the lactobin a and amylovorin l471 encoding genes are identical, and their distribution seems to be...

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International Journal of Food Microbiology 90 (2004) 93–106

The lactobin A and amylovorin L471 encoding genes are identical,

and their distribution seems to be restricted to the species

Lactobacillus amylovorus that is of interest for cereal fermentations

L. De Vuysta,*, L. Avontsa, P. Neysensa, B. Hosteb, M. Vancanneytb,J. Swingsb, R. Callewaerta

aResearch Group of Industrial Microbiology, Fermentation Technology and Downstream Processing,

Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, BelgiumbLaboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, BCCM/LMG Bacteria Collection,

Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Received 28 October 2002; received in revised form 22 April 2003; accepted 30 May 2003

Abstract

Lactobin A and amylovorin L471 are two bacteriocins produced by the phenotypically different strains Lactobacillus

amylovorus LMG P-13139 and L. amylovorus DCE 471, respectively. A 110-bp PCR fragment of the structural gene of lactobin

Awas obtained from total genomic DNA of L. amylovorus LMG P-13139, which was used as a probe to isolate a 3.6-kb HindIII

chromosomal fragment for sequencing. PCR amplification revealed that both the structural genes of both the bacteriocins

lactobin A and amylovorin L471 were identical. These bacteriocins will be further referred to as amylovorin L. Amylovorin L can

be defined as a small, strongly hydrophobic, antibacterial peptide consisting of 50 amino acids. It is synthesized as a precursor

peptide of 65 amino acids processed at a characteristic double-glycine proteolytic cleavage site. Amylovorin L hence belongs to

the class II bacteriocins. It has a narrow inhibitory spectrum, being most active towards Lactobacillus delbrueckii subsp.

bulgaricus LMG 6901T. Among 38 strains of the Lactobacillus acidophilus DNA homology group, another 6 L. amylovorus

strains were also inhibitory towards the L. delbrueckii subsp. bulgaricus LMG 6901T strain. The lactobin A or amylovorin L471

structural genes could be detected in the genomes of three of these L. amylovorus strains, but only after extensive PCR

amplification, indicating that the inhibitory substances were slightly different. The bacteriocins were characterized as small

(approximately 4800 Da), heat-stable peptides that were active in a wide pH range (2.2–8.0). Finally, preliminary experiments

indicated that the production of amylovorin L by L. amylovorusDCE 471 took place during a natural rye fermentation, indicating

its potential importance in the development of a functional (probiotic) starter culture for cereal fermentations.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Amylovorin L471; Lactobin A; Lactobacillus amylovorus; Lactobacillus acidophilus; Bacteriocin distribution

0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0168-1605(03)00298-8

Abbreviations: LBN, lactobin; AMY, amylovorin.

* Corresponding author. Tel.: +32-2-629-32-45; fax: +32-2-

629-27-20.

E-mail address: [email protected] (L. De Vuyst).

1. Introduction

Lactic acid bacteria (LAB) are well known for

their antagonistic activity against a variety of micro-

organisms. Bacteriocin production is one of the

L. De Vuyst et al. / International Journal of Food Microbiology 90 (2004) 93–10694

properties responsible for the antibacterial activity

against closely related species, but some Gram-pos-

itive food spoilers and pathogens are often inhibited

as well (De Vuyst and Vandamme, 1994; Klaenham-

mer, 1988). Bacteriocins are low-molecular-mass

peptides or high-molecular-mass proteins that are

mostly hydrophobic and thermostable or hydrophilic

and heat sensitive, respectively (De Vuyst and Van-

damme, 1994; Klaenhammer, 1993). Bacteriocin pro-

duction has been found among almost all genera of

LAB (De Vuyst and Vandamme, 1994; Wood and

Holzapfel, 1995).

The structural genes of a bacteriocin produced by a

strain belonging to a certain species are often wide-

spread among strains within this species. This has

been revealed through PCR amplification for instance

for the genes of nisin A/Z in Lactococcus lactis (De

Vos et al., 1993; Martınez et al., 1995; Meghrous et

al., 1999; Rodrıguez et al., 1995a; Ward et al., 1994),

lactocin S in Lactobacillus spp. (Rodrıguez et al.,

1995b), enterocin AS-48 in Enterococcus spp. (Joos-

ten et al., 1997; Rodrıguez et al., 1998), and pediocin

AcH/PA-1 in Pediococcus acidilactici (Rodrıguez et

al., 1997). Furthermore, it frequently happens that the

same bacteriocin is isolated from cultures of strains

from different species. Usually, this becomes clear

when the amino acid or gene sequence have been

elucidated. Numerous examples are available. For

instance, the bacteriocins sakacin A from Lactobacil-

lus sakei Lb706 (Holck et al., 1992), curvacin A from

Lactobacillus curvatus LTH 1174 (Tichaczek et al.,

1992), and sakacin K from L. sakei CTC 494

(Remiger et al., 1996) are identical. Further, among

different strains, variants of a bacteriocin are often

found, which differ only in one or a few amino acids.

The most common example is nisin A and nisin Z

produced by L. lactis subsp. lactis, which only differ

in one amino acid (Mulders et al., 1991). Further-

more, acidocin 1229 from Lactobacillus acidophilus

JCM 1229 (Tahara and Kanatani, 1996), gassericin

B3 from Lactobacillus gasseri JCM 2124 (Tahara et

al., 1997), and acidocin J1132 from L. acidophilus

JCM 1132 (Tahara et al., 1996) also only differ in

one or two amino acids. Therefore, bacteriocins seem

to be genus specific. Until now, only one exception

has been reported: pediocin AcH/PA-1 is naturally

produced by both P. acidilactici and Lactobacillus

plantarum strains (Ennahar et al., 1996).

Lactobacillus amylovorus, an obligate homofer-

mentative strain and a member of the L. acidophilus

DNA homology group, plays an important role in

several cereal fermentation processes such as type II

sourdoughs based on rye (Muller et al., 2001; Vogel

et al., 1999), and many traditional African and Asian

fermented food products, e.g. Sudanese Kisra pro-

duction from sorghum (Hamad et al., 1992), and

Indian idli made from rice and black gram dough

(Chavan and Kadam, 1989). Furthermore, strains of

L. amylovorus deserve special attention as they have

the capability to adhere to human intestinal cells

(Muller et al., 1998). The combination of adhesion

and formation of antagonistic compounds, in partic-

ular, bacteriocins, are especially tempting for the

development of probiotic strains from cereal origin

(Vogel et al., 1999).

In earlier reports, we described the isolation of

two bacteriocinogenic peptides produced by L. amy-

lovorus LMG P-13139 (Contreras et al., 1997) and

L. amylovorus DCE 471, two strains that differ

phenotypically as well as in their total cell protein

pattern (Callewaert et al., 1999). Isolation and puri-

fication of the antibacterial peptides revealed two

bacteriocins with estimated molecular masses of 4.5

and 6.0 kDa (Contreras et al., 1997), and 4.8 and 5.8

kDa (De Vuyst et al., 1996a), respectively. In both

cases, the smallest peptide was sequenced and des-

ignated as lactobin A and amylovorin L471, respec-

tively (Callewaert et al., 1999; Contreras et al.,

1997). The N-terminal part of 35 amino acids

determined so far for amylovorin L471 is identical

to the N-terminal amino acid sequence of lactobin A

(Callewaert et al., 1999). However, amylovorin L471

differs in molecular mass from lactobin A in 2 Da,

indicating the presence of a disulphide bond in

amylovorin L471 (Callewaert et al., 1999). Despite

this strong similarity, differences in inhibitory spec-

trum are observed in that amylovorin L471 inhib-

its more target bacteria than lactobin A (Callewaert

et al., 1999).

The aim of this paper was to assess the distri-

bution of the amylovorin structural gene among

strains of the L. acidophilus DNA homology group.

First, the structural genes of the bacteriocins lacto-

bin A and amylovorin L471 were sequenced. Next,

the distribution of the amylovorin L471 structural

gene was surveyed through PCR amplification in 38

L. De Vuyst et al. / International Journal of

other Lactobacillus strains of the L. acidophilus

DNA homology group. Further, the bacteriocins

from three other L. amylovorus strains were isolated

and characterized. Finally, based on its compe-

titiveness in rye fermentation, the potential impor-

tance of bacteriocin production by L. amylovorus is

underlined.

2. Material and methods

2.1. Bacterial strains and media

L. amylovorus LMG P-13139 (Contreras et al.,

1997) and L. amylovorus DCE 471 (De Vuyst et al.,

1996a) were used as the lactobin A and the amylo-

vorin L471 producer, respectively. Lactobacillus del-

brueckii subsp. bulgaricus LMG 6901T was used as

the bacteriocin-sensitive indicator organism (De

Vuyst et al., 1996a). The strain Enterococcus fae-

cium CTC 492 was used as a negative control in the

PCR reactions (Aymerich et al., 1996). The latter

strain was kindly provided by Dr. Marta Hugas

(IRTA, Monells, Spain). The strains L. plantarum

LMG 6907T and L. sakei subsp. carnosus LMG

17302T were used as a control in the rep-PCR anal-

ysis. Other LAB strains used are listed in Table 1.

Escherichia coli Sure2 was provided by Stratagene

(La Jolla, CA).

All strains were stored at � 80 jC in their ap-

propriate cultivation medium plus glycerol (final

concentration of 25.0%, v/v): MRS (Oxoid, Basing-

stoke, UK) for lactobacilli and E. faecium, and Luria

Bertani (LB) medium (Sambrook et al., 1989) for E.

coli. Before experimental use, the cultures were

propagated twice at 37 jC for 12 h, along with

shaking at 250 rpm in the case of E. coli, to obtain

fresh cultures; the transfer inoculum was 1.0% (v/v).

Lactobacillus iners was cultivated microaerophili-

cally (controlled atmosphere of 10% oxygen, 10%

carbon dioxide, and 80% nitrogen) in Columbia

medium (Oxoid) supplemented with 5% (v/v) horse

blood. MRS medium adjusted to pH 6.5 was used for

bacteriocin production. Bottom and overlay agar

media were prepared by addition of 15 and 7 g of

granulated agar (Oxoid), respectively, to 1 l of me-

dium. The media were sterilized by heating at 121

jC for 20 min.

2.2. DNA isolation from L. amylovorus LMG P-

13139, PCR-mediated isolation of the structural gene

of lactobin A, and cloning and sequencing of the PCR

fragments

Total genomic DNA from L. amylovorus LMG P-

13139 was prepared by the method described by Boot

et al. (1993). Two 35-mer degenerate lactobin A-

specific primers for PCR were deduced from the N-

and C-terminal amino acid sequence of lactobin A,

respectively (Contreras et al., 1997). The sense primer

(5VAAT/C G C I TAT/C A/T C I G C I G C I T/C T G

G G I T G 3V) correlated with the lactobin A DNA

sequence coding for the amino acids at positions 5–13,

whereas the antisense primer (5VC G TA I C C I G C I

A G I G C G C A I A C I G C 3V) stretched between theamino acids at positions 47 and 39. PCR reactions

were performed according to the standard procedures

in a total volume of 50 Al in the presence of 0.2 pmol

Al� 1 of sense primer, antisense primer, and dNTP mix.

Briefly, 100 ng of template DNA and 1 U Taq DNA

polymerase (Amersham Biosciences, Uppsala, Swe-

den) were added to each reaction. Before cycling,

template DNA was denatured at 95 jC for 3 min. In

each cycle, template DNA was denatured again at 95

jC for 30 s, annealing took place at 50 jC. Polymer-

ization was performed at 72 jC for 2 min. PCRs

consisted of 35 cycles and were followed by an extra

polymerization step at 72 jC for 5 min. Samples were

kept at 4 jC after the reactions were completed. Two

different PCR fragments of approximately 90 and 110

bp were generated, cloned in pBSK+ (2.9-kb vector;

Stratagene), and sequenced in both directions using T3

and T7 primers.

2.3. Cloning and sequencing of the structural gene of

lactobin A

Chromosomal DNA of L. amylovorus LMG P-

13139 was digested with HindIII according to the

manufacturer’s instructions (Boehringer Mannheim,

Mannheim, Germany). The 110-bp PCR fragment

was used as a homologue probe in digoxigenin-

labeled, nonradioactive, and random a-32P-dATP-la-

beled Southern hybridizations, carried out according

to the manufacturers’ instructions (Boehringer Man-

nheim; Amersham Biosciences). A selected DNA

fragment of 3.6 kb was isolated from the complete

Food Microbiology 90 (2004) 93–106 95

Table 1

Strains used for detection of the structural genes of lactobin A and

amylovorin L471

Strain Origin PCR I PCR II

Lactobacillus acidophilus

LMG 11430

human � �

L. acidophilus LMG 11467 human � �L. acidophilus LMG 11469 rat intestine � �L. acidophilus LMG 11472 unknown � �Lactobacillus amylovorus

DCE 471

corn steep liquor + +

L. amylovorus LMG P-13139 corn steep liquor + +

L. amylovorus LMG 9434 pig’s small

intestine

+/� +/�

L. amylovorus LMG 9496T cattle waste–corn

fermentation

� �

L. amylovorus LMG 13049 cattle waste–corn

fermentation

+/� +/�

L. amylovorus LMG 13135 cattle waste–corn

silage

+/� +/�

L. amylovorus LMG 18179 swine intestine � �L. amylovorus LMG 18192 human faeces � �L. amylovorus LMG 18197 pig faeces � �L. amylovorus LMG 18198 calf faeces � �Lactobacillus crispatus

LMG 9479Teye � �

L. crispatus LMG 12004 human urine � �L. crispatus LMG 18191 chicken faeces � �L. crispatus LMG 18199 human faeces � �Lactobacillus gallinarum

LMG 14751

chicken faeces � �

L. gallinarum LMG 14753 chicken faeces � �L. gallinarum LMG 14754 chicken faeces � �L. gallinarum LMG 14755 chicken faeces � �Lactobacillus gasseri

LMG 11413

human saliva � �

L. gasseri LMG 13134 vaginal tract � �L. gasseri LMG 18176 human intestine � �L. gasseri LMG 18203 human faeces � �Lactobacillus helveticus

LMG 13522

unknown � �

L. helveticus LMG 18182 dairy products � �L. helveticus LMG 18183 unknown � �L. helveticus LMG 18225 unknown � �Lactobacillus iners

LMG 18913

human vaginal

discharge

� �

L. iners LMG 18914 human urine � �L. iners LMG 18915 unknown � �L. iners LMG 18916 unknown � �Lactobacillus johnsonii

LMG 9436Thuman blood � �

L. johnsonii LMG 18175 human intestine � �L. johnsonii LMG 18204 mouse faeces � �L. johnsonii LMG 18206 pig faeces � �Enterococcus faecium

CTC 492

fermented

sausage

� �


HindIII digest of chromosomal DNA separated by

agarose gel electrophoresis, cloned in the unique

HindIII site of the pBSK+ cloning vector, and trans-

formed into competent E. coli Sure2 cells, using

general protocols (Sambrook et al., 1989). Nucleotide

sequence analysis was performed by sequencing dou-

ble-stranded DNA in two orientations by the dideoxy-

terminator chain method of Sanger et al. (1977) using

the nonradioactive Taq Dye Deoxy Terminator Cycle

sequencing kit (Applied Biosystems, Foster City, CA)

and a Model 377 A Sequencer (Applied Biosystems).

2.4. DNA isolation from, and primers and PCR

conditions, for detection of the lactobin A structural

gene

Total genomic DNA was isolated by use of the

Puregene kit (Gentra Systems, Minneapolis, MN).

DNA was checked on 2% (m/v) agarose gels and

quantified by the Dynaquant kit (Amersham Bioscien-

ces) with calf thymus DNA as standard (DQ 202,

Sigma, St. Louis, MO).

Three primers were chosen from the DNA se-

quence of the coding part of the lactobin A structural

gene (Fig. 1). The first primer (5V TGG ACT AAT

GCATAC AGC GC 3V), named (LBN) lactobin sense

primer, corresponded to the N-terminal end of lacto-

bin A (Contreras et al., 1997). The second primer (5VCCA ATTACA GCA CCC CATAC 3V), named LBN

antisense primer, corresponded to the C-terminal part

of lactobin A (Contreras et al., 1997). The third primer

(5VCTT TAC GAA CAT AAC CCG CC 3V), named

amylovorin (AMY) antisense primer, corresponded to

the C-terminal part of the known N-terminal amylo-

vorin L471 amino acid sequence, which is identical to

the N-terminal lactobin A amino acid sequence (Call-

ewaert et al., 1999).

A Perkin Elmer GeneAmp PCR System 9600

(Applied Biosystems) was used. The PCR reaction

Notes to Table 1:

CTC, Centre de Tecnologia de la Carn (Institut de Recerca i Tec-

nologia Agroalimentaries, Monells, Spain); DCE, Department of

Chemical Engineering (Vrije Universiteit Brussel, Brussels, Bel-

gium); LMG, Laboratorium Microbiologie Gent Culture Collection

(Ghent University, Gent, Belgium).

PCR I: LBN sense and LBN antisense primer set; PCR II: LBN

sense and AMY antisense primer set: +, amplification; � , no am-

plification; +/� , very weak amplification.

Fig. 1. Nucleotide sequence of the lactobin A structural gene of L. amylovorus P-13139 and primer selection for the detection of the lactobin A

and amylovorin L471 structural genes. The amino acid sequence of lactobin A including the leader is displayed in the one-letter code. The

double-glycine (� 1/� 2), leader-processing site is underlined. The 35 amino acids identical to amylovorin L471 are displayed in bold

(Callewaert et al., 1999). The nucleotide sequences corresponding to the primers are in italic and underlined. They are named the LBN sense,

AMY antisense, and LBN antisense primer, respectively (from left to right). The possible ribosome-binding site is in bold and underlined. The

terminator codon is indicated by an asterisk. Only part of the sequenced 3.6-kb HindIII fragment is shown.

L. De Vuyst et al. / International Journal of Food Microbiology 90 (2004) 93–106 97

was done in a volume of 50 Al of Gold PCR buffer

(Applied Biosystems) containing 2.5 mM of MgCl2,

0.2 mM of dNTPs, 0.6 AM of each primer, 1.25 U of

AmpliTaq Gold DNA polymerase (Applied Biosys-

tems), and 5 ng of template DNA (unless stated

otherwise). The amplification was started by a DNA

denaturation and an enzyme activation step at 95 jCfor 10 min. The amplification consisted of 35 cycles of

denaturation and enzyme activation at 95 jC during 30

s, and annealing and extension at 67 jC during 30 s

(unless stated otherwise). Final annealing and exten-

sion occurred at 67 jC for 12 min. At the end, the

temperature was decreased to 4 jC to stop the reaction.

First, the PCR conditions for detection of the

lactobin A structural gene were optimized. PCR ampli-

fications were carried out with 1, 5, 10, and 100 ng of

template DNA at an annealing and elongation temper-

ature of 63, 66, 67, 68, and 69 jC. The lactobin A

producer, L. amylovorus LMG P-13139, and the amy-

lovorin L471 producer, L. amylovorus DCE 471, were

used as positive controls; the enterocin A producer, E.

faecium CTC 492, was used as negative control. Total

genomic DNA was used as template.

Amplified DNA fragments were visualized on a

4.5% (m/v) LSI MP agarose gel (Boehringer Man-

nheim) after staining with ethidium bromide. A

øX174/HinfI DNA marker (726 to 24 bp; Stratagene)

was used to determine the length of the amplified

fragments. The gel was run at 150 V for 150 min.

Based on the nucleotide sequence of the lactobin A

structural gene (Fig. 1), a 144-bp DNA fragment and a

96-bp DNA fragment was expected to be amplified

with the LBN sense and LBN antisense primer set

(PCR I), and the LBN sense and AMY antisense

primer set (PCR II), respectively.

Cleaning of the PCR products to be sequenced was

done by use of the QIA quick spin PCR purification

kit (Qiagen, Hilden, Germany). The sequencing reac-

tion was carried out with 4 Al of Big Dye (Applied

Biosystems), 3 Al of PCR product, and 3 Al of primer

(LBN sense and LBN antisense or AMY antisense) of

20 ng Al� 1. The sequence of double-stranded DNA in

two orientations was determined on an ABI PRISM

377 DNA Sequencer (Applied Biosystems).

To study the distribution of the lactobin A structural

gene among strains of the L. acidophilus DNA ho-

mology group, two PCR experiments were performed,

one with the LBN sense and antisense primers (PCR I)

and another with the LBN sense and AMY antisense

primers (PCR II). Total DNA from L. amylovorus

LMG P-13139 and E. faecium CTC 492 were used

as positive and negative controls, respectively.

2.5. Bacteriocin isolations and assay of bacteriocin

activity and inhibitory spectrum

Lactobin A, amylovorin L471, and the bacteriocins

from L. amylovorus LMG 9434, L. amylovorus LMG

9496T, L. amylovorus LMG 13049, and L. amylovorus

LMG 13135 were produced and isolated as described

previously (Callewaert et al., 1999; De Vuyst et al.,

1996a). The material thus obtained was considered as

a purified bacteriocin preparation and could be stored

at � 80 jC (Callewaert et al., 1999).

Bacteriocin activity was measured by an agar spot

assay as described previously (De Vuyst et al., 1996a).

Briefly, twofold serial dilutions of cell-free culture

supernatant containing bacteriocin were spotted (10

Al) onto fresh lawns of the sensitive indicator organ-

ism L. delbrueckii subsp. bulgaricus LMG 6901T.


These lawns were prepared by propagating fresh

cultures to an optical density at 600 nm (OD600)

between 0.40 and 0.45, and adding 100 Al of the cell

suspension to 3.5 ml of overlay agar. Overlaid agar

plates were incubated at 37 jC for at least 12 h. The

bactericidal activity was defined as the reciprocal of

the highest dilution demonstrating complete inhibition

of the indicator lawn and was expressed in activity

units (AU) per milliliter of culture medium. To assay

the spectrum of activity of a bacteriocin towards other

lactobacilli, the antimicrobial activity of a purified

bacteriocin preparation was examined against the

target bacteria (Table 2) in at least two separate tests.

2.6. Characterization of the bacteriocins

For a characterization of the bacteriocins of L.

amylovorus LMG 9434 (solution of 50 AU ml� 1),

L. amylovorus LMG 13049 (3200 AU.ml� 1), and L.

amylovorus LMG 13135 (1200 AU.ml� 1), compared

to L. amylovorus DCE 471 (6400 AU ml� 1) and L.

amylovorus LMG P-13139 (1600 AU ml� 1), purified

bacteriocin was treated with several proteases in the

Table 2

Inhibitory spectra of the purified bacteriocin preparations from L.

amylovorus strains DCE 471, LMG P-13139, LMG 13135, LMG

13049, LMG 9496T, and LMG 9434

Bacteriocin

preparation

Indicator strain

DCE

471

LMG

P-13139

LMG

13049

LMG

13135

LMG

9496TLMG

9434

L. delbrueckii subsp.

bulgaricus LMG

6901T

+ + + + + +

L. amylovorus LMG

9434

+ + + + + �

L. amylovorus LMG

9496T+ + + � � �

L. amylovorus LMG

13135

+ + � � � �

L. amylovorus LMG

13049

+ + � � � �

L. amylovorus LMG

P-13139

+ � � � � �

L. amylovorus DCE

471

� � � � � �

The activities against L. delbrueckii subsp. bulgaricus LMG 6901T

of all preparations were 6400 AU ml� 1, except for the purified

bacteriocin preparation from L. amylovorus LMG 9434 that was

200 AU ml� 1.

+, inhibition; � , no inhibition.

appropriate buffer and final concentration indicated:

proteinase K, 1 mg ml� 1, 50 mM phosphate buffer, pH

7.0, 37 jC; trypsin, 2 mg ml� 1, 50 mM phosphate

buffer, pH 7.0, 37 jC; a-chymotrypsin, 5 mg ml� 1, 50

mM phosphate buffer, pH 7.5, 37 jC; and pepsin, 1 mg

ml� 1, 200 mM citrate buffer, pH 2.2, 37 jC. All

enzymes were purchased from VWR International

(Darmstadt, Germany). The residual activity of the

bacteriocin solutions was tested towards L. delbrueckii

subsp. bulgaricus LMG 6901T as described above. The

appropriate buffers and enzyme/buffer solutions were

used as controls. The activity was further tested after

various heat treatments (15, 30, and 60 min at 60 and

100 jC, and autoclaving during 15 min at 121 jC);nonheat-treated samples were used as a control. The

influence of pH on activity was tested in the following

buffers: 200 mM citrate buffer for pH 2.2, 3.0, 4.0, 4.5,

5.0, 5.5, and 6.0, and 100 mM sodium phosphate

buffer for pH 6.5, 7.0, and 8.0. The activity was

measured after 1 h and after 24 h of incubation at 25

jC. Buffer solutions were used as a negative control.

To estimate the molecular mass of the bacteriocins,

tricine–SDS–PAGE was carried out as described

previously (De Vuyst et al., 1996a).

2.7. Rye fermentation with L. amylovorus DCE 471

To test its suitability for rye fermentation, a freshly

prepared inoculum of L. amylovorus DCE 471 was

added to a mixture of rye (2.5 kg) and water (7.5 l) in

a Biostat C fermentor (B. Braun Biotech International,

Melsungen, Germany). The water was sterilized in the

fermentor at 121 jC for 20 min; after cooling to 37

jC, the rye was added to the fermentor, immediately

followed by inoculation. The fermentation was carried

out at a controlled temperature of 37 jC. The pH was

monitored on line. The stirring speed was 600 rpm; it

was decreased to 400 rpm after 12 h as the sourdough

viscosity decreased during the fermentation. Samples

were aseptically removed from the fermentation ves-

sel after 0, 3, 6, 12, 15, and 24 h, and analyzed for

total titratable acidity, total bacteria and yeast counts,

and soluble bacteriocin activity. Total titratable acidity

(TTA) was determined as the amount (ml) of 0.1 M

NaOH to titrate an aliquot of 10 g of a sample blended

with 100 ml of distilled water to a final pH of 8.5.

Total cell counts (CFU ml�1) of fresh samples were

determined on plates of Sourdough Simulation Medi-


um (SSM) agar adjusted to pH 5.4 (Messens et al.,

2002). Soluble bacteriocin activity of culture super-

natants obtained after microcentrifugation (13,000

rpm, 20 min) was tested by bioassay towards L.

delbrueckii subsp. bulgaricus LMG 6901T as indicator

organism as described above. Finally, to verify strain

domination of the added culture of L. amylovorus

DCE 471, four bacterial colonies from an agar plate

with approximately 10–100 colonies were randomly

picked up. The authenticity of these colonies was

identified by rep-PCR according to the method of

Gevers et al. (2001). The ability to produce bacterio-

cin was tested by overlaying the agar plates with soft

agar containing the sensitive indicator strain L. del-

brueckii subsp. bulgaricus LMG 6901T and observing

the plates for inhibition zones around the colonies.

Fig. 2. Visualization of the amplified PCR fragments. PCR am-

plification reactions were done on a blank sample (lanes 2 and 6)

and on total DNA of the following strains: E. faecium CTC 492

(lanes 3 and 7), L. amylovorus LMG P-13139 (lanes 4 and 8), and L.

amylovorus DCE 471 (lanes 5 and 9). Lanes 1 and 10 contain DNA

molecular mass markers (726 to 24 bp, top to bottom). In lanes 6 to

9, the LBN sense and LBN antisense primer set was used for the

amplification of a 144-bp fragment of the lactobin A structural gene.

In lanes 2–5, the LBN sense and AMY antisense primer set were

used for the amplification of a 96-bp fragment of the lactobin A

structural gene corresponding to the known N-terminal amino acid

sequence of amylovorin L471.

3. Results

3.1. PCR-mediated isolation of the structural gene of

lactobin A, cloning and sequencing of the PCR

fragments, and cloning and sequencing of the

structural gene of lactobin A

The 110-bp PCR fragment encoded a part of the

amino acid sequence of lactobin A, produced by L.

amylovorus LMG P-13139, and was located between

amino acids 5 and 47 (Fig. 1). The smaller 90-bp PCR

fragment concerned a 5Vtruncated form of the 110-bp

fragment due to nonspecific downstream hybridiza-

tion of the sense primer.

L. amylovorus LMG P-13139 contained only one

plasmid of approximately 30 kb that could be cured

from the strain by three consecutive overnight incu-

bations in the presence of 200 Ag of novobiocin per

ml without loss of bacteriocin production (results not

shown). The plasmid did not hybridize with chromo-

somal DNA of L. amylovorus LMG P-13139 in

Southern blot experiments, excluding the possibility

of chromosomal incorporation (results not shown).

Partial sequencing of the 3.6-kb HindIII chromo-

somal DNA fragment revealed the complete nucleotide

sequence of the structural gene of lactobin A, lbnA. A

relevant part of the sequencing results is represented in

Fig. 1. The structural gene encodes a prebacteriocin of

50 amino acids with a 15 amino acid leader peptide and

a G(� 2)G(� 1) proteolytic processing site, character-

istic of class II bacteriocins. Upstream of lbnA, a

possible ribosome-binding site is located.

3.2. PCR conditions for detection of the lactobin A

structural gene, sequencing of the lactobin A and

amylovorin L471 PCR amplicons, and detection of the

lactobin A and the amylovorin L471 structural genes

among other strains of the L. acidophilus DNA

homology group

An annealing and extension temperature of 67 jCand an amount of 5 ng of template DNAwere optimal

for the detection of the 144-bp and 96-bp DNA

fragments. At an annealing and elongation tempera-

ture of 63 jC, similar amplifications were obtained

with 1, 5, and 10 ng of template DNA. Increasing the

amount of template DNA to 100 ng decreased the

amplification. Increasing the annealing and elongation

temperature from 63 to 66, 67, 68, and 69 jC also

resulted in a decreasing amplification.

Both amplicons were detected in total DNA of the

lactobin A producer as well as in total DNA of the

Fig. 3. Tricine–SDS–PAGE gel showing the zones of inhibition of the purified bacteriocins from L. amylovorus LMG 13135 (a solution of

1200 AU ml� 1 was applied), L. amylovorus LMG 13049 (3200 AU ml� 1), L. amylovorus DCE 471 (6400 AU ml� 1), and L. amylovorus LMG

P-13139 (1600 AU ml� 1). After electrophoresis, the gels were placed on an MRS agar plate and overlaid with MRS soft agar containing L.

delbrueckii subsp. bulgaricus LMG 6901T as indicator organism. The right lane shows standard proteins stained with Coomassie blue.


amylovorin L471 producer but not in the enterocin A

producer (Fig. 2). This indicates that the amplification

was specific for lactobin A. It further indicates that the

overlapping coding parts of the structural genes of

lactobin A and amylovorin L471 were identical. In

both strains, two smaller DNA fragments were also

weakly detected with both primer sets. This could be

explained by a weak, nonspecific, downstream hy-

bridization of the LBN sense primer.

DNA sequencing of the four amplicons obtained

with the two sets of primers and the total DNA of the

lactobin A and amylovorin L471 producers revealed

that the structural genes of lactobin A and amylovorin

L471 were indeed identical (Fig. 1).

The 144-bp and 96-bp PCR fragments could not be

detected in the 38 closely related lactobacilli tested, of

Fig. 4. Growth (expressed as colony-forming units, CFU ml� 1, x), pH (nrye flour fermentation at a constant temperature of 37 jC inoculated with

which 8 were L. amylovorus strains (Table 1). Yet, a

very weak signal was observed with three L. amylo-

vorus strains (LMG 9434, LMG 13049, and LMG

13135), whose effect could be slightly enhanced after

extensive PCR amplification. This indicates that pos-

sibly a similar bacteriocin gene was present. Since no

differences in the quality of template DNA isolated

from the different strains were seen, slight differences

in sequence at the primer sites might explain the

inefficiency of the latter PCR reactions.

3.3. Determination of the inhibitory spectrum of the

isolated bacteriocins

Purified bacteriocin preparations from the six L.

amylovorus strains tested displayed inhibitory activity

), total titratable acidity (�), and bacteriocin production (.) during a

L. amylovorus DCE 471.


against L. delbrueckii subsp. bulgaricus LMG 6901T

(Table 2). L. amylovorus DCE 471 showed an activity

against all L. amylovorus strains tested. The other L.

amylovorus strains could be ranked in order of increas-

ing ability to inhibit other L. amylovorus strains. For

instance, L. amylovorus LMG 13049 inhibited two out

of six of the L. amylovorus strains tested, while L.

amylovorus LMG 9434 did not inhibit any of the L.

amylovorus strains tested. The same order also corre-

Fig. 5. Rep-PCR fingerprints using the (GTG)5 primer of randomly picked

12 h (lanes 13–16), 15 h (lanes 17–20), and 24 h (lanes 21–24) of ferment

fermentor (lane 25), L. plantarum LMG 6907T (lane 26) and L. sakei sub

sponded with a decreasing sensitivity against each

other. The most sensitive strain was L. amylovorus

LMG 9434, followed by L. amylovorus LMG 9496T, L.

amylovorus LMG 13135, L. amylovorus LMG 13049,

L. amylovorus LMG P-13139, and L. amylovorusDCE

471. Bacteriocin preparations from L. amylovorusDCE

471 and L. amylovorus LMG P-13139 displayed sim-

ilar inhibiting activities against other L. amylovorus

strains (Table 2). It was confirmed that the DCE 471

up colonies after 3 h (lanes 1–4), 6 h (lanes 5–8), 9 h (lanes 9–12),

ation; the preculture of L. amylovorus DCE 471 used to inoculate the

sp. carnosus LMG 17302T (lane 27). M: molecular mass marker.

Fig. 6. Bacteriocin production of colonies of L. amylovorus DCE

471 after 3 h (dilution, 10� 6, plate 1), 6 h (dilution, 10� 6, plate 2),

9 h (dilution, 10� 7, plate 3), 12 h (dilution, 10� 7, plate 4), and 15 h

(dilution, 10� 7, plate 5) of fermentation of rye flour that was

inoculated with a culture of L. amylovorus DCE 471 at a constant

temperature of 37 jC. The plates were overlayed with the indicator

strain L. delbrueckii subsp. bulgaricus LMG 6901T.


bacteriocin preparation inhibited L. amylovorus LMG

P-13139 as well; in contrast, the LMG P-13139 prep-

aration did not inhibit L. amylovorus DCE 471 (Call-

ewaert et al., 1999).

3.4. Characterization of the bacteriocins

The inhibitory activity of the purified bacteriocin

preparations of L. amylovorus LMG 9434, L. amylo-

vorus LMG 13049, L. amylovorus LMG 13135, and

of L. amylovorus DCE 471 and L. amylovorus LMG

P-13139 was lost after incubation with the proteolytic

enzymes proteinase K, trypsin, a-chymotrypsin, and

pepsin. This confirmed that an active protein moiety

was responsible for the inhibition of the indicator

strain. Further, the compounds retained full activity

after various heat treatments, including 60 min at 60

jC and 15 min at 100 jC for all L. amylovorus strains

tested. They were only partially inactivated after 60

min at 100 jC in the case of L. amylovorus LMG P-

13139 and L. amylovorus DCE 471 (a decrease with a

factor two) or after autoclaving during 15 min at 121

jC (a decrease with a factor four to eight). All

bacteriocin preparations were active over a wide range

of pH (2.2–8.0). All these data contribute to the

bacteriocinogenic nature of the antibacterial substan-

ces. Finally, tricine–SDS–PAGE showed that all L.

amylovorus strains produced a bacteriocin that dis-

played a molecular mass of approximately 4800 Da,

comparable to that of amylovorin L471 (Fig. 3). In the

case of L. amylovorus LMG 13049, two antibacterial

peptides were detected with a molecular mass of 4800

and 5800 Da, respectively. The purified bacteriocin of

L. amylovorus LMG 9434 was also run on this gel but

did not give an inhibition zone because of its too low

activity (200 AU ml� 1).

3.5. Rye fermentation with L. amylovorus DCE 471

Fig. 4 displays the growth and bacteriocin produc-

tion of L. amylovorus DCE 471 during a rye flour

fermentation at a constant temperature of 37 jC. Theinitial pH value of the dough mixture was 6.3

corresponding with a TTA of 1.4. Samples taken

immediately after inoculation showed a bacterial count

of 2.5� 105 CFU g� 1. Exponential growth of L.

amylovorus DCE 471 started after about 3 h and lasted

till 12 h of fermentation to reach a bacterial count of

3.5� 109 CFU g� 1. This coincided with a fast acidi-

fication of the medium as indicated by the observed pH

drop and increasing TTAvalue. An acidification rate of

0.13 h� 1 could be calculated. The growth was paral-

leled with amylovorin L471 production, underlining its

growth-associated kinetics and potential contribution

in competitiveness of the strain (De Vuyst et al., 1996b;

Messens et al., 2002). Maximum bacteriocin titers (150

AU ml� 1) were observed at the end of the exponential

growth phase, followed by a decrease most probably

due to adsorption to cells, flour particles, etc. (De Vuyst

et al., 1996b). The bacterial count dropped to 2.9� 108

CFU g� 1 after 24 h of fermentation. Then, a maximum

TTA of 18.4 was reached, which coincided with a final

pH value of 3.5. The dominance and competitiveness


of the added L. amylovorus DCE 471 strain has been

shown in two manners. First, identical rep-PCR finger-

prints were found for the preculture and the 24 samples

of four bacterial colonies randomly picked up after 3, 6,

9, 12, 15, and 24 h of rye fermentation (Fig. 5a,b).

Second, all colonies were shown to produce amylo-

vorin L471 throughout the fermentation as revealed by

their inhibition zones towards L. delbrueckii subsp.

bulgaricus LMG 6901T in an overlay assay (Fig. 6).

4. Discussion

The phenotypically different strains L. amylovorus

LMG P-13139 and L. amylovorus DCE 471 have

been identified as producers of a bacteriocin, called

lactobin A and amylovorin L471, respectively, with a

small difference in molecular mass and an identical N-

terminal amino acid sequence (Callewaert et al., 1999;

Contreras et al., 1997). Analysis of the amplified PCR

fragments from the structural bacteriocin genes iso-

lated from total DNA of both strains revealed that

lactobin A and amylovorin L471 are identical bacter-

iocins. They will be further referred to as amylovorin

L. The bacteriocin amylovorin L can be defined as a

small, strongly hydrophobic peptide, consisting of 50

amino acids, that is synthesized as a precursor peptide

of 65 amino acids processed at a characteristic double-

glycine proteolytic cleavage site, and hence belongs to

the class II bacteriocins (Nes et al., 1996). It has a

narrow inhibitory spectrum, being most active to-

wards L. delbrueckii subsp. bulgaricus LMG 6901T

among the strains tested (De Vuyst et al., 1996a;

Callewaert et al., 1999). The production of amylo-

vorin L seems to be associated with only some L.

amylovorus strains since the structural genes were not

detected in other strains of the L. acidophilus DNA

homology group. Nevertheless, almost all L. amylo-

vorus strains (LMG 13135, LMG 13049, LMG 9496T,

and LMG 9434) used in this study displayed antago-

nistic activity against L. delbrueckii subsp. bulgaricus

LMG 6901T. Their antibacterial peptides could be

concentrated and purified by ammonium sulphate

precipitation and chloroform/methanol extraction as

was done for amylovorin L471, lactobin A, and other

small, hydrophobic bacteriocins (De Vuyst et al.,

1996a; Zamfir et al., 1999; Foulquie Moreno et al.,

2003). The sensitivity of all these antibacterial pep-

tides towards several proteases further indicates their

bacteriocin nature. Hence, our data strongly indicate

that all L. amylovorus strains examined produced a

small bacteriocin that is characterized by a narrow

inhibitory spectrum. The latter property seems to be

common for bacteriocins from L. acidophilus (Bare-

foot and Klaenhammer, 1983).

Bacteriocin production and resistance among L.

amylovorus strains seems to be very common. L.

amylovorus DCE 471 showed a bactericidal activity

against all L. amylovorus strains tested. It was resis-

tant against the bactericidal compounds produced by

the other L. amylovorus strains. In contrast, L. amy-

lovorus LMG 9434, the most sensitive strain, dis-

played bactericidal activity only against L. delbrueckii

subsp. bulgaricus LMG 6901T. The other L. amylo-

vorus strains studied varied in bactericidal activity and

resistance. These data could suggest the production of

multiple bacteriocins and their corresponding immu-

nity proteins by each strain. Bacteriocin immunity

proteins specifically protect the bacteriocin producer

against the production of its own bacteriocin (Quadri

et al., 1995). In addition, bacteriocin production may

render bacteria more resistant against other bacterio-

cins (Eijsink et al., 1998). This observation was

suggested to be responsible for the specific action of

bacteriocins within closely related bacteria. It may

play an important role in the competitiveness of the

strains in a particular ecosystem. Moreover, the adapt-

ability of the strain in a food matrix is a very

important criterion in the selection procedure of a

suitable starter strain (Leroy et al., 2002).

All L. amylovorus strains used were isolated from a

similar corn source, except for L. amylovorus LMG

9434 that was isolated from the small intestines of a

pig (Table 1). L. amylovorus LMG 9496T, L. amylo-

vorus LMG 13135, and L. amylovorus LMG 13049

originated from a cattle waste–corn fermentation. L.

amylovorus DCE 471 (De Vuyst et al., 1996a) and L.

amylovorus LMG P-13139 (Contreras et al., 1997)

were isolated from corn steep liquor. Their cereal

origin may indicate the usefulness of L. amylovorus

strains in such an environment. Since an in situ

bacteriocin activity from L. amylovorus DCE 471

was detected during a rye fermentation inoculated

with this culture, and apparently being dominated by

the strain, the bacteriocin production may be respon-

sible for an improved competitiveness of this strain in


a cereal environment. This opens the way for the use

of bacteriocin-producing L. amylovorus strains in both

rye and sorghum fermentations (Messens et al., 2002).

An ecosystem of increasing importance is the

gastrointestinal tract, in particular, with respect to

the use of probiotic bacteria. It is well known that

probiotic LAB contribute to the stabilization of the gut

microflora (Salminen et al., 1996), thereby avoiding

the colonization of pathogenic bacteria through for

instance the production of antimicrobials like organic

acids (Ibrahim and Bezkorovainy, 1993), and preven-

tion of adherence to the epithelial cells through

competitive exclusion (Bernet et al., 1993; Chauviere

et al., 1992) or inhibition of adhesion (Coconnier et

al., 1993). Also, bacteriocins produced by LAB may

play an important role in this complex ecosystem. For

instance, from the 96 strains of the L. acidophilus

group isolated from human intestines, 62 displayed

bacteriocinogenic activity, indicating that bacteriocin

production is widespread among these bacteria

(Kawai et al., 1997). Indeed, almost all members of

the L. acidophilus group produce bacteriocins: L.

acidophilus (e.g. acidocin A, Kanatani et al., 1995),

L. amylovorus (e.g. amylovorin L471, De Vuyst et al.,

1996a), Lactobacillus crispatus (e.g. crispacin A,

Tahara and Kanatani, 1997), L. gasseri (e.g. gassericin

KT7, Zhu et al., 2000), and Lactobacillus johnsonii

(e.g. lactacin F, Fremaux et al., 1993). Interestingly,

the L. acidophilus group encompasses important pro-

biotic strains (Klein et al., 1998). However, up to now,

only bacteriocins from cultures of LAB strains isolat-

ed from faeces have been purified (Kawai et al., 1998,

2000, 2001). Remarkably, several of these bacterio-

cins as well as other bacteriocins from the L. acid-

ophilus DNA homology group share the same primary

sequence such as gassericin T (Kawai et al., 2000) and

lactacin X (Fremaux et al., 1993), lactobin A (Con-

treras et al., 1997) and amylovorin L471 (Callewaert

et al., 1999), and gassericin A (Kawai et al., 1998) and

acidocin B (Leer et al., 1995). They may therefore

play an important role in this complex ecosystem.

As a conclusion, it is likely that bacteriocin forma-

tion by L. amylovorus is interesting for the develop-

ment of probiotic LAB strains of cereal origin, cereals

becoming of growing importance in the development

of functional foods (Charalampopoulos et al., 2002).

It may contribute to a controlled development of the

cereal microflora during cereal fermentation, simulta-

neously involving the addition of a starter culture with

potential health-promoting properties.

Acknowledgements

The research presented was financially supported by

the Research Council of the Vrije Universiteit Brussel

(VUB) and the Fund for Scientific Research—

Flanders. Supports from Dr. Hilde Revets from the

Research Group of Cellular Immunology (VUB), from

Dr. Bruno Pot and Karen Lefebvre from the Laboratory

of Microbiology of Ghent University (RUG), and from

Dr. Danielle Janssens from the BCCM/LMG Bacteria

Collection (RUG) are gratefully acknowledged.

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the lactobin a and amylovorin l471 encoding genes are identical, and their distribution seems to be...

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