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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 4; Year 2015; Page: 257 - 263

Research Article

EFFECT OF Lactobacillus lactis AND Lactobacillus acidophilus CRUDE

BACTERIOCIN ON THE INHIBITION OF DAIRY PRODUCT SPOILAGE
BACTERIAL STRAINS
J. Bhuvaneswari* and D. Kanchana,
Department of Microbiology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India.
Abstract
Various food preservation methods are used in preservation and combining various lightly
preservation procedures to inhibit growth of food spoilage causing microorganisms. The effect of crude
bacteriocin of Lactobacillus lactis and Lactobacillus acidophilus on the inhibition of growth of five dairy
spoilage bacterial strains was studied in the present research. The diameter of the inhibition zone of all the
bacterial strains was increased with increase in concentration of culture filtrate from 20 to 100 g ml-1.
Highest zone of inhibition was observed against Listeria monocytogenes, Staphylococcus aureus, Yersinia
enterocolitica and Pseudomonas aeruginosa. Lowest zone of inhibition was recorded in Campylobacter
jejuni. The Minimum inhibitory concentration (MIC) of crude bacteriocin of Lactobacillus lactis against all
the dairy spoilage bacterial strains was assessed. Among the five dairy spoilage bacteria, Campylobacter
jejuni recorded lower MIC followed by Pseudomonas aeruginosa (60 l), Yersinia enterocolitica (70 l),
Staphylococcus aureus (80 l) and Listeria monocytogenes (90 l) was inhibited. The MIC of crude
bacteriocin of Lactobacillus acidophilus against dairy spoilage bacterial strains was studied. The
Campylobacter jejuni recorded lower MIC (50 l) followed by Pseudomonas aeruginosa (60 l), Yersinia
enterocolitica (70 l), Staphylococcus aureus (80 l) and Listeria monocytogenes (90 l) was inhibited.
Key words: Dairy product, Bacterial spoilage,
Bacteriocin, Lactobacillus lactis and Lactobacillus
acidophilus.

Article History
Received : 24.04.2015
Revised : 02.05.2015
Accepted : 15.05.2015
1. Introduction

Milk is the common universal product

with more commercial demand, regardless of the
source, adding important protein content (casein)
to the various age peoples for their diet. The
microbiological characteristics and quality of the
milk and milk products are very important to the
consumer. The growth and activity of food
spoilage microorganisms is mostly described and
studied as function of substrate base and of
various parameters such as temperature, pH, water
* Corresponding author: J. Bhuvaneswari
E-mail: jbhuvann@gmail.com

activity and atmospheric pressure. Food spoilage

is one of the most important issues which were
mainly faced by the food and dairy industries. In
fact, food borne illnesses are a global problem,
even in developing countries which is mainly
caused by spore forming and toxin producing
bacteria. Spoilage of dairy product is
predominantly caused by the microbial growth in
variety of dairy products. Many pathogenic
microorganisms, including Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus,
Listeria monocytogenes, Campylobacter jejuni,
Yersinia enterocolitica, Candida albicans,
Fusarium oxysporum, Aspergillus flavus, Rhizopus

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J. Bhuvaneswari/ Life Science Archives (LSA), Volume 1, Issue 4, Page 257 - 263, 2015
stolonifer,
Penicillium
chrysogenum
and
Salmonella typhi have been identified as the
causing agents of the major food borne diseases,
food borne illness and food spoilage (Betts et al.,
1999; Solomakos et al., 2008).
A number of microorganisms belongs to
Lactobacillus species are able to produce
biologically active antimicrobial peptides which is
a proteinaceous substance named bacteriocins
which have a broad spectrum activity, target
attained mode of action, appropriate molecular
weight and biochemical properties. Bacteriocins
are bacteriostatic or bactericidal to pathogenic
bacteria, especially those are close in
taxonomically related, but also bacteria confined
within the food spoilage causing group. The
producer strain is usually immune to the produced
bacteriocin (Abee et al., 1995; OKeeffe et al.,
1999; Van Reenen et al., 2002). Bacteriocins
produced by food-associated microorganisms such
as lactic acid bacteria, in particular Lactobacillus
lactis and Lactobacillus acidophilus is attracting
increasing attention as food biopreservatives
(Abee et al., 1995). Because of its protein nature,
bacteriocins are readily degraded by the protein
degrading enzyme protease in the gastrointestinal
tract of humans and most of the bacteriocin
producing lactic acid bacteria has Generally
Regarded as Safe (GRAS) status. Because of that
reasons bacteriocins are considered as the natural
biopreservatives (Schillinger et al., 2001;
Aymerich et al., 2000). Bacteriocins may also be
used as part of a multiple hurdle preservation
system (Cleveland et al., 2001), the bacteriocin
produces may be applied as bacteriocinogenic
cultures to non-fermented foods or even be used as
starter culture for fermented foods facilitate to the
improvement of quality and safety and to control
the bacterial spoilage or pathogenic bacterial
isolates (Zhang et al., 1999; Nilsson et al., 2000).
Several activities of bacteriocins have been
reported by various researchers and they proposed
its potential in the food industry (Cleveland et al.,
2001). However, it was important that applied
studies be done to confirm the effectiveness of the
addition of bacteriocins to food systems as it has

258

been shown that they are not effective in all food

systems (Ganzle et al., 1999; Cleveland et al.,
2001). Patented applications of bacteriocins as
food preservatives include the use of a
combination of nisin (produced by Lactococcus
lactis
subsp.
Lactis
and
Lactobacillus
acidophilus), a chelating agent and a surfactant to
inhibit the growth of both Gram positive and
Gram negative pathogenic microorganisms in
various dairy products like milk, cheese, butter,
butter milk, ghee and yoghurt (Blackburn et al.,
1998). Streptococcus and Pediococcus derived
bacteriocins in combination with a chelating agent
were successfully used to protect the dairy food
products against Listeria monocytogenes and other
milk spoilage causing microorganisms (Wilhoit,
1996).
2. Materials and Methods
2.1. Culture Collection
The strain Lactobacillus lactis (MTCC 4464) and Lactobacillus acidophilus (MTCC 4725) were obtained from MTCC (Microbial type
culture collection); Chandigarh was grown in
sterilized MRS broth (pH 6.0) for 2 days at 30 C.
The broth was centrifuged and the cell free
culture filtrates were collected.
2.2. Preparation of bacteriocin
MRS broth was prepared in 200 ml
quantities in 500 ml Erlenmeyer flask and
inoculated the culture of Lactobacillus lactis and
Lactobacillus acidophilus. After the incubation
period, cultured broth was centrifuged at 5000 rpm
for 15 min at 4oC. The supernatant was filtered
through 0.22 l filters (Hi-media; India) and
adjusted to pH 6.0 with 1M NaOH, to eliminate
the inhibitory effect caused by the decrease of pH.
This was followed by treatment with catalase to
remove the inhibitory action of hydrogen peroxide
and dissolved in phosphate buffer at pH 7.0 at 1
mg/ml final concentration and incubated for 30
min at room temperature. Supernatant was
concentrated by evaporation (Lyoplization) and
crude bacteriocin was prepared, used for further
assays (Ganzle et al., 1999). One gram of crude
bacteriocin was dissolved in 10 ml of sterile

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J. Bhuvaneswari/ Life Science Archives (LSA), Volume 1, Issue 4, Page 257 - 263, 2015
distilled water for 10% concentration and stored
in refrigerator. The antagonistic activity of crude
bacteriocin from Lactobacillus lactis and
Lactobacillus acidophilus was tested by Well
diffusion assay and Minimum inhibitory
concentration test was followed by Tagg and
McGiven (1971) and Darwina et al. (2012).
2.3. Effect of Lactobacillus lactis and
Lactobacillus acidophilus crude bacteriocin on
the inhibition of growth of the dairy product
spoilage bacterial strains by Well diffusion
assay method
One ml of the cell suspension (107 cell ml) of the dairy product spoilage bacterial strains
viz., Listeria monocytogenes (MPB - 1), Yersinia
enterocolitica (MPB - 2), Campylobacter jejuni
(MPB - 3), Staphylococcus aureus (MPB - 4) and
Pseudomonas aeruginosa (MPB - 5) were
prepared separately, mixed with the 100 ml
Mueller Hinton agar medium (seeded medium)
and plated. On the surface of the medium, well
were made by using sterile cork borer (6 mm in
size). Ten percent of crude bacteriocin at different
levels viz., 10, 20, 30, 40 and 50 l mixed with
sterile water to make upto total volume to 100 l
was discharged into the well. The well with sterile
water served as control. The plates were incubated
for 2 days at 30C. The inhibition zone (in mm)
was measured around the well using antibiotic
zone scale. Three replications were maintained in
each treatment. The test for antagonistic activity
was performed as per by Tagg and McGiven
(1971) and Jageethadevi et al. (2012).
1

2.4. Effect of Lactobacillus lactis and

Lactobacillus acidophilus crude bacteriocin on
the inhibition of dairy product spoilage
bacterial strains by Minimum inhibitory
concentration (MIC) method
Bacterial cell suspension of one ml was mixed
with 100 ml Nutrient broth. The seeded Nutrient
broth (along with the inoculum) was poured
separately into the test tube and ten percent of
crude bacteriocin (aqueous solution) at different
levels viz., 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8,
2.0, 2.2, 2.4, 2.6, 2.8 and 3.0 l, were added to the

259

test tubes and without Bacteriocin served as

control. The test tubes were incubated for 2 days
at 300C and the growth or inhibition was observed
by turbidity developed in the test tubes. The MIC
is the lowest concentration in the serial dilution
that inhibits the growth of the test organism.
Minimum inhibitory concentration test were
determined as followed by Tagg and McGiven
(1971).
3. Results and Discussion
Most lactic acid bacteria produce diacetyl
(2, 3-butanedione) during the stationary growth
phase by metabolizing the pyruvate accumulated
during the exponential growth phase. Some citrate
fermenting bacteria such as Lactococcus lactis and
Leuconostocs species produced diacetyl through
the fermentation of citrates. Lactic acid bacteria
showed broad spectrum antibacterial activity,
antifungal activity and antiprotozoal activity (Ray
and Daeschel, 1992; Saranraj and Geetha, 2012).
The effect of Lactobacillus lactis culture
bacteriocin on the inhibition of bacterial strains
viz.,
Listeria
monocytogenes,
Yersinia
enterocolitica,
Campylobacter
jejuni,
Staphylococcus aureus and Pseudomonas
aeruginosa was studied. The diameter of the
inhibition zone of all the bacterial strains was
increased with increase in concentration of culture
bacteriocin from 20 to 100 g ml-1. Highest zone
of inhibition was observed against Listeria
monocytogenes (23.00 mm), Staphylococcus
aureus (22.00 mm), Yersinia enterocolitica (21.00
mm) and Pseudomonas aeruginosa (18.66 mm).
Lowest zone of inhibition was recorded in
Campylobacter jejuni (19.33 mm). Many
researchers proposed that the lactic acid bacterial
isolates like Lactobacillus lactis and Lactobacillus
acidophilus possesses antagonistic activity against
infectious pathogens (Table 1). The Minimum
inhibitory concentration (MIC) of crude
bacteriocin of Lactobacillus lactis against all the
dairy spoilage bacterial strains was assessed and
the results were given in Table - 2.

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J. Bhuvaneswari/ Life Science Archives (LSA), Volume 1, Issue 4, Page 257 - 263, 2015

260

Table 1: Inhibitory effect of Lactobacillus lactis crude bacteriocin on Dairy product spoilage bacteria
by Agar well diffusion method
Concentration of
Diameter of zone of inhibition (mm)
Lactobacillus
Listeria
Yersinia
Campylobacter
Staphylococcus
lactis crude
monocytogenes
enterocolitica
jejuni
aureus
bacteriocin (l)
Control
NZ
NZ
NZ
NZ
10
13.66
12.00
10.00
13.00
20
14.66
13.00
11.66
14.00
30
16.00
14.66
12.00
15.33
40
17.66
15.00
13.66
16.66
50
23.00
21.00
18.33
22.00
S.Ed
1.64
1.56
1.42
1.57
CD (P = 0.05)
3.29
3.13
2.85
3.15
NZ No zone of inhibition; *Values are average of three replications

Pseudomonas
aeruginosa
NZ
11.66
12.66
13.00
14.00
19.66
1.41
2.83

Table 2: Minimum inhibitory concentration (MIC) of Lactobacillus lactis crude bacteriocin on

Dairy product spoilage bacteria
Concentration of
Lactobacillus
Minimum inhibitory concentration (l)
lactis crude
Listeria
Yersinia
Campylobacter
Staphylococcus
bacteriocin (l)
monocytogenes
enterocolitica
jejuni
aureus
10
+++
+++
+++
+++
20
+++
+++
++
+++
30
+++
+++
+
+++
40
+++
+++
+
+++
50
+++
++
+++
60
+++
+
++
70
++
+
80
+
90
100
+++ = More growth; ++ = Moderate growth; + = Growth; - = No growth

Pseudomonas
aeruginosa
+++
+++
++
+
+
-

Table 3: Inhibitory effect of Lactobacillus acidophilus crude bacteriocin on Dairy product spoilage bacteria
by Agar well diffusion method
Concentration of
Lactobacillus
Diameter of zone of inhibition (mm)
acidophilus crude
Listeria
Yersinia
Campylobacter
Staphylococcus
bacteriocin (l)
monocytogenes
enterocolitica
jejuni
aureus
Control
NZ
NZ
NZ
NZ
10
14.78
13.10
11.18
14.06
20
15.69
14.25
12.73
15.18
30
17.22
15.78
13.20
16.42
40
18.73
16.08
14.80
17.60
50
24.15
22.16
19.40
23.26
S.Ed
1.65
1.56
1.40
1.60
CD (P = 0.05)
3.04
3.13
2.81
3.03
NZ No zone of inhibition ; *Values are average of three replications

Pseudomonas
aeruginosa
NZ
12.70
13.58
14.26
15.18
20.72
1.41
2.83

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J. Bhuvaneswari/ Life Science Archives (LSA), Volume 1, Issue 4, Page 257 - 263, 2015

261

Table 4: Minimum inhibitory concentration (MIC) of Lactobacillus acidophilus crude bacteriocin on

Dairy product spoilage bacteria
Concentration of
Lactobacillus lactis
Minimum inhibitory concentration (l)
crude bacteriocin
Listeria
Yersinia
Campylobacter
Staphylococcus
(l)
monocytogenes
enterocolitica
jejuni
aureus
10
+++
+++
+++
+++
20
+++
++
++
+++
30
+++
++
+
++
40
++
++
+
++
50
++
++
++
60
++
+
+
70
+
+
80
+
90
100
+++ = More growth; ++ = Moderate growth; + = Growth; - = No growth

The increase in the level of bacteriocin of

Lactobacillus lactis from 10 to 100 l increased
the inhibition of growth of all the dairy spoilage
bacteria. Among the five dairy spoilage bacteria,
Campylobacter jejuni recorded lower MIC
followed by Pseudomonas aeruginosa (60 l),
Yersinia enterocolitica (70 l), Staphylococcus
aureus (80 l) and Listeria monocytogenes (90 l)
was inhibited.
The inhibitory effect of crude bacteriocin
of Lactobacillus acidophilus on the inhibition of
growth of five dairy spoilage bacterial strains viz.,
Listeria monocytogenes, Yersinia enterocolitica,
Campylobacter jejuni, Staphylococcus aureus and
Pseudomonas aeruginosa was studied and the
results were presented in Table 3. The diameter
of the inhibition zone of all the bacterial strains
increased with increase in concentration of culture
filtrate from 10 to 50 g ml-1. Among the five
bacterial strains, Listeria monocytogenes recorded
the highest mean inhibition (24.15 mm) followed
by Staphylococcus aureus, Yersinia enterocolitica,
Pseudomonas aeruginosa and Campylobacter
jejuni. The bacterial strain Listeria monocytogenes
recorded significantly more zone compared to
other dairy spoilage bacterial strains. The
Minimum inhibitory concentration (MIC) of crude
bacteriocin of Lactobacillus acidophilus against
all the dairy spoilage bacterial strains was assessed
and the results were given in Table - 4.

Pseudomonas
aeruginosa
+++
++
++
+
+
-

The increase in the level of bacteriocin of

Lactobacillus lactis from 10 to 100 l increased
the inhibition of growth of all the dairy spoilage
bacteria. Among the five dairy spoilage bacteria,
Campylobacter jejuni recorded lower Minimum
inhibitory concentration (MIC) (50 l) followed
by Pseudomonas aeruginosa (60 l), Yersinia
enterocolitica (70 l), Staphylococcus aureus (80
l) and Listeria monocytogenes (90 l) was
inhibited.
Ogunbanwo (2008) proposed that the lactic
acid bacteria Lactobacillus acidophilus which was
isolated from milk products and their study was
based on its ability to produce a bioactive protein
bacteriocin with inhibitory activity against food
spoilage and food borne pathogenic bacteria such
as Staphylococcus aureus, Listeria denitrificans,
Listeria monocytogenes, Campylobacter jejuni,
Yersinia enterocolitica, Pseudomonas aeruginosa,
Escherichia coli, Enterococcus faecalis and
Micrococcus luteus.
Lactic acid bacteria like Lactobacillus
lactis and Lactobacillus acidophilus isolated from
various milk products was able to show its
inhibitory action against Bacillus cereus, Bacillus
subtilis, Staphylococcus aureus, Micrococcus
luteus, Listeria monocytogenes and Gram negative
bacteria including Escherichia coli, Pseudomonas
fragi, Pseudomonas aeruginosa, Vibrio cholerae,

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J. Bhuvaneswari/ Life Science Archives (LSA), Volume 1, Issue 4, Page 257 - 263, 2015
Yersinia enterocolitica, Campylobacter jejuni,
Flavobacterium sp., Shewanell sp. and
Enterobacter aerogenes (Dilabaghi and Sharma,
1996).
The
biopreservative
activity
of
Lactobacillus acidophilus strains was reported by
Kanatani et al. (1995). They reported that
Lactobacillus acidophilus produced bacteriocin
that was active against different species of
Enterococcus casseliflavus, Lactobacillus lactis,
Lactobacillus acidophilus, Pediococcus sp.,
Streptococcus
pyogenes
and
Listeria
monocytogenes. The inoculation of Listeria
monocytogenes in dairy foods as starter culture
can be used to prevent the proliferation of dairy
product spoilage bacteria which may cause
degradation of milk products that was exposed to
high temperature.
The inhibitory effect of heat stability of
crude bacteriocin (extracted from Lactobacillus
lactis and Lactobacillus acidophilus) at various
time intervals and different temperatures against
dairy product spoilage bacteria were studied. The
inhibitory activity was unaffected by heating upto
120 C for 40 min and 70 % of activity retained
after heat treatment at 120 C for 15 min. More
inhibition was noted in 15 min time interval at 20
C followed by 40 C, 60 C, 80 C, 100 C and
120 C. Savitha Janakiraman et al. (2012) reported
that the activity of crude bacteriocin in
Lactobacillus acidophilus and Lactobacillus lactis
after heat treatment at 121 0C for 15 min.
Therefore, it could be grouped under heat stable
low molecular weight bacteriocin. The finding of
the present research was matched with the results
which were proposed by Savitha Janakiraman et
al. (2012).
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