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Applied and Environmental Microbiology, September 1998, p. 3512-3514, Vol. 64, No. 9
Instituto de Productos Lácteos de
Asturias (IPLA-CSIC),
Received 7 November 1997/Accepted 5 July 1998
Plantaricin C, a bacteriocin synthesized by Lactobacillus
plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30°C, 150 rpm, and a dilution rate of 0.05 h During a screening for bacteria with
good technological properties, Lactobacillus plantarum LL441
was isolated from homemade cheese whey. It produces one bacteriocin,
plantaricin C (6), a 3.5-kDa peptide that is resistant to
harsh environmental conditions and active at pH values from 2.0 to 7.0. It is bactericidal, and in some cases bacteriolytic, to a range of
gram-positive organisms that include members of the genera
Clostridium, Bacillus, and Staphylococcus and also to Listeria monocytogenes
(6). Plantaricin C induces the formation of pores in the
plasma membrane of sensitive cells, resulting in the dissipation of the
proton motive force and in leakage of cytoplasmic solutes. No receptor
or energized membranes are needed for its action, explaining, in part,
its wide range of susceptible bacteria (7). All these
properties allow plantaricin C to be considered a potential food
preservative. To achieve this objective, one of the first requirements
is mass production of the bacteriocin under standardized conditions to ensure a regular supply for the different tests to be performed and,
eventually, for the market. We chose continuous culture as a first step
towards this goal.
L. plantarum LL441 was grown in MRS (4) broth to
stationary phase, concentrated 10-fold in fresh MRS containing 20%
(vol/vol) glycerol, and stored at Biomass was measured as the absorbance at 600 nm
(A600). Dry-weight determinations were carried
out after triplicate culture samples (3 ml each) were passed through
0.22-µm pore-size filters and dried at 85°C overnight. A
correlation between dry weight and A600 was
established to calculate the biomass concentration from the absorbance
readings.
Plantaricin C production was tested by the agar well diffusion test
(17), with Lactobacillus sake CECT 906 as the
indicator strain. The titer was defined as the reciprocal of the
highest dilution showing definite inhibition of growth of the indicator lawn and was expressed in arbitrary activity units per milliliter.
Glucose level was determined with the glucose (Trinder) kit (Sigma
Chemical Co., St. Louis, Mo.). Other sugars, lactate, acetate, and
formate were determined by high-pressure liquid chromatography with a
Beckman System Gold Liquid Chromatograph as previously described
(8).
Influence of pH on the production of plantaricin C.
To
establish the optimal pH value for plantaricin C production, a
chemostat was run at a dilution rate of 0.15 h Growth of L. plantarum LL441 in the chemostat.
In
order to determine the maximum productivity of the strain, steady
states were obtained at different dilution rates, with glucose as the
carbon source and the pH kept at 5.0 (Fig.
1). Calculation of the critical dilution
rate (0.62 h Influence of the dilution rate and carbon source on the production
of plantaricin C.
Plantaricin C production was tested at different
dilution rates, with glucose, sucrose, or fructose as a growth-limiting
substrate; all were fed at the time zero concentration of 0.5%
(wt/vol).
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Copyright © 1998, American Society for Microbiology. All rights reserved.
Chemostat Production of Plantaricin C By
Lactobacillus plantarum LL441
ABSTRACT
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References
1 when
glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h1 when sucrose or fructose was used instead. Production
was abolished at high dilution rates, i.e., when the cells grew rapidly
in all carbon sources.
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20°C. Aliquots (15 ml each) of
this culture were used every time the fermentor was inoculated. Unless
otherwise stated, the culture medium (BRFS) contained (in grams per
liter) yeast extract, 10; MgSO4 · 7H2O,
0.05; (NH4)2HPO4, 2.5;
MnSO4, 0.005; and the carbon source (glucose, sucrose, or
fructose), 5. In experiments during medium development, the growth rate
increased up to sugar concentrations of 20 g/liter (higher
concentrations were not tested), indicating that it was unlikely that
other medium components would be limiting when the carbon source was
used at 5 g/liter. Incubation was done in a chemostat Biostat B (Braun, Melsungen, Germany) at 30°C, with a working volume of 700 ml. The pH
was maintained by the automatic addition of 2 N NH4OH. The
cultures were stirred at 150 rpm. Cultures were started in batch mode.
When the population in the vessel reached the exponential phase,
continuous cultures were established by pumping in fresh medium,
starting at the lowest dilution rate. Steady-state growth, as assessed
by biomass, and glucose and lactate concentrations, was reached after
three to four residence times of fresh medium passed through the
chemostat. Steady-state conditions were maintained for at least two
more volume passages, and only afterwards was data collection started.
All values shown in the text are averages of triplicates that differed
by less than 5%. The dilution rates could be varied upwards or
downwards, resulting in constant product concentrations once steady
states were reestablished.
1, which
corresponds to about 20% of the maximal growth rate. The growth-limiting substrate, glucose, was fed at a concentration of 5 g/liter at time zero. The production of plantaricin C was evaluated in
steady state at pH values of 4.5, 5.0, 5.5, and 6.0. Plantaricin C was
detected only at pH 5.0. The pH seems to be a factor that affects
bacteriocin production by lactic acid bacteria (3, 5, 13,
14), possibly indicating that it has a regulatory effect driven
by the ionic conditions of the medium or that it may exert a general
effect on the cell envelope, since variations in the osmolarity of the
media (18) or in the concentrations of ethanol
(13) and Tween 80 (15) were shown to influence the levels of production of other bacteriocins.
1) corresponding to the maximal growth rate
was done by the method of Pirt and Callow (16). Maximum
biomass productivity (0.47 g [dry weight] liter1
h1) corresponded to a dilution rate of 0.33 h1, around 50% of the maximal growth rate
(µmax = 0.62 h1). This behavior seems to be
characteristic of lactobacilli (1, 9, 10). Lactate was the
only fermentation product that was detected among those we looked for
(lactate, acetate, and formate), and the fermentation balances were
close to 100% carbon recovery (data not shown).
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FIG. 1.
Effects of the dilution rate on biomass, glucose, and
lactic acid concentrations, and biomass productivity by L. plantarum LL441 grown in BRFS medium with 0.5% glucose at 30°C,
a pH of 5.0, and 150 rpm.
1), when the
bacteria were growing very slowly. This finding is in agreement with a
previous observation (6) in which plantaricin C was produced
at the end of the exponential growth phase in MRS broth when,
presumably, the substrate levels were declining, which in turn would
provoke a decrease in the growth rate of the cultures. Generally, it is
assumed that bacteriocins are produced during the exponential growth
phase; hence, their synthesis would be considered growth linked
(5, 14). If this was true, production would have resulted in
a line parallel to biomass concentration and productivity would be
maximal near the µmax. However, plantaricin C production
was observed only at very low dilution rates, i.e., when the cultures
grew slowly; consequently, production would probably be best considered
to be typical of a product synthesized during the
exponential/stationary growth transition phase, as was already
anticipated by batch experiments (see above). This situation might be
more general; for example, de Vuyst and Vandamme (5) found
that there is a lag in nisin production with respect to growth in
cultures of Lactococcus lactis incubated with sucrose as a
carbon source. Furthermore, the production of pediocin AcH by
Pediococcus acidilactici H (3) might follow
secondary metabolite kinetics although, as the pH was not controlled,
the data might be misleading, since pH presumably influences production
of the bacteriocin. On the other hand, production of lactococcin 140 by
L. lactis seems to be growth linked (14).
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[in a new window]
FIG. 2.
Effects of the dilution rate on biomass concentration
and plantaricin C production (arbitrary activity units) by L. plantarum LL441 grown in BRFS medium with 0.5% of different
carbon sources at 30°C, a pH of 5.0, and 150 rpm. AU, arbitrary
units.
1) double those determined to give significant
production with glucose, although further increases in the dilution
rate resulted in the abolition of bacteriocin synthesis. It was also
determined that the bacteriocin titers of the culture supernatants were
similar under optimal conditions for the three carbon sources tested, i.e., the yield was doubled in media with sucrose or fructose with
respect to glucose. The apparent difference between plantaricin C
production in media with glucose, sucrose, and fructose was confirmed
by analysis of variance (12), which showed significant differences (P < 0.01). However, no significant
differences were found between the production levels of these last two
sugars.
All homofermentative lactic acid bacteria examined so far possess
phosphoenolpyruvate-sugar phosphotransferase systems (2) and, consequently, glucose would inhibit the uptake of sugars via
phosphorylation of the heat-stable protein of the
phosphoenolpyruvate-sugar phosphotransferase systems system
(19). The availability of glucose increases with dilution
rate, which could lead to increased levels of phosphorylated products
which may trigger regulatory mechanisms, leading to a reduced glucose
intake and to the switching off of catabolite-sensitive operons. This
may be the case of plantaricin C, because media with sucrose or
fructose as carbon sources still supported its synthesis at
concentrations that did not allow synthesis when the sugar used was
glucose.
From the point of view of the producer strain, the synthesis of
antimicrobial substances would be advantageous precisely when nutrients
become limiting, because under these conditions, the bacteriocins
produced would antagonize potential competitors for the exhausting
nutrients, a concept discussed in early reviews on antibiotic
production (11). This situation reflects what occurs in
chemostats at low dilution rates, when ATP levels in the cell are low
and the regulatory mechanisms mediated by phosphorylated proteins would
not be activated.
Finally, the supernatant of the harvest bottle (collected in the cold
for 24 h) had the same titer as the medium in the chemostat, indicating that downstream processing can be made batchwise at least
every 24 h.
The results presented here raise the possibility of scaling up
plantaricin C production experiments to pilot plant assays. The
starting conditions would probably be a batch-fed system with a limited
carbon source, a controlled pH, and feed rates corresponding to 10 to
20% of the µmax.
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ACKNOWLEDGMENTS |
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This work was supported by grants ALI93-0873-CO2 and BIOT-CT96-0402 from the Comisión Interministerial de Ciencia y Tecnología of Spain and the BIOTECH Program of the European Union, respectively. F.S. was the recipient of a visitor's grant from the University of Oviedo. We thank the Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT) and UNESCO on its 50th anniversary.
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FOOTNOTES |
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* Corresponding author. Mailing address: Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, c/ Julián Clavería s/n, 33006 Oviedo, Spain. Phone: 34 8 510 35 59. Fax: 34 8 510 31 48. E-mail: jsuarez{at}sauron.quimica.uniovi.es.
Present address: PROIMI, 4000 San Miguel de Tucumán,
Argentina.
Present address: PROIMI and Cátedra de Microbiología
Superior, Facultad de Bioquímica, Química y Farmacia,
Universidad Nacional de Tucumán, 4000 San Miguel de
Tucumán, Argentina.
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Applied and Environmental Microbiology, September 1998, p. 3512-3514, Vol. 64, No. 9
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