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early 1980s has alerted the general public to the

potential hazard of this mycotoxin. The tolerable

daily intakes of DON for adults and infants were

estimated to be 3 and 1.5 mgkg

1

, respectively. A

tolerance level of 1 p.p.m. for DON in grains for

human consumption has been set by a number of

countries, including the USA. In Canada, the guide-

line for DON in the uncleaned soft wheat used for

nonstaple foods is 2 p.p.m., but 1 p.p.m. for the infant

foods. Although inadequate storage may lead to the

production of some TCTC mycotoxins, infestation of

Fusarium spp. in wheat and corn in the field is of most

concern for the DON problem.

0041 TCTCs may also be involved in the so-called ‘sick

building syndrome’ (SBS) in humans. Stachybotrys

atra was isolated from a badly water-damaged

Chicago suburban home, in which the occupants

complained about headaches, sore throats, hair loss,

flu symptoms, diarrhea, fatigue, dermatitis, and gen-

eral malaise. Several TCTCs (verrucarins B and J,

satratoxin H, trichoverrins A and B) were found in

the S. atra-contaminated materials of this home. T-2

toxin, DAS, roridine A, and T-2 tetraol were isolated

from the dust samples from the air ventilation system

of another suspected sick building syndrome in three

urban Montreal office buildings. S. chartarum,an

indoor mold, has been associated with pulmonary

hemorrhage cases in the Cleveland, Ohio, area. Al-

though Stachybotrys spp. are consistently found in

these buildings, other molds and mycotoxins might

also be present. In a survey of buildings with moisture

problems in Finland, scientists have identified sterig-

matocystin (24%), TCTC (19%), and citrinin (4%)

in 79 samples analyzed. The trichothecenes found

included satratoxin G or H (five samples), DAS (five

samples), and 3-acetyl-DON, DON, verrucarol, or

T-2-tetraol in an additional five samples. Aspergillus

versicolor was present in most sterigmatocystin-

containing samples, and Stachybotrys spp. were pre-

sent in the samples where satratoxins were found.

Thus, it is important to identify both the mold and

mycotoxins for the cause of SBS. A remediation

program involving removal of all contaminated wall-

board, paneling, and carpeting in the water-damaged

areas of the home, as well as spraying a sodium

hypochlorite to all surfaces during remediation,

appeared to be effective. Air samples taken from post-

remediation buildings showed no detectable levels of

S. chartarum or related toxicity.

Other Selected Mycotoxins

0042 Other than those mycotoxins discussed above, sev-

eral naturally occurring mycotoxins with typical tox-

icoses may also be potentially hazardous to human

and animal health.

0043Mycotoxins produced by Aspergillus other than AF

and OA Sterigmatocystin (ST), structurally related

to AFB1 and a precursor for biosynthesis of AFB1, is

another naturally occurring hepatotoxic and carcino-

genic mycotoxin. Although the carcinogenicity of ST

is less than that of AFB1 in test animals (10–100 times

less), this mycotoxin has been found to be mutagenic

and genotoxic, and has been found in foods in certain

high esophageal cancer incidence regions of China

and Mozambique. Toxigenic fungi have been isolated

from patients with esophageal cancer, and these

strains are capable of producing ST in many commod-

ities. Although the role of ST in human carcinogenesis

appears to be indirect and inclusive, it acts mechanis-

tically similar to AFB in carcinogenesis through the

formation of DNA adducts and mutation of p53 sup-

pressor gene. The carcinogenic effects of ST seem to

be mediated by failure of p53-mediated G1 check-

point because exposure of cells to ST resulted in

failure of G1 arrest.

0044Aspergillus terreus and several other fungi

(A. flavus and A. fumigatus) produce the tremorgenic

toxins territrem A, B, and C, aflatrem, and fumitre-

morgin. Some of these, bound to acetylcholinesterase,

cause irreversible inhibition of the enzyme. Gliotoxin,

an epipolythiopiperazine-3,6-dione antibiotic con-

taining sulfur produced by piperazines antibiotic

produced by A. terrus, A. fumigatus, and Tricho-

derma viride, exerts immunosuppressive effects in

animals. The toxic effect is due to its covalent binding

with -SH enzymes. It induces apoptosis in spleen cells

with a dose-related simultaneous increase in cAMP

levels and markedly inhibits both perforin-dependent

and Fas ligand-dependent cytotoxic T-lymphocyte

(CTL)-mediated cytotoxicity. In addition, A. flavus,

A. wentii, A. oryzae, and Penicillium atraovenetum

are capable of producing nitropropionic acid (NPA),

a mycotoxin causing apnea, convulsions, congestion

in lungs and subcutaneous vessels, and liver damage

in test animals. This toxin was also identified as

an etiological agent for the ‘deteriorated sugar-

cane poisoning (SP),’ a fatal food poisoning that

occurred in China. However, the fungi involved in

the contamination in the sugarcane and NPA produc-

tion were Arthrinium sacchari, A. saccharicola and

A. phaeospermum.

0045Other mycotoxins produced by Penicillium Other

than OA, Penicillium spp. produce many mycotoxins

with diversified toxic effects. Cyclocholortine, luteo-

skyrin (LS), and rugulosin (RS) have long been con-

sidered to be possibly involved in the yellow rice

disease during World War II. They are hepatotoxins

and also produce hepatomas in test animals. How-

ever, incidences of food contamination with these

4104 MYCOTOXINS/Toxicology

toxins have not been well documented. LS inhibits

RNA polymerase through binding with the enzyme

and also forms a three-component complex, i.e.,

DNA–Mg

2þ

–luteoskyrin, with DNA. LS and several

related anthraquinone-type mycotoxins are capable

of inducing free radicals leading to the formation

of 8-hydroxydeoxyguanosine. Several other myco-

toxins, including patulin (PT), penicillic acid (PA),

citrinin (CT), cyclopiazonic acid (CPA), citreoviridin,

and xanthomegnin, which are produced primarily by

several species of Penicillium, have attracted some

attention because of their frequent occurrence in

foods. PT and PA are hepatotoxins and teratogens,

but PT is not an immunotoxin. They inhibit dehydro-

genases reversibly through interaction of the toxin

with the -SH group in the enzyme active center. The

a,b-unsaturated double bonds conjugated in lactone

ring of the toxin were considered to be sites for the

interaction with the –SH and –NH

groups of pro-

teins through addition or substitution reactions. In

the model systems, PT forms the same type of adducts

with glutathione and N-acetyl-l-cysteine (NAC).

However, free cysteine formed markedly different

adducts, including mixed thiol/amino-type adducts

involving the a –NH

group. PT–cysteine adduct(s)

still have a teratogenic effect. Due to its highly react-

ive double bonds that readily react with –SH groups

in foods, patulin is not very stable in foods containing

these groups. Nevertheless, PT is considered a health

hazard to humans. Many countries have regulatory

limits, most commonly at a level of 50 mgkg

1

, for PT

in various foods and juices.

0046 Frequently associated with the natural occurrence

of OA is CT, which is also a nephrotoxin. In addition

to Penicillium and Aspergillus spp., a recent discovery

of the ability of Monocuus ruber and M. purpureus

(two molds frequently used in the preparation of

certain oriental foods) to produce CT reiterated the

potential health hazard of CT to human health. One

of the mycotoxins closely associated with the natural

occurrence of AF in peanuts is CPA, which causes

hyperesthesia and convulsions as well as liver, spleen,

pancreas, kidney, salivary gland, and myocardial

damage. CPA alters Ca

2þ

hemostasis and induces

charge alterations in plasma membranes and mito-

chondria. The reversible inhibition of reticulum

2þ

-dependent ATPase is the primary cause of this

effect. Penicillium rubrum and P. purpurogenum pro-

duce two highly toxic hepatotoxins (LD50, 3.0 mg

1

in mice, I.P.) called rubratoxins A (minor) and

B (major), which are complex nonadrides fused with

anhydrides and lactone rings. Rubratoxin B has

been shown to have synergistic effects with AFB1. It

induced apoptosis in several cell systems. A lack

of extracellular Ca

2þ

ion accelerates RB-induced

DNA fragmentation of HL-60 cells. Neither protein

synthesis nor nucleic acid interaction was related to

the RB-induced APT, but proteases are likely to be

involved. p53 has also been found not to be involved

in the RB-induced APT signal transduction. Secalonic

acid D, a cleft palate-inducing mycotoxin produced

by P. oxalicum and other fungi, reduces palatal cAMP

levels. It inhibits the binding of the cAMP response

elements to the binding protein, alters phosphoryl-

ation, and leads to altered expression of genes

involved in cell proliferation, an event critical for

normal palate development.

0047In addition to the above hepatotoxins and nephro-

toxins, Penicillium spp. produce many mycotoxins

with strong pharmacological effects on neurosystems.

For example, the indoloditerpenes, penitrem A–F are

a series of temorgenic mycotoxins. Penitrem A, the

major toxin in this group, causes tremorgenic effects

in mice at a level of 250 mgkg

1

. Roquefortines A–C

(C is most toxic), produced by P. roqueforti and

several other Penicillium spp., have neurotoxic effects

in animals. Tremorgens in the paspalitrem group

(paspalicine, paspalinine, paspalitrem A and B, pas-

paline and paxilline) are produced by Claviceps pas-

pali and some Penicillium spp. Paxilline, a reversible,

noncompetitive inhibitor of the cerebellar inosital

1,4,5-triphosphate (InsP-3) receptor, inhibits the

InP-3-induced Ca

2þ

release.

0048Other mycotoxins produced by Fusarium Other

than TCTCs and Fm, some Fusarium spp. also pro-

duce other mycotoxins. Zearalenone (ZE), also called

F-2, is a phytoestrogen that has hyperestrogenic

effects and causes reproductive problems in animals,

especially swine. It interacts with estrogenic receptors

and stimulates protein synthesis by mimicking the

action of the hormone. Contamination with this

mycotoxin, sometimes together with DON, in

feed may result in a large economic loss in the swine

industry. It has been postulated that ZE may contrib-

ute to the overall estrogen load of women because of

its frequent natural occurrence. ZE stimulates cyto-

kine production. It also stimulates the growth of es-

trogen receptor-positive human breast carcinoma cell

line MCF-7 and functions as an antiapoptotic agent

by increasing the survival of MCF-7 cell cultures

undergoing apoptosis caused by serum withdrawal.

The mitogen-activated protein kinase signaling

cascade is required for ZE’s effects on cell-cycle

progression in MCF-7 cells. Fusarium moniliforme

also produces several other mycotoxins, including

fusarins A–F, moniliformin, fusarioic C, fusaric acid,

and beauvericin, in addition to Fms. Although the

impact of these mycotoxins on human health is

still not known, fusarin C (FC) has been identified

MYCOTOXINS/Toxicology 4105

as a potent mutagen. Moniliformin, which causes

cardiomyopathy in test animals, may be involved in

Keshan disease in humans in regions where dietary

selenium deficiency is also a problem. It binds

strongly with pyruvate dehydrogenase, thus affecting

the enzymes in the TCA cycle. Fusarium moniliforme

was also found to be most effective among a group of

several fungi tested in reducing nitrates to form

potent carcinogenic nitrosamines. Thus, the contam-

ination of foods with this fungus could be one of the

etiological factors involved in human carcinogenesis

in certain regions of the world. Fusarochromanones,

a group of mycotoxins produced by some Fusarium

equiseti isolates, cause tibial dyschondroplasia in

broiler chicks, turkeys, and ducks and were con-

sidered as the possible cause of Kashin-Beck disease

in China.

0049 Mycotoxins produced by other species Alternaria

alternata and other Alternaria spp. species are

capable of producing dibenzo-a-pyrone types of

mycotoxins (alternariol, alternariol monomethyl

ether (AME), altenuene, isoaltenuene and altenuisol),

tetramic acid metabolites, tenuazonic acid (TzA) and

related compounds, and perylene derivatives alter-

toxins (ATX) I, II (also called stemphyltoxin II), III

and stemphyltoxin. Although most of those com-

pounds are relatively nontoxic, AME has been

shown to be positive in Ames tests at relatively high

concentrations. TzA, a protein synthesis inhibitor

that interacts with peptidyltransferase in ribosomes,

is more toxic than others. It can chelate metal ions

and is capable of formation of nitrosamines. It is also

produced by Phoma sorghina and Pyricularia oryzae,

and may be related to ‘Onyalai,’ a hematological

disorder in humans in the south of Sahara in Africa.

Although no extensive survey has been conducted to

determine the occurrence of these mycotoxins in

human foods, limited studies indicate that the inci-

dence of some of these mycotoxins, such as AME and

TzA, could be high in apple and tomato products. A

recent discovery of the structural and functional simi-

larity between fumonisins and AAL toxins, a series of

phytotoxins produced by Alternaria, further shows

the importance of mycotoxins produced by fungi in

the Alternaria family.

0050 Sporidesmine, a group of hepatotoxins produced

by Pithomyces chartarum and Sporidesmium char-

tarum, causes facial eczema and liver damage in

animals. Contamination of these mycotoxins in

feeds can result in great economic loss to the sheep

industry. Slaframine, an interesting mycotoxin pro-

duced by Rhizoctonia leguminicola (in infested

legume forage crops), causes excessive salivation in

ruminants as result of the blockage of acetylcholine

receptor sites.

Role of Metabolism and Dietary

Modification

Role of Metabolism

0051Because metabolism plays a key role in the activation

and detoxification of mycotoxins, the biological

effects of mycotoxins are greatly affected by the

metabolism of the toxins in different animals. AFB1

is metabolically activated before the formation of

the AFB–DNA adducts, which exerts carcinogenic

effects. However, activated AFB1 can also be conju-

gated to proteins and interact with glutathione and

then be excreted from the body. Thus, glutathione

S-transferase serves as a key enzyme in the detoxifi-

cation process for AFB1. Mice are resistant to the

carcinogenic effects because they constitutively ex-

press an a-class glutathione S-transferase (mGSTA3-

3). Rats do not constitutively express a GST with a

high AFB1-epoxide conjugating activity and thus

are sensitive to AFB1-induced hepatocarcinogenesis.

Constitutively expressed human hepatic a-class GSTs

have little or no AFB1-epoxide-detoxifying activity.

In contrast to rodents, the v-class GSTs are respon-

sible for most of the AFB–epoxide-conjugating activ-

ity in the liver. Other types of cytochrome P450 are

also capable of metabolizing AFB1 to various hydro-

xylated metabolites, including AFM1 and AFQ1.

Demethylase is capable of converting AFB1 to

AFP1. The cytosolic steroid reductase can convert

AFB1 reversibly to aflatoxicol, which serves as a

reservoir for AFB1.

0052The importance of metabolism for other myco-

toxins is generally related to the detoxification pro-

cesses. For example, metabolic deacetylation and

deepoxidation of trichothecence mycotoxins lead

to the formation of hydroxylated derivatives and

deepoxide-TCTCs that are less toxic. Hydrolysis of

OA by proteolytic enzymes leads to the production of

nontoxic metabolites. Hydroxylated OA, formed by

the action of cytochrome p450, is also less toxic; no

evidence of OA metabolism to form glutathione con-

jugate or other adducts was found. Hydroxylation of

T-2 at the C-3

and OA at the C-4 position by micro-

somal enzymes has also been demonstrated. Most of

the hydroxylated mycotoxins and metabolites may be

excreted directly or form conjugates of glucuronide

or sulfate, which are then excreted. Thus, various

factors affecting the kinetics of formation of adducts

and detoxified metabolites greatly affect the toxicity

of mycotoxins. A number of factors, including the sex

4106 MYCOTOXINS/Toxicology

and species of the animal (genetics), environmental

factors, nutritional status, and mycotoxin synergism,

etc., can either directly or indirectly modulate the

toxic effects.

Dietary Modifications

0053 As discussed elsewhere in this encyclopedia, dietary

modification greatly affects carcinogenesis in experi-

mental animals and in humans. Without exception,

the carcinogenic and other toxic effects of myco-

toxins, especially aflatoxins, are also affected by

nutritional factors, dietary additives, and anticarcino-

genic substances. Since the manifestation of carcino-

genic/toxic effects of AFB1 depends on its ability to be

absorbed and metabolized to an active form and on

its subsequent interaction with DNA to form AFB1–

DNA adducts, dietary modifications on any or all of

these steps would lead to an increase or decrease in

the carcinogenic effects of aflatoxin. Mycotoxins

have a high affinity for the hydrated sodium calcium

aluminasilicate (or NovaSil) and other related prod-

ucts. Diets containing NovaSil and related absorbers

are effective in preventing absorption of AFB1 and

several other mycotoxins in animals. Chemoprotec-

tive agents and antioxidants such as ascorbic acid,

BHA, BHT, ethoxyquin, oltipraz, pentaacetyl genipo-

side, Kolaviron biflavonoids, and even green tea have

also been found to inhibit carcinogenesis caused by

AFB1 in test animals. Dietary administration of the

naturally occurring chemopreventive agents, ellagic

acid, coumarin or a-angelicalactone has been shown

to cause an increase in the activity of glutamate–

cysteine ligase, a key enzyme for the synthesis of

glutathione. The mechanism of such protective effects

was found to be the shifting of metabolism to a

detoxification route by formation of a AFB1–

glutathione conjugate rather than the formation of

AFB1–DNA adducts. Ebselen exerts a potent protect-

ive effect against aflatoxin B-1-induced cytotoxicity;

such a protective effect may be due to its strong

capability in inhibiting intracellular reactive oxygen

species formation and preventing oxidative damage.

0054 Other absorbents such as zeolite, bentonite, and

superactive charcoal are also effective in decreasing

the toxicity of mycotoxins such as T-2 toxin. The

toxicity of OA in test animals is minimized when

antioxidants such as vitamins C and E are added to

the diet. Aspartame, which has been found to be

partially effective in decreasing the nephrotoxic and

genotoxic effects of OA, may compete with OA for

binding to serum albumin. l-Phenylalanine was

found to protect the toxic effects of OA; administra-

tion of this amino acid prevented the OA’s inhibitory

effect on some of the enzymes discussed earlier.

Vitamin E was found to efficiently prevent cytotoxi-

city induced by FB1. (See Aspartame.)

Concluding Remarks

0055Mycotoxins can cause both acute and chronic effects

in prokaryotic and eukaryotic organisms, including

humans. Most of their effects are organ-specific, but

some mycotoxins may affect on many organs. Induc-

tion of cancer by some mycotoxins through initiation

and promotion processing, in animals and possibly in

humans, is one of the major concerns for their chronic

effects. Modulation of immune systems by some

mycotoxins is another concern. Interaction with

marcomolecules through noncovalent or covalent

binding, or both, is the basis of their mode of action.

With some mycotoxins, such as aflatoxins, metabolic

activation is necessary for their binding with specific

macromolecules, but with others, metabolic acti-

vation is not necessary. Nevertheless, metabolism

plays a key role in modulating their toxic effects

because metabolism can produce either activated or

nontoxic metabolites for subsequent conjugation and

excretions. Thus, factors affecting the metabolism of

mycotoxins greatly affect the toxicity of mycotoxins.

Recent studies have shown that many mycotoxins

modulate apoptosis, and investigation of the mechan-

isms involved has become an effective tool for under-

standing the mode of action of mycotoxins.

0056Almost all of the mycotoxins are very stable; only a

limited number of detoxification methods are currently

available, and some of these are not economically feas-

ible. From a toxicological point of view, dietary modi-

fications can reduce the risk to some degree. Because

most of these control measures are not very effective,

rigorous programs for preventing human and animal

exposure to the contaminated foods and feed are im-

portant, and the availability of effective methods for

monitoring the toxin levels in the foods is essential.

Although a risk assessment has been carried out for

some mycotoxins, more epidemiological data for

human exposure are needed for establishing toxico-

logical parameters and safe doses in humans. The

mycotoxin problem may be with us for a long time,

and many mycotoxicoses are not well characterized. It

is unrealistic to eliminate the problem. Only through

multiple approaches can we minimize the problem and

enhance human and animal health.

See also: Aflatoxins; Alkaloids: Properties and

Determination; Toxicology; Carcinogens: Carcinogenic

Substances in Food: Mechanisms; Mushrooms and

Truffles: Classification and Morphology; Mycotoxins:

Classifications; Occurrence and Determination;

Nitrosamines; Spoilage: Fungi in Food – An Overview

MYCOTOXINS/Toxicology 4107

Further Reading

Beasley VR (1989) Trichothecene Mycotoxicosis, Patho-

physiologic Effects, vols I and II. Boca Raton, FL: CRC

Press.

Bhatnagar D, Lillehoj EB and Arora DK (eds) (1991)

Handbook of Applied Mycology, vol. V, Mycotoxins in

Ecological Systems. New York: Marcel Dekker.

Bondy GS and Pestka JJ (2000) Immunomodulation by

fungal toxins. Journal of Toxicology and Environmental

Health B Critical Reviews 3: 109–143.

Bucci TJ and Howard PC (1996) Effect of fumonisin myco-

toxins in animals. Journal of Toxicology (Toxin

Reviews) 15: 293–302.

Busby WF, Jr. and Wogan GN (1979) Food-borne myco-

toxins and alimentary mycotoxicoses. In: Riemann H

and Byran FL (eds) Food-borne Infections and Intoxica-

tions. New York: Academic Press.

CAST (1989) Mycotoxins, Economic and Health Risks.

Task Force Report No. 116. Ames, IA: Council of

Agricultural Science and Technology.

Chu FS (1997) Trichothecene mycotoxicosis. In: Dulbecco

R (ed.) Encyclopedia of Human Biology (2nd edn. vol.

8), pp. 511–522. New York: Academic Press.

Chu FS (1998) Mycotoxins – occurrence and toxic effects. In:

Encyclopedia of Food and Nutrition – Food Contamin-

ants (2nd edn, pp. 858–869. New York: Academic Press.

Cole RJ and Cox RH (1981) Handbook of Toxic Fungal

Metabolites. New York: Academic Press.

Dragan YP, Bidlack WR, Cohen SM et al. (2001) Implica-

tions of apoptosis for toxicity, carcinogenicity, and risk

assessment: Fumonisin B-1 as an example. Toxicologic

Science 61: 6–17.

Eaton DL and Gallagher EP (1994) Mechanisms of afla-

toxin carcinogenesis. Annual Review of Pharmacology

and Toxicology 34: 135–172.

Galvano F, Piva A, Ritieni A and Galvano G (2001) Dietary

strategies to counteract the effects of mycotoxins: A

review. Journal of Food Protection 64: 120–131.

Harris CC (1996) The 1995 Walter Hubert lecture – Mo-

lecular epidemiology of human cancer: Insights from the

mutational analysis of the p53 tumour-suppressor gene.

British Journal of Cancer 73: 261–269.

Jackson L, DeVries JW and Bullerman LB (eds) (1996)

Fumonisins in Food. New York: Plenum.

Miller JD and Treholm HL (1994) Mycotoxins in Grain:

Compounds Other Than Aflatoxin. St Paul, MN: Eagan

Press.

Pitt JI (2000) Toxigenic fungi: which are important?

Medical Mycology 38: 17–22.

Reiley RT, Voss KA, Yoo HS, Gelderblom WCA and

Merrill AH, Jr. (1994) Mechanism of fumonisin toxicity

and carcinogenesis. Journal of Food Protection 57:

528–535.

Sharma RP and Salunkhe DK (1991) Mycotoxin and

Phytoalexins in Human and Animal Health. Boca

Raton, FL: CRC Press.

Shier WT (2000) The fumonisin paradoxa review of re-

search on oral bioavailability of fumonisin B-1, a myco-

toxin produced by Farium moniliforme. Journal of

Toxicology – Toxin Reviews 19: 161–187.

Smela ME, Currier SS, Bailey EA and Essigmann JM (2001)

The chemistry and biology of aflatoxin B-1 from muta-

tional spectrometry to carcinogenesis. Carcinogenesis

22: 535–545.

Ueno Y (1986) Toxicology of microbial toxins. Pure and

Applied Chemistry 58: 339–350.

Van Egmond HP (1989) Current situation on regulations

for mycotoxins: Overview of tolerances and status of

standard methods of sampling and analysis Food Addi-

tives and Contaminants 6: 139–188.

Wild CP and Hall AJ (2000) Primary prevention of hepato-

cellular carcinoma in developing countries. Mutation

Research (Reviews in Mutation Research) 462:

381–393.

Wogan GN (1992) Aflatoxins as risk factors for hepato-

cellular carcinoma in humans. Cancer Research (supple-

ment) 52: 2114s–2118s.

4108 MYCOTOXINS/Toxicology

NATAMYCIN

L V Thomas and J Delves-Broughton, Danisco

Innovation, Beaminster, Dorset, UK

Introduction

0001 Natamycin (also known as pimaricin) is a polyene

macrolide antimycotic produced by Streptomyces

natalensis. It is marketed commercially as the Nata-

max

family of products by Danisco, and as the

Delvocid

family of products by DSM. Natamycin

is used worldwide as a food preservative; its primary

application is surface treatment of cheese and pro-

cessed meat (e.g., dry sausages) by dipping, spraying,

or in emulsion coatings. It has several advantages as

a preservative, including broad activity spectrum,

efficacy at low concentrations, lack of resistance,

and activity over a wide pH range. Due to its low

solubility, natamycin does not migrate from the sur-

face into the food, and thus does not affect the

organoleptic properties. It has no effect on bacteria,

including those used as starter cultures or to promote

ripening. It is chemically stable and has prolonged

effectiveness. Moreover, it is easy to apply and has a

proven safety record. Its general characteristics are

summarized in Table 1.

History

0002 Natamycin was first discovered in 1955, when it was

isolated from culture filtrates of an Actinomycetes

bacterium isolated from soil near the town of Pieter-

maritzburg, in the Natal province of South Africa.

The antimycotic was first called pimaricin, a name

derived from this place of origin. Its current name

natamycin, as well as the name of its producer

organism Streptomyces natalensis, was similarly

derived from Natal. Natamycin has also been pro-

duced from closely related Streptomyces strains

isolated from other locations and has been given vari-

ous names (e.g., tennecetin, antifungal A-5283) and

trade names. Its potential as a food preservative was

recognized shortly after its discovery, when its effi-

cacy against molds and yeasts was investigated in

foods including fruit juice, carbonated drinks, fresh

strawberries and raspberries, dressed poultry, saus-

ages, cottage cheese, and hard cheese.

Structure

0003Natamycin has the empirical formula C

and a molecular weight of 665.7. The structure was

first determined in 1958, and later revised. It belongs

to the group of polyene macrolide antifungals, com-

pounds characterized by possession of a macrocyclic

ring of carbon atoms closed by lactonization, and

four conjugated double bonds. This classes it as a

tetraene antimycotic (Figure 1). The structure is

closely related to other antimycotics such as nystatin,

rimodicin, and amphotericin. The molecule is ampho-

teric due to one basic and one acid group and has an

isoelectric point of 6.5.

Stability and Solubility

0004Natamycin is a white/cream-colored crystalline

powder with no taste and little odor. It is usually

found as the stable trihydrate formulation. No signifi-

cant loss of activity occurs for several months if the

powder is stored in the dark at room temperature.

Aqueous suspensions are less stable, particularly if

exposed to light, certain oxidants, and heavy metals,

but remain sufficiently stable during practical use.

Although solutions are more unstable in acid or alka-

line conditions, the pH of food products is not nor-

mally at levels that would cause problems. For

instance, natamycin stored at 30



C for 21 days re-

portedly retained 100% activity at pH 5–7, retained

c. 85% at pH 3.6, and 75% at pH 9.0. In acid

solutions, the molecule undergoes hydrolysis at the

glycosidic linkage, forming mycosamine and apona-

tamycin and subsequently other compounds. Aque-

ous suspensions at neutral pH are stable for 24 h at



C and show little reduction in activity at this

temperature for several days and for shorter periods

at 100



0005Natamycin is poorly soluble in water (30–100

p.p.m. at room temperature) and almost insoluble

in nonpolar solvents, but shows good solubility in

strongly polar organic solvents (Table 1). To be

active, natamycin must be in solution. However,

solubility is not normally a limiting factor as nata-

mycin is usually effective at relatively low concen-

trations. In solution, natamycin has an ultraviolet

absorption spectrum with minima at 250, 295.5,

and 311 nm, and maxima at 220, 290, 303, and

318 nm.

Mode of Action and Antimicrobial Effect

Mechanism of Action

0006Natamycin acts against yeasts and molds but is inef-

fective against bacteria. It combines with ergosterol

and other sterols such as 24, 28-dehydroergosterol

and cholesterol, compounds present in the cell mem-

branes of yeast and molds but (with rare exceptions)

not present in bacteria. The irreversible binding of

natamycin to sterols, in particularl ergosterol, dis-

rupts the cell membrane integrity and increases

membrane permeability. This results in leakage of

essential cellular constituents including cations, caus-

ing a rapid drop in intracellular pH and ultimately

cell lysis. A study of laboratory-forced natamycin-

resistant mutants of Aspergillus flavus has confirmed

this theory. These either had reduced levels of ergos-

terol or none at all (and consequently much slower

growth rates – critical to survival in vivo). A second-

ary mode of action is the inhibition of glycolysis and

respiration.

Antimicrobial Spectrum

0007Natamycin is active against an extensive range of

yeasts and molds (Table 2). The preservative is

usually effective at concentrations between < 1 and

10 p.p.m. In general, yeasts are more sensitive than

molds: the minimum inhibitory concentration (MIC)

of yeasts is usually < 5 p.p.m., whereas that of molds

can be at least 10 p.p.m. Examples of MICs are shown

COOH

fig0001 Figure 1 The structure of natamycin.

tbl0001 Table 1 General characteristics of natamycin

Characteristic Description

Names Natamycin, pimaricin (pimafucin),

tennecetin, antifungal A-5283

(trade names: Natacyn, Myprozine)

Commercial products Natamax

, Delvocid

Producer organism Streptomyces natalensis

EU number E235

Formula C

Molecular weight 665.7 Da

Structure Polyene macrolide antimycotic

compound with a macrocyclic ring of

carbon atoms closed by

lactonization, and four conjugated

double bonds

Properties Amphoteric. Isoelectric point: 6.5

Solubility in different Water: 30–100 p.p.m.

solvents n-Butanol: 50–120 p.p.m.

Glycerol: 15 000 p.p.m.

Methylpyrrolidone: 120 000 p.p.m.

Glacial acetic acid: 185 000 p.p.m.

Absorption spectrum Maxima: 290, 303, 318 nm

Minima: 250, 295.5, 311 nm

Antimicrobial spectrum Most molds and yeasts

(MIC: < 5–20 p.p.m.)

JECFA ADI 0.3 mg kg

1

body weight

MIC, minimum inhibitory concentration; JECFA, Joint Food and

Agriculture Organization/World Health Organization Expert Committee on

Food Additives; ADI, acceptable daily intake.

tbl0002Table 2 Antimicrobial spectrum: examples of yeast and molds

sensitive to natamycin

Absidia Penicillium camemberti

Alternaria P. chrysogenum

Aspergillus chevalieri P. digitatum

A. clavatus P. expansum

A. flavus P. glabrum

A. nidulans P. islandicum

A. niger P. notatum

A. ochraceus P. roqueforti var. punctatum

A. oryzae Rhizopus oryzae

A. penicilloides Rhodotorula gracilis

A. roquefortii Saccharomyces bailii

A. versicolor S. bayanus

Botrytis cinerea S. cerevisiae

Brettanomyces bruxellensis S. exiguus

Candida albicans S. ludwigii

C. guilliermondii S. rouxii

C. vini S. sake

Cladosporium cladosporioides Sclerotina fructicola

Fusarium Scopulariopsis asperula

Gloeosporium album Torulopsis candida

Hansenula polymorpha T. lactis var. condensi

Kloeckera apliculata Wallenis sebii

Mucor mucedo Zygosaccharomyces barkeri

M. racemosus

4110 NATAMYCIN

in Table 3. Less susceptible species include Verticil-

lium cinnabarinum, Botrytis cinerea, and Penicillium

discolour, and also occur among the genera Aspergil-

lus, Fusarium, Penicillium, and Trichophyton. The

outcome of antifungal activity is usually cidal, in

contrast to sorbate, which is fungistatic. Natamycin

can inhibit aflatoxin synthesis, furthermore overall

control of fungi leads to control of aflatoxins.

0008 Resistance to polyene antimycotics such as nata-

mycin does not seem to occur naturally and it has also

proved difficult to generate resistant mutants in the

laboratory. For example, in a survey of factories of

natamycin-producers, no detectable difference in

natamycin sensitivity was found for isolates com-

pared to other factories. Similarly, in both a cheese

warehouse and sausage factory where natamycin had

been used for some time, no resistant strains were

isolated. This lack of resistance may be partly ex-

plained by the fact that natamycin occurs as micelles

in solution. Thus if a cell comes into contact with

natamycin in solution, the antimycotic concentration

is high and consequently lethal. Furthermore, the

site of activity is an essential component of the cell

membrane.

Factors Affecting Efficacy

0009 The antimycotic activity of natamycin is affected by

several factors, which may also affect its stability,

such as pH, temperature, light, oxidants, and heavy

metals. Natamycin is active over a wide pH range (pH

3–9); pH does not appear to affect its antimycotic

activity but does affect stability. Compounds such as

peroxides or chlorides, often used as cleaning/

disinfecting agents, should be used with care in the

proximity of natamycin.

Methods of Assay

0010 The natamycin content of food products can be

determined by microbiological, immunochemical,

spectrophotometric, and liquid chromatographic

(LC) procedures. The agar diffusion bioassay using

Saccharomyces cerevisiae can also be used for quan-

titative assessment. Spectrophotometric and LC

methods are commonly used for routine analysis.

High-performance liquid chromatography (HPLC)

with ultraviolet detection is considered one of the

most sensitive and accurate methods, with a detection

limit of 0.5 mg kg

1

.(See Chromatography: High-

performance Liquid Chromatography.)

0011International Dairy Federation standard 140A:

1992 specifies the method for determining the nata-

mycin contents of cheese rind and cheese adjacent to

the rind. In this method a known quantity of sample is

extracted with methanol, which is then diluted with

water and cooled to between 15 and 20



Cto

congeal and precipitate the majority of fat, followed

by filtration. The natamycin content can then be de-

termined in the filtrate by either a spectrometric or

HPLC method. For the spectrometric measurement

the spectrum of both a natamycin standard solution

and the test sample is recorded in the range 300–

340 nm. Absorbance is measured at the maximum of

approximately 318 nm, at the minimum of approxi-

mately 311 nm, and then at 329 nm. For measure-

ment by HPLC, ultraviolet detection should be at

308 nm and it is recommended that the mobile

phase of the column should comprise methanol/

water/acetic acid, 60 þ40 þ5 (v/v/v). Before analysis

of test samples, a standard natamycin solution is

first injected to determine retention time and for

calibration.

0012More recently, a rapid method using derivative

spectrophotometry has been described. In this

method cheese is extracted with acidified aqueous

acetonitrile, and natamycin content is directly quan-

tified in filtered extracts on the basis of the depth

of the trough at 322.6 nm after third-derivative

processing of the normal ultraviolet spectrum. An

enzyme immunoassay involving the use of a natamy-

cin–protein conjugate has also been reported, in

which natamycin coupled to horseradish peroxidase

was used as the labeled ligand. Detection limits

were 200–2000 pg ml

1

, which enabled the determin-

ation of concentrations as low as 0.005 mg dm

2

(0.1 mg kg

1

) in cheese rinds.

Toxicology and Legislation

0013Toxicology studies have been undertaken using mice,

rats, rabbits, and guinea pigs. Natamycin was least

toxic if administered orally (LD50 ¼1500 mg kg

1

rats and mice) or subcutaneously (LD50 ¼5000 mg

1

in rats), and most toxic if administered intraven-

ously (LD50 ¼5–10 mg kg

1

). No natamycin was

tbl0003 Table 3 Examples of sensitivity of yeasts and molds to

natamycin

Strain Minimal inhibitory concentration

(p.p.m.)

Aspergillus chevalieri 4298 0.1–2.5

Saccharomyces cerevisiae H 0.15

Penicillium chrysogenum 0.6–1.0

Aspergillus niger 1.0–1.8

Saccharomyces bailii 1.0

Candida albicans 1–2.5

Mucor mucedo 1.2–5.0

Penicillium notatum 4640 5.0

Saccharomyces rouxii 0562 5.0

Rhizopus oryzae 4758 10.0

NATAMYCIN 4111

absorbed from the human intestinal tract after 7 days’

feeding of up to a maximum of 500 mg day

1

. Feed-

ing studies have been conducted in rats, rabbits,

and dogs. The acceptable daily intake was set at

0.3 mg kg

1

body weight per day in 1976.

0014 Specification of natamycin in the USA (21 CFR

172.155) requires purity of the anhydrous compound

to be 97% + 2%, containing < 1 p.p.m. arsenic and

not more than 20 p.p.m. heavy metals.

0015 Natamycin is approved for use as an antimycotic in

various cheeses and processed meats in 32 countries

worldwide. A more general use as a food additive

is allowed in a few countries such as South Africa

(Table 4). In the European Union (EU) natamycin

(designated E235) is permitted for surface treatment

of hard, semihard, and semisoft cheese as well as dry

sausages. The maximum surface coverage permitted

in the EU is 1 mg dm

2

, and penetration is restricted

to 5 mm from the surface. In the USA, natamycin is

permitted only if the cheese standard allows the use of

‘safe and suitable’ antimycotics. Table 4 is a guide to

food legislation. This list is not comprehensive – one

should be aware that legislation for food additives

is under constant review. The reader is advised to

check the current legal situation with the appropriate

authorities.

tbl0004 Table 4 Food legislation on the use of natamycin

Country Food in which natamycin is permitted Maximum permitted level

Algeria Cheese rinds Used in suspension at 2.5 g l

1

Argentina Surface treatment of hard and semihard

paste cheeses

Limit of 1 mg dm

2

. Penetration limit of 2 mm

Australia Surface treatment of cheese rind Limit of 15 mg/kg

Uncooked fermented manufactured

meat products

Penetration limit of 3–5 mm

Bahrain Permitted food preservative

Brazil Surface treatment of cheese Limit of 2 mg dm

2

Bulgaria Cheese rind 500 mg kg

1

Canada Surface treatment of 47 listed cheeses 20 mg kg

1

based on total weight

Grated/shredded cheese (0.5% sodium

lauryl sulfate prohibited as dispersant)

10 mg kg

1

Chile Surface treatment of hard cheese

(prohibited in wine)

China Surface treatment of cheese, processed

meat products, moon cakes, baked

goods, fruit juices, and processing

utensils for easily moldy foods

Application by spraying or dipping in 200 – 300 mg kg

1

to leave a residue of <10 mg kg

1

Colombia Cheese 12.5 mg kg

1

Czech Republic Dairy and meat products – as EU

regulations (contact authorities for

further information)

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Cyprus Surface treatment of specified cheeses

Egypt Surface treatment of cooked cheese

(dehydrated, semidehydrated, and

semisoft cheese)

Limit of 2 mg 100 cm

2

(1 mg dm

2

)

European Union (EU) Surface treatment of specified cheese

and sausage

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Hungary Surface treatment of hard and semihard

cheese, dried, cured sausage

Limit of 1 mg dm

2

Iceland Surface treatment of ripened and whey

cheese

Limit of 2 mg dm

2

India Surface treatment of hard cheese Maximum application level: 2 mg dm

2

. Maximum residual

level in finished cheese: 1 mg dm

2

Penetration limit of 2 mm

Israel Surface treatment of specified cheese

Kuwait Permitted food additive

Lithuania Surface treatment of hard, semihard, and

semisoft cheese

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Mauritius Surface treatment of hard, semihard,

and semisoft cheese and dried cured

sausage

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Continued

4112 NATAMYCIN

Country Food in which natamycin is permitted Maximum permitted level

Mercosur Surface treatment of cheese Limit of 1 mg dm

2

. Maximum application of 5 mg kg

1

Penetration limit of 2 mm

Mexico Cheese surfaces Limit of 0.002 %

Norway Maximum level of suspension: 2 g kg

1

Surface treatment of hard, firm, and

semifirm cheese, dried, cured sausage

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Oman Surface treatment of specified cheese

Philippines Surface treatment of specified cheese

Poland Permitted in colored and uncolored soft

wax and polyvinyl acetate for

application to skin of hard cheese

No limits

Surface treatment of smoked, dried sausage Limit of 1 mg dm

2

Saudi Arabia Permitted as a mold inhibitor in

foodstuffs but controlled by standards

of composition

Slovakia Surface treatment of cheeses and dried,

cured sausage

Limit of 1 mg dm

2

South Africa Wine, alcoholic fruit beverages, and

grape-based liquors

30 mg l

1

Fresh fruit pulp 5 mg kg

1

Fruit juice (blackcurrant, pineapple, etc.) 5 mg kg

1

Fish sausages 6 mg kg

1

to be applied to the outer inedible casing only

Manufactured fish products, fish pastes,

fish roe and spawn, with the exception

of frozen fish, salted snoek,

and canned fish products

6mgkg

1

Lobsters (quick frozen) 6 mg kg

1

Edam, Gouda, Tilsiter, Limburger,

Cheddar, Cheshire

2mgkg

1

in rind without plastic coating;

500 mg kg

1

in a plastic coating; 10 mg kg

1

for

application to the surface of the cheese only

Cottage cheese, cream cheese Limit of 10 mg kg

1

Process or blended cheese, including

cheese spread, process cheese

preparations, and soft cheese

10 mg kg

1

for application to the surface of the

cheese only

Yogurt 10 mg kg

1

Canned foods 6 mg kg

1

Manufactured meat products Limit of 500 mg kg

1

on casing or 6 mg kg

1

in contents

Canned chopped meat, canned corned

beef, cooked cured luncheon meat,

cooked cured pork shoulder,

Biltong, frozen cooked-meat pie fillings

Limit of 6 mg kg

1

Tunisia Surface treatment of hard, semihard and

semisoft cheese; dried, cured sausage

Limit of 1 mg dm

2

Turkey Surface treatment of hard and semihard

cheese, dried cured sausages, salami,

and hot dogs

Limit of 1 mg dm

2

. Penetration limit of 5 mm

Ukraine Surface treatment of cheese Limit of 1 mg dm

2

. Penetration limit of 5 mm

United Arab Emirates Permitted food additive

USA Surface treatment of cuts and slices of

cheese

Limit of 20 mg kg

1

Nonstandard of identity yogurt Limit of 7 mg kg

1

Nonstandard of identity cream cheese Limit of 7 mg kg

1

Cottage cheese Limit of 7 mg kg

1

Sour cream Limit of 7 mg kg

1

Soft tortillas Limit of 20 mg kg

1

Nonstandard of identity salad dressing Limit of 20 mg kg

1

Venezuela Surface treatment of specified cheeses

and sausages

Maximum 0.5% suspension

Table 4 Continued

NATAMYCIN 4113