bacteriocins as a natural antimicrobial agent in food...

PAK. J. FOOD SCI., 24(4), 2014: 244-255

ISSN: 2226-5899

Pakistan Journal of Food Sciences (2014), Volume 24, Issue 4, Page(s): 244-255

244

Bacteriocins as a natural antimicrobial agent in food

preservation: A review

Muhammad Saeed, Wahab Ali Khan, Muhammad Asim Shabbir, Muhammad Issa Khan, Muhammad Atif

Randhawa and Iqra Yasmin

National Institute Of Food Science & Technology, University of Agriculture Faisalabad-Pakistan

*Corresponding Author: [email protected]

Abstract

Bacteriocins are ribosomally synthesized peptides or proteins with antimicrobial activity, produced by different

groups of bacteria. Many lactic acid bacteria (LAB) produce bacteriocins with rather broad spectra of inhibition.

Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is

currently the only bacteriocin widely used as a food preservative. Several LAB bacteriocins offer potential

applications in food preservation, and the use of bacteriocins in the food industry can help to reduce the addition

of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally

preserved and richer in organoleptic and nutritional properties. This can be an alternative to satisfy the increasing

consumers demands for safe, fresh-tasting, ready-to-eat, minimally-processed foods and also to develop “novel”

food products (e.g. less acidic, or with a lower salt content).

Key words: Bacteriocins, lactic acid bacteria, food preservative, antimicrobial activity

Introduction

Bacteriocins are antimicrobial compounds which are

synthesized ribosomally by many members of

Lactic Acid Bacteria (LAB) and other different

bacterial species (Garneau et al., 2002). Some of

LAB bacteriocins are effective against food spoilage

species i.e. faecalis, Enterococcus and Bacillus

species. Bacteriocins inhibit not only closely related

species of bacteria and also effective against food

borne pathogens such as Staphylococcus aureus,

Listeria monocytogenes Clostridium botulinum

(Delves-Broughton, 1990). Van Belkum and Stiles

(2000) reported that a heterogenous group of

antibacterial compound having size of molecules in

range of thousand Daltons to complex proteins

structures that might contains lipid moieties and

carbohydrates are the bacteriocins of LAB. Eijsink

et al., (2002) also defined bacteriocins the chief

peptides typically consisting of 20 to 60 amino

acids. There is large variation among the peptides,

e.g. in terms of amino acid sequence, length,

composition and post translational modifications,

secretion and processing machinery, antimicrobial

activity (alone or in combination with other

peptides). Most all bacteriocins at neutral or slightly

acidic pH have net positive charge and they

normally have stretches of sequence that are

amphiphilic and /or hydrophobic.

Classification of bacteriocins

Bacteriocins are commonly divided into four groups

(Ennahar et al., 2000) as shown in Table 2. They are

I) Lantibiotics; II) small hydrophobic heat-stable

peptides (< 13,000 Da); III) large heat-labile

proteins (> 30,000 Da) and IV) complex

bacteriocins showing the complex molecule of

protein with lipid and/or carbohydrate (Ouwehand,

1998).

1. Class I bacteriocins

Class I bacteriocins (lantibiotics) contains post-

translationally modified peptides (Van Belkum and

Stiles, 2000; O’Sullivan et al., 2002) which are

small (<5kDa) peptides containing β-methyllanthine

(MeLan), unusual amino acids lanthionine (Lan) and

a number of dehydrated amino acids (McAuliffe et

al., 2001) as shown in Table 1. In general, class I

peptides have typically from 19 to more than 50

amino acids (Cleveland et al., 2001).

2. Class II bacteriocins

Class II bacteriocins are ribosomally synthesized as

in active prepeptides that are modified by post

translational cleavage of the N-terminal leader

peptide generally at a double glycine (-2, -1) (Van

Belkum and Stiles, 2000).Class II bacteriocins

comprise a very large group of heat stable

unmodified peptide bacteriocins (O’Sullivan et al.,

2002) with molecular masses smaller than 10 kDa

(Oscariz and Pisabarro, 2001). None of the

bacteriocin in this class displays any

posttranslational modification beyond the cleavage

of an 18-21 amino acid leader region from the pro-

bacteriocin molecule. All are small, between 36 and

57 amino acids after loss of leader peptides (Hill,

1995). Member of this class can be further

subdivided into 3 groups: (1) Group IIa includes

listeria-active peptides that conserved N-terminal

sequence Try-Gly-Asn-Gly-Val and two cysteines

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forming a S-S (disulfide bridges) in their N-terminal

half of the peptide. (2) Group IIb consists of pore-

forming complexs requiring two petides for their

activity. (3) Group IIc includes thiol-activated

peptides which require reduced cysteine residues for

activity (O’Sullivan et al., 2002).

a) Class IIa

Characteristically, class IIa bacteriocins, like other

low molecular mass bacteriocins, are first formed as

ribosomally synthesized precursors or pre-peptides,

which appear not to be biologically active and

contain an N-terminal extension or leader sequence.

Subsequent cleavage of the pre peptide at a specific

processing site removes the leader sequence from

the antimicrobial molecule concomitantly with its

export to the outside of the cell. The leader peptide’s

removal during translocation is accomplished by the

same protein that is associated with the bacteriocin

transport. One important feature of the majority of

these leaders is the presence of two glycine residues

in the C-terminus of leader peptide, at positions -2

and -1 relative to the processing site, though this is

not distinctive of the class IIa. These leaders are

believed to serve as signal peptides for the

processing and the secretion of class IIa bacteriocins

by a dedicated transport system involving two

distinct proteins: an ABC type translocator and an

accessory protein. The two conserved glycine

residues may serve as a recognition signal for this

sec-independent transporter system (Ennahar et al.,

2000). In addition, class IIa bacteriocins are

characterized by occurrence of a

YGNGVXCXXXXCXV sequence motif in their N-

terminal half, including two cysteines that form

disulfide bridge (Eijsink et al., 1998). Another

shared characteristic of these bacteriocins is their

strong antagonistic effect on Listeria monocytogens.

Class IIa bacteriocins have been encountered in a

great variety of LAB belonging to the genera

Enterococcus, Lactobacillus, Pediococcus,

Leuconostoc and Carnobacterium (Eijsink et al.,

2002). Examples of class IIa bacteriocins are shown

in Table 3.

b) Class IIb

Group IIb consists of pore-forming complexes

requiring two peptides for their activity. These two

peptides can be either individually active but

synergistic when acting together (Enterocins L50A

and L50B), or they may both be necessary for their

antimicrobial activity (lactococcin Gα and Gβ,

lactococcin M and N, plantaricin EF and plantaricin

JK) (Oscariz and Pisabarro, 2001). Abp 118

composed of abp 118 α and abp118 β, which

exhibited the antimicrobial activity (Flynn et al.,

2002). It is important to note that one peptide

bacteriocins may display synergistic effects when

applied in combination the term two-peptide

bacteriocins (class IIb) refer only to sets of peptides

whose genes are in the same operon (Eijsink et al.,

2002). Examples of class IIb bacteriocins are shown

in Table 4.

c) Class IIc

Group IIc includes all class II bacteriocins that do

not fall into groups IIa or IIb (Table 5). Two types

of bacteriocins can be found within this group: (a)

antibiotics with one or two cysteine residues

(thiolbiotics and cystibiotics, repectively), and (b)

bacteriocins without cysteine (lactococcin A and

acidocin B). Some bacteriocins belonging to class II

c are exported via a sec-dependent pathway,

whereas others are exported by a sec-independent

mechanism (Oscariz and Pisabarro, 2001).

3. Class III bacteriocins

This group consists of bacteriocins that are heat-

labile proteins with a molecular mass larger than 30

kDa (Van Belkum ad Stiles 2000; Oscariz and

Pisabarro, 2001). They are usually inactivated

within 30 minutes by temperature of 100 °C or less

(Dodd and Gasson, 1994). Most of them are

produced by bacteria of the genus Lactobacillus

(Ouwehand 1998; Oscariz and Pisabarro, 2001), as

shown in Table 6.

4. Class IV bacteriocins

This class contains complex bacteriocins

(Ouwehand 1998) as shown in Table 7. Lipoid or

carbohydrate moieties appear to be necessary for

activity (Van Belkum and Stiles, 2000). The

existence of class IV is not generally accepted as it

may include regular peptide bacteriocins that have

not been properly purified (Nes et al., 1996).

Detection and assay of bacteriocin activity

There are many techniques for detecting bacteriocin

production. Most are based on the diffusion of

bacteriocins through solid or semisolid culture

media to inhibit growth of target strains (Lewus and

Montville, 1991). The methods frequently used to

detect bacteriocin activity involve (i) deferred

antagonism or indirect methods include the flip

streak and the spot-on-lawn-assays. In the flip-streak

method, the putative bacteriocin-producing strain is

streaked on a medium, incubated and a bacteriocin-

sensitive organism is streaked perpendicular to it on

the reverse side of the agar (which must be flipped)

(Harris et al., 1989; Lewus and Montville, 1991). In

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the spot-on-lawn method, the recognized bacteriocin

producing strain is spotted on an agar medium and

sensitive bacteria applied on resulted colony

(Ivanova et al., 2000); (ii) Well-diffusion assay is

direct assay. In this method well are cut into seeded

with a sensitive microorganisms and cell free

supernatant is placed in these wells (Harris et al.,

1989).

Purification of LAB bacteriocins

Most of purification techniques initiate with

separation of cell free supernatant then

concentration of culture supernatant like in salt

precipitation (e.g. extraction with organic solvents,

ammonium sulphate, acid precipitation (Muriana

and Luchansky, 1993) adsorption of bacteriocins

onto the producing cells at pH 5.5-6.8 or

hydrophobic matrix such as amberlite XAD-16

(Cintas et al., 2000). Subsequently, several

chromatographic steps including size exclusion (gel

filtration), adsorbtion (ion-exchange), or

hydrophobic interaction (reverse-phase)

chromatography have been used to achieve

significant purification of bacteriocins (Wu et al.,

2004). Todorov et al., (2004) summarized the

purification method used by others researchers as

follow: (1) ammonium sulphate precipitation, and

cation exchange-SP-sepharose, reversed-stationary-

phase (octyl-sepharose-CL-4B), stationary-phase

C2/C18 chromatography.(2) anion-exchange

chromatography (DEAE-Sephadex A-25) and

reverse-phase HPLC cation-exchange

chromatography (SP-sepharose fast-flow cation

exchange column), C2/C18 reverse-phase

chromatography and hydrophobic interaction

chromatography (phenyl-sepharose CL-4B column)

(3) ammonium sulphate precipitation (40%), and

cation exchange-SP-sepharose (4) ammonium

sulphate precipitation (55%), hydrophobic

interaction (C8), cation exchange chromatography

Mono S cation exchange column (phamacia,

Biotech). (5) ammonium sulphate precipitation

(80%).

Characterization of bacteriocins

Maximum LAB bacteriocins are hydrophobic,

amphiphilic molecules and cationic composed of

twenty to sixty residues of amino acid (Chen and

Hoover, 2003). In addition, Oscariz and Pisabarro

(2001) supported that cationic and highly

hydrophobic are two basic principles which almost

maximum of bacteriocins should fulfill. Maximum

bacteriocins which are small in size are dynamic

towards broad pH range i.e. 3.0-9.0 and while at

extreme pH resistance is shown at 11.0 (bavaricin

A), 1.0 (acidocin B) and has been observed, at pH

7.0 maximum of these bacteriocins are cationic e.g

lactocin S which have net charge of -1 at neutral pH

being the exception. Bacteriocins have higher

isoelectric point which permits them to interact at

physiological pH level with the bacterial membranes

surface which is anionic. This interaction can be

sufficient in the case of wide inhibitory spectrum

bacteriocins and make possible in the case of

receptors requiring compound addition of the

hydrophobic moiety into the membrane of bacteria.

A physical, rapid and sensitive method for detection

of bacteriocins could be a functional to track

purification actions to identify production of

bacteriocin during experiments concerning genetic

exploitation and to identify bacteriocins in food.

In culture or food products presence of bacteriocins

is confirmed by method of searching appropriate

molecular weight compounds. Matrix assisted laser

desorption/ionization time-of-flight mass

spectrometry (MALDI-TOF MS) appears to have

potential as one such method (Rose et al., 1999). For

determination of masses of purified and partially

purified bacteriocins of class I and II and effective

for proteins and peptides of molecular masses in

range of 0.5-30 kDa is determined by MALDI-TOF

MS (Hindre et al., 2003). In addition, molecular

weight of SA-FF22 bacteriocin like substance was

analyzed by ESI-MS (Jack et al., 1996). Moreover,

an extra ordinary powerful tool for determination of

biological samples’ molecular mass is electrospray-

ionization mass spectrometry (ESI-MS) (Walk et

al., 1999).

The last most important data required for complete

characterization are their sequence of amino acids is

determined by using automated protein sequencer

by Edman degradation (Jack et al., 1996). However,

Walk et al. (1999) reported that during automated

protein sequencing numerous troubles might arise of

compounds containing nonproteinogenic amino

acids. Complete amino acid sequence was

determined by using the coupled sequenator-ESI-

MS system. To get a complete sequence of

bacteriocin, the DNA sequence encoding the

bacteriocin can be analyzed (Zendo et al., 2003).

Moreover, studies should be focused on gene cluster

analyses which are taking part in immunity and

production of bacteriocins (Folli et al., 2003).

Biosynthesis of bacteriocins

The bacteriocins production is normally associated

with growth due to the reason that most of

production occurs for the period of middle of

exponential phase and at end of exponential phase,

it boost up to a maximum level till establishment of

the early-stationary phase (Cheigh et al., 2002). In

addition, other reports supported that this loss in

bacteriocin activity may be due to degradation by

endogenous protease induced during the growth

phase and/or the adsorption of bacteriocin on the

surface of the producer (Onda et al., 2003). Genes

coding for active bacteriocins are usually in operon

clusters (Cleveland et al., 2001). The genes

encoding bacteriocin production and immunity are

usually organized in operon clusters (McAuliffe et

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al., 2001). Normally at least four different genes are

necessary to accomplish the bacteriocins production

of LAB: (i) a devoted immunity gene (ii) a

prepeptide structural encoding of gene (iii) a

transporter ABC- encoding is dedicated and (iv)a

gene encoding the secretion machinery (Garneau et

al., 2002; Chen and Hoover, 2003). Bacteriocin

production genes encoding can be located on the

chromosome or encoded in a plasmid or transposon

(Chen and Hoover, 2003). In Bacteriocins synthesis,

first prepeptide and precursor which are biologically

inactive and have an N-terminal expansion sequence

of leader. Subsequence breakage of this prepeptide

at a particular processing site breaks the leader

sequence from the antimicrobial molecule

concurrently with its expulsion to the outside of the

cell. The mature bacteriocin’s translocation through

cytoplasmic membrane is mediated by ABC-

transpoter and accessory protein. The 3-component

regulatory system typically includes histidine

proteinase (HPK), response regulator (RR) and

induction factor (IF), that is needed for indication to

induce the transcription of target genes. The

immunity proteins provide total immunity towards

the bacteriocin producer’s strains (Eijsink et al.,

2002). This process described in Figure 1.

Bacteriocins Inhibitory Spectrum

LAB bacteriocins shows broad spectrum of activity

toward of gram-positive bacteria especially closely

related species (Messens and De Vuyst, 2002). The

primary target for bacteriocin action on gram

positive bacteria is their cytoplasmic membrane

(Garneau et al., 2002). The outer membrane of Gram

negative bacteria provides them permeability barrier

due to which these species are normally insensitive

to bacteriocins from LAB strains. The non thermal

preservation methods high hydrostatic pressure and

pulsed electric field could be applied for sub lethal

injury for gram negative bacteria (Caplice and

Fitzgerald, 1999). Bacteriocin affects Gram negative

bacteria when their outer membrane is impaired

(Abee et al., 1995). In addition, citrate and ethylene-

diamine-tetra-acetic acid (EDTA) are food grade

chelating agents can act to bind magnesium ions in

gram negative bacteria’s lipopolysaccharides outer

membrane so that these species could be susceptible

to bacteriocin attack. Fungi and Yeast are not

retarded by LAB 28 bacteriocins. The strains which

producer bacteriocins are normally immune towards

own bacteriocin (Messens and De Vuyst, 2002).

Mechanism of action of bacteriocins

Bacteriocins have huge variation in their chemical

structures. They influence various necessary

functions of the living cell i.e. translation,

transcription, replication, and biosynthesis of cell

wall, exceptionally most of bacteriocins act by

forming pores that break up energy potential of

sensitive cells and membrane channels (Oscariz and

Pisabarro, 2001). Basic mechanism of action of

these cationic peptides is to target cytoplasmic

membrane of sensitive cells. They forms pores in

cytoplasmic membrane and scatter the proton

motive force (PMF) and so deprive an essential

energy source of cell (Montville and Bruno., 1994;

McAuliffe et al., 2001). The PMF is composed of an

electrical component (the membrane potential; ∆ψ)

and chemical component (the pH gradient: ∆pH) and

drive synthesis of ATP and the accumulation of

other metabolites and ions throughout PMF-driven

transport systems in the membrane subside of the

PMF induced by bacteriocin action, leads to cell

death through cessation of energy-requiring

reactions (Ennahar et al., 2000; McAuliffe et al.,

2001; Chen and Hoover, 2003). The majority of

bacteriocins belongs to class I have demonstrated a

wide spectrum of inhibition. They not only restrain

closely related bacterial species as like species from

the genera Lactobacillus, Enterococcus,

Streptococcus, Leuconostoc, Pediococcus, and

Lactococcus but also Clostridium botulinum,

Bacillus cereus, and Staphylococcus aureus. In this

class a number of bacteriocins like thermophilin 13

and nisin prevent out-growth of spores of C.

botulinum and B. cereus (Chen and Hoover, 2003).

Application of LAB and bactriocins

1. Food biopreservation

Food spoilage and food born pathogenic

microorganisms are effected by bacteriocins

produced by LAB (Montville et al., 1995) and

bacteriocins could play a unique role in hurdle

preservation technology designing. In present time

attentions in these compounds has grown

significantly because of their prospective worth as

natural alternate for chemical food preservatives in

the production of foods with better safety and

keeping quality (Soomro et al., 2002). Previous

researches have clearly demonstrated that NaCl if

present enhanced the antimicrobial properties of

bacteriocins such as enterocin AS-48, leucocin F10,

nisin and others (Ananou et al., 2004). Bacteriocins,

produced by LAB, may be considered natural

preservative or biopreservatives. Biopreservation

refers to the use of antagonistic microorganisms or

their metabolic products to inhibit or destroy

undesired microorganisms in food to enhance food

safety and extend shelf life. Application of

bacteriocins for food biopreservation three

approaches are mostly employed (i) addition of LAB

which produce bacteriocin in product (ii) addition of

semi-purified or purified or semi-purified as food

preservatives (iii) previously fermented food used as

ingredient

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Table 1: Examples of lantibiotics produced by LAB

Lantibiotic Producing strain

Nisin Z L. lactis N8, NIZO22186

Nisin A L. lactis NIZOR5, 6F3,NCFB894, ATCC11454

Lactocin S Lb. sake 145

Lacticin 481 Lactococcuslactis CNRZ481, ADRIA85LO3

Lactococcin Lb. lactis ADRI85L030 CytolysinE. faecalis DS16

Lacticin 3147 Lc. lactisDPC3147

Variacin 8 Micrococcus varians MCV8

Table 2: Classification of bacteriocins from Gram-positive bacteria

Class Subclass Description

Class I Lantibiotics Ribosomally produced peptides that undergo

Extensive post-translational modification.

Ia Lantibiotics Small (<5kDa) peptides containing lanthionine

and β-methyllanthionine

Ib Globular peptides with no net negative charge

Class II Small (<10kDa), moderate (100° C) to high

(121°C) heat stable non-lanthionine containing membrane-

active peptides

II a Listeria-active peptides with –Y-G-N-G-V-X- C near the amino

terminus

II b Two-peptide bacteriocins

II c Thiol-activated peptides

a. Antibiotics with one or two cysteine residues

(thiobiotics and cystibiotics, respectively)

b. Antibiotics without cysteine.

Class III Lage (>30kDa) heat labile proteins

Class IV Complex bacteriocins protein with lipid and or Carbohydrate

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Table 3: LAB producing class IIa bacteriocins and their origins

Bacteriocin Producer strain

Mundticin E. mundtii AT06

Mesentericin Y105 Leuconostocmesenteroides Y105

Piscicocin 126 Carnobacteriumpiscicola JG126

Sakacin P Lb. sake LTH 673

PediocinAcH Pediococcusacidilactici H

Divercin V41 C. divergen V41

Bavaricin MN Lb. sake MN

Enterocin A E. faeciumCTC 492/T136

Piscicocin V1b C. piscicola V1

Enterocin P E. faeciumP13

Curvacin A Lb. curvatus LTH 1174

Carnobacteriocin B2 C. piscicola LV17A

Sakacin A Lb. sake LB 706

with a bacteriocin-producing strain in food

processing (Chen and Hoover, 2003). The genetics,

biosynthesis, structure, and application of LAB

bacteriocins in foods have been recently reviewed

(Deegan et al., 2006). In skim milk and against fish

spoilage at 25°C for at least 15 days Nisin is active

in opposition to L.monocytogenes ATCC 15313

(Elotmani and Assobhei ,2004).

In food processing and preservation high hydrostatic

pressure is an inventive technique that causes killing

and injury of bacterial cell (Ray, 2002). Bacteriocins

could be used synergistically with heat treatment to

reduce the intensity of heat treatments in foods

without compromising microbial inactivation. Heat

and Nisin could act synergistically against L.

monocytogenes and L. plantarum (Ueckert et al.,

1998), in milk heat resistance of L. monocytogenes

is reduced and in cold-pack lobster meat resistance

of L. monocytogenes may also be reduced (Budu-

Amoako et al., 1999). Though bacteriocins are

normally not active against Gram-negative bacteria

but in combination with high hydrostatic pressure

(HHP) which transiently sensitizes and damages

outer membrane of Gram-negative bacteria,

increasing the food preservation. Bacteriocins have

many application possibilities (Black et al., 2005).

The effect of bacteriocins is enhanced by

combination of chelators such as disodium

pyrophosphate, EDTA, trisodium phosphate, hexa

meta phosphate in opposition to Gram-negative

bacteria has been confirmed for nisin both in foods

and under laboratory conditions (Fang and Tsai,

2003).

2. Combination of bacteriocins with chemical

substances and natural Antimicrobials Presence of NaCl enhanced the antimicrobial action

of bacteriocins such as nisin, leucocin F10, enterocin

AS-48 and others (Ananou et al., 2004). However,

nisin activity was also antagonized by low

concentration of NaCl (Bouttefroy et al., 2000).

Sodium chloride may also induce conformational

changes of bacteriocins (Lee et al., 1993) or changes

in the cell envelope of the target organisms

(Jydegaard et al., 2000). Reduction of nitrite content

by addition of bacteriocins may be beneficial in the

food industry. The combinations of nisin and nitrite

delayed botulinal toxin formation in meat systems

and showed increased activity on clostridial

endospores outgrowth and also on Leuconostoc

mesenteroides and L. monocytogenes (Gill and

Holley, 2003). Addition of nitrite also increased the

anti-listeria activity of bacteriocinogenic lactobacilli

in meat and the activities of enterocin EJ97 against

L. monocytogenes, Bacillus coagulans and Bacillus

macroides (Garcia et al., 2004).

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Table 4: outline of known nonlantibiotic two peptide bacteriocins (Class IIb)

Bacteriocin Producer strain

Enterocin 1071A and 1071B E. faecalis BFE1071

ABP118 (Abp 118 α and β) Lb. salivarius UCC118

Enterocin L50A and L50B E. faecium L50

Lactocin 705 α and β Lb. casei CRL505

Lactacin F (LafX and LafA) Lb. johnsonii VPI11088

Lactocoocin Gα and β Lc. Lactis LMG2081

Plantaricin E and F Lb. plantarum C-11

Lactocoocin M and N Lc. Lactis subsp. cremoris 9B4

Plantaricin J and K Lb. plantarum C-11

Thermophilin 13 A and B S. Thermophilus SFi13

Plantaricin Sα and β Lb. plantarum PLCO10

Lacticin 3147 A1 and A2 Lc. Lactis DPC3147

Plantaricin Wα and β Lb. plantarum LMG2379

Table 5: Class IIc bacteriocins

Bacteriocin Producer strains

Cerein 7/8 Bacillus cereus Bc7

Enterocin B E. faeciumT136 ,E. faecium CECT 492

Lactococcin A Lactococcuslactis LMG 2130

Lactococcin B Lactococcuscremoris, Lactococcuslactis WM4

Divergicin A Carnobacteriumdivergens LV13

Acidocin B Lb. acidophilus M46

Table 6: Class III bacteriocins produced by LAB

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AcidophilucinA Lb. acidophilus

Caseicin 80 Lb. casei B80

Helviticin J Lb. heviticus

Helviticin V-1829 Lb. heviticus

Lacticin A Lb. delbrueckii

Lacticin B Lb. delbrueckii

Table 7: Class IV bacteriocins produced by LAB


Lactocin 27 Lb. helveticus

Leuconocin S Leuconostocparamesenteroides

Pediocin SJ-1 Pediococcusacidilactici

Figure 1: Biosynthesis of Class IIa bacteriocins

Figure 2: Combination of bacteriocins with chemical substances and natural antimicrobials

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Organic acids and their salts can potentiate the

activity of bacteriocins greatly, while acidification

enhances the antibacterial activity of both organic

acids and bacteriocins (Jack et al., 1995). The

increase in net charge of bacteriocins at low pH

might facilitate translocation of bacteriocin

molecules through the cell wall. The solubility of

bacteriocins may also increase at lower pH,

facilitating diffusion of bacteriocin molecules. It

describes sensitivity of L. monocytogenes to nisin

(400 IU/ml) increased in combination with lactate.

Further reports have confirmed the increased

antibacterial activity of nisin in combination with

sodium lactate in several food systems. A nisin-

sorbate combination showed increased activity

against Listeria, and B. licheniformis.

Chelating agents permeate the outer membrane

(OM) of Gram negative bacteria by extracting Ca2+

and Mg2+ cations that stabilize lipopolysaccharides

of this structure, allowing bacteriocins to reach the

cytoplasmic membrane. The enhanced effect of

chelators such as EDTA, disodium pyrophosphate,

trisodium phosphate, hexameta phosphate or citrate

and bacteriocins against Gram-negative bacteria has

been demonstrated for nisin both under laboratory

conditions and in foods (Fang and Tsai, 2003). Other

antimicrobial compounds such as ethanol can act

synergistically with nisin to reduce the survival of L.

monocytogenes (Brewer et al., 2002). Sub-lethal

concentrations of nisin (30 IU/ml) and monolaurin

(100μg/ml) in combination acted synergistically on

B. licheniformis vegetative cells and spore

outgrowth in milk. Synergism was also observed for

the sucrose fatty acid esters sucrose palmitate and

sucrose stearate and nisin against several strains of

L. monocytogenes, B. cereus (cells and spores), L.

plantarum and Staphylococcus aureus, but not

against Gram-negative bacteria. Reuterin also

showed a significant synergistic effect on L.

monocytogenes and a slight additive effect on S.

aureus after in combination with nisin (100 IU/ml),

although the antimicrobial effect of reuterin against

Gram-negative pathogens was not enhanced

(Arques et al., 2004).

Essential oils and their active components, the

phenolic compounds are also attractive natural

preservatives. When used in combination with

bacteriocins, the dose of added phenolic compounds

could be lowered thereby decreasing their impact on

the food flavour and taste. Nisin acted

synergistically with carvacrol, eugenol or thymol

against B. cereus and/or L. monocytogenes.

Combinations of nisin with carvacrol, eugenol, or

thymol resulted in synergistic action against

Bacillus subtilis and Listeria innocua, while nisin

and cinnamic acid had synergistic activity against L.

innocua, but only additive against B. subtilis.

Carvacrol (0.5 mM) was used to enhance the

synergy found between nisin and a pulsed electric

field treatment (PEF) against vegetative cells of B.

cereus in milk (Pol et al., 2001). The combination of

nisin and cinnamon accelerates death of Salmonella

Typhimurium and Escherichia coli O157:H7 in

apple juice. The natural variant nisin Z also acted

synergistically with thymol against L.

monocytogenes and B. subtilis. The antimicrobial

activity of enterocin AS-48 against S. aureus cell in

vegetable sauces was potentiated significantly in

combination with the phenolic compounds

carvacrol, geraniol, eugenol, terpineol, caffeic acid,

p-coumaric acid, citral and hydrocinnamic acid

(Grande et al., 2007).

Conclusions A large number of bacteriocins from LAB have been

characterized to date, and many different studies

have indicated the potential usefulness of

bacteriocins in food preservation. Bacteriocins are a

diverse group of antimicrobial proteins/peptides,

and therefore are expected to behave differently on

different target bacteria and under different

environmental conditions. Since the efficacy of

bacteriocins in foods is dictated by environmental

factors, there is a need to determine more precisely

the most effective conditions for application of each

particular bacteriocin. However, the combined

application of many other technologies (such as

ultrasonication, irradiation, microwave and ohmic

heating, or pulsed light) still remains unexplored.

Bacteriocinogenic cells may also act as living

factories in foods. The antimicrobial effects of

bacteriocins and bacteriocinogenic cultures in food

ecosystems must be understood in terms of

microbial interactions.

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