Nisin- An Antimicrobial Peptide and Its Applications in Food Industry a Review

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Puttalingamma V et al., IJSID, 2013, 3 (1), 23-33

International Journal of Science Innovations and Discoveries, Volume 3, Issue 1, January-February 2013

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NISIN- AN ANTIMICROBIAL PEPTIDE AND ITS APPLICATIONS IN FOOD INDUSTRY: A REVIEW

Puttalingamma .V

Defence Food Research Laboratory, Mysore-570011. Karnataka, India.

INTRODUCTION

INTRODUCTION

INTRODUCTION

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries An International pee

Review Journal for Scienc

Review Article Available online through www.ijsidonline.info

Received: 16-01-2013

Accepted: 10-02-2013

*Corresponding Author

Address:

Name:

Dr. Puttalingamma.V

Place:

Karnataka, India

E-mail:

[email protected]

ABSTRACT

Modern consumers demand food products without preservatives having fresh

quality even after storage. This review indicates that contamination occurring in plant and

animal associated environments contributes to human pathogens survival and can be

correlated with food borne infections. In recent years, there has been a growing interest in

the use of natural antimicrobials, especially nisin, a biopreservative in food as well as

packaging applications. Generally they are active against food spoilage and foodborne

pathogenic microorganisms including Bacillus cereus, Clostridium perfringens,

Staphylococcus aureus, and Listeria monocytogenes. Nisin is a ribosomally synthesized

peptide that has broad-spectrum antibacterial activity including many bacteria that are

food-spoilage pathogens.

Key words: Natural antimicrobials compound, nisin, biopreservative, food borne

pathogens and biocoating films.





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INTRODUCTION

The bacteriocin nisin is produced by some strains of Lactococcus lactis sub sp., Lactis ( Thomas et al., 2000; Haiping

Li et al ,2002) and is the first one to be characterized and most thoroughly studied. Its molecular formula is

C143H230N42O37S7. Enterococcus sp. strain N12ß was constructed by conjugation of the nisin-encoding transposon Tn5307

from L. lactis ATCC 11454 into Enterococcus sp. strain S12ß (Broadbent, et al 1995). Nisin is a member of the class of

antimicrobial substances known as so called lantibiotics, because they contain the unusual amino acid lanthionine. The fact

that lantibiotics are gene-encoded peptides synthesized by transcription and translation allows structural variants to be

generated by mutagenesis. Bacteriocin producing lactic acid bacteria (LAB) strains protect themselves against the toxicity of

their own bacteriocins through expression of a specific immunity protein which is generally encoded in the bacteriocin

operon. Since it is a natural antimicrobial peptide it has been approved as a natural food preservative by more than 50

countries and considered as generally recognized as safe (GRAS) by Food and Agriculture Organization (FAO)/World Health

Organization (WHO) and the European Union. The Nisaplin brand of nisin is certified as GRAS. Different bacterial genera

producing antimicrobial compound called bactericin are listed in table-1.

Table .1 Bacterial genera that produces bacteriocin

Acetobacter Corynebacterium Pediococcus Shigella Actinobacillus Enterococcus Psedomonas Streptococcus

Bacillus Erwinia Salmonella Propinobacterium

Brevibaacterium Lactococcus Serratia Staphylococcus

Closteridium Lactobacillus Yersinia

Haemophilus Leuconostock

Haloferax Listeria

Suma [1998], Balasubramanium (1995).

Table 2. Metabolic products elaborated by LAB and their antimicrobial properties

Products Target organisms

Organic acids

Lactic acid,

Acetic acid

Putrefactive effect on Gram- positive and negative bacteria,

Clostridia, fungi and yeast.

Hydrogen peroxide Pathogens and spoilage organisms in milk, meat and their

products.

Enzymes

Lactoperoxidase with H2O2 Pathogens and spoilage bacteria-Milk and dairy products.

Lysozyme Spoilage microorganisms, mainly Gram-positive bacteria.

Low molecular metabolites –

Diacetyle

Reuterin [3-oH –propionaldhyde]

Gram positive and negative bacteria, yeast and moulds, protozoa

and pathogens.

Bacteriocin- Nisin Food and waterborne pathogens, Gram-positive bacteria andspore formers. Antagonistic effect on LAB.

Others Gram positive bacteria, antimicrobial spectrum according to

producer strains and bacteriocin type.

Charumati Mishra et al., (1996).





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Table. 3 Nisin : Origin, Characteristics and Use

Characteristic Description

History 1928 Discovery

1947 First isolation

1951 First use in food

1962-65 Development of NisaplinRecognized as food preservative by FAO/WHO

1971 Primary structure determined

1998 Granted GRAS status [USFDA]

Producer organism Lactococcus lactis sub sp., lactis

Variants Nisin A, Nisin Z

Molecular weight 3353 Daltons [for the monomer]

Structure 33 amino acid peptide with characteristic lanthionine rings contains unusual amino

acids : ß methyl lanthionine, and Dehydrated serine and threonine.

Properties Cation with 3+ - [2+ for Nisin Z] Ampiphilic hydrophobic at N-terminus and

hydrophilic at C- terminus

Solubility 56 mg ml1 at pH 2.2, 3 mg/ ml at pH 5, 1mg/ml at pH 11.

Stability Optimum at pH 3.0 [<5% loss after 1150C for 20 min]

Activity Bacteriostatic and bacteriocidal against spores of gram positive bacteria. Sporostatic

against endospores formers [Bacillus and Closteridium]

Methods of

applications

In solution or as a dry powder, mixed in to food, often in combination with heat

treatment. Also surface treatment by spray immersion or in packaging, casing

materials.

Applications in foods Processed cheese and spreads, dairy desserts, cheeses [ricotta, cottage] Milk,

vegetable protein milk, liquid eggs, canned foods, fresh soups, Crumpets, beer and

wine, dressings and sauces.

Potential

applications

Pasteurized fruit juices stored at ambient temperature, meat and meat product, fish

products, dehydrated infant formula, vegetarian foods.

Thomas et al., 2000.

Classification of bacteriocins:

LAB-bacteriocins comprise a heterogeneous group of physicochemically diverse ribosomally-synthesized peptides

or proteins showing a narrow or broad antimicrobial activity spectrum against Gram-positive bacteria. Bacteriocins are

classified into separate groups such as the lantibiotics (Class I); the small (<10 kDa) heat-stable post-translationally

unmodified non-lantibiotics (Class II), further subdivided in to the pediocin-like and anti-Listeria bacteriocins (subclass IIa),

the two-peptide bacteriocins (subclass IIb), and the sec-dependent bacteriocins (subclass IIc); and the large (>30 kDa) heat-

labile non-lantibiotics (Class III). Lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though, many

LAB bacteriocins have been characterized biochemically and genetically but certain aspects of these compounds are still

unknown.

Nisin is a 34-amino-acid peptide produced by Lactococcus lactis subsp. lactis (ATCC 11454), which has emerged as

an important prototype for the study of the novel antibacterial properties and structure-activity relationships characteristic

of the lantibiotics. (Breukink, et al., 2006.). Lactococcus lactis is one of the most important micro-organisms involved in the

dairy industry. It is a non-pathogenic bacterium that is critical for manufacturing dairy products like buttermilk, yogurt and

cheese. The details about nisin, its discovery, characteristics and application in foods, method o of application are presented

in Table 3.





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Nisaplin: Composition

Appearance : Free flowing white powder

Average composition

2.5% 90% of sodium chloride

4% of protein

1.5% of carbohydrate

2% of moisture

Shelf life is 2 years at 4°C to 25°C

Joss Delves- Broughton .2007.

Lantibiotics

Lantibiotics are a class of peptide antibiotics that contain polycyclic thioether amino acids as well as the

unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. These characteristic cyclic thioether amino acids are

composed of either lanthionine or methyllanthionine. Lantibiotics are produced by a large number of Gram positive bacteria

such as Streptococcus and Streptomyces to attack other Gram positive bacteria and as such they are considered a member of

the bacteriocins. Lantibiotics are well studied because of the commercial use of these bacteria in the food industry for

making dairy products such as cheese. Bacteriocins are classified according to their extent of posttranslational

modifications. The lantibiotics are more extensively modified bacteriocins, also called Class I. Bacteriocins for which

disulfide bonds are the only modification to the peptide are Class II bacteriocins. Most bacteriocins are biologically active

single-chain peptides.

The realization of Nisin as a food preservative:

The first application of nisin was to prevent browning problems in semi-hard ripe cheese such as Emmenthal and

Gouda due to growth of C. butyricum and C. tyrobutyricum. Delves-Broughton(2005) had reported that heat resistant spores

of Bacillus spp. are able to survive . Pasteurized soup with Nisin at the levels of 2.5–5.0 mg/L is effective at preventing or

delaying outgrowth of psychroduric spoilage by Bacillus spp. during prolonged storage. And Nisin is also used in canned

dairy puddings containing semolina and tapioca. Uses of nisin to control spoilage lactic acid bacteria have been identified in

beer, wine, alcohol production and low pH foods such as salad dressings.

Nisin was used as a food preservative because:

(1) Nisin is non-toxic

(11) The producer strain L. lactis is regarded as safe (food-grade)

(111) There is no apparent cross-resistance related to therapeutic antibiotics

(1V) It is degraded immediately during digestion

(V) It is heat stable at low pH

Nisin is heat stable and active at low pH, which makes it a good candidate for a natural food preservative. Indeed, it

is used in this capacity in many different food products worldwide, in which it is particularly effective at preventing the

development of clostridial spores, which is a concern in many processed foods. These foods include meats, salad dressings,

canned vegetables, pasteurized liquid eggs (Delves-et al , 1996). Nisin is used in canned foods mainly for the control of

thermophilic spoilage. It is mandatory in most countries that low acid canned foods (pH>4.5) receive a minimum heat

process of F0 = 3 to ensure the destruction of C. botulinum spores. (Delves-Broughton (2005).





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Dairy products

Nisin is listed as a "natural preservative" in chemical dictionaries. In addition, Aplin & Barrett lists a benefit of

Nisaplin-brand nisin as "replacement or partial replacement of chemical preservatives." Nisin was awarded the Generally

Regarded as Safe (GRAS) designation in the U.S. Federal Register of April, 1988 and is approved as a natural food

preservative in the United States. Nisaplin brand nisin is a natural antimicrobial agent used worldwide since 1953 to control

bacterial spoilage in both heat-processed and low-pH foods.

Nisin was incorporated into packing materials using lacteriocins, Lacticin 3147 and Nisaplin to control Listeria

innocua and Staphylococcus aureus. Amalia et al., [2000] has reported that nisin along with EDTA will inactivate Gram

negative bacteria under neutral pH. Pathogens in ham and bologna cheese was controlled by treating with lysozyme, nisin

and EDTA. L. mesenteroids and B. thermosphacta, L. monocytogenes were also inhibited by nisin and crude bacteriocins of

LAB. (Puttalingamma et al ., 2006).

MODE OF ACTION OF NISIN

Nisin works by attaching to the plasma membrane of target cells and create pores in the cytoplasmic membrane

leading to cell lysis. It also alters membrane permeability and decreases the proton motive force (required to make ATP).

Autolysis of LAB is prevented by a similar mechanism in that the operon that codes for lantibiotic production also carries a

gene for resistance to the lantibiotic so that cell producing the lantibiotic does not kill itself. Nisin is predominantly

sporostatic rather than sporocidal, the more the spores are heat damaged the more they are susceptible to nisin, and nisin

appears to bind to sulphydryl groups on the spore surface.

Nisin kills bacteria by

(i) Forming voltage-dependent pores in the cell membrane (Héchard and Sahl 2002)

(ii) (ii) Preventing murein synthesis (Reisinger et al . 1980),

(iii) Inducing autolysis of susceptible cells (Bierbaum and Sahl 1985).

Autolysis of cells is a consequence of the release of two cell wall hydrolyzing enzymes, N-acetylmuramoyl-L-alanine amidase

(Bierbaum and Sahl 1985) and N -acetylglucosaminidase (Bierbaum and Sahl 1987). Nisin has also been shown to reduce the

thermal resistance of Bacillus spores (Beard et al .1999), and to prevent the germination of Bacillus (Nissen et al. 2001) and

Clostridium (Thomas et al. 2002) spores. In normal circumstances, Gram-negative bacteria are usually resistant to nisin

mainly due to their impermeable outer membranes. For pore formation and inhibition of cell wall synthesis, nisin binds at

nanomolar concentrations to the peptidoglycan precursor molecule undecaprenyl-pyrophosphoryl- MurNAc-

(pentapeptide)-GlcNAc, shortly lipid II. The N-terminus of nisin binds in 1:1 stoichiometry to lipid II, followed by the

interaction of C-terminus with the membrane.

A “wedge-model” for lipid II -independent pore formation has been proposed (Driessen et al. 1995). The formation

of the nisin pore results in leakage of the cell and loss of the proton motive force, which leads to cell death. Since murein is of

vital importance for the vast majority of prokaryotes and is restricted to these organisms, the inhibitors of murein synthesis

exhibit generally a high degree of selective toxicity. The synthesis of peptidoglycan is the target of a large number of

antibiotics.

Mechanism of action of nisin

(1). Acts on vegetative cells

(11). Nisin adsorbs to the cytoplasmic membrane where it forms transient pores





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(111). Low molecular weight compounds leak from the cell causing loss of energy

(1V). The pH gradient across the membrane becomes dissipated

(V). Collapse of the proton motive force (which drives ATP synthesis- the cell’s energy).

Mode of action of nisin against the cytoplasmic membrane

Nisin binds to the carbohydrate moiety of the cell wall precursor lipid II, using it as a docking molecule prior to pore

formation.

Nisin: mode of action against bacterial spores

Inhibition of pre-emergent swelling of spores

Stability and solubility

Nisaplin is an extremely stable product, showing no loss of activity over two years when stored under dry

conditions in the dark, below 25°C. Nisin shows increased solubility in an acid environment and becomes less soluble as the

pH increases. However, owing to the low level of nisin used in food preservation, solubility does not present a problem.

Nisin solutions are most stable to autoclaving (121°C for 15 min) in the pH range 3.0–3.5 (<10% activity loss). At pH values

below and above this range, there is marked decrease in activity (>90% loss at pH 1 or 7). Greater nisin retention occurs at

lower temperatures. In cold processed foods, proteolytic enzymes can affect nisin stability.

Application of nisin and its preservative effect

Nisin was the first bacteriocin derived from fermentation of a lactic-acid bacterium and was approved by the FDA in

April 1988 to use in prevention of Botulinum spores in pasteurized process-cheese spreads (Hirsch, 1951). It is the only

bacteriocin approved by the regulatory agencies of more than fifty countries to be used in milk products and canned

vegetables (Delvis, 1990; Thompkinson et al., 2000). It has attracted much attention in recent years due its success as a food

preservative. Fig-5. Nisin as a permitted additive in different countries and many countries like Australia, Franc, Peru and

Uk have not specified any limitation for the use of this bacteriocin ie nisin in any of the food products.table-4 and Fig- 1.

Table 4 Nisin as a permitted additive in different countries

Country Food in which nisin is permitted Max level [IU/g]

Australia Cheese, processed cheese, canned tomato No limit

France Processed cheese No limit

Peru Any food No limit

UK Cheese, canned foods, clotted creams No limit

US Pasteurized processed cheese spreads 10,000

Russia Processed cheese, canned vegetables 8000

Netherlands Factory cheese, processed cheese, cheese powder 800

Italy Cheese 500

Mexico Any food 500

Argentina Processed cheese 500

Belgium Cheese 100India Cheese, Tender coconut water 10 mg / Lit 1500 mg / Kg

Ref no –127 gazette notification

Nisin belongs to class I bacteriocins, it contains dehydroalanine and dehydrobutyrine residues. It exhibits

characteristics of class I bacteriocin in its bactericidal action. Nisin was first used in Swiss cheese to reduce browning

(Hirsch, 1951; Liu et al., 1990). Nisin is commercialized under brand name Nisaplin, it is a natural antimicrobial agent

(bacteriocin) used worldwide since 1953 to control bacterial spoilage in both heat-processed and low-pH foods. In 1950's,





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Aplin & Barrett pioneered the technique for the production of nisin (the active ingredient in Nisaplin) by the controlled

growth of Lactococcus Lactis, a bacterium which occurs naturally in milk. Today, Aplin & Barrett is the world's leading

manufacturer and supplier of nisin for use as natural food preservative. Nisin inhibit many food borne pathogens like

Listerira spp, Salmonell spp , Pseudomonas and Aeromonas etc.(Puttalingamma, et al, 2006 ) . They had incorporated nisin

into wax emulation and applied on the vegetable surface to extend the shelf life of fresh vegetables. Typical target organisms

and Level of nisin (mg/kg or mg/L) to inhibit their growth is presented in table-5. Puttalingamma, et al ( 2006) had applied

nisin and other lactic acid bacteria. Nisin a known bactericidal compound from lactic acid bacteria exhibited antagonistic

effect against all the selected pathogens. The mean zone of inhibition varied from 30± 0.01 to 34± 0.01 mm. Maximum AMA

was noticed with nisin since; this compound is a pure bacteriocin evidently exhibited higher effects than those observed

with crude extracts of LAB. L. lactis strain is known to produce Nisin; our observations have indicated that, antagonistic

effect seen with L. lactis was markedly higher than those from other strains of LAB. This confers the efficacy of the

bacteriocin from L. lactis as potent antimicrobial agent. However, if the activity of L. lactis (crude extract) against foodborne

pathogens was compared to those of Nisin, Nisin exhibited considerably high activity. Undoubtedly, the differences in the

activities may be due to the differences in purity of the bacteriocins.

Mean reduction in pathogenic cells from vegetable surface treated with LAB and nisin were seen. Pathogens were

inoculated with standard LAB cultures and pure bacteriocin nisin, they were incubated and tested for microbial load by pour

plated method. Nisin can inhibit completely with in 72 hours. L. lactis and L. plantarum one log in 72 hours. Bacterial counts

compared with the control due to the rapid antimicrobial action of nisin against

Bacteriocins into packaging films to control pathogenic Organisms.

The Bacteriocins are inhibitory peptides produced by many species of lactic acid bacteria. To control food

contamination and quality loss, edible coating or biodegradable packaging has been recently introduced in food processing.

The packaging can serve as a carrier for antimicrobial and antioxidant compounds, their presence could avoid moisture loss

during storage, reduce the rate of rancidity causing lipid oxidation and brown coloration, and reduce the pathogen

undesirable microorganism on the surface of foods.

Edible coatings and films prepared from polysaccharides, proteins and lipids have a variety of advantages such as

biodegradability, edibility, biocompatibility, appearance and barrier properties. They have been developed in order to

reduce and or inhibit the growth of microorganisms on the food surface. One is to incorporate bacteriocins directly into

polymers such as incorporation of nisin into biodegradable protein films. Two packaging film-forming methods, heat-press

and casting were generally used. Cooksey (2000) had studied by using antimicrobial packaging films for inhibition of

selected microorganism in Low density polyethylene (LDPE) film was successfully coated with nisin using methylcellulose

(MC)/ hydroxypropyl. Nisin was found to be effective in suppressing S. aureus and L. Monocytogenes. The production of a

nisin containing cellophane based coating was used in the packaging of chopped meat. The developed bioactive cellophane

reduced significantly the growth of the total aerobic bacteria through 12 days of storage at 4° C and would result in an

extension of the shelf life of chopped meat under refrigeration temperatures Ugur Geogeus et al ., (2004) investigated the

effectiveness of yogurt treatment followed by nisin and coating of and reported that 5YN application showed the most

significant inactivation of Salmonella with 1.97 log reduction while the control had the highest Salmonella count (p < 0:01)

on the 6th day of incubation at 4 0C.





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EDTA being metal chelator exerts an indirect effect on growth of microbes by limiting the availability of important

metals. The production of a nisin containing cellophane based coating was used in the packaging of chopped meat. The

developed bioactive cellophane reduced significantly the growth of the total aerobic bacteria through 12 days of storage at

4° C, would result in an extension of the shelf life of chopped meat under refrigeration temperatures Ugur Geo_geus. et al .,

(2004) investigated the effectiveness of yogurt treatment followed by nisin and coating of and reported that 5YN

application showed the most significant inactivation of Salmonella with 1.97 log reduction while the control had the highest

Salmonella count (p < 0:01) on the 6th day of incubation at 4 _C.

EDTA being metal chelator exerts an indirect effect on growth of microbes by limiting the availability of important

antagonistic effect than that seen with nisin alone. Research evidences also suggests that EDTA with crude bacteriocin or

nisin effectively inhibit the growth of foodborne and food spoilage organisms (Iikka and Tiina, 2000). Chi-Zhang et al . (2004)

suggested that the combination of packaging material containing nisin used in conjunction with nisin containing foods will

inhibit L. monocytogens growth.

Beneficial effects of Nisin as a preservative:

Enhances product quality, reduces processing temperatures, formulated at higher pH and therefore reduce acidity.

Reduce manufacturing and distribution costs, protects against temperature abuse of chilled products. Lower processing

times and temperatures.

In certain cases (eg. processed cheese products) require ambient temperature instead of chilled for storage and distribution.

Meets consumer demand for foods preserved with natural ingredients.

Replacement or partial replacement of chemical preservatives ensures food safety.

Control pathogenic / food poisoning organisms such as Listeria, Bacillus cereus and Clostridium botulinum as part of

an overall safe processing system. Fig,2,3 and 4.





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Fig 2: Tony Jin, et al ,2009.

Fig-3 (Puttalingamma et al .: 2006.)

The maximal nisin concentration migrating m was equivalent to 1000 IU mL)

of BHI broth. PLA film coated with nisin (PLA + nisin) or pectin ⁄PLA film without nisin had little or no effect on the growth

of L. monocytogenes as shown in Fig. 1, which was further confirmed by the agar diffusion test. The major potential food

applications of antimicrobial films include meat, fish, poultry, bakery goods, cheese, fruits and vegetables this needs to be

investigated to identify the types of food that can benefit the most from such antimicrobial packaging materials.

CONCLUSION

Nisaplin, it is a natural antimicrobial agent (bacteriocin) used worldwide since 1953 to control bacterial spoilage in

both heat-processed and low-pH foods. The literature reveals that the bacteriocin nisin has exhibited good antimicrobial

properties against Salmonella spp, Listeria monocytogenes, Pseudomonas spp, Staph aureus and A. hydrophila. Several

workers have also films coated with nisin and processed to extend shelf life of food. By the present scenario it can be





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concluded that nisin can be substituted for synthetic and chemical preservatives which are harmful for human beings and

also reduces manufacturing and distribution costs.

ACKNOLODGEMENT

I am thanking to Director DFRL Dr. H.V. Batra, for providing the necessary facility and encouragement.

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Nisin- An Antimicrobial Peptide and Its Applications in Food Industry a Review