BACTERIOCIN PRODUCED BY LACTIC ACID BACTERIA: A PROBIOTIC Review Article

Chemical preservatives, usually used during the long period, to protect the food materials by controlling undesirable bacteria and harmful spoiler, have been proved as toxic to human health. Conscious consumers have serious awareness to purchase safe foods without chemical additives. In the recent years, bio-preservation is gained increasing attention to harmonize consumer demands along with standard food quality. Various attempts are growing on the use of micro-organisms or their antimicrobial metabolites for the protection of food products. The bacteriocins produced by lactic acid bacteria (LAB) have a relatively broad antimicrobial spectrum against variety of food-borne pathogenic and spoilage bacteria. Bacteriocin-producing lactic acid bacteria or bacteriocins can be used in foods as bio-preservatives. The review is focused on bacteriocin produced by lactic acid bacteria.


INTRODUCTION
Natural micro-biota is traditionally used in the preservation of foods through occurring fermentations over the millennia.Lactic acid bacteria (LAB) have long been employed as starter cultures for the preservation of foods in the history of mankind [1].These are well known as 'food grade' microorganisms and widely used as protective cultures for the industrial processing of fermented dairy, meat, vegetables and cereal products [2].The probiotic starter cultures are able to produce antimicrobial metabolites.Bacteriocins are antimicrobial peptides or proteins produced by different genera of LAB, have a relatively broad antagonistic activity against variety of microorganisms.The use of LAB or its bacteriocin as biopreservative is a green ecological alternative strategy of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products.The bacteriocins are nontoxic, generally recognized as safe substances, usually pH and heattolerant, become inactivated by digestive proteases [3,4].

Lactic acid bacteria
LAB produce lactic acid as the major end-product during the fermentation of carbohydrates.These are abundant in nature.The microorganisms are found in milk, meat, green plants, grains and fermenting vegetables.LAB have been isolated from mucosal surface of animals [5], sourdoughs [6], vacuum-packaged refrigerated beef [7] and traditional Indian fermented foods such as appam batter and vegetable pickle [8].LAB are included in diverse genera of organisms [9]; those are Lactobacillus, Pediococcus, Lactococcus, Carnobacterium, Enterococcus, Lactosphaera, Leuconostoc, Melissococcus, Oenococcus, Streptococcus, Tetragenococcus, Vagococcus, Weissella and Bifidobacterium itc.These are the most important probiotic known to have beneficial effects on human gastrointestinal (GI) tract.LAB can be effective in preventing gastrointestinal disorders like diarrhea [10,11].The bacteria cause lowered blood cholesterol, increased immune response.Some LAB strains are even associated with anti-carcinogenic action and tumor control [12].Lactobacillus species may colonize on the mucosal surface of duodenum and stomach [13].

Probiotics
The term "probiotics", which literally means "for life", has been introduced to describe health-promoting bacteria [14].Probiotics defined as 'live microorganisms that beneficially affect the host's health by improving its microbial balance.The adaptations of useful microbes are possible in human intestine [15].
There are over 400 types of microbes present in human intestine that may be either harmful or beneficial.The beneficial ones assist in the breakdown of food and also manufacture vitamins that essential to the body.The microbes can break down and destroy some toxic chemicals that may have been ingested with the food.Under both healthy and sick conditions, several types of bacteria compete or fight with each other to establish dominance in the warm and moist environment of the alimentary canal that serves as an ecosystem for their survival and propagation.Potential probiotics species differ in terms of their bioavailability, metabolic activity and mode of action.In addition, probiotics must survive the transition to the target niche and protect the host against infection by pathogenic microorganisms [22].
Over the few past years, the research has been rapidly focused on the use of probiotic bacteria as supplements in food medicine with a growing commercial interest [23,24].Probiotics have been incorporated into a wide range of foods, including dairy products (cheese, yogurt and ice cream) and non-dairy products (chocolate, juices and cereals) [25].For use in foods, probiotics microorganisms should be capable of surviving passage through the digestive tract, serving to protect the host against infection by pathogenic microorganisms.The organism must be resistant to gastric juices and be able to grow in the presence of bile under conditions in the intestines [22].
A large range of microorganisms are considered as probiotics must be non-pathogenic and non-toxic such as LAB (e. g.Lactococcus lactis, Pediococcus acidilactici, Enterococcus faecium, etc.), non-lactic acid bacteria (e. g.Escherichia coli) and some yeasts (e. g.Saccharomyces boulardii, Saccharomyces cerevisiae etc.).

Bacteriocin
Bacteriocins are ribosomally synthesized peptides or proteins produced by different genera of bacteria.Bacteriocins offer several desirable properties that make them suitable for the preservation of food.These are nontoxic, generally recognized as safe substances, usually pH and heat-tolerant, become inactivated by digestive proteases; show a bactericidal mode of action, usually acting on the bacterial cytoplasm membrane [3,4].
Bacteriocins are extracellular proteinaceous compound produced by different LAB species.These have antibacterial activity against undesirable microorganisms, specifically closely related species of Gram-positive bacteria [27].About 100 y ago, a bacteriocin was first identified by Gratia as colicin, produced by Escherichia coli V [28].Colicin-producing strain E. coli is Gram Gram-negative bacteria.Both Gram negative and Gram-positive bacteria produce bacteriocins [29,30].E. coli strains produce another bacteriocin, known as microcins, which is smaller than colicins and has more similar properties with the bacteriocins produced by Gram-positive bacteria [31,32].Besides E. coli, other bacteriocins producing Gram-negative bacteria are Salmonella enterica, Enterobacter cloacae, Pseudomonas aeruginosa, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumoniae, and Hafnia alvei [30,33].
Bacteriocins from Gram-positive bacteria have lower molecular weight than colicins have a much broader range of antimicrobial activity against target bacteria [34].Usually, these are hydrophobic, cationic and membrane-permeabilizing small peptides in the range of size from 2 to 6 kDa [29,35], although there are exceptions [36].The bacteriocins produced by LAB are potential peptides which can kill or inhibit pathogenic bacteria that compete for the same ecological niche or nutrient pool [37,38].These are very active against food-borne pathogens such as Listeria monocytogenes, Clostridium botulinum, Staphylococcus aureus and spoilage microorganisms, including Bacillus sp. and Enterococcus faecalis [39].Moreover, the use of bacteriocins are considered as the great advantage for food and feed producers since these are non-toxic, active even at low concentrations and generally regarded as safe (GRAS status) [38].So the bacteriocins produced by LAB have received most considerable attention as antagonistic natural biopreservativs to inhibit the survival of pathogens in foods and to enhance food safety [27].The bacteriocins are considered to be safe bio-preservatives since they are easily degraded by proteases present in gastrointestinal tract [40].These are effectively used as natural preservatives in meat and milk [15].Only bacteriocin, namly nisin produced by Lactococcus lactis, is approved as a commercially available food additive in most major food-producing countries [40,41].Another important bacteriocin is pediocin that attracts research interest and will likely be the next to be used in the food industry [42], which has anti-listerial property [43,44], produced by Pediococcus strains.Nisin (Nisaplin TM ) and pediocin (Alta TM ) are used commercially as food additives to improve quality and safety by means of a natural ingredient.Apart from nisin and pediocin, other bacteriocins, such as lactocin S (produced by Lactobacillus sake L45) and curvacin A (produced by Lactobacillus curvatus), achieve bacterial killing [45].The limitation of bacteriocins from Gram-positive bacteria is that these are not active against Gramnegative bacteria.Because the outer membrane of Gram-negative bacteria acts as an impermeable barrier for the cells [46].
The bacteriocins of Gram-positive bacteria are generally produced in log phase to stationary phase.The production of bacteriocin is growth-associated because production occurs during the midexponential phase and increase to reach a maximal level at the end of the exponential phase or the beginning of the early-stationary phase [51].For example, nisin production starts in the mid-log phase and increases to a maximum as the growth of cells enter stationary phase [52].
The antibiotic therapy for some illnesses destroys the microbial flora (both the useful and the targeted harmful microbes) present in the digestive tract; where bacteriocin have no cross-resistance with antibiotics without affecting harmless microbiota.Bacteriocins are promising substitute for therapeutic antibiotics.The introduction of probiotic bacteria to the gastrointestinal tract is an important concern regarding the potential use of bacteriocin-producing strains, which will produce bacteriocins.Bacteriocins differ from most of conventional antibiotics that these are ribosomally synthesized peptides and have a relatively narrow killing spectrum [46,53] where antibiotics are secondary metabolites, have broad range of inhibitory activity.In addition, bacteriocins, proteinaceous and easily digested by proteases in the human digestive tract.Although some antibiotics are enzymatically synthesized are composed of amino acids, such as Vancomycin [40].Bacteriocins possess antibiotic properties, but these are not termed as antibiotics.Only therapeutic antibiotics can potentially illicit allergic reactions in humans and other medical problems in humans [54].
Lantibiotics and pediocin-like bactericins can able to kill a broad spectrum of Gram-positive bacteria, including important pathogens.The commercially available bacteriocins are nisin and pediocin PA-1/AcH.The main differences between bacteriocins and antibiotics are summarized in table 1. 5% nisin, the balance consisting of milk and milk solids derived from the fermentation of a modified milk medium by nisin-producing strains of L. lactis.The product is standardized to an activity of one million international units per gram.The assay method in most common use involves measuring zones of inhibition in agar seeded with the test organism [56,57].It is predominantly used in canned foods and dairy products, processed and natural cheeses [38].

Pediocin
The bacteriocin produced by Pediococcus species is known as pediocin that shows broad inhibition activity against pathogenic and food spoilage bacteria such as Listeria monocytogenes, Enterococcus faecalis, Staphylococcus aureus and Clostrium perfringes [3].Pediocin is small, heat-stable, non-lantibiotic peptide.Its molecular weight is approximately 3.5 kDa and is resistant to heat treatment at 121 °C for 10 min [58].Pediococcus acidilactici MM33 is the first pediocinproducing strain reported and could be used as probiotic in human to prevent enteric pathogen colonization [59].Pediocin like bacteriocins are sakacin P, leucocin A, and curvacin A [60,61].Various reported bacteriocins from Pediococcus species are shown in table 2.

Determination of bacteriocin activity
The antimicrobial activity of bacteriocin is usually determined against test organism by agar plate diffusion assay [64].The inhibition zone in agar medium is measured by the inhibited growth of indicator strain.The activity is expressed in arbitrary units (AU) ml −1 .One AU was defined as the reciprocal of the highest serial twofold dilution, showing a clear zone of growth inhibition of the indicator strain [65].

Classification of bacteriocins
Bacteriocins produced by Gram-positive bacteria are divided in four classes on the basis of physico-chemical properties such as antimicrobial activity, molecular weight, stability, immunity, mode of action etc [55,66].

Class I
Class I is comprised of modified bacteriocins, known as lantibiotics (lanthionine-containing peptides antibiotic), generally produced by LAB [67,68].Lantibiotics are ribosomally synthesized compounds that target a broad range of Gram-positive bacteria.Class I bacteriocin is being further subdivided into two classes, namely, Ia and Ib.Class Ia bacteriocins, such as nisin, are relatively elongated, flexible, cationic and hydrophobic peptides (2-5 kDa).Generally, they act through the formation of pores in the cytoplasmic membrane of target bacteria [69].Class Ib bacteriocins, which are globular peptides, such as mersacidin [68], have no net charge or a net negative charge; antimicrobial activity is related to the inhibition of specific enzymes [38,70].Examples of Class I bacteriocins are summarized in table 3. Class IIb bacteriocins composed by a complex of two distinct peptides, have little or no activity form pores in the membrane of their target cells [76].
Examples of class IIb bacteriocins are shown in table 5. Lactobacillus salivarius UCC118 Class IIc has been proposed as sec-dependent secreted bacteriocins [35,77].Two types of bacteriocins can be found within this group: (a) antibiotics with one or two cysteine residues (thiolbiotics and cystibiotics) and (b) bacteriocins without cysteine (lactococcin A and acidocin B).
Examples of class IIc bacteriocins are shown in table 6.

Class III
The class III bacteriocins are heat-labile proteins of large molecular weight larger than 30 kDa [78,79].They are usually produced by bacteria of Lactobacillus [73,78], e. g. helveticin J, acidophilucin A, lacticin A and B, caseicin80 [80,81].Examples of Class III bacteriocins are given in table 7.

Class IV
This group is comprised of complex bacteriocins carrying an undefined mixture of proteins, lipids or carbohydrates [29,82].

Mode of action
The cytoplasmic membrane of Gram-positive bacteria is the primary target for the action of bacteriocins [85].Bacteriocins are generally positively charged molecules with hydrophobic patches, where, Gram-positive bacteria have a high content of negatively charged lipids (phosphate groups) in the cytoplasmic membrane.Hydrophobic patches of bacteriocins electro-statically interact with the hydrophobic membrane of target cell.The hydrostatic portion easily inserts into the membrane and forms discrete pores [40,86].Pores in the cytoplasmic membrane dissipate proton motive force (PMF) and clearly affect the essential energy source of the cell.The PMF, which is composed of a chemical component (the pH gradient: ΔpH) and an electrical component (trans-membrane potential; Δψ), drive ATP synthesis and the accumulation of ions and other metabolites through PMF-driven transport systems in the membrane.Collapse of the PMF, induced by bacteriocin action, leads to cell death through cessation of energy-requiring reactions [87].All class I bacteriocins (lantibiotics), particularly nisin dissipate the proton motive force (PMF) to kill target cells by forming pores in the membrane in the leakage of cellular materials [88].
Gram-negative bacteria are generally insensitive to bacteriocins from LAB strains because of their outer membrane providing them with a permeability barrier.The sensitivity of Gram-negative bacteria can be increased by sub-lethal injury of the cells, using for instance high hydrostatic pressure and pulsed electric field as nonthermal methods of preservation [89].Bacteriocin affects Gramnegative bacteria when their outer membrane is impaired [90].

Immunity
Bacteriocin producer strains can protects themselves against their own antimicrobial substances is referred as immunity [71].The resistance of P. acidilactici H to pediocin AcH/PA-1 has been reported as specific immunity of strain [90].The composition of membrane lipid and the constitution of cell wall are responsible for the protection of cell against bacteriocin action [91].The inhibitory activity of bacteriocin depends on species of different genera, growth cultures and environmental conditions [92].

CONCLUSION
Application of LAB or bacteriocin as bio-preservative is a great strategy for preservation of functional food through killing of pathogenic and spoilage microorganisms.Variety of foods, especially dairy products supplemented with probiotics have been reported broadly.The beneficial microbes stimulate the human intestine flora as well as immunity through regular consumption of the food products.Another effort is going on to incorporate bacteriocin in packaging matrix.Bioactive packaging is able to extend shelf life and protect food with nutritional quality.The design of edible antimicrobial film or coatings is also developed for safety of food products.Some bioactive compounds are combined with bacteriocin to enhance the inhibition of bacteria.

Table 1 : Comparison of bacteriocins and antibiotics
In 1969, nisin was approved as food additive (234) by the Joint FAO/WHO Expert Committee.The bacteriocin is permitted for use as a safe natural food preservative in food in more than 50 countries, including the US and Europe.Nisin has been sold under the trade name of Nisaplin®, which contains approximately 2.
[55]isinNisin produced by Lactococcus lactis subsp.Lactis is a lantibiotic with a small peptide (molecular weight < 5 kDa).L. lactis strains are regarded as safe (food-grade).The bacteriocin exhibits antimicrobial activity towards a wide range of Gram-positive bacteria like Listeria sp and Micrococcus sp, especially prevents heat-resistant spore-forming spoilage like Bacillus and Clostridium[55].It is suitable as a food preservative since it is natural, toxicologically safe and quickly digested by proteases in GI tract.