A large number of ribosomally synthesized bacteriocins or bacteriocin-like substances are produced by lactic acid bacteria (LAB) have been identified and characterized in recent years due to their antimicrobial activity against food-borne pathogenic as well as spoilage bacteria. The bacteriocins are not antibiotics and the major difference between bacteriocins and antibiotics is that bacteriocins restrict their activity to genetically closely related species of the producing species and particularly to strains of the same species (Klaenhammer, 1993; Topisirovic et al., 2006). On the contrary, antibiotics have a wider activity spectrum and does not show any privileged effect on closely related strains (Savadogo et al., 2006; De vuyst and Leroy, 2007;
Zacharof and Lovitt, 2012). The antibacterial spectrum of bacteriocin from LAB frequently includes spoilage organisms, oral pathogens and food-borne pathogens such as Listeria monocytogenes, Escherichia coli, Streptococcus oralis and Staphylococcus aureus (De vuyst and Leroy, 2007; Zoumpopoulou et al., 2013). For these reasons there has been increased interest in bacteriocins for their application in food preservation. This helps in reduction of use of chemical preservatives, heat and other physical treatments, thus satisfying the demands of consumers for better taste of foods. Bacteriocin can be added to foods in the form of concentrated preparations as food preservatives, shelf-life extenders, additives and ingredients ( ex-situ) or they can be produced in situ by bacteriogenic starters, adjunct or protective cultures (Galvez et al., 2007; O‘Shea et al., 2013). In situ bacteriocin production offers several
advantages compared to ex situ production regarding both legal aspects and costs.
Bacteriocins are usually inactivated by low pH, heat and from digestive enzymes such as proteases (Todorov et al., 2011; O‘Shea et al., 2013).
Bacteriocin production is often proposed as an advantageous characteristic of probiotic bacteria as it contribute to the colonisation resistance of the host and its protection against gastrointestinal pathogens (Bourlioux, 1997; Fooks and Gibson, 2002; Avonts et al., 2004). In addition bacteriocin production by probiotic lactic acid bacteria increases stability of the food product during its storage and shelf-life (Salminen et al., 1996; Avonts et al., 2004). Nisin, the bacteriocin from some strains of Lactococcus lactis subsp. lactis, which was accorded GRAS (Generally recognized as safe) status and approved for food use by the U.S. Food and Drug Administration, has already found a variety of applications in food preservation (Twomey et al., 2002;
Cotter, 2012). Till date only nisin and pediocins have been used as biopreservatives in food systems (Rodríguez et al., 2002; Cotter, 2012). Bacteriocins have been isolated from the commercial probiotic strains Lactobacillus casei and Lactobacillus johnsonii La1 (Avonts et al., 2004).
Immobilized bacteriocins can also find application for development of bioactive food packaging (Galvez et al., 2007). The effectiveness of bacteriocins requires careful testing in the food systems for which they are intended to be applied against the target bacteria. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level (De vuyst and Leroy, 2007). The broad industrial applications of bacteriocin from LAB are shown in Fig.
1.6. Although, the industrial application of bacteriocins becomes restricted by several limiting factors such as; little or inconsistent production levels, high production costs, a non-ideal antimicrobial spectrum and potency, the risk of the emergence of resistance and the poor/lack of growth of some producing strains in particular foods (O‘Shea et al., 2013).
Fig. 1.6 Potential applications of bacteriocins from Lactic acid bacteria.
1.7.1 Characterization and nomenclature of bacteriocin
By definition, bacteriocins are small proteins with bactericidal or bacteriostatic activity against genetically closely related species (Topisirovic et al., 2006).
Bacteriocin production in LAB is growth associated and the antimicrobial activity is found in the growth medium between the late exponential phase and early exponential phase of the growth. LAB-bacteriocins comprise a heterogeneous group of physic- chemically diverse ribosomally-synthesized peptidesor proteins showing a narrow or
broad spectra of inhibition against both gram positive and gram negative bacteria.
Several in vitro and in vivo experiments are carried out on antagonism effect of different LAB strains against Helicobacter pylori, Clostridium difficile, Campylobacter jejuni, E. coli and Listeria monocytogenes. Bacteriocins from LAB are small peptides, 3-10 kDa, in size (Nes et al., 1996), although there are exceptions (Jorger and Klaenhammer, 1990). The mode of action of bacteriocins from LAB is of two types bacteriostatic and bactericidal. In bacteriostatic mode of action, the bacteriocin retard the reproducibility of the pathogenic strain and after the exclusion of bacteriocin, the pathogenic strain starts to grow again. In bactericidal mode the bacteriocin completely destroyed the pathogenic cell by pore formation, degradation of cellular DNA, disruption through specific cleavage of 16S rRNA and inhibition of peptidoglycan synthesis (Heu et al., 2006; Todorov and Dicks, 2009).
The classification of bacteriocin was first proposed by Klaenhammer, 1993 who classified into four main classes based on their chemical and genetic properties.
Class I, the lantibiotics (<5 kDa), contain the characteristic polycyclic thioether amino acids lanthionine or methyllanthionine, as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid; Class II, the small heat stable non lanthionine containing membrane active peptides (<10 kDa); Class III, large heat labile bacteriocins (>30 kDa); and Class IV, bacteriocin composed of an undefined mixture proteins, lipids or carbohydrates. Class II bacteriocin is again subdivided into three groups, Class IIa or pediocin like bacteriocin, Class IIb or two component bacteriocin and Class IIc or thiol activated bacteriocin. The other classification system was proposed by Cotter et al., (2005) and it contained only two class; Class I (the lantibiotics) and Class II (the non-lantibiotic bacteriocins). Later the Heng and Tagg,
(2006) proposed a universal scheme for classification of bacteriocin as shown in Fig.
1.7. According to this classification the bacteriocin is divided into four groups and Group I and Group II are subdivided in to three individual groups and Group III is subdivided into two individual groups.
Fig. 1.7 Universal classification scheme of bacteriocin (Heng and Tagge, 2006).
1.7.2 Purification of Bacteriocin
Various strategies for purification of bacteriocin from LAB have been used as shown in Fig. 1.8. Most frequently applied techniques for purification of bacteriocin is salt precipitation followed by various combinations of chromatography (De vuyst and Leroy, 2007; Todorov and Dicks, 2009; Zacharof and Lovitt, 2012). Purification of bacteriocin from LAB can be achieved by adsorption-desorption method, in which bacteriocin is initially adsorbed to the producer cells at neutral pH and then released after being treated at low pH (between 2-2.5). Pediocin ACCEL from P. pentosaceus
ACCEL and pediocin NV5 from P. acidilactici LAB5 were purified by this method (Mandal et al., 2008). Bacteriocins can also be purified by organic solvent extraction method, in which bacteriocin is extracted using an organic solvent, butanol. Pediocin A from P. pentosaceus FBB61 was purified using this method (Piva and Headson, 1994).
Fig. 1.8 Purification strategies of bacteriocin from Lactic acid bacteria.