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Previous Article | Next Article 
Applied and Environmental Microbiology, August 2000, p. 3427-3431, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Factors Affecting Exocellular Polysaccharide
Production by Lactobacillus delbrueckii subsp.
bulgaricus Grown in a Chemically Defined
Medium
Sandrine
Petry,1,*
Sylviane
Furlan,1
Marie-Jeanne
Crepeau,2
Jutta
Cerning,1 and
Michel
Desmazeaud1
Unité de Recherches Laitières et
Génétique Appliquée, INRA, 78352 Jouy-en-Josas
Cedex,1 and Unité de Recherche
sur les Polysaccharides, leur Organisation et Interaction, INRA,
44316 Nantes Cedex,2 France
Received 23 December 1999/Accepted 16 May 2000
 |
ABSTRACT |
We developed a chemically defined medium (CDM) containing lactose
or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of
Lactobacillus delbrueckii subsp. bulgaricus.
The factors found to affect EPS production in this medium were oxygen,
pH, temperature, and medium constituents, such as orotic acid and
the carbon source. EPS production was greatest during the
stationary phase. Composition analysis of EPS isolated at different
growth phases and produced under different fermentation conditions
(varying carbon source or pH) revealed that the component sugars were
the same. The EPS from strain L. delbrueckii subsp.
bulgaricus CNRZ 1187 contained galactose and glucose, and
that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the
relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus
strains produced as much EPS in the CDM as in milk. Furthermore, the
relative proportions of individual monosaccharides of EPS produced in
pH-controlled CDM or in milk were very similar. The CDM we developed
may be a useful model and an alternative to milk in studies of EPS production.
| |
INTRODUCTION |
Production of exopolysaccharides
(EPS) by lactic acid bacteria in milk is an important factor in
assuring the proper consistency and texture of fermented food
(14). These heteropolysaccharides are composed of linear and
branched repeating units varying in size from tetra- to
heptasaccharides. The final EPS of high molecular mass (1 × 106 to 2 × 106 Da) is formed by
polymerization of several hundred to a few thousand of these repeating units.
Using milk as a fermentation medium, EPS yields range from 50 to 425 mg/liter (2, 3, 4, 10, 16). Although milk medium is relevant
to the food industry, EPS isolation from such a complex medium is
tedious and time-consuming. Furthermore, EPS purification is hindered
by glycohydrolases present in the crude preparations that are capable
of degrading EPS. MRS, the usual medium for laboratory fermentation
using Lactobacillus delbrueckii subsp.
bulgaricus, contains compounds (e.g., beef extract, peptone, yeast extract) that interfere with the analysis of EPS (12). Semidefined medium developed by Kimmel and Roberts (12) and chemically defined medium (CDM) developed by Grobben et al.
(11) circumvent the problem of interference, but the ability
of these media to support growth and EPS production of various strains of L. delbrueckii subsp. bulgaricus has not been
evaluated, and no comparison between EPS produced in milk and in the
defined medium is available.
In this work, we developed a CDM for two strains of L. delbrueckii subsp. bulgaricus for which EPS
production and composition in milk have been previously studied. We
examined the influence of medium constituents and fermentation
parameters on growth, EPS yield, and sugar composition. Our results
show that relative monosaccharide ratios in EPS are affected by pH and
carbon sources. As expected, results were strongly strain
dependent. In addition, we describe CDM conditions under which
the relative monosaccharide composition of EPS is very similar to that
of EPS extracted from milk.
| |
MATERIALS AND METHODS |
Bacterial strains.
EPS-producing L. delbrueckii subsp. bulgaricus strains CNRZ 1187 and CNRZ 416 were obtained from the freeze-dried culture collection of
the Institut National de la Recherche Agronomique (Jouy-en-Josas,
France). CNRZ 1187 is a natural isolate of Greek homemade fermented
milk; CNRZ 416 was initially isolated from a commercial starter. These
strains were transferred to 10 ml of litmus milk and stored at
20°C.
CDM.
CDM contains (per liter of distilled water) 30 g
of lactose or glucose; 4.0 g of sodium acetate; 1.0 g of
tri-ammonium citrate; 2.0 g of KH2PO4;
2.0 g of K2HPO4; 0.5 g of
MgSO4 · 7H2O; 0.05 g of
MnSO4 · 1H2O; 0.02 g of
FeSO4 · 7H2O; 0.2 g of
CaCl2; 20 mg of adenine; 40 mg of xanthine; 0.4 g of
cysteine; 0.3 g of aspartic acid; 0.3 g of glutamic acid;
0.2 g of each the following amino acids: alanine, arginine,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophane, tyrosine, and
valine; 0.5 g of orotic acid; 0.5 mg of p-aminobenzoic
acid; 0.5 mg of folic acid, 2.0 mg of nicotinic acid; 2.0 mg of
Ca-pantothenate; 1.0 mg of biotin; 2.0 mg of pyridoxal; 2.0 mg of
riboflavin; and 1.0 mg of vitamin B12. The medium was autoclaved at 110°C for 10 min.
Fermentation conditions.
Batch fermentations without pH
control were carried out in N2-flushed 100-ml screw-cap
flasks with 80 ml of CDM for 96 h of incubation at 42°C. The
fermentations controlled at pH 6.0 (the pH was maintained at 6.0 by the
addition of 10 N NaOH) were carried out in a 2-liter fermentor (model
SET002M; NBS SARL-IncelTech; Toulouse, France) containing 1,500 ml of
CDM under slight agitation (150 t/min) for 40 h of incubation at
42°C. Cultures were inoculated with an 8-h preculture in CDM (1:20,
vol/vol). For tests with 5, 10, 20, 30, and 40 g of glucose or
lactose per liter without pH control, samples were taken in the
stationary phase after 48 h of incubation at 42°C.
Samples were examined (as relevant) for growth, pH determination,
glucose utilization, and EPS isolation. After incubation the cultures
were kept on ice, and cell numbers were determined microscopically by
counting individual cells after methylene blue staining
(15). The resulting values were expressed as direct microscopic counts of cells per milliliter. The pH was measured with a
pH meter equipped with an Ingold electrode. Glucose consumption was analyzed with the glucose-oxidase, peroxidase
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) kit
(Boehringer Mannheim, Meylan, France).
For fermentations in milk medium, reconstituted nonfat dry milk (10%,
wt/vol) was sterilized at 110°C for 10 min. Eighty-milliliter aliquots contained in 100-ml screw-cap flasks were inoculated with an
8-h preculture in milk (1:100, vol/vol) and incubated over different
periods at 42°C.
Protein determination.
Contaminating protein was analyzed by
the Pierce test with a bicinchonic acid kit (Pierce, Rockford, Ill.)
(data not shown). EPS extracts derived from CDM cultures in which the
carbon source and time of harvesting were varied contained between 20 and 57% protein and between 13 and 64% EPS. (In contrast, the EPS
yield represents only ~1% of similar extracts when a milk medium is used [unpublished data].) Presumably, Lactobacillus
strains secrete proteins that accumulate in the CDM. However, as
protein contamination does not disturb phenol sugar determination and
gas-liquid chromatography of alditol acetates, the presence of protein
in the initial extract is readily overcome.
EPS isolation and total sugar determination.
EPS was
isolated by ethanol precipitation as described (4), but the
precipitation method was simplified for the culture in CDM. After
removal of cells by centrifugation (16,000 × g for 10 min), the crude EPS was precipitated at 4°C by addition of 2 volumes
of ethanol (100%). The resulting precipitate was collected after
centrifugation (16,000 × g for 15 min) and redissolved
in water. The crude EPS solution was dialyzed at 4°C. The total sugar concentration was determined by the phenol-sulfuric acid method using
glucose as a standard (8). Results are expressed as
milligrams of glucose per liter.
Monosaccharide composition of isolated EPS.
For quantitative
sugar analysis, EPS was hydrolyzed with 4 N trifluoroacetic acid at
110°C for 16 h. When a precipitate (corresponding to protein and
salt not completely eliminated by dialysis) appeared upon
alkalinization, it was eliminated by centrifugation before derivation
of the component sugars to alditol acetates (1). The alditol
acetate contents were determined (using inositol as an internal
standard) by gas-liquid chromatography on a fused-silica capillary
column (30 m by 0.32 mm). The column temperature was 210°C, the
injector temperature was 210°C, the detector temperature was 240°C,
the split rate was 60 to 80 ml/min, and the carrier gas was hydrogen
maintained at a pressure of 0.7 × 105 Pa. Results are
expressed as a percentage of the total EPS.
| |
RESULTS |
Adaptation of CDM for EPS production by L. delbrueckii subsp. bulgaricus strains.
L.
delbrueckii subsp. bulgaricus strains show
significant variation in their ability to grow in different defined
media. For this reason, Morishita et al. (13) previously
developed four basal media, each adapted to a different
Lactobacillus species (L. plantarum,
L. casei, L. helveticus, and L. acidophilus). Starting with basal media adapted for L. helveticus, we modified and further adapted the medium for
growth and EPS synthesis of L. delbrueckii subsp.
bulgaricus strains CNRZ 1187 and CNRZ 416. Note that,
unlike the CDM of Grobben et al. (11), our CDM does not
contain oligo-elements, thiamine, lipoic acid, guanine, uracil, or
Tween 80; however, it does contain orotic acid, which is
naturally present in milk (80 to 100 mg/liter).
The effects of different additives were tested, and results are
summarized as follows. Flushing the CDM with nitrogen had a stimulating
effect on both EPS production (resulting in a twofold increase) and
growth. The addition of calcium chloride increased growth, and addition
of orotic acid increased EPS yield almost threefold. Thiamine (2 mg/liter) added to CDM had a negative effect on both growth and EPS
yield. Uracil and guanine did not effect growth or EPS production,
while the addition of adenine and xanthine stimulated both growth and
EPS synthesis. The optimal growth temperature of 42°C was also
optimal for EPS yield (results not shown). The CDM was modified to
optimize EPS production and is used as described in Materials and
Methods in experiments presented below.
Growth characteristics, glucose consumption, and EPS production in
CDM without pH control.
Under the conditions tested (see Materials
and Methods), growth of L. delbrueckii subsp.
bulgaricus CNRZ 1187 attained a maximum population of
109 cells/ml after 24 h, with a 16-h lag phase.
L. delbrueckii subsp. bulgaricus CNRZ 416 attained 9.5 × 108 cells/ml after 38 h, with a
20-h lag phase. Cultures of strain CNRZ 1187 were nearly fully
acidified at 48 h (pH 3.6) and reached a final pH of 3.5 at
96 h. The pH of the medium with strain CNRZ 416 was higher than
that of CNRZ 1187 after 48 h (pH 4.0), and a final pH of 3.7 was
reached after 96 h (Fig. 1). Under
these fermentation conditions, glucose utilization was slightly higher (11.5 g/liter) by strain CNRZ 1187 than by strain CNRZ 416 (10.0 g/liter) (Fig. 1). During exponential growth, glucose consumption was
only 3.5 g/liter for strain CNRZ 1187 and 2.0 g/liter for CNRZ 416 (Table 1).
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FIG. 1.
Growth, pH, EPS production, and glucose
utilization by L. delbrueckii subsp.
bulgaricus. Strains CNRZ 1187 and CNRZ 416 were grown in
non-pH-regulated CDM (30 g of glucose per liter) at 42°C. Each value
represents the average of three measurements. The maximal deviation
between the three measurements was 5% for growth and for glucose
utilization and 10% for EPS production. Symbols:  , growth; ×, pH;
 , EPS production;  , glucose utilization.
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|
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TABLE 1.
Glucose consumption and EPS production in L. delbrueckii subsp. bulgaricus strains under
various conditions
|
|
The timing of EPS synthesis was examined for the two strains, according
to growth phase and medium. EPS production was almost 40 mg/liter after
72 h of fermentation for strain CNRZ 1187, while the same EPS
yield was reached after 48 h of fermentation for strain CNRZ 416 (Fig. 1). In CDM, half of the total EPS was produced during the
exponential phase for strain CNRZ 1187, compared to only one-third in
milk (2). Strain CNRZ 416 produced one-third of the total
EPS during the exponential phase, compared to only one-quarter in milk
(Table 1). Despite these differences, it appears that for both strains,
a majority of EPS is produced during the stationary phase.
Effect of glucose concentration and pH control on EPS
synthesis.
For strain CNRZ 416 without pH-controlled conditions,
10 g of glucose per liter seemed to be the optimal carbon source
concentration for the highest EPS yield. The EPS yield of strain CNRZ
1187 appeared not to be affected by the sugar concentration of the
medium (Fig. 2). Furthermore, the EPS
yield of the two strains was higher with glucose than with lactose
in the medium (results not shown).
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FIG. 2.
Effect of initial glucose concentration on EPS produced
by L. delbrueckii subsp. bulgaricus strains grown
in non-pH-regulated CDM. Each value represents the average of three
measurements. The maximal deviation between the three measurements was
10%. Solid bars, strain CNRZ 1187; open bars, strain CNRZ 416.
|
|
Growth under pH-controlled conditions resulted in a
higher yield of EPS than that under non-pH-regulated
conditions. Under pH-controlled conditions, the EPS yield of
strain CNRZ 1187 was about 110 mg/liter, which is almost three times
more than the amount obtained without pH control. For strain CNRZ 416, the EPS yield was 175 mg/liter, which is about four times more than the amount obtained without pH control (Fig.
3). For both strains, the EPS yields in
CDM under pH-controlled conditions (pH 6) were about the same as those
in milk. Strikingly, most EPS was produced at the end of growth, and
95% (CNRZ 1187) and 85% (CNRZ 416) of the glucose was consumed in the
stationary phase (Table 1). This correlation suggests that the glucose
consumed at this late phase is used for the production of EPS. Taken
together, the above results indicate that a majority of EPS is produced
during stationary phase, regardless of the medium.
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FIG. 3.
Growth, EPS production, and glucose utilization by
L. delbrueckii subsp. bulgaricus. Strains CNRZ
1187 and CNRZ 416 were grown in CDM (30 g of glucose per liter) under
pH-controlled (pH 6.0) fermentation conditions at 42°C. Each value
represents the average of three measurements. The maximal deviation
between the three measurements was 5% for growth and for glucose
utilization and 10% for EPS production. Symbols: , growth; , EPS
production; , glucose utilization.
|
|
Monosaccharide analysis of EPS.
The EPS produced by
L. delbrueckii subsp. bulgaricus
strains CNRZ 1187 and CNRZ 416 are heteropolysaccharides composed
mainly of galactose and glucose. Rhamnose was only found in the EPS
from strain CNRZ 416. Mannose and arabinose were sometimes present, but
in much smaller proportions. The presence of these sugars was
independent of the medium and carbon source used for growth. The
proportions of the different monosaccharides expressed as a percentage
of the total were, however, quite different (Table 2).
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TABLE 2.
Relative monosaccharide composition of EPS produced by
L. delbrueckii subsp. bulgaricus strains under
various conditions
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|
For example, for strain CNRZ 1187, the proportions of
monosaccharides in EPS produced either in milk or in CDM without pH control varied as a function of the growth phase but not of the carbon
source. In CDM with the pH controlled at 6.0, the monosaccharide proportions remained constant for EPS produced during the exponential and stationary phases and were very similar to those of EPS produced in
milk (Table 2). For strain CNRZ 416, the monosaccharide proportions of
EPS produced in CDM or in milk were influenced by the growth phase but
also by the carbon source in CDM without pH control when glucose or
lactose was used as the carbon source. The monosaccharide proportions
of EPS in CDM with the pH controlled at 6.0 gave results similar to
those obtained in milk (Table 2). The above results show that
conditions can be determined for CDM in which the L. delbrueckii subsp. bulgaricus EPS monosaccharide
composition is similar to that in milk medium.
| |
DISCUSSION |
In this study, we designed a CDM that allows growth of several
L. delbrueckii subsp. bulgaricus strains and
allows high levels of EPS production when the pH is controlled.
Previous studies showed that L. delbrueckii subsp.
bulgaricus strain CNRZ 1187 (2) and CNRZ
416 (4) produce EPS during milk fermentation. The same
strains also produce EPS in CDM with glucose or lactose as the carbon
source, but in lower amounts if the pH is not controlled. A lower EPS
yield in CDM compared to that milk was observed for L. delbrueckii subsp. bulgaricus strain NCFB 2772 by
Grobben et al. (11). We proposed that the higher EPS yields
in milk during fermentation are due to small amounts of peptides
liberated from milk proteins by the action of bacterial proteases
(7). Our results show that when the pH was maintained at
6.0, EPS yields were the same as those obtained in milk for both
strains tested. This CDM may prove useful for biochemical analysis or
for screening L. delbrueckii subsp.
bulgaricus strains for the capacity to produce EPS. We noted
that even after dialysis, the crude EPS preparations isolated from CDM
contain protein produced by the bacteria, but in lesser amounts than
that isolated from milk. In CDM, this contamination did not interfere
with EPS measurements (see Materials and Methods), so the determination
of monosaccharide composition was considerably simplified.
Our results are consistent with previous reports (10, 11)
showing that L. delbrueckii subsp. bulgaricus
needs maximum bacterial growth for optimal EPS yields. EPS production
by a strain of L. delbrueckii subsp. bulgaricus
was previously reported as being growth related, but no EPS
synthesis was observed after cell growth had ceased (11). In
sharp contrast to those previous results, most of the EPS synthesis and
glucose utilization of the two strains tested here occurred in the
stationary phase regardless of culture medium. For mesophilic lactic
acid bacteria, growth at temperatures below 30°C is known to enhance
EPS production (5, 6, 17). This is consistent with a
proposed mechanism in which slow-growing cells exhibit much slower cell
wall synthesis and accordingly have a decreased need for isoprenoid
phosphate. As a consequence, more isoprenoid phosphate could be
available for EPS synthesis (18). Thus, growth and EPS
production may be in competition for lipid carriers. A possible
interpretation of our results in thermophilic lactic acid bacteria is
that isoprenoid phosphate carriers are primarily needed for cell wall
synthesis growth. Upon cessation of growth, there is a greater
availability of this intermediate for EPS synthesis. This may explain
our finding that EPS production is increased in stationary phase cells.
It has been shown with L. casei (6) and L. rhamnosus (9), both mesophilic acid bacteria, that the
presence of excess sugar in the medium (at concentrations between 10 and 20 g/liter) had a stimulating effect on EPS production,
although growth was apparently reduced. The L. delbrueckii
subsp. bulgaricus strains in this study did not show this
effect. For strain CNRZ 416, the optimal glucose or lactose
concentration for both growth and EPS production is 10 g/liter, while
strain CNRZ 1187 showed no influence of sugar concentration on EPS
yield. Furthermore, the two strains did not show the same behavior with
two different carbon sources; for strain CNRZ 416, the proportion
of the sugar constituents varied as a function of the carbon source,
while for the strain CNRZ 1187, it did not. These differences may
highlight variations in EPS regulation and synthesis in different strains.
It is important to know for future studies whether the EPS produced
from a particular strain of L. delbrueckii subsp.
bulgaricus in CDM is the same as that produced in milk. For
this, different characteristics must be considered, including
identification of the component monosaccharides, determination of their
relative proportions, and determination of the physicochemical
properties of the final EPS. From the present study, it can be
concluded that independently of medium, carbon source, and fermentation parameters, the same monosaccharides are present in the different EPS
extracted for one strain, i.e., galactose and glucose for strain CNRZ
1187 and galactose, glucose, and rhamnose for strain CNRZ 416. However,
the relative proportions of monosaccharides do vary, indicating that
the primary structures of EPS repetitive units are different according
to the strain. The advantage of pH-controlled conditions is the
considerable increase in EPS yield, as well as the monosaccharide
proportions that are similar to what is obtained in milk medium.
Physicochemical properties of the different EPS are under investigation
to determine whether the different EPS produced in CDM can be compared
in terms of structure and thickening properties.
| |
ACKNOWLEDGMENTS |
We are grateful to Alexandra Gruss from the Unité de
Recherches Laitières et Génétique Appliquée,
INRA, Jouy-en-Josas, France, for her help in the reading of the
manuscript. We also thank the Unité de Recherches sur la viande,
INRA, Jouy-en-Josas, France, for lending of the 2-liter fermentor
(SET002M; NBS SARL-IncelTech) and Catherine Béal from the
Laboratoire de Génie et Microbiologie des Procédés
Alimentaires, INRA, Paris-Grignon, France, for her help in
pH-controlled experiments.
This research was supported by a scholarship to Sandrine Petry from the
Department of Chemistry of the University College Dublin, by Zeneca,
and by EU contract FAIR-CT97-3098.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Recherches Laitières et Génétique Appliquée,
INRA, 78352 Jouy-en-Josas Cedex, France. Phone: 33 1 34 65 21 66. Fax:
33 1 34 65 20 65. E-mail: petry{at}jouy.inra.fr.
This paper is dedicated to Jutta Cerning, who passed away on 3 October 1999.
| |
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Applied and Environmental Microbiology, August 2000, p. 3427-3431, Vol. 66, No. 8
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
