-603250-3606805166995-371475-188595-148590JIMMA UNIVERSITY COLLEGE OF NATURAL SCIENCE BIOLOGY DEPARTMENT PhD in Applied Microbiology

-603250-3606805166995-371475-188595-148590JIMMA UNIVERSITY
COLLEGE OF NATURAL SCIENCE
BIOLOGY DEPARTMENT
PhD in Applied Microbiology (Food Microbiology)
JIMMA UNIVERSITY
COLLEGE OF NATURAL SCIENCE
BIOLOGY DEPARTMENT
PhD in Applied Microbiology (Food Microbiology)

BACTERIOCIN AS A NOVEL NATURAL FOOD PRESERVATIVE
-1905240665PhD in Applied Microbiology (Food Microbiology) Seminar II (Biol. 812) Submitted to Department of Biology (Applied Microbiology), College of Natural Science, Jimma University.

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PhD in Applied Microbiology (Food Microbiology) Seminar II (Biol. 812) Submitted to Department of Biology (Applied Microbiology), College of Natural Science, Jimma University.

-19052540By: Desalegn AmenuAdvisor: Ketema Bacha (PhD, Professor)
By: Desalegn AmenuAdvisor: Ketema Bacha (PhD, Professor)

41624250May 2018
Jimma University
May 2018
Jimma University

Table of Contents
TOC o “1-3” h z u LIST OF FIGURES PAGEREF _Toc515200908 h ivExecutive Summary PAGEREF _Toc515200909 h v1.INTRODUCTION PAGEREF _Toc515200910 h 12.CLASSIFICATION OF BACTERIOCINS PAGEREF _Toc515200911 h 32.1.Bacteriocin from Archaea PAGEREF _Toc515200912 h 32.2.Bacteriocin from Gram negative bacteria PAGEREF _Toc515200913 h 32.3.Bacteriocin Lactic Acid Bacteria PAGEREF _Toc515200914 h 42.3.1.Class I small posttranslationally modified peptides PAGEREF _Toc515200915 h 42.3.2.Class II: unmodified bacteriocins PAGEREF _Toc515200916 h 52.3.3.Class III(Large peptides) PAGEREF _Toc515200917 h 52.4.Bacteriocin Mode of Action PAGEREF _Toc515200918 h 63.BIOLOGY OF BACTERIOICN PAGEREF _Toc515200919 h 83.1.Bacteriocin biosynthesis PAGEREF _Toc515200920 h 83.2.Immunity Mechanism PAGEREF _Toc515200921 h 93.3.Bacteriocin Resistance Mechanism PAGEREF _Toc515200922 h 93.4.Bacteriocin Detection and Quantification PAGEREF _Toc515200923 h 103.5.Bacteriocins and recent advances in molecular biology and genome studies PAGEREF _Toc515200924 h 103.5.1.Bacteriocin bio engineering strategy for increased efficacy PAGEREF _Toc515200925 h 124.NOVEL APPROACHES TO PURIFYING BACTERIOCIN PAGEREF _Toc515200926 h 134.1.Bacteriocin Concentration methods PAGEREF _Toc515200927 h 134.1.1.Ammonium sulphate precipitation PAGEREF _Toc515200928 h 134.1.2.Acetone precipitation method PAGEREF _Toc515200929 h 134.2.Bacteriocin Purification methods PAGEREF _Toc515200930 h 144.2.1.Ion exchange chromatography (IEC) PAGEREF _Toc515200931 h 144.2.2.Affinity chromatography PAGEREF _Toc515200932 h 144.2.3.Size exclusion chromatography PAGEREF _Toc515200933 h 144.2.4.Capillary electrophoresis PAGEREF _Toc515200934 h 154.2.5.High performance liquid chromatography (HPLC) PAGEREF _Toc515200935 h 154.2.6.Reversed-phase high performance liquid chromatography (RP-HPLC) PAGEREF _Toc515200936 h 154.2.7.Thin layer chromatography PAGEREF _Toc515200937 h 164.2.8.Polyacrylamide gel electrophoresis (PAGE) PAGEREF _Toc515200938 h 164.2.9.UV–Visible spectrophotometry (UV–Visspectroscopy) PAGEREF _Toc515200939 h 165.BACTERIOCIN AND FOOD PRESERVATION PAGEREF _Toc515200940 h 185.1.Bacteriocins Potential for Food preservative PAGEREF _Toc515200941 h 185.2.Bacteriocin as promising natural food preservative PAGEREF _Toc515200942 h 185.3.Effectiveness of Bacteriocins in Food Systems PAGEREF _Toc515200943 h 205.4.Application of Bacteriocins as Novel Natural Food Preservation PAGEREF _Toc515200944 h 215.5.Challenges of using bacteriocin as natural food preservation PAGEREF _Toc515200945 h 225.6.Combining Bacteriocins with Other Hurdles PAGEREF _Toc515200946 h 226.CONCLUSION PAGEREF _Toc515200947 h 24REFERENCES PAGEREF _Toc515200948 h 25
LIST OF FIGURES TOC h z c “Figure” Figure 2. Mechanism of Action of Classes I, II and III Bacteriocins adopted from Alvarez-Sieiro et al. (2016). PAGEREF _Toc515710670 h 15Figure 3. Biosynthetic Pathway of Bacteriocins adopted from Chen and Hoover, 2003). PAGEREF _Toc515710671 h 17Figure 4. Bacteriocins Purification Methods PAGEREF _Toc515710672 h 25
Executive SummaryBacteriocin can be defined as a ribosomally synthesized antimicrobial polypeptides or a protein secreted by bacteria chiefly by lactic acid bacteria. They display potential antagonist activity against the intimately concomitant species of bacteria or unconnected groups of microorganism. Other terminology that should be consider here is a biological food preservation, it is the food preservation method that use naturally produced antimicrobial compound or normal-flora as a food preservative to prevent food spoilage or to food increase food shelf life. Currently due some ambitious rational motive like augmentative of demand consumers to get new fresh food products, as a result of potential health hazards of artificial food preservative and uncontrollable food additive while processing and preparation in needs advancement of alternative food preservation technologies maintenance of the freshness of the food products. So, application of Bacteriocin as natural food preservative can overcome this limitations and challenges. Therefore, Bacteriocin is emerging as the very likely natural alternative preservative against the antibiotic and now widely accepted as safe food preservative though out worldwide. Even though some microorganism; archaea and gram-negative bacteria produced Bacteriocin but majority of Bacteriocins is produced by lactic acid bacteria (LAB).Demand for new antimicrobial polypeptides; Bacteriocin safety potential and origin which is Generally Regarded as Safe (GRAS); increase of consumers’ demand and awareness on health effect and beneficial of fermented foods and government legislation attract Bacteriocin as novel natural food preservative and furthermore it searching the design of novel technologies used in food industry. In this review recent information on classification of bbacteriocins, bacteriocin mode of action, biology of bacteriocin, detection and purification techniques and current challenges and solution on application of bacteriocins as a novel natural food preservative will be discuss in detail.
Key words/phrases: Bacteriocin, food preservatives, Lactic Acid Bacteria, Novel natural food preservative.

INTRODUCTIONBacteriocins are defined as ribosomally synthesized polypeptides produced by different groups of prokaryotes bacteria specially gram negative bacteria, gram positive bacteria and arachea (Balciunas et al., 2013). While natural food preservative is the use of natural products from different sources(animal sources, plant sources, microbial sources …) or microflora or their products as promising food preservative to prevent food spoilage and pathogenic microorganism and thereby for enhancing the food shelf life (Simpson, 2015).
The first bacteriocin was discovered in 1925 by Gratia which was produced by Escherichia coli and the bacteriocin is called colicin(Gratia,1925).Even though different types of bacteriocins were discovered and identified so far, but until 1969 there was no any bacteriocin approved as safe to be used as food preservative. In 1969, nisin was discovered and its was the first bacteriocin approved as safe food preservative (Collins et al., 2010). Then after interest of bacteriocin produced by GRAS microorganism has increased and lead for discovering, detail characterization and application of bacteriocins in food industry (Collins et al., 2010). Furthermore, bacteriocins have got substantial interests for their GRAS status, because they are not challenging for human health since they are straightforwardly digested by human gastrointestinal tract, they do not produce toxin, not alter nutritional composition and quality of foods, their activity is not lost while using preservative and they are very effective in low concentration (Mills et al., 2011). Application of bacteriocin has play great roles in controlling and preventing food spoilage and pathogenic bacteria in the food sin addition to extending foods’ shelf life as well as establishing the populations of gut flora (Collins et al., 2010).
Identification and characterization of new and novel bacteriocin is not a new idea because just starting from traditional investigation, commonly new bacteriocins from different sources discovered by screening isolates for antimicrobial activity tracked by purification and identification of the bacteriocin and including genetic determinants and still now such activities are ongoing, recent examples is avicin A from Enterococcus avium (Birri et al., 2010), garvicin ML from Lactococcus garvieae (Borrero et al., 2011) and enterocin X from an Enterococcus faecium (Hu et al.,2010). It is possible to apply bacteriocin as food preservative using the following approaches: direct inoculation of purified or semi purified bacteriocins; addition of bacteriocins producers as food preservative and inoculation previous fermented products while food processing and storage (Chen and Hoover, 2003). Enormous numbers of bacteriocins have been isolated and discovered from gram positive and gram negative bacteria and bacteriocins produced by lactic acid bacteria are the most popular polypeptides used in food industry due to GRAS status and multipurpose important in pharmaceuticals, agriculture and vaccination and other huge industrial application(Mills et al., 2013). To our knowledge, currently more than 230 bacteriocins were studied, but only few of them screened to be tested as food preservatives (Alvarez-Sieiro et al., 2016). Greatly, now bacteriocins are attracting special responsiveness in worldwide for food productiveness (Desalegn Amenu, 2018).
Nowadays, consumers’ attitude and knowledge on beneficial health concern of natural fermented foods and side effects of chemically preserved foods and food additives are becoming more attractive. Thus, because of topical consumer demand for higher quality of fresh cutting food products and as well as of national and international governmental strict legislation on requirements to be satisfy for food safety and quality lead for the discovering of new and novel promising natural food preservative(Franz et al., 2010). So, use of antimicrobial peptides produced by lactic acid bacteria, bacteriocin as a natural food preservative will satisfy consumers’ demands for well fresh cut foods for a long period of time and safe foods without health impact food additive as well as governmental strict requirements and legislation. Therefore, the core idea of this assembly is to review and discuss the role and application of bacteriocin as a novel natural food preservative.

BACTERIOCINS CLASSIFICATION
Starting from the pioneer for bacteriocin classification Klaenhammer (1993), who was proposed four different types of bacteriocin classes to now (Célia et al., 2018) different scholar proposed different classes and sub classes of bacteriocin. Therefore there was no certain bacteriocin classification scheme and there was still a debate regarding to bacteriocin classification. Generally bacteriocin classification is basically depends on physic-chemical characteristics of bacteriocins, molecular weight, producers strains, bacteriocin structure, bacteriocin mode of action (Klaenhammer,1993), but recent classification system referring to biosynthesis mechanism and biological activity((Arnison et al., 2013). But, this review paper is attempt to classify bacteriocin according to classification proposed by Arnison et al. (2013), Alvarez-Sieiro et al. (2016) and (Célia et al., 2018) as they have proposed almost similar classification system specially focusing of novel bacteriocin classification. But before discussing bacteriocin let first discuss bacteriocin producing organism. Bacteriocin production is not restricted to only gram positive bacteria but also archaea and gram negative bacteria can produce bacteriocin. But this paper is mainly focusing bacteriocin produced by lactic acid bacteria because their GRAS potency and application good natural preservative.
Bacteriocin from ArchaeaLike other bacteria archae produced bacteriocin, this is called Archaeosin it has wider antimicrobial activity against large numbers of organism in extreme environment (Atanasova et al., 2013). The common example of this bacteriocin is halocin S8 produced by halobacteria, and there are two types of halocin (i) Protein halocins and Microhalocins (Shand, 2007) with the largest and smallest peptide respectively. Microhalocis halocin are more effective to be used as antimicrobial than protein because microhalocins halocin are highly resistance to environmental stress like low salt concentrations, heating and long-term storage while protein halocin less effective because they are more sensitive to environmental stress (Shand, 2007).

Bacteriocin from Gram negative bacteriaSimilarly gram negative bacteria produce can bacteriocins different types’ bacteriocin but most of the bacteriocin produced by these bacteria has narrow antimicrobial spectrum thus why it’s not widely use in the food industry. But the first bacteriocin was isolated and discovered from gram negative bacteria; the bacteriocins have relatively larger structure. Generally gram negative bacteriocins are categorized in the groups. These are; Microcins is the smallest bacteriocin (<20 kDa); colicin-like bacteriocins (20 to 90 kDa) this bacteriocin has medium size (Cascales et al. 2007) and tailocins is the third one and it’s the largest bacteriocin (Ghequire et al. 2014). Regarding to antimicrobial activity of these bacteriocin have ability to kill target organism either by cytotoxic activity by the action of nuclease or thorough pore formation lead cell death (Cascales et al. 2007).
Bacteriocin Lactic Acid Bacteria As already pointed out from discuss lactic acid bacteria bacteriocins are interest for the food industry due to safety status. As a result LAB have been used as food preservative for long period of time since they have ability to produced numerous antimicrobial activity in addition to bacteriocin (Egan et al., 2016). Due to bacterial all-encompassing use in natural fermented foods the greatest numbers of LAB are Generally Regarded as Safe (GRAS), granted by the American Food and Drug Agency (FDA). According to information obtained from EFSA (2007), European Food Safety Authority (EFSA) also granted the quality status LAB in food industry since they have and was proposed for QPS status (EFSA, 2007). As we discuss and summarized repeatedly LAB bacteriocins are effective to be used in food processing, production and preservation and they often efficiency over wide range of pH, overcome high temperatures effects also and have wide antimicrobial spectrum against enormous numbers food spoilage and pathogens (Ahmad et al., 2017). LAB bacteriocin are classified in to three basic category as evidence obtained above section these are; Class I,Class II and Class III(Arnison et al., 2013; Alvarez-Sieiro et al.,2016; Célia et al., 2018).

Class I These classes are commonly known as small posttranslational modified peptides because they undergo enzymatic modification throughout biosynthesis. This post modification activity is processed duet presence of uncommon amino acids and structures like lanthionine, heterocycles, head-to-tail cyclization, glycosylation. In additional they have leader peptide which functioned as enzyme recognition, transport, and keep continues inactivation of peptide inactive (Arnison et al., 2013; Medema et al., 2015). These groups are also known as lantibiotics and generally there are different genes responsible for lantibiotics biosynthesis they are arranged as operon and genes involved for maturation of bacteriocin are located on the same gene (Arnison et al., 2013; Alvarez-Sieiro et al., 2016). Ex. Nisin
Class IIUnlike class I bacteriocins which undergo posttranslational modified, these classes are non-posttranslational modified peptides. Mostly class II bacteriocin has comprehensive spectrum antimicrobials predominantly against food borne pathogens lie Listeria (Kjos et al., 2011). Class II bacteriocin peptides have three regions which are separated by flexible hinge that divide the peptides region to highly variable and conserved region (Haugen et al., 2008). They have N terminal leader that have attached C terminal. N terminal leader is cationic contains two cysteine residues combined by a disulfide bridge more conserved which have suggested to contribute in target interaction to killing mechanism of action (Cui et al., 2012). On other hands the C-terminus not highly conserved like N terminal therefore it’s less infective in target interaction specificity (Cui et al. 2012).

Class IIIThe unique property of this class is larger peptide, heat labile protein made of distinct domain of peptides. Contain N-terminal domain which called endopeptidase and a C-terminal domain it is substrate recognition (Nilsen et al., 2003; Lai et al., 2002). This class also contains non-lytic bacteriocins that display different mode of action, some of them inhibit target pathogens without killing and lysis target cell, while other class classes of this groups binds to the mannose or glucose, which prove sugar uptake of target organism some classes bacteriocins causes a membrane leakage of small molecules lead loss of essential molecules (Swe et al. 2009).
Table SEQ Table * ARABIC 1. LAB Bacteriocins Classification Adopted From Shih et al. (2016)
Classes Example Producers
Class I They are post-translationally modified, linear or globular peptides containing lanthionine, ?-methyl lanthionine and dehydrated amino acids. Nisin A Lactococcus lactissubsp.lactic
Nisin U Streptococcus uberis
Class II Heat stable, unmodified, non-lanthionine-containing bacteriocins, heterogeneous class
of small peptides Class II (pediocinPA-1like
bacteriocins) pediocin PA
carnobacteriocin Pediococcus acidilactici
Carnobacterium
Class III Large, heat unstable proteins Enterolisin A Enterococcus faecalis
Bacteriocin Mode of ActionAccording recent information revealed by Silva Sabo et al., 2014, bacteriocins have been shown different mechanism of actions which is basically depends of bacteriocin producers, as well as classes and subclasses of bacteriocin with some of them have bactericidal action those promote cell death but did not cause cell lysis, besides to this some of them inhibit the cell growth only. In case of gram negative bacteria they produced antimicrobial peptides that show antibactericidal by targeting the cell envelope with lipid II as targeting molecules (Cotter et al., 2013). This is true for almost in all lantibiotics and some class II bacteriocins those use Lipid II as dockling molecules to prevent the peptidoglycan biosynthesis by provoking peptidoglycan biosynthesis apparatus within the bacterial cell envelope (Breukink and de Kruijff, 2006). On other hands some bacteriocins use Lipid II as a cutting molecule to facilitate pore formation leading to cytoplasm membrane potential distraction and eventually, result cell death of the target organism (Machaidze and Seelig, 2003; Cotter et al., 2005).

Far from the above factors few bacteriocins need other receptor protein to attach and find target cell, therefore such bacteriocins used mannose phosphotransferase system (MPTS) to bind to the cell envelope in cell membrane following formation of pores (Cotter et al., 2013). Most likely some common bacteriocins provoke protein production and gene expression that help them simple to kill their target cells by inhibition of protein and gene expression (Parks et al., 2007; Vincent and Morero, 2009) (Metlitskaya et al., 2006). Antimicrobial action of some selected bacteriocins (Class I, II, and III) is shown in figure 2.

Figure SEQ Figure * ARABIC 2. Mechanism of Action of Classes I, II and III Bacteriocins adopted from Alvarez-Sieiro et al. (2016).BIOLOGY OF BACTERIOICNBacteriocin biosynthesis and Genetic regulation
Bacteriocins are a polypeptides produced by producers ribosomes and there are different genes responsible for bacteriocins biosynthesis and at some stages which are also important to regulate bacteriocin production and these genes are usually arranged as operon, they are located either on main chromosomes, on mobile gene and also on bacterial plasmids (McAuliffe et al., 2001), but the location and positions of genes are different among bacteria. Class I bacteriocins are encoded by structural genes which are called as LanA. Principally, after activation of structural genes lantibiotics are produced as biologically inactive peptides consisting of N-terminal leader pre-peptides attached with C terminal pro-peptides. Then later, leader peptide is removed and pro-peptide is modified to active lantibiotics. To remove leader peptides, pre-lantibiotics engaged proteolytic dispensation that leading for activation of the mature peptides (Ennahar et al., 2000). In contrast, in case of class II bacteriocins there is no post transitional modification reaction. Simply following the production of biological inactive pre-peptides, N –terminal leader repeatedly cleaved at specific processing site called Gly-Gly and disseminate from the cells via an enthusiastic ABC transporter with the help of its accessory protein (Ennahar et al., 2000). So this is clearly describing the main difference between lantibiotics and non lantibiotics bacteriocins. Some description regarding to bacteriocin biosynthesis s and regulation is illustrated in figure 3.
The two major elements involved in regulation and monitoring of bacteriocins biosynthesis and genetic regulation lantibiotics and non lantibiotics are histidine protein kinase (HPK) and cytoplasmic response regulator (RR). Histidine protein kinase regulated bacteriocins biosynthesis through process called autophosphorylation because HPK can sense a threshold level of bacteriocin in the environment. Therefore it’s possible to induce and enhance the production of bacteriocin in industrial production by adding bacteriocin to fermentation medium externally. After bacteriocins phosphorylated by HPK, Cytoplasmic response regulator use aspartic acid to changes some intracellular molecules that are serve as mediate for the transcription of genes responsible for bacteriocin production and regulation like structural gene, export genes and also the regulatory genes. Therefore, it’s the transcription of these genes that take place for the regulation and biosyhhesis of bacteriocins (Kuipers et al., 1998).

Bacteriocins producers are using immunity protection mechanism to prevent themselves from bacteriocins. There are some genes that mediate immunity and they are commonly ABC transport proteins (LanI and Lan FEG) (McAuliffe et al., 2001). LanI is attached to producer’s outer cell surface of cytoplasmic membrane to prevent pore formation this is to enhance for modulating interaction between bacteriocin and producers cells. Whereas The LanFEG employs its defending effect by transporting the bacteriocin by fastening to the producer cell’s surface on to the outside medium and retains the concentration of the bacteriocins attached to the cell surface at check and maintains the critical level of binding (Figure 3).

Figure SEQ Figure * ARABIC 3. Biosynthetic Pathway of Bacteriocins adopted from Chen and Hoover, 2003).Prebacteriocin and prepeptide of induction factor (IF), (2) Mature bacteriocin, (3) Regulation of bacteriocin (Histidine protein kinase, Cytoplasmic response regulator), (4) Response regulator activates transcription of the regulated genes and (5) Producer immunity mediation (Chen and Hoover, 2003).

Bacteriocin Resistance MechanismAlthough bacteriocin most promising natural food preservative, but there are some challenging factors that can hinder its efficiency in food system. In this respect, bacteriocin resistant is one challenges factors due to some spontaneous or induced mutation that cause some modifications in membrane and cell wall, 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). From all above mentioned, mutational changes is the most challenging factors specially which is resulted with application of low concentrations of bacteriocins as food preservative while bacteria will start develop adaptive mechanism (Riley et al., 2002).

Bacteriocin Detection and QuantificationCommonly bacteriocin detection and quantification employee the following three important methods: biological, genetic and immunological tests ((Martínez et al., 2000). To identify and isolate bacteriocin producers strain the first method should apply for searching their antagonistic activity is biological tests and which use bioassay methods like agar diffusion test and turbidometric methods. So by referring their inhibition characters for selected indicators it is possible to screen potential bacteriocin producers (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) after amplification of particular DNA extract of producers. But the draw backs of this method is its incapability the quantify and amount of bacteriocin. Finally, immunological test is also the methods universally used for bacteriocin detection and quantification from different medium contains bacteriocin producers trains, particular by identifying the occurrences of purified and semi purified bacteriocin either from cell free supernatant or crude bacteriocin (Leroy et al., 2002).

Bacteriocins and recent advances in molecular biology and genome studies Development of advanced molecular biology is novel valuable tools to study microorganism’s microbial ecology and genetic organization foods and food products which are very crucial idea for the identification of bacteriocinogenic potential and their capacity of proliferation and inhibition of unwanted bacteria as well as their potential adaptive mechanism to stress conditions. Molecular technique is used to determine the distribution of bacteriocin producing and regulating genes. PCR amplification is one technique which is capable to determine specific primers for bacteriocin genes in food fermentation (Urso et al., 2006; Matijašiæ et al., 2007). It is the best methods to solve problem of differentiation of closely related bacteria from mixed populations because with the application PCR it’s very simple to extra bacterial DNA from different community using this technique. Similarly, DNA-based technology is a new science that could study particular gene expression, environmental influence of bacterial gene expression and bacteriocin mechanism of action as well as responses of target bacteria to added or inoculated bacteriocin in the foods (Urso et al., 2006). Recent information regarding to bacteriocinogenic strains distribution in the food products, the heterogeneous response of bacterial populations to bacteriocins can be also study by fluorescence-based technology is mostly the recent advanced molecular biology technique (Fernández de Palencia et al., 2004; Hornbæk et al., 2006).
The most and challenging research areas in bacteriocin production and purification are identification and screening of particular bacteriocinogenic potential of LAB which is not easily understood by conventional methods. These may be due to several factors that can hinder bacteriocin production, bacteriocin purification and detection. These are: environmental influence bacteriocin producers; loss of production capacity, because bacteriocin productivity may be lost due to gene mutation, and inducible character of bacteriocins. Finally all these factors will affect bacteriocin production and decrease its effectiveness in food systems. However, these routine and complex ideas can be solved with the analysis of bacterial complete genome that reveals presence of potential bacteriocin genes and novel bacteriocins independently of the producer capacity of strains (Nes and Johnsborg, 2004). In addition, other authors also revealed that particular bacterial gene responsible for bacteriocin production may be omitted in the annotation process of bacterial genomes (De Jong et al., 2006). Not only this but also, it’s possible to develop appropriated methodology to study bacteriocin mechanisms of action and responses of target bacteria to specific bacteriocin based on bacterial genomic sequences and gene identification
In general, the developments of contemporary advances in molecular biology and genomic studies that are currently flourished are leading and tracking forward the development of technology toward isolation and screening of a novel potential of bacteriocinogenic strains for fascinating application in food industry and also the sciences is very important to improve our understanding on the health benefits, international and national aspects of bacteriocin application in the food preservation practices.
Bacteriocin bio engineering strategy for increased efficacyThe rational reasons behind application of bacteriocin engineering strategy are; to improve bacteriocin effectiveness and efficiency; to enhance antimicrobial effectiveness of bacteriocin and to increases stability and persistence of bacteriocin for future use as continuous and effective food preservative. This is done with then application of genetic engineering using molecular techniques for the discovery of novel bacteriocin (Gillor et al., 2005). As some authors shown that random mutagenesis is the most recent engineering strategy that was applied to generate bacteriocin (Rink et al., 2007; Field et al., 2008). Even though this technology has a potential capacity to generate a potent novel natural food preservative, due to consumer awareness and resistance and constricting governmental legislation, application and development of bioengineering in food system will certainly limit in near future. Nevertheless, as awareness regarding bioengineering microbes improved further than scientific community and customers’ demands increased for food processed with genetically modified microbes, bacteriocin bioengineering may become excellent technology in food safety and quality

NOVEL METHODS TO PURIFYING BACTERIOCINThere are different methods for production and purification of bacteriocin. The most widely used methods are bacteriocin concentration techniques (Ammonium sulphate and acetone precipitation) and bacteriocin purification techniques (different chromatography methods and electrophoreses) (Galvez et al., 2014). The generalized method used for the purification of bacteriocin is given in Fig. 4. Unless we are suffering for large scale production which needs at least two types of chromatography techniques, purification and production of bacteriocin employee simple techniques with a minimum processing step (Espitia et al., 2012). Although, there are many methods are employed for purification and isolation of bacteriocin we are focusing only few of them which are more commonly used. Here are some sort of discuss on novel methods of bacteriocin purification.

Bacteriocin Concentration methods
Ammonium sulphate precipitationAmmonium precipitation is the preliminary steps in bacteriocins purification and the most widely used approach for isolation of large content of bacteriocins. After appropriate cultivation of bacteriocin producers on MRS agar, the cell free supernatant is collected by centrifugation and crude bacteriocin is also obtained by ammonium salt saturation methods for protein precipitation. To collect crude bacteriocin first protein should be precipitate and separated by centrifugation, the pellet protein is dissolved in distilled water or buffer solution followed membrane filtration gel filtration for further purification of bacteriocin (Mohanty et al., 2016). As a result this step is known as partial purification since it is possible to use bacteriocin at this stage to apply as food preservative.

Acetone precipitation methodAs of ammonium sulphate precipitation, acetone precipitation is used for partial purification for bacteriocin since it applies salting out methods to precipitate protein. Almost follow the same procedures with ammonium sulphate precipitation. The only difference is bacteriocin purification needs application chromatographic techniques to reach final concentration (Biswas and Banerjee, 2016).

Bacteriocin Purification methodsIon exchange chromatography (IEC)Ion exchange chromatography purification is mainly depends of the interaction of opposite charges present on the surface of peptides and resin or it is the cationic or anionic exchange between bacteriocin and resin. Sodium chloride is the most commonly used salt for bacteriocin purification in this method (Colins et al., 2012). But for large scale production its high flow rate and strength cationic and anionic exchanger to intensification the efficiency of purification.

Affinity chromatographyAffinity chromatography is a techniques used to purify bacteriocin by based on specific interaction between peptides and ligand present of the chromatography matric it purify bacteriocin based on their affinity to specific ligand, the bacteriocin which bound to ligand is selected and unbounded one is washed away, but this method is applied for partial purification only and finally bacteriocin of interest is obtained with several modification of pH, ionic strength or polarity (Colins et al., 2012).

Capillary electrophoresisIt is multipurpose technique and its relies on migration of charges presents on bacteriocin, is solution and on electric filed, so based charge migration among these parameters its possible to purify bacteriocin simply (Colins et al. 2012).

High performance liquid chromatography (HPLC)HPLC is the most advantageous techniques since it has capacity to purify bacteriocin with their detail information like their source and its quantity (Colins et al., 2012). For purification of bacteriocin column and high pumping systems utilized. The charge present on the surface of stationary column and antimicrobial peptides are opposite and usually application of automatic and high pumping system are used to minimize limitation caused by manual operation (Mohanty et al., 2016; Sankar et al., 2012).

Polyacrylamide gel electrophoresis (PAGE)After purification, the molecular weight of bacteriocin is determined by Polyacrylamide gel electrophoresis and purified bacteriocin is treated with tricine sodium dodecyl sulphate polyacrylamide electrophoreis. Usually bacteriocin standard solutions are used as standard to estimate the molecular weight of bacteriocin with the application of gels (stacking solution and separating gel (Zhao et al. 2015; Upendra et al. 2016).

647704445Figure 4. Bacteriocins Purification Methods
BACTERIOCIN AND FOOD PRESERVATION
Bacteriocins Prospective for Food preservativeContinuously, consumers are really concerned about the health adverse effects regarding to chemical food preservatives and food additives. As result, consumers are more attractive to natural and fresh foods which have no any artificial food preservative. Therefore, consumers’ insight, stresses for processed foods with longer shelf life free of any food additive and convenience, convincing researchers and concerned body to dig a promising natural and novel food preservatives ADDIN EN.CITE ;EndNote;;Cite;;Author;Banerjee;/Author;;Year;2014;/Year;;RecNum;73;/RecNum;;DisplayText;(Banerjee, 2014);/DisplayText;;record;;rec-number;73;/rec-number;;foreign-keys;;key app=”EN” db-id=”zx5w5r95iddr05esze8xvsxydvswpfe95vd5″ timestamp=”1527037731″;73;/key;;key app=”ENWeb” db-id=””;0;/key;;/foreign-keys;;ref-type name=”Journal Article”;17;/ref-type;;contributors;;authors;;author;Arun K. Verma and Rituparna Banerjee;/author;;/authors;;/contributors;;titles;;title;Bacteriocins: Potential in Food Preservation;/title;;secondary-title;Encyclopedia of Food Microbiology, Second Edition, 2014, 180–186;/secondary-title;;/titles;;periodical;;full-title;Encyclopedia of Food Microbiology, Second Edition, 2014, 180–186;/full-title;;/periodical;;pages;180-186;/pages;;volume;01;/volume;;dates;;year;2014;/year;;/dates;;urls;;/urls;;electronic-resource-num;10.1016/B978-0-12-384730-0.00029-X;/electronic-resource-num;;/record;;/Cite;;/EndNote;(Banerjee, 2014). So, bacteriocin produced by GRAS lactic acid bacteria is considered as an exceptional or novel natural food preservative since it satisfy the consumers and governmental requirements for foods, in addition it has a potential capacity to prevent proliferation and spread of food spillage microorganism and food borne disease when used with other preservation methods and processing techniques ADDIN EN.CITE ;EndNote;;Cite;;Author;Banerjee;/Author;;Year;2014;/Year;;RecNum;73;/RecNum;;DisplayText;(Banerjee, 2014);/DisplayText;;record;;rec-number;73;/rec-number;;foreign-keys;;key app=”EN” db-id=”zx5w5r95iddr05esze8xvsxydvswpfe95vd5″ timestamp=”1527037731″;73;/key;;key app=”ENWeb” db-id=””;0;/key;;/foreign-keys;;ref-type name=”Journal Article”;17;/ref-type;;contributors;;authors;;author;Arun K. Verma and Rituparna Banerjee;/author;;/authors;;/contributors;;titles;;title;Bacteriocins: Potential in Food Preservation;/title;;secondary-title;Encyclopedia of Food Microbiology, Second Edition, 2014, 180–186;/secondary-title;;/titles;;periodical;;full-title;Encyclopedia of Food Microbiology, Second Edition, 2014, 180–186;/full-title;;/periodical;;pages;180-186;/pages;;volume;01;/volume;;dates;;year;2014;/year;;/dates;;urls;;/urls;;electronic-resource-num;10.1016/B978-0-12-384730-0.00029-X;/electronic-resource-num;;/record;;/Cite;;/EndNote;(Banerjee, 2014). Thus, bacteriocins have many application in food industry while production, processing and storage to fork which lack numerous blocks or hurdles to the growth of pathogenic and spoilage bacteria. Thus, the application of bacteriocins food industry as food preservative provides to extend the foods shelf life by decreasing food contamination while endorsing good flavor, aroma and texture as well as keeping foods nutritional value.

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. Even if, modern advances in technology has been developing in food production and processing, but still the way word is preserving and keeping foods is still in under questions and economic loss and health problems due to lack of effective food preservation is still flourishing. Newly discovered and more effective chemical preservatives have been identified and successfully applied in various food processing and production. As we have repeatedly pointed at several sections in this paper, again and again , consumers’ interest and appreciation of natural food product since they are not new for the health concerns of chemical food additives make naturally produced antimicrobial peptides more attractive than others. Consequently, Bacteriocins have a great potential to meet this request in the food industries as a promising natural food preservative (Gálvez et al., 2007, Gálvez et al., 2008; Balciunas et al., 2013).
In the food preservation, bacteriocins are the supreme natural food preservative due to the followings reasons; they are produced by GRAS microorganism, they have relatively broad antimicrobial activity, they show versatile bactericidal mode of action by emphasizing cytoplasmic membrane which affect their genetic manipulation ad organization (Gálvez et al., 2008). As many studies indicates that the applications of bacteriocins with the above mentioned property has the crucial roles in food industries because of their subsequent rewards such as incensement of food shelf life in stress environments, reduced transmission of food-borne pathogens, reduce economic losses due to food spoilage and food borne disease decease food additives and chemical preservatives, healthier preservation of food nutrients and vitamins, as well as organoleptic properties of foods, permit the marketing of novel types of foods(Gálvez et al., 2007;Gálvez et al., 2008; Balciunas et al., 2013) as a generally they increase industrial application of natural food preservatives. From different natural food preservatives discovered and proposed, bacteriocin has caught the attention of many food scientists to be applied as novel natural food preservative. Now day’s bacteriocin is developing as the very promising natural food preservative which is alternative to chemical preservatives and ahead commercial importance worldwide. So, bacteriocin is the most potent antimicrobial peptides serve as novel natural food preservatives
Currently bacteriocins are used as promise food preservatives, because; bacteriocins approved as safe in food as they are not complicated with proteases in the gastrointestinal tract (Elayaraja et al. 2014), nontoxic and broad-spectrum activity against foods spoiling microorganisms and show minimum inactivation when exposed to the protease enzymes(Balciunas et al. 2013; Galvez et al. 2014). In addition, bacteriocins can surely enhance the storage life of food products and they are ecofriendly antimicrobial peptides, besides to this they can diminish economic loss of food-spoilage and side effects of artificial food preservatives (Galvez et al. 2014).

Effectiveness of Bacteriocins in Food Products
Basically bacteriocins are inoculated to food following three basic methods: direct inoculation of purified and semi purified bacteriocins, inoculation of bacteriocins producer’s strains to the foods and producing the bacteriocin in situ by adjunct cultures( using previously fermented food as inoculants(Cotter et al., 2005; Deegan et al., 2006). Its efficiency and effectiveness is based of bacteriocin produces and particular food products. Due to some factors use of purified bacteriocin as food industry is not continuously good-looking and the most attractive one is if bacteriocins are incorporated in the food products (Deegan et al., 2006). In the main cases, the efficiency of bacteriocins is greatly depends on intrinsic and extrinsic factors like; interactions with food matric and some issue of precipitation; factor that may undermine the biological activity of bacteriocins and irregular dissemination of bacteriocin molecules in the food matrix (Coma, 2008). Solubility, stability, physical and chemical composition of foods, osmolality and fat content are some common challenging factors that have substantial influence on the activity of bacteriocin to be applied a good natural food preservative. One can overcome and reduce this limitation thorough hurdles technology.

Application of Bacteriocins as Novel Natural Food Preservative
The strategies for application of LAB bacteriocins as novel natural preservation in food systems are based on their beneficial properties on human heath besides to this it should fulfill the following criteria to be applied as a novel and safe natural food preservatives (1) should not causes damage to gastrointestinal tract and safe for consumers, (2) should have to show wide spectrum against wide range food spoilage microorganism, (3) should have to show resistance mechanism against enzymes present in the food metrics and (4) finally the bacteriocins must be able to show thermal stability against wide range of PH, high salt concentration and other factors unless and other wise it’s difficult to apply bacteriocin as safe natural food preservatives (Ananou et al.,2007; Galves et al ., 2014). Applications of LAB bacteriocins in food as novel natural food preservation have been developed either by using of LAB starter or protective, or may be back sloping of previously fermented food by bacteriocin-producing LAB in food processing and most of the time inoculation of purified bacteriocins directly as food preservative (Ananou et al., 2007). Application of all these methods are depend of the types of food and target LAB to be applied as food preservative. As a result of GRAS property LAB bacteriocins they are appropriate for industrial use and adequate to satisfy these expanding consumers’ demands for fresh-tasting, safe, naturally preserved and industry processed food products with improved nutritional value and some better organoleptic property of foods.

Challenges of using bacteriocin as natural food preservation
Even though there are different techniques of bacteriocins application as novel food preservative, still there are many challenges of bacteriocin application as novel food preservative. Special, in situ productions of bacteriocins in food system are affected by some factors which can hinder the bacteriocins application one is decline of bacteriocinogenic strains due to unfavorable pH, temperature fluctuations during processing and storage conditions like freezing, 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. Therefore, due to this application of bacteriocins as starter or protective culture may not be effective in these cases (Galvez et al., (2007; 2008; Eldin et al., 2017).

Apart from the emergence of resistant of some bacteriocin strains to bacteriocins, and genetic instability of the producer strains would also reduce the efficacy of bacteriocins preservative potentially by bacteriocinogenic cultures. In addition, all these factors are interconnected to each other and affect the food preservative potential of bacteriocins. Overall, efficacy of pure bacteriocins in the food systems are affected by the permeability and as well as the solubility of the bacteriocins in the food matrix, and the pH of the food matrix which influences the solubility of the bacteriocin and inactivation by the enzymes present in the food system are also some other challenges that can reduced bacteriocin efficiency in the foods (Galvez et al., (2007; 2008; Eldin et al., 2017). But all these limitation would be approved and solved with application of bacteriocin combining with hurdles technology and therefore hurdles technology is the most strategy to increase bacteriocins efficiency.

Combining Bacteriocins with Other HurdlesOne of the approaches to improve the protective action of bacteriocins is the combination with other hurdles such as chemical additives (such as EDTA, sodium lactate, potassium diacetate, and others), heating, and high-pressure treatments (Egan et al., 2016). Narayanan and Ramana (2013) observed that the use of pediocin in combination with eugenol incorporated into polyhydroxybutyrate films worked in synergized form and provided an effective hurdle preventing food contamination. Other researchers used successfully the mixture of bacteriocins and EDTA in the sensitization of Gram-negative bacteria (Prudêncio et al., 2015). Gram-negative bacteria become sensitive to bacteriocins if the permeability of their outer membrane is compromised with chelating agents, such as EDTA (Chen and Hoover, 2003). Several authors also observed the synergistic effect of bacteriocins after temperature treatments (Prudêncio et al., 2015). High pressure processing is a common technique for inactivating microorganisms at room temperature, but this treatment does not ensure the complete inactivation of microorganisms (Prudêncio et al., 2015). Several studies have demonstrated the synergistic effect of bacteriocins such as nisin with high pressure processing on the inactivation of food microorganisms (Garriga et al., 2002; Zhao et al., 2013). It is well documented that the use of bacteriocins in combination with these processing techniques enhances bacterial inactivation (Chen and Hoover, 2003). As an example, Rodriguez et al. (2005) demonstrated the efficacy of the application of reduced pressures combined with bacteriocin-producing LAB to improve cheese safety.

CONCLUSION
Bacteriocins from Generally Recognized as Safe (GRAS) LAB have continued to gain great interest among an increasing number of research groups due to their huge application potential in food, industries.
The potential of bacteriocins produced by lactic acid bacteria (LAB) as natural bio preservatives for food against resistant Gram-positive pathogens is huge. Once harnessed, this can result in the minimal use of antibiotics and chemical preservatives in foods, as preferred by well-informed consumers.
Moreover, due to their strong potency against antibiotic-resistant pathogens, bacteriocins may be a viable solution to the growing problem of multidrug-resistant pathogens. Nonetheless, more research still needs to be done in the isolation and characterization of bacteriocins to maximize their potential in food and pharmaceutical applications.

Since, there is a growing demand for food that is free of synthetic chemicals as preservatives, it is necessary to examine and identify alternative and safe approaches for controlling foodborne pathogens.
However, despite their potential, the use of natural antimicrobials in food systems remains limited mainly due to the side effects of undesirable flavor or aroma.

Therefore, further research is needed to determine the optimum levels of antimicrobials that can be safely applied in food systems without unduly altering any sensory characteristics.
An alternative approach would be to utilize one or more compounds that could produce synergistic effects at low concentrations without altering any sensory
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