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Antibacterial metabolites of lactic acid bacteria: Their diversity and properties

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The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.
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ISSN 00036838, Applied Biochemistry and Microbiology, 2012, Vol. 48, No. 3, pp. 229–243. © Pleiades Publishing, Inc., 2012.
Original Russian Text © L.G. Stoyanova, E.A. Ustyugova, A.I. Netrusov, 2012, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2012, Vol. 48, No. 3, pp. 259–275.
229
For more than a hundred years, lactic acid bacteria
(LAB) have been attracting the attention of research
ers. The effect of LAB on human health and their abil
ity to preserve food are of interest to many scientists.
The association between LAB and human health was
first pointed out by I.I. Mechnikov, who suggested that
the cause of many diseases is the negative effect on
human tissues caused by various toxins and metabo
lites produced by microorganisms that enter the body.
Searching for ways to prevent premature aging, he dis
covered the property of lactic acid bacteria to inhibit
the development of harmful microbes that inhabit the
gastrointestinal tract [1]. For a long time, this fact was
underrated. Only in the 1990s, after a series of success
ful research projects, scientists rekindled their interest
in Mechnikov’s idea about the significant role of LAB
in human health. These studies served as an impetus to
investigate their antimicrobial and probiotic proper
ties.
Since ancient times, mankind has been interested
in increasing the shelf life of food products. Table salt,
sometimes with acetic acid, was a common preserva
tive. These days, the food industry actively uses chem
ical preservatives and antibiotics, which have bacteri
cidal and antifungal properties. However, these preser
vatives cause alarm among consumers due to their
toxicity and the possibility of suppressing the natural
microbiota of the body. The use of LAB and their
metabolites that have antimicrobial properties is one
of the most actively developed alternative approaches
to food preservation. As is known, LAB is closely asso
ciated with food and have a GRAS (Generally Recog
nized As Safe) status, which defines them as absolutely
safe for human and animal health. The main antimi
crobial compounds produced by LAB are organic
acids formed in the process of sugar fermentation,
which results in a rapid acidification of the environ
ment and prevents the growth of other groups of
microorganisms [2]. Recent decades have seen a lot of
data on the production of LAB antimicrobials, which
belong to different classes of organic compounds and
are able to inhibit the growth of other microorganisms.
To extend the shelf life of food products, some of these
metabolites, particularly, bacteriocin nisin, have been
successfully used in industry. Nevertheless, the
demand for new antimicrobial substances is constantly
growing in the food industry, so the separation and
characterization of compounds with antimicrobial
properties produced by LAB is a promising direction
for future research of practical importance. The prop
erties of individual LABproduced antimicrobial
metabolites, their mechanism of action, as well as the
prospects for use as preservatives, will be discussed in
this review.
Properties of LAB.
LAB are a Grampositive,
asporogenic (with the exception of the genus
Sporo
lactobacillus
), catalasenegative, cytochromefree,
air and acidtolerant bacteria, which produce lactic
acid as a major metabolite. In nature, LAB are con
fined to nutrientrich habitats: milk, meat, vegetables;
some species live on plants, whereas others are part of
normal human and animal gastrointestinal microbiota
[3]. Historically, bacteria belonging to the
Lactococcus,
Streptococcus, Pediococcus, Leuconostoc
, and
Lactoba
cillus
genera [4] are the core of this group.
Lactic acid fermentation is an energy source for
LAB. There are two known main pathways of sugar
Antibacterial Metabolites of Lactic Acid Bacteria:
Their Diversity and Properties
L. G. Stoyanova, E. A. Ustyugova, and A. I. Netrusov
Biology Department, Moscow State University, Moscow, Russia
email: stoyanovamsu@mail.ru
Received June 16, 2011
Abstract
—The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bac
teria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes
data on lowmolecularweight antimicrobial substances, which are primary products or byproducts of lactic
fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by
LAB; their potential use as food preservatives has been discussed. The characteristics and classification of
bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the
example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium
Lactococcus lactis
subsp.
lactis
. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective
directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.
DOI:
10.1134/S0003683812030143
230
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
transformation by LAB: glycolysis (EmbdenMeyer
hofParnas pathway) and glucose6phosphate (War
burgDickensHorecker shunt) [5].
As a result of glycolysis, lactic acid is produced with
up to a 98% yield:
С
6
Н
12
О
6
2СН
3
СНОHСООН + E
.
Bacteria belonging to this group are called homolactic
LAB genera. The following LAB are responsible for
homofermentation:
Lactococcus lactis
subsp.
lactis,
L. lactis
subsp.
cremoris, Streptococcus thermophilus,
Lactobacillus delbrueckii
bv.
bulgaricus, L. acidophilus,
L. helveticus,
and
L. casei,
and
L. plantarum
.
Bacteria that utilize the glucose6phosphate path
way during lactic acid fermentation also produce con
siderable amounts of acetic acid, ethyl alcohol, carbon
dioxide, and other neutral byproducts (such as
diacetyl and acetoin). Up to 50% of fermentable hex
oses may be used for the production of these byprod
ucts. This type of fermentation is called heterofermen
tation:
Heterofermentation is a more complex process
than homofermentation. The group of heterofermen
tative LAB include some of rodshaped lactobacilli
(such as
L. brevis
and
L. fermentum
), their subgenus
Streptobacterium
, some cocci (such as
Streptococcus
acetoinicus
), and all species of
Leuconostoc
[5].
LOWMOLECULARWEIGHT
ANTIMICROBIAL LAB METABOLITES
A closer examination of LAB over the past decades
has revealed their ability to produce antimicrobial sub
stances of different structures [3]. Besides lactic acid,
many LAB strains produce a significant amount of
nonspecific lowmolecularweight compounds, such
as organic acids, hydrogen peroxide, diacetyl, reu
terin, etc., which define the spectrum of their antimi
crobial activity.
Organic acids.
As stated above, lactic acid and ace
tic acid are the main endmetabolites formed by LAB
during fermentation. When compared to lactic acid,
acetic acid has a broader spectrum of antimicrobial
activity. At the same time, it is known that a synergistic
effect exists between both acids: mixtures of acetic and
lactic acid suppress growth of the pathogenic Gram
negative enteric bacterium
Salmonella
typhimurium
[5]. It was noted that Llactate has higher inhibitory
activity than its Disomer [6]. Various microorganisms
react differently to the acidity of the environment. For
example, at pH below 5.0, lactic acid inhibits the
growth of sporeforming bacteria but does not affect
the growth of microscopic fungi and yeasts.
Hydrogen peroxide.
In the presence of oxygen,
LAB can also produce
Н
2
О
2
when subjected to
NADH oxidase and superoxide dismutase activities.
When heme is absent from the environment, LAB
C H O CH CHOHCOOH
+ CH CH OH or CH COOH)+CO E.
6126 3
32 3 2
(
+
does not produce catalase; this results in peroxide
accumulation. The peroxide effect can be amplified in
the presence of lactoperoxidase and thiocyanate,
which are present in natural LAB habitats, such as
milk [7]. The antimicrobial activity of hydrogen per
oxide is linked to the strong oxidizing effect. The
observed growth inhibition of
Lactococcus
and Gram
negative
Pseudomonas
spp., responsible for food con
tamination, was due to the accumulation of peroxides
by
Lactococcus
and
Lactobacillus
.
Pyrrolidone5carboxylic acid.
This acid is only
produced by certain types of LAB, such as
Lactobacil
lus casei
ssp.
casei
and
L. casei
ssp.
pseudoplantarum
,
and has bactericidal activity against
Bacillus subtilis
and
Enterobacter cloacae
[6].
Diacetyl.
It is the component responsible for the
characteristic aroma in butter. It is formed during
transformation of citrate via pyruvate. The maximum
formation of diacetyl is observed under slightly acidic
pH. It is mainly active against Gramnegative bacte
ria, belonging to the genera
Salmonella
,
Yersinia
,
Escherichia
, and
Aeromonas
, and also against Gram
positive bacteria belonging to the genus
Bacillus
[8].
Reuterin (
β
OHpropionic aldehyde).
This com
pound is formed under anaerobic conditions from
glycerol by
Lactobacillus reuteri
,
L. brevis
,
L. buchneri
,
L. collinoides,
and
L. corniformis
[9]. Reuterin exhibits
antagonistic activity against many pathogenic micro
organisms, including enterobacteria (
Salmonella
and
Shigella
); bacteria genera
Clostridium
,
Staphylococcus
,
and
Listeria
; yeasts of the genus
Candida
; and proto
zoa of the genus
Trypanosoma
. A wide range of activi
ties results from the ability of reuterin to bond with
SHgroups of enzymes, including ribonucleotide
reductase [7].
ANTIFUNGAL AGENTS OF LAB
Antifungal activity is not a characteristic physio
logical property of LAB. Many strains of micro
mycetes are sensitive to lactic and acetic acids pro
duced by LAB. However, a series of recent publica
tions has traced the production of specific antifungal
compounds by LAB strains. The largest number of
these strains was isolated from plants that were kept
under anaerobic conditions [9]. The following types of
LABproduced antifungal compounds are known:
diketopiperazine (circular dipeptides cyclo(GlyL
Leu), cyclo(LPheLPro) and cyclo(LPhe
trans
4OHLPro)); hydroxy derivatives of fatty acids (p
hydroxyphenyllactate); benzoic acid; methylhydan
toin; mevalonolactone; pentocin TV35b; and reuterin.
The first three types of compounds are described in
more detail in [10].
Diketopiperazines.
For a long ti me, it has b een con 
sidered that these compounds are products of protein
degradation. The mechanism of their formation still
remains unclear. Nevertheless, it was found that the
synthesis occurs via a nonribosomal pathway that uses
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
ANTIBACTERIAL METABOLITES OF LACTIC ACID BACTERIA 231
a multifunctional enzyme. Diketopiperazines can also
be formed from peptides in alkaline or acidic environ
ments. Diketopiperazines produced by some LAB
strains [11] are given in Table 1. Recently, a new com
pound with antifungal activity has been separated
from
Lactobacillus plantarum
AF1. It was identified as
3,6bis(2methylpropyl)2,5piperazinedione. This is
the only publication that mention the antifungal activ
ity of LAB, which resulted from the activity of a cyclic
compound (LeuLeu) that belongs to 2,5diketopip
erazines [12].
Hydroxy derivatives of fatty acids.
Some LAB pro
duce 2hydroxyhexane and 3hydroxyheptadecane
carboxylic acids, which belong to this class of com
pounds. The MiLABH strain of the genus
L. plan
tarum
produces several hydroxylated fatty acids with
strong antifungal activity: 3hydroxydecanoic acid,
3hydroxydodecanoic acid, 3hydroxytetradecanoic,
and 3hydroxy5cisdodecanoic acids. LAB produce
hydroxy derivatives of fatty acids from their unsatur
ated counterparts. All of the above unsaturated fatty
acids exhibit antibiotic activity against a broad range
of yeasts and mold [9]. The total inhibitory activity of
hydroxy fatty acids ranges from 10 to 100 mg/ml due
to their poor solubility in aqueous solutions.
3Phenyllactate.
The compound is a metabolite in
phenylalanine metabolism and can be formed in LAB
cells from phydroxyphenylpyruvic acid. This end
metabolite exhibits antibiotic activity against Gram
positive and Gramnegative bacteria and also affects a
wide range of microscopic fungi [13]. According to lit
erature,
L. plantarum
can produce several related
compounds: phenyllactic acid, 4hydroxyphenyllac
tic acid, and 3hydroxyphenyllactic acid; while
L. coryniformis
,
L. sakei
, and
Pediococcus pentosaceus
only produce phenyllactic acid [14]. Apart from lacto
bacilli, propionibacteria also synthesize phenyllactic
acid. Figure 1 shows its structural formula.
There are only a few literature references that con
tain information on lactococci possessing antifungal
activity. Specifically, the
Lactococcus lactis
LI4 strain
that inhibits the growth of
Candida albicans
DMST
5239 was isolated from cultured dairy products. The
activity of this strain was sustained in the pH range
2.0–4.0 and retained even after autoclaving [15]. Roy
et al. [16] isolated the antifungal peptide component
from an
L. lactis
culture. In addition, the inhibition of
growth and aflatoxin production by
Aspergillus flavus
fungi at cocultivation with lactococci were described.
In this case, the active component was a phosphogly
colipid with a low molecular weight of less than 500 dal
tons [7] and containing an aromatic ring. A thermostable
compound with a low molecular weight, which loses
activity during prolonged storage, was responsible for the
inhibition of aflatoxin production [17].
Lactococci strains with a broad range of antibiotic
action, including antifungal activity, were isolated at
the Department of Microbiology, Moscow State Uni
Table 1.
Diketopiperazines synthesized by lactic acid bacteria [11]
Diketopiperazines Producer Structural formula
Cyclo(LPheLPro)
Lactobacillus plantarum
MiLAB 393,
L. coryniformis
Si3
Cyclo(LPhe
trans
4OHLPro)
L. plantarum
MiLAB 393,
L. coryniformis
Si3
Cyclo(LPhe
cis
4OHDPro)
Pediococcus pentosaceus
MiLAB 170,
L. plantarum
MiLAB 14
N
NH
H
H
O
O
N
NH
H
H
O
O
H
HO
N
NH
H
H
O
O
H
HO
O
OH
HO H
Fig. 1.
The structure of 3phenyllactate [14].
232
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
versity. These strains inhibited the development of
fungi belonging to the genera
Aspergillus
and
Fusarium
and yeast genera
Candida
and
Rhodotorula
. They
included natural strains of
Lactococcus lactis
subsp.
lactis
isolated from fresh cow milk and cultured dairy
products, as well as strains produced by cell engineer
ing [18, 19]. The lactococci strains identified as
Lacto
coccus lactis
subsp.
lactis
synthesized alkyl ketones. These
compounds defined the antifungal activity of those
strains [20], suggesting that these lactococci can be
potentially used to prevent spoilage of fruits and vegeta
bles due to contamination by fungi and yeast [21–23].
BACTERIOCINS OF LAB, THEIR
CLASSIFICATION AND PROPERTIES
Bacteriocins are heterogeneous antimicrobial pep
tides with varied levels and spectra of activity, mecha
nisms of action, molecular weight, and physicochem
ical properties. It is known that many microorganisms
are able to synthesize bacteriocins, but LAB bacterio
cins are of the greatest interest as potential novel pre
servatives. Studies on bacteriocins produced by LAB
began in the 1930s, starting from investigations of nisin
produced by the lactococcus
L. lactis
subsp.
lactis
[24].
So far, a large number of bacteriocins synthesized as
Grampositive and Gramnegative bacteria have been
studied and fully characterized. The group of bacteri
ocinproducing LAB includes representatives of dif
ferent genera:
Lactococcus, Lactobacillus, Leuconos
toc, Pediococcus
, and
Streptococcus
. The synthesis of
bacteriocin is strainspecific. The greatest number of
bacteriocinproducing bacteria have been isolated
from fermented dairy and meat products and silage,
but a number of strains (
Enterococcus durans, Lacto
bacillus animalis, Leuconostoc
sp.) were isolated from
soil samples [25]. There is evidence that nisinproduc
ing strains of
L. lactis
subsp.
lactis
were isolated from
breast milk [26]. Bacteriocins differ from classical
antibiotics by three main properties: the synthesis of
bacteriocins occurs on ribosomes, bacteriocins have a
specific spectrum of activity, and each bacteriocin has
its own specialized immune protein [27].
According to the chemical structure, LAB bacteri
ocins are divided into several classes.
Class I includes lantibiotics, which are peptides
with modified amino acids (lanthionine,
β
methyl
lanthionine). This class contains two types of lantibi
otics:
1. Type A includes linear flexible peptides, which
form pores in the bacterial membrane, for example,
nisins [28].
2. Type B includes “hard” globular peptides, which
are either uncharged or negatively charged. Mersaci
din, actagardine, and cinnamycin are type B lantibiot
ics [29, 30].
Class II includes peptides, which do not contain
modified amino acids. It is divided into three main
groups:
1. Subclass IIa bacteriocins include peptides with
the specific conserved Nterminal sequence TyrGly
AsnGlyValXaaCys (Xaa denotes any amino acid).
Usually, they are thermostable and contain from 37
(leucocin A and mesentericin Y105) to 48 (carnobac
teriocin B2 and enterocin SEK4) amino acids [31]
and are characterized by high activity against patho
genic bacteria of the genus
Listeria
, which is often
found in raw foods.
2. Subclass IIb consists of twopeptide bacteriocins
with a double glycine–type leader sequence, which
includes lactococcin G, plantaricin E/F, lactacin F,
and thermophilin 13.
3. Subclass IIc is represented by circular bacterio
cins with covalently linked C and Nterminus [32].
Class III bacteriocins consist of large (with a
molecular mass of more than 30 kDa) nonlantibiotics
and heatlabile proteins, such as helveticin J and lact
acin B [33].
Class IV consists of complex bacteriocins contain
ing both proteins and lipids or carbohydrate compo
nents [34]. The information about this class of bacte
riocins is limited and contradictory.
One type of LAB, and even one strain, can produce
bacteriocins of different classes. For example,
Lacto
coccus lactis
subsp.
lactis
produces the following lanti
biotics: nisin
s А, В, С,
L, Z, Q, and F [35]; lacticins
481, and twopeptide lantibiotic lacticin 3147 [36].
Lactococci produce lactococcin 972 [37] and lacticin
QU 5 [38] out of secondclass bacteriocins. Lactocy
clin Q is a recently discovered circular bacteriocin pro
duced by
Lactococcus
sp. QU 12 [39].
As shown by a comparative study of strains con
ducted at the Department of Microbiology, Moscow
State University, the synthesis of bacteriocins is strain
specific [20]. The following strains were studied: natu
ral strains 194 and 119x, the traditional nisinproduc
ing MSU strain obtained by adaptive selection, and the
recombinant F116 strain obtained by protoplast fusion
of two congenic strains (729 and 1605). Strain 729 is a
natural strain isolated from milk; strain 1605 is a
mutant obtained via induced mutagenesis.
Physicochemical properties of bacteriocins, syn
thesized by the 119x and MSU strains, did not differ
from those of nisin (Fig. 1), but an antibiotic complex
was isolated from the F116 hybrid strain. This com
plex is a mixture of biologically active components
that consist of three separate fractions with different
physicochemical and biological properties (Table 2).
The main fraction of the antibiotic complex is a pep
tide fraction, which is only active against Gramposi
tive bacteria, including sporogenic acidresistant
В.
coagulans
that is dominant in canned and preserved
food products. That fraction was a polypeptide con
verted into two subunits with molecular mass of 3353
and 3376 Da under acidic conditions. The chemical
structure and antibiotic properties of that fraction are
similar to that of nisin. A chromatographically pure
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
ANTIBACTERIAL METABOLITES OF LACTIC ACID BACTERIA 233
antifungal component with a molecular mass of
506.9 Da was separated from the other fractions.
Based on the IR spectrum, it was assigned to a group
of alkylaromatic ketones, which also contain hydroxy
groups (Fig. 2, Table 2.). The third fraction is a minor
one with insignificant biological activity. The physico
chemical and biological properties of the selected
components of the antibiotic complex produced by
strain F116 are shown in Table 2. The data were ana
lyzed using the Biologically Active Natural Products
Database (BNPD) [40]. It led to the conclusion that
the main components of the peptide fraction (LGSH
and LGSH
1
) are nisins, and LGSH (with a molecu
lar mass of 3353 Da) is absolutely identical to nisin A.
The other two components, including the antifungal
component, have not previously been described in lit
erature and are new natural biologically active com
pounds [41].
Lantibiotics.
Lantibiotics (lanthioninecontaining
peptides) are polycyclic molecules produced as a result
of posttranslational modifications of precursor pro
teins. They contain modified amino acids [42]. Their
molecules contain sulfhydryl rings formed by sulfur
containing amino acids. LABproduced lantibiotics
vary in the number of thioether bonds (from two to
five) and molecular masses (Table 3) [43].
Nisin is the most elaborated lantibiotic. It is effec
tive against many strains of Grampositive bacteria,
including staphylococci, streptococci, bacilli, and
clostridia, and, to a lesser extent, it is also active against
mycobacteria [44]. Nisin at a concentration of 0.3 mg/ml
can inhibit the spore germination of bacteria belonging to
the genera
Bacillus
and
Clostridium
to the same extent as
heat treatment [8]. It was found that nisin interacts with
sulfhydryl groups in the spore membrane, thereby pre
venting its germination [45, 46].
Twopeptide lantibiotics were not allocated to a
separate class of bacteriocins. Plantaricin W is pro
duced by
Lactobacillus plantarum
, and lacticin 3147 is
produced by
Lactococcus lactis
. Twocomponent lan
tibiotic lacticin 3147 has a relatively broad spectrum of
activity. It is active against methicillinresistant strains
of
Staphylococcus aureus
, vancomycinresistant strains
of
Enterococcus faecalis
, penicillinresistant
Pneumo
coccus
, as well as
Propionibacterium acnes
,
Streptococcus
mutans, and foodborne pathogens (
Listeria monocyto
genes
,
Staphylococcus aureus
, and
Bacillus cereus
) [36].
Lacticin 3147 at a concentration of 20000 IU/ml com
pletely suppressed the pathogen population number
Table 2.
Physicochemical and biological properties of the components of a bacteriocinlike complex formed by a hybrid
strain of
Lactococcus lactis
subsp.
lactis
F116 [20]
Properties Component
LGSB LGSC LGSH
1
* LGSH* Nisin
A
Mol.
mass, (M + H)
+
,
m
/
z
, (MALDIMS) 506.9 3161.6 3353 3353**
UV spectrum,
λ
max
, nm, (solvent)
260 (С
2
Н
5
ОН) 215; 274 (С
2
Н
5
ОН) 215 (Н
2
О) 215 (Н
2
О) 215 (Н
2
О)
TLC (SiO
2
),
R
f
in the system:
methanolH
2
O (96 : 4)
0.75 0.43 0 0 0
Electrophoresis on paper** in electrolyte
:
1)
E
1
, pH = 2.4, 550
V, 2 h, cm
00119.59.5
2)
E
2
, pH = 1.1, 250
V, 3 h, cm
004.33.33.3
Biological spectrum of action bacteria and
fungi Weak e f fe c t
on Gram+ bacteria Gram+ bacteria, including heatre
sistant
B. coagulans
Notes: * The LGSH fractions were obtained by preparative paper electrophoresis in electrolyte
E
1
(550, 2.5 h). They were stained with
the Pauly reagent using bioautography with
B. subtilis
and
B. coagulans
as test organisms.
** The distance traveled by a substance from the starting line toward the cathode in cm: “0 cm” (at the start line), electrically neu
tral substance; “3.3–11 cm” (migration toward the cathode), basic substance.
729 1605 194 F119 MSU strain Nis
Fig. 2.
Thin layer chromatography of bacteriocinlike
complexes, produced by the
Lactococcus lactis
subsp.
lactis
F119 hybrid strain and its parental strains compared to
nisin, nisinproducing MSU strain, and natural strain 194
[20] in a methanol–water (96 : 4) system; bioautography
on Silufol plates; the test organism was
B. coagulans
.
234
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
ing at a rate of more than 10
4
cells in 2 h [47]. Both
peptides of lacticin 3147, as well as peptides of planta
ricin W, have bactericidal activity, but the greatest
activity was observed for the synergistic action of these
peptides when used in the ratio 1 : 1. Plantaricin W
contains unmodified cysteine and serine amino acid
residues, which is the only example among lantibiot
ics. Some authors propose to allocate these two bacte
riocins to a separate family [48]. Different genetically
diverse strains of
Lactobacillus salivarius
isolated from
the human intestine can synthesize twocomponent
bacteriocins. The bacterium
L. salivarius
is included
in probiotic compositions, because salivaricin P
reduces the number of bacteria of the family
Entero
bacteriaceae
and does not affect the total number of
lactobacilli in the gut [49].
Biosynthesis and formation of mature lantibiotic
molecules.
Lantibiotics are synthesized on ribosomes
as peptideprecursors and undergo an intensive post
translational modification [50]. Lantibiotic synthesis
can be divided into several main steps [51]: (1) riboso
mal synthesis; (2) dehydration of serine and threonine
with the formation of 2,3dehydroalanine and (Z)
2,3dehydrobutyrine, respectively; (3) stereoselective
addition of cysteine to dehydroalanine and dehy
drobutyrine with the formation of thioether rings of
lanthionine (Lan) and
β
methyllanthionine
(MeLan), respectively; (4) export of the fully modified
precursor by the ABCtransporter; (5) proteolytic
cleavage of the leader peptide [52].
Figure 3 shows a nisin biosynthesis diagram [53].
The NisA enzyme binds to the Nterminus, which is
called the leader peptide. NisB and NisC catalyze the
lactam formation: NisB is a dehydratase, which acts
on serine and threonine, and NisC is a cyclase, which
forms all thioether rings [54, 55]. Lanthionine bridges
that form ring structures serve as stabilizers of confor
mations, which are essential for biological activity and
lantibiotic resistance to proteases. The removal of the
leader peptide from a prepeptide is catalyzed by the
NisP enzyme and is the final processing stage [56]. A
leader peptide has a conserved sequence of 24–30
amino acid residues, and it is essential to tag the pre
cursor molecule and to prevent its activation within
the cell [42].
In the process of lacticin 481 maturation, the LctM
enzyme catalyzes both prepeptide modification reac
tions: the dehydration of serine and threonine and the
formation of three thioether cycles. The leader peptide
is cleaved by the Nterminal LctT domain (ABC
transporter), which also excretes a mature product.
Lantibiotic prepeptide conversion into mature mole
cules requires the release of energy from ATP and the
presence of Mg
2+
[57].
Lantibiotic biosynthesis genes are organized in
clusters. In addition to the structural peptide genes,
there are genes that are important for the manifesta
tion of the activity of bacteriocins. These genes encode
the following [51]:
(1) enzymes, which participate in a number of
modifications: LanC catalyzes the dehydration of
serine and threonine, while LanB is responsible for the
formation of thioether bonds between cysteine and
dehydrated amino acids;
(2) LanT are proteins, which export the prepeptide
from a cell;
(3) LanI are immune proteins, which confer the
resistance;
(4) LanP is a serine protease, which cleaves the
leading sequence during the release of lantibiotics;
(5) other regulatory proteins.
Synthesis of lantibiotic molecules is provided by
the structural
lan
gene. In many cases,
lanA
is the first
gene in an operon and encodes the precursor molecule
[42]. The genes responsible for the modification
(
lanB, lanC, lanM, land
, and
lanJ
), processing (
lanP
and
lanT
), transport (
lanT
), immunity (
lanI, lanEFG
,
and
lanH
), and regulation (
lanR, lanK, lanQ,
and
lanX
) are localized near the structural gene. The gene
clusters involved in the synthesis and regulation of
bacteriocins can be located on the chromosome or on
plasmids. Usually, the genes of sensory and regulatory
proteins are transcribed together in a locus associated
with the synthesis of lantibiotic; genes encoding sen
sory proteins follow the regulatory genes. Figure 4
shows nisin and lacticin 481 gene clusters [51].
The transcription of the structural
nisA
gene is
directly regulated by a secreted and fully modified
nisin molecule via a twocomponent signal transduc
tion regulatory system [58]. In this case, nisin acts as a
pheromone with antimicrobial properties [59]. The
initiation from the
nisA
gene promoter is associated
with the products of the
nisR
gene, which encodes a
response regulator, and the
nisK
gene, which encodes
a histidine kinase [60]. The required concentration of
nisin for induction is less than 14 ng/ml. At the first
stage, a NisK histidine kinase responds to the presence
of nisin molecules in its environment and then is sub
jected to autophosphorylation in the cytosol. Then the
phosphate group is transferred to NisR, which is a pro
teinactivator of transcription during nisin synthesis. At
Table 3.
Characteristics of some LABproduced lantibiot
ics [43]
Lantibiotic Molecular
mass, Da Number
of thioethers Producer
Nisin A 3353 5
Lactococcus
lactis
Nisin Z 3330 5
L. lactis
Lacticin 481 2901 3
L. lactis
Lacticin S 2764 3
Lactobacillus
sake
Carnocin U149 4635 2–3
L. pisicola
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
ANTIBACTERIAL METABOLITES OF LACTIC ACID BACTERIA 235
1
5
10
15
20
25
30
34
–5 –10 –15 –20
–1 *
COOH
NH
2
Nisin precursor A
NisB, NisC
*
*
*
**
****
**
*
1
5
10
15
20
25
30
34
–5 –10 –15 –20
–1
COOH
NH
2
1
5
10
15
20
25
30
34
COOH
H
2
N
S
SSS
S
AB C DE
SS
S
S
S
Ile
Arg
Pro
Ser
Ala
Gly Asp Lys Val Leu Asn Phe Thr Mat
HisCys
Ile
Pro
Ser Ser Ser Ser
Ser
Ser
Ser
Ser
Ala
Ala
Gly
Asp Lys Lys
Lys
Lys Val
Val
Ile
Lys
Leu
Leu
Asp
Asn
Leu
Thr
Thr
ThrThrThr Mat
Mat
His
Cys Cys Cys
Cys
Gly
Gly
Ile
Arg
Pro
Ser
Ala
Gly Asp Lys Val Leu Asn Phe Thr
HisAla
Ile
Pro
Ser Ser Ser Ser
Dha
Ser
Ala
Dha
Ala
Ala
Gly
Asp Lys Lys
Lys
Lys Val
Val
Ile
Lys
Leu
Leu
Asp
Asn
Leu
Abu
Abu
Abu
AbuDhb Mat
Mat
His
Ala Ala Ala
Ala
Gly
Gly
Ile HisAla
Ile
Pro
Dha
Ser
Ala
Dha
Ala
Ala
Gly
Lys
Lys
Val
Ile
Lys
Leu
Leu
Asn Abu
Abu
AbuAbuDhb Mat
Mat
His
Ala Ala Ala
Ala
Gly
Gly
Prenisin А
NisT, NisP
Nisin А
Fig. 3.
Schematic representation of nisin biosynthesis [53]; NisBdehydratase acts on serine and threonine; NisC cyclase forms
thioether rings; the NisT enzyme is fully engaged in the export of the modified precursor; and the NisP enzyme is responsible for
the removal of the leader peptide from the prepeptide.
236
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
nisA nisB nisT nisC nisI nisP nisR nisK nisF nisE nisG
lctA lctM lctT lctF lctE lctG
Fig. 4.
Gene clusters of nisin and lacticin [51]:
nisA
and
lctA
are the structural genes of nisin and lacticin, respectively;
nisB
and
nisC
are the genes responsible for the modification of nisin molecules;
nisP
and
nisT
are the genes responsible for the processing
and transport of nisin;
nisI
and
nisEFG
are the genes responsible for the immunity of cellproducers to nisin; and
nisR
and
nisK
are the genes involved in the regulation of production. Lacticin genes with identical functions have the same designation.
the third stage, the precursor is modified by the NisB and
NisC enzymes, and after that it is translocated across the
membrane by ABCtransporter NisT. The leader peptide
is cleaved by the NisP protease [53].
Production of lacticin 481 by some strains of
L. lac
tis
is regulated by extracellular pH. The acidification of
a medium with lactic acid stimulates the expression of
both promoters [42].
Mechanism of lantibiotic action.
Bacteriocins of
lactic acid bacteria are cations and act on the mem
branes of target cells, which results in pore formation
and dissipation of the transmembrane ion gradients
[61]. Lantibiotics do not require a specific receptor
protein, since they bind to lipid II (undecaprenyl
pyrophosphorylMurNAs(pentapeptide)GlcNAc)
at sites of cell wall synthesis. Lipid II participates in the
synthesis of peptidoglycan. It is a highly dynamic mol
ecule, which is present in all bacteria. It carries out the
transport of cell wall subunits across the cytoplasmic
membrane [62]. It is a bactoprenollinked cell wall
precursor that consists of a peptidoglycan head
(Nacetylmuramic acid pentapeptide (MurNAc)),
the basic building block of the cell wall (glutamine
Nacetylglucosamine (GlcNAc)), and the undeca
prenylpyrophosphate (PP) tail, which acts as a carrier
of peptidoglycan from the cytoplasm. Lipid II is com
posed of 11 polyisoprene residues, in which pyrophos
phate MurNAcpentapeptide is linked to GlcN
(glutamine Nacetylglucosamine). Lipid II is synthe
sized within the cell on the cytoplasmic membrane via
lipid I, which is a complex of UDPMurNAcpen
tapeptideGlcNAc with undecaprenyl pyrophos
phate. Subsequently, lipid II moves to the outer side of
the membrane, and two amino sugars get attached to
the cell wall. The process can be repeated; undecapre
nyl pyrophosphate can be transported back to the
outer side of the membrane after undecaprenyl pyro
phosphate is dephosphorilated; and the cycle can be
repeated.
The mechanism of lantibiotic action is most exten
sively studied for nisin. There are two factors, which
play an important role in the interaction between nisin
and the membranes of sensitive cells. They are the
presence of negatively charged lipids in the membrane
and the membrane potential [63, 64] and the thickness
of the cell wall and the availability of lipid II [65].
Nisin has a double effect on the target cell. When it
is linked to lipid II, it blocks the synthesis of cell walls
and, in some cases, may use lipid II to secure itself in
the membrane and to initiate pore formation. At high
concentrations of nisin, pores can be formed in the
absence of lipid II. In this case, the maximum activity
of nisin was observed at a 50–60% concentration of
negatively charged lipids in the membrane [66].
Figure 5 shows the suggested mechanism of nisin
action. At the initial stage, nisin binds to the pyro
phosphate of lipid II [66–68]. Then the Cterminus of
the molecule is transferred through the membrane and
appears on its inner side. At this stage, the most impor
tant thing is the mobility between clusters A, B, C, and
D, E in the nisin molecule. The interaction with lipid
II stabilizes the transmembrane orientation of the
peptide, which results in the formation of a stable pore
of 2 nm in diameter [69]. Eight molecules of nisin and
four molecules of lipid II are involved in the formation
of one pore [69, 70].
Different levels of bacterial sensitivity to nisin are a
result of different concentrations of lipid II in the
membrane. It is known that in
E.coli
, the content of
this molecule is much lower (
2
×
1
0
3
molecules per
cell), while in
Micrococcus flavus
it is 1
×
10
5
. In gen
eral, class I bacteriocins use lipid II for specific mem
brane binding [71].
The action mechanism of lantibiotics with similar
structures of A and B rings depends on lipid II [62]. In
particular, mersacidin and actagardine (type B lantibi
otics) also form a complex with lipid II, but this bind
ing only blocks the incorporation of lipid II into pep
tidoglycan, which leads to slow cell lysis [72]. It is
assumed that mersacidin blocks the peptidoglycan
synthesis at the level of transglycosylation, but in this
case pores are not formed in the membrane [42].
Microorganisms that produce bacteriocins have a
protection system from their own products; they use
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
ANTIBACTERIAL METABOLITES OF LACTIC ACID BACTERIA 237
the gene expression of immune proteins. The sensitiv
ity of the producer varies and depends on the level of
bacteriocin production [73]. Immune proteins exhibit
a considerable variability: different immune proteins
may correspond to nearly identical bacteriocins. NisI
is a known protein immune to nisin and has a lipopro
tein signal sequence encoded by the
nis
I gene of nisin
biosynthesis operon. The NisI protein is attached to
the outer membrane surface of the cellproducer,
where it ties up nisin, decreasing its local concentra
tion [42].
Properties and biosynthesis of class II bacteriocins.
The second class of bacteriocins consists of unmodi
fied peptides, which are subdivided into three sub
classes. Subclass IIa consists of pediocinlike bacteri
ocins, subclass IIb consists of twopeptide bacterio
cins, and subclass IIc includes circular bacteriocins.
Table 4 shows the main bacteriocins from subclasses
IIa and IIb and their producers. These bacteriocins
differ in molecular mass [74–99].
Many of these peptides are stable at low pH and
inhibit the growth of Grampositive bacteria, which
are responsible for food contamination (
Bacillus
cereus
,
Clostridium perfringens
,
Staphylococcus aureus
,
Listeria monocytogenes
). For example, the antibacterial
activity of lacticin Q produced by
L. lactis
subp.
lactis
QU 5 is simil ar to tha t of nisi n A. Nevertheless, l acticin Q
is more stable under basic conditions and more effective
against many Grampositive bacteria, while lacto
coccin A, lactococcin Q, and lactococcin 972 inhibit the
growth of only few lactococci strains [100].
All bacteriocins in the IIa subclass are cationic
peptides that consist of two structural units: a highly
conserved Nterminus with a typical sequence Tyr
GlyAspGlyVal and less conservative Cterminal
domain [101]. The Nterminus forms three antiparal
lel
β
layers supported by disulfide bridges; the Cter
minus forms one or two amphiphilic
α
helixes. In
class II bacteriocins, positively charged amino acids
required for the interaction with the membrane are
G
N
Pi
+
Nisin Pore formation
MGG
NN
Pi Pi
Pi
Pi
Pi
+
+
+
+
++
+
+
MM
C
CC
O
HO
HO
CH2
OH
NH
CO
CH
3
OO
O
CH2
OH
NH
CO
CH3
O
CH
CO
LAla
DGlu
LLys
DAla
DAla
PO
O
O
POO
O
82
NA, B, C D, E C
+
Nisin clusters
++
Lipid II
Fig. 5.
Model of lipid II–dependent pore formation by nisin [66]. G is glutamine Nacetylglucosamine; M is Nacetylmuramic
pentapeptide.
238
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
mostly located in the hydrophilic Nterminus. The
Cterminus of a bacteriocin is responsible for defining its
specificity and for the penetration of the membrane,
while the hydrophilic
β
folds of the Nterminal are
responsible for attaching to the membrane surface [31].
The structural genes of many bacteriocins that do
not contain lanthionine are located on plasmids,
except for divercin V41 [102], sakacin P, and carno
bacteriocin B2 [103]. In some cases, one plasmid can
carry genes for the synthesis of several bacteriocins,
such as plasmid r9V46, which encodes lactococcins
A, B and M, and the corresponding immune proteins.
In contrast, different individual plasmids from differ
ent strains may encode the same bacteriocin. For
example, to encode lactococcin À, there are three dif
ferent plasmids in two subspecies of
L. lactis
subsp.
lactis
. Usually, genes are organized into one or more
operons. In pediocin PA1 and plantaricin 423, all
four genes required for biosynthesis are localized in
one operon. In other cases, genes are distributed over
several operons: one operon carries the structural and
immunity genes, the second one carries the secretion
genes, and the third one carries the genes encoding the
regulators of biosynthesis [31].
Subclass IIa bacteriocins are synthesized as precur
sors with a signal sequence. Prepeptide molecules are
identical for sakacin A and curvacin A, for lantibiotic
lacticin 481 and lactococcin DR, and for sakacin 674
Table 4.
Class II bacteriocins and their producers
Subclass IIa bacteriocins
Bacteriocin Producer Molecular mass, Da Reference
Sakacin
A
Lactobacillus sakei
Lb 706 4300 [74]
Sakacin 674
L. sake
Lb764 4437 [75]
Lacticin Q
Lactococcus lactis
QU 5 5926 [38]
Lactococcin
A
L. lactis
subsp
. cremoris
LMG 2130 5778 [76]
Lactococcin 972
L. lactis
subsp.
lactis
IPLA 972 7500 [77]
Lactococcin DR
L. lactis
subsp.
lactis
ADRIA 85L030 3400 [78]
Carnobacteriocin
A
Carnobacterium piscicola
LV17A 5052 [79]
Carnobacteriocin B2
C. piscicola
LV17 4969 [80]
Curvacin
A
Lactobacillus curvatus
LTH1174 4309 [81]
Divercin V41
Carnobacterium divergens
4509 [82]
Divergicin M35
C. divergens
M35 4518 [83]
Lactococcin MMFII
L. lactis
MMFII 4144 [84]
Leucocin
A
Leuconostoc gelidum
UAL 187 39 30 [8 5]
Mesentericin Y105
L. mesenteroides
Y105 3446 [86]
Plantaricin 423
Lactobacillus plantarum
423 3500 [87]
Pediocin PA1
Pediococcus acidilactici
PAC 1.0 4629 [88]
Pedioci
AcH
P. acidilactici
AcH 4628 [89]
Sakacin
G
Lactobacillus sake
2512 3834 [90]
Bacteriocins
subclass IIb
Lactococcin G
L. lactis
LMGT2081 4346; 4110 [91]
Lactococcin MN
L. lactis
9B4 4325; 4377 [51]
Lactococcin Q
L. lactis
QU 4 4260; 4018 [92]
Plantaricin E/F
Lactobacillus plantarum
C11 2687; 2758 [93]
Plantaricin J/K
L. plantarum
C11 2921; 3508 [94]
Plantaricin NC8
L. plantarum
NC8 3587; 4000 [95]
Lactacin F
Lactobacillus johnsonii
VPI11088 2500; – [96]
Lactacin 705
Lactobacillus casei
CRL 705 3357; – [97]
Thermophilin 13
Streptococcus thermophilus
Sfi13 5776; 3910 [98]
Leucocin H
Leuconostoc
MF215B –* [99]
* The molecular mass is not determined due to the inconstancy of the peptide amino acid composition.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
ANTIBACTERIAL METABOLITES OF LACTIC ACID BACTERIA 239
and sakacin R. It is anticipated that the prepeptide
directs the polypeptide in the secdependent path of
excretion, which also includes the stabilization of the
prepeptide during translation, preservation of the
molecule in an inactive state, and participation in
translocation [51]. Class I and II dacteriocins are
secreted via ABCtransporters [34].
Twopeptide bacteriocins from the IIb subclass
have only relatively recently been identified. Table 4
presents the properties of genetically and biochemi
cally characterized bacteriocins from that subclass.
Subclass IIb bacteriocins have much in common
with subclass IIa bacteriocins. In particular, they are
also cations and have a hydrophobic or amphiphilic
molecule in the region. Usually, both peptides act syn
ergistically. However, individual peptides of lactacin F,
plantaricin E/F, and plantaricin J/K can also exhibit
antimicrobial activity, but to a lesser extent [104]. It is
known that in lacticin 705, only one peptide is respon
sible for the antibacterial activity, while the other one
(the molecular weight of which is not determined due
to the inconstancy of its amino acid composition) is
responsible for the recognition of the membrane
receptor of the target cell [105]. Genes responsible for
the synthesis of individual peptides of twopeptide
bacteriocins are located one after another in the same
operon, and each bacteriocin has only one gene for an
immune protein, suggesting that both peptides function
as a single complex [103]. In addition, it was found that
during an attack on the cell, there are direct interactions
between complementary peptides of lactococcin G,
plantaricin E/F, and plantaricin J/K [106].
A quorum sensing system is used to regulate the
production of class II bacteriocins. It consists of three
components and is expressed in a ternary regulatory
system: a pheromone peptide, histidine kinase as a
transmembrane protein (pheromone receptor), and a
cytosolic protein response. It is shown that the synthe
sis of thermophilin 13 by
Streptococcus thermophilus
LMD9, as well as the synthesis of plantaricins by
strains
L. plantarum
C11, E/F, and J/K, depends on
the growth phase and concentration of the phero
moneinduced factor [107].
Action mechanism of class II bacteriocins.
It was
suggested that the receptor molecule for bacteriocins
IIa subclass is the mannose transporter [108].
The protein belongs to the phosphotransferase
system (PTS) and is encoded in the
mpt
operon. PTS
is responsible for the transport and phosphorylation of
sugar within cells in both Grampositive and Gram
negative bacteria. Mannose PTS permeases consist of
four domains: IIA, IIB, IIC, and IID. Cytoplasmic
domains IIA and IIB are involved in phosphorylation,
while membrane domains IIC and IID are involved in
transport. Subunits IIC and IID, located within the
membrane, are the targets for the second class of bac
teriocins [31]. The Nterminus of bacteriocins is
responsible for binding to the membrane of target cells
Man
t
EII
Man
t
EII
through electrostatic interactions, whereas the tryp
tophancontaining Cterminal region is responsible
for the penetration of bacteriocins into the membrane
[109]. It has been well documented that in the case of
lactococcin À produced by
L. lactis
subsp.
lactis
the
presence of membrane components IIC and IID is
sufficient for sensitivity [107]. The proposed model of
lactococcin A action and the mechanism of protection
against it are shown in Fig. 6.
According to this scheme, a bacteriocin interacts
with the IIC and IID components of the mannose PTS
as with a cell surface receptor (Fig. 6, A; positions
1
and
2
). After the bacteriocin is bound, it increases the
permeability of the membrane (position
3
), which
leads to cell death. Perhaps, the pore in the membrane
is formed through oligomerization of the bacteriocin
molecules or due to the destruction of the mannose
PTS complex.
In immune cells that do not produce bacteriocins,
immune proteins are not associated with mannose
PTS (Fig. 6, B, position
1
'). In cells, which produce
bacteriocins, immune proteins are strongly bound
with receptor proteins (IIC and IID) to protect them
from death [107]. Probably, this model is also realized
for lactococcin B and some pediocinlike bacterio
cins.
The lactococcin Q mechanism of action differs
from that of subclass IIa bacteriocins. The presence of
a receptor molecule on the plasma membrane is not
required for its action. It is assumed that lactococcin Q
electrostatically binds to the negatively charged bacte
rial cell membrane and then embeds in it, forming
pores [110].
Twopeptide bacteriocins from class IIb increase
the cell membrane permeability to ions due to the for
mation of pores, in which both peptides are embedded
[111, 112]. Each bacteriocin forms pores, which are
only permeable for certain types of ions. Thus, lactococ
cin G increases the permeability for many monovalent
cations and choline with the exception of
H
+
, while plan
taricins E/F and J/K make the membrane permeable for
all monovalent cations, including
H
+
[113].
Due to the fact that LAB bacteriocins belonging to
class II have high antibiotic activity against the
Listeria
innocua
and
L. monocytogenes
pathogens, which grow
in foods and raw food products, those bacteriocins are
prospective for use as biopreservatives for longterm
storage [114]. Curvacin A [81], divercin V41 [82], lac
tocin MMFII [84], leucocin A [85], plantaricin 423
[87], pediocins
РА
1 and
АсH
[114–116], and
sakacins P and G [75, 90] are among these bacterio
cins.
At the present time, the interest of researchers in
LAB has increased dramatically due to their safety and
high enzymatic and antimicrobial activity. LAB are
the subject of basic research devoted to the develop
ment of new active probiotics and various antimicro
bial preservative agents.
240
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 48 No. 3 2012
STOYANOVA
et al.
LAB are producing a wide range of antimicrobial
metabolites, belonging to different classes of chemical
compounds. The bacteriocin group is the best studied
one. Because of their nontoxicity, bacteriocins are the
most promising leads for the development of a new
generation of antibiotic drugs with probiotic proper
ties [27, 117, 118]. Due to the fact that LAB have a
GRAS status, their bacteriocins are in demand in
industry as safe and specific biopreservatives [119,
120]. Many bacteriocins produced by this group of
bacteria are successfully proven to be biopreservatives
for meat products, fish, dairy products, vegetables,
and fruits [121, 122]. The main approaches to using
bacteriocins to increase the shelf life of foods involve
using different bacteriocins in combination, including
them in packing materials, and combining them with
other preservatives. The effectiveness of bacteriocins is
determined by the activity of their producers. Strains
from a variety of substrates are screened for high pro
ductivity; different genetic methods, including cell
engineering, are used for the same purpose [18, 20,
123–127].
On the other hand, bacteriocins can be used in
medicine as alternative antibiotics [128, 129].
There are significant differences in the structures of
molecules from various classes of bacteriocins, and,
therefore, there are differences in their stability, mech
anism, and the range of their antimicrobial activity.
Specifically, lantibiotics are characterized by a com
plicated process of posttranslational reactions that
result in the formation of lanthionine bridges in a mol
ecule, which are responsible for the stability of the
peptide in the external environment and for a wider
range of activity.
LABproduced compounds with untifungal activi
ties are of special scientific and practical value. This
diverse group of substances is poorly understood. Lac
tococci strains (identified as
L. lactis
subsp.
lacits
) syn
thesize alkyl ketones, which determine their antifun
gal activity. Many authors have noted the possibility of
using LAB strains to prolong the shelf life of fruits and
vegetables, which are prone to damage by microscopic
fungi [12, 13, 23]. Given that the need of the food
industry, medicine, and agriculture in antifungal
agents is growing every year and that the currently used
fungicides (chemicals) are toxic to humans and ani
mals and can be accumulated in the soil and water, the
search for new antifungal agents from nonpathogenic
forms of microorganisms is an urgent problem.
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... LAB are fermentative bacteria that produce antimicrobial compounds such as lactic acid [1,2]. LAB are divided into two groups: those that produce lactic acid as the main product of glucose fermentation are called homofermenters, and those that produce the same molar amount of lactate, carbon dioxide, and ethanol from hexose are called heterofermentative [3,4]. The LAB are known to improve immune-mediated health complications such as allergies, atopic dermatitis, rhinitis, oral tolerance, cancer, and inflammatory diseases [5]. ...
... Several applied studies have been carried out to determine the function of various LAB properties, including antibacterial activity with a broad spectrum [4,[8][9][10]. This function is due to the antimicrobial property that can inhibit the growth of other microorganisms and is promising as an alternative antibiotic for certain bacteria [2,8,[11][12][13]. ...
... Some important compounds, including organic acids, diacetyl, hydrogen peroxide, bacteriocin, and reuterin, have antimicrobial power with a variety of characteristics, such as specific or nonspecific molecular weights. Each of these compounds has a different antimicrobial object-level [4]. ...
Article
Full-text available
Objective: The purpose of this research is to detect the antibacterial properties of lactic acid bacteria (LAB) against pathogenic bacteria. Materials and methods: Isolation and determination of Lactobacillus spp. Testing of the antibacterial activity of LAB was conducted using filtrate and nonfiltrate forms. The lactic acid bacterial isolates were confirmed to be identified through Gram staining, cell shape, catalase testing, and motility testing. Results: The results of the analysis of the LAB inhibition zone using filtrate and nonfiltrate forms against the bacteria Bacillus cereus were included in the very strong category. The results of the analysis of the LAB inhibitory zone using filtrate and nonfiltrate forms and the agar well method against Staphylococcus aureus bacteria were classified into the very strong category. The results of the LAB inhibitory zone analysis using filtrate and nonfiltrate forms with the well method against Escherichia coli bacteria are included in the very strong category, whereas the results from the LAB inhibitory zone analysis using the filtrate and nonfiltrate forms with the agar diffusion method (disks) are included in the strong category. Conclusion: Based on the results, LAB isolated from Sumba mare's milk displayed antibacterial activity in the strong and very strong categories against pathogenic bacteria such as B. cereus, S. aureus, and E. coli.
... LAB are gram-positive microorganisms that produce lactic acid as the major product of their primary metabolism [10], and they exhibit antimicrobial properties [11]. Several metabolites are produced by LAB during their primary (lactic acid, acetic acid, hydrogen peroxide) and secondary metabolisms, including diacetyl, reutericyclin, 3-phenyllactic acid, benzoic acid, methylhydantoin, benzeneacetic acid, 2-propenyl ester, mevalonolactone, 2,6-diphenyl-piperidine, proteinaceous compounds, diketopiperazines and other lowmolecular weight compounds [12][13][14]. ...
... The mass spectra of the compounds are shown in Fig. 5. The three identified diketopiperazines have been reported to exhibit antifungal activity [10,27,28], while 9-octadecenoic acid is associated with antimicrobial activity [29]. Diketopiperazines are likely produced under these conditions as signalling molecules to modulate bacterial communication and protect against other microorganisms [30]. ...
Article
Full-text available
Mycotoxins may be present in nuts, coffee, cereals, and grapes, among other products. Increasing concerns about human health and environmental protection have driven the application of biological control techniques that can inhibit fungal contaminants. In this study, the growth inhibition of the ochratoxigenic fungus Aspergillus carbonarius Ac 162 was evaluated using 5 lactic acid bacteria (LAB). The LAB studied were Lactobacillus plantarum MZ801739 (J), Lactobacillus plantarum MZ809351 (31) and Lactobacillus plantarum MZ809350 (34), isolated in the Ivory Coast, and Lactobacillus plantarum MN982928 (3) and Leuconostoc citreum MZ801735 (23), isolated in Mexico. J, 31, 34, 3 and 23 are the internal strain codes from our laboratory. LAB were cultivated in De Man, Rogosa and Sharpe (MRS) broth, and different polyols (glycerol, mannitol, sorbitol, and xylitol) were added to the culture broth to stimulate the production of antifungal compounds. The fungal inhibition studies were performed using the poisoned food technique. The highest inhibition of A. carbonarius growth was obtained by cultivating L. plantarum MZ809351 in the presence of xylitol and glycerol. Under these conditions, 1 L of the L. plantarum MZ809351 cultures were used to identify antifungal compounds. The compounds were concentrated by solid-phase extraction and then characterized by GC–MS. In addition to 9-octadecenoic acid, 3 diketopiperazines or cyclic dipeptides were identified, including cyclo (Leu-Leu), cyclo (Pro-Gly) and cyclo (Val-Phe), which were compounds related to microbial antifungal activities. Xylitol and glycerol induced the production of these antifungal compounds against A. carbonarius Ac 162. On the other hand, adding xylitol and glycerol to the MRS broth reduced the Ochratoxin A (OTA) content to 56.8 and 54.7%, respectively. This study shows the potential for using L. plantarum MZ809351 as a biocontrol agent to prevent the growth of A. carbonarius and reduce the production of OTA in foods.
... ICD strains that form 2-hydroxyhexanoic and other hydroxy derivatives of fatty acids are described in the literature; all of them exhibit antibiotic activity against a wide range of yeasts and molds [11]. DOI In Kefir the proportion of long chain fatty acids to the total amount of FA is higher than in Amashi. ...
... These acids are metabolites of microorganisms included in starter cultures. Phenyllactic acid found in Kefir is one of the metabolites of phenylalanine metabolism and can be formed in the cells of lactic acid bacteria from p-hydroxyphenylpyruvate. Phenyllactic acid exhibits antibiotic activityagainst gram-positive and gram-negative bacteria, and also acts on a wide range of microscopic fungi[11]. This metabolite is formed by some lactobacilli and propionic acid bacteria. ...
Article
. Both Russia and South Africa have a long-standing history of fermented milk product consumption. Along with the products widely distributed around the world, such as yoghurts, in each of these countries there are a number of national products. An example of a widely demanded fermented milk product in Russia is Kefir.This productis used not only as a food source in the diet of children and adults, but also in medical institutions, since ithasa positive effect onhuman health when consumed regularly. South Africa is characterized by the consumption of products such as Amasi,which is produced commercially. Its consumption has also been shown to have beneficial effects on the digestive system. In this research, the metabolic profiles(fatty acid composition and volatile compounds) of these fermented milk products were analyzed and these showed significant differences. The results indicated that this metabolite composition reflected the different production protocols and microbial complexity of these dairy products. The functional properties of the studied drinks were also considered.The average content of L-leucine equivalents in Amasi was slightly higher (6.5-8.9mMol×L −1) than in Kefir (4.9-6.7mMol×L −1). Antioxidant and antihypertensive activity of the fermented products correlated with the depth of hydrolysis of the milk proteins. Amasishowed higher antioxidant and antihypertensive activities (600- 796µМolТE/ml and 1.3-1.5mg/ml, respectively) than Kefir (246-574µМolТE/ml and 2.0-4.3mg/ml, respectively). Keywords: fermented products, Kefir, Amasi,metabolic profile, antioxidant potential, antihypertensive properties
... The production of these compounds can extend the shelf life by limiting the growth of contaminating and pathogenic microorganisms. In this sense, the growth and subsequent disappearance of some bacteria is common due to intra-and interspecific competition, as well as competition regarding the substrate, hence involving production of organic acids (Stoyanova et al. 2012). In this way, the fluctuation of pH values is a (Di Cagno et al. 2011;Begunova et al. 2020). ...
Article
Full-text available
To prevent foodborne diseases and extend shelf life, antimicrobial agents may be used in food to inhibit the growth of undesired microorganisms. The present study was aimed to determine the antimicrobial and antifungal activities of the fermented medicinal plants extract using Lactobacillus acidophilus ATCC 4356. The fermentation kinetic parameters, biochemical composition and the volatile compounds of the fermented plant extract were assessed. The results showed that, the fermented plants extract exhibited high content in polyphenols, flavonoids, and tannins (152.7 mg AGE/L; 93.6 mg RE/L; and 62.1 mg CE/L, respectively) comparing to non-fermented the extract. The GC–MS headspace analyses showed the presence of 24 interesting volatile compounds. The richness of the fermented plants extracts in polyphenols and bioactive compound, such as Eucalyptol, Camphene, α-Phellandrene, α-Terpinene, improves their biological activity. In addition, the fermented plants extract exhibited a high antimicrobial potential against pathogenic bacteria and fungi determined by different methods. The maximum inhibition showed in the fermented plants extract against Escherichia coli 25922/3, Pseudomonas aeruginosa 27853 ATCC, Staphylococcus aureus 29213 ATCC, Enterococcus aerogenes 13048 ATCC, Phytophthora infestans P3 4/91 R + , P. infestans P4 20/01 R, P. infestans (GL-1). The obtained results support the hypothesis of using lactic fermentation as a functional ingredient to improve food preservation. The bioprocesses of fermentation technology enhance antimicrobial and antifungal activities which could be used in different industrial applications.
... Lactic acid bacteria (LAB), a type of probiotic, are generally recognized as safe and often used in fermented foods. LAB strains could produce several metabolites (organic acids, hydrogen peroxide and bacteriocins, etc.) to inhibit the growth of foodborne pathogens and degrade nitrite (Stoianova et al., 2012). Kim et al. (2021) showed that inoculation of Limosilactobacillus fermentum J2 increased ferulic acid content in fermented rice bran and no visible growth of foodborne pathogens were found during fermentation. ...
Article
Full-text available
The potential of Lactiplantibacillus plantarum ZJ316 (ZJ316) as a starter culture for quality improvement and microbial community regulation in pickled mustard fermentation was elucidated in this study. Our results show that ZJ316 can deter the occurrence of nitrite peaks and maintain the nitrite content of pickled mustard at a low level (0.34 mg/kg). The headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry results indicate that ZJ316 gives a good flavor to pickled mustard. According to the 16S rDNA results, Firmicutes were the predominant microbiota after inoculation with ZJ316, and the abundances of Citrobacter, Enterobacter, and Proteus decreased simultaneously. In addition, antibacterial activity analysis showed that the supernatant of pickled mustard inoculated with ZJ316 had a significant inhibitory effect on Staphylococcus aureus D48, Escherichia coli DH5α, and Listeria monocytogenes LM1. In conclusion, L. plantarum ZJ316 has potential for use as an ideal starter in the process of vegetable fermentation.
... For 'example' LAB showed significant antagonistic potential against phytopathogenic bacteria and is widely accepted by the United States Food and Drug Administration (FDA) as healthy (GRAS), rendering it suitable for the production of bioprotective agents in fresh fruits and vegetables [24]. LAB produces a number of antimicrobial substances, including antifungal diketopiperazines, derivatives of fatty acid hydroxide, 3-phenylactate, antibacterial bacteriocins and bacteriocin-like compounds, and general antimicrobials such as organic acids, hydrogen peroxide, diacetyl and reuterine (b-OH-propionic aldehyde) pyrrolidone-5-carboxylic acid [25]. Further, Seo, et al. [26] confirmed the vital role of natural organic acids produced by LAB in controlling root-knot nematodes. ...
Article
Full-text available
Root-knot nematodes are economically important obligate parasites of plant root that parasitize more than 3000 species of plant. Lactic Acid Bacteria (LAB) are well known as strong producers for wide range of organic acids that have been suggested recently to possess a remarkable lethal effect on root-knot nematodes. In the present 'study' 37 LAB strains were isolated from soil and agricultural wastes by using MRS medium and screened for their nematicidal activity under lab conditions. Four bacterial isolates, recorded lethal effect on second stage juveniles by more than 94%, were selected and identified by molecular method. Further, the nematicidal activity at two concentrations (i.e 50 and 10%) of the most promising ten LAB cultures was screened. The nematicidal activity of LAB was positively correlated to the concentration of their suspension cultures.2S4 (Pediococcus pentosaceus MW558270), 2S5 (Pediococcus pentosaceus MW558883), 3S1 (Pediococcus pentosaceusMW558885) and 1A3 (Pediococcus pentosaceus MW558152) isolates showed significant mortality effect by 88.42, 87.37, 81.05 and 85.96%at the low concentration (10%), respectively. In order to confirm the relationship between organic acid production by LAB and their nematicidal activity, an artificial succinic acid, lactic acid, malic acid, acetic acid, and their mixtures were tested at concentration of 1% for their ability to inhibit juvenilesvitality. The mixture of acids induced the maximum mortality effect by 100% followed by lactic acid which recorded 99.66% mortality effect. The microscopic studies and the malformation pattern of the juveniles indicating that the nematicidal activity of LAB may be derived mainly from their natural organic acids mainly lactic acid.
... Insights into genetic motifs, patterns, and elucidation of generic pathways decipher the functional dimensions of a particular isolate of interest. Armed with this knowledge, the so-called difficult to detect novel secondary metabolites and other antimicrobial substances can be easily determined by adaptation of these advanced tools (Corr et al. 2007;Stoyanova et al. 2012). This was further exemplified by the detection of various gene clusters of mucus binding pili responsible for intestinal adherence (Douillard et al. 2013), detection of epigenetic alterations (Casadesus and Low 2006), and motifs responsible for coding resistance to crucial antibiotics (Proença et al. 2017) of different Lactobacillus species. ...
Book
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This reprint presents a print version of the Special Issue of the journal {Foods} dedicated to new insights into food fermentation. Food fermentation has been used for thousands of years for food preservation. At present, fermented foods remain appreciated by consumers thanks to the high-quality standards achieved and the improvements in terms of nutritional and organoleptic characteristics. The production processes, type of raw material, microbial cultures, etc., can affect these products’ quality and safety characteristics. A vast array of microorganisms can be found in fermented foods, and microbial succession during fermentation, as well as during ripening, contributes to the desired properties of these foods. In addition to the sensory and safety aspects, microorganisms present in fermented foods can positively affect people’s health due to their potential probiotic nature and the production of benefcial metabolites such as vitamins and antioxidant compounds. The goal of this Special Issue was to broaden the current knowledge on advanced approaches concerning food fermentation, gathering studies on conventional and unconventional food matrix fermentation, functional compounds obtained through fermentation, fermentations increasing quality and safety standards, as well as papers presenting innovative approaches shedding light on the microbial community that characterizes fermented foods. In the 13 papers collected in this volume, interested readers will fnd a collection of scientific contributions providing a sample of the state-of-the-art and forefront research in food fermentation. Among the articles published in the Special Issue, the geographic distribution of the studies is wide enough to attract the interest of an international audience of readers. The editors would like to thank the authors for their collaboration and commitment to publishing their high-quality scientifc articles.
Article
1. This study aimed at the effects of a novel Lactobacillus bulgaricus (L. bulgaricus) strain and Enterohemorrhagic Escherichia coli (E. coli) O157: H7 on intestinal flora and growth performance of broilers, and the protective effect of L. bulgaricus on broilers in challenged experiment by E. coli O157: H7.2. In vitro bacteriostatic test showed that the cell-free supernatant (CFS) of L. bulgaricus isolate had obvious inhibitory effect on E. coli O157: H7.3. Eighty 1-day-old male broilers were randomly assigned into 4 treatment groups with 4 replicate per treatment. All group received basic diet in addition to the specific treatments: NC group, gavage with normal saline; In LBP group, gavage with L. bulgaricus isolate (1×109 CFU/mL) during the whole process, and challenged with E. coli O157: H7 (3×109 CFU/mL); EC group, gavage with E. coli O157: H7 (3×109 CFU/mL); LB Group, gavage with L. bulgaricus isolate. At the age of 21 days, broilers were weighed and feed conversion ratio (FCR) was calculated. Cecum and cecal contents, ileum and feces samples were taken after slaughter.4. The challenge of E. coli O157: H7 resulted in an increase in TLR-4, NF-κB and IL-8 mRNA in cecal tissue, a decrease in Villus: crypt ratio in ileum, a decrease in overall diversity of intestinal microflora and a poor FCR.5. The L. bulgaricus isolate decreased the mRNA expression of TLR-4, NF-κB and IL-8 induced by E. coli O157: H7, reduced the content of E. coli O157: H7 in the cecum of broilers, increased the Villus: crypt ratio, increased the abundance of beneficial bacteria and overall diversity of intestinal microflora, made good FCR.6. The L. bulgaricus isolate can maintain the intestinal health, improve the growth performance of broilers and reduce the colonization of E. coli O157:H7 in the cecum.
Article
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Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II–targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.
Article
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Sensitivity of Listeria monocytogenes to the bacteriocin mesentericin Y105 was previously shown to be dependent on the sigma(54) subunit of the RNA polymerase. This points towards expression of particular sigma(54)-dependent genes. The present study describes first, ManR, a new sigma(54)-associated activator, and second, Ell(t)(Man), a new sigma(54)-dependent PTS permease of the mannose family, both involved in sensitivity to mesentericin Y105, since interruption of their corresponding genes led to resistance of L. monocytogenes EGDe. Ell(t)(Man) is likely composed of three subunits encoded by the mpt operon (mptA, mptC and mptD genes). Interruption of either the proximal (mptA) or distal (mptD) gene led to resistance, supporting results obtained in Enterococcus faecalis. Accordingly, such PTS permeases of the mannose family should be involved in sensitivity of different target strains to mesentericin Y105. In L. monocytogenes, expression of the mpt operon is shown to be controlled by sigma(54) and ManR and to be induced by both glucose and mannose. The latter result indicates that these sugars are transported by the Ell(t)(Man) permease. Moreover, these sugars correlatively induce sensitivity of L. monocytogenes to mesentericin Y105, strongly favouring the primary role of Ell(t)(Man). MptD, a membrane subunit of Ell(t)(Man), presents an additional domain compared to most IID(Man) subunits described in data banks. An in-frame deletion of this domain in mptD led to resistance of L. monocytogenes, showing its connection with sensitivity and suggesting that it could be directly involved in the recognition of the target cell by mesentericin Y105. Taken together, the results of this work demonstrate that Ell(t)(Man) is prominent in sensitivity to mesentericin Y105 and could be a receptor for subclass IIa bacteriocins.
Book
While lactic acid-producing fermentation has long been used to improve the storability, palatability, and nutritive value of perishable foods, only recently have we begun to understand just why it works. Since the publication of the third edition of Lactic Acid Bacteria: Microbiological and Functional Aspects, substantial progress has been made in a number of areas of research. Completely updated, the Fourth Edition covers all the basic and applied aspects of lactic acid bacteria and bifidobacteria, from the gastrointestinal tract to the supermarket shelf. Topics discussed in the new edition include: • Revised taxonomy due to improved insights in genetics and new molecular biological techniques • New discoveries related to the mechanisms of lactic acid bacterial metabolism and function • An improved mechanistic understanding of probiotic functioning • Applications in food and feed preparation • Health properties of lactic acid bacteria • The regulatory framework related to safety and efficacy Maintaining the accessible style that made previous editions so popular, this book is ideal as an introduction to the field and as a handbook for microbiologists, food scientists, nutritionists, clinicians, and regulatory experts.
Article
The morphological, cultural, physiological, and biochemical properties of novel efficient nisin-producing strains of Lactococcus lactis subsp. lactis were studied. These strains, which were obtained by fusion of the protoplasts of allied parental strains with low nisin-producing activities, turned out to be similar to their parents in morphology and cultural properties but differed from them in carbohydrate fermentation patterns, requirements for growth factors (amino acids, purines, pyrimidines, and vitamins), and sensitivity to antibiotics. The novel strains enriched the collection of efficient producers of nisin, a widely used food preservative.
Article
Efficient nisin producers were derived by fusing the protoplasts of two allied strains 729, Lactococcus lactis subsp. lactis and 1605, with low nisin-synthesizing abilities. Conditions promoting protoplast formation, fusion, and regeneration were elaborated. The process of protoplast fusion was studied by electron microscopy. Some physiological and biochemical properties of the parent strains were preliminarily studied to develop selective media suitable for selecting the required clones. The effect of various selective agents in the regeneration medium on the segregation of recombinant clones was studied with respect to nisin production. The most potent producers of nisin were 10 to 14 times more efficient than the parent strains.
Article
Several metabolic properties of lactic acid bacteria (LAB) serve special functions, which directly or indirectly have impact on processes such as improved quality and safety and flavour development in the malting and brewing industry. LAB are widely distributed in nature and in spontaneous fermentations, often they are found to be the dominating microflora resulting in both the inhibition of spoilage bacteria and organisms. This review describes the applications of LAB in malting and brewing. Mycotoxins are naturally occurring toxic secondary metabolites of fungi that may be present in cereals. Several of these mycotoxins have been associated with human and animal diseases and are known to survive the brewing process. LAB have been shown to restrict the growth of the most important toxigenic fungi thereby reducing the formation of these harmful toxins. The occurrence of mycotoxins in cereals is discussed and their effect in beer is reviewed. The main features of this review are: (I) LAB starter cultures in malting and brewing (II) production of acid malt; (III) biological acidification of mash and wort in brewing; (IV) bacteriocins produced by LAB in brewing; (V) LAB and antifungal activity; (VI) mycotoxins in cereals.
Article
Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105.
Article
The production of sakacin K byLactobacillus sakeiCTC494 at different temperatures and pH was evaluated before its application as a bioprotective culture againstListeriain different meat-based food packaging systems, that is, oxygen-permeable film, under vacuum and under a modified atmosphere (20% CO2: 80% O2) and stored at 7°C. TheLactobacillusculture produced the bacteriocin at a range of temperatures from 4°C to 30°C and at initial pH from 5·5 to 6·5.Listeriainhibition in raw minced pork, poultry breasts and modelized cooked pork could not be achieved by the sole application of vacuum or a modified atmosphere. Inoculation ofLb. sakeiCTC494 or sakacin K inhibited the growth ofListeriato different extents in all the products studied in each system; the greatest inhibition being observed in the vacuum packaged samples of poultry breasts and cooked pork, and in the modified atmosphere packaged samples of raw minced pork. Addition of sakacin K resulted in immediate bactericidal action againstListeriain every product and atmosphere studied.