram of CDM per
hour]) for growth of L. sake CTC 494 in MRS broth at
different temperatures.
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Influence of pH.
When temperature is kept at a constant value,
it is possible to investigate the effect of the acidity level on cell
growth and bacteriocin production. Table 2 and Fig.
5 show the values of the different
parameters used for modeling (µmax,
YX/S, mS, Xmax,
kb, and kinact) and of
the highest bacteriocin titer (Bmax) obtained. The fermentations were performed at a constant temperature of
30°C.
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FIG. 5.
Influence of pH on the biomass production
(YX/S [] in grams of CDM per gram of
glucose, mS [] in grams of glucose per gram
of CDM per hour, and Xmax [] in grams of
CDM per liter) (a) and the bacteriocin production
(Bmax [] in arbitrary units per milliliter,
kb [] in arbitrary units [in thousands]
per gram of CDM, and kinact [] in liters
per gram of CDM per hour) (b) of L. sake CTC 494 in MRS
broth at a constant temperature of 30°C. Lines drawn are according to
the model.
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Maximal cell yield was obtained between pH 5.5 and 6.5. Sakacin K
production was maximal at pH 5.0, but the acidity range
for optimal
bacteriocin production was rather narrow. With a fermentation
temperature of 30°C, bacteriocin titer appeared to be fairly low
at
pH values above 5.5 and undetectable at pH 4.5. The bacteriocin
inactivation rate increased when pH increased, probably because
of the
higher degree of adsorption to the
cells.
As shown in Fig.
6, cells grew fastest
between pH 6.0 and 6.5 while the theoretical minimum and maximum pHs
for growth were
4.06 and 8.40, respectively.
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FIG. 6.
Influence of pH on the maximum specific growth rate (per
hour) of L. sake CTC 494 in MRS broth at 20°C (),
25°C (), and 30°C (). Lines drawn are according to the
model.
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Model.
An empirical model was obtained from the experimental
data by combining the temperature and pH profiles. The model allows calculation of parameters for cell growth and bacteriocin production for a range of physiological pH and temperature (T) values
(20°C 
T 35°C; 4.5 pH 6.5). Eventually, calculated values that are negative have to be
set to 0.
where
e is the base of the natural logarithm. In
addition, equation 7 yielded a formula for the estimation of
µ
max:
The model gives satisfactory correlations (
r2 
0.93) with experimental values for all parameters (Table
3). The validation
of the model through
fermentations at 25°C with pH 5.5 and 23°C
with pH 5.0 was
successful, as predicted values corresponded well
with the experimental
measurements. However, the predicted value
for the specific bacteriocin
production rate,
kb, for the fermentation
at
23°C and pH 5.0 was far below expectations (2,950 instead of
8,010 kAU g of CDM
1). Apparently,
kb had
reached its upper limit and higher values
could not be achieved when
acidity and temperature were varied
under the given set of
environmental conditions.
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TABLE 3.
Comparison of calculated and experimental values for
µmax, YX/S, mS,
Xmax, Bmax,
kb, and kinact for
L. sake CTC 494 at a given combination of pH
and temperaturea
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DISCUSSION |
Lactobacilli and pediococci are naturally involved in various meat
fermentations and are consequently used as starter cultures (6). Most studies about the in situ behavior of bacteriocins against Listeria monocytogenes have been done with
bacteriocinogenic strains of Pediococcus acidilactici. This
species is the main starter culture used in the manufacture of
American-style fermented meat products. In Europe, fermented
sausages are manufactured with starter cultures containing
mainly L. sake and L. curvatus and,
to a lesser extent, Lactobacillus plantarum. Few
papers about the inhibition of Listeria monocytogenes during
meat fermentations by a bacteriocinogenic strain of L. sake have been published (22, 38). In addition, only
the competitiveness and inhibitory capacity towards Listeria
have been examined. There was no examination of bacteriocin production kinetics.
This paper describes a model for the production of biomass and
bacteriocin by the bacteriocin-producing meat isolate L. sake CTC 494. The modeled growth (determined with the logistic
equation) and sakacin K activity curves fit well with experimental
data. The assumption that bacteriocin titer is dependent on cell
growth was confirmed experimentally. Activity increases while
cells are growing exponentially and decreases when cell mass
production begins to level off. This leveling off occurs when
cell growth becomes inhibited due to the accumulation of lactic acid
and the exhaustion of sugar and possibly of essential amino acids
(26).
A combined model was elaborated from temperature and pH profiles in
order to predict both cell growth and bacteriocin production for any
combination of temperature and acidity level (between 20 and 35°C and
pH 4.5 and 6.5, respectively). The model was validated successfully by
two extra fermentations for all parameters (µmax, YX/S, YL/S, mS,
Xmax, and kinact) except for
the specific bacteriocin production rate (kb).
The experimental value (2,950 kAU g of CDM1) was far
below the predicted peak value (8,010 kAU g of CDM1)
(Fig. 7). Apparently, this is the highest
value one can practically achieve when the pH and temperature of
classical MRS broth are varied. This may be due to a limited immunity
of bacteriocin-producing cells. A modification might be made in the
model by introducing a cutoff value for kb of
2,950 kAU g of CDM1 as an upper limit.
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FIG. 7.
Surface model showing the influence of pH and
temperature (in degrees Celsius) on kb (in
arbitrary units [in thousands] per gram of CDM) before (a) and after
(b) introduction of an upper limit for this parameter.
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The parameters used in the model all appeared to be strongly influenced
by pH and temperature (Fig. 7 to
9).
However, in all such experiments, as the total amount of glucose is
quantitatively converted into lactate, the yield coefficient,
YL/S, for lactate production can be set equal to
1 g g1 for any combination of temperature and pH.
This means that L. sake CTC 494 is a homofermentative
LAB, which is a desirable feature for sausage starter cultures. Another
consequence is that apparently no glucose is used for the building of
the cell material, which is due to the facts that LAB metabolize sugars
predominantly to generate a necessary flux of biochemical energy but
that cell material is synthesized from nitrogen-containing organic
matter (7).
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FIG. 8.
Surface model showing the influence of pH and
temperature (in degrees Celsius) on
Xmax (in grams of CDM per liter) (a) and
µmax (per hour) (b).
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FIG. 9.
Surface model showing the influence of pH and
temperature (in degrees Celsius) on kinact (in
liters per gram of CDM per hour).
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The production of biomass and sakacin K is obviously related to
temperature. Maximal values are achieved at temperatures between 20 and
25°C. Interestingly, the fermentation of European sausages is
performed at similar temperatures (34). Schillinger obtained comparable results for L. sake Lb 706, stating that
optimal growth and sakacin A production occur at temperatures between
20 and 30°C (37). Sakacin K titer drops significantly as
temperature exceeds 25°C. This is due to the combined effect of a
lower specific productivity (kb) (Fig. 7) and a
higher bacteriocin degradation rate (kinact)
(Fig. 9). Although cells grow faster, cell yield also decreases with
higher temperatures (Fig. 8), because the energy needed for maintenance
becomes more important. Maintenance-generating processes, such as
turnover of macromolecules and maintenance of proton gradients, are
indeed strongly growth dependent (29). The maintenance
coefficient (mS) seems to be correlated with the inverse of the cell yield coefficient (YX/S).
The optimal pH for growth rate and biomass formation (pH 6.0) does not
correspond to the optimal pH for sakacin K production. This is because
the tendency to produce sakacin K (specific bacteriocin production) is
maximal at pH 5.0 (Fig. 7) and because the sakacin K inactivation rate
increases with pH due to greater adsorption to the cells (Fig. 9).
Adsorption is pH dependent (50) and nonspecific and probably
involves lipoteichoic acids (3). At 30°C, sakacin K
activity is highest at pH 5.0, 0 at pH 4.5, and rather low at pH 5.5, meaning that the pH range for good sakacin production is narrow. This
range, however, corresponds to the pH drop observed with the
fermentation of sausages (normally from a pH of about 5.8 to a final
value of approximately 4.8).
These results indicate that satisfactory sakacin K activity can be
expected when L. sake CTC 494 is used as a sausage
starter culture. Microbial growth and bacteriocin bioavailability in
situ will also be dependent on the influence of several other factors, such as the presence of (curing) salts (16), a limited
diffusion of both nutrients and bacteriocin in the sausage matrix, and
a lowered water activity of the microbial environment. This knowledge can be used to develop new protective and/or starter organisms with the
potential to improve both the hygienic status of the food and their
competitiveness in food fermentations. Hugas et al. have indeed
demonstrated a diminishment of Listeria number by 1.25 log
units in sausages fermented with L. sake CTC 494 compared to that of sausages fermented with a nonbacteriocinogenic
control strain (22). Considering both the antilisterial
capacities of the strain and the good sensorial quality of the final
product, one can postulate that L. sake CTC 494 has
high potential for industrial application as a novel
bacteriocin-producing sausage starter culture. In addition, previous
studies have shown that the addition of original isolates to the
sausage mixture results in the inhibition of staphylococcal growth,
mainly due to acid production (28). The combined action of
organic acids and bacteriocin might be effective against undesirable
opportunistic bacteria in fermented meat products.
In this paper, an attempt was made to model the production of biomass
and bacteriocin by the bacteriocin-producing isolate L. sake CTC 494. It was shown that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K. Although the experiments were carried out with in vitro cultures, the
research will contribute to a better understanding of the behavior of
the strain in the meat environment. Further work is in progress to
investigate the influence of the particular conditions prevailing in
fermented sausages (salt, curing agents, limited diffusion in the
matrix, low water activity, etc.) on sakacin K production and
L. sake CTC 494 growth.
| |
ACKNOWLEDGMENTS |
This work was financially supported by the Research Council of
the Vrije Universiteit Brussel, the Fund for Scientific
Research
Flanders, and the European Community (grant FAIR-CT97-3227).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division
of Industrial Microbiology, Fermentation Technology and Downstream
Processing (IMDO), Department of Applied Biological Sciences,
Vrije Universiteit Brussel, 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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Abstract
Introduction
