Food Control 18 (2007) 359–363

www.elsevier.com/locate/foodcont

Inhibitor activities of two Lactobacillus strains, isolated

from sourdough, against rope-forming Bacillus strains
a,*
Özay Mentesß , Recai Ercan a, Mustafa Akçelik b

a
Department of Food Engineering, Faculty of Engineering, Ankara University, Diskapi Campus, Diskapi, 06110 Ankara, Turkey
b
Department of Biology, Faculty of Science, Ankara University, Tandogan, Ankara, Turkey

Received 3 May 2005; received in revised form 26 October 2005; accepted 30 October 2005

Abstract

The effect of two different sourdoughs, produced with Lactobacillus plantarum LMO25 and Lactobacillus alimentarius LMO7, with
antimicrobial activities on inhibition of rope-forming Bacillus strains in wheat bread was studied. Addition of 15% or 20% low pH
(pH 3.5–4.0) sourdough to bread dough, which were produced by using two strains (Lb. plantarum LMO25 and Lb. alimentarius
LMO7) separately, prevented the generation of visual rope caused by Bacillus subtilis and Bacillus licheniformis. However, adding
10% sourdough was not enough to prevent the generation of visual rope. When repeated with sourdoughs with a higher pH
(pH > 4), additives at 10% or 15% did not prevent the generation of rope, whereas additives at 20% prevented the generation of visual
rope caused by both B. subtilis and B. licheniformis.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Rope spoilage; Sourdough; Bacteriocin; Lactobacillus; Bacillus

1. Introduction extracellular, slimy polysaccharides (Rosenquist & Hansen,

All over the world, microbial attacks on bread cause
very important losses in baking industry. Main reasons
for these losses are the suitable conditions in bread and
other flour based foods for microbial growth, such as aw
and pH. The frequent problems, occurring most frequently
in baking industry, are mould contamination and rope
spoilage (Jenson, 1998).
Rope spoilage of bread is usually caused by Bacillus
spp., especially B. subtilis and B. licheniformis. Rope can
become noticeable within 12–24 h after loaf is taken from
the oven. The spoilage is initially noticed as an unpleasant
odour, followed by a discoloured, sticky soft bread crumb
caused by the breakdown of starch and proteins by micro-
bial amylases and proteases, and by the production of
*
Corresponding author. Tel.: +90 312 317 0550x1175; fax: +90 312 317
8711.
E-mail address: mentes@eng.ankara.edu.tr (Ö. Mentesß).
1995; Von Holy & Allan, 1990; Voysey & Kaur, 1987).
When the cell counts of B. subtilis and B. licheniformis
reach over 105 cfu/g, they present a potential risk of food
borne illnesses (Kirschner & Von Holy, 1989). In bread
production, to avoid the growth of these and other micro-
bial contaminants, organic acids or approximately 15%
sourdough can be added to common dough (Voysey &
Hammond, 1993). The antimicrobial activity of sourdough
arises from lactic acid, acetic acid, carbon dioxide, diacetyl,
ethanol, hydrogen peroxide and bacteriocins produced by
lactic acid bacteria during fermentation. Bacteriocin-pro-
ducing Lactobacillus strains are preferred as sourdough
starter cultures because of their strong antimicrobial activ-
ity by bacteriocins (Rosenquist & Hansen, 1998; Vogel
et al., 1999; Voysey & Hammond, 1993). As well as the
antimicrobial effects of sourdough, its addition improves
dough properties, bread texture and flavour, retards the
staling process and extends the mould free shelf life. Sour-
dough addition is a promising procedure to protect bread
from spoilage, since it is in agreement with the consumer

0956-7135/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2005.10.020
360 Ö. Mentesß et al. / Food Control 18 (2007) 359–363
demand for natural and additive-free products (Messens &
De Vuyst, 2002).
The aim of this study was to investigate the effect of Lac-
tobacillus strains, which have antibacterial activity (Lb.
plantarum LMO25 and Lb. alimentarius LMO7), in two
different sourdoughs against growth of B. subtilis and
B. licheniformis.
2. Materials and methods
2.1. Bacterial strains and media
Lb. plantarum LMO25 and Lb. alimentarius LMO7
strains, which have antimicrobial activities against B. sub-
tilis and B. licheniformis, were isolated from sourdough
(Mentesß, Akçelik, & Ercan, 2004). Rope-forming Bacillus
strains were supplied from Department of Biotechnology
and Biochemistry, Agricultural University of Norway. Lac-
tobacillus strains were grown in MRS medium (De Man,
Rogosa, & Sharpe, 1960) at 37 C for 24 h. Bacillus strains
were grown in Nutrient Broth (Merck) at 30 C for 24 h
with shaking.
2.2. Physicochemical analyses
Frozen bread and sourdough samples were defrosted
overnight in a refrigerator. The pH values were measured
according to AOAC Method No.: 981.12 (AOAC, 1990).
For the determination of total titratable acidity (TTA),
10 g sourdough or bread sample were blended with 5 mL
acetone and 50 mL distilled water were added to this mix-
ture. This suspension was transferred into a beaker
(200 mL) by washing 45 mL distilled water and titrated
against 0.1 N NaOH to a final pH of 8.5. Total titratable
acidity was expressed as the amount of NaOH used (mL)
(Uluöz, 1965).
2.3. Preparation of spore suspensions
The Bacillus strains were inoculated into Nutrient Agar
(Merck) supplemented with 10 mg/L of MnSO4 · H2O and
incubated at 30 C for 6 d. Spores were collected with pep-
tone buffered saline (PBS) and the cultures were heated at
85 C for 15 min in a water bath then rapidly cooled to
room temperature with cold water. The spore suspensions
were centrifuged (7000g, 20 min, 20 C) and resuspended
in PBS. The spore counts were determined by plating on
plate count agar (PCA) at 30 C for 72 h (Katina, Sauri,
Alakomi, & Sandholm, 2002).
2.4. Preparation of cultures for sourdoughs
Lb. plantarum LMO25 and Lb. alimentarius LMO7
strains were grown in MRS broth overnight at 37 C. An
inoculum from overnight culture (1%) was inoculated into
10 mL MRS broth and incubated for a further 3 h. Cells
were harvested by centrifugation, washed once with PBS
and resuspended in PBS. Bacterial counts were determined
by plating on MRSA (72 h, 37 C). Fresh cells were added
to sourdough at a level of 107 cfu/g (Rosenquist & Hansen,
1998).
2.5. Preparation of sourdough
Sourdough I [pH 3.5–4.0 and total titratable acidity
(TTA) > 12]: An inoculum of lactic acid bacteria was
added to the mixture of 700 g of water and 300 g of wheat
flour. The mixture was allowed to ferment at 35 C for 22–
24 h without agitation before inoculation of sourdough at
10 g, 15 g and 20 g/100 g bread dough.
Sourdough II [pH > 4.0 and total titratable acidity
(TTA) 3–12]: An inoculum of lactic acid bacteria
(107 cfu/g) was added to the mixture of 700 g of water
and 300 g of wheat flour. The mixture was allowed to fer-
ment at 35 C for 12–14 h without agitation before inocu-
lation of sourdough at 10 g, 15 g and 20 g/100 g bread
dough. Samples of 50 g were frozen for measurements of
pH and TTA (Katina et al., 2002).
2.6. Baking procedures
Breads were made according to Rapid-Mix Test. An
amount of 5.0% pressed yeast, 1.5% salt, 1.0% sugar,
1.0% fat and water, adjusted according to the water
absorption determined by a farinograph, was added to
the white wheat flour. Concentrations are based on flour.
In control bread I, B. subtilis or B. licheniformis spores
were added to the bread recipe described above at 100
and 250 spores/g dough, respectively. To determine the
ability of selected lactic acid bacteria to inhibit the growth
of rope-forming Bacillus strains in wheat bread, sourdough
were added to the bread recipe in addition to Bacillus
spores. The amounts of water and flour were identical in
control breads and sourdough fermented bread. All ingre-
dients were mixed for 1 min (1400 rpm/min). After proof-
ing at 32 C for 20 min, the dough was degassed and
proofed for further 10 min. Then it was scaled into four
400 g portions, rounded manually and kept at 32 C and
80 ± 5% rh for 30 min. At the end of the proofing time
dough pieces were baked at 225 ± 5 C for 25 min and then
cooled in aseptic conditions for 2 h. The loaves were cut
with sterilized knives into slices and packed individually
in polyethylene bags. Individually packed bread slices were
stored up to 7 d at 30 C. Two slices of bread were frozen
( 20 C) for later measurements of pH and TTA (Anony-
mous, 1971; Pepe, Blaiotta, Moschetti, Greco, & Villani,
2003). The wheat flour used in this study contained 14%
moisture; protein (N · 5.7), 12.7% of dry matter (dm);
ash, 0.63% of dm; and wet gluten, 29.6%.
2.7. Detection of rope formation in wheat bread
Individually packed bread slices were checked daily for
possible rope symptoms. On the day of visible roping,
 Ö. Mentesß et al. / Food Control 18 (2007) 359–363 361

bread slices (20 g) were homogenized in PBS with orbital
shaker and number of Bacillus cells was determined by
plating serial dilutions onto Plate Count Agar. Bacillus like
colonies were counted after incubation at 30 C for 48 h.
Bacillus spore numbers were determined on the day of
visual roping in control bread I (Thompson, Waites, &
Dodd, 1998).
3. Results and discussion
It was previously reported that lactic acid bacteria are
predominant microorganisms in sourdough, and in many
cases yeasts are present in significant numbers (Vogel
et al., 1999). Sourdough is an essential ingredient for ensur-
ing baking properties of doughs and preserving bread from
spoilage (Messens & De Vuyst, 2002). In this study, we
investigated the inhibitory activities of two Lactobacillus
strains, Lb. plantarum LMO25 and Lb. alimentarius
LMO7, against B. subtilis and B. licheniformis. Sourdoughs
prepared with two Lactobacillus strains were added to
bread dough at 10, 15, 20 g sourdough/100 g bread dough
and tested for their inhibitory activities in low (3.5–4.0) or
high pH (>4.0) against B. subtilis or B. licheniformis which
were inoculated to bread dough at 100 or 250 spores/g
dough, respectively. Two controls, bread I without sour-
dough addition and bread II without B. subtilis and B.
licheniformis as well as sourdough addition were used in
each baking experiment.
Initially the effect of Lactobacillus strains in low pH
sourdough were investigated. In control bread I, which
was inoculated with 100 spores/g of B. subtilis, rope spoil-
age was observed after 4 d storage at 30 C and the number
of B. subtilis in bread was found as 4.5 · 108 cfu/g. Nega-
tive control bread II did not show any rope symptoms dur-
ing 7 d storage. Although addition of 10% sourdough
produced either with Lb. plantarum LMO25 or Lb. alimen-
tarius LMO7 were insufficient to prevent the generation of
visible rope the number of B. subtilis were reduced to 1.6–
2.3 · 102 cfu/g. When the level of sourdough addition was
increased to 15%, both Lactobacillus strains inhibited rope
formation and recovered numbers of B. subtilis were
decreased to 1.9–3.5 · 102 cfu/g. Addition of 20% sour-
dough inhibited both rope spoilage and growth of B. sub-
tilis (Table 1).
B. licheniformis inoculation (250 spores/g) to bread
dough did not cause obvious visual roping in the control
bread I until 7 d of storage and the numbers of B. licheni-
formis recovered (1.8 · 108 cfu/g) was lower than B. sub-
tilis. Low number of B. licheniformis might cause the
reduced rope-forming. Although addition of 10% sour-
dough produced with Lb. plantarum LMO25 or Lb. alimen-
tarius LMO7 did not prevent rope formation, both
addition of 15% and 20% sourdough reduced the number
of B. licheniformis to 102 and <10 cfu/g, respectively and
inhibited rope formation (Table 2).
When the test bakings were repeated with sourdough
with higher pH (>4), additions at 10% or 15% did not pre-
vent the development of rope. However, 20% sourdough
addition prevented the generation of visual rope caused
by both B. subtilis and B. licheniformis (Tables 3 and 4).
These results showed that the optimum amount of sour-
dough addition, whether in low or high pH sourdoughs,
produced with Lb. plantarum LMO25 or Lb. alimentarius
LMO7 to prevent rope spoilage caused by B. subtilis or
B. licheniformis is 20%. These data are consistent with
another study which showed the rope spoilage of wheat
bread was avoided by adding 20–30% of sourdough which
was produced by Lactobacillus plantarum VTT E-78076,
Pediococcus pentosaceus VTT E-90390 or Lactobacillus
brevis (commercial strain) (Katina et al., 2002).
The pH values of the bread prepared with different
amounts of low pH and high pH sourdoughs varied in

Table 1
The effect of Lactobacillus strains in low pH (pH 3.5–4.0) sourdough against growth of Bacillus subtilis (100 spores/g dough)
LAB used as starter Amount of sourdough pH of TTA of pH in TTA in Number of B. subtilis Visual rope
culture in sourdough g/100 g bread dough sourdough sourdough bread bread (cfu/g) after 4 d storage prevention
Control Ia – – – 5.78 2.5 4.5 · 108
Control IIb – – – 5.92 2.7 <10
LMO7 10 3.81 12.7 5.62 3.5 2.3 · 104
LMO25 10 3.78 14.3 5.55 3.2 1.6 · 104
Control Ia – – – 5.78 2.5 4.5 · 108
Control IIb – – – 5.92 2.7 <10
LMO7 15 3.81 12.7 5.20 3.7 3.5 · 102 +
LMO25 15 3.78 14.3 5.05 3.5 1.9 · 102 +
Control Ia – – – 5.78 2.5 4.5 · 108
Control IIb – – – 5.92 2.7 <10
LMO7 20 3.81 12.7 4.95 4.3 <10 +
LMO25 20 3.78 14.3 4.91 4.0 <10 +
LMO7: Lb. alimentarius LMO7.
LMO25: Lb. plantarum LMO25.
TTA: Total titratable acidity, 0.1 N NaOH, mL.
a
Sourdough not added.
b
Not inoculated with B. subtilis, sourdough not added.
362 Ö. Mentesß et al. / Food Control 18 (2007) 359–363

Table 2
The effect of Lactobacillus strains in low pH (pH 3.5–4.0) sourdough against growth of Bacillus licheniformis (250 spores/g dough)
LAB used as starter Amount of sourdough pH of TTA of pH in TTA in Number of B. licheniformis Visual rope
culture in sourdough g/100 g bread dough sourdough sourdough bread bread (cfu/g) after 7 d storage prevention
Control Ia – – – 5.76 2.5 1.8 · 108
Control IIb – – – 6.00 2.6 <10
LMO7 10 3.76 12.2 5.70 2.9 7.7 · 104
LMO25 10 3.68 14.3 5.50 3.2 2.4 · 104
Control Ia – – – 5.76 2.5 1.8 · 108
Control IIb – – – 6.00 2.6 <10
LMO7 15 3.76 12.2 5.29 3.6 1.2 · 102 +
LMO25 15 3.68 14.3 5.00 3.5 2.6 · 102 +
Control Ia – – – 5.76 2.5 1.8 · 108
Control IIb – – – 6.00 2.9 <10
LMO7 20 3.76 12.2 4.90 4.2 <10 +
LMO25 20 3.68 14.3 4.70 4.0 <10 +
LMO7: Lb. alimentarius LMO7.
LMO25: Lb. plantarum LMO25.
TTA: Total titratable acidity, 0.1 N NaOH, mL.
a
Sourdough not added.
b
Not inoculated with B. licheniformis, sourdough not added.

Table 3
The effect of Lactobacillus strains in high pH (pH > 4.0) sourdough against growth of Bacillus subtilis (100 spores/g dough)
LAB used as starter Amount of sourdough pH of TTA of pH in TTA in Number of B. subtilis Visual rope
culture in sourdough g/100 g bread dough sourdough sourdough bread bread (cfu/g) after 4 d storage prevention
Control Ia – – – 5.76 2.5 3.4 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 10 4.35 7.0 5.64 3.7 1.6 · 105
LMO25 10 4.32 6.2 5.61 3.5 2.5 · 105
Control Ia – – – 5.76 2.5 3.4 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 15 4.35 7.0 5.58 3.8 4.6 · 104
LMO25 15 4.32 6.2 5.55 3.5 2.3 · 104
Control Ia – – – 5.73 2.5 3.4 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 20 4.35 7.0 5.50 4.0 <10 +
LMO25 20 4.32 6.2 5.48 3.7 <10 +
LMO7: Lb. alimentarius LMO7.
LMO25: Lb. plantarum LMO25.
TTA: Total titratable acidity, 0.1 N NaOH, mL.
a
Sourdough not added.
b
Not inoculated with B. subtilis, sourdough not added.

the range of 4.70–5.70 and 5.48–5.64, and the TTA values
varied in the range of 2.9–4.3 and 3.4–4.0, respectively
(Tables 1–4). According to the many references the final
pH of the bread is a major factor in controlling rope
(Quintavalla & Gola, 1993; Quintavalla & Parolari,
1987). Rosenquist and Hansen (1998) suggested that final
pH value in bread should be below 4.8 to prevent rope for-
mation. On the contrary, our data indicated that 15% low
pH or 20% high pH sourdough addition could prevent rope
spoilage despite the final pH of the bread was above 4.8.
We have previously demonstrated that strains used in
this study, Lb. plantarum LMO25 and Lb. alimentarius
LMO7, produce bacteriocins with different host range
activities including B. subtilis and B. licheniformis (Mentesß,
Ercan, & Akçelik, 2005). Production of bacteriocins may
explain the rope inhibition in breads with final pH values
above 4.8.
Test baking results also clearly showed that low pH (pH
3.5–4.0) sourdoughs exhibited stronger inhibitor activity
against B. subtilis and B. licheniformis than high pH
(>4.0) sourdoughs. This data is also consistent with our
prior data which shows the bacteriocins produced by Lac-
tobacillus strains have optimal activities at pH 3.0–4.0
(Mentesß et al., 2005). Acidity influences the antimicro-
bial production and many bacteriocins display greater anti-
bacterial activity at low pH values (Corsetti, Gobbetti, &
Smacchi, 1996; Jack, Tagg, & Ray, 1995).
In this study, we observed that bacteriocin-producing
Lactobacillus strains can play a significant role in prevent-
ing rope formation in bread by the addition of sourdough
 Ö. Mentesß et al. / Food Control 18 (2007) 359–363 363

Table 4
The effect of Lactobacillus strains in high pH (pH > 4.0) sourdough against growth of Bacillus licheniformis (250 spores/g dough)
LAB used as starter Amount of sourdough pH of TTA of pH in TTA in Number of B. licheniformis Visual rope
culture in sourdough g/100 g bread dough sourdough sourdough bread bread (cfu/g) after 7 d storage prevention
Control Ia – – – 5.73 2.5 1.3 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 10 4.40 7.0 5.64 4.0 6.5 · 106
LMO25 10 4.36 6.2 5.61 3.4 3.7 · 106
Control Ia – – – 5.73 2.5 1.3 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 15 4.40 7.0 5.60 3.9 4.2 · 104
LMO25 15 4.36 6.2 5.58 3.4 2.9 · 104
Control Ia – – – 5.73 2.5 1.3 · 108
Control IIb – – – 5.90 2.7 <10
LMO7 20 4.40 7.0 5.50 4.0 <10 +
LMO25 20 4.36 6.2 5.50 3.4 <10 +
LMO7: Lb. alimentarius LMO7.
LMO25: Lb. plantarum LMO25.
TTA: Total titratable acidity, 0.1 N NaOH, mL.
a
Sourdough not added.
b
Not inoculated with B. licheniformis, sourdough not added.

prepared by these strains. Further investigations need to be
done on identification of these bacteriocins which affect
bacterial growth and spore germination.
Acknowledgements
This study was supported by Ankara University Scien-
tific Research Council (Project No. 2003-07-11-079). The
authors thank to Dr. Çaðla TÜKEL (Department of Med-
ical Microbiology and Immunology, College of Medicine,
Texas A&M University) for the critical reading of the
manuscript.
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