FEMS Microbiology Letters 17 (1983) 13-17
Published by Elsevier Biomedical Press
13
Lactose and sucrose utilization by
Streptococcus thermophilus
T e r e n c e D. T h o m a s a n d V a u g h a n L. C r o w
New Zealand Dairy Research Institute, Palrnerston North, New Zealand
Received and accepted 1 September 1982
1. I N T R O D U C T I O N
Sugar fermentation by Streptococcus thermophilus has not been extensively studied despite the
use of these organisms in some important milk
fermentations, such as in the manufacture of
yoghurt and certain Swiss and Italian cheese varieties where relatively high temperatures are used.
This is in contrast with the commercially important mesophilic lactic streptococci (S. cremoris
and S. lactis). The available data for S. thermophilus indicate that disaccharides (lactose and
sucrose) are fermented more readily than their
component monosaccharides [1-4] and that
marked strain variation occurs [5]. Most strains do
not possess phospho-fl-galactosidase activity [4,6],
suggesting that lactose transport does not involve
a phosphotransferase system. During lactose fermentation, galactose accumulates in the medium
[3,4].
Out of 15 strains of S. thermophilus studied, two
underwent rapid autolysis after growth at 42°C in
broth containing either lactose, glucose or galactose [7]. In contrast, no autolysis was observed
after growth on sucrose [7]. The present study was
undertaken to examine aspects of disaccharide
utilization in batch cultures and the involvement
of sugars in autolysis of S. thermophilus.
2. MATERIALS A N D M E T H O D S
S. thermophilus strains were obtained from the
following sources: CNRZ strains 368 and 391
from the National Agricultural Research Institute,
Jouy-en-Josas, France; N C D O strains 573 (which
is the type strain [8]) and 821 from the National
Institute for Research in Dairying, Reading, England; strain TS 2 from CSIRO Dairy Research
Laboratory, Melbourne, Australia. The culture
medium was Ts broth [9] which contained 75 mM
phosphate buffer and had an initial pH of 6.8. For
some experiments phosphate buffer was deleted
and the pH adjusted to 6.8. Sugar solutions were
filter-sterilized before addition to autoclaved broth.
Organisms were adapted to growth on the appropriate sugar by subculture at 37°C. After inoculation (1% unless stated otherwise), experimental
cultures were incubated at 42°C under static conditions. Bacterial density was measured turbidimetrically at 600 nm (Spectronic 20, Bausch and
Lomb, Rochester, NY) and bacterial dry weight
was obtained by membrane filtration of culture
samples. For enzyme assay, culture samples were
chilled at 4°C, centrifuged (20000 x g, 1 min) and
the supernatant immediately assayed for fl-galactosidase and lactate dehydrogenase (LDH). flGalactosidase was assayed at 42°C using 1 mM
o-nitrophenyl-fl-galactopyranoside (ONPG) in 50
mM phosphate buffer (pH 7.2). L D H was assayed
at 42°C using 0.25 mM N A D H and 10 mM pyruvate in 50 mM triethanolamine hydrochloride
buffer (pH 6.9). One unit of fl-galactosidase or
L D H activity was defined as the amount of enzyme which catalysed ONP release and N A D H
oxidation, respectively, at rates of 1 t~mol/h. For
sugar analyses, supernatant samples were immediately placed in a boiling water bath for 5 min
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies
14
which inactivated fl-galactosidase. Enzymatic
analyses were used for determination of lactose
[10], galactose [10], sucrose [11] and fructose [12],
whereas glucose was determined' with Statzyme
reagents (Worthington Diagnostics, Freehold, N J).
Cells were disrupted to estimate the total enzyme
concentration in cultures. Procedures for cell-free
extract preparation and protein determination have
been described previously [13].
3. RESULTS
3.1. Growth on lactose
During growth of S. thermophilus TS 2 in broth
containing 14 m M lactose, glucose and galactose
accumulated in the medium reaching concentrations of 1 mM and 13 raM, respectively, in a
typical experiment (Fig. 1A). Growth stopped
when lactose was exhausted, the maximum turbidity corresponding to 0.43 mg (dry weight)
bacteria/ml. Glucose and galactose were slowly
utilized after growth had stopped. Cytoplasmic
enzymes (fl-galactosidase and L D H ) were released
from TS 2 cells during growth (Fig. 1A). Cessation
of the total cellular enzyme content was released in
a typical experiment (Fig. 1A). At most, only
about half of the free glucose concentration in the
TS 2 culture (Fig. IA) could be accounted for by
the extracellular fl-galactosidase activity. This was
determined by adding known concentrations of
fl-galactosidase, in the form of crude extracts from
TS2, t o growth medium at 42°C containing different lactose concentrations and measuring the rate
of glucose release at pH 6.8 and 6.0. Therefore it is
apparent that glucose, as well as galactose, was
released from the cells. Strains 368, 391 and 821
all gave results similar to those shown for TS z in
Fig. 1A. In different experiments with strain TS 2,
the maximum concentration of cytoplasmic enzymes released corresponded to between 0.1% and
0.8% of the total. This variation may have led to
the different free glucose concentrations and maxim u m cell densities also observed in these experiments.
Growth of S. thermophilus 573 on lactose also
resulted in galactose and glucose accumulation
although glucose reached higher concentrations,
approx. 4.5 mM in a typical experiment (Fig. 1B),
than with the other strains. After growth stopped,
due to lactose exhaustion, glucose metabolism continued and culture turbidity declined. In contrast
to the other four strains, 573 did not release cytoplasmic enzymes during exponential growth. As
expected, enzyme release commenced with autolysis and the cell-free enzymes reached much higher
concentrations (Fig. 1B) than with the other (nonautolytic) strains (see Fig. IA). The accumulation
of galactose and glucose during the first 3 h of
incubation (Fig. 1B) was largely due to extracellular lactose hydrolysis by fl-galactosidase carried
over in the inoculum. Therefore with 573 growing
on limited lactose, the maximum cell density
reached will depend upon the initial fl-galactosidase concentration which is in turn influenced by
the inoculum percentage and by factors affecting
the fi-galactosidase concentration in the inoculum
culture (e.g. extent of autolysis and enzyme stability). The rate and extent of autolysis with 573
varied in different experiments.
3.2. Growth on sucrose
During growth of strain T S 2 o n 8 mM sucrose,
fructose accumulated in the medium to a maxim u m concentration of 1.7 mM (Fig. 2). Growth
stopped due to sucrose exhaustion at a maximum
cell density of 0.50 mg (dry weight)/ml. Fructose
also accumulated in the 573 culture but, in contrast to TS2, this sugar was rapidly utilized after
sucrose depletion (Fig. 2). As with lactose, strain
573 underwent autolysis after growth stopped due
to sucrose exhaustion. No glucose was detected in
the T S 2 and 573 cultures at any stage. Fructose
accumulation also occurred with the other three
strains (368, 391 and 821) but with only one strain
(821) did the concentration equal that of the
sucrose used.
The doubling times during exponential growth
at 42°C of the five strains on either lactose or
sucrose were all approx. 25 mih. With lactose and
sucrose the apparent molar growth yields for strain
TS 2 were, respectively, 37 and 65 g (dry weight)
b a c t e r i a / m o l disaccharide utilized.
15
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~lactosex
I
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I
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S. thermophi/us TS 2
20
,
10 - 1
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~
13
turbidity
LDH
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galectose
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8
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Time (h)
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8 ' 24
Fig. 1. Growth of S. thermophilus strains TS 2 (A) and 573 (B) in broth containing 14 m M lactose (2% inoculum) showing turbidity
(O), lactose (e) utilization, galactose (rn) and glucose (ll) accumulation, and release of /3-galactosidase (,8-gal, z~) and lactate
dehydrogenase (LDH, A) into the medium. The culture pH values after 8 h were 5.6 (A) and 5.1 (B). Note that the scale for cell-free
enzyme concentration in (B) is ten times that in (A).
10
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3. 3. Effect of pH on autolysis
•
=
When S. thermophilus 573 (the type strain) was
grown in broth containing varying initial concentrations of either lactose or sucrose, autolysis
occurred in the culture where disaccharide was
exhausted while the culture pH was > 5 (i.e. in the
cultures with low initial disaccharide concentrations). This behaviour is illustrated in Fig. 3A with
sucrose as the disaccharide (with lactose, which is
only partially fermented, about twice the disaccharide concentration was required for the same
effect). At low initial disaccharide concentrations,
autolysis was inhibited when the buffer was deleted from the medium (Fig. 3A). The effect of pH
•
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t
~,a
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-,= . . . .
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turbidity
/
z/
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O
0.01
01
2
4
6
Time (h)
8 ''
24
Fig. 2. Growth of S. thermophilus strains TS 2 (solid symbols)
and 573 (open symbols) in broth containing 8 m M sucrose
showing turbidity increase (o, O), sucrose utilization ( l l , D )
and fructose concentrations (A, za; broken lines). No free
glucose was detected in the cultures. The culture pH values
after 5 h were 5.9 and 5.7 for strains TS 2 and 573, respectively.
16
100
m
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r
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Time (h)
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6
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pH
Fig. 3. Effect of pH on autolysis of S. thermophilus573. (A) Terminal pH in batch cultures. Initial sucrose concentrations were either 6
mM (.) or 15 mM (m). In one culture, with an initial sucrose concentration of 6 mM (©), phosphate buffer was omitted from the
medium. Culture pH values after 4.5 h are given (after 24 h the values had decreased by 0. l to 0.2 pH units). (B) Buffer pH in cell
suspensions. Cell growing exponentially on either lactose (zx)or sucrose (A) were harvested and resuspended in 40 mM phosphate/20
mM citrate buffers (pH 4.0 to 7.0) at 42°C for 2 h. Lysis was determined by the decrease in turbidity and is expressed as the
percentage decrease in the initial turbidity.
on the autolysis of cells s u s p e n d e d in buffer is
shown in Fig. 3B. Extensive autolysis occurred
within 2 h i n c u b a t i o n p e r i o d except at p H values
<5.
4. D I S C U S S I O N
T h e present s t u d y indicates that d i s a c c h a r i d e
f e r m e n t a t i o n b y S. thermophilus growing in batch
c u l t u r e has some u n u s u a l aspects. D u r i n g growth
on lactose the galactose m o i e t y was used only
sparingly, as p r e v i o u s l y r e p o r t e d [3,4]. However, as
well as galactose a c c u m u l a t i o n in the m e d i u m ,
glucose also a p p e a r e d . T o our knowledge, there
have been no previous reports of glucose a c c u m u l a t i o n in S. thermophilus cultures. W i t h four of the
five strains e x a m i n e d , c y t o p l a s m i c enzymes (inc l u d i n g fl-galactosidase) were released d u r i n g
g r o w t h while no further release occurred after
g r o w t h s t o p p e d . In contrast, the o t h e r strain (573)
d i d not release c y t o p l a s m i c enzymes d u r i n g g r o w t h
but autolysis and enzyme release did occur after
g r o w t h s t o p p e d p r o v i d e d the culture p H r e m a i n e d
a b o v e 5. W h e n a p a r t i a l l y autolysed culture of 573
was transferred to fresh m e d i u m , the cell-free fig a l a c t o s i d a s e carried over resulted in extensive
extracellular lactose hydrolysis which a c c o u n t e d
for the high glucose c o n c e n t r a t i o n in the m e d i u m .
However, with the other four strains (e.g. TS z) the
a m o u n t of fl-galactosidase transferred was small
and, although this e n z y m e was released d u r i n g
growth, its activity could not account for all of the
glucose which a p p e a r e d . This suggests that glucose, as well as galactose, was released from the
cells.
D u r i n g growth of all strains (except 821) on
sucrose, b o t h glucose and fructose moieties were
utilized s i m u l t a n e o u s l y although some fructose did
a p p e a r in the m e d i u m . Strain 821 d i d not utilize
the fructose moiety. Therefore with most strains
the sucrose molecule was m o r e c o m p l e t e l y utilized
t h a n the lactose molecule. This m a y explain why
previous investigators o b s e r v e d autolysis with 14
17
mM lactose but not with 14 mM sucrose [7] since
lower pH values would have resulted in the sucrose
cultures. In our laboratory, the medium [9] used
for routine subculture of S. cremoris and S. lactis
contains a limiting lactose concentration (14 mM,
final pH 5.6) to minimize the injury suffered by
cells exposed to low pH values [14]. On the other
hand, use of culture media containing excess
lactose (e.g. milk) will prevent autolysis of S. thermophilus strains such as 573 because of the resulting low culture pH values.
ACKNOWLEDGEMENT
We gratefully acknowledge J.S. Cleland for excellent technical assistance.
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