Fluorescence-Based Comparative Evaluation of BactericidalPotency and Food Application Potential of Anti-listerialBacteriocin Produced by Lactic Acid Bacteria Isolatedfrom Indigenous Samples

Atul Kumar Singh • Sandipan Mukherjee •

Manab Deb Adhikari • Aiyagari Ramesh

Published online: 3 April 2012

� Springer Science + Business Media, LLC 2012

Abstract The aim of the present study was to ascertain

the potency of anti-listerial bacteriocin produced by lactic

acid bacteria (LAB) isolated from indigenous samples

of dahi, dried fish, and salt-fermented cucumber. A total of

231 LAB isolates were obtained from the samples, of

which 51 isolates displayed anti-listerial activity. The anti-

listerial LAB were identified by PCR as Lactobacillus sp.,

Pediococcus sp., Enterococcus sp., and Lactococcus sp.

PCR also enabled the detection of Class IIa bacteriocin-

encoding genes such as enterocin A, pediocin, and plan-

taricin A in some of the LAB isolates. The culture filtrate

from anti-listerial LAB isolates demonstrated bacteriocin-

like inhibitory substance (BLIS) against common Gram-

positive pathogenic bacteria such as Staphylococcus aur-

eus, Enterococcus faecalis, and Bacillus cereus, and partial

characterization of BLIS confirmed the production of

bacteriocin by the LAB isolates. Sensitive fluorescence-

based assays employing specific probes indicated the

comparative potencies of the bacteriocin and clearly

revealed the membrane-targeted anti-listerial activity of the

purified bacteriocin produced by selected LAB isolates.

The food application potential of plantaricin A produced by

a native isolate Lactobacillus plantarum CRA52 was evi-

denced as the bacteriocin suppressed the growth of Listeria

monocytogenes Scott A inoculated in paneer samples that

were stored at 8 �C for 5 days.

Keywords Lactic acid bacteria � Bacteriocin �Membrane damage � Listeria monocytogenes �Paneer

Introduction

Lactic acid bacteria (LAB) play a critical role in food

processing and spontaneous fermentation and are used in a

wide range of fermented food. LAB contribute significantly

to flavor, aroma, and texture development [10, 24]; have

tremendous potential in improving the shelf-life of food

products; and ensure food safety by producing bacteriocins

[8, 19, 29]. Bacteriocins are essentially ribosomally syn-

thesized antimicrobial peptides that are considered to be

safe natural biopreservatives as they are sensitive to pro-

teases in the gastrointestinal tract and are effective in

controlling foodborne pathogens [9]. Nisin is the only

FDA-approved bacteriocin, which is widely used for

preservation of pasteurized processed cheese [11]. How-

ever, the limited activity spectrum of nisin with respect to

pH and its inherent insolubility has underscored the need

for additional bacteriocins that demonstrate superior sta-

bility over a wide range of pH and are suitable for food

fermentation and preservation processes.

Among the foodborne pathogens, Listeria monocytoge-

nes has been a serious cause of concern as it is ubiquitous

and can contaminate food at pre-and post-harvest stages of

production. Food safety issues are compounded as Listeria

is psychrotrophic and tolerant to stresses caused by low pH

and high acid content [22]. The use of bacteriocin-pro-

ducing (Bac?) LAB to combat Listeria is especially

attractive as reports suggest that bacteriocins from LAB,

such as nisin and pediocin-like Class IIa bacteriocins,

exhibit significant anti-listerial activity [13, 25]. A large

number of reports have demonstrated the application

potential of anti-listerial LAB or the bacteriocin produced

by such strains to inhibit the growth of Listeria in fer-

mented food samples [2, 3, 18]. In recent years, research

efforts have been focused toward isolation of anti-listerial

A. K. Singh � S. Mukherjee � M. D. Adhikari � A. Ramesh (&)

Department of Biotechnology, Indian Institute of Technology

Guwahati, Guwahati 781 039, Assam, India

e-mail: aramesh@iitg.ernet.in

123

Probiotics & Antimicro. Prot. (2012) 4:122–132

DOI 10.1007/s12602-012-9100-4

LAB from various food samples including traditionally

fermented food [1, 5]. It may be envisaged that indigenous

fermented food are likely to constitute a unique ecological

niche to screen bacteriocin-producing LAB strains. This

formed the basis of the present investigation wherein we

report the isolation of anti-listerial bacteriocin-producing

LAB from samples of dahi, dried fish, and fermented

cucumber. The anti-listerial activity of the bacteriocin

produced by the native LAB strains was compared by

sensitive fluorescence-based mechanistic studies that

clearly revealed a prominent membrane-targeted activity.

Furthermore, the efficacy of an anti-listerial bacteriocin to

inhibit the growth of L. monocytogenes in paneer (soft

cheese) samples, which is a widely consumed perishable

dairy product in India is demonstrated.

Materials and Methods

Bacterial Strain and Growth Conditions

All LAB and non-LAB strains were propagated and

maintained under the conditions mentioned in our previous

investigation [31].

Growth Media, Fluorescent Dyes, and Reagents

Brain Heart Infusion (BHI) broth and de Man, Rogosa and

Sharpe (MRS) broth were obtained from HiMedia, Mum-

bai. 5 (and 6) carboxyfluorescein diacetate succinimidyl

ester (cFDA-SE), propidium iodide (PI), N-phenyl naph-

thylamine (NPN), 3, 30-dipropylthiadicarbocyanine iodide

(diSC35), polymixin B, and valinomycin were purchased

from Sigma-Aldrich, USA. HEPES buffer was procured

from Sisco Research Laboratories (SRL), Mumbai, India.

Isolation of Anti-listerial LAB

Anti-listerial LAB were isolated from dahi (a lactic cul-

tured milk product obtained from domestic source), dried

fish, and salt-fermented cucumber. A total of 15 samples

were analyzed from each source. Dried fish sample con-

sisted of freshwater fish Corica soborna also known as the

Ganges river sprat. The commercial designation of the fish

is ‘Keski’ and it is sold as sun-dried whole fish. Dahi and

dried fish samples were added to MRS broth at 5 % (w/v)

level and enriched for 48 h at 37 �C. The enriched samples

were pour plated on MRS agar medium at various dilutions

and incubated at 37 �C for 24–48 h to obtain presumptive

LAB colonies. In case of salt-fermented cucumber samples

enriched in MRS, isolation of presumptive LAB was per-

formed as reported earlier [30]. All putative LAB colonies

were subjected to microscopic observation to determine

cell shape, gram-staining, and catalase activity test. Anti-

listerial LAB isolates were identified by overlaying the

colonies with BHI broth–soft agar medium (0.85 % agar)

seeded with L. monocytogenes Scott A and by observing

zone of inhibition (ZOI) around the colonies.

Identification of Potent Anti-listerial LAB

and Detection of Bacteriocin Gene

Potent anti-listerial LAB isolates were subjected to stan-

dard tests to establish their taxonomic identity [15]. Sugar

fermentation tests were conducted using the HiCarbohy-

drateTM kit (HiMedia, Mumbai) following the manufac-

turer’s instructions. PCR-based genus identification of the

LAB isolates was accomplished by using 16S rRNA gene-

specific primers [12, 30]. For isolates CRA21, CRA51, and

DF14, the PCR amplicons were sequenced and subjected to

BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

PCR-based detection of bacteriocin-encoding genes in the

antagonistic LAB isolates was accomplished by using

primers specific for nisin, pediocin, plantaricin A, and

enterocin A [30]. Bacteriocin gene amplicons from isolates

CRA21, CRA51, and DF14 were sequenced and subjected

to BLAST analysis.

Bacteriocin-Like Inhibitory Substance (BLIS)

LAB isolates exhibiting a ZOI of 10 mm or more against L.

monocytogenes Scott A in the colony overlay assay were

grown in MRS broth at 37 �C for 18 h, and the culture

filtrate was recovered by centrifugation at 8,8329g for

10 min at 4 �C. The pH of the culture filtrate was adjusted

to 7.0 with 2.0 N NaOH, followed by filter sterilization

using a 0.22-lm membrane filter (Millipore, India). The

resulting solution was referred to as bacteriocin-like

inhibitory substance (BLIS). Antimicrobial activity of

BLIS against select Gram-positive pathogenic bacteria

Staphylococcus aureus MTCC 96, Enterococcus faecalis

MTCC 439, and Bacillus cereus MTCC 1305 was mea-

sured by agar well diffusion assay [30]. Preliminary char-

acterization of BLIS was accomplished by studying the

effect of enzymatic treatment, dialysis, pH, and heat

treatments on the anti-listerial activity of BLIS.

Minimum Inhibitory Concentration (MIC)

and Minimum Killing Concentration (MKC) of Purified

Bacteriocin

A cell-adsorption method was adopted for purification of

the bacteriocin produced by potent anti-listerial LAB iso-

lates [15]. Bacteriocin titer in the purified sample was

ascertained against L. monocytogenes Scott A by a spot-on-

Probiotics & Antimicro. Prot. (2012) 4:122–132 123

123

lawn assay [14], and activity was expressed as arbitrary

units per ml (AU ml-1). Purity of the bacteriocin prepa-

ration was analyzed by Tricine-SDS-PAGE [28], and

determination of bacteriocin activity in the gel was

accomplished by the method described earlier [6]. MIC and

MKC of purified bacteriocin from select anti-listerial LAB

isolates were determined by a standard method [27].

Fluorescence-Based Comparative Assessment

of Bacteriocin Activity

The comparative potency of bacteriocin produced by native

LAB strains was evaluated using fluorescence-based

mechanistic studies on target cells of L. monocytogenes

Scott A. Essentially the studies included the following:

Membrane Damage

Purified bacteriocin samples from native LAB Lactoba-

cillus plantarum CRA21, Pediococcus pentosaceus

CRA51, and Enterococcus faecium DF14 were added at

varying concentrations (400, 800, 1,600, 3,200, and

6,400 AU ml-1) separately to L. monocytogenes Scott A

cells in PBS, which were pre-labeled with cFDA-SE by a

standard method [23]. Following the addition of bacterio-

cin, the labeled target cells were incubated at 37 �C for 6 h

in a circulating water bath (MultiTemp III, Amersham

Biosciences). Subsequently, the samples were centrifuged

at 8,8329g for 5 min, and leakage of dye from target cells

treated with bacteriocin was determined by measuring

fluorescence of the cell-free supernatant at an excitation

wavelength of 488 nm and an emission wavelength of

518 nm in a spectrofluorimeter (FluoroMax-3, HORIBA).

The fluorescence measurements were normalized by sub-

tracting the fluorescence of effluxed dye from control cells.

Fluorescence measurements were taken for three indepen-

dent samples and expressed as mean values.

Membrane damage of bacteriocin-treated target cells

was also determined by measuring uptake of PI. A stock

solution of PI was prepared in sterile MilliQ water at a

concentration of 1.5 mM. Cells of L. monocytogenes Scott

A (106 CFU) were treated with varying concentrations of

the purified bacteriocin preparation as mentioned before.

Subsequently, cells were washed with PBS, and PI was

added at a final concentration of 30 lM. After incubation

for 10 min, samples were centrifuged and washed in sterile

MilliQ water to remove excess dye. PI fluorescence in cells

was measured at an excitation wavelength of 535 nm and

an emission wavelength of 617 nm. Fluorescence data for

each sample were normalized with the optical density at

617 nm. The data points obtained for untreated cells were

subtracted from all experimental values.

Loss in Membrane Permeability

Loss of membrane permeability in L. monocytogenes Scott

A was determined using the fluorescent probe NPN as

described earlier [17], with slight modifications. Briefly,

target cells of L. monocytogenes Scott A cells were grown

in BHI broth at 37 �C in a shaker incubator to achieve the

desired growth (A600 of 0.5). The cells were centrifuged at

8,0009g for 3 min and washed twice with 5 mM HEPES

buffer (pH 7.4), followed by resuspension in the same

buffer. NPN was added at a final concentration of 10 lM to

1.0 ml cells taken in a cuvette. Varying concentrations of

purified bacteriocin obtained from native LAB strains were

added separately to the cells. Increase in fluorescence

intensity of NPN was measured as a function of time fol-

lowing the addition of bacteriocin. Cells treated with

polymixin B was used as a positive control. All fluores-

cence measurements were taken in a spectrofluorimeter

(FluoroMax-3, HORIBA) at an excitation and emission

wavelength of 350 and 420 nm, respectively. Fluorescence

measurements were taken for three independent samples.

Effect on Transmembrane Potential of Target Cells

The membrane depolarizing activity of the bacteriocins

(plantaricin A, pediocin, and enterocin A) from native LAB

strains on L. monocytogenes Scott A was evaluated using a

membrane potential-sensitive dye DiSC35 [36]. Valino-

mycin (30 lM) was used as a positive control, and blank

samples consisting of only cells and dye were included to

eliminate the background effect.

Anti-listerial Activity of Bacteriocin in Food Sample

Paneer was chosen as a model food sample to study the

effect of plantaricin A obtained from Lact. plantarum

CRA52 on the growth of L. monocytogenes Scott A inoc-

ulated in the product. Paneer is an Indian heat-acid coag-

ulated product of milk similar to tofu. Fresh paneer

samples purchased from commercial outlet were chosen,

and storage experiments were conducted to study the effect

of varying concentration of plantaricin A on the growth of

L. monocytogenes Scott A inoculated at two levels (6.3 and

4.3 log10 CFU g-1 of paneer). The detailed scheme for the

paneer experiments is outlined in Fig. 1.

Nucleic Acid Sequence

The GenBank accession numbers for partial 16S rRNA

gene sequence of isolates CRA21, CRA51, and DF14 were

FJ424061, FJ424059, and FJ424060, respectively. Bacte-

riocin gene amplicons from isolate CRA21, CRA51, and

DF14 were sequenced and their GenBank accession

124 Probiotics & Antimicro. Prot. (2012) 4:122–132

123

numbers were FJ424063, EU616745, and FJ424062,

respectively.

Results

Anti-listerial Bacteriocin-producing LAB Isolates

A total of 231 isolates were obtained cumulatively from

enriched indigenous samples, which encompassed 112

isolates from salt-fermented cucumber, 75 isolates from

dahi, and 44 isolates from dried fish. The isolates were

Gram-positive, catalase-negative and were either rod- or

cocci-shaped and were thus presumptive LAB isolates. In

the colony overlay assay, 51 colonies revealed anti-listerial

activity. The maximum number of anti-listerial LAB iso-

lates were obtained from fermented cucumber (26 nos.)

followed by dahi (14 nos.) and dried fish samples (11 nos.).

PCR experiments indicated the presence of a vast majority

of Lactobacillus sp. (11 nos.) in dahi samples, whereas 3

isolates were recognized as Lactococcus sp. In dried fish

samples, PCR revealed the predominance of Lactobacillus

sp. (10 nos.). In fermented cucumber samples, Lactoba-

cillus sp. (17 nos.) and Pediococcus sp. (9 nos.) could be

detected by PCR. A representative figure of an agarose gel

demonstrating the presence of specific amplicons corre-

sponding to various LAB genera is shown in Fig. 2a.

Phenotypic and sugar fermentation tests identified the

potent anti-listerial LAB isolates as Lact. plantarum (iso-

lates DF9, CRA21, CRA52, and CRA61), Ent. faecium

(isolate DF14), Ped. acidilactici (isolate CRA28), and Ped.

pentosaceus (isolate CRA51). These results were further

substantiated by partial 16S rRNA gene sequencing for

isolates DF14, CRA21, and CRA51 (GenBank accession

nos. FJ424060, FJ424061, and FJ424059, respectively).

PCR could account for the presence of only two plantaricin

A producers (CDRA27 and CDRA57) in dahi samples. In

dried fish samples, plantaricin A (isolate DF9) and

Fig. 1 Scheme of experiment

to study the effect of plantaricin

A produced by Lactobacillusplantarum CRA52 on the

growth of Listeriamonocytogenes Scott A

inoculated in paneer samples

Probiotics & Antimicro. Prot. (2012) 4:122–132 125

123

enterocin A (isolate DF14) producers could be detected by

PCR. In fermented cucumber, an overwhelming majority

were pediocin producers (11 nos.) while a small number of

plantaricin A producers (5 nos.) were also discernible. The

representative amplicons of the bacteriocin gene obtained

from select LAB isolates are shown in Fig. 2b. Bacteriocin

gene amplicons from isolate CRA21, CRA51, and DF14

were sequenced and their GenBank accession numbers

were FJ424063, EU616745, and FJ424062, respectively.

Antimicrobial Spectrum and Salient Features of BLIS

Among the LAB isolates, the anti-listerial activity of BLIS

present in the culture filtrate of isolates DF14, CRA21,

CRA51, and CRA52 was comparatively higher (ZOI of

16–18 mm in agar well diffusion assay). Isolate CRA52

revealed the highest average ZOI of 18 mm. BLIS from

native LAB isolates DF14, CRA21, CRA51, CRA52, and

CDRA27 also displayed activity against other gram-posi-

tive pathogenic bacteria such as Staph. aureus MTCC 96,

Ent. faecalis MTCC 439, and B. cereus MTCC 1305. The

anti-listerial activity of BLIS from select LAB isolates was

completely abolished after treatment with trypsin. How-

ever, activity was not affected by catalase treatment. It was

also observed that the activity was retained in a 1.0-kDa

dialysis bag and was abolished when the dialysate from a

12.0-kDa dialysis bag was tested. Results from additional

experiments provided evidence for retention of activity of

BLIS at various pH and heat treatments.

Purification of Bacteriocin, MIC, and MKC

Purification of bacteriocin produced by select anti-listerial

LAB isolates by cell-adsorption method resulted in

appreciable yield and purification fold. Bacteriocin activity

in the purified sample for the isolates ranged from 102,400

to 204,800 AU ml-1. The purified peptides were detected

by Tricine-SDS-PAGE analysis. A representative result for

purified pediocin produced by Ped. pentosaceus CRA51 is

shown in Fig. 3. It is evident that Tricine-SDS-PAGE

analysis revealed a single band corresponding to an

approximate molecular size of 4.6 kDa (Fig. 3a), with

retention of activity against L. monocytogenes Scott A

(Fig. 3b). The average MIC of purified bacteriocin from

the anti-listerial LAB isolates varied from 83.3 to

266.6 AU ml-1, whereas MKC ranged from 133.3 to

533.3 AU ml-1.

Comparison of Bactericidal Potency of Class IIa

Bacteriocins by Fluorescence-Based Assay

cFDA-SE-labeled target cells of L. monocytogenes Scott A

were treated with 400–6,400 AU ml-1 of purified

Fig. 2 Representative PCR amplicons obtained from native LAB

isolated from enriched indigenous samples with (a) 16S rRNA-based

genus-specific primers, Lb: Lactobacillus; Lc: Lactococcus; Ped:

Pediococcus; Leu: Leuconostoc; Lane M: k DNA EcoRI/HindIII

double digest size marker; b Bacteriocin gene-specific primers, P:

pediocin; N: nisin; M: mesentericin; E: enterocin A; PL: plantaricin

A; Lane M: 100 bp DNA ladder

Fig. 3 a Tricine-SDS-PAGE for purified pediocin produced by

Pediococcus pentosaceus CRA51; b Gel overlay assay of purified

pediocin showing anti-listerial activity. Lane M is a low molecular

weight marker (Sigma-Aldrich, USA). Arrow ‘1’ indicates purified

pediocin, and arrow ‘2’ depicts anti-listerial activity of purified

pediocin

126 Probiotics & Antimicro. Prot. (2012) 4:122–132

123

bacteriocin from LAB strains Lact. plantarum CRA21,

Ped. pentosaceus CRA51, and Ent. faecium DF14, and

leakage of the dye from treated cells was measured to

quantify the anti-listerial activity of the bacteriocins. The

results of the experiments are shown in Fig. 4a–c. It is

quite evident from the figure that leakage of cFDA-SE

increased as a function of increasing dose of bacteriocin.

Additional evidence of membrane damage in target cells of

L. monocytogenes Scott A was ascertained by uptake of PI,

which exhibited a dose-dependent pattern akin to leakage

of cFDA-SE, as observed in Fig. 4d–f.

Membrane permeabilization of L. monocytogenes Scott

A following exposure to the bacteriocin produced by the

native LAB isolates was determined by the NPN uptake

assay. A time-dependent increase in NPN fluorescence was

evident for cells treated with various Class IIa bacteriocins.

On the basis of the end-point NPN fluorescence (16 min

following exposure to bacteriocin), it was apparent that

plantaricin A produced by native strain of Lact. plantarum

CRA52 exhibited the highest membrane permeabilization

activity, with an end-point NPN fluorescence of 1,404,679

counts per second (cps), whereas pediocin from isolate

CRA51 and enterocin A from isolate DF14 revealed a

corresponding NPN fluorescence of 1,398,650 and

1,386,325 cps, respectively. The kinetics of increase in

NPN fluorescence for representative Class IIa bacteriocins

indicates that a rapid increase in NPN fluorescence

occurred in the first 7 min followed by a plateau in case of

L. monocytogenes Scott A cells treated with plantaricin A

and pediocin (Fig. 5a, b). In case of cells treated with en-

terocin A, the plateau was reached in about 5 min (Fig. 5c).

An increase in NPN fluorescence obtained for bacteriocin-

treated cells also revealed a dose-dependent pattern and

this trend was unequivocally observed for every bacterio-

cin. It was also observed that for all the samples, NPN

fluorescence obtained from cells treated with 1.0 lg ml-1

polymixin B (positive control) was higher compared to

bacteriocin-treated samples.

The membrane depolarization activity of Class IIa

bacteriocins obtained from LAB isolates on L. monocyt-

ogenes Scott A was determined by a fluorimetric method

using the membrane potential-sensitive dye DiSC35. Fig-

ure 5d–f shows the time course of DiSC35 fluorescence

obtained from cells treated with varying concentrations of

Class IIa bacteriocin and valinomycin (positive control).

It is quite clear from the figure that considerable increase

in DiSC35 fluorescence signal is observed following

treatment of Listeria cells with bacteriocin and this trend

was observed for all three bacteriocins. Further, treatment

of cells with higher dose of bacteriocin resulted in a

Fig. 4 Fluorescence-based assessment of membrane damage in L.monocytogenes Scott A following treatment with varying concentra-

tions of representative Class IIa bacteriocins produced by native LAB

strains. The measurements included cFDA-SE dye leakage (a–c) and

PI uptake (d–f) in bacteriocin-treated target cells

Probiotics & Antimicro. Prot. (2012) 4:122–132 127

123

corresponding increase in DiSC35 fluorescence. Compara-

tive analysis of DiSC35 end-point fluorescence (400 s fol-

lowing exposure to bacteriocin) indicated that the

membrane depolarization activity of plantaricin A obtained

from isolate CRA52 was superior (8,923,432 cps) com-

pared to pediocin from isolate CRA51 and enterocin A

from isolate DF14, which showed an end-point fluores-

cence of 7,989,110 and 6,366,130 cps, respectively.

Application of Anti-listerial Bacteriocin in Paneer

Samples

On the basis of antimicrobial activity, it was apparent that

plantaricin A produced by Lact. plantarum CRA52

exhibited broad-spectrum activity and exhibited the most

potent anti-listerial activity (ZOI of 18 ± 0.6 mm in agar

well diffusion assay). The strong anti-listerial activity of

this bacteriocin was also corroborated by low MIC and

MKC values (83.3 and 133.3 AU ml-1, respectively) and

strong membrane permeabilization and membrane depo-

larization activity. Hence, in the food application studies,

the potential of plantaricin A produced by isolate CRA52

to inhibit the growth of L. monocytogenes Scott A

inoculated in commercially available paneer samples was

tested at two different inoculum levels of the target path-

ogen (approximately 6.3 and 4.3 log10 CFU g-1) and

varying bacteriocin concentrations (2,560 and

1,280 AU g-1). It can be observed from Fig. 6a that the

levels of L. monocytogenes Scott A increased steadily from

an initial 6.3 log10 CFU g-1 to around 8.0 log10 CFU g-1

in the control samples after 5 days of incubation at 8 �C.

Application of 1,280 AU g-1 plantaricin A extract led to a

continuous decrease in the levels of Listeria till 60 h of

incubation, and a viable count of 5.53 log10 CFU g-1 was

obtained (Fig. 6a). The final cell number obtained in the

product at the end of 5 days of storage period at 8 �C was

5.74 log10 CFU g-1. It is also evident from Fig. 6a that at

higher bacteriocin concentration of 2,560 AU g-1, the

inhibitory effect on L. monocytogenes Scott A was marked,

and a progressive decline in the viable cell population

could be observed till 72 h of incubation, resulting in a

viable cell count of 4.89 log10 CFU g-1. The final cell

number of the pathogen after 5 days of storage at 8 �C was

4.93 log10 CFU g-1. In case of the experiments with lower

inoculum levels of L. monocytogenes (Fig. 6b), it was

observed that for control samples, an increase of viable

Fig. 5 Fluorescence-based detection of membrane permeabilization

and membrane depolarization in L. monocytogenes Scott A following

treatment with varying concentrations of representative Class IIa

bacteriocins produced by native LAB strains. The measurements

included NPN uptake (a–c) and DiSC35 fluorescence (d–f) in

bacteriocin-treated target cells

128 Probiotics & Antimicro. Prot. (2012) 4:122–132

123

Listeria cells by more than one log cycle (5.92 log10

CFU g-1) was evident within 48 h, and after 5 days of

storage at 8 �C, the viable counts culminated at 6.14 log10

CFU g-1. However, in presence of 1,280 AU g-1 plan-

taricin A in the paneer samples, the growth of the pathogen

was impeded, and a final viable count of the pathogen was

estimated to be 2.87 log10 CFU g-1. At a higher bacte-

riocin concentration of 2,560 AU g-1, the viable count of

Listeria in paneer revealed a progressive decline to reach a

level of 2.07 log10 CFU g-1.

Discussion

In the present study, potent anti-listerial LAB strains were

isolated from various indigenous samples (dahi, dried fish,

and fermented cucumber) enriched in MRS medium. The

isolates obtained from the samples were either rod- or

cocci-shaped and were initially attributed to LAB based on

the premise that they were Gram-positive and catalase-

negative. The prevalence of LAB isolates in fermented

cucumber and dahi agrees with our previous results [30,

31]. We could also obtain LAB, albeit in lesser numbers, in

dried fish samples. A colony overlay assay could identify

anti-listerial LAB isolates obtained from the samples. We

could isolate LAB from dried fish samples, which dis-

played considerable anti-listerial activity. This finding is

significant in light of only a few reports that indicate the

presence of antagonistic LAB in fish samples [32, 34]. PCR

facilitated rapid genus identification of the anti-listerial

LAB isolates. Based on standard phenotypic and sugar

fermentation tests, potent anti-listerial isolates were iden-

tified as Lact. plantarum (isolates DF9, CRA21, CRA52,

and CRA61), Ent. faecium (isolate DF14), Ped. acidilactici

(isolate CRA28), and Ped. pentosaceus (isolate CRA51).

16S rRNA gene sequencing further substantiated species-

level identification of the LAB isolates CRA21 (FJ424061),

CRA51 (FJ424059), and DF14 (FJ424060). It has been

previously demonstrated that PCR-based screening of bac-

teriocin gene is a rapid and convenient tool to characterize

antagonistic LAB isolates [20, 30]. Among the anti-listerial

LAB isolates obtained in the present investigation, PCR

experiments revealed a large number of Class IIa bacte-

riocin producers (plantaricin A, pediocin, and enterocin A),

which was further substantiated by partial nucleic acid

sequence of the bacteriocin gene from isolates CRA21

(FJ424063), CRA51 (EU616745), and DF14 (FJ424062).

The anti-listerial activity of the neutralized culture fil-

trate from select LAB isolates could be attributed to BLIS

and is in accordance with the well-established fact that

pediocin-like Class IIa bacteriocins are known to possess

high anti-listerial activity [13]. Inhibition of the growth of

pathogens such as Staph. aureus, Ent. faecalis, and B.

cereus by BLIS obtained from isolates DF14, CRA21,

CRA51, CRA52, and CDRA27 supports earlier reports that

indicate inhibition of Gram-positive pathogenic bacteria by

bacteriocins produced by LAB strains [1, 19]. Trypsin-

mediated inactivation of anti-listerial activity of BLIS from

select LAB isolates indicated the proteinaceous nature of

the anti-listerial compound. The role of hydrogen peroxide

as one of the components of BLIS was negated as catalase

treatment failed to abolish its antimicrobial activity.

Additional results clearly established the small molecular

size, pH, and heat stability of BLIS. Collectively, these

results provide strong evidence for bacteriocin production

by the anti-listerial isolates and support earlier research

Fig. 6 Effect of plantaricin A produced by Lactobacillus plantarumCRA52 on the growth of L. monocytogenes Scott A inoculated in

commercial paneer samples and stored at 8 �C for 5 days. Initial

levels of L. monocytogenes Scott A inoculated in paneer samples were

a 6.3 log10 CFU g-1 and b 4.3 log10 CFU g-1

Probiotics & Antimicro. Prot. (2012) 4:122–132 129

123

work that reports similar cardinal features of bacteriocin

produced by LAB [15, 33].

Purification of bacteriocin produced by select anti-lis-

terial LAB isolates (DF9, DF14, CRA21, CRA28, CRA51,

CRA52, and CRA61) by cell-adsorption method resulted in

appreciable yield and purification fold. Bacteriocin, being a

cationic peptide, could be readily adsorbed on producer

cells at pH 6.0 where the bacterial cell surface bears a net

negative charge. Subsequently, bacteriocin molecules

could be selectively desorbed at an acidic pH [15, 37]. In

the course of the investigation, the major aim was to

conduct a comparative study on the efficacy of select anti-

listerial bacteriocin from native LAB isolates and monitor

the target cell damage as a function of bacteriocin con-

centration. To this end, fluorescence-based assays provided

a strong platform to evaluate the relative potencies of

prototype Class IIa anti-listerial bacteriocins plantaricin A,

pediocin, and enterocin A. Experiments conducted with the

fluorescent dyes cFDA-SE and PI could ubiquitously

establish dose-dependent membrane damage in target cells

of L. monocytogenes Scott A. cFDA-SE is a cell-permeant

dye, and following uptake of the dye, the succinimidyl

group conjugates with aliphatic amines of intracellular

proteins. Fluorescence is detected due to the accumulation

of the fluorescent form of the dye, following cleavage of

the ester by intracellular esterase activity [16]. The use of

cFDA-SE-labeled target cells in bacteriocin assay rendered

a distinct advantage as the cross-linking of cFDA-SE with

intracellular bacterial proteins minimized the leakage of

the dye from intact cells, and pore formation in the mem-

brane of target cells following treatment with bacteriocin

could be clearly deciphered by measuring efflux of the dye

from damaged cells. Additional evidence for progressive

membrane damage of target cells as a function of bacte-

riocin concentration was obtained by measuring the uptake

of PI in bacteriocin-treated cells. PI is used as an indicator

of membrane integrity and has been used to determine

membrane damage [35]. PI is able to enter cells only if the

membrane is permeabilized or compromised. Upon entry

into cells, PI binds to single- and double-stranded nucleic

acids, yielding an intense red fluorescence. On the basis of

cFDA-SE and PI fluorescence, the membrane-targeted

dose-dependent anti-listerial activity of select Class IIa

bacteriocins was established and was in accordance with

earlier studies on mode of action of bacteriocins [4, 7, 21].

Experiments conducted to measure the uptake of the

hydrophobic probe NPN clearly suggested appreciable

membrane permeabilization of Listeria cells by the bacte-

riocins even at low concentrations of 350 and 450 AU.

Among the bacteriocins tested, plantaricin A was appar-

ently most potent as indicated by the magnitude of NPN

uptake in target cells, followed by pediocin and enterocin A.

Class IIa bacteriocins are known to induce permeabilization

of the target cell, possibly by forming ion-selective pores,

which results in the dissipation of the proton motive force

and depletion of intracellular ATP, culminating in the col-

lapse of transmembrane potential [13]. In the present study,

a progressive increase in DiSC35 fluorescence clearly sug-

gested rapid membrane depolarization in bacteriocin-trea-

ted target cells of L. monocytogenes Scott A. In contrast to

conventional microbiological assays such as agar well dif-

fusion assay or spot-on-lawn assay, which are less sensitive

and which fail to reflect the mode of action of bacteriocins,

it is quite evident from the present study that the fluores-

cence-based assays could not only provide an insight into

the mechanism of action of the bacteriocins produced

by native LAB strains but also rank the anti-listerial

potency of the bacteriocins based on the sensitivity of the

measurements.

Conventional as well as fluorescence-based bacteriocin

assays clearly established that plantaricin A produced by

the native Lact. plantarum CRA52 exhibited highest anti-

listerial activity. Hence, the promise of this bacteriocin as

an anti-listerial agent was ascertained in paneer, which is a

widely consumed non-fermented dairy product devoid of

any protective culture and is thus vulnerable to post-pro-

cessing contamination. Further, the commercially available

product is stored under refrigerated conditions, and hence,

contamination with psychrotrophic bacterial pathogens

such as L. monocytogenes could pose serious health con-

cerns. From the present investigation, it was evident that

paneer without any added bacteriocin and stored under

refrigeration was conducive to the growth of Listeria. This

may be attributed to the psychrotrophic nature of the

pathogen and the absence of any competitive or protective

microbial culture in paneer. Interestingly, in case of paneer

samples treated with plantaricin A, it was encouraging to

observe that the growth of L. monocytogenes was inhibited.

It was also apparent that the growth inhibition of L. mon-

ocytogenes in paneer was a function of both the concen-

tration of bacteriocin used and the initial inoculum level of

the target pathogen. Additional factors such as binding of

the bacteriocin to the paneer matrix resulting in depleted

bioavailability of the molecule for target cell interaction

and emergence of bacteriocin-resistant variants of L.

monocytogenes, as a consequence of continuous exposure

to bacteriocin as reported earlier [26], may also influence

the anti-listerial activity of the bacteriocin in the food

sample.

Conclusion

In the present investigation, anti-listerial bacteriocin-pro-

ducing LAB were isolated from enriched indigenous

samples. The application of PCR facilitated genus

130 Probiotics & Antimicro. Prot. (2012) 4:122–132

123

identification as well as detection of Class IIa bacteriocin

producers among the isolates. Phenotypic, sugar fermen-

tation tests, and 16S rRNA gene sequencing facilitated

species-level identification of potent anti-listerial LAB

isolates. Interestingly, culture filtrates from many of the

isolates exhibited a broad-spectrum antimicrobial activity

against common Gram-positive pathogenic bacteria. Partial

characterization of the antimicrobial compound in the

culture filtrate corroborated the presence of bacteriocin. A

combination of sensitive fluorescence-based assays could

clearly suggest the probable mode of action of bacteriocins

from select LAB isolates as well as identify a candidate

bacteriocin with the most potent anti-listerial activity. A

significant outcome of the present investigation was to

demonstrate the promise of plantaricin A, an anti-listerial

bacteriocin produced by strain Lact. plantarum CRA52, in

mitigating the growth of L. monocytogenes in a perishable

sample like paneer which was stored under refrigerated

conditions. It is anticipated that once the technological

attributes of the potent anti-listerial LAB strains isolated in

the present investigation are ascertained, some of the

strains are likely to find niche applications in future, par-

ticularly in food fermentation processes both as starter and

bioprotective cultures.

Acknowledgments We thank the Council of Scientific and Indus-

trial Research (CSIR), New Delhi, Government of India for a research

grant [No. 38(1251)/10/EMR-II]. We thank the National Facility of

Automated DNA Sequencing, Department of Biochemistry, Delhi

University, South campus for their support in nucleic acid sequencing.

References

1. Albano H, Pinho C, Leite D, Barbosa J, Silva J, Carneiro L,

Magalhaes R, Hogg T, Teixeira P (2009) Evaluation of a bacte-

riocin-producing strain of Pediococcus acidilactici as a biopre-

servative for ‘‘Alheira’’, a fermented meat sausage. Food Control

20:764–770

2. Albano H, Oliveira M, Aroso R, Cubero N, Hogg T, Teixeira P

(2007) Antilisterial activity of lactic acid bacteria isolated from

‘‘Alheiras’’ (traditional Portuguese fermented sausages): In situ

assays. Meat Sci 76:796–800

3. Allende A, Martinez B, Selma V, Gila MI, Suarez JE, Rodriguez

A (2007) Growth and bacteriocin production by lactic acid bac-

teria in vegetable broth and their effectiveness at reducing Lis-teria monocytogenes in vitro and in fresh-cut lettuce. Food

Microbiol 24:759–766

4. Atrih A, Rekhif N, Moir AJG, Lebrihi A, Lefebvre G (2001)

Mode of action, purification and amino acid sequence of plan-

taricin C19, an anti-listeria bacteriocin produced by Lactobacillusplantarum C19. Int J Food Microbiol 68:93–104

5. Belgacem BZ, Ferchichi M, Prevost H, Dousset X, Manai M

(2008) Screening for anti-listerial bacteriocin-producing lactic

acid bacteria from ‘‘Gueddid’’ a traditionally Tunisian fermented

meat. Meat Sci 78:513–521

6. Bhunia AK, Johnson MC, Ray B (1987) Direct detection of an

antimicrobial peptide of Pediococcus acidilactici in sodium

dodecyl sulphate-polyacrylamide gel electrophoresis. J Ind

Microbiol 2:319–322

7. Budde BB, Rasch M (2001) A comparative study on the use of

flow cytometry and colony forming units for assessment of the

antibacterial effect of bacteriocins. Int J Food Microbiol 63:

65–72

8. Castellano P, Belfiore C, Fadda S, Vignolo G (2008) A review of

bacteriocinogenic lactic acid bacteria used as bioprotective cul-

tures in fresh meat produced in Argentina. Meat Sci 79:483–499

9. Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bac-

teriocins: safe, natural antimicrobials for food preservation. Int J

Food Microbiol 71:1–20

10. Coolbear T, Crow V, Harnett J, Harvey S, Holland R, Martley F

(2008) Developments in cheese microbiology in New Zealand—

use of starter and non-starter lactic acid bacteria and their

enzymes in determining flavour. Int Dairy J 18:705–713

11. Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing

innate immunity for food. Nat Rev Microbiol 3:777–788

12. Deasy BM, Rea MC, Fitzgerald GF, Cogan TM, Beresford TP

(2000) A rapid PCR based method to distinguish between Lac-tococcus and Enterococcus. Syst Appl Microbiol 23:510–522

13. Drider D, Fimland G, Hechard Y, Mcmullen LM, Prevost H

(2006) The continuing story of Class IIa bacteriocins. Microbiol

Mol Biol Rev 70:564–582

14. Halami PM, Ramesh A, Chandrashekar A (2000) Megaplasmid

encoding novel sugar utilizing phenotypes, pediocin production

and immunity in Pediococcus acidilactici C20. Food Microbiol

17:475–483

15. Halami PM, Ramesh A, Chandrashekar A (2005) Fermenting

cucumber, a potential source for the isolation of pediocin like

bacteriocin producers. World J Microbiol Biotechnol 21:1351–

1358

16. Hoefel D, Groobya WL, Monisa PT, Andrews S, Saint CP (2003)

A comparative study of carboxyfluorescein diacetate and car-

boxyfluorescein diacetate succinimidyl ester as indicators of

bacterial activity. J Microbiol Methods 52:379–388

17. Ibrahim HR, Sugimoto Y, Aoki T (2000) Ovotransferrin anti-

microbial peptide (OTAP-92) kills bacteria through a membrane

damage mechanism. BBA 1523:196–205

18. Jagannath A, Ramesh A, Ramesh MN, Chandrashekar A, Va-

radaraj MC (2001) Predictive model for the behavior of Listeriamonocytogenes Scott A in Shrikhand, prepared with a biopre-

servative pediocin K7. Food Microbiol 18:335–343

19. Jones RJ, Hussein HM, Zagorec M, Brightwell G, Tagg JR

(2008) Isolation of lactic acid bacteria with inhibitory activity

against pathogens and spoilage organisms associated with fresh

meat. Food Microbiol 25:228–234

20. Knoll C, Divol B, du Toit M (2008) Genetic screening of lactic

acid bacteria of oenological origin for bacteriocin-encoding

genes. Food Microbiol 25:983–991

21. Kristiansen PE, Fimland G, Mantzilas D, Nissen-Meyer J (2005)

Structure and mode of action of the membrane-permeabilizing

antimicrobial peptide pheromone plantaricin A. J Biol Chem

280:22945–22950

22. Le Marc Y, Hucket V, Bourfeois CM, Guyonnet JP, Mafart P

(2002) Modelling the growth kinetics of Listeria as a function of

temperature, pH and organic acid concentration. Int J Food

Microbiol 73:219–237

23. Lee YK, Ho PS, Low CS, Arvilommi H, Salminen S (2004)

Permanent colonization by Lactobacillus casei is hindered by the

low rate of cell division in mouse gut. Appl Environ Microbiol

70:670–674

24. Leroy F, De Vuyst L (2004) Lactic acid bacteria as functional

starter cultures for the food fermentation industry. Trends Food

Sci Technol 15:67–78

Probiotics & Antimicro. Prot. (2012) 4:122–132 131

123

25. Montville TJ, Chen Y (1998) Mechanistic action of pediocin and

nisin: recent progress and unresolved questions. Appl Microbiol

Biotechnol 50:511–519

26. Naghmouchi K, Kheadra E, Lacroix C, Fliss I (2007) Class

I/Class IIa bacteriocin cross-resistance phenomenon in Listeriamonocytogenes. Food Microbiol 24:718–727

27. Pucci MJ, Vedamuthu ER, Kunka BS, Vandenbergh PA (1988)

Inhibition of Listeria monocytogenes by using bacteriocin PA-1

produced by Pediococcus acidilactici PAC 1.0. Appl Environ

Microbiol 54:2349–2353

28. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-

polyacrylamide gel electrophoresis for the separation of proteins

in the range from 1 to 100 kDa. Anal Biochem 166:368–379

29. Settani L, Corsetti A (2008) Application of bacteriocin in vege-

table food biopreservation. Int J Food Microbiol 121:123–138

30. Singh AK, Ramesh A (2008) Succession of dominant and

antagonistic lactic acid bacteria in fermented cucumber: insights

from a PCR-based approach. Food Microbiol 25:278–287

31. Singh AK, Ramesh A (2009) Evaluation of a facile method of

template DNA preparation for PCR-based detection and typing of

lactic acid bacteria. Food Microbiol 26:504–513

32. Thapa N, Pal N, Tamang JP (2006) Phenotypic identification and

technological properties of lactic acid bacteria isolated from

traditionally processed fish products of the Eastern Himalayas. Int

J Food Microbiol 107:33–38

33. Todorov SD, Dicks LMT (2006) Screening for bacteriocin-pro-

ducing lactic acid bacteria from boza, a traditional cereal bev-

erage from Bulgaria. Comparison of the bacteriocins. Process

Biochem 41:11–19

34. Tome E, Teixeira P, Gibbs PA (2006) Anti-listerial inhibitory

lactic acid bacteria isolated from commercial cold smoked sal-

mon. Food Microbiol 23:399–405

35. Virto R, Manas P, Alvarez I, Condon S, Raso J (2005) Membrane

damage and microbial inactivation by chlorine in the absence and

presence of a chlorine-demanding substrate. Appl Environ

Microbiol 71:5022–5028

36. Vooturi SK, Cheung CM, Rybak MJ, Firestine SM (2009)

Design, synthesis, and structure-activity relationships of benzo-

phenone-based tetraamides as novel antibacterial agents. J Med

Chem 52:5020–5031

37. Yang R, Johnson MC, Ray B (1992) Novel method to extract

large amounts of bacteriocins from lactic acid bacteria. Appl

Environ Microbiol 58:3355–3359

132 Probiotics & Antimicro. Prot. (2012) 4:122–132

123