The molecular and technological characterization of lactic acid bacteria in einkorn sourdough: effect on bread quality

Abstract

This study aimed to investigate the characterization of lactic acid bacteria strains (LABs) in spontaneous sourdough produced from einkorn flour as a new sourdough source and to determine some quality characteristics of sourdough bread. Thirty-two different LABs were isolated from spontaneously fermented einkorn sourdough under laboratory conditions. Seven different lactic acid bacteria strain (LAB) species, Lb. crustorum (dominant species 10), Pediococcus (4), Lb. brevis (6), Lb. paraplantarum (1), Lb. plantarum (5), Lb. fermentum (4), and Lb. curvatus (2) were identified by PCR (16S). Lb. paraplantarum and P. acidilactici showed high antibacterial activity against B. subtilis ATCC6633, B. cereus ATCC11778, and E. coli ATCC25922, and Lb. crustorum MN047 and L. brevis R-1 showed an antifungal effect on P. carneum, A. flavus, and A. niger. At the same time, all strains showed an acid-tolerant effect, because they showed a survival ratio higher than 70% at pH 2.5. The highest phytase activity was observed from L. paraplantarum 2815 and L. plantarum AAS3. The study aimed to investigate Lb. brevis R-1 and Lb. paraplantarum 7285 starter culture effects and bread quality parameters on dough made solely with einkorn or with a mixture of 25%, 50%, or 75% einkorn flour, compared to a solely wheat flour sourdough. The sourdough bread containing 25% and 50% einkorn flour had better volume, specific volume, and textural properties than bread containing 75% and 100% einkorn flour; they were scored as good as 100% wheat dough bread in terms of sensory properties. This study recommends that einkorn sourdough could be utilized as a beneficial source, having a diverse range of LABs and strong technological properties.

Full text

Vol:.(1234567890)
Journal of Food Measurement and Characterization (2020) 14:1646–1655
https://doi.org/10.1007/s11694-020-00412-5
1 3
ORIGINAL PAPER
The molecular andtechnological characterization oflactic acid
bacteria ineinkorn sourdough: eect onbread quality
ElifÇakır1· MuhammetArıcı1· MuhammedZekiDurak1· SalihKarasu1
Received: 20 September 2019 / Accepted: 10 February 2020 / Published online: 19 February 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
This study aimed to investigate the characterization of lactic acid bacteria strains (LABs) in spontaneous sourdough pro-
duced from einkorn flour as a new sourdough source and to determine some quality characteristics of sourdough bread.
Thirty-two different LABs were isolated from spontaneously fermented einkorn sourdough under laboratory conditions.
Seven different lactic acid bacteria strain (LAB) species, Lb. crustorum (dominant species 10), Pediococcus (4), Lb. brevis
(6), Lb. paraplantarum (1), Lb. plantarum (5), Lb. fermentum (4), and Lb. curvatus (2) were identified by PCR (16S). Lb.
paraplantarum and P. acidilactici showed high antibacterial activity against B. subtilis ATCC6633, B. cereus ATCC11778,
and E. coli ATCC25922, and Lb. crustorum MN047 and L. brevis R-1 showed an antifungal effect on P. carneum, A. flavus,
and A. niger. At the same time, all strains showed an acid-tolerant effect, because they showed a survival ratio higher than
70% at pH 2.5. The highest phytase activity was observed from L. paraplantarum 2815 and L. plantarum AAS3. The study
aimed to investigate Lb. brevis R-1 and Lb. paraplantarum 7285 starter culture effects and bread quality parameters on dough
made solely with einkorn or with a mixture of 25%, 50%, or 75% einkorn flour, compared to a solely wheat flour sourdough.
The sourdough bread containing 25% and 50% einkorn flour had better volume, specific volume, and textural properties than
bread containing 75% and 100% einkorn flour; they were scored as good as 100% wheat dough bread in terms of sensory
properties. This study recommends that einkorn sourdough could be utilized as a beneficial source, having a diverse range
of LABs and strong technological properties.
Keywords Lactic acid bacteria (LAB)· PCR· Sourdough· Einkorn bread
Introduction
Due to an increase in consumer demand for high quality,
healthy food, the importance of cereals with good nutrition
and sensory properties has also increased [1]. The trend
to use sourdoughs in bread production has increased due
to the high sensorial and nutritional quality and shelf-life
properties of the sourdough bread. The use of wheat or rye
flour in producing sourdough has a long tradition in bread
technology, but in recent years, the interest in other flours in
sourdough fermentation has increased. Other flours are used
in sourdough preparation mainly due to improved functional
properties and nutritional values. Another reason for using
other flours instead of wheat flour in sourdough production
is special diets, for example those that require gluten-free
bread [2].
Einkorn (Triticum monococcum L.) promises growth
thanks to its specific quality and high nutritional value.
Einkorn is a diploid species of wheat grown in the Mediter-
ranean and in small areas of Europe [3]. Compared with
other types of wheat, einkorn wheat contains more protein,
lipids (unsaturated fatty acids), phosphorus, potassium,
pyridoxine, lutein, and beta carotene [4]. Einkorn also has a
lower content of beta-amylase and lipoxygenase to prevent
the decay of compounds such as carotenoids, tocopherols,
conjugated phenolics, and phytosterols and to protect the
nutritive properties of food.
Sourdough can be produced both spontaneously and
commercially. It has been reported that sourdough having
single- or multiple-stage fermentation processes improves
the texture, volume, and shelf life of bread [5–7]. Spontane-
ous sourdough, depending on the microflora of the flour,
* Muhammet Arıcı
muarici@yildiz.edu.tr
1 Department ofFood Engineering, Chemical
andMetallurgical Faculty, Yıldız Technical University,
34210Esenler, Istanbul, Turkey

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The molecular andtechnological characterization oflactic acid bacteria ineinkorn sourdough:…
1 3
is started by the fermentation of the flour and water mix-
ture. LABs and yeasts become the dominant species in the
sourdough microbiota after it is fed a few times throughout
the week [8, 9]. Commercial sourdough is produced using
starter cultures that consist of pure LABs and yeasts [7].
The origin of the flour, the storage conditions, the hygienic
conditions, and the technological factors of the fermentation
process affect the microflora of the spontaneous sourdough
[7, 10].
While there are many studies on the nutritional value of
the einkorn flour products [11–13], this is the first study
to determine lab species in fermented Turkish einkorn
sourdough. The primary objective of this research was to
characterize LABs in spontaneous sourdough produced
from einkorn flour and to explore the diversity of the LABs.
Another aim of this study was to explore the technological
characteristics of LABs and to investigate the potential of
making sourdough bread formulated with 25%, 50%, 75%,
and 100% einkorn flours by preparing a starter culture from
bacteria with the best properties.
Materials andmethods
Materials
Einkorn flour was provided by the Istanbul Halk Ekmek
(İstanbul, Turkey). All chemicals used in this study were
obtained from Merck (Darmstadt, Germany).
Preparation ofspontaneous sourdough
Spontaneous sourdough was formed by fermenting a flour
and water mixture without the addition of an external starter
culture. Einkorn wheat flour and water were mixed by a
dough blender (KitchenAid, Antwerp, Belgium) at a ratio
of 1:1 to form sourdough. The dough was kneaded for 4min
for homogenization and was left to ferment at 28°C for
24h. Every 24h, 100g of flour and 100mm of water were
added to the dough; the fermentation process was continued
for 4days.
Isolation andidentication ofLABs fromsourdough
samples
LABs were isolated and counted using ModMRS, MRS,
Rose Agar, and MRS-5 supplemented with cycloheximide.
For each sourdough batch, serial tenfold dilutions were com-
monly performed using peptone solutions for sourdough
samples. They were plated to the corresponding agar using
the spread-plate procedure. After the plating, different colo-
nies were selected from the plates containing the highest
sample dilutions (10−6). Stock cultures were prepared from
selected colonies and were stored at − 80°C with glycerol,
as described by [14]. FTIR and sequence identifications of
the isolates were also performed.
Identication ofLABs byFTIR
LABs were grown on APT agar under anaerobic conditions
at 34°C for 48h. The suspension prepared by a growing
colony and 100 μL sterile distilled water was transferred
to an Eppendorf tube. Of the resulting solution, 25 μL was
transferred to the slices on the ZnSe FTIR plate and dried at
40°C for 45min. Measurements were conducted using an
HTS-XT FTIR spectrometer in the region between 400 and
4000cm−1 (Bruker, Karlsruhe, Germany). For data process-
ing, OPUS software v.6 (Bruker) was used [15].
DNA extraction
For total DNA isolation, the colonies were transferred to
sterile Eppendorf tubes (1.5mL) from the isolates that were
purified in the appropriate agar in the previous step. After
centrifugation at 13,000×g for 10min at 4°C, pellets were
frozen at − 80°C for 15min and were resuspended in 95ml
of 1X PCR buffer [16].
Bacterial identication by16S rDNA gene sequencing
PCR was performed on LABs selected from different
groups according to comparison results from FTIR. Iden-
tification was performed with 16S rDNA gene sequencing.
Genes of isolates were amplified using three different prim-
ers, PEU7-DG74, p806-p8FPL, and 27F-1492R. A PCR
mixture within the 25 μL final reaction volume contained
10 × PCR buffer, 2.5mM of dNTP mixture, 0.5μM each
of forward and reverse primers (PEU7 and DG74), and 5
U of Taq DNA polymerase (Sigma, USA) [17]. PCR was
performed using the following cycling conditions: 95°C for
5min (initial denaturation), 30 cycles at 94°C for 15s/52°C
for 30s/72°C for 2min, and a final extension at 72°C for
5min; p806-p8FPL cycling conditions were 94°C for 5min,
35 cycles at 94°C for 1min/75s at 56°C/75s at 72°C, and
a final extension for 10min at 72°C. For the 27F-1492R
primer pairs, DNA was denatured at 94°C for 2min and
was amplified for 35 cycles at 94°C for 40s, at 45°C for
50s, and at 72°C for 50s, with a final extension applied for
2min at 72°C [18].
The images of gels displaying PCR products were as
taken after 1.5h of electrophoresis at 100V on 1.2% gel.
Obtained sequences were searched in the Basic Local Align-
ment Search Tool (BLASTn) database, and the matching
microorganism sequence records with their respective simi-
larity percentages were determined [19].

1648 E.Çakır et al.
1 3
pH andTTA analysis
The pH value of the sourdough samples was measured by
a pH meter (HANNA Instruments, Italy). Titratable acid
analysis (TTA) was conducted according to the method
described by [20].
Technological capacity characterization ofLABs
Bile, pH, potassium sorbate, andNaCl tolerance
LABs were activated in the broth using the method described
by [21], and their optical density (OD) on a 600nm spec-
trophotometer was set to the value of 1. After their OD was
set, the LAB bacteria were inoculated in an MRS broth com-
posed of a pH value of 2.5 and 0.3% bile.
For K-sorbate and NaCl, LABs were inoculated to an
MRS broth with 6% NaCl and on an MRS broth containing
100ppm potassium sorbate; they were incubated at 37°C
for 24h. The agar spot method was employed for plating
on MRS agar. OD values were compared with those of the
control group; the culture was counted after 24h to calculate
the surviving percentage as follows:
where, A: Bile salt/pH broth bacteria count after 24h and
B: Initial bacteria count
where, A: Potassium sorbate/NaCl broth bacteria count after
24h and B: Initial bacteria count.
Antimicrobial activity
The antimicrobial activity of the LABs was determined
by the diffusion agar method. Pathogens were activated
in the broth for 24h at 37 °C. Pathogenic bacteria (B.
subtilis ATCC6633, B. cereus ATCC11778, and E. coli
ATCC25922, which were approximately 105–107cfu/mL)
were spread on nutrient agar. The LABs were activated at
37°C and were inoculated in the amount of 1% into 10mL
of the MRS broth. After being centrifuged at 14,000×g for
5min, 20 μL of the resulting supernatant was diffused into
nutrient agar wells with a 2mm diameter. Inhibition zones
were measured after 24h incubation at 37°C. Diameters
were measured in millimeters.
Antifungal activity
The antifungal activities of the sourdough isolates were
determined against Penicillium carneum, Aspergillus
Bile salt∕pH −tolerant bacteria count (%)=A∕B×100
Potassium sorbate∕NaCl −tolerant bacteria count (%)=A∕B×100
flavus, and Aspergillus niger. Molds were grown in potato
dextrose agar for 7days at 30°C. LABs were activated,
scratched on the MRS agar, and grown for 24h on MRS
broth. The spot was diffused on 10 μL of MRS agar and
incubated for 24h at 34°C. Then 10mL of malt extract
soft agar was overlaid and incubated aerobically for 48h at
30°C. The forming inhibition zones were examined [22].
Phytase activity
The ability of LABs to produce phytase was determined in
the medium containing calcium phytate or sodium phytate.
Pikovskaya’s agar was prepared, and the LABs were inoc-
ulated via the drop method and were then incubated at
30°C for 7days. Subspecies around which transparent
zones were observed were classified as phytase-positive.
Subspecies around which no transparent zones observed
were classified as phytase-negative. Zone diameters were
measured in millimeters [23].
Preparation ofsourdough
LAB and yeast isolates showing the best technological
characteristics were selected to use in sourdough fermenta-
tion. Accordingly, the strains stocked at − 80°C were first
activated for 24h on a solid medium (MRS agar), followed
by the incubation of lactic acid bacteria in a 10ml MRS
broth medium for 24h. Subsequently, the cultures were
transferred to 50mL of liquid media for a second time and
were again incubated for 24h, and the cell pellets were
washed by centrifugation at 4°C. After incubation pel-
lets were obtained by centrifuging at 6000×g for 15min,
microbial cell pellets were used in preparations of einkorn
and wheat dough.
Bread production
Einkorn flour was used to prepare blends with wheat flour
in wheat flour/einkorn flour ratios of 0/100, 25/75, 50/50,
75/25, and 100/0. The water absorption of these flours was
determined primarily on the Brabender farinograph. The
farinograph values of these flours were 61%, 58.3%, 56%,
55%, and 52.5%, respectively. The sourdough samples were
added to comprise 20% of the dough weight and salt (1.5%
of dry matter). After 20min at room temperature, the dough
was fermented for 3h at 30°C and at 85% relative humid-
ity in a fermentation cabinet (Nuve TK252, Turkey). The
dough was baked in an electric oven (Fimak, 161 Turkey) for
40min at a 190°C top temperature. The bread was cooled
at room temperature for 2h before analysis.

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The molecular andtechnological characterization oflactic acid bacteria ineinkorn sourdough:…
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Sourdough analysis, bread dough andbread analyses
After the produced pieces of bread reached room tempera-
ture, weight (g) measurements were made, and volume
(mL) values were determined based on displacement with
millet seeds. Specific volume (mL/g) values were found by
proportioning the obtained volume values to the weight.
To determine the textural properties of the bread sam-
ples, each sample was sliced to 1.25mm thickness; it was
equipped with a 5kg load cell and a 36mm diameter
cylindrical compression probe and was subjected to a
compression test using a texture analyzer (SMS TA.XT2
Plus, UK). Hardness properties were determined. Also, the
crumb and breadcrust color, Hunter L, a and b color values
were determined by a color determination device (CR-100
Konica Minolta, Japan) [24].
Sensory analysis ofbread samples
In the sensory analysis, crust and breadcrumb color, pore
structure, taste, smell, chewability, and general acceptance
were evaluated by 12 trained panelists, placing the samples
in a score range from 1 (very bad) to 7 (very excellent).
Statistical analyses
One-way analysis of variance (ANOVA) was performed
using JMP version 9 to determine important differences
between the significance with reference to the meas-
ured properties. Significant differences were declared at
p < 0.05. The mean and standard deviation of three repli-
cates were also calculated.
Results anddiscussions
pH, acidity andmicrobiological analysis
The pH and total titratable acidity values of the einkorn
sourdough were obtained during 4days of fermentation.
The TTA and pH of einkorn sourdoughs during the fer-
mentation period were significantly changed (p < 0.05).
The pH decreased from 6.18 to 3.81 for 4days, and the
TTA increased from 0.16 to 1.87%. In this study, the maxi-
mum values of LAB count varied according to agar types.
LABs isolated from ModMRS, MRS-5, MRS, and Rose
agars ranged from 8.56 to 9.61 log (cfu)/g. Max and min
values of LAB count were obtained from ModMRS and
MRS-5, respectively.
Identication oflactic acid bacteria
FTIR mathematically enables the identification of micro-
organisms by computer data processing, with the analysis
and comparison of the whole spectra [25]. In total, 75 lactic
acid bacteria strains isolated from einkorn sourdough were
selected on MRS, ModMRS, Rose, and MRS-5 agars, and 42
isolates were first subjected to FTIR analysis and clustered
to select genotypic identification. Thus, in our study, accord-
ing to the W5-W4-W3 spectral regions, the first derivative
method was applied to the spectra of the LAB analysis of the
cluster analysis (Fig.1a). According to the cluster analysis,
32 isolates were selected and were then identified genotypi-
cally. LABs isolated from einkorn sourdough were identi-
fied via 16S rDNA sequencing; the sequencing results are
shown in Table1. The sequence alignments were used to
build a phylogenetic tree, using the neighbor-joining method
(Fig.1b). Six main groups were clustered as Pediococcus,
Lb. brevis, Lb. curvatus, Lb. fermentum, Lb. crustorum, and
Lb. plantarum. The dominant species was Lb. crustorum.
Bile andpH tolerance
A concentration of 0.3%, close to human bile concentra-
tion, is accepted as a critical value [26]. Thirteen subspe-
cies of LABs isolated from einkorn survival percentages
are illustrated (Table2). All subspecies exhibited high tol-
erance to bile salts, with a survival ratio of over 85%. Lb.
brevis qp109, Lb. fermentum 1949, P. acidilactici A-4, Lb.
crustorum MN047, Lb. crustorum B-481, Lb. brevis R-1, Lb.
plantarum FM02, Lb. plantarum 1838, and Lb. plantarum
GM1403 showed the highest tolerance, with a survival rate
of over 90%.
Another critical property of probiotic microorganisms
was the acid tolerance of LAB subspecies. These subspecies
survived the acidic environment of the stomach and reached
the intestinal system and formed colonies to compete with
the pathogenic bacteria [27].
Thirteen subspecies tested were incubated for 24h at 2.5
pH, and their survival rates were calculated (Table2). The
highest survival rates were observed in Lb. paraplantarum
72,815, Lb. fermentum 1949, Pediococcus acidilactici A-4,
Lb. crustorum MN047, Lb. crustorum B-481, Lb. brevis R-1,
and the Lb. plantarum GM1403 subspecies, with a survival
rate of over 90%.
In the study, the three strains of Lb. plantarum SW03,
Lb. plantarum SW07, and P. acidilactici SW05 showed the
highest survival capacity when they were exposed to gastric
acid, with a pH of 2–2, 5–3 [28]. In the study of [29], kefir
isolates were deemed to be acid tolerant, as they survived
at a rate of 70%. By using this ratio as a reference, the LAB
subspecies isolated from einkorn could also be deemed to
be acid tolerant.

1650 E.Çakır et al.
1 3
Potassium sorbate andNaCl
All isolates were observed to be tolerant to 6% NaCl con-
centration. As shown in Table2, isolates exhibited a survival
ratio of over 85%. Lb. brevis qp109, Lb. fermentum 1949,
Pediococcus acidilactici A-4, Lb. paraplantarum, Lb. crus-
torum MN047, Lb. crustorum B-481, Lb. plantarum AAS3,
Lb. plantarum FM02, Lb. plantarum 1403, and Lb. brevis
R-1 demonstrated a survival rate over 90%.
P. acidilactici A-4 had the lowest survival rate for
K-sorbat, while the other subspecies exhibited a survival
ratio of over 85%. Survival rates for K-sorbat over 90% were
observed for Lb. brevis qp109, L. paraplantarum, L. fer-
mentum 1949, L. crustorum MN047, L. plantarum AAS3,
L. crustorum B-481, L. plantarum FM02, and L. fermentum
1301.
Potassium sorbate and salt, which are used as pre-
servatives in fermented products, are important for bread
and fermented cereal products as well. Salt, used both as
a preservative and for taste, as well as potassium sorbate
have great importance for bread and cereal products. These
chemical additives used in fermented products to prevent
the growth of pathogenic microorganisms may also inhibit
the growth of starter culture; thus, species tolerant of such
preservatives are preferred [30]. For this reason, the percent
vitality of LABs isolated from einkorn wheat was measured
(Table2). In their study, [31] reported that no meaningful
difference was observed between isolates at 15°C in 2%,
4%, or % NaCl, and a significant decrease in alive cell count
was observed for isolate Lb. lactic subsp. lactis UTNFa38 in
6% NaCl. In this study, the maximum ratio of salt used was
determined as 6%, due to the observation of the LAB-killing
effect of 6% NaCl.
Antimicrobial activity
There has been great interest in potential pathogen-antago-
nizing LABs in food processing applications, as LABs were
accepted as food-grade ingredients [32, 33]. In this study, the
antimicrobial activity of isolated LABs against pathogenic
1122
333333 444555
66 777777777888
9999
10
111111 12
131313131313 141414
15 16
0
0.02
0.04
0.06
0.08
0.1
0.12
Heterogeneity
2AB .0
SB3 _H7. 0
SB26 _A7. 0
SB7 _D3. 0
SB73 _G3.0
SB18 _B11. 2
SB51_A1 -. 1
18AB _E9. 1
SB34 _E9. 1
SB63 _A9. 0
SB24 _A11. 0
SB71 _B1. 1
SB17 _D3. 1
SB22 _H9. 0
SB20 _D11.0
SB64 _A5. 0
SB68 _B5. 1
5AB _A5.0
SB10 _C7. 1
SB12 _C7 - .1
SB11 _B9. 1
SB23 _B9. 1
SB4 _F5.1
SB44 _F12.0
SB5 H5.1
SB13 H5.1
SB15 _G7.0
SB53 _F3.0
17AB _G1.0
SB16 _C12.0
SB62 _E11. 0
SB19 _E7. 1
SB67 _E7. 1
SB25 _E5. 1
SB14 _H11.0
SB74 _D1. 1
SB76 _C9. 0
SB21 _A9. 0
SB75 _D3. 1
SB6 _E11.0
SB66 _F9.0
SB38 _B1. 0
Data preprocessing: First derivative
Ward's algorithm Frequency Ranges =
Standard (Euclidean distance) 1500 - 698 /cm
Method File = TEMPVIEW.cla
Date: 16/07/2019
a
Fig. 1 a The FTIR fingerprinting of 42 LAB strains. Using Ward’s
algorithm method to form a hierarchical clustering of LAB strains.
Ward; Spectral windows: W4 (1200–900cm−1), W5 (900–700cm−1),
W3 (1500–1200cm−1). b Neighbor-joining relationship of 16S rDNA
sequence of einkorn sourdough LAB isolates

1651
The molecular andtechnological characterization oflactic acid bacteria ineinkorn sourdough:…
1 3
bacteria B. subtilis ATCC6633, B. cereus ATCC11778, and
E. coli ATCC25922 was observed (Table3).
Since B. subtilis causes rope spoilage in bread, antibacte-
rial activity against B. subtilis is very important to increase
the shelf life of bread. All LAB subspecies isolated from
einkorn sourdough showed an antibacterial effect on B.
subtilis. Lb. paraplantarum 72,815, and P. acidilactici A-4,
while Lb. crustorum MN047 exhibited a strong resistance
against B. subtilis, B. cereus, and E. coli. All subspecies
were observed to exhibit antimicrobial activity against B.
subtilis ATCC6633. Eight LAB subspecies showed antimi-
crobial activity against E. coli ATCC 25,922. The study of
[34] isolated LAB B. cereus from baby feces, and the isolate
displayed a high inhibition against Lb. rhamnosus and Lb.
curvatus; in the same study, L. curvatus was found to be
high.
Fungal spoilage is one of the basic problems for bread and
other bakery products, causing economic losses and health
problems [35]. In this study, the antifungal activity of LAB
subspecies against Penicillium carneum, Aspergillus flavus,
and Aspergillus niger was examined. As shown in Table3,
Lb. paraplantarum 72,815, Lb. crustorum MN047, Lb.
crustorum B-481, and Lb. brevis R-1 displayed antifungal
activity against Penicillium carneum, Aspergillus flavus, and
Aspergillus niger molds. Lb. brevis Qp109, Lb. curvatus LII,
Lb. fermentum 1949, Lb. fermentum1301, and Lb. plantarum
GM1403 showed no antifungal activity.
Phytase activity
The phytase activity of the LAB isolates is shown in Table3.
Some LAB isolates showed phytase activity, while others
did not, with phytase activity significantly changing accord-
ing to the LAB isolates (p < 0.05). The highest phytase activ-
ity was measured in Lb. paraplantarum 72,815 and Lb.
plantarum AAS3 (14mm), and the lowest was observed
in Lb. brevis qp109 and Lb. plantarum GM1403 (6mm)
(Table3). Previous studies have shown that most minerals
form a complex bond with phytic acid to produce phytates,
Fig. 1 (continued)
Table 1 Genotypic identification by 16S rDNA sequence analysis of LAB isolated during einkorn sourdoughs fermentation
Isolate number LAB Identity (%) Isolate number LAB Identity (%)
1 SB3 Lb. brevis 0138 99 17 SB21 Lb.plantarumGM1403 99
2 SB4 Lb. plantarum AAS3 99 18 SB22 Lb. paraplantarum 72,815 99
3 SB5 Lb. curvatus LII 99 19 SB23 Lb. crustorum B-481 99
4 SB6 Lb. plantarum FM02 99 20 SB24 Lb. crustorum B-481 99
5 SB7 Lb. crustorum MN047 99 21 SB25 Lb. fermentum IMDO 130,101 99
6 SB10 Lb. crustorum B-481 99 22 SB26 Lb. crustorum B-481 99
7 SB11 Lb. brevis 0138 99 23 SB34 Lb. brevis 0138 99
8 SB12 Lb. crustorum B-481 99 24 SB53 Lb. plantarum 1838 99
9 SB13 Lb. LII curvatus 99 25 SB63 Lb. brevis qp109 100
10 SB14 Lb. crustorum B-481 99 26 SB67 Pediococcus acidilactici A-4 100
11 SB15 Lb. crustorum B-481 99 27 SB66 Pediococcus acidilactici A-4 100
12 SB16 Lb. crustorum B-481 99 28 SB68 Lb. brevis R-1 99
13 SB17 Lb. plantarum AAS3 99 29 2AB Pediococcus acidilactici A-4 100
14 SB18 Lb. crustorum MN047 99 30 5AB Lb. fermentum NRIC1949 100
15 SB19 Lb. fermentum NRIC 1949 100 31 17AB Lb. crustorum B-481 99
16 SB20 Pediococcus pentosus 0123 99 32 18AB Lb. fermentum NRIC 1949 100

1652 E.Çakır et al.
1 3
Table 2 The viable counts of 13 strains in MRS supplemented with 6% NaCl, 0.3% bile, 100mg/kg potassium sorbate (pH 2.5)
Viable cell counts of 13 strains isolated from einkorn sourdough after incubation for 24h in MRS broth at 6% NaCl, 0.3% bile, and 100mg/kg
potassium sorbate (pH 2.5). But, the control was only incubated in MRS broth for 24h. Values for each pH at 24h not sharing the same letters
are significantly different from each other (p < 0.05)
LAB strains Log CFU/
mL
Log CFU/
mL
(%6 NaCl) Log CFU/
mL
%0.3 Bile Log CFU/
mL
100mg/kg
K-sorbate
Log CFU/
mL
pH 2.5
Control 6% NaCl Survival (%) 0.3% Bile Survival (%) 100mg/kg
p. sorbate
Survival (%) pH 2.5 Survival (%)
L. brevis
qp109
9.14 ± 0.3d8.26 ± 0.2d90d8.35 ± 0.2c91bc 8.56 ± 0.3b94a7.38 ± 0.7ef 81f
L. curvatus
TW
9.26 ± 0.2c8.25 ± 0.5d89d7.86 ± 0.3h85f8.20 ± 0.3e89e7.86 ± 0.3cd 85e
L. praplan-
tarum 9.03 ± 0.3e8.11 ± 0.5e90d8.07 ± 0.3ef 89cd 8.6 ± 0.4g89e8.40 ± 0.2b93b
L. fermen-
tum 1949
9.06 ± 0.2e8.39 ± 0.2bc 93c8.36 ± 0.3c92b8.35 ± 0.6d92b7.84 ± 0.3cd 91d
P. acidilactic
A-4
8.62 ± 0.2g8.11 ± 0.5e95b8.12 ± 0.8de 94a7.21 ± 0.5j84g 7.90 ± 0.2cd 95a
L. crustorum
MN047
8.28 ± 0.5ı7.88 ± 0.5f95ab 7.90 ± 0.6h 95a7.58 ± 0.2ı92bc 7.49 ± 0.3e91d
L. plantarum
AAS3
9.26 ± 0.5c8.34 ± 0.2cd 90d8.05 ± 0.5efg 87e8.76 ± 0.5a95a7.22 ± 0.2g 78g
L. crustorum
B-481
8.38 ± 0.5h8.06 ± 0.2e96a7.91 ± 0.3gh 94a7.52 ± 0.3ı90d7.76 ± 0.2d93bc
L. brevis R-1 8.93 ± 0.2f8.44 ± 0.3bc 95b7.94 ± 0.5fgh 90d7.90 ± 0.2h 88e8.53 ± 0.2ab 92bcd
L. fermen-
tum 1301
9.20 ± 0.3cd 7.87 ± 0.6f86e8.21 ± 0.5d89cd 8.47 ± 0.3c92b7.81 ± 0.5cd 85e
L. plantarum
FM02
9.26 ± 0.2c8.45 ± 0.6b91c8.74 ± 0.2a94a8.40 ± 0.5d91cd 7.25 ± 0.5fg 78g
L. plantarum
1838
9.47 ± 0.3a8.11 ± 0.5e86e8.49 ± 0.5b90cd 8.19 ± 0.2ef 86f7.93 ± 0.1c84e
L. plantarum
GM1403
9.39 ± 0.2b8.81 ± 0.2a94b8.81 ± 0.2a94a8.14 ± 0.3f87f7.54 ± 0.2a91cd
Table 3 Antimicrobial and phytase activity of 13 identified species
The different lower case letters in the same column show statistical significance (p < 0.05)
Diameter of antibacterial inhibition zones (mm): + +˃ 10mm; + < 9mm; antifungal positive ( +), not determined ( −), Phytase (-) no activity
LAB strain B. subtilis B. cereus E. Coli Aspergillus Penicillium Aspergillus Phytase (mm)
ATCC 6633 ATCC 11,778 ATCC25922 niger carneum flavus
Lb. brevis Qp109 ++ − − − − − 6 ± 0.1e
Lb.curvatus LII + + − − − − −
Lb. paraplantarum 72,815 ++ ++ ++ + + + 14 ± 0.1a
Lb. fermentum 1949 ++ + + − − − 9 ± 0.1d
P. acidilactic A-4 ++ ++ ++ − + + 10 ± 0.1c
Lb. crustorum MN047 ++ + + + + + −
Lb. plantarum AAS3 ++ ++ + + − − 14 ± 0.0a
Lb. crustorum B-481 ++ − − + + + 12 ± 0.1b
Lb. brevis R-1 ++ ++ + + + + −
Lb. fermentum 1301 ++ + − − + − −
Lb. plantarum FM02 ++ − − − + − −
Lb. plantarum 1838 ++ ++ − − + − −
Lb. plantarum GM1403 ++ ++ − − − − 6 ± 0.0e

1653
The molecular andtechnological characterization oflactic acid bacteria ineinkorn sourdough:…
1 3
and the bioavailability of these minerals is reduced [36,
37]. Reduction of phytates in bread production is achieved
via enzymatic degradation and endogen phytase activity
increase or LABs, yeasts, and other microorganisms [38].
This study concluded that the phytase activity of isolates
should be investigated to increase the bioavailability of the
minerals in sourdough bread.
Properties ofsourdough
To determine the changes in the doughs due to the rise of
acidity, pH values and microbial load were measured after
kneading and after the fermentation process. For sourdough
BS1 (wheat sourdough), the measured pH decreased from
5.59 to 3.47, corresponding to a rise in acidity from 0.21%
to 1.21%, while for the sourdough SS1 (einkorn sourdough),
the pH values were 5.6 and 3.35, and the corresponding
acidity was 0.26% and 1.43%, respectively. All samples were
found to be statistically different (p < 0.05). For sourdough
made of 100% einkorn flour, SS1, the post-fermentation pH
was measured to be lower than that in the sourdough made
of 100% wheat flour, BS1; however, the TTA was higher.
At the beginning of fermentation, all sourdough showed
LABs in the range of 7.90 ± 0.7 log cfu/g BS1 and 7.88 ± 0.3
log cfu/g SS1; after fermentation, the LAB count was found
to be a range of 8.51 ± 0.4 log cfu/g BS1 to 9.60 ± 0.6
log cfu/g. The beginning of the fermentation yeast count
was found to be in the range of 6.0 ± 0.6 log cfu/g to
6.02 ± 0.9 log cfu/g; after fermentation, the yeast number
was 6.65 ± 0.7 log cfu/g to 6.55 ± 0.4 log cfu/g (p < 0,05).
Einkorn sourdough was found to contain more LABs than
wheat sourdough, while the yeast contents were found to
be statistically close. Lactic acid bacteria in the dough fer-
mented glucose to produce lactic acid, which caused a reduc-
tion in dough pH.
Properties ofbread
The weight, specific bread volume, hardness, and color
values of the sourdough bread samples were as shown in
Table4. The bread volume varied to between 295 ± 7.0
cm3 and 172 ± 3.5 cm3; the specific volume ranged from
2.16 ± 0.1 cm3/g to 1.37 ± 0.1 cm3/g; and the hardness was
in the range of between 3.79 ± 0.4N and 1569 ± 0.3N.
The volume and specific volume decreased as the ratio of
einkorn flour increased. The volume of SYE4 bread (made
from 100% einkorn flour) was found to be statistically simi-
lar to the volume of SYE3 bread (containing 75% einkorn
flour) (p > 0,001), while the specific volume values were
found to be lowest in the SYE4 bread samples. The high-
est specific volume was found in the control bread SKE1
Table 4 Volume, hardness and color properties of the bread samples
Significant differences in volume specific volume hardness and color values of einkorn bread containing different amounts of wheat flour are shown in different letters (p < 0.05)
Bread code Flour type Volume (cm3)S.Volume (cm3/g) Hardness (N) Crust clour Crumb clour
L* a* b* L* a* b*
SKE1 %100 Wheat (control) 288 ± 1.25a2.13 ± 0.03a3.79 ± 0.40e68.34 ± 0.91a6.01 ± 0.52d30.97 ± 0.82a70.50 ± 0.20a0.29 ± 0.11e18.01 ± 0.50b
SYE1 %25 Einkorn %75 wheat 249 ± 4.00b1.86 ± 0.01b6.53 ± 0.01d64.82 ± 0.70b8.32 ± 0.40c27.84 ± 0.50b62.66 ± 0.10b2.45 ± 0.00d17.89 ± 0.50b
SYE2 %50 Einkorn %50 Wheat 234 ± 1.75c1.73 ± 0.02c9.70 ± 0.10c60.34 ± 0.30c8.59 ± 0.44c26.31 ± 0.30c61.00 ± 0.00c3.98 ± 0.30c20.13 ± 0.23a
SYE3 %75 Einkorn %25 Wheat 214 ± 4.53d1.55 ± 0.04d12.53 ± 0.43b58.73 ± 0.11d9.91 ± 0.20b25.59 ± 0.77cd 58.65 ± 0.20d5.29 ± 0.25b20.35 ± 0.32a
SYE4 %100 Einkorn 206 ± 1.51d1.47 ± 4.95e15.69 ± 0.30a53.88 ± 0.12e11.82 ± 0.14a25.43 ± 0.50cd 55.02 ± 0.03e6.41 ± 0.20a20.7 ± 0.41a

1654 E.Çakır et al.
1 3
(containing 100% wheat flour). The specific volume of SYE1
bread was closest to that of the control bread. The volume
of the SYE2 bread was lower than the volume of the control
bread and 19% higher than that of the SYE3 bread.
The use of the starter culture consisting of SS1 sour-
dough increased the volume of the SYE4 bread, made of
100% einkorn flour. Texture analysis (Table4) showed hard-
ness value of the breads decreased. The least hardness was
observed for the bread SYE1, which contained 25% einkorn
flour. The effect of the gluten network formed by adding
wheat flour to the einkorn flour and starter culture was to
improve the volume and textural properties of the bread.
The brightness of the crust and crumb color was observed
to be highest for ordinary wheat flour, and bread with SYE1
added was observed to have a similar brightness. As redness
increased on the crusts and in the crumbs of SYE2, SYE3,
and SYE4 bread, due to the ratio of einkorn flour, yellow-
ness was reduced on the crust and increased in the crumb.
Einkorn flour contains more lutein than wheat flour [39],
which causes einkorn bread to be yellower. Wheat bread was
brighter, while einkorn bread was observed to be yellower
and redder.
The sensory properties of the bread samples were as
shown in Fig.2. Bread samples produced from SYE1 and
SYE2 were close to the reference (control) bread in terms
of sensory score (p > 0.05). Regarding taste, sourdough
bread with 75% (SYE3) and 100% (SYE4) einkorn flour
content showed lower scores compared to SYE1 and SYE2.
Although in their study, [40] reported to have observed low
volume, thick walls in the crumb, and irregular texture for
bread made of 100% einkorn wheat, in this study, sourdough
bread made of 100% einkorn flour and fermented with the
starter was observed to have no thick wall structure either on
the crust or in the crumb. When evaluated by the panelists,
the texture was deemed statistically close to sourdough
bread with 75% and 100% einkorn flour, and it was scored
as acceptable.
Conclusion
The biodiversity of lactic acid bacteria in einkorn sourdough
was determined. Thirteen different LAB strains isolated
from einkorn sourdough were identified. The dominant
species was Lactobacillus crustorum. LABs play an impor-
tant role in sourdough fermentation and also have probiotic
potential, thanks to their functional and technological prop-
erties. Lb. brevis R-1 and Lb. paraplantarum 7285 showed
the highest technological activity and showed the possibil-
ity of being used as starter cultures in the preparation of
einkorn bread. The Lb. brevis R-1 and Lb. paraplantarum
7285 combinations of starter culture and the addition of
wheat flour with added proportions to the einkorn improved
the sourdough structure and bread quality of the einkorn
bread. Einkorn sourdough allowed the isolation of promis-
ing microorganisms. Our findings suggested that LABs from
einkorn dough could potentially be considered for use in the
bakery industry.
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