Comparison of the microbial dynamics and
biochemistry of laboratory sourdoughs prepared with
grape, apple and yogurt
Abstract
The microbiological culture-dependent characterization and physicochemical characteristics of
laboratory sourdough prepared with grape (GS) were evaluated and compared with apple (AS)
and yogurt (YS), which are the usual Spanish sourdough ingredients. Ripe GS took longer than
AS and YS to reach the appropriate acidity and achieved lower values of lactic acid. In all
sourdoughs, the lactic acid bacteria (LAB) increased during processing and were the dominant
microorganisms (>1E+8 CFU/g). GS, as well as AS, had high diversity of LAB species. In ripe
YS Pediococcus pentosaceus was the only species identified; in GS and AS, several
Lactobacillus were also found, Lb. plantarum, Lb. brevis, and Lb. sakei; in addition, in GS
Weisella cibaria also appeared. Regarding the yeast population, non-Saccharomyces yeasts
from GS and AS showed a very high specific population (> 1E+7 CFU/g), but this was reduced
in ripe sourdough (<1E+4 CFU/g). Finally, the Saccharomyces group dominated in all
sourdoughs. Starting ingredients or raw material provided microbiological specificity to
sourdoughs, and grape could be considered one of them.
Keywords
Sourdough, grape, yeast, lactic acid bacteria, ingredients
1
Introduction
Sourdough was introduced in middle of the 19th Century and has been used in bread production
since ancient times, because it was necessary for dough leavening (Hansen and Schieberle,
2005). The tradition of making wheat bread with the addition of sourdough is still widely used
in some European countries. Spanish artisan bakeries, especially in Catalonia (northeast Spain),
are interested in promoting and encouraging the consumption of these types of longfermentation breads again. Their preparation is more labour-intensive but it allows these
bakeries to be different and to respond to the demand of the consumers for these types of breads,
as well as enabling them to fend off competition from industrial bread manufacturers.
In Spain, sourdough is called ‘mother-dough’ and sometimes the name of the distinguishing
ingredient is added (apple sourdough, lactic sourdough). The basic ingredients are wheat flour
(if necessary, supplemented with malted barley flour), whole wheat or rye flour, and water. In
order to increase the microbiological diversity of sourdough, fermented apple juice or a little
milk derivative such as yogurt are frequently added during preparation. The use of lightly
fermented grape must could provide, as in wine, greater diversity of the non-Saccharomyces
yeasts group and may contribute to give specificity to the sensory characteristics of sourdough.
Specific ingredients can also be added to part of the ripe sourdough in the final stages of
propagation, in order to transform it and impart a special aroma and flavour to the bread
(Bellsolà, 2010). The development of the sourdough is performed as a multi-stage process
which controls factors like the incubation temperature, pH and aroma. The bakeries have
propagated the same spontaneous sourdough for many years, as in earlier generations (Barriga,
2003), and they don’t add commercial starters at any time during the spontaneous sourdough
process.
The impact of endogenous factors such as the chemical composition and microbiology of the
dough during the continuous propagation of the sourdough produces a dynamic balance among
the microorganisms present such as LAB and yeasts (De Vuyst and Neysens, 2005). Between
these microorganisms, there are specific and stable associations, and their metabolic activity can
contribute to the production of aroma and flavour compounds. LAB contributes specifically to
the process of acidification of the sourdough since they are the main microbial group
responsible for the production of lactic and acetic acids. Yeast is considered to be the main
factor responsible for CO2 production and the increase in dough volume, especially if baker’s
yeast is not used for bread making. In general, in ripe sourdough, the LAB counts are already
1E+9 CFU/g of dough, and 1E+6-1E+7 CFU/g of dough to yeasts (Arendt et al., 2007). The
level and intensity of the factors that simultaneously affect the lactic and alcoholic fermentation
of the sourdough will influence acid formation, volatile compound production, and carbon and
nitrogen degradation, ultimately determining the overall quality of the bread.
2
The aim of this study is to evaluate the behaviour of white grape must (variety Xarel·lo) lightly
fermented, for 48 h at 20ºC, as a new starting ingredient in sourdough preparation. The
microbial dynamics of LAB and yeasts, as well as changes in biochemical parameters (pH,
acidity, and other organic compounds), will be evaluated during sourdough preparation. These
characteristics, together with the results of molecular identification of LAB species, will be
compared with the sourdoughs elaborated with yogurt and apple juice fermented which are
usually used in artisan bread-making.
Materials and methods
Sourdough process preparation using apple, yogurt, and grape in laboratory conditions
Three types of sourdough were prepared using a multiphase protocol provided by an artisan
bakery (‘Fleca Parés’, Vilafranca del Penedès, Catalonia, Spain) (Table 1). This method, with
some variations, is one recommended by the Association of Bakers of Barcelona (Spain). The
sourdoughs were prepared by starting each of them with different ingredients such as organic
apple (AS), plain yogurt (YS) and organic grape (GS).The grape sourdough studied was
prepared using white Xarel.lo variety from the Valldolina vineyard. The sourdoughs
elaborations were duplicated, and Table 1 shows the process scheme for the three sourdoughs.
The dough was prepared in a spiral dough mixer (Kenwood KM 336) at 200 rpm for 5 min,
conditions which allowed good mixing of the ingredients so as to obtain the correct consistency.
The sourdough process comprises three phases: the first phase is the preparation of the liquid
starter (apple juice fermented and grape must lightly fermented). The fermentation of small
pieces of apple mixed in water and honey for 5 days at 40ºC, and stirred twice a day, was
performed. The grapes were pressed and the must was fermented 2 days at 20ºC. For the yogurt
sourdough, a plain commercial product was used, and according to the guidelines
concentrations of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophillus
(≥ 1.0E+06 CFU/g of each species) were expected.
A second phase is the pre-sourdough preparation, and in this the growth microorganisms,
fermentation and microbial stabilization would take place. This phase lasts 5 days at two
different temperatures; for the first three days, the pre-sourdough is kept at 22°C, facilitating the
growth of LAB and yeast; the remaining two days storage is at 5°C to select and stabilize the
existing microbiota. In this phase, samples to be analysed were taken every 24 or 48 h before
the next mixing (samples named P1, P2, P3, P4).
Later on, the third phase is the propagation and maintenance of the ripe solid sourdough. The
liquid sourdough becomes solid modifying the dough/flour/water relationship. Daily process,
known as refreshment, is performed three times a day, at 9:00 a.m., 1:00 p.m., and 5:00 p.m.
3
Before each refreshment step at 9:00 h (every 24 h) samples were taken from sourdough to be
analyzed (samples named R1, R2, R3, R4, R5).
Microbiological analysis
Control of yeast (total and non-Saccharomyces) and LAB. Every 24 or 48 h, a microbiological
analysis was performed depending on preparation phase. For the analysis, 10 g of sourdough
was homogenized with 90 mL of saline solution ringer ¼ (Scharlab, Spain) using a Stomacher
apparatus (BagMixer® 400P, Interscience, Paris) for 2 min. Decimal dilutions were made using
the same solution, and microbiological seeding was performed in duplicate on agar plates: i)
total yeasts count on WL agar supplemented by 0.5 g/L chloranphenicol (Scharlab, Spain), ii)
non-Saccharomyces yeasts on lysine agar (Scharlab, Spain), and iii) lactic acid bacteria on MRS
agar (Scharlab, Spain) supplemented by 15% tomato juice, 15% grape juice, and 75 mg/L
cycloheximide (pH 5.5). The yeasts were incubated for 3 to 5 days at 27°C, and lactic bacteria
were incubated for 5 days at 30°C. The countable plates (between 30 and 300 colonies in MRS
agar and 15 and 150 colonies in WL or Lysine agar) were used to quantify the microbial
population. Yeast colonies were classified depending on their morphology, colour and colony
size. Based on these differences, the percentage of different types of yeast colonies that
appeared throughout the study was determined.
Isolation and identification of LAB by PCR-sequence of the 16S rDNA gene. Three strains
LAB were isolated per sample in order to pick colonies of different appearance. The isolates
were characterized by Gram staining, and catalase testing. Homofermentative and
heterofermentative tests were performed to determine CO2 production from glucose in paraffinsealed test-tubes.
For LAB identification 10 mL Elliker broth (Scharlab, Spain) was inoculated with isolated
bacteria for testing. Cells were grown for 3-4 days at 30°C, until sufficient population was
achieved for the purification of the DNA. Cells from the culture were harvested by
centrifugation at 5000 g for 15-30 min at 4°C, and washed with 10 mL of PBS. The bacteria
pellet was then suspended in 1.5 mL of PBS and recovered in a microtube by centrifugation at
5000 g for 5 min at 4°C. The pellet was suspended in 100 L of buffer P1 (0.05 mol/L TrisBase, pH 8.0, 4 g/L EDTA, and 100 mg/L RNAase); this suspension was stored at 4°C for 5
min. Subsequently, 150 L of lysis buffer P2 (8 g/L NaOH, and 10 g/L SDS) and 60 L of 10
mg/mL Lysozyme (prepared in 10 mM Tris, pH 8) were added, and the mixture was kept at
37°C for 1 h. The reaction was then neutralized using 200 L buffer P3 (294.5 g/L potassium
acetate, pH 5.5), and the suspension was centrifuged at 10000 g for 5 min. The supernatant was
obtained using absolute ethanol to precipitate the DNA. The DNA quality was determined by
the ratio A260/A280.
4
The 16S ribosomal gene was amplified by PCR and sequenced to the molecular identification of
the lactic acid bacteria (De Vuyst et al., 2002; De Vuyst and Vancanneyt, 2007; Scheirlinck et
al., 2008). To this end, universal primers 8F (5’-AgAgTTTgATCCTggCTCAg-3’) and 1510R
(5’-ggTTACCTTgTTACgACTT-3’) were used in the PCR to amplify a product of 1.5 Kb
approximately. The PCR was performed at 40 cycles, applying the following steps: DNA
denaturation for 1 min at 94°C, annealing for 30 s at 55°C, and DNA polymerization for 2 min
at 72°C. The PCR was finished with an extension for 5 min at 70°C. Finally, amplification
products were analysed on 1.5% agarose gels. These amplicons were then used as templates for
the sequencing reaction (BigDye, Quiagen, Germany), and the DNA fragments were
commercially sequenced (Macrogen, Korea). Sequences were analysed by performing a BLAST
(Basic Local Alignment Search Tool) search of the National Center for Biotechnology
Information nonredundant DNA sequence database (National Center for Biotechnology
Information, U.S. National Library of Medicine, Bethesda, MD).
Physical and chemical analysis
The changes in biochemical characteristics during the multistage sourdough process were
determined from an analysis of the pH, the total titratable acidity (TTA), the sugars (maltose,
sucrose), the organic acids (lactic and acetic acid), and ethanol. 60 g samples were taken from
the different sourdough stages and kept frozen at -20°C in 10 g aliquots prior to analysis. All
determinations were performed with two replicates and results were expressed as mean values
jointly with their standard errors.
Determination of pH and total titratable acidity (TTA). The pH values were determined in 10 g
samples blended with 90 mL distilled water. The measurements were taken using a pH-meter
(Crison Instruments, S.A., Spain). Then the same suspensions were titrated to an end point of
pH 8.5 using 0.1 mol/L NaOH (Lefebvre et al, 2002). TTA was expressed as the spent NaOH
volume.
Determination of sugars, organic acids, and ethanol. The organic component extraction
method was adapted from Meignen et al. (2001). The 10 g dough samples were blended with 90
mL of distilled water. The sample was kept in an ultrasonic bath for 15 min, and then agitated at
room temperature for a further 45 min. The dough extracts were centrifuged at 4000 g at 15°C
for 15 min. The concentrations of maltose, sucrose, lactic acid, acetic acid, and ethanol were
analysed by HPLC (McFeeters et al., 1984). HPLC separation was performed at room
temperature using a reversed phase C18 column (250 mm x 4.6 mm) eluted with 0.05 M
phosphoric acid (pH 2.5, adjusted with concentrated NH4OH) and at a flow rate of 1.2 mL/min.
Before injection, 40 µL of the sample was mixed with an equal volume of elution buffer (2X),
and filtered using a nylon syringe filter (0.45 µm). The samples were automatically injected into
5
a chromatograph (110B Model, Beckman) with a refractive index detector (RID 156 Model,
Beckman).
Statistical analysis
With the physical and chemical determinations of laboratory sourdough, taking into account the
last values when the stabilization of the three sourdoughs was achieved, the one-way analysis of
variance and multiple comparisons tests (Tukey's method) were used in order to compare the
different groups of data (Minitab, 2007). The probability level of significance was set at 0.05.
Results and discussion
Microbiological results
Microbiological count and population dynamics in laboratory prepared sourdough. The timecourse of mean values and corresponding standard errors of the populations of microorganisms
in sourdoughs prepared (GS, AS, YS) are shown in Figure 1(a) to (c). In the three sourdoughs,
the viable LAB increased throughout the process, while the total yeasts and non-Saccharomyces
yeasts decreased in different percentages according to the sourdough. The ripe sourdough
microorganisms were largely dominated by LAB (> 1E+08 CFU/g), and yeasts were found in a
lower proportion. The ratio LAB/yeasts was close to 10000/1 in GS sourdough while in AS and
YS this ratio was 1000/1. The LAB/yeast ratio obtained across the three sourdoughs was greater
than others described in the literature (Wlodarczyk, 1985; Vera et al., 2009). It is known that
several factors influence the results of development and control of LAB sourdough and the ratio
of values could change (Van der Meulen et al., 2007), for example, sourdough preparation
conditions (such as ingredients, temperature, time backslopping) and LAB control methodology
(such as media, incubation conditions -aerobic, anaerobic, microaerophilic-, culture-independent
methods). In our study the culture-dependent method and aerobic conditions were selected, as
done by many other authors (Van der Meulen et al., 2007; Vranckren et al., 2011; Zhang et
al.,2011; Lattanzi et al., 2013). The methodology used means that some LAB strains could be
lost, and the same could happen in other conditions. It was estimated that the colony numbers
isolated from aerobically and anaerobically incubated agar plates were generally 10 times higher
than on anaerobically grown plates (Viiard et al., 2013).
The main differences in viable LAB counts among the three sourdoughs prepared were detected
throughout the pre-sourdough phase. The initial LAB count in GS was 1.5E+03 CFU/g, which
was 2 and 3 exponential units lower than those found in YS and AS counts, respectively. LAB
concentration in GS increased at the beginning of the ripe sourdough phase when the count was
1E+08 CFU/g, a value which was maintained in a stable manner throughout the rest of the
process. In contrast, the initial LAB counts in the AS and YS pre-sourdough phase were higher,
6
reaching 1E+08 CFU/g at the end of this phase. During the ripe sourdough phase, LAB
increased slightly in a similar manner to AS and YS, reaching a population of 1E+09 CFU/g.
The total yeast counts at the beginning of the preparation for the three pre-sourdoughs were also
different. For this microbial group, GS behaved more like AS. GS had an initial yeast count of
3.65E+07 CFU/g (i.e. P1, Figure 1(a)), one order of magnitude higher than AS and nearly 6
units higher than YS. High initial total yeast counts in GS and AS are the result of both
sourdoughs, being prepared using grape must lightly fermented and apple juice fermented, and
consequently containing a population of proliferative yeast. By contrast, YS showed a low total
yeast count due to dilution of the yeast found in wholemeal flour when mixed with the yogurt.
According to the literature, the total yeast counts in flour were around 1E+02 – 1E+04 CFU/g
(Berghofer et al., 2003; Eglezos, 2010). Nevertheless, the total yeast population in the yogurt
pre-sourdough after 72 h (i.e. P3) was similar to that for the apple pre-sourdough. In the three
sourdoughs, the maximum total yeast population was achieved at some point during the presourdough stage. The controls performed at the ripe sourdough stage showed that the total yeast
population in GS and YS decreased, stabilizing at the end of the study. In AS, the total yeast
population increased and stabilized at the end of the ripe sourdough phase, but without reaching
the high counts found in the pre-sourdough phase.
The non-Saccharomyces counts at the initial preparation of the sourdough (i.e., P1,) were higher
in GS (1E+07 CFU/g), followed by AS (1E+06 CFU/g), and lower concentrations were
observed in YS (1E+01 CFU/g). Yeast non-Saccharomyces counts carried out during the ripe
sourdough phase showed a large decrease in all three sourdoughs. In the GS and AS presourdoughs, the non-Saccharomyces yeast count coincided with the total yeast count. Thus, in
GS and AS, the majority of the yeasts were different from the Saccharomyces genera. In the YS
sourdough there is a difference of about 3 or 4 exponential units between total yeast and nonSaccharomyces yeast count. It seems that in the YS sourdough there was less diversity of yeasts,
with most belonging to the Saccharomyces group. The lowest concentration of nonSaccharomyces yeasts may be related to the yogurt used as the starting ingredient. Yogurt does
not contain yeast, unlike grape must lightly fermented and apple juice fermented. During the
preparation of GS and AS it was introduced and multiplied the yeasts found in their own starter
sourdough, as well as those present in the flour itself. A wide variety of yeast species of
Saccharomyces and non-Saccharomyces yeasts have been described in connection with apple
fruit (Pelliccia et al., 2011; Vadkertiová et al., 2012) and in the cider-making process; where the
frequency of non-Saccharomyces in the first phase of fermentation varied with the harvest
(Pando et al., 2010). As in cider apple, non-Saccharomyces yeasts are also found predominantly
in grapes and in freshly processed must. Even though S. cerevisiae is the most important yeast
for wine production, the role of non-Saccharomyces yeasts in wine fermentation is receiving
increasing attention by wine microbiologists (Jolly et al., 2006). Finally, the results in GS and
7
AS of the non-Saccharomyces yeasts highlight the importance of the initial ingredient used in
the preparation of sourdoughs. Changes in procedure such as starting a new sourdough more
often or incorporating the differential ingredient at the last refreshment could ensure the activity
of non-Saccharomyces yeasts.
Figure 2 shows the frequency of different types of yeasts isolated in the preparation of
sourdough. As described in the Materials and methods section, the yeasts were also
characterized according to colour colonies and morphology on WL and Lysine agar. They were
differentiated by two types of non-Saccharomyces yeast (Type 1.1 and Type 1.2 differentiated
by morphology on Lysine) and two types of Saccharomyces yeast (Type 2.1 and Type 2.2, only
differentiated by colour on WL). The GS pre-sourdough and the first days of ripe sourdough
were dominated by a type of non-Sacchararomyces yeast different from that of AS. Lysine agar
was shown to be useful to differentiate and quantify the non-Saccharomyces yeast group. Thus,
Lysine agar is currently employed by yeast producers to detect contamination and to carry out
quality controls on wines (Gordún et al, 1991; Hansen et al., 2001; Tofalo et al., 2011) and the
brewing industry (Kühle and Jespersen, 1998). It is known that the environment of propagation
has an influence on the microbiota composition, but in contrast to other studies (Vrancken et al.,
2010; Minervini et al., 2012), Saccharomyces yeast was dominant in all ripe sourdough
propagated in laboratory. It is necessary to identify the isolated yeasts involved in the successive
populations during the development of the sourdough. In any case, the ingredients included in
the raw materials influence the type of yeast present.
Identification of LAB. Table 2 presents the results of the identification of LAB. A total of 35
strains were identified from laboratory sourdoughs. Some strains of LAB were non-culturable
after their initial isolation, or growth was very low, and so the extraction of DNA was deficient.
The medium used for strain maintenance was the same as for isolation (MRS agar enriched with
vitamins from grape juice and tomato). Various authors have proposed modifying the MRS agar
according to the genera to be isolated (Vera et al., 2009). MRS supplemented with maltose and
fructose as well as amino acids and vitamins with lowered pH are successful for the isolation of
sourdough LAB (De Vuyst and Vancanneyt, 2007). Nevertheless, the medium does not seem to
be the best suited for the maintenance of some acid lactic strains. Also, for this reason it is
necessary to continue the search for new LAB species, focusing the investigation on improving
the formulation of the growth medium and the growth parameters (De Vuyst and Vancanneyt,
2007).
The identification of LAB allowed differentiation between AS and YS. The AS presented
greater bacterial diversity than YS. YS showed the presence and the domain of Pediococcus
pentosaceus from the beginning of the pre-sourdough until the end. In YS there was no detected
characteristic LAB from plain yogurt (as Lb. delbrueckii subsp. bulgaricus and S.
thermophillus) and this may be due to the temperature of the sourdoughs process in which the
8
maximum microbiological growth occurred as in the first part of the second phase. It was
performed at 22ºC according to the recipe. Yogurt bacteria needs higher growth temperature and
this may have caused the loss of these species.
In AS, the same specie dominated throughout the process, together with other Lactobacillus
species like Lb. plantarum, Lb. brevis and Lb. sakei. The GS was similar to AS in terms of
diversity. The novelty was that Weisella cibaria appeared, which had not been encountered in
AS or YS. Unlike grape must lightly fermented, apple juice fermented is an important source of
LAB because the malolactic and alcoholic fermentations start at the same time. In the traditional
cider process, a great diversity of LAB has been described, fundamentally heterofermentative
bacteria; being Lactobacillus species the most abundant and in low proportion Pediococcus
(Dueñas et al., 1994; Sánchez et al., 2010); other dominant LAB as Leuconostoc (Salih and
Drilleau, 1988) and Oenococcus (Sanchez et al., 2012) have been mentioned.
In cereal sourdough, in general, microbiota is also clearly dominated by lactobacilli, and more
than 55 Lactobacillus species have been identified. Other LAB species may also be found,
including members of the genera Weissella, Pediococcus, Leuconostoc, Lactococcus,
Enterococcus and Streptococcus (Huys et al., 2013). Some species like Lb. sanfranciscensis
seem to be specific to the sourdough environment, but others, such as Lb. brevis and Lb.
plantarum, have also been found in many other food ecosystems (De Vuyst et al., 2009). The
use of different ingredients with their own microflora may have influenced the diversity of
LAB, single species in YS and more complex communities in GS and AS. It is not frequent to
find a sole LAB specie, but occasionally it has been described for Lb. sanfranciscensis
(Zapparoli et al., 1996, 1998 and Foschino et al., 1999) and other species. In these sourdoughs
processed for 10 days, Pediococcus pentosaceus was present in all phases, and in other
sourdough studies the microarray data also revealed the dominance of Pediococcus pentosaceus
in most of the fermentations during the whole period (Weckx et al., 2010).
Physical and chemical determinations of laboratory sourdough
The results of pH and TTA are shown in Figure 3(a) and (b), respectively. In the measuring of
pH at the mature phase (R4-R5), statistically significant differences between GS and AS-YS
were detected (p-value<0.001), but not in the TTA (p-value=0.769). GS takes four days to reach
a pH of 4, following the recipe, which is the ripe sourdough phase. However, some days before
finishing the preparation or during the pre-sourdough stage, both AS and YS already had pH
values of around 4. In all three types of sourdough, the pH value stabilized at pH 4. Final pH,
which ranges from 3.5 to 4.3, is considered as an index of a well-developed sourdough
fermentation (Collar et al., 1994). TTA of the three ripe sourdoughs stabilized at values close to
6 mL, but GS took several days longer than AS and YS to reach this acidity. Monitoring of both
pH and TTA parameters indicates that GS matures more slowly than AS and YS, although
9
ultimately similar pH and TTA values are reached. The evaluation of the change of pH and TTA
during the preparation of the sourdough allows the establishment of the time when the
sourdough ripens; something which does not always coincide with expectations from the recipe,
especially when new ingredients are included, as in this study. In P3 and P4 stages, according to
the recipe, flour and water were added and kept for 2 h at 22°C and 22 h at 5°C. The latter
condition affected the decrease of the enzymatic activity of microorganisms present. Therefore,
there was a dilution of the pH and TTA accumulated and there was not enough fermentation
time to recover these two parameter values, especially in GS, and less in AS and YS. The lower
LAB counts in the GS pre-sourdough phase (around 1E+05 CFU/mL while in AS and YS was
close to 1E+8 CFU/mL) was the main difference that may have influenced the delay in reaching
the optimum pH and TTA. Several studies have revealed the substantial influence of the LAB
evolution (counts and composition) on acidification behaviour (Collar et al., 1994, Gatto and
Torriani, 2004; Corsetti et al., 2007; Bartkiene et al., 2013). Equally high concentrations (≥
1E+08 CFU/mL) were reached by LAB in the ripe phase of the three sourdoughs.
Other parameters quantified during the processing of the sourdough were lactic acid and acetic
acid; the results are shown in Figure 3(c) and Table 3 respectively. In the measurement of lactic
acid at the mature phase (R4-R5), significant differences between GS and AS-YS were detected
(p-value=0.001). The acetic acid, in some cases, was not quantified due to the lack of precision
of the analytical method. It was not possible to establish a correlation between acetic acid
concentration and the number of heterolactic strains despite the heterofermentative LAB being
the main producer. The concentration of lactic acid in the GS was considerably lower than in the
AS and YS sourdoughs, both in the pre-sourdough and ripe sourdough phases (Figure 3(c)). The
average concentration of lactic acid in the final phase of ripe sourdough (last 5 days) was 0.4
g/100 g for GS and 1.0 g/100 g for AS and YS. The production of acetic acid in GS was lower
than in AS (Table 3); the ripe AS (last 5 days) produced 0.27 g/100 g. As expected, acetic acid
production was lower than lactic acid and the fermentation ratio between lactic and acetic acids
(molar ratio between these acids) to AS was 2.5.
The results relating to maltose and ethanol in the sourdough are shown in Table 3. There were
no statistically significant differences in the amount of maltose in the three ripe sourdoughs (pvalue=0.494). In the sourdough prepared during the pre-sourdough and ripe sourdough phases,
maltose was available for the different microorganisms, which indicates that the amount of
maltose produced from hydrolysed starch by the amylase enzymes was greater than the maltose
metabolized by different microorganisms. In the final phase of the ripe sourdough (last 2 days),
the concentration of maltose was lower and stabilized for all the sourdough with values close to
0.4 g/100 g. Ethanol production by the microorganisms was detected during the processing of
the sourdough, and there were significant differences between GS and AS-YS in the mature
phase (p-value<0.001); maximum values were reached in this final phase of the ripe sourdough
10
(1.03 g/100 g, 1.89 g/100 g and 2.46 g/100 g for GS, AS, and YS respectively), showing a
Saccharomyces group population close to 1E+05 for GS, and close to or higher than 1E+06 for
AS and YS. Ethanol is the main metabolite produced by yeasts of the Saccharomyces group
and, in a smaller quantity, by heterofermentative LAB, nevertheless in some sourdoughs ethanol
concentration could be attributed to heterofermentative LAB (Van der Meulen et al, 2007).
During the sourdough preparation the ethanol concentration showed some variations with an
increase at the end of the study, coinciding with the dominance of the Saccharomyces group
over the non-Saccharomyces group, and with an increase of their metabolic activity due to the
fermentation temperature, 29ºC (higher than the previous phase, 22ºC), and the use of nutrients
from the three refreshments per day. In YS (pre-sourdough and ripe sourdough) ethanol
concentrations were higher than in GS sourdough, coinciding with a reduced presence of nonSaccharomyces group yeasts and higher concentrations of the Saccharomyces group, which are
more resistant to this metabolite. The rate of alcoholic fermentation of S. cerevisiae was
dependent on the temperature used (Gobetti et al., 1995), especially at 22ºC yeast activity
decreased compared to 25, 28 or 31ºC (Häggman and Salovaara, 2008), and the inoculum yeast
size (Akdogan and Ozilgen, 1992). Other factors may have favoured the ethanol production,
such as the interaction between yeast and LAB (Guerzoni et al., 2007).
Conclusions
When comparing sourdoughs made with the three different ingredients, it can be stated that
grape must lightly fermented and apple juice fermented offer microbiological specificities that
are different from yogurt. They provide their own microbiota, which does not always remain in
ripe sourdough, and they obstruct or retard the development of the indigenous microorganisms
in the flour, with an effect that could modify the organoleptic properties.
In this study, a new sourdough prepared from grape must lightly fermented was evaluated and it
proved to be a good alternative to the traditional ingredients used in artisan bread-making. The
grapes correspond to local indigenous variety, specifically the Xarel.lo, which was processed by
fermentation over a 48 h period at 20°C. From a physicochemical standpoint, and following this
recipe, GS requires more time than AS and YS to attain a suitable pH. And microbiologically,
GS shows a population and a diversity of microorganisms similar to AS. It would be interesting
in future studies to optimize the preparation of the grape sourdough process and determine the
influence of the grape must on the fermentation time, the sourdough microbiota, and the longterm stability of the microbiota, especially the group of non-Saccharomyces, as well as the
overall flavour produced.
Acknowledgements
11
Thanks to Marc and Gabriel Parés of the centennial artisan bakery Fleca Parés, for providing us
with the recipes and sourdough ingredients, and motivating us to carry out this research.
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16
Table 1. Sourdoughs process, preparation and refreshments of ripe sourdoughs prepared
with grape (GS), or apple (AS), or yogurt (YS).
First phase: Preparation of differential ingredient
AS
YS
GS
0.3 Kg of apples (with skin)
0.3 L of white grape must
0.3 L of water + 10 g of honey
just pressed
5 days, 40ºC.
2 days, 20ºC
Second phase: Pre-sourdough
0.3 Kg of preceding apple
0.3 Kg of plain
0.3 Kg of preceding grape
juice fermented
yogurt
must lightly fermented
+ 0.1 Kg Whole wheat flour, 48 h at 22ºC
P1
Similar process for AS, YS, GS
Preceding sour (Kg)
Wheat flour (Kg) Water at 28ºC (L) Time and temperature
0.1
0.5
0.6
24h, 22ºC
P2
0.3
0.5
0.5
2h, 22ºC + 22h, 5ºC
P3
0.4
1
1
2h, 22ºC + 22h, 5ºC
P4
Third phase: Ripe sourdough (3 daily refreshments: at 9 a.m., 1 p.m. and 5 p.m.)
Preceding sour (Kg)
Wheat flour (Kg) Water at 28ºC (L) Time and temperature
0.4
0.4
0.2
4 h, 29ºC (at 9 a.m.)
R*
0.4
0.4
0.2
4 h, 29ºC (at 1 p.m.)
0.2
0.4
0.2
16 h, 29ºC (at 5 p.m.)
R*: Refreshment ripe sourdough was propagated for 5 days in this study (R1, R2, R3, R4, R5)
P (P1 to P4) and R (R1 to R5) are the sourdough sampling codes.
17
Table 2. Identification of sourdough LAB isolates by means of 16S rDNA sequencing.
Sourdoughsa
Identificationb
Specie
Fermentationc
Lactobacillus sunkii
AB366385.1 (95%)1
HE-F
2
Pediococcus pentosaceus
EU180605.1 (92%)
HO
AS/ First phase
Pediococcus pentosaceus
HM130536.1 (96%)2
HO
Pediococcus pentosaceus
AB494722.1 (95%)2
HO
Leuconostoc mesenteroides JN863681.1 (95%)3
HE
2
Pediococcus pentosaceus
EU180605.1 (85%)
HO
AS/ Second
Pediococcus pentosaceus
AB494722.1 (98%)2
HO
phase
Pediococcus pentosaceus
EU080993.1 (97%)2
HO
Lactobacillus brevis
HM067023 (98%)2
HE
2
Pediococcus pentosaceus
HM130536.1 (99%)
HO
Lactobacillus sakei
GU125609.1 (98%)3
HE-F
Lactobacillus sakei
GU125609.1 (98%)3
HE-F
AS/ Third phase
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Lactobacillus plantarum
GU195643.1 (98%)4
HE-F
Pediococcus pentosaceus
AB494722 (99%)2
HO
Lactobacillus plantarum
GU138610.1 (98%)2
HE-F
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
YS/ Second
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
phase
Lactobacillus sakei
GU125609.1 (99%)3
HE-F
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Pediococcus pentosaceus
AB481102.1 (98%)2
HO
Pediococcus pentosaceus
AB481102.1 (98%)2
HO
Pediococcus pentosaceus
AB494722.1 (93%)2
HO
YS/ Third phase
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
2
Pediococcus pentosaceus
AB481102.1 (99%)
HO
GS/Second phase Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Pediococcus pentosaceus
AB481102.1 (99%)2
HO
Lactobacillus sakei
GQ222408.1 (98%)3
HE-F
Weisella cibaria
GU138616.1 (99%)4
HE
3
GS/ Third phase
Lactobacillus plantarum
GU195643.1 (98%)
HE-F
Pediococcus pentosaceus
AB550294.1 (99%)2
HO
Lactobacillus plantarum
GU195643.1 (98%)3
HE-F
Lactobacillus brevis
AB494718.1 (96%)2
HE
a
Sourdough: type of sourdough/ elaboration phase)
b
GenBank Acces (Maximum identity, %). (1) Watanabe et al., 2009; (2) Iacumin et al., 2009;
(3)
Catzeddu et al., 2006; (4) Corsetti et al., 2003.
c
Fermentation: (HE) Heterolactic, (HE-F) Heterolactic-Facultative, (HO) Homolactic.
18
Table 3. Mean values (and their corresponding standard errors) of some organic
compounds, maltose, acetic acid, and ethanol, during the evolution of the sourdoughs
prepared with grape (GS), apple (AS) and yogurt (YS).
Sample
(Days)
P3
P4
R1
R2
R3
R4
R5
GS
1.51
1.35
1.19
1.61
1.68
0.47
0.47
Maltose (g/100 g)
AS
YS
(0.03)
(0.03)
(0.06)
(0.02)
(0.03)
(0.16)
(0.02)
2.55
2.27
3.05
3.27
3.16
0.46
0.43
(0.01)
(0.06)
(0.13)
(0.08)
(0.10)
(0.07)
(0.12)
0.24
1.61
3.11
2.94
0.25
0.41
0.33
(0.02)
0.07)
(0.06)
(0.14)
(0.05)
(0.10)
(0.07)
Acetic acid (g/100 g)
GS
AS
YS
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ: Not quantificable
19
0.20
0.23
0.25
0.32
0.33
0.23
0.23
GS
Ethanol (g/100 g)
AS
YS
(0.02) 0.18 (0.01) 0.94 (0.05) 0.15 (0.03) 1.70
(0.00) 0.20 (0.02) 0.42 (0.02) 0.03 (0.03) 0.26
(0.01) 0.22 (0.01) 0.20 (0.03) 0.14 (0.02) 0.59
NQ
(0.03)
0.25 (0.02) 0.17 (0.02) 0.95
NQ
(0.03)
0.50 (0.01) 0.20 (0.02) 1.89
NQ
(0.02)
0.89 (0.13) 1.93 (0.08) 2.72
NQ
(0.02)
1.16 (0.26) 1.84 (0.08) 2.20
(0.20)
(0.06)
(0.14)
(0.20)
(0.39)
(0.16)
(0.30)
Figure captions
Figure 1. Time-course of the populations of microorganisms in sourdoughs prepared with a)
grape (GS), b) apple (AS), and c) yogurt (YS). Count (CFU/g of sourdough) in P1, P2, P3, and
P4 stages of the pre-sourdough. Count (CFU/g of sourdough) in R1, R2, R3, R4, R5 stages of
ripe sourdough. Mean values and their corresponding standard errors.
Figure 2. Frequency of different yeast types in laboratory sourdough phases (P, pre-sourdough,
R, ripe-sourdough) prepared with different ingredients (apples, yogurt and grapes). The data are
based on cell and colony color and morphology on WL agar. Type 1 is non-Saccharomyces
yeast (grown on WL and Lysine agar): Type 1.1, group of white, creamy convex colonies and
Type 1.2, group of pale green, creamy flat colonies. Type 2 Saccharomyces yeast (only grew on
WL agar): Type 2.1 group of white, creamy colonies with a peaked center and Type 2.2 like 2.1
but green in color.
Figure 3. Physical and chemical characteristics of sourdoughs prepared with grape (GS), apple
(AS), and yogurt (YS). a) pH changes in the processing of sourdough. b) Total titratable acidity
(TTA) of sourdoughs; TTA was expressed as mL Na0H 0.1 mol/L used to titrate a 10 g sample
(blended with 90 ml) to pH 8.5. c) Lactic acid (g/100 g) in sourdoughs. Mean values and their
corresponding standard errors.
20
Figure 1.
GS (CFU/g)
a)
1,E+10
1,E+09
1,E+08
1,E+07
1,E+06
1,E+05
1,E+04
1,E+03
1,E+02
1,E+01
1,E+00
LAB
Tot Yeast
Non-Saccha
P1
AS (CFU/g)
b)
1,E+10
1,E+09
1,E+08
1,E+07
1,E+06
1,E+05
1,E+04
1,E+03
1,E+02
1,E+01
1,E+00
LAB
Tot Yeast
Non-Saccha
P1
YS (CFU/g)
c)
P2 P3 P4 R1 R2 R3 R4 R5
Sample (Days)
1,E+10
1,E+09
1,E+08
1,E+07
1,E+06
1,E+05
1,E+04
1,E+03
1,E+02
1,E+01
1,E+00
P2 P3 P4 R1 R2 R3 R4 R5
Sample (Days)
LAB
Tot Yeast
Non-Saccha
P1
P2 P3 P4 R1 R2 R3 R4 R5
Sample (Days)
21
Figure 2.
Type 1.1
Type 1.2
Type 2.1
Type 2.2
100
60
40
Grape
Apple
22
P1
P2
P3
P4
R1
R2
R3
R4
0
P1
P2
P3
P4
R1
R2
R3
R4
20
P1
P2
P3
P4
R1
R2
R3
R4
Frequency (%)
80
Yogurt
Figure 3
7
a)
GS
AS
6
pH
YS
5
4
3
P3
b)
P4
R1 R2 R3
Sample (Days)
R4
R5
10
Titratable acidity (mL)
8
6
4
GS
AS
2
YS
0
P3
1,5
c)
R1 R2 R3 R4
Sample (Days)
R5
R1 R2 R3 R4
Sample (Days)
R5
GS
AS
1,2
Latic acid (g/100g)
P4
YS
0,9
0,6
0,3
0
P3
P4
23