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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Review Article

LANTIBIOTIC NISIN: NATURAL PRESERVATIVE FROM LACTOCOCCUS LACTIS Suganthi.V1, E. Selvarajan2, C.Subathradevi3, V. Mohanasrinivasan4*

1Research Associate, Division of Environmental Biotechnology, School of Bio Sciences and Technology, VIT University, Vellore-632 014 India

2Research Associate, Division of Environmental Biotechnology, School of Bio Sciences and Technology, VIT University, Vellore-632 014 India

3Assistant Professor Senior, Division of Industrial Biotechnology, School of Bio Sciences and Technology, VIT University, Vellore-632 014 India

4Assistant Professor Senior, Division of Environmental Biotechnology, School of Bio Sciences and Technology, VIT University, Vellore-632 014 India

Article Received on: 09/11/11 Revised on: 18/12/11 Approved for publication: 02/01/12

*Email: v.mohan@vit.ac.in

ABSTRACT The increasing demand for high quality safe foods that are not extensively processed has created a niche for natural food preservative. Studies confirm that food allergies due to chemical preservatives affect as much as 2.5% of the population. Recent research had suggested bacteriocins (Nisin) are the ideal biological food preservative. Nisin was proteinaceous antibacterial substances produced by Lactococcus lactis, a homofermentative bacterium. Naturally nisin occurs in two different forms nisin A and nisin Z. Nisin has wide range of inhibitory mode of action on Gram negative bacteria and food borne pathogens. Food preservation is a continuous war against the microorganisms spoiling the food or making it unsafe. So, nisin is actually the only lantibiotic bacteriocins used as a food preservative. This review paper will discuss about the Lactococcal strain used for the production of nisin, different forms of nisin, the mode of action of nisin, the cost reductive methods for the production and purification of nisin. So that it can be used in large scale industry for the high yield of nisin and the wide application of nisin in food industries. KEY WORDS Nisin, Lactococcus lactis, Mode of action, Biopreservation, Bacterial inactivation & Bacterial proteins INTRODUCTION The increasing concerns of consumers about the possible adverse health effects from the presence of chemical additives in food .The need for the industry to preserve the shelf life of food and safety of food products have increased the research interest in finding new natural effective food preservative. Food preservation is a continuous war against the microorganisms spoiling the food or making it unsafe. On talking about food preservation the much attention is associated with the presence of lactic acid bacteria (LAB) in food products. Lactic acid bacteria are the microbes that humans have used for many years to make a variety of processed foods. Later it has been identified that the preservation effect result from the antimicrobial action of bacteriocins as well as metabolites , such as lactic acid and hydrogen peroxide, produced by Lactic acid bacteria1 .The bacteriocins produced by Gram positive bacteria like LAB are made up of small pepetides,3-6KDa,in size2. Nisin is the most extensively characterized bacteriocin of antimicrobial proteins produced by lactic acid bacteria3. Nisin Nisin is a bacteriocin composed of 34 amino acids with a molecular mass of 3354KDa and is produced by certain strains of Lactococcus lactis subsp lactis4,5 .It is an effective bactericidal agent against several related and unrelated bacteria and occurs naturally in many dairy products6.Historically, interest in nisin was based on the first observation that led to the discovery of bacteriocins were made by 7in England when they discovered that certain Lactococcus strains had an inhibitory effect on the growth of other Lactic acid bacteria. Nisin is the only bacteriocin to have found a widespread application in the food industry. It is

permitted as a food additive in at least 46 countries, particularly for the inhibition of Clostridium species in processed cheese, dairy products, and canned products8. Nisin exhibits a broad spectrum of inhibitory activity against Gram-positive bacteria including their spore forms8. Nisin is a heat stable and contains 34 amino acids and is synthesized by post translational processing of ribosomally synthesized precursors9. Lactococcus lactis Lactococcus lactis was the first bacterium that was isolated in pure culture in 1873 by Joseph Lister. It is one of the main ingredients in milk fermentations (in Latin lac) to produce butter, buttermilk and various soft and hard cheeses18Lactococcus lactis is a homofermentative bacterium. Its primary function is rapid lactic acid production from lactose. The characteristics of Lactococcus include carbon metabolism, extracellular and intracellular proteolytic system. This species have two subspecies and a biovar: Lactococcus lactis subsp lactis; Lactococcus lactis subsp cremoris; Lactococcus lactis subsp. lactis biovar diacetylactis19,20. The most important development as the result of the molecular genetics investigation of Lactococcus lactis is the opening up of its use in areas outside the long standing traditional food production, such as the expression of proteins for pharmaceutical use, the expression of membrane proteins, the production of live and oral vaccines and the delivery of pharmaceutical proteins to the human gut21. Lactococcus lactis is a safe microorganism that has been consumed over thousands of years. Later, specific strains have been developed that has the ability to survive only in the host organism and not in the environment. Therefore, it is used to produce in situ therapeutic substances

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or act as a delivery vehicle. Strains that produce human IL-10 in the human gut are currently being tested in clinical trials for the treatment of Crohn’s disease22.The antigens originate e.g. from viruses (SARS), bacteria (Helicobacter pylori), parasites (malaria) and Pollen (birch). Various gene expression systems for constitutive and regulated expression are available for Lactococcus lactis23.The most important and widely used regulated gene expression system is the NICE system – Nisin Controlled gene expression24. Structure And Chemical Characteristics Of Nisin The molecular weight of nisin is approximately 3350 Daltons. Nisin is soluble and highly stable at acidic solution; at aqueous solution of pH 2, the solubility of nisin is 57 mg/ml, and nisin retains it biological activity even if it is autoclaved25. However, at alkaline pH the solubility decreases dramatically and nisin becomes biologically inactive, probably due to chemical modifications2. Many classifications, essentially based on their molecular weight and mechanism of action have been proposed for nisin26, 6.Nisin is a 34-amino acid peptide with a molecular mass of 3.5 KDa. Naturally nisin can be categorized into two forms, nisin A and nisin Z. The variants A and Z, differing by one amino acid9six natural variants have been identified and characterized in (Table 2). Structure Of Nisin There are only three natural variants of Nisin were discovered; nisin A, nisin Z and nisin Q. Nisin A, comprise 34 amino acids with five intermolecular sulfide bridges (rings A-E)33. Nisin Z was found to be widely distributed contains asparagine instead of histidine at position 2734. Interestingly, nisin Z was shown to have antimicrobial activity, membrane insertion35, and pore forming ability36 similar to nisin A.On the other hand, nisin Q was discovered recently in only one producer31. Mode Of Action Of Nisin The inhibiting mode of action towards vegetative cells consists of several phases it was active against a broad spectrum of Gram-positive bacteria; Escherichia coli and other Gram-negative bacteria are only affected when their outer membranes are weakened or disrupted by treatment with EDTA or osmotic shock37. Nisin has a dual activity against spore-forming bacteria it inhibits the outgrowth of spores and kills cells in the vegetative state. The 2, 3 didehydroamino acid residues in nisin are thought to act against spores by interacting with the membrane sulfhydryl groups of germinating spores38. The primary target of nisin in vegetative cells is the cytoplasmic membrane. It dissipates the membrane potential of whole cells, cytoplasmic membrane vesicles and artificial membrane vesicles (liposomes) 39, 40. Earlier studies with nisin demonstrated that it inhibited peptidoglycan biosynthesis 41,9and that it interacted with either lipid I or lipid II42. It was later found that nisin caused pore formation in the membranes of sensitive bacteria43. More recently, it was shown that nisin interacts with a docking molecule, lipid II, which is a membrane-bound precursor for cell wall biosynthesis. Indeed, in the absence of this precursor, significantly higher concentrations of nisin are required to form pores36, 44 this mechanism is shown in (Fig.1). The mode of action described a voltage- dependent depolarization of the membrane by nisin. The membrane

potential is not essential, but that the total proton motive force stimulates the action of nisin45. Nisin and pep 5 induce cellular autolysis in Staphylococci46, 47. Membrane disruption is believed to result from the incorporation of nisin into the cytoplasmic membrane to form an ion channel or pore. The efficiency of insertion of nisin into liposomes depends on the phospholipids composition of the liposomes. This may account for the differences in sensitivity seen between bacterial species or strains, as permeabilization only occurs in liposomes that contain zwitterionic phospholipids48, 49. The activity nisin can be significantly reduced by divalent and trivalent cations, and so the activity can be prevented by gadolinium (Gd+3), a lanthanide which is known to inhibit various channels in eukaryotic and prokaryotic cells50. The ions get binds and neutralize the negatively charged groups of phospholipids and it makes the lipid condense, which result in a more rigid membrane, which probably decreases the efficiency through which nisin inserts and form spores. Nisin inactivates endospores by preventing post-germination swelling and subsequent spore outgrowth51, 52. Interaction Of Nisin With Food Material The limitation to bacteriocin usage in food was their interaction with food constituents, which can affect their activity53. Food structure, food composition such as nutrients, ingredients and additives, buffering capacity of food products and their physiochemical characters such as pH, temperature, water activity (aW), atmosphere (O2, CO2), redox potential, and load of microbes can influence nisin activity in food products. Nisin has been Granted Generally Recognized as Safe (GRAS) status by the Food and Drug Administration (FDA). In addition, nisin used in combination with surfactants, chelators and adjuvants is also effective against both Grams - negative and resistant Gram-positive bacteria. Many lactic acid bacteria produce proteinaceous antimicrobials bacteriocins, some of which could provide valuable alternatives to traditional therapeutic antibiotics for the treatment of infectious diseases54. Bacteriocin activity was inactivated by proteolytic enzymes, but not by other non-proteolytic enzymes. Mitomycin C and UV light did not affect the activity of the nisin while chloroform extraction completely destroyed their activity55 Nisin has the characteristics to tolerate acid, high temperature and low temperature storage and these makes the nisin to be widely used in milk and milk products, meat, egg products and canned food. The use of nisin in a preservation of meat has gained success due to the interface of meat components such as phospholipids 56and high pH 57in this case nisin becomes extremely less soluble and also ineffective 9. Therefore, this indicates the limitation of the use of nisin. Production And Purification Bacteriocin production is influenced by several environmental factors such as pH, temperature, concentration of nitrogen and carbohydrate sources and the presence of essential elements such as vitamins and oligo-elements58.The major limiting factor, which is obtaining pure bacteriocin, should not be a problem. General methods of bacteriocin purification as based on their biochemical properties: cationic and amphiphilic peptides. The general process for bacteriocin purification often involves a straight-forward four step process59. The methods most frequently used for isolation, concentration and purification include salt precipitation of bacteriocin from culture supernatant, cationic exchange

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chromatography and reverse- phase high performance liquid chromatography60. There are some limitation in the nisin production such as low yields, low product concentration, and high medium cost61. But there is a growing consumer demand for natural food additives, so there is a need for most cost effective methods to produce nisin. For this purpose material from dairy products for example agriculture waste such as potato hydrolysate and fermented barley extracts have been used for the production of nisin62, 63. Use of food grade nisin could also be enabled by using food grade production of nisin64. Nisin production was carried out using a readily available plant product such as soy whey may help in minimal processing requirement and easy barriers in using food for nisin production65.Various strategies for the purification of bacteriocins from complex cultivation broths have developed for the cationic and hydrophobic characteristics66. Usual methods for bacteriocins extraction are based on their affinity to organic solvents, their variation in solubility in concentrated salt solutions and at a given pH value. The presence of hydrophobic regions in bacteriocin molecules is essential for their activity against sensitive bacteria, since inactivation of microorganisms by bacteriocins depends on the hydrophobic interaction between the bacterial cells and bacteriocin molecules67. Complex growth medium such as de Man Rogoso Sharpe (MRS) medium commonly used for cultivation of fastidious Lactic Acid Bacteria interfere in bacteriocin purification68. Since bacteriocins are secreted into the culture medium, most strategies start with a step to concentrate bacteriocins from the culture supernatant, using for example diatomite calcium silicate 69or ammonium sulfate precipitation 70. Further steps was carried out by using preparative isoelectric focusing or multiple chromatographic separations, including cation exchange, gel filtration, hydrophobic interaction and reverse-phase liquid chromatography are necessary to achieve significant purification of bacteriocins71. Lactococci are widely employed in the dairy industry. Bacteria from this group are involved in the production of the lantibiotic nisin, lactococcins, lactostrepcins, diplococcins and others72. A single-step method for the purification of nisin, based on an immunoafinity chromatography was developed73. A simple one step purification method, was developed using expanded bed ion exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A16466. Applications Of Nisin Biopreservative effect of Nisin Lantibiotics are potent post-translationally modified antimicrobial peptides produced by many Gram-positive bacteria74. Nisin is produced by some strains of Lactococcus lactis subsp. lactis. It is a pentacyclic peptide containing three unusual amino acids in its structure, dehydroalanine, lanthionine and ß-methyl-lanthionine, and has a molecular weight of 3510 Da. The structure of nisin is shown in (Fig 2) and was first reported by Gross E 33. The presence of 5 internal thioether rings formed by lanthionine (Ala-s-Ala) and ß- methyl lanthionine (Abu-S-Ala) group is responsible for the conformation of nisin. It is inactivated by a-chymotrypsin, but resistant to treatments with pronase, trypsin, and heat under acidic conditions 6 . Nisin is effective against Gram-positive pathogens and prevents outgrowth of Clostridium and Bacillus spores. Nisin

has been approved for use in the United States as the antibotulinal agent in processed cheese spreads. Considerable research has been carried out on the antilisterial properties of nisin in foods and a number of applications have been proposed. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production and low pH foods such as salad dressings75. Production of highly purified nisin preparations and enhancement by chelators has led to interest in the use of nisin for ulcer therapy in human, and mastitis control in cattle75. Nisin activity against Listeria.monocytogenes is decreased in the presence of increasing fat concentration but inactivation of nisin in the presence of fat was decreased with addition of a nonionic emulsifier such as Tween 80, but not by an anionic emulsifier such as lecithin 76. Application of Nisin in the Preservation of Dairy Products Use of nisin in food was as a preservative in processed cheese products and this continuous to be one of the major applications of nisin to this day77. The ingredients used in the manufacture of these products are raw cheese, butter, skim milk powder, often various added flavors, phosphate or citrate emulsifying salts, and added water. Nisin A is also an effective inhibitor of Listeria monocytogenes and growth of this pathogen was effectively inhibited by Nisin A in camembert 78 and in cottage cheese at 4°C as well as 37°C. The application of nisin in dairy foods which require lactic acid starter bacteria presents a problem because the wide spectrum of inhibition associated with nisin includes lactic acid bacteria themselves. The bacteriocin is produced in milk and is unaffected by the heat treatment. This milk was mixed with fresh milk and used for cheese making. The lactococcal strains were shown to grow and produce acid normally in the milk, whereas Listeria monocytogenes introduced in at the same time was rapidly killed. Application of Nisin in the Preservation of Meat Products Concern on high levels of nitrite in cured meat has leaded various workers to consider alternative preservation systems, which include a reduction in nitrite levels, and these have included nisin79.54. The use of these bacteriocins alone was not successful, promising results were obtained when it was combined with reduced levels of nitrite: 100-250 ppm nisin A combined with 120 ppm nitrite was more effective than the conventional 156 ppm nitrite 80,81 Listeria monocytogenes is a food-borne pathogen which is ubiquitous in the environment and can be isolated from foods of different origin, including meat and meat products. Nisin and butylated hydroxyl anisole (BHA) were added to study the shelf life meat sausage 82. Application of Nisin in the Preservation of Fish The application of nisin A in the preservation of fish products has been studied by 80Nisin treatment of cod, herring, and smoked mackerel fillets inoculated with Clostridium botulinum spores brought about a delay in toxin production of 5 days at 10°C, but only by half a day at 26°C. Nisin treatment did nor interferes with growth of non-pathogenic bacteria and in all samples botulinum toxin was formed before spoilage was evident. The effects of nisin Z, carnocin U149 and bavaricin on bacterial growth and shelf life of brined shrimp was recently evaluated and compared with those of a benzoate-sorbate solution and a control with no added preservatives83. Crude or purified nisin Z was applied to the same material the shelf life was extended to 31 days.

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Such results offer clear perspectives for the biopreservation of certain fish products with nisin Z. Application of Nisin in the Preservation of Alcoholic Bevarages Nisin is used in distilled alcohol production, both for beverages and industrial production. When added to fermentation mashes that are naturally contaminated with Lactic acid bacteria, the latter’s activity can be controlled and cause increased alcohol yield by allowing the yeast less competition for substrate21. Nisin is introduced during fermentation although the spoilage of lactic acid bacteria is sensitive to nisin, the yeasts are shown to be completely unaffected. In the brewing industry nisin is added to fermenters for controlling and preventing contamination, reducing pasteurization process and increasing the shelf life of unpasteurized or bottle conditioned beers. Similar applications also occur in the wine industry. However, nisin cannot be used during fermentation of wine that depends on desirable molalactic acid fermentation. CONCLUSION Lactic acid bacteria have been recognized as safe, and Bacteriocins (Nisin) produced by these microorganisms act as a good solution to the problem of resistant strains to antibiotics. During the last decade, a large number of Lactic acid bacteria bacteriocins have been identified, and in some cases, biochemically and genetically characterized. The development in bacteriocins research has been favored by the recognition of the role that these bacteriocins producing bacteria may play in the hygienic quality assurance of food and as feed supplements 88. This may be partially due to the fact that newly discovered broad-spectrum bacteriocins have yet to be fully characterized and officially approved. Thus nisin is the only lantibiotic licensed as a good food grade preservative. Convincing evidence of inhibition of pathogens and spoilage bacteria is required to stimulate commercial interest in bacteriocins as agents for bio preservation. From the present review paper it is evident that the nisin is currently being used as a effective biopreservative for several food materials. ACKNOLEDGMENT The authors are grateful to the Management, VIT University, Vellore for providing the facilities and constant encouragement for this work. REFERENCES 1. Caplice, E, Fitzgerald, GF. Food fermentation Role of microorganism in food production and preservation. Int.J.Food.Microbiology.1999; 50:131-1492. 2. Nes ,IF, Bao Diep ,D, Havarstein, LS , Brurberg, MB , Eijsink,V , Holo, H. Biosynthesis of bacteriocins of lactic acid bacteria. Antonie van Leeuwenhoek.1996; 70: 113-128. 3. Jack, RW, Tagg, JR, Ray, B. Bacteriocins of Gram–positive bacteria. Microbiol. Rev.1991; 59: 171–200. 4. Jung, G.Lantibiotics: Asurvey. In Nisin and Novel Antibiotics ed. Leiden: ESCOM Science publishers.1991; 1-34. 5. Murugesh,S, Mohanasrinivasan,V, Subathra Devi,C ,Mahesh,N, Manivannan,S.Bio- preservation using Bacteriocin (NISIN) Produced from Lactococcus lactis subsp lactis. Indain.J. Applied Microbiology.2003; 3(1):23-26. 6. Hurst, A.Nisin.Adv.in.Appl.Microbiology.1981; 27:85-123. 7. Rogers, LA and Whitter,EO. Limitic factors in the lactic fermentation.J.Bacteriology.1928; 16(4):211-229. 8. Delves-broughton, J. Nisin and its uses as a food preservative. International J of Food Technology.1990; 43:73-76. 9. Hansen, JN and Liu, W. Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 1990. 2551–8.

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Fig 1: Shows the mode of action of nisin against gram positive and food borne pathogens

Wiedemann et al., (2001).J.Biol.Chem. 276(1772-1779).

Fig. 2 The structure of nisin

Table1: Reported Effects on the applications of Nisin for bacterial inactivation BACTERIOCIN OBSERVAED EFFECTS REFERENCES

NISIN Increased inactivation of Bacillus and clostridium spores

Increased bactericidal activity and spectrum (E.coli, S. aureus)

Increased inactivation of bacteria associated with milk

Increased sensitivity of pressure resistant E.coli

Increased inactivation of B.cereus spores and inhibition of the surviving fraction in cheese

Increased inactivation of L. monocytogenes scott A in cheese inoculated with a

nisin-producing strain

Improved bactericidal effect on spores and aerobic mesophilic bacteria in cheese

Increased inactivation of E. coli and L. innocua in liquid whole egg

10

11

12

13

14

15

16

17

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Table 2: The variants of Nisin with the production strain

VARIANTS MOLECULAR WEIGHT

PRODUCTION STRAIN REFERENCES

Nisin A 3353 Lactococcus lactis 27,28

Nisin Z 3330 Lactococcus. lactis 29

Nisin Q 3327 Lactococcus. Lactis 30

Nisin F ND Lactococcus. Lactis 31

Nisin U 3029 Streptococcus uberis 32

Nisin U2 3029 Streptococcus uberis 32

Table.3: Examples of Effective use of Nisin in food FOOD PRODUCTS TARGET ORGANISM EFFECTIVE NISIN CONCENTRATION (IU/ml or IU/g) REFERENCES

Canned beans C. thermosacchrolyticum 200 84

Cottage cheese L. monocytogenes 2,000 85

Ricotta cheese L. monocytogenes 100 86

Skim milk B. cereus spores 4,000 87

Source of support: Nil, Conflict of interest: None Declared