Bacteriocins and Their Applications in Food Preservation.

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Bacteriocins and Their Applications in Food Preservation a

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a

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Ramith Ramu , Prithvi S Shirahatti , Aishwarya T Devi , Ashwini Prasad , Kumuda J. , a

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Lochana M. S. , Zameer F. , Dhananjaya B. L. & Nagendra Prasad M. N.

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Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570 006, India b

Department of Microbiology, Faculty of Life Sciences, JSS University, Mysore, India

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Mahajana Life Science Research Laboratory, Department of Biotechnology, Microbiology and Biochemistry, Pooja Bhagavat Memorial Mahajana Post Graduate Centre, Metagalli, Mysore - 570 016, Karnataka, India.

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Toxinology/Toxicology and Drug Discovery Unit, Center for Emerging Technologies, Jain Global Campus, JainUniversity, Kanakapura Taluk, Ramanagara-562112, Karnataka, India. Accepted author version posted online: 20 Jul 2015.

To cite this article: Ramith Ramu, Prithvi S Shirahatti, Aishwarya T Devi, Ashwini Prasad, Kumuda J., Lochana M. S., Zameer F., Dhananjaya B. L. & Nagendra Prasad M. N. (2015): Bacteriocins and Their Applications in Food Preservation, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2015.1020918 To link to this article: http://dx.doi.org/10.1080/10408398.2015.1020918

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ACCEPTED MANUSCRIPT Bacteriocins applications Bacteriocins and their applications in food preservation Ramith Ramu1, Prithvi S Shirahatti1, Aishwarya T Devi1, Ashwini Prasad2, Kumuda J1, Lochana MS1, Zameer F3, Dhananjaya BL4, Nagendra Prasad MN1,* 1

Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570 006,

Downloaded by [University of Otago] at 03:49 21 July 2015

India 2

Department of Microbiology, Faculty of Life Sciences, JSS University, Mysore, India

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Mahajana Life Science Research Laboratory, Department of Biotechnology, Microbiology and

Biochemistry, Pooja Bhagavat Memorial Mahajana Post Graduate Centre, Metagalli, Mysore 570 016, Karnataka, India. 4

Toxinology/Toxicology and Drug Discovery Unit, Center for Emerging Technologies, Jain

Global Campus, JainUniversity, Kanakapura Taluk, Ramanagara-562112, Karnataka, India. *

Corresponding Authors Dr. M.N. Nagendra Prasad, Sri Jayachamarajendra College of

Engineering&JainUniversity, Karnataka, India, Phone: +91 9886480528 Fax: +91821 2548290, Email: [email protected], [email protected] ABSTRACT Bacteriocins are ribosomally-synthesized antimicrobial peptides or proteinaceous compounds produced by bacterial strains. They are generally effective in inhibiting the growth of similar or closely related bacterial strains. A high diversity of various bacteriocins is produced by many lactic acid bacteria (LAB) and is found in numerous fermented and non-fermented foods. Several bacteriocins from LAB extend potential applications in food preservation, thus help foods to be

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ACCEPTED MANUSCRIPT naturally preserved and richer in organoleptic and nutritional properties. Though chemical preservatives for the preservation of food are successful to some extent, their quality is not as satisfying as fresh food. Hence, an alternative is required and bacteriocins serve the purpose. Nisin is currently the only bacteriocin widely used as a food preservative. Numerous bacteriocins have been characterized chemically, biochemically, genetically and also at the molecular level to understand their basic mode of action. This article gives an overview of classification of


bacteriocins, isolation & characterization, and mode of action. Besides, article highlights the optimized parameters for growth of bacteria in the production of bacteriocins and various bioassays for their determination. Special emphasis has been provided on explaining the beneficial aspects of nisin. Keywords Bacteriocin; Nisin; Lactic acid bacteria (LAB); Lantibiotics; Bio-preservation.

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ACCEPTED MANUSCRIPT INTRODUCTION All organisms produce organic compounds that are not directly involved in their normal growth, development, or reproduction and are termed as secondary metabolites (Fraenkel 1959). They are generally low-molecular-mass products that include antibiotics, pigments, toxins, effectors of ecological competition and symbiosis, pheromones, enzyme inhibitors, immunomodulating agents, receptor antagonists, agonists and so on (Demain 1992).


Likewise, bacteriocins are produced by bacterial ribosomes as secondary metabolites. Antibiotics are also produced by some bacteria and, hence, along with bacteriocins they should be considered as different antimicrobial compounds (Davidson and Hoover 1993). A large group of functionally diverse antimicrobial compounds found in all major lineages of bacteria and archaea constitute the bacteriocin family (Riley and Wertz 2002). While some possess a narrow bactericidal spectrum, many others exhibit a broader activity against some of the distantly related species (Klaenhammer 1993; Adam and Moss 1995). Over-all, specific immunity proteins responsible for this are encoded in the bacteriocin operon (McAuliffe et al., 2000; Cintas et al., 2001). Majority of them exert their action through the formation of transitory poration complexes or ionic channels which cause reduction or dissipation of the proton motive force leading to the formation of pores in the cytoplasmic membrane of sensitive cells (Cintas et al., 1998; Herranz et al., 1999). They also employ other killing mechanisms like cell wall interference and nuclease activity (Braun et al., 1994; Smarda and Smajs, 1998). Considering the antibacterial property of bacteriocins, some researchers speculate on considering them and antibiotics under the same class but since bacteriocins are bactericidal peptides, while antibiotics are produced by multi-enzyme complexes, there remains a demarcation between the

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ACCEPTED MANUSCRIPT two bactericidal agents. Most bacteriocins kill a narrow spectrum of bacteria, as compared to the traditional antibiotics (Tu et al., 2002). Some bacteriocins produced by lactic acid bacteria (LAB) are called lantibiotics and they have potential applications in the food industry. Currently existing synthetic food preservatives raise some concern with regard to the quality of food and are associated to several health hazards. In this view, bacteriocins prove safer alternatives and constitute a revolutionary breakthrough in the food preservation. Hence, bacteriocins are


efficiently produced from isolated and purified bacteria. Bacteriocins are categorized and discussed in detail as shown in Figure 1. They are characterized based on different principles viz., chemical, biochemical, molecular, and genetic characteristics (Mian et al., 2012). Classification Bacteriocins are an important part of the defence system of bacteria and are produced by both Gram-positive and Gram-negative species. Bacteriocins from Gram-positive bacteria are broadly classified into 4 groups. The presence of sulfide (such as lanthionine) and disulfide bonds has been taken as the major distinguishing factor since it also entails their spectrum of activity (Jack et al., 1995). However, the molecular mass, thermo-stability, enzymatic sensitivity, presence of post translational modified amino acids, and mode of action also play a role, especially in the classification into sub-groups (Klaenhammer 1993). The groups may be stated as: i. Class I-Bacteriocins ii. Class II-Bacteriocins iii. Class III-Bacteriocins iv. Class IV-Bacteriocins Class I-Bacteriocins

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ACCEPTED MANUSCRIPT Class I bacteriocins are post-translationally modified peptides containing unusual amino acids, such as lanthionine or methyl lanthionine residues and are called lantibiotics (Nissen and Nes 1997). They are produced by LAB to attack other Gram-positive bacteria and are smaller than 5 kDa (Boman 1995). Nisin, subtilin, cytolysin and variacin 8 are a few examples (Gross and Morell, 1971; Gross and Kitz, 1973; Gilmore et al., 1994). Class II-Bacteriocins


Class II bacteriocins are heat-stable, unmodified peptides, and do not contain the unusual amino acid and hence they are called non-lantibiotics. They are small peptide molecules which are smaller than 10 kDa and produced by LAB (Bierbaum and Sahl 2009). Class II bacteriocins are further divided into 3 sub-classes, namely IIa, IIb, and IIc (Ennahar et al., 2000). Pediocin-like bacteriocins, two-component bacteriocins, and thiol-activated bacteriocins are grouped under the IIa, IIb, and IIc sub-classes, respectively. Some of the examples are pediocin PA-1, lactacin F, enterocin AS-48 (Nissen et al., 2009). The major difference between Class I and Class II bacteriocins is that Class I bacteriocins undergo post-translational modifications and thus give rise to the unusual amino acid lanthionine, whereas Class II bacteriocins do not undergo any such modifications. Class III-Bacteriocins Class III bacteriocins are heat-labile and larger (>10 kDa) protein molecules (Savadogo et al., 2006). They are sub-classified into 2 sub-classes: Class IIIa or bacteriolysins and Class IIIb or non-lytic bacteriocins. Lysostaphin, a 27-kDa peptide that is produced from Gram-positive Staphylococcus species, is the best-studied bacteriolysin (Bastos et al., 2010). Class IV-Bacteriocins

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ACCEPTED MANUSCRIPT Class IV bacteriocins are complex proteins that contain lipid or carbohydrate moieties (Oman et al., 2011; Upreti and Hinsdill 1975). This group was added after the observation that bacteriocin activities obtained in a cell free supernatant were abolished not only by protease treatments, but also by glycolytic and lipolytic enzymes (Jimenez et al., 1993; Tagg et al., 1976; Nes et al., 2000; Garneau et al., 2002). Plantaricin S and leuconocin S are major examples of Class IV bacteriocins (Vijay and John 2002).


Further, according to the scheme of Cotter et al., (2005), bacteriocins are classified into 2 major groups, namely lantibiotics and unmodified peptides. The authors eliminated classes IIa, III, and IV and restructured class II. Thus, the class of unmodified peptides includes pediocin-like peptides, two-component peptides, cyclic peptides, and miscellaneous bacteriocins. Considering both the schemes, a universal bacteriocin classification was established, as shown in Figure 1. While Gram-positive bacteria are classified as detailed in the above section, Gram-negative bacteriocins are categorized based on their size. These bacteriocins are produced from Gramnegative bacteria (arise mainly from enterobacteriaceae) and are heat-stable molecules. They are mainly classified into 2 classes based on their molecular mass: i.

High-molecular-mass peptides (30 kDa-80 kDa) named colicins

ii.

Low-molecular-mass peptides (1 kDa-10 kDa) called microcins (Drider and Ribuffat 2011).

Since the beginning of 21 stcentury, bacteriocins have been classified in several other ways, based on their specific adsorption and immunity patterns (Frederic 1957). Reeves named and grouped bacteriocins into 16 classes based on the species of the producing strain, like the colicins which are bacteriocins produced by E. coli, pyocins by Pseudomonas aeruginosa, arizonacins by

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ACCEPTED MANUSCRIPT Paracolobactrum arizonae, cloacins by Enterobacter cloacae, pneumocins by Klebsiella pneumoniae, pesticin by Yersinia pestis, monocin by Listeria monocytogenes, cerecin by Bacillus cereus, and staphylococcin by Staphylococcus aureus (Reeves 1965). Mode of action of bacteriocins Bacteriocins affect different essential functions of the living cell viz., transcription, translation, replication, and cell wall biosynthesis due to great variety of chemical structures, but most of


them destroy the energy potential of sensitive cells by forming membrane channels or pores (Juan et al., 2000; Ganzle 2004). The best-described mechanism is pore formation. The presence of molecular receptors in the membrane of the target cell is suggested by the relatively small action spectrum of some bacteriocins, although this has not been demonstrated (Van and Stiles 2000). A model demonstrated using nisin suggests that, using C-terminal, nisin primarily binds to phosphatidylglycerol, a universal receptor, present on the cytoplasmic membrane of target bacteria (Breukink and Kruijff 2006). Hence a complete loss of activity could be observed if the C-terminal is removed (Giffard et al., 1997). Binding of nisin is followed by insertion and penetration of a part of peptide into the cytoplasmic membrane. Fluorescence studies indicate a parallel orientation of nisin molecule with respect to the membrane surface having N-terminal inserted slightly deeper than C-terminal (Breukink et al., 1998). This initiates the process of membrane insertion and pore formation, leading to rapid cell death (Cotter et al., 2005; Breukink and Kruijff 2006). In general, the helical amphiphilic structure of class II peptides allows them to get inserted into the membrane of the target cell, leading to depolarization and death (Cotter et al., 2005; Drider et

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ACCEPTED MANUSCRIPT al., 2006). At the hydrophilic N-terminus of the peptides, the initial interaction takes place with the heads of the anionic membrane phospholipids. The C-terminus of the peptide is thought to be involved in hydrophobic interactions with the membrane as it is more hydrophobic than the Nterminal (Alonso et al., 2000).Class II bacteriocins allow the insertion of the peptide into the membrane of sensitive microorganism, using its amphiphilic structure, and causes depolarization and death (Brashears et al., 1998).


On the contrary, the large bacteriolytic proteins, such as lysostaphin (Class III bacteriocins), lead to death and cell lysis by directly acting on the cell wall of Gram-positive target cells (Cotter et al., 2005). Under the influence of various mechanisms, bacteriocins cause microbial cell killing either in an isolated or most consorted manner (Paker et al., 1989; Daw and Falkiner 1996). Unbalanced cytoplasmic membrane function, inhibition of nucleic acid synthesis, interference with protein synthesis and changing the cell translator mechanism lead to bacteriocins killing a microbial cell (Nes and Tagg 1996). Due to the development of specific immunity mediated by a protein, bacteriocin-producing cells are not affected by the action of their own bacteriocin (Hancock and Chapple 1999). Bacteriocins have been noteworthy in detections and causing fatality to competitor bacteria, with little or no damage to their producing cell (Tadashi and Schnneewind 1998). Microorganisms have competitive superiority when they dispute ecological niches in the environment where they are developing their metabolic activities (Stockewell et al., 1993). Based on this narrow action, bacteriocins play a role as intra-specific mediators or promoters of interactions among microbial populations (Jennifer et al., 2001). When 2 or more microorganisms are present in an environment and adversely interfere with growth and survival

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ACCEPTED MANUSCRIPT of other ones, antibiosis occurs. It is the antagonistic action exerted by bacteriocin-producing bacteria on the other bacteria in the same environment (Schillinger 1990; Ray 1996). Isolation of bacteriocinogenic strains from food systems The majority of bacteriocin producers are natural food isolates and hence they are suited for food applications (Raja et al., 2010). An increased health-conscious public now tries to avoid foods with chemical preservatives or which have undergone extensive processing. The most powerful


means for obtaining useful cultures for scientific and commercial purposes are isolation and screening of microorganisms from natural sources (Vanden et al., 1993). Isolation procedures employed by different researchers were relatively similar, but for the culture conditions and the growth media required they differed between different food sources. The principle involved in their purification remained the same. Isolation of LAB from milk products According to the procedure employed by Arokiyamary and Shivakumar (2011) on milk products, isolation of bacteriocins from LAB was standardized. The bacterial count was performed to make appropriate dilutions on the most accepted medium, MRS agar, to maintain optimal growth. The LAB grown in an aerobic environment, utilizing the nutrients from MRS agar, were isolated using bacteriological techniques. Isolation of bacteriocins from appam batter Vijai et al., (2005) standardized a procedure for the extraction of bacteriocins from South Indian special dosa (commonly known as appam) batter. Saline dilutions of appam batter, cultured on MRS agar anaerobically, was transferred to MRS broth and checked for purity. For the purpose

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ACCEPTED MANUSCRIPT of upgrading purity, sterilization of centrifuged supernatant using a filter membrane of 0.22 µm was utilized. Isolation of bacteriocins from chicken intestine Apart from food products, isolation of bacteriocins has been reported in marine environments, rock sediments, and chicken intestine. Narayanapillai et al., (2012) have designed an isolation method from chicken intestine. Briefly, pure cultures obtained from inoculation of crushed


pieces of chicken intestine were grown on MRS agar at optimum temperature to test the bacteriocin production. The strains from the pure culture were Gram-stained to examine bacteriocin-producing bacteria and further grown at optimum temperature. Bacteriocins, being an extremely heterogeneous group of substances, their purification to homogeneity become a very complex task. Although bacteriocins are generally extracted after centrifugation using the ammonium sulfate precipitation method, there are 3 other major methods of purification which have been widely accepted. The first is a conventional method and includes ammonium sulfate precipitation, ion exchange, hydrophobic interaction, gel filtration, and reverse phase high-pressure liquid chromatography (RP-HPLC). A second method is much simpler and generally used for the production of bacteriocins at laboratory-scale. The steps involve ammonium sulphate precipitation, solvent precipitation using chloroform/methanol and then RP-HPLC. The third bacteriocin purification method requires maximization of the bioavailability of bacteriocins by varying the pH of the fermentation medium. Further, a standard unit operation protocol, namely hydrophobic interaction gel or expanded bed adsorption, can be employed. This method is used for the production of bacteriocins at bulk scale (Eduardo et al.,

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ACCEPTED MANUSCRIPT 2013). Isolation of bacteriocins has also been done from a variety of food systems like dhokla, khaman, murabba, chyawanprash, kalakand, and rasgulla. Characterization principles of common bacteriocins By definition, bacteriocins can also be designated as antibiotics due to their bactericidal mode of action against closely related species (De Man et al., 1960; Klement et al., 1990; Kuryolowcz 1991; Davis 1999). The most abundant and diversified molecules with antibiotic activity


produced by different bacteria including Gram-positive and Gram -negative species are bacteriocins (Riley 1998). A major difference between bacteriocins and antibiotics is found in their activity of inhibiting specific microorganisms. Bacteriocins restrict their activity to strains of related species whereas antibiotics have a wider activity spectrum and do not show any preferential effect on closely related strains (Jennifer et al., 2001). Based on their molecular size, antibacterial spectrum, stability, physical and chemical properties, and action mode, bacteriocins are formed into heterogeneous groups (Davidson and Hoover 1993; De Vuyst and Vandamme 1994, b). Most bacteriocins of low molecular weight are cationic and have greater antimicrobial activity at low pH (Sinell 1989; Bendjeddou et al., 2012). Adsorption of bacteriocins to Gram-positive cell surfaces is dependent on pH and shows maximum adsorption at or above pH 6. The mode of action, effect of heat, pH, proteolytic enzymes, salt, and detergents on bacteriocin activity, determination of molecular mass, amino acid composition and sequence, and determination of the genetic organization, production, and secretion are included in the characterization of bacteriocins (Svetoslav 2009). Biochemical characterization of bacteriocin BacUB9

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ACCEPTED MANUSCRIPT According to the experiments conducted by Maja et al., (2010) bacteriocin BacUB9 is a heatstable molecule. It is produced from a Gram-positive bacterium, Lactobacillus paracasei subsp. paracasei BGUB9.Among the 2 bacteriocins characterized from this bacterium, BacUB9 is the one which has a very narrow anti-microbial spectrum. Its molecular mass is 4 kDa and it is susceptible to the activity of proteolytic enzymes. Its activity decreases at 100ºC for 60 min and its activity is completely abolished at 100ºC for 120 min. Bacteriocin BacUB9 retains its activity


in the pH range of 1-10 when it is present in a cell-free supernatant of the producer strain. Antimicrobial activity is lost at pH 11. Treatment of bacteriocin UB9 with various proteolytic enzymes (pepsin, trypsin, α-chymotrypsin, pronase E, and proteinase K) results in the loss of its antibacterial activity. Bacteriocin UB9 shows that it is of a proteinaceous nature and it is strongly suggested that it belongs to the class II bacteriocins. Molecular characterization of bacteriocin Carocin D Carotovoricin and carocin S1 are 2 different bacteriocins which are found in the Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum, causing soft-rot disease in various plants (Eunjung et al., 2010). Carocin D is the third bacteriocin found in the same producing species. An antibacterial activity of carocin D against the indicator strain Pcc3 is carried by a particular strain Pcc21 (Chan et al., 2009). Carocin D is encoded by the caroDK gene located in the genomic DNA, which is an immunity protein providing protection. The Edman degradation process determined the N-terminal amino acid sequences of purified carocin D. In a comparison with the amino acid sequence of caroDK, it was found that 8 amino acids are missing at the N terminus. Mora et al., (2008) and Bendjeddou et al., (2012) proved that 8 amino acids are removed from the N terminus of carocin D during maturation and that it is synthesized as a

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ACCEPTED MANUSCRIPT precursor peptide. Bacteria became resistant to carocin D, when caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3. This bacteriocin is slight resistant to heat but not to proteases. CarocinD sensitive, non-pathogenic bacteria readily express this bacteriocin, which has great potential as a biological control agent. Chemical and genetic characterization of bacteriocins Carnobacteriocins BM1 and B2 According

to

observations

made

by Quadri (1994), the

Gram-positive

bacterium


Carnobacterium piscicola LV17B produces carnobacteriocins BM1 and B2, which are thermo stable class II bacteriocins. The purification of these bacteriocins includes hydrophobic interaction, size exclusion, and reversed-phase high-performance liquid chromatography (Pillet et al., 1995). Using the information from the N-terminal amino acid sequences for the purified bacteriocins, probes were synthesized to locate structural genes for the carnobacteriocins (Saucier et al., 1995). A HindIII fragment of 1.9-kilobase (kb) from a plasmid (pCP40) of 61-kb contains the carnobacteriocin B2 structural gene. Presence of the 61-kb plasmid is necessary for the expression of chromosomal bacteriocin and its immunity function. These bacteriocins are synthesized as pre-bacteriocins (Jack et al., 1995). Mature 43-amino acid carnobacteriocin BM1 (4524.6 kDa) and the mature 48-amino acid carnobacteriocin B2 (4969.9 kDa) are obtained by the post-translational cleavage of an 18-amino acid N-terminal extension at a Gly-Gly (positions -2 and -1). These 2 peptides show significant amino acid homology to each other and with those class II bacteriocins containing the YGNGV amino acid motif near the N terminus. Biochemical and genetic characterization of bacteriocin Nisin Z Nisin is the first lantibiotic (Class I - Bacteriocins) mentioned in the scientific literature; it is produced by certain strains of Lactococcus lactis (Jung 1991; Murugesh 2003). It has a

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ACCEPTED MANUSCRIPT molecular weight of 3354 and is composed of 34 amino acids and displays a bactericidal mode of activity and is said to be nontoxic to animals and humans (Hurst 1983). Rogers and Whittier observed its activity for the first time in 1928, and it was studied as a discrete antimicrobial substance in 1944 (Mattick and Hirsch 1947). Nisin is the best-studied bacteriocin produced by some Lactococcus lactis strains and has been used for food preservation in over 50 countries. The corresponding study describes characterization of nisin Z-producing L. lactis strain isolated


from Boza traditional cereal-based fermented food from Turkey. In this study, bacteriocin by Lactococcus lactis subsp. lactis GYL32 strain inhibited not only related strains but also Grampositive bacteria, such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. Treatment with proteinase K and α-chymotrypsin inactivates the antimicrobial substance. It is heat-stable when treated at 100 ºC for 20 min. Different pH, enzyme, and heat treatments concluded that the bacteriocin produced by GYL32 is nisin Z. The sequence analysis result showed that it is nisin Z, and its genetic determinants are encoded on genomic DNA. The molecular weight of nisin Z was determined as 6,700 Da by Tricine-SDS-PAGE analysis. The results of this study suggested that nisin Z producer L. lactis subsp. lactis GYL32 strain may be used as a starter culture for improving the safety of fermented products (Gozde and Yasin 2012). Optimization Different ecological niches from which strains of bacteriocin have been isolated, including meat, fish, fruits, vegetables, milk, and cereal products (Ammor et al., 2006). Factors affecting the bacteriocin production include type of medium used, glucose concentration, temperature, pH, and agitation (De Vuyst and Vandamme 1994, b); Sanchez et al., 2002). These bacteriocins differ widely in molecular weight, pH, presence and number of particular groups of amino-acids.

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ACCEPTED MANUSCRIPT However, the differences in antimicrobial activity cannot be solely attributed to the amino acids or its sequences present (Svetoslav 2009). Some of the widely used bacteriocins have been well researched and their production has been well optimized. For example, in the production of nisin and pediocin PA-1 from P. acidilactici, Yang and Ray (1994) identified that high cell density, optimal pH, and specific nutrients in the media resulted in an increased yield. Specifically for nisin, the pH optimum was identified to be


pH 4 – 6 and temperature optimum at 150C. Also, Coventry et al., (1996) established that significant protein-free production of bacteriocins was obtained using food-grade diatomite calcium silicate and several desorbing agents in comparison to ammonium sulfate precipitation. Universally, Class I and Class II bacteriocins are stable at acidic pH. Rodriguez et al., (2002) and Jack et al., (1996) effectively demonstrated this for pediocin PA-1 and piscicolin 126 which remained stable at pH range between pH 4-6 and pH 2-3, respectively. According to Usmiati (2009), the optimum conditions for bacteriocin production on S. typhimurium, E. Coli, and L. monocytogenes (Holo et al., 1991; Sullivan 2002) are at 330 C and pH 5. The trials involved 2 steps: i. Lactobacillus isolates are selected based on their potential in producing bacteriocins. ii. Bacteriocin production process is optimized (Bromberg et al., 2004). The parameters to be observed are turbidity of bacterial culture in nutrient broth and the number of activated colonies of the isolate (Vaughan et al., 2001). Svetoslav et al.(2012) observed that, bacteriocin ST22Ch belonging to Class II group, had a narrow spectrum of activity, was heatresistant and stable at pH 2-10. He also observed that different levels of bacteriocin ST22Ch are produced during the stationary phase of fermentation in the presence of 2% (w/v) glucose and a

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ACCEPTED MANUSCRIPT combination of tryptone, meat extract, and yeast extract. These findings determined the varying requirements for an optimal production of bacteriocins. Assays for the detection of bacteriocins and their activity For the detection of bacteriocin production and its activity, many methods have been described. It is a fact that bacteriocins can diffuse in solid or semisolid culture media, which can be subsequently inoculated with a suitable indicator strain. Making use of this fact, authors Kekessy


and Piguet (1970) conducted experiments on the detection of bacteriocins using agar. Indicator and producing strains of bacteria were used for the screening purpose (Nicolle and Prunet 1964). Micro-titration method for bacteriocin activity assay Using the micro-titration method involving polystyrene micro-plate wells containing MRS broth, Daba et al., (1994) made serial 2-fold dilutions of bacteriocin preparations. The diluted culture was incubated at optimum temperature and the activity was further detected. For the experiment, P. acidilactic bacteria were utilized. The ELISA test Bouksaim et al., (1999) utilized ELISA for the detection of bacteriocins. They mainly used affinity-purified anti-nisin immunoglobulin for the binding of nisin and anti-nisin peroxidise which was linked with the substrate. A carbonate-bicarbonate buffer of basic pH was used for incubation. To reduce the nonspecific binding, the microtiter wells were coated with a blocking reagent, Tween-20. The bound enzyme was determined which implied the amount of bacteriocins. ELISA and agar diffusion assays, even though were more sensitive for pure nisin, they failed to differentiate nisin from subtilin and also the closest structural analogue of nisin (Fowler et al., 1975; Falahee et al., 1990). Later, competitive direct ELISA assays were

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ACCEPTED MANUSCRIPT published by Suarez et al., (1996) where the detection limit for nisin was based on polyclonal mouse antibodies, and the above limit for nisin was measured utilizing the monoclonal mouse antibodies. Turbidimetric assay for nisin It was used for detection of nisin by Simone et al., (2003) by the use of a special media containing meat extract, yeast extract, tryptone, glucose, NaCl, Na 2HPO4 with neutral pH. At


specific time intervals, samples were collected, their optical densities were measured spectrophotometrically and the growth curve of the test microorganism was obtained. To stop the further growth of microorganism, the samples were injected with thiomersalate solution. Electrophoretic method The only electrophoretic method for nisin quantification has been published by Rossano et al., (1998). Their capillary zonal electrophoretic assay was used to analyze nisin from milk. The bioluminescence genes from Xenorhabdus luminescens species were placed under the nisin inducible promoter in the same plasmid and were transformed into the non-nisin producer L. lactis. This led to the extra cellular nisin stimulation and a subsequent addition of substrate for the luciferase, resulted in bioluminescence by the indicator strain (Wahlstrom and Saris 1999). Very recently Immonen and Karp (2007) introduced an improved luciferase assay. Chemiluminescence-assisted Immunodot assay for nisin It was described by Bouksaim et al., (1999) in which serum from rabbit immunized with nisin Z was used to detect nisin. Dadoudi et al., (2001) developed the first method to distinguish between nisin A and nisin Z. The monoclonal antibodies raised against nisin Z could be used to quantify

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ACCEPTED MANUSCRIPT nisin Z with detection limits in pure solution, in fermentation broth, in milk and in whey and the antibodies developed this way did not cross-react with nisin A. Detection using agar It is a fact that bacteriocins can diffuse in solid or semi-solid culture media, which can be subsequently inoculated with suitable indicator strain like phage (FredericQ 1957). Before proceeding to any steps of assay, chloroform vapour was used to ensure the sterility of agar


surface. As chloroform vapours attack plastic, the assay does not allow the use of plastic Petridishes (Nicolle and Prunet 1964). Kekessy and Piguet (1970) described a new technique based on the tri-dimensional diffusion of bacteriocins. The detection of bacteriocins was done on the reverse side of agar plate which serves as culture surface for the indicator strain. There is no direct contact between producer and indicator strains of bacteria. As in the case of other methods, where it is not easy to decide whether a zone of inhibition is due to either producing strain or indicator strain, in this method, no inhibition area due to indicator strain are formed. Beneficial aspects of bacteriocins In context to food, it isn’t enough to only improve production but it is also necessary to store the produce for distribution and later use. But there are many criteria considered, in order to safely store food products (Brul and Coote 1999). The currently existing preservatives are not entirely satisfactory to the consumers, food laws or the producers. Food preservation basically requires the maintenance of the functional properties of the product, and also other physico-chemical properties so that it remains appealing to the consumer. However, higher the preservative treatment, higher is the damage caused to the food product (Ray 1992). Thus, continued treatment with the preservatives could effectively nullify their use.

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ACCEPTED MANUSCRIPT Bacteriocins as biopreservatives In recent times, there is a continuous raise of awareness in the public regarding the amount of chemical intake, which curbs the amount of chemical preservatives that may be used in food (Harris et al., 1997). Thus, barely any variety exists without controversies of the multifarious health problems associated with them. In view of the above problems, bio-preservatives are in very high commercial demand at present, in the form of protective cultures or their metabolites


i.e. enzymes and bacteriocins (Holzapfel et al., 1995). Bio-preservation is defined as extension of shelf life and food safety by use of natural or controlled microbiota and/or their antimicrobial compounds. The destruction of the pathogens depends on improved food safety by search of more efficient chemical preservative and drastic physical treatment (high temperature treatment). These have various drawbacks such as toxicity of chemicals used, nutritional alteration of food and to overcome these disadvantages, LAB and their bacteriocins are being popularized. Bacteriocins from LAB Lactic acid bacteria have been widely used for thousands of years in the production of fermented products. Since they are present in several consumed products, they have been deemed safe for human consumption and been provided the GRAS (Generally Regarded As Safe) status (Jeevaratnam et al., 2005). Due to this, most of the research on bacteriocins so far has been concentrated exclusively from LAB. The bacteriocins from food grade Lactic Acid Bacteria (LAB) qualify as an ideal food bio preservative primarily because: i. It is proven non-toxic to humans ii. Does not alter the nutritional properties of the food product iii. Effective at low concentration

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ACCEPTED MANUSCRIPT iv. Active under refrigerated storage (Sinell 1989; Daeschel and Ray 1992; Gould 1995). FDA approved bacteriocin–nisin Most of the Gram-positive bacteria are especially susceptible to Nisin (Class I-Bacteriocin), but have little or no effect on Gram-negative bacteria, yeast or fungi (De Vuyst and Vandamme 1994, a). In the year 1969, nisin was stated to be safe and natural food additive by FAO/WHO expert committee on food additive (FAO/WHO 1969). Nearly 15 years later, nisin was used


commercially in around 39 countries (Hurst 1983). It was given the designation E234 in 1983 after being incorporated to the EEC food additive list (EEC 1983). Nisin was GRAS (Generally Recognized As Safe) certified in USA in 1988 (Federal Register 1988) and by Food and Drug Administration in the year 2001 (FDA 2001). It was allowed as food additive in more than 50 countries including Europe, China and the US by the year 1996 (Delves-Broughtan et al., 1996). Its potential application was first demonstrated in 1981 for food preservation and elaborated its use since 1990 (Delves-Broughtan 1990). Nisin concentrate was commercialized as ‘Nisaplin’ by Alpin and Barrett which is now used as a preservative in dairy products, cured meat, canned food and other fermentation industry divisions. Nisin was first discovered in 1928 as a result of difficulties faced in cheese making where storage of milk had supported the growth of contaminant organisms to produce the inhibitor (Hirsch et al., 1951). A partially purified form of nisin was commercially made (De Vuyst and Vandamme 1994 , a; Twomey et al., 2002) and a saleable bacteriocin-containing fermented powder is available as a food additive (Rodríguez et al., 1999). Bacteriocins have been found to inhibit food spoilage by pathogenic bacteria such as Staphylococcus aureus (Chang and Chang 2011), Escherichia coli (Foulds and Shemin 1969),

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ACCEPTED MANUSCRIPT Bacillus cereus (Kabore et al., 2013), Listeria monocytogenes (Pucci et al., 1988) and Clostridium perfringens (Gamal 2006) which are recalcitrant to food preservation methods. The production and application of bacteriocin could effectively combat nearly all of the problems of the food industry apropos preservation. Bacteriocins are well suited for this purpose due to their thermo-stability and pH tolerance. Bacteriocins from traditional products


There are so many different types of food ranging from spicy ethnic food to down home favourites. The options are endless, even within a particular recipe or type of food. India being the country of diversified geographical and cultural origin is also known for its many traditional cuisines. Many of these products, apart from their cultural heritage also have been known to possess many beneficial qualities. Few examples of such products are dosa (Mallesha et al., 2010), idli (Vijayendra et al., 2010), kokum (Khurana and Kanawjia 2007) and lassi (Amit et al., 2012). They are traditionally known for their effects such as cooling the body, aiding digestion, probiotic effects, protection against certain microbial attacks and so on. Indian traditional products provide a promising array of sources for obtaining bacteriocin producing bacteria. Lactic acid bacteria isolates from appam batter, capable of producing good amount of bacteriocins have been identified by Vijai et al., (2005). Isolates of bacteriocin producing microorganisms were also isolated from curd, dosa batter and idli batter and the bacteria were identified as a species of Lactobacillus while the effect of physical and cultural parameters were tested on them (Sourav and Arijit 2010). Lactobacillus species was also recently isolated from different milk products such as curd, milk peda, butter and ghee and the bacteriocin from them were found effective against many common food pathogens (Arokiyamary and

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ACCEPTED MANUSCRIPT Shivakumar 2011). So it is sufficiently established that Indian traditional products are rich in lactic acid bacteria that produce bacteriocins with the potential to be used as food preservatives. However, most of the work was started only very recently and there are still many unexplored traditional products that may provide invaluable bacteriocins. CONCLUSION Bacteriocins are a diverse group of antimicrobial proteins or peptides ribosomally encoded


covering a broad range of applications in the food and medical industry. They employ a variety of killing mechanisms that contain genes which are either chromosomally or plasmid encoded with certain toxins. Bacteriocins possess relatively narrow killing spectrum that includes cytoplasmic membrane pore formation, cell wall interference and nuclease activity that alters the cell membrane permeability properties. Most of the bacteriocins were originally isolated from organisms which are involved in food fermentation. Considering its wide applications, optimization of the production of bacteriocins requires much attention. LAB produced bacteriocins also known as Lantibiotics, were regarded as GRAS and hence is one of the most researched types of bacteriocins. The most potent lantibiotic is nisin which is produced by Lactococcus lactis and has been regarded as the only bacteriocin used as a food preservative that has been approved by the FDA. Various bioassays are designed most of which are with regard to Nisin which also finds applications in pharmaceuticals. Bacteriocins are potentially used in food, human and animal health applications. The use of bacteriocins is of great interest in the food system since they are recognized as safe and can also be used as bio-preservatives. Thus it is desirable to expand the understanding of the activity of bacteriocins in order to determine their efficacy more accurately for future applications in food model systems. Food safety is deemed as

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ACCEPTED MANUSCRIPT the paramount of international concern. Since bacteriocins are considered as natural products, usage of bacteriocins in food systems will surely gain good acceptance from customers who demand for more natural and safer food products that are free from chemicals. Declaration of Interest The authors declare there is no conflict of interest ACKNOWLEDGEMENTS


Ramith Ramu thanks TEQIP New Delhi, India for awarding the Research fellowship. The authors are grateful to the Principal, Sri Jayachamarajendra College of Engineering, Mysore, and the Head of the Department of Biotechnology for their encouragement and support. DBL thanks Jain University for constant encouragement and support given to the progress in research.

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ACCEPTED MANUSCRIPT Table. 1: Various bacteriocins and their target bacteria in food systems

Producing Strain

Group

Bacteriocin

Target Bacteria

Clostridium Class I

Nisin

botulinum, Listeria


monocytogens

Source

References

Camambert,

Hirsch and

ricotta and

others 1951;

manchego

Sulzer and

cheese.

Busse 1991

Enterocin AS-48, Class II

Enterocin A, Pediocin

Gram

Listeria monocytogens

Meat

Ananou and others 2005

ACH

positive

Staphylococcus Class III

Lysostaphin

aureus and Staphylococcus epidermidis

Pasteurized milk, cheese, sausages

Wu and others 2003; Maria and others 2004 Carr and

Class IV

Plantaricin S

L. paraplantarum

Milk and

others 2002;

and L. pentosus

cheese

Pepe and others 2004 Brenda, 2007;

Colicins

Colicin E1

E.coli O157:H7

Meat

Gram

Obi and Campbell, 1978

negative Microcins

Microcin S

E. coli E2348/69

Probiotic

Anke and

drug

others 2012

Symbioflor 2

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Fig 1: Classification of Bacteriocins

41

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Fig 2: Mechanism of Pore Formation by Nisin

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Bacteriocins: Recent Trends and Potential Applications.

Trends in utilization of agro-industrial byproducts for production of bacteriocins and their biopreservative applications.

Essential oils: extraction, bioactivities, and their uses for food preservation.

Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications.

Potential herbs and herbal nutraceuticals: food applications and their interactions with food components.

Oxygen absorbers in food preservation: a review.

Nanocomposites in food packaging applications and their risk assessment for health.

Applications of ultrasound in food and bioprocessing.

Beneficial Effects of Spices in Food Preservation and Safety.

Antimicrobial edible coatings and films from micro-emulsions and their food applications.

Metabolic stimulation of plant phenolics for food preservation and health.

Polysaccharide-Based Membranes in Food Packaging Applications.

Applications of power ultrasound in food processing.

Effect of Hurdle Technology in Food Preservation: A Review.

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat.

Cytokines: applications in domestic food animals.

Nanostructured Biomaterials and Their Applications.

Model animals and their applications.

Magnetic Nanomaterials and Their Applications.

Uroplakins and their potential applications in urology.

Analytical methods in lipidomics and their applications.

Protein engineering and its applications in food industry.

Sugar ester surfactants: enzymatic synthesis and applications in food industry.

Silk Fibroin as Edible Coating for Perishable Food Preservation.