Centro de Referencia para Lactobacilos (CERElA), Chacabuco 145,. S. M. de Tucuman, Tucum&-r, 4000- Argentina. 2. Catedra de Microbiologia Superior, ...
BIOTECHNOLOGY LETTERS Volume 18 No.4 (April 1996) p.435-439 Received as revised 19th February.
EXOPOLYSACCHARIDE
PRODUCTION
BY LACTOBAClLLUS
CONTROLLED
Fernanda
Mozzil,
Graclela
CASE/ UNDER
PH
S. de Glorll g2, G. Oliver1 and Graclela de Valdezl
F.
sp
1. Centro de Referencia para Lactobacilos (CERElA), Chacabuco S. M. de Tucuman, Tucum&-r, 4000- Argentina
145,
2. Catedra de Microbiologia Superior, Facultad de Bioquimica, Quimica y Farmacia, Universidad National de Tucuman, Argentina. SUMMARY The exopolysaccharide (EPS) production and growth characteristics of Lactobacillus casei CRL 87 under pH control were studied. Maximum polymer synthesis (488 mg/l) and cell viability (2.4~10~~ cfu/ml) occurred when L. casei was cultured at a constant pH of 6.0 and 30°C for 24 h. However, the optimum specific EPS production (3.9x10m5 g EPS/g cell dry weigt) and EPS yield (4.3%) were found at a pH of 4.0.
INTRODUCTION Some strains of lactic acid bacteria, as well as other microorganisms, under
certain
environment
culture
conditions,
during fermentation.
consistency
exocellular
polysaccharides
are capable
of synthesizing
(EPS) that are released
The presence of these polymers in fermented
and viscosity of the final product and decreases the susceptibility
to the
milks improves
to syneresis
the
(Cerning,
1990). The amount culture conditions. carbohydrates
of microbial
Exopolysaccharide
and low temperatures
also affect the EPS production. on the polysaccharide
whether
on the slime-producing
is generally
(Sutherland,
favoured
strain and the
by the excess
of nutrient
1977; Kojic et al., 1992). Mineral requirements
by L. casei CRL 87 (Mozzi et al., 1995).
effect
Culture pH is also a
(1958) reported that pH values near neutrality are the best ones for most of the EPS synthesis by a strain of L. sake on the pH was
by Van der Berg et al. (1995).
It was previously production
formation
strains. The dependence
clearly demonstrated
depends
It has recently been reported that MnS04 exerts a stimulatory
production
critical factor. Wilkinson slime-producing
EPS produced
observed in our laboratory that the optimal initial pH for exopolysaccharide
by L. casei CRL 87 was 6.0-6.5 (Mozzi et al., 1994). The question has been raised as to or not controlling
polysaccharide
synthesis.
the pH of the culture medium will have a beneficial
effect on the
In this work the findings from the EPS production as well as the growth
435
characteristics
of L. casei CRL 87 when it was cultured at different values of constant pH will be
reported.
MATERIALS AND METHODS Microorganism: Lacfobacillus casei CRL 87 used in this study was obtained from the CEREIA culture collection and was previously isolated from a regional cheese. Stock cultures were maintained in 10% sterile non-fat-skim milk at -20°C. Growth conditions: L. casei CRL 87 was grown in a suitable culture medium for EPS synthesis designated MSE which contained peptone, 15%; Tryptone, 1.0%; galactose, 2.0%; MnS04.4H20, 0.005%; CaCl2, 0.35%; Tween 80, 0.1%. The pH was adjusted to 6.0 after sterilization at 121’C for 15 min. MnS04.4H20, CaC12 and galactose were separately sterilized and added to the broth to reach the right final concentration. A 4% (v/v) inoculum and 24 h incubation at 30°C were the culture conditions used. Each culture was subcultured at least three times just prior to experimental use. Fermentations were performed in a 2.0 I fermenter containing 1.8 I of the culture medium described above. The temperature was maintained at 3O”C, the pH was continuously adjusted to 6.0, 5.0 or 4.0 using sterile 1 M DL lactic acid and/or sterile 1M ammonium hydroxide. No air was added and agitation speed was maintained at 20 rpm The culture medium was inoculated with a 16 h active culture at the rate of 4% (v/v) (about lo8 cfu/ml), and fermentation was allowed to proceed for 72 h. Samples were aseptically drawn after 24, 48 and 72 h and cooled in ice water before the assays. Cell viability:
Cell viability was determined by the plate dilution method using the culture medium mentioned above supplemented with 1.2% agar (sterilized at 121°C for 15 min). Serial dilutions of each sample were plated in duplicate and plates were incubated at 37°C for 48 h. Results were expressed as colony forming units (cfu)/ml. A correlation between cfu and biomass produced was determined by measuring cell dry weight (cell dry wt of the culture at different stages of the growth curve. The calculated conversion factor was 3.2~10~A.
Exoceiiuiar poiysaccharide (EPS) isolation: The soluble EPS from the cell-free supernatant obtained by centrifugation (16,000 x g, 30 min at 4°C) was precipitated by adding 3 volumes of cold 95” ethanol followed by an overnight storage at 4°C. The precipitates, collected by filtration, were redissolved in distilled water, dialysed against the same solution at 4°C during 24 h to eliminate residual sugars from the culture medium (water was changed four times during that period) and then freeze-dried and stored at 4°C. Total polysaccharides were estimated by the phenolsulphuric method (Dubois et al., 1956) using glucose as a standard. The total EPS production was expressed as mgA, the specific EPS production (Ypjx), calculated by dividing the amount of EPS produced by the cell dry weight, was expressed as g EPSIg cell dry wt and the EPS yield (VP/& the amount of EPS produced divided by the amount of sugar consumed, was expressed as %. Residual determined
sugar determination:
The amount of residual sugars by the phenol sulphuric method (Dubois et a/., 1956).
in the culture
medium
was
Repeatability: all results presented in this paper are the mean of 3 replicate assays. RESULTS AND DISCUSSION Figure 1 shows the EPS production and the growth characteristics to the pH. The maximum which remained observations
constant
polysaccharide
synthesis was obtained at pH 6.0 with a value of 488 mg/l,
until the end of the fermentation
made during fermentations
of L. casei CRL 87 with respect
without
436
(72 h) (Fig. la). This differs from the
pH control where the amount
of polymer
was
reduced when the incubation time extended beyond 24 h (Mozzi et al., 1994). This would indicate a higher EPS stability, synthesized
under a controlled pH.
Fig. 1. Exopolysaccharide
production
of L. casei under
and growth
characteristics
pH control.
a
C
b
48
-24
time
log
72
u 24
48
(d)
time
time (d) .
The kinetics of polysaccharide
72
48
24
72
(d)
pH 6.0; v pH 5.0 ; v pH 4.0
synthesis at pH 5.0 and 4.0 was notably different from what
was found at pti 6.0. At pH 5.0 the polymer production was highest (231 mg/l) after 48 h, which decreased
to a third after 72 h of incubation.
On the contrary, at pH 4.0, L. casei synthesized
315
mg/l after 48 h, without showing a reduction in the amount of EPS formed till the end of the studied period. The reduction in EPS observed at pH 5.0 might be due to the activation of certain degrading agents (glycohydrolases) and Townsley When fermentations maximum Similar
that are capable of degrading the polymer, as it was suggested
by Macura
(1984) and Cerning et a/. (1988). comparing without
the polysaccharide
production
of studies
with a similar
initial pH in
pH control (data not shown) and with pH control, it was found that the
amount of EPS formed in the latter ones was 3.6 times higher than in the former ones. results
have
been
reported
for Xanthomonas
campestris
and
Pseudomonas
strains
(Moraine and Rogovin, 1973; Marques et a/., 1986). Figure 1b-c shows the results of cell viability and residual sugar when L. casei CRL 87 was grown at different stationary conditions
pH values. A pH of 6.0 and an incubation time of 24 h were the optimal
for the growth of L. casei (2.4~10’~
cells decreased
gradually
cfu/ml). From this moment, the number of viable
until 48 h, reaching a minimum value of 1.6x1 0’ o cfu/ml. This behaviour
437
could be due to the nearly complete consumption incubation,
of the carbon source (91%) during the first day of
thus becoming a limiting growth factor (Fig. lc).
The count of viable cells at pH 5.0 was 50% less than that corresponding h, while the sugar consumption
to pH 6.0 after 24
was similar at both pH values. From that moment onwards only the
number of cells at pH 5.0 decreased
until 72 h.
At pH 4.0, the highest colony count (3.3~10~ cfu/ml) was obtained after 48 h. This could be explained
by the fact that, under these culture conditions,
themselves
to the acidity
phenomenon
of the environment,
was reflected
which
the microorganisms
determines
a slower
in the high amount of residual sugar observed
should
adapt
cell growth.
This
in the fermentation
medium (12.35 g/l after 24 h), which decreased during the incubation time (1.61 g/l after 72 h). When working under controlled pH, the amount of colonies increased with respect to that obtained in free fermentations neutralization
of the lactic acid, accumulated
the amount of cells. Stadhouders
one logarithmic
unit
(data not shown). This was due to the fact that
in the culture medium, resulted in a multiplication
of
et a/. (1969) also found that it was possible that the biomass,
obtained for certain strains of streptococci,
was multiplied by a factor of 10 by controlling
the pH of
the culture medium. Table 1 shows the values for the efficiency of EPS calculated (Yph) and the substrate consumption
c/,/s).
in function of the cell viability
The specific EPS production
the culture medium was adjusted tp pH 4.0, reaching the maximum which was due to a lower cell development highest efficiency
of the conversion
pH, but after a fermentation polysaccharide observations
synthesis
observed
(VP/,) was higher when
value after 72 h (3.9x10e5),
under those culture conditions
(Fig. 1b). The
of sugars into polymer (VP/,= 4.3%) was also obtained at this
of 24 h. These
results
is highest under unfavourable
indicate
that the cell efficiency
in the
conditions for the cell growth (pH 4.0). Our
do not agree with those found by Heald and Kristiansen
(1985) who have shown that
an increase of the pH from 3.5 to 6.3 resulted in a higher polysaccharide
yield by Aureobasidium
pulhlans.
Table 1. Specific production and yield of exopolysaccharide
by Lactobacillus
casei CRL 87 grown
under controlled pH.
Yp/x time 24 48 72
(h)
6.0
x 10-6 PH 5.0
4.0
5.9 9.1 9.1
3.7 12.5 4.7
15.9 30.0 39.0
Yph: Specific EPS production
=p/s
(g EPS/g cell dry wt)
VP/,: EPS yield (g EPS x 100/g consumed sugar)
438
(8)
6.0
PI-' 5.0
4.0
3.4 3.0 3.5
1.0 1.6 0.5
4.3 2.7 2.3
In spite of a higher polymer production by L. caseiCRL with respect to that determined
in free fermentations
(Maui
87 obtained under a controlled et al. 1994) the maximum
(Ypjs) was similar in both cases. Moraine and Rogovin (1971) established of the fermentation Xanthomonas decrease
medium,
campestris
in the polymer
efficiency
due to the deviation
However,
efficiency
that by controlling the pH
the amount of the usable glucose and the xanthan
NRRL B-1459 can be duplicated.
pH
production
by
these authors observed
of the carbon source towards
a
a higher
biomass production. The results obtained let us conclude, that at a constant pH of 6.0, the EPS production caseiCRL polymer
87 is linked to the biomass while at lesser pH values (4.0) a higher cell efficiency synthesis
exopolysaccharide
is observed. production
Future
work will
be focused
and yield by this microorganism
on the
improvement
to use this polymer
by L. in the of the
as a possible
food additive.
ACKNOWLEDGEMENTS The authors supported (CONICEl),
by PID-BID
thank Mr Oscar Peinado for technical N” 314 from Consejo
National
assistance.
de lnvestigaciones
This study was partially Cientfficas
y Tdcnicas
Argentina.
REFERENCES Cerning, J. (1990) FEMS Microbial. Rev. 87, 113-130. Cerning, J., Bouillane, C., Desmazeaud, M. and Landon, M. (1988) Biotechnol. Lett. 10, 255260. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Anal. Chem. 28, 350356. Heald, P.J. and Kristiansen, B. (1985) Biotechnol. Bioeng. 27, 1516-l 520. Kojic, M., Vujcic, M., Banina, A., Cocconcelli, P., Cerning, J. and Topisirovi C, L. (1992) Appl. Environ. Microbial. 58, 4086-4068. Macura, D. and Townsley, P.M. (1984) J. Dairy Sci. 87, 735-744. Marques, A.M., Estariol, I., Alsina, J., Fuste, C., Simon-Pujol, D., Guinea, J. and Congregado, F. (1986) Appl. Environ. Microbial. 52, 1221-1223. Moraine, R.A. and Rogovin, P. (1971) Biotechnol. Bioeng. 13, 381. Moraine, R.A. and Rogovin, P. (1973) Biotechnol. Bioeng. 15, 229-238. Maui, F., S. de Giori, G., Oliver, G., F. de Valdez, G. (1994) Milchwissenschaft49, 667-670. Mozzi, F., S. de Giori, G., Oliver, G., F. de Valdez, G. (1995) Milchwissenschaft 50, 186-188. Stadhouders, J., Jansen, L.A. and Hup, G. (1969) Netherlands Milk Dairy 23, 182-199. Sutherland, I.W. (1977) Bacterial exopolysaccharides-their nature and production. In: Surface Carbohydrates of the Prokaryotes ce//, I. W. Sutherland, ed. Academic Press, New York. Van den Berg, D.J.C., Robjin, G.W., Janssen, A.C., Giuseppin, M.L.F., Vrekker, R., Kamerling, J.P., Vliegenthart, J.F.G., Ledeboer, A.M. and Verrips, CT. (1995) Appl. Environ. Microbial. 61, 2840-2844. Wilkinson, J.F. (1958) Bacterial. Rev. 22, 46-69.
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