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

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sorbic acid to hexadienol by certain strains of lactic

acid bacteria. This product can react with ethanol to

form 1-ethoxy-2,4-hexadiene and 2-ethoxy-3,5-hexa-

diene, which give rise to a geranium-type odor, occa-

sionally detectedin wines treated with thepreservative.

Sulfur Dioxide and Sulfites

0012 Molecular sulfur dioxide passes into yeast cells by

passive transport and possibly into some other micro-

bial cells by active transport. As with carboxylic

acids, it interferes with membrane transport pro-

cesses. However, sulfites are much more chemically

reactive than any of the carboxylic acids referred to

above. Thus, the sulfite ion acts as a powerful nucleo-

phile causing the cleavage of disulfide bonds of

proteins; it reacts with coenzymes (NAD

), cofactors,

and prosthetic groups (flavin, thiamin, heme, folic

acid, and pyridoxal). Consequently, a broad range

of metabolic enzymes are inactivated, and structural

proteins that contain disulfide bonds may be de-

natured. Prior to the death of yeast cells treated with

sulfite, there is a rapid decrease in ATP content. This

has been attributed to the inactivation of glyceralde-

hyde-3-phosphate dehydrogenase by sulfite species.

The additive also reacts with carbonyl constituents

of the metabolic pool, to form hydroxysulfonates.

When treated with sublethal concentrations of sulfite,

yeasts tend to excrete increased amounts of acetalde-

hyde. This is due to the trapping of this metabolic

intermediate as the particularly stable hydroxysulfo-

nate, thereby preventing its conversion to ethanol.

Glycerol is formed instead of ethanol by reduction of

glyceraldehyde-3-phosphate to glycerol-3-phosphate,

which is subsequently dephosphorylated.

Nitrite Ion

0013 Nitrite ion is believed to exert its action on the phos-

phoroclastic system of enzymes, which causes the

conversion of pyruvate to acetate and is a source

of ATP. Evidence for this involvement comes from

the fact that in the presence of nitrite, the ATP

concentration in the cell rapidly decreases, and pyru-

vate is excreted. Nitrite ion is in equilibrium with

nonionic forms, including NO. This is a good ligand

for iron, and the mechanism of inhibition is likely to

be the reaction of NO with the nonheme iron-con-

taining pyruvate:ferredoxin. In aerobic bacteria, the

heme iron of cytochrome oxidase is also a likely

target for NO. Inhibition of enzymes with sulfhydryl

groups as a result of the formation of S-nitroso

products is also possible at relatively high nitrite con-

centrations.

0014 When nitrite-containing bacteriological media

are heated, they become more inhibitory towards

Clostridium botulinum than media that have been

heated prior to the addition of the preservative. This

so-called Perigo effect can be modeled using mixtures

of cysteine, nitrite, and iron(II) salts. When heated,

such mixtures give rise to iron–sulfur-bridged com-

plexes, of which Roussin’s black salt is a well-known

example. These compounds have been shown to be

effective inhibitors of clostridial spores.

Nisin

0015Nisin is a polypeptide (molecular weight 3500) that

usually exists as a dimer. It is produced by Lacto-

coccus lactis and may be formed naturally in

cheese. The polypeptide chain contains l-amino

acids and the unusual sulfur-amino acids lanthionine

and b-methyl-lanthionine. A specific enzyme, nisi-

nase, has been widely reported, and its formation is

possibly one reason why many lactic acid bacteria

inactivate nisin. The peptide is also susceptible to

a-chymotrypsin but not to pepsin, trypsin, and car-

boxypeptidase A, among other proteolytic enzymes.

Chemical Reactivity of Preservatives

towards Food Components

0016Chemical reactions between food preservatives and

components of microbial cells, or with food com-

ponents where there are implications with regard to

antimicrobial action, have been described above.

However, some food preservatives, and particularly

sorbic acid, sulfur dioxide, sulfites, and nitrite ions

are capable of more extensive reactivity with food

components. This may lead to the formation of

reaction products of toxicological importance and

a reduction in the concentration of available

preservative.

Sulfur Dioxide and Sulfites

0017It is well known that the concentration of sulfite

species in a food decreases with time, and the shelf-

life may be limited by this reactivity. The conversion

of sulfite to sulfate through autoxidation is catalyzed

by transition metal ions and involves the formation of

oxidizing free radicals. There is evidence to suggest

that the facile autoxidation of ascorbic acid, which is

less easily controlled by antioxidants, may promote

the oxidation of sulfite in foods containing the vita-

min. The free-radical intermediates (



OH,





)in

sulfite autoxidation are known for their ability

to cause the oxidation of unsaturated organic

compounds in the absence of suitable antioxidants.

Free-radical sulfonation of unsaturated organic com-

pounds by





is also possible. An important

nucleophilic reaction of the sulfite ion is its addition

PRESERVATIVES/Classifications and Properties 4775

to the a,b-unsaturated carbonyl moiety of 3,4-

dideoxyosulos-3-enes formed as reactive intermedi-

ates in Maillard and ascorbic acid browning. This

reaction can cause a considerable depletion of the

additive in foods susceptible to these forms of none-

nzymatic browning.

Nitrite

0018 The reaction of secondary amines with nitrosating

species derived from nitrite to give N-nitroso com-

pounds is very well known and has been given much

publicity on account of the possible carcinogenicity of

the products. The reaction proceeds at a maximum

rate in the pH range 2.25–3.4 and is catalyzed by

weakly acid anions, notably thiocyanate. In meat

products, the reaction is most probably associated

with the adipose tissue, and it is suggested that non-

ionic amine and the nitrosating species N

parti-

tion into the nonaqueous phase where a facile

reaction takes place. A similar explanation has

been suggested for the catalytic effect of surfactants

and cell membranes on nitrosation, but in this in-

stance, the reaction takes place within the micellar

environment. C-Nitrosation of phenolic components

of food, particularly in smoked products, leads to

the formation of nitrosophenols, which can readily

oxidize to the corresponding nitro compounds. The

C-nitrosation of activated methylene groups is also

established; suitable reactants in food include the 3-

deoxyosuloses formed as intermediates in Maillard

and ascorbic acid browning reactions, when the prod-

uct is an oxime. S-Nitroso compounds are readily

formed by the nitrosation of thiols and represent a

reversibly bound form of the preservative.

Chemical Interactions between

Preservatives

0019 Sulfite species, nitrite, and sorbic acid are chemically

incompatible. Sulfite ion reacts readily with nitrite

ion to form the imidodisulfonate ion. The mechanism

of the reaction between sorbic acid and sulfite species

depends on whether or not oxygen is present. Under

aerobic conditions, a sulfite-mediated oxidation of

sorbic acid takes place. Under anaerobic conditions,

a much slower nucleophilic addition of sulfite ion

gives 5-sulfo-3-hexenoic acid. Mixtures of sorbic

acid and nitrite ion give rise to ethylnitrolic acid and

1,4-dinitro-2-methylpyrrole, both of which give a

positive result in the Ames mutagenicity test.

See also: Acids: Properties and Determination; Natural

Acids and Acidulants; Nisin; Nitrates and Nitrites;

Preservatives: Analysis

Further Reading

Branen LA, Davidson PM, Salminen S and Thorngatwe III

JH (eds) (2002) Food Additives. New York: Marcel

Dekker.

Davidson PM and Branen LA (eds) (1993) Antimicrobials

in Foods. New York: Marcel Dekker.

Gould GW (ed.) (1989) Mechanisms of Action of Food

Preservation Procedures. London: Elsevier Applied

Science.

Lueck E (1980) Antimicrobial Food Additives. Characteris-

tics, Uses, Effects. Berlin: Springer-Verlag.

Wedzicha BL (1984) Chemistry of Sulphur Dioxide in

Foods. London: Elsevier Applied Science.

Wedzicha BL (1988) Distribution of low-molecular weight

food additives in dispersed systems. In: Dickinson E and

Stainsby G (eds) Advances in Food Emulsions and

Foams, pp. 329–371. London: Elsevier Applied Science.

Food Uses

B L Wedzicha, University of Leeds, Leeds, UK

Factors Affecting Choice

General Considerations

0001The type of preservative applicable to a particular

need is determined by the composition of the food,

the type of microbial spoilage that takes place, and

the desired shelf-life. Important compositional vari-

ables include pH, since the efficacy of a given preser-

vative generally decreases with increase in pH. The

maximum pH at which a preservative is useful is

often quoted. Some components of food can also

potentiate the action of preservatives, e.g., chelating

substances such as ethylenediaminetetraacetic acid or

critic acid can render preservatives more effective and

at the same time protect the food from other forms

of spoilage. Salt is a common food component that

acts synergistically with several food preservatives.

Ideally, the use of preservatives should be regarded

within a ‘preservative systems‘ approach to include

interactions, rather than in terms of individual sub-

stances. This concept is extended to include other

methods of food preservation, e.g., the use of thermal

processes in conjunction with chemical preservatives,

to optimize the use of each. Such effects of food

components and processing on antimicrobial activity

also make theoretical predictions of the efficacy of a

given preservative unreliable, and the levels of appli-

cation are usually decided upon empirically.

4776 PRESERVATIVES/Food Uses

0002 Preservatives are also chosen for their specific phys-

ical properties such as solubility in particular foods

and ease of handling. Calcium sorbate, for instance, is

sparingly soluble in water (1.2 g per 100 g water) and

is suitable for the surface treatment of foods such as

cheeses as it does not quickly dissolve in surface mois-

ture or migrate into the cheese. Chemical stability is

important with regard to the handling of sulfites; the

disulfites (metabisulfites) tend to be the most stable

sulfite species towards autoxidation and dissolve in

water to give solutions with pH values in the middle

of the pH range of foods.

0003 Cost may be an important consideration; sulfites are

the cheapest to use, whilst sorbic acid, sorbates and the

p-hydroxybenzoate esters are the most expensive.

Spectrum of Antimicrobial Action

0004 In general, benzoic acid, its salts and esters of

p-hydroxybenzoic acid have the broadest spectrum

of antimicrobial activity and are useful against many

spoilage bacteria, fungi, and yeasts. Benzoic acid

cannot, however, be relied upon to preserve foods

against bacteria effectively because, whilst food

poisoning and spore-forming bacteria are inhibited

at normal levels of use in low pH foods, many spoil-

age bacteria are more resistant. Benzoic acid is most

suitable for foods at pH 2.5–4, whilst the esters of p-

hydroxybenzoic acid may be effective to pH 7 or

higher. However, the latter tend to be less effective

against bacteria and particularly Gram-negative or-

ganisms. Thus, in relatively acidic products where

yeasts and molds are the greater causes of spoilage,

benzoic acid and its salts offer an effective means of

preservation.

0005 Sorbic acid and its salts are useful additives against

yeasts and molds but are less effective against bac-

teria. They provide antimicrobial activity up to pH

6.5 and are good preservatives for foods with a high

fat content and pH, e.g., low-fat spreads, processed

cheeses; the p-hydroxybenzoate esters tend to be too

soluble in the nonaqueous component of such foods,

whilst benzoate is relatively ineffective at the higher

pH values.

0006 Sulfur dioxide and sulfites also represent broad-

spectrum antimicrobial agents in acidic foods, par-

ticularly beverages. In general, this preservative is

more effective against bacteria than molds and yeasts,

but Gram-positive bacteria are less susceptible than

Gram-negative bacteria. The effect against yeasts is

greatest around pH 3.5 and falls off markedly as the

pH is raised above this value. There is a tendency for

desirable fermentation yeasts to show a greater resist-

ance to this preservative than undesirable ‘wild

yeasts‘ and lactic acid bacteria. This is exploited in

the use of sulfites during wine-making. Despite the

relatively high pH associated with meat products

such as the British sausage (often pH 6–6.5), sulfites

tend to inhibit enterobacteriaceae, and particularly

salmonellae, in this environment. In some situations,

sulfites are added also to inhibit chemical spoilage,

notably enzymic browning caused by oxidation of

o-diphenols, other forms of oxidative spoilage, and

nonenzymatic browning of reducing sugars and

ascorbic acid.

0007Propionic acid and its salts are effective against

molds despite the fact that some species of Penicil-

lium can grow on media containing as much as 5%

propionic acid. Yeasts and most bacteria are generally

less affected. However, a specific target for propionic

acid is the rope-forming Bacillus subtilis in bread,

which is effectively controlled at pH 6. The preserva-

tion of a wide range of foods with vinegar (acetic

acid) has been known since ancient times; acetic

acid is more effective against yeast and bacteria than

molds. Its effect on bacteria is, however, predomin-

antly due to the associated reduction in pH. Aceto-

bacter and some lactic acid bacteria are unaffected by

this preservative. Lactic acid is similarly a preserva-

tive associated with traditionally prepared and par-

ticularly fermented foods. As with acetic acid, its

antimicrobial action is mainly due to a reduction of

pH to below that at which bacteria can grow. It does,

however, inhibit spore-forming bacteria at pH 5.0 but

is ineffective against yeasts and molds.

0008The most important effect of nitrite ion in food is

its inhibition of Clostridium botulinum in cured meat

products. Whilst it also has an inhibitory effect

towards a wide range of different species of bacteria,

some, particularly Salmonella, lactobacilli, and Clos-

tridium perfringens, tend to be more resistant. Nitrite

and nitrate are essential for the development of cured

meat color and flavor.

0009Thiabendazole, orthophenylphenol, and biphenyl

inhibit fungi and are suitable only for surface appli-

cation.

Specific Applications

Beverages

0010Nonalcoholic beverages, including fruit juices, are

usually preserved with benzoic acid and its salts,

esters of p-hydroxybenzoic acid, sorbic acid and its

salts, or the sulfites. Typical concentrations of benzoic

acid required are in the range of 500–1000 mg kg

1

In the event that these levels lead to a noticeable taste,

benzoic acid is replaced wholly or partly by mix-

tures of methyl and propyl esters of p-hydroxyben-

zoic acid (usually 2:1, methyl:propyl) at a combined

PRESERVATIVES/Food Uses 4777

concentration of 300–500 mg kg

1

, or sorbic acid and

its salts at 200–1000 mg kg

1

. Whilst sulfites are ef-

fective antimicrobial agents in beverages at concen-

trations of 50–500 mg of SO

per kilogram, they tend

to undergo significant oxidation when beverage con-

tainers are repeatedly opened and air admitted. A

combination of sulfite with another preservative,

e.g., sorbic or benzoic acid, is frequently used for

fruit juices such that the sulfite acts to control forms

of chemical spoilage, and lactic and acetic acid fermen-

tation, whilst the second preservative acts as a longer-

lasting agent against yeasts and molds. A significant

disadvantage of adding sulfites to beverages colored

with anthocyanins is that these are decolorized, even at

low concentrations of preservative, and additional

sulfite-stable food-color agents are required. Fruit

juice concentrates used for the manufacture of

normal-strength juice may contain any of the preser-

vatives used in the final beverage. In practice, it is not

feasible to add sufficiently high concentrations of the

carboxylic acid or ester preservatives to the concen-

trate to provide an acceptable level of additive in the

finished product. In general, such concentrates need to

be protected against browning, which demands high

levels (e.g., 2.5–10 g of SO

per kilogram) of sulfite;

this also acts as a preservative. In any case, the concen-

tration of preservative in the finished product is a

result of further addition after dilution and blending.

0011 Sulfites are the major preservatives for alcoholic

beverages. Their most important function is the con-

trol of the so-called wild yeasts in grape juice, and

after fermentation, the preservative is required at

levels of 50–150 mg of SO

to inhibit further micro-

bial action. A specific problem associated with the use

of sulfites to preserve wine, cider, and perry is that

fruit that has been affected by molds may contain

significant amounts of acetaldehyde, pyruvic acid,

a-ketoglutaric acid, and other aldehydes and ketones,

which are able to bind the preservative reversibly as

hydroxysulfonate adducts. When present in this

form, sulfite is unable to act as a preservative; how-

ever, the concentration of hydroxysulfonates is in-

cluded in the analytical concentration of the

preservative in the beverage. When grapes affected

by Botrytis are used for wine-making, as much as

80% of the total preservative can be found as

hydroxysulfonate adducts. During fermentation in

the presence of sulfites, yeasts form sulfite-binding

compounds and particularly acetaldehyde. Sorbic

acid is also a good preservative for wine, and despite

its chemical reactivity towards sulfite, mixtures of the

two are advocated; in such a situation, sorbic acid

serves to prevent further fermentation, whilst the

sulfite protects against chemical and bacterial spoil-

age. However, depletion of sulfite through chemical

reactions could result in malolactic bacteria metabol-

izing sorbic acid to intermediates, which ultimately

lead to off-flavors. In general, potassium sorbate is

the preferred salt on account of its higher solubility

than that of the sodium salt. In some wines, however,

potassium tartrate may precipitate, in which case, the

sodium salt is used.

Fruit Products

0012Sulfur dioxide is an effective fumigant in the control

of postharvest decay of grapes caused by the fungus

Botrytis cinerea. Raspberries may be similarly treated

for Botrytis and Cladosporium. Another unique

application of sulfite is for the storage, as pulp, of

soft fruit to be used for jam manufacture. A typical

‘sulfite liquor‘ would contain up to 3000 mg of SO

per kilogram of solution and is capable of preserving

fruit for up to 2 years with a high retention of ascor-

bic acid. Whilst the anthocyanins are bleached in this

process, the color of the fruit reappears as the preser-

vative is ‘lost‘ during jam-making. An endopolygalac-

turonase enzyme from Rhizopus sexualis is not

inactivated by sulfite and can cause the breakdown

of strawberries in pulp. Jam and related products,

e.g., fruit purees and pie fillings, may be success-

fully preserved with benzoic acid (1000 mg kg

1

p-hydroxybenzoate ester (methyl:propyl ester, 3:1;

700 mg kg

1

), sorbic acid (800–1500 mg kg

1

), and

sulfite (100 mg of SO

per kilogram in jam).

0013Dried fruits are frequently prepared with the aid of

sulfite as an antibrowning agent. In such products the

concentration of additive may be as high as 2500 mg

of SO

per kilogram, and this will also effectively

preserve the product. When a relatively moist unsul-

fited product is desired, a preservative is necessary,

and a potassium sorbate dip or spray to give 200–

500 mg of sorbic acid per kilogram, or similar appli-

cation of sodium benzoate at 1000 mg of benzoic acid

per kilogram is effective.

Vegetable Products

0014One of the most common traditional methods of

preserving vegetables is by pickling with vinegar,

acetic acid contributing to the reduction of pH, a

specific antimicrobial effect and a characteristic

taste. Typically, raw vegetables are immersed in 0.5–

3% acetic acid solution, but some yeasts and molds

are still capable of causing long-term spoilage. The

combined use of acetic acid with benzoic acid,

p-hydroxybenzoate ester mixtures (methyl:propyl,

2:1) or sorbic acid (all up to 1000 mg kg

1

) allows

lower acetic acid concentrations and safeguards agai-

nst spoilage. In sweet relishes, sorbic acid is more

effective than benzoic acid on account of the relatively

4778 PRESERVATIVES/Food Uses

high pH. The selective control of yeasts, molds, and

putrefactive bacteria is the reason why sorbic acid is

useful at levels of 500–2000 mg kg

1

in vegetable

fermentations, where it allows the growth of the

desired lactic acid-producing organisms.

0015 Fresh vegetables are generally dipped in sulfite

solutions to prevent enzymatic browning, though this

also protects against microbial spoilage. It is feasible

to preserve vegetarian burger-type products with

sulfite. However, dehydrated vegetables are

microbiologically stable but require sulfite as an anti-

browning agent for the dehydration process and sub-

sequent storage, at a final level of some 2500 mg of

per kilogram of dried product.

Baked Products

0016 Propionic acid is widely used in the making of bread

and cakes to inhibit surface mold and Bacillus sub-

tilis, which causes rope. The sodium salt is used

chiefly in confectionery products, whilst the calcium

salt is used for bread. Levels of propionic acid up to

3000 mg kg

1

may be found in such applications.

Sorbic acid is also an effective preservative for cereal

products, at concentrations similar to those of propi-

onic acid, and inhibits the mold Trichosporon found

in rye bread and cakes. Unfortunately, sorbic acid

also inhibits yeast. A potential solution could be to

use sorboyl palmitate, which has no antimicrobial

activity but degrades on heating, liberating sorbic

acid. Mixtures of esters of p-hydroxybenzoic acid

(methyl:propyl, 3:1) at 300–600 mg kg

1

are useful

preservatives for cakes (particularly fruit cakes) but

inhibit yeasts and cannot be used in bread. Some

components of flour confectionery, e.g., icing, fillings,

and marzipan, can be preserved with benzoic acid,

esters of p-hydroxybenzoic acid, and sorbic acid.

Dairy Products

0017 Apart from the small-scale use of hydrogen peroxide

to preserve milk by reducing the severity of heat

treatment required for sterilization, the major use of

preservatives in dairy products is for cheese. Propi-

onic acid is formed naturally during the ripening of

certain cheeses but may be used as an added preser-

vative for surface treatment to prevent mold growth.

Nitrite ion, which is formed from added nitrate in

cheese, inhibits fermentations caused by clostridia

and coliforms such as Enterobacter, which are un-

desirable because they may give rise to the production

of carbon dioxide with resultant blowing of the

cheese. Nitrite ion is to be avoided in cheeses where

the production of propionic acid is important

because nitrite also inhibits the bacteria required for

its formation. Nisin is a good antimicrobial agent

for cheese because it is particularly effective against

clostridia such as Cl. tyrobutyricum in cheeses with a

higher pH. The egg white antimicrobial enzyme, lyso-

zyme, is employed for a similar purpose. Cheese-

making represents a major use of sorbic acid, where

it serves two purposes: to inhibit surface mold

growth, achieved by dipping, dusting (preferably

with calcium sorbate), or incorporation of the preser-

vative into wax coatings or packaging films, or to

protect packs of processed cheese after opening, by

incorporation of the preservative into the cheese at

levels of 500–700 mg of sorbic acid per kilogram.

Meat and Fish Products

0018The curing of meat is probably the best known use of

nitrites and nitrates. They are used in conjunction

with salt, which has the effect of reducing the water

activity sufficiently to reduce spoilage by Pseudo-

monas and related organisms, whilst nitrite prevents

the growth of germinating spores. Curing is regarded

as protection against food poisoning caused by

Clostridium botulium. Eventual spoilage in cured

meat is caused by lactic acid bacteria if the salt con-

tent is low, or by micrococci and vibrios if it is high

together with a variety of molds and yeasts. Off-

flavors are associated with hydrolysis of fat. The

storage life of cured meat is extended by smoking as

a result of additional antimicrobial agents from the

smoke; again, when the meat ultimately spoils, it is

dominated by lactic acid bacteria. Typical concentra-

tions of sodium nitrite and nitrate in cured meat are

50–200 and 500 mg kg

1

, respectively.

0019Sulfites are added to a small number of commin-

uted meat products (sausages, burgers) at levels of

600 mg of SO

per kilogram, to achieve a maximum

of 450 mg of SO

in the finished product. Whilst the

total sulfite content of such a product falls slightly

during storage, a large proportion becomes reversibly

bound to carbonyl compounds of microbial origin.

The success of sulfite in this application is the result of

its inhibition of salmonellae, whilst spoilage of sul-

fited meat is confined to Gram-positive microflora

consisting of lactobacilli and Brochothrix thermo-

sphacta. The result is that the spoilage is associated

with a sour odor, unlike the spoilage of untreated

meat, which gives rise to a ‘putrid‘ smell. The use of

sulfites in meat products leads to the destruction

of thiamin and is not permitted in important meat

sources of the vitamin. Mold growth on the surface of

dry sausage, during the drying period, may be con-

trolled by dipping the casings in a solution of potas-

sium sorbate prior to stuffing.

0020Fish may be preserved in acetic acid (10–30 g kg

1

)

by the traditional process of marinating. Whilst this

PRESERVATIVES/Food Uses 4779

provides basic protection against pathogenic micro-

organisms, lactobacilli may grow, and the use of

acetic acid should be combined with a mild thermal

process or other antimicrobial agents. Benzoic acid

is slightly more effective than sorbic acid in this

application (1000 mg kg

1

), and esters of p-hydroxy-

benzoic acid (300–600 mg kg

1

) may also be used.

Fat Products

0021 Although benzoic acid had been used for some time for

the preservation of margarine at concentrations

of 800–1500 mg kg

1

, it is far from ideal for this appli-

cation owing to the relatively high pH of the food.

However, it is a good preservative for mayonnaise,

though improved performance against acid-producing

bacteria can be obtained by using mixtures of benzoic

and sorbic acids. Margarine and low-fat spreads can be

preserved most effectively by means of sorbic acid.

Miscellaneous Uses

0022 Food ingredients that are normally manufactured in

the form of solutions, or have a water activity suffi-

cient to sustain microbial growth, may be treated

with preservatives. Examples include flavoring prep-

arations, gelatin, malt extracts, and antifoam agents.

Carbon Dioxide

0023 Carbon dioxide is not a preservative in the usual

meaning of the word. However, in most of the modi-

fied-atmosphere-packaged foods, it does help to pre-

serve the food. It specifically inhibits the growth

of the typical fast-growing oxidative Gram-negative

bacteria that quickly spoil food, e.g., meat and fish.

Slower-growing, less obnoxious microorganisms,

such as lactic acid bacteria, often then take over, but

the shelf-life can be substantially extended. The

extent of such inhibition by CO

is greatly enhanced

as the temperature is reduced. It has been speculated

that this results mainly from the increased solubility

of the gas at low temperatures. The shelf-life increases

for meat and fish can be by two to three times if chill

control is effective.

See also: Alcohol: Properties and Determination; Bread:

Chemistry of Baking; Cakes: Chemistry of Baking; Fish:

Miscellaneous Fish Products; Jams and Preserves:

Chemistry of Manufacture; Meat: Preservation; Extracts;

Preservatives: Analysis

Further Reading

Branen LA, Davidson PM, Salminen S and Thorngatwe III

JH (eds) (2002) Food Additives. New York: Marcel

Dekker.

Davidson PM and Branen LA (eds) (1993) Antimicrobials

in Foods. New York: Marcel Dekker.

Hayes PR (1985) Food Microbiology and Hygiene.

London: Elsevier Applied Science.

Lueck E (1980) Antimicrobial Food Additives. Characteris-

tics, Uses, Effects. Berlin: Springer-Verlag.

Wedzicha BL (1984) Chemistry of Sulphur Dioxide in

Foods. London: Elsevier Applied Science.

Analysis

B L Wedzicha, University of Leeds, Leeds, UK

Background

0001Food preservatives constitute a group of compounds

of widely different molecular structures; they are or-

ganic and inorganic substances with different func-

tional groups and tendencies to form ions. There are

no procedures that are generally applicable to the

analysis of preservatives as a class of food additive;

the procedures are specific to the preservative being

analyzed. The lowest concentrations of commonly

used preservatives are of the order of a few milligrams

per kilogram of food, and, with few exceptions,

recommended or statutory methods of analysis are

designed to give a good accuracy at levels of 10 to

> 1000 mg of preservative per kilogram of food. The

question of the lower limit of detection is rarely

an issue, unless it is desired to use small sample sizes,

e.g., < 1 g, or to determine whether or not a food or its

ingredients had been treated with a preservative. For

solid foods, small sample sizes often lead to non-

representative sampling and should be avoided. Not

all the procedures described constitute official

methods of analysis. Frequently, for routine analysis,

a food manufacturer would use a rapid or cheap

analytical technique standardized against an official

method. The official status of given procedures varies

from country to country.

0002Organic and inorganic acid preservatives may be

added in the form of the undissociated acid or a

variety of salts. In food, the ionic composition is

determined largely by concentration and pH, but it

is generally impossible to predict this accurately for

any given situation. In order to avoid complications

with the specification of the amount of preservative

in a food, this is usually referred to as the weight-

for-weight concentration of the undissociated acid,

e.g., benzoic acid, sorbic acid, or sulfur dioxide. Ni-

trite and nitrate levels are expressed in terms of the

weight of the sodium salt.

4780 PRESERVATIVES/Analysis

0003 There are, of course, a very large number of pos-

sible analytical procedures available for each preser-

vative. Those given here represent a selection to

illustrate the variety of methods recommended for

use on food samples.

Carboxylic Acids and Esters

Extraction

0004 All the carboxylic acid and ester preservatives are

steam-volatile from acidified food samples, and

steam distillation offers an effective means of separ-

ating them from the sample. The nonionic nature of

these preservatives in acid solution also allows extrac-

tion with a variety of organic solvents, e.g., diethyl

ether, and dichloromethane. Such organic solvents

may also be used to isolate, or concentrate, preserva-

tives obtained in large volumes of steam distillate.

The subsequent detection and determination of pre-

servatives depend on the substance to be analyzed.

(See Acids: Properties and Determination.)

Analysis of Extracts and Distillates

0005 Rapid screening of extracts and distillates for sorbic

and benzoic acid and the esters of p-hydroxybenzoic

acid is possible by thin-layer chromatography (TLC)

on silica gel. If separated on plates containing an F

254

(fluorescent indicator), these acids may be readily

visualized under UV light. Acetic and propionic

acids can be identified by paper chromatography

using an acid–base indicator for visualization of

spots.

0006 The total acidity in steam distillates is an accept-

able measure of the concentration of an acid preser-

vative provided that only one such preservative is

used and that a blank for the food sample is known.

Propionic and acetic acids can be separated using a

silica gel column with butanol–chloroform solvent

and the eluate titrated for quantitation. Solvent ex-

tracts of food samples can only be subjected to direct

spectrophotometric analysis with caution. For

example, benzoic acid, extracted into diethyl ether

from tomato products, jams, jellies, and soft drinks,

may be determined by measuring its absorbance at

267.5, 272, and 276.5 nm, and using the average of

the highest and lowest wavelength readings as the

background value for the absorbance at 272 nm. In

general, solvent extracts and steam distillates can be

analyzed directly by reverse-phase high-performance

liquid chromatography (HPLC) or by gas–liquid

chromatography (GLC) after forming trimethyl-

silyl or methyl esters. Internal standards for chroma-

tography are chosen to have extraction and

chromatographic behavior similar to that of the

preservative being analyzed. Standards are generally

added to the food before extraction, and any deriva-

tization is carried out on both the analyte and the

standard. Examples of internal standards include

phenylacetic and caproic acids for gas chro-

matographic analysis of benzoic and sorbic acids,

respectively. (See Chromatography: Thin-layer Chro-

matography; High-performance Liquid Chromatog-

raphy; Gas Chromatography.)

0007Esters of p-hydroxybenzoic acid are determined

in steam distillates or extracts either by reversed-

phase HPLC or by direct spectrophotometry as

p-hydroxybenzoic acid after saponification.

0008Oxidation of sorbic acid to malonaldehyde by

means of dichromate and subsequent reaction with

thiobarbituric acid (TBA) offers a specific nonchro-

matographic assay of this preservative in steam distil-

lates from all types of food. Frequently, crude solvent

extracts can be analyzed in the same way without any

undue interference from other TBA-reactive sub-

stances either present in food or formed during

dichromate oxidation.

Enzymatic Methods

0009Specific enzymatic procedures for the determination

of acetic and formic acids are available. Acetic acid

is determined by conversion to pyruvate (using ATP

and coenzyme A in the presence of acetylcoenzyme A

synthetase), which reacts in turn with oxaloacetate

(citrate synthetase) obtained by the reduction of

malate by NAD

(malate dehydrogenase). The rate

of utilization of NAD

is a measure of acetic acid

concentration. Formic acid is determined more

simply by measuring the rate of utilization of

NAD

during the oxidation of the acid to hydrogen

carbonate (formate dehydrogenase). In general,

sample preparation for enzymatic methods involves

the preparation of aqueous extracts of solid foods,

separating fat, precipitating protein (perchloric acid),

decolorizing strongly colored samples (charcoal), and

adjusting to pH 7–8.

Sulfur Dioxide and Sulfites

Classification

0010Sulfur dioxide and sulfite are considered to exist in

food in two forms: free and bound. The former

includes all species of sulfur in oxidation state þ4,

i.e., SO

, HSO



,SO

2

, and S

2

. The latter term

is used to describe that preservative which is in the

form of hydroxysulfonate (carbonyl-HSO



) adducts.

Legislation requires that total (free þbound)

preservative be determined. In food processing,

the concentration of free additive determines its

PRESERVATIVES/Analysis 4781

antimicrobial effect and is frequently measured. A

specific problem that exists with this preservative is

that itsmeasured concentration falls with time as a

result of reactions with food components, and as

a result of autoxidation after packaged foods are

opened and exposed to air. Samples must be analyzed

without delay and exposure to air minimized.

0011 A broad classification of analytical methods for

sulfur dioxide and sulfites is based on whether or

not a separation procedure is involved. Methods are

usually referred to as direct, when a specific analytical

reaction is applied to the whole food sample, and

indirect, when the additive is recovered before deter-

mination.

Direct Methods of Analysis

0012 The simplest analysis of sulfites in foods involves

iodimetric titration after allowing for a blank repre-

senting the reactivity of the food sample itself towards

iodine. This is normally found by adding an aldehyde

or ketone (formaldehyde, acetaldehyde, acetone) to

combine with free sulfite before the blank titration.

The free sulfite content of a food is determined by

acidifying the sample and titrating with iodine. The

total (free þbound) sulfite may be determined by

raising the pH of the sample to pH 10 to decompose

any hydroxysulfonates before acidifying and titra-

tion. Iodimetric titration is applicable to beverages

and aqueous extracts/homogenates of solid foods.

Dark-colored beverages may be titrated using electro-

metric detection of the end point.

0013 Sulfites in solution may be determined spectropho-

tometrically by reaction with tetrachloromercurate

þpararosaniline þHCHO reagent, 5,5

-dithiobis(2-

nitrobenzoic acid) (DTNB), or malachite green.

These reactions depend on the nucleophilic behavior

of the sulfite ion and suffer interference from nucleo-

philes in foods, particularly thiols. Despite this, the

pararosaniline method has been used widely for

direct determination of the preservative in beverages,

solutions of sugars, and aqueous extracts or homo-

genates of solid foods including fruits and vegetables,

and has been adapted successfully for an ‘autoanaly-

zer‘ system.

0014 Few chromatographic methods of analysis of

sulfites in foods have gained acceptance, mainly

because of the problems associated with detection

of the species. Ion-exclusion chromatography of

the sulfite ion with amperometric detection is the

best-established chromatographic method available

and can be applied directly to beverages and aqueous

extracts of foods.

0015 Other direct analyses include the use of polarog-

raphy and sulfur dioxide-sensitive electrodes but are

not in widespread use. Enzymatic methods of analysis

make use of the oxidation of sulfite to sulfate þH

(sulfite oxidase) and assay of H

with NADH

(NADH-peroxidase). The rate of formation of NAD

is used to determine the sulfite concentration. Sulfite

oxidase may also be immobilized at the tip of an

oxygen-measuring electrode for a direct indication

of the rate of utilization of oxygen by the enzyme in

sulfite oxidation. The pH requirement for these en-

zymatic analyses is slightly alkaline (pH 7.5–8), and

the method can give only the total additive concen-

tration.

Indirect Methods of Analysis

0016All indirect methods of analysis of sulfur dioxide and

sulfites in foods involve the conversion of the ionic

forms to gaseous SO

as the method of separating the

additive from the food. The Monier–Williams distil-

lation technique, described in 1927, is still the

standard by which other methods are evaluated.

This method, and variants of it, involve the distilla-

tion, under reflux, of the food sample acidified with

HCl, H

,orH

, in a gentle stream of an inert

gas. SO

is trapped in H

or iodine. Some vari-

ations of this technique include the codistillation of

with water with a downward sloping condenser,

to speed up the procedure. The most serious interfer-

ence arises from the presence of volatile compounds,

which are oxidized by H

to acid, or react with

iodine. Such interfering components are found in

onions, leeks, and cabbage. Small samples may

be analyzed by replacing the conventional trapping

agents by pararosaniline reagent or DTNB, which

allow spectrophotometric determination of SO

at a

much greater sensitivity than is possible by titration.

Polarographic analysis of the distillate offers a good

alternative. Desorption of SO

from cold acidified

samples enables free preservative to be determined;

prolonged boiling (e.g., 1.75 h) or pretreatment with

alkali causes the decomposition of hydroxysulfonates

and gives the total preservative present. In general,

distillation methods do not require any sample prep-

aration and are equally applicable to solid and liquid

foods.

0017An automatic distillation unit ahead of an ‘auto-

analyzer‘ with spectrophotometric detection provides

an effective automatic indirect method of analysis

for liquid samples and aqueous extracts. An ingenious

approach to separating dissolved SO

from an

acidified food sample is by diffusion through

polytetrafluoroethylene membrane. This may be

used in a flow injection arrangement where the diffu-

sion cell carries streams of acidified liquid food or

extract solution on one side of the membrane and a

suitable reagent for spectrophotometric analysis on

the other.

4782 PRESERVATIVES/Analysis

0018 Analysis of gaseous SO

in the headspace above

acidified liquid foods or homogenates of solid foods,

or transfer of SO

through the headspace from the

sample to a reagent for spectrophotometric analysis

in a microdiffusion cell, offers an alternative indirect

method of analysis.

Nitrites and Nitrates

0019 The most important uses of nitrite and nitrate as food

preservatives are in meat products, and the majority

of analytical procedures for food have been devised

especially for this application. There are, however,

numerous published procedures for the determin-

ation of the ions in water, and soil, and those

occurring naturally in plant material. (See Nitrates

and Nitrites.)

0020 Since nitrites and nitrates are readily soluble in

water, aqueous extracts from homogenates of the

food at neutral or weakly alkaline pH are sufficient.

A low extraction pH leads to the loss of nitrite. Apart

from pH, important variables in extraction are time

and the addition of heavy-metal salts (e.g., BaCl

CdCl

, HgCl

). A proportion of nitrite present in

food may exist in bound form as nitrosothiols, nitro-

sylmyoglobin, etc.; methods of extraction that in-

volve the heavy-metal ions and long extraction times

(e.g., 1–4 h) cause the release of such bound additives.

Techniques based on short extraction periods (e.g.,

10 min) at neutral pH measure mainly the free nitrite.

Nitrosyl hemoproteins may be decomposed to species

convertible to nitrite if the extracting medium con-

tains 80% acetone.

0021 Extracts generally contain dissolved and suspended

protein. This is removed, and solutions are clarified

by means of zinc acetate þpotassium ferrocyanide,

alumina cream, potash alum or zinc sulfate þborax.

In general, the reagents allow recoveries of nitrite of

> 98% with standard deviations no greater than

0.75%, from meat samples, with the exception of

the procedure involving the use of alumina cream,

for which the recovery is slightly lower at 95%.

0022 Nitrate may be determined directly using a reaction

in which it is used as a nitrating agent for variety

of organic substrates and particularly the xyle-

nols (dimethylphenols); the nitro-compounds so

formed may be determined spectrophotometrically.

This reaction can also be used to determine the

concentration of nitrite if it is first oxidized to nitrate

with permanganate. Alternatively, nitrate may be

reduced to nitrite by spongy cadmium or enzymati-

cally with NADPH (nitrate reductase). The rate of

formation of NADP in the enzymatic procedure

may be used to measure nitrate concentration

directly. The most widely used reaction for the

determination of nitrite is that with an aromatic pri-

mary amine (usually sulfanilic acid) in acid solution

to form a diazonium salt, which is coupled to

an aromatic amine or hydroxy compound to give an

azo dye. This can be determined spectrophoto-

metrically. Typical coupling agents have included

1-naphthylamine and 1-naphthol, which are carci-

nogenic, and 1-naphtholsulfonic acid and N-1-

naphthylethylenediamine dihydrochloride are now

frequently recommended. Both oxidizing and redu-

cing agents interfere; ascorbic acid is a particular

problem.

0023The sample blank can represent a significant error

in analysis. There is no completely satisfactory way of

preparing this; suggestions have included the passing

of the deproteinized sample through an ion-exchange

resin to remove nitrate and nitrite before determining

the blank.

Other Preservatives

Biphenyl

0024Steam distillation of citrus peel homogenates and

extraction of distillate with heptane provides a

sample that may be analyzed for biphenyl by TLC

on silica gel with heptane as the developing solvent.

Quantitative analysis is by extracting biphenyl from

the TLC medium with ethanol and measuring the

absorbance of resulting solutions at 248 and 300 nm.

The absorbance at 300 nm is used to correct for back-

ground absorbance. GLC methods of analysis are also

available.

Thiabendazole

0025Thiabendazole may be extracted from food with acid

(HCl), in which it is subsequently reduced with zinc in

30% glycerolþphenylenediamine. Subsequent oxida-

tion with ferric ions gives rise to a blue complex,

which is extracted into butanol for spectrophotomet-

ric measurement.

Lactic Acid

0026Standard methods for the determination of lactic acid

involve its conversion to the Fe(III) complex for spec-

trophotometric measurement, or by GLC after com-

plete methylation. In an enzymatic procedure, lactic

acid is converted to pyruvate by NAD

(lactate dehy-

drogenase); the product is trapped by reacting with

glutamate (glutamate–pyruvate transaminase) in

order to displace the lactic acid–pyruvate equilibrium

to the right. The rate of utilization of NAD

is used to

measure lactic acid concentration. Sample prepar-

ation is generally as described for carboxylic acids

above.

PRESERVATIVES/Analysis 4783

Hydrogen Peroxide

0027 Spot tests for the presence of hydrogen peroxide in

milk use either a solution of vanadium pentoxide

in H

(pink or red color) or KI/starch (blue color).

0028 Quantitative analysis is with peroxidase and a

suitable substrate. The frequently recommended

o-dianisidine is carcinogenic, and substrates such as

guaiacol or 2,2

-azinobis (3-ethylbenzthiazolidine-6-

sulfonic acid) are preferred. Assays on milk samples

are carried out after precipitation of the protein by

adjusting to pH 4.5 with HCl.

Nisin

0029 The complexity of procedures for isolating and chem-

ically quantifying nisin has led to the development of

microbiological assays as normal analysis procedures.

One international unit of nisin activity is the extent of

inhibition of a test microorganism caused by approxi-

mately 25 ng of pure nisin. A suitable test microor-

ganism is Streptococcus agalactiae. A particular assay

procedure is an adaptation of the methylene blue

reduction test; the delay in decolorization of the dye

is proportional to nisin concentration over a 10-fold

concentration range. A new method of analysis

involves the enzyme-linked immunosorbent assay,

which is much more straightforward than the micro-

biological assay and should become popular.

See also: Chromatography: High-performance Liquid

Chromatography; Gas Chromatography;

Immunoassays: Radioimmunoassay and Enzyme

Immunoassay; Nitrates and Nitrites; Preservatives:

Classifications and Properties