Food Bioscience 38 (2020) 100791

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Thermophilic lactic acid bacteria affect the characteristics of sourdough

and whole-grain wheat bread
Dalia Cizeikiene a, *, Jolita Jagelaviciute a, Mantas Stankevicius b, Audrius Maruska b
a
Department of Food Science and Technology, Kaunas University of Technology, Kaunas, Lithuania
b
Vytautas Magnus University, Department of Biology / Environmental Research Centre, Kaunas, Lithuania

A R T I C L E I N F O A B S T R A C T

Keywords: Application of selected starter cultures for sourdough propagation may ensure stable lactic acid bacteria (LAB)
Lactobacillus diversity and defined properties of sourdough that affect quality. The impact of Lactobacillus delbrueckii ssp.
Volatile aroma compounds bulgaricus MI, L. rossiae GL14 and L. acidophilus DSM 20079 as starter cultures were investigated for spoilage
Whole-grain wheat sourdough
prevention and whole-grain wheat and sourdough bread properties. Whole-grain wheat sourdough propagated
Triticum aestivum L
with these strains showed high phytase, amylase and xylanases activities. The highest phytase activity was
obtained in sourdough prepared with L. bulgaricus MI. The results of gas chromatography–mass spectrometry
using solid-phase microextraction showed that the compositions of volatile compounds in sourdough and whole-
grain wheat bread depended on the LAB starter. In bread with L. acidophilus, 3-octen-2-ol and n-hexadecane were
found, whereas those compounds were not found in other bread samples or in sourdough. Tetrahydrofurfuryl
acetate was found only in bread prepared with L. bulgaricus MI sourdough. N-Pentadecane was found only in
bread prepared with L. rossiae sourdough and bread prepared with spontaneous sourdough. The application of
thermophilic sourdough increased bread porosity, elasticity, crumbliness and moisture content, but did not in­
fluence crumb hardness. Moreover, the fungal spoilage on the bread crust surface was suppressed using sour­
dough prepared with thermophilic LAB. The strain of LAB used for sourdough preparation influenced the shelf-
life of bread.

1. Introduction
Sourdough is an ingredient in the production of bread. It is a complex
biological system based on lactic acid and alcoholic fermentation that
depends on the microflora and fermentation conditions (Hansen, 2012).
Many bakeries use sourdough as the leavening agent at an industrial
scale to improve bread quality (Hansen, 2012). Ensuring sourdough
quality is difficult with long propagation times because it strongly de­
pends on the microflora present in the flour and the temperature in
bakeries, which may change during different seasons. Therefore, sour­
dough preparation using selected cultures and heat-treated flour for
fermentation may ensure stable Lactobacillus diversity, defined proper­
ties of sourdough and ensure consistent quality. Implementation of
technological objectives resulted in the development of fermentation
technologies for sourdough and starter cultures with better biochemical
properties. Bread attributes of aroma and dominant volatile compounds
depended on the fermentative microflora and raw material used for
bread production (Corona et al., 2016). Therefore, the application of
selected lactic acid bacteria (LAB) strains for sourdough production
might lead to more desirable products.
Although whole-grain breads have been recommended because of
their high contents of dietary fiber and minerals, many minerals in ce­
reals are bound to phytic acid as phytate, consequently reducing their
bioavailability (Kumar et al., 2010). This problem is especially relevant
in the production of wheat-baked goods that use sped up production
processes during the fermentation so that minerals are not released from
the phytate complexes (Kumar et al., 2010). Therefore, the application
of biological tools for increasing mineral bioavailability from
whole-grain products may be desirable. Enzymatic activities of LAB
improve mineral bioavailability, increase arabinoxylans production by
xylanolytic enzymes and increase starch hydrolysis and proteolysis
(Hansen, 2012). The search for new LAB strains with favorable enzyme
activities to incorporate into whole-grain wheat medium may improve
sourdough quality.
Another relevant issue is bread manufacturing without synthetic
preservatives. Sourdough LAB contributes to the development of the

* Corresponding author. Department of Food Science and Technology, Kaunas University of Technology, Radvilenu rd. 19, LT-50254, Kaunas, Lithuania.
E-mail address: dalia.cizeikiene@ktu.lt (D. Cizeikiene).

https://doi.org/10.1016/j.fbio.2020.100791
Received 9 October 2019; Received in revised form 15 October 2020; Accepted 16 October 2020
Available online 19 October 2020
2212-4292/© 2020 Elsevier Ltd. All rights reserved.
D. Cizeikiene et al.
sensory characteristics in the final product and increases bread shelf-life
(Elsanhoty et al., 2017). The properties of sourdough strongly depend on
the LAB strains used for sourdough preparation (Hadaegh et al., 2017).
Modifications of LAB microbiota influence the biochemical features of
some sourdoughs (Minervini et al., 2018).
During sourdough fermentation, lactic and acetic acids are the main
products. Since elevated levels of D-lactate are harmful to humans (Kang
et al., 2006), L-(+)-lactic acid is the preferred isomer in food. Therefore,
the search for starter LAB cultures producing pure L-isomer or only small
amounts of the D-isomer is preferable. Obligate homofermentative
Lactobacillus acidophilus and L. delbrueckii subsp. bulgaricus produce D,
L-lactic acid from hexoses via the Embden-Meyerhof-Parnas pathway
(Hansen, 2012).
Sourdough fermentation is often evaluated using pH, total titratable
acidity (TTA) and microflora composition (Chavan & Chavan, 2011);
however, other biochemical properties such as acetic and lactic acid
contents and enzymatic activity should be analyzed as well to ensure
high quality bread. Sensory properties of bread such as taste, porosity,
crumbliness, hardness and smell are also important for consumer
acceptance (Chavan & Chavan, 2011).
Industrial sourdough, referred to as type II sourdoughs, are domi­
nated by thermophilic and acid-tolerant heterofermentative lactobacilli
(Hansen, 2012). In type II sourdoughs fermented at elevated tempera­
tures (>35 ◦ C), yeast are essentially absent (Hansen, 2012). To develop
thermophilic sourdough production technologies, new sourdough
starter culture applications with defined biochemical properties are
needed. The aim of this study was to evaluate the influence of three
starter cultures, L. bulgaricus MI, L. rossiae GL14 and L. acidophilus DSM
20079, on the biochemical characteristics such as pH, TTA, volatile
acidity, enzyme activity and the content of lactic acid in thermophilic
sourdough and its influence on the quality and flavor profile of
whole-grain wheat bread.
2. Materials and methods
2.1. Sourdough preparation
Sourdough starters for whole-grain wheat bread baking were pre­
pared using a single culture of each LAB strain. L. delbrueckii ssp. bul­
garicus (L. bulgaricus) MI, L. rossiae GL14, and L. acidophilus DSM 20079
strains were previously propagated in DeMan, Rogosa and Sharpe (MRS)
broth (CM 0359, Oxoid Ltd., Basingstoke, Hampshire, UK) at 40 ◦ C for
24 h. L. bulgaricus MI previously isolated from dairy was obtained from
the collection of the Food Institute of Kaunas University of Technology
(Kaunas, Lithuania). L. rossiae GL14 had been previously isolated from
Lithuanian liquid whole-grain rye sourdough, which was propagated in
the bakery at 40 ◦ C. L. acidophilus DSM 20079 was previously isolated
from the human intestinal tract and purchased from the Leibniz Institute
DSMZ-German Collection of Microorganisms and Cell Cultures GmbH
(Braunschweig, Germany). Whole-grain wheat flour (Malsena Plius Ltd.,
Vievis, Lithuania) (Triticum aestivum L, flour composition: 2.30 g fat,
0.50 g saturated fatty acids, 70.9 g carbohydrates, 1.30 g sugars, 11.9 g
crude protein, and 0.20 g salt; country of origin - Lithuania) was mixed
with water and a 4-stage fermentation process was used.
For the first stage, 5.5 ml (LAB count was in the range of 2.1–3.6⋅109
CFU ml− 1) of fresh single culture propagated 24 h in MRS broth was
mixed with flour (10 g), tap water (20 ml) and fermented for 4 h. After
fermentation, flour (20 g) and water (40 ml) were added and the mass
was fermented for 20 h (stage 2). Later, flour (35 g) and water (70 ml)
were added and fermented for another 24 h (stage 3). For the fourth
stage, flour (70 g) and water (66 g) were added, mixed and fermented for
an additional 24 h. All 4 steps were done at 40 ◦ C. Spontaneous sour­
dough fermentation followed the 4-stages without starter LAB at 40 ◦ C
for 72 h.
2
Food Bioscience 38 (2020) 100791
2.2. Determination of total LAB count
Total LAB counts in sourdough were calculated using standard
counting techniques (ISO 4833, 2003) with some modifications. For LAB
cultivation, MRS agar (Liofilchem, Roseto degli Abruzzi, Italy) was used.
The Petri plates were incubated for 72 h at 40 ◦ C in a jar using anaerobic
atmosphere generation bags (Sigma Aldrich Co., St. Louis, MO, USA).
The total number of viable LAB was expressed as colony forming units
(CFU) g− 1.
2.3. The preparation of whole-grain wheat bread
Wheat dough was prepared from 1 kg of the whole-grain wheat
(Triticum aestivum L.) flour, 15 g sea salt with iodine (Droga, Soline
Pridelava soli d.o.o., Portorose, Slovenia), 20 g fresh compressed
Saccharomyces cerevisiae yeast (Lallemand Baltic Ltd., Panevėžys,
Lithuania), 150 g sourdough (60% moisture content) and 617 ml tap
water (to obtain 46% dough moisture content). The dough was then
mixed (3 min slowly and 8 min fast) in a Diosna SP25 mixer (Diosna
Dierks & Söhne GmbH, Osnabrük, Germany). The dough was proofed at
30 ◦ C and 85% relative humidity for 30 min in a proofer (Miwe Michael
Wenz GmbH, Arnstein, Germany). The dough was divided into pieces of
450 g, shaped into a loaf and proofed at 30 ◦ C and 85% relative humidity
for 40 min in the proofer. The bread was baked at 220 ◦ C for 45 min in a
deck oven (Miwe Michael Wenz GmbH). As a control sample, the wheat
dough was prepared with a spontaneous sourdough starter. For each
treatment, three breads were made.
2.4. Determination of TTA and pH
TTA of the sourdough starter, dough or bread was measured ac­
cording to the method described in AACC 02–31.01 (2000). Sample (10
g) was suspended in distilled water (90 ml) and homogenized in labo­
ratory porcelain mortar. The TTA value was expressed in ml of 0.1 mol
l− 1 NaOH solution 10 g− 1 of sample to obtain pH = 8.5. Sourdough pH
was determined using a portable pH meter with a puncture electrode
(Delta Track Inc., Pleasanton, CA, USA) according to method AACC
02–52.01 (2000). Analyses were carried out in triplicate.
2.5. Determination of volatile acidity and lactic acid in sourdough
For determination of volatile acidity, sourdough sample (25 g) was
poured into a distillation flask with distilled water (50 ml) and 2%
sulfuric acid (7.5 ml). An appropriate distillation program (80% power,
540 s duration) was started using a Behr S4 Distillation unit (Behr Labor-
Technik GmbH, Düsseldorf, Germany). The distillate was titrated with
0.1 mol l− 1 NaOH solution to obtain pH = 8.5. The amount of 0.1 mol l− 1
NaOH solution used for titration was recalculated to the amount of 1
mol l− 1 NaOH required to neutralize the volatile acid compounds in a
100 g sample (Juodeikiene et al., 2017).
A K-DLATE 08/11 enzymatic test (Megazyme Ltd., Wicklow, Ireland)
was used for total lactic acid and D/L-lactate determination according to
manufactures recommendation. For sample extract preparation, sour­
dough (1 g) was mixed with distilled water (80 ml) and was filtered
through a Whatman’s filter paper No. 1 (Sigma Aldrich). Filtrate was
diluted with distilled water to 100 ml. Lactic acid isomers were deter­
mined using three reactions. In the first reaction 1.5 ml distilled water,
0.1 ml sample, 0.5 ml solution 1 (glycylglycine buffer, pH = 10), 0.1 ml
solution 2 (nicotinamide-adenine dinucleotide solution) and 0.02 ml
suspension 3 (D-glutamate-pyruvate transaminase) were added. For the
blank sample preparation, water was used instead of sample. After 3 min
0.02 ml D-lactate dehydrogenase suspension was added. The absorbance
was measured at 340 nm in a spectrophotometer (Model Genesys 10,
Thermo Electron LED GmbH, Langenselbold, Germany) after 3 min (A1).
In the second reaction, 0.02 ml D-lactate dehydrogenase was added. The
absorbance was measured at 340 nm after 5 min (A2). In the third
D. Cizeikiene et al.
reaction, 0.02 ml L-lactate dehydrogenase was added. The absorbance
was measured at 340 nm after 10 min (A3). The D/L-lactic acid standard
at different concentrations was used to obtain a linear regression
equation. The absorbance differences (A2-A1 and A3-A2) were deter­
mine for both blank and sample. The absorbance difference of the blank
was subtracted from the absorbance difference of the sample. The cor­
rected A2-A1 = ΔAD-lactic acid and A3-A2 = ΔAL-lactic acid. Lactic acid
content was calculated using the following equation Eqn 1:
V × MW
c= × ΔAD− lactic acid or ​ ΔAL− lactic acid (1)
ε×d×v
where: c = D-or L-lactic acid concentration (g l− 1), V = final volume
(ml), MW = molecular weight of lactic acid (90.1 g mol− 1), ε =
extinction coefficient of nicotinamide adenine dinucleotide reduced
form at 340 nm = 6300 l x mol− 1 x cm− 1, d = light path (1 cm), and v =
sample volume (ml).
2.6. Determination of enzymatic activities in sourdough
Amylase activity was determined using the starch-iodine assay ac­
cording to Xiao et al., 2006 with some modifications. One unit of
amylase activity for the starch-iodine assay is defined as the disap­
pearance of an average of 1 mg of iodine-binding starch min− 1 in the
assay at pH 7.0 and 30 ◦ C. Sourdough (0.1 g ml− 1) was prepared in 0.1
mol l− 1 sodium phosphate buffer (pH 7.0). Sample extract was obtained
after filtration through Whatman’s No. 1. The reaction mixture of tested
sample consisted of 1 ml sample extract and 1 ml starch (Sigma Aldrich)
solution (1 mg ml− 1). For the control sample assay, 1 ml phosphate
buffer was mixed with 1 ml starch solution (1 mg ml− 1). For blank
sample, 1 ml phosphate buffer was mixed with 1 ml sample extract.
Enzymatic hydrolysis was carried out at 30 ◦ C for 30 min. The reactions
were stopped with 0.5 ml 1 mol l− 1 HCl, and then 2.5 ml 1 mol l− 1 iodine
solution (5 mol l− 1 I2 and 5 mol l− 1 KI) and 5 ml distilled water were
added. The absorbance of samples (Asample and Acontrol) were measured
at 580 nm immediately using a blank sample. The absorbance difference
of Asample and Acontrol was used for the starch equivalents value (SEV)
calculations from the equation of the standard curve. The standard curve
was prepared using a starch solution (1 mg ml− 1) and 0.1 mol l− 1 sodium
phosphate buffer (pH 7.0) to obtain final starch concentrations from 0 to
1 mg ml− 1. Amylase activity was calculated using the following equation
Eqn 2:
SEV × DF × 2.5
U= × 10 (2)
1 × 30 × 2.5
where: U = amylase activity g− 1 sourdough (units g− 1), SEV (mg ml− 1),
DF = sample dilution factor, 2.5 = total volume of the assay (2.5 ml), 1
= volume of sourdough sample extract used (1 ml), 30 = time of assay
(30 min), 2.5 = volume used with the colorimetric determination (2.5
ml), and 10 = coefficient to calculate amylase activity g− 1 sourdough.
Phytase activity was determined according to Nuobariene et al.
(2011). One unit of phytase activity is defined as the amount of phytase
that liberates 1 μmol inorganic phosphate min− 1 from a 3 mmol potas­
sium phytate solution at pH 5.5 and 30 ◦ C. Sourdough sample extract
(0.1 g ml− 1) was prepared in 0.2 mol l− 1 sodium acetate buffer (pH 5.5).
The reaction mixture consisted of 0.8 ml 0.2 mol l− 1 sodium acetate
buffer (pH 5.5) containing 3 mmol l− 1 potassium phytate (Sigma
Aldrich) and 0.2 ml sample extract. For blank sample, 0.8 ml 0.2 mol l− 1
sodium acetate buffer (pH 5.5) without potassium phytate was mixed
with 0.2 ml sample extract. Enzymatic hydrolysis was carried out at
30 ◦ C for 30 min. The reactions were stopped with 1 ml 10% tri­
chloroacetic acid. The reaction mixture (0.2 ml) was mixed with color
reagent (1.6 ml, 10 mmol l− 1 (NH4)6Mo7O24⋅4H2O:2.5 mol l− 1 H2SO4:
acetone at a 1:1:2 ratio). The absorbance was measured after 20 min at
355 nm using the blank sample.
The absorbance was used for the liberated inorganic phosphate
3
Food Bioscience 38 (2020) 100791
equivalents (LIPE) calculations from the equation of the standard curve.
The phosphate standard curve was prepared using KH2PO4 (Sigma-
Aldrich), dissolved in 0.2 mol l− 1 sodium acetate buffer (pH 5.5) to
obtain final phosphate concentrations from 0 to 2 μmol ml− 1 of inor­
ganic phosphate. Phytase activity was calculated using the following
equation Eqn 3:
LIPE × DF × 2
U= × 10 (3)
30 × 0.2
where: U = phytase activity g− 1 sourdough (units g− 1), LIPE (μmol
ml− 1), DF = sample dilution factor, 2 = total volume of assay (2 ml), 0.2
= volume of sourdough sample extract used (0.2 ml), 30 = time of assay
(30 min), and 10 = coefficient to calculate phytase activity g− 1
sourdough.
Xylanase activity was determined by assessing the total content of
reducing sugars according to Cizeikiene et al. (2018). One unit of
xylanase activity is defined as the amount of enzyme that releases 1 μmol
of xylose equivalent from xylan substrate min− 1. Sourdough sample
extract (0.1 g ml− 1) was prepared in 0.2 mol l− 1 sodium acetate buffer
(pH 5.5). The reaction mixture consisted of 0.1 ml sourdough sample
extract, 0.1 ml xylan (5 mg ml− 1) from birch wood (Sigma Aldrich) and
0.8 ml 0.05 mol l− 1 sodium acetate buffer (pH 5.5). For the blank
sample, 0.2 mol l− 1 sodium acetate buffer (pH 5.5) was used instead of
xylan. Enzymatic hydrolysis was carried out at 30 ◦ C for 30 min. For
xylose equivalent evaluation, the reaction mixture was mixed with 1 ml
3.5-dinitrosalicylic acid regent (1 g 3.5-dinitrosalicylic acid and 30 g
sodium potassium tartrate dissolved in 100 ml 0.4 mol l− 1 NaOH) in
equal parts and boiled for 5 min, then cooled. The absorbance was
measured immediately at 540 nm using the blank sample. The absor­
bance was used for liberated xylose equivalents (XE) calculations from
the equation of the standard curve. The standard curve was prepared
using xylose solution (1 μmol ml− 1) and 0.2 mol l− 1 sodium acetate
buffer to obtain final xylose concentrations in range from 0 to 6.66 μmol
ml− 1 with the same conditions as the enzyme sample. Xylanase activity
was calculated using the following equation Eqn 4:
U=
XE × DF ​
× 10 (4)
0.1 × 30
where: U = xylanase activity g− 1 sourdough (units g− 1), XE (μmol ml− 1),
DF = sample dilution factor, 0.1 = volume of sourdough sample extract
used (0.1 ml), 30 = time of assay (30 min), and 10 = coefficient to
calculate xylanase activity g− 1 sourdough.
Non-specific protease activity was measured using casein as the
substrate (Cupp-Enyard, 2008). One unit of protease activity is the
amount of tyrosine equivalents (μmol) released from casein min− 1 at pH
7.5 and 37 ◦ C. Sourdough sample extract (0.1 g ml− 1) was prepared in
10 mmol l− 1 sodium acetate buffer with 5 mmol l− 1 calcium chloride
(pH 7.5). The enzymatic reaction mixture consisted of 1 ml sample
extract and 5 ml 0.65% casein solution (pH 7.5 at 37 ◦ C). After 10 min
the reaction was stopped with 5 ml 110 mmol l− 1 trichloroacetic acid
solution. For the blank 1 ml sample extract was added after the tri­
chloroacetic acid solution. The solutions were incubated at 37 ◦ C for 30
min. After incubation, the test solution and the blank were filtered using
a 0.45 μm polyethersulfone syringe filter (Sigma Aldrich). Two ml
filtrate, 5 ml 0.5 mol l− 1 sodium carbonate solution and 1 ml 0.5 mol l− 1
Folin & Ciocalteu’s solution (Sigma Aldrich) were mixed and incubated
for 30 min. After incubation, the solutions were filtered through a
Whatman’s No. 1 and the absorbance was measured at 660 nm using the
blank. The absorbance was used for liberated L-tyrosine equivalents (TE)
calculations from the equation of the standard curve. The standard curve
was prepared using L-tyrosine (Sigma-Aldrich) solution (1.1 mmol), 5 ml
0.2 mol l− 1 sodium carbonate, 1 ml 0.5 mol l− 1 Folin & Ciocalteu’s so­
lution and water to obtain the final L-tyrosine concentrations from 0.05
to 0.55 μmol. Protease activity was calculated using the following
equation Eqn 5:
D. Cizeikiene et al.
TE × 11
U= × 10 (5)
1 × 10 × 2
where: U = protease activity g− 1 sourdough (units g− 1), TE (μmol), 11 =
total volume of assay (11 ml), 1 = volume of sourdough sample extract
used (1 ml), 10 = time of assay (10 min), 2 = volume used in colori­
metric determination (2 ml), and 10 = coefficient to calculate protease
activity g− 1 of sourdough.
2.7. Analysis of volatile compounds using gas chromatography and mass
spectrometry (GC-MS)
Analysis of volatile compounds using GC-MS was carried out
following Juodeikiene et al. (2013).
Samples of breadcrumb were prepared using solid-phase micro­
extraction (SPME), using a SPME device with Stableflex ™ (grey color)
(Sigma Aldrich) and a 50/30 μm layer of PDMS/CAR/DVB fiber
(Supelco Inc., Bellefonte, PA, USA). During extraction, sample (0.5 g)
was incubated in gas-tight bottles with a septum (Sigma Aldrich) at
50 ◦ C for 10 min. Adsorption to the fiber was carried out at 50 ◦ C for 15
min. Thermal desorption was at 280 ◦ C for 1 min in the GC.
A gas chromatograph model GC-2010 (Shimadzu, Corp. Kyoto,
Japan) and a single quadrupole mass spectrometer GCMS-QP2010
(Shimadzu, Corp.) were used for a GC-MS analysis. An RTX-5MS
(Restek, Co., Bellefonte, PA, USA) column with dimensions of 30 m of
length, film thickness 0.25 μm and inner diameter 0.25 mm was used for
separation. Carrier gas helium 99.999% (AGA, Ryga, Latvia) was used at
1.7 ml min− 1. The injector temperature was 280 ◦ C. Desorption was
carried out using split mode 1:10. The temperature gradient started from
35 rising to 180 ◦ C at 5 ◦ C min− 1, then to 280 ◦ C at 20 ◦ C min− 1 and
maintained for 3 min.
MS conditions used: Ion source temperature was 220 ◦ C and interface
temperature was 260 ◦ C. An electron ionization detector at 70 eV ioni­
zation energy was used for detection of the compounds, scanning mass
range 40–600 m/z. Compounds were determined from the mass spectra
and comparing to the highest similarity in the electronic NIST v14.1
library (The National Institute of Standards and Technology (NIST),
Gaithersburg, MD, USA). The relative percentage of molecules of each
extract constituent was determined by dividing its peak area by the total
area of all the peaks and multiplying by 100 assuming that the detector
response was independent of the ions. Thus always giving a sum of all
compounds of 100%.
2.8. Determination of specific volume and porosity of the bread
Bread mass was measured using a digital balance. Bread volume was
determined using a modification of the AACC Method 10–05.01 (2000)
rapeseed replacement method using millet grit (Skanėja, Vilnius,
Lithuania) instead of rapeseeds. The bread was placed in a container of
known volume (VC) and the basin filled to the brim with millet, bread
was removed and the volume of the millet (VM) was measured using a
measuring cylinder. The specific volume (SV) of bread was calculated
using the following equation Eqn 6.
VC − VM
SV = (6)
BM
where: SV (cm3 g− 1), BM = bread mass (g), VC (cm3), and VM (g).
Bread porosity was determined using the Zhuravlev device (Biomer Ltd.,
Krasnoobsk, Russia) (Kulushtayeva et al., 2019; Smolnikova et al., 2020),
which consist of the following parts:
1) a metal cylinder with an inner diameter of 3 cm and a pointed edge
on one side;
2) wooden sleeve;
3) a wooden tray with a transverse wall and a slot with 1.5 cm deep
positioned at a distance of 3.8 cm from the wall.
4
Food Bioscience 38 (2020) 100791
A slice of bread with a width of at least 7–8 cm was cut with a knife
from the center of the bread. From the crumb of the slice at a distance
of at least 1 cm from the bread crust, using the Zhuravlev, a cylindrical
shape piece of bread crumb was cut out exactly at 27 cm3. For this, the
cylinder is penetrated into a piece of crumb by rotation. The cylinder
filled with the crumb is placed on the tray so that its rim fits snugly
into the slot on the tray. Then the bread crumb is pushed out of the
cylinder with the wooden sleeve for 1 cm and cut off at the edge of the
cylinder with a sharp knife. The cut off slice of crumb is removed. The
crumb left in the cylinder is pushed out to the wall of the tray and also
cut off at the edge of the cylinder. To determine the porosity of wheat
bread, 3 cylindrical cuts of 27 cm3 each are made. The cut-off slices of
the crumb are weighed with an accuracy of 0.01 g. Bread porosity was
calculated using the following equation Eqn 7.
( )
V − Gp
x= × 100 (7)
V
where: x = bread porosity (%), V = total volume of cut-off crumbs
(27 cm3), G = weight of cut-off crumb (g), and p = the relative density
of non-porus crumb, which is 1.31 for wheat flour baked goods
(Kulushtayeva et al., 2019; Smolnikova et al., 2020).
2.9. Screening for anti-mold activities on the bread surface
For anti-mold activity screening, bread samples were stored at 25 ±
1 ◦ C for 9 days in the dark in closed polythene bags (Vigdomus, Vilnius,
Lithuania) 18 h after baking. The loaves were examined externally for
the presence of visible mold daily.
2.10. Sensory analysis
Sensory analysis of the breads was carried out using 15 trained stu­
dents and staff of Kaunas University of Technology (Kaunas, Lithuania)
(7 males and 8 females aged between 22 and 35) 18 h after baking ac­
cording to the ISO 6658 method (2005), using a 7-point line scale for 5
bread attributes (porosity, elasticity, hardness, moisture content and
crumbliness) where a value of 1 corresponded to the lowest intensity
and a value of 7 corresponded to the highest intensity of the attribute.
Whole-grain wheat bread samples were evaluated for overall accept­
ability using a 7-point hedonic scale where point 1 was ‘dislike
extremely’ and point 7 was ‘like extremely’ although such a trained
panel may not be representative of consumers so these results need to be
considered with caution. Blinded samples without crust were served in
booths. Water was provided for rinsing between samples. Panelists, who
had completed 40 h of training about sensory evaluation of food prod­
ucts, were asked to directly mark their response for each parameter on
the questionnaire.
2.11. Statistical analysis
All experiments were carried out in three independent replicates.
Results were expressed as the average of three replicated measurements.
The STATISTICA software package version 10.0 (StatSoft Corp. Krakow,
Poland) was used to determine if variables differed among the control
and tested samples using the Duncan post-hoc test (p < 0.05). Parame­
ters were compared through correlation. Triplicate data were evaluated
using two-way ANOVA.
3. Results and discussion
3.1. Microbiological analysis of thermophilic sourdough
The results confirmed that tested strains were adapted in the whole-
grain wheat sourdough medium. The highest content of LAB were in
D. Cizeikiene et al.
sourdough with L. rossiae starter culture (2.4⋅109 CFU g− 1) as a single
strain used for fermentation. The least number of LAB was in sourdough
prepared with L. bulgaricus starter (7.1⋅108 CFU g− 1), whereas the
number of LAB was 2.0⋅109 CFU g− 1 in sourdough with L. acidophilus
starter. The number of LAB, which were used for fermentation as a single
culture were similar to those of regular sourdough (Nuobariene et al.,
2015). During the fermentation specifically adapted LAB were found at
108–109 CFU g− 1, which depends on starter cultures, temperature and
flour type (Hansen, 2012). LAB are responsible for acidification of the
sourdough, sensory attributes, valuable nutritional components, texture
traits and aroma of the baked goods (Giannone et al., 2018).
3.2. pH and TTA of thermophilic whole-grain wheat sourdough
The most important parameters observed during sourdough prepa­
ration were pH, TTA, acetic and lactic acid contents, and count of viable
LAB. The kinetics of pH and TTA during the sourdough propagation
indicated a successful acidification process for all trials. During 72 h of
fermentation, the lowest pH of all thermophilic LAB sourdough were
observed after 24 h, whereas after 12 h of fermentation the more rapid
decreasing pH were observed in sourdough with L. bulgaricus and
L. acidophilus compared with the pH of sourdough fermented with
L. rossiae (Fig. 1). The TTA of sourdough during propagation slowly
increased during the first 12 h, whereas during the next 12 h rapid in­
creases were observed with all thermophilic LAB (Fig. 1). Sourdough pH
should be < 4.5 and it should contain >5⋅108 metabolically active CFU
g− 1 (Lönner & Preve-Åkesson, 1988). Typical TTA values of wheat
sourdough have been reported to be 8–13 (Katina et al., 2002), while in
Fig. 1. The influence of LAB strain on total titratable acidity and pH
of sourdough.
5
Food Bioscience 38 (2020) 100791
whole-grain sourdough the TTA values have been reported to be 16–22
(Hansen, 2012). White-wheat sourdough starter with higher pH
(4.77–5.17) and low TTA (3.47–4.5) were reported by Nisa et al. (2016).
In whole-grain wheat sourdoughs, TTA has been reported to be 12–22,
depending on LAB strain used for sourdough propagation (Cizeikiene
et al., 2015; Hansen, 2012). Results confirmed that LAB strains could be
applied for whole-grain wheat sourdough production.
3.3. Lactic acid content and volatile acidity of sourdough
Lactic acid content and volatile acidity in thermophilic sourdough
are shown in Fig. 2. The highest volatile acidity was found in whole-
grain wheat sourdough fermented with L. acidophilus, whereas signifi­
cantly less volatile acidity were found in sourdough fermented with
L. rossiae and L. bulgaricus compared with L. acidophilus. The highest
total lactic acid content was determined in sourdough with L. acidophilus
with the highest D(− ) isomer content. Fermentation breaks down car­
bohydrates to form acetic and lactic acids, resulting in decreased sugar
in sourdough. Lactobacillus species have the potential for only L(+) lactic
acid production (Broadbent et al., 2014); however, the LAB used for
sourdough preparation produced a mixture of L(+) and D(− ) lactic
acids, and the predominant isomer depended on the LAB strain as a
starter. In some cases, high content of D(− ) isomer may be influenced by
wild LAB strains present in the flour. Microbial production of L(+) and D
(− ) lactic acids proceeds either separately or as a mixture of two isomers
in different proportions, depending on the microorganism, substrate and
growth conditions (Tanyıldızı et al., 2012). It was once supposed that
the relatively small amount of D-lactate normally present in the blood of
humans (concentration 5–20 μmol l− 1 in healthy adults compared to
1000 μmol l− 1 for L-lactate) is solely derived from exogenous sources
such as diet and carbohydrate-fermenting bacteria normally present in
Fig. 2. Volatile acidity (a) and lactic acid (b) content in thermophilic LAB
sourdough. Mean values within a column with different letters (a–f) are
significantly different (p < 0.05).
D. Cizeikiene et al.
the gastrointestinal tract (Talasniemi et al., 2008). However, accumu­
lation of D-lactate ≥3 mmol l− 1 in serum may cause D-lactic acidosis
(Kang et al., 2006); therefore, the use of starter cultures to produce a
small amount of D-isomer or pure L-isomer is preferred for sourdough
production. Much higher concentrations of D-lactic acid (0.244 g 100
g− 1) were reported in sourdough bread compared with reference bread
prepared with bakery yeast (0.003 g 100 g− 1) (Maioli et al., 2008).
Therefore, bread producers should pay attention to the concentrations of
D-lactic acid in fermented products. The low content of D-isomer pro­
ducing L. bulgaricus MI strain could be a potential benefit for thermo­
philic sourdough production.
3.4. Enzymatic activity in sourdough
Phytase activity in sourdough with L. acidophilus and L. rossiae were
19.1 and 22.3% less, respectively, than L. bulgaricus sourdough
(Table 1). Xylanase activity in sourdough with L. rossiae and L. bulgaricus
were 68.8 and 54.3% less, respectively, than L. acidophilus sourdough.
Amylase and xylanase have a significant role in bread dough prepara­
tion, and phytase-positive LAB are preferred in bread production espe­
cially in whole-grain wheat bread production due to their ability to
increase nutritional value. Only a few strains of LAB, e.g., L. amylovorus
and L. plantarum, showed consistent extracellular phytase activity with
values of 125–146 U ml− 1. However, Reale et al. (2007) were unable to
confirm these results using the same L. amylovorus strain with identical
growth conditions. Furthermore, only a few strains of LAB isolated from
sourdough, e.g., L. plantarum, L. brevis, L. curvatus, L. sanfranciscensis, L.
fermentum and L. plantarum were shown to express intracellular (cyto­
plasmic and/or cell-wall bound) phytase activity in phytate-rich me­
dium. However, in this study results showed that the phytase activities
of LAB strains isolated from sourdough were extracellular consistent
with Nuobariene et al. (2015). However, LAB strains produced higher
phytase activity in comparison to Nuobariene et al. (2015). On the other
hand, Zamudio et al. (2001) found that L. fermentum and P. pentosaceus
at pH 4.5 and 37 ◦ C showed low extracellular phytase activity (<6.3 mU
ml− 1).
Little protease activity was found in all the whole-grain wheat
sourdoughs. L. delbrueckii ssp. bulgaricus and L. acidophilus had increased
proteolytic activity in reconstituted skim milk supplemented with large
peptides, showing that proteolytic activity strongly depends on the
medium and its supplements (Gandhi & Shah, 2014).
Amylase activity was similar between LAB strains. Amapu et al.
(2016) reported maximum amylase activity between 0.38–1.10 U ml− 1
for LAB isolates from milled cereals, cassava flour and fruits after 24 h of
incubation.
In LAB, xylanases were expressed by L. brevis (Hu et al., 2011),
however, these enzymes are mainly found in different species of Asper­
gillus and Trichoderma (Butt et al., 2008). The results showed that
whole-grain wheat sourdough prepared using L. acidophilus had signif­
icantly higher xylanase activity in comparison with other starter strains;
therefore, it can be potentially used to enrich bread with arabinoxylan
oligosaccharides from wheat bran.
Table 1
Enzymatic activities (U g− 1) in thermophilic LAB sourdough.
LAB strain Amylase Protease Phytase Xylanase
activity activity activity activity
L. acidophilus 1.33±0.004a (5.5±0.1)⋅ 10±0.2ac 370±10a
DSM 20079 10− 3a
L. bulgaricus MI 1.35±0.002a (5.5±0.1)⋅ 12.9±0.2b 170±10b
10− 3a
L. rossiae GL14 1.30±0.003a (5.4±0.1)⋅ 10.4±0.3c 120±10c
10− 3a
Data expressed as a mean value (n = 6) ± SD; SD, standard deviation.
Mean values within a column with different letters (a–c) are significantly
different (p < 0.05).
6
Food Bioscience 38 (2020) 100791
3.5. Sourdough bread quality evaluation
The addition of thermophilic sourdough increased the TTA of whole-
grain wheat dough and bread (Fig. 3a). The addition of L. acidophilus,
L. bulgaricus and L. rossiae sourdough increased bread acidity by 42.9,
37.4 and 31.7%, respectively, in comparison to whole-grain bread pre­
pared with spontaneous sourdough. The addition of thermophilic LAB
sourdough increased bread porosity and specific volume by 22.7 and
17.1%, respectively, in comparison to bread prepared with spontaneous
sourdough (Fig. 3b). There were no significant differences between
thermophilic LAB starters for specific volume and porosity of bread.
Strong positive correlations were found between TTA of dough and
specific volume of bread (R2 = 0.925) and TTA of dough and porosity of
bread (R2 = 0.940). A strong negative correlation was found between
total lactic acid in sourdough and specific volume of bread (R2 =
− 0.999), and a weak negative correlation was found between volatile
acidity and specific volume of bread (R2 = − 0.367). Meanwhile, a
moderate negative correlation was found between volatile acidity and
porosity of bread (R2 = − 0.656). The application of sourdough has been
reported to either increase or decrease bread volume (Barber et al.,
1992). A positive influence of sourdough on bread volume might be due
to increased baker’s yeast metabolic activity which produces more CO2.
Another reason could be that the appropriate acidity of the sourdough
protein network might enhance the ability of gluten to retain CO2
(Habibi Najafi et al., 2016). Corsetti et al. (2008) reported that accu­
mulation of water-soluble pentosanes might increase volume.
Sensory properties increased with increased bread porosity, elastic­
ity, crumbliness and moisture, but were not influenced by crumb
hardness (Fig. 4). Panelists indicated that whole-grain wheat bread
made with sourdough previously prepared with L. rossiae was more
acceptable than bread prepared with spontaneous sourdough, or
L. acidophilus and L. bulgaricus (Fig. 4a).
LAB fermentation provided precursor compounds for the formation
of volatile compounds during sourdough propagation and baking as well
as substrates for microbial conversion of amino acids to flavor precursor
compounds (Thiele et al., 2002). Therefore, the application of sour­
dough in wheat bread has increased as a means to improve the quality
and flavor of bread. Application of thermophilic sourdough produced
using L. rossiae had significant positive effects on the sensory properties
of fresh bread samples.
3.6. Volatiles from sourdough and sourdough bread
Most aroma compounds are created during the baking of bread
(Winters et al., 2019), but sourdough volatiles are also formed during
fermentation (Pétel et al., 2017). Yeast and LAB fermentations lead to
the formation of specific volatile compounds. LAB fermentation needs
>12 h to produce adequate amount of volatiles, whereas this is achieved
in a few h for yeast fermentation (Pétel et al., 2017). The GC-MS/SPME
analysis found differences in compositions of volatile compounds be­
tween spontaneous sourdough (reference) and starter LAB-fermented
sourdough prepared from whole-grain wheat flour (Table 2). Both
quantitative and qualitative differences have been observed. Fermented
products mainly differed in the accumulation of higher alcohols. The
compositions of volatile compounds in whole-grain wheat sourdough
and in whole-grain wheat bread depended on the LAB strain used as a
starter. There were different compounds in the reference sample than in
samples fermented with thermophilic starter cultures. The main com­
pounds found in the headspace of spontaneous sourdough were
ethyl-hexanoate, n-hexanol and n-heptadecane. Ethyl-octanoate was
found in all sourdough samples fermented with thermophilic LAB,
whereas this compound was not detected in spontaneous sourdough.
Limonene and ethyl-octanoate were observed in all sourdoughs with
starter LAB, whereas those compounds were not found in spontaneous
sourdough. Although ethyl-hexanoate and ethyl-octanoate were domi­
nant in sourdough, those compounds have not been found in bread. In
D. Cizeikiene et al.
Fig. 3. Whole-grain wheat sourdough influence on attributes of dough and bread: acidity (a), and porosity and specific volume (b). Mean values within a column of
the same color with different letters (a–c) are significantly different (p < 0.05).
all sourdoughs with starter LAB, ethyl-hexanoate was observed, while in
bread this compound was not found. In bread with L. acidophilus sour­
dough, 3-octen-2-ol and n-hexadecane were found, whereas they were
not found in other bread samples or in sourdough. Thermophilic sour­
dough prepared with L. acidophilus was different from the others due to
the highest xylanase activity and the highest volatile acidity. Tetrahy­
drofurfuryl acetate was found only in bread prepared with L. bulgaricus
sourdough, which had the highest phytase activity and the lowest con­
tent of D(− ) lactic acid. N-Pentadecane was found only in bread pre­
pared with L. rossiae sourdough and bread prepared without starter
sourdough. Moreover, n-heptanal and phenethyl alcohol were identified
only in sourdough prepared with L. rossiae. Ethyl-hexanoate, terpinolene
and ethyl-palmitate were found only in bread with spontaneous sour­
dough, whereas these compounds were not found in bread with sour­
dough using specific LAB. Various precursor molecules to aroma
compounds were in connection with the metabolic pathways involved in
sourdough and bread aroma. The non-metabolic activities such as cell
lysis are also responsible for bread volatiles (Smid & Kleerebezem,
2014). Lysis of LAB cells is caused by bacteriophages or by peptido­
glycan hydrolases and it results in the release of cytoplasmic enzymes
into the matrices of the fermentable cereal compounds (Smid & Kleer­
ebezem, 2014). LAB are able to produce aroma compounds from amino
acids (Helinck et al., 2004). They reported that L. bulgaricus produced
7
Food Bioscience 38 (2020) 100791
large amounts of alcohols and/or aldehydes. Formation of aldehydes,
alcohols and acids from α-keto acids by L. bulgaricus mainly results from
the action of α-keto acid decarboxylase, which produces aldehydes
which are then oxidized or reduced to acids or alcohols (Helinck et al.,
2004). On the other hand, α-keto acid dehydrogenase produces acyl
coenzymes A during α-keto acid conversion to acids in LAB (Helinck
et al., 2004). Organic acids affect the starch and protein proportions by
increasing amylase and protease activities through a pH reduction
(Clarke et al., 2002; Thiele et al., 2002). Therefore, organic acids are
responsible for the formation of taste and odor compounds in sourdough
(Lahtinen, 2012). In addition, Thiele et al. (2002) showed that dough
acidifying is an important factor for inducing proteolysis and has a major
role in the formation of flavor and odor in sourdough.
3.7. Inhibition of bread spoilage
Bread prepared with thermophilic LAB starter sourdough molded
much slower. Control whole-grain wheat bread prepared with sponta­
neous sourdough started to mold after 3 days of storage. Compared to
control bread prepared with spontaneous sourdough, fungal spoilage on
the bread crust surface was suppressed by 1–2 days when sourdough was
prepared with L. rossiae, by 2–3 days when sourdough was prepared
with L. acidophilus and by 3–4 days when sourdough was prepared with
D. Cizeikiene et al.
Fig. 4. Thermophilic sourdough influence on sensory attributes (a) and overall acceptability (b) of whole-grain wheat bread. Mean values within a column of the
same color with different letters (a–c) are significantly different (p < 0.05).
L. bulgaricus. Lavermicocca et al. (2000) showed that LAB sourdough can
also serve as a natural replacement for synthetic food preservatives to
suppress bread molding. The shelf-life of bread was not related to dough
TTA (R2 = 0.145), sourdough volatile acidity (R2 = 0.127), lactic acid
content in sourdough (R2 = 0.265) and bread TTA (R2 = 0.250). Fungal
spoilage is the main cause of economic loss in bakeries and in the food
industry (Saranraj & Geetha, 2012) and is regarded as a source of my­
cotoxins, causing human health problems (Legan, 1993). As an alter­
native for the reduction of microbial contamination, LAB with high
antimicrobial activities may be considered as a bio-preservative. It is
therefore important to find LAB with positive sensory properties and a
wide spectrum of antimicrobial activities to be used to prolong bread
shelf life.
4. Conclusion
Starter cultures L. delbrueckii ssp. bulgaricus MI, L. rossiae GL14 and
8
Food Bioscience 38 (2020) 100791
L. acidophilus DSM 20079 could be adapted for whole-grain wheat
sourdough production. Biochemical characteristics such as pH, TTA,
volatile acidity, enzymes activity (phytase and xylanase) and D-lactic
acid content in thermophilic whole-grain wheat sourdough depended on
the LAB strain used as a starter. The three strains (L. bulgaricus MI,
L. rossiae GL14 and L. acidophilus DSM 20079) applied to whole-grain
wheat sourdough influenced the composition of volatile compounds in
the sourdough and the bread. Application of selected strains for bread
production increased acidity, porosity and overall acceptability of
whole-grain wheat bread. L. acidophilus DSM 20079 is recommended for
the production of whole-grain wheat sourdough with high xylanase
activity. L. delbrueckii ssp. bulgaricus MI is recommended for sourdough
with low D(− ) lactic acid content and for manufacturing bread with
reduced phytate content. Informed selection of starter cultures for
whole-grain thermophilic wheat sourdough preparation can produce
desired properties of baked goods.
D. Cizeikiene et al. Food Bioscience 38 (2020) 100791

Table 2
The composition of volatiles from sourdough and bread prepared from whole-grain wheat and thermophilic sourdough, the relative percentage.
Compound name Sourdough Bread
− 1
RT min Spontaneous sourdough With starter LAB With spontaneous sourdough With starter sourdough

L.a. L.b. L.r. L.a. L.b. L.r.

n-hexanal 4.63 6.32 3.18 10.5 4.92 6.22 6.50 – 10.6

n-hexanol 6.81 17.5 5.23 8.27 4.10 – – – –
n-heptanal 7.35 – – – 2.77 13.9 7.27 8.52 7.38
2-pentyl-furan 9.82 2.6 – – – 10.2 11.2 13.7 14.9
ethyl-hexanoate 10.11 37.0 28.8 31.2 25.0 5.19 – – –
delta-3-carene 10.12 – – – – 5.51 5.63 5.44 7.13
limonene 10.75 – 4.23 5.09 7.54 14.1 16.5 20.3 18.2
terpinolene 12.69 – – – – 3.35 – – –
delta-2-carene 12.71 – – – – – 1.90 1.81 5.12
phenethyl alcohol 15.09 – – – 7.54 – – – –
ethyl-benzoate 15.69 6.05 7.18 3.20 6.24 – – – –
n-dodecane 15.99 – – – – 22.1 24.2 17.4 20.7
ethyl-octanoate 16.10 – 32.2 30.1 36.7 – – – –
n-tridecane 18.88 – – – – 14.8 21.4 25.7 16.0
n-decane 18.98 – 1.88 2.62 – – – – –
ethyl-decanoate 21.84 5.92 6.71 3.96 – – – – –
undecanol 24.25 – 1.69 – – – – – –
3-octen-2-ol 26.70 – – – – – 1.59 – –
tetrahydrofurfuryl acetate 26.72 – – – – – – 2.37 –
n-heptadecane 28.95 12.8 – – 3.54 – – – –
n-pentadecane 29.06 – 5.89 4.69 – 3.04 – 4.70 –
n-hexadecane 29.07 – – – – – 3.84 – –
ethyl-palmitate 33.20 7.39 3.02 0.35 1.62 1.58 – – –
(7Z)-tetradecenal 34.46 4.51 – – – – – – –

– not determined; RT – retention time; L.a. – L. acidophilus; L.b. – L. bulgaricus; L.r. – L. rossiae.

Declaration of competing interest
The authors have no conflicts of interests to report.
Acknowledgment
The authors are very thankful to anonymous reviewers for comments
and suggestions on the earlier versions of this paper. The authors wish
sincerely express their profound gratitude to the Kaunas University of
Technology and Vytautas Magnus University for financially supporting
this project.
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