Food Control 21 (2010) 7681

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus

sake C2, isolated from traditional Chinese fermented cabbage
Yurong Gao, Shiru Jia *, Qiang Gao, Zhilei Tan
Tianjin Key Laboratory of Industry Microbiology, School of Biological Engineering, Tianjin University of Science and Technology, No. 29 of the 13th Avenue, TEDA, Tianjin 300457, China

a r t i c l e

i n f o

Article history:
Received 1 October 2008
Received in revised form 1 April 2009
Accepted 11 April 2009

Keywords:
Lactobacillus sakei
Bacteriocin
16S rDNA sequence
Molecular weight
Traditional Chinese fermented cabbage

a b s t r a c t
By excluding the effects of organic acids and hydrogen peroxide, strain C2 producing a bacteriocin
strongly inhibited Staphylococcus aureus ATCC 63589 and Escherichia coli ATCC 25922 was selected from
300 lactic acid bacteria isolated from traditional Chinese fermented cabbage. Strain C2 was identied as
Lactobacillus sakei by phenotypical and physiological tests and 16S rDNA identication. This bacteriocin,
which was designated sakacin C2, was puried by cold ethanol and Sephadex G50 column chromatography for further studies. The molecular weight of sakacin C2 was 5.5 kDa by Tris-Tricine SDSPAGE, which
was the largest among all known sakacins. Sakacin C2 exhibited a wide range of antimicrobial activity,
strong heat stability (15 min at 121 C) and pH stability (pH 3.08.0). Sakacin C2 was sensitive to protease
but insensitive to lipase, a-amylase and b-amylase. These characters suggested that sakacin C2 was a
novel Class II bacteriocin with a broad inhibitory spectrum and might broaden the application range of
bacteriocins from LAB in the food industry.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Bacteriocins are dened as antimicrobial peptides or proteins
generally active against closely related species (Cleveland, Montville, Nes, & Chikindas, 2001). In terms of safety, bacteriocins from
lactic acid bacteria (LAB) have attracted more interest than those
from other resources, because most of LAB are related to fermented
foods (Osmanagaoglu, 2007). From various foods such as fermented sausage, yoghourt, fermented vegetables, etc., many novel
bacteriocin-producing LAB have been isolated and the characteristics of these bacteriocins have been investigated extensively (Ross,
Morgan, & Hill, 2002).
The bacteriocins produced by LAB have been classied in four
groups according to their biochemical characteristics (Klaenhammer, 1993). Small and heat-stable peptides containing thioether
amino acids are classied to Class I bacteriocins (lantibiotics)
(molecular weight 6 5 kDa) (Nes et al., 1996). Small, heat-stable,
non-lantibiotic peptides are Class II bacteriocins (molecular
weight 6 10 kDa) (Nes & Holo, 2000). Some large, heat-stable proteins belong to Class III bacteriocins (molecular weight P 10 kDa)
and Class IV are large, complex bacteriocins containing lipid or carbohydrate groups (Joerger & Klaenhammer, 1990). Class I and Class
II bacteriocins are most likely to be used as bio-preservatives in the
food industry to substitute for chemical preservation in all these

* Corresponding author. Tel.: +86 22 60601598; fax: +86 22 60602298.

E-mail address: jiashiru@tust.edu.cn (S. Jia).
0956-7135/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2009.04.003

four groups (Elotmani, Revol-Junelles, Assobhei, & Milliere, 2002).

However, presently industrialized bacteriocins, e.g. nisin, exhibit
certain limitations. On the one hand some bacteriocins are ineffective against Gram-negative spoilage and pathogenic bacteria,
yeasts and molds; on the other hand they do not inhibit all foodborne Gram-positive spoilage and pathogenic bacteria. Therefore,
those disadvantages limit the application range of bacteriocins in
the food industry. To date, screening of LAB producing novel bacteriocins with a broad antimicrobial spectrum and identifying those
novel bacteriocins have attracted the most attention.
Fermented vegetables contain many species of LAB. Some bacteriocin-producing LAB such as Lactobacillus pentosus B96 (Delgado, Brito, Peres, No-Arroyo, & Garrido-Fernndez, 2005),
Lactobacillus plantarum C19 (Atrih, Rekhif, Milliere, & Lefebvre,
1993), Enterococcus faecium BFE 900 (Franz, Schillinger, & Holzapfel, 1996), L. plantarum LPCO10 (Jimnez-Daz, Rios-Snchez, Desmazeaud, Ruiz-Barba, & Piard, 1993) and Lactobacillus brevis P319 (Li, Sun, Yang, & Hou, 2008) were successively isolated from
fermented vegetables. Traditional Chinese fermented cabbage is a
widely used traditional China food made from green cabbage and
its production is a spontaneous fermentation process by a mixed
microbial population mainly composed of LAB. However, to date,
there has been no report about bacteriocin-producing LAB isolated
from traditional Chinese fermented cabbage.
In the present paper, we report the selection, identication and
primary characteristics of a novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2 from traditional
Chinese fermented cabbage. Further, this paper is the rst report

Y. Gao et al. / Food Control 21 (2010) 7681

on the selection of bacteriocin-producing Lactobacillus sakei strain

from fermented vegetables to date.
2. Materials and methods
2.1. Traditional Chinese fermented cabbage samples
The liquor samples of traditional Chinese fermented cabbage
were collected from the pilot plant of Food College of Heilongjiang
August First Land Reclamation University (Daqing, China).
2.2. Screening of LAB producing bacteriocin with a broad inhibitory
spectrum from traditional Chinese fermented cabbage samples
The liquor samples (0.1 ml, 104106 dilution) were spread directly on the surface of MRS agar containing 5 g l1 calcium carbonate and then were incubated at 30 C for 12 days. Colonies
with clear zones around on MRS agar plates were regarded as
acid-producing bacteria and they were randomly selected and
puried by re-plating on MRS agar plates. The puried colonies
were primarily identied by Gram staining and catalase tests. Only
both Gram-positive and catalase-negative strains were selected
and stored in MRS broth with 20% (v/v) glycerol at 20 C.
The selected strains were inoculated into the tubes containing
10 ml MRS broth each and statically incubated at 30 C for 18 h.
Then 1% (v/v) of those cultures were inoculated into 250 ml MRS
broth individually and incubated at 30 C for 24 h without agitation. Bacterial cells were removed by centrifugation (5000g,
15 min, 4 C) and supernatants were examined by the diameters
of inhibition zones using agar diffusion assay method (Ennahar,
Sashihara, Sonomoto, & Ishizaki, 2000) with Staphylococcus aureus
ATCC 63589 and Escherichia coli ATCC 25922 as indicator strains.
Briey, 100 ll of cell-free supernatants were placed into wells
(7.80 mm in diameter) on MRS agar plates seeded with the above
indicator strains. After incubation at 30 C for 18 h, the diameters
of inhibitory zones were determined.
The pH value of the fermented supernatants was adjusted to 6.0
with NaOH (4 mol l1) to eliminate the effect of low pH value and
the pH of MRS broth was also adjusted to the same value using
either lactic acid or acetic acid as control, respectively. After adding
beef liver catalase (50 U ml1, Sigma), the cell-free supernatants
(pH 6.0) were incubated at 37 C for 12 h to eliminate the effect
of hydrogen peroxide and the same supernatants without catalase
were used as control (Gurban Oglu Gulahmadov et al., 2006).
Diameters of inhibition zones were measured again for nal
selection.
To determine the possible protein nature of the detected antimicrobial substances, after eliminating hydrogen peroxide and
low pH effects, the bacterial cell-free supernatants (pH 6.0) were,
respectively, incubated at 37 C overnight with pepsin (Sigma),
trypsin (Sigma) or protease K (Sigma) at a nal concentration of
3 mg ml1 individually and those without enzyme treatment were
used as control. Diameters of inhibition zones were recorded using
S. aureus ATCC 63589 as indicator (Ennahar et al., 2000).

77

nology (Dalian, China) Co. Ltd. At rst, PCR was carried out using a
TaKaRa 16S rDNA Bacterial Identication PCR Kit (Code No. D310).
The PCR primers are as follows: forward primer: 50 -AGAGTTTGATCCTGGCTCAG-30 , reverse primer: 50 -GTGTGACGGGCGGTGTGTAC-30 (Takara Bio., Dalian, China). PCR conditions included
denaturing at 94 C for 5 min followed by 30 cycles of denaturing
at 94 C for 1 min, annealing at 5055 C for 1 min, polymerizing
at 72 C for 1.5 min and at last polymerizing at 72 C for 5 min.
The PCR products were puried with TaKaRa Agarose Gel DNA
Purication Kit Ver. 2.0 (Code No. DV805A) and sequenced by
Takara Bio. (Dalian, China). Sequence homologies were examined
by comparing the obtained sequences with those in the DNA Databases (http://www.ncbi.nlm.nih.gov/BLAST) (Chen, Yanagida, &
Shinohara, 2005). A bootstrap consensus tree (1000 copies) was
obtained by multiple sequence alignment with the Neighbor-Joining method using Mega software, version 4.2 (Kumar, Tamura, &
Nei, 2004; Thompson, Higgins, & Gibson, 1994).
2.4. Purication of sakacin C2
One liter of MRS broth was inoculated with 1.5% (v/v) overnight
seed culture of isolated strain C2 and statically cultivated at 30 C
for 24 h. After removing the cells by centrifugation (5000g, 15 min,
4 C), the cell-free supernatant adjusted to pH 6.0 with 4 mol l1
NaOH was concentrated to one-tenth of the original volume in a
rotary vacuum evaporator (RE-3000, Yarong Biochemical Instrument, China) and cooled down to below 4 C in 4-fold volume cold
ethanol. Precipitation was removed by centrifugation (10,000g,
20 min, 4 C) and the supernatant was subjected to rotary vacuum
evaporation to remove ethanol and concentrated to one-fth of the
volume before precipitation.
After a Sephadex G50 column (1.0 cm  160 cm) chromatography system was equilibrated with deionized water, the sample
was eluted at a ow rate of 0.5 ml min1 for 8 h. The fractions were
collected and detected for the bacteriocin activity using S. aureus
ATCC 63589 as indicator. Briey, 2-fold serial dilutions of sakacin
C2 preparations above were prepared in sterile PBS buffer (pH
6.0). One hundred microliters of the diluted samples were transferred into wells on MRS agar plates seeded with S. aureus ATCC
63589 indicator. After incubation at 30 C for 18 h, bacteriocin
activity (AU ml1) of sakacin C2 preparations was detected using
the described denition (Xiraphi et al., 2006). The fraction with
most antibacterial activity was scanned using an ultraviolet-visible
all-wave-length scanner (UV-2550, Shimadzu, Japan) and the
wave-length displaying the highest absorbance was recorded.
The absorbance of each portion was measured at this wave-length.
The bacteriocin-containing fractions were pooled together and
concentrated by vacuum lyophilization.
Folin phenol method was applied to determine protein concentrations of various purication preparations of sakacin C2 and bovine serum albumin (Sigma) was used as standard (Lowry,
Rosebrough, Farr, & Randall, 1951).
2.5. Determination of molecular weight by Tris-Tricine SDSPAGE
analysis

2.3. Strain identication

In this work, one perspective strain C2 from traditional Chinese
fermented cabbage was rst determined by phenotypical and
physiological tests including Gram staining, cell morphology,
growth ability, respectively, at 10 C, 15 C and 45 C, also in 3%,
6% and 9% NaCl as well as carbohydrate fermentation patterns
(Kandler & Weiss, 1986).
Then strain C2 was nally identied by 16S rDNA sequence
analysis. All the PCR processes were carried out by TaKaRa Biotech-

The molecular weight of sakacin C2 preparations were determined by the Tris-Tricine SDSPAGE with 5% stacking gel and
16% separating gel (Schagger & Von Jagow, 1987). The puried sakacin C2 preparations along with low molecular weight markers
(Sigma) were run together in one gel at 200 V for 7 h. Half of the
gel was stained with Coomassie brilliant blue R-250 for molecular
weight determination, meanwhile another half gel for antimicrobial activity assay was washed in sterile water, overlaid with nutrient soft agar seeded with 1% (v/v) S. aureus ATCC 63589 and

78

Y. Gao et al. / Food Control 21 (2010) 7681

incubated at 30 C for 18 h (Yanagida, Chen, Onda, & Shinohara,

2005).

Table 1
The antimicrobial activity of the primarily selected strains after eliminating organic
acid effect.

2.6. Antimicrobial spectrum of sakacin C2

Strain number

Antimicrobial activity
against
Staphylococcus
aureus (mm)a

Antimicrobial activity
against Escherichia
coli
(mm)a

The control of lactic acid (pH 6.0)

The control of acetic acid (pH 6.0)
G14
S16
S7
C2
C10
C24
C27
C33
C36
C39

11.28
10.60
13.14
15.10
15.22
11.62
15.80
11.20

12.34

Partially puried sakacin C2 preparations from cold ethanol

precipitation were used to determine the antimicrobial spectrum
against indicator organisms (Table 3). The indicatory LAB were
statically incubated in MRS broth at 30 C for 24 h. All the other
indicatory bacteria were cultivated overnight in nutrient broth at
37 C with gentle agitation for 12 h. Saccharomyces cerevisiae ACCC
20036 was grown in yeast extract peptone dextrose (YEPD) broth
(120 r min1, 30 C, 18 h) and Aspergillus niger ACCC 30005 in wort
broth (120 r min1, 30 C, 24 h). Diameters of inhibition zones
were also measured by agar diffusion assay method (Ennahar
et al., 2000).
2.7. Effects of enzymes, temperature and pH on activity of sakacin C2
The partially puried sakacin C2 preparations from cold ethanol
precipitation were, respectively, treated with a-amylase
(1 mg ml1), b-amylase (1 mg ml1), lipase (2 mg ml1), trypsin
(3 mg ml1), protease K (3 mg ml1) and pepsin (3 mg ml1) and
incubated at 37 C for 12 h. In another test, partially puried sakacin C2 preparations were incubated, respectively, in either thermostatic water bath at 80, 90 and 100 C for 30 and 60 min or in an
autoclave at 121 C for 15 min. By adjusting the pH value in a range
from 2.0 to 11.0 and keeping for 12 h, the effect of pH was tested.
For all the experiments here, S. aureus ATCC 63589 was used as
indicator and controls were maintained without any treatment
(Foulqui Moreno, Callewaert, Devreese, Van Beeumen, & De Vuyst,
2003). Bacteriocin activity (AU ml1) of all samples was assayed as
described (Xiraphi et al., 2006).

a
Values were mean of three independent experiments carried out in duplicate; ,
not detected; mm, the diameter of inhibition zone including that of Oxford cup
(7.80 mm).

cell-free supernatants of 10 strains with distinct antimicrobial

activity against both S. aureus ATCC 63589 and E. coli ATCC
25922 indicators were obtained. After eliminating low pH effect,
only cell-free supernatant of strain C2 still exhibited distinct inhibitory activity against both indicators (Table 1). Further, after catalase treatment, its antimicrobial activity was still the same as the
control (data not shown).
After treatment by all the three kinds of protease, the antimicrobial activity of cell-free supernatant of strain C2 disappeared.
It indicated that the substance with strong antimicrobial activity
was sensitive to protease. Therefore, the protein nature of this antimicrobial substance produced by strains C2 was veried.

3. Results and discussion

3.2. Strain identication

3.1. Screening of LAB producing bacteriocin with a broad inhibitory

spectrum from traditional Chinese fermented cabbage samples

Cells of strain C2 were Gram-positive, like coccus-rod shaped

and without spore formation. It could grow at 10 C and 15 C
but not at 45 C, as well as in 3% and 6% but not in 9% NaCl. Strain
C2 could produce CO2 from glucose but not ammonia from arginine. Moreover, it could metabolize L-arabinose, glucose, lactose,

A total of 300 acid-producing bacteria isolated from traditional

Chinese fermented cabbage were cultivated in MRS broth and the

Fig. 1. Phylogenetic trees drived from 16S rDNA sequence of strain C2. All the sequences used here were from LAB type strains. Numbers at the nodes indicated the bootstrap
values on neighbor-joining analyses of 1000 resampled data sets. Bar 0.2 represent sequence divergence.

79

Y. Gao et al. / Food Control 21 (2010) 7681

Table 2
Purication and activity of sakacin C2.
Purication step

Volume
(ml)

Bacteriocin activity
(AU ml1)a

Protein concentration
(mg ml1)a

Specic activity
(AU mg1)a

Yield
(%)a

Purication (fold)a

Crude
Ethanol extract
Column
chromatography

100
10
3

105
843
2280

11.7
26.8
19.2

8.90
31.45
118.75

100.0
80.2
65.3

1.00
3.53
13.34

Values were mean of three independent experiments carried out in duplicate.

successively isolated from fermented vegetables (Atrih et al.,

1993; Delgado et al., 2005; Franz et al., 1996; Jimnez-Daz et al.,
1993; Li et al., 2008). However, to our knowledge, this is the rst
report on a bacteriocin-producing strain of L. sake isolated from
fermented vegetables.

45 kDa
35 kDa
27 kDa
20 kDa

3.3. Purication of sakacin C2

14.4 kDa
9.5 kDa
6.5 kDa
4.1 kDa

Fig. 2. Tris-Tricine SDSPAGE of sakacin C2. Lane 1: low molecular weight markers;
lane 2, 3: sakacin C2; Lane 4: gel overlaid with nutrient broth soft agar surface
seeded with 1% (v/v) Staphylococcus aureus ATCC 63589.

melibiose, cellobiose, saccharose, gluconate, maltose, D-mannose,

ribose, dulcitol, salicin and D-fructose, but not D-xylose, D-turanose,
rafnose, rhamnose and sorbitol.
For 16S rDNA molecular identication of strain C2, the nucleotide sequence of a 1495-bp fragment was amplied from its genome DNA. Phylogenetic tree was drawn from sequence alignment
and comparison (Fig. 1). It revealed that the 16S rDNA nucleotide
sequence of strain C2 was of 99% similarity with that of L. sakei
DSM 20017 (AM113784). Thus strain C2 was identied as L. sake
and the GenBank access number is EU586177.
Fermented vegetables are good sources of LAB. Many bacteriocin-producing LAB such as L. pentosus B96, L. plantarum C19, E. faecium BFE 900, L. plantarum LPCO10 and L. brevis P-319 were

The sample by cold ethanol precipitation was applied to a

Sephadex G50 column chromatography system and the fractions
with distinct antibacterial activity were mainly from No. 50 to
No. 58. Among these fractions, the highest activity fraction (No.
54) was diluted to 50-fold and applied to all-wave-length scanning.
The highest absorbance was found at 210 nm.
For various purication techniques, the yield, activity and purication fold of sakacin C2 obtained were summarized in Table 2.
The column chromatography technique provided a fairly pure
preparation (13.34-fold), while 80% cold ethanol precipitation gave
comparatively low purication (3.53-fold) (Table 2).
3.4. Molecular weight determination of sakacin C2 by Tris-Tricine
SDSPAGE
Determined by relative mobility, the molecular weight of sakacin C2 was approximately 5.5 kDa (Fig. 2). After Tris-Tricine SDS
PAGE, the single band of sakacin C2 was still active against the
indicator strain S. aureus ATCC 63589 (Fig. 2, lane 4). Therefore,
that the 5.5 kDa band was the antimicrobial peptide (sakacin C2)
was veried.
From meat products and malted barley, several bacteriocin-producing L. sake strains have been isolated since 1992. These bacteriocins are as follows: sakacin A (4.3 kDa) from L. sake Lb706 (Holck,
Axelsson, Birkeland, Aukrust, & Blom, 1992), sakacin G (3.8 kDa)

Table 3
Antimicrobial spectrum of sakacin C2.
Indicator strains
Lactobacillus plantarum B1
Lactobacillus plantarum B2
Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus delbrueckii subsp. delbrueckii
Streptococcus thermophilus
Lactobacillus acidophilus
Bacillus subtilis
Staphylococcus aureus
Sarcina ava
Listeria innocua
Bacillus cereus
Escherichia coli
Salmonella typhimurium
Shigella exneri
Saccharomyces cerevisiae
Aspergillus niger

Sourcea
Fermentation cabbage
Fermentation cabbage
ACCC 11057
ACCC 11046
CICC 06038
ATCC 4356
ACCC 11060
ATCC 63589
CMCC 29001
ATCC 10297
CMCC 63301
ATCC 25922
CMCC 47729
CMCC 51606
ACCC 20036
ACCC 30005

G+/G
+

G
G+
G+
G+
G+
G+
G+
G+
G+
G+
G+
G
G
G

Diameter of inhibition zoneb

++
++
++
+++
++
+++

+++
+++
+++
+
++
++
++

a
ATCC, American type culture collection; ACCC, agricultural culture collection of China; CMCC, China center of medicine culture collection; CICC, China center of industrial
culture collection.
b
+, Diameter of inhibition zone: 8.0012.00 mm; ++, 12.0016.00 mm; +++, more than 16.00 mm; , no inhibition zone.

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Y. Gao et al. / Food Control 21 (2010) 7681

from L. sake 2512 (Simon, Fremaux, Cenatiempo, & Berjeaud, 2002),

sakacin M (4.6 kDa) from L. sake 148 (Sobrino et al., 1992), sakacin
P (4.4 kDa) from L. sake LTH673 (Tichaczek, Nissen-Meyer, Nes, Vogel, & Hammes, 1992) as well as sakacin P, sakacin 5X (4.3 kDa)
and sakacin 5T (4.1 kDa) produced by L. sakei 5 (Vaughan, Eijsink,
OSullivan, OHanlon, & Van Sinderen, 2001). The measured molecular mass of sakacin C2 (5.5 kDa) suggested that it may be a novel
bacteriocin produced by L. sake, since the molecular masses of all
the bacteriocins produced by other L. sakei species are in the range
of 3.84.6 kDa.
3.5. Antimicrobial spectrum of sakacin C2
Sakacin C2 exhibited a broad antimicrobial activity (Table 3). It
could signicantly inhibit Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus acidophilus, Sarcina ava, Listeria innocua
and S. aureus, moderately against L. plantarum B1, L. plantarum
B2, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus,
E. coli, Salmonella typhimurium and Shigella exneri, less against
Bacillus cereus but not against Bacillus subtilis, S. cerevisiae and A.
niger.
From this result, we can see that sakacin C2 has inhibitory activity against not only a large range of LAB but also many Gram-positive and Gram-negative bacteria (Table 3). This activity against
many Gram-negative bacteria was not frequently seen in bacteriocins from LAB. The inhibitory spectra of the known bacteriocins
from L. sakei were quite narrow. For example, the inhibition spectrum of sakacin G appeared to be limited to L. sakei and Pediococcus
cerevisiae strains tested, as far as LAB are concerned (Simon et al.,
2002). Among three kinds of bacteriocins (sakacins P, 5X and 5T)
by L. sakei 5, only sakacin 5X inhibited many Gram-positive beer
spoilage organisms, such as L. brevis, Enterococcus facealis, etc.,
but their inhibitory activity to Gram-negative bacteria has not
been found yet, while sakacin P and sakacin 5T only could inhibit
two out of thirteen LAB strains (Vaughan et al., 2001). Sakacin A
showed a narrow inhibitory spectrum only to some other lactobacilli (Schillinger & Lcke, 1989), and sakacin M only inhibited some
Gram-positive bacteria (Sobrino et al., 1992). From all reports on
the antimicrobial characteristics of the known bacteriocins by L.
sakei strains, we only can see their antimicrobial activity on a
few Gram-positive bacteria, but not on Gram-negative bacteria.
Table 4
Effects of enzymes, temperature and pH on the bacteriocin activity of sakacin C2.
Residual bacteriocin activity (%)a

Treatment
Enzymes

Heat (C)

pH

a-Amylase
b-Amylase
Lipase
Trypsin; pepsin; protease K

100.0
100.0
100.0
0.0

80 (30 min)
80 (60 min)
90 (30 min)
90 (60 min)
100 (30 min)
100 (60 min)
121 (15 min)

100.0
100.0
96.3
91.5
88.2
84.1
80.9

2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0

56.5
80.9
84.7
86.0
100.0
91.9
82.1
75.3
74.7
50.2

a
Values were mean of three independent experiments carried out in duplicate.
The bacteriocin activity of the untreated sample was 320 AU ml1.

3.6. Effects of enzymes, temperature and pH on sakacin C2

As shown in Table 4, after treatment by proteolytic enzymes,
the antibacterial activity of sakacin C2 was abolished, while amylase and lipase had no effect. This result showed that sakacin C2
was a kind of peptide not containing lipid or carbohydrate groups.
Sakacin C2 was heat-stable even after autoclaved at 121 C for
15 min. Also, its bacteriocin activity remained stable until incubation for 12 h at pH values between 3.0 and 8.0. So, sakacin C2
showed both strong heat stability and pH-stability, further, this
property was the same to some other Class II bacteriocins (Luchansky, 1999). This may offer an essential promise for its application in
the processing and conservation of various foods.
4. Conclusions
From 300 LAB strains, strain C2 which produced a bacteriocin
strongly inhibits S. aureus ATCC 63589 and E. coli ATCC 25922
was isolated from traditional Chinese fermented cabbage. This
strain was identied as L. sake by phenotypical and physiological
tests and 16S rDNA identication. This is the rst bacteriocin-producing strain of L. sake isolated from fermented vegetables.
After purication by cold ethanol and Sephadex G50 column
chromatography, the molecular weight of sakacin C2 was 5.5 kDa
by Tris-Tricine SDSPAGE. Sakacin C2 exhibited a wide range of
antimicrobial activity against not only a large range of LAB but also
many food borne spoilage and pathogenic Gram-positive and
Gram-negative bacteria. And it has strong heat stability and pH
stability. Sakacin C2 was sensitive to protease but insensitive to lipase, a-amylase and b-amylase. The molecular mass, heat stability
and antimicrobial spectrum of sakacin C2 suggested that it was a
novel broad-spectrum Class II bacteriocin produced by L. sake.
To date, L. sakei species have been generally used as starter in
fermented sausage, and is regarded as safe (RAS) strains used in
the food industry (Hammes, Bantleon, & Min, 1990; Ross et al.,
2002). L. sake C2 can produce a large amount of lactic acid and
the pH value of its fermented MRS broth can be below 4.0 (data
not shown), thus it might be applied to many fermented foods as
producing starter.
These studies might hopefully lay the groundwork for future
application of sakacin C2 and its producer, and broaden the application range of L. sake as producing starter and bio-preservative in
the food industry.
The further work is to determine the DNA and amino acid sequences and action modes of sakacin C2, and to research the effects
of L. sake C2 on food quality and the production of this novel sakacin C2 in various fermentation conditions.
Acknowledgement
This research was funded by China National 863 Program
(2006AA10Z347).
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