Accepted Manuscript

Effect of a novel Lactobacillus paracasei starter on sourdough bread quality

Ioanna Mantzourani, Stavros Plessas, Maria Odatzidou, Athanasios

Alexopoulos, Alex Galanis, Eugenia Bezirtzoglou, Argyro Bekatorou

PII: S0308-8146(18)31344-X
DOI: https://doi.org/10.1016/j.foodchem.2018.07.183
Reference: FOCH 23298

To appear in: Food Chemistry

Received Date: 18 October 2017

Revised Date: 23 April 2018
Accepted Date: 25 July 2018

Please cite this article as: Mantzourani, I., Plessas, S., Odatzidou, M., Alexopoulos, A., Galanis, A., Bezirtzoglou,
E., Bekatorou, A., Effect of a novel Lactobacillus paracasei starter on sourdough bread quality, Food Chemistry
(2018), doi: https://doi.org/10.1016/j.foodchem.2018.07.183

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Effect of a novel Lactobacillus paracasei starter on sourdough bread quality

Ioanna Mantzourani1, Stavros Plessas1,*, Maria Odatzidou1, Athanasios Alexopoulos1, Alex

Galanis2, Eugenia Bezirtzoglou1, Argyro Bekatorou3

1
Laboratory of Microbiology, Biotechnology and Hygiene, Faculty of Agriculture

Development, Democritus University of Thrace, 68200, Orestiada, Greece

2
Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus

University of Thrace, Dragana, 68100, Alexandroupolis, Greece

3
Department of Chemistry, University of Patras, 26500, Patras, Greece

* Corresponding author:

Tel: +30 2552041141, Fax: +30 2552041141

E-mail: splessas@agro.duth.gr (S. Plessas)

1
ABSTRACT

The novel Lactobacillus paracasei K5 strain, recently isolated from Greek cheese, was

evaluated as potential sourdough bread starter. Breads were made using different amounts of

L. paracasei sourdoughs as well as traditional sourdough for comparison. Quality

characteristics of the breads (acidity and rising) were examined, as well as rope spoilage

through macroscopic observations and molecular analysis (PCR-DGGE). The highest acidity

levels (3.15 g lactic acid and 1.13 g acetic acid per Kg of bread) and better resistance to rope

spoilage were observed when bread contained 30% w/w L. paracasei K5 sourdough. Spoilage

in the L. paracasei K5 breads was observed at 15-16 days, 5 days later than the control

breads. In addition, L. paracasei K5 sourdough improved the bread sensory properties, as

reflected by consumer preference and GC/MS analysis of aroma volatiles. Therefore, L.

paracasei K5 can be successfully used for sourdough bread making with good quality and

extended shelf-life.

Keywords: Lactobacillus paracasei K5, sourdough, quality, rope spoilage, PCR-DGGE

Chemical compounds studied in this article

Lactic acid (PubChem CID: 61503); Acetic acid (PubChem CID: 176); Butan-1-ol (PubChem

CID: 263); 2-Phenylethanol (PubChem CID: 6054); Benzaldehyde (PubChem CID: 240);

Ethanol (PubChem CID: 702); Ethyl acetate (PubChem CID: 8857); Furfural (PubChem CID:

7362); Isoamyl alcohol (PubChem CID: 31260); Isobutyl alcohol (PubChem CID: 6560)

2
1. Introduction

Bread making is one of the oldest food bioprocesses, practiced for thousands of years

(Shewry & Hey, 2015). Bread is still an essential food for humans, and a basic daily diet

component, especially in the developing world. It is estimated that in Europe the annual bread

consumption is approximately 58 Kg per capita, and about half that amount is made with

sourdough (Cappelle, Guylaine, Gänzle & Gobbetti, 2013). Sourdough is dough fermented

with naturally occurring or selected lactic acid bacteria and yeasts. The use of sourdough in

bread making offers several advantages, such as better flavour, improved nutritional value,

and extended shelf-life in terms of both sustaining good texture and flavour characteristics

(delayed staling), as well as resistance to microbial spoilage (Cavallo et al., 2017). In

addition, the use of sourdough may allow reduction or avoidance of chemical preservatives in

bread, therefore it is of great technological and nutritional importance. These advantages have

intensely attracted interest from food bioprocessing scientists to develop novel products with

improved properties, mainly based on the use of selected or novel sourdough starters (Simsek,

Hilmi Con & Tulumoglu, 2006; Arendt, Ryan & Dal Bello, 2007; Plessas, Fischer, Koureta,

Psarianos, Nigam & Koutinas, 2008; Plessas et al., 2011; Plessas, Alexopoulos, Bekatorou,

Mantzourani, Koutinas, & Bezirtzoglou, 2011; Silow, Axel, Zannini & Arendt, 2016).

The microbiological environment of a typical traditional (wild) sourdough is complex,

and the stability of sourdough microbiota depends on a number of parameters such as flour

microbiota, flour composition (carbohydrates, free amino acids, enzymes, etc.), fermentation

time and temperature, the number of sourdough refreshment steps, the fermentation time

between refreshments, and the use of starters and/or baker's yeast (Pontonio et al., 2017;

Mivervivi et al., 2012; Minervini, Lattanzi, De Angelis, Celano & Gobbetti, 2015; Van

Kerrebroeck, Maes & De Vuyst, 2017). Therefore, sourdough standardization is very difficult,

3
and this is a significant drawback for food industries, which require stability and

reproducibility of product quality. This problem can be overcome by using defined single or

mixed starter cultures for sourdough production, and many relative scientific works have been

published (Plessas et al., 2011; Plessas, Fischer, Koureta, Psarianos, Nigam & Koutinas,

2008; De Vuyst et al., 2014; Valerio, De Bellis, Lonigro, Visconti & Lavermicocca, 2008;

Katina, Sauri, Alakomi & Mattila-Sandholm, 2002; Mantzourani et al., 2014; Axel et al.,

2016; Reale et al., 2016).

Recently, a novel Lactobacillus paracasei strain, namely K5, was isolated from Greek

Feta-type cheese (Plessas et al., 2017). L. paracasei K5 displayed probiotic potential in a

series of established in vitro tests. L. paracasei is usually found in the human intestinal tract,

and has also been isolated from natural habitats, such as fermented dairy products and plant

material. Some L. paracasei strains with probiotic properties are used in functional food

production (Canani et al., 2017; Toh et al., 2013). In a recent study, the use of L. paracasei

K5, free or immobilized on wheat bran (to form a synbiotic biocatalyst), was reported for

industrial production of soft white cheese (Terpou et al., 2018). The investigation focused on

the effect of L. paracasei K5 on cheese quality and shelf-life along with monitoring the

viability of the strain during storage. The results of that study were very promising for the

commercial application of L. paracasei K5 for food production with potential probiotic

properties.

In the present study, the objective was to evaluate the suitability of L. paracasei K5 strain

for sourdough bread making. The work focused on the evaluation of quality characteristics,

and the shelf-life of breads produced with various amounts of L. paracasei K5 sourdough, as

well as on the antimicrobial effect of the novel starter against Bacillus spp. (rope spoilage),

compared with traditional sourdough.

4
2. Materials and methods

2.1 Microorganisms

The novel potential probiotic strain L. paracasei K5 was recently isolated from Greek Feta-

type cheese (Plessas et al., 2017) and is maintained at −80 °C at the Democritus University of

Thrace in de Man, Rogosa, and Sharpe (MRS) broth (Fluka, Buchs, Switzerland)

supplemented with glycerol (20%, v/v; Sigma–Aldrich, St. Louis, USA). For the bread

making experiments, it was grown in MRS broth at 37°C for 24 h and the culture was

maintained at 4°C in the broth, which was periodically refreshed with MRS broth. Further cell

mass production was also carried out in MRS broth at 37°C (Terpou et al., 2018). The culture

was harvested by centrifugation at 5000 rpm for 10 min, and the harvested cell mass was used

for sourdough bread making. Baker’s yeast was a commercial Saccharomyces cerevisiae

strain obtained in the form of pressed blocks (70% w/w moisture), manufactured by S.I.

Lesaffre, France.

2.2 Sourdough bread making using L. paracasei K5

A commercial soft white flour was used for bread making (Hellenic Biscuit CO S.A., Greece),

with the following composition (% w/w): protein 11.0, carbohydrates 72.0, fat 1.5, fibre 2.2

and moisture 12.0. During bread making, mixing of ingredients was performed mechanically

and the dough was moulded manually in 1.5 L baking pans.

Mother sponge was prepared by mixing 300 g wheat flour, 160 ml tap water and 30%

w/w (on flour basis) of L. paracasei K5 (wet weight) for 15 min. The sponge was incubated at

30oC for 24 h. Sourdough was prepared by mixing 250 g of fermented mother sponge with

5
300 g wheat flour and 160 ml tap water for 15 min, and it was incubated at 30oC for 24 h

(Sourdough A). Finally, breads containing approximately 20% and 30% w/w (on flour basis)

of Sourdough A were produced. The dough of the first type of bread (L1) contained 100 g

sourdough A, 500 g wheat flour, 270 ml tap water and 4 g salt. The dough of the second type

of bread (L2) contained 150 g Sourdough A, 500 g wheat flour, 270 g tap water and 4 g salt.

In all cases an amount of 1% w/w (on flour basis) of pressed baker’s yeast was added. All

doughs were fermented at 30oC for 2 h, proofed at 40oC for 60 min in order for the bread to

reach its highest volume before baking (Cauvain, 2015), and then it was baked at 230oC for

40 min.

Control trials were carried out with sourdoughs prepared with traditional, wild microflora

sourdough (Sourdough B), provided by a local bakery. Two sourdough breads were produced

containing 20% and 30% (on flour basis) of Sourdough B (W1 and W2, respectively), at the

same conditions as described above for the L. paracasei K5 sourdoughs. All trials were

carried out in triplicate.

2.3 Detection of rope spoilage by molecular identification of Bacillus spp.

2.3.1 Bread samples

Samples were received from all 4 types of sourdough breads, approximately 2 h after baking.

The samples were sliced aseptically to portions of 10 g and were stored at room temperature.

They were observed every day for quality characteristics (aroma, taste, overall quality) and

rope appearance (sweet rope odour, discoloration of the crumb, and sticky threads). At the

moment that spoilage was observed macroscopically all bread samples were subjected to

PCR-DGGE analysis as described below.

6
2.3.2 DNA extraction

At the moment that spoilage was observed macroscopically, the bread sample was

homogenized in sterilized ¼ Ringer’s solution (Sigma-Aldrich) and the suspensions were

subjected to DNA extraction using a DNeasy Tissue Kit (Qiagen) according to the

manufacturer’s protocol.

2.3.3 PCR and DGGE analysis

PCR and DGGE analysis were performed as described before (Mantzourani et al., 2014).

Briefly, bacterial DNA was amplified with primers V3f (5’-CCTACGGGAGGCAGCAG-3’)

and V3r (5’-ATTACCGCGGCTGCTGG-3’) (Pepe, Blaiotta, Moschetti, Greco & Villani,

2003). A GC clamp (5’-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG

CCCG-3’) was added to primer V3f. The reverse primer (V3r) was fluorophore-labeled and

synthesized with the addition of a 5’-terminal 6-carboxyfluorescein (FAM). Amplifications

were carried out in a thermal cycler (Mastercycler, Eppendorf, Germany), at a final volume of

50 μl, by using an amplification cycle characterized by an initial touchdown step in which the

annealing temperature was lowered from 65 to 55°C in intervals of 2°C every two cycles, and

10 additional cycles were done with annealing at 55°C. The template DNA was denatured for

5 min at 94°C and extension was performed at 72°C for 3 min. A final extension of 72°C for

10 min ended the amplification cycle. The PCR products were analyzed by DGGE by using

an INGENYphorU DGGE system (Ingeny). After the electrophoresis, the gels were scanned

with a fluorescent imager (Molecular Imager FX, BioRad) and the bands of interest were

excised, the DNA was eluted and reamplified using the V3f primer without the GC clamp,

and the V3r primer without the addition of a 5’-terminal 6-carboxyfluorescein (FAM). The

PCR products were purified, and sent for sequencing to VBC-Biotech, Austria. Searches in

the GenBank with the BLAST program were performed to determine the closest known

7
relatives of the partial 16S rDNA sequences obtained. DGGE analyses were performed at

least twice.

2.4 Organic acids analysis

Amounts of 10 g of bread were mixed with 90 ml of sterile distilled water for 2 min through a

Stomacher Blender 400 (Seward Laboratory, London, UK). The bread extracts were

centrifuged at 20,000 rpm and organic acids (lactic and acetic) were determined by ion-

exchange liquid chromatography as described before (Plessas, Fischer, Koureta, Psarianos,

Nigam & Koutinas, 2008). In brief, a Shimadzu HPLC system was used consisting of a Shim-

pack ICA1 column, a LC-10AD pump, a CTO-10A oven (40°C) and a CDD-6A conductivity

detector. Phthalic acid (2.5 mM) and tris(hydroxymethyl)aminomethane (2.4 mM; pH 4.0)

was used as mobile phase (1.2 ml/min). The sample dilution was 5% v/v and the injection

volume was 60 μl. Determinations of all organic acid concentrations were carried out by

means of standard curves of lactic and acetic acids (Fluka), and valeric, isovaleric, and

hexanoic acids (Sigma-Aldrich) (Plessas, Fischer, Koureta, Psarianos, Nigam & Koutinas,

2008).

2.5 Determination of pH and total titratable acidity

The sourdough and sourdough bread pH values were determined using a Sentron pH-meter,

type Argus. For the determination of total titratable acidity (TTA), 10 g of breadcrumb were

blended with 90 ml of deionised water and the suspension was titrated with 0.1 N NaOH to a

final pH 8.5. The TTA was expressed as the volume (ml) of NaOH consumed.

8
2.6 Determination of specific loaf volume

After cooling, the loaves were weighed and the loaf volume was measured by the rapeseed

displacement method (Hallen, Ibanoglu & Ainsworth, 2004). Each loaf was placed in a

container and was covered with rapeseed to totally fill the container. After the removal of the

loaf, the volume of the rapeseed was noted. The loaf volumes were calculated by deducting

the rapeseed volume from the container volume. The specific loaf volume was calculated as

ml/g.

2.7 Analysis of aroma volatiles

Gas chromatography/mass spectrometry (GC/MS) analysis of volatile compounds was carried

out using the headspace solid-phase microextraction (SPME) sampling technique as described

before (Plessas, Fischer, Koureta, Psarianos, Nigam & Koutinas, 2008; Plessas, Alexopoulos,

Bekatorou, Mantzourani, Koutinas, & Bezirtzoglou, 2011). In brief, 2 g of sample were

placed in a 20 ml glass vial sealed with a rubber septum and immersed in a water bath at

60oC. The SPME needle was introduced through the septum and the fibre (2 cm, 50/30 mm

DVD/Carboxen/PDMS Stable Flex Supelco, Bellefonte, PA, USA) was exposed to the

headspace for 60 min. Desorption of volatiles compounds took place into the injector port of

the gas chromatograph for 5 min at 280°C in splitless mode. The GC/MS analysis was

performed on a Shimadzu GC-17A gas chromatograph coupled to a GCMS-QP5050A mass

spectrometer. A Supelco CO WAX-10 column (0.25 μm film thickness; 60 m×0.32 mm i.d.)

was used. The column temperature program was: 35°C, held for 5 min; increased by 5°C/min

to 50°C, held for 5 min; increased by 5.5°C/min to 230°C, held for 5 min. The total run time

was 51.73 min. The carrier gas was He (2 ml/min). The scan mode was used to detect all

9
compounds in the range m/z 33–200 and mass spectra were recorded by electronic impact (EI)

at 70 eV. Identification of volatile compounds was done by comparison with standard

compounds (Sigma-Aldrich) and MS data with those in NIST107, NIST21 and SZTERP

libraries. The identification of volatiles was done by comparison of the MS data with those of

standard compounds and those in NIST107, NIST21 and SZTERP libraries. For semi-

quantitative analysis of volatiles, 4-methyl-2-pentanol (Sigma–Aldrich) diluted in pure

ethanol was used as the internal standard (IS) at various concentrations (4, 40 and 400 μg/g of

sample). The volatile compounds were quantified by dividing the peak areas of the

compounds of interest by the peak area of the IS and multiplying this ratio by the initial

concentration of the IS (expressed as μg/g) (Plessas, Alexopoulos, Bekatorou, Mantzourani,

Koutinas, & Bezirtzoglou, 2011). Bread samples of similar shape and size were cut from the

same loaf of bread and kept in closed containers. One piece of bread was analyzed every day

for a total period of 5 days. All assays were carried out in triplicate.

2.8 Consumer sensory evaluations

The sourdough breads were daily evaluated at a local bakery for a total of 15 days.

Specifically, 25 untrained testers (consumers) scored the produced breads according to a

preference protocol based on a 0 (unacceptable) to 10 (excellent) scale (Plessas, Fischer,

Koureta, Psarianos, Nigam & Koutinas, 2008). At least 3 samples were provided to each

tester. In addition, the testers were advised to express their opinion regarding the appearance

of spoilage by evaluation of the main spoilage characteristics (distinct flavour of ripe

cantaloupe, discolouration, and sticky texture) (Pepe, Blaiotta, Moschetti, Greco & Villani,

2003). The results were recorded as average scores plus standard deviations for aroma, taste

and overall quality (volume, texture, colour and flavour).

10
2.9 Statistical analysis

The data obtained from physicochemical characteristics, aroma volatile compounds and

sensorial analysis of the various breads were analysed for their mean differences with the

Analysis of Variance (ANOVA) procedure followed by Duncan’s post hoc multiple range test

to extract the specific differences between the various treatments. Analysis was performed by

using IMB SPSS v20 (IBM Corp.) at an alpha level of 5%.

3. Results and discussion

In a recent study the L. paracasei K5 strain was screened among 45 lactic acid bacteria

isolates from Greek Feta type cheese, for its probiotic potential based on a series of

established in vitro tests (tolerance to low pH, pepsin, pancreatin, and bile salts, susceptibility

against common antibiotics, etc.) (Plessas et al., 2017). The performance of strain K5 was

similar or even better than the reference probiotic strain L. plantarum ATCC 14917 regarding

results obtained from international in vitro tests (Plessas et al., 2017). In that study, the

technological performance of L. paracasei K5, for possible use in functional food production,

was also evaluated in fermentation of pomegranate juice. A novel multiplex PCR assay, based

on random amplified polymorphic DNA (RAPD) analysis was also developed for accurate

detection of the strain in foods. Recently, the suitability of the strain was also confirmed in

soft white cheese production (Terpou et al., 2018). In the present study, the potential of L.

paracasei K5 strain as a starter culture for sourdough bread making was examined.

Specifically, sourdough breads were prepared using sourdough containing either the L.

paracasei K5 strain or traditional sourdough (wild microflora). The breads were made with

different amounts of added sourdough (20 and 30% w/w on flour basis) (Katina, Sauri,

11
Alakomi & Mattila-Sandholm, 2002; Plessas, Alexopoulos, Bekatorou, Mantzourani,

Koutinas, & Bezirtzoglou, 2011), and were analysed for pH, TTA and specific loaf volume

(Table 1 and Figure 1), organic acids (Table 1) and aroma volatile compounds (Table 2). The

breads were also subjected to consumer sensory evaluations (Table 3). Finally, the appearance

of rope spoilage was examined through macroscopic observations as well as molecular

analysis (PCR-DGGE) (Table 4 and Figure 2).

3.1 Sourdough bread quality characteristics

The final moisture of all breads was 31-35%, and rising was good in all cases (above 2 ml/g)

(Plessas, Fischer, Koureta, Psarianos, Nigam & Koutinas, 2008; Plessas, Pherson, Bekatorou,

Nigam & Koutinas, 2005), while pH values were lower compared to the control sourdough

breads. The same was observed for the TTA values, which were higher (statistically

significant) in the case of the sourdough breads prepared with L. paracasei K5 (L2: 8.6 ml

NaOH N/10). This was expected since the initial load of acidity was statistically higher in

Sourdough A (TTA 13.1 ml NaOH N/10 and pH 3.8) prepared with L. paracasei K5 than in

the control Sourdough B (TTA 12.1 ml NaOH N/10 and pH 4.1) (Figure 1). These findings

are very good taking into account other investigations that define as proper acidification

properties the TTA values above 6 ml NaOH N/10 and pH values under 5. For example,

addition of 20–30 g of sourdough fermented with selected lactic acid bacteria per 100 g of

wheat dough (leading to pH values of sourdoughs below 4.0 and TTA values above 12),

inhibited the growth of Bacillus spp. (Katina, Sauri, Alakomi & Mattila-Sandholm, 2002).

Similar results were obtained in the case of individual organic acids analysis (Table 1).

Both sourdough breads containing L. paracasei K5 (L1 and L2) had higher concentrations of

all the analysed organic acids (lactic, acetic, isovaleric, pentanoic and hexanoic acid). Bread

12
made with 30% w/w sourdough containing L. paracasei K5 (L2) had the highest (statistically

significant) acidity regarding lactic acid production (3.15 g lactic acid/Kg of bread) compared

to all the other sourdough bread samples while in the case of acetic production, sourdoughs

samples L2 and W2 contained the higher amounts and 1.13 g acetic acid/Kg of bread and 1.01

g acetic acid/Kg of bread respectively) compared to the other sourdough bread samples (L1

and W1). In particular, lactic acid concentration determined in L2 sample was 22.5% higher

than in W2 and 16% higher than L1, while acetic acid concentration determined in L2 sample

was 10.6 % higher than in W2 and 26.5% higher than L1. Regarding the other organic acids

determined (isovaleric, pentanoic and hexanoic acid) higher concentrations were observed for

bread samples L2 and W2. Similar results were obtained in a previous study regarding the use

of kefir for sourdough bread production (Mantzourani et al., 2014). In that case, the levels of

both lactic and acetic acid in the sourdough breads prepared with kefir grains were

approximately 41–82% higher (2.22–2.85 g/Kg bread) than the control samples (wild

microflora), while acetic acid concentration alone was about half- to one-fold higher (0.77–

1.00 g/Kg bread), respectively.

3.2 Aroma volatiles and sensory evaluations

Regarding the semi-quantitative determination of aroma-related compounds (μg/g) extracted

from the sourdough bread samples by SPME and analysed by GC/MS, the results are

presented in Table 2. Various volatile compounds were identified including alcohols, esters

and carbonyl compounds that can potentially influence bread flavour (Hansen & Schieberle

2006; Plessas, Alexopoulos, Bekatorou, Mantzourani, Koutinas, & Bezirtzoglou, 2011;

Plessas et al., 2011). Specifically, key volatile compounds (Pétel, Onno & Prost, 2017) such

as heptanol, 2-phenylethyl acetate, 1-octen-3-ol, benzaldehyde, 2- nonenal and furfural, were

13
in most cases identified or determined at higher concentrations in the L. paracasei K5

sourdough breads L1 and L2.

Even though the presence or absence of a volatile compound might not exhibit a

detectable effect on the flavour of bread, it is noteworthy that the sourdough bread sample

(L2) had a more complex composition of headspace volatile compounds (more compounds

identified and at higher concentrations) (Table 2). According to the literature, the higher the

complexity of volatile compounds, the better the aroma of bread is (Pétel, Onno & Prost,

2017; Aslankoohi, Herrera-Malaver, Rezaei, Steensels, Courtin & Verstrepen, 2016;

Gassenmeier & Schieberle, 1995; Plessas, Alexopoulos, Bekatorou, Mantzourani, Koutinas,

& Bezirtzoglou, 2011). Specifically, the sourdough bread L2 contained 6.52 μg/g of alcohols,

0.98 μg/g of esters and 4.35 μg/g of carbonyl compounds, which were much higher compared

to the other sourdough bread samples (L1, W1 and W2). This is in accordance with the

preliminary sensory evaluation conducted by the non-trained testers (consumers) (Table 3),

showing a similar pattern of preference among the displayed sourdough breads (better scores

for the L2 samples).

3.3 Appearance of rope spoilage

The appearance of rope spoilage in all bread samples was examined through macroscopic

observations and sensorial tests (Table 3). The results showed that the L. paracasei K5

sourdough breads (L1 and L2) resisted to spoilage longer than the control breads (W1 and

W2). Specifically, spoilage in the sourdough bread samples L1 and L2 was obvious at the 15th

and 16th day of storage, respectively, compared to the control breads W1 and W2 that were

developed spoilage at the 10th and 11th day of storage, respectively (Table 3). In addition, the

14
sourdough bread sample L2, received the best preference scores during the consumer

evaluation (Table 3).

At the moment that spoilage was observed macroscopically, all bread samples were

subjected to PCR-DGGE analysis to further verify these observations. DGGE is a commonly

used methodology for the identification of microbial species in complex populations. It is a

rapid and affordable method, allowing multiple samples to be processed simultaneously

(Green, Leigh & Neufeld, 2015; Mantzourani et al., 2014). The results showed that Bacillus

subtilis and Bacillus cereus were detected at the 16th and 15th day of storage in sourdough

breads L2 and L1, respectively (Figure 2, Table 4). In the same manner, B. subtilis spoilage in

the control sourdough bread samples W1 and W2 was detected at the 10th and 11th day,

respectively. Of note, at earlier days of storage, Streptococcus salivarius is usually present in

the traditional sourdough microflora (Corsetti, Settanni, Valmorri, Mastrangelo & Suzzi,

2007; Robert, Gabriel & Fontagné-Faucher, 2009). The extended shelf-life of sourdough

breads L1 and L2 can be attributed to the higher concentrations of organic acids, especially

lactic and acetic acids, as it has been explained before since they both exhibit antimicrobial

activities and are effective against rope spoilage (Simsek, Hilmi Con & Tulumoglu, 2006;

Mantzourani et al., 2014). Last but not least, the higher levels of the other minor organic acids

(isovaleric, pentanoic and hexanoic acid) in sourdough bread sample L2 may also exhibit

antimicrobial effect since, as it has been reported in the past, they may act in a synergistic

way protecting the sourdough bread from microbial spoilage (Katina, Sauri, Alakomi &

Mattila-Sandholm, 2002; Mantzourani et al., 2014).

4. Conclusions

15
The results obtained in this study indicate that the L. paracasei K5, recently isolated from

Greek Feta type cheese, can be successfully used for sourdough bread making. The produced

sourdough breads displayed good quality in terms of rising and acidity, as well as improved

sensory properties, as indicated by consumer preference evaluations and GC/MS analysis of

aroma volatiles. Moreover, macroscopic observations and PCR-DGGE analysis showed that

rope spoilage was delayed compared to the control (traditional sourdough) breads. Therefore,

L. paracasei K5 can be used for good quality and extended shelf-life sourdough bread

making.

Conflict of interest

The authors declare that there are no conflicts of interest in this research article.

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Figure Captions

Figure 1. TTA and pH values determined in sourdoughs A (L. paracasei K5) and B (wild

microbiota).

Figure 2. Bacterial DGGE profile of DNA extracted from bread. Lane 1 corresponds to bread

sample W1 spoiled at the 10th day, lane 2 to bread sample W2 spoiled at the 10th day, lane 3 to

bread sample L1 spoiled at 15th day, and lane 4 to bread sample L2 spoiled at the 16th day of

storage. Bands indicated by letters were excised and, after reamplification, subjected to

sequencing.

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 Table 1. Physicochemical characteristics (acidity, rising, and organic acids content) of breads

made with 20% and 30% w/w (on flour basis) L. paracasei K5 sourdough A (L1 and L2,

respectively), and with equal amounts of control sourdough B (W1 and W2, respectively).

Starter TTA (ml Organic acids (g/Kg bread)

Sourdough
Amount pH NaOH SLV (ml/g)
starter Lactic Acetic Isovaleric Pentanoic Hexanoic
(%w/w) N/10)

L. paracasei K5

L1 20 4.7±0.1a 6.8±0.3b 2.4±0.1a 2.65±0.18b 0.83±0.12b 0.04±0.01b 0.05±0.01a 0.03±0.01b

L2 30 4.5±0.1b 8.6±0.35a 2.5±0.1a 3.15±0.25a 1.13±0.11a 0.07±0.01a 0.06±0.01a 0.05±0.01a

Control

W1 20 5.2±0.1c 5.9±0.3c 2.5±0.1a 1.73±0.13c 0.58±0.11c 0.04±0.01b Tr Tr

W2 30 4.8±0.1a 6.5±0.3b 2.5±0.1a 2.44±0.09b 1.01±0.06a 0.08±0.01a 0.03±0.01b 0.03±0.01b

TTA: total titratable acidity; SLV: specific loaf volume; Tr: Traces (<0.01 g/Kg)

Different superscript letters in a column indicates statistically significant differences (ANOVA, Duncan’s multiple

range test, p<0.05)

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 Table 2. SPME GC/MS analysis of aroma-related compounds (μg/g) extracted from

sourdough bread samples produced using L. paracasei K5 sourdough (L1 and L2) and control

sourdough (W1 and W2).

Concentration (μg/g)
KI Compound RI
L1 L2 W1 W2

Alcohols

832 Ethanol a 4.15±0.15a 4.07±0.17a 4.19±0.12a 4.13±0.18a

1012 Isobutyl alcohol a 0.28±0.05a 0.19±0.05b 0.11±0.04b 0.12±0.03b

1120 Isoamyl alcohol a 0.48±0.04a 0.54±0.07a 0.14±0.03b 0.18±0.05b

1160 1-Butanol a 0.18±0.05ab 0.27±0.04a 0.14±0.05b 0.18±0.05ab

1230 1-Pentanol b 0.25±0.07a 0.19±0.04a nd 0.10±0.03b

1257 1-Hexanol a 0.15±0.08b 0.25±0.05a 0.07±0.01c 0.09±0.02c

1435 1-Heptanol b Tr 0.09±0.02 nd nd

1466 1-Octanol a 0.07±0.04b 0.15±0.05a nd nd

1480 2-Heptanol a 0.05±0.05a 0.09±0.03a nd nd

1540 1-Octen-3-ol b 0.15±0.05b 0.16±0.05a nd nd

1670 Benzyl alcohol a 0.11±0.05b 0.19±0.04a 0.08±0.02b 0.12±0.04ab

1812 2-Phenyl ethanol a 0.24±0.05b 0.33±0.05a 0.09±0.03c 0.19±0.04b

Esters

<800 Ethyl acetate a 0.18±0.04a 0.22±0.05a 0.05±0.01b 0.09±0.02b

1107 Butyl acetate a 0.07±0.02ab 0.10±0.04a Tr 0.05±0.01b

1162 Hexyl acetate b 0.04±0.01a 0.06±0.01a nd 0.03±0.01a

1250 Ethyl pentanoate b 0.06±0.02a 0.09±0.01a 0.03b 0.03±0.01b

1395 Ethyl exanoate b 0.09±0.01a 0.12±0.01a nd nd

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 1438 Ethyl octanoate b 0.03±0.01a 0.05±0.01a nd nd

1445 Ethyl heptanoate a 0.07±0.01a 0.09±0.02a nd nd

1590 Isobutyl acetate b 0.05±0.02a 0.08±0.02 a nd nd

1848 Ethyl dodecanoate b 0.06±0.01a 0.09±0.01a nd nd

1850 2-Phenyl-ethyl acetate b Tr 0.04±0.01 nd nd

2410 Ethyl octadecanoate b nd 0.04±0.01 nd Tr

2429 Ethyl 9-octadecenoate b nd 0.03±0.01 nd Nd

Carbonyl compounds

<800 Acetaldehyde b 0.23±0.04b 0.32±0.05a 0.08±0.01c 0.09±0.01c

812 Butanal, 2-methyl b 0.09±0.01b 0.11±0.02a 0.04±0.01c 0.04±0.01c

986 Butanal, 3-methyl a 0.38±0.05a 0.39±0.05a 0.03±0.01b 0.06±0.01b

1002 Hexanal a 0.08±0.01a 0.10±0.03a 0.04±0.01b 0.04±0.01b

1080 Heptanal a Tr 0.07±0.01 nd Tr

1100 2,3 Butanedione b nd 0.06±0.01 nd nd

1334 Furfural a 0.19±0.04c 0.32±0.05a 0.18±0.05c 0.25±0.05b

1358 Nonanal b 0.08±0.01b 0.27±0.05a Nd Tr

1448 Butyrolactone b 1.25±0.05b 1.89±0.15a 0.53d 0.92±0.11c

1458 Benzaldehyde a 0.29±0.05b 0.41±0.07a 0.10c 0.18±0.01c

1541 2-Nonenal b 0.08±0.0b 0.13±0.05a nd 0.05±0.01b

1582 5- Methyl-furfural b 0.14±0.04b 0.28±0.02a nd 0.05±0.01c

1. KI: Kovats Index; RI: Reliability of identification; a: Positive identification by MS data and retention times
and those of standard compounds. b: Positive identification by MS data only. Tr: Compound present at
<0.01 μg/g bread (traces); nd: not detected.
2. Different superscript letters in a row indicates statistically significant differences (ANOVA, Duncan’s
multiple range test, p<0.05)

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Table 3. Preliminary sensory evaluation test during a 16-day storage of breads made with L.

paracasei K5 sourdough A and with the control sourdough B.

Storage time (days) Bread sample Aroma Taste Overall quality

1 L1 8.5 ± 0.2ab 8.7 ± 0.1b 9.2 ± 0.2ab

1 L2 8.8 ± 0.2a 8.9 ± 0.1a 9.2 ± 0.1a

1 W1 8.4 ± 0.1b 8.5 ± 0.1c 8.9 ± 0.2abc

1 W2 8.5 ± 0.1ab 8.6 ± 0.1bc 8.9 ± 0.3ac

3 L1 8.2 ± 0.2b 8.3 ± 0.1a 8.0 ± 0.2a

3 L2 8.3 ± 0.1b 8.4 ± 0.1a 8.3 ± 0.2a

3 W1 7.7 ± 0.1c 7.9 ± 0.1b 8.1 ± 0.2a

3 W2 9.0 ± 0.3a 8.0 ± 0.1b 8.2 ± 0.1a

5 L1 7.5 ± 0.1a 7.1 ± 0.1b 7.3 ± 0.2b

5 L2 7.8 ± 0.2a 7.5 ± 0.1a 7.7 ± 0.1a

5 W1 6.9 ± 0.3b 7.0 ± 0.1b 7.1 ± 0.1b

5 W2 7.0 ± 0.3b 7.0 ± 0.2b 7.3 ± 0.1b

8 L1 6.7 ± 0.2a 7.1 ± 0.1ab 6.7 ± 0.3ab

8 L2 6.9 ± 0.1a 7.3 ± 0.2a 7.0 ± 0.3a

8 W1 6.5 ± 0.3a 6.8 ± 0.1b 6.4 ± 0.1b

8 W2 6.6 ± 0.3a 6.9 ± 0.2b 6.4 ± 0.1b

10 W1 SPOILED (discoloured and sticky)

10 W2 5.7 ± 0.3b 5.8 ± 0.2b 5.4 ± 0.1b

10 L1 6.3 ± 0.1a 6.1 ± 0.3ab 5.8 ± 0.1ab

10 L2 6.5 ± 0.1a 6.4 ± 0.1a 6.3 ± 0.3a

11 W2 SPOILED (discoloured and sticky)

25
11 L1 5.9 ± 0.2a 5.7 ± 0.1a 5.5 ± 0.2b

11 L2 6.1 ± 0.1a 6.1 ± 0.3a 6.0 ± 0.2a

15 L1 SPOILED (discoloured and sticky)

15 L2 5.7 ± 0.1 5.5 ± 0.3 5.7 ± 0.3

16 L2 SPOILED (discoloured and sticky)

L1 and L2: breads made with 20% and 30% (w/w on flour basis) L. paracasei K5

sourdough A, respectively. W1 and W2: breads made with 20% and 30% (w/w on flour

basis) control sourdough B, respectively. Different superscript letters (per storage day)

indicate statistically significant differences (ANOVA, Duncan’s multiple range test,

p<0.05).

Different superscript letters (per storage day) indicates statistically significant

differences (ANOVA, Duncan’s multiple range test, p<0.05)

26
Table 4. Sequencing results of bands cut from the DGGE gel.

a b
Band Most closely related species Identity (%) Accession Number

A Bacillus subtilis 99 KT007498.1

B Bacillus cereus 99 KP100400.1

Bacillus subtilis 99 JQ311931.1

C Bacillus subtilis 98 KT007498.1

D Bacillus cereus 98 GQ226038.1

Bacillus subtilis 98 JQ311992.1

E Streptococcus salivarius 98 GU425980.1

Streptococcus salivarius 98 GU425969.1

F Streptococcus salivarius 99 GU425980.1

a
Bands are lettered as indicated on DGGE gel shown in Figure 2; bAccession numbers of

sequences of most closely related species found with Blast search.

27
Fig. 1

28
Fig. 2

29
Highlights

 Novel, potentially probiotic, Lactobacillus paracasei K5 used as sourdough starter

 The K5 breads had good quality (rising, acidity, organic acids, consumer preference)

 The K5 breads had more complex volatile profiles as shown by SPME-GC/MS

 The appearance of rope spoilage was delayed (macroscopic and PCR-DGGE analysis)

 L. paracasei K5 can be used for good quality & extended shelf-lifesourdough bread

30