FEMS Microbiology Letters 109 (1993) 237-242
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
237
FEMSLE 05432
Utilisation of maltose and glucose by lactobacilli
isolated from sourdough
Peter Stolz, Georg B6cker, Rudi F. Vogel and Walter P. Hammes
Institut fiir Lebensmitteltechnologie, Universitdt Hohenheim, Stuttgart, FRG
(Received 18 February 1993; revision received 2 March 1993; accepted 8 March 1993)
Abstract." The utilisation of glucose and maltose was investigated with Lactobacillus strains isolated from sourdough starters. These
preparations have been in continuous use for a long period to produce sourdough from rye, wheat and sorghum. The major
metabolic products formed by resting cells from glucose or maltose were lactate, ethanol and acetate. Upon fermentation of
maltose, resting cells of Lactobacillus sanfrancisco, L. reuteri, L. fermentum and Lactobacillus sp. released up to 13.8 mM glucose
after 8 h. The ratio of released glucose per mol of utilised maltose was up to 1 : 1. Glucose formation was high when starved cells of
L. sanfrancisco and Lactobacillus sp. were used. This is consistent with maltose utilisation via maltose phosphorylase which
phosphorylates maltose without the expenditure of ATP and thus allows the cell to waste glucose in the presence of abundant
maltose. The glucose formed may be utilised by the lactobacilli or other microorganisms, e.g. yeasts. However, the release of
glucose into the medium by sourdough lactobacilli prevents competitors from utilising the abundant maltose by glucose repression.
In strains of L. sanfrancisco, maltose utilisation was very effective and not subject to glucose repression. Therefore, they overgrow
other microorganisms sharing this habitat. Wild isolates of L. sanfrancisco were initially unable to grow on glucose. Upon growth
on maltose such strains required adaptation times of up to 150 h to grow on glucose. After subsequent transfer of glucose-grown
cells to fresh medium the strains resumed growth both on glucose or maltose. They readily lost their ability to grow on glucose
upon exposure to maltose. L. sanfrancisco exhibited biphasic growth characteristics on media containing glucose, maltose or both
carbon sources. Evidence is provided that biphasic growth and metabolite formation are dependent on the redox potential.
Key words: Lactobacillus sanfrancisco; Sourdough fermentation; Maltose metabolism; Glucose utilisation; Ecology
Introduction
Sourdough starter preparations are traditionally p r e p a r e d u n d e r n o n - a s e p t i c b u t w e l l - d e f i n e d
conditions. During consecutive cycles of the respective process the fermenting organisms are
Correspondence to: R.F. Vogel, Institut fOr Lebensmitteltechnologie, Universit~it Hohenheim, Garbenstr. 25, W-7000
Stuttgart 70, FRG.
continuously propagated and strains are selected
w h i c h a r e m o s t a d a p t e d t o t h i s e n v i r o n m e n t [1].
T h e s e s t r a i n s f o r m e r l y i d e n t i f i e d as Lactobacillus
brevis ssp. lindneri [2,3] w e r e a l l o t t e d t o L. sanfrancisco [4], w h i c h w a s d e s c r i b e d a s a n e w s p e c i e s
p r e v a i l i n g i n w h e a t s o u r d o u g h [5]. P r e v i o u s s t u d ies r e v e a l e d t h a t o n l y t w o s t r a i n s o f L. sanfrancisco a n d o n e s t r a i n o f L. brevis r e p r e s e n t e d
9 9 . 9 % o f t h e m i c r o f l o r a o f r y e s o u r d o u g h a t cell
c o u n t s o f a p p r o x . 2 × 10 9 [6]. T h e s e l a c t o b a c i l l i
s h a r e t h e h a b i t a t w i t h t h e y e a s t Candida milleri
238
which was p r e s e n t at cell counts of < 10 6 cfu
g-1. T h e c o m b i n a t i o n o f L. sanfrancisco a n d
Candida milleri was also f o u n d in w h e a t sourd o u g h [7]. S i m i l a r m i c r o b i a l a s s o c i a t i o n s w e r e
d e t e c t e d in s o r g h u m s o u r d o u g h for t h e p r o d u c tion o f Kisra which is t h e m a j o r staple food in
S u d a n . T h e m a i n o r g a n i s m s p r e s e n t in this type
of s o u r d o u g h w e r e few strains o f L. amylovorus,
L. reuteri a n d L. fermentum a n d o n e strain o f
Candida krusei [8]. This c o m b i n a t i o n o f m a l t o s e fermenting lactobacilli and maltose-negative
yeasts was found, a l t h o u g h the c o n d i t i o n s p r e s e n t
in rye a n d wheat, o r s o r g h u m d o u g h a r e d i f f e r e n t
with r e s p e c t to e n v i r o n m e n t a l factors (e.g. t e m p e r a t u r e , w a t e r activity, r e d o x p o t e n t i a l a n d
availability o f substrates). T h e m e t a b o l i c activities
o f the a s s o c i a t e d o r g a n i s m s e.g. s t a r c h d e g r a d a tion a n d m a l t o s e f e r m e n t a t i o n allow synergism
[8]. T h e m e t a b o l i c p r o d u c t s f o r m e d by t h e n u m e r ically d o m i n a t i n g lactobacilli m a i n l y d e t e r m i n e
t h e quality o f t h e s o u r d o u g h a n d thus t h e nutritional value, shelf life, t e x t u r e a n d flavour o f t h e
b r e a d . T h e s e m e t a b o l i t e s result f r o m t h e f e r m e n t a t i o n o f low m o l e c u l a r sugars p r e s e n t in the
d o u g h o f which m a l t o s e is p r e v a l e n t . In this comm u n i c a t i o n , t h e utilisation was i n v e s t i g a t e d o f
glucose a n d m a l t o s e by lactobacilli i s o l a t e d f r o m
sourdough. Lactobacilli from other environments
w e r e u s e d for c o m p a r i s o n . A g e n e r a l p r i n c i p l e is
p r o p o s e d which is causative for the ability o f
t h e s e o r g a n i s m s to c o m p e t e in this e n v i r o n m e n t .
Materials and Methods
Microorganisms and culture conditions
T h e strains o f m i c r o o r g a n i s m s u s e d d u r i n g this
study a r e listed in T a b l e 1. Lactobacillus sp.
LTH1735 a n d L. reuteri L T H 3 1 2 0 w e r e grown in
sanfrancisco m e d i u m ( S M ) [6]. A l l o t h e r strains
w e r e grown in m M R S c o n t a i n i n g t h e following
c o m p o n e n t s (g 1-1): t r y p t o n e (10), yeast extract
(10), m a l t o s e (20), K 2 H P O 4 . 3 H 2 0 (2), cysteineHC1 (0.5), M g S O a . 7 H 2 0
(0.1), M n S O 4 . 4 H 2 0
(0.05), T w e e n 80 (1). T h e p H was a d j u s t e d to 6.2.
A l l i n c u b a t i o n s w e r e p e r f o r m e d in screw c a p
b o t t l e s at 30°C. T h e ability o f strains o f L. sanfrancisco to grow o n glucose o r m a l t o s e as sole
c a r b o n source o r on a c o m b i n a t i o n o f b o t h was
i n v e s t i g a t e d in m M R S c o n t a i n i n g a t o t a l o f 20 g
1-1 o f t h e r e s p e c t i v e sugars. T h e p r e s e n c e o f
oxygen in t h e m e d i u m was i n d i c a t e d by a c o l o u r
c h a n g e o f t h e r e d o x i n d i c a t o r r e s a z u r i n e which
was a d d e d at a c o n c e n t r a t i o n o f 1 mg 1-1.
Preparation o f resting cells
Cells w e r e grown to t h e late e x p o n e n t i a l p h a s e ,
h a r v e s t e d by c e n t r i f u g a t i o n a n d w a s h e d in PBS-
Table 1
Major metabolites formed by resting cells of lactobacilli upon fermentation of maltose
Strain
L. sanfrancisco
L. sanfrancisco
L. sanfrancisco
L. reuteri
Lactobacillus sp.
Lactobacillus sp.
Lactobacillus sp.
Lactobacillus sp.
L. fermentum
L. casei
L. plantarurn
L. sake
ATCC 27651
LTH 1729
LTH 2581
LTH 3120
LTH 3125
LTH 1731
LTH 1735
LTH 2585
ATCC 14931
DSM 20011
DSM 20174
LTH 677
Maltose
Lactate
Acetate
Ethanol
Glucose
- 17.6
- 17.2
-22.0
- 11.7
- 8.2
-31.6
-9.0
- 11.4
- 16.1
- 7.9
-6.0
- 5.7
32.7
28.5
30.1
6.7
28.6
51.4
5.7
8.4
16.2
21.2
20.0
21.0
1.8
2.3
0.8
2.8
1.3
3.1
4.7
4.4
2.3
0.0
0.5
1.4
31.0
27.2
32.6
5.3
3.0
47.3
3.6
7.3
15.9
0.0
0.0
0.0
0.4
4.6
6.9
12.5
1.6
5.8
4.5
2.8
13.8
2.3
0.2
0.2
Molar ratio
glc/mal
0.02
0.27
0.32
1.10
0.20
0.19
0.50
0.25
0.86
0.26
0.03
. 0.04
glc, glucose; mal, maltose. The concentrations of the sugars are given in mmol 1-1. The negative values for maltose indicate maltose
consumption.
239
maltose at p H 6.0 consisting of 700 ml solution I
containing (g 1-1): NaCI (8), KCI (0.2), N a 2 H P O 4
• 2 H 2 0 (2.2), K H z P O 4 (12); 100 ml solution II:
CaC12 (0.1); 100 ml solution III: M g C l z - 6 H 2 0
(0.2); 100 ml solution IV: maltose (20). All solutions were mixed after autoclaving separately.
Subsequently, the cells were suspended in PBS
containing 50 mM maltose to an OD578 of 4.0.
For the preparation of starved resting cells, PBS
without maltose was used in the washing procedure, i.e. the starvation time was approximately 1
h.
Determination of sugars and organic acids
Resting cells were incubated anaerobically at
30°C. Samples (1 ml) were taken for H P L C analysis after 0.5, 1, 3, 5, 8 and 22 h. The cells were
removed by centrifugation and the proteins present in the supernatant were precipitated during
more than 2 h after addition of 50 ~1 of 60%
perchloric acid. The proteins were removed by
centrifugation and the sugars and organic acids in
the supernatant were analysed by H P L C as described previously [8].
Results
Release of glucose by sourdough lactobacilli
The metabolism of glucose and maltose was
investigated of strains of L. sanfrancisco and
Lactobacillus sp. isolated from rye and wheat and
L. reuteri isolated from sorghum sourdough. Lactobacillus sp. strains were identified as a taxonomically separate cluster of strains which represented up to 80% of the microflora in some
sourdoughs. The major metabolites formed from
maltose by resting cells of sourdough lactobacilli
upon 8 h fermentation of maltose are listed in
Table 1. L. casei DSM 20011, L. fermentum
ATCC 14931, L. plantarum DSM 20174 and L.
sake L T H 677 were used for comparison. L.
casei, L. plantarurn, L. sake and Lactobacillus sp.
L T H 3125 produced mainly lactate, whereas the
major products formed by L. sanfrancisco, L.
reuteri, Lactobacillus sp. L T H 1731, 1735 and
2585 were lactate and ethanol in addition to
varying amounts of acetate. Glycerol, 1,3-pro-
60
0
=E
g
40
o
"=
30
g=
¢= 20
~
0
0
=,..:--:
10
0
I
0
5
I
I
15
10
time (h)
I
I
20
25
Fig. 1. Time course of maltose utilisation and release of
glucose by resting cells of L. sanfrancisco L T H 2581 and L.
reuteri L T H 3120. Concentration of m a l t o s e / g l u c o s e in fermentations with L. sanfrancisco (t3, II) L. reuteri (©, e).
pane-diole, and succinate, were detected as minor metabolites. In addition to these metabolites
some strains formed significant amounts of glucose from maltose and released it into the buffer
medium. L. reuteri L T H 3120 and L. fermentum
ATCC 14931 formed nearly equimolar amounts
of glucose per maltose utilised. No glucose formation was observed with L. sanfrancisco ATCC
27651, L. plantarurn DSM 20174 and L. sake
L T H 677. The time course of maltose utilisation
and release of glucose by resting cells of L.
sanfrancisco L T H 2581 and L. reuteri L T H 3120
is depicted in Fig. 1. All strains of L. sanfrancisco
and Lactobacillus sp. L T H 1735 exhibited an
increased release of glucose upon maltose fermentation when starved cells were used (Table
2). This response was not detected with starved
Table 2
Release of glucose by starved resting cells of lactobacilli upon
fermentation of maltose
Strain
L, sanfrancisco
L. sanfrancisco
L, sanfrancisco
Lactobacillus sp.
L. reuteri
Maltose Glucose Molar ratio
glc/mal
A T C C 27651 - 20.2
L T H 1729
-19.8
L T H 2581
- 7.8
L T H 1735
-9.6
L T H 3120
- 10.2
13.8
18.0
6.9
7.2
8.3
0.68
0.91
0.88
0.75
0.81
glc, glucose; mal, maltose. T h e concentrations of the sugars
are given in mmol 1-1. The negative values for maltose
indicate maltose consumption.
240
10'
E
c
1
o
0,1
O,Ol
,
20
0
~
40
,
60
,
80
~
1 O0
,
120
,
140
~
160
,
180
,
200
time (11)
Fig. 2. Adaptation of strains of L. sanfrancisco to glucose. The time course is depicted of the increase of OD57s. OD578 of the
culture of L. sanfrancisco strains growing on maltose/glucose as carbon source: ATCC 27651 (A, • ) , LTH 1729 ([3, • ) , LTH
2581 (©, o).
cells of L. reuteri L T H 3120 which revealed reduced release of glucose and a balanced metabolite formation from maltose which was in contrast
to an unbalanced metabolite formation observed
with starved cells of L. sanfrancisco (data not
shown). Under these conditions not yet identified
peaks were observed during H P L C analysis which
were also responsible for the stoichiometrically
unbalanced ratio of utilised maltose to produced
metabolites with non-starved cells of Lactobacil/us sp. L T H 1735 and 2585 (Table 1). Release of
glucose was also observed with growing cells of
L. sanfrancisco, L. fermentum and Lactobacillus
sp. on mMRS containing maltose, at values of
0.5-5 mmol 1-1.
Growth of L. sanfrancisco on maltose or glucose
Strains of L. sanfrancisco were precultured
overnight on mMRS containing maltose or glucose and transferred to mMRS containing maltose or glucose, respectively. In Fig. 2, the time
course is depicted of the increase of OD578 upon
growth of L. sanfrancisco in these media. The
strains grew well on maltose, whereas a lag phase
of 20, 110 and 150 h was observed on glucose
with L. sanfrancisco LTH 2581, L T H 1729 and
A T C C 27651, respectively. When the strains had
acquired the ability to ferment glucose they were
able to resume growth on maltose and glucose
when transferred to fresh media containing one
of these sugars (Fig. 3). However, they lost their
10
•
E
lumnlam
•
•
nn
n - -
1
0
0,1
0,01
20
40
60
80
1O0
120
140
160
180
200
220
240
260
time (h)
Fig. 3. Growth of L. sanfrancisco ATCC 27651 on mMRS containing maltose, maltose and glucose or glucose. The time course is
depicted of the increase of OD57s. The arrows indicate the transfer of cells to fresh medium. Growth on medium containing
maltose ( • ), glucose(~), maltose and glucose ( • ).
241
3,00
10,00
2,50
....---""'""--"
•~ 2,00
O
1,00
c
~
1,50
,oo
ditions prevailed, whereas oxygen was present
during the initial phase of fermentation in screw
cap bottles. U p o n transfer of cells from the stationary phase into fresh medium the same biphasic growth was observed. After inoculation at
higher cell densities the metabolic switch was
observed after a shorter time (Fig. 3).
o, oO
Discussion
0,50
0,00
0,01
0
20
40
60
time (h)
Fig. 4. Biphasic growth of L. sanfrancisco ATCC 27651 on
mMRS containing maltose, and the concomitant change in
the molar ratio of acetate/ethanol. The ratio of acetate to
ethanol is depicted as grey bars; growth on mMRS containing
maltose (10 g-t) (i). After 30 h the fermentation conditions
were strictly anaerobic.
ability to grow on glucose upon exposure to maltose.
Biphasic growth of L. sanfrancisco on glucose and
maltose
The growth of L. sanfrancisco A T C C 27651
was investigated with maltose, glucose or both
carbon sources, and the metabolites formed were
determined. The time course of growth and
metabolite formation is depicted in Figs. 3 and 4.
The cell yield measured by the maximum OD578
obtained with cells growing on glucose as sole
carbon source or glucose and maltose was 50% of
the value obtained with maltose when the quantity of glucose moieties was equal in both media
(Fig. 3). In fermentations containing maltose and
glucose, both sugars were utilised simultaneously,
and no preference for glucose or maltose was
observed. Nevertheless, growth of the culture exhibited biphasic characteristics irrespective of the
carbon source. During the fermentation the redox potential decreased and a change was observed in the metabolic products formed (Fig. 4).
In the initial phase mainly acetate and lactate
were formed. Subsequently, lactate and ethanol
were formed. At this stage strictly anaerobic con-
The maltose metabolism of Lactobacillus
strains isolated from sourdough revealed some
peculiar features which can affect the microbial
ecology in the dough and the sensorial properties
of the bread. By tradition, bakers consider the
ratio of lactate to acetate as a characteristic criterion for bread quality [1]. This ratio is controlled
by technological measures, e.g. the formula, temperature and batch size. During dough mixing,
the entry of oxygen leads to an increase of the
redox potential. The present investigation indicates an influence of the redox potential on
growth and metabolite formation of sourdough
lactobacilli. The biphasic growth characteristics
observed with L. sanfrancisco was concomitant
with a change in the metabolites formed. No
diauxic growth was observed in media containing
both glucose and maltose. At the start of the
fermentation, residual oxygen is present in the
medium which is reduced, and anaerobic conditions are established. The change from aerobic to
anaerobic conditions is visualised by the addition
of resazurine to the medium. As long as sufficient
oxygen is present acetate is formed in addition to
lactate. The ethanol formed in the later fermentation phase (strictly anaerobic) may be necessary
to regenerate reduction equivalents, i.e. NAD.
When cells were used at a higher inoculation
density the metabolic switch occurred earlier because the oxygen was consumed faster.
In strains of L. sanfrancisco, maltose fermentation was not repressed by glucose. In contrast,
twice the cell yield was obtained with 20 g 1-1
maltose compared with 20 g 1-1 glucose, i.e. with
the same number of glucose residues. Thus, utilisation of maltose is more effective than utilisation
of glucose. The higher cell yield on maltose and
242
the release of glucose at ratios of up to 1:1
glucose per consumed maltose is consistent with
maltose utilisation via maltose phosphorylase. The
presence of this enzyme was demonstrated in
strains of L. brevis isolated from spoiled beer
[9,10]. It phosphorylates and cleaves maltose
without the expenditure of ATP to form glucose
and glucose-l-phosphate which is further metabolised. Thus, the cell can afford 'wasting' of glucose in the presence of abundant maltose, which
is the major low-molecular carbon source in beer
and in sourdough. Part of the glucose was released into the medium, whereas it was simultaneously metabolised or accumulated intracellulary resulting in a ratio of released glucose to
utilised maltose lower than 1. Simultaneous utilisation of glucose and maltose was also observed
with growing ceils of L. sanfrancisco. The reason
for the lag phase in the growth of L. sanfrancisco
ATCC 27651 and LTH 1729 during adaptation to
glucose in the absence of maltose remains to be
elucidated. It may be caused by the presence of
different transport systems for maltose and glucose which are regulated by the sugars or by the
redox potential.
Increasing levels of glucose were detected during the fermentation of sorghum [8] and rye sourdough (data not shown). The apparently futile
release of glucose may become an ecological advantage as it prevents maltose positive competitors from utilising the abundant maltose. The
release of glucose by lactobaciUi is consistent with
'control' of the growth of bacteria and yeasts
competing for maltose by feeding or glucose repression. This is supported by the observation
that a Saccharomyces cerevisiae strain disappeared from the microbial population of a sourdough during consecutive fermentations even after initial inoculation at high cell densities [ll].
The disappearance may be caused by repression
of genes involved in maltose fermentation by the
glucose released by sourdough lactobacilli. The
release of glucose by L. sanfrancisco prevents
competitors from utilising maltose and thus affects the microbial ecology in sourdough. Furthermore, the ability of L. sanfrancisco to compete is supported by effective utilisation of the
maltose, which is only available for strains exhibiting this mechanism.
Acknowledgement
This work was supported by the Commission
of the European Communities Contract No.
BIOT-CT91-0263 (SSMA).
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