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Applied and Environmental Microbiology, August 2000, p. 3519-3527, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Correlation of Activities of the Enzymes
-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose
Pyrophosphorylase with Exopolysaccharide Biosynthesis by
Streptococcus thermophilus LY03
Bart
Degeest and
Luc
De Vuyst*
Research Group of Industrial Microbiology,
Fermentation Technology and Downstream Processing, Department of
Applied Biological Sciences, Vrije Universiteit Brussel, B-1050
Brussels, Belgium
Received 18 February 2000/Accepted 30 May 2000
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ABSTRACT |
The effects of different carbohydrates or mixtures of carbohydrates
as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two
heteropolysaccharides with the same monomeric composition (galactose
and glucose in the ratio 4:1) but with different molecular masses.
Lactose and glucose were fermented by S. thermophilus
LY03 only when they were used as sole energy and carbohydrate sources.
Fructose was also fermented when it was applied in combination with
lactose or glucose. Both the amount of EPS produced and the
carbohydrate source consumption rates were clearly influenced by the
type of energy and carbohydrate source used, while the EPS monomeric
composition remained constant (galactose-glucose, 4:1) under all
circumstances. A combination of lactose and glucose resulted in the
largest amounts of EPS. Measurements of the activities of enzymes
involved in EPS biosynthesis, and of those involved in sugar nucleotide
biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that
the levels of activity of 
-phosphoglucomutase, UDP-galactose
4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated
with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in
either the rhamnose nucleotide synthetic branch of the EPS biosynthesis
or the pathway leading to glycolysis was observed for S. thermophilus LY03.
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INTRODUCTION |
Food hydrocolloids play an important
role in the rheological properties of food products (29,
34). Examples are plant carbohydrates such as modified starch and
guar gum, animal proteins like gelatin and casein, and microbial
exopolysaccharides (EPS) such as xanthan and gellan.
The addition of food additives is not always allowed, so natural
yoghurts and fermented milks are dependent on the EPS-producing
capacities of the starter strains used. EPS are microbial
polysaccharides that are secreted extracellularly and are either
associated with the cell surface in the form of capsules or
released into the medium in the form of slimes (11). The
thermophilic yoghurt strains Lactobacillus delbrueckii
subsp. bulgaricus and Streptococcus
thermophilus produce heteropolysaccharides that consist of a
repeating unit of neutral sugars (glucose, galactose, and rhamnose) in
different ratios (1, 3, 6, 11, 13, 15, 17-21, 24,
32; G. T. Lamothe, F. Stingele, J.-R. Neeser, and B. Mollet, Abstr. Am. Soc. Microbiol. Conf. Streptococcal Genet., abstr.
no. 2A-16, p. 67, 1998). Some of these polysaccharides display
pseudoplastic and thixotropic properties (4, 5, 6, 13). It
has been postulated that their rheological properties are influenced
not only by the amount of EPS but also by its structure. Recently, the
structures of the repeating units of some EPS produced by L. delbrueckii subsp. bulgaricus (21) and
S. thermophilus (3, 13, 15, 24, 32) have
been elucidated. However, there is still a lack of knowledge of the
physiological aspects of EPS production. Although EPS production is
growth associated in thermophilic lactic acid bacterium strains
(11, 12), the EPS biosynthesis pathway is very complex.
Contradictory results have been reported in the literature concerning
the influence of the carbohydrate source present in the medium on
monomeric composition and hence the structure of the EPS produced.
Grobben et al. showed that the proportions of glucose and fructose as carbohydrate sources influenced both the amount and monomeric composition of the EPS produced by L. delbrueckii subsp.
bulgaricus NCFB 2772 (19). This was reflected in
the activities of the enzymes involved, namely, UDP-glucose
pyrophosphorylase, dTDP-glucose pyrophosphorylase, and the rhamnose
nucleotide synthetic enzyme system. Escalante et al. found that
UDP-glucose pyrophosphorylase was correlated with EPS production in a
ropy strain of S. thermophilus but not, although the
enzyme was present, in a nonropy strain and that the Leloir enzyme
UDP-galactose 4-epimerase was not correlated with EPS biosynthesis in
any strain (14). We need more knowledge of the nutritional
factors influencing the activities of the enzymes involved in the
biosynthetic pathway of EPS and the regulation of the energy and carbon
flows between the Embden-Meyerhof-Parnas (EMP) pathway and the
biosynthesis of EPS.
The yoghurt strain S. thermophilus LY03 produces a
heteropolysaccharide consisting of galactose and glucose in the ratio
4:1. Its production is growth associated, and both physical and
chemical conditions have been optimized to obtain maximum EPS
production (8). In this paper, we report the differences in
EPS production and monomeric composition between cultures of
S. thermophilus LY03 grown on different carbohydrates
or mixtures of carbohydrates. The effects of growth conditions on the
activities of 10 enzymes involved in the EMP pathway and the
biosynthesis of sugar nucleotides as the precursors for EPS
biosynthesis were evaluated for the first time. A correlation between
the activities of EPS precursor-forming enzymes and the amounts
of EPS produced was unambiguously demonstrated.
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MATERIALS AND METHODS |
Bacterial strains.
S. thermophilus LY03, an
industrial yoghurt starter culture, was used as the EPS-producing
strain throughout this study (12). S. thermophilus NR, another industrial starter culture, was used as a
non-EPS-producer. Both strains were kindly provided by V. Marshall (The
University of Huddersfield, Huddersfield, United Kingdom). The strains
were stored at 
80°C in MRS broth (Oxoid, Basingstoke, England)
containing 25% (vol/vol) glycerol. Before experimental use, the
bacteria were propagated twice in MRS broth at 42°C for 12 h.
Growth on different carbohydrate sources.
A customized MRS
broth was used as the basic EPS production medium; it contained 30 g of peptone (Oxoid) per liter, 12 g of yeast extract (Merck) per
liter, 8 g of Lab Lemco (Oxoid) per liter, 2 g of
K2HPO4 per liter, 5 g of sodium acetate
per liter, 2 g of triammonium citrate per liter, 0.2 g of
MgSO4 · 7H2O per liter, 0.038 g of
MnSO4 · H2O per liter, and 1 ml of Tween
80 per liter (7). Different initial concentrations of
specific energy and/or carbohydrate sources were tested: 0.22 and 0.29 M for lactose, 0.42 M for glucose, and 0.06, 0.11, 0.17, 0.22, 0.28, and 0.42 M for galactose. Furthermore, equivalent amounts of fructose
(0.06, 0.11, 0.17, 0.22, 0.28, and 0.42 M), rhamnose (0.06, 0.12, 0.18, 0.24, 0.30, and 0.46 M), maltose (0.03, 0.06, 0.09, 0.12, 0.15, and
0.22 M), and sucrose (0.03, 0.06, 0.09, 0.12, 0.15, and 0.22 M) were
applied. Also, additions of (i) 0.14 M glucose, galactose, or fructose
and 0.15 M rhamnose to 0.15 M lactose; (ii) 0.14 M fructose to 0.22 M
lactose, and (iii) 0.07 M lactose, 0.14 M fructose, and 0.15 M rhamnose
to 0.28 M glucose were examined.
Fermentation conditions.
For all fermentations but one (0.22 M lactose plus 0.14 M fructose) the optimal initial
carbon/complex-nitrogen ratio as determined earlier was applied
(7). For the control strain, S. thermophilus NR, lactose (0.22 M) was used as the sole energy and carbohydrate source. The pH was controlled at 6.2 ± 0.1 by automatic addition of 10 N NaOH. The pH and the amount of base added were monitored online. Temperature was kept constant at 42 ± 0.1°C. To keep
the fermentation broth homogenous, agitation was performed at 100 rpm
with a stirrer composed of three standard impellers.
The fermentor inoculum was always prepared in two steps. First, 10 ml
of customized MRS broth was inoculated with 100 µl of a freshly
prepared S. thermophilus LY03 culture. After 12 h
of incubation at 42°C, it was used to inoculate 100 ml of production medium. After 12 h of growth at 42°C, this second preculture was used to inoculate the fermentor (10 liters). All fermentations were
done in 15-liter laboratory fermentors (Biostat C; B. Braun Biotech
International, Melsungen, Germany), with a working volume of 10 liters.
They were computer controlled (MicroMFCS for Windows NT software), and
were sterilizable in situ. Sterilization was performed at 121°C for
20 min. The energy and carbohydrate source was sterilized separately
(20 min at 121°C) and aseptically pumped into the fermentor. Samples
were aseptically withdrawn from the fermentor to determine cell number
(CFU), biomass (cell dry mass [CDM]), EPS production (polymer dry
mass [PDM]), lactic acid concentration, galactose concentration, and
residual substrate (carbohydrate) concentration (lactose, glucose,
fructose) as described elsewhere (12). Isolation of both
high-molecular-mass EPS (HMM-EPS) and low-molecular-mass EPS (LMM-EPS)
and monomer analysis were done as previously described (7).
Averaged standard deviations of 20.0 and 5.0% were observed for EPS
isolation and monomer analysis, respectively.
Samples of 50 ml were taken to prepare cell extracts for measuring
enzyme activity (see below) at three or four time points:
once or twice
during the exponential growth phase, at the end
of the exponential
growth phase when EPS production reached its
maximum, and during the
stationary phase or beyond the EPS maximum
production
level.
The maximum specific growth rate (µ
max, per hour) was
calculated as the maximum slope from the linearized values of the
biomass
(grams of CDM per liter) as a function of fermentation time
(hours).
The maximum carbohydrate consumption rates
(
rmax, per hour) were
calculated from the
residual concentration of the carbohydrate
in the medium. To confirm
the reliability of the experiments,
one fermentation, which was chosen
at random, was repeated twice
and yielded comparable results for all
parameters (see also Table
1).
Preparation of cell extracts.
Samples, freshly taken from
the fermentation broth (50 ml), were centrifuged at 20,000 × g for 20 min at 4°C, and the supernatant fluid was decanted.
The cell pellet was washed with potassium phosphate buffer (0.05 mol
liter
1, pH 7.0) and then resuspended in 6 ml of phosphate
buffer and held on ice. The chilled cells were lysed using a Vibra-Cell
sonicator (Sonic & Materials Inc., Danbury, Conn.) with a microtip
setting (sonic power, 375 W; output control, 5) for 6 min and a 50%
duty cycle consisting of 30 s of sonication pulses and 30 s
of rest (to cool the suspension). Cell debris was removed by
centrifugation (20,000 × g for 20 min at 4°C), and
the supernatant fluid (the cell extract) was stored at 80°C and
used as a source of enzymes.
For the optimization of the cell lysis, preliminary experiments were
done, based on measurements of the intracellular lactate
dehydrogenase
activity. They indicated that sonication (6 min,
50% duty cycle),
compared with the results of a glass-bead treatment
carried out
according to the method of Hansen et al. (
22), gave
the most
reproducible and optimum results for release of proteins.
These results
were obtained within the limited time required to
avoid great losses of
enzymatic activities due to extensive heating
during sonication. The
reaction mixture for the measurement of
the lactate dehydrogenase
activity contained (for 1 ml) 10 mM
dithiothreitol, 0.5 mM NADH, 50 mM
sodium pyruvate, and 50 mM
3-morpholinopropane sulfonic acid, dissolved
in 950 µl of ultrapure
water, plus 50 µl of cell extract. Reaction
mixtures were incubated
at 30°C, and the oxidation of NADH was
monitored spectrophotometrically
at 340
nm.
Enzyme assays.
Enzyme assays were done at 37°C in a volume
of 1.0 ml (unless otherwise indicated), and the formation or
disappearance of NAD(P)H was monitored by measuring the absorbance at
340 nm (
340 = 6,220 M1 · cm1). The protein concentration of cell extracts was
determined using a commercial kit (DC protein assay; Bio-Rad
Laboratories, Hercules, Calif.) that is based on the method of Lowry et
al. (25). In all of the assays, the reaction velocity was
linearly proportional to the amount of cell extract. All enzyme
activity measurements were done in triplicate and expressed as mean
values and standard deviations. Specific activities are expressed as
nanomoles of substrate converted into product during 1 min for 1 mg of
total cell protein.
The
-phosphoglucomutase reaction mixture contained 179 µmol of
glycylglycine (pH 7.4), 0.67 µmol of
-NAD phosphate, 0.02
µmol of glucose 1,6-diphosphate, 30 µmol of MgCl
2, 43 µmol of
L-cysteine, 1 U of glucose 6-phosphate
dehydrogenase, and cell
extract. The reaction was initiated by adding
5.0 µmol of
-glucose
1-phosphate (
28).
The reaction mixture for
-phosphoglucomutase was exactly the same as
that for
-phosphoglucomutase, except that
-glucose
1-phosphate
was used as the
substrate.
The UDP-glucose pyrophosphorylase forward reaction mixture contained 50 µmol of Tris · HCl (pH 7.5), 8 µmol of MgCl
2,
1.58
mg of cysteine hydrochloride (pH 7.5), 0.5 µmol of NAD, 1.25 µmol
of UTP, 25 µg of UDP-glucose dehydrogenase, and cell extract.
The reaction was initiated by adding 1 µmol of
-glucose
1-phosphate
(
19).
The UDP-galactose 4-epimerase reaction mixture contained 40 µmol of
glycylglycine-NaOH (pH 8.5), 5 µmol of MgCl
2, 0.6 µmol
of NAD, 25 µg of UDP-glucose dehydrogenase, and cell extract.
The
reaction was initiated with 0.2 µmol of UDP-galactose (
14,
19).
The dTDP-glucose pyrophosphorylase forward reaction mixture contained
50 µmol of Tris · HCl (pH 7.5), 8 µmol of MgCl
2,
1.58
mg of cysteine hydrochloride (pH 7.5), 1.25 µmol of dTTP, and
cell extract. The reaction was initiated by adding 1 µmol of
-glucose
1-phosphate and stopped by adding 50 µl of 1 M HCl. The
samples
were assayed by high-pressure liquid chromatography as
described
for monomer analysis (
7,
19).
The activity of dTDP-glucose 4,6-dehydratase was assayed in a reaction
mixture (0.5 ml) containing 25 µmol of sodium phosphate
(pH 7.0),
0.05 µmol of NAD, and cell extract. The reaction was
initiated with
0.25 µmol of dTDP-glucose. At different time intervals,
samples (50 µl) were withdrawn, added to 750 µl of 0.1 N NaOH,
and reincubated
for 15 min (at 37°C). Formation of
dTDP-6-deoxy-
D-xylo-4-hexulose
(
320 = 4,600 M
1 · cm
1) was determined
spectrophotometrically at 320 nm (
2,
36).
The dTDP-rhamnose synthetic enzyme (
26), which is
responsible for the conversion of
dTDP-6-deoxy-
D-xylo-4-hexulose to
dTDP-
L-rhamnose,
was assayed by monitoring NADPH oxidation
in a mixture of 0.5
µmol of NADPH, 50 µmol of Tris · HCl (pH
8.0), and cell extract.
The reaction was initiated by the addition of
0.3 µmol of dTDP-glucose
and stopped by adding 300 µl of 0.5 M
NaOH. The mixture was then
reincubated (at 37°C) for 10 min
(
19).
The phosphoglucose isomerase reverse reaction mixture contained 50 µmol of potassium phosphate (pH 6.8), 5 µmol of MgCl
2,
0.4 µmol of NADP, 0.01 ml of glucose 6-phosphate dehydrogenase
(180 U · ml
1), and cell extract. The reaction was
initiated by adding 2.5
µmol of fructose 6-phosphate (
19,
30).
The reaction mixture for the 6-phosphofructokinase assay contained 50 µmol of Tris · HCl (pH 7.5), 5 µmol of MgCl
2, 50 µmol
of KCl, 0.15 µmol of NADH, 1.25 µmol of ATP, 50 µg of
aldolase,
20 µg of triosephosphate isomerase-glycerol phosphate
dehydrogenase,
and cell extract. The reaction was initiated by adding 1 µmol
of fructose 6-phosphate (
19,
35).
The fructose 1,6-bisphosphatase reaction mixture contained 50 µmol of
glycylglycine (pH 8.5), 5 µmol of MgCl
2, 0.4 µmol
of
NADP, 25 µg of phosphoglucose isomerase, 25 µg of glucose
6-phosphate
dehydrogenase, and cell extract. The reaction was initiated
by
adding 2 µmol of fructose 1,6-bisphosphate (
19,
35).
Statistical significance of correlations.
Statistical
significance of correlations between enzyme activities and amounts of
EPS is based on a correlation test. It consists of a simple test of
hypothesis that decides whether the correlation is significantly
different from 0 or not. The test is performed by seeing how the
absolute correlation value (r) compares to a critical value
according to a chosen level of probability (P value). If the
correlation is significant, then it can be concluded that the two
factors are indeed associated, but if the correlation is found to be
insignificant, it is concluded that the two factors might not be
related at all. In the latter case, the correlation might be due to
sampling randomness.
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RESULTS |
Effect of the energy and carbohydrate source on bacterial
growth.
Different initial concentrations of specific
energy sources and/or carbohydrate sources, in particular,
lactose, glucose, galactose, fructose, rhamnose, maltose, and sucrose,
were first tested as potential substrates for S. thermophilus LY03. S. thermophilus LY03 displayed
a homofermentative metabolism and grew on both lactose and glucose.
Galactose, fructose, rhamnose, maltose, and sucrose as sole energy
sources were not fermented by S. thermophilus LY03 and
neither was the galactose moiety of lactose. Fructose was fermented by
the bacterium when it was used in combination with lactose or glucose.
When rhamnose was applied as an additional carbohydrate source, it was
not fermented and could not be found in the EPS produced. Therefore,
fermentations with additional rhamnose were not further studied.
Fermentations were then done with lactose and glucose as the sole
substrate or in combination with other carbohydrates (Table
1). From these experiments, it was clear
that
S. thermophilus LY03 grew well on glucose and
lactose and consumed glucose and
lactose efficiently. This is reflected
in the µ
max values of 1.3
and 1.1 h
1,
respectively, the maximum substrate consumption rates
(
rmax)
of 0.3 and 0.3 h
1,
respectively, and the maximum attainable biomass concentrations
of 4.7 and 4.5 g of CDM · liter
1, respectively. Both
the µ
max values and the maximum attainable
biomass
concentrations were comparable in all other cases, i.e.,
approximately
0.93 h
1 and 4.6 g of CDM · liter
1, respectively, except that the maximum
biomass values were higher
than 5.0 g of CDM · liter
1 for the experiments with a combined energy source
of glucose
and fructose or glucose and galactose. When grown on lactose
as
the sole energy source,
S. thermophilus LY03 used
only the glucose
moiety and galactose accumulated in the medium.
In contrast, galactose
was converted to lactic acid upon prolonged
fermentation with
lactose as the sole energy source as well as during
fermentations
with the following carbohydrate combinations: 0.15 M
lactose plus
0.14 M galactose, 0.28 M glucose plus 0.14 M galactose,
0.22 M
lactose plus 0.14 M fructose, and 0.28 M glucose plus 0.07 M
lactose.
In all cases, approximately 0.02 M galactose was converted
into
lactic acid from the end of the exponential growth phase and
beyond.
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TABLE 1.
Influence of the carbohydrate source on cell growth and
EPS production during S. thermophilus
LY03 fermentationsa
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When cultures were grown on both lactose and fructose, glucose and
fructose, and lactose and glucose, both carbohydrate sources
were
consumed simultaneously during growth. They were converted
into
lactic acid homofermentatively. However, the
rmax varied
when different carbohydrate
combinations were used. Indeed, explicit
increases in
rmax were seen from averages of 0.4 to 1.0 h
1 for both glucose and fructose, when we used the
combinations
of 0.15 M lactose plus 0.14 M glucose and 0.15 M lactose
plus
0.14 M fructose, respectively. Lactose was only slowly consumed
(
rmax = 0.1 h
1) in the
experiment with an increased initial energy source concentration
of
0.22 M lactose plus 0.14 M fructose and in the fermentation
with the
carbohydrate combination of 0.15 M lactose plus 0.14
M glucose,
indicating possible substrate inhibition or catabolite
repression by
easily fermentable
substrates.
Effect of the energy and carbon source on EPS production.
For each of the fermentations mentioned above, the amount of
EPS produced (both the HMM-EPS and LMM-EPS fractions), as well as
the monomeric composition of the polysaccharide material, was determined (Table 1).
For all experiments, the monomeric compositions of both LMM-EPS and
HMM-EPS were determined for each sample taken. No variation
in EPS
composition was observed; galactose and glucose were observed
in all
cases in a 4:1 ratio. Both the total maximum amount of
EPS and the
maximum amount of EPS from each fraction independently
were
clearly influenced by the nature of the carbohydrate source.
Greater amounts of each EPS fraction and of the total EPS were
observed with 0.22 M lactose as the sole carbohydrate source,
and the
greatest amounts of HMM-EPS and total EPS were found with
the
combination of 0.15 M lactose plus 0.14 M glucose, compared
to amounts
in the other fermentations. When glucose was used as
the sole
carbohydrate source, both HMM-EPS and the total amount
of EPS were
significantly lower than for all other fermentation
conditions
tested.
Activities of enzymes involved in the EMP pathway, sugar nucleotide
biosynthesis, and EPS biosynthesis.
To determine whether the
differences in EPS production between cultures of S. thermophilus LY03 grown on different carbohydrates as substrate
sources could be related to precursor availability, the activities of
10 enzymes involved in the EMP pathway and in the biosynthesis and
interconversion of sugar nucleotides were determined. For comparison,
the same enzyme activities were also measured for S. thermophilus NR, a strain that does not produce EPS. Samples
were taken at three or four time points (see Materials and Methods)
during the different fermentations described above.
Enzyme activities and EPS production during fermentation of
S. thermophilus LY03 on lactose, on a combination of
lactose and
another carbohydrate source, on glucose, or on
a combination of
glucose and another carbohydrate source and of
the non-EPS-producer
S. thermophilus NR are shown in
Tables
2 through
6.
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TABLE 2.
Amounts of HMM, LMM, and total EPS measured in fermented
medium of the EPS-producing strain S. thermophilus LY03a
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TABLE 3.
Activities of enzymes involved in the EMP pathway and the
pathway leading to the biosynthesis of sugar nucleotides and EPS in
cell extracts of the EPS-producing strain S. thermophilus LY03a
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TABLE 4.
Amounts of HMM, LMM, and total EPS measured in fermented
medium of the EPS-producing strain S. thermophilus LY03a
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TABLE 5.
Activities of enzymes involved in the EMP pathway and the
pathway leading to the biosynthesis of sugar nucleotides and EPS in
cell extracts of the EPS-producing strain S. thermophilus LY03a
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TABLE 6.
Activities of enzymes involved in the EMP pathway and the
pathway leading to the biosynthesis of sugar nucleotides and EPS
in cell extracts of
S. thermophilus NRa
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The activity of
-phosphoglucomutase was linked to EPS biosynthesis
and the amount of EPS produced. Indeed, a maximum amount
was reached
for all fermentations at the end of the exponential
growth phase. For
all carbohydrate sources used, the specific
activity paralleled the
maximum total amount of EPS. A correlation
(
r) of 0.85 was found, which was statistically significant at
a
P of
<0.01 (Fig.
1). For the
non-EPS-producing strain, a very
low specific activity of
-phosphoglucomutase was detected. Neither
of the strains
displayed
-phosphoglucomutase activity.
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FIG. 1.
Relationship between the activities of the enzymes
-phosphoglucomutase ( ; y = 0.3716x + 354.76;
r = 0.85), UDP-galactose 4-epimerase ( ; y = 0.1764x + 157.53; r = 0.81), and UDP-glucose
pyrophosphorylase ( ; y = 0.1005x + 86.01; r = 0.80) and the total amount of EPS produced (total sample size
was 10).
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The same trend of activities during fermentation was observed for both
UDP-glucose pyrophosphorylase (
r = 0.80;
P < 0.01)
and UDP-galactose 4-epimerase (
r = 0.81;
P < 0.01) (Fig.
1). All
other relationships between enzyme
activities and the amounts
of EPS gave a correlation coefficient,
r, of less than 0.70 (
P > 0.05) (Fig.
2). This result indicates a decreased
level of or
no significance for the involvement of the measured enzyme
activities
in EPS production compared to those of
-phosphoglucomutase, UDP-glucose
pyrophosphorylase, and
UDP-galactose 4-epimerase.
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FIG. 2.
Relationship between the activities of the enzymes
6-phosphofructokinase (primary axis) (; y = 0.8912x + 1426; r = 0.61), phosphoglucose isomerase (primary axis)
(; y = 1.0579x + 998; r = 0.69), and
dehydratase (secondary axis) ( ; y = 0.005x + 15.46;
r = 0.13) and the total amount of EPS produced (total sample
size was 10).
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For the rhamnose nucleotide synthetic branch of EPS biosynthesis,
a very low enzyme activity was measured for dTDP-glucose
4,6-dehydratase. The dTDP-glucose pyrophosphorylase and the
dTDP-rhamnose
synthetic enzyme system displayed almost no activity in
cell extracts
of both
strains.
There was no substantial specific activity of fructose
1,6-bisphosphatase for any of the carbohydrate combinations
tested
for either strain. Furthermore, a clear decrease in the
specific
activities of both phosphoglucose isomerase and
6-phosphofructokinase
was observed when galactose was present as an
additional carbohydrate
source during fermentation. Also, for the
non-EPS-producing strain,
the specific activities of these enzymes were
comparable with
the values observed for the
S. thermophilus LY03
strain.
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DISCUSSION |
The yoghurt strain S. thermophilus LY03 produces a
heteropolysaccharide consisting of galactose and glucose in the ratio
4:1. In this study, fermentation experiments were carried out, using different carbohydrates or combinations of carbohydrates as substrates for bacterial growth and EPS production.
Lactose and glucose were taken up by S. thermophilus
LY03 and metabolized homofermentatively. Galactose was not used as
energy source. Indeed, S. thermophilus metabolizes
lactose in a homofermentative way and takes up lactose using a
lactose-galactose antiport system that excretes the galactose moiety
into the medium (10). S. thermophilus does
not ferment galactose (23). However, for some fermentation
experiments, very small amounts of galactose were also converted into
lactic acid towards the end of the exponential growth phase. This may
be explained by the fact that non-galactose-fermenting strains contain
intact genes for the Leloir pathway. However, these genes are usually
not transcribed (9).
The variations observed for the maximum carbohydrate consumption rates
can be explained by substrate inhibition and/or catabolite repression
of the more quickly metabolized energy sources. Indeed, de Vos and
Vaughan have stated that the lac genes (lactose metabolism) may be under (glucose) catabolite repression control (10).
For the one experiment carried out with a higher initial carbohydrate concentration, the low maximum carbohydrate consumption rates may also
be explained by the fact that the carbohydrate/nitrogen ratio was not
optimal for bacterial growth in this particular case (7).
The monomeric composition of the EPS produced by S. thermophilus LY03 was not affected by any of the carbohydrate
sources used in the fermentation experiments. This is an important
conclusion, given the contradictory reports in the literature
concerning the influence of the carbohydrate source on EPS composition.
The main reasons are most likely analytical artifacts encountered
during composition analysis and carbohydrate quantification
(11). Our results are in agreement with those of Escalante
et al., who did not observe any variation in monomeric composition when
using either lactose or glucose as an energy source for S. thermophilus (14).
Unlike the monomeric composition of the EPS, the amount of EPS produced
was clearly influenced by the carbohydrate source. When
glucose was used as the sole carbohydrate source, the
amount of EPS produced by S. thermophilus LY03
was significantly lower while the maximum cell density was
unchanged. Some authors also report the stimulation of EPS production
without a concomitant increase of the biomass concentration by
varying the carbohydrate source or increasing its concentration. For
instance, L. delbrueckii subsp. bulgaricus NCFB
2772 produced considerably larger amounts of EPS when it was grown on
glucose or lactose than when it was grown on fructose (19).
The amount of EPS produced seems to be correlated with the activities
of three enzymes: -phosphoglucomutase, UDP-galactose 4-epimerase,
and UDP-glucose pyrophosphorylase. Some authors have reported the
specific activities of enzymes involved in the biosynthesis of EPS from
lactic acid bacteria (14, 16, 19, 27, 31). A relationship
between EPS biosynthesis and the activity of the enzyme
-phosphoglucomutase was reported for L. lactis
(31). Our results clearly showed that none of the
S. thermophilus strains displayed
-phosphoglucomutase activity. The correlation that we observed
between the amount of EPS and the activity of UDP-glucose pyrophosphorylase was also reported by Escalante et al.
(14). They observed that UDP-glucose pyrophosphorylase was
correlated with EPS production in a ropy S. thermophilus strain but not in a nonropy strain. Grobben et
al. found that cultures of L. delbrueckii subsp.
bulgaricus NCFB 2772 grown on glucose displayed a higher UDP-glucose pyrophosphorylase activity than cultures grown on fructose
(19). They also observed that cells grown on a mixture of
glucose and fructose showed enzyme activities similar to those of cells
grown on glucose alone, while glucose was preferentially consumed as
the energy source. Because cultures of L. delbrueckii subsp.
bulgaricus NCFB 2772 grown on fructose produced EPS with a
lower level of galactose and even though the activity of UDP-galactose 4-epimerase was only slightly lower in these cells, the authors concluded that this enzyme does not play an important role in the
composition of the EPS produced. However, a relationship between the
activity of the enzyme UDP-galactose 4-epimerase and EPS production was
found for L. lactis subsp. cremoris
(16). In contrast, the UDP-glucose pyrophosphorylase
activity was inversely correlated with EPS production in the latter
strain (16). UDP-galactose 4-epimerase activity was
not correlated with EPS biosynthesis in any of the S. thermophilus strains tested by Escalante et al. (14).
Consequently, glucose or the glucose moiety from lactose hydrolysis
seems to be the source of carbohydrate for heteropolysaccharide biosynthesis in lactic acid bacteria. Indeed, a high UDP-glucose pyrophosphorylase activity was always found in our experiments. In addition, the Leloir pathway enzyme UDP-galactose 4-epimerase, necessary to convert UDP-galactose to UDP-glucose, displayed a very high activity in the galactose-negative S. thermophilus LY03 strain examined, in particular when the
EPS-producing and non-EPS-producing strains of S. thermophilus were compared. This indicates that the Leloir enzyme
UDP-galactose 4-epimerase is involved in the biosynthesis of precursors
for EPS production in EPS-producing galactose-negative S. thermophilus strains. This finding might also explain why
accumulation of UDP-glucose in cells of S. thermophilus leads to the production of EPS and why UDP-galactose 4-epimerase activity increased upon accumulation of UDP-galactose going hand in
hand with a decreased galactokinase activity (33).
The fact that rhamnose is not present in the EPS produced by
S. thermophilus LY03 corresponds with the very low
activities that we observed for the enzymes involved in the
biosynthesis of dTDP-rhamnose. This finding is in agreement with the
work of Grobben et al., who detected almost no activity for this enzyme system in cultures grown on fructose, which resulted in rhamnose-free EPS, and who detected high activities in cultures grown on glucose, which led to rhamnose-containing EPS (19).
The enzyme fructose 1,6-bisphosphatase did not display a substantial
specific activity for any of the carbohydrate combinations or for the
control strain, indicating that the flux of carbon to EPS production
does not follow the route from fructose 1,6-bisphosphate through
fructose 6-P to glucose 6-P and that all fructose 6-P is converted into
lactic acid for energy generation. Finally, a clear decrease of the
specific activities of both phosphoglucose isomerase and
6-phosphofructokinase was observed for S. thermophilus LY03, when galactose was present as an additional carbohydrate source
during fermentation. These results suggest inhibition or repression of
these enzymes by galactose and hence limitation of the carbon flux
towards glycolysis, without a significant change in the amount of EPS.
Because the -phosphoglucomutase enzyme is responsible for the
linkage between sugar catabolism and sugar anabolism, it will be
important to increase the carbon flux via phosphoglucomutase to a
sufficiently high level to improve EPS production. This might be done
via genetic engineering or process engineering. The same might be
accomplished by overexpression of UDP-galactose 4-epimerase and/or
UDP-glucose pyrophosphorylase.
| |
ACKNOWLEDGMENTS |
This work benefited from financial support from the Institut
Yoplait International. We further acknowledge financing from the
European Commission (grants FAIR CT98-4267 and INCO-Copernicus IC15-CT98-0905), the Flemish Institute for the Encouragement of Scientific and Technological Research in the Industry (IWT), the Fund
for Scientific Research (FWO
Flanders), and the Research Council of
the Vrije Universiteit Brussel (VUB).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Research Group
of Industrial Microbiology, Fermentation Technology and Downstream
Processing (IMDO), Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 32 2 6293245. Fax: 32 2 6292720. E-mail: ldvuyst{at}vub.ac.be.
| |
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Abstract
Introduction
