International Journal of Food Microbiology 81 (2003) 137 – 145

www.elsevier.com/locate/ijfoodmicro

Isolation of nisin-producing Lactococcus lactis WNC 20 strain

from nham, a traditional Thai fermented sausage
W. Noonpakdee a,*, C. Santivarangkna a, P. Jumriangrit a, K. Sonomoto b, S. Panyim a
a
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
b
Laboratory of Microbial Science and Technology, Division of Bioscience and Biotechnology, Graduate School of Bioresource and
Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Received 21 January 2002; received in revised form 14 May 2002; accepted 29 May 2002

Abstract

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One
Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne
pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus.
Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 jC for 15 min) and
was active over a wide pH range (2 – 10). The bacteriocin was inactivated by a-chymotrypsin and proteinase K but not other
proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The
gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing
of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at
position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the
fermented product.
D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Nisin Z; Lactococcus lactis; Nham; Thai fermented sausage

1. Introduction of several undesirable Gram-positive bacteria in the

Lactic acid bacteria (LAB) are widely used in food
fermentation including dairy, meat, vegetable and
bakery products (Marrug, 1991). They are known to
produce many different antibacterial substances
including bacteriocins, which can inhibit the growth
*
Corresponding author. Tel.: +66-2-2015600; fax: +66-2-
2480375.
E-mail address: scwnp@mahidol.ac.th (W. Noonpakdee).
genera Bacillus, Enterococcus, Listeria, Clostridium
and Staphylococcus (Tagg et al., 1976; Klaenhammer,
1993). Many bacteriocins have been isolated and there
is increasing interest in using these bacteriocins as
natural food preservatives (Jack et al., 1995). The
bacteriocin nisin, which is produced by Lactococcus
lactis subsp. lactis, has been extensively studied and
used as a food preservative because of its lethal action
and wide spectrum of activity (Hurst, 1981; Delves-
Broughton et al., 1996). Two nisin variants, which
differ only by one amino acid substitution at position

0168-1605/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 2 1 9 - 2
138 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145
27, have been studied extensively. Nisin A contains
histidine and nisin Z asparagine (Mulders et al.,
1991). Usually, nisin producer strains are isolated
from dairy products (Rauch et al., 1994), some from
vegetables (Cai et al., 1997; Choi et al., 2000; Harris
et al., 1992) and very few from fermented meat
(Rodriguez et al., 1995).
Nham is a Thai traditional fermented sausage
made from ground pork with garlic, pepper, salt
and cooked rice. It can be consumed raw or cooked.
This fermented meat product has developed to full-
scale commercialization, but the technology of fer-
mentation is still mediated by indigenous bacteria
rather than added starter cultures. The bacteria found
most commonly in Thai fermented meat products are
lactobacilli, pediococci and micrococci, but the pre-
cise role of these bacteria in the quality of the
products is not known (Thiravattanamontri et al.,
1998). Developments in this meat fermentation have
been focusing on application of defined starter cul-
tures to increase process control and product consis-
tency (Campbell-Platt, 1995). In addition to
difficulties in obtaining product consistency, patho-
gens in raw fermented sausage still cause food safety
problems. Listeria monocytogenes, which has been
frequently recovered from fermented sausage (John-
son et al., 1990; Aymerich et al., 1998), is of
particular concern because it results in a high mortal-
ity rate and of its ability to grow at low pH (Gahan
et al., 1996).
The use of bacteriocin-producing strains as starter
cultures or protective cocultures in the in situ control
of food pathogens is one of the possible ways to
improve food safety (Kim, 1993; Holzapfel et al.,
1995; Stiles, 1996; Caplice and Fitzgerald, 1999;
Hugas, 1999). Cultures used in fermentation of meat
are mostly Lactobacillus (Sobrino et al., 1991; Gar-
riga et al., 1993; Hugas et al., 1995; Swetwiwatthana
et al., 1999) and Pediococcus (Bhunia et al., 1988;
Harris et al., 1989). However, the isolation of nisin-
producing Lactococcus strains from dry fermented
sausage (Rodriguez et al., 1995) indicated the poten-
tial use of lactococci in meat fermentation systems.
More recently, the use of bacteriogenic Lactococcus
strains in salami and sausage production has been
successfully developed (Coffey et al., 1998; Scannell
et al., 2001). This study reports the detection and
isolation of a nisin Z-producing Lc. lactis strain
(WNC 20) isolated from a Thai fermented pork
sausage (nham).
2. Materials and methods
2.1. Bacterial strains and media
Bacterial strains used in this study are listed in
Table 1. All LAB isolates and indicator strains were
grown in MRS broth (Merck) at 30 or 37 jC. Listeria,
Salmonella, Bacillus were grown in tryptic soy broth
(Difco) at 30 jC. Enterococcus and Staphylococcus
were grown in nutrient broth (Difco). Escherichia coli
was grown in Luria – Bertani (Difco) medium at 37
jC. Clostridium was grown under anaerobic condi-
tions in reconstituted Clostridium medium (E. Merck)
at 30 jC. All cultures were maintained as frozen
stocks held at 80 jC in appropriate broth contain-
ing 20% glycerol (wt/vol). Throughout the experi-
ments, strains were subcultured every 2 weeks on agar
media and kept at 4 jC. Before use in experiments,
cultures were propagated twice in broth overnight.
Soft agar media were prepared by adding 0.6% (wt/
vol) agar to liquid media. Catalase at a final concen-
tration of 50 U ml 1(Boehringer Mannheim) was
added to soft agar overlays to eliminate the potential
inhibitory effect of H2O2 produced by colonies.
2.2. Isolation of bacteriocin-producing bacteria and
bacteriocin assay
Bacteriocin-producing bacteria were isolated by
the direct plating method (Coventry et al., 1997). A
10% food sample in diluent (0.85% NaCl) was
homogenized and 10-fold serially diluted. Pour plates
of serial dilutions (1-ml aliquots) in media MRS were
incubated under anaerobic conditions (anaerobic gen-
eration kit; Merck) for 48 h at 30 jC. Multiple plates
of serial dilution (three to five plates providing a total
of approximately 1000 colonies) were overlaid with a
set of four indicators and incubated at 30 jC for
another 18– 24 h. The four indicator strains used were
Lactobacillus curvatus ATCC 25601, Lactobacillus
plantarum TISTR 850, Pediococcus pentosaceus
TISTR 374 and Propionibacterium freudenreichii
TISTR 446. Colonies suspected to be producing zones
of growth inhibition in the indicator lawn were
 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145 139

Table 1 was tested by spotting 10 Al of the supernatant onto

Antibacterial spectrum of bacteriocins produced by Lc. lactis subsp. soft agar lawn (0.6%) seeded with 0.1 ml of an
lactis WNC 20 and Lc. lactis subsp. lactis DL 11
overnight grown indicator strain and incubated over-
Indicator straina Sensitivityb DL 11c
night. Cultures producing an inhibitor in broth were
WNC 20
then purified by streaking from the broth and restreak-
Bacillus pumilus TISTR 061 ++d ++
ing for single colony isolates two to three times.
Bacillus subtilis A-48 ++ ++
B. cereus IFRPD 2037 ++ + For a semiquantitative assay of the bacteriocins,
B. cereus ATCC 11778 ++ ++ twofold serial dilutions of the supernatant were tested
Lactobacillus sp. A28 +++ +++ using Lb. plantarum TISTR 850 as the indicator
Lb. plantarum TISTR 850 +++ +++ strain. The activity was expressed in arbitrary units
Lactobacillus sake TISTR 911 +++ +++
(AU ml 1). One AU was defined as the reciprocal of
Lactobacillus pentosus LP-711 +++ +++
Lb. curvatus ATCC 25601 +++ +++ the highest serial twofold dilution, which did not show
Leuconostoc cremoris ATCC 19254 ++ ++ inhibition of the indicator strain per milliliter (Schil-
Leuconostoc mesenteroides ATCC 10830 ++ ++ linger et al., 1993).
Lis. monocytogenes IFRPD 2068 ++ ++
Lis. monocytogenes DMST 7995 ++ ++
2.3. Identification of the bacteriocin-producing isolate
Lis. monocytogenes DMST 2871 ++ +
Listeria inocua IFRPD 2071 ++ ++
Micrococcus luteus TISTR 884 +++ +++ Only one isolate (designated WNC 20) of the
Pediococcus acidilactici ATCC 8081 +++ +++ 14,020 screened stably produced bacteriocin in broth
P. pentosaceus TISTR 374 +++ +++ media. Strain WNC 20 was therefore further charac-
Pr. freudenreichii TISTR 446 ++ ++
terized and identified on the basis of Gram stain,
Staphylococcus aureus TISTR 118 ++ ++
Staphylococcus aureus IFRPD 2034 ++ ++ catalase reaction and other identification tests (Schil-
Enterococcus faecalis TISTR 927 ++ ++ linger and Lucke, 1987). Carbohydrate fermentation
Enterococcus faecium TISTR 928 + + was determined using the API 50 CHL system (Bio-
Clostridium butyricum A-4 + + Merieux, France). Salt tolerance was tested using 4%
Clostridium perfringens DMST 11147 + +
and 6.5% NaCl in MRS medium. Total soluble whole
Salmonella sp. TISTR 101
Salmonella derby A-2 cell proteins were analyzed by sodium dodecyl sul-
Escherichia coli ATCC 25922 phate polyacrylamide gel electrophoresis (SDS-
E. coli ATCC 43895 PAGE), and the pattern was compared with that of
a
ATCC, American Type Culture Collection; TISTR, Thailand reference LAB (Elliot et al., 1991).
Institute of Scientific and Technological Research; DMST, Depart-
ment of Medical Science Thailand; IFRPD, Institute of Food 2.4. Determination of antimicrobial spectrum
Research and Product Development.
b
All indicators were tested for inhibition of growth as deter-
mined by a zone of growth inhibition in the indicator lawn culture. Cell-free supernatant was used to determine the
c
DL 11 is Lc. lactis subsp. lactis, a nisin-producing strain, a antimicrobial spectrum of activity. Cells were grown
gift from Dr. A.J. Hillier (Commonwealth Scientific and Industrial in MRS broth for 20 h at 30 jC and supernatant was
Research Organization, Division of Food Science and Technology, prepared as described above. The spectrum of activity
Australia).
d of the supernatant was tested against a wide range of
Inhibition zone (mm): +++ , 10 – 15 mm; ++ , 5 – 9 mm; +, 1 –
4 mm; , no inhibition. indicator strains comprising LAB and food-borne
pathogens as shown in Table 1. The antimicrobial
spectrum of the nisin-producing strain Lc. lactis
randomly selected and removed using a sterile Pasteur subsp. lactis DL 11 was compared with that of the
pipette. The agar plug (from Pasteur pipette) was bacteriocin-producing strain isolated from nham.
inoculated into broth media and incubated anaerobi-
cally for 48 h at 30 jC. Culture supernate was 2.5. Effects of enzymes, pH and heat treatment
obtained by centrifugation at 12,000 rpm for 20 min
at 12 jC and then was adjusted to pH 6.5 with 5 M The effect of various enzymes on bacteriocin
NaOH and filtered through a 0.45-Am filter. Inhibition activity was determined by incubating 200 Al of
140 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145

filter-sterilized cell-free supernatant with 20 Al of the and 2 bp downstream of the coding region (Dodd et
following enzyme solutions at a final concentration of al., 1990; Mulders et al., 1991). The restriction sites
1 mg/ml: proteinase K (pH 7.0; Sigma), trypsin (pH EcoRI and KpnI were added at 5Vend, respectively, for
7.0; Sigma), pepsin (pH 3.0; Merck), a-chymotrypsin cloning purpose in other studies elsewhere. Forward
(pH 7.0; Sigma), papain (pH 6.0; Sigma), a-amylase and reverse primers were as followed:
(pH 7.0; Sigma) and lipase (pH 7.0; Sigma). After 2 h
of incubation at 37 jC, enzyme activity was terminated primer 1: 5V primer 5V CCG GAA TTC ATA AGG
by heating at 100 jC for 5 min. Untreated samples AGG CAC TCA AAA TG 3V
were used as control. The residual bacteriocin activity primer 2: 3V primer 5V CGG GGT ACC TAC TAT
was assayed against indicator strain Lb. plantarum CCT TTG ATT TGG TT 3V.
TISTR 850. The sensitivity of the active substances to
different pH values was estimated by adjusting the pH The amplified PCR products was purified from low
of the supernatant samples between 2 and 10 using 5 M melting point agarose and the nucleotide sequences
NaOH or 5 M HCl. After 2 h of incubation at room were determined using a Dye Terminator Sequencing
temperature, the residual activity was assayed. Sam- kit (Perkin-Elmer) and ABI PRISM 377 DNA
ples treated with proteinase K before pH adjustment sequencer (Perkin Elmer) as described by the instruc-
were used as control to correct inhibition due to pH. To tions of the manufacturer.
evaluate the effect of heat and pH on bacteriocin
activity, the cell-free supernatant samples were heated
at 100 jC for 30 min or 121 jC for 15 min under 3. Results
different pH conditions. The residual activity was then
assayed against the indicator strain. 3.1. Isolation and identification of bacteriocin-pro-
ducing strains
2.6. Production studies
A total of 14,020 colonies from 12 nham samples
The kinetics of bacteriocin production was deter- were examined for potential inhibitory substance
mined by inoculating strain WNC 20 at 1% vol/vol
solution into 200 ml of M17 broth (Oxoid) and
incubating at 30 and 37 jC under uncontrolled pH Table 2
Effect of enzyme and pH on the bacteriocin activity of strain WNC
conditions. The initial pH of the culture broth was 6.8. 20
At appropriate intervals, 10-ml samples were removed
Enzyme treatment Bacteriocin activity
from the cultures and analyzed for cell growth and (AU ml 1)
bacteriocin activity. The growth of cells was followed
Control 12,800
by measuring the optical density (OD) at 600 nm. Proteinase K 0
a-Chymotrypsin 0
2.7. DNA extraction, polymerase chain reaction Papain 12,800
(PCR) and DNA sequencing Pepsin 12,800
Trypsin 12,800
a-Amylase 12,800
Genomic DNA was extracted by a modification of Lipase 12,800
the method of Engelke et al. (1992). The polymerase pH
chain reaction analysis followed procedure described 2 12,800
by Horn et al. (1991) with some modifications. The 3 12,800
4 12,800
PCR amplification was carried out in a 50-Al mixture
5 12,800
in a DNA thermo cycler (Perkin Elmer Cetus Model 6 12,800
TCI). The conditions consisted of 30 cycles of 90 jC 7 12,800
for 1 min, 55 jC for 1 min and 72 jC for 2 min. The 8 6400
primers were designed from nisin A structural gene, 9 3200
10 3200
which were complementary to regions 17 bp upstream
 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145 141

Table 3
Heat stability of bacteriocin produced by strain WNC 20
1
pH Residual bacteriocin activity (AU ml )
No heat 100 jC 121 jC
(control) for 30 min for 15 min
3.0 12,800 12,800 12,800
5.0 12,800 6400 400
7.0 12,800 1600 0

against the four indicator strains. A total of 28

colonies were recorded as positive, producing inhib-
ition clear zones on agar media (detection rate 0.2%).
Of these colonies, only one designated WNC 20
stably secreted inhibitory substance into culture broth.
This bacteriocin-producing strain WNC 20 was a
Gram-positive, catalase-negative coccus, nonmotile
and produced no gas from glucose. The strain hydro-
lyzed arginine grew at 10, 15 and 40 jC at pH 4 and
9 and in media containing NaCl at 4% but not at
6.5%. Based on these characteristics and the anal-
ysis of the carbohydrate fermentation pattern by the
API CHL kit, WNC 20 was potentially identified as
Lc. lactis subsp. lactis. This identification was
confirmed by comparing whole cell protein pattern Fig. 2. Agarose gel electrophoresis of PCR products with nisin
gene-specific primer. Genomic DNA used were from: lane 1: Lc.
of strain WNC 20 on SDS-PAGE with that of
lactis subsp. lactis DL 11 (positive control); lane 2: Lc. lactis subsp.
reference strain Lc. lactis subsp. lactis DL 11. Both lactis WNC 20; lane 3: P. pentosaceus TISTR 374 (nonnisin-
had the identical protein patterns (data not shown). producing strain); lane 4: water as a negative control. Lane M was
loaded with 100-bp ladder DNA markers (Gibco).
3.2. Characterization of the bacteriocin
bacteriocin exhibited inhibitory activity against a broad
The inhibitory spectrum of the bacteriocin produced range of closely related bacteria in the genera Lacto-
by Lc. lactis WNC 20 is presented in Table 1. The bacillus, Pediococcus, Leuconostoc and some patho-

Fig. 1. Growth (.) and bacteriocin production (n) of Lc. lactis subsp. lactis WNC 20 in MRS broth with uncontrolled pH condition at 30 jC.
142 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145

genic strains of Bacillus, Staphylococcus, Listeria and
Clostridium. The bacteriocin was not effective against
Gram-negative bacteria. Nisin A produced by Lc. lactis
DL 11 was used as an experimental control and showed
an inhibitory spectrum identical to that of Lc. lactis
WNC 20. The degree of inhibition of bacteriocin
produced, however, showed slight differences. Inhib-
ition zones obtained from WNC 20 strain in agar
diffusion assay were larger than those obtained from
Lc. lactis DL 11 especially towards Bacillus cereus
IFRPD 2037 and Lis. monocytogenes DMST 2871.
The effect of enzyme and pH on the activity of the
bacteriocin produced by strain WNC 20 is presented
in Table 2. Protease sensitivity assays demonstrated
that the antimicrobial substance produced by WNC 20
strain was a bacteriocin since its inhibitory activity
was completely eliminated by treatment with enzyme
proteinase K and a-chymotrypsin. The activity was,
however, not inactivated by other proteases including
trypsin, papain, pepsin and nonprotease enzyme
including a-amylase and lipase. These characteristics
were similar to those of nisin. The bacteriocin was
active over a wide pH range between 2 and 10, but the
activity was much lower in basic pH.
The effect of heat and pH on the bacteriocin acti-
vity is shown in Table 3. Inhibitory activity was not
destroyed by exposure to elevated temperature at pH
3 even at autoclaving temperature (121 jC for 15
min). The inhibitory activity was, however, des-
troyed if heated at higher pH value, a characteristic
similar to nisin. Based on these results, it appeared
that Lc. lactis WNC 20 produced a nisin-like bacter-
iocin.
Cell growth and bacteriocin production properties
of Lc. lactis WNC 20 were examined in flask cultures
at 30 and 37 jC in the noncontrolled pH condition.
The production of bacteriocin was higher at 30 jC
(data not shown). The bacteriocin was produced
maximally in the early stationary phase, and the
activity declined during the late stationary phase as
shown in Fig. 1.
3.3. Confirmation that WNC 20 is a nisin-producing
strain
To prove that the bacteriocin produced by Lc. lactis
WNC 20 was nisin, PCR analysis using the published
sequences of the nisin structural gene (Dodd et al.,

Fig. 3. Nucleotide sequence and deduced amino acid sequence of the nisZ gene isolated from Lc. lactis subsp. lactis WNC 20. The amino acid
sequence is shown below the coding sequence. The nucleotide in the nisZ sequence that differs with that in the nisA gene sequence is in bold and
italic. Primers are in bold and underline. Stop codon is shown by asterisk.
 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145
1990) was performed. Two primers complementary to
sequences occurring proximal to the 3V and 5V ends of
the nisin A structural gene were used to amplify nisin
gene from the genomic DNA of Lc. lactis DL 11 and
Lc. lactis WNC 20. The result is shown in Fig. 2. A
227-bp fragment was amplified from the genomic
DNA of Lc. lactis WNC 20 (lane 2), which was
identical to that amplified from a nisin-producing
strain of Lc. lactis DL 11 (lane 1). No DNA fragment
was amplified using genomic DNA extracted from P.
pentosaceus TISTR 374, which did not produce
bacteriocin (lane 3), or no DNA was present (lane
4). The amplified PCR product of Lc. lactis WNC 20
was subsequently sequenced as shown in Fig. 3.
Results indicated that sequences were 100% identical
to that of nisin A except for a C-to-A transversion at
position 148. This resulted in an asparagine (AAT)
residue at position 27 of the nisin peptide, instead of
histidine (CAT). This indicates that the bacteriocin
produced by Lc. lactis WNC 20 is nisin Z.
4. Discussion
The aim of this current study was to isolate and
characterize bacteriocin-producing LAB from nham, a
traditionally fermented pork sausage which is usually
consumed without cooking in various parts of Thai-
land. This fermented meat product is typically pro-
duced on a small scale or at household levels and is
sometimes associated with problems such as product
short shelf-life and poor hygiene. The use of starter
cultures is not a common practice, resulting in product
failure and inconsistency. Moreover, the presence of
food spoilage and pathogenic bacteria in nham some-
times poses problems (Swetwiwatthana et al., 1999).
The bacteriocin-producing Lc. lactis WNC 20 could
prove to be useful as a starter culture or protective
culture in fermented meat and other fermented food
products as well.
Few strains of Lc. lactis have been reported to be
isolated from meat substrates even though meat envi-
ronments have been considered adequate for the
growth of lactococci (Garver and Muriana, 1993;
Rodriguez et al., 1995; Barakat et al., 2000). Garver
and Muriana (1993) isolated Lc. lactis FS92 from
meat products and Rodriguez et al. (1995) isolated
two nisin-producing Lc. lactis strains from dry fer-
143
mented sausages from different regions of Spain.
Recently, Barakat et al. (2000) isolated one strain of
Lc. lactis from cooked, modified atmosphere-pack-
aged, refrigerated, poultry meat. Our isolation of
nisin-producing Lc. lactis WNC 20 and those two
strains from Rodriguez et al. (1995) suggests that
nisin-producing Lc. lactis strains may be more wide-
spread in meat products than previously reported. The
detection rate of bacteriocin-producing strains from
total number of colonies tested in this study was about
0.2%, which was comparable with previous report by
Rodriguez et al. (1995) where less than 0.5% of 4608
LAB examined was a bacteriocin producer and iden-
tified as Lc. lactis. Both results indicate that in meat
substrates, this species may only be present in very
low number.
The antimicrobial substance produced by Lc.
lactis WNC 20 strain had similar enzymatic pH
sensitivity and heat insensitivity pattern to nisin.
The identical antimicrobial spectrum and larger
inhibition zones in agar diffusion assay of bacterio-
cin obtained from WNC 20 as compared with those
from DL 11, a nisin A-producing strain, suggested
that the bacteriocin produced by WNC 20 was nisin
Z (De Vos et al., 1993). This was confirmed by
sequence analysis of nisin structural gene of WNC
20 (Fig. 3), which contained an asparagine at posi-
tion 27 instead of histidine as in nisin A (Mulders et
al., 1991). This finding that WNC 20 strain pro-
duced nisin Z is different from the previous report
by Rodriguez et al. (1995) where both Lactococcus
strains isolated from dry fermented sausages produced
nisin A.
Many studies have shown that the properties of
fermenting sucrose and nisin production were closely
linked and the genes involved on these traits might be
located on the conjugative transposon on the chromo-
some (Horn et al., 1991; Rauch and De Vos, 1992;
Olasupo et al., 1999). The capacity of strain WNC 20
to ferment sucrose suggested that this nisin Z gene
might be located on a conjugative transposon on the
chromosome.
The relative sensitivity of Lc. lactis to acid would
suggest that these organisms have a minor role in
meat fermentation. However, they could be used at
early stages of meat fermentation or together with
other more acid-tolerant, nisin-resistant meat starter
cultures. Moreover, Lc. lactis WNC 20 could become
144 W. Noonpakdee et al. / International Journal of Food Microbiology 81 (2003) 137–145
more acid-tolerant as a result of a pH adaptation
process similar to those reported for enteric bacteria
(Foster and Hall, 1990). At present, studies are in
progress to determine the properties of this WNC 20
strain as a starter culture in meat fermentation sys-
tem.
Acknowledgements
This work was partially supported by a grant
from Thailand Research Fund granted to Prof. Dr.
Sakol Panyim. We would like to thank Dr. Hillier
for providing Lc. lactis subsp. lactis DL 11. We
also gratefully acknowledge the support from the
Japan Society for the Promotion of Science (JSPS)
and the National Research Council of Thailand
(NCRT).
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