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Appl Environ Microbiol, February 1998, p. 659-664, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Optimization of Exopolysaccharide Production by
Lactobacillus delbrueckii subsp. bulgaricus
RR Grown in a Semidefined Medium
Stacy A.
Kimmel,
Robert F.
Roberts,* and
Gregory R.
Ziegler
Department of Food Science, The Pennsylvania
State University, University Park, Pennsylvania 16802
Received 11 August 1997/Accepted 25 November 1997
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ABSTRACT |
The optimal fermentation temperature, pH, and Bacto-casitone (Difco
Laboratories, Detroit, Mich.) concentration for production of
exopolysaccharide by Lactobacillus delbrueckii subsp.
bulgaricus RR in a semidefined medium were determined by
using response surface methods. The design consisted of 20 experiments,
15 unique combinations, and five replications. All fermentations were
conducted in a fermentor with a 2.5-liter working volume and were
terminated when 90% of the glucose in the medium had been consumed.
The population of L. delbrueckii subsp.
bulgaricus RR and exopolysaccharide content were measured
at the end of each fermentation. The optimum temperature, pH, and
Bacto-casitone concentration for exopolysaccharide production were
38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions
was 354 mg of exopolysaccharide/liter, which was within the 95%
confidence interval (217 to 374 mg of exopolysaccharide/liter). An
additional experiment conducted under optimum conditions showed that
exopolysaccharide production was growth associated, with a specific
production at the endpoint of 101.4 mg/g of dry cells. Finally, to
obtain material for further characterization, a 100-liter fermentation
was conducted under optimum conditions. Twenty-nine grams of
exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.
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INTRODUCTION |
Interest in exopolysaccharide
(EPS)-producing lactic acid bacteria has increased recently because
these food grade organisms produce polymers important in determining
the rheological properties of dairy products and may have application
in nondairy foods. When added to food products, polysaccharides
function as thickeners, stabilizers, emulsifiers, gelling agents, and
water binding agents (24). Evaluation of the functional
attributes of potentially useful EPSs in foods requires that the
material be available in sufficient quantities. This generally requires
scale-up from bench to pilot scale fermentations once optimal
conditions have been identified.
Optimization of the growth environment is important to achieving
maximal EPS production by organisms such as Xanthomonas, Pseudomonas, and Rhizobium spp. (2, 19, 21,
23). Published efforts to optimize EPS production in lactic acid
bacteria have included studies evaluating the effects of such
environmental conditions as temperature and pH (6, 9, 10, 15, 16, 20, 28). A number of these studies examined the effect of temperature by using various EPS-producing strains of
Lactobacillus delbrueckii subsp. bulgaricus.
Garcia-Garibay and Marshall (9) found that specific polymer
production (equivalent milligrams of dextran per CFU) by strain NCFB
2772 grown in skim milk was greater at a temperature (48°C) higher
than the optimum for growth (37 to 42°C). Greater specific polymer
production (milligrams per gram of cells [dry weight]) at a higher
temperature (45°C) was also found by Grobben et al. (10),
who examined EPS production by the same strain in defined medium. Mozzi
et al. (16) found a correlation between the optimum growth
temperature (37 to 42°C) and maximum polymer production by strain CRL
870. In contrast, Schellhaass (20) reported higher polymer
production by strain RR at temperatures below the optimum for growth.
Van den Berg et al. (28) conducted a study evaluating the
effects of temperature and pH on EPS production by L. sake
0-1. They found, by using a one-variable-at-a-time (OVAT) approach, that optimal EPS production occurred at 20°C and pH 5.8. Another study by Mozzi et al. (15) found that maximum polymer
synthesis (488 mg/liter) by L. casei CRL 87 at 30°C
occurred at pH 6.0. However, optimum specific production (EPS produced
per gram [dry weight] of cells) and EPS yield (grams of EPS × 100/grams of sugar consumed) were found at pH 4.0.
For some EPS-producing bacteria, such as Xanthomonas,
Pseudomonas, and Rhizobium spp.,
nitrogen-limiting conditions result in increased EPS production
(3, 25). The effect of nitrogen concentration on EPS
production by lactobacilli has not been examined.
L. delbrueckii subsp. bulgaricus RR is a common
EPS-producing strain. The effect of using L. delbrueckii
subsp. bulgaricus RR as a starter culture on the rheological
properties of milk gels (yogurt) has been examined under various
conditions (12, 20, 26, 27). In addition, the monosaccharide
composition and repeating structure of the EPS produced by L. delbrueckii subsp. bulgaricus RR have been determined
(11). However, characterization of the interaction between
milk proteins and the EPS produced by L. delbrueckii subsp.
bulgaricus RR, as well as evaluation of the functional
effects of the EPS in nonfermented food systems, has been hampered by
the unavailability of sufficient quantities of the polymer.
The objectives of the present work were to determine the optimum
temperature, pH, and Bacto-casitone (nitrogen; Difco Laboratories, Detroit, Mich.) concentration for production of EPS by L. delbrueckii subsp. bulgaricus RR in a semidefined
medium by using a response surface experimental design and to use these
conditions to produce EPS for further characterization.
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MATERIALS AND METHODS |
Bacterial strain and culture preparation.
L.
delbrueckii subsp. bulgaricus RR was originally
obtained from the laboratory of H. A. Morris (University of
Minnesota, St. Paul) and maintained in MRS broth (Difco). Stock
cultures were prepared by mixing a pure culture grown for 12 to 16 h at 42°C in MRS broth with an equal volume of a 10% glycerol
solution and storing the mixture at 
75°C. Working cultures were
prepared by transferring 0.5 ml of the frozen stock culture to 10 ml of MRS broth and incubating it for 12 to 18 h at 40°C. The working culture of strain RR was transferred (1% [vol/vol]) to 20 ml of MRS
broth and incubated for 12 to 18 h at 40°C. This preculture was
centrifuged at 8,000 × g and 4°C for 10 min and
washed twice with 20 ml of sterile distilled water. The resulting cell
suspension (in sterile distilled water) was used to inoculate larger
volumes (2.0 liters) of semidefined medium (SDM).
Culture medium.
The SDM used as a base for EPS experiments
had the following composition (in grams per liter): dextrose, 20; Tween
80, 1; ammonium citrate, 2; sodium acetate, 5; MgSO4
· 7H2O, 0.1; MnSO4, 0.05;
K2HPO4, 2; yeast nitrogen base without ammonium
and amino acids (Difco), 5; Bacto-casitone (Difco), 10. The pH of all
media was adjusted to 6.5 ± 0.2 prior to sterilization by heating
for 15 min at 121°C. Glucose was sterilized separately and
aseptically added to the medium. Previous work in our laboratory
indicated that generation times for strain RR in MRS and SDM did not
differ significantly at 42°C and that the medium provided minimal
interference with the assays used to quantify EPS (data not shown).
Experimental design.
To determine the optimum conditions for
EPS production by strain RR, a response surface experimental design
using a quadratic model was created by using ECHIP (ECHIP, Inc.,
Hockessin, Del.) (29). Three variables were included in the
model: the temperature (35 to 45°C), pH (4 to 6), and Bacto-casitone
(nitrogen) concentration (10 to 30 g/liter) of the growth medium. The
design contained 20 experiments, 15 unique combinations, and five
replications (see Table 1).
Fermentations.
All optimization fermentations were conducted
in a 2.5-liter working volume Bio-flow III (New Brunswick, Edison,
N.J.) fermentation vessel. After inoculation, the pH declined from 6.5 to the set point for that experiment due to lactic acid production. The
pH was then maintained at the set point by addition of 5 N NaOH. Agitation was maintained at 200 rpm throughout the fermentation. Immediately prior to inoculation, the medium was sparged with nitrogen
for 20 min at a rate of 1 liter/min. Samples (approximately 30 ml) were
removed at regular intervals and analyzed for growth, glucose, and EPS
as described below. All fermentations were terminated when 90% of the
initial glucose had been consumed.
Measurement of growth.
Growth was monitored
spectrophotometrically (650 nm) and by plating suitable dilutions on
MRS agar, followed by anaerobic incubation for 48 h at 42°C. For
the profile experiment, cell dry weight determinations were conducted
on duplicate 10-ml samples of fermentation broth. Each sample was
centrifuged (8,000 × g for 10 min) and washed twice
with distilled water. The pellet was resuspended in 5 ml of distilled
water, dried at 97°C for 5 h, and subsequently weighed.
Glucose analysis.
The glucose content of the medium during
fermentation was monitored by high-performance liquid chromatography.
The chromatographic system consisted of a Waters 510 solvent delivery
system (Waters Corp., Milford, Mass.), a Rheodyne 7125 sample injector
(20-µl loop; Rheodyne Inc., Cotati, Calif.), a Bio-Rad IG Cation H
guard column (Bio-Rad, Hercules, Calif.), an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad), and a Waters 410 differential refractometer (Waters Corp.). The column and detector were maintained at 35°C. The
solvent, 0.005 M sulfuric acid, was delivered at a flow rate of 0.6 ml/min. Data were collected by using the SSI Vision IV data collection
system (Scientific Systems Inc., State College, Pa.). Glucose was
quantified by relating the peak area to a standard curve.
Quantification of EPS.
The procedure used for EPS
quantification was based on that described by Gancel and Novel
(8). Approximately 10 g of culture medium was
accurately weighed into a 50-ml centrifuge tube and then heated in a
boiling water bath for 10 min to inactivate enzymes potentially capable
of polymer degradation (4, 5). Samples were cooled to room
temperature, 100 µl of a 5% (wt/vol) pronase E (EC 3.4.24.31; Sigma
Chemical Co., St. Louis, Mo.) solution was added, and the mixture was
incubated for 1 h at 37°C in a water bath shaker. Following
protein digestion, 250 µl of 80% (wt/vol) trichloroacetic acid was
added and samples were mixed well and stored at 4°C for a minimum of
30 min and then centrifuged (8,000 × g for 20 min) to
remove cells and protein. The supernatant was decanted into dialysis
tubing (molecular weight cutoff, 6,000 to 8,000) and dialyzed for
48 h against four changes of distilled water. All assays were
performed in duplicate, and the same procedure was done with
uninoculated medium. The carbohydrate concentration of the retentate
was determined by using the phenol-sulfuric acid method (7).
The carbohydrate assay was calibrated by using a mixture of
D-galactose, D-glucose, and
L-rhamnose (5:1:1) (11), and results are
reported as milligrams of carbohydrate per liter. The values shown for
EPS were calculated by subtracting the amount of background
interference in uninoculated medium (approximately 44 mg of
carbohydrate/liter) from the amount in fermented broth.
Large-scale production and recovery of EPS.
Large-scale
production of EPS was conducted in a 100-liter (working volume)
fermentation vessel (Bio-Service, Allentown, Pa.). Agitation was
maintained at 100 rpm, and the vessel was flushed with nitrogen during
sterilization to obtain an anaerobic environment. Temperature,
agitation, and pH were monitored and controlled with a Macintosh
computer (Apple Computer Inc., Cupertino, Calif.) and a program
developed in- house by using the Lab View development system (National
Instruments, Austin, Tex.). A model 2700 Select Biochemistry Analyzer
(YSI Biochemical Products, Yellow Springs, Ohio) was used to monitor
glucose utilization throughout fermentation and recovery. At the
endpoint (90% of glucose utilized), agitation was increased to 350 rpm
and the temperature of the vessel was raised to 100°C. After 15 min
at 100°C, the vessel was cooled to 37°C and 100 ml of a 5%
(wt/vol) pronase E (EC 3.4.24.31; Sigma Chemical Co.) solution was
added. After 60 min at 37°C, 250 ml of 80% (wt/vol) trichloroacetic
acid was added and the vessel was cooled to 17°C. After treatment,
fermentation broth was pumped to a Sharples A316Y centrifuge (Sharples
Inc., Warminster, Pa.) and centrifuged at 16,800 rpm to remove cells
and protein. EPS was concentrated by ultrafiltration of the clarified
fermentation broth by using a regenerated cellulose membrane (PLGC;
50-ft2 surface area; molecular weight cutoff, 10,000;
Millipore Corp., Bedford, Mass.) in a 50 RL Process Ultrafiltration
System (Millipore Corp.). Low-molecular-weight solutes were removed by
diafiltration of the EPS-containing retentate with distilled water
until the conductivity of the permeate approached that of distilled
water (YSI model 34 conductance-resistance meter). The EPS-containing retentate was placed in a 20-liter glass carboy, and the EPS was precipitated by adding 2 volumes of chilled 95% ethanol and then stored for 2 days at 4°C. The top layer of liquid was decanted, and
the bottom layer, which contained the precipitated EPS, was placed in
bottles and centrifuged at 8,000 × g and 4°C for 20 min. The pellet was removed, resuspended in distilled water, and lyophilized (40°C). The protein content of the dried material was
measured by the Bradford method (1), the total carbohydrate concentration was determined by the phenol-sulfuric acid method (7), and the moisture content was measured by weighing
material before and after freeze-drying (40°C).
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RESULTS |
Optimization of EPS production.
A response surface
experimental design was used to determine the optimum temperature, pH,
and Bacto-casitone concentration for EPS production by strain RR.
Results obtained from all of the experiments in the design are
presented in Table 1. Run 18 (trial 9)
was excluded from the data analysis because growth conditions were too
stringent and the endpoint (90% of glucose utilized) was not reached
within 168 h. The optimum temperature, pH, and Bacto-casitone
concentration for EPS production were determined to be 38°C, 5, and
30 g/liter, respectively (Fig. 1), with a
predicted optimum production of 295 mg of EPS/liter. The actual yield
under these conditions was 354 mg of EPS/liter, which was within the 95% confidence interval predicted (217 to 374 mg of EPS/liter). A plot
of the results indicated that higher EPS production might be obtained
if the Bacto-casitone concentration was greater than 30 g/liter (Fig.
2). To investigate, an experiment was
conducted at the optimum temperature (38°C) and pH (5.0) with a
Bacto-casitone concentration of 40 g/liter. The EPS production under
these conditions was found to be 324 g/liter, lower than the amount of
EPS produced under optimum conditions (Table 1). When this result was
included in the analysis, the predicted optimum and the significant
relationships among growth conditions and response variables did not
change. Table 2 provides the coefficients
of the statistical models derived from response surface methods, as
well as the statistical significance of each term.
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TABLE 1.
Results of fermentation experiments done to optimize EPS
production by L. delbrueckii subsp.
bulgaricus RR
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FIG. 1.
Contour plot of EPS production by L. delbrueckii subsp. bulgaricus RR at 30 g of
Bacto-casitone per liter as a function of temperature and pH. Contour
lines with numbers are significantly different (P < 0.05). The diagonal line is the design boundary; i.e., experimental
conditions below this line were not included in the design.
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FIG. 2.
Three-dimensional plot of EPS production by L. delbrueckii subsp. bulgaricus RR at pH 5.0 as a
function of temperature and Bacto-casitone concentration.
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To profile the optimum conditions for EPS production by strain RR, an
experiment was conducted at 38°C, pH 5, and 30 g of
Bacto-casitone per liter. Growth and EPS production were monitored
throughout the fermentation. The profile
showed that EPS production
was growth associated, i.e., EPS production
followed the growth
of the organism, with a specific production at the
endpoint of
101.4 mg of EPS/g of dry cells and a specific production
rate
during growth of 0.472 mg of EPS/g of cells/h (see Fig.
4).
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FIG. 3.
Contour plot of productivity (product) with 30 g of
Bacto-casitone per liter as a function of temperature and pH. Contour
lines with numbers are significantly different (P < 0.05). The diagonal line is the design boundary; i.e., experimental
conditions below this line were not included in the design.
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FIG. 4.
Profile of EPS production, cell dry weight, and glucose
utilization by L. delbrueckii subsp. bulgaricus
RR grown under optimum conditions for EPS production (38°C, pH 5.0, 30-g/liter Bacto-casitone).  , EPS;  , glucose;  , cell dry
weight.
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Isolation and recovery of EPS.
To obtain material for further
characterization, a 100-liter fermentation was conducted by using the
optimum conditions for EPS production (38°C, pH 5, 30 g of
Bacto-casitone per liter). Following ultrafiltration and diafiltration
to concentrate the EPS and remove low-molecular-weight materials, ca. 5 liters of EPS-containing retentate was obtained. Following ethanol
precipitation of the retentate and freeze-drying, 29.2 g of
semipure polysaccharide was obtained (Table
3).
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DISCUSSION |
Optimization of EPS production.
This study was designed to
examine three growth conditions (temperature, pH, and Bacto-casitone
concentration) likely to affect EPS production. The influence of the
carbon source on EPS production was not examined because other studies
have shown, in general, that glucose (10 to 20 g/liter) provides the
highest yield of EPS (6, 14, 17).
The optimum temperature for EPS production was within the optimum
growth range for strain RR (37 to 45°C) (
13). Published
work examining the effect of temperature on EPS production by
lactic
acid bacteria has been contradictory. Some reports found
greater
amounts of EPS produced at temperatures within the optimum
growth range
(
9,
10), while others have suggested that more
EPS is
produced at growth temperatures that are less than the
optimum
(
20). Discrepancies in the literature exist for a variety
of
reasons, including different ways of measuring EPS, different
growth
media, conditions and times of measurement, lack of pH
control, and
various means of expressing EPS production (milligrams
of EPS,
milligrams of EPS per liter, milligrams of EPS per CFU).
Little work has been done examining the effect of pH on EPS production
by lactobacilli, and none has been conducted with the
organism used in
this study. Mozzi et al. (
15) measured maximum
polysaccharide synthesis (488 mg/liter) and the highest cell numbers
at
a constant pH of 6.0 for
L. casei CRL 87. In addition, the
amount of EPS (milligrams per liter) was 3.6 times as high in
fermentations with pH control as in those without pH control.
In a
study conducted with
L. sake 0-1, the optimum pH for EPS
production was 5.8 but higher cell numbers were achieved at pH
6.2 (
28). From this result, the authors concluded that
conversion
of sugar to EPS is more efficient at pH 5.8 but sugar is
more
efficiently converted to biomass at pH 6.2.
For some EPS-producing bacteria, such as
Xanthomonas,
Pseudomonas, and
Rhizobium spp., nitrogen
limitation results in increased
EPS production (
3,
25). This
was not the case for strain
RR. Optimum EPS production occurred in a
growth medium containing
three times as much Bacto-casitone (30 g/liter) as that needed
to support growth (10 g/liter). Increasing the
Bacto-casitone
content in SDM to 40 g/liter did not result in an
increased level
of EPS production (Table
1). This may have been because
other
nutrients became limiting in batch culture.
A response surface experimental design employing a quadratic model was
used in this study. This type of model is logical because
factors that
affect microbial growth often show a single maximum.
Statistical
analysis provided information about the effect of
growth conditions on
the response variables (Table
2). A significant
relationship was found
between temperature (temperature and temperature
squared) and EPS
production. In general, the relationship between
temperature and the
growth of microorganisms shows a single maximum
but is asymmetric
(
22), as was the relationship observed here.
The
relationship between pH and microbial growth often shows a
broad
maximum (
22). In this study, it is likely that pH did
not
significantly affect EPS production because the range used
in this
study (4 to 6) was on the relatively flat part of the
curve. In fact,
Fig.
1 suggests that the optimum temperature and
pH conditions for EPS
production exist within the ellipse bounded
by 36 to 39°C and pH 4.5 to 5.5, rather than at a specific point.
Finally, there was a
significant relationship between the Bacto-casitone
concentration and
EPS production, which is shown in Fig.
2. A
number of growth conditions
and their interrelationships were
significantly related to the number
of CFU per milliliter and
time to endpoint. It is generally known that
growth conditions
such as those examined in this study have an effect
on these variables.
Productivity, defined as production of the maximum amount of a product
in a minimum amount of time, is an important variable
from an
industrial perspective. To determine conditions for optimum
productivity, a combined response variable, designated productivity,
was calculated as the weighted sum of the individual responses
EPS
concentration and time to endpoint (Table
1). By using the
EChip
optimization routine (
29), individual responses are scaled
so they can be fairly combined, and the maximum of the combined
response is the simultaneous optimum of the individual responses.
Individual responses are subjectively weighted to reflect their
relative importance. In this case, EPS concentration and time
to
endpoint were equally weighted. Optimum productivity was found
at
39°C, pH 5.6, and 30 g of Bacto-casitone per liter (Fig.
3).
Under these conditions, the model predicted that 236 mg of EPS/liter
would be produced in 12.45 h. In the case of expensive medium
components, laborious preparation procedures which result in a
high
ratio of preparation time to fermentation time, or high residual
processing costs, it would be appropriate to weight EPS production
higher than time to endpoint, thereby reducing the residual substrate
(at the cost of longer fermentation times).
Previous reports examining the effect of more than one condition on EPS
production have utilized an OVAT approach (
28).
An OVAT
approach assumes no interaction among the variables being
investigated.
Response surface methodology allows simultaneous
evaluation of many
growth conditions and also examines interaction
among the variables
(Table
2).
Profile under optimum growth conditions.
A profile of the
fermentation conducted by using the optimum conditions for EPS
production showed that EPS production was growth associated (Fig. 4).
Similar results were obtained by Grobben et al. (10) by
using strain NCFB 2772. This result suggests that optimizing conditions
for growth of the EPS-producing organism would result in maximal
production of EPS. Growth association of EPS production does not occur
with some other EPS-producing strains, such as Xanthomonas
and Alcaligenes spp. (25).
Isolation and of recovery of EPS.
A large-scale fermentation
conducted under optimum conditions for production yielded 29.2 g
of crude EPS. Even on scale-up, the amount of EPS produced by strain RR
(367.84 mg of EPS/liter; Table 3) was within the 95% confidence
interval (216 to 374 mg of EPS/liter) predicted by the response surface
model. This is significant, since scale-up is often difficult due to
changes in surface-to-volume ratios. In this case, scale-up may have
been more straightforward, since oxygen transfer is not the problem under anaerobic conditions that it may be under aerobic conditions.
The greatest loss during recovery of crude EPS was probably due to the
method of harvesting the EPS, in which only the bottom
layer of
precipitated material was centrifuged following ethanol
precipitation
(Table
3). The remaining EPS may have been present
as fine particles
dispersed throughout the ethanol-retentate mixture.
A filtration step
to recover the EPS might result in higher recovery
rates than the
process used in this study.
The protein content of crude material was determined to give an
estimate of purity. The material was found to contain 1.42%
(0.43 ± 0.11 g) protein. It is likely that the protein was carried
over
from medium ingredients during recovery and is not tightly
bound to
EPS. Oda et al. (
18) also found a small amount of protein
contaminating polysaccharide isolated from
L. helveticus
var.
jugurti.
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ACKNOWLEDGMENTS |
This research was supported in part by grants from the
Pennsylvania Dairy Promotion Program and the American Dairy Association and Dairy Council Mid East.
We thank Mark Signs for his assistance with fermentation scale-up.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science, The Pennsylvania State University, 111 Borland
Laboratory, University Park, PA 16802. Phone: (814) 863-2959. Fax:
(814) 863-6132. E-mail: RFR3{at}psu.edu.
| |
REFERENCES |
| 1.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 2.
|
Breedveld, M. W.,
L. P. T. M. Zevenhuizen,
H. C. J. Canter Cremers, and A. J. B. Zehnder.
1993.
Influence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum.
Antonie Leeuwenhoek
64:1-8.
|
| 3.
|
Cerning, J.
1990.
Exocellular polysaccharides produced by lactic acid bacteria.
FEMS Microbiol. Rev.
87:113-130.
|
| 4.
|
Cerning, J.,
C. Bouillanne, and M. J. Desmazeaud.
1988.
Extracellular polysaccharide production by Streptococcus thermophilus.
Biotechnol. Lett.
10:255-260.
|
| 5.
|
Cerning, J.,
C. Bouillanne,
M. Landon, and M. Desmazeaud.
1992.
Isolation and characterization of exopolysaccharides from slime-forming mesophilic lactic acid bacteria.
J. Dairy Sci.
75:692-699[Abstract].
|
| 6.
|
Cerning, J.,
C. M. G. C. Renard,
J. F. Thibault,
C. Bouillanne,
M. Landon,
M. Desmazeaud, and L. Topisirovic.
1994.
Carbon source requirements for exopolysaccharide production by Lactobacillus casei CG11 and partial structure analysis of the polymer.
Appl. Environ. Microbiol.
60:3914-3919[Abstract/Free Full Text].
|
| 7.
|
Dubois, M.,
K. A. Gilles,
J. K. Hamilton,
P. A. Rebers, and F. Smith.
1956.
Colorimetric method for determination of sugars and related substances.
Anal. Chem.
28:350-356.
|
| 8.
|
Gancel, F., and G. Novel.
1994.
Exopolysaccharide production by Streptococcus salivarius ssp. thermophilus cultures. 1. Conditions of production.
J. Dairy Sci.
77:685-688[Abstract].
|
| 9.
|
Garcia-Garibay, M., and V. M. E. Marshall.
1991.
Polymer production by Lactobacillus delbrueckii ssp. bulgaricus.
J. Appl. Bacteriol.
70:325-328.
|
| 10.
|
Grobben, G. J.,
J. Sikkema,
M. R. Smith, and J. A. M. de Bont.
1995.
Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 grown in a chemically defined medium.
J. Appl. Bacteriol.
79:103-107.
|
| 11.
|
Gruter, M.,
B. R. Leeflang,
J. Kuiper,
J. P. Kamerling, and F. G. Vliegenthart.
1993.
Structural characterisation of the exopolysaccharide produced by Lactobacillus delbrueckii subspecies bulgaricus rr grown in skimmed milk.
Carbohydr. Res.
239:209-226[Medline].
|
| 12.
|
Hess, S. J.,
R. F. Roberts, and G. R. Ziegler.
1997.
Rheological properties of nonfat yogurt stabilized using Lactobacillus delbrueckii ssp. bulgaricus producing exopolysaccharide or using commercial stabilizer systems.
J. Dairy Sci.
80:252-265[Abstract].
|
| 13.
|
Kandler, O., and N. Weiss.
1986.
Regular, nonsporing gram positive rods, p. 1208-1233. In
J. G. Holt, M. E. Sharpe, N. S. Mair, and P. H. A. Sneath (ed.), Bergey's manual of systematic bacteriology, vol. 2.
The Williams & Wilkins Co., Baltimore, Md.
|
| 14.
|
Kojic, M.,
M. Vujcic,
A. Banina,
P. Cocconcelli,
J. Cerning, and L. Topisirovic.
1992.
Analysis of exopolysaccharide production by Lactobacillus casei CG11, isolated from cheese.
Appl. Environ. Microbiol.
58:4086-4088[Abstract/Free Full Text].
|
| 15.
|
Mozzi, F.,
G. S. de Giori,
G. Oliver, and G. F. de Valdez.
1996.
Exopolysaccharide production by Lactobacillus casei under controlled pH.
Biotechnol. Lett.
18:435-439.
|
| 16.
|
Mozzi, F.,
G. Oliver,
G. S. de Giori, and G. F. de Valdez.
1995.
Influence of temperature on the production of exopolysaccharides by thermophilic lactic acid bacteria.
Milchwissenschaft
50:80-82.
|
| 17.
|
Mozzi, F.,
G. Savoy de Giori,
G. Oliver, and G. Font de Valdez.
1995.
Exopolysaccharide production by Lactobacillus casei. II. Influence of the carbon source.
Milchwissenschaft
50:307-309.
|
| 18.
|
Oda, M.,
H. Hasegawa,
S. Komatsu,
M. Kambe, and F. Tsuchiya.
1983.
Anti-tumor polysaccharide from Lactobacillus sp.
Agric. Biol. Chem.
47:1623-1625.
|
| 19.
|
Sanford, P. A.
1979.
Exocellular microbial polysaccharides.
Adv. Carbohydr. Chem. Biochem.
36:265-313[Medline].
|
| 20.
|
Schellhaass, S. M.
1983.
.
Ph.D. thesis.
University of Minnesota, St. Paul.
|
| 21.
|
Shu, C., and S. Yang.
1990.
Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris.
Biotechnol. Bioeng.
35:454-468.
|
| 22.
|
Sinclair, C. G.
1987.
Microbial process kinetics, p. 75-131. In
J. Bulock, and B. Kristiansen (ed.), Basic biotechnology.
Academic Press, London, England.
|
| 23.
|
Souw, P., and A. L. Demain.
1979.
Nutritional studies on xanthan production by Xanthomonas campestris NRRL B1459.
Appl. Environ. Microbiol.
37:1186-1192[Abstract/Free Full Text].
|
| 24.
|
Sutherland, I. W.
1990.
Food usage of polysaccharides, p. 117-125. In
I. W. Sutherland (ed.), Biotechnology of microbial polysaccharides.
Cambridge University Press, Cambridge, England.
|
| 25.
|
Sutherland, I. W.
1990.
Physiology and industrial production, p. 70-88. In
I. W. Sutherland (ed.), Biotechnology of microbial exopolysaccharides.
Cambridge University Press, Cambridge, England.
|
| 26.
|
Tamime, A. Y.
1977.
The behavior of different starter cultures during the manufacture of yogurt from hydrolysed milk.
Dairy Ind. Int.
42:7-11.
|
| 27.
|
Teggatz, J. A., and H. A. Morris.
1990.
Changes in the rheology and microstructure of ropy yogurt during shearing.
Food Struct.
9:133-138.
|
| 28.
|
van den Berg, D. J. C.,
G. W. Robijn,
A. C. Janssen,
M. L. F. Giuseppin,
R. Vreeker,
J. P. Kamerling,
J. F. G. Vliegenthart,
A. M. Ledeboer, and C. T. Verrips.
1995.
Production of a novel extracellular polysaccharide by Lactobacillus sake 0-1 and characterization of the polysaccharide.
Appl. Environ. Microbiol.
61:2840-2844[Abstract].
|
| 29.
|
Wheeler, B.,
R. Betsch, and T. Donnelly.
1993.
.
EChips user's guide version 6.0 for Windows.
EChip, Inc., Hockessin, Del.
|
Appl Environ Microbiol, February 1998, p. 659-664, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
