Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
ISSN: 2319-7706 Volume 3 Number 6 (2014) pp. 269-276
http://www.ijcmas.com
Original Research Article
Antimicrobial Activity of Lactobacillus acidophilus that
carry the Bacteriocin Gene
Anwar A. Abdulla*
Department of Biotechnology, College of Sciences, Babylon University, Iraq
*Corresponding author
ABSTRACT
Keywords
Lactobacillus
acidophilus,
antibacterial
activity,
Bacteriocin
gene,
PCR.
A total of six isolates of Lactobacillus acidophilus isolated from different yoghurts.
These isolates were identified by phenotypic and biochemical methods. Using agar
well diffusion methods, the cell-free extract of growth culture of the six
Lactobacillus acidophilus isolates demonstrated wide antibacterial spectrum
against indicator pathogenic strains. Adjustment of the pH of the growth
supernatants to 6.5 did not affect the inhibitory activity of the supernatants of the
tested isolates on the indicator bacteria suggesting the inhibitory activity is not
attributable to the lactic acid and other organic acids produced by the tested
isolates. Using PCR, a bacteriocin gene of 4000 bp was amplified by specific
primers from all of the six isolates.
Introduction
Lactobacillus are member of lactic acid
bacteria (LAB), a broadly defined group
characterized by the formation of lactic
acid as a sole or main end product of
carbohydrate metabolism (Satokari et al.,
2005). In raw milk and dairy products
such as yoghurts, cheese and fermented
milks, lactobacilli are naturally present or
added intentionally due to their heath
benefit for the consumer (Vernoux et al.,
2003). Exploitation of LAB as a
preservative agent is advantageous not
only in improving the microbial safety of
food but also as a probiotic in animals and
humans to improve the balance of
microflora and to inhibit pathogenic
bacteria in intestinal tract (Soomro et al.,
2002).
The health benefits offered by LAB can
be nutritional or therapeutic including
production
of
vitamins,
immunomodulation, reduction in the risk
of diarrhea, and mutagenic activity and a
decrease in serum cholesterol (Briand et
al., 2006; Myllyluoma et al.,2007;
Linsalata et al., 2010). Bacteriocins are
proteinaceous antibacterial compounds
that exhibit bactericidal activity against
species closely related to the producer
strain. Several important types of
bacteriocins such as nisin, diplococcin,
acidophilin, and bulgaricin are identified
(Signoretto et al., 2000). Increased use of
antibiotics is a key factor in the
emergence
of
antibiotic
resistant
pathogens.
Probiotics
such
as
Lactobacillus spp are reported to have
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
inhibitory activity against common human
pathogens through their ability to produce
antibacterial
substances
such
as
bacteriocins which have potential to be
used in therapeutics and as food biopreservatives (Mccatez et al., 2008).
Several bacteriocins from Lactobacillus
spp. have been characterized with respect
to their protein sequence, molecular mass,
biochemical properties and antimicrobial
activity spectrum. Bacteriocins that have
all D-amino acids have antibacterial
activity but exhibit more resistance to
proteolytic enzymes and are less cytotoxic
compared with bacteriocins that have all
L-amino acids (Pangsomboon et al.,
2006).
yoghurt brands and to assess their anti
bacterial activity against some pathogenic
bacteria, and verification of the presence
of bacteriocin genes in the tested isolates.
Materials and Methods
Isolation and Identification
Twenty two samples of different brands
of traditional yoghurts were collected
from a local market. Samples were
serially diluted in the range of (10-1- 10-6).
One hundred µl from each dilution were
added to MRS broth. Test tubes were
incubated at 37 for 24 h under anaerobic
condition in the presence of 5% CO2. A
loopful of each culture was streaked on to
the MRS agar. Plates were incubated at
37C for 48 h under anaerobic condition in
the presence of 5% CO2 (Sneath et al.,
2009).
Genes that encode bacteriocins are found
exclusively on the chromosome, and it is
bacterial
ribosomallysynthesized
peptides or proteins with antimicrobial
activity (Sano et al., 1990). Additionally,
the gene may be a part of a transposons as
in the case of nisin and lacticin 481
(Dufour et al. 2000). Nisin was the first
bacteriocin to be isolated and approved
for use in foods, specifically to prevent
the outgrowth of Clostridium botulinum
spores in cheese spreads in England
(Montaville and Winkowski, 1997).
Bacterial isolates were identified based on
the colony characteristics and biochemical
characterization
(Gram
staining,
production of catalase, indole, starch
hydrolysis, casein hydrolysis, gelatinase,
growth at 15°C and 45°C in MRS broth.
The identification of the genus
Lactobacillus was confirmed to the
species level based on their ability to
ferment sugars (MacFaddin, 2000).
Molecular methods used for the detection
of bacteriocins have some improved
aspects over the traditional methods. The
PCR technique has been used to rapidly
identify lactobacilli that may produce
well-characterized bacteriocins, and have
been used to detect genes responsible for
bacteriocin production and regulation in
bacterial cultures (Sonomoto and Yokot,
2011).
Preparation of cell free supernatant
The bacterial strains were grown in MRS
broth that contained 1.5% glucose for 24h
at 30°C. The cultures were harvested by
centrifugation at 6000 rpm/ 15 min/ 4°C)
to obtain a cell free supernatant.
Supernatants were filter-sterilized by
passing through a sterile 0.2 µl pore size
filter. The pH of the supernatants was
adjusted to 6.5 with 10 N of NaOH
(Dunne et al., 2001).
The aim of the present study was to
isolate and characterize Lactobacillus
acidophilus from traditional different
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
pre-mix (SolGent 2x Taq PCR Pre
Mix, SolGent Co., Ltd.), and 200ng
genomic DNA. The reaction mixture was
amplified in a GTC thermal cycler
(Cleaver
Scientific,
UK).
Initial
denaturation was carried out at 95°C for 3
min and the target DNA was amplified in
35 cycles. Subsequently, each cycle
consisted of denaturation at 95°C for 30
sec, followed by annealing at 61°C for 40
sec. Elongation was carried out at 72°C
and the extension time at 1 min. The final
extension step was performed at 72°C for
5 min and the holding temperature was 10
sec.
Detection of antimicrobial activity of
the supernatants
Screening for antimicrobial activity in the
tested supernatants against several
indicator bacterial spp was then
performed. On E.coli, Pseudomonas
aerogenosa, Aeromonas hydrophila,
Proteus vulgaris, Staphylococcus aureas,
Staphylococcus
epidermidis,
Streptococcus pyrogenes, and Bacillus
subtilis. An agar-well diffusion assay was
used (Ogunbanwo et al., 2003). Aliquots
of 50 µl of the sterile supernatant were
placed in 4 mm diameter wells on MullerHinton-agar plates previously seeded with
the respective indicator bacteria. After 18
h of incubation at 37°C, the diameters of
the zones of growth inhibition were
measured.
Gel Electrophoresis
The amplified PCR products were
checked for the expected size on 1% (w/v)
agarose gel and visualized after staining
with ethidium bromide under ultraviolet.
A DNA molecular weight marker (Gene
aid/ Korea) was used to measure the
weight of the fragments (Sambrook et al.,
2001).
Genotype-identification
DNA isolation
DNA extraction from the selected
bacterial isolates was performed by the
wizard genomic DNA purification kit
(Promega / USA) according to the
instructions
of
the
manufacturer.
Concentrations of DNA were measured
by using Nano Drop-spectrophotometer.
Results and Discussion
Isolation
and
Identification
Lactobacillus acidophilus
of
Out of 22 yoghurt samples, 6
Lactobacillus acidophilus were isolated.
The isolates were purified and
characterized by morphological and
biochemical tests according to the
methods of MacFaddin (2000), which
include Gram positive, rod- shaped, nonmotile, indol production-negative, MR
test-negative, VP test-positive, nitrate
reduction-negative,
catalase-negative,
gelatinase- negative, casein hydrolysisnegative, growth at -15°C negative and
growth at -45°C
positive, the
carbohydrates fermentation patterns of the
isolates are listed in table (1 & 2).
Primer design and PCR condition
The sequence of the primer set bacteriocin
forward was 5 - AAG AGT TTG ATC
CTG GCT CAG - 3 and the primer set
bacteriocin reverse was 5 - CTA CGG
CTA CCT TGT TAC GA - 3 (ventura et
al., 2001).
The specific designed primers were
ordered for synthesis from AccuOligo /
Bioneer /Korea. The PCR reaction was
performed in a total volum 30 µl containg
10 p mole/µl of each primer, 2x Taq PCR
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
Table.1 Biochemical test of identification for Lactobacillus acidophilus
Test
Gram stain
Motility
Catalase
Indole
MR
VP
Nitrate reduction
Gelatinase
Casein hydrolysis
Growth at 15°C
Growth at 45°C
LB1
LB2
+
+
LB3
+
LB4
+
LB5
+
LB6
+
-
-
-
-
-
-
+
+
+
+
+
+
-
-
±
-
-
±
-
±
+
+
±
+
+
+
+ : positive; - : negative; ± : variable
Table.2 Carbohydrates fermentation by Lactobacillus acidophilus
Test
Glucose
Fructose
Ribose
Trehalose
xylose
Lactose
Sorbitol
Raffinose
Rhamnose
Mannitol
Galactose
Sucrose
LB1
LB2
LB3
LB4
LB5
LB6
+
+
+
+
+
+
+
+
+
-
-
-
+
+
±
-
±
+
+
+
+
+
+
±
-
±
±
±
±
+
+
+
+
+
+
-
-
-
-
-
-
+
+
+
+
+
+
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
-
-
+ : positive; - : negative; ± : variable
Figure.1 Antimicrobial activity of Lactobacillus acidophilus against 4 indicator
bacterial spp.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
Figure.2 Antimicrobial activity of Lactobacillus acidophilus against
4 indicator bacterial spp.
Figure.3 Amplified PCR products from Lactobacillus acidophilus with bacteriocin primer.
Lane (1- 6) PCR products amplified from 6 Lactobacillus acidophilus. Lane 7: negative
control. Lane M: 10kb markers.
Proteus vulgaris, Staphylococcus aureas,
Staphylococcus
epidermidis,
Streptococcus pyogenes, and Bacillus
subtilis. The highest inhibitory activity
observed was against Bacillus subtilis,
Pseudomonas aerogenosa, Streptococcus
pyogenes,
Proteus
vulgaris,
Staphylococcus
aureas,
Aeromonas
Detection of antimicrobial activity
The results of present study, suggest that
the cell-free-supernatants exerted varying
inhibitory effect on the indicator
pathogenic strains. Inhibitory activity was
assessed against E.coli, Pseudomonas
aerogenosa,
Aeromonas
hydrophila,
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
hydrophila. Weaker activity was observed
against Staphylococcus epidermidis and
E.coli Fig (1 & 2). This indicates that the
Lactobacillus acidophilus possessing of
antibacterial activity.
inhibit the growth of Gram-positive
pathogenic , spoilage bacteria as well
yeasts and also inhibit the growth of some
Gram-negative species, therefore , such
these LAB can be used as probiotic and as
biopreservative (Lozo et al., 2004;
Topisirovic et al., 2006).
Detection of bacteriocin gene by PCR
The strains (LB1, LB2, LB3, LB4, LB5,
and LB6) were amplified with the
bacteriocin primers, producing a product
4000bp Fig 3.
The growth-inhibiting activity of LAB to
other bacterial spp has generally been
attributed to the fact that Lactobacillus spp
lower the pH and / or produce lactic acid,
for example strains of L.acidophilus ,L.
casei subsp, rhammnosus and L.
bulgaricus inhibited the growth of clinical
isolates of H.pylori (Midolo et al., 1995).
The addition of 10 N NaOH to adjust the
pH of the cell-free-supernatant to 6.5 was
for the elimination the inhibitory effects of
organic acids including : propionic acids,
lactic acetic and formic acid (Dunne et al.,
2001). Taale et al., (2013) tested 9
isolates of for bacteriocin gene. He
demonstrated that 8 out of the 9 tested
isolates produced the 3500- 4000 bp in the
PCR products. Garde et al. (2001)
detected the genes necessary for the
synthesis of lacticin 481 and nisin using
PCR techniques with specific probes on an
isolate of L. lactis subsp. lactis.
Antimicrobial activity is one of the most
important selection criteria for probiotics.
Lactic acid bacteria are produced some
substances such as organic acids hydrogen
peroxide, carbon dioxide and bacteriocins
(Dunne et al., 2001). Most of the
bacteriocins from LAB are cationic,
hydrophobic, or amphiphilic molecules
composed of 20 to 60 amino acid residues
(Nes and Holo 2000). These bacteriocins
are commonly classified into 3 groups that
also include bacteriocins from other Grampositive bacteria (Nes et al. 1996). Joshil
et al., (2006) reported that bacteriocin
from L. plantarum CA44 was activated
against, S. aureus, Bacillus cereus and
E.coli. Mkrtchyana et al., (2009) described
that Lactobacillus acidophilus n.v. Er
317/402 strain Narine produces a small
bacteriocin with a molecular weight of 1.1
kDa, designated acidocin LCHV. The
possible mechanism of bacteriocin
resistance of Gram-negative and some
Gram-positive bacteria has been suggested
to be associated with the barrier properties
of the outer membrane and the cell wall
(Stevens et al. 1991).
The present study, suggest that the six
Lactobacillus acidophilus can produce
antimicrobial compounds which can be
similar to bacteriocins and since the
isolates possessed bacteriocin gene, it is
conceivable that the ingredient in the
tested
supernatants
could
include
bacteriocin because they have bacteriocins
genes.
Bacteriocins have been reported to be
inhibitory against several other bacteria,
most of bacteriocins produced by Gram
positive bacteria are from lactic acid
bacteria (Ennahar et al. 2000; Garneau et
al., 2002). Some LAB bacteriocins can
In conclusion, Lactobacillus acidophilus
isolates that harbor the gene for
Bacteriocin exerted inhibitory effect on the
growth of a group of indicator bacterial
pathogens. It is conceivable that
bacteriocin is among the ingredients of the
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6) 269-276
supernatants prepared from the tested
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activity assay.
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