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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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