Food Control 23 (2012) 547e551

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Food Control
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Partial characterisation of bacteriocins produced by lactic acid bacteria isolated

from Thai fermented meat and fish products
Onanong Pringsulaka*, Narumon Thongngam, Nuttika Suwannasai, Wisrutta Atthakor,
Kajeenart Pothivejkul, Achariya Rangsiruji
Department of Biology, Faculty of Science, Srinakharinwirot University, Wattana, Bangkok 10110, Thailand

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

Article history: Lactic acid bacteria (LAB) from Thai fermented meat and fish products were isolated. From a total of 93
Received 25 March 2011 samples, 152 isolates of lactic acid bacteria were obtained. Antimicrobial activity screening was per-
Received in revised form formed using the agar spot test and the agar well diffusion method. Of the six isolates which produced
6 August 2011
antimicrobial activities against Weissella confusa N31, only isolate N23 was identified as Weissella cibaria
Accepted 13 August 2011
(GenBank accession number AB494716.1) with 99% similarity by 16S rDNA sequence analyses. Complete
inactivation of antimicrobial activity produced by W. cibaria N23 was observed after treatment of the
Keywords:
bacteriocins with trypsin, actinase, protease XIII, ficin, trypsin from porcine pancreas, a-chymotrypsin
Weissella cibaria
Bacteriocins
and pepsin. In addition, the inhibitory activities were not affected by the addition of catalase. Taken
Lactic acid bacteria together, these results confirmed that the inhibitory compounds produced by this strain were protein-
Thai fermented meat and fish products aceous in nature and possessed typical characteristics of bacteriocins. The highest yield of bacteriocin
produced by W. cibaria N23 was recorded at 20 h. The bacteriocin N23 remained stable after 2 h of
incubation at pH values between 2.0 and 8.0, and also for 15 min at 121
C. The bacteriocin produced by
W. cibaria N23 was found to have a narrow antibacterial spectrum, being able to inhibit only W. confusa
N31. In addition, bacteriocin N23 did not adhere to the surface of the producer cells. The results produced
from this study will contribute to the existing body of knowledge and enhance the databases of
bacteriocin-producing Weissella.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Throughout Thailand’s history, fermented meats, including

At present, the trend towards greater food safety is attracting
a significant amount of interest. The use of chemical preservatives
to prevent food spoilage and inhibit food-borne pathogens is being
questioned due to lack of consumer acceptance and potential
health risks. Since consumers are becoming increasingly health
aware, the application of bacteriocins as a natural preservative in
food has recently received considerable attention (Papagianni &
Anastasiadou, 2009).
Thai fermented meats are produced naturally, using raw mate-
rials available locally in each area of Thailand. Common to all Thai
fermented products, salt and other ingredients such as rice bran,
pepper, minced garlic and chilies are usually added to the meat. The
mixture is then left in a jar or wrapped tightly in banana leaves or
a plastic bag and allowed to ferment for several days or months
depending on the kind of products.
* Corresponding author. Tel.: þ66 (2)649 5000x8517; fax þ66 (2)260 0127.
E-mail addresses: opringsulaka@gmail.com, onanong@swu.ac.th (O. Pringsulaka).
Nham (Thai fermented pork), Sai Krok Preaw (fermented pork
sausage) and Plaa-ra (Thai fermented fish), have been, and remain,
widely consumed by the Thai people. Generally, such fermented
meats are carried out naturally by lactic acid bacteria (LAB).
Through natural fermentation, the products become acidic (pH
approximately 4.5) and develop a sour flavour. Like most other
meat fermentations, Nham fermentation relies on indigenous lactic
acid bacteria; however, the development of pilot- and large-scale
fermented food manufacture requires a lactic starter culture for
high productivity.
Lactic acid bacteria (LAB) are a diverse group of microorganisms
that produce lactic acid as the primary end-product of the
fermentation of carbohydrates (Carr, Chill, & Maida, 2002). LAB are
Gram positive, non-spore forming, catalase-negative, and acid-
tolerant. They have a low GC content, and are either facultatively
anaerobic or microaerophilic. LAB produce a variety of bacteriocins,
most of which can be grouped into the classes described by
Klaenhammer (1993). Since bacteriocins produced by LAB are
considered to be Generally Recognized as Safe microorganisms

0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.08.029
548 O. Pringsulaka et al. / Food Control 23 (2012) 547e551
(Anastasiadou, Papagianni, Filiousis, Ambrosiadis, & Koidis, 2008;
Elegadob, Kima, & Kwon, 1997; Nettles & Barefoot, 1993), they are
receiving much attention. Typically, LAB species which produce
bacteriocin belong to the genera Lactobacillus, Lactococcus, Strep-
tococcus, Pediococcus, Oenococcus, Enterococcus, Leuconostoc and
Carnobacterium (BACTIBASE: http://bactibase.pfba-lab-tun.org/
main.php). These bacteriocin-producing bacteria are probably
amongst the most promising natural food biopreservatives
(Atanassova, Meindl, & Ring, 2001; Leroy & De Vuyst, 2003).
The genus Weissella, formerly classified as the genera Leuco-
nostoc and Lactobacillus, is found in a variety of meat products,
including fresh and vacuum packaged meats and poultry, as well as
processed and fermented meat products (Holzapfel, 1998;
Holzapfel & Schillinger, 1992; Reuter, 1975; Von Holy & Holzapfel,
1989). The morphology of weissellas varies from spherical or
lenticular cells to irregular rods (Björkroth & Holzapfel, 2006). In
Thailand, weissellas have been isolated from traditional fermented
meats, such as Plaa-ra, Plaa-Som (Kopermsub & Yunchalard, 2010;
Srionnual, Yanagida, Lin, Hsiao, & Chen, 2007; Tanasupawat, Shida,
Okada, & Komagata, 2000; Wongsuphachat, Kittikun, & Maneerat,
2010), Saikrork-preaw, Mum (Chavasirikunton, Vatanyoopaisarn,
& Phalakornkule, 2006e2007) and Nham (Pringsulaka,
Patarasinpaiboon, Suwannasai, Atthakor, & Rangsiruji, 2011).
However, only a few strains of Weissella have been reported to
produce an antimicrobial substance (Chavasirikunton et al.,
2006e2007; Pal & Ramana, 2010; Srionnual et al., 2007).
Research on bacteriocin from Thai fermented foods has been
reported elsewhere because Thailand is home to a variety of fer-
mented food products in every region. However, little information
is available on the characterisation of bacteriocin-producing Weis-
sella species. Interestingly, only 3 species of Weissella have been
reported to produce bacteriocin (Weissella cibaria, Weissella confusa
and Weissella paramesenteroides). The objective of this study was to
isolate and characterise a new bacteriocin produced by W. cibaria
N23, which was isolated from Thai fermented meat and fish
products.
2. Materials and methods
2.1. Bacterial strains and culture media
The bacteriocin producer W. cibaria N23 was isolated from
Nham (Thai fermented pork). The bacterial strains used for the
inhibitory tests were: E. coli JM 109, Listeria innocoa ATCC 33090,
Lactobacillus sakei JCM 1157, Lb. plantarum ATCC 8014, Lactococcus
lactis JCM 7638, L. lactis subsp. cremoris TUA 1344L, Leuconostoc
mesenteroides JCM 6124, Pediococcous pentosaceus JCM 5885,
P. pentosaceus JCM 5890, Streptococcus salivarius JCM 57077,
W. confusa N31, W. confusa N33 and W. cibaria N22. All strains of LAB
stock cultures were stored at 20
C in MRS broth (Difco Labora-
tories, Detroit, MI) containing 10% (v/v) glycerol whereas E. coli and
Listeria innocua were propagated in BHI broth and maintained as
frozen stocks at 20
C in BHI broth (Difco Laboratories, Detroit,
MI) containing 10% (v/v) glycerol. Before use, frozen cultures were
plated onto MRS agar or BHI agar (Difco Laboratories, Detroit, MI).
2.2. Lactic acid bacteria (LAB) isolation
Ninety-three samples of Thai fermented meat and fish prod-
ucts were purchased from local markets in northern, north
eastern and central Thailand. All samples were diluted 10-fold
with a sterile 0.85% NaCl solution and mixed thoroughly. Then,
0.1 ml of each dilution was spread onto MRS agar (Oxoid Ltd.,
Basingstoke, U.K.) with 0.1% CaCO3 and incubated anaerobically
for 24e48 h at 30
C. Clear halo surrounded colonies were
selected and re-streaked on MRS agar plates to obtain pure
cultures. Bacteriocin-producing strains were preliminarily char-
acterised by Gram staining, catalase testing and other identifica-
tion tests (Schillinger & Lücke, 1987). Carbohydrate fermentation
was analysed using API 50 CHL (BioMerieux, France) according to
the supplier’s recommendations. The 16S ribosomal DNA (16S
rDNA) was amplified using standard PCR protocol and the
universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and
1492R (50 -TACGGYTACCTTG TTACGACTT-30 ) to obtain 1500 bp PCR
amplicons (Erko and Michael, 1991). PCR protocols were carried
out as described by Pringsulaka et al. (2011). The PCR products
were visualised by gel electrophoresis on a 0.6% (w/v) agarose gel
and UV irradiation after staining with ethidium bromide (1 mg/
ml). The 16S rDNA fragments were purified with a MinElute Gel
Extraction kit (Qiagen) according to the manufacturer’s protocol
and used as sequencing templates.
2.3. Antibacterial activity determination
Bacteriocin activity was determined by agar spot test and agar
well diffusion assay. The agar spot test was performed using the
method described by Schillinger and Lücke (1989). Overnight
cultures of the strains to be tested for antagonistic activity were
spotted onto the surface of MRS agar plates containing 1.5% agar
and incubated for 24e48 h at 30
C and 37
C until the colonies
developed. About 107 cfu/ml of indicator strains, W. confusa N31,
were inoculated into soft MRS agar containing 0.5% agar and
immediately poured onto previously prepared MRS agar plates.
The plates were incubated at 30
C and 37
C for 24 h in a jar
under anaerobic conditions. The agar well diffusion assay was
used to confirm the antimicrobial activity. Cell-free culture
supernatants were obtained by centrifuging the cultures at
8000 rpm at 4
C for 10 min, and were then adjusted to pH 6
with 1 M HCl or NaOH and filtered through 0.45 mm membrane
filters. The supernatant (100 ml) was placed in wells cut in MRS
agar plates (20 ml) seeded (1% v/v) with the W. confusa N31. The
plates were incubated at 30
C for 24 h and the diameters of the
zones of growth inhibition were then measured. The activities of
bacteriocin were quantified by serial twofold dilutions and
expressed in arbitrary units per millilitre (AU/ml). Arbitrary units
were defined as the reciprocal of the highest dilution which gave
a distinct inhibition zone.
2.4. Characterisation of antimicrobial substance
Aliquots of the filtered supernatant (0.45-mm Millipore filter) of
an overnight culture were treated with catalase (1000 U ml1),
trypsin, actinase, protease XIII, ficin, trypsin from porcine pancreas,
a-chymotrypsin and pepsin at a final concentration of 10 IU/mg in
phosphate buffer (pH 7.0). The supernatants were incubated with
these enzymes at 37
C for 2 h, after which the retention of
bacteriocin activity in treated samples was determined by an agar
well diffusion assay as described above.
2.5. Time course of production of the bacteriocin and growth curve
of W. cibaria N23
The growth of W. cibaria N23 in 1000 ml MRS broth and the
production of bacteriocin during this growth were monitored. For
analyses, 10 ml samples were aseptically taken from the culture at
4, 8, 12, 16, and 24 h. The growth of the strain was monitored by
measuring its optical density at 600 nm at 30
C at the afore-
mentioned time intervals during the 24 h period. Bacteriocin
activity was detected by agar well diffusion assay and expressed
as AU/ml.
 O. Pringsulaka et al. / Food Control 23 (2012) 547e551
2.6. Heat and pH stability of bacteriocin
The influence of pH on the activity of bacteriocin in the
membrane-filtered superanatant was investigated at pH values of
2.0, 4.0, 6.0, 7.0, 8.0 and 10.0 at room temperature (30  2
C) for
2 h. After the pH tests, the samples were readjusted to pH 7.0. The
activity of bacteriocin was then determined using the agar well
diffusion.
To test for heat sensitivity, a separate batch of samples from the
supernatant were extracted and heated to 60, 80, 100 and 121
C for
10, 20, 30 and 60 min each. Following the heat sensitivity tests, the
samples were readjusted to pH 7.0. An agar well diffusion assay was
then carried out with W. confusa N31 as the sensitive strain. All
experiments were done in triplicate.
2.7. Inhibitory spectrum
The inhibitory spectrum of bacteriocin was assessed by testing
for indicator strains, including E. coli JM 109, L. innocua ATCC 33090,
L. sakei JCM 1157, Lb. plantarum ATCC 8014, L. lactis JCM 7638,
L. lactis subsp. cremoris TUA 1344L, L. mesenteroides JCM 6124,
P. pentosaceus JCM 5885, P. pentosaceus JCM 5890, S. salivarius JCM
57077, W. confusa N31, W. confusa N33 and W. cibaria N22. An agar
well diffusion assay with W. confusa N31 as the indicator strain was
carried out at 37
C as described above.
2.8. Effect of carbohydrate on the production of bacteriocin N23
One millilitre cell suspension of W. cibaria N23 (OD600 ¼ 0.1)
was inoculated into 10 ml MRS broth. The effect of various carbo-
hydrates on bacteriocin activity was determined by substituting
glucose in the MRS broth with sucrose and lactose, all of which
were incubated at 30
C for 18e20 h. An agar well diffusion assay
with W. confusa N31 as the indicator strain was carried out at 37
C
as described above.
2.9. Adsorption of bacteriocin onto producer cells
Adsorption of bacteriocin onto producer cells was carried out
using the method described by Yang, Johnson, and Ray (1992).
After 18 h of growth, the culture broth was adjusted to pH 7.0 and
further incubated at 4
C for 30 min. After centrifugation, the
activity of the bacteriocin (AU/ml) in the cell-free supernatant
was assayed while the cell pellets were washed with 0.1 M
phosphate buffer (pH 6.5), centrifuged, resuspended in 100 mM
NaCl (pH 2.0) and stirred for 1 h at 4
C. The bacterial culture was
centrifuged again, neutralized to pH 7.0 and then tested for
antibacterial activity.
Table 1
Some characteristics of the 6 bacteriocin-producing strains.
Isolate Catalase test Gas from glucose Growth at
Temperature
(๐C)
10 45
A8 e þ e þ
N8 e þ e þ
N23 e þ e þ
11 e þ e þ
14 e þ e þ
24 e þ e þ
þ ¼ positive,  ¼ negative.
549
3. Results and discussion
3.1. Isolation of bacteriocin-producing strains
Out of 93 Thai fermented meat and fish samples screened, 152
lactic acid bacteria with clear zones on the MRS agar supplemented
with CaCO3 were isolated on the basis of Gram stain, catalase and
oxidase tests. They were all screened for their ability to inhibit the
growth of W. confusa N31 using the agar spot test and agar well
diffusion assay. Six isolates, namely A8, N8, N23, 11, 14 and 24, in
which zones of inhibited growth appeared were selected for further
studies.
3.2. Identification of the bacteriocin-producing strains
Partial identification tests were performed as described by
Smibert and Krieg (1994). Six of the isolates (A8, N8, N23, 11, 14 and
24) were heterofermenters and able to grow at 45
C. Some of their
characteristics are shown in Table 1. Further identification to species
level was carried out on these six isolates, and was based on carbo-
hydrate fermentation reactions using API 50 CHL kits and confirmed
by 16S RNA sequence analyses. Of the six, five (A8, N8, 11, 14 and 24)
were identified as Lactobacillus plantarum with 100% similarities
(GenBank accession number AB494717.1, EU789400.1, GU451063.1,
GU253892.1 and FJ542291.1, respectively). Only isolate N23 was
identified as W. cibaria with 99% similarity (GenBank accession
number AB494716.1). Because many bacteriocin-producing strains of
Lactobacillus have previously been isolated and characterised, only
W. cibaria N23 was selected for further study.
3.3. Growth and bacteriocin production
Detectable levels of bacteriocin produced by W. cibaria N23 at
30
C were recorded after 4 h of growth, and at subsequent 4 h
intervals during the continuous bacteriocin production. Maximum
activity yield against W. confusa N31 was observed at 20 h of
growth (300 AU/ml) and remained stable, as shown in Fig. 1. The
graph in Fig. 1 illustrates that bacteriocin production occurs mostly
during the exponential phase of the growth curve and maximal
bacteriocin production parallels the growth rate. Maximum
bacteriocin activity of strain N23 was achieved at the end of the
exponential phase, which is characteristic of primary metabolites.
Similar results were reported for weissellicin 110 (Srionnual et al.,
2007). However, when incubated at 37 and 40
C, both cell
growth and bacteriocin production were found to be lower than at
30
C (data not shown). Regarding the inhibitory spectra of bacte-
riocin produced by W. cibaria N23, it was found that W. cibaria N23
inhibits only W. confusa N31; as such, it is characterised as having
a narrow spectrum of inhibition.
Growth at NaCl (%) Growth at pH
4.0 6.5 8 18 4.4 7.0 8.5 9.6
þ þ e e e þ þ þ
þ þ þ e e þ þ þ
þ þ e e e þ þ þ
þ þ þ e þ þ þ þ
þ þ þ e þ þ þ þ
þ þ þ e þ þ þ þ
550
4 500
3.5
400
3
2.5
OD 600 nm
300
2
200
1.5
1
100
0.5
0 0
4 8 12 16 20 24
time (h)
Fig. 1. Growth and bacteriocin production of W. cibaria N23.
3.4. Effect of enzymes, temperature and pH on activity of
bacteriocins
Bacteriocins produced by W. cibaria N23 were completely
inactivated after treatment with the proteolytic enzymes trypsin,
actinase, protease XIII, ficin, trypsin from porcine pancreas, a-
chymotrypsin and pepsin, confirming their proteolytic nature. The
exposure of bacteriocins to different pH values showed that
bacteriocin N23 remained fully active in the pH range of 2.0e8.0;
however, at pH 10.0 its activity was completely lost. At higher
temperatures, bacteriocin was completely stable after heat treat-
ment at 60e100
C for 20 min, and maintained 50% of the activity
when subjected to 100
C for more than 30 min or to a 121
C for
15 min (data not shown).
3.5. Effect of carbohydrate on activity of bacteriocins
In comparison to glucose, the use of lactose and sucrose resulted
in lower yields of bacteriocin. Maximal levels of bacteriocin
produced by the N23 strain (400 AU/ml) occurred in the MRS
medium with 2% glucose after incubation for 20 h. Other concen-
trations of glucose (1, 3 and 5%) yielded lower bacteriocin activity
levels, at 200 AU/ml, 300 AU/ml and 300 AU/ml, respectively (data
not shown).
3.6. Adsorption to producer cells
Bacteriocins produced by W. cibaria N23 showed no increase in
activity after the treatment of the producer cells with 100 mM NaCl
at pH 2.0 (data not shown), suggesting that the bacteriocins did not
adhere to the surface of the producer cells. Similar results were
reported in other bacteriocin-producing lactic acid bacteria
(Ivanova, Kabadjova, Pantev, Danova, & Dousset, 2000; Todorov &
Dicks, 2005; Todorov et al. 1999).
4. Conclusion
W. cibaria N23, isolated from Nham (Thai fermented pork), was
found to produce a bacteriocin against closely related Weissella
strains. Among Weissella species, only W. cibaria 110,
W. paramesenteroides DFR-8 and W. confusa CP3-1 were reported as
bacteriocin producers (Chavasirikunton et al., 2006e2007; Pal &
Ramana, 2010; Srionnual et al., 2007). Although bacteriocin
produced by strain N23 was found to have a narrow inhibition
spectrum, the knowledge gained from the study of this strain will
O. Pringsulaka et al. / Food Control 23 (2012) 547e551
provide valuable information and enhance existing databases of
bacteriocin-producing Weissella species.
Acknowledgement
bacteriocin activity
(AU/ml)
This work was supported by the annual government statement
OD 600 nm of expenditure, Srinakharinwirot University. The Faculty of Science,
Srinakharinwirot University also provided a partial support grant
for Ms. Narumon Thongam in the form of a graduate fund, which
was received with gratitude.
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