Chapter 7

Physiology and Biochemistry

of Lactic Acid Bacteria

Michael Gänzle and Marco Gobbetti

7.1 Introduction

In the past decades, studies on the physiology and biochemistry of sourdough

lactic acid bacteria provided insight into the microbial ecology of sourdough as
well as the effect of the metabolic activity of lactic acid bacteria on flavor, texture,
shelf-life, and nutritional properties of leavened baked goods. Lactic acid bacteria
are the dominant microorganisms of sourdough. Their metabolic versatility favors
adaptation to the various processing conditions and the metabolic interactions with
autochthonous yeasts determine mechanisms of proto-cooperation during sour-
dough fermentation [1–3]. Lactobacillus species are most frequently found in
sourdough fermentations although species belonging to the genera Pediococcus,
Enterococcus, Lactococcus, Weissella and Leuconostoc were also identified ([4–6],
see Chap. 5). A large number of Lactobacillus species were first identified from
sourdoughs or fermentation processes of cereals [5]. This chapter gives an over-
view of the general growth and stress parameters, carbohydrate and amino acid
metabolism, synthesis of exopolysaccharides and antimicrobial compounds, and
the conversion of phenolic compounds and lipids of lactic acid bacteria during
sourdough fermentation.

M. Gänzle (*)
Department of Agricultural, Food and Nutritional Science, University of Alberta
Edmonton, Canada
e-mail: mgaenzle@ualberta.ca
M. Gobbetti
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy

M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, 183

DOI 10.1007/978-1-4614-5425-0_7, © Springer Science+Business Media New York 2013
184 M. Gänzle and M. Gobbetti

7.2 General Growth and Stress Parameters

The large diversity of lactic acid bacteria associated with sourdough fermentation
(Chap. 5), is matched by a comparable diversity of general growth and stress parame-
ters. Different processes of sourdough fermentation select for organisms with different
growth parameters. Generally, type I sourdoughs, which are characterized by fre-
quent back-slopping to achieve leavening without addition of baker’s yeast, select
for organisms growing rapidly in cereal substrates. Under these conditions,
Lactobacillus sanfranciscensis is often found as the predominant organism. Type II
sourdoughs, which are characterized by long fermentation times and high fermenta-
tion temperatures, select for acid-tolerant organisms and L. pontis, L. fermentum,
L. reuteri and related organisms are frequently found [6–8].
This effect of fermentation parameters on sourdough microbiota is reflected by
the response of the growth of sourdough lactic acid bacteria to pH, temperature, and
NaCl concentration. Mathematical models for the growth of sourdough lactic acid
bacteria were developed for L. sanfranciscensis [9], L. pontis [10], L. amylovorus
[10, 11] and L. plantarum [12]. The optimum temperature for growth is species
specific; mesophilic organisms grow optimally between 30 and 35°C, while thermo-
philic organisms do not grow at ambient temperature (25°C) and grow optimally
between 40 and 45°C [9–11, 13]. Traditional sourdough fermentations in Europe
are typically carried out in the temperature range of 25–35°C (Chap. 4) and are thus
dominated by mesophilic lactic acid bacteria. Many industrial processes and cereal
fermentations in tropical climates are conducted at higher temperatures and accord-
ingly select for thermophilic lactic acid bacteria [7, 8].
The optimum pH of sourdough lactic acid bacteria is typically between 5.0 and
6.0 [9–11], matching the pH of sourdough after inoculation with 5–20% of a
previous batch of sourdough. Remarkably, the pH of wheat flour, about 6.2–6.5, is
close to the maximum pH permitting growth of L. sanfranciscensis, pH 6.7 [9]. The
minimum pH of growth of L. sanfranciscensis and L. pontis was determined as pH
3.9 and pH 3.5, respectively. Growth and lactic acid production by L. plantarum
continues until a pH of 3.1 is reached [12]. The higher pH tolerance of L. pontis
corresponds to its competitiveness in type II sourdoughs, which select for acid-tol-
erant lactic acid bacteria. The acid tolerance of L. pontis and related organisms
adapted to type II sourdoughs is dependent on conversion of arginine and glutamate,
which consume intracellular protons, and on the formation of exopolysaccharides.
These metabolic pathways are discussed in more detail below.
Lactic acid bacteria tolerate concentrations of undissociated acetic and lactic
acids that exceed by far those concentrations that are typically encountered in sour-
dough [9–11]. Growth of L. sanfranciscensis and L. pontis is observed at lactate
concentrations of up to 300 and 500 mmol/L, respectively [9, 10]; both organisms
also tolerate high concentrations of acetic acid. This high tolerance for organic acids
contrasts with the response of yeasts, which are inhibited by undissociated organic
acids but not by low pH. Moreover, because the pH but not the organic acid concen-
tration limits the growth of lactic acid bacteria in sourdough, the selection of cereal
7 Physiology and Biochemistry of Lactic Acid Bacteria 185

substrates with a high buffering capacity, for example whole wheat flour or bran,
allows the production of sourdough or sourdough products with a high concentration
of organic acids and a corresponding high total titrable acidity.
The response of sourdough lactic acid bacteria to high salt concentrations is also
species specific. Generally, obligate heterofermentative lactobacilli are more sensi-
tive to NaCl in comparison to other lactobacilli. For example, the heterofermentative
L. sanfranciscensis and L. pontis are inhibited by 4% NaCl whereas L. plantarum
and L. amylovorus tolerate up to 6% NaCl [9, 10, 12].

7.3 Metabolism of Carbohydrates

Lactic acid bacteria belong to three metabolic categories: (1) obligately homofer-
mentative organisms, which ferment hexoses through the EMP (Embden-Meyerhof-
Parnas) pathway to lactate as the major end product of carbohydrate metabolism
(Fig. 7.1). Pentoses are not fermented. Examples of obligately homofermentative
lactobacilli occurring in sourdough include L. delbrueckii, L. acidophilus,
L. farciminis, L. amylovorus, and L. mindensis. (2) Obligately heterofermentative
organisms, which ferment hexoses and pentoses through the 6-PG/PK
(6-phosphogluconate/phosphoketolase) pathway and synthesize equimolecular
amounts of lactate and ethanol or acetate. CO2 is additionally produced from hexo-
ses (Fig. 7.2). Key examples of obligately heterofermentative lactobacilli in sour-
dough are L. sanfranciscensis , L. rossiae , L. brevis , L. pontis , and L. fermentum.
(3) Facultatively heterofermentative organisms, which ferment hexoses through the
EMP pathway, and pentoses and gluconate through the 6-PG/PK pathway. Examples
of facultatively homofermentative lactobacilli occurring in sourdough include L. plan-
tarum, L. alimentarius, L. paralimentarius, and L. curvatus [13].
Homofermentative metabolism of hexoses under anaerobic conditions yields
2 mol ATP per mol hexose. In contrast, heterofermentative metabolism of hexoses
under anaerobic conditions yields only 1 mol ATP per mol hexose unless co-substrates
are present (Fig. 7.1). Contrarily to other fermented foods where obligately homo-
fermentative species have the major role, obligately heterofermentative lactic acid
bacteria are dominant in sourdough fermentations [6]. Several factors determine the
dominance of heterofermentative strains: (1) the metabolism of maltose via maltose
phosphorylase activity, simultaneous fermentation of hexoses and pentoses through
the 6-PG/PK pathway, and the use of fructose and other substrates as an external
acceptor of electrons; (2) the optimal pH and temperature which often coincides
with the values of sourdough fermentation; (3) the capacity of showing alternative
phenotype responses and to markedly adapt under various environmental stresses;
and (4) the synthesis of a large spectrum of antimicrobial compounds [6]. The vari-
able sources of fermentable carbohydrates in sourdough determine phenotypic
responses that include the use of external acceptors of electrons, the preferential
and/or simultaneous use of nonconventional energy sources, and the interaction with
exogenous or endogenous enzymes from the flour.
186 M. Gänzle and M. Gobbetti

Glucose
Out
1 ATP
ADP
Glucose-6-P

2
In
Fructose-6-P
ATP
3
ADP
Fructose-1,6-DP
4
5
Glyceraldehyde-3-P Dihydroxyacetone-P
(2) Pi (2) NAD+
6
(2) NADH+2H+
(2) 1,3-Phosphoglycerate
ADP
7
ATP
(2) 3-Phosphoglycerate

8
(2) 2-phosphoglycerate

9 H2O
(2) Phosphoenolpyruvate
ADP
10
ATP
(2) Pyruvate
NADH+H+
11
NAD+
(2) Lactate

Fig. 7.1 Embden-Meyerhof-Parnas (EMP) pathway; homolactic fermentation. The final products
of glucose metabolism are in bold. (2) indicates the formation of two moles of each compound. 1
Glucokinase, 2 glucose-6-phosphate isomerase, 3 phosphofructokinase, 4 fructose 1,6-bisphos-
phate aldolase, 5 triosephosphate isomerase, 6 glyceraldehyde 3-phosphate dehydrogenase, 7
3-phosphoglycerate kinase, 8 phosphoglycerate mutase, 9 enolase, 10 pyruvate kinase, 11 lactate
dehydrogenase

7.3.1 Use of External Acceptors of Electrons

Because the energy yield of heterofermentative metabolism of hexoses is low,

the competitiveness of obligate heterofermentative lactobacilli depends on the
use of external electron acceptors. The practical relevance of the use of external
acceptors of electrons is represented by the substantial modification of the
7 Physiology and Biochemistry of Lactic Acid Bacteria 187

Sucrose Maltose
Glucose
18 H+
Fructose Out
+
H
Maltose
Pi
Glucose 1 Glucose-1-P
ATP
ADP
2 3 In
Glucose-6-P
NAD+
4
NADH + H+
6-P Gluconate
5 NAD+

CO2 NADH + H+
Ribulose-5-P

Xylulose-5-P
Pi

8 Acety l-P 7
Glyceraldehyde -3-P
Acetate CoA
ADP 9 Pi NAD+
ATP Pi 12
NADH+H+
Acetyl-CoA 1,3-Phosphoglycerate
ADP
NADH+H+ 10 13
CoA ATP
NAD+
3-Phosphoglycerate
Acetaldeide
NADH+H+ 14
11
NAD+ 2-Phosphoglycerate
Ethanol
15 H2O

Phosphoenolpyruvate
ADP
16
ATP
Pyruvate
NADH+H+
17
NAD
Lactate

Fig. 7.2 6-Phosphogluconate/phosphoketolase pathway (6-PG/PK); heterolactic fermentation.

The final products of glucose metabolism are in bold (Adapted from [3]). 1 Maltose phosphorylase,
2 hexokinase, 3 phosphoglucomutase, 4 glucose-6-phosphate dehydrogenase, 5 6-phosphogluconate
decarboxylase, 6 epimerase, 7 phosphoketolase, 8 acetate kinase, 9 phosphotransacetylase, 10
aldehyde dehydrogenase, 11 alcohol dehydrogenase, 12 glyceraldehyde 3-phosphate
dehydrogenase, 13 3-phosphoglycerate kinase, 14 phosphoglycerate mutase, 15 enolase, 16 pyru-
vate kinase, 17 lactate dehydrogenase, 18 levansucrase
188 M. Gänzle and M. Gobbetti

Fructose R=O GSSG O2, H2O2

NADH+H+ NADH+H+ NADH+H+ NADH+H+
1 2 3 4
NAD+ NAD+ NAD+ NAD+

Mannitol R-OH 2 GSH H2O2, H2O

Fig. 7.3 Examples of some reactions that allow NADH + H+ co-factor reoxidation (Adapted from
[3]). 1 Mannitol dehydrogenase, 2 alcohol dehydrogenase, 3 glutathione dehydrogenase, 4 NADH
oxidase. GSSG, oxidized glutathione; GSH reduced glutathione; R=O, aldehyde (e.g., hexanal);
R-OH, corresponding alcohol (e.g., hexanol)

fermentation quotient (see Chap. 4) which positively influences the sensory and
shelf-life characteristics of sourdough baked goods [2]. Additional ATP is syn-
thesized when acetyl-phosphate is employed for acetate synthesis through ace-
tate kinase (Fig. 7.2). The synthesis of acetate and ATP as alternative metabolites
from acetyl-phosphate requires the availability of co-substrates to oxidize
NADH that were generated in the upper branch of the 6-PG/PK pathway. When
external acceptors of electrons are available, the recycling of NADH is achieved
without the need to synthesize ethanol from acetyl-phosphate. Most heterofer-
mentative lactic acid bacteria are capable of fructose reduction to mannitol to
achieve co-factor regeneration (Fig. 7.3). Fructose is quantitatively converted to
mannitol by most heterofermentative lactic acid bacteria under acidic condi-
tions [3, 14]. When maltose-negative and maltose-positive sourdough lactic
acid bacteria are associated, fructose conversion may have a further role [2].
Most strains of Weissella spp. differ from other heterofermentative lactic acid
bacteria because they do not convert fructose to mannitol with concomitant
acetate formation [15]. The activity of the mannitol dehydrogenase of L. san-
franciscensis LTH2581 is optimal at 35°C and pH 5.8–8.0 [16]. Once synthe-
sized, mannitol could be further used as an energy source by strains of
L. plantarum. This occurs under anaerobiosis and in the presence of ketoacids
(e.g., pyruvate) as electron acceptors [17].
Oxygen is also used as an external electron acceptor and is reduced to H2O with
H2O2 as an intermediate ([2, 18]; Fig. 7.3). Aerobiosis also induced the expression
of a 12.5-kDa superoxide dismutase (SOD), probably Mn2+ dependent [18]. Overall,
lactic acid bacteria possess various enzymes that are involved in the detoxification
of oxygen radicals. NADH-peroxidases and the system involved in the transport of
L-cysteine are specifically used by sourdough lactobacilli to detoxify H2O2 [19].
The latency phase of growth and cell yield of L. sanfranciscensis CB1 are positively
influenced by traces of oxygen and Mn2+ [18]. When aldehydes are available in the
environment, L. sanfranciscensis showed R-specific activity by NADH-dependent
alcohol dehydrogenase [20, 21] (Fig. 7.3). For instance, the reduction of hexanal to
hexanol activates the acetate kinase pathway and the synthesis of acetic acid. Also
the reduction of oxidized glutathione (GSSG) into reduced glutathione (GSH), via
glutathione dehydrogenase, protects against oxidative stress, and allows the
synthesis of acetic acid [22] (Fig. 7.3).
7 Physiology and Biochemistry of Lactic Acid Bacteria 189

Acetate H2O
Oxaloacetate 3
Citrate Malate Fumarate
1 6
NADH+H+
NADH+H+ NAD+ 5 7
2
CO2 NAD+
CO2
4
Pyruvate Lactate Succinate

NADH+H+ NAD+

Fig. 7.4 Citric acid metabolism (Adapted from [3]). 1 Citrate lyase, 2 oxaloacetate decarboxylase,
3 malate dehydrogenase, 4 lactate dehydrogenase, 5 malolactic enzyme, 6 fumarase. 7 succinate
dehydrogenase

7.3.2 Metabolism of Organic Acids

Sourdough lactic acid bacteria exhibit strain-dependent metabolism of citrate, malate,

and fumarate; a few species are also capable of anaerobic lactate metabolism. Citrate,
malate, and lactate conversion consumes intracellular protons and thus increases the
acid tolerance of lactic acid bacteria. The initial steps of citrate metabolism by lactic
acid bacteria are transport by citrate permease and the reaction catalyzed via citrate
lyase (Fig. 7.4). Two alternative destinations are possible for oxaloacetate: the first
allows the synthesis of succinic acid; the second proceeds via decarboxylation into
pyruvate [23]. Lactococcus lactis converts part of the pyruvate into a-acetolactate.
This reaction takes place when external acceptors of electrons (e.g., citrate) are avail-
able, resulting in a surplus of pyruvate with respect to the amount needed to regenerate
NADH through lactate dehydrogenase. a-Acetolactate is reduced to acetoin or nonen-
zymatically converted to diacetyl, an important flavor compound of the bread crumb
[24]. Diacetyl formation is not observed in obligate heterofermentative lactobacilli and
conversion of citrate to succinate is the most common route for other sourdough lactic
acid bacteria. Nevertheless, the synthesis of diacetyl was found in sourdoughs fer-
mented with L. plantarum, L. farciminis, L. alimentarius and L. acidophilus [25].
Lactobacillus sanfranciscensis uses the route of the pyruvate to convert citrate into
lactate and acetate, also including the co-fermentation with maltose [26, 27]. The
regeneration of the co-factor during the reaction catalyzed by the lactate dehydroge-
nase allows the additional synthesis of acetate. Lactate formation from citrate does not
cause a decrease of the value of pH, and, therefore, the citrate metabolism of L. san-
franciscensis during sourdough fermentation is not limited by the low pH [3]. The use
of citrate during sourdough fermentation thus favors an increase of lactate and acetate
concentrations. Malate and fumarate are also converted to lactate by L. sanfranciscen-
sis [3, 26] (Fig. 7.4). In sourdough, the synthesis of pyruvate and lactate may also result
from the catabolism of nonconventional substrates (e.g., amino acids). For instance,
serine could be deaminated into ammonium and pyruvate. This latter may be, in turn,
reduced to lactate. Pyruvate may be synthesized directly (e.g., alanine) or indirectly
(e.g., aspartic acid) through the reaction of transamination [17].
190 M. Gänzle and M. Gobbetti

Lactate conversion has been described for L. parabuchneri, an isolate from ting, a
lactic-fermented sorghum sourdough [28]. Lactate conversion to propanediol proceeds
through NADH-dependent reduction of lactate to lactaldehyde and 1,2 propanediol.
The reduction of two NADH to NAD+ allows the concomitant formation of acetate
from lactate [29]. Lactate conversion to 1,2-propanediol occurs mainly in the
stationary phase of growth and the consumption of lactate improves the stationary
phase survival of L. parabuchneri [30].
The practical relevance of the use of nonconventional energy sources is more
complex and less diffuse within the population of sourdough lactic acid bacteria. In
some cases, the addition (e.g., citrate) is needed for conditioning of the microbial
metabolism. In other cases (e.g., deamination or transamination of amino acids), it
is possible to note a large phenotype variability depending on the biotypes.

7.3.3 Preferential and/or Simultaneous Use of Energy Sources

Lactic acid bacteria use various sources of energy according to hierarchical features
that are determined through mechanisms of global control, mainly regulated at the
transcription level [31]. When bacteria are subjected to a mixture of energy sources,
they preferentially use the substrate that ensures the highest cell yield. When
growing on glucose-containing mixtures, L. amylovorus DCE 471 always consumed
glucose most rapidly, which seemed to steer growth during the early phase. Maltose
consumption started only when low levels of glucose were reached [32]. The pres-
ence of glucose repressed the fermentation of fructose, maltose, and sucrose of
L. paralimentarius and Weissella cibaria [33]. However, maltose, sucrose and fruc-
tose metabolism is not repressed by glucose in the sourdough-adapted species
L. reuteri and L. sanfranciscensis. Levansucrase, the only enzyme responsible for
sucrose metabolism in L. sanfranciscensis and a major contributor to sucrose
metabolism in L. reuteri is constitutively expressed or regulated independent of the
carbohydrate supply [35, 39]. In L. reuteri, sucrose phosphorylase is induced by
sucrose or raffinose but not repressed by glucose [40]. The efficient and preferential
metabolism of maltose and sucrose by several obligate heterofermentative lactoba-
cilli from sourdough likely reflects adaptation to cereal substrates where sucrose
and raffinose are the major carbon sources in the resting grain while maltose is the
major carbon source that is liberated by cereal enzymes during fermentation.
Metabolism via maltose phosphorylase or sucrose phosphorylase activity allows
a higher energy yield because the chemical energy of the glycosidic bond is employed
for ATP synthesis [34, 35]. Both enzymes are frequently found in heterofermentative
lactic acid bacteria from sourdough. Expression of maltose phosphorylase in L. san-
franciscensis and L. reuteri is constitutive and not repressed by glucose, in contrast,
hexokinase activity is observed only if glucose is available. During growth on malt-
ose, L. sanfranciscensis phosphorylyses maltose and accumulates glucose in the
environment, according to the molar ratio of ca. 1:1 (Fig. 7.2) [34, 36]. Glucose
accumulated by L. sanfranciscensis is available for maltose-negative yeasts and lac-
tic acid bacteria. Nevertheless, the release of glucose from the cell takes place only
7 Physiology and Biochemistry of Lactic Acid Bacteria 191

when the activity of the enzyme hexokinase is not induced [37]. The accumulation of
glucose was not found for some strains of L. sanfranciscensis that were cultivated in
the presence of maltose and fructose. Therefore, it was hypothesized that once liber-
ated the glucose is used through the activity of the enzyme hexokinase, which is in
turn induced by the presence of fructose [38].
Some strains of L. sanfranciscensis have the capacity to express b-glucosidase
activity, but this activity is repressed by glucose [41]. Lactobacilli from sourdough
also show simultaneous rather than consecutive fermentation of pentoses and hexo-
ses. Compared to growth on maltose as the only energy source, L. alimentarius
15 F, L. brevis 10A, L. fermentum 1 F e L. plantarum 20B showed the highest
growth and acidification rates, and the highest cell yield when cultivated in the pres-
ence of xylose, ribose, and arabinose [42]. Other sourdough lactic acid bacteria
showed the highest performance in terms of growth and synthesis of acetic acid
when cultivated in the presence of a mixture of pentose carbohydrates [43]. The
presence of pentoses induces the synthesis of the phosphoketolase enzyme in facul-
tatively heterofermentative lactic acid bacteria (e.g., L. plantarum) and allows the
second half of the 6-PG/PK pathway to proceed (Fig. 7.2). The selection of lactoba-
cilli with the capacity to ferment pentose carbohydrates is thus a suitable alternative
to sucrose addition to increase acetate formation.

7.4 Proteolysis and Catabolism of Free Amino Acids

7.4.1 Proteolysis

Lactic acid bacteria are characterized by multiple amino acid auxotrophies. Lactic
acid bacteria depend on substrate-derived proteases or on the activity of their
proteolytic system to satisfy the nitrogen metabolism [44]. The proteolytic sys-
tems of lactic acid bacteria includes serine cell-envelope-associated proteinase
(CEP – PrtP) which is associated to the cell wall, oligopeptide and amino acid
transporters, and a large number of intracellular peptidases [45] (Fig. 7.5).
Contrary to lactic acid bacteria in dairy fermentations, L. sanfranciscensis ATCC
27651T and most other sourdough lactobacilli do not possess a cell-envelope-
associated proteinase and depend on cereal-associated proteases [46, 47]. The
comparison between sourdoughs and chemically acidified doughs showed that the
degradation of the native proteins is mainly due to the activity of cereal endoge-
nous proteinases [46, 48, 49]. The acidification by sourdough lactic acid bacteria
favors the activation of aspartate-proteinases from cereals, which have an optimal
pH of activity that ranged between 3.0 and 4.5 [50]. Nevertheless, selected strains
of sourdough lactobacilli showed the capacity to hydrolyze albumins, globulins,
and gliadins during fermentation [51–53]. Oligopeptides (4–40 amino acids) are
transported inside the bacterial cell and hydrolyzed through a complex system of
peptidases. An overview of the intracellular peptidases of L. sanfranciscensis is
shown in Fig. 7.6. Several intracellular peptidases of L. sanfranciscensis CB1
were biochemically characterized: a 65-kDa metal-dipeptidase (PepV), with
192 M. Gänzle and M. Gobbetti

protein

acid
PrtP +
alkaline
biosynthesis -

oligopeptides ATP
Amino acids

H+

Fig. 7.5 Proteolytic system of lactic acid bacteria (Adapted from [44]). PrtP, Cell-envelope-
associated proteinase (CEP)

PepO PepE
Phe Ser
Endopeptidase
PepF

PepN (general aminopeptidase)

PepC (general aminopeptidase)
Glu/Asp PepA (narrow specificity aminopeptidase)
Pro Prolyl-oligopeptidase
Pro PepP (aminopeptidase P)
Leu PepL (leucyl aminopeptidase)
Pro PepI (proline iminopeptidase) Exopeptidase
PepV (general dipeptidase)
Carboxypeptidase
Pro
Carboxypeptidase P / Prolyl-carboxypeptidase
Pro
PepX (X- prolyl dipeptidyl aminopeptidase)
Gly Pro Gly Ile
Dipeptidyl-peptidase IV
Leu Pro Gly
Dipeptidyl-peptidase II
Pro
PepQ (prolidase)
Pro
PepR (prolinase)
PepT (tripeptidase)

Fig. 7.6 Peptidase system of lactic acid bacteria (e.g. Lactobacillus sanfranciscensis) and substrate
specificity (Adapted from [50])
7 Physiology and Biochemistry of Lactic Acid Bacteria 193

Gliadins ( ),
Glutenins disulfide bonds:
, glutenins LMW (Low Molecular Weight),
, glutenins y-HMW (High Molecular Weight), Primary proteolysis by cereal proteinases
, glutenins x-HMW (High Molecular Weight), Microbial reduction of disulfide bonds
, hydrolysis products,
, peptides

Secondary proteolysis by
microbial enzymes, amino acids
catabolism Addition of enzymes
(fungal or malt-derived)

- Peptides hydrolised to free amino acids

- Production of volatile compounds
responsable for the aroma
- Comple degradation of proteins to small
peptides and free amino acids

Fig. 7.7 Proteolytic scheme that occurs during sourdough fermentation. Figure shows substrates,
enzymes and the activities of primary and secondary proteolysis (Adapted from [50])

elevated specificity towards dipeptides containing hydrophobic amino acids; a

75-kDa general aminopeptidase (PepN); and an X-prolyl-dipeptidyl aminopepti-
dase (PepX), which exclusively hydrolyzes substrates with the N-terminal
sequence X-Pro [51, 54]. The capacity to hydrolyze oligopeptides adjacent to
proline residues was shown in selected sourdough lactobacilli. However, the pro-
teolytic system of lactic acid bacteria does not cleave peptides with the sequence
motif XPP [55]. Overall, sourdough fermentation increases amino acid concentra-
tions compared to doughs that are chemically acidified [48, 52]. Proteolysis dur-
ing sourdough fermentation proceeds in two stages (Fig. 7.7): (1) primary
proteolysis that releases oligopeptides, and is mainly operated by cereal endoge-
nous proteinases that are activated at low pH and by accumulation of thiols; and
(2) secondary proteolysis that releases free amino acids and small-sized peptides,
and is exclusively operated through peptidase activity of lactic acid bacteria.
194 M. Gänzle and M. Gobbetti

The degradation of the cereal proteins has a fundamental importance for the
rheology and sensory features of leavened baked goods. High molecular weight
glutenins (HMWG) and gliadins, following the decreasing order of solubility of
fractions g, a, and w, are hydrolyzed into alcohol-soluble oligopeptides [47]. The
partial hydrolysis of glutenins during sourdough fermentation results in the depo-
lymerization and solubilization of the glutenin macropolymer (GMP), thus affect-
ing the viscoelastic properties of the dough. The degree of polymerization of GMP
is also influenced by reducing agents. The reduced glutathione (GSH) is the most
important reducing agent in wheat dough. GSH may be subjected to an exchange
reaction with the thiol groups of the gluten proteins, thus reducing the intermolecu-
lar disulfide bond and favoring the decrease of the molecular mass of the GMP [50].
Sourdough heterofermentative lactic acid bacteria possess glutathione reductase
activity (see Sect. 3.1), which reduces the extracellular oxidized GSSG to GSH. The
continuous recycling of GSSG into GSH keeps the level of SH groups in the dough
elevated and increases the number of SH groups in the gluten proteins [56].
The proteolytic system of sourdough lactic acid bacteria contributes to the accu-
mulation of bioactive peptides in sourdough. Fermentation of rye malt sourdoughs
with L. reuteri resulted in the accumulation of angiotensin-converting-enzyme inhib-
itory peptides [55]; the formation of peptides with antioxidant activity was also dem-
onstrated [57]. Sourdough fermentation in combination with fungal enzymes was
also demonstrated to decrease the concentration of gluten to levels of less 10 ppm,
which are tolerated by celiac patients [52, 58]. This hydrolyzed wheat flour was used
for the manufacture of baked goods and administered to celiac patients for 60 days.
As shown by hematology, immunology and histological analyses, the consumption
of 10 g of equivalent gluten per day was absolutely safe for celiac patients [59, 60].
Nine peptidases were partially purified from the pooled cytoplasm extract of the
above-selected sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the
most common immunogenic peptide generated during digestion of Triticum species.
At least three peptidases, general aminopeptidase type N (PepN), X-prolyl dipeptidyl
aminopeptidase (PepX), and endopeptidase (PepO) were necessary to detoxify the
33-mer without generation of related immunogenic peptides. After 14 h of incuba-
tion, the combination of at least six different peptidases totally hydrolyzed the 33-mer
into free amino [61]. Peptidase activities of sourdough lactobacilli show consider-
able diversity at the strain level [55, 62] and the expression of genes coding for
peptidases and peptide transport is under control of the concentration of peptides that
are present during sourdough fermentation [46].

7.4.2 Amino Acid Metabolism

Peptides and free amino acids represent the substrates for microbial conversion, or
are transformed during baking to volatile flavor compounds (Table 7.1). Catabolism
of free amino acids by sourdough lactic acid bacteria not only has sensory
implications but also increases acid resistance and the microbial energy yield under
7 Physiology and Biochemistry of Lactic Acid Bacteria 195

Table 7.1 Examples of amino acid precursors and derived carbonylic compounds, which are gen-
erated during sourdough fermentation and/or during baking
Amino acid precursor Derived carbonylic compound
Leucine 3-Methylbutanol
Isoleucine 2-Methylbutanol
Valine 2-Methylpropional
Alanine Acetaldehyde
Methionine Methional
Phenylalanine Phenylacetaldehyde
Threonine 2-Hydroxypropional

starvation conditions [63, 64]. Major pathways for amino acid conversion by lactic
acid bacteria include decarboxylation reactions, transamination, and metabolism by
lyases (for review, see [65]). These pathways lead to the synthesis of ketoacids,
aldehydes, acids and alcohols, which are important flavor compounds of baked
goods (Table 7.1) [66]. Major pathways that are relevant in sourdough fermentation
are outlined in more detail below.
The catabolism of arginine (Arginine Deiminase, ADI) was studied in depth on
sourdough lactic acid bacteria (Fig. 7.8). Three enzymes are involved, arginine
deaminase (ADI), ornithine carbamoyl transferase (OTC), and carbamate kinase
(CK). A fourth protein, located at the cell membrane which acts as transporter,
allows the antiporter exchange between arginine and ornithine [67]. The activity of
ADI, OTC, and CK enzymes is a species-specific property of several obligately
heterofermentative species, including L. rossiae, L. reuteri, L. brevis, L. hilgardii,
L. fermentum, L. pontis, and L. fructivorans [68]. Generally, arginine is quantita-
tively converted to ornithine by ADI-positive lactic acid bacteria during sourdough
fermentation [48]. The activity of ADI, OTC, and CK of L. rossiae CB1 is well
adapted to the acidity (pH 3.5–4.5) and temperature (30–37°C) conditions of sour-
dough fermentation [69]. The ADI pathway in L. fermentum IMDO 130101 is up-
regulated in response to temperature and salt stress conditions [70]. During
sourdough fermentation, the expression of the ADI pathway favors: (1) microbial
growth and survival, which determines a constant composition of the microbial
population in the sourdough; (2) the enhanced tolerance of lactic acid bacteria to
acidity by contributing to homeostasis of the intracellular pH; and (3) an increased
synthesis of ornithine which is converted, during baking, into 2-acetyl-1-pyrroline,
the compound responsible for the typical flavor of the bread crust.
Glutamine is the most abundant amino acid of wheat proteins; L. sanfranciscen-
sis and other sourdough lactobacilli convert glutamine into glutamate (Fig. 7.9).
Glutamine conversion to glutamate improves the adaptation of lactobacilli to sour-
dough acidity due to consumption of protons and liberation of ammonia which
increases the extracellular pH. The synthesis of glutamate also has a positive effect
on the sensory properties of leavened baked goods [71]. Glutamate is alternatively
decarboxylated to g-aminobutyrate (GABA), or converted to a-ketoglutarate (aKG)
by glutamate dehydrogenase (EC 1.4.1.3). Heterofermentative lactobacilli preferably
196 M. Gänzle and M. Gobbetti

Fig. 7.8 Arginine deiminase 2-acetyl-pyrroline

catabolism (ADI) in
sourdough lactic acid baking
bacteria (Adapted from [3]).
1 Arginine-ornithine ornithine arginine
1
antiporter, 2 arginine Out
deaminase, 3 ornithine
transcarbamylase, 4
carbamate kinase In
arginine
H+
2
NH4+
citrulline
3

Carbamoyl-phosphate
ornithine
H+ + ADP
4
ATP
CO2+ NH4+

Out In
pH < 4.4 pH = 7

glutamine glutamine

H2O
1
NH3
NH3
glutamate glutamate α
α-ketoglutarate
3
H+ NADH + H+
2 4
NAD+
CO2
γ-aminobutyrate γ-aminobutyrate α-hydroxyglutarate

Fig. 7.9 Glutamine and glutamate metabolism of sourdough lactic acid bacteria. 1 Glutaminase,
2 glutamate decarboxylase, 3 glutamate dehydrogenase, 4 alcohol dehydrogenase

employ aKG as an electron acceptor to regenerate NADH [20]. aKG also acts as
the preferred amino acceptor in transamination reactions with leucine, phenylala-
nine, and other amino acids [72]. After peptide transport, the transamination reac-
tions are the second limiting factor for the conversion of amino acids by lactic acid
bacteria [73]. Consequently, the addition of a-ketoglutarate into the food matrix or
the selection of glutamate dehydrogenase positive strains considerably increases the
7 Physiology and Biochemistry of Lactic Acid Bacteria 197

benzaldehyde
3
1 2
phenylalanine phenylpyruvate phenyllactate
4
5

phenylacetate 6 phenylacetaldehyde 7 phenylethanol

Fig. 7.10 Phenylalanine catabolism in Lactobacillus plantarum and Lactobacillus sanfranciscen-

sis. The most abundant metabolic products are underlined, those responsible for the aroma com-
pounds are in bold (Adapted from [3]). 1 Transaminase, 2 dehydrogenase, 3 chemical oxidation,
4 multienzyme complex, 5 decarboxylase, 6 and 7, dehydrogenase

conversion of amino acids. Glutamate dehydrogenase activity is variable depending

on the biotype of lactic acid bacteria [73]. This enzyme is also dependent on NADH
+ H+, which links the catabolism of glutamate of L. sanfranciscensis DSM20451 to
the regeneration of NADH during the central metabolism of carbohydrates (Fig. 7.9)
[74]. Glutamate decarboxylase (GAD) activity was also found in sourdough lactic
acid bacteria [75, 76]. GAD converts L-glutamate to GABA, through a single-step
a-decarboxylation. GABA, a four-carbon nonprotein amino acid, has several
functional activities. Sourdough fermentations with L. plantarum, and Lc. lactis or
L. reuteri allowed the synthesis of GABA in concentrations of up to 9 g/kg, compa-
rable to those found in functional preparations [75, 76]. Glutamate decarboxylase-
mediated acid resistance was shown to contribute to the persistence of L. reuteri in
type II sourdough fermentations [64].
The catabolism of phenylalanine by L. sanfranciscensis and L. plantarum is shown
in Fig. 7.10. Leucine, isoleucine, and valine undergo a similar mechanism. Alternative
pathways for leucine metabolism were proposed but remain to be validated by enzy-
matic and genetic analyses [77]. Phenyl-lactate or phenyl-acetate are the main end
products of the catabolism of phenylalanine. For L. plantarum TMW1.468, a gluta-
mate-dehydrogenase negative strain, the synthesis of phenyl-lactate is increased
through addition of aKG but not with glutamate and citrate added. Conversely, for
L. sanfranciscensis DSM20451, a glutamate-dehydrogenase positive strain, the
synthesis of phenyl-lactate is increased through addition of aKG, glutamate and pep-
tides containing glutamate. However, aKG and glutamate favor the conversion of
phenylalanine only when fructose or citrate are also present, probably owing to the
co-factor dependence of glutamate dehydrogenase [74]. During sourdough fermenta-
tion, L. plantarum TMW1.468 and L. sanfranciscensis DSM20451 synthesize ca.
0.35–1.1 mM of phenyl-lactate; the concentration of 1.5 mM inhibits the growth of
L. sanfranciscensis DSM20451 during growth in mMRS at pH 5.3 [3].
Cystathionine lyase activities are also diffuse within sourdough lactic acid bac-
teria (Fig. 7.11) [78]. A homotetrameric 160-kDa cystathionine g-lyase was purified
and characterized from L. reuteri DSM 20016 [79]. The enzyme is active towards a
large number of amino acids and amino acid derivatives, including methionine, and
allows the synthesis of ammonia, a-ketobutyrate, and low-molecular-weight volatile
sulfur compounds.
198 M. Gänzle and M. Gobbetti

1 2
NH3 + α-ketobutyrate + cysteine cystathionine homocysteine + pyruvate + NH3
3

Fig. 7.11 Cystathionine metabolism (Adapted from [169]). 1 Cystathionine-g-lyase, 2 cystathio-

nine-b-lyase, 3 cystathionine-b-synthetases

7.5 Synthesis of Exopolysaccharides

The formation of exopolysaccharide by lactic acid bacteria during sourdough

fermentation improves culture survival and stress resistance, impacts dough rheology
and bread texture, and allows production of bread with specific nutritional functionality.
EPS from lactic acid bacteria can be categorized on the basis of their composition or
their biosynthesis. EPS composed of only one type of constituting monosaccharide are
classified as homopolysaccharides (HoPS). Examples include levan, which is composed
of fructose, or dextran, which is composed of glucose. Heteropolysaccharides (HePS)
are composed of two or more constituting monosaccharides. Lactic acid bacteria employ
two alternative routes for EPS synthesis, intracellular synthesis from sugar nucleotides
by glycosyltransferases, and extracellular synthesis from sucrose by glucansucrases or
fructansucrases. EPS produced by glucansucrase or fructansucrase activity are invari-
ably HoPS composed of glucose or fructose, respectively [80, 81]. EPS produced by
intracellular glycosyltransferases predominantly are HePS with glucose, galactose,
N-acetylglucosamine, N-acetylgalactosamine, rhamnose, or fucose as constituting
monosaccharides [82–84]. Noticeable exceptions of HoPS produced by intracellular
glycosyltransferases include a galactan from Lc. lactis [85] and b-glucan produced by
Pediococcus parvulus and Oenococcus oeni [86].
Large-scale screening of strain collections of sourdough lactic acid bacteria
revealed that very few isolates are capable of HePS formation [39, 87–89]; only one
report documents formation of HePS in sourdough [90]. In contrast, HoPS forma-
tion is a frequent trait of sourdough lactic acid bacteria and any sourdough is likely
to contain at least one HoPS-forming strain [39, 88]. Moreover, the in situ formation
of HoPS during sourdough fermentation as well as the effect of HoPS on bread
quality is well documented ([91, 92]), for a review see [93] and Chap. 8). Subsequent
paragraphs will hence only briefly discuss HePS formation and focus on HoPS
formation and applications.

7.5.1 EPS Biosynthesis and HoPS Structure

HePS formation by lactic acid bacteria is mediated by large gene clusters that are
encoded on plasmids or the chromosome (for reviews, see [82–84, 94]). EPS gene
clusters generally code for proteins regulating EPS biosynthesis (EpsA in
Streptococcus thermophilus Sfi6), polymerization, export, and chain length
determination (EpsB, EpsC, and EpsD in S. thermophilus Sfi6), and one or several
7 Physiology and Biochemistry of Lactic Acid Bacteria 199

Enzyme properties: - sucrose

- pH and temperature optimum - fructose, glucose
- polymer length, linkage type, - maltose
Strain properties: and degree of branching - other acceptor sugars
- general growth parameters - hydrolysis to transferase ratio (pentoses)
- glucansucrase /
fructansucrase expression Substrate:
- formation of lactate and - buffering capacity (pH profile of
acetate (utilization of fermentation)
fructose as electron - concentration of acceptor
acceptor) III Hydrolysis carbohydrates and release during
- other metabolic properties fermentation.
relevant for bread quality - presence of other structure forming
polymers (e.g. pentosans, gluten)

II Oligosaccharide Process conditions:

lactate, formation
acetate, - time, temperature of fermentation
ethanol, CO2 - Level and type of sucrose addition
(mannitol) (batch / fed-batch)
I Exopolysaccharide formation

Influence on bread quality: texture, shelf life, and content of dietary fibre

Fig. 7.12 Factors influencing yield of exopolysaccharides and the influence of exopolysaccha-
rides on bread quality. Glucansucrases catalyze three alternative reactions: (I) polymerization of
glucose or fructose to exopolysaccharides, (II) glycosyl transfer to acceptor carbohydrates to form
oligosaccharides, (III) sucrose hydrolysis to glucose and fructose. EPS properties and yield are
additionally dependent on the concentration of substrate-derived acceptor carbohydrates, and the
ambient pH and temperature

phosphor-glycosyltransferases or glycosyltransferases (EpsE, EpsF, EpsG, and EpsI

in S. thermophilus Sfi6). EPS is synthesized by glycosyltransferases with sugar
nucleotides derived from glucose-6-phosphate as substrates. The repeating unit of
the polysaccharide, consisting of two to eight monosaccharides, is assembled by
sequential addition of monosaccharides to the membrane-bound lipid carrier undec-
adeprenyl pyrophosphate. The repeating unit is exported and polymerized extracel-
lularly. HePS biosynthesis requires energy-rich sugar nucleotides as precursors, and
the assembly and export of the repeating units competes with the peptidoglycan
biosynthesis; therefore, HePS yields are relatively low, typically well below 1 g/L.
HoPS synthesis is mediated by extracellular cell-wall associated or soluble glu-
cansucrases or fructansucrases [80, 81]. Both groups of enzymes are classified as
retaining glycosyl hydrolases and use sucrose as substrate. Fructansucrases but not
glucansucrases also employ raffinose, stachyose, or verbascose as substrate [40].
Catalysis by glucansucrases and fructansucrases proceeds through a covalently
linked glucosyl-enzyme or fructosyl-enzyme intermediate, respectively, with subse-
quent transfer of the glycosyl moiety to a growing polymer chain, a suitable acceptor
carbohydrate, or water (Fig. 7.12). Sucrose hydrolysis or oligosaccharide formation
are thus alternative reactions to polysaccharide synthesis [80, 95–97]. The ratio of
sucrose hydrolysis to oligo- or polysaccharide synthesis depends on the enzyme
structure as well as the substrate concentrations [98, 99]. Because the catalytic mech-
anism retains the chemical energy of the glycosidic bond of the substrate, HoPS
synthesis does not require energy-rich substrates or co-factors. Several recent reviews
200 M. Gänzle and M. Gobbetti

provide an excellent overview on structure-function relationships of glucansucrases

and fructansucrase [80, 81, 100].
HoPS produced from sucrose are composed only of fructose or glucose but never-
theless have a large diversity with respect to linkage type, or degree of branching, and
molecular weight. Dextran is mainly composed of a−(1 → 6) linked glucose molecules
with a varying degree of a−(1 → 3) or a−(1→ 2) linkages and a−(1 → 3,6) branching
points. Dextran is produced by many strains of Leuconostoc spp. and Weissella spp.
but dextran-forming Lactobacillus spp. were also described [80, 81, 88]. Mutan is
mainly composed of a−(1 → 3) linked glucose moieties and is produced by
Streptococcus spp. and L. reuteri [80, 81]. Glucans with alternating a−(1 → 6) and
a−(1 → 3) or alternating a−(1 → 4) and a−(1 → 6) linkages are referred to as alternan
and reuteran, respectively. Reuteran formation has to date exclusively been observed
in strains of L. reuteri [81]. The current knowledge on structure-function relationships
of glucansucrases allows the manipulation of the linkage type [101] as well as the
molecular weight and degree of branching [102]. Information on structure-function
relationships of glucansucrases has also been used for identification of wild-type glu-
cansucrases producing polymers with desired properties [103]. The linkage type in the
oligosaccharides formed by glucansucrases generally matches the linkage types found
in the corresponding polysaccharide [81, 97]. For example, L. reuteri 121 produces
a−(1 → 4) and a−(1 → 6) linked reuteran with a relative molecular weight of 8 × 107;
in the presence of glucose or maltose as acceptor carbohydrates, maltose and isomalt-
ose or isomaltotriose and panose are produced. Fructans produced by lactic acid bac-
teria include the predominantly b−(2 → 1) linked inulin and the predominantly
a−(2 → 6) linked levan; both polymers contain b−(2 → 1 → 6) branching points [80,
81, 98, 104]. A majority of fructan-producing sourdough isolates form levan.
Remarkably, levansucrases form predominantly inulin-type fructo-oligosaccharides
and hetero-oligosaccharides from sucrose [95, 98].

7.5.2 Ecological Function of HoPS Production and HoPS

Formation in Dough

The ecological function of glucansucrases and fructansucrases for cereal-associated

lactic acid bacteria was related to biofilm formation, sucrose and raffinose utilization,
and stress resistance. In oral streptococci, glucansucrases and fructansucrases are
primarily responsible for sucrose-dependent biofilm formation on the tooth enamel,
and are considered major virulence factors of these organisms [105]. Analogous to
the biofilm formation by oral streptococci, colonization and biofilm formation by
L. reuteri in nonsecretory epithelia of the proximal intestinal tract of animals was
found to be dependent on HoPS formation. The reuteransucrase of L. reuteri
TMW1.106 was a major contributor to formation of the extracellular biofilm matrix,
the levansucrase in the same organism functions as a glucan-binding protein. Both
enzymes are required for colonization of reconstituted lactobacilli-free mice by
L. reuteri TMW 1.106 [106].
7 Physiology and Biochemistry of Lactic Acid Bacteria 201

Glucansucrases and fructansucrases use one of the two constituting monosaccharides

of sucrose for poly- or oligosaccharide synthesis, the other monosaccharide, fructose
and glucose, respectively, accumulates in the fermentation substrate. Levansucrases also
release the a-galactosides melibiose, manninotriose, and manninotetraose from
raffinose, stachyose, and verbascose, respectively [40]. In L. sanfranciscensis LTH2590,
levansucrase is the only enzyme capable of sucrose and raffinose hydrolysis [40, 98]. In
L. reuteri and Lc. mesenteroides, levansucrase, glucansucrase and sucrose phosphory-
lase constitute alternative pathways for sucrose (and raffinose) metabolism. Metabolism
by extracellular levansucrases is the preferred metabolic route for raffinose, stachyose,
and verbascose, presumably because the resulting a-galactosides have a smaller degree
of polymerization, which facilitates transport across the cytoplasmic membrane [40].
Glucansucrase activity invariably accumulates fructose and thus supports acetate forma-
tion by heterofermentative lactic acid bacteria [98]. Because high quantities of acetate
have detrimental effects on the structure of wheat bread [107], dextran-producing
Weissella spp. that do not convert fructose to mannitol with concomitant acetate forma-
tion exhibited superior performance in baking applications when compared to
L. reuteri or L. sanfranciscensis [91, 92, 108].
A contribution of HoPS formation to stress resistance of lactic acid bacteria has
particularly been demonstrated for L. reuteri. Here, HoPS or fructo-oligosaccharides
increase survival at acid conditions during the stationary phase [103], and improves
survival of freeze-dried L. reuteri during storage [109]. The protective effects of
levan and fructo-oligosaccharides are partially attributable to their specific interac-
tion with phospholipids of biological membranes [109, 110]. However, protective
effects are not limited to levan and fructo-oligosaccharides; dextran and b-glucan
also improved acid resistance and stationary phase survival [86, 111].
Lactic acid bacteria producing HoPS from sucrose in laboratory medium
generally also produce HoPS during growth in sourdough [39]. HoPS concentrations
typically range from 1 to 8 g/kg dough [39, 91, 92, 98, 112]; more than 10 g/kg
were reported in optimized, pH-controlled fermentations [113]. Glucansucrases
and fructansucrases are extracellular enzymes, thus, their activity is governed by
ambient conditions rather than intracellular pH and substrate concentrations
(Fig. 7.12). The optimum pH of levansucrases and glucansucrases of sourdough
lactobacilli ranges from 4.5 to 5.5, matching the pH profile of sourdough fermenta-
tions [98, 99]. HoPS yields in sourdough were maximized by pH-controlled fer-
mentations at a pH of 4.7 [113]. Because sucrose acts both as glycosyl-donor and
glycosyl-acceptor in HoPS formation, the sucrose concentration has a decisive
influence on the ratio of sucrose hydrolysis, oligosaccharide formation, and poly-
saccharide formation. Below 10 g/kg sucrose, sucrose hydrolysis is the predominant
reaction [98, 114]. Increasing sucrose concentrations initially increase the forma-
tion of levan over hydrolysis. After a further increase of the sucrose concentration,
sucrose increasingly acts as a glycosylacceptor and oligosaccharide formation is the
predominant reaction catalyzed by glucansucrases or fructansucrases [98, 114, 115].
In sourdough fermentation with 100 g/kg sucrose, L. sanfranciscensis LTH2590
produced 11 g/kg fructo-oligosaccharides but only 5 g/kg levan [98]. High reuteran
yields from L. reuteri TMW1.106 were achieved in pH static fed-batch fermentations
202 M. Gänzle and M. Gobbetti

that maintained sucrose concentrations at 10% throughout fermentation [113]. The

addition of acceptor carbohydrates other than sucrose also influences HoPS yields
in sourdough fermentations. Maltose is a stronger acceptor for glucansucrases
compared to glucose [97, 115]. Correspondingly, dextran yields in wheat sourdoughs,
characterized by high maltose concentrations throughout fermentation, were
substantially lower compared to yields by the same strains in sorghum fermentations,
which are characterized by low maltose and high glucose concentrations [15].
Maltose, xylose, and arabinose also act as alternative acceptor carbohydrates for
levansucrase activity [95, 96].
Exopolysaccharide-producing sourdough starter cultures have found industrial
application to improve the textural properties of bread [116]. In addition to the func-
tion of HoPS as hydrocolloids in baking applications, specific HoPS were found to
prevent pathogen adhesion to eukaryotic cells [117] and to exhibit prebiotic activity
[92, 118]. HoPS-producing lactic acid bacteria thus enable the formulation of baked
goods with specific health properties.

7.6 Antimicrobial Compounds from Sourdough

Lactic Acid Bacteria

Leavened baked goods can become contaminated by spores of the genus Bacillus, which
survive baking, yeasts, mainly belonging to the genera Pichia and Zygosaccharomyces,
which colonize the surface and negatively affect the sensory properties, and, especially,
by moulds, mainly belonging to the genera Penicillium, Aspergillus and Cladosporium
which alter the color and the sensory properties, and, in some cases, synthesize myco-
toxins. More than 40 species of fungi were described as contaminants of baked goods.
Although chemical preservatives (e.g., sorbate and propionate, ethanol) are routinely
used for preventing the contamination of leavened baked goods, sourdough lactic acid
bacteria show a number of natural bio-preservative features that are complementary to
chemical preservatives, or can even substitute their use. Antimicrobial compounds from
sourdough lactic acid bacteria were additionally shown to influence sourdough micro-
biota, and to contribute to the stability of individual strains.
The inhibitory activity of sourdough lactic acid bacteria is generally attributable
to rapid consumption of oxygen and fermentable carbohydrates, and the formation
of lactate with concomitant reduction of the pH. Additional metabolites with specific
antimicrobial activity include diacetyl, hydrogen peroxide, acetate and other short-
chain fatty acids, and reuterin. Acetate formation by heterofermentative lactobacilli
in sourdough is readily adjusted by addition of sucrose or pentoses [39], and con-
tributes to shelf-life extension of bread (see below). The odor threshold of diacetyl,
butyrate, and caproate is substantially lower than the concentrations required for
antimicrobial activity; these compounds can thus not be accumulated in bread with-
out adverse effects on the sensory bread quality. Although individual sourdough
lactic acid bacteria are capable of reuterin synthesis, reuterin formation has not been
achieved in cereal fermentations [119].
7 Physiology and Biochemistry of Lactic Acid Bacteria 203

7.6.1 Antifungal Compounds from Sourdough

Lactic Acid Bacteria

Antifungal compounds synthesized by sourdough lactic acid bacteria include

diacetyl, hydrogen peroxide, acetate, propionate, caproate, 3-hydroxy fatty acids,
phenyllactate, cyclic dipeptides, reuterin, and fungicidal peptides [120]. Numerous
reports describe a substantial increase of the mould-free shelf life of bread owing to
antifungal activities of sourdough lactic acid bacteria [121, 122]. However, metab-
olites of lactic acid bacteria responsible for the antifungal effect remain in most
cases unknown. Acetate inhibits fungal growth only at concentrations that
significantly impair the sensory and textural quality of bread [30, 107, 123].
Phenyllactate and 4-hydroxyphenyllactate were initially characterized as antifun-
gal metabolites from L. plantarum ITM21B [124]. Lactobacilli capable of produc-
ing phenyllactate and hydroxyphenyllactate delayed the growth of Aspergillus
niger and Penicillium roqueforti for up to seven days of bread storage [125]. The
concentration of phenyllactate accumulated by lactobacilli in sourdough fermenta-
tion, however, is about 100 fold lower than its minimal inhibitory concentration
[74, 126] and the antifungal effect of the sourdough is thus not attributable to phe-
nyllactate accumulation. Likewise, the levels of antifungal cyclic dipeptides in
bread are 1,000 fold lower than their MIC, but above the threshold imparting a
metallic and bitter taste [127]. Accordingly, the antifungal effect of sourdough lac-
tic acid bacteria is attributed to a synergistic activity of several compounds. The
antifungal effect of L. reuteri, L. plantarum, and L. brevis in the presence of Ca
propionate (0.2%, w/w) [128] delayed fungal growth by 8 days compared to the
bread started with baker’s yeast alone. It was hypothesized that a synergistic activ-
ity between acetic acid and phenyllactate with chemical preservatives was respon-
sible for this effect. The antifungal effect of L. buchneri and L. diolivorans against
growth of four moulds on bread was attributed to a combination of acetate and
propionate [30]. The preservative effect of L. amylovorus [121, 122] was attributed
to the synergistic activity of more than ten antifungal compounds, including phe-
nyllactate, phenolic acids, fatty acids, and cyclic dipeptides [129].
In addition to the antifungal metabolites of lactic acid bacteria, inhibitory pep-
tides derived from the substrate may also contribute to the preservative effect.
A water-extract from beans in combination with sourdough fermented with L. brevis
AM7 contained three natural inhibitory compounds, two phaseolins (NCBI n. gi
130169, gi 403594) and one lectin (NCBI n. gi 130007) [130]. Antifungal peptides
were also identified from the water-extract of sourdough. The combined activity of
the above compounds determined a delay in fungal growth of up to 21 days, lead-
ing to a shelf life for the bread that was comparable to that found when using Ca
propionate (0.3% w/w). A very extensive bread shelf life was also achieved by
using sourdough lactic acid bacteria and amaranth flour or wheat germ [131, 132].
Sourdough fermented with the nonconventional yeast Wickeramomices anomalus
LCF1695 and L. plantarum was shown to exhibit strong antifungal activity based
on synergistic activities between the inhibitory peptides liberated by L. plantarum
204 M. Gänzle and M. Gobbetti

and ethyl-acetate produced by W. anomalus [133]. Fungal contamination was

delayed by 28 days under pilot plant bakery conditions. Although indubitable
progress has been achieved in the making and storage of baked goods, fungal con-
tamination remains one of the main problems in terms of long-term shelf life of
bakery products. The search for novel methods of bio-conservation appears to be
one of the most promising tools in this regard, even though a combination of vari-
ous inhibitory compounds appears to be inevitable to markedly extend the storage
of leavened baked goods.

7.6.2 Antibacterial Compounds from Sourdough

Lactic Acid Bacteria

Antibacterial metabolites from lactic acid bacteria include reuterin and the organic
acids described above. However, the emphasis of research related to antibacterial
activities was placed on bacteriocins, ribosomally synthesized peptides with anti-
bacterial activity against closely related organisms, and reutericyclin. The pre-
vention of bread spoilage by rope-forming bacilli does not require the selection of
specific protective cultures. Growth of endospores of Bacillus spp. is readily inhib-
ited by modest acidification as is characteristic for sourdough bread [134–136].
Acetate and propionate are more effective against rope-forming bacilli than lactic
acid [134]. Remarkably, the bacteriocins nisin and pedicoin, either included as
additives, or generated in situ by bacteriocin-producing lactic acid bacteria, were
ineffective [134].
Bacteriocin formation by sourdough lactic acid bacteria was described particu-
larly for strains of L. sakei, L. plantarum and L. amylovorus (for a review, see [137,
138]). Bacteriocins appear not to be suitable for extending the shelf life of bread but
formation in sourdough fermentation was shown to enhance the stability of sour-
dough microbiota. Lactococcus lactis M30, a lacticin 3147-producing strain [139],
produced a bacteriocin during fermentation. The inhibitory activity persisted under
the low values of pH of sourdough and after thermal treatments that corresponded
to baking temperatures. Similarly, the bacteriocin-producing strain L. amylovorus
DCE471 persisted for a long time during sourdough propagation [140]. The com-
petitiveness of L. pentosus 2MF8, which synthesized a bacteriocin-like inhibitory
substance [141] and Lc. lactis subsp. lactis M30, which synthesized lacticin 3147
[139] were studied during sourdough propagation. Lactococcus lactis subsp. lactis
M30 showed a larger spectrum of inhibition compared to L. pentosus 2MF8, and did
not inhibit the growth of L. sanfranciscensis. After 20 days of back-slopping, the
persistence of Lc. lactis subsp. lactis M30 inhibited the indicator strain L. plan-
tarum 20, without interference in the growth of L. sanfranciscensis CB1. The above-
described features of bacteriocins, together with the demonstration of the in situ
inhibitory activity, encourage the use of antimicrobial compounds to facilitate the
persistence of the starter cultures and the conditioning of the microbial interactions
that occur during sourdough fermentation.
7 Physiology and Biochemistry of Lactic Acid Bacteria 205

Reutericyclin is a tetramic acid derivative with a broad spectrum of activity

against Gram-positive bacteria, including rope-forming bacilli [142, 143]. To date,
all reutericyclin-producing strains were isolated from an industrial rye sourdough.
Reutericyclin is produced to active concentrations in sourdough and the persistence
of L. reuteri strains during long-term propagation of a rye sourdough was attributed
to the synthesis of reutericyclin (for a review, see [144]). The use of reutericyclin-
producing L. reuteri as a sourdough starter also caused a delay in the growth of
Bacillus sp. during bread storage [144].

7.7 Metabolism of Phenolic Compounds and Lipids

The major phenolic compound in wheat and rye is ferulic acid bound to cell wall
polysaccharides [145]. Changes in the ferulic acid content during sourdough
fermentation are predominantly the result of oxidation reactions and cereal enzymes
[146, 147]. In contrast, cereal grains used in African cereal fermentations, particularly
sorghum and millet, are rich in phenolic compounds, including phenolic acids, phe-
nolic acid esters, desoxyanthocyanidins, and tannins [148, 149]. In these cereal
grains, metabolism of phenolic compounds influences product properties, and the
content of antimicrobial phenolic compounds influences the microbial ecology of
sorghum sourdough fermentations [150]. A review on the metabolism of food phe-
nolics by lactic acid bacteria, based predominantly on isolates from wine and veg-
etable fermentations, is provided by Rodriguez et al. [151].
Conversion of phenolic compounds is based on glycosyl hydrolases, for example
b-glucosidase and a-rhamnosidase, which convert flavonoid glycosides to the cor-
responding aglycones [152, 153], esterase degrading methyl gallate, tannins, or
phenolic acid esters [154], and decarboxylases and reductases with activity on phe-
nolic acids [151]. The strain-specific metabolism of phenolic compounds is particu-
larly well described for L. plantarum [151, 155]. Decarboxylation of hydroxyl
cinnamic acids generates the corresponding vinyl derivatives [156]. Reductases
hydrogenate the double bond of hydroxyl cinnamic acids or their decarboxylated
vinyl derivatives [151, 156]. The spectrum of activities is strain specific; organisms
capable of conversion of hydroxy cinnamic acids harbor reductase activity, decar-
boxylase activity, or both [150, 151, 156]. Analysis of phenolic compounds during
sorghum sourdough fermentation revealed that strain-specific glycosyl hydrolase,
decarboxylase, and phenolic acid reductase contributed to the conversion of pheno-
lic compounds [151].
Phenolic compounds, including phenolic compounds from sorghum and mil-
let, exhibit strong antimicrobial activity [148, 150]. Particularly the activity of
hydroxy benzoic and hydroxy cinnamic acids is well characterized [157]. The
resistance of lactic acid bacteria towards phenolic acids is highly strain specific
and the strain-specific capability for phenolic acid conversion corresponds to
resistance [157, 158]. Because the conversion of hydroxy cinnamic acids by
reductase- or decarboxylase activities reduced the antimicrobial activity of caffeic
206 M. Gänzle and M. Gobbetti

acid two to fivefold, metabolism was recently identified as a mechanism of

detoxification [157]. To date, lactic acid bacteria capable of conversion of pheno-
lic compounds were predominantly isolated from fermented beverages (wine,
whisky, beer), or African sorghum fermentations. These organisms are likely a
suitable source for starter cultures of phenolic-rich cereals and pseudocereals
used in gluten-free baking [159].
Lipid metabolism in sourdoughs is poorly described and particularly lipase activ-
ity of sourdough lactic acid bacteria appears not to be relevant. However, sourdough
fermentation influences lipid oxidation and the influence of lipid oxidation products
on bread flavor. During flour storage and during dough mixing, chemical oxidation
and cereal-derived lipoxygenase, respectively, convert free linoleic acid to lipid per-
oxides. Peroxides are chemically converted to lipid aldehydes, i.e., (E)-2-nonenal
and (E,E)-2,4-decadienal, with a strong influence on the flavor of the bread crumb
[21]. Heterofermentative lactobacilli reduce these aldehydes to the corresponding
alcohols with a much lower flavor threshold through alcohol reductase activity.
Homofermentative lactobacilli may increase lipid oxidation through formation of
hydrogen peroxide [21]. Baker’s yeast also reduced flavor-active aldehydes result-
ing from lipid oxidation, but slower and through different metabolic pathways [21].
Thiol accumulation by sourdough lactic acid bacteria [56] may additionally influence
lipid oxidation. Cysteine and related thiol compounds reduce linoleic acid perox-
ides to the corresponding hydroxy fatty acids [160], and thus interrupts the reaction
cascade leading to flavor-active aldehydes.

7.8 Cell-to-Cell Communication

Bacteria synthesize, release, sense, and respond to small signaling molecules that
are defined as auto-inducers. The signaling molecules accumulate in the environ-
ment and lead to a series of physiological and biochemical responses when the
quorum (threshold concentration) is reached. The term quorum sensing is derived
from this consideration, and is mostly used to describe cell-to-cell communication
[161]. Overall, the mechanisms of intraspecies communication include the use of
acyl-homoserine lactones (AHL) and auto-inducing peptides (AIP) for Gram-
negative and -positive bacteria, respectively [162, 163]. The interspecies communi-
cation is mainly based on signaling molecules such as furanone derivatives. The
mechanisms involve the LuxS protein or AIP molecules and the three-component
regulatory system (3CRS) [161]. Several studies considered the microbial dynamics
during sourdough fermentation [164]. Within this complex food ecosystem, an
understanding of the mechanisms of interspecies communication should give new
insights into the physiological response of sourdough lactic acid bacteria and the
interactions in an heterogeneous microbial community that govern growth and
metabolism. The mechanism of cell communication was studied in L. sanfrancis-
censis CB1 co-cultivated with other sourdough lactic acid bacteria [165]. The high-
est number of dead and/or damaged cells of L. sanfranciscensis CB1 was found
7 Physiology and Biochemistry of Lactic Acid Bacteria 207

when co-cultured with L. plantarum DC400 or L. brevis CR13. The co-cultivation

with L. rossiae A7 did not interfere with survival compared to the mono-culture.
The analysis of the proteome revealed the induction of several cytoplasm proteins
of L. sanfranciscensis CB1, especially when associated with L. plantarum DC400.
The majority of the induced proteins had a key role in the mechanisms of response
to environmental stresses, leading to the hypothesis that co-cultivation with some
species of lactic acid bacteria could be considered like an environmental stress
which promoted cell communication. A central role for the LuxS protein was estab-
lished, while also furanone derivatives were identified as presumptive signaling
molecules. The synthesis of volatile compounds and the activity of peptidases also
decreased when L. sanfranciscensis CB1 was cultivated together with L. plantarum
DC400. The mechanisms of cell communication were also studied in L. plantarum
DC400 [166]. The co-cultivation of this strain with other sourdough lactic acid bac-
teria did not interfere with survival compared to the mono-culture. Nevertheless, the
analysis of the proteome revealed the induction of proteins involved in the response
to environmental stresses and cell communication. Lactobacillus plantarum DC400
synthesized the pheromone plantaricin A (PlnA) at variable concentrations that
depended on the associated microbial species [167]. The co-cultivation with L. pen-
tosus, L. brevis and other sourdough lactic acid bacteria species did not modify the
synthesis of PlnA compared to the mono-culture conditions and did not show effects
on the viability and survival of the other species. On the contrary, the co-cultivation
with L. sanfranciscensis increased the synthesis of PlnA and caused a decrease of
the viability of this species. The same effect was found during growth of L. sanfran-
ciscensis in the presence of the chemically synthesized PlnA. Under these environ-
mental conditions, the analysis of the proteome revealed the over-expression of
proteins that had a central role in the energy metabolism, catabolism and biosynthesis
of proteins and amino acids, stress responses, homeostasis of the redox potential, and
programmed cell death. Notwithstanding antimicrobial activities due to substrate
competition and synthesis of inhibitory compounds (e.g., phenyl lactic acid), and the
elevated capacity to adapt to changing environments, it could be hypothesized that
the dominance of L. plantarum during sourdough fermentation [168] may relate to
the synthesis of the pheromone/bacteriocin PlnA as stimulated by mechanisms of
quorum sensing.

References

1. Gobbetti M, De Angelis M, Corsetti A, Di Cagno R (2005) Biochemistry and physiology of

sourdough lactic acid bacteria. Trends Food Sci Technol 16:57–69
2. Gobbetti M (1998) The sourdough microflora: interactions of lactic acid bacteria and yeast.
Trends Food Sci Technol 9:267–274
3. Gänzle MG, Vermeulen N, Vogel RF (2007) Carbohydrate, peptide and lipid metabolism of
lactic acid bacteria in sourdough. Food Microbiol 24:128–138
4. Hammes WP, Gänzle MG, Hammes WP, Gänzle MG (1998) Sourdough bread and related
products. In: Wood BJB (ed) Microbiology of fermented foods, vol 199. Blackie Academic
and Professional, London
208 M. Gänzle and M. Gobbetti

5. Ehrmann MA, Vogel RF (2005) Molecular taxonomy and genetics of sourdough lactic acid
bacteria. Trends Food Sci Technol 16:31–42
6. De Vuyst L, Neysens P (2005) The sourdough microflora: biodiversity and metabolic
interactions. Trends Food Sci Technol 16:43–56
7. Vogel RF, Knorr R, Müller MRA, Steudel U, Gänzle MG, Ehrmann MA, Vogel RF, Knorr R,
Müller MRA, Steudel U, Gänzle MG, Ehrmann MA (1999) Non-dairy lactic fermentations.
The cereal world. Antonie van Leeuwenhoek 76:403–411
8. Meroth CB, Walter J, Hertel C, Brandt MJ, Hammes WP (2003) Monitoring the bacterial
population dynamics in sourdough fermentation processes by using PCR- denaturing gradi-
ent gel electrophoresis. Appl Environ Microbiol 69:475–482
9. Gänzle MG, Ehmann MA, Hammes WP (1998) Modelling of growth of Lactobacillus
sanfranciscensis and Candida milleri in response to process parameters of the sourdough
fermentation. Appl Environ Microbiol 64:2616–2623
10. Wolfrum G (2003) Wachstum und Physiologie der Mikroflora in Getreidefermentationen.
Dissertation, Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt der Technischen Universität München
11. Messens W, Neysens P, Vansieleghem W, Vanderhoeven J, De Vuyst L (2002) Modeling
growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in response to tem-
perature and pH values used for sourdough fermentation. Appl Environ Microbiol
68:1431–1435
12. Passos FV, Fleming HP, Ollis DF, Felder RM, McFeeters RF (1994) Kinetics and modeling of
lactic acid production by Lactobacillus plantarum. Appl Environ Microbiol 60:2627–2636
13. Hammes WP, Vogel RF (1995) The genus Lactobacillus. In: Wood BJB, Holzapfel WH (eds)
The genera of lactic acid bacteria. Blackie Academic and Professional, London, p 19
14. Vrancken G, Rimaux T, De Vuyst L, Leroy F (2008) Kinetic analysis of growth and sugar
consumption by Lactobacillus fermentum IMDO 130101 reveals adaptation to the acidic
sourdough ecosystem. Int J Food Microbiol 128:58–66
15. Galle S, Schwab C, Arendt E, Gänzle M (2010) Exopolysaccharide forming Weissella strains
as starter cultures for sorghum and wheat sourdoughs. J Agric Food Chem 58:5834–5841
16. Korakli M, Vogel RF (2003) Purification and characterization of mannitol dehydrogenase
from Lactobacillus sanfranciscensis. FEMS Microbiol Lett 220:281–286
17. Liu SQ (2003) Practical implication of lactose and pyruvate metabolism by lactic acid bacte-
ria in food and beverage fermentation. Int J Food Microbiol 83:115–131
18. De Angelis M, Gobbetti M (1999) Lactobacillus sanfranciscensis CB1: manganese, oxigen,
superoxide dismutase and metabolism. Appl Microbiol Biotechnol 51:358–363
19. De Angelis M, Gobbetti M (2004) Environmental stress response in Lactobacillus: a review.
Proteomics 4:106–122
20. Zhang C, Gänzle MG (2010) Metabolic pathway of a-ketoglutarate in Lactobacillus sanfran-
ciscensis and Lactobacillus reuteri during sourdough fermentation. J Appl Microbiol
109:1301–1310
21. Vermeulen N, Czerny M, Gänzle MG, Schieberle P, Vogel RF (2007) Reduction of (E)-2-
nonenal and (E, E)-2,4-decadienal during sourdough fermentation. J Cereal Sci 45:78–87
22. Vermeulen N, Kretzer J, Machalitza H, Vogel RF, Gänzle MG (2006) Influence of redox-
reactions catalysed by homo- and heterofermentative lactobacilli on gluten in wheat sour-
doughs. J Cereal Sci 43:137–143
23. Ferain T, Schanck AN, Delcour J (1996) 13 C nuclear magnetic resonance analysis of glucose
and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum.
J Bacteriol 178:7311–7315
24. Hansen Å, Schieberle P (2005) Generation of aroma compounds during sourdough fermenta-
tion: applied and fundamental aspects. Trends Food Sci Technol 16:85–94
25. Damiani P, Gobbetti M, Cossignani L, Corsetti A, Simonetti MS, Rossi J (1996) The sour-
dough microflora. Characterization of hetero- and homofermentative lactic acid bacteria,
yeast and their interactions on the basis of the volatile compounds produced. Z Lebensm Wiss
Technol 29:63–70
7 Physiology and Biochemistry of Lactic Acid Bacteria 209

26. Stolz P, Böcker G, Hammes WP, Vogel RF (1995) Utilization of electron acceptors by
lactobacilli isolated from sourdough I Lactobacillus sanfrancisco. Z Lebensm Unters Forsch
201:91–96
27. Gobbetti M, Corsetti A (1996) Co-metabolism of citrate and maltose by Lactobacillus brevis
subsp. lindneri CB1 citrate-negative strain: effect on growth, end-products and sourdough
fermentation. Z Lebensm Unters Forsch 203:82–87
28. Sekwati-Monang B, Gänzle M (2011) Microbiological and chemical characterisation of ting,
a sorghum-based sourdough product from Botswana. Int J Food Microbiol 150:115–121
29. Oude Elferink SJWH, Krooneman J, Gottschal JC, Spoelstra SF, Faber F, Driehuis F (2001)
Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus
buchneri. Appl Environ Microbiol 67:125–132
30. Zhang C, Brandt MJ, Schwab C, Gänzle MG (2010) Propionic acid production by cofermen-
tation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough. Food Microbiol
27:390–395
31. Titgemeyer F, Hillen W (2002) Global control of sugar metabolism: a gram-positive solution.
Antonie Van Leeuwenhoek 82:59–71
32. Leroy F, De Winter T, Adriany T, Neysens P, De Vuyst L (2006) Sugars relevant for sour-
dough fermentation stimulate growth of and bacteriocin production by Lactobacillus amylo-
vorus DCE 471. Int J Food Microbiol 112:102–111
33. Paramithiotis S, Sofou A, Tskalidou E, Kalantzopulos G (2007) Flour carbohydrate catabo-
lism and metabolite production by sourdough lactic acid bacteria. World J Microbiol
Biotechnol 23:1417–1423
34. Stolz P, Böcker G, Vogel RF, Hammes WP (1993) Utilization of maltose and glucose by
lactobacilli isolated from sourdough. FEMS Microbiol Lett 109:237–242
35. Schwab C, Walter J, Tannock GW, Vogel RF, Gänzle MG (2007) Sucrose utilization and
impact of sucrose on glycosyltransferase expression in Lactobacillus reuteri. Syst Appl
Microbiol 30:433–443
36. Gobbetti M, Corsetti A, Rossi J (1994) The sourdough microflora. Interactions between lactic
acid bacteria and yeasts: metabolism of carbohydrates. Appl Microbiol Biotechnol
41:456–460
37. Neubauer H, Glaasker E, Hammes WP, Poolman B, Konings WN (1994) Mechanisms of
maltose uptake and glucose excretion in Lactobacillus sanfrancisco. J Bacteriol
176:3007–3012
38. De Vuyst L, Schrijvers V, Paramithiotis S, Hoste B, Vancanneyt M, Swingis J, Kalantzopoulos
G, Tsakalidou E, Messens W (2002) The biodiversity of lactic acid bacteria in greek tradi-
tional wheat sourdoughs is reflected in both composition and metabolite formation. Appl
Environ Microbiol 68:6059–6069
39. Tieking M, Korakli M, Ehrmann MA, Gänzle MG, Vogel RF (2003) In situ production of
EPS by intestinal and cereal isolates of lactic acid bacteria during sourdough fermentation.
Appl Environ Microbiol 69:945–952
40. Teixeira JS, McNeill V, Gänzle MG (2012) Levansucrase and sucrose phoshorylase contrib-
ute to raffinose, stachyose, and verbascose metabolism by lactobacilli. Food Microbiol
31:278–284
41. De Angelis M, Gallo G, Settanni L, Corbo MR, McSweeney PLH, Gobbetti M (2005)
Purification and characterization of an intracellular family 3 b-glucosidase from Lactobacillus
sanfranciscensis CB1. Ital J Food Sci 17:131–142
42. Gobbetti M, Lavermicocca P, Minervini F, De Angelis M, Corsetti A (2000) Arabinose fer-
mentation by Lactobacillus plantarum in sourdough with added pentosans and a-L-arabino-
funarosidase: a tool to increase the production of acetic acid. J Appl Microbiol 88:317–324
43. Gobbetti M, De Angelis M, Arnault P, Tossut P, Corsetti A, Lavermicocca P (1999) Added
pentosans in breadmaking: fermentations of derived pentoses by sourdough lactic acid bacte-
ria. Food Microbiol 16:409–418
44. Kunji ER, Mierau I, Hagting A, Poolman B, Konings WN (1996) The proteolytic system of
lactic acid bacteria. Antonie Van Leeuwenhoek 70:187–221
210 M. Gänzle and M. Gobbetti

45. Guèdon E, Renault P, Ehrlich SD, Delorme C (2001) Transcriptional pattern of genes coding
for the proteolytic system of Lactococcus lactis and evidence for coordinated regulation of
key enzymes by peptide supply. J Bacteriol 183:3614–3622
46. Vermeulen N, Pavlovic M, Ehrmann MA, Gänzle MG, Vogel RF (2005) Functional charac-
terization of the proteolytic system of Lactobacillus sanfranciscensis DSM20451T during
growth in sourdough. Appl Environ Microbiol 71:6260–6266
47. Wieser H, Vermeulen N, Gaertner F, Vogel RF (2007) Effect of different Lactobacillus and
Enterococcus strains and chemical acidification regarding degradation of gluten proteins dur-
ing sourdough fermentation. Eur Food Res Technol 226:14
48. Thiele C, Gänzle MG, Vogel RF (2002) Contribution of sourdough lactobacilli, yeast, and
cereal enzymes to the generation of amino acids in dough relevant for bread flavour. Cereal
Chem 79:45–51
49. Loponen J, Mikola M, Katina K, Sontag-Strohm T, Salovaara H (2004) Degradation of HMW
glutenins during wheat sourdough fermentation. Cereal Chem 81:87–93
50. Gänzle MG, Loponen J, Gobbetti M (2008) Proteolysis in sourdough fermentations: mecha-
nisms and potential for improved bread quality. Trends Food Sci Technol 19:513–521
51. Gobbetti M, Smacchi E, Corsetti A (1996) The proteolytic system of Lactobacillus sanfran-
ciscensis CB1: purification and characterization of a proteinase, a dipeptidase, and an amino-
peptidase. Appl Environ Microbiol 62:3220–3226
52. Di Cagno R, De Angelis M, Lavermicocca P, De Vincenzi M, Giovannini C, Faccia M,
Gobbetti M (2002) Proteolysis by sourdough lactic acid bacteria: effects on wheat flour pro-
tein fractions and gliadin peptides involved in human cereal intolerance. Appl Environ
Microbiol 68:623–633
53. Pepe O, Villani F, Oliviero D, Greco T, Coppola S (2003) Effect of proteolytic starter cultures
as leavening agents of pizza dough. Int J Food Microbiol 84:319–326
54. Gallo G, De Angelis M, McSweeney PLH, Corbo MR, Gobbetti M (2005) Partial purification
and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus sanfranci-
scensis CB1. Food Chem 9:535–544
55. Hu Y, Stromeck A, Loponen J, Lopes-Lutz D, Schieber A, Gänzle MG (2011) LC-MS/MS
quantification of bioactive antiotensin I-converting enzyme inhibitory peptides in rye malt
sourdoughs. J Agric Food Chem 59:11983–11989
56. Jänsch A, Korakli M, Vogel RF, Gänzle MG (2007) Glutathione reductase from Lactobacillus
sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol-exchange reac-
tions in wheat sourdoughs. Appl Environ Microbiol 73:4469–4476
57. Coda R, Rizzello CG, Pinto D, Gobbetti M (2012) Selected lactic acid bacteria synthesize
antioxidant peptides during sourdough fermentation of cereal flours. Appl Environ Microbiol
78:1087–1096
58. Rizzello CG, De Angelis M, Di Cagno R, Camarca A, Silano M, Losito I, De Vincenzi M, De
Bari MD, Palmisano F, Maurano F, Gianfrani C, Gobbetti M (2007) Highly efficient gluten
degradation by lactobacilli and fungal proteases during food processing: new perspectives for
celiac disease. Appl Environ Microbiol 73:4499–4507
59. Di Cagno R, Barbato M, Di Camillo C, Rizzello CG, De Angelis M, Giuliani G, De Vincenzi
M, Gobbetti M, Cucchiara S (2010) Gluten-free sourdough wheat baked goods appear safe
for young celiac patients: a pilot study. J Ped Gastroent Nutr 51:777–783
60. Greco L, Gobbetti M, Auricchio R, Di Mase R, Landolfo F, Paparo F, Di Cagno R, De Angelis
M, Rizzello CG, Cassone A, Terrone G, Timpone L, D’Aniello M, Maglio M, Troncone R,
Auricchio S (2011) Safety for patients with celiac disease of baked goods made of wheat
flour hydrolyzed during food processing. Clin Gastroenterol Pathol 9:24–29
61. De Angelis M, Cassone A, Rizzello CG, Gagliardi F, Minervini F, Calasso M, Di Cagno R,
Francavilla R, Gobbetti M (2010) Gluten-free pasta made of Triticum turgidum L. var. durum:
mechanisms of epitopes hydrolysis by peptidases of sourdough lactobacilli. Appl Environ
Microbiol 75:50–518
62. De Angelis M, Di Cagno R, Gallo G, Curci M, Siragusa S, Crecchio C, Parente E, Gobbetti
M (2007) Molecular and functional characterization of Lactobacillus sanfranciscensis strains
isolated from sourdoughs. Int J Food Microbiol 114:69–82
7 Physiology and Biochemistry of Lactic Acid Bacteria 211

63. Christensen JF, Dudley EG, Pederson JA, Steele JL (1999) Peptidases and amino acid
catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 76:217–246
64. Su MSW, Schlicht S, Gänzle MG (2011) Contribution of glutamate decarboxylase in
Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation. Microb
Cell Factories 10(suppl1):S8
65. Fernandez M, Zuniga M (2006) Amino acid catabolic pathoways of lactic acid bacteria. Crit
Rev Microbiol 32:155–183
66. Kieronczyk A, Skeie S, Olsen K, Langsrud T (2001) Metabolism of amino acids by resting
cells of non-starter lactobacilli in relation to flavour development in cheese. Int Dairy J
11:217–224
67. Tonon T, Bourdineaud JP, Lonvaud-Funel A (2001) The arcABC gene cluster encoding the
arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene.
Res Microbiol 152:653–661
68. Hammes WP, Hertel C (2006) The genera Lactobacillus and Carnobacterium. Prokaryotes
4:320–403
69. De Angelis M, Mariotti L, Rossi J, Servili M, Fox PF, Rollàn G, Gobbetti M (2002) Arginine
catabolism by sourdough lactic acid bacteria: Purification and characterization of the arginine
deiminase (ADI) pathway enzymes from Lactobacillus sanfranciscensis CB1. Appl Environ
Microbiol 68:6193–6201
70. Vrancken G, Rimaux T, Wouters D, Leroy F, De Vuyst L (2009) The arginine deiminase
pathway of Lactobacillus fermentum IMDO 130101 responds to growth under stress condi-
tions of both temperature and salt. Food Microbiol 26:720–727
71. Vermeulen N, Gänzle MG, Vogel RF (2007) Glutamine deamidation by cereal-associated
lactic acid bacteria. J Appl Microbiol 103:1197–1205
72. Weingand-Ziadé A, Gerber-Dé Combay C, Affolter M (2003) Functional characterization of
a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus. Enzyme Microb
Technol 32:862–867
73. Tanous C, Kieronczyk A, Helinck S, Chambellon E, Yvon M (2002) Glutamate dehydroge-
nase activity: a major criterion for the selection of flavour-producing lactic acid bacteria
strains. Antonie Van Leeuwenhoek 82:271–278
74. Vermeulen N, Gänzle MG, Vogel RF (2006) nfluence of peptide supply and co-substrates on
phenylalanine metabolism of Lactobacillus sanfranciscensis DSM20451T and Lactobacillus
plantarum TMW1.468. J Agric Food Chem 54:3832–3839
75. Coda R, Rizzello CG, Gobbetti M (2010) Use of sourdough fermentation and pseudo-cereals
and leguminous flours for the making of a functional bread enriched of g-aminobutyric acid
(GABA). Int J Food Microbiol 37:236–245
76. Stromeck A, Hu Y, Chen L, Gänzle MG (2011) Proteolysis and bioconversion of cereal pro-
teins to glutamate and g aminobutyrate in rye malt sourdoughs. J Agric Food Chem
59:1392–1399
77. Serrazanetti DI, Ndagijimana M, Sado-Kamdem SL, Corsetti A, Vogel RF, Ehrmann M,
Guerzoni ME (2011) Acid-stress mediated metabolic shift in Lactobacillus sanfranciscensis
LSCE1. Appl Environ Microbiol 77:2659–2666
78. Curtin ÁC, De Angelis M, Cipriani M, Corbo MR, McSweeney PLH, Gobbetti M (2001)
Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH,
temperature and NaCl by quadratic response surface methodology. J Appl Microbiol
91:312–321
79. De Angelis M, Curtin ÁC, McSweeney PLH, Faccia M, Gobbetti M (2002) Lactobacillus
reuteri DSM 20016: purification and characterization of a cystathionine g-lyase and use as
adjunct starter in cheese-making. J Dairy Res 69:255–267
80. Korakli M, Vogel RF (2006) Structure/function relationship of homopolysaccharide produc-
ing glycansucrases and therapeutic potential of their synthesised glycans. Appl Microbiol
Biotechnol 71:790–803
81. van Hijum SAFT, Kralj S, Ozimek LK, Kijkhuizen L, van Geel-Schutten IGH (2006)
Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic
acid bacteria. Microbiol Molec Biol Rev 70:157–176
212 M. Gänzle and M. Gobbetti

82. de Vuyst L, de Vin F, Vaningelem F, Degeest B (2001) Recent development in the biosynthesis
and applications of heteropolysaccharides from lactic acid bacteria. Int Dairy J 11:687–708
83. Boels IC, van Kranenburgt R, Hugenholtz J, Kleerebezem M, de Vos WM (2001) Sugar
catabolism and its impact on the biosynthesis and engineering of exopolysaccharide produc-
tion in lactic acid bacteria. Int Dairy J 11:723–732
84. Broadbent JR, McMahon DJ, Welker DL, Oberg CJ, Moineau S (2003) Biochemistry,
k genetics, and applications of exopolysaccharide production from Streptococcus thermophi-
lus: a review. J Dairy Sci 86:407–423
85. Gruter M, Leeflang BR, Kuiper J, Kamerling JP, Vliegenthart JF (1992) Structure of the
exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a
defined medium or skimmed milk. Carbohydr Res 231:273–291
86. Dols-Lafargue M, Lee HY, le Marrec C, Heyraud A, Chambat GP, Lonvaud-Funel A (2008)
Characgterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus
and Oenococcus oeni, two bacterial species commonly found in wine. Appl Environ Microbiol
74:4079–4090
87. Van der Meulen R, Grosu-Tudor S, Mozzi F, Vaningelgem F, Zamfir M, Font de Valdez G, De
Vuyst L (2007) Screening of lactic acid bacteria isolates from dairy and cereal products for
exoplysaccharide production and genes involved. Int J Food Microbiol 118:250–258
88. Bunaix M-S, Gabriel V, Morel S, Robert H, Rabier P, Remaud-Siméon M, Gabriel B,
Fontagné-Faucher C (2009) Biodiversity of exopolysaccharides produced from sucrose by
sourdough lactic acid bacteria. J Agric Food Chem 57:10889–10897
89. Palomba S, Cavella S, Torrieri E, Piccolo A, Mazzei P, Blaiotta G, Ventorino V, Pepe O
(2012) Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of
wheat sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-
producing starter culture. Appl Environ Microbiol 78:2737–2747
90. Galle S, Schwab C, Arendt EK, Gänzle MG (2011) Structural and rheological characterisa-
tion of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sour-
dough. Food Microbiol 28:547–553
91. Di Cagno R, De Angelis M, Limitone A, Minervini F, Carnevali P, Corsetti A, Gänzle M,
Ciati R, Gobbetti M (2006) Glucan and fructan production by sourdough Weisella cibaria
and Lactobacillus plantarum and their effect on bread texture. J Agric Food Chem
54:9873–9881
92. Schwab C, Mastrangelo M, Corsetti A, Gänzle MG (2008) Formation of oligosaccharides
and polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10 M in sor-
ghum sourdoughs. Cereal Chem 85:679–684
93. Galle S, Arendt EK Exopolysaccharides from sourdough lactic acid bacteria. A review. Crit
Rev Food Sci Nutr (in press)
94. Jolly L, Stingele F (2001) Molecular organization and functionality of exopolysaccharide
gene clusters in lactic acid bacteria. Int Dairy J 11:733–746
95. Tieking M, Kühnl W, Gänzle MG (2005) Evidence for formation of heterooligosaccharides
by Lactobacillus sanfranciscensis during growth in wheat sourdough. J Agric Food Chem
53:2456–2461
96. Beine R, Moraru R, Nimtz M, Na’amineh S, Pawlowski A, Buchholz K, Seibel J (2008)
Synthesis of novel fructooligosaccharides by substrate and enzyme engineering. J Biotechnol
138:33–41
97. Dols M, Remaud Simeon M, Willemot R-M, Vignon MR, Monsan PF (1998) Structural char-
acterization of the maltose acceptor-products synthesized by Leuconostoc mesenteroides
NRRL B-1299 dextransucrase. Carbohydr Res 305:549–559
98. Tieking M, Ehrmann MA, Vogel RF, Gänzle MG (2005) Molecular and functional character-
ization of a levansucrase from Lactobacillus sanfranciscensis. Appl Microbiol Biotechnol
66:655–663
99. Kralj S, Stripling E, Sanders P, van Geel-Schutten GH, Dijkhuizen L (2005) Highly hydrolytic
reuteransucrase from probiotic Lactobacillus reuteri strain ATCC 55730. Appl Environ
Microbiol 71:3942–3950
7 Physiology and Biochemistry of Lactic Acid Bacteria 213

100. Seibel J, Buchholz K (2010) Tools in oligosaccharide synthesis: current research and application.
Adv Carbohydr Chem Biochem 63:101–138
101. Kralj S, van Leeuwen SS, Valk V, Eeuwema W, Kamerling JP, Dijkhuizen L (2008) Hybrid
reuteransurase enzymes reveal regions important for glucosidic linkage specificity and the
transglucosylation/hydrolysis ratio. FEBS J 275:6002–6010
102. Irague R, Rolland-Sabaté A, Laurence Tarpuis L, Doublier JLO, Moulis C, Monsan P, Remeaud-
Siméon M, Potocki-Véronèse G, Buléon A (2011) Structure and property engineering of a–D–
glucans synthesized by dextransucrase mutants. Biomacromolecules 13:187–195
103. Kaditzki SJ, Behr J, Stocker A, Kaden P, Gänzle MG, Vogel RF (2008) Influence of pH on the
formation of glucan by Lactobacillus reuteri TMW 1.106 exerting a protective function
against extreme pH values. Food Biotechnol 22:398–418
104. Anwar MA, Kralj S, van der Maarel MJEC, Dijkhuizen L (2008) The probiotic Lactobacillus
johnsonii NCC533 produces high-molecular-mass inulin from sucrose by using an inulosu-
crase enzyme. Appl Environ Microbiol 74:3426–3433
105. Cvitkovitch DG, LI YH, Ellen RP (2003) Quorum sensing and biofilm formation in strepto-
coccal infections. J Clin Invest 112:1626–1632
106. Walter J, Schwab C, Loach DM, Gänzle MG, Tannock GW (2008) Glucosyltransferase A
(GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggrega-
tion, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract.
Microbiology 154:72–80
107. Kaditzky S, Seitter M, Hertel C, Vogel RF (2008) Performance of Lactobacillus sanfrancis-
censis TMW1.392 and its levansucrase deletion mutant in wheat dough and comparison of
their impact on bread quality. Eur Food Res Technol 227:433–442
108. Galle S, Schwab C, Dal Bello F, Coffey A, Gänzle M, Arendt E (2012) Influence of in situ
synthesised exopolysaccharides on the quality of gluten-free sorghum sourdough bread. Int J
Food Microbiol 155:105–112
109. Schwab C, Vogel RF, Gänzle MG (2007) nfluence of oligosaccharides on the viability and
membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying. Cryobiology
55:108–114
110. Vereyken IJ, Chupin V, Demel RA, Smeekens SCM, De Kruijff B (2003) Fructans insert
between the headgroups of phospholipids. Biochim Biophys Acta 1510:307–320
111. Kim D-S, Thomas S, Fogler HS (2000) Effects of pH and trace minerals on long-term starva-
tion of Leuconostoc mesenteroides. Appl Environ Microbiol 66:976–981
112. Katina K, Maina NH, Juvonen R, Flander F, Johansson L, Virkki L, Genkanen M, Laitila A
(2009) In situ production and analysis of Weissella confusa dextran in wheat sourdough. Food
Microbiol 26:734–743
113. Kaditzky S, Vogel RF (2008) Optimization of exopolysaccharide yields in sourdoughs fer-
mented by lactobacilli. Eur Food Res Technol 228:291–299
114. Waldherr FW, Meissner D, Vogel RF (2008) Genetic and functional characterization of
Lactobacillus panis levansucrase. Arch Microbiol 190:497–505
115. Rodrigues S, Lona LMF, Franco TT (2005) The effect of maltose on dextran yield and molec-
ular weight distribution. Bioprocess Biosyst Eng 28:9–14
116. Decock P, Capelle S (2005) Bread Technology and sourdough technology. Trends Food Sci
16:113–120
117. Wang Y, Gänzle MG, Schwab C (2010) EPS synthesized by Lactobacillus reuteri decreases
binding ability of enterotoxigenic Escherichia coli to porcine erythrocytes. Appl Environ
Microbiol 76:4863–4866
118. Bello FD, Walter J, Hertel C, Hammes WP (2001) In vitro study of prebiotic properties of
levan-type exopolysaccharides from lactobacilli and non-digestible carbohydrates using
denaturing gradient gel electrophoresis. Syst Appl Microbiol 24:232–237
119. Gänzle MG, Zhang C, Sekwati-Monang B, Lee V, Schwab C (2009) Novel metabolites from
cereal-associated lactobacilli – Novel functionalities for cereal products? Food Microbiol
26:712–719
120. Schnürer J, Magnusson J (2005) Antifungal lactic acid bacteria as preservatives. Trends Food
Sci Technol 16:70–78
214 M. Gänzle and M. Gobbetti

121. Dal Bello F, Clarke CI, Ryan LAM, Ulmer H, Schober TJ, Ström K, Sjörgen J, van Sinderen
D, Schnürer J, Arendt EK (2007) Improvement of the quality and shelf life of wheat bread by
fermentation with the antifungal strain Lactobacillus plantarum FST1.7. J Cereal Sci
45:309–218
122. Ryan LAM, Dal Bello F, Arendt EK (2008) The use of sourdough fermented by anifungal
LAB to reduce the amount of calcium propionate in bread. Int J Food Microbiol
125:274–278
123. Drews E (1959) Der Einfluß gesteigerter Essigsäurebildung auf die Haltbarkeit des
Schrotbrotes. Brot Gebäck 13:113–114
124. Lavermicocca P, Valerio F, Evidente A, Lazzaroni S, Corsetti A, Gobbetti M (2000)
Purification and characterization of novel antifungal compounds from the sourdough
Lactobacillus plantarum strain 21B. Appl Environ Microbiol 66:4084–4090
125. Lavermicocca P, Valerio F, Visconti A (2003) Antifungal activity of phenyllactic acid against
moulds isolated from bakery products. Appl Environ Microbiol 69:634–640
126. Ryan LAM, Dal Bello F, Czerny M, Koehler P, Arendt EK (2009) Quantification of phenyl-
lactic acid in wheat sourdough using high resolution gas chromatography – mass spectrom-
etry. J Agric Food Chem 57:1060–1064
127. Ryan LAM, Dal Bello F, Arendt EK, Koehler P (2009) Detection and quantitation of 2,5-dike-
topeperazines in wheat sourdough bread. J Agric Food Chem 57:9563–9568
128. Gerez CL, Torino MI, Rollán G, de Valdez GF (2009) Prevention of bread mould spoilage by
using lactic acid bacteria with antifungal properties. Food Control 20:144–148
129. Ryan LAM, Zannini E, Dal Bello F, Pawlosksa A, Koehler P, Arendt EK (2011) Lactobacillus
amylovorus DSM19280 as a novel food-grade antifungal agent for bakery products. Int J
Food Microbiol 146:276–283
130. Coda R, Rizzello CG, Nigro F, De Angelis M, Arnault P, Gobbetti M (2008) Long-term
fungal inhibitory Activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and
sourdough lactic acid bacteria during bread storage. Appl Environ Microbiol
74:7391–7398
131. Rizzello CG, Cassone A, Coda R, Gobbetti M (2011) Antifungal activity of sourdough fer-
mented wheat germ used as an ingredient for bread making. Food Chem 127:952–959
132. Rizzello CG, Coda R, De Angelis M, Di Cagno R, Carnevali P, Gobbetti M (2009) Long-term
fungal inhibitory activity of water-soluble extract from Amaranthus spp. seeds during storage
of gluten-free and wheat flour breads. Int J Food Microbiol 131:189–196
133. Coda R, Cassone A, Rizzello CG, Nionelli L, Cardinali G, Gobbetti M (2011) Antifungal
activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fer-
mentation: identification of novel compounds and long-term effect during storage of wheat
bread. Appl Environ Microbiol 77:3484–3492
134. Rosenquist H, Hansen A (1998) The antimicrobial effect of organic acids, sour dough and
nisin against Bacillus subtilis and B. Licheniformis isolated from wheat bread. J Appl
Microbiol 85:621–631
135. Katina K, Sauri M, Alakomi H-L, Mattila-Sandholm T (2002) Potential of lactic acid bacgte-
ria to inhibit rope spoilage in wheat sourdough bread. Lebensm Wiss u-Technol 35:38–45
136. Pepe O, Blaiotta G, Moschetti G, Greco T, Villani F (2003) Rope-producing strains of
Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria. Appl
Environ Microbiol 69:2321–2329
137. Messens W, De Vuyst L (2002) Inhibitory substances produced by Lactobacilli isolated from
sourdoughs – a review. Int J Food Microbiol 72:31–43
138. Settanni L, Corsetti A (2008) Application of bacteriocins in vegetable food biopreservation.
Int J Food Microbiol 121:123–138
139. Hartnett DJ, Vaughan A, van Sinderen D (2002) Antimicrobial-producing lactic acid bacteria
isolated from raw barley and sorghum. J Inst Brew 108:169–177
140. Leroy F, De Winter T, Foulquié Moreno MR, De Vuyst L (2007) The bacteriocin producer
Lactobacillus amylovorus DCE 471 is a competitive starter culture for type II sourdough
fermentations. J Sci Food Agric 87:1726–1736
7 Physiology and Biochemistry of Lactic Acid Bacteria 215

141. Corsetti A, Settanni L, Van Sinderen D (2004) Characterization of bacteriocin-like inhibitory

substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in
situ activity. J Appl Microbiol 96:521–534
142. Gänzle MG, Höltzel A, Walter J, Jung G, Hammes WP (2000) Characterization of reutericy-
clin produced by Lactobacillus reuteri LTH2584. Appl Environ Microbiol 66:4325–4333
143. Hurdle JG, Heathcott AE, Yan B, Lee RE (2011) Reuter;icyclin and related analogues kill
stationary phase Clostridium difficile at achievable colonic concentrations. J Antimicrob
Chemother 66:1773–1776
144. Gänzle MG (2004) Reutericyclin: biological activity, mode of action, and potential applica-
tion. Appl Microbiol Biotechnol 64:326–332
145. Andreasen MF, Christensen LP, Meyer AS, Hansen A (2000) Content of phenolic acids and
ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties. J Agric Food Chem
48:2837–2842
146. Piber M, Koehler P (2005) Identification of dehydro-ferulic acid-tyrosine in rye and wheat:
evidence for a covalent cross-link between arabinoxylans and proteins. J Agric Food Chem
53:5276–5284
147. Boskov Hansen H, Andreasen MS, Nielsen MM, Larsen LM, Bach Knudsen KE, Meyer AS,
Cristensen LP, Hansen A (2002) Changes in dietary fibre, phenolic acids, and activity of
endodenous enzymes during rye bread-making. Eur Food Res Technol 214:33–42
148. Taylor JR, Schober TJ, Bean S (2006) Novel and non-food uses for sorghum and millets.
J Cereal Sci 44:252–271
149. Dykes L, Rooney LW (2006) Sorghum and millet phenols and antioxidants. J Cereal Sci
44:236–251
150. Svensson L, Sekwati Monang B, Lopez-Lutz D, Schieber A, Gänzle MG (2010) Phenolic
acids and flavonoids in non-fermented and fermented red sorghum (Sorghum bicolor (L.)
Moench). J Agric Food Chem 58:9214–9220
151. Rodriguez H, Curiel JA, Landete JM, de las Rivas B, de Felipe FL, Gómez-Cordovés C, Mancheno
JM, Munoz R (2009) Food phenolics and lactic acid bacteria. Int J Food Microbiol 132:79–90
152. Marazza JA, Garro MS, de Giori GS (2009) Aglycone production by Lactobacillus rhamno-
sus CRL981 during soymilk fermentation. Food Microbiol 26:333–339
153. Avila M, Jaquet M, Moine D, Requena T, Pelaez C, Arigoni F, Jankovic J (2009) Physiological
and biochemical characterization of the two a-L-rhamnosidases of Lactobacillus plantarum
NCC245. Microbiology 155:2739–2749
154. Curiel JA, Rodriguez H, Acebron I, Mancheno JM, delas Rivas B, Munoz R (2009) Pdoruction
and physiochemical properties of recombinant Lactobacillus plantarum tannase. J Agric
Food Chem 57:6224–6230
155. De las Rivas B, Rodriguez H, Curiel JA, Landete JM, Munoz R (2009) Meolcular screening
of wine lactic acid bacteria degrading hydroxycinnamic acids. J Agric Food Chem
57:490–494
156. Van Beek S, Priest FG (2000) Decarboxylation of substituted cinnamic acids by lactic acid
bacteria isolated during malt whisky fermentation. Appl Environ Microbiol 66:5322–5328
157. Sanchez-Maldonado AF, Schieber A, Gänzle MG (2011) Structure-function relationships of
the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria. J Appl
Microbiol 111:1176–1184
158. Campos FM, Couto JA, Figueiredo AR, Toth IV, Rangel AOSS, Hogg T (2009) Cell mem-
brane damage induced by phenolic acids on wine lactic acid bacteria. Int J Food Microbiol
135:144–151
159. Moroni AV, Dal Bello F, Arendt EK (2009) Sourdough in gluten-free bread-making: an acient
technology to solve a novel issue? Food Microbiol 26:676–684
160. Elshof MBW, Veldink GA, Vliegenthart JFG (1998) Biocatalytic hydroxylation of linoleic
acid in a double-fed batch system with lipoxygenase and cysteine. Fett-Lipid 246–251
161. Fuqua C, Winans SC, Greenberg EP (1996) Census and consensus in bacterial ecosystems:
the LuxR-LuxI family of quorum-sensing transcriptional regulators. Ann Rev Microbiol
50:727–751
216 M. Gänzle and M. Gobbetti

162. Fuqua C, Parsek MR, Greenberg EP (2001) Regulation of gene expression by cell-to-cell
communication: acyl-homoserine lactone quorum sensing. Ann Rev Genet 35:439–68
163. Nakayama J, Cao Y, Horii T, Sakuda S, Akkermans AD, de Vos WM, Nagasawa H (2001)
Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum
sensing in Enterococcus faecalis. Mol Microbiol 41:145–154
164. Gobbetti M, De Angelis M, Di Cagno R, Minervini F, Limitone A (2007) Cell–cell
communication in food related bacteria. Int J Food Microbiol 120:34–45
165. Di Cagno R, De Angelis M, Limitone A, Minervini F, Simonetti MC, Buchin S, Gobbetti M
(2007) Cell-cell communication in sourdough lactic acid bacteria: a protomic study in
Lactobacillus sanfranciscensis CB1. Proteomics 7:2430–2446
166. Di Cagno R, De Angelis M, Coda R, Gobbetti M (2009) Molecular adaptation of sourdough
Lactobacillus plantarum DC400 under co-cultivation with other lactobacilli. Res Microbiol
20:1–9
167. Di Cagno R, De Angelis M, Calasso M, Vicentini O, Vernocchi P, Ndagijimana M, De
Vincenzi M, Dessì MR, Guerzoni ME, Gobbetti M (2010) Quorum sensing in sourdough
Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with
other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells. Proteomics
10:2175–2190
168. Siragusa S, Di Cagno R, Ercolini D, Minervini F, Gobbetti M (2009) Taxonomic structure
and monitoring of the dominant population of lactic acid bacteria during wheat flour sour-
dough type I propagation using Lactobacillus sanfranciscensis starters. Appl Environ
Microbiol 75:1099–1109
169. Gobbetti M, De Angelis M, Di Cagno R, Rizzello CG (2007) The relative contributions of
starter cultures and non-starter bacteria to the flavour of chees. In: Weimer BC (ed) Improving
the flavour of cheese. Woodhead publishing limited, Cambridge, England, pp 121–156