Chapter 4

Technology of Sourdough Fermentation

and Sourdough Applications

Aldo Corsetti

4.1 Definition of Sourdough

Sourdough technology is widely used in bread making and cake production as it

confers distinctive characteristics, high sensory properties and shelf life to the
resulting products (Table 1). Sourdough is “a mixture of wheat and/or rye flour
and water, possibly with added salt, fermented by spontaneous (from flour and
environment) lactic acid bacteria and yeasts which determine its acidifying and
leavening capability. These activities are obtained and optimized through con-
secutive refreshments (or re-buildings, replenishments, backslopping)” (3–5).
The term refreshment deals with the technique by which a dough made of flour,
water and possibly other ingredients ferments spontaneously for a certain time
(possibly at a defined temperature) and it is subsequently added as an inoculum to
start the fermentation of a new mixture of flour and water (and possibly other
ingredients).
When applied for a defined interval of time such a process provides a sourdough
with constant and repeatable leavening and acidifying performances reliant on the
growth of lactic acid bacteria and yeasts that are well adapted to the environment.
After the preparation of the sourdough, the refreshment technique is aimed at main-
taining the metabolic activity of the microbial communities at all times (6).
When the sourdough is added to a mixture of water and flour to start consecutive
propagations (refreshments) to obtain the final mass or full sour to be used as the
leavening agent, it can be designated the “mother sponge” (2, 4). Generally, a sour-
dough contains a variable number of lactic acid bacteria and yeasts, ranging from
107 to 109 cfu/g and 105 to 107 cfu/g, respectively, with a ratio of about 100:1 (7).

A. Corsetti (*)
Department of Food Science, University of Teramo, Teramo, Italy
e-mail: acorsetti@[Link]

M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, 85

DOI 10.1007/978-1-4614-5425-0_4, © Springer Science+Business Media New York 2013
86 A. Corsetti

Table 4.1 Characteristics of sourdough bread versus baker’s yeast bread (adapted from (1, 2))
Characteristics Sourdough bread Baker’s yeast bread
pH 3.8–4.6 5.3–5.8
Lactic acid 0.4–0.8% 0.005–0.04%
Acetic acid 0.10–0.40% 0.005–0.04%
Bread volume 0.22–0.30 £0.20
Flavour Complex aroma and flavour
Staling Slow Rapid
Shelf life Good protection against microbial High sensibility to bacteria and
contaminations mould spoilage
Some nutritional Optimal phytase activity and Low phytase activity, decalcifying
aspects hydrolysis of phytic acid effect
responsible for ion (Ca2+, Fe2+,
Mg2+ etc.) binding
Free amino acid concentration Free amino acid concentration
increase similar to that of flour
Decrease of glycemic index

The main role of lactic acid bacteria (mainly obligately and facultatively
heterofermentative lactobacilli) is in the acidification process while yeasts mainly
account for the leavening of the dough by releasing CO2 (4).

4.2 Sourdough Preparation and Storage

Sourdough (mother sponge) preparation can be fulfilled through many different

protocols. The main objective is to obtain a leavening agent that contains well-
adapted resident microorganisms. Such microorganisms have to produce sufficient
CO2 to leaven the dough, and organic acids and other metabolites to provide rye
or wheat bread with good texture and sensory properties, and extended shelf life
(3). Two classical procedures, for example the French and American systems, are
discussed below.

4.2.1 The French System

Mother sponge preparation for obtaining the French “pain au levain” begins
with a quite firm wheat flour dough (dough yield, DY of 150–152, see Sect. 4.6.2)
with addition of salt and malt. This dough undergoes a first fermentation step
lasting ca. 24 h. This corresponds to the early fermentative activity of flour-
resident yeast and lactic acid bacteria, which results in a low CO2 and organic
acid release (2). The decrease of pH induces the activity of flour endogenous
proteases which together with bacterial hydrolytic enzymes act on gluten and
4 Technology of Sourdough Fermentation and Sourdough Applications 87

lead to a lower dough firmness. The second step begins with the first refreshment
which is aimed at introducing oxygen and new fermentable carbohydrates into
the mixture to stimulate microbial growth and activity. The refreshment is
obtained by adding a quantity of flour corresponding to the weight of the
previous fermented dough and a quantity of water to bring the DY to a value
(e.g. 148) lower than the previous one. This dough ferments quickly and repre-
sents the starting dough for the next refreshment. By applying such a procedure
a sourdough with a steady fermentative and leavening capability is obtained.
During the last step, each refreshment is carried out at a regular interval of time
(e.g. 7–8 h), with the aim of maintaining an equilibrium in the ratio between
microbial communities (2). According to Calvel (2), when the dough volume
increases by three to fourfold with respect to the initial dough, a new refresh-
ment should be performed. The mother sponge (levain chef), which is obtained
following the above procedure, represents the dough used to prepare the full
sour needed to leaven the bread-dough.

4.2.2 The American System

The American system relies for the mother sponge preparation on a mix of water
and wheat and/or rye flours. In contrast to the French system, the value of DY
ranges from 225 (liquid dough with a ratio water:flour of 1.25:1) to 250 (liquid
dough with a ratio water:flour of 1.5:1). These values of DY remain unaltered
throughout the consecutive refreshments (8). The time and temperature of fer-
mentation are strictly controlled during each phase. In the first step, the water-
flour mixture ferments at 32–35°C for 24 h in order to acidify the dough. At the
end of this step, the first refreshment is obtained by adding flour and water to the
previous fermented dough, without changing the value of DY. After 8 h of fer-
mentation at 32–35°C, the second refreshment is carried out and the dough fer-
ments for another 16 h. After the above procedure is applied, refreshments are
carried out every 8–16 h between which the dough is allowed to ferment at
24–27°C. With the aim of calculating the amount of water and flour to be added
at each replenishment stage a multiplicative factor of 4 is considered. Under
these conditions, the weight of the fermented dough is multiplied by 4 every
two refreshments. This allows one to determine the quantity of water and flour
to be used for the next refreshment and the ratio between the two ingredients is
maintained to 1.25:1. By using this system the value of DY remains constant
until a sourdough with a pH value of 3.6–3.8 and TTA (total titratable acidity)
of 16–20 ml NaOH/20 g of dough (as calculated following the American sys-
tem) is obtained. A last fermentation of 8 h at 24–27°C is needed to confer to the
sourdough the sensory and leavening performances of the mother sponge.
Generally, a sourdough with the above characteristics is obtained in approxi-
mately 5 days, after which it is maintained in an active state by storage at low
temperature (e.g. 4°C) and subjected to refreshment at least once a day (8).
88 A. Corsetti

4.2.3 Sourdough Storage

The liquid sourdough (DY 225–250) that is frequently used by bread manufacturers
in the United States is stored at 1–2°C, after rapid refrigeration, or at 4–5°C. It is
used to start a new fermentation within 2–3 days, without the refreshment step. In
the case of prolonged storage (10 days) one or two refreshments are needed to
activate the metabolism of the lactic acid bacteria and yeasts. A prolonged storage
of the sourdough for a few months at 4–5°C is possible when the ratio between
water and flour is reduced by adding flour at ca. 0.43:1 (30% water: 70% flour).
In this case, a firm sourdough, with DY of 143, is produced. Such a storage type
necessarily requires sourdough reactivation (at least two refreshments) before use
(8). In many artisan bread preparations, different and often empirical storage
techniques are applied. Generally, as a consequence of the daily schedule of bread
manufacture, a portion of the sourdough is refreshed at least one time before its
use. Nevertheless, by applying a separate storage protocol, part of the mother
sponge can be stored at low temperature (4–6°C) for some weeks after putting it
in a cloth bag tied with string (9). Refreshments are needed before reusing such a
sourdough in a bread-making protocol. In some cases sourdough can be frozen
and reused after refreshment.

4.3 Classification of Sourdoughs

Sourdough bread making is an ancient biotechnological process and various proto-

cols for its use are applied in many countries. On the basis of the technology applied,
sourdoughs have been grouped into three types (10), to which a fourth type, named
sponge-dough, can be added.

4.3.1 Type I Sourdough

Traditional sourdoughs whose microorganisms are kept metabolically active through

daily refreshments are included in this group. Type I sourdoughs are generally suit-
able for achieving dough leavening without addition of baker’s yeast; the dough
propagation described above for French and U.S. sourdoughs are examples of Type
I sourdoughs. Generally, a three-stage protocol is applied relying on three refresh-
ments over 24 h in order to obtain the leavened dough to bake. Each step is charac-
terized by a given DY as well as fermentation temperature and time. At the end of
the last step of fermentation the sourdough is used as the leavening agent; thus it can
be considered as a natural starter culture containing many microbial strains (11). In
wheat and/or rye flour sourdoughs, dominating strains belong to the species
Lactobacillus sanfranciscensis which can co-exist with other obligately
4 Technology of Sourdough Fermentation and Sourdough Applications 89

heterofermentative lactic acid bacteria such as L. pontis, L. brevis, L. fermentum,

L. fructivorans and with the yeasts Candida milleri, C. holmii, Saccharomyces
cerevisiae and S. exiguus (recently renamed Kazachstania exigua).

4.3.2 Type II Sourdough

Sourdoughs obtained through a unique fermentation step of 15–20 h followed by

storage for many days belong to this group. Type II sourdoughs are generally not
suitable for achieving dough leavening but are used for dough acidification, and as
dough improvers. These sourdoughs are generally liquid (DY of ca. 200) and they
are produced at the industrial level using bioreactors or tanks at a controlled tem-
perature that exceeds 30°C. Such a protocol aims at shortening the fermentation
process (12). During storage, a portion of the mature sourdough can be used as the
inoculum with the aim of acidifying the dough and enriching it with aroma and
flavour compounds which are characteristics of sourdough baked goods. On the
basis of the long fermentation time, high DY, and temperature of fermentation, lac-
tobacilli such as L. panis, L. reuteri, L. johnsonii, and L. pontis, which are resistant
to low pH, dominate these sourdoughs (10, 13). Spontaneous flour yeasts are inhib-
ited and, consequently, the leavening of the final dough is obtained by adding com-
mercial baker’s yeast.
Through a combined approach consisting of culture-dependent and culture-inde-
pendent systems, Meroth et al. (14, 15), by using an experimental model based on a
laboratory scale-fermentation, showed a different prevalence of lactic acid bacteria
and yeasts in type I and type II rye-based sourdough, started with a mixture of com-
mercially available sourdough starters and baker’s yeast. In particular, L. sanfranci-
scensis, L. mindensis and C. humilis dominated the type I sourdough. L. crispatus,
L. pontis, L. fermentum and S. cerevisiae prevailed in the type II sourdough propa-
gated at 30°C; the same sourdough propagated at 40°C showed a dominance of
L. crispatus, L. panis and L. frumenti as well as the disappearance of all the yeasts
that had been added with the starter mixture. Finally, L. johnsonii, L. reuteri and
C. krusei characterized a type II sourdough fermented at high temperature (40°C),
but produced with rye bran instead of rye flour.

4.3.3 Type III Sourdough

Type II liquid sourdoughs, which are dried/stabilized after preparation, are named
type III sourdoughs (12). They are mainly used at the industrial level as their quality
is more constant compared to type I sourdough, and they are simpler to manage and
less time consuming. On the basis of the preparation method, type III sourdoughs
are dominated by drying-resistant lactic acid bacteria such as Pediococcus pentosa-
ceus, L. plantarum and L. brevis (10). A detailed description of this type of sour-
dough and its industrial application are reported in Sect. 4.8.
90 A. Corsetti

4.3.4 Sponge Dough

The sponge dough is aimed at acclimatizing baker’s yeast (S. cerevisiae) and
improving swelling of the flour components, loaf volume, taste and flavour of the
bread and its shelf life. It is an indirect process which, from a technological and
microbiological point of view, could be considered as an intermediate procedure
between straight-dough (or a direct process in which just baker’s yeast is used to
start the fermentation) and sourdough (an indirect process in which fermentation
starts without the addition of baker’s yeast). Sponge dough is obtained in two steps:
in the first dough (pre-dough) the baker’s yeast is mixed with a part of the flour and
water of the recipe, while the second dough is obtained by adding the rest of the
flour, water and possibly other ingredients to the fully fermented pre-dough.
Depending on the type of bread, the length of pre-dough fermentation can vary from
3 to 20 h and as a consequence, various percentages of flour and yeast, besides dif-
ferent combinations of DY and temperature can be applied in this step (16, 17). As
in the case of longer fermentations lactic acid bacteria present as contaminants from
either baker’s yeast or flour grow in the dough reaching typically more than 108 cfu/g
and contribute to the overall quality of baked goods (4), the sponge dough can be
included in the category of sourdoughs.

4.4 Examples of Sourdough Applications

Once produced, the sourdough is generally submitted to various refreshments either

to activate the microbial metabolism or to increase the dough mass (Fig. 4.1).
Different types of sourdough (e.g. type I, II or III) are used for the manufacture
of various leavened baked products, which range from traditional Italian or French
wheat bread, white pan bread, rye bread, San Francisco bread to traditional cakes
famous throughout the world such as Panettone. Examples of those sourdough
applications are given below.

4.4.1 A Traditional Italian Sourdough Bread:

The Altamura Bread

The Altamura bread is the first European bread that received the PDO (Protected
Denomination of Origin) status. It is manufactured in the Apulia region (Italy) using
specific cultivars of durum wheat (Triticum durum) flour and the technology is
based on type I sourdough. The full sour is obtained by a three-stage procedure in
order to gradually increase the amount of leavened dough. At each step, water and
durum wheat flour are mixed with a previously fermented dough, which is added at
the proportion of ca. 20% based on flour weight. On the basis of the ratio between
4 Technology of Sourdough Fermentation and Sourdough Applications 91

1st refreshment –
Water fermentation at 25°C Flour
for 5 - 6 h

2nd refreshment –
Water fermentation at 28°C Flour
for 7 - 8 h

3rd refreshment –
Water fermentation at 25°C Flour
for 2 - 3 h

Full sour

Leavening agent for

Mother sponge stored till using bread production
for the next bread-making

Fig. 4.1 Representative flow sheet to obtain a full sour from a mother sponge using a three-step
procedure. Fermentation time and temperature given in the example can change depending on
bread-making protocol (Adapted from (1))

the ingredients that are indicated in the original recipe (100 kg of durum wheat
flour + 20 kg of sourdough + 2 kg NaCl + 60 l of water) and assuming a full sour with
a DY of 160, which contains 12.5 kg of flour, the final dough should have a DY of
162. As in many traditional productions, the time and temperature used at each stage
are not defined but variously determined based on the bread-makers experience.

4.4.2 French Bread (Pain au levain)

Although various protocols based on one or two steps are used to obtain an adequate
quantity of full sour (levain tout point), a three-stage system is usually applied to
prepare the traditional wheat flour sourdough French bread, according to the type I
92 A. Corsetti

sourdough procedure. The first step consists of the mixing of a part of the mother
sponge (levain chef) with flour and water to achieve around four times the initial
mass. This dough (DY 160) ferments for 1.5–2 h at ca. 25°C and is named levain de
première (fresh sour). It is used as a starter to obtain a dough with a DY of ca. 185,
which ferments for 7–8 h at a temperature slightly higher than the previous one.
Those parameters, including the long mixing time to oxygenate the dough, stimu-
late the growth of yeasts and the leavening capability of the levain de seconde (basic
sour). By using this dough as the starter for the third refreshment, the last dough or
levain tout point (full sour) is obtained after a short fermentation time (ca. 2 h) with
the aim of controlling the hydrolytic activities of the dough and of preserving the
gas-retaining and bread-making capabilities. The levain tout point is used at the
proportion of 25% with respect to the mass of the final dough and the dough is ready
for baking after 30 min of leavening (2).

4.4.3 Rye Sourdough Bread

As reported for French bread, the rye or wheat/rye flour sourdough bread is obtained
using single or multi-step protocols, depending on the type of bread making and
product. The “three-stage sourdough process, basic-sour overnight” requires ca.
15–20 h and, as described by Spicher and Pomeranz (17), represents a good refer-
ence model for type I sourdough application in rye-flour-based bread making. The
first stage leads to the anfrischsauer (fresh sour), which, in turn, is started by the
anstellgut (mother sponge), a commercial starter or a part (ca. 10–15% of the total
dough weight) of a full sour (vollsauer) from a previous bread making. The
anfrischsauer has a DY of 200–250 and ferments for ca. 6 h at 25–26°C. Afterwards,
it contains a high number of yeasts and it is used to start a new dough based on rye
flour and water (DY of about 160–180). After 5–8 h of overnight fermentation at
26–30°C, lactic acid bacteria grow and the grundsauer (basic sour) is obtained.
A further refreshment (3 h at 30–33°C), using the basic sour as the starter for a mix
of rye flour and water (DY 180–200), leads to the vollsauer (full sour), which has
values of pH and TTA of 4.0 and 10.5 ml NaOH 0.1 N/10 g of dough, respectively.
A part (40%) of this sourdough represents the natural starter for the final dough (DY
170), which contains rye flour and an equivalent amount of wheat flour in the case
of a rye/wheat flour bread preparation. The dough is baked after dough resting of
10 min at 28°C, followed by proofing.

4.4.4 White Pan Bread

The white pan bread is one of the most diffused breads in the United States and
represents a good example of bread that is manufactured using type II sourdough.
As reported by Kulp (8), the manufacture of this bread relies on a “one-stage system”
4 Technology of Sourdough Fermentation and Sourdough Applications 93

where just one refreshment is needed to mix the liquid full sour with the other
ingredients of the recipe. The quite soft dough (DY 175) ferments in two separate
steps: the first at room temperature (ca. 25–26°C) for 25–30 min and the second at
35°C for ca. 75–100 min. Before baking, the dough shows values of pH of 4.3–4.4
and values of TTA of 9–11 ml NaOH 0.1 N/20 g of dough. Overall, the liquid starter
(full sour) represents 37% of the final dough but different percentages can be used,
which depend on the recipe and industrial or artisan levels. In the last case, a lower
quantity of starter is used, which requires a longer fermentation period and different
working schedule. Because of the high acidity of the sourdough starter, a short mix-
ing time is required due to the increased solubility of the gluten proteins, which
decreases the time for development of the gluten network.

4.4.5 Panettone Cake

Panettone is a traditional Italian cake consumed over Christmas and famous through-
out the world. Panettone has a soft structure, with regular holes, and characteristic
flavour, which is derived from dough ingredients (water, flour, butter, sugar, eggs,
salt, and others) and processing. Both at artisan and industrial levels, the sourdough
biotechnology is traditionally based on many refreshments (e.g. type I sourdough)
and an increased concentration of sugar is added during the last steps (9, 12).
Comparable to most other type I sourdoughs, sourdough microbiota are predomi-
nantly composed of L. sanfranciscensis. During traditional manufacture, a sour-
dough with a low fermentation quotient (FQ, see Sect. 4.6.3) is used and time and
temperature of fermentation are strictly controlled. In particular, the dough tem-
perature does not exceed 30°C and the temperature of water and other ingredients
has to be not lower than 20–22°C. Various studies have attributed the prolonged
softness and shelf life of Panettone cake (it is manufactured several months before
consumption) to the presence of dextran producing Leuconostoc mesenteroides in
the sourdough, which uses sucrose as an exo-polysaccharide (EPS) precursor (12).
On the basis of such a finding a new protocol relying on type III sourdough has
recently been proposed as an alternative to the traditional manufacture (see Sect. 4.8).
A sourdough containing 25% (on dry matter) of dextran, which is stabilized by
refrigeration, pasteurization or drying, has been used to shorten the time to obtain-
ing a product with similar characteristics to the traditional Panettone cake (18).

4.4.6 San Francisco Bread

San Francisco bread is traditionally manufactured using the very famous San
Francisco sourdough, which possesses a typical microbial community, mainly
consisting of L. sanfranciscensis and S. exiguus (renamed to K. exigua). Traditional
bread making relies on the type I sourdough. Various refreshments and long
94 A. Corsetti

fermentation times at low temperature are used to increase the concentration of

acetic acid synthesized by the obligately heterofermentative L. sanfranciscensis.
Recently, a liquid San Francisco sourdough, which is stabilized through pasteuriza-
tion, has been introduced to the market. Thus, also the type III sourdough is used as
an acidifying and flavouring agent for the manufacture of the San Francisco bread
in ca. 3 h (no-time-dough process) (see Sect. 4.7) (12).

4.5 Stability of the Sourdough Microbiota During

Propagation and Use

In order to keep microorganisms active, type I sourdoughs are, generally, propa-

gated daily. Thus, problems of microbiota stability during propagation and in the
use of such a type of “biological starter” arise.
In general, it is recognized that in a sourdough ready to be used (e.g. the mother
sponge—see Sect. 4.2) for bread and other baked goods production the following
basic characteristics occur:

– The prevalence of a microbial community different from that of the flour used as
raw material (3, 19, 20);
– A stable ratio among lactic acid bacteria and yeasts (to the order of 100:1 or
higher) (7);
– A predominance of facultatively and, especially, obligately heterofermentative
lactobacilli over other lactic acid bacteria (3).

As reported by Vrancken et al. (21), laboratory sourdoughs based on wheat,

rye, or spelt that are backslopped daily, reach equilibrium through a three-step
process: (1) prevalence of sourdough-atypical lactic acid bacteria, (2) prevalence
of sourdough-typical lactic acid bacteria, and (3) prevalence of highly adapted
sourdough-typical lactic acid bacteria (21). In a recent paper dealing with the
comparative analysis of seven mature sourdoughs of type I backslopped for
80 days at artisan and laboratory levels under constant technological parameters,
Minervini et al. (22) showed that, while the cell density of presumptive lactic
acid bacteria and related biochemical features were not affected by the environ-
ment of propagation, all sourdoughs harboured a certain number of species and
strains, which were dominant throughout time and, in several cases, varied
depending on the environment of propagation (21). Moreover, besides stable
species and strains, other lactic acid bacteria temporarily contaminated the sour-
doughs with differences between artisan and laboratory levels. Interestingly, this
occasional succession of strains and species only slightly affected the kinetic of
sourdough acidification; nevertheless, it could influence the sensory properties
of the resulting leavened baked product. Overall, as a stable microbiota during
sourdough propagation and use is essential in order to obtain standard and repeat-
able final products, the problem of microbial stability in terms of species and
4 Technology of Sourdough Fermentation and Sourdough Applications 95

Fig. 4.2 Persistence of strains of Lactobacillus plantarum (DB200, --♦--; 12H1, --l--; 2MF8,
--£--; G10C3, --D--) and Lactobacillus sanfranciscensis (LS6, -£-; LS41, –n–; LS48, —◊—;
LS3, —X—) after sourdough propagation at 30°C for 6 h during ten subsequent days (Adapted
from (24) and from (23))

strain composition in type I sourdough propagated by applying different endog-

enous (e.g. type of flour, quantity of water) and exogenous (e.g. temperature/time
of fermentation) parameters should be carefully addressed, both in terms of
identification and typing of dominant and sub-dominant microorganisms (see
Sect. 4.6.1) as well as to select robust, well-adapting and competitive starter strains
(23). The robustness of sourdough lactobacilli varies depending on the species
and on the strains. While the majority of L. sanfranciscensis strains showed quite
a low robustness during daily backslopping performed at the laboratory level (24),
selected strains of L. plantarum seemed to share several phenotypic traits that
determined the capacity to outcompete the contaminating lactic acid bacterium
biota (23) (Fig. 4.2).

4.6 Methods to Evaluate the Performance of the Sourdough

Both microbiological and physico-chemical parameters are used to evaluate the

performance of a sourdough. The level of complexity is different and depends on the
purpose of the analyses. The microbiological aspect essentially deals with the assess-
ment of the community of lactic acid bacteria and yeasts, as those microorganisms are
dominant in a good quality sourdough and are generally present at the ratio of approx.
100:1 (7). Nevertheless, an array of both phenotypic and genotypic methods is neces-
sary to identify the species/strain composition of the dominant and sub-dominant
microbiota of the sourdough. An overview of those systems is given below.
96 A. Corsetti

4.6.1 Determination of the Number of Lactic Acid Bacteria

and Yeasts

The standard plate count is used to estimate the cell density of both lactic acid bacteria
and yeasts. Suitable culture media to assess the number of sourdough lactic acid bacteria
include SDB (Sour Dough Bacteria) medium, as originally described by Kline and
Sugihara (25); or MRS (de Man, Rogosa, Sharpe) medium (51), with modifications
concerning the pH and the nutrient composition, particularly to include maltose and
fructose as carbon sources and electron acceptors, respectively, and cysteine as reducing
agent (26–28); Homohiochii medium, especially recommended to enumerate obligately
heterofermentative lactic acid bacteria (29); SFM (San Francisco medium), containing
wheat or rye bran especially useful for lactic acid bacteria as well as lactobacilli (30).
To all the above media cycloheximide (100 ppm) can be added to inhibit the
growth of yeast when requested.
Yeast are generally enumerated on WL medium (31); YPDA medium (32, 33);
Sabouraud dextrose agar (34, 35); YGC agar (14); and Malt extract agar (36).
To inhibit bacterial growth, all the above media generally contain 50–100 ppm of
chloramphenicol.

4.6.2 Phenotypic and Genetic Analyses to Identify and Type

Lactic Acid Bacteria and Yeasts

Sourdough lactic acid bacteria and yeasts are generally identified using a polyphasic
approach, combining both phenotypic and genotypic assays.
Besides morphological and physiological tests, specific biochemical assays are
available to identify at species level Weissella spp., Pediococcus spp. and
Enterococcus spp. as well as the predominant sourdough lactic acid bacteria,
which are often members of the genus Lactobacillus (19). In that case, the minia-
turized commercial test (e.g. API 50 CHL, Biomerieux, France) or automated
assay (e.g. Biolog system) are frequently applied after a preliminary evaluation
based on cell morphology, Gram stain, catalase test, growth at 15°C and 45°C,
CO2 production from glucose, and NH3 release from arginine (37). Nevertheless,
the high biochemical diversity in the above-mentioned genus, means that the
identification scheme must include some chemotaxonomic analysis (e.g. SDS-
PAGE, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis of total or
cell-wall associated proteins) (38, 39) or, mainly, genotypic tests such as 16S
rRNA gene sequencing (30). Moreover, on the basis of specifically designed prim-
ers targeting species-specific sequences, many rapid identification systems rely-
ing on PCR (e.g. multiplex-PCR, Intergenic Spacer Region-based PCR) have
been recently applied to sourdough lactic acid bacteria identification (27, 40, 41).
Among genotypic systems, PCR-RAPD (Random Amplified Polymorphic DNA)
as well as RFLP (Restriction Fragment Length Polymorphism) or PFGE (Pulsed
Field Gel Electrophoresis) have been successfully applied for the evaluation of
intra-specific differences (e.g. strain typing) (38, 42, 43).
4 Technology of Sourdough Fermentation and Sourdough Applications 97

On the basis of a similar identification scheme as described above, sourdough

yeasts can be preliminarily identified by a phenotypic approach consisting of cell
and colony morphology, presence and number of spores, carbohydrates, organic
acids, alcohols and nitrite fermentation/assimilation tests using commercial kits
(e.g. ID32C, API 20C AUX, RapID Yeast Plus System, Rapid Yeast Plus), growth at
different temperatures and salt concentrations (44). As for lactic acid bacteria,
chemotaxonomy based on fatty acids, proteins and polysaccharides can be success-
fully applied to yeast identification. The genotypic approach is often based on the
sequencing of various ribosomal genes (e.g. 18S, 5,8S, 26S, 5S, and spacer frag-
ments) (45), as well as on the PCR-RFLP of the rDNA 5,8S-ITS (Internal Transcribed
Spacers) (34). As for lactic acid bacteria, yeast strain typing mainly relies on the
PCR-RAPD technique.
Both lactic acid bacteria and yeasts can be identified and monitored during sour-
dough fermentation by culture-independent systems, for example by PCR-DGGE
(Denaturing Gradient Gel Electrophoresis), generally directed toward the
amplification of specific variable regions of 16S or 26S rRNA genes of lactic acid
bacteria and yeasts, respectively. A similar approach was applied by Meroth et al.
(14, 15) to evaluate the effect of type I and type II sourdough fermentation on micro-
bial population dynamics.

4.6.3 Determination of the Physico-Chemical Parameters

The balance between the communities of lactic acid bacteria and yeasts and the
composition of the microbial communities in terms of species and strains markedly
influences the sourdough performances and the overall quality of related leavened
baked goods. The microbial community is influenced by some sourdough
characteristics and, in turn, it modifies the chemical composition and physical
parameters of the sourdough, which is reflected in the overall quality of the prod-
ucts (Fig. 4.3). An overview of the physico-chemical characteristics of the sour-
dough, which are related to its performance, is determined through the evaluation
of the following parameters:
– Dough Yield (DY)
– Dough Acidity (pH and Total Titratable Acidity, TTA)
– Fermentation Quotient (FQ).
Dough Yield (DY). The ratio between water and flour in the dough is indicated as
dough yield and it deals with the dough consistency. Considering that different
flours have different capabilities to absorb water, doughs of various consistency are
obtained having the same DY. DY is calculated as follows: DY = (flour weight + water
weight) × 100/flour weight. To consider other ingredients in the formula, the expres-
sion is modified as follows: DY = dough weight × 100/flour weight.
Overall, a firm wheat flour sourdough has a DY of 150–160. A liquid sourdough
shows values close to 200. Intermediate values of DY indicate soft dough (12). It
has been observed that both DY and temperature of fermentation markedly influence
98 A. Corsetti

Endogenous factors

Carbohydrates
Nitrogen sources
Minerals
Lipids, free fatty acids
Enzymatic activities (e.g. proteases,
amylases) Lactic acid bacteria
Raw materials/ Environment
Microbiota Product quality
composition, flavour
ratio, texture
performances shelf-life
nutritional value
Inoculum Yeasts Contaminating microbiota
Exogenous factors
Temperature
Dough Yield (aw)
Oxygen (redox potential)
Fermentation time
Number of refreshments

Fig. 4.3 Endogenous and exogenous factors impacting on sourdough characteristics and
performances as well as on the overall quality of the final product (Adapted from (4))

the aroma of the sourdough and, especially, the molar ratio between lactic and acetic
acids (FQ). Overall, more acetic acid is present in firm dough fermented at 25–30°C,
while more lactic acid is found in soft dough fermented at 35–37°C (12, 46).
Obviously, on the basis of microbial adaptation to the various environmental fac-
tors, the combination of DY and temperature during refreshments markedly
influences sourdough microbiota and its performance.
Acetic acid plays an important role by influencing many bread properties (flavour,
rope inhibition, shelf life), and its content in sourdough can be increased by fructose
addition or by aeration in the presence of heterofermentative lactobacilli (47). In
many cases, the effect of temperature on the acetic acid content of type I sourdoughs
(generally propagated at a temperature of about 25°C), can strictly depend on the
yeast activity: at low temperature, yeasts grow and hydrolyze kestose and other
fructo-oligosaccharides, with the fructose being available as electron acceptors for
a higher production of acetic acid by L. sanfranciscensis via the acetate-kinase path-
way (47). At a high temperature (more than 32°C), growth of yeasts is inhibited,
fructo-oligosaccharides remain un-hydrolyzed, and L. sanfranciscensis has less
fructose available for acetate production (48).
pH. The final pH, which ranges from 3.5 to 4.3, is usually considered as an index of
a well-developed sourdough fermentation (50). The pH of the sourdough influences
the values of pH of the final dough and bread, depending on the amount of full sour
that is used as the inoculum. In the case of a standard inoculum of 20% (referred to
the dough weight), values of pH that range from 4.7 to 5.4 are usually found in the
final dough (50).
TTA (Total Titratable Acidity). The value of TTA is a measure of the total organic
acids synthesized during sourdough fermentation. TTA is expressed as ml of NaOH
4 Technology of Sourdough Fermentation and Sourdough Applications 99

0.1 N/10 g of dough. The values of TTA range from 30 to 150 ml NaOH 0.1 N/10 g
for liquid to 40–220 NaOH 0.1 N/10 g for dried sourdoughs. Nevertheless, the
optimal value of TTA for the sourdough depends on the type of bread. Overall,
sourdough with high TTA are preferred for bread making with rye flour (48).
Fermentation Quotient (FQ). FQ indicates the molar ratio between lactic and acetic
acids during sourdough fermentation. After the determination of the concentration
of lactic and acetic acids by enzymatic or chromatographic methods, the FQ is cal-
culated as follows: FQ = (g of lactic acid in 100 g of dough/molecular weight of
lactic acid): (g of acetic acid in 100 g of dough/molecular weight of acetic acid).
This parameter is strictly related to the type of lactic acid bacteria dominating
the fermentation and markedly varies depending on the balance between homo-
and hetero-fermentative lactobacilli. In turn, this balance depends on exogenous and
endogenous factors which prevail during fermentation (e.g. fermentable sugar and
oxygen concentration, DY, time and temperature, etc.).

4.7 The Use of Stabilized Sourdoughs Versus

Active Sourdoughs

On the basis of the research progress in the field of bread-making biotechnology,

novel types of sourdough have recently been developed with the main aim of
improving the flavour of leavened baked products without using traditional protocols
of manufacture based on active sourdough. Currently, various commercial
preparations of stabilized sourdough are available on the market. They are used for
making traditional breads and are based on a mix of tailor-made aroma compounds.
Different types of stabilized sourdough are obtained starting from an accurate
selection and mix of raw materials (e.g. flour from T. aestivum or T. durum wheat, rye
or other cereals) and microbial strains. Dried sourdough is a type of stabilized prod-
uct useful for this purpose. Different drying protocols can be applied, for example
freeze-drying, spray granulation, fluidized bed drying, spray drying and drum dry-
ing, among which the latter two can be cited here as the most common for type III
sourdough production (12, 48). In both cases, the higher the DY of the starting type
II sourdough the higher the resulting TTA value of the derived type III sourdough,
which also increases due to water evaporation. In the spray-drying process, the liquid
sourdough is dried by using a warm air flux that removes water until the humidity
becomes less than 10%. In the drum-drying system, the vapour of the warmed drums
removes water from the thin-layered liquid sourdough during contact. On the basis
of various combinations of time and temperature and on the extent of the Maillard
reaction, different type III sourdoughs are obtained, which show different degrees of
caramelization or toasting and, as a consequence different flavouring activities. Since
many volatile compounds (especially acetic acid) are missing due to the evaporation,
even if to a different extent depending on the drying technique, pasteurization, cool-
ing or salting can be applied to obtain a stabilized liquid or pasty sourdough (12, 48).
With the exception of cooling, all the other stabilization systems lead to microbiota
inactivation and stop gas and/or acid production (48), giving a sourdough which can
100 A. Corsetti

be classified as type III. Especially liquid sourdough represents an advantage for

industrial applications because it can be pumped and easily dosed, while showing
constant quality (12). Generally, the use of a stabilized sourdough is quite simple. It
can be stored at room temperature for a long time (30–60 days) and directly added to
the final dough at a proportion of 5–10%. Because of the low cell density of lactic
acid bacteria and yeasts, baker’s yeast is generally added to leaven the dough for
bread production. Examples of applications of type III sourdough in the modern
bakery industry are the use of a stabilized liquid rye sourdough for rye bread produc-
tion; dextran-containing sourdough (produced by the use of selected L. mesenteroi-
des strains) stabilized by cooling, pasteurization, or drying for the production of
Panettone, rye bread and toast bread; pasteurized San Francisco sourdough in liquid
form for the production of San Francisco bread (12).
The selection of microbial strains represents the current challenge for sourdough
bread-making industries and relies on several metabolic traits which are related to
the most important flavour compounds. Overall, strains isolated from raw materials
or sourdoughs are in vitro and in situ selected and evaluated mainly based on the
kinetics of acidification, proteolytic activity and nitrogen metabolism and synthesis
of aromatic compounds. Recently, fermentation processes based on selected lactic
acid bacteria and liquid sourdough have been evaluated with the aim of improving
the biosynthesis of specific chemical compounds (49).

4.8 Use of Pure Cultures of Sourdough Starters Versus

Stabilized Sourdoughs

As for other fermented food biotechnologies (dairy, oenology, meat) the interest in
using a commercial starter culture is rapidly increasing for sourdough breadmaking.
In this context, some researches have been devoted to the use of freeze-dried or
frozen preparations of lactic acid bacteria (4, 20). Nevertheless, only a few applica-
tions of selected lactic acid bacteria starters are currently documented both at arti-
san and industrial levels. Difficulties are mainly related to the possibility of obtaining
highly concentrated lactic acid bacteria preparations as well as to the performance
of the selected lactic acid bacteria in situ. From an applicative point of view starter
cultures should be pre-cultivated in a mix of flour and water to obtain metabolically
active strains before use as an inoculum. Nevertheless, it has been found that many
strains, which were in vitro selected for interesting traits, show some difficulties in
dominating the sourdough ecosystem and overcoming the endogenous lactic acid
bacteria throughout refreshments. Recently, Siragusa et al. (24) demonstrated that, after
ten consecutive refreshments, only three out of nine L. sanfranciscensis strains,
which were in vitro selected for the optimal rate of acidification, proteolytic activity
and release of flavour compounds, were able to persist during sourdough fermenta-
tion due to the dominance of autochthonous L. plantarum strains. Thus, the capabil-
ity to adapt to the sourdough ecosystem seems to be an essential trait for selecting
strains in order to obtain a sourdough with a constant microbial composition and
4 Technology of Sourdough Fermentation and Sourdough Applications 101

performance. Currently, this seems the main limitation in the use of pure cultures as
sourdough starters and the most innovative applications mainly deal with the use of
stabilized sourdoughs.

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