Food Control 31 (2013) 499e507

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Food Control
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Molecular cloning and antimicrobial activity of enterolysin A and helveticin J

of bacteriolysins from metagenome of Chinese traditional fermented foods
Tiantian Zhang a, Yingjie Pan a, Bailin Li a, Jie Ou a, Jianmin Zhang a, Yourong Chen a, Xu Peng b,
Lanming Chen a, *
a

Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), China Ministry of Agriculture,
College of Food Science and Technology, Shanghai Ocean University, 999 Hu Cheng Huan Road, Shanghai 201306, PR China
Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, 2200 N Copenhagen, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 August 2012
Received in revised form
21 October 2012
Accepted 3 November 2012

Expansion of food industry and international trade in foodstuffs, together with increasing worldwide
attention to food safety heightened the need of exploring natural preservatives such as bacteriocins to
prevent food contamination and spoilage. In this study, we uncovered a signicant diversity of enterolysin A and helveticin J gene sequences belonging to bacteriolysins in popular Chinese traditional fermented foods using culture-independent metagenomic approach. The CODEHOPs targeting conserved
motifs of the enterolysin A and helveticin J genes from Lactobacillales were designed to amplify
respective gene sequences from six metagenomic DNA extracts by PCR. The amplicons were evaluated by
sequence clone libraries and phylogenetic analyses. All translated enterolysin A sequences (77e80 amino
acids) derived from four libraries were grouped into one cluster with 84e96% identity to homologs from
Lactobacillus in GenBank database. Deduced helveticin J sequences (169e196 amino acids) derived from
ve libraries were classied into two distinct clusters, which shared 58e100% identity with public
homologs in the database. Based on the obtained sequences, one of intact enlA and hlvJ genes was
amplied by genome walking TAIL-PCR, and cloned into the expression vector pET-28a in Escherichia coli
BL21, respectively. The recombinant EnlA (DFE5) producing a 30.6-kDa protein displayed antimicrobial
activity against three bacterial genera, including human pathogens Listeria monocytogenes and Staphylococcus aureus. The expressed HlvJ (RS9) encoding a 249-aa protein was observed to inhibit Lactobacillus
helveticus. This study demonstrated the validity of the metagenomic approach used in this study for
identifying natural variants directly from complex food samples, but also provided the evidence that
Chinese traditional fermented foods could be a valuable bacteriocin gene source for further exploration
in food industry.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Bacteriocins
Diversity
Antimicrobial activity
Metagenome
Fermented foods
Food safety

1. Introduction
There are many kinds of fermented foods made of traditional
manufacturing procedures for hundreds or thousands of years in
China, such as milk cake (known as Rubing in China), milk slice
(known as Rushan in China), Qula cheese, stinky tofu, and sweet
Chinese rice wine (known as Jiuniang, or Jiang Mijiu in China).
Among them, Rubing and Rushan are special kinds of cheeses made
from fresh goats or cows milk mainly by ethnic minorities living in
Yunnan Province of the Southwest China, while Qula cheese is
made from yaks milk by the people living in the Tibetan plateau

* Corresponding author. Tel.: 86 0 21 61900504; fax: 86 0 21 61900365.

E-mail address: lmchen@shou.edu.cn (L. Chen).
0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2012.11.015

with an average altitude of over 4000 m in China (Duan et al.,

2008). Traditionally, raw goats milk is boiled, cooled, and then
mixed with a fermentation starter which is usually made from
a sort of pickled local vine (known as Naiteng in China). The
resulting acidic water is ltered through a gauze dressing, and
curds are pressed to form the white soft goats milk cake (known as
Yang Rubing in China). It is a traditional method to store goats milk
mainly by people of the ethnic Bais living in mountainous region.
Milk slice is a type of rm cheese made from fresh cows milk. Via
air-drying method, it can be stored for several months and exported
to the Southeast Asia. As a well known and popular traditional
fermented Chinese snack, stinky tofu is a kind of fermented tofu,
which is made from soybeans and has been one of the main protein
sources in the East since ancient times (Chao, Tomii, Watanabe, &
Tsai, 2008). The history of making sweet Chinese rice wine can

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T. Zhang et al. / Food Control 31 (2013) 499e507

also be dated back to the Shang Dynasty (1600BCe1046BC) in

China. It is produced from steamed glutinous rice with a starter
(known as Qu in China). After fermentation, the unltered Sweet
Chinese rice wine tastes sweet, triggered with a wine aroma, and
has been consumed throughout East and Southeast Asia.
Lactic acid bacteria (LAB) play an essential role in food
fermentation. One of the most important contributions of these
microorganisms is to extend shelf life of fermented products (Xie
et al., 2011). Growth of spoilage and pathogenic bacteria in these
foods is inhibited mainly due to the presence of starter-derived
inhibitors such as lactic acid, hydrogen peroxide and bacteriocins
(Nishie, Nagao, & Sonomoto, 2012; Vandenbergh, 1993). Bacteriocins are ribosomally synthesized proteinaceous compounds, which
exhibit broad or narrow spectrum of bactericidal or bacteriostatic
activities primarily against other bacteria sharing the same
ecological niche (Diep & Nes, 2002). For example, bacteriocin nisin,
discovered from LAB in 1933 and rst marketed in England in 1953,
has been approved by the Joint Food and Agriculture Organization/
World Heath Organization (WHO) Expert Committee, the European
Union (EU), and the US Food and Drug Agency (FDA) for use as food
additives in over 48 countries (Cotter, Hill, & Paul, 2005). The major
classes of bacteriocins produced by LAB include: lanthioninecontaining lantibiotics (class I), such as nisin; the nonlanthionine-containing bacteriocins (class II); and the large, heatlabile murein hydrolases (formerly class III bacteriocins), designated as bacteriolysins (Cotter et al., 2005). To our knowledge, the
bacteriolysins were the least investigated bacteriocins, with only
ve members have been characterized genetically to date. They
were Millericin, Enterolysin A (EnlA), Zoocin A, Lysostaphin and
Helveticin J (HlvJ), produced by Streptococcus milleri (Beukes,
Bierbaum, Sahl, & Hastings, 2000), Enterococcus faecalis (Nilsen,
Nes, & Holo, 2003), Streptococcus zooepidemicus 4881 (Simmonds,
Simpson, & Tagg, 1997), Staphylococcus aureus (Kumar, 2008), and
Lactobacillus helveticus (Joerger & Klaenhammer, 1990), respectively, all of which belong to Lactobacillales. Some kinds of fermented foods have been assessed to possess a very diverse and rich
microora (Gonzlez et al., 2007). For example, a wide variety of
LAB strains (4.2  103e1.2  106 CFU/ml) belonging to 7 genera and
32 species were identied in the fermentation of stinky tofu brines
(Chao et al., 2008). Therefore, microbial communities of fermented
foods could serve as a gene pool coding for bacteriocins, the
potential of which, however, has largely been untapped. In this
study, we employed a culture-independent metagenomic approach
to investigate the bacteriolysins in six kinds of popular traditional
fermented foods in China. Our results provided the rst evidence of
a signicant diversity of enterolysin A and helveticin J gene
sequences in the microbial communities of traditional fermented
foods. In addition, based on the sequences obtained from metagenomic DNA extracts, one of enlA and hlvJ genes was cloned and
expressed in Escherichia coli, respectively, and their biological
activities were characterized.
2. Materials and methods
2.1. Bacteria strains, plasmids and growth media
E. coli TOP10 [genetype: F mcrA D(mrr-hsdRMS-mcrBC) F80
lacZ DM15 DlacX74 recA1 araD139 D(ara-leu)7697 galU galK
rpsL(Strr) endA1 nupG] (TianGen Biotech Co. Ltd. Beijing, China) was
used as a host strain for transforming recombinant vectors in the

cloning. E. coli BL21(DE3) [genotype: F ompT hsd SB(r
B mB ) gal
dcm(DE3) ] (Tiangen) was employed as a host for expression of
recombinant proteins. The plasmid pGM-T (TianGen) and pET-28a
(Merck Millipore, Darmstadt, Germany) was used as TA cloning
and gene expression vectors, respectively. Lactobacillus strains were

routinely grown in MRS medium (Difco), and Listeria monocytogenes in BHI medium (Oxoid). E. coli and other bacteria tested in
this study were grown in LuriaeBertani (LB) medium (Sambrook &
Russell, 2001), while recombinant E. coli strains were grown in LB
medium in both liquid broth and agar plates supplemented with
ampicillin (100 mg/ml) or kanamycin (30 mg/ml).
2.2. Isolation of metagenomic DNA from traditional fermented
foods
Samples of goats milk cake, cows milk cake (known as Niu
Rubing in China, made from cows milk) and milk slice were collected
from Dali, Yunnan Province in China in May 2011, while Qula cheese
was from Lasa, Tibet in September 2011, stinky tofu and sweet
Chinese rice wine were from Shanghai, and Chengdu, Sichuan
Province in April and May 2011, respectively. Each sample (20 g) was
homogenized in 4 volumes of sterile Milli-Q water (Millipore Billerica, Massachusetts, USA) by using lab blender BagMixer (Interscience, Paris, France). The instant ltration in BagFilter was
centrifuged at 380 g (CT14RD, Techcomp, Shanghai, China) for 2 min
to remove impurities and food debris. Microbial cells in supernatant
were collected by centrifugation at 5000 g for 3 min, and cell pellet
was washed twice with Milli-Q water for metagenomic DNA isolation. According to the manufactures instructions, metagenomic
DNA was prepared using Biospin bacteria genomic DNA extraction
Kit (BIOER Technology, Hangzhou, China). DNA concentration was
determined using a SmartSpec plus SOP (Bio-RAD, California, USA).
2.3. Designing of degenerate primers for PCR
Based on public enterolysin A and helveticin J amino acid
sequences in the National Center for Biotechnology Information
(NCBI) sequence database (http://www.ncbi.nlm.nih.gov/Entrez/),
multiple sequence alignments were performed using the ClustalW2
software
(http://www.ebi.ac.uk/Tools/msa/clustalw2/)
(Larkin et al., 2007). The resulting conserved blocks of amino acid
residues in ClustalW2 format were used as input to load into the
program iCODEHOP (https://icodehop.cphi.washington.edu/icodehop-context/Welcome) (Boyce, Chilana, & Rose, 2009), by
which a set of CODEHOPs were designed. A CODEHOP is a hybrid
primer consisting of a 30 -degenerate core and 50 -non-degenerate
clamp region (Rose, Henikoff, & Henikoff, 2003). The sequences of
the CODEHOPs which successfully generated PCR products in this
study were listed in Table 1. Oligonucleotide primers were
synthesized by Shanghai Sangon Biological Engineering Technology
and Services Co., Ltd. (Shanghai, China).
2.4. PCR amplication and cloning of enlA and hlvJ gene fragments
PCR amplication was performed in a 20 ml reaction volume
containing 1  Premix Ex Taq version 2.0 (Japan TaKaRa Biotechnology (Dalian) Co. Ltd.), 5 mM each of the oligonucleotide primers,
and 5e10 ng of template DNA. The temperature gradient PCR was
carried out to amplify enterolysin A gene sequences under the
following conditions: initial denaturation of 95  C for 5 min was
followed by 30 cycles consisting of denaturation at 94  C for 30 s,
primer annealing at a temperature gradient from 45.3 to 65.1  C for
30 s, and elongation at 72  C for 30 s, followed by nal elongation at
72  C for 5 min. The same method was used to amplify helveticin J
sequences at the temperature gradient from 40.4 to 60.1  C and
elongation time at 72  C for 40 s. All amplications were performed
in a Peter Thermal Cycler (Eppendorf AG22331, Hamburg, Germany).
The desired PCR products were puried and recovered from
agarose gels using the Axygen gel extraction clean-up kit (Axygen,
California, USA). The puried PCR fragments were ligated into the

T. Zhang et al. / Food Control 31 (2013) 499e507

Table 1
Oligonucleotide primers used in this study.
Primers

Sequences (50 -30 )

Targeted regions
or genes

HJC-1F
HJJ-9R
EF3-F
EG5-R
DFE5-5F-SP3
DFE5-5FSP2
DFE5-5FSP1
DFE5-5SSP3
DFE5-5SSP2
DFE5-5SSP1
DFE5-3-SP3
DFE5-3-SP2
DFE5-3-SP1
DFE5-F
DFE5-R
DFE5-M-F
DFE5-M-R
RS9-5-SP3
RS9-5-SP2
RS9-5-SP1
RS9-3-SP3
RS9-3-SP2
RS9-3-SP1
RS9-F
RS9-R
RS9-M-F
RS9-M-R

TTCGGCCACACncaracntggg
GGTCTCGCCCcanggdawytt
GCCTGACAATTATGTCGAArtntaycarga
CTTGTATGATTTTCATCGGGttnarccangt
GGATAGCCCCAGATGTCCTTTT
GGCGAAACCTTCCTGGTAGACT
TGACCGTGCTTGCTGATGTAGT
GGCGAAACCTTCCTGGTAGACT
GGTCTTGGTGATCCCCAGGTGC
GATTTTCATCGGGTTCAGCCAC
CGTGGCTGAACCCGATGAAAAT
GCAAGCACGGTCACCCTTACAA
TCAAGCTGGGCAATAAGATCGG
ATGAGCAAGAATAAGAAAAATAAT
CTATTTGATAAATGACTTCAAGTAG
CCGGAATTCaATGAGCAAGAATAAG
CCGCTCGAGbTTTGATAAATGACTT
AGCATATTCCCAAGTCTGAGTGTG
GTACTCTAGCAATTTGGGTAGTCCAC
CGTTTCAAGTCAGCACCAGCAT
GAAGCTGCTGTTTCTCCAGACTATC
GCTGGTTCTCAACAAGGCATCA
GTGGACTACCCAAATTGCTAGAGTAC
ATGGATGGTCAAGGTAGC
TTACCAACTAATCTTATAAATTCTA
CCGGAATTCaATGGATGGTCA
CCGCTCGAGbCCAACTAATCTT

Internal region
of hlvJ
Internal region
of enlA
50 -terminal region
of enlA (DFE5)

a
b

50 -terminal region
of enlA (DFE5)
30 -terminal region
of enlA (DFE5)
enlA (DFE5)
enlA (DFE5)
50 -terminal
region of hlvJ (RS9)
30 -terminal region
of hlvJ (RS9)
hlvJ (RS9)
hlvJ (RS9)

EcoRI restriction site.

XhoI restriction site.

pGM-T vector. According to the manufactures instructions, ligation

was performed in a 20 ml reaction volume with a molar ratio of
vector: insert of 1:3, and incubated at 16  C overnight. After incubation, the ligation DNA was transformed into E. coli TOP10
competent cells by heat-shock method (Sambrook & Russell, 2001).
Transformation culture was spread on LB-ampicillin agar plates,
and then incubated at 37  C overnight.
2.5. Sequencing and phylogenetic analyses
About 25e30 positive white colonies from each library were
randomly picked for plasmid preparation using MiniBEST plasmid
purication kit version 2.0 (TaKaRa). Automated DNA sequencing
was carried out using ABI 3730XL capillary sequencer (Applied
Biosystems, California, USA) and BigDye terminator version 3.1
cycle sequencing kit (PerkineElmer, Massachusetts, USA) at the
China Human Genome Center (Shanghai, China). The intact enterolysin A and helveticin J gene sequences obtained in this study have
been deposited in the GenBank sequence database under the
accession no. JX944507eJX944508.
Sequencing analysis was performed using the DNASTAR software (version 7.1, http://www dnastar.com). Putative functions
were inferred using the Basic Local Alignment Search Tool (BLAST)
(http://www.ncbi.nlm.nih.gov/BLAST) against the NCBI database.
The neighbor-joining method in the molecular evolutionary genetic
analysis software package MEGA (version 4.0) (Tamura, Dudley,
Nei, & Kumar, 2007) was used to construct a phylogenetic tree,
based on multiple sequence alignments produced by the ClustalW2. A bootstrap analysis with 1000 replicates was carried out to
check the reliability of the tree.
2.6. Cloning of intact enlA and hlvJ genes by genome walking
TAIL-PCR
Several enlA and hlvJ sequences with different similarities were
chosen for further analysis, of which the intact enlA (DFE5) and hlvJ

501

(RS9) genes that have already been cloned and expressed were
presented in this study. Their full length genes were amplied
using genome walking thermal asymmetric interlaced PCR (TAILPCR) approach with the genome walking kit (TaKaRa) as described
by the manufacturer. Based on the enlA (DFE5) sequence obtained
in this study, two sets of specic primers, DFE5-5F-SP3, DFE5-5FSP2 and DFE5-5F-SP1; DFE5-5S-SP3, DFE5-5S-SP2 and DFE5-5SSP1 (Table 1), were designed using the software Primer 5.0 (http://
www.PremierBiosoft.com) to obtain the 50 -terminal region of the
gene for the rst and second round genome walking, respectively.
Similarly, the specic primers DFE5-3-SP3, DFE5-3-SP2 and DFE53-SP1 (Table 1) were designed to obtain the 30 -terminal sequence of
the enlA (DFE5). The TAIL-PCR was performed in a 50 ml reaction
volume containing 1  LA PCR Buffer II (TaKaRa), 2.5 U TaKaRa LA
Taq, 8 ml of 2.5 mM each of dNTP, 1 ml of AP Primer (100 pmol/ml)
(TaKaRa), 1 ml of specic primer (10 pmol/ml), and 1 ml template
DNA. The TAIL-PCR was performed according to the instructions of
the manufacture. The metagenomic DNA prepared from traditional
fermented stinky tofu samples was used as DNA template in the
rst-round amplication, while the rst and second round amplicons were used in the second and third round reactions with corresponding AP and SP primers, respectively.
Based on the obtained sequences by genome walking, the intact
enlA (DFE5) gene was amplied with the specic primers DFE5-F
and DFE5-R (Table 1) under the conditions described above,
except annealing at 50  C for 30 s, and elongation at 72  C for 1 min.
The amplicon was puried and cloned into the pGM-T vector as
described above. Recombinant vector, pGM-T-enlA(DFE5), was
veried by sequencing, and used for further analyses in this study.
Similarly, based on the hlvJ (RS9) sequence obtained in this
study, the 50 and 30 -terminal region of the gene was obtained by
genome walking using the specic primer sets of RS9-5-SP3, RS9-5SP2 and RS9-5-SP1; RS9-3-SP3, RS9-3-SP2 and RS9-3-SP1, respectively (Table 1). The intact hlvJ gene was amplied with the primers
RS9-F and RS9-R (Table 1), and cloned into the pGM-T vector, which
yielded the recombinant plasmid pGM-T-hlvJ(RS9).
2.7. Expression of the enlA and hlvJ in E. coli
The intact enlA gene was amplied using the primers DFE5-M-F
and DFE5-M-R (Table 1) designed with restriction endonuclease
digest sites of EcoRI and XhoI at 50 end, respectively. The amplicon
including a putative leader peptide was puried, and digested with

Table 2
Inhibitory spectra of the recombinant EnlA (DFE5) and HlvJ (RS9).
Indicator strains

Sourcea

EnlA (DFE5)

HlvJ (RS9)

Escherichia coli

ATCC43889,
O157:H7
AS CMCC50041
AS1.88
AS1.103
ATCC15009
AS1.551

e
e
e

e
e
e

ATCC19115
ATCC13932
ATCC19116
ATCC33091
CDCAB91093

e
e
e
e
e

Salmonella enteritidis
Bacillus subtilis
Lactobacillus acidophilum
Lactobacillus helveticus
Lactobacillus delbrueckii
subsp. bulgaricus
Listeria monocytogenes
L.isteria monocytogenes
L.isteria monocytogenes
Listeria innocua
Staphylococcus aureus

Inhibition zonesb

a
ATCC, American Type Culture collection; AS, Institute of Microbiology, Chinese
Academy of Science; CDC, Center for Disease Control and Prevention, Shanghai,
China.
b
Inhibition zone (diameter): , >14 mm and over; , 11e14 mm; , 0.5e10
mm; e, no zone.

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T. Zhang et al. / Food Control 31 (2013) 499e507

Fig. 1. Alignments of enterolysin A (A) and helveticin J (B) amino acid sequences from homologs in GeneBank database and representative novel sequences recovered in this study
by PCR amplication of metagenomic DNA extracts from the Chinese traditional fermented foods with newly designed degenerate primers. The numbering of sequences is indicated
above the alignments. The starting and ending residue numbers of each protein sequence used in this study are indicated before and after each sequence. Conserved amino acid
residues are highlighted and shown below each alignment, and those for designing CODEHOPs are boxed with solid lines. The enterolysin A and helveticin J sequences displayed in
the alignments are as following: (A) L. delbrueckii subsp. lactis DSM20072 (EGD28130.1; L.DSM20072), L. acidophilus 30SC (ADZ07395.1; L.30SC), L. amylovorus GRL1112

T. Zhang et al. / Food Control 31 (2013) 499e507

the two enzymes (Promega, Wisconsin, USA) at 37  C for 3 h.

Restriction fragment was then puried, and ligated into the
expression vector pET-28a at the EcoRI and XhoI cloning sites.
Ligation DNA was transformed into E. coli BL21(DE3) competent
cells by heat-shock method as described above. Transformants
grown on LB agar plates supplemented with 30 mg/ml kanamycin
were identied by colony PCR assay. Plasmid DNA of positive
colonies were isolated and digested with EcoRI and XhoI. One
recombinant vector, pET-28a-enlA(DFE5), was used for recombinant protein expression induced by isopropyl-b-D-thiogalactopyranoside (IPTG) as described by Chen, Tian, et al. (2012). Briey,
E. coli BL21 containing pET28a-enlA(DFE5) was inoculated into
50 ml of LB-Kanr medium, and cultured at 37  C to OD600 0.3e0.5.
IPTG was then added to a nal concentration of 0.6 mM, and the
cells were incubated at 20  C for 20 h. Subsequently, the cells were
harvested by centrifugation at 10,000 g for 10 min at 4  C, resuspended in 5-fold of BugBuster protein extraction reagent (Merck
Millipore, Darmstadt, Germany), and incubated at 25  C for 20 min.
The supernatant containing the expressed target protein was isolated by centrifugation at 16,000 g for 20 min at 4  C, and analyzed
by SDS-PAGE electrophoresis with 12% separation gel and 5%
stacking gel according to the standard protocol.
Similarly, the intact hlvJ (RS9) gene was amplied using the
primers RS9-M-F and RS9-M-R (Table 1), and cloned into the pET28a, which yielded the recombinant plasmid pET-28a-hlvJ(RS9).
The expression of hlvJ gene in E. coli BL21 was induced and assayed
as described above.
2.8. Antimicrobial activity assay
The antimicrobial activity of the expressed recombinant proteins
was determined by the agar diffusion method described by Chen,
Yan, et al. (2012). The indicator strains, listed in Table 2, were used
in the assay. Briey, a 200 ml of cell free extract was added into each
Oxford cup on the top layer of agar (0.5%), which was seeded with
overnight culture of an indicator stain, and overlaid on 1.5% agar
prepared with proper medium. The plates were placed at 4  C
overnight, and then incubated at 37  C. After 20e24 h of incubation,
the diameters of the zones of inhibition were measured. Inhibition
was scored positive if the width of the clear zone around the Oxford
cups was 0.5 mm (Millette, Dupont, Archambault, & Lacroix, 2007).
All assays were performed in triplicate and the data was expressed as
a mean. The cell-free extract of E. coli (pET-28a) was used as a control.
3. Results and discussion
3.1. Cloning of enterolysin A and helveticin J gene sequences from
the metagenomic DNA extracts
The enterolysin A amino acid sequences were searched in the
NCBI database, and produced 16 hits. Eleven of these, ranging from
207 to 343 amino acids from six different Lactobacillus and one
Enterococcus species, were used for designing degenerate primers
(Fig. 1, A). Similarly, total 17 helveticin J amino acid sequences were
obtained by database searches, 10 of which, ranging from 325 to

503

350 amino acids from Lactobacillus, were used for designing

primers to amplify helveticin J gene fragments (Fig. 1, B).
A multiple sequence alignment was performed with each set of
known sequences using ClustalW2. Two blocks of highly conserved
motifs Y-Q-E and T-W were identied within the enterolysin A
sequence alignment (Fig. 1, A), while the G-H-T-Q-T-W and P-W-G
were identied within the helveticin J alignment (Fig. 1, B). Based on
the conserved motifs, degenerate primers were designed by the
iCODEHOP. Two primer pairs were selected to amplify helveticin J
sequences from the metagenomic DNA extracts, but only one pair
HJC-1F and HJJ-9R (Table 1) successfully yielded PCR products with
an expected length of 625 bp, while the primer pair EF3-F and EG5-R
(Table 1) was chosen to amplify enterolysin A sequences of 240 bp.
The temperature gradient PCR was performed using the degenerate primers EF3-F and EG5-R with the metagenomic DNA extract
from goats milk cake as template, and the best annealing temperature
was established at 50  C. Single amplicon of around 0.25 kb was obtained from goats milk cake, milk slice, Qula cheese and stinky tofu
samples, respectively. No PCR product was detected from the other
two samples (data not shown). Similarly, with the degenerate primers
HJC-1 and HJJ-9, the best annealing temperature was established at
53  C. A desired amplicon of approximately 0.6 kb was yielded from all
kinds of samples except stinky tofu (data not shown).
Individual amplicon was puried, ligated into the pGM-T vector
and transformed into E. coli TOP10 competent cells, which
produced four enterolysin A sequence clone libraries, designated
GCE, RSE, QCE and DFE representing goats milk cake, milk slice,
Qula cheese and stinky tofu sample, respectively. Similarly, ve
helveticin J libraries were yielded, designated GC, NRB, RS, QC and
RW representing goats milk cake, cows milk cake, milk slice, Qula
cheese and sweet Chinese rice wine sample, respectively. White
colonies from each of the nine libraries were randomly selected for
plasmid preparation and sequencing. More than 95% of recombinant vectors of the nine libraries carried inserts in size similar to
that of their corresponding amplicons as revealed by agarose gel
electrophoresis (data not shown).
3.2. Diversity of enterolysin A sequences derived from the Chinese
traditional fermented foods
Although based on a limited number of enterolysin A sequences
available in the GenBank database, a total of fty seven unique
enterolysin A amino acid sequences of high quality were derived
from the GCE, RSE, QCE and DFE libraries, which showed 84%e96%
identity to the homologs in the GenBank database, particularly to
the enterolysin A from Lactobacillus delbrueckii subsp. lactis
DSM20072 (EGD28130.1). As an important microorganism for
industrial fermentations, it was predominantly found in fermented
milk products and used as starter cultures for yogurt and cheese
production (Moschetti et al., 1997). Based on the translated amino
acid sequences of 77e80 amino acids, a phylogenetic tree was
constructed by the MEGA4.0 (Fig. 2, A), and it revealed that all
sequences derived from the four libraries fell into a single cluster,
designated a, together with the public enterolysin A sequences
used for designing the CODEHOPs, except the E. faecalis
(AF249740_1) from Entercoccus. The sequences in cluster a were

(YP_004032074.1; L.GRL1112), L. helveticus H10 (ADX70076.1; L.H10), L. crispatus ST1(YP_003601667.1; L.ST1), L. crispatus JV-V01 (ZP_03996602.1; L.JV-V01-2), L. acidophilus NCFM
(YP_194074.1; L.NCFM), L. acidophilus ATCC4796 (ZP_04021930.1; L.ATCC4796), L. ultunensis DSM16047 (ZP_04010362.1; L.DSM16047), L. crispatus JV-V01 (ZP_03996619.1; L.JV-V011), E. faecalis (AF249740.1; E. faecalis). (B) L. amylovorus GRL1118 (AEA31374.1; L.GRL1118), L. amylovorus GRL1112 (YP_004031202.1; L.GRL1112), L. acidophilus 30SC (YP_004292739.1;
L.30SC), L. helveticus H10 (ADX69752.1; L.H10), L. helveticus DSM20075 (ZP_05753421.1; L.DSM.2007), L. crispatus ST1 (YP_003602006.1; L.ST1), L. crispatus CTV-05 (ZP_07791463.1;
L.CTV-05), L. acidophilus ATCC4796 (EEJ75201.1; L.ATCC4796), L. acidophilus NCFM (AAV43383.1; L.NCFM), L. gasseri JV-V03 (EFJ70693.1; L.JV-V03). The DFE5 (accession no.
JX944507), DFE20 and DFE48; GCE11 and GCE13; QCE2 and QCE25; RSE5 and RSE21 were representative enterolysin A gene sequences obtained from the four libraries, respectively
in this study, while the GC2 and GC3; RS9 (accession no. JX944508), RS13 and RS28; QC3 and QC24; NRB19 and NRB26; RW3 and RW23 were representative helveticin J gene
sequences recovered from the ve libraries, respectively. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

504

T. Zhang et al. / Food Control 31 (2013) 499e507

A
DFE: stinky tofu
QC: Qula cheese

RS: milk slice

21

17

15

GC: goats milk cake

: public enterolysin A sequences

11
20

41

9
48

27

10

22

99

31

46
39
35

19
15
58

78

21
34
99
82

98
47

69

92

90
99

14
34
62
0
1
3

0
32

21

12

12

13

14

31

15
17
8

17

13

11

17

14

DFE21
DFE48
DFE31
DFE19
DFE1
DFE49
DFE3
DFE16
DFE4
DFE47
DFE6
DFE13
DFE11 subcluster I
DFE5
DFE12
DFE7
DFE41
L.DSM20072
RSE22
DFE8
RSE12
RSE10
QCE2
DFE45
RSE19
RSE18
RSE21
DFE20
DFE24
L.ST1
L.JV-V01-2
GCE10
GCE11
GCE23
L.H10
L.30SC
L.GRL1112
L.DSM16047
L.JV-V01-1
L.NCFM
L.ATCC4796
GCE29
GCE13
RSE5
RSE11
GCE1
GCE32
GCE21
GCE26
GCE15
QCE15
subcluster II
RSE4
RSE6
QCE4
QCE8
GCE4
GCE2
QCE18
GCE3
QCE21
QCE25
RSE28
GCE16
QCE13
RSE17
QCE7
QCE16
E.faecalis

cluster

Fig. 2. Phylogenetic trees showing evolutionary relationships of deduced unique enterolysin A and helveticin J amino acid sequences obtained from the Chinese traditional fermented foods in this study. Based on the fty-seven enterolysin A (A) and one hundred and eight helveticin J (B) sequences, the neighbor-joining phylogenetic trees were constructed by using MEGA4.0, together with the public enterolysin A and helveticin J homologs in the GenBank database, respectively. Bootstrap percentages are shown at nodes.

grouped into two distinct subclusters, I and II. All sequences yielded
from the stinky tofu library fell into the subcluster I, while the
majority (90% and 79%) of sequences produced from the goats milk
cake and Qula cheese libraries fell into the subcluster II, and the
sequences from the milk slice library were distributed into the two
subclusters. Although relatively shorter in comparison to the helveticin J sequences obtained in this study, there was also a signicant number of amino acid residues conserved in the multiple
sequence alignment with all enterolysin A sequences in the cluster,
which also included the two highly conserved motifs for the
CODEHOPs design (Fig. 1, A). The sequences were more closely
related to each other than to the public enterolysin A sequences, for
example, sequences from the stinky tofu library shared 92e98%
identity to each other at the amino acid level, suggesting that less
abundant enterolysin A-producing LAB bacteria existed in the fermented stinky tofu samples. In addition, no PCR product was
amplied from cows milk cake and sweet Chinese rice wine
samples with the EF3-F and EG5-R primers. The different physical
and chemical characteristics of the fermented foods, such as

percentage of water and sugar, might effect on the growth of

bacteriocins-producing LAB on different foods.
3.3. Diversity of helveticin J sequences derived from the Chinese
traditional fermented foods
With our newly designed CODEHOPs HJC-1 and HJJ-9, in total one
hundred and eight unique helveticin J gene sequences in high quality
were effectively recovered from the goats milk cake, cows milk cake,
milk slice, Qula cheese and sweet Chinese rice wine libraries. There
was a signicant number of amino acid residues conserved in the
multiple sequence alignment within these sequences, which included
the two highly conserved motifs for the CODEHOPs design (Fig. 1, B).
Based on their deduced amino acid sequences ranging from 169 to 196
amino acid residues, a neighbor-joining phylogenetic tree was constructed by using the MEGA4.0, and it revealed two distinct clusters,
designated b and g (Fig. 2, B). Cluster b consisted of sequences derived
from the ve libraries, while cluster g included sequences from four of
these libraries except the Qula cheese library. Individual traditional

T. Zhang et al. / Food Control 31 (2013) 499e507

NRB: Niu rubing

0

QC: Qula cheese

11

15

RS: milk slice

17

RW: sweet Chinese rice wine

48

26

GC: goats milk cake

: public helveticin J sequences

13

55
42

45
15

13

44

3
32

34

89

22

99
30

99

40

99
18
80

34

67

37

56
49

21
99
15

15

59

39

88

34

95
94

58

87
85

11
11

56
1

9
99

49
82
99

98

35
30
55

30

26
23
14

0
0

3
99
48

99

9
13
85

35

18

19
43

57

44
46

10
66

13
51

45

14

57

22
36

22

16

14

13

18

1
0

505

NRB11
NRB12
NRB22
NRB6
NRB17
QC28
NRB18
NRB15
NRB21
NRB5
NRB2
NRB23
NRB4
NRB20
NRB26
NRB13
QC16
QC9
QC3
QC21
RS19
QC8
QC12
QC14 subcluster I
QC2
QC22
QC6
QC26
QC11
QC5
QC10
QC13
RW12
RW23
QC7
NRB1
NRB24
RS1
RS9
QC1
QC23
QC25
QC27
RW27
RS11
RS30
RS31
RS18
RS7
QC17
RS3
RS5
RS6
RS20
RS25
L.JV-V03
L.GRL1118
L.GRL1112
QC4
L.ST1
L.NCFM
L.ATCC4796
GC11
L.CTV-05
NRB8
subcluster II
RS12
RS10
GC27
NRB9
L.DSM.20075
GC22
NRB16
GC14
RS28
GC2
NRB19
QC15
L.30SC
NRB10
QC24
L.H10
GC23
RW29
RW17
NRB25
RW6
RW24
RW16
RW3
RW1
RW10
RW20
RW2
RW5
RW22
RW30
RW26
RW18
RW15
cluster
RW21
RW25
GC7
GC28
GC32
GC15
GC31
GC1
RS14
GC19
GC8
RS13
RS23
GC17
GC3
GC5
RS8
GC9
GC18

cluster

Fig. 2. (continued).

fermented food often possessed unique helveticin J sequences associated with it, nevertheless, clades of these sequences were clustered
according to the kinds of foods, rather than the different geographic
locations where the foods were manually processed. For example, all
sequences derived from the Qula cheese library exclusively fell into
the large cluster b, together with the majority of sequences from the
cows milk cake library (95.6%) and milk slice library (80%). All these
sequences were yielded from foods made from cows or yaks milk,
given that Qula cheese was made in Tibetan plateau, China, whereas
cows milk cake and milk slice were in Yunnan province. Similarly, 86%
of the sequences derived from the sweet Chinese rice wine library fell

into the cluster g, which was less related to known helveticin J

homologs in the NCBI database, based on the phylogenetic analysis
(Fig. 2, B). In addition, sequences yielded from the milk slice library fell
into all clusters throughout the phylogenetic tree, implying potentially more diverse helveticin J-producing LAB present in the microbial community of the milk slice than in other food samples.
A total of 65.7% of all helveticin J sequences fell into the cluster b,
which shared 58e100% identity at the amino acid level to known
homologous in the database. Two sequences RS1 and QC15 were
100% identical to the helveticin J from L. helveticus DSM20075
(ZP_05752561.1),
and
Lactobacillus
amylovorus
GRL1118

506

T. Zhang et al. / Food Control 31 (2013) 499e507

(AEA32497.1), respectively. L. helveticus has been used as starter

culture or adjunct culture in the food and fermentation industries
for a long time (Kilpi, Kahala, Steele, Pihlanto, & Joutsjoki, 2007),
while L. amylovorus was abundant in the intestine of piglets that
contains several potential probiotic properties, such as antimicrobial activity against enteric pathogens (Lhteinen et al., 2009).
Within this cluster, only 11.3% of the sequences were derived from
the sweet Chinese rice wine and goats milk cake libraries. The
cluster b was further grouped into two distinct subclusters, I and II.
In the subcluster I, the majority (94.5%) of sequences was produced
from the cows milk cake, Qula cheese and milk slice libraries. Only
three sequences from the sweet Chinese rice wine library were
clustered into this subcluster. All ten know helveticin J sequences
used for designing the CODEHOPs were distributed into a well
supported subclass II within the cluster b, which was comprised of
22.5% of sequences in the cluster b, produced from four of the ve
libraries except the sweet Chinese rice wine library. Moreover, all
sequences in the subclass II were very closely related to each other
with identities ranging from 98% to 100% at the amino acid level.
The majority (86%) of the helveticin J sequences derived from
sweet Chinese rice wine and milk slice libraries fell into the cluster
g, which included only a single sequence from the cows milk cake
library, and four from the milk slice. Interestingly, phylogenetic
analysis revealed that the sequences derived from the goats milk
cake library were more closely related to the sequences from the
sweet Chinese rice wine than to those from milk slice. All sequences
in the cluster g shared 65e70% similarity at the amino acid level to
the known helveticin homologs in the database. Moreover, none
public homologs fell into this cluster. This clustering suggested that
the cluster g may represent a unique helveticin J type or novel LAB
niches may exist in the microbial communities of the four kinds of
fermented foods.
3.4. Cloning and expression of intact enlA and hlvJ genes in E. coli
To assess the biological activities of the enterolysin A and helveticin J present in the Chinese traditional fermented foods, the
intact enlA and hlvJ genes were cloned by TAIL-PCR based on the
DFE5 (235 bp) and RS9 (532 bp) sequences, respectively. The 50 terminal region of enlA (DFE5) gene was obtained by two-round
genome walking, which produced approximately 0.4 and 0.3 kb
amplicons, respectively, while a 0.5 kb amplicon was produced for
the 30 -terminal region by one-round genome walking. The DNA
sequences of the amplicons were determined and assembled. The
intact EnlA (DFE5) open reading frame (ORF) consisting of 269
amino acid residues was identied within a 1.2-kb fragment by
BLAST analysis. Downstream of the EnlA ORF, the 30 -terminal region
of a conserved hypothetical protein fragment was found. The 807bp sequence containing the intact enlA (DFE5) gene was amplied
from the metagenomic DNA prepared from traditional fermented
stinky tofu samples, and cloned into the pGM-T vector, which
yielded the recombinant vector, pGM-T-enlA(DFE5). The cloned
DNA fragment was veried by DNA sequencing. Database searches
revealed that the enlA (DFE5) displayed 95% identity at the amino
acid level (in a 256 aa stretch) to a putative enterolysin A from
Lactobacillus delbrueckii subsp. lactis DSM20072 (EGD28130.1), and
only 35% identity (in a 168 aa stretch) to the known enterolysin A
(AAG29099.1) that was cloned and characterized from E. faecalis
(Nigutov, Serencov, Piknov, Javorsk, & Pristas, 2008).
Heterologous expression of functionally active enterolysin A
from E. faecalis in E. coli was reported previously (Nigutov et al.,
2008). In this study, the obtained enlA (DFE5) gene was cloned
into the expression vector pET28a, yielding the recombinant
plasmid pET28a-enlA(DFE5) as described in the materials and
methods. Expression of the recombinant EnlA (DFE5) in E. coli BL21

was induced by 0.6 mM IPTG at 20  C for 20 h, and assayed by SDSPAGE (data not shown). Sequence analysis also revealed that the
recombinant EnlA (DFE5) with a calculated molecular mass of
30.6 kDa contains the conserved N-terminal endopeptidase domain
belonging to peptidase family M23 with typical motifs HXXXD
(103e107 aa) and HXH (183e185 aa) (Hickey, Twomey, Ross, & Hill,
2003; Malinicova, Dubikova, Piknova, Pristas, & Javorsky, 2012;
Nilsen et al., 2003). The EnlA (DFE5) in the soluble cellular fraction
was used in further bacteriocin activity studies.
The same strategy was employed for cloning and expression of
hlvJ gene in E. coli BL21. The 50 and 30 -terminal regions of hlvJ (RS9)
gene were obtained by genome walking TAIL-PCR, and approximately 0.2 and 0.6 kb amplicons were produced, respectively. DNA
sequences of the amplicons were determined and assembled,
which produced a 750-bp intact hlvJ ORF. The full-length of hlvJ
(RS9) gene was amplied and veried as described in the materials
and methods. The intact hlvJ (RS9) gene (747-bp, not including the
stop codon) was subsequently cloned into the expression vector
pET28a, which yielded a recombinant vector pET28a-hlvJ(RS9).
Expression of recombinant HlvJ (RS9) in E. coli BL21 was induced
and assayed as described above. The HlvJ (RS9), encoding 249 aa
with a calculated molecular mass of 28.1 kDa, displayed 99%
identity at the amino acid level over its entire length to a putative
helveticin J from L. helveticus MTCC5463 (EGF35161.1), and 42%
identity (in a 241 aa stretch) to the known helveticin J (AAA63274.1)
cloned from L. helveticus 481 (Joerger & Klaenhammer, 1990).
3.5. Inhibitory spectra of the EnlA and HlvJ
It has been reported that enterolysin A has a broad inhibitory
spectrum against several bacterial genera, including Enterococcus,
Lactobacillus, Lactococcus, Bacillus, Staphylococcus, Propionibacterium, Pediococcus and Listeria (Hickey et al., 2003; Nigutova et al.,
2007; Nilsen et al., 2003). In this study, antimicrobial activity of
the recombinant EnlA (DFE5) expressed in E. coli BL21 was determined by the agar diffusion method. As presented in Table 2, the
EnlA (DFE5) in the soluble cellular fraction displayed inhibitory
activities against six of the ten indicator strains tested, evidenced
by clear zones of inhibited growth when compared with that of the
control. Of these sensitive strains, L. monocytogenes ATCC19115 and
ATCC13932 belonging to serotype 4b are human food-borne
pathogens associated with clinical human listeriosis cases and
outbreaks (Orsi, Den Bakker, & Wiedmann, 2011), while S. aureus
CDCAB91093 is also a human pathogen. An antilisterial effect of
enterolysin A was also observed by Nigutova et al. (2007), where
the enterolysin A from rumen E. faecalis strain was effectively
against Listeria innocua 2710. In addition, inhibition of growth was
also detected with the indication strain Bacillus subtilis AS1.88.
However, none of the Lactobacillus strains tested was inhibited by
the EnlA (DFE5). Moreover, the gram negative human pathogens,
E. coli ATCC43889 (O157:H7) and Salmonella enteritidis AS
CMCC50041, were observed resistant against the EnlA (DFE5), this
result correlates with previous reports in that enterolysin A was
found being active against gram positive bacteria (Hickey et al.,
2003; Nigutova et al., 2007; Nilsen et al., 2003), probably because
the specic receptor of the EnlA (DFE5) could be absent or modied
on cell walls of these indicator strains (Malinicova et al., 2012).
It was reported that the cloned and expressed helveticin J from
L. helveticus 481 displayed bacteriocin activity against several
closely related species including L. helveticus, Lactobacillus jugurti,
Lactobacillus bulgaricus and Lactococcus lactis (Joerger &
Klaenhammer, 1990). In this study, a narrow inhibitory spectrum
of the HlvJ (RS9) was detected, except for biologically active against
the L. helveticus ATCC15009, nine of the ten strains tested were
sensitive to the bacteriocin.

T. Zhang et al. / Food Control 31 (2013) 499e507

Along with the expansion of food production, processing and

international trade in foodstuffs, there is increasingly intensied
pressure to prevent food contamination and render contaminated
food safe (Kferstein & Abdussalam, 1999). Even though chemical
preservatives effectively controlled growth of spoilage and pathogenic bacteria in foods, and have widely been applied in food
industry, particularly in developing countries, the increasing
concern about the potential threats of chemical additives to human
health substantially heightened the need of exploring new natural
preservatives. LAB are the worlds recognized safe food-grade
microorganisms. Bacteriocins produced by them can safely be
used as natural food preservatives directly, providing great potential for research and food industry. It is reasonable to assume that
numerous bacteriocins exist in environment, but they can be
difcult to detect and identify (Wieckowicz, Schmidt, Sip, & Grajek,
2011). Culture-dependent assays were used to detect new bacteriocins (Gonzlez et al., 2007; Yi, Zhang, Tuo, Han, & Du, 2010),
which was both time and labor consuming. Compared with traditional methods using specic primers to detect public described
bacteriocins (Belgacem et al., 2010; Choho et al., 2008), the cultureindependent metagenomic approach applied in this study was
demonstrated to be rapid and effective for identifying natural
variants of enterolysin A and helveticin J with biological activity
from complex traditional fermented food samples, which could be
valuable for further investigation.
4. Conclusion
To our knowledge, this study constituted the rst investigation
of bacteriolysin diversity in microbial communities of fermented
foods. The enlA (DFE5) and hlvJ (RS9) genes were cloned, expressed
and characterized, respectively. The results not only demonstrated
the validity of the metagenomic approach in this study for identifying novel bacteriocins directly from complex fermented food
ecosystem, but also provided the evidence that Chinese traditional
fermented foods could be a rich bacteriocin gene source valuable
for further exploration in food industry.
Acknowledgments
This work was supported by Grants No.09320503600 and
No.10391902300 from Shanghai Municipal Science and Technology
Commission, a Grant No.B-9500-10-0004 and a Leading Academic
Discipline Project (No. J50704) from Shanghai Municipal Education
Commission.
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