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

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species such as C. pasteurianum, B. macerans,and

B. coagulans. Nisin addition levels of 1.25–2.50 mg

1

are used in high-acid tomato-based products.

Meat Products

0030 Concern regarding the high levels of nitrite in cured

meat has resulted in research investigating the use of

nisin as a partial replacement for nitrite. Results indi-

cated that only high (and uneconomic) levels of nisin

achieved good control of C. botulinum. Further work

is necessary before a case for such an application is

demonstrated. More encouraging results have been

obtained in vacuum-packed cooked continental-type

sausages where lactic acid bacteria can cause spoilage

by production of gas, off-odors, and slime. Addition

of nisin into the sausage mix at levels of 1.25–

6.25 mg kg

1

or dipping the cooked sausage into

nisin solutions of 5.0–25.0 mg l

1

has proved effect-

ive in increasing shelf-life at storage temperatures of

6–12



C. Investigations have shown that nisin is more

inhibitory against lactic acid bacteria in sausages with

lower fat levels. It was also shown that nisin had a

greater effect in sausages containing diphosphate

compared to those with orthophosphate.

Fish and Shellfish

0031 Relatively few studies have been carried out using

nisin in fresh fish mainly because the predominant

flora tends to be Gram-negative bacteria. The poten-

tial hazard of botulism in both vacuum-packed and

modified-atmosphere-packed fish has led to work at

the Torry Research Station in the UK on the use of

nisin as an antibotulinal agent. Application of nisin

by spray to fillets of cod, herring, and smoked mack-

erel inoculated with C. botulinum type E spores

resulted in a significant delay in toxin production

at 10 and 26



C. Another problem in smoked fish

is the presence and growth of the psychroduric

pathogen L. monocytogenes, especially in fresh and

lightly preserved products. Nisin has been shown

to be an effective antilisterial agent in smoked

salmon, especially when packed in a carbon dioxide

atmosphere.

0032 L. monocytogenes can also be a problem in shell-

fish, particularly crabs and lobsters, as their meat can

only be lightly heat-processed without significant

product damage occurring. Nisin, in combination

with a reduced heat process, that does not cause

product damage of lobster meat, has been shown to

achieve a Listeria kill that is significantly better than

either heat or nisin used alone. An effective nisin level

for this application is 25 mg kg

1

. Washing crabmeat

with nisin has been shown to reduce levels of

L. monocytogenes.

Salad Dressings

0033The development of cold blended salad dressings with

reduced acidity can improve the flavor of many var-

ieties that are considered to have an over-acid taste.

Using reduced levels of acetic acid and raising the pH

from 3.8 to 4.2 can make salad dressings prone to

lactic acid bacterial spoilage during ambient storage.

Such growth has been successfully controlled by the

addition of nisin at 2.5–5.0 mg l

1

Natural Cheese

0034The first application of nisin was to prevent blowing

problems in semihard ripe cheese such as Emmenthal

and Gouda due to growth of C. butyricum and

C. tyrobutyricum. Although promising results were

obtained, a problem with inhibition of the starter

cultures and consequent delay of the ripening process

led to the work being discontinued. However, the

increase in knowledge of lactic acid bacterial genetics

and the need to devise methods for the control

of L. monocytogenes has resulted in fresh interest

into the use of nisin in natural cheese. To achieve

success, nisin-resistant starters must be used in con-

junction with nisin to insure successful development

of the cheese. Natural nisin-producing, nisin-resistant

strains tend to lack important properties associated

with cheese starter cultures such as good flavor pro-

duction, eye formation, acidifying characteristics,

and bacteriophage resistance. Using the food-grade

genetic transfer technique of conjugation it has been

possible to develop nisin-producing, nisin-resistant

starter cultures with the above-desired properties.

Cheeses have been made with sufficient nisin content

to provide protection against growth of Clostridium

spp., Staphylococcus aureus, and Listeria monocyto-

genes.

Yogurt

0035The addition of nisin to stirred yogurt postproduction

has an inhibitory effect on the starter culture (a mix-

ture of Lactobacillus delbrueckii subsp. bulgaricus

and Streptococcus thermophilus strains), thereby pre-

venting subsequent overacidification of the yogurt.

Thus an increase in shelf-life is obtained by maintain-

ing the flavor of the yogurt (less sour) and preventing

syneresis. Typical addition levels for this application

are 0.5–1.25 mg kg

1

Alcoholic Beverages

0036Acid-tolerant lactic acid bacteria of the genera Lacto-

bacillus, Pediococcus, and Leuconostoc can spoil

beer and wine due to growth along with production

of off-flavors, off-odors, slime, or haze. At levels of

0.25–2.5 mg l

1

, nisin is effective in preventing such

4134 NISIN

spoilage. Yeasts are unaffected by nisin, thus the pre-

servative can be added during the fermentation. Nisin

can be added to fermenters to prevent or control

contamination and can also be used to increase the

shelf-life of unpasteurized and bottle-conditioned

beers. Furthermore, nisin can be used in the pitching

yeast wash as an alternative to acid washing for the

control of lactic acid bacteria. Unlike nisin treatment,

acid washing can have an adverse effect on yeast

viability and performance. Typical levels for this ap-

plication are 25.0–37.5 mg l

1

. Similar applications

occur in the wine industry with the limitation that

nisin cannot be used in wines that depend upon a

desirable malolactic fermentation as the bacteria re-

sponsible are usually nisin-sensitive. This latter prob-

lem has been overcome by developing nisin-resistant

strains of Leuconostoc oenos that can grow and

maintain malolactic acid fermentation in the presence

of nisin. In the production of distilled spirits, nisin can

inhibit the lactic acid bacteria that compete with the

yeast for substrate in the fermentation mash, thus

resulting in increased alcohol yield in the final distil-

late. Alcohol yield has been increased by over 10%

using this method.

Potential Future Applications

0037 Research on nisin as a food additive continues as the

demand increases for convenient long shelf-life and

safe food preserved by safe preservatives with a nat-

ural connotation. Current research includes the use of

nisin in combination with novel food preservation

systems such as ultra high pressure, electroporation,

pulsed electric fields, and nanothermosonication. It is

evident that, like the classical use of heat preservation

processes, bacterial spores are more resistant to these

novel processes than bacterial vegetative cells and

nisin in combination with these processes is synergis-

tic against bacterial cells and surviving bacterial

spores. Nisin in combination with other safe food

additives that provide synergistic effects against

Gram-positive bacteria or widen the antimicrobial

spectrum to include Gram-negative bacteria, yeasts,

and molds are also objectives of many research teams.

Incorporation of nisin into packaging and edible films

may also provide new areas of application. Finally,

emerging food spoilage bacteria such as Alicyclo-

bacillus acidoterrestris (an acid-tolerant, spore-

forming bacteria that can grow at pH 2.5 and is a

potential problem in pasteurized fruit juice) and

B. sporothermodurans (a mesophilic, spore-forming

bacteria whose spores can survive ultra heat-treated

(UHT) processing) have been shown to be nisin-sen-

sitive. New applications to control such bacteria are

currently being realized.

See also: Alcohol: Properties and Determination;

Canning: Principles; Cheeses: Chemistry and

Microbiology of Maturation; Processed Cheese;

Clostridium: Occurrence of Clostridium botulinum;

Dressings and Mayonnaise: The Products and Their

Manufacture; Chemistry of the Products; Eggs: Use in the

Food Industry; Fish: Processing; Listeria: Properties and

Occurrence; Meat: Sausages and Comminuted Products;

Milk: Processing of Liquid Milk; Preservation of Food;

Shellfish: Contamination and Spoilage of Molluscs and

Crustaceans; Spoilage: Bacterial Spoilage

Further Reading

Delves-Broughton J (1990) Nisin and its uses as a food

preservative. Food Technology 44: 100–117.

Delves-Broughton J and Gasson MJ (1994) Nisin. In: Dillon

VM, Board RG (eds) Natural Antimicrobial Systems and

Food Preservation, p. 99. Wallingford: CAB Inter-

national.

De Vuyst L and Vandamme EJ (1994) Nisin, a lantibiotic

produced by Lactococcus lactis subsp. lactis: properties,

biosynthesis, fermentation and applications. In: De

Vuyst, Vandamme EJ (eds) Bacteriocins of Lactic A Bac-

teria: Microbiology, Genetics and Applications, p. 151.

London: Blackie Academic & Professional.

Hurst A (1991) Nisin. Advances in Applied Microbiology

27: 85–123.

Hurst A and Hoover DG (1993) Nisin. In: Davidson PM,

Branen AL (eds) Antimicrobials in Foods, p. 369. New

York: Marcel Dekker.

Thomas LV, Clarkson MR and Delves-Broughton J (2000)

Nisin. In: Naidu AS (ed) Natural Food Antimicrobial

Systems. CRC Press, USA, pp. 463–524.

NISIN 4135

NITRATES AND NITRITES

M J Dennis and L A Wilson, Central Science

Laboratory, Sand Hutton, York, UK

Background

0001 Nitrate is a normal component of plant tissues,

whereas nitrite is not normally found in significant

quantities unless microbiological spoilage occurs.

Both nitrate and nitrite are permitted food additives

for use in curing meat. Their original function was to

confer microbiological safety, but they were also

found to contribute useful color and flavor character-

istics, which, with the advent of refrigeration, are

now of greater technological significance. There

have been considerable concerns over the safety of

nitrate and nitrite in foods. Nitrate is largely unreact-

ive but can be reduced to nitrite, which can then react

with secondary amines to form nitrosamines (many of

which are carcinogens). Equally, it is becoming ap-

parent that oxides of nitrogen play an important role

in human physiology. Hence, in this review, we will

cover the occurrence of nitrate and nitrite in food,

the legislation governing their use as food additives,

and legislation governing their occurrence in vege-

tables, their human dietary intake, their potential

health benefits, and concerns.

Occurrence in Foods

0002 Nitrate is found as a naturally occurring compound

in foods such as vegetables, fruit, cereals, fish, milk,

and dairy products, and is also found in water as a

consequence of agricultural practices such as the use

of nitrogen-containing fertilizers and from animal

waste. Low levels are generally found from these

sources, except in the case of some vegetables. Nitrate

and nitrite are also permitted as food additives in

some foods, primarily as protection against botulism.

Vegetables and Fruit

0003 Nitrate in the soil is taken up by plants for use as a

nitrogen source in the formation of proteins. Protein

production occurs as a result of photosynthesis, but

when light levels fall, the rate of photosynthesis de-

creases, and nitrate accumulates in cell fluids and

sap. The levels of nitrate in vegetables grown under

low light conditions are thus correspondingly higher

than those grown under bright light, as shown in

Figure 1. Overall, nitrate accumulation in plants

is determined by genotype, growing conditions,

especially light levels and soil temperature, and

nitrogen fertilization.

0004Nitrate content varies considerably according to

species, with vegetables such as spinach and lettuce

often containing up to 2500 mg kg

1

, whereas those

such as asparagus have levels as low as 13 mg kg

1

Table 1 lists the concentrations of nitrate found in

some vegetables and fruits, and compares the levels

found in different countries. The natural levels of

nitrate in vegetables generally are high when com-

pared with other food groups. It is estimated that

75–80% of the total daily intake comes from vege-

tables, compared with only about 5–10% from drink-

ing water. Cooking has been shown to decrease the

concentration of nitrate in foods, dependent on the

cooking technique used (see Table 2).

0005Nitrite levels in vegetables and fruit are low,

usually below 2 mg kg

1

, except where there has

been damage or improper storage leading to the

microbiological reduction of nitrate to nitrite.

Meat and Meat Products

0006Fresh meat normally contains low levels of nitrate

and nitrite, estimated at < 4–7mgkg

1

and < 0.4–

0.5 mg kg

1

, respectively, in the UK in 1997, although

higher concentrations of about 10–30 mg kg

1

nitrate are found in cured meats such as ham and

salami, where nitrite and nitrate have been incorpor-

ated as a permitted additive. Nitrite salts have

January

March

May

July

Month

September

November

Concentration of nitrate

(mg/kg nitrate)

500

1000

1500

2000

2500

3000

3500

4000

4500

fig0001Figure 1 Seasonal variation in nitrate concentrations of lettuce

(mg per kilogram of nitrate) (Denmark). From Petersen A and

Stoltz S (1999) Nitrate and nitrite in vegetables on the Danish

market: content and intake. Food Additives and Contaminants 16(7):

291–299, with permission.

4136 NITRATES AND NITRITES

customarily been added to a range of meats for pre-

servation purposes, especially as an antimicrobial

agent with regard to Clostridium botulinum,topro-

duce the pink color of cured meats via the formation

of a nitric oxide–myoglobin complex and to give the

traditional flavor expected from these products. Ni-

trite is preferred as a curing agent, as it reacts more

quickly than nitrate, and less is required for color

stabilization, but nitrate may be added to act as a

reservoir in case the nitrite level is depleted during

curing. Measures have been taken to reduce the

amounts of nitrite used during meat curing processes

owing to concern over the formation of nitrosamines.

These restrictions have led to significant reductions in

the nitrate and nitrite contents of cured meats. Nitrite

is now frequently added to meat products at much

lower levels, commonly 80–160 mg kg

1

, in conjunc-

tion with sodium ascorbate or sodium erythorbate.

It is considered that the benefits of adding nitrite to

products of this type far outweigh the potential risks.

(See Clostridium: Botulism.)

Cheese

0007Potassium nitrate is permitted as an additive in cheese

manufacture in order to control microbiological con-

tamination, which may produce early gassing, and

thus to improve the quality of the finished product.

Natural concentrations in cheese were found at

1–8mgkg

1

of nitrate, with concentrations rising to

4–27 mg kg

1

of nitrate and < 0.6–1mgkg

1

of nitrite

when nitrate was used as an additive.

Fish and Fish Products

0008Nitrite is permitted in some countries as an additive

to some smoked and cured fish and fish products,

when it is used as a preservative and color fixative.

Levels of both nitrate and nitrite are low in the fresh

product, but a recent Japanese survey (1998) ana-

lyzed nitrite levels in different salted and processed

tbl0002Table 2 Effects of cooking on nitrate concentrations in

vegetables (MAFF)

Vegetable Cooking

method

Meannitrate concentration

(mgper kilogram of nitrate)

Percentage

change

Beetroot Fresh 1211

Boiled 906 25

Cabbage Fresh 338

Boiled 114 66

Carrot Fresh 97

Boiled 71 27

Cauliflower Fresh 86

Boiled 46 47

Onion Fresh 48

Fried 44 8

Potato Fresh 167

Boiled 85 49

Fried 136 19

Baked 194 þ16

Spinach Fresh 1631

Boiled 468 71

Sprout Fresh 59

Boiled 24 59

Swede Fresh 118

Boiled 72 39

Tomato Fresh 17

Fried 27 þ59

Data from: Ysart G, Miller P, Barrett G, Farrington D, Lawrance P and

Harrison N (1999) Dietary exposures to nitrate in the UK. Food Additives

and Contaminants 16(12): 521–532. All data used with the author’s

permission.

tbl0001 Table 1 Comparison of average nitrate levels determined in

vegetables and fruits (mg per kilogram of nitrate in fresh product)

by different countries

Vegetable/fruitAustraliaDenmark

ItalyUKUSA

Artichoke 16

Asparagus 13 60

Aubergine 370

Bean 265 450 466

Beetroot 2124 1490 1211 3288

Broccoli 310 400 1014

Brussels sprout 44 7–12 59 164

Cabbage 1062 342 240 338 712

Carrot 66 170–210 97 274

Cauliflower 221 37 86 658

Celery 1305 3151

Cucumber 151

Endive 1780

Kale 1096

Leek 308 700

Lettuce 943 2600 1051 2330

Melon 4932

Mushroom 219

Onion 22 80 48 235

Parsley 973 1380

Pea 66 57 40

Pepper (sweet) 88 78 165

Potato (sweet) 44 65

Potato 177 432 110 155 150

Pumpkin 177 550

Radish 1735 2600

Spinach (fresh) 1783 2100 1631 2470

Spinach (frozen) 680

Tomato 44 17 80

Turnip (greens) 9040

Turnip (root) 970 535

Average for 1993, 1994 and 1995/1996.

Data from Lyons DJ, Rayment GE, Nobbs PE and McCallum LE (1994)

Nitrate and nitrite in fresh vegetables from Queensland. Journal of the

Science of Food and Agriculture 64: 279–281; Petersen A and Stoltze S (1999)

Nitrate and nitrite in vegetables on Danish market: content and intake.

Food Additives and Contaminants 16(7): 291–299; Santamaria P (1997)

Contributo degli ortaggi all’assunzione giornaliera di nitrato, nitrito e

nitrosamine. Industrie Alimentari XXXVI (Novembre): 1329–1333; Ysort G,

Miller P, Barrett G, Farrington D, Lawrance P and Harrison N (1999)

Dietary exposures to nitrate in the UK. Food Additives and Contaminants

16(12): 521–532; Walker R (1990) Nitrates, nitrites and

N-nitrosocompounds. A review of the occurrence in food and diet and the

toxicological implications. Food Additives and Contaminants 7(6): 717–768.

All data have been used with the author’s permission.

NITRATES AND NITRITES 4137

fish products and found that, although the mean con-

centrations were about 30–40% of the permitted

limit, some products contained nitrite at levels of up

to 32 mg kg

1

Beverages

0009 Beverages make a significant contribution to nitrite

intake due to high consumption. Although the con-

centrations found are low (< 0.4–0.8 mg l

1

), the

mean nitrite intake in the UK from this source is

0.34 mg per person per day.

Water

0010 Nitrate is ubiquitous in potable water. Its presence is

due to a number of factors: significant sources are

agricultural activity, for example, the use of nitrogen-

ous fertilizers; sewage effluents and urban run-off and

the geology of the area. It is formed when nitrogenous

organic sources, such as urea and proteins, or fertil-

izers containing inorganic nitrogen are decomposed

by microorganisms in water or soil to form ammonia,

which is then oxidized to nitrate and nitrite. Up to

5mgl

1

of nitrate have been found in rainwater in

European urban areas, although levels are somewhat

lower in rural areas. However, levels rise as a result of

human activities, and groundwater with concentra-

tions of up to 1500 mg l

1

have been found in an

agricultural area in India. Nitrate is very soluble in

water and easily moves through the soil into aquifers

and into the drinking water supply. Nitrite is readily

oxidized to nitrate and is consequently found in water

at much lower levels, especially after water treatment

involving chlorination. Water obtained from public

water companies tends to contain lower levels of

nitrate, usually less than 10 mg l

1

in most countries,

but private wells throughout the world, especially in

agricultural areas, may have concentrations often

exceeding 50 mg l

1

. However, the exposure to water

from the latter source is less common; for example,

it is estimated that only about 2% of the European

population draw their water from private wells.

Legislation

0011 Maximum permitted levels of nitrate in potable water

have been set at levels intended to prevent the occur-

rence of methaemoglobinemia in infants and other

susceptible sections of the population. The second

edition of the WHO Guidelines for drinking-water

quality recommended a guideline value of 50 mg l

1

for nitrate and 3 mg l

1

for nitrite levels for water.

The US Federal Government has set a similar

maximum contaminant level of 45 mg l

1

0012 Concern over the in vivo formation of nitrosamines

from nitrites has led to a reduction in the levels

permitted when they are used as food additives. In

the UK, the current residual concentration of nitrite

(as sodium nitrite) in cured bacon is limited to

175 mg kg

1

and 100 mg kg

1

for other cured meats.

The maximum residual concentration of nitrate (as

sodium nitrate) in all cured meats is 250 mg kg

1

. The

use of potassium nitrate in cheese is limited to that

giving a residual concentration of 50 mg kg

1

. Regu-

lations in other countries are similar: Australia and

New Zealand set a maximum of 125 mg kg

1

sodium nitrite for cured meats and 50 mg kg

1

total nitrate/nitrite in cheeses such as Gouda, Edam,

and Havarti.

0013In Europe, Commission Regulation No. 194/97 sets

the maximum levels for certain contaminants in food-

stuffs, including nitrate in lettuce and spinach. By the

use of specific measures intended to provide better

control of the sources of nitrate and good agricultural

practices, the levels in spinach should not exceed

2500 mg kg

1

(summer crop) and 3000 mg kg

1

(winter crop) and in lettuce, levels should not exceed

3500 mg kg

1

(summer crop except open-grown

lettuce where the limit is 2500 mg kg

1

from May

to September) and 4500 mg kg

1

(winter crop).

Dietary Intake of Nitrate and Nitrite

0014The acceptable daily intake (ADI) is defined as the

maximum amount of a chemical that can be ingested

daily over a lifetime with no appreciable health risk,

and is based on the highest intake that does not give

rise to observable adverse effects. The European

Commission’s Scientific Committee for Food set the

ADI for nitrate at 0–3.65 mg per kilogram of body-

weight (equivalent to an intake of 219 mg day

1

for a

60-kg person) and 0–0.06 mg per kilogram of body-

weight (equivalent to an intake of 3.6 mg day

1

for a

60-kg person) for nitrite. These ADIs are similar to

those set by the joint FAO/WHO experts committee.

The ADIs apply to all sources of intake, although the

use of nitrite as an additive in baby food intended for

infants below the age of 3 months is not permitted.

The nitrite ADI does not apply to infants under

3 months.

0015Dietary exposure to food components may be

assessed by the use of several approaches, including

the use of market basket studies, food diary records,

per capita estimations using consumption data with

the concentrations of nitrate and nitrite in foods, and

urinary biomarkers. Intake studies have shown that

the ADIs are not exceeded by the general population

across the world, as shown in Table 3. The question

of whether some subgroups of the population are

consuming high levels of nitrate was addressed by

the UK’s Food Standards Agency, who carried out a

study into the nitrate intake of vegetarians. This

4138 NITRATES AND NITRITES

showed that, although there was a slightly higher

intake, dietary exposure was similar to other con-

sumers and was well below the ADI.

0016 In summary, food is the main source of nitrate,

especially potatoes, green vegetables, and other vege-

tables. Water also makes a significant contribution.

Estimates of nitrite intake suggest that 1–3 mg day

1

is typical and occurs mainly from beverages, due to

high consumption.

Microbiological Nitrate Reduction

0017 A detailed investigation of the reduction of nitrate

to nitrite by microbiological contaminants of the

brewing process has been undertaken. Concerns

over the presence of nitrosamines in beer have led to

detailed studies of the mechanism of formation of

these compounds in order to prevent their formation

as far as technologically feasible. Nitrosodimethyl-

amine was formed in malt from nitrogen oxides pre-

sent in kiln gases. Concentrations of this contaminant

were greatly reduced by modifications to kilning

conditions. Apparent total N-nitroso compounds

(ATNC) provide an estimate of the total N-nitroso

compounds present in a sample. The brewing indus-

try sought to minimize ATNC and set a target of

20 mg (N-NO) kg

1

. It was established that the

major source of nitrosating agent was from microbial

reduction of nitrate. Hence, care in ensuring that

microbiological contamination was minimized played

an important role in meeting this target.

0018 Microorganisms can be differentiated into assimi-

latory and dissimilatory nitrate reducers. The former

reduce nitrate to nitrite and ammonia for use as their

source of cell nitrogen. The latter use nitrate as a

terminal electron acceptor for energy production

(oxygen is the usual terminal electron acceptor under

aerobic growth conditions). Nitrite, nitric oxide, ni-

trous oxide, and sometimes nitrogen may all be pro-

duced by dissimilatory nitrate reduction. A number of

different bacteria and yeasts are capable of nitrate

reduction. The effect is not merely to produce nitrite

that subsequently undergoes the well-known acid-

catalyzed chemical nitrosation reactions. Rather, the

nitrosation reactions appear to be catalyzed by the

nitrate and nitrite reductase enzymes. This mechanism

enables nitrosation to occur at pH 6–9 which cannot

happen in the chemical reaction at alkaline pH.

0019However, the significance of microbiological re-

duction of nitrate is wider than that caused by the

microbiological contamination of food. Nitrate-

reducing bacteria can be found in the mouth and the

colon. Nitrate from the diet (or that produced meta-

bolically) is transferred from the blood to the saliva in

the salivary glands. It has been suggested that 25% of

orally ingested nitrate may be secreted in saliva.

Twenty per cent of this nitrate can then be reduced

by oral bacteria to nitrite, which is swallowed. Hence,

it is apparent that saliva makes the major contribu-

tion to dietary nitrite intake. Saliva nitrite levels rise

at night and are higher in older people, especially

men. Salivary nitrate and nitrite vary little from day

to day, but vary more over a 5-year period.

0020Bacterial nitrosation may occur at any site in the

body where there is infection, and these may be linked

to cancers at these sites. However, the colon provides

a site rich in microorganisms at all times. It is appar-

ent from measurements of fecal ATNC that bacterial

nitrosation reactions occur at this site. ATNC forma-

tion is influenced by the nature of the organisms

present, the transit time of the fecal mass through

the colon and to consumption of nitrate and (surpris-

ingly) red meat. Since dietary nitrate is absorbed by

the small intestine, it is difficult to appreciate how the

nitrosation reaction may occur. It is possible that

there may be diffusion of nitrate from the colonic

epithelium. Alternatively, red meat might influence

fecal ATNC by carrying nitric oxide into the colon

as nitrosyl myoglobin, by favoring the growth of

nitrate-reducing bacteria in the colon, or possibly by

nitrosyl myoglobin acting as a nitrosating agent itself.

These possibilities require further study.

Concerns over Nitrate and Health

0021A considerable body of evidence has been collected

concerning the direct effects on health of the nitrate

and nitrite ion. There is no evidence of carcino-

genicity from nitrate consumption in experimental

animals or through human epidemiological studies.

Long-term animal studies on nitrite do not indicate

carcinogenicity unless combined with amine intake

tbl0003 Table 3 Comparison of daily dietary exposures to nitrate in

different countries

Country Dietary exposure (mg day

1

)

UK 52

Finland 77

The Netherlands 52

The Netherlands 78–91

Basque Country, Spain 60

Belgium 154 (vegetables)

Egypt 296 (nitrate and nitrite)

USA 300

Poland 85 (duplicate hospital diets)

Poland 65 (estimate from hospital diets)

India 78

EU 18–131 (vegetables)

From Ysart G, Miller P, Barrett G et al. (1999) Dietary exposures to nitrate

in the UK. Food Additives and Contaminants 16(12): 521–532.

NITRATES AND NITRITES 4139

leading to the formation of carcinogenic nitros-

amines. The latter studies usually involved nitrite

concentrations far higher than those commonly en-

countered in the diet. Nitrite has demonstrated muta-

genic activity in in vitro assays. There is considerable

evidence of the safety of substantially higher expos-

ures to nitrite during clinical use than those normally

encountered from the diet.

0022 The most important direct toxic effect of nitrite (or

nitrate after its reduction) is the condition known as

methemoglobinemia. Absorbed nitrite ion is rapidly

oxidized to nitrate by reaction with oxyhemoglobin

to produce methemoglobin. The latter can not carry

oxygen around the body, and although the reaction

can be reversed enzymatically, if large quantities of

methemoglobin are produced, fatality can result.

Infants are deficient in this repair enzyme and hence

are especially at risk if they receive unusually high

intakes of nitrate, for example from contaminated

well water. This may be exacerbated by concurrent

infections resulting in endogenous nitrate synthesis.

0023 Another concern is that consumption of nitrite (or

nitrate reduced by oral bacteria to nitrite) may result

in the nitrosation of amines to produce carcinogenic

nitrosamines in the acid conditions of the stomach.

Such an effect has been found in a human study after

consuming a meal consisting of vegetables high in

nitrate and fish (to provide a source of amines). Simi-

lar studies in animals have been shown to induce

cancer. Such nitrosation reactions may be enhanced

by bacterial nitrosation in the achlorhydric stomach.

0024 Volatile nitrosamines are not normally found in

urine (unless a urinary infection is present) or in

feces. However, feces do contain ATNC, although

the toxicological significance of this measurement is

not understood. The formation of ATNC appears to

require an active microbiological flora in the colon

capable of nitrate reduction. Hence, ATNC would

appear to be, at least, a biomarker of nitrosation

reactions occurring in the colon. It is possible for the

colonic bacteria to make a more subtle contribution

to colon cancer than just the production of potential

carcinogens. For example, nitrate-reducing enteric

fermentative bacteria lead to the production of more

acetate and less butyrate from substrate. Butyrate is

considered to have a protective effect against cancer,

since it promotes apoptosis (programmed cell death),

so any selection for bacteria producing acetate may

favor colonic epithelial cell proliferation.

0025 There are a number of medical conditions other

than cancer that have been associated with nitrate/

nitrite intake. In Finland, a significant correlation has

been found between the occurrence of type 1 diabetes

and the nitrite intake of both the children and their

mothers. No association was found for nitrate intake.

Since most nitrite intake arises from saliva, it seems

likely that the association with nitrite is in fact a proxy

for preformed dietary nitroso compounds, some of

which (e.g., N-nitrosomethylurea) have well estab-

lished toxic effects on pancreatic beta cells. Studies

in Iceland and Sweden have also found similar

correlations.

0026An association has been observed between mater-

nal periconceptional exposure to nitrate and an

increased risk of anencephalopathy (but not spina

bifida). The lack of a quantitative association be-

tween nitrate intake and risk may indicate that some-

thing other than nitrate is the causative factor. It

should be noted that not all studies have been able

to demonstrate such a link, but nitrosocompounds

have been shown to cause defects in the central

nervous system of experimental animals.

0027Nitrate acts as a competitive inhibitor of iodine

uptake by the thyroid and thyroid hypertrophy has

been demonstrated experimentally in rats given

drinking water containing large amounts of nitrate.

There is some epidemiological evidence that popu-

lations exposed to high nitrate concentrations in

drinking water show an average increase in thyroid

volume, although it cannot be excluded that the

effects are due to other compounds.

Potential Health Benefits of Nitrate

0028Nitric oxide plays a crucial role in human metabol-

ism. It is synthesized from arginine by two related

enzyme systems – one constitutive and one inducible.

The constitutive form provides continuous vasodila-

tion and inhibits platelet adhesion and aggregation;

hence, it can help to prevent hypertension. The indu-

cible isozyme is generated in response to bacterial

lipopolysaccharide and hence is considered to play

an important role in prevention of bacterial disease.

However, NO production is also greatly increased in

inflammatory bowel disease (e.g., Crohn’s disease).

NO synthesis also occurs in the central nervous

system, but its physiological role in this tissue remains

unexplained. These mechanisms for nitric oxide

generation mean that man excretes more nitrate

than is ingested.

0029Dietary nitrate is readily absorbed, transported in

plasma, and concentrated 10-fold in the saliva by the

salivary gland. Although nitrate is excreted in the

urine, the kidney recovers some 80% of the nitrate.

Hence, this renal salvage and salivary concentration

suggest that nitrate is physiologically important.

Nitric oxide has widespread antimicrobial activity,

and hence, it is hypothesized that the salivary concen-

tration of nitrate and its microbial conversion to

nitrite are a symbiotic activity to generate quantities

4140 NITRATES AND NITRITES

of nitric oxide in the stomach for protection against

pathogens. Inhibition of gastric NO synthesis does

not have clinical relevance, but this could be due to

the high microbiological standards of contemporary

diets. Nitrate is also expressed in sweat where signifi-

cant amounts of nitrite and bacterially generated

nitric oxide can also be found and where a protective

effect against pathogens has also been postulated.

0030 Gastric juices provide a rich source of nitrosatable

substrates, which, some consider, far exceeds those

provided by food. Hence, since nitrate can be endo-

genously formed, carried in the blood, and excreted in

saliva, it would appear unavoidable that some gastric

nitrosation takes place. However, much more gastric

nitric oxide is produced than might be expected from

acid-catalyzed dissociation of nitrite to nitric oxide

and nitrogen dioxide. Thus, it would appear that

another reductant (e.g., ascorbic acid) is produced in

gastric juice in order to facilitate the generation of

NO. Therefore, it may be that man is metabolically

adapted to the possibility of gastric nitrosation. This

view is supported by the failure of a number of epi-

demiological studies to demonstrate a link between

nitrate intake and cancer in man.

See also: Curing; Escherichia coli: Occurrence and

Epidemiology of Species other than Escherichia coli;

Legislation: Contaminants and Adulterants; Meat:

Preservation; Nitrosamines; Smoked Foods: Principles;

Applications of Smoking; Water Supplies: Chemical

Analysis

Further Reading

Benjamin N (2000) Nitrates in the human diet – good or

bad? Annales De Zootechnie 49: 207–216.

Commission of the European Communities Commission

Regulation (EC) No. 466/2001 (2001) Setting maximum

levels for certain contaminants in foodstuffs. Official

Journal of the European Communities No. L77: 1–13.

Croen LA, Todoroff K and Shaw GM (2001) Maternal

exposure to nitrate from drinking water and diet

and risk neural tube defects. American Journal of

Epidemiology 153(4): 325–331.

Hill MJ (1996) Factors controlling endogenous N-nitrosa-

tion. European Journal of Cancer Prevention 5(supple-

ment 1): 71–74.

Hughes R, Cross AJ, Pollock JRA and Bingham S (2001)

Dose-dependent effect of dietary meat on endogenous

colonic N-nitrosation. Carcinogenesis 22(1): 199–202.

Hunt J and Turner MK (1994) A survey of nitrite concen-

trations in retail fresh vegetables. Food Additives and

Contaminants 11(3): 327–332.

Ishiwata H, Nishijima M, Fukasawa Y, Ito Y and Yamada T

(1998) Evaluation of the inorganic food additive (nitrite,

nitrate and sulfur dioxide) content of foods and estima-

tion of daily intake based on the results of Official

Inspection in Japan in the fiscal year 1994. Journal of

the Food Hygiene Society of Japan 39(2): 78–88.

Lyons DJ, Rayment GE, Nobbs PE and McCallum LE

(1994) Nitrate and nitrite in fresh vegetables from

Queensland. Journal of the Science of Food and Agricul-

ture 64: 279–281.

Ministry of Agriculture, Fisheries and Food (1998a) MAFF

UK – Survey of nitrite and nitrate in bacon and cured

meat products. Food Surveillance Information Sheet

No. 142.

Ministry of Agriculture, Fisheries and Food (1998b) MAFF

UK – 1997 Total Diet Study – Nitrate and nitrite. Food

Surveillance Information Sheet No. 163.

Ministry of Agriculture, Fisheries and Food (1998c) MAFF

UK – Duplicate diet study of vegetarians – nitrate analy-

sis. Food Surveillance Information Sheet No. 165.

Mirvish SS, Reimers KJ, Kutler B et al. (2000) Nitrate and

nitrite concentrations in human saliva for men and

women at different ages and times of the day and their

consistency over time. European Journal of Cancer

Prevention 9: 335–342.

Parham NJ and Gibson GR (2000) Microbes involved in

dissimilatory nitrate reduction in the human large intes-

tine. FEMS Microbiology Ecology 31: 21–28.

Perner A and Rask-Madsen J (1999) Review article: the

potential role of nitric oxide in chronic inflammatory

bowel disorders. Alimentary Pharmacology and Thera-

peutics 13: 135–144.

Petersen A and Stoltze S (1999) Nitrate and nitrite in vege-

tables on the Danish market: content and intake. Food

Additives and Contaminants 16(7): 291–299.

Rowland IR, Granli T, Bockman OC, Key PE and Massey

RC (1991) Endogenous N-nitrosation in man assessed

by measurement of apparent total N-nitroso compounds

in faeces. Carcinogenesis 12(8): 1395–1401.

Santamaria P (1997) Contributo degli ortaggi all’assun-

zione giornaliera di nitrato, nitrito e nitrosamine. Indus-

trie Alimentari XXXVI (Novembre): 1329–1334.

Smith NA (1994) Cambridge Prize Lecture: Nitrate reduc-

tion and N-nitrosation in brewing. Journal of the Insti-

tute of Brewing 100: 347–355.

van Maanen JMS, van Dijk A, Mulder K et al. (1994)

Consumption of drinking water with high nitrate levels

causes hypertrophy of the thyroid. Toxicology Letters

72: 365–374.

Virtanen SM, Jaakkola L, Rasanen L et al. (1994) Nitrate

and nitrite intake and the risk for type 1 diabetes in

Finnish children. Diabetic Medicine 11: 656–662.

Walker R (1990) Nitrates, nitrites and N-nitrosocom-

pounds: a review of the occurrence in food and diet

and the toxicological implications. Food Additives and

Contaminants 7(6): 717–768.

World Health Organization (1998) Guidelines for drinking

water quality, 2nd edn. Addendum to vol. 2 Health

criteria and other supporting information, pp. 64–80.

Ysart G, Miller P, Barrett G, Farrington D, Lawrance P

and Harrison N (1999) Dietary exposures to nitrate in

the UK. Food Additives and Contaminants 16(12):

521–532.

NITRATES AND NITRITES 4141

NITROSAMINES

R A Scanlan,OregonStateUniversity,

Corvallis,OR,USA

Introduction

000 1 Investigations during the past 30 years on the occur-

rence of N-nitrosamines and the etiology of these

compounds in human cancer began with two import-

ant discoveries. In 1956, the UK scientists David

Barnes and Peter Magee reported N-nitrosodimethyl-

amine (NDMA) as a carcinogen in experimental

animals. In Norway in the early 1960s, NDMA

proved to be the causative agent in the death of

agricultural animals which had been fed nitrite-pre-

served fish meal. The nitrite had reacted with amines

in the fish meal to produce lethal amounts of NDMA.

Since nitrite is added to human foods, particularly

cured meats, and amines occur commonly in most

foods, this raised the question as to whether carcino-

genic nitrosamines might also form in human foods.

This article summarizes the results of the many inves-

tigations which have addressed this important issue.

Chemistry of

-Nitrosamine Formation

Basic Reactions

000 2 N-Nitrosamines are formed by a chemical reaction

between a nitrosating agent and a secondary or a

tertiary amine. Primary amines react with nitrosating

agents to form unstable N-nitroso derivatives which

degrade to olefins and alcohols. The structures of

several nitrosamines which occur in foods are

shown in Figure 1.

0003Oxides of nitrogen (NO

) in which the nitrogen is

in a þ 3or þ 4 oxidation state can serve as nitrosating

agents. A much studied nitrosating agent which par-

ticipates in nitrosamine formation in foods is nitrous

anhydride (N

). Nitrous anhydride forms readily

from nitrite in aqueous acidic solution, as shown in

eqn (1).

2NO



þ 2H

Ð N

þ H

O ð1Þ

0004Nitrous anhydride combines with the unshared

pair of electrons on unpronated secondary amines

through a nucleophilic substitution reaction to form

N-nitrosamines, as depicted in eqn (2).

NH þ N

! R

N  N ¼ O þ HNO

ð2Þ

0005The rate of nitrosation is pH-dependent and is

governed by the concentrations of amine and nitrite,

as shown in eqn (3):

Rate ¼ k½amine ½nitrite 

ð3Þ

0006Since the nitrous anhydride reacts with unproto-

nated amine, the rate of nitrosation for secondary

amines is inversely proportional to amine basicity.

The pH optimum for nitrosation of most second-

ary amines is between 2.5 and 3.5. This is due to

the counteracting effects of acidity on nitrous anhyd-

ride concentration, which increases at low pH, and

the concentration of unprotonated amine, which

increases at high pH.

0007The pH optimum has a number of implications.

Although most foods are less acidic than pH 2.5–

3.5, many foods are sufficiently acidic to allow

nitrosation, albeit at rates slower than maximal. Fur-

thermore, the 2.5–3.5 pH range is sufficiently

close to the acidity of the human stomach to allow

nitrosamine formation, providing that amines

and nitrosating agents are present.

0008Tertiary amines react with nitrosating agents in

acidic aqueous solution through a mechanism called

nitrosative dealkylation to form nitrosamines. Nitro-

sative dealkylation involves conversion of a tertiary

amine to a secondary amine, which subsequently

reacts with nitrosating agent to form a nitrosamine.

Nitrosation Inhibitors

0009Nitrosation can be influenced by a wide range of

catalysts and inhibitors. Ascorbic acid, a-tocopheral,

and sulfur dioxide are used to inhibit nitrosamine

formation in foods. Ascorbic acid reduces nitrous

anhydride to nitric oxide, as shown in eqn (4).

NDEA

NDMA

NPYR

NPRO

HOOC

fig0001 Figure 1 Structure of some nitrosamines which occur in foods.

NDMA, N-nitrosodimethylamine; NPYR, N-nitrosopyrrolidine;

NDEA, N-nitrosodiethylamine; NPRO, N-nitrosoproline.

4142 NITROSAMINES

Ascorbic acid þ N

!Dehydroascorbic acid

þ 2NO þ H

O ð4Þ

Ascorbic acid is not completely effective in blocking

nitrosation, since under oxidative conditions the

nitric oxide can be reconverted to nitrous anhydride.

Presumably a-tocopheral reacts with nitrous anhyd-

ride through a similar redox process to inhibit nitro-

sation. Both ascorbic acid and a-tocopheral are added

to cured meats, and sulfur dioxide is used in process-

ing barley malt to inhibit nitrosation.

Carcinogenicity

0010 Although N-nitroso compounds have been shown to

be acutely toxic, mutagenic, and teratogenic, their

carcinogenic properties are of grave concern and

have been studied extensively. Over 300 N-nitroso

compounds, including many nitrosamines, have

been demonstrated to be carcinogens in experimental

animals. More than 50 animal species, including

higher primates, are susceptible to N-nitroso com-

pound-induced carcinogenesis. It has been observed

that tumors induced by nitrosamines in experimental

animals show similar morphological properties to

tumors in corresponding human organs. Few scien-

tists doubt that nitrosamines are capable of inducing

cancer in humans. However, the amount of a given

nitrosamine which is required to induce cancer in a

human population is unknown. (See Cancer: Car-

cinogens in the Food Chain.)

Analytical Methodology

0011 For purposes of analysis, nitrosamines are classified

as either volatile or nonvolatile. Volatile nitrosamines

are relatively nonpolar, low-molecular-weight com-

pounds, such as NDMA and N-nitrosopyrrolidine

(NPYR). They possess sufficient vapor pressure to

allow removal from a food matrix by distillation

and subsequent separation by gas chromatography.

Chemiluminescent detectors designed to be relatively

specific for nitrosamines allow quantitative analysis

of volatile nitrosamines in foods and other biological

materials at submicrogram per kilogram levels. Con-

firmation of nitrosamine identity is accomplished

by mass spectrometry. (See Chromatography: Gas

Chromatography; Mass Spectrometry: Principles

and Instrumentation.)

0012 Nonvolatile nitrosamines such as N-nitrosoproline

(NPRO) tend to be of higher molecular weight, or

more polar than volatile nitrosamines, and hence

possess relatively low vapor pressures. These

properties have impeded development of analytical

methodology. Although procedures are available for

determination of some nonvolatile nitrosamines,

mainly N-nitrosated amino acids and amino acid

derivatives, analytical methodology generally is less

well developed for this class of compounds.

0013A procedure usually referred to as apparent total

N-nitroso compound determination has been

developed and used for analyses of foods and body

fluids. Presumably when using this procedure all

N-nitroso compounds in the sample are measured

as a single entity. A limitation is that information

regarding the identity of individual N-nitroso

compounds is not provided by this methology. It is

therefore impossible to access the carcinogenicity of

compounds detected by this procedure.

Formation and Occurrence

Exogenous Formation

0014Foods During the past 25 years many foods have

been analyzed for volatile nitrosamines. In general,

foods in most western diets have been examined more

extensively than foods from Asia, Africa, and South

America. Although approximately 20 volatile nitros-

amines have been identified in a variety of foods and

beverages, NDMA and NPYR have been found most

commonly. Most of the other volatile nitrosamines

which occur in foods do so infrequently. Table 1 pre-

sents a condensation of many reports of the occur-

rence of volatile nitrosamines in foods from around

the world. The summary is not comprehensive;

rather, it is intended to show the foods in which

nitrosamines occur most commonly.

0015Nitrosamines form in foods because under certain

circumstances the precursors, amines and nitrosating

agents, occur in foods. Amines occur commonly

in food, and are formed biosynthetically and by

tbl0001Table 1 Volatile nitrosamines which occur most commonly in

foods

Food Nitrosamine Range

(mgkg

1

)

Occurrence

Fried bacon NDMA, NPYR 1–100 Consistent

Cured meats NDMA, NPYR, NPIP ND–50 Sporadic

Beer NDMA ND–5 Sporadic

Cheese NDMA ND–5 Sporadic

Cooked fish NDMA, NDEA ND–50 Sporadic

Salt-dried fish NDMA, NDEA ND–1000

Nonfat dry milk NDMA ND–1 Consistent in

direct-fire

dried product

The range is intended to encompass data in most reports since 1980. In

general, most positive samples contained nitrosamines at the lower end of

the range. ND, not detectable.

NDMA, N-nitrosodimethylamine; NPYR, N-nitrosopyrrolidine; NDEA,

N-nitrosodiethylamine; NPIP, N-nitrosopiperidine.

NITROSAMINES 4143