Bacteriocin

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely
related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this
has been debated [1] They are phenomenologically analogous to yeast and paramecium killing factors,
and are structurally, functionally, and ecologically diverse. Bacteriocins were first discovered by A.
Gratia in 1925.[2][3] He was involved in the process of searching for ways to kill bacteria, which also
resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few
years. He called his first discovery a colicine because it killed E. coli.

Classification of bacteriocins
Bacteriocins are categorized in several ways, including producing strain, common resistance
mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only
phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins [4],
the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins
(formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a
diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example
the bacteriocins produced by Staphylococcus warneri, are called as warnerin[5] or warnericin. In fact,
one of the oldest known so-called colicins was called colicin V and is now know as microcin V. It is
much smaller and produced and secreted in a different manner than the classic colicins. The bacteriocins
of lactic acid-fermenting bacteria are called lantibiotics. This naming system is problematic for a number
of reasons. First, naming bacteriocins by what they putatively kill would be more accurate if their killing
spectrum were contiguous with genus or species designations. The bacteriocins frequently possess
spectra that exceed the bounds of their named taxa and almost never kill the majority of the taxa for
which they are named. Further, the original naming is generally derived not from the sensitive strain the
bacteriocin kills, but instead the organism that produces the bacteriocin. This makes the use of this
naming system a problematic basis for theory; thus the alternative classification systems.

Methods of classification

Alternative methods of classification include: method of killing (pore forming, dnase, nuclease, murein
production inhibition, etc), genetics (large plasmids, small plasmids, chromosomal), molecular weight
and chemistry (large protein, polypeptide, with/without sugar moiety, containing atypical amino acids
like lanthionine) and method of production (ribosomal, post ribosomal modifications, non-ribosomal).

One method of classification fits the bacteriocins into Class I, Class IIa/b/c, and Class III. [6]

Class I bacteriocins

The class I bacteriocins are small peptide inhibitors and include nisin.

Class II bacteriocins

The class II bacteriocins are small heat-stable proteins. The class IIa bacteriocins (pediocin-like
bacteriocins) are the largest subgroup and contain an N-terminal consensus sequence -Tyr-Gly-Asn-Gly-
Val-Xaa-Cys across this group. The C-terminal is responsible for species-specific activity, causing cell-
leakage by permeabilizing the target cell wall. Class IIa bacteriocins have a large potential for use in
food preservation as well medical applications, due to their strong anitlisterial activity, and broad range
of activity. The class IIb bacteriocins (two-peptide bacteriocins) require two different peptides for
activity. Other bacteriocins can be grouped together as Class IIc (circular bacteriocins). These have a
wide range of effects on membrane permeability, cell wall formation and pheromone actions of target
cells.

Class III bacteriocins

Large, heat-labile protein bacteriocins.

Abstract

A total of 220 strains of LAB isolated from 32 samples of traditional fermented food from Senegal were
screened for bacteriocin production. Two bacteriocin producers, Lactococcus lactis subsp. lactis and
Enterococcus faecium, were identified from 12 bacteriocin-producing isolates on the basis of phenotypic
analyses and 16S rDNA sequence. Both bacteriocins produced by new isolates show antimicrobial
activity against Listeria monocytogenes and Bacillus coagulans whereas only that produced by
Lactococcus lactis has an activity against Bacillus cereus. Bacteriocin-producing Lactococcus lactis
strains were found in a variety of traditional foods indicating a high potential of growth of this strain in
variable ecological complex environment. Partial 16S rDNA of the two bacteriocin producers obtained
in this study has been registered to Genbank databases under the accession number AY971748 for
Lactococcus lactis subsp. lactis (named CWBI-B1410) and AY971749 for Enterococcus faecium
(named CWBI-B1411). The new bacteriocin-producing Lactococcus lactis subsp. lactis strain has been
selected for identification and application of the bacteriocin to food preservation.

Keywords : bacteriocin, food preservation, lactic acid bacteria

1. Introduction

Lactic acid bacteria (LAB) are the biological basis for the production of a great multitude of fermented
foods (Lasagno et al., 2002). The most important contribution of these bacteria to fermented products is
to preserve the nutritive qualities of the raw material and inhibit the growth of spoilage and pathogenic
bacteria (Matilla-Sandholm et al., 1999). This inhibition may be due to the production of many
metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, diacetyl and bacteriocins
(Ennahar et al., 2000 ; Lasagno et al., 2002). Some bacteriocins kill only bacteria belonging to the same
species as producer whereas other bacteriocins kill a broad range of Gram positive bacteria (Conventry
et al., 1997 ; Ennahar et al., 2000 ; Mc Auliffe et al., 2001 ; Garneau et al., 2002). The incorporation of
these compounds as biopreservative ingredient into model food has been shown to be effective in the
control of pathogenic and spoilage micro-organisms (O'Sullivan et al., 2002). They have attracted
considerable interest in recent years and several works have focused on the isolation and development of
new strains of bacteriocin-producing bacteria. The detection rate of bac+ strains from LAB isolates can
be as low as 0.2% and therefore needs a large number of isolates from food sources (Conventry et al.,
1997).

The preservation of foods by lactic fermentation has a long history of use in Africa. The multitude of
products can be an appropriate ecological habitat for holding wild strains of LAB capable of producing
bacteriocin. The aim of the current study was to select bacteriocin-producing LAB from such products in
order to use these proteinaceous inhibitors to improve the microbial quality and safety of foods.

2. Materials and methods

2.1. Culture and media

MRS1 agar (MRS with 0.1% glucose, and 50 µg/ml of cycloheximide) and M17m agar (0.5% glucose
and 50 µg/ml cycloheximide) were utilised for isolation of bacteria from food sources. Different food-
borne pathogens and Gram+ bacteria including Escherichia coli, Salmonella infantis, Salmonella
typhimurium, Staphylococcus aureus, Listeria monocytogenes and Lactobacillus curvatus from CWBI
collection were used as sensitive. Strains of Lactobacillus curvatus are found to be the most suitable
indicator for the quantification of antimicrobial effects of all bacteriocins investigated in both agar and
broth system (Conventry et al., 1997).

2.2. Food sources

Strains were isolated from a total of 32 samples of traditional foods from Senegal (Table 1).

2.3. Detection of antimicrobial activity

Lactic acid bacteria were isolated from samples, by direct plating on MRS1 or M17m. A 10% (w/v) food
sample in diluent [0.1% (w/v) peptone] was homogenized and 10-fold serially diluted. Plates of serial
dilution in MRS1 and M17m media were incubated anaerobically (BD, BBL Campypak microaeroplilic
systems Envelopes, Sparks, USA) for 48 h at 30°C. Plates providing a total of 300 colonies were
overlaid with a set of 6 indicators and incubated at 37°C for 12 h. Colonies producing zones of growth
inhibition in the indicator lawn were isolated from within the agar, inoculated into broth media (MRS1
or M17m) and incubated for 24 h at 30°C. Culture supernatant was prepared as follows: an overnight
culture of each isolate was centrifuged at 8,000 r.p.m. The resulting supernatant was neutralized (pH 6.5)
with NaOH 5N, sterilized by filtering with acrodisc (pore size 0.22 µm) and assayed for the presence of
an inhibitor in the broth following the Agar well diffusion assay (WDA) technique (Barefoot et al.,
1983) as follows. Molten agar was first seeded with indicator organism (110 µl of overnight culture per
20 ml of agar) in sterile Petri dishes, and after solidification, dried for 15 min. under flow hood. Wells of
uniform diameter (6 mm) were bored in the agar. Aliquots (60 µl) of the cell-free supernatant (CFS)
were dispensed in wells, and plates were incubated overnight at 37°C. Inhibition of growth was
determined by an area of inhibition surrounding each agar well.

2.4. Bacteriocin assay

To confirm the bacteriocin effect, catalase (65 UI/ml) was added to CFS and the technique was repeated.
The effect of various enzymes and heat treatment of CFS activity were also investigated. Units and MIC
(Minimum inhibitory concentration): the activity present in the neutralized (pH 6.5) cell-free supernatant
of producing cultures was determined by twofold serial dilution of the supernatant in sterile phosphate
buffer pH 6 (Barefoot et al., 1983). Activity units per milliliter (AU/ml) were determined as the inverse
of the last dilution at which growth inhibition was still detectable following the agar WDA. To
determine the effects of enzymatic treatments, samples (180 µl of twice the minimum inhibitory
concentration corresponding to the supernatant from the cell-free culture) were incubated with 20 µl
portion of following enzyme solutions, P3911 (16.6 UI/ml), type XIV (7.9 UI/ml), type XVIII
(0.66 UI/ml), proteinase K (59.2 UI/ml), chymotrypsin (700 UI/ml) at 37°C for 1 h 30 min. (Jack et al.,
1996) and the residual activity was measured following the WDA. Positive controls were incubated with
20 µl of 50 mM phosphate buffer (pH 6.5). To determine the effect of temperature and pH on the
stability of the inhibitor sample corresponding to a dilution of ¼ of neutralized cell-free supernatant
(pH 6.5) in 50 mM phosphate buffer at pH 5, 7 and 9, and aliquots of each were subsequently heated to
60, 70, 80, 90, 100, 110 and 121°C for 10 min. (Ryan et al., 1996). The remaining activity was assayed
and compared to activity at each pH prior to heat treatment.

2.5. Bacterial identification

Selected isolates were examined microscopically for cellular morphology and Gram stain phenotype.
Catalase activity was tested by spotting colonies with 3% hydrogen peroxide. Fermentation of different
sugars was determined by API 50 CHL (Biomerieux). PCR was used to amplify the 16S rRNA gene of
bacteriocin-producing strains. The 16S rDNA sequence was determined by direct sequencing. Total
DNA was isolated by using Wizard genomic DNA purification kit (Promega, Madison, USA). Primers
used for PCR and DNA sequencing are presented in table 2. The PCR amplification was performed with
the primer pair SPO/SP6 targeted against regions of 16S rDNA (Ventura et al., 2001). Amplification of
DNA was performed in a Mastercycler personal thermal cycler (Eppendorf). PCR conditions included a
hot start at 96°C (5 min.), 25 cycles consisting of hybridation at 50°C (1 min), polymerisation at 72°C
(2 min.), denaturation at 96°C (1 min) and a final extension at 72°C (10 min.). PCR products were
resolved by electrophoresis in 1% (w/v) agarose gel and visualized by ethidium bromide (1 µl/10 ml)
staining. 16S rDNA PCR amplicons were purified following the microcon YM-100 kit (Bedford, MA,
USA) and sequenced using the Big Dye Terminator V3.0 kit as specified by the supplier with primers
described in table 2 while automated sequencing of both strands of the PCR products was done on an
ABI 3100 automated gene sequencer (ABI, Forster, USA). The sequences obtained (350–500 bp) were
then assembled in silico (Vector NTI) using overlapping zones between the various sequences to form
the contiguous sequence. Phylogenetic analysis was realised by an alignment of sequence consensus of
the 16S rDNA genes collected in an international database (Genebank). The results were then expressed
in percentage of homology between the submitted sequence and the sequences resulting from the
database.

3. Results and discussion

3.1. Detection of antimicrobial activity and bacteriocin assay

A total of about 70000 colonies isolated from food samples were examined for detection of antibacterial
activity against a set of 6 indicators. A total of 340 (0.6% detection rate) colonies producing a growth
inhibition area in the indicator lawn were recorded. Staphylococcus aureus demonstrated the highest
detection rate among indicator bacteria. Two hundred and twenty colonies which displayed antibacterial
activity against the indicator lawn were randomly isolated and purified; 20 of these strains produced
antibacterial activity in the neutralized cell-free supernatant, whereas only 12 confirmed the activity
when the CFS was treated with catalase (65 UI/ml). The activity was either completely or partially
inactivated by proteolytic enzymes (Figure 1) but was resistant to heat (Figure 2). These results
demonstrated that antimicrobial compounds produced by our 12 isolates were heat stable protein or
peptide indicating bacteriocin-like substances. Four food samples (12.5% incidence rate) yielded
bacteriocin-producing strains. The distribution of bacteriocin producers in food samples is presented in
table 2. Our observations could be correlated with similar studies reported these last years. Garver et al.
(1993) have reported 13% of products yielding bacteriocin-producers by direct plating and 21% by
enrichment while Conventry et al. (1997) obtained 43% by direct plating and 46% by enrichment.
Lasagno et al. (2002) identified two bacteriocin-producers among 206 isolates selected on the basis of
the inhibition of Lactobacillus plantarum by the CFS using the WDA. Four of the twelve bacteriocin-
producing isolates obtained (designated CWBI-B1410, CWBI-B1411, CWBI-B1427 and CWBI-B1428)
were selected for further study on the basis of their food source, indicator used for detection, activity
against seven indicators and stability of activity upon repeated subcultures.
3.2. Inhibitory spectra

The sensivity of 33 bacterial strains from different genera to the bacteriocin-like substances produced by
the four selected isolates are presented in table 3. Neutralised cell-free supernatant from CWBI-B1410,
CWBI-B1427 and CWBI-B1428 isolates demonstrated similar spectra of activity broader than the one
produced by the CWBI-1411 isolate. This one showed a reasonably diverse spectrum. All bacteriocins
produced by the four isolates showed antimicrobial activity against Bacillus coagulans involved in food
spoilage, and the food-poisoning bacterium Listeria monocytogenes, whereas only bacteriocins
produced by CWBI-B1410, CWBI-B1427 and CWBI-B1428 isolates inhibited Bacillus cereus.
3.3. Bacteriocin activity

Activity units per ml (AU/ml) of bacteriocins was determined following WDA assay and presented in
table 4. Bacillus coagulans showed the more sensivity to bacteriocins. The level of activity against this
strain, (104 to 105 AU/ml), can be correlated with similar studies reported these last years. Flynn et al.
(2002) have reported 106 AU/ml, using a bacteriocin produced by Lactobacillus salivarius subsp.
salivarius, whereas Garcia et al. (2003) reported 105 AU/ml with enterocin EJ97 produced by
Enterococcus feacalis EJ97. Bacteriocins produced by CWBI-B1410, CWBI-B1427 and CWBI-B1428
isolates also demonstrated an activity of 103 AU/ml against Pediococcus pentosaseus whereas that
produced by CWBI-B1411 is more active against Listeria monocytogenes (data not shown). CWBI-
B1410 showed the highest production of bacteriocin.

3.4. Identification of bacteriocin-producers

Bacteriocin-producing isolates were catalase negative, and Gram positif cocci (CWBI-B1410, CWBI-
B1427 and CWBI-B1428) or ovoid (CWBI-B1411). Fermentation of different carbohydrates was
performed using the API 50 CHL system (API Biomerieux). 22 reactions (sugar fermentation) were
determined and 6 of them provided a mean of discriminating them (Table 5). Identification made by the
API database correlation indicated that all the four strains were Lactococcus lactis subsp. lactis.
However, a low percentage of similarity (91%) was obtained for CWBI-B1411 (Table 6).
Nucleotide sequences of 16S rDNA of the four bacteriocin-producing isolates were carried out to
confirm or infirm biochemical species identification. The determined 16S rDNA sequences of isolates
were compared directly with the Genebank database. A high level of similarity of 16S ribosomal DNA
nucleotide sequences (99% of matches) of CWBI-B1410, CWBI-B1427 and CWBI-B1428 strains was
observed with the sequences of Lactococcus lactis subps. lactis strains whereas the sequence of CWBI-
B1411 strain matched best with that of Enterococcus faecium strains (Table 6). The closest matches for
CWBI-B1411 strain and Enterococcus faecium were different from the identification determined by API
method (Table 6). However, we prioritized genetic identification because of it accordance with
morphology analysis, and the low percentage of similarity obtained by biochemical analysis which could
be due to the inadequacy of API CHL 50 for well identification of Enterococcus strains. Partial 16S
rDNA of the two bacteriocin-producing isolates Lactococcus lactis subsp. lactis (CWBI-B1410) and
Enterococcus faecium (CWBI-B1411) have been registered to Genebank databases respectively under
the accession numbers AY971748 and AY971749. Due to the high amount of bacteriocin produced in the
culture supernatant, the Lactococcus lactis subsp. lactis strain (named CWBI-B1410) was selected for
further investigations.

4. Conclusion

Traditional fermented foods from Senegal provide an appropriate ecological habitat for wild bacteriocin-
producing LAB. Bacteriocin producers mainly belong to Lactococcus lactis subsp. lactis group.
Bacteriocin-producing Lactococcus lactis strains were found in a variety of fermented products
indicating a high potential of growth of this strain in different ecological complex environment. The
produced bacteriocins showed a broad spectrum of activity including spoilage microorganisms and
pathogens associated with food such as Bacillus coagulans, Listeria monocytogenes and Bacillus
cereus. These results show the potential usefulness of these bacteriocins justifying a more in depth
investigation for their identification and application as food biopreservatives.

Production, Purification, Stability and Efficacy

of Bacteriocin from Isolates of Natural Lactic Acid
Fermentation of Vegetables
Vinod Kumar Joshi1*, Somesh Sharma1 and Neerja S. Rana2
1Fermentation Technology Laboratory, Department of Postharvest Technology
2Department of Vegetable Crops, Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, 173230 Solan (H.P.), India
Received: November 11, 2005
Accepted: March 14, 2006
Summary
The antimicrobial activity of partially purified bacteriocin produced during natural
lactic acid fermentation of carrot, radish and cucumber was assessed and characterized.
Out of ten strains, the isolated strain CA 44 of Lactobacillus genus from carrot fermentation
produced bacteriocin with maximum antimicrobial activity against Escherichia coli, Staphylococcus
aureus and Bacillus cereus, though it was more effective against E. coli than others.
Bacteriocin was stable at up to 100 °C but its activity declined compared to that at 68 °C
and was completely lost at 121 °C. The maximum antimicrobial activity was retained within
the pH range of 4–5, but it was adversely affected by the addition of papain. Bacteriocin
was also effective against B. cereus in different fruit products (pulp, juice and wine) indicating
its potential application as a biopreservative in fruit products.
Key words: antimicrobial, bacteriocin, lactic acid fermentation, Lactobacillus, Staphylococcus,
Bacillus cereus, E. coli, pathogenic microorganism, stability, biopreservative
Introduction
Preservation of vegetables by lactic acid fermentation
is an ancient practice involving lactic acid bacteria
(LAB), which predominantly produce lactic acid besides
certain compounds such as bacteriocin, which has antimicrobial
activity against other groups of microorganisms.
The antimicrobial activity of bacteriocins produced
by LAB has been detected in foods such as dairy products,
meats, barley, sourdough, red wine, fermented vegetables,
etc. (1–5). Therefore, the strains of lactic acid
bacteria have also potential to act as a biopreservative or
natural food preservative (6–8). The bacteriocins produced
inhibited food spoilage and pathogenic bacteria
such as Staphylococcus aureus, Escherichia coli, Bacillus cereus,
B. subtilis, Listeria monocytogenes and Clostridium perfringens
which are recalcitrant to traditional food preservation
method (9). The use of bacteriocins or the microorganisms
that produce them is attractive to the food industry
in the face of increasing consumer demand for
natural products and the growing concern about foodborne
diseases. It has also necessitated the need to exploit
the biologically derived antimicrobial substances
produced by LAB. It is not clear if any bacteriocin is
produced in the vegetables fermented by LAB in natural
or inoculated fermentation. The bacteriocin produced by
the strains isolated from naturally fermented vegetables
has neither been characterized nor checked for its efficacy
in various food products. Therefore, keeping in view
the above objectives the present investigations were carried
out and the results obtained are discussed here.
Materials and Methods
Fermented vegetables
Vegetables (carrot, radish and cucumber) procured
from the markets were washed, peeled and grated/sliced.
V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006) 435
*Corresponding author; Fax: ++91 (0)1792 252 242; E-mail: vkjoshipht@rediffmail.com
The grated carrot and radish were fermented with dry
salt 2 % (by mass) at 27 °C, whereas sliced cucumbers
were fermented in 3 % (by mass per volume) brine at 32
°C. Predominant microflora were isolated from these
samples.
Pathogenic bacterial cultures
Standard bacterial cultures, viz. Escherichia coli (0165),
Staphylococcus aureus (B-43-5) and Bacillus cereus procured
from Central Research Institute (CRI), Kasauli, were used
in bacteriocin screening procedures and all the cultures
were maintained as per the recommended practices.
Isolation and identification of bacteriocin
producing bacteria
The bacteriocin producers from naturally fermented
carrot, radish and cucumber were isolated by pour plate
method technique as per the conventional method (10)
using MRS agar. After incubation for 24–48 h at 32 °C,
typical colonies were isolated and purified. The isolates
were differentiated on the basis of their morphological,
cultural and physiological characteristics such as oxidase
test, utilization of citrate as a sole carbon source and catalase
test (10,11), and accordingly were tentatively identified
up to the genus level (12).
Screening of isolates for antimicrobial activity
Antimicrobial activity of the bacterial isolates against
all the pathogenic microorganisms was determined by
well diffusion method (13–16) under aerobic conditions.
Agar plates were inoculated with 100 mL of each target
microorganism after growing them in a broth and diluting
appropriately. Wells (3 mm) were cut into the plates
and 100 mL of cell-free culture supernatant fluid of the
isolated strain was placed into each well. The inhibitory
activity against E. coli was tested on EMB agar whereas
Staphylococcus aureus and Bacillus cereus were tested on
nutrient agar. Plates were kept at cool temperature for 2 h
and then incubated at 37 °C for 24 h. The antimicrobial
activity was determined by measuring the diameter of
the inhibition zone around the wells. The bacterial isolate
showing the widest zone of inhibition against the target
microorganism was selected for further studies.
Partial purification of bacteriocin
Isolated strain having maximum antimicrobial zone
was grown in MRS broth at 37 °C for 24 h. After incubation,
the broth was centrifuged at 5000 × g for 10 min
and the cells were separated out. Supernatant was used
as a crude bacteriocin. Different concentrations of ammonium
sulphate were added to the supernatant. After
stirring on a magnetic stirrer, it was kept undisturbed at
4 °C overnight. Precipitates formed were collected by centrifugation
at 10 000 × g for 10 min and redissolved in 20
mmol sodium phosphate buffer with pH=6.0. Inhibition
zone of different fractions was recorded in comparison
with the crude bacteriocin.
Characterization of bacteriocin
Heat stability
A volume of 5 mL of bacteriocin in different test
tubes was overlaid with paraffin oil to prevent evaporation
and then heated at 68 and 100 °C for 10 and 20 min,
respectively, and at 121 °C for 15 min under pressure.
The heat-treated bacteriocin samples were then assayed
for antimicrobial activity as described earlier.
Effect of pH
A 5-mL aliquot of partially purified bacteriocin was
taken in test tubes and the pH values of the contents
were adjusted to 2–9 individually, using either diluted
NaOH or HCl (1 M NaOH or 1 M HCl solution). After
allowing the samples to stand at room temperature for 2 h
the activity was assayed as described earlier.
Effect of proteolytic enzyme (papain)
A 5-mL aliquot of bacteriocin preparation was taken
in test tubes and treated with papain (100 TU) 1 mg/mL
at pH=7. The test tubes with and without the enzyme
(control) were incubated for 2 h at 37 °C and heated for
3 min at 100 °C to denature the enzyme. Both the control
and the samples were assayed for antimicrobial activity
by using well diffusion method.
Determination of preservative effect of bacteriocin
The food products, viz. juice (apple), pulp (apricot)
and prepasteurized wine (plum) were sterilized and inoculated
with Bacillus cereus at 108 CFU/mL. Initial count of
inoculated samples was recorded and bacteriocin supernatant
at a concentration of 0.05 to 0.5 % was added. After
24 and 72 h, the plate count was recorded and compared
with the control (without bacteriocin).
Results and Discussion
Based on morphological and biochemical tests, all the
isolates were identified as belonging to lactic acid bacteria
(LAB) group except RA33, which was identified as
yeast. The isolate CA44 (giving maximum antimicrobial
activity) was Gram-positive, rod shaped, negative for
catalase and peroxidase test, having circular and white
colonies on the MRS media. The strain was also positive
for galactose, arabinose, mannitol, sorbitol, sucrose, glucose,
trehalose, lactose, raffinose and negative for maltose,
citrate and arginine test. Isolate CA44 from carrot
produced the maximum inhibition zone against all the
tested microorganisms and was maximum against E. coli.
The best conditions for bacteriocin production by Lactobacillus
plantarum in batch fermentation were the salt
concentration ranging from 2.3 to 2.5 % and temperature
ranging from 22–27 °C (17). Lactobacillus plantarum strain
isolated from fermented carrots which produced bacteriocin
with antibacterial activity against Staphylococcus
aureus and spheroplasts of Gram-negative bacteria (18)
and Lactococcus lactis ssp. cremoris was also isolated from
radish fermentation (1).
An increase in antimicrobial activity after partial purification
of crude bacteriocin by ammonium sulphate precipitation
took place (Fig. 1). The fraction with the highest
bacteriocin activity was precipitated with 20–30 %
436 V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)
(by mass per volume) ammonium sulphate. The antimicrobial
activity (in terms of inhibition zone diameter) increased
from 12 to 23 mm. There was 1.91-fold increase
in the partially purified bacteriocin activity than that of
crude bacteriocin. Earlier, the inhibitory activity of bacteriocin
isolated from malted barley was precipitated from
cell free supernatant using 40 % ammonium sulphate saturation,
and resuspended in 2 mmol sodium phosphate
buffer, pH=6.0 and purified using chromatography (19).
Partially purified bacteriocin was found to be stable
at 68 °C for up to 20 min. At 100 °C for 10 min it could
retain 55 % of antimicrobial activity, while at the same
temperature for 20 min, only 28 % of activity could be
retained (Table 1). However, after incubation for 15 min
at 121 °C, the complete loss of activity took place. Compared
to the earlier reports on bacteriocin, residual activity
was lower in our study than reported earlier (20).
Furthermore, since tolerance of bacteriocin to heat is
known to depend on the stage of purification, pH, presence
of culture medium, other protective components,
etc. that might have influenced the antimicrobial activity
in our findings too. The heat stability of bacteriocin discussed
here indicates that it could be used as biopreservative
in combination with thermal processing to preserve
the food products. Furthermore, when comparatively
low temperature is employed for processing compared
to high temperature being used at present, the retention
of nutrients would be higher. However, more studies on
these aspects are needed.
The partially purified bacteriocin showed maximum
activity against the target microorganisms at pH=5.0
(Fig. 2), but after pH=5.0 the activity of the bacteriocin
gradually but continuously decreased. At pH=9.0, the
antimicrobial activity was drastically reduced to more
than 2.5 times that of the control. Thus, the bacteriocin
was found active over a wide pH range with the highest
activity at low pH range of 4–5. Earlier, the bacteriocin
produced by a newly isolated Bacillus species strain 8A
was found active over a pH range of 5–8 but was inactivated
when incubated outside these limits (9). Another
bacteriocin produced by Lactococcus lactis D53 and 23 was
active over a wide pH range with the highest activity
shown at low pH range of 3–5 (13), as was the case with
the bacteriocin from Pediococcus sp. (21). Bacteriocin activity
was completely lost when treated with proteolytic
enzyme (papain), which is in agreement with the earlier
report (22). The bacteriocin pediocin ACH from Pedicoccus
acidilacti was sensitive to proteolytic enzymes and was
completely inactivated by several proteolytic enzymes
(22,23). The stability of bacteriocin to different conditions
reflects that such compounds can withstand the
conditions normally encountered in food processing, so
would remain effective during processing.
The partially purified bacteriocin from isolate CA44
was also tested for preservative effect against B. cereus
(Table 2), and clearly the preservative effect in juice, wine
and pulp increased with the increase in the concentration
of bacteriocin. Maximum reduction of Bacillus cereus
population of 92 % was observed in wine followed by
juice (87 %) and pulp (63 %) at a concentration of 0.5 %.
V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006) 437
0
5
10
15
20
25
Crude 0–20 20–30 30–40 40–50 50–60 60–70 70–80
Inhibition zone diameter/mm
bacteriocin
m/V(ammonium sulphate)/%
Fig. 1. Increase in antimicrobial activity of bacteriocin from
Lactobacillus sp. isolate (CA44) using ammonium sulphate fractionation
Table 1. Effect of temperature on antimicrobial activity of partially purified bacteriocin from isolated Lactobacillus sp. (CA44)
Temperature/°C t/min
Inhibition zone diameter/mm
E. coli B. cereus S. aureus
68 10 23 (100) 19 (100) 20 (95)
20 22 (95) 19 (100) 20 (95)
100 10 15 (65.21) 13 (68.42) 11 (55)
20 10 (43.47) 9 (47.36) 6 (28.57)
121 15 0 0 0
Control (without heat treatment) – 23 19 21
Values in parentheses represent retention of antimicrobial activity (in %)
5.0
7.0
9.0
11.0
13.0
15.0
17.0
19.0
21.0
23.0
25.0
Inhibitionzone diameter/mm
E. col i
B. cereus
S. aureus
pH
Control 2 3 4 5 6 7 8 9
Fig. 2. Effect of pH on antimicrobial activity of partially purified
bacteriocin from Lactobacillus sp. isolate (CA44)
However, in control (without bacteriocin), no reduction
was observed in the count of B. cereus. The results (Fig.
3) further revealed that microbial count drastically decreased
in wine and the same pattern was followed in
juice too. In pulp, only a concentration of bacteriocin
above 0.2 % drastically decreased the microbial count.
Highest antimicrobial activity of bacteriocin against the
target microorganism in wine could partly be attributed
to inhibitory effect of ethanol. Briefly, the results indicate
that bacteriocin possessed several desirable characteristics
of a biopreservative.
Conclusion
The study revealed that bacteriocin from Lactobacillus
sp. isolated from natural lactic acid fermentation of
vegetables possesses a wide spectrum of inhibitory activity
against Escherichia coli, Staphylococcus aureus and
Bacillus cereus. Therefore, it has a potential for application
as a biopreservative in different food products as
such or in combination with other preservation methods.
Since lactic acid fermentation is employed mostly
for development of products, especially for flavour and
taste of the fermented products, the production of bacteriocin
in such products assumes more significance as
biopreservative apart from imparting probiotic effect to
the product.

Screening and application of bacteriocin-producing thermotolerant

lactic acid bacteria
J. Nakayama1, S. Nitisinprasert2, T. Zendo1, P. Wilaipun3, A. Swetwiwathana4, W. Noonpakdee5,
W. Malaphan6, H. Matsusaki7, K. Doi1, K. Sonomoto1, 8
1Faculty of Agriculture, Graduate School, Kyushu Univ., 2Faculty of Agro-Industry, Kasetsart Univ., 3Faculty
of Fisheries, Kasetsart Univ., 4Faculty of Agricultural Technology, King Mongkut’s Institute of Technology
Ladkrabang (KMITL), 5Faculty of Science, Mahidol Univ., 6Faculty of Science, Kasetsart Univ., 7Faculty of
Environmental and Symbiotic Sciences, Prefectural Univ. of Kumamoto, 8Bio-Architecture Center, Kyushu
Univ.
Objective: Bacteriocins produced by lactic acid bacteria (LAB) have attracted special interests
from the aspect of their potential use as safe and natural antimicrobials which can be applied as
food preservatives, fine chemicals or post-antibiotic pharmaceuticals. Since bacteriocins are
relatively small peptides whose molecular weights are ranging from two thousands to seven
thousands in most cases, they are generally thermostable and heat tolerant. This feature would give
an advantage for the practical use in food and other industries. On the other hand, LAB are
generally mesophilic and their bacteriocin biosyntheses are even more susceptible to high
temperature than primary metabolism. This would be disadvantageous when bacteriocin-producing
LAB are applied into natural fermented food as a starter culture or into animals as probiotics.
Especially in tropical countries, this would be more serious. To overcome this problem, we
screened thermotolerant LAB (TLAB) which can efficiently produce bacteriocin at more than 40oC
from Thai natural resources.
Results and Discussion
Screening: To aim bacteriocin-producing TLAB, we have performed a large scale screening on our
LAB collections. LAB strains have been isolated from Thai traditional fermented foods such as
Pla-ra (fermented fish) and Nham (fermented meat) or other Thai natural resources such as silage
and chicken intestine. TLAB were evaluated according to the growth at temperatures ranging from
40oC to 50oC. Bacteriocin activity and spectrum was examined by a conventional plate assay with
twelve indicator strains including genera of Bacillus, Micrococcus, Listeria, Lactobacillus,
Enterococcus, Leuconostoc, Pediococcus, and Lactococcus. Antimicrobial activity against
gram-negative bacteria is occasionally examined by using Escherichia coli and Salmonella sp.
After the screening under normal temperature, we examined bacteriocin productivity at
temperatures higher than 40oC.
During the course of screening, we found a number of bacteriocin-producing LAB which could
grow well at higher than 40oC but failed to produce bacteriocin at the same conditions. However,
some strains listed in Table 1 showed a significant bacteriocin activity at the temperatures higher
than 40oC. Especially, it is noticeable that Lactobacillus reuteri KUB-AC5, Pediococcus
pentosaceus TISTR 536 and Pediococcus acidilactici KUB-L0026 showed significant bacteriocin
activity even at 50oC.
As a result, more than ten bacteriocin-producing TLAB strains belonging to four genera,
Enterococcus, Lactobacillus, Lactococcus and Pediococcus, were found with a variety of
antimicrobial spectra. It should be noted that Lactobacillus reuteri KUB-AC5 isolated from
chicken intestine showed antimicrobial activity against both gram-negative and gram-positive
bacteria. Pediococcus acidilactici KUB-L0026 also showed antimicrobial activity against
gram-negative bacteria and Bacillus cereus but not against LAB tested.
Peptide purification and structural analysis: As a result of purification, some strains were found
to produce more than one peptides having bacteriocin activity. Enterococcus faecium NKR-5-3
isolated from Pla-ra produced at least four peptides having bacteriocin activity. One of them was
identified as a known bacteriocin, brochocin A, while the other three were found to have novel
amino acid sequences. Enterococcus faecium KU-B5 isolated from sugar apple produced three
peptides with bacteriocin activity and two were identified as known bacteriocin, enterocin A and B,
while the other one named enterocin X was found to have a novel amino acid sequence.
Lactobacillus salivarius AC21 isolated from chicken intestine also produced two peptides named
SalAα and SalAβ, which were suggested to link with each other by disulfide bridge. SalAα is
identical to known bacteriocin Abp118α while SalAβ has a new amino acid sequence. This
two-peptide bacteriocin was named salivacin K21.
Application: Pediococcus pentosaceus TISTR 536 isolated from Nham produces known
bacteriocin pediocin PA-1 which shows strong anti-liesteria activity. TISTR 536 was applied as a
starter culture for Nham and compared to nisin Z-producing strain. TISTR 536 exhibited higher
productivity of both lactic acid and bacteriocin, and its pediocin PA-1 produced during the
fermentation period was more stable than nisin Z produced under the same condition. TISTR 536
applied to Nham showed strong antagonism against Salmonella anatum which is commonly
contaminated Salmonella species. This effect was significantly higher than that by non-bacteriocinproducing
control strain, suggesting a synergism between lactic acid and pediocin PA-1. This data
suggests the potential use of TISTR 536 as a starter for Salmonella-free Nham production.
The other bacteriocin-producing TLAB found in this project would be also applicable for certain
purposes. Lactobacillus reuteri KUB-AC5 could be applied as probiotics with anti-Salmonella
activity for broiller chicken. E. faecium NKR-5-3 could be applied as a starter for Pla-ra. E.
faecalis K-4 could be applied as a starter culture for silage. Feeding back of bacteriocin-producing
TLAB to original source would be good way for the biocontrol of natural microflora.
References: (1) S. Nitisinprasert, N. Nilphai et al., Screening and identification of effective thermotolerant
lactic acid bacteria producing antimicrobial activity against Escherichia coli and Salmonella sp. resistant to
antibiotics. Kasetsart Journal (Nat. Sci.). 34, 387-400 (2000). (2) P. Wilaipun and T. Zendo et al., The
two-synergistic peptide bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thai
fermented fish (Plara). ScienceAsia. 30, 115-122 (2004). (3) A. Swetwiwathana, N. Lotong et al., Maturation
of Nham - a Thai fermented meat product. Fleisch Wirtschaft International. 22, 46-49 (2007).
Table. 1 Bacteriocin-producing thermotolerant LAB found in this project
Producer strain Source
Tolerant
temp.(ºC)*
Bacteriocin
Antimicrobial
spectrum**
Main researcher
E. faecium NKR-5-3 Pla-ra 40 Peptide A, B, C, D Broad P. Wilaipun
E. faecalis NKR-4-1 Pla-ra 40
Two-peptide
lantibiotic
Broad P. Wilaipun
E. raffinosus KU822 Ornament fish 40 Peptide A, B Moderate P. Wilaipun
Lb. plantarum PMU33 Somfak 45 Plantaricin W Broad W. Noonpakdee
Lc. Lactis WNC20 Nham 40 Nisin Z Nisin type W. Noonpakdee
Lb. fermentum onil2 Nham 40 Peptide 1256da Narrow W. Noonpakdee
P. pentosaceus WNK19 Nham 40 Pediocin PA-1 Class IIa type W. Noonpakdee
Lc. Lactis KU-T1 Tofu’s whey 41 Unknown Narrow W. Malaphan
E. faecium KU-B5 Sugar apple 41-43 Enterocin A, B, X Class IIa type W. Malaphan
Lb. reuteri KUB-AC5 Chicken intestine 50 Peptide KAC5 E. coli, Salmonella S. Nitisinprasert
Lb. salivarius AC21 Chicken intestine 45 Salivacin K21 Broad H. Matsusaki
P. pentosaceus TISTR 536 Nham 50 Pediocin PA-1 Class IIa type A. Swetwiwathana
Lc. lactis N100 and N190 Nham 40 Nisin Z Nisin type A. Swetwiwathana
P. acidilactici KUB-L0026 Silage 50 Peptide KPA26
B. cereus, E.. coli,
Salmonella
S. Nitisinprasert
E. faecalis K-4 Silage 45 Enterocin SE-K4 Class IIa type K. Doi
* Highest temperature at which bacteriocin was produced.
** Activity in culture supernatant. Nisin type, showing strong activity against wide range of Gram-positive bacteria;
Class IIa type, showing strong activity especially against Listeria sp.

Determine maximum antimicrobial activity of

bacteriocins.
Microbial Update International • June, 2007 •
Preserving vegetables using lactic acid fermentation involves lactic acid bacteria (LAB) that predominantly produce
lactic acid in addition to bacteriocins. The antimicrobial activity of bacteriocins produced by LAB has been detected in
foods, such as dairy products, meats, barley, sourdough, red wine and fermented vegetables.

Researchers indicate that strains of LAB have potential to act as a biopreservative or natural food preservative. The
bacteriocins are able to inhibit spoilage and pathogenic bacteria, such as S. aureus, E. coli, B. cereus, B. subtilis, L.
monocytogenes and C. perfringens. These bacteria are not responsive to traditional food preservation methods.

The use of bacteriocins or the microorganisms that produce them is attractive to the food industry in the face of
increasing consumer demand for natural products and the concerns over foodborne diseases. It has also led to the need
to exploit the biologically derived antimicrobial substances produced by LAB.

It is not clear if any bacteriocin is produced in vegetables fermented by LAB in natural or inoculated fermentation. The
bacteriocin produced by strains isolated from naturally fermented vegetables has neither been characterized nor
checked for efficacy in various food products. So, Indian scientists decided to characterize the antimicrobial activity of
partially purified bacteriocin produced during the natural lactic acid fermentation of carrot, radish and cucumber.

Out of 10 strains, the isolated strain CA 44 of the Lactobacillus genus from carrot fermentation produced a bacteriocin
that had strong antimicrobial activity against E. coli, S. aureus and B. cereus, although it was more effective against E.
coli than other bacteria. The bacteriocin was stable up to 100 C, but its activity had declined compared to bacteriocin at
68 C. Its stability was completely lost at 121 C.

The maximum antimicrobial activity was retained within a pH range of 4 to 5. But this activity was adversely affected
by the addition of papain, a cysteine protease present in papaya, and which is useful in tenderizing meat and other
proteins. Bacteriocins were also effective against B. cereus in different fruit products--pulp, juice and wine--indicating
their potential application as biopreservatives in these types of products.