**3. Bacteriocins: genetics and biosynthesis**

Bacteriocins produced from LAB are synthesized by genes encoded by either chromosomes or plasmids. For example, Plantaricin 423 [24] is plasmid encoded, while genes for enterocin A, sakacin P, divercin V41 and carnobacteriocin B2 and BM1 [25, 26] are localized on bacterial chromosomes. Plasmids concerned with the production of bacteriocins differ significantly in size. Some of them have been identified to carry the genetic determinants for numerous bacteriocins [3, 27].

#### **3.1 Genetic constitution of bacteriocin operons**

The genes accountable for synthesis of bacteriocin are often localized in single or multiple operons and are individually transcribed [28]. Most commonly, two types of genes are involved in production of bacteriocins: (i) structural genes and (ii) genes which encode for immunity protein. However, in some cases, apart from these two genes, other specific export machinery and regulatory genes are also required, thus making the bacteriocin operon much more intricate [29, 30].

#### *3.1.1 Class I bacteriocin operon*

Class I bacteriocin operon can be localized either on the bacterial chromosome or the plasmid. Few examples of lantibiotics with plasmid genetic determinants include: lacticin 481 [31] encoded by genes localized on 70 kb plasmid and the two-component lacticin 3147 [32] encoded on 63 kb plasmid, both of which are produced by Lactobacillus lactis. Class I bacteriocins are considered to be more complex than class II non-lantibiotics as they require supplementary enzyme encoding genes for post-translational modifications. The biosynthesis of lantiobiotics involves the translation of pre-peptide which undergoes few modifications, and then the modified pre-peptide moves onto the other side of the membrane, and the amino-terminal signal peptide is cleaved via proteolytic enzymes present in the cytoplasm. The best described lantibiotic is nisin, whose genetic determinants are reported to be localized on conjugative transposon Tn5276 contained by the bacterial chromosomes. Genes which aid in nisin production and immunity are transferred conjugally and it is reported to be situated in a nucleotide segment of size 8.5 kb. The gene cluster of nisin is designated as nisABTCIPRKFEG, and contains eleven genes which include structural genes, immunity protein encoding self-defensive genes, transporter genes and response regulator genes [3, 30]. Biosynthesis of nisin involves following steps: (a) firstly nisin A undergoes translation to form pre nisin A (b) The partially formed nisin A is then changed to form precursor nisin A via the proteins encoded by both nisB and nisC and (c) lastly, the precursor nisin is transported extracellularly by the nisT and nisP gene products by cleaving the leader peptide at the same time to obtain the end product, Nisin A (**Figure 3a**) [3, 33].

#### *3.1.2 Class II bacteriocin operon*

The genes encoding for structural proteins of class II bacteriocins are mostly localized on plasmids, for example, pediocin PA-1 and pediocin AcH [4] extracted from Pediococcus acidilactici strains. Bacteriocins belonging to class II usually require 2 to 8 genes for their production. The genes concerned with their production, secretion and immunity are: structural gene, immunity gene, ATP-binding cassette (ABC) protein encoding genes and its accessory proteins. Pediocin AcH, the bacteriocin representing class IIa is organized in a gene segment of 3500 bp

**147**

(**Figure 3b**).

*operon. P: Promoter region.*

**Figure 3.**

exchanging the leader peptide [29].

**3.2 Biosynthesis of bacteriocins**

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens*

having four genes designated as (i) papA, (ii) papB, (iii) papC and (iv) papD [3]

*Schemmatic structure of genes concerned with the synthesis of bacteriocins. (a) Nisin operon (b) Pediocin AcH* 

Bacteriocins or antimicrobial peptides are primarily biosynthesized as prepeptides which are biologically non-active, having amino-terminal signaling peptide which remains linked to carboxyl-terminal pro-peptide [13, 36]. This pre-pro-peptide is encoded by the structural genes. The pre-peptide is directed towards the maturation

The genes involved in synthesis generally produce inactive pre-pro peptide having an amino-terminal leader peptide [29, 34]. The gene which imparts immunity is transcribed in parallel, along with the bacteriocin precursor. The ABC transporter and its accessory proteins which aid in the export of bacteriocins are encoded by a group of genes typically localized on operon present in closer proximity to immunity genes [3, 12, 29]. The leader peptide sequence of nearly all the bacteriocins of class IIa is of double glycine-type (GG-type), though, several of them have also been found to possess a sec-type amino-terminal signal sequence, for example, bacteriocin 31, listeriocin 743A, and enterocin P [35]. These bacteriocins are understood to be released out by sec-dependent exporting system. The ABC tranposters are assumed to aid in the recognition of GG-type leader peptides [29]. ABC transporters during transmembrane translocation consequently remove the GG-leader sequence and the fully active bacteriocin is afterwards secreted. Class IIa bacteriocin can be expressed heterologously through another secretion system by

*DOI: http://dx.doi.org/10.5772/intechopen.95747*

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens DOI: http://dx.doi.org/10.5772/intechopen.95747*

**Figure 3.**

*Biomimetics*

**3. Bacteriocins: genetics and biosynthesis**

**3.1 Genetic constitution of bacteriocin operons**

*3.1.1 Class I bacteriocin operon*

thus making the bacteriocin operon much more intricate [29, 30].

Bacteriocins produced from LAB are synthesized by genes encoded by either chromosomes or plasmids. For example, Plantaricin 423 [24] is plasmid encoded, while genes for enterocin A, sakacin P, divercin V41 and carnobacteriocin B2 and BM1 [25, 26] are localized on bacterial chromosomes. Plasmids concerned with the production of bacteriocins differ significantly in size. Some of them have been identified to carry the genetic determinants for numerous bacteriocins [3, 27].

The genes accountable for synthesis of bacteriocin are often localized in single or multiple operons and are individually transcribed [28]. Most commonly, two types of genes are involved in production of bacteriocins: (i) structural genes and (ii) genes which encode for immunity protein. However, in some cases, apart from these two genes, other specific export machinery and regulatory genes are also required,

Class I bacteriocin operon can be localized either on the bacterial chromosome or the plasmid. Few examples of lantibiotics with plasmid genetic determinants include: lacticin 481 [31] encoded by genes localized on 70 kb plasmid and the two-component lacticin 3147 [32] encoded on 63 kb plasmid, both of which are produced by Lactobacillus lactis. Class I bacteriocins are considered to be more complex than class II non-lantibiotics as they require supplementary enzyme encoding genes for post-translational modifications. The biosynthesis of lantiobiotics involves the translation of pre-peptide which undergoes few modifications, and then the modified pre-peptide moves onto the other side of the membrane, and the amino-terminal signal peptide is cleaved via proteolytic enzymes present in the cytoplasm. The best described lantibiotic is nisin, whose genetic determinants are reported to be localized on conjugative transposon Tn5276 contained by the bacterial chromosomes. Genes which aid in nisin production and immunity are transferred conjugally and it is reported to be situated in a nucleotide segment of size 8.5 kb. The gene cluster of nisin is designated as nisABTCIPRKFEG, and contains eleven genes which include structural genes, immunity protein encoding self-defensive genes, transporter genes and response regulator genes [3, 30]. Biosynthesis of nisin involves following steps: (a) firstly nisin A undergoes translation to form pre nisin A (b) The partially formed nisin A is then changed to form precursor nisin A via the proteins encoded by both nisB and nisC and (c) lastly, the precursor nisin is transported extracellularly by the nisT and nisP gene products by cleaving the leader peptide at the same time to obtain the end product, Nisin A

The genes encoding for structural proteins of class II bacteriocins are mostly localized on plasmids, for example, pediocin PA-1 and pediocin AcH [4] extracted from Pediococcus acidilactici strains. Bacteriocins belonging to class II usually require 2 to 8 genes for their production. The genes concerned with their production, secretion and immunity are: structural gene, immunity gene, ATP-binding cassette (ABC) protein encoding genes and its accessory proteins. Pediocin AcH, the bacteriocin representing class IIa is organized in a gene segment of 3500 bp

**146**

(**Figure 3a**) [3, 33].

*3.1.2 Class II bacteriocin operon*

*Schemmatic structure of genes concerned with the synthesis of bacteriocins. (a) Nisin operon (b) Pediocin AcH operon. P: Promoter region.*

having four genes designated as (i) papA, (ii) papB, (iii) papC and (iv) papD [3] (**Figure 3b**).

The genes involved in synthesis generally produce inactive pre-pro peptide having an amino-terminal leader peptide [29, 34]. The gene which imparts immunity is transcribed in parallel, along with the bacteriocin precursor. The ABC transporter and its accessory proteins which aid in the export of bacteriocins are encoded by a group of genes typically localized on operon present in closer proximity to immunity genes [3, 12, 29]. The leader peptide sequence of nearly all the bacteriocins of class IIa is of double glycine-type (GG-type), though, several of them have also been found to possess a sec-type amino-terminal signal sequence, for example, bacteriocin 31, listeriocin 743A, and enterocin P [35]. These bacteriocins are understood to be released out by sec-dependent exporting system. The ABC tranposters are assumed to aid in the recognition of GG-type leader peptides [29]. ABC transporters during transmembrane translocation consequently remove the GG-leader sequence and the fully active bacteriocin is afterwards secreted. Class IIa bacteriocin can be expressed heterologously through another secretion system by exchanging the leader peptide [29].

### **3.2 Biosynthesis of bacteriocins**

Bacteriocins or antimicrobial peptides are primarily biosynthesized as prepeptides which are biologically non-active, having amino-terminal signaling peptide which remains linked to carboxyl-terminal pro-peptide [13, 36]. This pre-pro-peptide is encoded by the structural genes. The pre-peptide is directed towards the maturation and transportation of the protein by the signal peptide which behaves as recognition spot and keeps the producing strain protected by being in an inactive state within the bacterial cells. Additionally, the signal peptide interacts with pro-peptide domain to make sure that it is in the proper state for further interface with modification machinery [24, 32]. The structural genes play an important role in bacteriocin biosynthesis and are generally succeeded by the immunity genes.

To defend the bacteriocin producing strains from the killing action of their self- bacteriocins, a self-defensive system known as immunity has been evolved by these bacteria. The protection from bacteriocins is gained via production of specific immunity proteins via immunity genes. These genes encoding immunity protein lie closer to the structural and accessory genes of bacteriocins [19]. Dissimilarity in expression and presence of these genes accounts for huge disparity in sensitivity demonstrated by LAB for bacteriocins. The size of immunity proteins lie in the range of 51 to 150 amino acids. An immunity gene encodes the immunity protein which lies downstream to the structural protein coding genes usually in the same operon. In the bacterial cell, transcription of both the bacteriocins and their immunity genes are regulated in parallel as defensive system can be stopped concurrently. Immunity of LAB depends on the definite immunity protein, Lan I [19], which basically remains bound to the exterior side of the plasma membrane. It grants protection to the producer strains by shutting off the membrane pores formed by the bacteriocin molecules and transferring them to the neighboring medium, hence, regulating the amount of bacteriocin present on the cell surface upto a critical level.

Bacteriocins are transported extracellularly with the aid of transporter proteins, which are basically from ABC transporter family. In prokaryotic and eukaryotic organisms, ABC transporters assist in the oozing of an ample range of products. These transporters include (i) bacterial importers, which aid in transportation of vitamins, oligopeptides, sugar, phosphate, amino acids and metallic ions, (ii) eukaryotic exporters, which enable the transportation of lipotropic drugs, pigments and peptides, and (iii) bacterial exporters, which carry large toxic protein, polysaccharides, heme molecules, and high and low molecular weight antibiotics [34]. The bacteriocin ABC transporters facilitate the exclusion of substrate from the signal peptide and its transportation across the cytoplasmic membrane hence, performing dual functions. This aids in effectively preventing the active and fully formed bacteriocins from enduring inside the cytoplasm.

The bacteriocin synthesis is regulated via multi-component regulatory system that involves (i) a signal peptide (ii) a cell surface bound receptor to which signal peptide binds and (iii) a response regulator. The signal peptide binds with histdine-kinase receptor and the binding leads to phosphorylation, signaling surge that finally targets the phosphate residue towards the response regulator that then induces the gene expression and production of bacteriocins by binding with the promoter region [34].

### **4. Bacteriocins: mode of action**

Bacteriocins are recognized for restraining the growth of pathogenic microorganisms by forming pores in the cell envelope, and are known to be highly effective against Gram positive bacteria. Their activity against Gram negative bacteria is reported to be very less because of the outer wall of bacteria, which might possibly block the site of bacteriocin action [37]. Bacteriocins are proposed to exert their inhibitory effect using different mechanisms which include (i) a change in enzymatic activity (ii) restricting spore germination (iii) anionic carrier inactivation and (iv) with the aid of selective and non-selective pore formation in cell

**149**

bacteria [5].

molecules (**Table 2**) [5, 29].

**4.1 Bactericidal mode of class I bacteriocins**

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens*

membrane [14, 37]. However, most of the bacteriocins follow the fourth model and

Formation of pore in the cell membrane induced by the bacteriocins has been expected to take place by three models [38]: (i) a wedge-like model (ii) a barrelstave like model and (iii) carpet model. A wedge-like model involves insertion of nisin and lipids via proton motive force. In barrel-stave like model, upon insertion in the membranes, bacteriocins arrange themselves to make a bunch of α helical peptides. The interior wall of the pore is formed by the inner hydrophilic faces of these peptides and the outer hydrophobic surface faces the membrane lipids. On the other hand, in carpet model, the pore is induced by peptides. In this model, an individual peptide might get assembled analogous to the cell surface and hinders the bilipid organization of the cell membrane and hence results in transient permeability due to strong phospholipid mobilizing activity [41]. For the induction of pore formation, class I bacteriocins are thought to act by means of wedge-like model, while in bacteriocins belonging to class II, pore formation may be either permitted via barrel-stave like model, or the carpet model [38]. Pore formation in class IIa bacteriocins is induced through barrel-stave like model probably because of the putative transmembrane helices in the peptide structure of these bacteriocin

Bacteriocins belonging to class I are known to act by dual killing mechanisms, both of which have the same end results. Nisin is most researched antimicrobial peptide of this class. It exerts the bactericidal mode of action since it can diffuse easily through the anionic lipid membrane. It causes pore formation in the target membrane by coming in contact with the lipid II; a peptidoglycan precursor. The

The primary interface connecting the bacteriocins and the target cell membrane involves either of the following two mechanisms: (a) attachment of bacteriocin to the membrane bound receptor molecule and/or (b) interface amidst the positively charged amino acids of bacteriocins and negatively charged phospholipid molecules on the cell membrane [29, 38]. In the first mechanism, the bacteriocins often need a receptor molecule on the surface of the target organism which varies among different species and sub-species [39]. The second mechanism involves three basic steps for the bactericidal action: binding, insertion and pore formation. In the binding and insertion step, when the bacteriocins come in contact with the target membrane, their C-terminal consisting of hydrophobic amino acids penetrates the hydrophobic region of the bacterial membrane and binds with the mannose phosphotransferase permease which ultimately leads to the leakage of the membrane. The interactions among phospholipids (negatively charged) in the target cell membrane and groups of amino acids (positively charged) in the bacteriocin are chiefly involved in the binding and insertion step. The bacteriocin finally creates pore in the target cell and allows the outflow of rather large molecules. Formation of pores eventually leads to the ionic imbalance, disturbing nucleic acid content and leakage of inorganic phosphates [19, 40]. The preliminary interruptions stimulate the dispersion of the proton motive force (PMF) that ultimately leads to the disturbance of pH and transmembrane potential of the cell. But this mode of action does not work for negatively stained bacteria as their bacterial membrane consists of lipopolysaccharide as an outer membrane which differs from Gram positive bacteria. Some researchers have reported that when bacteriocins are combined with compounds that have the capability to disrupt the outer membrane such as surfactants like Triton X-100 or EDTA, they can be rendered active against Gram negative

*DOI: http://dx.doi.org/10.5772/intechopen.95747*

act by targeting the cell envelope.

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens DOI: http://dx.doi.org/10.5772/intechopen.95747*

membrane [14, 37]. However, most of the bacteriocins follow the fourth model and act by targeting the cell envelope.

The primary interface connecting the bacteriocins and the target cell membrane involves either of the following two mechanisms: (a) attachment of bacteriocin to the membrane bound receptor molecule and/or (b) interface amidst the positively charged amino acids of bacteriocins and negatively charged phospholipid molecules on the cell membrane [29, 38]. In the first mechanism, the bacteriocins often need a receptor molecule on the surface of the target organism which varies among different species and sub-species [39]. The second mechanism involves three basic steps for the bactericidal action: binding, insertion and pore formation. In the binding and insertion step, when the bacteriocins come in contact with the target membrane, their C-terminal consisting of hydrophobic amino acids penetrates the hydrophobic region of the bacterial membrane and binds with the mannose phosphotransferase permease which ultimately leads to the leakage of the membrane. The interactions among phospholipids (negatively charged) in the target cell membrane and groups of amino acids (positively charged) in the bacteriocin are chiefly involved in the binding and insertion step. The bacteriocin finally creates pore in the target cell and allows the outflow of rather large molecules. Formation of pores eventually leads to the ionic imbalance, disturbing nucleic acid content and leakage of inorganic phosphates [19, 40]. The preliminary interruptions stimulate the dispersion of the proton motive force (PMF) that ultimately leads to the disturbance of pH and transmembrane potential of the cell. But this mode of action does not work for negatively stained bacteria as their bacterial membrane consists of lipopolysaccharide as an outer membrane which differs from Gram positive bacteria. Some researchers have reported that when bacteriocins are combined with compounds that have the capability to disrupt the outer membrane such as surfactants like Triton X-100 or EDTA, they can be rendered active against Gram negative bacteria [5].

Formation of pore in the cell membrane induced by the bacteriocins has been expected to take place by three models [38]: (i) a wedge-like model (ii) a barrelstave like model and (iii) carpet model. A wedge-like model involves insertion of nisin and lipids via proton motive force. In barrel-stave like model, upon insertion in the membranes, bacteriocins arrange themselves to make a bunch of α helical peptides. The interior wall of the pore is formed by the inner hydrophilic faces of these peptides and the outer hydrophobic surface faces the membrane lipids. On the other hand, in carpet model, the pore is induced by peptides. In this model, an individual peptide might get assembled analogous to the cell surface and hinders the bilipid organization of the cell membrane and hence results in transient permeability due to strong phospholipid mobilizing activity [41]. For the induction of pore formation, class I bacteriocins are thought to act by means of wedge-like model, while in bacteriocins belonging to class II, pore formation may be either permitted via barrel-stave like model, or the carpet model [38]. Pore formation in class IIa bacteriocins is induced through barrel-stave like model probably because of the putative transmembrane helices in the peptide structure of these bacteriocin molecules (**Table 2**) [5, 29].

#### **4.1 Bactericidal mode of class I bacteriocins**

Bacteriocins belonging to class I are known to act by dual killing mechanisms, both of which have the same end results. Nisin is most researched antimicrobial peptide of this class. It exerts the bactericidal mode of action since it can diffuse easily through the anionic lipid membrane. It causes pore formation in the target membrane by coming in contact with the lipid II; a peptidoglycan precursor. The

*Biomimetics*

and transportation of the protein by the signal peptide which behaves as recognition spot and keeps the producing strain protected by being in an inactive state within the bacterial cells. Additionally, the signal peptide interacts with pro-peptide domain to make sure that it is in the proper state for further interface with modification machinery [24, 32]. The structural genes play an important role in bacteriocin biosynthesis

To defend the bacteriocin producing strains from the killing action of their self- bacteriocins, a self-defensive system known as immunity has been evolved by these bacteria. The protection from bacteriocins is gained via production of specific immunity proteins via immunity genes. These genes encoding immunity protein lie closer to the structural and accessory genes of bacteriocins [19]. Dissimilarity in expression and presence of these genes accounts for huge disparity in sensitivity demonstrated by LAB for bacteriocins. The size of immunity proteins lie in the range of 51 to 150 amino acids. An immunity gene encodes the immunity protein which lies downstream to the structural protein coding genes usually in the same operon. In the bacterial cell, transcription of both the bacteriocins and their immunity genes are regulated in parallel as defensive system can be stopped concurrently. Immunity of LAB depends on the definite immunity protein, Lan I [19], which basically remains bound to the exterior side of the plasma membrane. It grants protection to the producer strains by shutting off the membrane pores formed by the bacteriocin molecules and transferring them to the neighboring medium, hence, regulating the amount of bacteriocin present on the cell surface upto a critical level. Bacteriocins are transported extracellularly with the aid of transporter proteins, which are basically from ABC transporter family. In prokaryotic and eukaryotic organisms, ABC transporters assist in the oozing of an ample range of products. These transporters include (i) bacterial importers, which aid in transportation of vitamins, oligopeptides, sugar, phosphate, amino acids and metallic ions, (ii) eukaryotic exporters, which enable the transportation of lipotropic drugs, pigments and peptides, and (iii) bacterial exporters, which carry large toxic protein, polysaccharides, heme molecules, and high and low molecular weight antibiotics [34]. The bacteriocin ABC transporters facilitate the exclusion of substrate from the signal peptide and its transportation across the cytoplasmic membrane hence, performing dual functions. This aids in effectively preventing the active and fully formed

The bacteriocin synthesis is regulated via multi-component regulatory system that involves (i) a signal peptide (ii) a cell surface bound receptor to which signal peptide binds and (iii) a response regulator. The signal peptide binds with histdine-kinase receptor and the binding leads to phosphorylation, signaling surge that finally targets the phosphate residue towards the response regulator that then induces the gene expression and production of bacteriocins by binding with the

Bacteriocins are recognized for restraining the growth of pathogenic microorganisms by forming pores in the cell envelope, and are known to be highly effective against Gram positive bacteria. Their activity against Gram negative bacteria is reported to be very less because of the outer wall of bacteria, which might possibly block the site of bacteriocin action [37]. Bacteriocins are proposed to exert their inhibitory effect using different mechanisms which include (i) a change in enzymatic activity (ii) restricting spore germination (iii) anionic carrier inactivation and (iv) with the aid of selective and non-selective pore formation in cell

and are generally succeeded by the immunity genes.

bacteriocins from enduring inside the cytoplasm.

**148**

promoter region [34].

**4. Bacteriocins: mode of action**


**151**

**Bacteriocin** Enterolisin A

**Producing strain**

*Enterococcus faecalis*

**Protein sequence**

(340 amino acids)

MKNILLSILG VLSIVVSLAF SSYSVNAASN EWSWPLGKPY AGRYEEGQQF GNTAFNRGGT YFHDGFDFGS AIYGNGSVYA VHDGKILYAG WDPVGGGSLG AFIVLQAGNT NVIYQEFSRN VGDIKVSTGQ TVKKGQLIGK FTSSHLHLGM

TKKEWRSAHS SWNKDDGTWF NPIPILQGGS TPTPPNPGPK NFTTNVRYGL RVLGGSWLPE VTNFNNTNDG FAGYPNRQHD MLYIKVDKGQ MKYRVHTAQS GWLPWVSKGD KSDTVNGAAG MPGQAIDGVQ LNYITPKGEK LSQAYYRSQT TKRSGWLKVS ADNGSIPGLD SYAGIFGEPL DRLQIGISQS NPF

*Class III bacteriocins*

Helveticin J

*Lactobacillus*

(330 amino acids)

MKHLNETTNV RILSQFDMDT GYQAVVQKGN VGSKYVYGLQ LRKGATTILR GYRGSKINNP ILELSGQAGG HTQTWEFAGD RKDINGEERA GQWFIGVKPS KIEGSKIIWA KQIARVDLRN QMGPHYSNTD FPRLSYLNRA GSNPFAGNKM

THAEAAVSPD YTKFLIATVE NNCIGHFTIY NLDTINEKLD EKGNSEDVNL ETVKYEDSFI IDNLYGDDNN SIVNSIQGYD LDNDGNIYIS SQKAPDFDGS

YYAHHKQIVK IPYYARSKES EDQWRAVNLS EFGGLDIPGK HSEVESIQII

GENHCYLTVA YHSKNKAGEN KTTLNEIYEL

*General overview of few important bacteriocins belonging to Lactic acid bacteria, their protein sequence and killing mechanism (Data obtained from BACTIBASE database of bacteriocins* 

SWN

**Table 2.**

*[http://bactibase.hammamilab.org]).*

Generally show bactericidal mode of action.

*helveticus*

**Killing mechanism**

Cell wall degrading enzyme shows bacteriolytic mode of action.

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens*

*DOI: http://dx.doi.org/10.5772/intechopen.95747*


*General overview of few important bacteriocins belonging to Lactic acid bacteria, their protein sequence and killing mechanism (Data obtained from BACTIBASE database of bacteriocins [http://bactibase.hammamilab.org]).*

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens DOI: http://dx.doi.org/10.5772/intechopen.95747*

*Biomimetics*

**Killing mechanism**

Dual killing mechanism leading to cell death.

Inhibits peptidoglycan formation.

Interaction with the cell membrane leads to the

pore formation even at minimal concentrations.

Form transmembrane pores in the target bacterial

cytoplasmic membrane.

Pore formation by barrel stave model.

Form cell membrane channel by barrel stave

mechanism.

Membrane leakage and cell death.

Causes scattering of proton motive force and by

pore formation.

Show killing action and inhibit the growth of

*Listeria monocytogenes*

Pore formation (effective inhibitor of *Listeria* 

*monocytogenes*)

**150**

**Bacteriocin** *Class I bacteriocins*

Nisin Lacticin 481 Lacticin 3147

Lactocin-S

*Lactococcus* 

(30 amino acids)

TTPATPAISI LSAYISTNTC PTTKCTRAC

*lactis* subsp. *lactis*

Lactobacillus

(40 amino acids)

STPVLASVAV SMELLPTASV LYSDVAGCFK YSAKHHC

sakei

L45

Plantaricin A Acidocin J1132 β

*Class II bacteriocins*

Plantaricin J Acidocin 8912

Bavaricin-A Pediocin PA-1

*Pediococcus acidilactici*

(50 amino acids)

*Lactobacillus sakei*

(50 amino acids)

KYYGNGVHXG KHSXTVDWGT AIGNIGNNAA ANXATGXNAG G

KYYGNGVTCG KHSCSVDWGK ATTCIINNGA MAWATGGHQG NHKC

*Lactobacillus acidophilus*

(30 amino acids)

KTHYPTNAWK SLWKGFWESL RYTDGF

*Lactobacillus plantarum*

(30 amino acids)

GAWKNFWSSL RKGFYDGEAG RAIRR

*Lactobacillus acidophilus*

(30 amino acids)

GNPKVAHCAS QIGRSTAWGA VSGA

*Lactobacillus plantarum*

(30 amino acids)

AYSLQMGATA IKQVKKLFKK WGW

*Lactococcus lactis*

(30 amino acids)

KGGSGVIHTI SHECNMNSWQ FVFTCCS

*Lactococcus lactis subsp.* 

(40 amino acids)

ITSISLCTPG CKTGALMGCN MKTATCHCSI

HVSK

*Lactis*

**Producing strain**

**Protein sequence**

#### **Figure 4.**

*Two-way killing mechanism of class I bacteriocins. (a) Nisin forms complex with the lipid II molecule and halts the synthesis of cell wall. (b) In second mechanism, nisin forms complex with lipid II molecules and initiates formation of pore, ultimately leading to cell death.*

existence of lipid II increases nisin activity thereby causing membrane depolarization, disruption of bilayer organization and outflow of limited metabolites like, ions, nucleotides and amino acids, hence halting all the biosynthetic pathways and causing cell death [41]. Nisin is very effective against its target strain and has been reported to show antimicrobial activity even at nanomolar concentrations. When present in lower concentration, nisin gets attached to lipid II molecule and halts cell wall formation by enzyme activity which causes cell death. Furthermore, when nisin is present in higher amount, the nisin- lipid II complex causes sudden cell death by inducing pores in the cell membrane of bacteria (**Figure 4**). Hence, this complex assists in two modes of killing, involving both blockage of cell wall synthesis, and formation of pores (**Figure 4**).

### **4.2 Bactericidal mechanism of class II bacteriocins**

Bacteriocins of class II (non-lantibiotics) show a killing action against related organisms and cause depolarization and cell death by easy insertion into the cell membrane due to their amphiphilic nature. These bacteriocins are capable of disturbing the pH gradient and transmembrane potential of cells which results in scattering of proton motive force; it is reported that this dispersal of proton motive force is believed to be their key action for exerting the fatal activity [42, 43]. The killing mechanism of class II bacteriocins varies among subclasses (**Table 3**) [39]. Class IId and class IIe mechanisms are still under study and are poorly understood till now.

## **5. Potential applications of bacteriocins in food sector**

Bacteriocins produced by LAB have promising applications in the health, pharmaceutical and food sector. However, this section basically reviews the applications of bacteriocins in food safety. Currently, in food industries, bacteriocins are

**153**

**S.No.**

1.

**Subclass**

IIa

**Killing mechanism**

i.

These bacteriocins are highly potent against *L.monocytogenes.*

They act by binding to the mannose phosphotransferase system proteins which are present in the non-polar core of the cell membrane.

ii. iii.

The conserved amino-terminus and carboxyl-

terminus region is accountable for the inhibiotory activity against *L.monocytogenes* and few other desired organisms.

i.

They are found to exert their killing effect against target organisms only when both the peptides show

[44]

synergistic activity.

ii.

Killing mechanism of this subclass involves

permeabilization of the target membrane which

causes leakage of small cytoplasmic molecules.

They create comparatively small and specific pores

than bacteriocins of class IIa.

iii.

> 3.

**Table 3.**

*Tabulated overview of Killing mechanism of subclass of class II bacteriocins.*

IIc

i. pathogens.

ii.

They act similar to the other bacteriocins in case

of mode of action towards target organism, i.e, by

causing membrane permeabilization, efflux of ions

and leading to cell death.

They show wide antimicrobial spectrum against food

[45]

2.

IIb

**References**

[28, 42]

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens*

*DOI: http://dx.doi.org/10.5772/intechopen.95747*


*Tabulated overview of Killing mechanism of subclass of class II bacteriocins.*

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens DOI: http://dx.doi.org/10.5772/intechopen.95747*

*Biomimetics*

**Figure 4.**

existence of lipid II increases nisin activity thereby causing membrane depolarization, disruption of bilayer organization and outflow of limited metabolites like, ions, nucleotides and amino acids, hence halting all the biosynthetic pathways and causing cell death [41]. Nisin is very effective against its target strain and has been reported to show antimicrobial activity even at nanomolar concentrations. When present in lower concentration, nisin gets attached to lipid II molecule and halts cell wall formation by enzyme activity which causes cell death. Furthermore, when nisin is present in higher amount, the nisin- lipid II complex causes sudden cell death by inducing pores in the cell membrane of bacteria (**Figure 4**). Hence, this complex assists in two modes of killing, involving both blockage of cell wall synthe-

*Two-way killing mechanism of class I bacteriocins. (a) Nisin forms complex with the lipid II molecule and halts the synthesis of cell wall. (b) In second mechanism, nisin forms complex with lipid II molecules and* 

Bacteriocins of class II (non-lantibiotics) show a killing action against related organisms and cause depolarization and cell death by easy insertion into the cell membrane due to their amphiphilic nature. These bacteriocins are capable of disturbing the pH gradient and transmembrane potential of cells which results in scattering of proton motive force; it is reported that this dispersal of proton motive force is believed to be their key action for exerting the fatal activity [42, 43]. The killing mechanism of class II bacteriocins varies among subclasses (**Table 3**) [39]. Class IId and class IIe mechanisms are still under study and are poorly understood

Bacteriocins produced by LAB have promising applications in the health, pharmaceutical and food sector. However, this section basically reviews the applications of bacteriocins in food safety. Currently, in food industries, bacteriocins are

sis, and formation of pores (**Figure 4**).

*initiates formation of pore, ultimately leading to cell death.*

**4.2 Bactericidal mechanism of class II bacteriocins**

**5. Potential applications of bacteriocins in food sector**

**152**

till now.

#### *Biomimetics*

extensively used as biological preservatives. Both Gram positive and Gram negative bacteria are efficient in producing the bacteriocins, but those originating from LAB have a greater importance in the food sector [46]. Bacteriocins produced by LAB apart from being regarded as safe also have QPS (qualified presumption of safety) status, and show deleterious effects against food pathogens even in nanomolar range [47, 48].

Bacteriocins are known to have potential preservative properties either when used alone or when combined with other preservation methods in the form of hurdle technology [4, 30, 36]. Bacteriocins as natural preservatives offer following advantages: (a) extended shelf life of food, (b) protection from economic loss due to food spoilage, (c) preservation of the nutrients and vitamins of food and thus maintenance of the flavor and taste of food (d) satisfaction of consumer demands and (e) stability at variable temperatures and heat. Although, the use of purified form of bacteriocin is the usually applied approach, however, the direct addition of LAB has also been found to be effective [36]. The bacteriocins can be applied by at least three different ways to advance the safety and quality of food, (i) by addition of purified or partially purified preparation of bacteriocins in food items, (ii) by addition of the product formerly fermented with a producer strain [19].

One of the essential applications of bacteriocin is bioactive packaging which protects the food from exterior contaminants. Bioactive packaging generally involves the assimilation of producer strains into the packaging substance that is expected to directly interact with food, thereby, helping to improve the storage life and food safety by restricting the growth of food spoiling organisms, mainly in meat and cheese [49]. The interaction of food surface with the packaging film allows easy diffusion of bacteriocins into the food making this method advantageous over drop-wise addition and sprinkling of bacteriocins on food material; as in the latter cases, the food components may hinder the antibacterial activity of bacteriocins [50–53]. Antimicrobial packaging films are able to be developed by straight-away inclusion of the antimicrobial peptide in the packaging of foodstuff, or by an addition of a packet having bacteriocin in the ready-to-eat packed food, which would be later released in the food product during storage period. Research on nisin coated packaging is increasingly being encouraged since the past few years. Neetoo et al. [54] studied the use of nisin-coated synthetic films on vacuum-packed cold-smoked salmon, and observed that the coating when carried out at a specific storage temperature resulted into a remarkable decrement in the survival rate of *L. monocytogenes*. This method is considered to be better than other methods of bacteriocin application as discussed before the bacteriocins can be deactivated, lose its activity by coming directly in contact with food components [55].

Bacteriocins of LAB are normally found to be potent inhibitors of pathogenic Gram positive bacteria, but when nisin and few other bacteriocins are treated with surfactants, they show wide activity against Gram negative bacteria as well. Nisin is the most researched bacteriocin and has been approved for its commercial production for use in cheese, canned vegetables, egg products etc. [56]. It is often used in acidic foods and also has been used in inhibiting undesirable microorganisms in beer and wine [1]. Nisin is also reported to be conjugated with few other preservative procedures such as heat or other types of bacteriocins. This process/technique is found to be more effective in eradicating food spoiling bacteria due to the increased antimicrobial spectrum. Nisin is preferably applied in aqueous form, as the powder form may have improper dispersal issues. Sea food industry is also being benefited from nisin in restricting the expansion of food spoiling microorganisms. A research by Bakkal et al. [57] showed the delayed growth of *L. monocytogenes* in cold smoked salmon when treated with nisin. A similar study carried out by Pei et al. [58] discussed the inhibitory action by nisin on the spoilage bacteria present in tangerine

**155**

*Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens*

wine. Nisin in amalgamation with lactases has been found to be highly competent to restrict the flourishment of *L. monocytogenes*, this may be due to synergistic activity [59]. Use of nisin for non-acidic food and dairy products is very limited; therefore the need arises to identify new bacteriocins having ability to retain stability in dif-

Comparatively, only a few studies have been carried out on the applications of pediocin in the food industry. Pediocin PA- 1/AcH which is accepted for commercial use in meat products has been found to efficiently inhibit the development of *Listeria* species present in ice cream mixture, ground beef and sausage mix [60]. The addition of pediocins as preservatives in the food system aids in efficiently inhibiting the pathogenic food bacteria thereby guaranteeing the extended storage life of food, and safety of consumers [1, 4]. However, only a few bacteriocins have been approved at commercial level. The reason might be that, the newly identified antimicrobial peptides yet remain to be fully characterized, and further studies need to be carried out to gain a complete insight in their molecular mechanism

Bacteriocins are effective in inhibiting several other food spoiling pathogenic bacteria also. Chang and Chang [61] reported the restriction of growth of *Staphylococcus aureus* and *Escherichia coli* by Kimchi made with *Leuconostoc citreum* GJ7, a bacteriocin producing strain. Starter culture of LAB was also found to restrict the *Bacillus cereus* growth in rice fermentation [62] and growth of *Escherichia coli* and *Clostridium perfringens* were also reported to be inhibited by *Lactobacillus plantarum* and *Lactobacillus salivarius* on chicken feed media [63]. These are known to be the major trouble-causers to the food preservation industry. Although the use of bacteriocins has now started becoming popular in the food industry but their full potential has not yet been realized. They still suffer from several limitations as narrow antimicrobial spectrum, high dosage necessity, high production expenditure and low yield. To combat these limitations, research is now-a-days heading in the direction towards combining two or more classes of bacteriocins that aids in checking their efficacy as an improved/better preservative. Additionally, bacteriocins are also being conjugated with nanoparticles to increase their antimicrobial spectrum and for efficient delivery in target cells [30, 40, 64, 65]. These advancements are bound to contribute more towards new inventions and applications in food sector.

Bacteriocins have proven themselves to be potent antimicrobials produced by bacteria. Their applications in various sectors encourage for carrying out research for investigating different applications of bacteriocins in diverse areas. Currently, there is a critical need to combat the limitations of bacteriocins, most significantly, the narrow antimicrobial spectrum. For their effective use against various Gram-negative and food-borne pathogens, the already existing techniques need to be supplemented with new methods. There is a need to develop cost-effective methods for purification

Studies conducted using nanotechnology techniques for enhancing antimicrobial spectrum and lowering the dosage requirement of bacteriocins are also encouraged. After that, evaluating the safety of nanoconjugates using various toxicity tests both *in-vivo* and *in-vitro* should be conducted to authorize their use in both food and pharmaceutical sectors. Furthermore, studying the interaction between bacteriocins and nanoparticles can promote the use of bacteriocins in future. Moreover, there is a need to introduce and design new open-access bacteriocin database, having information about newly discovered bacteriocins; their properties structure, and applications.

*DOI: http://dx.doi.org/10.5772/intechopen.95747*

implicated in bacteriocin production.

**6. Future perspective**

of bacteriocins and enhancing their production.

ferent food systems and is also thermally stable.

#### *Bacteriocins of Lactic Acid Bacteria as Potent Antimicrobial Peptides against Food Pathogens DOI: http://dx.doi.org/10.5772/intechopen.95747*

wine. Nisin in amalgamation with lactases has been found to be highly competent to restrict the flourishment of *L. monocytogenes*, this may be due to synergistic activity [59]. Use of nisin for non-acidic food and dairy products is very limited; therefore the need arises to identify new bacteriocins having ability to retain stability in different food systems and is also thermally stable.

Comparatively, only a few studies have been carried out on the applications of pediocin in the food industry. Pediocin PA- 1/AcH which is accepted for commercial use in meat products has been found to efficiently inhibit the development of *Listeria* species present in ice cream mixture, ground beef and sausage mix [60]. The addition of pediocins as preservatives in the food system aids in efficiently inhibiting the pathogenic food bacteria thereby guaranteeing the extended storage life of food, and safety of consumers [1, 4]. However, only a few bacteriocins have been approved at commercial level. The reason might be that, the newly identified antimicrobial peptides yet remain to be fully characterized, and further studies need to be carried out to gain a complete insight in their molecular mechanism implicated in bacteriocin production.

Bacteriocins are effective in inhibiting several other food spoiling pathogenic bacteria also. Chang and Chang [61] reported the restriction of growth of *Staphylococcus aureus* and *Escherichia coli* by Kimchi made with *Leuconostoc citreum* GJ7, a bacteriocin producing strain. Starter culture of LAB was also found to restrict the *Bacillus cereus* growth in rice fermentation [62] and growth of *Escherichia coli* and *Clostridium perfringens* were also reported to be inhibited by *Lactobacillus plantarum* and *Lactobacillus salivarius* on chicken feed media [63]. These are known to be the major trouble-causers to the food preservation industry. Although the use of bacteriocins has now started becoming popular in the food industry but their full potential has not yet been realized. They still suffer from several limitations as narrow antimicrobial spectrum, high dosage necessity, high production expenditure and low yield. To combat these limitations, research is now-a-days heading in the direction towards combining two or more classes of bacteriocins that aids in checking their efficacy as an improved/better preservative. Additionally, bacteriocins are also being conjugated with nanoparticles to increase their antimicrobial spectrum and for efficient delivery in target cells [30, 40, 64, 65]. These advancements are bound to contribute more towards new inventions and applications in food sector.

## **6. Future perspective**

Bacteriocins have proven themselves to be potent antimicrobials produced by bacteria. Their applications in various sectors encourage for carrying out research for investigating different applications of bacteriocins in diverse areas. Currently, there is a critical need to combat the limitations of bacteriocins, most significantly, the narrow antimicrobial spectrum. For their effective use against various Gram-negative and food-borne pathogens, the already existing techniques need to be supplemented with new methods. There is a need to develop cost-effective methods for purification of bacteriocins and enhancing their production.

Studies conducted using nanotechnology techniques for enhancing antimicrobial spectrum and lowering the dosage requirement of bacteriocins are also encouraged. After that, evaluating the safety of nanoconjugates using various toxicity tests both *in-vivo* and *in-vitro* should be conducted to authorize their use in both food and pharmaceutical sectors. Furthermore, studying the interaction between bacteriocins and nanoparticles can promote the use of bacteriocins in future. Moreover, there is a need to introduce and design new open-access bacteriocin database, having information about newly discovered bacteriocins; their properties structure, and applications.

*Biomimetics*

range [47, 48].

extensively used as biological preservatives. Both Gram positive and Gram negative bacteria are efficient in producing the bacteriocins, but those originating from LAB have a greater importance in the food sector [46]. Bacteriocins produced by LAB apart from being regarded as safe also have QPS (qualified presumption of safety) status, and show deleterious effects against food pathogens even in nanomolar

Bacteriocins are known to have potential preservative properties either when used alone or when combined with other preservation methods in the form of hurdle technology [4, 30, 36]. Bacteriocins as natural preservatives offer following advantages: (a) extended shelf life of food, (b) protection from economic loss due to food spoilage, (c) preservation of the nutrients and vitamins of food and thus maintenance of the flavor and taste of food (d) satisfaction of consumer demands and (e) stability at variable temperatures and heat. Although, the use of purified form of bacteriocin is the usually applied approach, however, the direct addition of LAB has also been found to be effective [36]. The bacteriocins can be applied by at least three different ways to advance the safety and quality of food, (i) by addition of purified or partially purified preparation of bacteriocins in food items, (ii) by

addition of the product formerly fermented with a producer strain [19].

its activity by coming directly in contact with food components [55].

Bacteriocins of LAB are normally found to be potent inhibitors of pathogenic Gram positive bacteria, but when nisin and few other bacteriocins are treated with surfactants, they show wide activity against Gram negative bacteria as well. Nisin is the most researched bacteriocin and has been approved for its commercial production for use in cheese, canned vegetables, egg products etc. [56]. It is often used in acidic foods and also has been used in inhibiting undesirable microorganisms in beer and wine [1]. Nisin is also reported to be conjugated with few other preservative procedures such as heat or other types of bacteriocins. This process/technique is found to be more effective in eradicating food spoiling bacteria due to the increased antimicrobial spectrum. Nisin is preferably applied in aqueous form, as the powder form may have improper dispersal issues. Sea food industry is also being benefited from nisin in restricting the expansion of food spoiling microorganisms. A research by Bakkal et al. [57] showed the delayed growth of *L. monocytogenes* in cold smoked salmon when treated with nisin. A similar study carried out by Pei et al. [58]

discussed the inhibitory action by nisin on the spoilage bacteria present in tangerine

One of the essential applications of bacteriocin is bioactive packaging which protects the food from exterior contaminants. Bioactive packaging generally involves the assimilation of producer strains into the packaging substance that is expected to directly interact with food, thereby, helping to improve the storage life and food safety by restricting the growth of food spoiling organisms, mainly in meat and cheese [49]. The interaction of food surface with the packaging film allows easy diffusion of bacteriocins into the food making this method advantageous over drop-wise addition and sprinkling of bacteriocins on food material; as in the latter cases, the food components may hinder the antibacterial activity of bacteriocins [50–53]. Antimicrobial packaging films are able to be developed by straight-away inclusion of the antimicrobial peptide in the packaging of foodstuff, or by an addition of a packet having bacteriocin in the ready-to-eat packed food, which would be later released in the food product during storage period. Research on nisin coated packaging is increasingly being encouraged since the past few years. Neetoo et al. [54] studied the use of nisin-coated synthetic films on vacuum-packed cold-smoked salmon, and observed that the coating when carried out at a specific storage temperature resulted into a remarkable decrement in the survival rate of *L. monocytogenes*. This method is considered to be better than other methods of bacteriocin application as discussed before the bacteriocins can be deactivated, lose

**154**

This will help the researchers to study already characterized bacteriocins in a simpler way. Additionally use of newly developed techniques viz., mass spectroscopy, bioinformatics studies will aid in understanding and characterizing the bacteriocins in more efficient ways.
