Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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0041 In general, NAD

is involved as an electron acceptor

in energy-yielding metabolism, being oxidized by the

mitochondrial electron transport chain, while the

major coenzyme for reductive synthetic reactions is

NADPH. An exception here is the pentose phosphate

pathway (hexose monophosphate shunt), which re-

duces NADP

to NADPH, and is the principal meta-

bolic source of reductant for fatty acid synthesis.

Role of NAD in ADP-ribosylation of Proteins

0042 ADP-ribosyltransferases catalyze the transfer of

ADP-ribose from NAD

on to arginine, lysine, or

asparagine residues in acceptor proteins, to form

N-glycosides. In addition to endogenous ADP-

ribosyltransferases, a number of bacterial toxins, in-

cluding diphtheria and cholera toxins, Escherichia

coli enterotoxin LT and Pseudomonas aeruginosa

exotoxin A, also have ADP-ribosyltransferase activ-

ity, which is not subject to the same regulation as the

enzymes in tissues.

0043ADP-ribosylation (Figure 5) is a reversible modifi-

cation of proteins, and there are specific hydrolases

which cleave the N-glycoside linkage. A variety of

guanine nucleotide-binding protein (G protein)

tbl0001 Table 1 Urinary excretion of niacin metabolites as an index of niacin nutritional status

Elevated Adequate Marginal Deficient

-methylnicotinamide

mmol 24 h

1

>48 17–47 5.8–17 <5.8

mg g

1

creatinine >4.4 1.6–4.3 0.5–1.6 <0.5

mmol mol

1

creatinine >4.0 1.3–3.9 0.4–1.3 <0.4

Methylpyridone carboxamide

mmol 24 h

1

>18.9 6.4–18.9 <6.4

mg g

1

creatinine >4.0 2.0–3.9 <2.0

mmol mol

1

creatinine >4.4 0.44–4.3 <0.44

Ratio of methyl pyridone carboxamide to N

-methylnicotinamide 1.3–4.0 1.0–1.3 <1.0

NAD

Nicotinamide Nicotinic acid

Nicotinamide

deamidase

N-methyltransferase

Aldehyde

dehydrogenase

Nicotinamide N-oxide

N-methylnicotinamide

N-methylpyridone

4-carboxamide

N-methylpyridone

2-carboxamide

(N-methylnicotinic acid)

Trigonelline

(nicotinoyl glycine)

Nicotinuric acid

ONH

COOH

fig0003 Figure 3 Metabolites of nicotinamide and nicotinic acid.

4124 NIACIN/Physiology

a-subunits involved with the regulation of adenylate

cyclase activity are substrates for ADP-ribosylation,

either activating the stimulatory G protein or

inactivating the inhibitory G protein. The result of

ADP-ribosylation by either mechanism is increased

adenylate cyclase activity, and hence an increase in

intracellular cAMP and the opening of membrane

calcium channels. Other proteins that are regulated

by ADP-ribosylation include the elongation factor(s)

in protein synthesis and integrin and other cyto-

skeletal proteins.

0044 Poly(ADP-ribose) polymerase (Figure 5) is primar-

ily a nuclear enzyme, which is present in relatively

large amounts – up to 1 mol per 1000 base pairs

in DNA. It is a zinc finger protein that binds to a

nick in DNA caused by strand breakage or excision

of an incorrect base. When bound to DNA the

enzyme undergoes an autocatalytic poly(ADP-ribosy-

lation), adding branched chains of up to 200 ADP-

ribose monomers at each of multiple sites on the

enzyme. This poly(ADP-ribosylated) protein then

interacts with the a-helical tail of histones, and

displacing histones from DNA binding, so leaving

the site clear for the DNA repair enzymes to act.

There is then slow hydrolysis of the poly(ADP-

ribose), permitting the histones to return to bind the

repaired DNA.

0045 There is some evidence that poly(ADP-ribose)

polymerase may also be involved in DNA replication,

since it copurifies with the replication fork and

enzymes of DNA replication.

NAD Glycohydrolase and Intracellular Calcium

Regulation

0046A cell membrane-bound NAD glycohydrolase cata-

lyzes a base exchange between NAD

and nicotinic

acid, yielding nicotinic acid adenine dinucleotide, as

shown in Figure 6. The same enzyme also catalyzes

exchange of the nicotinamide of NAD

with a variety

of other compounds, including histamine; there is

some evidence that this may provide a mechanism

for rapid sequestration, and hence inactivation, of

histamine.

0047The enzyme-bound ADP-ribose that is the inter-

mediate in this base exchange reaction may also

undergo either hydrolysis to yield ADP-ribose (so

that the enzyme catalyzes overall hydrolysis of

NAD

to nicotinamide and ADP-ribose), or internal

cyclization to release cyclic ADP-ribose.

0048Both nicotinic acid adenine dinucleotide and cyclic

ADP-ribose act to release intracellular stores of cal-

cium. They appear to act as second messengers in

transmembrane signal transduction, although the

extracellular ligand has not been identified. There is

evidence that all-trans-retinoic acid stimulates the

cyclase activity of the enzyme, although it is not

clear whether this is by a cell surface action or as

a result of nuclear actions. In b-islet cells of the

pancreas, glucose acts to stimulate the cyclase activ-

ity, and the resultant cyclic ADP-ribose causes a

calmodulin-dependent mobilization of calcium and

increased secretion of insulin.

Requirements and Recommendations

0049In view of the central role of the nicotinamide nucle-

otides in energy-yielding metabolism, and the fact

that, at least under normal conditions, the nicotina-

mide released by ADP-ribosyltransferase and poly-

(ADP-ribose) polymerase is available to be reutilized

for nucleotide synthesis, niacin requirements are

usually expressed per unit of energy expenditure.

0050Depletion and repletion studies suggest that, on the

basis of urinary excretion of N

-methylnicotinamide,

the average niacin requirement is 5.5 mg per

1000 kcal (1.3 mg per MJ). Allowing for individual

variation, recommended dietary allowances (RDAs)

in most countries are set at 6.6 mg niacin equivalents

(preformed niacin plus 1/60 of the dietary trypto-

phan) per 1000 kcal (1.6 mg per MJ). When energy

intakes are very low, it is assumed that expenditure

will not fall below about 2000 kcal, and this is the

basis for the calculation of RDAs for adults with low

energy intakes.

0051There is little requirement for preformed niacin in

the diet, since average intakes of protein (at least in

developed countries) will provide enough tryptophan

Nicotinamide adenine dinucleotide (NAD)

OP P

OH OH

CONH

Phosphorylated in NADP

Oxidized coenzyme

NAD

or NADP

Reduced coenzyme

NADH or NADPH

CONH

fig0004 Figure 4 Oxidation and reduction of the nicotinamide

coenzymes.

NIACIN/Physiology 4125

to meet requirements. Assuming that the diet pro-

vides some 15% of energy from protein, and this

protein provides 14 g of tryptophan per kg, this im-

plies an intake of 37.5 g of protein (525 mg of trypto-

phan) per 1000 kcal. Since 60 mg of tryptophan is

equivalent to 1 mg of dietary niacin, this suggests

that an average diet provides 8.75 mg niacin equiva-

lents per 1000 kcal (2 mg per MJ) from tryptophan

alone.

Groups at Risk of Niacin Deficiency (Pellagra)

0052The tryptophan–niacin deficiency disease pellagra

has been a problem in areas of the world where

maize or sorghum is the dietary staple, and intakes

of both tryptophan and niacin are inadequate. The

problem may be compounded by deficiency of

riboflavin or vitamin B

, both of which are required

for the synthesis of NAD from tryptophan.

Arginine

Nicotinamide

ADP-ribosyltransferase

Poly(ADP-ribose)

polymerase

Nicotinamide adenine dinucleotide (NAD)

Glutamate

OHOH

fig0005 Figure 5 The reactions of adenosine diphosphate (ADP)-ribosyltransferase and poly(ADP-ribose) polymerase.

4126 NIACIN/Physiology

0053 A number of mycotoxins and chemotherapy agents

used in the treatment of cancer activate poly(ADP-

ribose) polymerase; exposure may result in significant

depletion of NAD, and hence contribute to the

development of pellagra.

0054 Pellagra may arise as a result of drug-induced

inhibition of tryptophan metabolism, e.g., by the anti-

tuberculosis drug isoniazid (iso-nicotinic acid hydra-

zide) and the anti-parkinsonian drugs benserazide and

carbidopa. Massively increased synthesis of 5-hydro-

xytryptamine, as occurs in the carcinoid syndrome,

results in the development of pellagra, as a result of

diversion of tryptophan away from NAD synthesis.

0055 Pellagra also arises as a result of rare inborn errors

of tryptophan metabolism or the impairment of

tryptophan absorption (Hartnup disease).

Toxicity

0056Nicotinic acid has been used clinically in large doses (of

the order of 1–3 g per day) as a hypolipidemic agent. It

reduces both triglycerides and total cholesterol by

about 20%, acting as an inhibitor of cholesterol syn-

thesis. It has a more marked effect on cholesterol in

low-density and very-low-density lipoproteins, and in-

creases high-density lipoprotein cholesterol.

0057Nicotinic acid in such doses caused a marked vaso-

dilatation, with flushing, burning, and itching of the

skin; after a large dose there may be sufficient vaso-

dilatation to cause hypotension. After the adminis-

tration of 1–3 g of nicotinic acid daily for several

days, the effect wears off to a considerable extent.

0058At intakes in excess of 1 g of niacin per day there are

ultrastructural changes in the liver, and changes in liver

Nicotinamide adenine dinucleotide (NAD)

Nicotinamide

Nicotinic

acid

Nicotinic acid adenine dinucleotide (NAAD)

Base exchange

enzyme

ADP-ribose

Cyclic ADP-ribose

fig0006 Figure 6 The reactions of nicotinanide adenine dinucleotide (NAD) glycohydrolase and formation of nicotinic acid adenine

dinucleotide and cyclic adenosine diphosphate (ADP)-ribose.

NIACIN/Physiology 4127

function tests, carbohydrate tolerance, and uric acid

metabolism, which are reversible on withdrawal of

niacin. Sustained-release preparations are associated

with more severe liver damage than simple prepar-

ations, and may cause clinical liver failure, presumably

because they permit more prolonged maintenance of

high blood and tissue concentrations of the vitamin.

0059 Supplements of several grams of tryptophan per

day have been used with some success in the treat-

ment of depressive diseases, apparently without

ill effect. However, a potentially fatal eosinophilia–

myalgia syndrome associated with the use of trypto-

phan supplements has been reported, with more than

1200 cases reported to the US Center for Disease

Control in 1989. It seems most likely that the prob-

lem was due to a trace contaminant (ethylidene

bis-tryptophan) in a single batch of tryptophan,

rather than toxicity of tryptophan per se.

See also: Cereals: Dietary Importance; Coffee: Analysis

of Coffee Products; Enzymes: Functions and

Characteristics; Pellagra; Vitamins: Overview;

Determination

Further Reading

Bender DA (1983) Biochemistry of tryptophan in health

and disease. Molecular Aspects of Medicine 6: 101–197.

Bender DA (2003) Nutritional Biochemistry of the

Vitamins, 2nd edition. New York: Cambridge University

Press.

Bender DA and Bender AE (1986) Niacin and tryptophan

metabolism: the biochemical basis of niacin require-

ments and recommendations. Nutrition Abstracts and

Reviews (Series A) 56: 695–719.

De Murcia G and de Murcia JM (1994) Poly(ADP-ribose)

polymerase: a molecular nick-sensor. Trends in Bio-

chemical Sciences 19: 172–176.

Desnoyers S, Shah GM, Broche G, Hoflack JC, Verreault A

and Poirier GG (1995) Biochemical properties and func-

tion of poly(ADP-ribose) glycohydrolase. Biochimie 77:

433–438.

Frei B and Richter C (1988) Mono-(ADP-ribosylation) in

rat liver mitochondria. Biochemistry 27: 529–535.

Galione A and White A (1994) Ca

2þ

release induced by

cyclic ADP-ribose. Trends in Cell Biology 4: 431–436.

Moss J and Vaughan M (1988) ADP-ribosylation of guanyl

nucleotide binding regulatory proteins by bacterial

toxins. Advances in Enzymology 61: 303–379.

Okamoto H, Takasawa S and Tohgo A (1995) New aspects

of the physiological significance of NAD, poly ADP-

ribose and cyclic ADP-ribose. Biochimie 77: 356–363.

Rose DP (1972) Aspects of tryptophan metabolism in

health and disease. Journal of Clinical Pathology 25:

17–25.

Ueda K and Hayaishi O (1985) ADP-ribosylation. Annual

Review of Biochemistry 54: 73–100.

Nicotinic Acid See Niacin: Properties and Determination; Physiology

NISIN

G C Williams and J Delves-Broughton, Danisco

Innovation, Beaminster, Dorset, UK

Introduction

0001 Nisin is a polypeptide bacteriocin that exhibits anti-

bacterial activity against a wide range of Gram-

positive bacteria and is particularly effective against

bacterial spores. It shows little or no activity against

Gram-negative bacteria, yeasts, and molds. Nisin is

produced by certain strains of Lactococcus lactis

subsp. lactis. Commercial preparations of nisin are

widely used as food preservatives throughout the

world. It is recognized as both a toxicologically safe

and natural substance.

History

0002Nisin was discovered in England in 1928 when prob-

lems arose in cheese-making. Batches of milk became

contaminated with a nisin-producing strain of L. lac-

tis, and as a result of nisin’s inhibitory properties, the

growth of the cheese starter cultures was inhibited.

Nisin was subsequently isolated and characterized. Its

name was derived from Group N (Streptococcus)

inhibitory substance. The discovery of nisin predated

that of penicillin, so therefore it is not surprising

that initial research focused on its potential use for

4128 NISIN

therapeutic purposes in medical and veterinary appli-

cations. It was found to be unsuitable for such pur-

poses, mainly because of its limited antimicrobial

spectrum in addition to its poor solubility and stability

in body fluids. The first interest in its potential as a

food preservative came in the 1950s when it was evalu-

ated for the control of gas-producing Clostridium

spp. in Swiss cheese. However, problems occurred

due to the fact that the cheese starter cultures were

inhibited, which adversely affected the cheese-

ripening process. This problem has now been largely

overcome by the development of nisin-resistant

cheese starter cultures. The next milestone was its

development as a preservative in processed cheese in

the mid-1950s. During this period Aplin & Barrett

Ltd were experiencing unacceptable spoilage prob-

lems due to clostridial growth in processed cheese

spreads. This problem was eliminated by the inclu-

sion of nisin-rich curd into the processed cheese mix.

With this success the company went on to develop

the commercial concentrated preparation of nisin

termed Nisaplin. Nisaplin has a nisin content of

25 mg g

1

and is currently used worldwide as a food

preservative. Recently Chr. Hansen from Denmark

has introduced a similar product of the same potency,

termed Chrisin.

Units of Activity

0003 The first definition was that of a reading unit (later

known as an international unit), which was defined as

the amount of nisin necessary to inhibit one cell of

Streptococcus agalactiae in 1 ml of broth. It was so

termed in recognition that much of the early work on

nisin was carried out at the National Institute for

Research in Dairying, Shinfield (Reading), England.

Since then an international reference preparation of

nisin has been established by the World Health Or-

ganization Expert Committee in Biological Standard-

ization. This reference preparation contains, like the

commercial preparations Nisaplin and Chrisin, 25 mg

(1 million international units) of pure nisin per gram.

Throughout this article all levels of nisin are ex-

pressed as mg pure nisin per kilogram or liter.

Structure and Biosynthesis

0004Two natural nisin molecules exist, termed nisin A and

nisin Z. The structure of the nisin A molecule was

elucidated in 1971 and is presented in Figure 1.Itisa

34-amino-acid polypeptide with amino and carboxyl

endgroups, and five internal ring structures involving

disulfide bridges. It possesses three unusual amino

acids: dehydroalanine, lanthionine, and b-methyl-

lanthionine. Lanthionine appears to be a common

feature in a number of more recently characterized

bacteriocins that are collectively known as lantibio-

tics. Nisin Z differs from nisin A by the substitution of

asparagine for histidine at position 27. Nisin Z has a

similar antimicrobial activity to nisin A, although

nisin Z shows greater diffusion in agar gels. Japanese

workers have successfully synthesized the nisin A

molecule and confirmed its basic structure. Nisin

A has a molecular weight of 3354 Da. There is

evidence that nisin can exist as both dimers and

tetramers.

0005A precursor molecule to nisin has been identified

and its structural gene isolated and characterized. The

ILEU

LYS

DHB

ALA

ILEU

DHA

LEU

ALA

ABA

PRO

GLY

ALA

ABA

GLY

ALA

LEU

MET

GLY

ALA

MET

ABA

ALA

ABA

ALA

HIS

ALA

SER

ILEU

HIS

VAL

DHA

COOH

fig0001 Figure 1 The structure of nisin A. ABA, aminobutyric acid; DHA, dehydroalanine; DHB, dehydrobutyrine (b-methyldehydroalanine);

ALA-S-ALA, lanthionine; ABA-S-ALA, b-methyllanthionine.

NISIN 4129

nisin determinant of L. lactis is a component of a

large transmissible gene block that also encodes for

sucrose metabolism and nisin immunity. The gene

block is located in the chromosome as opposed to

being in a plasmid. The ability of L. lactis to synthe-

size nisin is a conjugally transmissible property that

has been transferred to negative phenotype recipients.

Mode of Action and Antimicrobial Effect

0006 Nisin works in a concentration-dependent manner

both in terms of the amount of nisin applied and the

number of vegetative cells or spores that need to be

inhibited or killed. The primary site of action in sen-

sitive Gram-positive cells is the cytoplasmic mem-

brane where insertion of nisin into the membrane

causes disruption of membrane function. An initial

step is the docking of nisin on to lipid II, which is

followed by the insertion of the nisin II lipid into the

membrane where it forms pores. Through these

pores, essential cell constituents leak from the inside

to the outside of the cell and cause depletion of the

proton motive force. Gram-negative bacteria are nor-

mally protected from nisin by their outer cell wall

acting as an impermeable barrier to the nisin mol-

ecule. However, if the outer wall of Gram-negative

cells is made permeable to nisin by partial or complete

disruption, the cells can then become sensitive to

nisin. This indicates that their cytoplasmic mem-

branes are sensitive to nisin. Recent work has demon-

strated that yeast protoplasts and spheroblasts are

also sensitive to nisin. Gram-negative bacteria can be

sensitized to nisin by sublethal heat treatment, freeze–

thaw cycling, and exposure to chelating agents that

remove divalent ions from their cell walls, thus

making them more permeable to nisin. Good effects

can be demonstrated against Gram-negative bacteria

by the combined use of nisin and a chelating agent

such as ethylenediaminetetraacetic acid (EDTA) in a

simple buffer system. In food systems, however, such

good results are not usually observed as the chelating

agent will preferentially bind with the divalent ions

that are more readily available in the food matrix.

0007 The action of nisin against spores is predominantly

sporostatic as opposed to sporicidal. Nisin affects

the postgermination stages of spore development. It

inhibits preemergent spore swelling, and thus the

outgrowth and formation of vegetative cells. The

active sites in spores are thought to be membrane-

bound sulfhydryl groups. An important factor in re-

lation to heat-processed foods is that progressive heat

damaging of spores results in them becoming increas-

ingly sensitive to nisin.

0008 The nisin sensitivity of both cells and spores can

vary between genera and even between strains of the

same species. Nisin action against vegetative cells can

either be bactericidal or bacteriostatic depending on

a number of factors, such as nisin concentration,

bacterial population size, physiological state of the

bacteria, and the conditions of growth. Bactericidal

effects against vegetative cells are enhanced under

optimal growth conditions when the bacteria are in

an energized state. In contrast, bacteriostatic effects

are enhanced when nisin forms part of a multipreser-

vation system in which growth conditions are non-

optimal and other inhibitory factors are exerted

(hurdle technology). The fact that different condi-

tions are required to insure either bacterial destruc-

tion or inhibition needs to be taken into consideration

when applying nisin in a food preservation system.

Nisin efficacy in foods is dependent on an effective

level of nisin being maintained throughout the whole

shelf-life of the food.

Solubility and Stability

0009Nisin is most soluble in acid substrates and becomes

progressively less soluble as the pH increases. Thus

at pH 2.2 the solubility is 56 000 mg l

1

,atpH5itis

3000 mg l

1

and at pH 7 it is 1000 mg l

1

. In practical

food preservation situations the level of nisin

treatment is unlikely to exceed 50 mg l

1

and thus

solubility is never a problem. Commercial powder

preparations of nisin contain residual solids from

the fermentation process that are insoluble. This can

produce cloudy suspensions in water but has no det-

rimental effect on the efficacy of nisin.

0010Commercial preparations of nisin are remarkably

stable. They show no loss of activity over a 2-year

period providing they are stored under dry condi-

tions, in the dark, and at temperatures below 25



Nisin solutions are most stable to autoclaving (121



for 15 min) in the pH range 3.0–3.5 (< 10% activity

loss). Values below and above this pH range result in

a marked loss of activity, especially those furthest

removed from the range (> 90% activity loss at pH

1 or pH 7 in a buffer system). Losses at typical pas-

teurization temperatures used in foods are signifi-

cantly less and food components can protect nisin

during heat processing.

0011The stability of nisin in a food system is dependent

on three factors: incubation temperature, length of

storage, and pH. In a nonheat-processed food a

fourth factor will be the possible presence of protease

enzymes. Nisin retention at warm ambient tempera-

tures above 25



C will be far less than at cooler tem-

peratures. On a practical level this means that higher

nisin addition levels are required for the preservation

of foods in warmer climates. Retention of nisin activ-

ity in an acidic food product will be better than in a

4130 NISIN

more neutral food stored under similar conditions.

Nisin can be inactivated by many nonspecific prote-

ases and by any proteolytic enzyme that can cleave

the histidine-valine bond (residues 31–32) or the

dehydroalanine bond (residues 33–34), e.g., thermo-

lysin, chymotrypsin, subtilisin, ficin, papain, and bro-

melain. Elastase, pepsin, and leucine aminopeptidase

have no action on nisin, and trypsin has a reversible

action. A variety of bacteria can produce the enzyme

‘nisinase’ which specifically inactivates nisin. Some of

the bacterial species reported as being able to produce

nisinase include Lactobacillus plantarum, Streptococ-

cus thermophilus, and Bacillus cereus.

0012 The food additives, and related sulfiting agents

sodium metabisulfite (an antioxidant, bleaching, and

broad-spectrum antimicrobial agent) and titanium

dioxide (a whitening agent) can also cause nisin

degradation.

Methods of Assay

0013 A number of bioassay methods have been devised.

These include dye reduction methods using resazurin

or methylene blue with a sensitive lactic acid bacter-

ium in a milk-based medium, turbidometric growth

measurement assay, horizontal agar plate diffusion

assay, the bioluminescent measurement of released

adenosine triphosphate (ATP) from Lactobacillus

casei, and an enzyme-linked immunoabsorbent

assay (ELISA). The method in most common use is

the horizontal agar plate diffusion assay employing

the test organism Micrococcus luteus. The lower limit

of detection in this assay is about 0.025 mg l

1

recently described novel technique has been the de-

velopment of a strain of L. lactis that can sense nisin

and transduce the signal into bioluminescence with as

little as 0.0125 ng ml

1

nisin being detectable by this

method.

0014 Quantitative analysis of nisin can also be achieved

by high-performance liquid chromatography (HPLC)

analysis. A nisin-containing liquid is assayed chroma-

tographically on a hydrophobic (C18) narrow-base

HPLC column by gradient elution. Calculations

are based on the peak height and quantification

done by comparison with a standard nisin prepar-

ation. The lower limit of detection by HPLC is

around 10 mg l

1

Toxicology and Legislation

0015 Toxicity studies carried out in laboratory animals

with levels of nisin far in excess of those used in

foods have shown that nisin is nontoxic and is not

carcinogenic. Nisin is rapidly inactivated in the intes-

tine by digestive enzymes and cannot be detected in

the saliva of human beings 10 min after consumption

of liquid containing 5 mg l

1

nisin. There is no evi-

dence of sensitization (allergy problems) and micro-

biological studies have not shown any cross-

resistance problems that may affect the efficacy of

therapeutic antibiotics. It is important that nisin and

other bacteriocins are not classified as antibiotics as

this could hamper their future acceptance as food

preservatives.

0016In 1969, the Joint Food and Agriculture Organiza-

tion/World Health Organization (FAO/WHO) Expert

Committee on Food Additives reviewed the toxico-

logical data for nisin and recommended its use as a

food preservative, with an acceptable daily intake

(ADI) of 0.825 mg kg

1

of body weight per day. At

present its use is permitted in approximately 60 coun-

tries, including the US, the former USSR countries,

and China. In the EU it has the food additive number

E234.

Preservation of Foods using Nisin

0017Although nisin use as a food preservative originated

with processed cheese products, other application

areas have since been identified. Many of these are

in products that, by their nature, are pasteurized

during production but are not fully sterilized. In

such foods Gram-negative bacteria, yeasts, and

molds are destroyed by the heat treatment, and the

surviving microflora consists of Gram-positive spore-

forming bacteria that can be controlled by the use of

nisin. The effectiveness of nisin in such products is

only complete if postprocessing contamination is

eliminated or minimized. Nisin is also used in canned

vegetables to control thermophilic spoilage, in non-

heated acidic products to control lactic acid bacteria,

and in products that are at risk from the psychroduric

food-poisoning bacterium, Listeria monocytogenes.

0018Table 1 summarizes the major categories of food in

which nisin is used, and the typical spoilage or patho-

genic bacteria in these products that are controlled by

nisin.

Processed Cheese Products

0019Processed cheese products cover a wide range, includ-

ing block cheese (approximately 44–46% moisture),

slices (46–50% moisture), spreads (52–60% mois-

ture), and sauces and dips (56–65% moisture). All

are heat-processed and contain emulsifying salts.

Product innovation in the industry is considerable

and formulations can be of low fat or reduced sodium

chloride content and may contain various flavor

additives such as herbs, fish, shellfish, and meat. All

these factors, along with bacterial quality of the

raw ingredients, severity of the melt process, filling

NISIN 4131

temperature, and shelf-life requirement can affect the

microbial stability of processed cheese products and

hence the requirement for nisin and the level at which

it needs to be applied.

0020 The ingredients used in the manufacture of these

products are raw cheese, butter, skimmed milk

powder, whey powder, phosphate, or citrate emulsi-

fying salts and water. Spores of anaerobic clostridial

species are often present in some of these ingredients,

particularly the cheese, and are able to survive the

heat process of 85–105



C for 6–10 min commonly

used in the heat process. The composition of pro-

cessed cheese in terms of the relatively high pH

(5.6–6.0) and moisture content combined with low

redox potential (anaerobic conditions) can result in

spore germination and growth, which may result

in subsequent spoilage due to production of gas,

off-odors, and digestion of the cheese. Clostridium

spp. often associated with the spoilage of proces-

sed cheese are C. sporogenes, C. butyricum, and

C. tyrobutyricum. Trials with processed cheese prod-

ucts have been carried out in the UK using a cocktail

of spores of the aforementioned Clostridium spp. at

inoculation levels of approximately 200 spores per

gram. Spoilage was prevented during storage at



C by 6.25 mg kg

1

nisin. Partial control was

achieved with 2.5 mg kg

1

whilst control samples

that did not contain nisin readily became spoiled.

0021The potential for growth and toxin production by

C. botulinum in processed cheese products, particu-

larly spreads, is of considerable significance. Trials in

the US have indicated that, in processed cheese

spreads, nisin is effective in delaying or preventing

the growth and subsequent toxin production by in-

oculated spores of C. botulinum types A and B. These

studies indicated that the use of nisin as an effective

preservative in processed cheese spreads should form

part of a multicomponent food preservation system.

Levels of moisture, sodium chloride, phosphate emul-

sifier salt, and pH are all factors important in deter-

mining the necessary level of nisin to provide the

required shelf-life. Facultative aerobic Bacillus spp.

can also cause spoilage problems in processed cheese

products and these organisms are also controllable

using nisin. Levels used to prevent spoilage are 5–

20 mg kg

1

, whereas levels used to provide protection

against C. botulinum are 12.5 mg kg

1

and above.

Other Pasteurized Dairy Products

0022Other pasteurized dairy products, such as dairy des-

serts, cream, clotted cream, and mascarpone cheese,

often cannot be subjected to full sterilization without

damaging their organoleptic properties, texture, and/

or appearance, and are thus sometimes preserved

with nisin to extend their shelf-life. For example,

tests on a chocolate dairy dessert resulted in a

tbl0001 Table 1 Typical addition levels of nisin (and Nisaplin) in examples of food applications

Typeof food/application

Additionlevel of nisin

(mg kg

1

or mg l

1

)

Additionlevel of Nisaplin

(mg kg

1

or mg l

1

) Typical target organisms

Processed cheese 5–15 200–600 Bacillus spp.

Clostridium spp.

Pasteurized chilled dairy desserts 1.25–3.75 50–150 Bacillus spp.

Pasteurized milk and milk products 0.25–10.0 10–400 Bacillus spp.

Clostridium spp.

Pasteurized liquid egg products 1.25–5.0 50–200 Bacillus spp.,

e.g., B. cereus

Pasteurized chilled soups 2.5–5.0 100–200 Bacillus spp.

Crumpets 3.75–6.25 150–250 Bacillus cereus

Canned foods (low acid) 2.5–5.0 100–200 Bacillus stearothermophilus

C. thermosaccharolyticum

Canned foods (high acid) 1.25–2.5 C. pasteurianum

B. macera ns

B. coagulans

Canned lobster 25.0 1000 Listeria monocytogenes

Ricotta cheese 5.0 200 Listeria monocytogenes

Continental-type cooked sausage:

Added to mix 1.25–6.25 50–250 Lactic acid bacteria

Dipping 5.0–25.0 200–1000

Salad dressings 1.25–5 50–200 Lactic acid bacteria

Beer:

Pitching yeast wash 25–37.5 1000–1500 Lactic acid bacteria, e.g.,

Lactobacillus spp.

During fermentation 0.63–2.5 25–100 Pediococcus spp.

Postfermentation 0.25–1.25 10–50

4132 NISIN

20-day increase in shelf-life with 3.75 mg kg

1

nisin

at 7



C. The same nisin level gave a 30-day increase

in shelf-life for a cre

me caramel dessert stored at



0023 The addition of nisin to pasteurized milk is permit-

ted in countries that may experience shelf-life prob-

lems due to high ambient temperatures, long-distance

transport, and inadequate refrigeration. Trials at

Reading University, UK, with nisin added at 1 mg l

1

before pasteurization at 72



C for 15 s, 90



C for 15 s,

or 115



C for 2 s all resulted in significant shelf-life

extension of the milk when stored at 10



C. Similar

benefits to shelf-life have also been demonstrated

with pasteurized flavored milk.

Pasteurized Liquid Egg Products

0024 Pasteurized liquid egg products (whole, yellow, and

white) and value-added egg products (e.g., omelets,

scrambled eggs, pancake mixes) receive heat treat-

ments designed to insure the destruction of Salmon-

ella. In the UK, for instance, liquid whole egg must be

pasteurized for at least 2.5 min at a temperature of

64.4



C. However, such heat treatment is insufficient

to kill bacterial spores and the more heat-resistant

nonspore-forming Gram-positive bacteria such as

Enterococcus faecalis. Many of these surviving bac-

teria are capable of growth at refrigerated tempera-

tures and pasteurized egg products often have a short

shelf-life. Application of nisin at levels of 2.5–

5mgl

1

has been shown to act as an effective preser-

vative, giving significant increases in shelf-life and

providing protection against the growth of the psy-

chroduric food-poisoning bacteria B. cereus and

L. monocytogenes.

Pasteurized Soups

0025 A recent trend in soup manufacture has been a move

towards the production of fresh pasteurized products

with a relatively limited chilled shelf-life. Heat-resist-

ant spores of Bacillus spp. are able to survive the

pasteurization treatment and may be capable of

growing and causing spoilage under conditions of

chill abuse storage. Trials with nisin at levels of 2.5–

5.0 mg l

1

have been found to be very effective at

preventing or delaying the outgrowth of psychroduric

spoilage Bacillus spp. during prolonged shelf-life stor-

age of these pasteurized soup products.

High-moisture Hot Plate Products

0026 Crumpets are high-moisture flour-based products

that are popular in the UK, Australia, and New Zea-

land. Crumpets are produced on a hot plate from a

flour batter and contain yeast, an aerating agent or

both to give them a raised profile and open texture.

They are toasted before eating. Crumpets have a non-

acid pH (pH 6), high moisture (48–54%) and high

water activity (0.95–0.97). The product is sold at

ambient temperature and has a shelf-life of 5 days.

There have been a number of food-poisoning out-

breaks due to the growth of B. cereus in crumpets,

particularly in Australasia. Flour used in the manu-

facture of crumpets invariably contains low numbers

of B. cereus spores that are not killed during the hot

plate cooking process. During the 3–5-day ambient

shelf-life of the product, levels of B. cereus can in-

crease from undetectable levels to > 10

cfu g

1

– suf-

ficient bacteria to cause food poisoning. Addition of

nisin to the batter mix at 3.75 mg kg

1

prevents the

growth of B. cereus to these potentially dangerous

levels. Such use of nisin has received regulatory ap-

proval in Australia and New Zealand.

Canned Foods

0027Nisin 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) re-

ceive a minimum heat process of F

¼3 to insure the

destruction of C. botulinum spores, i.e., the minimum

botulinum cook. Low-acid foods processed at F

3 are susceptible to spoilage from surviving heat-

resistant spores of thermophilic bacterial species of

B. stearothermophilus (cause of flat sour spoilage)

and C. thermosaccharolyticum (cause of can swells).

Thus nisin addition can facilitate prolonged storage

of canned vegetables at warm ambient temperatures

by inhibiting spore outgrowth of these thermophilic

spoilage organisms. The use of nisin can also allow a

reduction in the F

process down to the minimum of

3 without increasing the potential risk of thermo-

philic spoilage. Other advantages are reduced heat

damage to the foods as well as potential saving in

energy consumption. Nisin usage levels in low-acid

canned vegetables are 2.5–5.0 mg kg

1

. Residual

nisin levels in canned foods after high temperature

processing can be as low as 2% of the addition level.

However, the fact that heat-resistant thermophilic

spores are highly sensitive to nisin combined with

the heat damage enhancing their sensitivity means

that extremely low levels of residual nisin can still

be effective in this application. Preacidification of

the brine with citric acid improves nisin retention

with minimal effect on the pH of the vegetables

after processing.

0028Examples of use are canned peas, carrots, peppers,

potatoes, mushrooms, okra, baby sweetcorn, and as-

paragus. Nisin is also used in canned dairy puddings

containing semolina and tapioca.

0029Bacterial spoilage of canned high-acid foods (pH

below 4.5) is restricted to nonpathogenic spoilage

NISIN 4133