LWT - Food Science and Technology 43 (2010) 1320e1324

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LWT - Food Science and Technology

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Protocols for the isolation and detection of lactic acid bacteria

with bacteriocinogenic potential
Paula Mendonça Moraes, Luana Martins Perin, Maria Beatriz Tassinari Ortolani,
Anderson Keizo Yamazi, Gabriela Nogueira Viçosa, Luís Augusto Nero*
Departamento de Veterinária, Campus Universitário, Universidade Federal de Viçosa, Viçosa, MG, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to evaluate culture media and methodologies for isolation and detection
Received 2 December 2009 of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese
Received in revised form were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35
C/48
5 May 2010
h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of
Accepted 6 May 2010
them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with
catalase (BHI þ C) and M17 (both at 35
C/24 h). The proteinaceous nature of the antagonistic cultures
Keywords:
was verified using: spot-on-the-lawn (MRS, 25
C/24 h, under anaerobiosis) and well-diffusion (cultures
Lactic acid bacteria
Bacteriocins
amplified on modified MRS broth at 25
C/24 h, and then neutralized using NaOH). Listeria mono-
Analytical methods cytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from
Spot-on-the-lawn MRS (83 by M17 and 65 by BHI þ C) in comparison to KFS (24 by M17 and 15 by BHI þ C). The spot-on-
Well-diffusion assay the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was
considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like
substances, activity that was better identified using the spot-on-the-lawn methodology.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Pilet, Gigout, Prévost, & Leroi, 2009). Specifically, bacteriocins

Lactic acid bacteria (LAB) are widely used by food industries in
the production of fermented products and as tools for food safety
(Adams & Mitchell, 2002; Leroy & de Vuyst, 2004; Ross, Morgan, &
Hill, 2002). These microorganisms naturally produce several
substances with an antimicrobial potential, such as organic acids,
CO2 and hydrogen peroxide (Carr, Chill, & Maida, 2002; de Martinis,
Santarosa, & Freitas, 2003). Some LAB strains are capable of
producing bacteriocins (Cotter, Hill, & Ross, 2005; Riley & Wertz,
2002), polypeptides with specific antimicrobial activity against
a group of bacteria of the same or different species (Cotter et al.,
2005; Nes & Johnsborg, 2004).
With the increase in demand by consumers for products that
are less processed and free of preservatives (Álvarez-León,
Román-Viñas, & Serra-Majem, 2006), antagonistic LAB or their
bacteriocins have been increasingly used for controlling patho-
gens in certain foods (Gong, Meng, & Wang, 2010; Holzapfel,
Geisen, & Schillinger, 1995; Huang et al., 2009; Matamoros,
* Corresponding author. Tel.: þ55 31 38991463.
E-mail address: nero@ufv.br (L.A. Nero).
represent an attractive option for the food industry because they
do not alter the taste or smell of the final products (Nes &
Johnsborg, 2004). Some already characterized bacteriocins have
an antimicrobial activity against several spoilage and pathogenic
microorganisms (Gao, Jia, Gao, & Tan, 2010; Gong et al., 2010;
Schillinger, Becker, Vignolo, & Holzapfel, 2001; Sobrino-López &
Martín-Belloso, 2008).
The use of these microbiological safety tools by the food
industry has increased the isolation of new LAB cultures which
are able to produce bacteriocins or bacteriocin-like substances.
The tests normally used for initial identification of the antago-
nistic activity are based on the diffusion of the antimicrobial
substances in the culture media and on the inhibition of
a sensitive microorganism. The techniques most frequently used
for this purpose are variations of two protocols originally
described in the 1970s, the spot-on-the-lawn (Fleming, Etchells, &
Costilow, 1975) and the well-diffusion assay (Tagg & McGiven,
1971).
The objective of this study was to compare different protocols
and culture media used to detect the antagonistic activity of LAB
naturally present in raw milk and soft cheese, and to identify
their ability of producing of bacteriocins or bacteriocin-like
substances.

0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2010.05.005
 P.M. Moraes et al. / LWT - Food Science and Technology 43 (2010) 1320e1324
2. Materials and methods
2.1. LAB isolation
Thirty-three samples of raw milk and eighteen samples of soft
cheese made with raw milk were collected directly from dairy
farms of the Viçosa region, Minas Gerais State, Brazil. All samples
were ten-fold diluted on NaCl solution (0.85 g/100 ml) and pour
plated on de ManneRogosaeSharpe (MRS, Difco, Loveton Circle
Sparks, MD, USA) and KangeFungeSol (KFS, which is MRS sup-
plemented with phenylethanol at 0.175 ml/100 ml, and lactic acid
at 0.2 ml/100 ml, Kim, Fung, & Kang, 2001), in duplicates, and
incubated at 35
C for 48 h under anaerobiosis (GasPakÔ Plus
Anaerobic System, BBL, Franklin Lakes, NJ, USA). After incubation,
389 and 356 colonies were randomly isolated from MRS and KFS,
respectively, purified by successive streaks on MRS plates (incu-
bation at 35
C). The purified cultures were classified according the
Gram staining and catalase production (Downes & Ito, 2001), and
stored in MRS broth with glycerol (20 ml/100 ml) at 80
C.
2.2. Verification of antagonist activity
All LAB cultures isolated from MRS and KFS were submitted to
an initial screening to verify the presence of antagonist activity
using the spot-on-the-lawn technique (adapted from Fleming et al.,
1975) using M17 (Oxoid Ltd., Basingstoke, Hampshire, England)
supplemented with lactose (10 g/100 ml) and brain-heart infusion
agar (Oxoid) supplemented with 100 IU/ml catalase (C1345, Sigma,
St. Louis, MO, USA) (BHI þ C). An aliquot of 2 ml of each LAB culture
previously amplified in MRS broth (incubation at 25
C for 24 h)
was spotted on plates containing 10 ml of one of the two culture
media and was incubated at 35
C for 24 h. After incubation, the
plates were overlayed with 8 ml of BHI semi-solid agar (0.8 g/
100 ml of bacteriologic agar, Merck, Darmstadt, Germany) inocu-
lated with 105 CFU/ml of a culture of Listeria monocytogenes ATCC
7644 (used as indicator). The plates were then incubated at 35
C
for 24 h. The presence of a distinct inhibition zone around the spots
was considered a positive antagonistic effect. Lactobacillus sakei 2a
was used as positive control of antagonism activity due to the
production of bacteriocin (de Martinis & Franco, 1998). For this step,
9 cm diameter plates were used and 8 LAB cultures were tested per
plate, spotted in equal distances.
Using the antagonistic cultures from the initial screening, new
antagonism tests were carried out using three culture media
(BHI þ C, M17 and modified MRS with 0.5 g/100 ml dextrose e
mMRS) to confirm the antimicrobial activity. Tests were conducted
as previously described using the spot-on-the-lawn protocol, with
incubation at 35
C for 24 h (BHI with catalase and M17) and at
25
C for 24 h under anaerobiosis (mMRS). In this evaluation, the
diameter of the inhibition halos was measured in order to allow the
comparison between the tested culture media.
2.3. Verification of the proteinaceous nature of the antimicrobial
substances
The production of bacteriocins or bacteriocin-like substances
was confirmed using proteolytic enzymes in 2 different protocols:
spot-on-the-lawn (Fleming et al., 1975; Harris, Daeschel, Stiles, &
Klaenhammer, 1989) and well-diffusion assay, which uses the
neutralized supernatant of LAB cultures (Harris et al., 1989; Tagg &
McGiven, 1971). In both protocols, L. monocytogenes ATCC 7644 was
used as indicator, along with the following proteolytic enzymes (all
from Sigma, at 20 mg/ml): a-chymotrypsin (C4129), proteinase K
(P8044), and trypsin TPCK (T1426). Lb. sakei 2a was used as
a positive control due its bacteriocin production (de Martinis &
1321
Franco, 1998). For proteolytic enzymes, MilliQ water was used as
a negative control.
Using the spot-on-the-lawn protocol, each LAB culture identi-
fied as antagonistic was recovered in MRS broth (25
C for 24 h) and
an aliquot of 2 ml was spotted onto plates containing 10 ml of mMRS
agar and incubated at 25
C for 24 h under anaerobiosis. After
incubation, wells with a 3 mm diameter were cut adjacent to the
colony and inoculated with 20 ml of each enzyme. After absorption
at room temperature (10e15 min), the agar was covered with an
overlayer of semi-solid BHI (8 ml) inoculated with 105 CFU/ml of
the indicator and incubated at 35
C for 24 h. The absence of an
inhibition zone in the proximity of the well indicates sensitivity to
this specific protease, thus confirming the proteinaceous nature of
the antagonistic substance. For this step, 9 cm diameter plates were
used and 2 LAB cultures were tested per plate, spotted in equal
distances. For each culture, 4 spots were inoculated (3 proteolytic
enzymes and the negative control). In the center of each plate the
bacteriocin producer was inoculated in 2 spots (1 for proteinase K
and 1 for the negative control).
Using the well-diffusion-assay protocol, LAB cultures identified
as antagonists were recovered in 50 ml of mMRS broth (25
C for
24 h) and centrifuged at 6800 g for 20 min at 4
C. The superna-
tants obtained from this process were neutralized with NaOH at
1 mol equi/l (to eliminate the inhibitory effect due to acid produc-
tion) and sterilized by filtration (Millex SLGP033RS, 0.22 mm, Mil-
lipore, Bedford, MA, USA). Plates containing 20 ml of semi-solid BHI
(0.8 g/100 ml of bacteriologic agar) previously inoculated with
105 CFU/ml of the indicator were prepared. Adjacent wells with
a diameter of 5 and 3 mm were cut on these plates, and 40 ml of the
supernatant and 20 ml of each enzyme, respectively, were depos-
ited. After absorption (4
C for 2 h), the plates were incubated at
35
C for 24 h. For this step, 9 cm diameter plates were used and 2
LAB cultures inoculated in equidistance cut wells were tested per
plate. For each culture, 4 wells of 5 mm were cut (3 proteolytic
enzymes and the negative control). In the center of each plate 2
wells of 5 mm were cut for the bacteriocin producer supernatant
inoculation (1 for proteinase K and 1 for the negative control).
A schematic flow chart of the conducted laboratorial procedures
is presented on Fig. 1.
2.4. Data analysis
The LAB cultures identified as antagonists and potential
producers of bacteriocins or bacteriocin-like substances were
grouped by the culture media used for the initial antagonism
screening and the protocols used to verify the proteinaceous nature
of the produced antimicrobial substances. The frequencies obtained
in each situation were compared using a chi-square test (c2,
P < 0.05) with XLStat software version 2009.1.02 (Addinsoft, New
York, NY, USA).
3. Results and discussion
All the cultures selected for the antagonism tests from MRS and
KFS presented typical LAB characteristics (Gram-positive cocci and
rods, without catalase production; Axelsson, 2004) with
a predominance of Gram-positive cocci. Regardless of the culture
media considered during the isolation stage and the initial
screening of antagonism, 111 LAB cultures were identified as
potential producers of substances with antimicrobial activity. A
higher frequency of antagonists was observed in cultures isolated
from MRS (87) than in those obtained from KFS (24), being signif-
icantly distinct (c2 ¼ 35.79, degrees of freedom ¼ 1, P < 0.0001).
The frequencies of cultures identified as antagonists categorized
by the methods of isolation used during the initial screening are
1322 P.M. Moraes et al. / LWT - Food Science and Technology 43 (2010) 1320e1324

Samples/Steps Laboratorial procedures

33 raw milk samples
18 fresh cheese samples
Pour plated on MRS and KFS
Random isolation of LAB
cultures to be tested
Purification on MRS
Gram staining and catalase production test
Identification of antagonistic activity using spot-on-lawn method,
using BHI+C and M17 (Listeria monocytogenes ATCC 7644 as
target microorganism)
Confirmation of antagonistic activity using spot-on-the-lawn method,
using M17, BHI+C and mMRS (Listeria monocytogenes ATCC
7644 as target microorganism)
Identification of antagonistic
activity of LAB cultures
Identification of proteinaceus nature of antimicrobial substances by:
spot-on-the-lawn and well-diffusion assay (Listeria monocytogenes
ATCC 7644 as target microorganism)
Identification of proteinaceous
nature of the antimicrobial
substances produced by the
antagonistic LAB cultures

Fig. 1. Flow chart of procedures employed to identify the antagonistic activity of the tested lactic acid bacteria and the proteinaceous nature of the produced antimicrobial
substances. LAB: lactic acid bacteria, MRS: de ManneRogosaeSharpe agar, KFS: KungeFungeSol agar, BHI þ C: brain-heart infusion agar supplemented with catalase (100 IU/ml),
mMRS: modified de ManneRogosaeSharpe agar, with dextrose (at 0.5 g/100 ml).

presented in Table 1. No significant differences were observed in
the comparison between the results obtained from the BHI þ C and
M17. Nevertheless, considering all antagonist cultures, the
frequency obtained from M17 was statistically higher than that
obtained using BHI þ C (P < 0.05). This indicates that the M17
culture media presents a better performance for the detection of
LAB with antagonistic activity (Table 1). Furthermore, 31 LAB
cultures presented antagonism only using M17, while only four
presented this action when BHI þ C was used, indicating a greater
specificity of M17 in the detection of antimicrobial activity.
The higher frequency of antagonistic cultures observed using
M17 is probably caused by the composition of this culture media
and the aerobic incubation that allow the production of diverse
antimicrobial substances, as well as bacteriocins and bacteriocin-
like substances. This culture medium is frequently used for the
detection of antagonistic LAB activity that is naturally present in
foods (Benkerroum, Houatwi, Sandine, & Tantaoui-Elaraki, 1993;
Benkerroum, Oubel, Zahar, Dlia, & Filali-Maltouf, 2000;
Benkerroum et al., 2002), but it contains a high concentration of
lactose (10 g/100 ml), which determines the production of organic
acids. As such, culture media with low levels of carbohydrate, like
BHI (2% glucose), present an alternative for the detection of cultures
with antimicrobial potential due to the production of substances
with a proteinaceous nature, such as bacteriocins (de Martinis,
Públio, Santarosa, & Freitas, 2001; Harris et al., 1989; Lewus,
Kaiser, & Montville, 1991; Lewus & Montville, 1991).
In addition to minimizing the production of acids, the BHI used
in this study received a solution of catalase to hydrolyze the
hydrogen peroxide that may have been produced by the tested LAB
cultures (Moreno, Lerayer, & Leitão, 1999; Schillinger & Lücke,
1989). Hydrogen peroxide is another substance with antimicro-
bial potential produced by LAB (Carr et al., 2002) and may interfere
with the identification of antimicrobial activity from the produc-
tion of bacteriocins or bacteriocin-like substances. Incubation of
LAB in an anaerobic environment can also inhibit the production of
hydrogen peroxide by LAB (de Martinis et al., 2001; Lewus et al.,
1991; Lewus & Montville, 1991; Schillinger & Lücke, 1989).
Considering these characteristics, using mMRS incubated under
anaerobiosis enhanced the formation of inhibition halos (Table 2),
which facilitates the identification of antimicrobial activity. The use
of mMRS under anaerobiosis for the detection of antagonistic
activity restricts the production of other antimicrobial substances
naturally produced by LAB such as organic acids and hydrogen
peroxide. Despite the easier visualization of inhibition halos during
the antagonism test using mMRS, some LAB cultures previously
identified as antagonistic showed difficulties in growing and
forming halos resulting in the reduction or absence of halos. The
transitory loss of antimicrobial LAB activity has been previously
described and can occur due to a number of factors such as the
interference of environmental conditions and the non-induction of
the expression of genes coding antimicrobial substances, such as
bacteriocins (Gálvez, Abriouel, López, & Omar, 2007).
As a result of the performance presented in the confirmation of
antimicrobial activity (Table 2), mMRS was adopted as the culture
media used to verify the proteinaceous nature of antimicrobial
substances produced by antagonistic LAB. The spot-on-the-lawn
 P.M. Moraes et al. / LWT - Food Science and Technology 43 (2010) 1320e1324 1323

Table 1
Frequencies of lactic acid bacteria (LAB) cultures isolated from raw milk and soft cheese isolated from de ManneRogosaeSharpe agar (MRS) and KangeFungeSol (KFS) agar
that had antagonistic activity against Listeria monocytogenes ATCC 7644, as identified by the spot-on-the-lawn method using brain-heart infusion agar with catalase (BHI þ C)
and M17 agar, and the evaluation of the proteinaceous nature of the antimicrobial substances confirmed by the spot-on-the-lawn and well-diffusion-assay methods.

Culture media for Initial antagonism screening Evaluation of proteinaceous nature Statistical testb
LAB isolation
Culture media Antagonistic cultures Spot-on-the-lawn Well-diffusion-assay
MRS BHI þ C 65 50 13 c2 ¼ 42.16, df ¼ 1, P < 0.0001
M17 83 60 12 c2 ¼ 56.51, df ¼ 1, P < 0.0001
Statistical testa c2 ¼ 2.7, df ¼ 1, P ¼ 0.1 c2 ¼ 0.41, df ¼ 1, P ¼ 0.522 c2 ¼ 0.80, df ¼ 1, P ¼ 0.372
KFS BHI þ C 15 13 2 c2 ¼ 16.13, df ¼ 1, P < 0.0001
M17 24 15 2 c2 ¼ 15.39, df ¼ 1, P < 0.0001
Statistical testa c2 ¼ 2.2, df ¼ 1, P ¼ 0.138 c2 ¼ 2.66, df ¼ 1, P ¼ 0.103 c2 ¼ 0.25, df ¼ 1, P ¼ 0.617
Total BHI þ C 80 63 15 c2 ¼ 57.64, df ¼ 1, P < 0.0001
M17 107 75 14 c2 ¼ 71.58, df ¼ 1, P < 0.0001
Statistical testa c2 ¼ 4.5, df ¼ 1, P ¼ 0.035 c2 ¼ 1.77, df ¼ 1, P ¼ 0.183 c2 ¼ 1.12, df ¼ 1, P ¼ 0.290
c2 ¼ chi-square test, df ¼ degrees of freedom, P ¼ level of significance.
a
Comparison between the frequencies obtained by the different culture media in the initial antagonism screening.
b
Comparison between the frequencies obtained by the different methodologies in the evaluation of proteinaceous nature.

technique presented a higher performance with frequencies of
antagonist cultures that produce bacteriocins or bacteriocin-like
substances statistically that are higher than those obtained by the
well-diffusion-assay (P < 0.05, Table 1). Considering each culture
media for the isolation of LAB and for the detection of antagonist
activity individually, significant differences were not observed
(P > 0.05).
The spot-on-the-lawn method is widely used to detect the
proteinaceous nature of antimicrobial substances produced by LAB
(Benkerroum et al., 2000; de Martinis et al., 2001; García-
Almendárez, Cann, Martin, Guerrero-Legarreta, & Regalado, 2008;
Lewus & Montville, 1991). This method is advantageous in that
proteinaceous substances can be identified even in cultures that
produced small inhibition halos. Using this technique, 61 of the 80
antagonistic cultures were identified as being bacteriocinogenic or
bacteriocinogenic-like during the first screening with BHI þ C,
while 57 of the 106 antagonistic cultures were identified as such
with M17. On the other hand, the well-diffusion assay allowed the
enzymatic confirmation of LAB cultures that presented evident
antagonism during the second screening with the formation of
inhibition halos larger than 16 mm in mMRS. Using this method,
the proteinaceous nature of 24 antagonistic cultures was confirmed
in BHI þ C, while 24 were confirmed in M17. The formation of small
halos using the well-diffusion-assay technique may be explained by
the insufficient concentration of antimicrobial substances in the
supernatant of the cultures (Harris et al., 1989; Lewus & Montville,
1991). This may limit the use of well-diffusion assays for detection
of cultures with antimicrobial potential.
The initial identification of the inhibitory activity of LAB isolated
from foods is fundamental for further studies about their
Table 2
Frequencies of lactic acid bacteria cultures isolated from raw milk and fresh cheese
with antagonistic activity against Listeria monocytogenes ATCC 7644. Cultures were
grouped by the inhibition halo diameter identified by spot-on-the-lawn using three
culture media: modified de ManneRogosaeSharpe (mMRS), M17 and brain-heart
infusion with added catalase (BHI þ C).
Inhibition halo diameter (mm) Culture media
mMRS BHI þ C M17
<5 13 64 50
6e10 51 24 44
11e14 7 12 11
15e18 10 2 3
>19 30 9 3
Total 111 111 111
antimicrobial activity. After antagonist activity is detected,
complementary tests are required to confirm the proteinaceous
nature of the produced substances, to identify the spectrum of
other sensitive microorganisms, to verify the factors that interfere
with the antimicrobial activity and to verify the pathogenicity of
the isolated cultures (de Martinis et al., 2003; Harris et al., 1989;
Lewus et al., 1991; Lewus & Montville, 1991; Maragkoudakis et al.,
2009; Rodriguez, Gonzalez, Gaya, Nunez, & Medina, 2000). Patho-
genicity testing with the cultures and bacteriocins produced is also
fundamental for applications in foods for human consumption
(Belgacem et al., 2010; Gomes et al., 2008; Yoon, Kim, & Hwang,
2008). Despite L. monocytogenes being the most frequent target
(de Martinis et al., 2003; García-Almendárez et al., 2008; Harris
et al., 1989; Lewus & Montville, 1991; Singh & Ramesh, 2008),
further studies must consider distinct pathogens.
4. Conclusion
Considering the results, MRS for the isolation of antagonistic
LAB cultures performed better and may be used with BHI þ C or
M17 for the detection of antimicrobial activity. An alternative to this
screening would be the use of mMRS under anaerobiosis, which
would allow for a more evident production of bacteriocins or
bacteriocin-like substances by LAB. In addition, the spot-on-the-
lawn method confirmed the proteinaceous nature of the substances
produced by the antagonistic LAB in a higher proportion than the
well-diffusion-assay method.
Acknowledgments
L. A. Nero received financial support from FAPEMIG (Fundação
de Amparo à Pesquisa do Estado de Minas Gerais, PPM-00093-09).
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