Z Lebensm Unters Forsch (1995) 201:402 410
9 Springer-Verlag 1995
Original paper
Utilization of electron acceptors by lactobacilli isolated from sourdough
II. Lactobacillus pontis, L. reuteri, g amylovorus, and L. fermentum
Peter Stolz 1, Rudi F. Vogel 2, Walter P. Hammes 1
1 Institut ffir Lebensmitteltechnologie,Garbenstrasse 25, D-70599 Stuttgart, Germany
2 Lehrstuhl fiir Technische Mikrobiologie, Technische, Universit/it Mi.inchen,D-85350 Freising-Weihenstephan,Germany
Received: 13 September 1994/Revised version: 24 November 1994
Abstract. The metabolism of maltose and the use of elec-
tron acceptors has been investigated in strains of lac-
tobacilli which are known to be stable elements in sour-
doughs, which, traditionally, have been used for a long
time. The metabolic features of LactobacilIus sanfrancisco
have been described by us in a previous communication.
Similar principles have been detected for the compet-
itiveness of L. pontis, L. reuteri, L. fermentum and
L. amylovorus, as well as species-sPecific characteristics.
Based on these findings the metabolic key reactions have
been identified and the use of electron acceptors present in
sourdough are presented in a schematic overview. In con-
trast to L. sanfrancisco, these species can not use oxygen as
an electron acceptor, and the length of their lag phase was
not affected by agitation. Malate and fumarate were re-
duced to succinate, and fructose was used, depending on
the species, as an electron acceptor, carbon source or both.
All heterofermentative sourdough lactobacilli efficiently
split maltose using maltose phosphorylase. Glucose was
excreted, which induced glucose repression in competing
indigenous micro-organisms, without affecting the mal-
tose metabolism of sourdough lactobacilli. Lactobacilli
generate additional adenosine 5'-triphosphate (ATP) from
acetyl phosphate in the presence of electron acceptors.
These special features are suggested to represent a general
principle which accounts for the prevalence of specific
heterofermentative lactobacilli which are propagated over
long periods present in sourdough fermentations.
Introduction
Lactobacilli are predominant specialists in fermenting
food substrates, although they are known to be micro-
organisms which have a reduced metabolic capacity. The
factors decisive for the competitiveness of the dominant
strains are still poorly understood. For control of the
Correspondence to: R.F. Vogel
zeitschriftfur
fermentation process, however, this knowledge is of para-
mount importance as fermentation proceeds under non-
aseptic conditions and over long periods of time, as is the
case with sourdough, an intermediate product obtained in
breadmaking, which is maintained by reiterative inocula-
tion with part of the previous batch [-1]. It has been
demonstrated that the microbial flora consists mainly of
characteristic and highly competitive lactobacilli which
are present at concentrations ranging between 108 and 109
colony forming units (cfu) per gram [,1-4]. These include
strains of Lactobacillus sanfrancisco and strains formerly
allotted to L. brevis. Some of the strains initially classified
as L. brevis belong to a new species designated as L. pontis
[-5]. These strains can constitute up to 80 % of the micro-
bial flora of rye or wheat sourdough. Similarly, strains of
L. reuteri, L. fermentum and L. amylovorus can be found at
high cell counts in sorghum sourdough, a batter which is
propagated at tropical temperatures of 30-40~ over
long periods and used for Kisra production [6].
In doughs prepared without an inoculum, a vast spec-
trum of additional bacteria, especially lactic acid bacteria
may occur [7-9]. These organisms are usually also present
in traditionally treated sourdoughs, however at concen-
trations three orders of magnitude below those deter-
mined for the competitive strains El, 2, 4, 10].
In sourdoughs prepared from wheat or rye, as well as
in fermented batters from sorghum, yeasts were found to
be a second group of micro-organisms present at cell
counts of up to 10 6 cfu/g. Although the composition of the
substrates and the fermentation parameters are different
in sourdoughs prepared in traditional ways, from wheat,
rye or sorghum, a characteristic combination is built up
consisting of maltose-fermenting lactobacilli and maltose-
negative yeasts; these include strains of Candida milleri
or C. krusei [-3, 4, 6, 11]. The yeasts utilize glucose and
fructose present or generated in the flour [,,12, 13].
Remarkably, the presence of maltose-fermenting yeasts
in sourdough has also been reported [8, 10, 14, 15].
Sourdough is rich in starch and polyfructosanes,
which are enzymatically degraded to the fermentable
carbohydrates maltose, fructose and some glucose [,16].
The sourdough lactobacilli effectively metabolize maltose,

which is a key feature of their competitiveness in this
environment ([17] P. Stolz, R.F. Vogel, W.P. Hammes,
unpublished data). Upon maltose fermentation, glucose is
released by the lactobacilli into the medium in amounts
the maximum of which are equimolar with the fermented
maltose and glucose-l-phosphate formed by maltose
phosphorylase without expenditure of adenosine 5'-
triphosphate (P. Stolz, R.F. Vogel, W.P. Hammes, unpub-
lished data (ATP) [-19]). As suggested by Stolz et al. ([18],
unpublished data) this glucose affects the growth of the
competing lactobacilli and yeasts by feeding and can fur-
ther prevent their consumption of maltose via a glucose
repression. Thus, the ability to ferment maltose does not
appear to be of relevance for the indigenous yeasts in
sourdough.
Furthermore, it has been demonstrated that strains of
L. sanfrancisco readily consume fructose by its preferential
use as an electron acceptor when maltose is available in
excess [20]. Under the latter conditions the cells
do not form ethanol, instead they gain an additional
ATP by formation of acetate from acetyl phosphate. This
reaction becomes possible as nicotinamide adenine dinuc-
leotide (NAD) is regenerated by reducing fructose to
mannitol.
In the preceding communication [20] the unique
metabolic adaptation of L. sanfrancisco to sourdough
fermentation was described. It was the purpose of this
study to investigate if, or to what extent the findings with
L. sanfrancisco represent a common principle in the
microbiology of traditionally treated sourdoughs. For this
purpose strains of species were investigated that are of
similar technological importance in sourdoughs as L. san-
francisco.
Materials and methods
Bacterial strains and media. The strains used were L. pontis LTH
2587, LTH 1731 and LTH 1735, L. amylovorus LTH 3122, L.
fermentum LTH 3125 and L. reuteri LTH 3120. The cultures were
grown in a modified MRS medium (mMRS) as described by Stolz
et al. [20].
Culture conditions and determination of metabolites. Cultures
were grown and metabolites determined according to Stolz et al.
E203.
Results
The effect of agitation on growth and metabolite
formation of anaerobic cultures
L. pontis L T H 2587, L T H 1731 and L T H 1735, L. reuteri
L T H 3120, L. amylovorus L T H 3122 and L. fermentum
L T H 3125 were grown as agitated cultures anaerobically
on mMRS plus 5 g/1 fructose. The addition of fructose
was necessary because L. pontis strains L T H 1731
and L T H 1735 exhibited only poor growth on mMRS, as
is shown in Fig. la-f. The lag phases of the strains
were not affected by agitation, however, the cells gener-
ally grew better. The ratio of acetate to ethanol was
unchanged.
403
The effects of aeration and strictly anaerobic conditions
on growth
L. pontis L T H 2587, L T H 1731 and L T H 1735, L.
amylovorus L T H 3122, L. fermentum L T H 3125 and L.
reuteri L T H 3120 were grown on mMRS plus fructose
(5 g/l) under strictly anaerobic conditions as well as in
aerated cultures. The kinetics of growth and the metab-
olites formed were determined. The effects of agitation on
anaerobic growth are included in Fig. la-f. All strains
grew well under strictly anaerobic conditions, reaching
a final optical density at 578 nm (0D578) of approxim-
ately 3, with the exception of L. amylovorus L T H 3122, the
culture of which grew to an ODsv8 of only 2. Aerated
cultures ofL. reuteri L T H 3120 did not grow at all and the
other strains exhibited only poor growth.
The effect of electron acceptors on L. pontis
Cells of L. pontis L T H 2587, L T H 1731 and L T H 1735
were grown under anaerobic conditions without agitation
in the presence of various substrate combinations. The
metabolic products formed during the fermentation were
determined. The results obtained with samples drawn
after 50 h of fermentation serve as a representative
example and are listed in Table 1.
Remarkably, the amounts of mannitol formed were
not equimolar with the amount of fructose consumed:
25retool fructose were reduced to approximately
20 mmol mannitol and concomitantly formed 10 mmol
acetate. A fraction of 5 mmol fructose was metabolized to
lactate and ethanol.
The growth characteristics in media containing mal-
tose or maltose plus fructose are depicted in Fig. 2a-c. In
the presence of fructose, all strains of L. pontis exhibited
a significantly reduced lag phase. In addition, L. pontis
L T H 1731 and L T H 1735 exhibited strongly increased
growth rates and final cell yields in the presence of maltose
plus fructose. With maltose alone L. pontis L T H 1735
reached a final ODsv8 of 1.5 after 70 h, whereas L. pontis
L T H 1731 reached an OD578 of only 1.1 after 115 h. At
this stage all maltose had been consumed by L. pontis
L T H 1735, whereas strain L T H 1731 had consumed only
7.5 mmol of the maltose present in the medium. This
indicates that the latter strains ofL. pontis require fructose
for effective formation of ATP.
Citrate was not used as an electron acceptor by any
strain of L. pontis, whereas malate and fumarate served
as electron acceptors for L. pontis L T H 1731 exclusively.
Succinate was formed in amounts which were equimolar
with the amounts of malate or fumarate consumed.
Effect of electron acceptors on L. reuteri L T H 3120,
L. amylovorus L T H 3122 and L. fermentum L T H 3125
Cells ofL. reuteri L T H 3120, L. amylovorus L T H 3122 and
L. fermentum L T H 3125 were grown under anaerobic
conditions, without agitation, in the presence of various
substrate combinations. The metabolic products formed
during the anaerobic fermentation were determined and
404

3,0 a 3,0
d
2,5 2,5

2,0 2,0
E
e- E
t-

~ 1,5 ~ 1,5
e~
o 0 1,0
1,0

0,5 0,5

0,0 I I I I I I 0,0 I I I I I
0 5 10 15 20 25 30 0 5 10 15 20 25 30
time (h) time (h)
3,0 b 3,0 e

2,5 2,5

2,0 2,0
S E
~ 1,5
//
1,5
in
O r
O 1,0 0 1,0

0,5 0,5

0,0 , , , , , , 0,0 ' , , , ,

0 5 10 15 20 25 30 0 5 10 15 20 25 30
time (h) time (h)
3,0 C 3,0 f
2,5 2,5

E 2,0 9 E 2,0

~1,5 j,
O 1,0 81,o

0,5 ~.~...,, 0,5

fm
0,0 I 0,0 " - - " C F : ~ Q 'm
"/ ml" I I I I
0 5 10 15 20 25 30 0 5 10 15 20 25 30
time (h) time (h)
Fig. la-f. The effect of agitation on the growth of sourdough lactobacilli. The time course is depicted of the increase in the optical density at
578 nm (ODs78) of cultures of: a L. pontis LTH 2587, b L. pontis LTH 1731, e L. pontis LTH 1735, d L. reuteri LTH 3120, e L. amylovorus LTH
3122, f L. fermentum LTH 3125. The cells were grown on mMRS maltose (27.75 mmol/1) plus fructose (27.75 retool/i). The cultures were
incubated in anaerobic conditions with agitation ([~) at 150 rpm, and without agitation (ll)

their concentrations present in samples taken after 50 h
are listed in Table 2. L. reuteri L T H 3120 exhibited equal
growth rates when grown on maltose or maltose plus
fructose (Fig. 2d). After 31 h, this strain started to metab-
olize fructose when maltose was depleted, and mannitol
was not produced from fructose. In addition, increased
amounts of acetate were detected under these conditions.
Similarly, L. amylovorus L T H 3122 exhibited biphasic
growth characteristics, but used fructose preferentially as
a carbon source in media containing maltose plus fructose
(Fig. 2e). This homofermentative organism produced
mainly lactate from fructose. In fermentation media con-
taining fructose, L.fermentum L T H 3125 exhibited a fruc-
tose-stimulated growth pattern and used fructose as an
electron acceptor, forming stoichiometric amounts of
mannitol. The increase in the formation of acetate corre-
sponded to half the amount of mannitol formed.
The effect of fructose on the lag phase ofL. amyIovorus
L T H 3122, L. fermentum L T H 3125 and L. reuteri L T H
3120 is depicted in Fig. 2d-f. With the exception of
T a b l e 1. M a j o r m e t a b o l i t e s f o r m e d b y g r o w i n g cells o f Lactobacillus pontis ( f r o m r y e s o u r d o u g h ) . T h e c o n c e n t r a t i o n s listed a r e p r e s e n t in c u l t u r e s u p e r n a t a n t s a f t e r 5 0 h o f f e r m e n t a t i o n o f
m a l t o s e p l u s a p o t e n t i a l e l e c t r o n a c c e p t o r u n d e r a n a e r o b i c c o n d i t i o n s . T h e v a l u e s a r e g i v e n in m m o l / 1 . T h e c o n c e n t r a t i o n s o f m a l t o s e a n d f r u c t o s e w e r e 2 7 . 7 5 m m o l / 1 e a c h , t h e
c o n c e n t r a t i o n s o f c i t r a t e , m a l a t e a n d f u m a r a t e w e r e 1 g / t e a c h , i.e. 4.4, 7.5 a n d 6.3 m m o l / 1 , r e s p e c t i v e l y , a t t h e b e g i n n i n g o f t h e f e r m e n t a t i o n . N e g a t i v e v a l u e s i n d i c a t e c o n s u m p t i o n o f
substrates. Positive values indicate products formed. Ac/EtOH, Ratio of acetate to ethanol; -, not detected

Metabolites

Strain Substrate Maltose Glucose a Fructose Mannitol Citrate Malate Fumarate Succinate Lactate Ethanol Acetate Ac/EtOH

Maltose - 22.36 2.51 - 42.04 46.28 1.59 0.034

L. pontis Maltose + fructose - 22.75 - 0.48 - 24.96 20.36 - 49.54 44.69 10.20 0.228
LTH 2587 Maltose + citrate - 24.55 0.49 - 0.00 51.05 48.59 3.44 0.071
Maltose + malate - 9.07 0.76 - 0.00 - 0.00 18.98 18.13 1.27 0.070
Maltose + fumarate - 11.97 0.68 - 0.00 0.00 22.08 23.73 3.18 0.134
Maltose - 7.44 8.14 - - 6.12 6.76 1.93 0.286
L. pontis Maltose + fructose -- 21.42 2.34 - 25.36 21.87 - - 44.58 38.21 10.48 0.274
LTH 1731 Maltose + citrate - 22.91 - 0.80 -- 0.00 - - 45.15 49.85 0.37 0.007
Maltose + malate - 22.92 0.79 - - 5.01 - 5.51 44.69 47.05 3.83 0.081
Maltose + fumarate - 22.87 0.37 - 3.46 4.75 45.50 47.84 2.55 0.053

L. pontis Maltose 23.34 0.49 - - 45.73 48.37 1.61 0.033

LTH 1735 Maltose + fructose - 21.53 0.35 25.11 20.20 - 47.41 35.76 11.10 0.310

a Glucose was present as a component in t h e m e d i u m in concentrations u p t o 0.7 m m o l / 1

T a b l e 2. M a j o r m e t a b o l i t e s f o r m e d b y g r o w i n g cells o f l a c t o b a c i l l i ( f r o m s o r g h u m s o u r d o u g h ) . T h e c o n c e n t r a t i o n s listed a r e p r e s e n t i n c u l t u r e s u p e r n a t a n t s a f t e r 50 h o f f e r m e n t a t i o n o f
m a l t o s e p l u s a p o t e n t i a l e l e c t r o n a c c e p t o r u n d e r a n a e r o b i c c o n d i t i o n s . T h e v a l u e s a r e g i v e n in retool/1. T h e c o n c e n t r a t i o n s o f m a l t o s e a n d f r u c t o s e w e r e 2 7 . 7 5 m m o l / 1 e a c h , t h e
c o n c e n t r a t i o n s o f c i t r a t e , m a l a t e a n d f u m a r a t e w e r e 1 g/1 e a c h , i.e. 4.4, 7.5 a n d 6.3 m m o l / 1 , r e s p e c t i v e l y , a t t h e b e g i n o f t h e f e r m e n t a t i o n . N e g a t i v e v a l u e s i n d i c a t e c o n s u m p t i o n o f s u b s t r a t e s .
P o s i t i v e v a l u e s i n d i c a t e p r o d u c t s f o r m e d , n.d., N o t d e t e r m i n e d ( h o m o f e r m e n t a t i v e Lactobacillus)

Metabolites

Strain Substrate Maltose Glucose Fructose Mannitol Citrate Malate Fumarate Succinate Lactate Ethanol Acetate Ac/EtOH

Maltose 25.20 - 0.74 - - 51.67 55.90 3.60 0.065

L.fermentum Maltose + fructose - 21.41 0.96 - 27.04 25.78 - - 42.23 40.73 12.76 0.313
LTH 3125 Maltose + citrate - 24.96 - 0.95 - - 1.02 - - 50.05 54.71 0.08 0.000
Maltose + malate - 25.63 - 0.73 - - - 4.24 5.34 49.10 51.88 3.63 0.070
Maltose + fumarate -- 24.96 -- 0.69 - - -- 4.82 5.55 48.89 52.72 3.82 0.072

Maltose -- 24.13 1.44 . . . . . . 47.54 50.83 1.08 0.027

L. reyteri Maltose + fructose -- 23.14 0.10 - - 12.13 0.00 - - 57.27 59.08 7.74 0.131
LTH 3120 Maltose + citrate - 26.03 0.91 0.00 - 47.24 51.79 8.87 0.171
Maltose + malate - 25.17 1.23 - 7.32 6.09 45.29 52.40 3.57 0.068
Meltose + fumarate - 24.77 1.32 - 4.63 4.36 44.07 50.26 5.93 0.118

Maltose - 15.80 1.26 - - 57.92 1.64 - n.d.

L. amylovorus Maltose + fructose 1.50 0.67 - 27.23 0.00 - - 61.70 4.93 0.21 n.d.
LTH 3122 Maltose + citrate - 21.05 0.73 - - - 1.79 - 88.67 0.51 3.41 n.d.
Maltose + malate - 21.73 0.88 - - 2.62 - 2.89 86.96 0.90 1.63 n.d.
Maltose + fumarate - 21.13 0.85 5.08 3.92 85.51 0.61 2.22 n.d.

a Glucose was present as a component in the medium

406

3,5
3,5 d
3,0 3,0

2,5 2,5

~ 2,0 E
= 2,0
r 1,5 r 1,5 fructose
O o m fermentation starts
1,0 1,o

0,S 0,5
0,0 i
o,o I I I 4
0 10 20 30 40 5O 0 10 20 30 40 50
time (h) time (h)
3,5 b 3,5 e

3,0 3,0
/ fructose utilized,
/ maltose
2,5 2,5 tarmentation starts

~ 2,0

1,5

1,0
t'~ 1,5
o
9 1,0

0,5 9 0,5
r.l-~ '~ll ,
0,0 m l 0,0 : ! I
10 20 30 40 50 115 10 20 30 40 50
time (h) time (h)
3,5 C 3,5 f
3,0

2,5 2,5
E 2,0 _E 2,0

1,5 m~,,, ~'~ 1,5

o o
1,0 9 1,0

0,5

0,0 I ' I I I ~ / : 0,0 I I I I I

10 20 30 40 50 70 0 10 20 30 40 50
time (h) time (h)
Fig. 2a-f. Effect of fructose on the growth of sourdough lactobacilli. The time course is depicted of the increase in ODsv8 of cultures of: a L.
pontis LTH 2587, b L. pontis LTH 1731, e L. pontis LTH 1735, d L. reuteri LTH 3120, e L. amylovorus LTH 3122, f L.fermentum LTH 3125.
The cells were grown in mMRS maltose (27.75 retool/l) and mMRS maltose (27.75 mmol/l) plus fructose (27.75 retool/l). The cultures were
incubated without agitation in: maltose without fructose (11), or maltose plus fructose ([]). The concentrations of the metabolic products
formed after 50 h of fermentation are listed in Table 2

L. reuteri, the presence of fructose resulted in a reduced lag
phase when compared to the growth of cultures on
maltose alone.
Citrate was not, or only poorly, used by L. reuteri
L T H 3120, L. fermentum L T H 3125 and L. amyIovorus
L T H 3122. All strains used malate and fumarate as elec-
tron acceptors, as indicated by the formation of succinate
(Table 2). The fermentation balance exhibited minor
deviations in some of these experiments, indicating the
utilization of other (unknown) compounds present in the
complex medium.
It is remarkable that in the course of the fermentation
of maltose without an added electron acceptor, L. pontis
L T H 1731, L T H 1735, L. reuteri L T H 3120 and L.fermen-
turn L T H 3125 released glucose as an intermediate prod-
uct, into the medium at concentrations of 8, 3.5, 3.5 and
5.5 mmol/1, respectively. This glucose was metabolized in
the successive fermentation phases.
 407

(a) glucose maltose H+

maltose H+ cytoplasm

glucose glucose-1P

ADP
/J 2

glucose-6P
4~ -NAD+
"-~NADH+H +
t
CO2@ 5
NAD+
NADH+H+

A'm acetate
xylulose-5P ADP P~

glycerinaldehyde-3p acetylphosphate
I fructose __malate,fumarate 02
Pi ~ NAD+
15~'~lNADH+H+~'~18 NADH+
citrate+_~
H 19, NADH+H+~ 26
NAD+r
~ADP acetaldehyde~.. mannitol lactate H202
16 ~ _ NADH+H+
t0 ~'-~A~ see Fig. 3b
t ~.,~NAD+
ethanol

13 ~f-_ ADP
t ~-~ATP

14~ NAD+
NADH+H+
lactate
408

H=O
NAD+
fructose fumarate malate
19
21 22"~c~ acetate
oxaloacetate
NAD+ -'f~[ NAD+ co,J
@ pyruvate
mannitol succinate lactate
co2
NADH+H+
NAD+

02 glucose-l-P NADH+H+NAD+
~ NADH+H+ II
26 2 erythrose-4-P 29 " ~ erythritol
NAD+ .
Pi
H202 glucose-6-P 2-~ fructose-6-P- ~
ATP
27 ~ NADH+H+ 3
acetyI-P ~-----P-acetate
--._ NAD+ ATP 17
glucose
2 H20 2NADH+H+'~ 15,16
='~
(b) ethanol

Fig. 3a, b. Metabolic key reactions of sourdough lactobacilli, a Metabolism of maltose, b fate of potential electron acceptors upon maltose
fermentation. The enzymes and organisms involved are given in the following list:

Maltosephosphorylase: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581, L. pontis LTH 2587, LTH 1731, LTH 1735, L.
reuteri LTH 3120, L. fermentum LTH 3125:
Phosphoglucomutase: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581, L. pontis LTH 2587, LTH 1731, LTH 1735, L. reuteri
LTH 3120, L.fermentum LTH 3125:
3 Hexokinase: all lactic acid bacteria:
4-8 Enzymes of the phosphogluconate pathway: all heterofermeutative lactic acid bacteria:
8 Phosphoketolase: all heterofermentative lactic acid bacteria:
9-14 Enzymes of the Emden-Meyerhof glycolytic pathway: all lactic acid bacteria:
15 Phosphotransacetylase: all heterofermentative lactic acid bacteria:
16 Alcohol dehydrogenase: all heterofermentative lactic acid bacteria:
17 Acetate kinase: all heterofermentative lactic acid bacteria:
18 Mannitol dehydrogenase: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581, L. pontis LTH 2587, LTH 1731, LTH 1735, L.
fermentum LTH 3125:
19 Fumarase: L. sanfrancisco ATCC 27651, LTH 1729, L. pontis LTH 1731, L. reuteri LTH 3120, L. amylovorus LTH 3122, L.
fermenturn LTH 3125:
20 Succinate dehydrogenase: L. pontis LTH 1731, L. reuteri LTH 3120, L. amylovorus LTH 3122, L. fermentum LTH 3125:
21 Malolactic enzyme: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581:
22 Malate enzyme: not found:
23 Citrate lyase: L. salfrancisco ATCC 27651, LTH 1729, L. amylovorus LTH 3122, L. fermenturn LTH 3125:
24 Oxaloacetate decarboxylase: L. sanfrancisco ATCC 27651, LTH 1729, L. amylovorus LTH 3122, L. fermentum LTH 3125:
25 Lactate dehydrogenase: all lactic acid bacteria:
26 NADH-H202-oxidase: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581:
27 NADH peroxidase: L. sanfrancisco ATCC 27651, LTH 1729, LTH 2581:
28 Glucosephosphate isomerase: all lactic acid bacteria:
29 Erythritol dehydrogenase and erythrose-4-P-phospnotransferase: L. sanfrancisco LTH 1729, LTH 2581.

The erythritol formation (reactions 3, 8, 15-17, 28, 29) is analogous to that decribed by Veiga-Da-Cunha et al. [32] for Leuconostoc oenos.
Possibly reaction 29 is catalysed by a glycerol dehydrogenase. L. sanfrancisco LTH 1729 and LTH 2581 (resting cells only). Facilitated
diffusion (fac. dif.) and symport according to Neubauer et al. [33]

Discussion
The aptitude of sourdough for the production of baked
goods exhibiting the desired sensorial properties depends
on its fermentative florae and their metabolic products.
The fermentation of sourdough is based on the function of
a variety of homo- and heterofermentative lactobacilli and
yeasts. A number of investigations into the sourdough
microflora have dealt with its potential and the effect of
technology on sourdough fermentation. However, there is
still a lack of knowledge concerning the metabolic features
which cause a predominance of specific micro-organisms
in this habitat. In our investigations we used strains of
lactobacilli which are known to be stable elements in
sourdoughs which have a long tradition of use [-4, 6].
These lactobacilli are heterofermentative and are charac-
terized by a high competitive potential and by their
known reputation for producing an attractive product
with the desired organoleptic properties. In addition to
lactate they form ethanol and acetate. Reactions leading
to acetate formation, or those which prevent its formation
deserve special attention, as the ratio of lactate to acetate
in sourdough is considered by bakers to be a major
quality trait and should range from 2.0 to 2.7 [21]. This
ratio can be affected by influencing the production of
acetic acid, as has been described for wheat sourdoughs
[22, 23]. Furthermore, acetate formation generates addi-
tional ATP from acetyl phosphate by acetate kinase [24].
In the study presented by Stolz et al. [20] and in this
paper, mechanisms have been elucidated which affect
acetate formation and the competitiveness of sourdough
lactobacilli. These are consistent with an overview on the
metabolic reactions presented in Fig. 3.
In contrast to homofermentative lactobacilli, hetero-
fermentative species produce only 1 mol ATP from
glucose under anaerobic conditions via the phospho-
gluconate pathway. This apparent disadvantage can be
compensated for by the use of electron acceptors and the
maltose phosphorylase reaction ([18, 20], P. Stolz, R.F.
Vogel, W.P. Hammes, unpublished observations).
Metabolic energy can be saved by cleavage of maltose
by maltose phosphorylase which produces glucose and
glucose-1-phosphate without the expenditure of ATP. Ad-
ditional ATP is formed by heterofermentative lactobacilli
in the presence of oxygen or biochemical electron accep-
tors such as fructose, citrate, fumarate and malate, which
are present in the flour [13, 17, 20, 25] and allow the
regeneration of NAD. However, the concentrations of
these compounds vary with respect to the kind and origin
of the cereal as well as with the milling process. Therefore,
the ratio of acetate/ethanol depends on the oxidation-
reduction potential of the system [26]. L. sanfrancisco
gained additional ATP, supposedly by an oxygen-insensi-
tive mechanism of acetate synthesis (reaction 17 in
Table. 3) which was described for Leuconostoc species by
Condon [27], and grew better in aerated cultures than
under anaerobic conditions [20]. This additional capacity
to regenerate NADH was not observed in strains investi-
gated in this study. These strains did not grow, or only
poorly so, in the presence of oxygen.
In further contrast to L. sanfrancisco, L. pontis and
L.fermentum which preferably used fructose as an electron
409
acceptor, L. reuteri and L. amylovorus used fructose as
a carbon source. This indicates a different role of fructose
for these two sets of organisms isolated from different
environments. Furthermore, the homofermentative L.
amyIovorus does not increase ATP production. A growth-
promoting effect of fructose suggested by Stamer and
Stoyla [28] for L. brevis was observed with L. sanfran-
cisco, L. pontis and L.fermentum, which use fructose as an
electron acceptor. This resulted in a reduced lag phase, as
observed in anaerobic cultures ([20] this study). Addition-
ally, L. pontis LTH 1735 needed fructose for a more
effective maltose metabolism. L. arnylovorus also exhibited
a reduced lag phase in the presence of fructose; this was
due to preferential fermentation of fructose over maltose.
Citrate was used as an electron acceptor in minor
amounts by two maltose-fermenting strains of L. sanfran-
cisco [20], L. amylovorus and L. fermentum. However,
L. sanfrancisco did not produce succinate from malate or
fumarate as observed with L. pontis LTH 1731, L.fermen-
turn, L. reuteri and L. amylovorus and, thus, could not use
these compounds as electron acceptors. Nevertheless,
malate and fumarate were metabolized by L. sanfrancisco
to lactate [20].
The competitiveness of heterofermentative sourdough
lactobacilli is improved not only by an increased yield of
ATP in the presence of electron acceptors, but also by the
seemingly wasteful production of glucose in the presence
of excess maltose. Glucose is accumulated in the medium
upon maltose fermentation by resting ceils of L. sanfran-
cisco, L. pontis, L. reuteri and L. fermentum [18] and is
produced as an intermediary product by growing cells of
these organisms ([20] this study). These limited amounts
of glucose may be used by competing micro-organisms,
but they also prevent the competing micro-organisms
from utilizing the abundant maltose via a mechanism of
glucose repression. In sourdough lactobacilli the utiliza-
tion of maltose is not repressed by glucose, as observed
with homofermentative and facultatively heterofermen-
tative lactobacilli, e.g.L, plantarum LTH 2605 isolated
from sourdough and L. sake LTH 677 isolated from meat
(data not shown), or yeasts [29].
We suggest that the effectiveness of the sum of these
special features is generally responsible for the prevalence
of specific heterofermentative lactobacilli in sourdough
fermentations which are propagated over long periods
[-2, 4]. From spontaneous wheat and rye sourdough fer-
mentations a variety of lactic acid bacteria and yeasts
were isolated [-7, 15]. Many of these organisms may be
mere contaminants from the environment and can not be
considered as sourdough specific as they disappear upon
prolonged propagation of these preparations [3]. A sim-
ilar finding was reported for sorghum fermentations,
where a different set of organisms was found in
spontaneous fermentations [30] as compared to the
micro-organisms isolated-from sourdoughs propagated
for long periods [6].
The sensorial properties of traditionally prepared
sourdough bread are highly appreciated [31]. However
modern concepts of sourdough production involve chem-
ical acidification or application of starters containing
organisms which do not belong to the competitive flora in
traditionally treated doughs. Their application is tempting
410
from a t i m e - s a v i n g e c o n o m i c aspect. A n i m p r o v e d
k n o w l e d g e of the m e t a b o l i c p o t e n t i a l of the typical
s o u r d o u g h lactobacilli i n v e s t i g a t e d b y us ([20], this study)
m i g h t allow m o r e a t t e n t i o n to be p a i d to the d e m a n d s of
the c o n s u m e r s a n d the b r e a d - m a k i n g industry.
Acknowledgements. This work was supported by the BRIDGE pro-
ject of the European Community (Contract No. BIOT-CT91-0263-
SSMA).
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