Letters in Applied Microbiology 2004, 38, 251–256 doi:10.1111/j.1472-765X.2004.01478.

Bacteriocin-like substance production by Bacillus

licheniformis strain P40

F. Cladera-Olivera, G.R. Caron and A. Brandelli

Laboratório de Bioquı´mica e Microbiologia Aplicada, Departamento de Ciência de Alimentos, ICTA, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brasil

2003/0807: received 10 September 2003, revised 22 October 2003 and accepted 18 November 2003

ABSTRACT
F . C L A D E R A - O L I V E R A , G . R . C A R O N A N D A . B R A N D E L L I . 2004.
Aims: To investigate the production of bacteriocin-like compounds by Bacillus spp. isolated from the Amazon
basin.
Methods and Results: An antimicrobial substance produced by Bacillus licheniformis strain P40 was inhibitory to a
broad range of indicator strains, such as Listeria monocytogenes, Bacillus cereus and clinical isolates of Streptococcus
spp. The compound was stable at 100
C, but lost its activity when treated at 121
C/103Æ5 kPa for 15 min. It
was resistant to the proteolytic action of trypsin and papain but sensitive to pronase E and was stable within a wide
range of pH (3–11). The substance was bactericidal and bacteriolytic to L. monocytogenes.
Conclusions: An antibacterial peptide produced by Bacillus licheniformis was characterized, presenting a broad
spectrum of activity against pathogenic and spoilage organisms.
Significance and Impact of the Study: The identification of a substance active against important pathogens
addresses an important aspect of food safety.

Keywords: Amazon, Bacillus, bacteriocin, fish bacteria, Listeria monocytogenes.

genus to investigate for antimicrobial activity as Bacillus

INTRODUCTION
Bacteriocins are bacterial peptides that inhibit or kill
microorganisms that are usually, but not always, closely
related to the producer strain (Klaenhammer 1993). Those
produced by lactic acid bacteria (LAB) are largely studied
with the perspective to search for safe and food-grade
preservatives of biological origin (O’Sullivan et al. 2002).
Although the research is mainly dedicated to LAB, these
antimicrobial peptides are produced by several other classes
of bacteria, such as enterococci (Parente and Ricciardi 1994),
staphylococci (Oliveira et al. 1998) and corynebacteria
(Motta and Brandelli 2002).
The genus Bacillus includes a variety of industrially
important species and has a history of safe use in both food
and industry (Paik et al. 1997). Bacillus is an interesting
Correspondence to: A. Brandelli, Laboratório de Bioquı´mica e Microbiologia
Aplicada, Departamento de Ciência de Alimentos, ICTA, Universidade Federal do
Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-970 Porto Alegre, Brazil
(e-mail: abrand@vortex.ufrgs.br).
ª 2004 The Society for Applied Microbiology
species produce a large number of peptide antibiotics
representing several different basic chemical structures
(von Döhren 1995). The production of bacteriocins or
bacteriocin-like substances (BLS) has been described for
many Bacillus species such as B. thuringiensis (Paik et al.
1997), B. subtilis (Zheng et al. 1999) and B. cereus (Bizani
and Brandelli 2002).
The purpose of this study was to evaluate the antimicro-
bial activity produced by Bacillus licheniformis strain P40
isolated from the teleost fish Leporinus sp. originated from
the Amazon basin.
MATERIALS AND METHODS
Microorganisms
The producer strain P40 was given by Universidade Federal
do Amazonas (Manaus, Brazil). The organism was isolated
from the intestinal contents of the teleost fish Leporinus sp.
252 F . C L A D E R A - O L I V E R A ET AL.

of Amazon basin, at central Amazonia, near Manaus, Brazil
(3
06¢S, 60
01¢W). The indicator strains listed in Table 1
are laboratory stocks obtained from different sources and
were kept frozen in 20% (v/v) glycerol at )20
C. The
organisms were propagated in appropriate media and
temperature, as indicated in Table 1.
Bacterial identification
The strain P40 was analysed according to morphological
examinations and biochemical tests (Clauss and Berkeley
1986; MacFaddin 2000). Additionally, an API 50CH kit was
used and the data was submitted to automated interpretation
using the APILAB Plus software (BioMérieux, Marcy-
l’Etoile, France).
Preparation of antimicrobial substance
The strain P40 was grown in 100 ml of Brain Heart Infusion
(BHI) medium (Oxoid, Basingstoke, UK) at 30
C in a rotary
shaker at 125 cycles per min for 48 h. Cells were harvested
by centrifugation at 10 000 g for 15 min, and culture
supernatants were sterilized with 0Æ45 lM filter membranes
and stored at 4
C until utilization.

Table 1 Antimicrobial activity spectrum of

Temperature Inhibition
the antimicrobial substance
Indicator organism Media (
C) zone (mm)*

Actinomyces sp. (food isolate) MH 30 0

Bacillus cereus ATCC 14579 BHI 37 11
Bacillus cereus (food isolate) BHI 37 10
Bacillus subtilis (food isolate) BHI 37 10
Brochothrix thermosphacta ATCC 11509 BHI 37 0
Corynebacterium fimi NCTC 7547 BHI 37 10
Enterococcus faecalis (clinical isolate) BHI 37 7
Lactobacillus acidophilus ATCC 4356 MRS 30 11
Listeria monocytogenes ATCC 7644 BHI 37 12
Listeria inoccua (food isolate) BHI 37 11
Rhodococcus sp. (soil isolate) MH 37 15
Staphylococcus aureusATCC 25923 BHI 37 0
Staphylococcus aureus (food isolate) BHI 37 0
Staphylococcus aureus 4059 (clinical isolate) BHI 37 0
Staphylococcus aureus 5089 (clinical isolate) BHI 37 0
Staphylococcus epidermidis (clinical isolate) BHI 37 0
Staphylococcus intermedius (clinical isolate) MH 37 7
Streptococcus sp. (b-haemolytic) MH 37 11
Streptococcus sp. (clinical isolate) MH 37 13
Streptococcus sp. (clinical isolate) MH 37 11
Aeromonas hydrophila (clinical isolate) MH 37 9
Aeromonas sp. (clinical isolate) MH 37 10
Enterobacter aerogenes (food isolate) BHI 37 15
Erwinia carotorovora (food isolate) BHI 25 15
Erwinia carotorovora 309 (food isolate) BHI 25 8
Erwinia carotorovora A325 (food isolate) BHI 25 15
Escherichia coli ATCC 25922 BHI 37 0
Escherichia coli (food isolate) BHI 37 0
Pasteurella haemolytica (clinical isolate) MH 37 12
Pseudomonas sp. (clinical isolate) BHI 37 0
Salmonella Enteritidis (food isolate) MH 37 0
Salmonella Gallinarium (clinical isolate) MH 37 9

*Diameter of the inhibition zone (mm) around the disc.

Zone of inhibition was hazy.
BHI, brain heart infusion; MH, Mueller-Hinton; MRS, deMan Rogosa Sharpe; ATCC, American
Type Culture Collection, Rockville, USA; NCTC, National Collection of Type Culture,
Colendale, UK; Clinical isolates were from Hospital de Clı́nicas Veterinárias (HCV/FAVET),
Porto Alegre, Brazil; Food isolates were from Laboratório Central do Estado (LACEN/RS), Porto
Alegre, Brazil.

ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 251–256, doi:10.1111/j.1472-765X.2004.01478.x

Antimicrobial activity assay
The antimicrobial activity against all indicator strains was
detected by agar disc diffusion assay (Motta and Brandelli
2002). An aliquot of 20-ll cell-free culture supernatant was
applied on discs (6 mM) on agar plates previously inoculated
with a swab submerged in each individual indicator strain
suspension, which corresponded to a 0Æ5 McFarland turbidity
standard solution. Plates were incubated at optimal tempera-
ture for the test organism. The amount of antibacterial activity
was determined by the serial 2-fold dilution method. The
reciprocal value of the highest dilution where an inhibition
was observed was taken as activity units ml)1 (AU ml)1).
Effects of proteolytic enzymes, heat, pH and
chemical substances on antimicrobial activity
Proteolytic enzymes. The effects of proteolytic enzymes
were tested on cell-free supernatant. Aliquots were treated
with papain, trypsin or pronase E (Sigma, St Louis, USA) at
final concentrations of 2 and 10 mg ml)1 at 37
C for 1 h. An
untreated cell-free supernatant and the enzyme in the buffer
alone served as controls.
Thermal stability. To analyse thermal stability, aliquots of
cell-free supernatant were exposed to temperatures ranging
from 30 to 100
C for 30 min, 100
C for 5–60 min, 121
C
(103Æ5 kPa) for 15 min and frozen for up to 30 days.
pH stability. Samples of the antimicrobial substance were
incubated at different pH values (pH 3–11) for 2 h at 25
C,
neutralized to pH 7Æ0, and tested for antimicrobial activity.
Effect of chemicals. Chemicals were added to the
substance and incubated for 1 h at 37
C before being tested
for antimicrobial activity. Organic solvents were used at the
working concentration of 50% (v/v). The detergents Tween
20 and Tween 80 were used at 10% (v/v) and sodium
deoxycholate was used at 1 mg ml)1 . EDTA and trichlo-
roacetic acid (TCA) were used at 10 mM and 0Æ61 M,
respectively. After treatment with TCA, samples were
centrifuged at 10 000 g for 5 min and the supernatant was
neutralized to pH 7Æ0 before testing for antimicrobial
activity. Chemicals diluted with 8Æ75 g l)1 NaCl and cell-
free supernatant diluted with 8Æ75 g l)1 NaCl were used as
controls.
After each treatment the samples were tested for antimi-
crobial activity against L. monocytogenes ATCC 7644.
Gel filtration chromatography
An aliquot (500 ll) of cell-free supernatant was loaded on a
column (0Æ8 · 30 cm) of Sephadex G-100 pre-equilibrated
ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 251–256, doi:10.1111/j.1472-765X.2004.01478.x
BACTERIOCIN OF BACILLUS LICHENIFORMIS 253
and eluted with 10 mM sodium phosphate pH 7Æ0 or 10 mM
sodium phosphate pH 7Æ0 containing 1Æ5 M NaCl. Fractions
of 1Æ0 ml were collected and monitored for antimicrobial
activity against L. monocytogenes ATCC 7644 and absorb-
ance at 280 nm.
Mode of action on Listeria monocytogenes ATCC
7644
The antimicrobial substance (final concentration
50 AU ml)1) was added to early exponential growth
phase cells of L. monocytogenes ATCC 7644 in 16 ml
BHI medium. Sterile BHI medium was added to control
tubes. Changes in the turbidity of the cultures were
recorded at an O.D. of 600 nm and the number of
colony-forming units (CFU) was determined by plating
the samples on BHI agar.
RESULTS
Characterization of producing strain
Microscopic observation of the isolate showed a straight
rod with endospores. The bacterium grew aerobically, was
strongly catalase positive, presented variable Gram-stain,
and was Gram-positive in the KOH test. Together with
additional biochemical tests and the use of an API 50CHB
kit, these characteristics indicated that the isolate belongs
to the genus Bacillus (Clauss and Berkeley 1986). The
analysis with the APILAB Plus software indicated 99Æ5%
identity with Bacillus licheniformis.
Growth and bacteriocin production
The strain P40 was aerobically incubated in BHI medium
at 30
C in a rotary shaker. Cell growth reached the
stationary phase after 12 h of cultivation and maximum
antibacterial activity was observed from 15 h (Fig. 1). It
was observed that pH values were nearly constant
(pH ¼ 7Æ0–7Æ5). A peak in the specific production rate
was observed at 12 h, coinciding with the late exponential
growth phase (Fig. 1).
Inhibitory spectrum
Cell-free supernatant of a culture of Bacillus licheniformis
strain P40 was tested for the presence of antimicrobial
activity against several strains. Inhibitory activity was
observed against several bacteria, including important
pathogenic and spoilage microorganisms such as B. cereus,
L. monocytogenes and clinical isolates of Streptococcus sp.
(Table 1).
254 F . C L A D E R A - O L I V E R A ET AL.

2 tested, excepting to 121
C and 103Æ5 kPa for 15 min

(Table 2). The substance lost its activity after treatment
16 with TCA and butanol, while all other chemicals used
600

Specific production rate

caused no effect on antimicrobial activity (Table 2). The

(AU log CFU–1 h–1)

Activity (AU ml–1)
12 antimicrobial substance was stable in all pH tested (3–11),
OD 600 nm

1 400 remaining about 100% of its initial activity.

8

200 Gel filtration chromatography

4
The antimicrobial activity produced by Bacillus licheniformis
0 0
P40 was analysed by gel filtration chromatography on
0
0 10 20 30 40 50 Sephadex G-100. The antimicrobial activity eluted at the
Time (h) void volume of the column, but when the elution was carried
out with high ionic strength buffer (1Æ5 M NaCl), the
Fig. 1 Production of antimicrobial activity during growth of Bacillus antimicrobial activity was detected within the included
licheniformis strain P40 in brain heart infusion medium at 30
C. volume of the column (Fig. 2).
Turbidity (black circles) and antibacterial activity (squares) were
monitored and the specific production rate (open circles) calculated at
the times indicated. Each point represents the mean of three Effect on Listeria monocytogenes ATCC 7644
independent experiments
The results of the inhibitory substance on the growth of
L. monocytogenes ATCC 7644 are shown in Fig. 3. A
dilution of the antimicrobial substance (50 AU ml)1 final
Effects of proteolytic enzymes, heat, pH and
concentration) added to about 107 CFU ml)1 of L. mono-
chemical substances on antimicrobial activity
cytogenes resulted in a rapid decrease in the number of viable
The antimicrobial substance (560 AU ml)1) was tested for counts, reaching zero over a period of 2 h (Fig. 3). In
sensitivity to several proteases, chemicals, heat and pH. The another experiment, a decrease in the O.D. readings from
results are summarized in Table 2. The antimicrobial
substance was sensitive to pronase E but not to papain and
trypsin. The compound was stable at all temperatures

1600
Table 2 Factors affecting antimicrobial activity of Bacillus
licheniformis P40
Bacteriocin activity (AU ml–1)

Residual
1200
Treatment activity (%)*

Enzymes
Trypsin 100
800
Papain 100
Pronase E 25
Heat
60
C/30 min 92 400
80
C/30 min 81
100
C/30 min 76
121
C/103Æ5 kPa/15 min 0
Chemicals 0
Acetone 100 0 2 4 6 8 10 12 14 16 18 20
1-Butanol 0 Fraction number
Chloroform 100
Methanol 100 Fig. 2 Elution profiles of bacteriocin-like substances (BLS) during gel
Toluene 100 filtration chromatography. Crude BLS was applied on a Sephadex
Trichloroacetic acid 0 G-100 column eluted with 10 mM sodium phosphate pH 7Æ0 (circles)
or 10 mM sodium phosphate containing 1Æ5 M NaCl (squares).
*Residual activity compared with antimicrobial activity before the Fractions were collected and assayed for antimicrobial activity. The
treatment. arrow indicates the void volume of the column (Vo)

ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 251–256, doi:10.1111/j.1472-765X.2004.01478.x

10
1·2
8 cin produced by a B. licheniformis isolated from buffalo
log CFU ml–1
OD 600 nm
6 0·8
4 crobial spectrum and is more heat-stable than other
0·4
2
0·0
0 2 4 6 8 10 12
Time (h)
Fig. 3 Effect of bacteriocin-like substances (BLS) on growth of
Listeria monocytogenes. Turbidity (open symbols) and viability (black
symbols) were monitored in control (circles) and treated (squares) cells
with a final concentration of 50 AU ml)1 . The arrow indicates the
time of BLS addition. Each point represents the mean of three
independent experiments
1Æ0 to 0Æ05 was observed in 15 min after the addition of the
substance to cell suspensions of L. monocytogenes.
DISCUSSION
B. licheniformis P40 produces an antimicrobial substance that
showed a broad inhibitory spectrum, including several
spoilage and pathogenic microorganisms, and Corynebacte-
rium fimi NCTC 7547, which is described as susceptible to
all bacteriocins tested (Oliveira et al. 1998).
Considering the properties of this antimicrobial com-
pound, i.e. relative insensitivity to pH and temperature and
the presence of an essential peptide moiety, it was charac-
terized as a bacteriocin-like substance (Klaenhammer 1993;
Hyronimus et al. 1998). This BLS was sensitive to pronase
E and its activity was lost with TCA treatment, indicating its
proteinaceous nature. However, it was resistant to papain
and trypsin, suggesting that this compound is more resistant
to proteolytic treatments than other BLS produced by
Bacillus spp. (Paik et al. 1997; Hyronimus et al. 1998). This
agrees with the fact that some Bacillus produce antimicro-
bials which are cyclic peptides containing unusual amino
acids, therefore more resistant to proteases (von Döhren
1995).
Maximum antimicrobial activity was coinciding with
maximum biomass, and maximum specific production rates
were observed at the exponential phase. As the BLS was
produced only during the exponential growth phase it can be
ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 251–256, doi:10.1111/j.1472-765X.2004.01478.x
BACTERIOCIN OF BACILLUS LICHENIFORMIS 255
considered a primary metabolite, like bacteriocins produced
by Enterococcus faecium (Parente and Ricciardi 1994) and
Lactobacillus amylovorus (De Vuyst et al. 1996). A bacterio-
rumen was characterized, however, it seems to be not related
to the BLS produced by strain P40 because the former is
produced anaerobically as a secondary metabolite and
appears to be a cell-bound bacteriocin (Pattnaik et al.
2001). In addition, the BLS P40 presents a broad antimi-
antimicrobial substances produced by Bacillus and lactoba-
cilli isolated from fish intestines (Sugita et al. 1998; Halami
et al. 1999).
The BLS eluted at the void volume of Sephadex G-100,
suggesting a molecular weight (MW) higher than 150 kDa.
However, in the presence of 1Æ5 M NaCl the activity eluted
within the included volume, corresponding to about 20 kDa.
This indicates that the BLS is secreted as large aggregates,
like linocin M18 produced by Brevibacterium linens (Valdés-
Stauber and Scherer 1994). Contradictory data on MW is
also described for cerein 7, an antibacterial peptide produced
by B. cereus (Oscáriz and Pisabarro 2000). The association of
bacteriocin molecules into large aggregates is possibly
because of the highly hydrophobic nature of the peptides.
The food-borne pathogen L. monocytogenes is involved in
outbreaks linked to the consumption of contaminated dairy
products or vegetables (Muriana 1996). The use of bacte-
riocins to inhibit L. monocytogenes in some foods has been
already reported (O’Sullivan et al. 2002). However, resist-
ance of L. monocytogenes strains to conventional bacteriocins,
such as nisin and pediocin, has been described (Rasch and
Knochel 1998). The BLS identified in this work was able to
kill a concentration of about 107 CFU ml)1 of L. monocy-
togenes, suggesting that the mode of action was bactericidal.
This study indicates the relevance of the BLS produced
by Bacillus licheniformis P40 as a natural biopreservative for
control of pathogenic and spoilage microorganisms.
ACKNOWLEDGEMENTS
We thank Dr S. Astolfi-Filho from Universidade Federal do
Amazonas for isolate P40. G.R. Caron and F. Cladera-
Olivera have received fellowships from FAPERGS and
CNPq, respectively.
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