Essay: Bacteriocins

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1. INTRODUCTION
Bacteriocins are generally defined as ribosomally synthesized peptides produced by bacteria that have bacteriostatic or bactericidal activity against other related and unrelated microorganisms (Balciunas et al., 2013). Preservatives are natural or synthetic substances that are added to fruits, vegetables, prepared food items, cosmetics and pharmaceuticals in order to increase their shelf life and maintain their quality and safety by inhibiting, retarding or arresting their fermentation, acidification, microbial contamination and decomposition Simpson (2015),while natural food preservative is the use of natural or controlled microflora or antimicrobials as a way of preserving food extending its shelf life (Baust and Baust, 2006).

Bacteriocin was firstly identified by Gratia (1925) as an antimicrobial protein produced by Escherichia coli and named colicin. The interest in bacteriocins produced by GRAS microorganisms has been leading to considerable interest for nisin discover, being the first bacteriocin to gain widespread attention and commercial application since 1969. As a result, the field has developed increasingly, resulting in the discovery and detailed characterization of a great number of bacteriocins from LAB in the last few decades (Collins et al., 2010). Bacteriocins have attracted considerable interest for their use as safe food preservatives; as they are easily digested by the human gastrointestinal tract, not toxin to eukaryotic organism, not alter nutritional composition of the foods, their activity is lost in the presence of preservative, effective in low concentration (Mills et al., 2011). When deliberately added, bacteriocins have been found to play a fundamental role in the control of pathogenic and undesirable flora, as well as in the establishment of beneficial bacterial populations of gut flora (Collins et al., 2010).

Traditionally, new bacteriocins have been identified by screening bacterial isolates for antimicrobial activity followed by purification and identification of the bacteriocin and its genetic determinants. Such a strategy is still fundamental for detection and identification of powerful bacteriocins of various subclasses, and recent examples of this include; avicin A that was identified from Enterococcus avium strains isolated from faecal samples of healthy human infants from Ethiopia and Norway (Birri et al., 2010), garvicin ML produced by a Lactococcus garvieae strain isolated from mallard duck (Borrero et al., 2011) and enterocin X isolated from an Enterococcus faecium strain from sugar apples (Hu et al.,2010).

The application of bacteriocins for bio preservation of foods usually includes the following approaches: inoculation of food with the bacteriocin-producer strain; addition of purified or semi-purified bacteriocin as food additive; and use of a product previously fermented with a bacteriocin-producing strain as an ingredient in food processing (Chen and Hoover, 2003). An increasingly number of bacteriocins have been isolated and identified from Gram-positive and Gram-negative microorganisms. Recently, the bacteriocins produced by lactic acid bacteria have been the most popular group due to the characteristics of their producers that have GRAS property and versatile usefulness in many industrial applications (Mills et al., 2013). As current information revealed that, recently over 230 bacteriocins produced by LAB have been isolated, reported and studied, some of purified or semi-purified bacteriocins have been tested in food systems, especially in dairy foods (Alvarez-Sieiro et al., 2016). More recently bacteriocins get special attention in food sector due to its potential natural food preservatives against spoilage and pathogenic bacteria (Desalegn Amenu, 2018).

Nowadays, consumers are aware of the health concerns regarding food additives; the health benefits of “natural” and “traditional” foods, processed without any addition of chemical preservatives, are becoming more attractive. Thus, because of recent consumer demand for higher quality and natural foods, as well as of strict government requirements to guarantee food safety, food producers have faced conflicting challenges (Franz et al., 2010). The use of bacteriocins as natural food preservatives fulfills consumer demands for high quality and safe foods without the use of chemical preservatives. In the next sections, we will present bacteriocin producing organisms, bacteriocin classification, their mode of action and structure, bacteriocin detection and purification, their biotechnological applications as novel natural food preservatives.

2. CLASSIFICATION OF BACTERIOCINS
For the past years, several classifications of bacteriocins have been proposed taking into consideration and the first classification proposed by Klaenhammer (1993). Recently, in order to classify novel bacteriocins, Alvarez-Sieiro et al. (2016) proposed an adjusted classification scheme based on the biosynthesis mechanism and biological activity in accordance with other proposals (Arnison et al., 2013). They propose three major classes: Class I – small post-translationally modified peptides; Class II – unmodified bacteriocins; and Class III – larger peptides (>10 kDa, thermo-labile), being each one subdivided into subclasses, this discuss the classification system proposed by (Arnison et al., 2013), Alvarez-Sieiro et al. (2016) and (Célia et al., 2018).

2.1. Bacteriocin from Archaea
Archaeosins are the bacteriocins produced by archaea. They are known to have wider spectrum of activity across species and domains present in the extreme environments (Atanasova et al., 2013). Halocin S8 from halobacteria, a short hydrophobic peptide with 36 amino acids is the first discovered archaeosin. They are generally divided into two groups (i) Protein halocins (30–40 kDa) like H1 and H4 ( Shand, 2007) and Microhalocins (smaller than 10 kDa) like H6/H7, R1, C8, S8 and U1 (Shand, 2007). Protein halocins are generally more sensitive to environmental stress. Microhalocins have the ability to withstand low salt concentrations, heating, long-term storage (Shand, 2007).

2.2. Bacteriocin from Gram negative bacteria
Gram negative bacteria produce bacteriocins which have narrow antibacterial activity spectrum. Colicins from E.coli, Klebicins of Klebsiella pneumonia, marcescins of Serratia marcescens, alveicins of Hafnia alvei, cloacins of Enterobacter cloacae and pyocins of Pseudomonads are the representatives of gram negative bacterial group. The bacteriocins have relatively larger structure. They are divided in to three types: (i) Microcins which are less than 20 kDa in size, (ii) colicin-like bacteriocins (CLBs) 20 to 90 kDa in size (Cascales et al. 2007) and (iii) tailocins which are high molecular weight bacteriocins with multi subunits resembling the tail like structure of bacteriophages (Ghequire et al. 2014).Thus the bacteriocins are bound and imported in to cell membrane and exercising the cytotoxic activity by nucleases/pore formation (Cascales et al. 2007). They are generally heat labile with the exception of Microcin V produced by E.coli.

2.3. Bacteriocin Lactic Acid Bacteria
Although there are several microorganisms that produce bacteriocins, those produced by the lactic acid bacteria (LAB) are of particular interest to the dairy industry (Egan et al., 2016). LAB have long been used in a variety of food fermentations by converting lactose to lactic acid, as well as producing additional antimicrobial molecules such as other organic acids, diacetyl, acetoin, hydrogen peroxide, antifungal peptides, and bacteriocins (Egan et al., 2016). As a result of their extensive use in traditional fermented products, most of the LAB are Generally Regarded as Safe (GRAS), granted by the American Food and Drug Agency (FDA). The European Food Safety Authority (EFSA) also granted the Qualified Presumption of Safety (QPS) status to most of the LAB genera, such as Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and some Streptococcus (EFSA, 2007). Nevertheless, species of the genus Enterococcus and some Streptococcus are pathogenic, thus, they do not have GRAS status and were not proposed for QPS status (EFSA, 2007). Lactic acid bacteria bacteriocins are often active across a range of pH values, resistant to high temperatures and active against a range of food pathogenic and spoilage bacteria (Ahmad et al., 2017). In addition, LAB bacteriocins are sensitive to digestive proteases such as pancreatin complex, trypsin and chymotrypsin, and thus do not impact negatively on the gut microbiota (Egan et al., 2016).

2.3.1. Class I small posttranslationally modified peptides
This class encompasses all the peptides that undergo enzymatic modification during biosynthesis, which provides molecules with uncommon amino acids and structures that have an impact on their properties (e.g., lanthionine, heterocycles, head-to-tail cyclization, glycosylation). They consist of a leader peptide which serves for enzyme recognition, transport, and keeping the peptide inactive, which is fused to a core peptide (Arnison et al., 2013). The key signatures for an appropriate and systematic definition of novel members of this class have been recently suggested (Medema et al., 2015). Lanthipeptides are peptides possessing unusual amino acids, such as lanthionine and/or (methyl) lanthionine (Arnison et al., 2013). Lanthipeptides undergo PTMs, and generally the genes involved in the maturation process are located in the same operon. Ex. Nisin and Mersacidin ((Alvarez-Sieiro et al., 2016).

2.3.2. Class II: unmodified bacteriocins
These class II bacteriocins are broad spectrum antimicrobials particularly active against Listeria (Kjos et al., 2011). The structure of peptides of class II can be divided different regions separated by a flexible hinge (Haugen et al., 2008). The cationic N-terminal half contains two cysteine residues joined by a disulfide bridge, and a conserved YGNGVXC motif, which has been suggested to participate in target interaction (Cui et al., 2012). The replacement of this disulfide bridge by hydrophobic interaction can still retain the activity (Sit et al., 2012). The C-terminus is less conserved and seems to be involved in the target cell specificity (Cui et al. 2012).

2.3.3. Class III(Large peptides)
Class III bacteriocins are large-molecular-weight and heat labile antimicrobial proteins usually composed of different domains. For instance, based on sequence analysis, enterolysin A consists of an N-terminal endopeptidase domain and a C-terminal substrate recognition domain similarly to zoocin A (Nilsen et al., 2003; Lai et al., 2002). Millericin B is a murein hydrolase. Its production depends on the expression of three genes encoding millericin B precursor (MilB), immunity protein (MilF), and transporter protein (MilT) (Beukes et al. 2000). Similarly, enterolysin A cleaves within the peptidoglycan of target cells between Lalanine and D-glutamic acid of the stem peptide and between L-lysine of the stem peptide and D-aspartic acid of the interpeptide bridge (Khan et al. 2013). On the other hand, non-lytic bacteriocins exhibit their bactericidal mode without causing concomitant cell lysis. For instance, dysgalacticin from S. pyogenes binds to the glucose- and/or Man-PTS, resulting in the inhibition of the sugar uptake, and also causes a membrane leakage of small molecules (Swe et al. 2009). Summary of Lactic acid bacteria are summarized in detain as figure 1.

Figure 1. Bacteriocins classification (Alvarez-Sieiro et al., 2016).
2.4. Bacteriocin Mode of Action
Bacteriocins have distinct mechanisms of action and can be divided into those that promote a bactericidal effect, with or without cell lysis, or bacteriostatic, inhibiting cell growth (da Silva Sabo et al., 2014). Most of the bacteriocins produced from LAB, in particular those inhibiting Gram-positive bacteria; exert their antibacterial effect by targeting the cell envelope-associated mechanisms (Cotter et al., 2013). Several lantibiotics and some class II bacteriocins target Lipid II, an intermediate in the peptidoglycan biosynthesis machinery within the bacterial cell envelope and, by this way they inhibit peptidoglycan synthesis (Breukink and de Kruijff, 2006). Other bacteriocins use Lipid II as a cutting molecule to facilitate pore formation resulting in variation of the cytoplasm membrane potential and ultimately, cell death (Machaidze and Seelig, 2003). Nisin, the most studied lantibiotic, is capable of both mechanisms (Cotter et al., 2005).

Some bacteriocins damage or kill target cells by binding to the cell envelope-associated mannose phosphotransferase system (MPTS) and subsequent formation of pores in the cell membrane (Cotter et al., 2013). Other bacteriocins can kill their target cells by inhibition of gene expression (Parks et al., 2007; Vincent and Morero, 2009) and protein production (Metlitskaya et al., 2006). Antimicrobial action of some selected bacteriocins(Class I,II, and III) is shown in figure 2.

Figure 2. Mechanism of Action of Classes I, II and III Bacteriocins ((Alvarez-Sieiro et al., 2016).¬

3. BIOLOGY OF BACTERIOICN
3.1. Bacteriocin biosynthesis and Genetic regulation
Since bacteriocins ribosomally synthesized protein and the genes necessary for their production and immunity protection of bacteriocin producers are usually arranged as operons (McAuliffe et al., 2001b) which are located on conjugative transposable elements, main chromosome and also on plasmids. The location of this gene is based on the producer’s bacteria. Lantibiotics are encoded by structural gene(s) generically referred to as lanA. Lantibiotics are synthesized as biologically inactive pre-peptides consisting of an N-terminal leader peptide attached to the C-terminal pro-peptide. During the maturation process, the leader peptide is removed and the C-terminal pro-peptide is modified to the active lantibiotic. Following the modification reactions, the modified pre-lantibiotics undergo proteolytic processing to remove the leader peptide, leading to activation of the mature peptide. Unlike the lantibiotics, class II bacteriocins do not undergo extensive post-translational modifications. Following synthesis of the biologically inactive pre-peptide, cleavage of the N-terminal leader sequence frequently occurs at a specific processing site Gly-Gly and is performed in tandem with export from the cell through a dedicated ABC transporter and its accessory protein (Ennahar et al., 2000). These ABC transporters contain an N-terminal domain that has proteolytic activity and thus resembles the hybrid LanT transporters. The two conserved glycine residues, which are also found in some class I lantibiotic leaders, may serve as a recognition signal for this sec-independent transporter system (Figure 3).

The two component regulatory system is usually involved in the regulation of the biosynthesis of both lantibiotics and non lantibiotics are composed of histidine protein kinase (HPK) which is membrane bound and a cytoplasmic response regulator. The regulation of bacteriocin biosynthesis is made by the autophosphorylation of the histidine residue (which is a highly conserved residue) by HPK in the intracellular domain on sensing a threshold level of bacteriocin in the environment. This is why bacteriocin is added externally to the fermentation medium to induce and enhance the production of bacteriocin in industrial production (Figure 3). The RR accepts the phosphorylated group through the conserved aspartic acid group present in them, which in turn causes some intramolecular changes triggering the response regulator mediated transcription of the structural gene, export genes and also the regulatory genes (Kuipers et al. 1998).

The immunity to the produced bacteriocins is conferred to the producer strains by means of immunity proteins. Like in the case of lantibiotics the immunity is mediated by LanI and LanFEG (belongs to ABC transport proteins). These two immunity proteins are coded on to multiple open-reading frames (McAuliffe et al., 2001).The LanI immunity protein prevents the formation of pores by the bacteriocins in the producer cells by attaching themselves to the outer surface of the cytoplasmic membrane and modulating the interaction of the bacteriocin with the producer cell’s surface. The LanFEG exerts its protective effect by transferring the bacteriocin molecules attaching to the producer cell’s surface on to the outside medium and keeps the concentration of the bacteriocins attached to the cell surface at check and maintains the critical level of binding (Figure 3).

3.2. Bacteriocin Resistance Mechanism
On the other hand, the bacteriocin resistance mechanism is completely different from the immunity. In this respect, resistant strains are mainly arisen from spontaneous or induced mutations that cause changes in membrane and cell wall, such as alterations in the bacteriocin receptors, electrical potential, fluidity, membrane lipid composition and load or cell wall thickness(Cintas et al., 2001; Riley et al., 2002;Riley et al., 2002). Although exact mechanism of the bacteriocin resistance has not understood yet, Van Schaik et al. underlined that the mutational changes on the cell surface of the resistant strain may occur following cell exposure to low concentrations of bacteriocins as part of an adaptive response to some other internal or external stress factors (Riley et al., 2002).

3.2. Bacteriocin Detection and Quantification
Techniques used to identify, detect and quantify bacteriocins can be divided into three main groups (Martínez et al., 2000): biological, genetic and immunological tests. Biological tests are the starting point in searching for bacteriocin-producing LAB. The most commonly used bioassays are the agar diffusion test and turbidometric methods, based on the inhibition of the growth of an indicator microorganism inoculated in the plate (Cintas et al., 2010). Polymerase chain reaction (PCR) or DNA-DNA hybridization (Southern blotting) are genetic tests that can determine if a bacterial strain has the genetic potential to encode a specific bacteriocin (Martínez et al., 2010). However, detecting the bacteriocin structural gene in a host organism does not provide information about or quantify its production. Immunological tests are the methods of choice for detecting and quantifying bacteriocins. Most of these tests are based on the transfer of an antigen to an inert surface so that, once fixed to this surface, a specific antibody can recognize it. In general, immunological assays allow bacteriocin detection and quantification in different substrates, whether they are found in culture media supernatants of producer microorganisms or in food (Leroy et al., 2002).

3.3. Bacteriocins and recent advances in molecular biology and genome studies
Advances in molecular biology and molecular microbial ecology have provided new valuable tools to study microorganisms in food ecosystems, such as the determination of their bacteriocinogenic potential, the capacity for proliferation and inhibition of unwanted bacteria, or the response to stress factors. The distribution of bacteriocin-encoding genes among food isolates is a common issue that can be easily be resolved by molecular techniques. PCR amplification with specific primers for bacteriocin genes may be used to follow the predominance of an inoculated strain in food fermentation, as shown for sakacin-P producing L. sakei during production of fermented sausages (Urso et al., 2006) and L. gasseri K7 in semi-hard cheese (Matijašiæ et al., 2007). In other cases, it could solve the problems of differentiation of closely related bacteria in mixed populations. Similarly, DNA-based technology could be used to follow the expression of bacteriocin genes in food systems, as well as the influence of environmental conditions on gene expression and the stress response of target bacteria to the produced or added bacteriocin in food. In a recent work, expression of sakacin P structural gene sspA by the L. sakei strain I151 in sausages was studied in order to determine the influence of the production procedure for fermented sausages on bacteriocin production (Urso et al., 2006).The use of fluorescence-based technology could also provide valuable information on the distribution of bacteriocinogenic strains within the food matrix (Fernández de Palencia et al., 2004) or the heterogeneous response of bacterial populations to bacteriocins (Hornbæk et al., 2006).

Another line of research on bacteriocin application may rise from genomic studies. Classical methods for detection of produced bacteriocins may underestimate the bacteriocinogenic potential of LAB due to several factors such as the influence of environmental conditions on bacteriocin production, the inducible character of many bacteriocins, and the loss of the production capacity (which may be caused by gene mutation, gene loss or genetic rearrangements). However, the analysis of complete genomes may reveal the presence of potential bacteriocin genes and new bacteriocins independently of the producer capacity of strains (Nes and Johnsborg, 2004). Many bacteriocins are encoded by small genes that are often omitted in the annotation process of bacterial genomes (De Jong et al., 2006). Based on the genome sequences and gene identification, it will be possible to develop adequate methodologies (such as microarray technology) to study the global response of bacteria to bacteriocins. Overall, the set of methodologies that have emerged in recent years provide an arsenal of barely unexplored tools that could expand the potential of bacteriocinogenic strains for food application and improve our understanding on the global effects of bacteriocins in food ecosystems, allowing a more rational application of these natural antimicrobial hurdles in foods.

3.3.1. Bacteriocin bio engineering strategy for increased efficacy
Bacteriocin bioengineering can be exploited to improve bacteriocin solubility and stability, increase the spectrum of bacteriocin inhibition, and enhance antimicrobial activity. The gene-encoded nature of bacteriocins renders these antimicrobial molecules ideal candidates for bioengineering strategies. Novel bacteriocins can be generated by either mutating bacteriocin-encoding genes or by fusing genes from different bacterial species (Gillor et al., 2005). A more recent study demonstrated that various activities of nisin can be engineered independently (Rink et al., 2007). Random mutagenesis was recently used to generate the largest bank of randomly mutated nisin derivatives reported to date (Field et al., 2008). This led to identification of a nisin-producing mutant with enhanced activity against the mastitic pathogen Streptococcus agalactiae as a result of an amino acid change in the hinge region. Although this type of technology has the potential to generate a limitless supply of potent naturally-derived food preservatives, consumer resistance to genetic engineering and restrictive legislation will undoubtedly limit its development and applications in the near future. However, as knowledge regarding genetically modified organisms expands beyond the scientific community and consumer demands for minimally processed foods increase, it is likely that engineered bacteriocins may enjoy a lucrative future in food safety.

4. NOVEL APPROACHES TO PURIFYING BACTERIOCIN
Bacteriocins are often ribosomally synthesized, extracellular released low molecular weight peptides usually with 30–60 amino acids, which show bactericidal effect on other closely related bacteria (Meza et al., 2015). More research has been carried out on the bacteriocin produced by Gram positive bacteria especially from lactic acid bacteria (Meza et al., 2015). Ammonium sulphate precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, capillary electrophoresis and phase high-performance liquid chromatography are various techniques used for the purification of bacteriocin (Galvez et al., 2014). The generalized method used for the purification of bacteriocin is given in Fig. 5. Purification technique should be easy and simple with minimum processing. For large scale purification at least two types of chromatographic techniques are used in combination such as ion exchange chromatography and gel permeation followed by HPLC (Espitia et al., 2012).

4.2. Bacteriocin Concentration methods
4.2.1. Ammonium sulphate precipitation
Ammonium precipitation method is used for the isolation of many types of bacteriocins. This technique is used for salting out of proteins or peptides. The crude extract of peptide is first centrifuged then cell free supernatant is cooled at 20 min at 10,000 rpm and 4℃. The pellet of is dissolved in distilled water or buffer solution and later membrane dialyzed. This step is followed by gel filtration for purification of the peptides (Mohanty et al., 2016).

4.2.2. Acetone precipitation method
Like ammonium sulphate precipitation, acetone precipitation is used for the salting out of protein or peptides. The crude extract of the bacteriocin is centrifuged to get the cell free extract. The supernatant is separated and the acetone is added in that cell free filtrate, then it is allowed to cooled and kept overnight in the freeze at 4℃, which is followed by the centrifugation at 10,000 rpm. The obtained pellet is dissolved in deionized water and can be purified by using other chromatographic techniques (Biswas and Banerjee, 2016).

4.3. Bacteriocin Purification methods
4.3.1. Ion exchange chromatography (IEC)
IEC is a separation technique, which depends on the interaction of the charge present on the surface of peptides and resin. It may be cationic or anionic exchange depending on charge of the peptide. For large scale purification of peptides high flow rate and strength exchanger are required, which can increase the efficiency of purification. The most commonly used salt is sodium chloride (Colins et al., 2012).

4.3.2. Affinity chromatography
Affinity chromatography depends on the interaction between proteins or peptide and a specific ligand present in chromatography matrix. Peptide of interest bound to ligand and unbounded material is washed away. The bound bacteriocin can be obtained by providing suitable conditions such as changing pH, ionic strength or polarity. This method is employed for the purification of peptides but unfortunately, it provides partial purification (Colins et al., 2012).

4.3.3. Size exclusion chromatography
It is also known as gel permeation or gel filtration method, which depends on the molecular weight of protein or peptides. Molecule separation of this technique depends on the size exclusion. Gel permeation technique is generally used for desalting of ions so; it is employed after the salt precipitation. In this method, resin acts as stationary phase; whereas, buffer is used as a mobile phase. Salt is present in the buffer solution which is responsible for the separation of proteins and beads (Colins et al., 2012). The drawback of this technique is that the capacity of column used for size exclusion chromatography is less and the flow rate is slow. Usually for the purification of antimicrobial peptides, Sephadex G-100 and 0.02 M sodium acetate buffer are used. Column elution is also conducted by using the same buffer. The collected fraction of the eluted sample is then tested by using SDS-PAGE to determine the molecular weight of the desired antimicrobial peptide (Jabeen and Khanum 2014; Mohanty et al., 2016).

4.3.4. Capillary electrophoresis
It is a versatile method used for the purification of peptides. It relies on the migration of the charges present in the peptides as per the charge present in the solution and also on the charge of electric field (Colins et al. 2012).

4.3.5. High performance liquid chromatography (HPLC)
HPLC is considered as one of the most important methods, which is used for isolation and purification of peptides irrespective of their source and its quantity (Colins et al., 2012). In this method, column and high pumping systems are utilized for the purification of peptides. The charge present on the surface of stationary column and antimicrobial peptides are opposite. Two solutions, such as 0.1 % trifluroacetic acid and 80 % acetonitrile are used for maintaining the charge, pH of the column and for the proper purification of peptides. Automatic and high pumping system is the major advantage of this technique, so the manual errors which take place by other methods can be minimized. Different flow rate and solvents can be used as per the purification of the desired peptide (Mohanty et al., 2016; Sankar et al., 2012).

4.3.6. Reversed-phase high performance liquid chromatography (RP-HPLC)
RP-HPLC is a well-known tool for the purification of the antimicrobial peptides. For the purification of peptides, the lypohilized peptide is dissolved in water and fractionated under reverse HPLC and passed through RP-C18 column. The eluted sample is collected at various times interval. Its absorbance is noted and purity or maximum content of peptides can be detected (Sankar et al., 2012). The stability and purity of peptide can be efficiently determined by reverse phase HPLC and hence, it is widely employed for large scale purification of peptides. In HPLC and Reverse phase HPLC, automated controller pumps are used. Handling device is also automated to enhance efficiency. Ion exchange chromatography and ammonium sulphate precipitation yield the partial purification of the peptides (Mohanty et al. 2016).

4.3.7. Thin layer chromatography
Thin Layer Chromatography or High Performance Thin Liquid Chromatography (HP TLC) is recently used by the researchers for the purification of the peptides. HP TLC methods have improved the purification of peptides. It has several advantages such as it is easy to perform; cheaper, small quantity of sample is required. It is performed on 40 % silica gel. Then the sample is loaded on the silica plate and it is subjected to different organic solvent system. Then separated bands or spots appeared on the plates and it is observed under UV-trans illuminator. The eluted spot of antimicrobial peptide is generally tested for its antimicrobial activity (Biswas and Banerjee 2016).

4.3.8. Polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis is one of the techniques used for determining the purity and the molecular weight. The purified antimicrobial peptide is subjected to tricine sodium do decyl sulphate polyacrylamide electrophoreis. Two gels are used for performing the electrophoresis, one is (5 %) stacking solution and another is separating gel (12 %). Polypeptide standard solution is used as the standard for estimating the molecular weight of the peptide. At the time of electrophoresis, two different voltages are used. When the samples are in stacking gel, the voltage is 30 V; whereas when the samples are in resolution gel the voltage is 150 V (Zhao et al. 2015; Upendra et al. 2016).

4.3.9. UV–Visible spectrophotometry (UV–Visspectroscopy)
The qualitative purification of antimicrobial peptide is determined with the help of UV–Vis spectrophotometry. It was investigated by the researchers that for determining the purity of bacteriocin, the UV Vis spectra range from 200 to 400 nm. After comparing the UV–Vis spectra of standard along with purified crude extract, purity of the crude extract can be estimated (Song et al. 2014; Zhao et al. 2015; Upendra et al. 2016). Currently, Upendra et al. (2016) reported the purity of crude extract of nisin by comparing the UV–Vis spectra of the standard nisin. It was found that pure nisin shows UV–Vis absorbance at 280–320 nm.

Figure 4. Bacteriocins Purification Methods

5. BACTERIOCIN AND FOOD PRESERVATION
5.2. Bacteriocins Potential for Food preservative
Consumers consistently have been concerned about possible adverse health effects from the presence of chemical additives and preservatives in their foods. This has resulted in consumers’ attention to natural and ‘fresher’ foods with no chemical preservatives added. This perception, coupled with the increasing demand for minimally processed foods with longer shelf life and convenience, has stimulated research interests in finding natural and effective novel preservatives. Bacteriocins, produced by LAB, may be considered an excellent natural preservative that fulfill these requirements. Bacteriocins act synergistically against spoilage and pathogenic microbes when used with other preservation methods and processing techniques (Banerjee, 2014).

Thus, they may have applications in minimally processed refrigerated foods, for example, vacuum- and modified atmosphere-packaged refrigerated meats and ready-to-eat meals, which lack multiple barriers or hurdles to the growth of pathogenic and spoilage bacteria. Thus, the application of bacteriocins in minimally processed foods helps to prevent damage to nutrients that may be decreased by extreme treatments.

5.3. Bacteriocin as promising natural food preservative
Although modern advances in technology have been developing day by day, the preservation of food is still a debated issue, resulting in economic losses due to food spoilage and undesirable effects on human health. In this respect, many chemical preservatives have been identified and successfully applied in various food processing applications. However, the growing awareness of consumers and the health concerns regarding chemical food additives make natural antimicrobials more attractive. Bacteriocins, especially produced by LAB, have a great potential to meet this request in the food industries (Gálvez et al., 2007, Gálvez et al., 2008; Balciunas et al., 2013).

In food preservation, the bacteriocins produced by LAB are generally regarded as safe substances (GRAS property) and have a relatively broad antimicrobial spectrum against many food-borne pathogenic and spoilage bacteria, show a bactericidal mode of action, usually acting on the bacterial cytoplasmic membrane and their genetic determinants are usually plasmid-encoded, facilitating genetic manipulation (Gálvez et al., 2008). As many studies indicates that the applications of bacteriocins with these features in food industries can extend shelf life of foods, provide extra protection during temperature abuse conditions, decrease the risk for transmission of food-borne pathogens through the food chain, ameliorate the economic losses due to food spoilage, reduce the application of chemical preservatives, permit the application of less severe heat treatments without compromising food safety: better preservation of food nutrients and vitamins, as well as organoleptic properties of foods, permit the marketing of novel types of foods( Gálvez et al., 2008)

The interest in bacteriocins produced by GRAS microorganisms has been leading to considerable interest for nisin, being the first bacteriocin to gain widespread commercial application since 1969. As a result, the field has developed increasingly, resulting in the discovery and detailed characterization of a great number of bacteriocins from LAB in the last few decades. Among bio preservatives, bacteriocin has caught the attention of food scientists to be used as a natural food bio preservative due to its antimicrobial activity against food spoilage and pathogenic bacteria. Now day’s bacteriocin is emerging as the very promising natural alternative against the antibiotic and chemical preservatives and gaining commercial importance worldwide. So, bacteriocin is the most potent antimicrobial peptides serve as novel natural food preservatives

Promise activity of bacteriocins as food preservatives
Although, the new technologies have been developed, the food preservation is still an important issue, as improper food preservation results in the health problems (Balciunas et al. 2013). There are several benefits of using bacteriocin (nisin) in food preservation, which is synthesized by lactic acid bacteria (Galvez et al. 2014): (i) it is approved as safe in food consumption because bacteriocins are sensitive to the proteases in the gastrointestinal tract and also active against foodborne pathogens (Elayaraja et al. 2014). (ii) It is not poisonous to eukaryotic cells. (iii)It possesses broad-spectrum activity against foods spoiling microorganisms. (iv) It is pH and heat resistant. (v) It shows minimum inactivation when exposed to the protease enzymes. Recent research demonstrated that due to many advantages of antimicrobial peptides over chemical preservatives, these can surely enhance the storage life of food products by preventing the growth of food-spoilage organisms. Moreover, antimicrobial peptides are ecofriendly. Addition of bio-preservatives helps in preventing economic loss of food-spoilage (Galvez et al. 2014).

5.4. Effectiveness of Bacteriocins in Food Systems
The application of bacteriocins in food preservation can offer several benefits: (i) an extended shelf-life of foods; (ii) provide extra protection during temperature abuse conditions; (iii) decrease the risk for transmission of food-borne pathogens through the food chain; (iv) ameliorate the economic losses due to food spoilage; (v) reduce the application of chemical preservatives; (vi) permit the application of less severe heat treatments without compromising food safety, better preserving food nutrients and vitamins, as well as organoleptic properties of foods; and (vii) they may serve to satisfy industrial and consumers demands (Gálvez et al., 2007).

Bacteriocins can be introduced into food in at least three different ways: (i) by adding the purified or semi-purified bacteriocin directly to food; (ii) by incorporating an ingredient based on a fermentate of a bacteriocin-producing strain into the food; or (iii) by producing the bacteriocin in situ by adjunct cultures (Cotter et al., 2005; Deegan et al., 2006). The use of purified/semi-purified bacteriocins is not always attractive to the food industry, as in this form they may be labelled as additives and require regulatory approval. The two other alternatives (fermented ingredient/in situ production) do not require regulatory approval or preservative label declarations. These options are frequently regarded as more attractive routes through which bacteriocins can be incorporated into a food (Deegan et al., 2006).

The efficacy of bacteriocins in foods will greatly depend on various food-related factors that in most cases involve: (i) interactions with food components (fat or protein particles); (ii) precipitation; (iii) inactivation by conditions that destabilize the biological activity, like proteolytic degradation or oxidation; and (iv) uneven distribution of bacteriocin molecules in the food matrix (Coma, 2008). The chemical composition and the physical conditions of food, e.g. pH, osmolarity and fat content, can have a significant influence on the activity of bacteriocins. For example, the solubility, stability and biological activity of nisin are dependent on the pH medium.

5.5. Application of Bacteriocins as Novel Natural Food Preservation
The strategies for application of LAB bacteriocins as novel natural preservation in food systems are based on their beneficial properties on human heath: a) they are GRAS substances like nisin; b) they are non-toxic on eukaryotic cells; c) they become inactivated by digestive proteases and have insignificant influence on the normal gut microflora; d) they are usually thermostable and pH-tolerant; e) they possess a wide antimicrobial spectrum against many food spoilage and pathogenic bacteria; f) they demonstrate a bactericidal mode of action by acting on bacterial cytoplasmic membrane that does not lead to cross resistance to conventional antibiotics (Ananou et al.,2007; Galves et al ., 2014). Applications of LAB bacteriocins in food as novel food preservation have been developed as follows: a) by inoculation of food with LAB – starter or protective cultures for in situ production of bacteriocins; b) use of previously fermented food by fermentative of a bacteriocin-producing strains as an ingredient in food processing; c) by addition of semi- or purified LAB bacteriocins (Ananou et al., 2007). The use of bacteriocins as ingredients or additives requires some new strategies for large scale production in suitable low-cost food-grade media (Garcia et al., 2010).

Moreover, the application of LAB bacteriocins in food biopreservation is important due to some other useful features: a) decreases the risk of food poisonings; b) decreases the risk of contamination with food-borne pathogens through the food chain; c) extends the shelf life of food products; d) reduces the intensity of physical treatments and protects the food during temperature abuse conditions; e) decreases economic losses caused by the food spoilage; f) reduces the adding of chemical preservatives and thus to avoid their harmful effects on human health; g) provides better preservation of the food nutrients, vitamins and improves the organoleptic properties of food; h) provides alternative preservation barriers for “novel” foods less acidic, with a lower salt content, and with a higher water content (Galves et al ., 2014). All of these beneficial properties make LAB bacteriocins suitable for industrial use and adequate to satisfy the increasing consumers’ demands for fresh-tasting, safe, naturally preserved and minimally-processed food products with improved organoleptic and nutritional characteristics.

5.6. Challenges of using bacteriocin as natural food preservation
The in situ production of bacteriocins in the food system is affected by reduced growth and/or production of bacteriocins by the bacteriocinogenic strains due to unfavorable pH, aw, temperature fluctuations during processing and storage like freezing, thawing, homogenization which impair the viability of the bacteriocin producing bacterial cells and inhibition by other food components like spices, additives, salt concentration, food structure, buffering capacity etc Apart from them the emergence of pathogenic strains resistant to the produced bacteriocins, and genetic instability of the producer strains would also reduce the efficacy of the bio preservation by bacteriocinogenic cultures. Thus these factors are interrelated to each other and affect the bio preservation technique. The efficacy of pure bacteriocins in the food systems are affected by the permeability of the bacteriocins in the food matrix, and the pH of the food matrix which influences the solubility of the bacteriocin, distribution in the food matrix, inactivation by the enzymes present in the food systems like meat products, interaction with food additives and food components like complex formation with glutathione (Galvez et al., (2007; 2008; Eldin et al., 2017)

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