Identification and Partial Characterization of a

Bacteriocin-Like Inhibitory Substance (BLIS) from

Lb. Bulgaricus K41 Isolated from Indigenous
Yogurts
Davood Zaeim, Sabihe Soleimanian-Zad, and Mahmoud Sheikh-Zeinoddin

Abstract: Forty-two strains of Lactobacillus bulgaricus isolated from locally made yogurts were examined and compared
for bacteriocin producing ability using spot on lawn assay which improved by taking photo and image processing.
Lb. bulgaricus K41 exhibited the highest inhibition level against indicators. K41 Bacteriocin-like inhibitory substance is
sensitive to proteolytic enzymes (proteinase K, pepsin, and trypsin) but α-amylase makes slight reduction in its activity
and it is resistant to lipase. This antibacterial peptide is extremely heat-stable (121 ◦ C for 15 min) and remains active over
a wide pH range (pH = 2 to 10); also nonionic detergents (Tween-20, Tween-80, and Triton X100) showed no effect
on its activity. The inhibitory spectrum is against Gram-positive bacteria (except Staphylococcus aureus) with extremely
antilisterial activity and it is almost ineffective against Gram-negative bacteria. The mode of its action was identified as
bactericidal against Listeria monocytogenes. The properties of K41 bacteriocin-like inhibitory substance add to its safety
as a biopreservative produced by a generally recognized as safe (GRAS) bacterium suggesting it can be used in hurdle
technology for ready-to-eat foods as one of the main sources of Listeria contaminations.

Keywords: bacteriocin-like inhibitory substance, image processing, L. monocytogenes, Lb. bulgaricus, photometric assay, spot

M: Food Microbiology
on lawn assay

& Safety
Practical Application: Antimicrobial substance, which is produced by Lb. bulgaricus K41, can be used as a biopreservative
for controlling Listeria in processed foods. Also Lb. bulgaricus K41 can be added as an adjunct flora to cheeses and some
ready-to-eat foods.

Introduction come completely inactive by gastrointestinal enzymes and have

Up to now, one of the best strategies for promoting safety
of foods and extending their shelf-life without any side effects
on consumer’s health and also maintaining the nutritious and
organoleptic characteristics of food products, is using biopreser-
vation with emphasizing on lactic acid bacteria (LAB) (Castellano
and others 2008). LABs like all other microorganisms produce
different antimicrobial compounds, such as organic acids, hydro-
gen peroxide, CO2 , diacetyl, low molecular weight antimicrobial
substances (for example, Reuterin and Reutericyclin) and bacte-
riocins (Ammor and others 2006). Bacteriocins are ribosomally
synthesized peptides or proteins which reveal antagonistic effects
against other species particularly closely related strains to producer
cell (Corsetti and others 2008). LAB’s bacteriocins have some ad-
vantages that nominate them as a good choice for biopreservation.
Many LAB’s bacteriocins are generally recognized as safe (GRAS)
by the U.S. FDA and have no antagonistic effects on eukaryotic
cells excepting cytolysin, produced by Enterococcus faecalis (Heng
and others 2007). Because of their proteinous nature, they be-
MS 20130447 Submitted 4/2/2013, Accepted 10/8/2013. Authors Zaeim and
Sheikh-Zeinoddin are with Dept. of Food Science and Technology, College of Agricul-
ture, Isfahan Univ. of Technology, Isfahan, 84156-83111, Iran. Author Soleimanian-
Zad is with Inst. of Biotechnology and Bioengineering, Isfahan Univ. of Technology, Isfa-
han, Iran. Direct inquiries to author Soleimanian-Zad (E-mail: soleiman@cc.iut.ac.ir).
slight effects on intestinal microbiota. Also, most of the LAB’s
bacteriocins are resistant to pH and heat so they can be utilized in
food industries (Gálvez and others 2007).
The preliminary term to find a novel proper bacteriocin is to
screen potential bacteriocin-producing LABs (Yi and others 2010).
One of the main sources of LABs is traditional dairy fermented
foods such as yogurt. Yogurt is a popular dessert in Middle East
which is known as an unsweetened product. For centuries, yogurt
has been a common dish in all social classes and also it is consumed
for medical remedies. These characteristics as well as the recent
listeriosis outbreaks (2011, associated with cantaloupe and 2012,
associated with cheese) persuade us to explore antilisterial bacte-
riocins on this local dairy product. The dominant microflora of
yogurt comprises of Streptococcus thermophilus and lactobacillus bul-
garicus. According to the literature, only few researches have been
carried out on Lb. bulgaricus bacteriocins compared to other lacto-
bacilli (such as Lb. plantarum and Lb. sakei) also their characteristics
have been described insufficiently (Abdel-Bar and others 1987;
Balasubramanyam and Varadaraj 1998; Akpinar and others 2011).
The objective of present study was to find and characterize a
novel bacteriocin with the capability of being used as a biop-
reservative. To achieve this aim, 42 strains of Lb. bulgaricus, already
isolated from indigenous yogurts, were screened in terms of bac-
teriocinogenic activity by spot on lawn method against 2 indi-
cators. The most effective strain was chosen and bacteriocin-like



C 2013 Institute of Food Technologists
R

doi: 10.1111/1750-3841.12314 Vol. 79, Nr. 1, 2014 r Journal of Food Science M67
Further reproduction without permission is prohibited
 Identification and characterization of . . .
Table 1–Indicator strains and their sensitivity to K41 BLIS.
Indicator Straina
Gram-positive bacteria
Listeria monocytogenes RITCC1293
Staphylococcus aureus PTCC 1113
Bacillus subtilis PTCC 1023
Bacillus cereus PTCC 1015
Sarcina ureae PTCC 1642
Gram-negative bacteria
Escherichia coli O157:H7 Native
Proteus vulgaris PTCC 1312
Klebsiella pneumonia PTCC 1053
Salmonella typhi PTCC 1609
Shigella dysenteriae PTCC 1188
a
RITCC, Razi Institute Type Culture Collection; PTCC, Persian Type Culture
Collection; E. coli O157:H7 strain was provided by Dr. M.H. Eskandari, Dept. of Food
Science and Technology, Shiraz Univ., Shiraz, Iran.
b
Inhibition level (1-R): +++, >0.5; ++, 0.3–0.5; +, 0.1–0.3; –, <0.1.
inhibitory substance (BLIS) production, sensitivity to enzymes,
detergents, heat and pH, also its inhibitory spectrum and mode of
activity were studied using photometric method.
Materials and Methods
Bacterial strains, culture, and storage conditions
M: Food Microbiology
Twenty samples of different indigenous yogurts were aseptically
collected from homemade producers in Isfahan zone (central area
& Safety
of Iran). All yogurts had already been made by back-sloping prac-
tice using no industrial starter cultures. Ten grams of each sample
was added into 90 mL 1/4-strength Ringer’s solution, serial deci-
mal dilutions were prepared, plated onto MRS agar (ScharlauTM ,
Spain) and incubated at 37 ◦ C in a microaerobic atmosphere for 48
h. Pure cultures were acquired by streaking onto the same medium
repeatedly. Forty-two strains of Lb. bulgaricus were identified with
morphological, biochemical (Chervaux and others 2000; Mannu
and others 2000), and Species-Specific PCR methods (Chagnaud
and others 2001) in a prior study (unpublished data). A single
primer pair which targets the proline iminopeptidase (pepIP) gene
of Lb. bulgaricus was employed in Species-Specific PCR (Torriani
and others 1999). All of the LAB stock cultures were stored in
MRS broth with 30% glycerol at –80 ◦ C in the culture collec-
tion of Microbiology Biotechnology laboratory, Dept. of Food
Science and Technologies, Isfahan Univ. of Technology, Isfahan,
Iran. Before experimental use, the cultures were propagated twice
in MRS by incubation at 37 ◦ C under microaerobic condition
using a CO2 incubator (BinderTM , Germany) for 24 h.
Indicator strains listed in Table 1 were maintained frozen in
BHI broth (MerckTM , Germany) containing 30% glycerol. Fresh
cultures were provided by inoculation of frozen stocks into BHI
broth followed by incubation at 37 ◦ C for 14 h, aerobically.
Assays for screening Lb. bulgaricus and detecting BLIS
Inhibitory activities of 42 Lb. bulgaricus strains, which isolated
from indigenous yogurts, were investigated against L. monocyto-
genes (as Gram-positive indicator) and Escherichia coli O157:H7
(as Gram-negative indicator) by spot on lawn assay as described
by Lewus and Montville (1991). To enhance the accuracy, plates
were shot by a digital camera (Fuji FilmTM – FinePix S2500HD,
4000×2248 pixels, ISO: 64, sharpness: hard, photometry: spot)
at the fixed angle (90◦ between lens axis and plate), distance (30 Cell growth, pH changes, and BLIS production
cm), and illumination in a photography box (a wooden box de-
signed in Engineering Laboratory, Dept. of Food Science and cubated at 30 ◦ C in a microaerobic atmosphere. Samples were
Technologies, Isfahan Univ. of Technology, 60×30×30 cm). Im- taken hourly to determine viable counts, OD600 and pH. After
M68 Journal of Food Science r Vol. 79, Nr. 1, 2014
Table 2–Details of image processing.
Sensitivityb Step 1: Filters > Sharpen > Smart sharpen Amount: 500%
Radius: 64.0 px
Step 2: Filters > Sketch > Torn edgea Image balance: 20
+++ Smoothness: 15
− Contrast: 15
++
++ a
Filter was exerted at default foreground and background colors.
++
ages were saved on memory card as JPEG files and transferred to an
+ AsusTM laptop computer (K50IN). After unifying scales of photos
+ (image size: 120×115 mm, resolution: 250 pixel/inch) by Adobe
−
−
PhotoshopTM CS5 (ME ver. 12.0) software, images were processed
− by the same program to increase clarity of coronas around the spots.
For image processing, 2 filters which are presented in details in
Table 2 were employed. Spot and corona diameters (mm) were
measured in 3 different axes by Ruler Tool in Photoshop software
(Figure 1). Inhibition level (I) was calculated as the ratio between
corona diameter and LAB spot diameter (average of 3 observations
in triplication).
Photometric assay was performed as described by Parente and
others (1995) with slight modifications. Briefly, 1% v/v of 18 h
LAB culture was inoculated into sterile MRS and incubated at 30
◦
C under microaerobic condition for 24 h. Crude supernatant was
obtained by centrifuging (8000g at 4 ◦ C for 10 min). The pH of
crude supernatant was adjusted to 6 ± 0.02 by adding 4N NaOH
for neutralizing acid inhibition, followed by filtration through 0.2
μm pore size cellulose acetate filter. To rule out the inhibitory
activity of H2 O2 , catalase (100 to 250U/mL, SigmaTM , U.S.A.)
was added and incubated at 37 ◦ C for 1.5 h aerobically. The
outcome was named treated supernatant (TS). Indicator culture
was adjusted to OD600 = 0.1 ± 0.02 and inoculated (5%v/v)
into BHI broth followed by dispensing in 1.8 mL amounts to
sterile screw caped tubes and 200 μL of TS (10% v/v) were
pipetted into each tube then incubated at 37 ◦ C aerobically for
14 h (RITCC1293, PTCC1113, and PTCC1642) or 8 h (other
strains). A quantity of 200 μL of sterile MRS was added as control.
Response (R) was calculated as the ratio between OD600 sample
and OD600 control (average of triplication) and inhibition level (I)
was obtained by 1.
 
OD600 Sample
I=1−R=1− (1)
OD600 Control
Quantitative evaluation for photometric assay
A modification of critical dilution assay (CDA) (Pucci and oth-
ers 1988; Parente and others 1995) was performed. TS was serially
diluted (2-fold) using sterile MRS broth and the antagonistic ac-
tivities were tested against Listeria monocytogenes as an indicator
strain by photometric assay (PA), and response (R) was calculated.
The critical dilution was described as the last dilution exhibit-
ing a visible inhibition compared to the control. The titer (T) of
the BLIS, in AU/mL, was calculated as (200/2000)1000D, where
D is the dilution factor. To obtain the titer of BLISs from R in
subsequent experiments, the R/log(T) curve was drawn and the
relationship between R and log(T) was modeled by a nonlinear
equation (Parente and others 1995) which is usable for acquiring
titer (AU/mL) from response.
An 18-h LAB culture was adjusted to OD600 = 0.05 and in-
Identification and characterization of . . .

10 h, secondary samples were taken every 2 h for determining inhibition level (I) was calculated as described in section “Assays
antimicrobial activity against L. monocytogenes by PA for 24 h. Also for screening Lb. bulgaricus and detecting BLIS.”
antimicrobial activities were investigated after 36 and 48 h (Powell
and others 2007; Ponce and others 2008; Jiang and others 2012). Mode of action of produced BLIS
OD600 of L. monocytogenes culture was adjusted to 0.05 and
dispensed between 2 flasks. After 2 h incubation at 37 ◦ C, TS
Initial studies on BLIS characterization (10% v/v of final concentration) was added to the test sample and
TS was adjusted to pH = 6.5 and incubated at 37 ◦ C for sterile MRS was used as control. OD600 and nr. of viable counts
2 h in the presence of 0.1 and 1 mg/mL pepsin (2500U/mL, were recorded hourly for 14 h and after 24 h (Powell and others
SigmaTM , U.S.A.), trypsin (7500U/mL, SigmaTM , U.S.A.), pro- 2007; Todorov and others 2011b).
teinase K (30U/mL, RocheTM , Germany), lipase (10U/mL,
SigmaTM , U.S.A.), and α-amylase (500U/mL, SigmaTM , U.S.A.). Statistical analysis and graphics
After incubation, enzymes were inactivated by boiling water for 3 All experiments were performed in triplicate and the repre-
min. Antimicrobial activities were determined by PA. The 50 mM sented data are their means. Statistical analysis were carried out
potassium phosphate buffer (pH = 7) containing no enzymes was with SASTM 9.1 software by using procedures General Linear
used as control (Gulahmadov and others 2009; Todorov 2010). Model (GLM). Significant differences between the means were
The effect of surfactants on BLIS was studied by incubating TS at estimated by the Student’s t-test at P ≤ 0.05 by using the same
37 ◦ C for 5 h in the presence of 1% and 2% detergents containing program. Graphs were generated by using Microsoft ExcelTM
Tween-20, Tween-80, and Triton X100. Antimicrobial activities software.
of treatments were determined by PA (Todorov and others 2007;
Todorov 2010). To determine thermostability of BLIS, TS samples Results and Discussion
were heated to 60, 80, and 100 ◦ C for 30 and 60 min, also 121
◦
C for 15 min and residual activity was examined by PA (Jiang Screening of Lb. bulgaricus strains for BLIS production
and others 2012). The pH crude supernatant was adjusted to 2, Inhibitory activities of 42 Lb. bulgaricus strains were investigated
4, 6, 8, and 10 by adding 4N NaOH and HCl. After 30 min, pH against 2 indicators by spot on lawn assay which was enhanced by

M: Food Microbiology
of samples were adjusted to 6.0 and followed by filtration through taking photo and image processing as shown in Figure 1. Spot on
0.2 μm cellulose acetate filter. H2 O2 inhibitory effect ruled out lawn is a convenient and reproducible method for detecting BLIS

& Safety
by catalase and residual activity was determined by PA (Todorov produced by LAB (Lewus and Montville 1991; Cadirci and Citak
and others 2011b). All experiments were performed in triplicate. 2005); also accuracy was increased by taking photo and measuring
diameters by software. By using TSAYE medium, acid inhibition
was eliminated and H2 O2 production was limited intensively by
Inhibitory spectrum of produced BLIS incubating all plates anaerobically (Lewus and Montville 1991), so
Inhibitory spectrum of BLIS was tested against 10 spoilage and residual antimicrobial activity was attributed to BLIS.
pathogenic bacteria (Table 1). Standard indicators were provided K41 strain exhibited the highest suppressive effect against both
by adjusting OD600 = 0.1 and determining nr. of viable counts. indicators with significant differences (P ≤ 0.05) and was selected
Standard indicators were inoculated into BHI broth (5% v/v) and for identifying its BLIS. All strains showed more inhibitory ac-
10% v/v (final concentration) of TS was pipetted into each tube tivity against L. monocytogenes than E. coli. Many researchers have
and incubated at 37 ◦ C aerobically. Sterile MRS was added as reported the same results that Gram-positive bacteria especially
control (Parente and others 1995; Gao and others 2010). After in- Listeria species are very sensitive to lactobacilli bacteriocins (Çon
cubation, the OD600 and nr. of viable counts were determined and and others 2001; Ammor and others 2006; Xie and others 2011).

Figure 1–Spot on lawn plate before and after image processing. Four Lb. bulgaricus strains (J11, Is71, K41, and J2) were spotted on TSAYE and incubated
at 30 ◦ C for 72 h anaerobically; plates were overlaid with soft BHI agar (0.75% agar) which had been seeded with L. monocytogenes (indicator). After
incubation (37 ◦ C for 24 h aerobically) images of plates were taken. a: spot diameter, b: corona diameter; 1, 2, 3: axis of measurements.

Vol. 79, Nr. 1, 2014 r Journal of Food Science M69

 Identification and characterization of . . .

According to Parente and others (1995), for maximizing the sensi- ity rose to maximum at early stationary phase when cell count
tivity of a method the most sensitive indicator should be selected, and pH were approximately constant. Results suggest that K41
hence L. monocytogenes was chosen as indicator for K41 BLIS iden- BLIS may be a secondary metabolite. Our results are supported by
tification. Balasubramanyam and Varadaraj (1998) which reported that Lb.
bulgaricus CFR2028 produce a bacteriocin between the late loga-
Quantitative assay for photometric method rithmic and early stationary phase. Similar results were reported for
For quantitative assay, R/log(T) curve (Figure 2) was generated bacteriocin ST8KF from Lb. plantarum (Powell and others 2007),
and according to Parente and others (1995), a nonlinear model is bacteriocin R1333 from Lb. sakei (Todorov and others 2011b),
suitable (R2 = 0.994) for acquiring titer (T) (AU/mL) of BLIS and a bacteriocin from Lb. lactis (Rajaram and others 2010).
from response (R) in photometric assay which showed in 2. No significant (P ≤ 0.05) change was recorded at inhibitory
T = −297. ln (R) + 2.139 (2) activity upon 24, 36, and 48 h further incubation. Our results
suggested that K41 BLIS was not decomposed after 48 h, also it
did not adsorb on the surface of the producer cells in contrast to
Lb. bulgaricus K41 Cell growth and BLIS production GM005 bacteriocin from Lactococcus sp. GM005 (Onda and others
Cell growth, pH changes, and BLIS production of Lb. bulgari- 2003).
cus K41 in MRS were investigated at 30 ◦ C under microaerobic
condition (Figure 3). Inhibitory activity of K41 TS was initially Effect of enzymes and detergents on K41 BLIS
detected after 12 h (20AU/mL) and surged to 132AU/mL after As shown in Table 3, Proteinase K as one of the most active
22-h incubation. According to K41 growth curve, production of endopeptidase with no pronounced cleavage site decreased K41
BLIS was started at late logarithmic phase and inhibitory activ- BLIS activity up to 96% after incubation. Pepsin which cleaves

Figure 2–R/log(T) curve of K41 BLIS. Response is

ratio between OD600 Sample and OD600 Control;
titer was resulted from (200/2000)1000D
M: Food Microbiology

formula, when D is the dilution factor.

& Safety

Figure 3–Culture specifications of Lb. bulgaricus K41 in MRS broth at 30 ◦ C under microaerobic condition. OD600 (•), Nr. of viable counts (), changes
in medium pH (−−) and BLIS production (column charts).

M70 Journal of Food Science r Vol. 79, Nr. 1, 2014

Identification and characterization of . . .

Table 3–Effects of enzyme, detergent, time and temperature antimicrobial components of Lb. sakei (Gao and others 2010) and
combination and pH on BLIS which was produced by Lb. bul- str. thermophilus (Ivanova and others 1998; Gao and others 2010).
garicus K41.
Incubation of 0.1 mg/mL α-amylase had no significant (P ≤
BLIS titer Residual 0.05) effect on inhibitory activity of K41 BLIS, while incubation
Treatment (AU/mL) ± SDa activity (%)b with 1 mg/mL α-amylase caused 17% reduction in its inhibitory
Enzymes activity at the same time. Sensitivity to α-amylase has been re-
Control (buffer added) 128.35 ± 6.86 100.0
Proteinase K (0.1 mg/mL) 5.78 ± 2.73 4.5 ported for BLIS from Lb. buchneri and Lb. pentosus and it was
Proteinase K (1 mg/mL) 5.42 ± 2.04 4.2 attributed to presence of carbohydrate in their structure (Gulah-
Pepsin (0.1 mg/mL) 11.54 ± 2.62 9.0 madov and others 2009). Detecting 17% reduction in BLIS ac-
Pepsin (1 mg/mL) 10.27 ± 1.93 8.0 tivity after incubation with 1 mg/mL α-amylase could be due to
Trypsin (0.1 mg/mL) 44.35 ± 8.23 34.6
Trypsin (1 mg/mL) 37.75 ± 2.25 29.4
employing photometric assay which is more accurate than other
Lipase (0.1 mg/mL) 121.96 ± 7.21 95.0 methods as mentioned by Parente and others (1995). It seems K41
Lipase (1 mg/mL) 122.7 ± 11.05 95.6 BLIS has proteinous essence but its maximal activity depends on
α-amylase (0.1 mg/mL) 121.94 ± 5.56 95.0 glycosylation, so needs both protein and glycoprotein portions.
α-amylase (1 mg/mL) 106.13 ± 2.98 82.7 Similar results were reported for a bacteriocin from Lb. plantarum
Surfactants and detergents LB-B1 (Xie and others 2011).
Control (no detergent added) 127.05 ± 0.5 100.0
Tween-20 (1%) 124.16 ± 6.3 97.7
The results showed that Tween-20, 80, and Triton X100 as non-
Tween-20 (2%) 126.47 ± 3.0 99.5 ionic detergents had no effect on K41 BLIS antimicrobial activity
Tween-80 (1%) 125.33 ± 6.6 98.6 in both concentrations. Similar results were reported for bacte-
Tween-80 (2%) 127.07 ± 4.1 100.0 riocin R1333 produced by Lb. sakei R1333 (Todorov and others
Triton X100 (1%) 126.03 ± 3.6 99.2 2011b). However, various results (Todorov and Dicks 2005; Powell
Triton X100 (2%) 127.05 ± 1.8 100.0
and others 2007; Todorov and others 2011b) were reported about
Time and temperature
Control (No thermal treatment) 130.31 ± 4.67 100.0
the effects of detergents on lactobacilli bacteriocins and according
60 ◦ C, 30 min 122.00 ± 9.42 93.6 to Todorov and others (2011a) the sensitivity to detergents seems
60 ◦ C, 60 min

M: Food Microbiology
119.44 ± 3.50 91.7 to be a bacteriocin-dependent characteristic.
80 ◦ C, 30 min 120.27 ± 3.72 92.3
80 ◦ C, 60 min 119.77 ± 3.08 91.9

100 ◦ C, 30 min 116.50 ± 2.30
100 ◦ C, 60 min 116.83 ± 3.20
121 ◦ C, 15 min 108.34 ± 3.17
pH
2 122.67 ± 2.30
4 125.68 ± 12.11
6 (control) 125.83 ± 2.33
8 125.83 ± 2.65
10 124.37 ± 7.41
a
Results are presented as the mean of triplicate experiments ± standard deviation.
b
Percent of remainder activity after treatment.
peptides through their hydrophobic bonds, preferably aromatic
amino acids, caused 92% reduction in BLIS activity and trypsin
which just cleaves peptide chains mainly at their carboxyl side of
the amino acids lysine or arginine reduced approximately 68% of
BLIS activity. By considering the information about the cleavage
sites and activity of the proteolytic enzymes, we can expound
our results and show proteinous nature of K41 BLIS, whereas
bacterial proteinous compounds with antagonistic activity against
closely related species are classified into bacteriocins category (Jack
and others 1995) and since the active substances in K41 TS have
not been described for its amino acid and nucleotide sequences
yet, they will be introduced as BLIS as supposed by Corsetti and
others (2008). Todorov and others (2007) reported the complete
inactivation or significant reduction in antimicrobial activity of
bacAMA-K (produced by Lb. plantarum AMA-K) after treatment
with proteolytic enzymes supports proteinous nature of bacAMA-
K. Similar results and deductions were presented by Deraz and
others (2005) for acidocin D20079, Todorov and others (2011b)
for bacteriocin R1333, and Xie and others (2011) for a bacteriocin
produced by Lb. plantarum LB-B1.
No significant change in BLIS activity after 2 h incubation with
lipase (consist of 0.1 and 1 mg/mL) demonstrated the absence
of lipid in BLIS structure. The same results were reported for
& Safety
Effect of heat and pH on K41 BLIS
89.4
89.7 K41 BLIS inhibitory activity was not decreased by thermal treat-
83.1 ment at 60, 80, or 100 ◦ C after 30 and 60 min significantly (P ≤
0.05). In addition, it sustained 83% of its activity after autoclaving
97.5 (121 ◦ C for 15 min) (Table 3). Two hours of incubation at pH
100.0
100.0
values from 2.0 to 10.0 also had no effect on K41 BLIS activity
100.0 and it remained stable completely (Table 3). Heat and pH-stability
98.8 of lactobacilli bacteriocins have been reported by many researchers
(Deraz and others 2005; Todorov and others 2007; Gao and others
2010; Xie and others 2011) as Gálvez and others (2007) pointed
to that as a desirable property for utilizing them in food biopreser-
vation. K41 BLIS by tolerating acidic condition and sterilization
process could be a proper choice for applying in hurdle technology.
Inhibitory spectrum of K41 BLIS
K41 TS showed antagonistic activity against Gram-positive bac-
teria including L. monocytogenes, Bacillus subtilis, B. cereus, and sarcina
ureae with the exception of Staphylococcus aureus which was com-
pletely resistant to K41 BLIS. Gram-negative bacteria were also not
inhibited generally, however E. coli O157:H7 and Proteus vulgaris
revealed slight sensitivity (Table 1). Insufficiency of Gram-positive
bacteria especially LAB bacteriocin-producing strains to inhibit
Gram-negative bacteria was reported frequently (Jimenez-Diaz
and others 1993; Jack and others 1995; Todorov and others 1999;
Héquet and others 2007); however several cases of Gram-negative
inhibition by LAB bacteriocin-producing strains were reported
recently (Ponce and others 2008; Gao and others 2010). Lb. sakei
exhibited antilisterial activity; however, it was not able to inhibit
Gram-negative bacteria and also revealed a slight inhibitory effect
on St. aureus (Ammor and others 2006). Four isolates of Entero-
coccus faecium (MMZ04, MMZ09, MMZ13, and MMZ17) inhibit
listerial species but they are not effective on St. aureus and Gram-
negative bacteria (Serratia marcescens CIP 5814 and Yersinia entero-
colitica CIP 814) (Ben Belgacem and others 2008). Outer mem-
brane by covering the cell wall and cytoplasmic membrane may
protect receptors to connect to bacteriocin’s active sites and cause

Vol. 79, Nr. 1, 2014 r Journal of Food Science M71

 Identification and characterization of . . .
Gram-negative bacterial resistance to LAB bacteriocins as reported
by other researchers (Ammor and others 2006; De Vuyst and Leroy
2007; Ben Belgacem and others 2008). Staphylococcal resistance
M: Food Microbiology
to cationic peptides such as bacteriocins was described properly
& Safety
by Peschel and Vincent Collins (2001). Our results are supported
by Akpinar and others (2011) who reported that among 25 Lb.
bulgaricus isolates, only 6 isolates showed inhibitory activity against
St. aureus, whereas other 18 isolates inhibited L. monocytogenes. St.
aureus was also resistant against pediocin LB-B1 produced by Lb.
plantarum LB-B1 (Xie and others 2011). According to the litera-
ture mentioned, K41 BLIS is a strong antilisterial peptide which
inhibits a spectrum of Gram-positive bacteria but is ineffective on
Gram-negative bacteria.
Mode of K41 BLIS activity
Growth curve of L. monocytogenes as an indicator is demon-
strated in Figure 4, in the presence and absence of K41 TS. OD600
of L. monocytogenes medium in the presence of K41 TS increased
from 0.05 (corresponding to 1.45×105 CFU/mL) to 1.1 (corre-
sponding 9.7×107 CFU/mL) over 14 h of incubation, whereas
OD600 of the control (absence of K41 TS) increased to 1.62 (cor-
responding 2.22×108 CFU/mL) over the same period. After 24
h of incubation, OD600 of control remained constant about 1.63
(corresponding 2.25×108 CFU/mL) but OD600 of sample de-
clined to 0.98 (corresponding 4.30×107 CFU/mL). Increasing of
L. monocytogenes OD600 and cell count after K41 TS addition may
be caused by the quantity of active agents and it seems that the
quantity of inhibitory component in the supernatant is too little
to suppress 2.3×105 CFU/mL of indicator bacteria which were
at the beginning of logarithmic growth. The decrease of OD600
and nr. of viable counts after 24 h in the presence of K41 BLIS
suggest that the L. monocytogenes cells were lysed and this result
demonstrates the bactericidal mode of K41 BLIS activity. Dif-
ferent reports are available about LAB bacteriocins which most
of them pointed to bactericidal mode of action. Ben Belgacem
and others (2008) reported reduction of optical density and viable
counts would be the evidence of bactericidal mode of action. Sim-
ilar reports were presented about Sakacin LSJ618 from Lb. sakei
LSJ618 (Jiang and others 2012), bacteriocin AMA-K from Lb.
M72 Journal of Food Science r Vol. 79, Nr. 1, 2014
Figure 4–Effect of Lb. bulgaricus K41
treated supernatant (TS) on L.
monocytogenes RITCC1293 growth. OD600
in the presence () and absence () of
K41 TS and Nr. of viable counts (CFU/mL)
in the presence (◦) and absence (•) of K41
TS.
plantarum (Todorov and others 2007), and Sakacin R1333 from
Lb. sakei R1333 (Todorov and others 2011b).
Conclusion
This study was an attempt to identify a bacteriocin-producing
Lb. bulgaricus and characterize its antimicrobial substance with em-
phasizing on application of accurate methods. Forty-two strains
of Lb. bulgaricus were screened by spot on lawn assay that was
enhanced by taking photo and image processing so as these en-
hancements increased the accuracy of the method considerably.
Lb. bulgaricus K41 revealed the most antagonistic activity against
indicators especially L. monocytogenes and the sensitivity to prote-
olytic enzymes confirmed the proteinous nature of antibacterial
substance, thus it was classified into BLIS. Heat stability (121 ◦ C
for 15 min), remaining active over a wide pH range (pH = 2 to
10), and antilisterial activity of K41 BLIS without any effect on
Gram-negative bacteria suggest that it can be used as an antiliste-
rial biopreservative for extending the shelf-life of some processed
foods. Also, we showed K41 BLIS has a bactericidal mode of activ-
ity against L. monocytogenes. Since Lb. bulgaricus K41 is a generally
recognized as safe (GRAS) bacterium, it can be used as a compet-
itive flora in hurdle technology for controlling L. monocytogenes in
particular dairy products such as cheeses and ready-to-eat foods.
To our knowledge, not much research has been done on Lb. bulgar-
icus bacteriocin’s identification and characterization and this study
is the first report about a glycoprotein containing antimicrobial
substance from Lb. bulgaricus. Further studies are required to deter-
mine the protein and genetic structure of BLIS from Lb. bulgaricus
K41.
Acknowledgments
The authors would like to acknowledge the Isfahan Univ. of
Technology for financial support of this research and Dr. M.H.
Eskandari, Shiraz Univ., for his cooperation. The authors declare
that there is no conflict of interests.
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