**2. Bacteriocins: antimicrobial peptide derivatives of lactic acid bacteria**

During exponential growth, a huge number of both Gram positive and Gram negative bacteria produce peptides with antimicrobial activities. A universal term "bacteriocin" is used for these protein-like substances with bactericidal activity restricted to related species [4]. However, bacteriocins derived from Gram positive bacteria possess a moderately broader spectrum of antimicrobial activity. These proteinaceous compounds are degraded in the gastrointestinal tract of humans by protease digestion; hence, they become inactive in the human gut and do not pose any harmful effect. Production of bacteriocins is considered as an advantage by the food producers as they are efficient in killing or restricting pathogenic bacteria that struggle for the same habitat in a sufficient amount [5, 6].

Although several bacterial species produce bacteriocins, but LAB group of bacteria include some of the most important genera responsible for an elevated amount of bacteriocin production effective in the food industry [7]. LAB are considered as a heterogeneous cluster of Gram positive, non-aerobic but aero-tolerant, non-sporulating, catalase-negative, acid-tolerant, bacteria having low GC-content, which produce lactic acid by fermentation of sugars. They are recognized to be obtained from an array of different sources, for instance, meat, milk products, vegetables [6], grains, plants [8, 9] mucosal surface of animals, beverages, and from various fermented foods [10, 11]. LAB produce various metabolic products with antimicrobial properties, but current research is more focused on the production of bacteriocins which are toxic proteinaceous compounds and are known to exhibit a strong bactericidal activity against closely-related species; even small doses of bacteriocins are reported to be highly efficient in eradicating food spoiling bacteria [12–14].

#### **2.1 Bacteriocins: classification**

Bacteriocins produced from LAB were earlier categorized in four general classes on account of their characteristics by Klaenhammer et al. [12]. However, a more recent modified scheme of classification of LAB has been formulated by Alvarez-Sieiro et al. [13] which is now more acceptable (**Figure 1**). It includes only three major classes: Class I which includes lantibiotics, Class II which includes nonlantibiotics and class III which are basically large peptides (**Table 1**).

**143**

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

Among all the three classes discussed in **Table 1**, the bacteriocins of class I and II have caught the attention of researchers; since these are more abundant and have been reported to have potential industrial applications including application in the food sector [15]. However, till date, only Nisin (class I) and Pedicon PA-1 (class II) are marketed commercially as a food additive. Nisin (marketed as Nispalin) is commercially approved to be used as a preservative in many food items in almost 50 countries in the world and Pediocin PA-1 (commercialized as Alto®2341) is autho-

Nisin is a 34 amino acid polypeptide having a molecular mass of 3354 Daltons. Synthesis of nisin is intricate, involving transcription, followed by post-translational modifications, secretion and final processing. It is synthesized as a pro-pre-peptide of 57 amino acid residues, where few residues located at terminal end function as the amino-terminal signal sequence of nisin molecule [1]. The other end of the nisin (carboxyl-terminal region of 34 residues) contains threonine, serine, and cysteine which are involved in the generation of reformed amino acids such as lanthionine, methyllanthionine, dehydroalanine and dehydrobutyrine, which are present in fully matured nisin [1] (**Figure 2a**). Nisin generally occurs in the form of a dimer (7 kDa), but may be present as a tetramer (14 kDa) at times. Nisin has two variants namely, nisin A and nisin Z. The difference between these two variants is because of a difference of one amino acid residue at the 27th position, histidine and asparagine being the 27th amino acid for nisin A and nisin Z, respectively. Nisin is predominantly used in dairy products and in canned food, and is particularly efficient when used in the production of cheese, restricting the growth of heat-tolerant sporulating strains [19]. It is also reported to be effective against mastitis-causing Gram positive organisms [20]. *Lactococci*, producer of nisin occur naturally in cheese and raw milk. Apart from

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

rized for its use in meat products [4].

*Classification of bacteriocins produced by lactic acid bacteria.*

**Figure 1.**

*2.1.1 Important commercialized bacteriocins: nisin and pediocin*

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

**Figure 1.**

*Biomimetics*

organisms. These are known to show preservative effect owing to their capability to produce hydrogen peroxide, organic acids, diacyls, and bacteriocins. Among all these, the antimicrobial compounds, bacteriocins, have received a greater attention as natural preservatives because nearly all of them are heat tolerant up to a particu-

The bacteriocins are known to be produced by both positively and negatively Gram stained bacteria, however, the highest number of bacteriocins studied and identified are reported to be the antimicrobial peptides of Gram positive bacteria. A freely available database, BACTIBASE, which is totally dedicated to bacteriocins, consists of almost 177 sequences of bacteriocins out of which 156 belong to Gram positive bacterial strains, and the remaining 18 belong to Gram negative [2]. Production of bacteriocin depends on their genetic determinants; genes for bacteriocin production are reported to be localized either on plasmid or chromosomes [3]. These antibacterial peptides are synthesized on ribosomes and are known to show a bactericidal activity against identical or approximately related species [1]. Bacteriocins confer their bactericidal mode of action by creating a pore in the target

**2. Bacteriocins: antimicrobial peptide derivatives of lactic acid bacteria**

During exponential growth, a huge number of both Gram positive and Gram negative bacteria produce peptides with antimicrobial activities. A universal term "bacteriocin" is used for these protein-like substances with bactericidal activity restricted to related species [4]. However, bacteriocins derived from Gram positive bacteria possess a moderately broader spectrum of antimicrobial activity. These proteinaceous compounds are degraded in the gastrointestinal tract of humans by protease digestion; hence, they become inactive in the human gut and do not pose any harmful effect. Production of bacteriocins is considered as an advantage by the food producers as they are efficient in killing or restricting pathogenic bacteria that

Although several bacterial species produce bacteriocins, but LAB group of bacteria include some of the most important genera responsible for an elevated amount of bacteriocin production effective in the food industry [7]. LAB are considered as a heterogeneous cluster of Gram positive, non-aerobic but aero-tolerant, non-sporulating, catalase-negative, acid-tolerant, bacteria having low GC-content, which produce lactic acid by fermentation of sugars. They are recognized to be obtained from an array of different sources, for instance, meat, milk products, vegetables [6], grains, plants [8, 9] mucosal surface of animals, beverages, and from various fermented foods [10, 11]. LAB produce various metabolic products with antimicrobial properties, but current research is more focused on the production of bacteriocins which are toxic proteinaceous compounds and are known to exhibit a strong bactericidal activity against closely-related species; even small doses of bacteriocins are reported to be highly efficient in eradicating food spoiling bacteria [12–14].

Bacteriocins produced from LAB were earlier categorized in four general classes on account of their characteristics by Klaenhammer et al. [12]. However, a more recent modified scheme of classification of LAB has been formulated by Alvarez-Sieiro et al. [13] which is now more acceptable (**Figure 1**). It includes only three major classes: Class I which includes lantibiotics, Class II which includes non-

lantibiotics and class III which are basically large peptides (**Table 1**).

lar temperature range and are amenable to proteolytic inactivation [1].

cell, thus helping in combating the food pathogens.

struggle for the same habitat in a sufficient amount [5, 6].

**142**

**2.1 Bacteriocins: classification**

*Classification of bacteriocins produced by lactic acid bacteria.*

Among all the three classes discussed in **Table 1**, the bacteriocins of class I and II have caught the attention of researchers; since these are more abundant and have been reported to have potential industrial applications including application in the food sector [15]. However, till date, only Nisin (class I) and Pedicon PA-1 (class II) are marketed commercially as a food additive. Nisin (marketed as Nispalin) is commercially approved to be used as a preservative in many food items in almost 50 countries in the world and Pediocin PA-1 (commercialized as Alto®2341) is authorized for its use in meat products [4].

### *2.1.1 Important commercialized bacteriocins: nisin and pediocin*

Nisin is a 34 amino acid polypeptide having a molecular mass of 3354 Daltons. Synthesis of nisin is intricate, involving transcription, followed by post-translational modifications, secretion and final processing. It is synthesized as a pro-pre-peptide of 57 amino acid residues, where few residues located at terminal end function as the amino-terminal signal sequence of nisin molecule [1]. The other end of the nisin (carboxyl-terminal region of 34 residues) contains threonine, serine, and cysteine which are involved in the generation of reformed amino acids such as lanthionine, methyllanthionine, dehydroalanine and dehydrobutyrine, which are present in fully matured nisin [1] (**Figure 2a**). Nisin generally occurs in the form of a dimer (7 kDa), but may be present as a tetramer (14 kDa) at times. Nisin has two variants namely, nisin A and nisin Z. The difference between these two variants is because of a difference of one amino acid residue at the 27th position, histidine and asparagine being the 27th amino acid for nisin A and nisin Z, respectively. Nisin is predominantly used in dairy products and in canned food, and is particularly efficient when used in the production of cheese, restricting the growth of heat-tolerant sporulating strains [19]. It is also reported to be effective against mastitis-causing Gram positive organisms [20]. *Lactococci*, producer of nisin occur naturally in cheese and raw milk. Apart from


 *Classification of bacteriocin produced by lactic acid bacteria.*

**145**

**Figure 2.**

*PA-1.*

classes as well.

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

nisin, other bacteriocins produced from genus *Lactococcus* are also of economic importance. Lacticin 3147 is an example of bacteriocin derived from *Lactococcus spp*. which works efficiently in a broad array of pH and comprises a wide antimi

*Structure of (a) Nisin, showing the existence of post-translationally modified amino acids and (b) Pediocin* 

In contrast, Pediocin PA-1 is derived from the genus *Pediococcus* which is natu

Although, nisin is approved by the Food and Drug Administration (FDA) for use as a food preservative, its use is limited to acidic foods only, because of its low solubility and constancy at high and neutral pH. This was the major reason for the search for bacteriocins from other species which led to the discovery of bacteriocins from *Pediococcus* species [21]. These bacteriocins are more effectual than nisin in inhibiting food spoiling pathogens, but their antibacterial spectrum is not as broad as nisin [22, 23]. Thus, although both nisin and pediocin play a significant role as a biological preservative in the food processing industry, their glaring limitations have caused the rise of research on bacteriocins from other

rally found in many plant sources [8]. Strains of *Pediococcus* have been used since a long time as inoculum for the manufacture of fermented foods of natural sources [19]. Pediocin PA-1 shows an inhibitory response against few Gram positive bacteria but is reported to show strong antimicrobial activity against *Listeria monocytogenes* which is one of the chief food spoiling bacteria [4]. Pediocin PA-1 comprises of 44 amino acids with no post-translational modifications and has a molecular weight is 4646.95 Daltons (**Figure 2b**). The genes responsible for pediocin PA-1 biosynthesis are positioned on a 3.5 kb DNA segment of plasmid, and comprise four genes; (i)

crobial action spectrum over Gram positive bacteria [5].

pedA (ii) pedB (iii) pedC and, (iv) pedD.



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

### *Biomimetics*

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

**Figure 2.**

*Biomimetics*

[6, 15]

**Major representatives**

Nisin A, Enterocin W

**References**

**144**

**Class** I (Lantibiotics)

Ia Ib charge.

**Subclass**

**Characteristic features**

Small, heat-tolerant, and post-translationally modified peptides.

Elongated, flexible, cationic peptides that generally perform their activity via pore

Normally compact, with globular structures, either having negative charge or no net

These are enzyme inhibitors, immunologically active and act by interrupting the

essential enzymatic reactions in the target species.

Heat-resistant antimicrobial peptides, devoid of any modified amino acid residues.

Pediocin-like, active against Listeria spp., also known as listeria-active peptides.

It consists of complex of two different inactive peptides, activated by complex

Mostly circular peptides, N-terminal and C-terminal covalently linked to each other to

form stable structure.

Non-pediocin, one-peptide, non-linear, sec-dependent bacteriocins.

Non-ribosomal having post-translational modifications at serine-rich C-terminal

Large in size, heat-sensitive, usually multi-domain with catalytic properties and have domain-type structure.

Different domains in their domain structure function in different manner:

i. binding to the receptor,

ii. translocation, and

iii. antimicrobial activity.

Group A Group B

**Table 1.**

*Classification of bacteriocin produced by lactic acid bacteria.*

Non-lytic proteins.

lytic enzymes that act by causing cell wall lysis.

Enterolysin A

Caseicin 80

formation of these two inactive peptides.

Pediocin PA-1/Ach Sakacin A

Carnobacteriocin X

Lactococcin G, Lactacin F Plantaricins

EF and Plantaricin JK

Acidocin B, Carnobacteriocin A,

Enterocin P

Epidermicin NI01

Microcin E492

[13, 18]

[13, 16, 17]

II (Nonlantibiotics) IIa IIb IIc IId IIe

> III

region.

formation and disruption of cell membrane.

*Structure of (a) Nisin, showing the existence of post-translationally modified amino acids and (b) Pediocin PA-1.*

nisin, other bacteriocins produced from genus *Lactococcus* are also of economic importance. Lacticin 3147 is an example of bacteriocin derived from *Lactococcus spp*. which works efficiently in a broad array of pH and comprises a wide antimicrobial action spectrum over Gram positive bacteria [5].

In contrast, Pediocin PA-1 is derived from the genus *Pediococcus* which is naturally found in many plant sources [8]. Strains of *Pediococcus* have been used since a long time as inoculum for the manufacture of fermented foods of natural sources [19]. Pediocin PA-1 shows an inhibitory response against few Gram positive bacteria but is reported to show strong antimicrobial activity against *Listeria monocytogenes* which is one of the chief food spoiling bacteria [4]. Pediocin PA-1 comprises of 44 amino acids with no post-translational modifications and has a molecular weight is 4646.95 Daltons (**Figure 2b**). The genes responsible for pediocin PA-1 biosynthesis are positioned on a 3.5 kb DNA segment of plasmid, and comprise four genes; (i) pedA (ii) pedB (iii) pedC and, (iv) pedD.

Although, nisin is approved by the Food and Drug Administration (FDA) for use as a food preservative, its use is limited to acidic foods only, because of its low solubility and constancy at high and neutral pH. This was the major reason for the search for bacteriocins from other species which led to the discovery of bacteriocins from *Pediococcus* species [21]. These bacteriocins are more effectual than nisin in inhibiting food spoiling pathogens, but their antibacterial spectrum is not as broad as nisin [22, 23]. Thus, although both nisin and pediocin play a significant role as a biological preservative in the food processing industry, their glaring limitations have caused the rise of research on bacteriocins from other classes as well.
