1395
Journal of Food Protection, Vol. 58, No. 12, Pages 1395-1404
Copyrighl©, International Association of Milk, Food and Environmental
Aspergillus flavus and Aspergillus parasiticus: Aflatoxigenic
Fungi of Concern in Foods and Feedst: A Review
HASSAN GOURAMA* and LLOYD B. BULLERMAN*
Department of Food Science and Technology, University of Nebraska, Lincoln, Nebraska 68583-0919
(MS# 91-230: Received 22 December 19911Accepted 15 May 1995)
ABSTRACT
Aspergillusflavus and the closely related subspeciesparasiticus
have long been recognized as major contaminants of organic and
nonorganic items. A. flavus, a common soil fungus, can infest a wide
range of agricultural products. Some A. flavus varieties produce
aflatoxins, which are carcinogenic toxins that induce liver cancer in
laboratory animals. A.flavus var.flavus, A.flavus subsp. parasiticus,
and A. nomius share the ability to produce aflatoxins. Identification
of the A. flavus species group is mainly based on the color and
macroscopic and microscopic characteristics of the fungus. A. flavus
growth and aflatoxin biosynthesis depend on substrate, moisture,
temperature, pH, aeration, and competing microflora. The growth of
A. flavus and aflatoxin production are sometimes unavoidable.
Aflatoxins are considered natural contaminants; the ideal control
approach is prevention of mold growth and aflatoxin production. The
detection of members of the A.flavus species group in foods and feed
is generally carried out by using plate techniques such as surface
spread or direct plating. Research on alternative fungal detection
methods is still in its infancy. Few immunoassay techniques have
been investigated in this regard. Aflatoxins are generally analyzed by
chemical methods, although immunochemical methods which use
antibodies are becoming common analytical tools for aflatoxins.
Key words: Aspergillus flavus, Aspergillus parasiticus, aflatoxins
Species of Aspergillus have long been known to be
common contaminants of human food and animal feeds.
Antonio Micheli in 1729 (84) was the first to name the genus
Aspergillus. Micheli's monograph revolutionized mycology
into a major science. Aspergillus species affect food products,
wood, leather, textiles, kerosine, paint, plastics, rubber, ce-
ment, and pharmaceutical items (114). They possess a high
metabolic versatility and a great ability to disperse their
spores. Micheli noticed that the spore chains rise radially from
the vesicle, which he thought resembled a holy-water sprin-
kler (aspergillum). Micheli's Latin descriptions of the mold
t Published as Paper No.1 1009 Journal Series, Agricultural Re-
search Division, Lincoln, Nebraska 68583. Research was con-
ducted under Project 16-042.
t Present Address: Food Science, Penn State Berks Campus,
Tulpehocken Road, P. O. Box 7009, Reading, PA 19610-6009.
JOURNAL
Sanitarians
USA
isolates are still used as specific names, e.g., Aspergillus
capitatus, Aspergillus niger, etc. (98). Aspergillus are com-
mon saprobic molds, which grow in a wide range of natural
substrates and climatic conditions. Austwick (8) reported that
one conidial head may produce up to 50,000 spores. Aspergil-
lus spp. are of particular importance to humans. Although
many Aspergillus species are considered pathogens or spoil-
age organisms, many others are beneficial. Some species are
used to prepare fermented foods (57). Aspergillus spp. can
also be allergenic, toxigenic, and pathogenic to humans and
animals. Pathogenic Aspergillus spp. represent a real hazard
to animal health: they can produce numerous diseases such as
avian aspergillosis and bovine mycotic abortion (2). Mold
spores can cause hypersensitivity reactions in sensitized pa-
tients, fibrosis, and hypersensitivity pneumonitis. Aspergillus
spp. also produce mycotoxins, which are formed as secondary
metabolites and cause mycotoxicoses in animals and humans
(29). Aflatoxins which are produced by some Aspergillus
jlavus species are considered potent carcinogens (12).
MORPHOLOGICAL DESCRIPTION OF
ASPERGILLUS FLA VUS
The production of phialides and foot cells demonstrates
the presence of Aspergillus species (95, 108). The production
of phialides alone is not enough to characterize aspergilli.
Although phialides are common to most Aspergillus spp. they
are also formed by Penicillium species. Raper and Fennell
(98) reported that the presence of foot cells is evidence that a
mold isolate is Aspergillus; however, the absence offoot cells
does not prove that the isolate does not belong to Aspergillus
group. Most morphological descriptions oftheA.jlavus group
were done on standard media such as Czapek's solution agar
and malt extract agar.
Conidial head
The conidial head characteristics such as color, shape,
and size are important key diagnostic criteria of the Aspergil-
lus group (94, 98). The shape of conidial heads varies from
columnar to radiate and globose. The arrangement of phialides
on the vesicle dictates the shape of the conidial head. The size
of the conidial head is determined by the size of the vesicle and
OF FOOD PROTECTION, VOL. 58, DECEMBER 1995
1396 GOURAMA
the length of the conidial chains. The color of the conidia
determines the color of the conidial head; thus the color of
A. jlavus is light to deep yellow green, or olive green.
Conidiophore
The conidiophore, which is also known as the stalk, is a
thick-walled branch which arises perpendicularly from the
foot cell (Fig. 1) (67). Generally conidiophores are unbranched
and are composed of three parts: the foot cell, stipe, and
vesicle. Although conidiophores are usually nonseptate,
septation may occur in certain species (98). The character of
the outer surface of the conidiophore can vary from smooth to
rough. These features constitute a key element for the identi-
fication of Aspergillus spp. The conidiophores in the A. jlavus
group are rough and hyaline (nonpigmented).
Figure 1. Conidiophores characteristic of Aspergillus spe-
cies, showing a single layer of phialides or sterigmata
(uniseriate) and double layer of cells, phialides and metulae
(biseriate). (Adaptedfrom Klich and Pitt (67); drawn by A. D.
Hocking).
Vesicle
The vesicle is the swollen apex of the conidiophore. The
shape can be globose, hemispherical, elliptical, or clavate
(club shaped). Vesicles in the A. jlavus group are elongated
when young and become globose as the culture ages. The
shape varies with the composition of the substrate. The
diameter varies from 10 to 65 ~m (98). These features of the
vesicle are important diagnostic characters for identification
of Aspergillus species (Fig. 1).
Sterigmata
Sterigmata (singular sterigma) are defined as specialized
conidiogenous cells which develop on the fertile area of the
vesicle. Sterigmata on the vesicle are uniseriate or biseriate,
depending on whether one (uniseriate) or two (biseriate)
layers of cells are present. The primary (first layer) sterigmata
are called metulae, while the secondary (second layer) ones
are called phialides (Fig. I). In the uniseriate species, the
phialides are produced directly from the vesicle, while for the
biseriate species, the phialides arise from the metulae. The
phialides are the conidia-bearing cells (each phial ide bears a
JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER
AND BULLERMAN
chain of conidia). Generally phialides are nonseptate. Al-
though most A. jlavus species are biseriate (two layered),
some species are uniseriate (single layered).
Spores (conidia)
Conidia (singular: conidium), also called spores, are
asexual reproductive structures. Conidia in Aspergillus spp.
are one-celled structures that can be uni- or multinucleate. The
phialide is the conidiogenous cell. At the initial stages of spore
formation, the tip ofthe phialide is ruptured and a cytoplasmic
mass moves to the neck of the phial ide, forming a bulbous
structure (54). New wall material is formed to close the new
structure which forms the first conidium. The new conidia are
formed beneath the earlier ones, so the oldest conidium is at
the apex of the chain while the youngest is at its base. After
mitosis in the phialide, the newly formed nuclei move into the
young conidium. The formation of an inner conidial mem-
brane severs the cytoplasmic connection. The conidia of the
Aspergillus group are found in various shapes and colors and
can be smooth or roughened.
Sclerotia
Certain species of the A. jlavus group produce sclerotia
(singular: sclerotium). These are compact, hard masses of
mycelia, which vary in size and shape. The color of sclerotia
varies from yellow to brown or black. Sclerotia are survival
structures which the fungus uses to overwinter in the soil.
Production of sclerotia is an important diagnostic clue for
some Aspergillus species (98).
Cleistothecia and Hulle cells
Hulle cells are thick-walled cells produced by some
Aspergillus species. Cleistothecia are ascocarps (fruiting bod-
ies) without an opening, that are involved in sexual reproduc-
tion. They are found in the teleopmorphs of A. nidulans and A.
amstelodami. They are not known to be produced in the A.
jlavus group (98).
CLASSIFICA TION AND IDENTIFICATION
Aspergillus taxonomy is mainly based on morphological
characteristics. Antonio Micheli applied the name Aspergillus
to the imperfect state (anamorph) of the fungus (81) . Later, the
perfect state (teleomorph) was discovered in some Aspergil-
lus species, which created confusion in the taxonomy of the
Aspergillus group. Raper and Fennell (98) classified the genus
Aspergillus in both the Eurotiacea and Moniliacea families.
They divided the genus into 18 groups and 132 species with 18
varieties. Minor revisions have been made since then (81).
Samson and Van Reenen-Hoekstra (101) introduced 42 addi-
tional taxa and produced a useful survey of species described
since 1945. Raper and Fennell (98) summarized the important
lines of demarcation into the following criteria: shape and
color of conidial heads, characteristics of conidiophores,
presence or absence of metulae, size of sterigmata, and the
presence or absence of Hulle cells.
The use of appropriate media is essential to achieve an
accurate identification of the Aspergillus species. Raper and
Fennell's keys (98) have been universally used for the last 30
years and described the species when it is grown on malt
1995
 ASPERG1ILUS FLA VUS AND A. PARAS1TlCUS
extract agar and Czapek agar. Generally potato dextrose agar
(PDA) may also be used; however, appearance on PDA is not
considered to be consistent enough to be useful diagnosti-
cally. Selective media such as Aspergillusjlavus-parasiticus
agar (AFPA) (91) can be used to identify A.jlavus species. The
major taxonomic references (67, 98, 112, 113) base the
identification keys on the color and morphology of the fungi,
which in many cases is not reliable. The characteristics can
change with media, growth conditions, and mutations.
Kozakiewicz (68) suggested the use of scanning electron
microscopy (SEM) to solve this problem. With the high
resolution of SEM, diagnostic keys of Aspergillus could be
based on the differences in the ornamentation of the conidia.
Kozakiewicz (68) demonstrated that the genetic stability of
the spore ornamentation is maintained under different condi-
tions of physiological and environmental stress. The conidium
ornamentation has been divided into nine categories that
varied from echinulate to smooth. In a more recent publication
by the same author (69), it was reported that based on conidial
ornamentation, A. jlavus species fall into two distinct catego-
ries, echinulate or lobate-reticulate. The roughened lobate-
reticulate type of conidia are evident in Fig. 2. Using SEM,
Kozakiewicz (69) found that many of the A. parasiticus
cultures obtained from different world collections were
misidentified. In addition, SEM is a useful tool to differentiate
between mixed related isolates such as A. parasiticus,
A. oryzae and A. jlavus. Kurtzman et al. (71) described
Aspergillus nomius as a new af1atoxigenic species that is
phenotypically similar to A. jlavus. The separation between
the two species was based on the presence of intermediate
sclerotia and low growth temperature.
DETECTION OF A. FLA VUS IN FOODS AND FEEDS
Generally, detection of A. jlavus in foods and feeds is
carried out by using traditional microbiological plating meth-
ods, either surface spread or direct plating of kernels and
seeds. Various media are used for this purpose: PDA, acidi-
fied PDA and PDA with antibiotics. Different antibiotics can
be used: chlortetracycline, chloramphenicol, oxytetracycline,
Figure 2. Electron micrograph of the conidial head of As-
pergillus f1avus showing lobate-reticulate conidia. (Courtesy
of Dr. R. A. Samson, Centraalbureau voor Schimmelcultures,
Baarn, The Netherlands).
JOURNAL OF FOOD PROTECTlON.
1397
gentamicin and streptomycin. Other media can be used, such
as malt extract agar and Czapek agar. None of these media are
selective forA. jlavus, as other mold genera such as Penicil-
lium and Fusarium also grow on these media. King et al. (65)
developed a medium using dichloran and rose bengal to make
dichloran-rose bengal-chloramphenicol agar (DRBC), which
restricts the spreading of fungal colonies without affecting
spore germination.
In 1974, Bothast and Fennell (23) developed a differen-
tial medium for A.jlavus species, called Aspergillus differen-
tial medium (ADM). The differential ingredient of ADM is
ferric citrate (0.05%), which reacts with fungal metabolites
such as kojic acid and aspergillic acid to produce a bright
orange-yellow pigment on the reverse side ofthe colony. Pitt
et al. (96) added dichloran and chloramphenicol to ADM to
make a new medium called Aspergillusjlavus and parasiticus
agar (AFPA). AFPA is composed of peptone, 10 g; yeast
extract, 20 g; ferric ammonium citrate, 0.5 g; chloramphenicol,
100 mg; agar, 15 g; distilled water, 11; and dichloran, 2 mg.
The final pH of this medium is ca. 6.2. Cultures on AFP A are
incubated at 30°C for 42 to 48 h. Dichloran inhibits spreading
of fungi, while chloramphenicol inhibits bacteria. A. jlavus
and A. parasiticus are identified on this medium by production
of typical yellow to olive green spores and a bright orange
reverse. Another advantage of this medium is that A. jlavus
and A. parasiticus grow rapidly because the medium is incu-
bated at 30°C, permitting identification within 3 days in most
cases. This medium was recommended for use in enumerating
A.jlavus species in nuts, com, spices and soil (95). Aspergillus
niger produces a yellow but not orange reverse color, and after
48 h of incubation A. niger starts to develop its dark'brown to
black conidia, which easily distinguish it from A. jlavus.
Aspergillus ochraceus grows slowly at 30°C and the yellow
color appears after 48 h (95). The use of AFPA shortens the
time required to isolate and identify A. jla vus species. Another
advantage is the isolation and identification of potentially
af1atoxigenic fungi. In many laboratories it is easier and more
economical to first look for af1atoxigenic fungi to identify
contaminated materials than to chemically test for af1atoxins.
Another potential detection method for A.jlavus species
is the use of immunoassays. Notermans and Heuvelman (87)
developed an enzyme-linked immunosorbent assay (ELISA)
to detect different mold species in foods. Molds produce
extracellular polysaccharides that are cell bound and immu-
nologically active. The antigens were found to be genus
specific and heat stable and not found in nonmoldy food
samples. Holland Biotechnology (HBT) in The Netherlands
has developed a mold latex immunoagglutination kit to detect
antigens produced by Aspergillus and Penicillium species.
More work needs to be done to develop specific kits for
mycotoxigenic species such as A. jlavus.
BIOLOGY AND HABITAT OF A. FLA VUS
Aspergillus jlavus species are present in soil and contami-
nate a wide variety of agricultural products in the field, storage
areas, and processing plants and during distribution. Ajlavus,
A.jlavus subsp. parasiticus, and A. nomius are the only molds
that have so far been shown to produce af1atoxins (71).
Aspergillus oryzae and Aspergillus tamarii were found to be
VOL. 58. DECEMBER 1995
1398 GOURAMA
nontoxic. A. flavus strains range from nontoxic to those that
produce aflatoxins B} and B2, whereas A. flavus subsp.
parasiticus produces aflatoxins BI' B2, GI, and G2• A. flavus
subsp. parasiticus tends to be more stable in producing
aflatoxins than A. flavus. All aflatoxin-producing fungi are
soil microorganisms, but there are some differences in the
pattern of occurrence (43,44). A.flavus spores occur more in
air than soil, and are generally found in temperate regions of
the world. A. flavussubsp. parasiticus is adapted to warmer
environments such as the tropical and subtropical regions, and
it has been found to be associated most often with soil (43).
Thus, A. parasiticus is the more common contaminant of
peanuts, while A. flavus is more common in corn. Wicklow et
al. (119, 120) reported that sclerotia are the primary inoculum
in corn fields. Klich and Pitt (66) examined more than 150
isolates of A. flavus, A. oryzae, A. parasiticus, A. sojae, and A.
tamarii for taxonomic criteria. Ornamentation of conidia was
found to be the most effective criterion for distinguishing A.
flavus andA. parasiticus. Conidia fromA.flavus isolates were
smooth to slightly roughened, while conidiafromA. parasiticus
were rough. Other criteria such as aflatoxin production are
needed to distinguish A. flavus from A. oryzae.
AFLATOXINS
Introduction
Aflatoxins, a group of toxins structurally related to sev-
eral secondary fungal metabolites, are produced by
A. flavus, A. flavus subsp. parasiticus and A. nomius (71).
Aflatoxins were discovered in 1960 after the toxic outbreak in
England that became known as "Turkey-X disease." In this
outbreak thousands of turkey poults died after consuming
contaminated Brazilian groundnut (peanut) meal (21). The
main microbial contaminant of the peanut meal was identified
as Aspergillusflavus. A chemical analysis of the peanut meal
yielded a series of toxic compounds that fluoresced under UV
light. These compounds were named aflatoxins (for A.flavus
toxin.) There are four main aflatoxins, BI, B2, GI and G2 (Fig.
3). The B group are bifurano coumarins fused to
cyclopentanone, and the G group are bifurano coumarins
fused to a lactone. The B group fluoresces blue in long-
wavelength ultraviolet light, while the G group fluoresces
green. The subscripts 1 and 2 designate the chromatographic
mobility (Rf values) pattern of the compounds on thin layer
chromatography (TLC) plates.
Dutton and Heathcote (46) characterized the hemiacetal
derivatives of aflatoxin B) (AFB)) and aflatoxin GI (AFG))
that were designated B2a and G2a (Fig. 3). Biotransformation
of aflatoxin in several animal species results in the production
of aflatoxin M1 (AFM) and aflatoxin M2 (AFM). These
aflatoxins (AFM1, AFM2) were first isolated from the milk
and urine of animals fed aflatoxins (3, 63). Later on, two
hydroxy derivatives of aflatoxin Gl and G2, aflatoxins GM}
and GM2, respectively, were isolated and characterized (56).
The four major aflatoxins (AFB I' AFB} AFG}, AFG) and the
minor aflatoxins (AFMI' AFM2, AFB2a, AFG2a, GM} and
GM2) are considered "naturally occurring" toxins.
B iosynthes is
The biosynthetic pathways for aflatoxins have been ex-
tensively investigated (16,17,18,73,74,75). Aflatoxins are
JOURNAL OF FOOD PROTECTION,
AND BULLERMAN
coP
o 0
1~
CCJCCCHS
o 0
liZ
o o o o
cc&
o
1~
H
CC;CCHS
(HZ ~CHZCH
o 0 o 0 -" OeHS
Aflatoxicol Parasitical
Figure 3. Chemical structures of aflatoxins.
considered secondary metabolites, which are defined as com-
pounds that are not essential for growth. The main precursors
of the secondary metabolites are acetate "polyketides." The
different aflatoxins are produced as a family of chemical
compounds from acetate and malonate building blocks that
are produced during the idiophase. The functional role of
aflatoxin in the producing fungus is not known. The four main
aflatoxins (AFB" AFB2, AFGI, and AFG2) are CI7 com-
pounds classified as nonaketides (115). Most of the aflatoxin
biosynthetic studies have used precursors that were isotopi-
cally labelled and precursor accumulating mutants which do
not form aflatoxins. Many investigators, by using isotope-
labeled acetate, concluded that aflatoxin BI, is derived from
acetate (7, 17, 97). The methoxy group in the aflatoxin
molecule is derived from methionine. The following com-
pounds have been determined to be intermediate compounds
in the biosynthesis of aflatoxin BI: norsolorinic acid, averantin,
averufin, versiconal hemiacetal acetate, versicolerin A and
sterigmatocystin (Fig. 4). Sterigmatocystin is naturally pro-
duced by Aspergillus versicolor (16). Singh and Hsieh (107)
reported that the mutant strain AVR -1 converts versicolorin A
and sterigmatocystin into AFBj' and AFGI.
Many investigators have proposed that all the other
aflatoxins are derived from AFB( (58, 79, 80). However, the
biosynthetic relationship between the AFB group and the
AFG group is controversial. Bhatnagar et al. (18) and Cleve-
VOL. 58, DECEMBER ]995
 ASPERGILLUS FLAVUS AND A. PARASlTICUS 1399

"'lo S r
0\1
~
toxins occurs after 4 to 7 days at 24°C (64). Moisture content
o ~ 0 -\10 ",I 10\1 of the substrate and relative humidity are also critical to
POLYKETIDE NORSOLORIN IC ACID
aflatoxin production. Diener and Davis (42) reported that
maximum aflatoxin production occurred in corn kernels with
a moisture content of 25% at 30°C. The minimum relative
0\100\1011 ~" 0\10011
I _ I 0 _ "I I 0 humidity (RH) for aflatoxin production varies between 83%
IO~ 110 .
o
"0 CII, 1I0~0A..--o
0
and 88%. Aflatoxin yields increase with increasing RH up to
AVERANlIN AVEIlUFIN VERSICONAL HEMIACETAL 99%. The degree of aeration is also important for aflatoxin
ACETATE
production, because mold growth and aflatoxin production
0\1 0 011
are aerobic processes. Many workers have reported that
0100
~rll" I
"O
higher yields of aflatoxin are produced in shaken flasks rather
.
II~O.J...O..p o r
o ~ 1I,c0 '"
I
0 0
I
~II,CO
I I than stationary flasks (64). Landers et al. (72) showed that
0 0
VERSICOLORIN A STER IGMATOCTSTIN AFLATOX IN 8. increasing CO2 concentration from 20% to 100% gradually
inhibited aflatoxin production. Various reports have stated
Figure 4. Biosynthetic pathway of aflatoxin Bland structures that initial pH does not significantly affect aflatoxin produc-
of known intermediates. tion, while other investigations have shown that higher yields
ofaflatoxins are obtained at acid pHs (4 to 6) (64). Buchanan
land et al. (34) reported that O-methylsterigmatocystin is an and Ayres (26) reported that pH values of less than 6 favored
intermediate between ST and AFB" Dutton et al. (47) re- aflatoxin B J and B2, while a pH higher than 6 favored the
ported that the AFBJ' and AFB biosynthetic pathways are production of aflatoxins G1 and Gr Basappa et al. (13) re-
. d 2 ported that maximal yields were obtained when the media
10 ependent. Yabe et al. (122) found that aflatoxins contain-

ing dihydrobisfuran (AFB J' AFG) are produced from dihydro were buffered at pH 5 to 6. There are conflicting reports
sterigmatocystin and that both pathways seem to be catalyzed concerning the final pH in aflatoxin production; these varia-
by the same enzymes. tions are mainly due to the medium used (64). Substrates
Many substances have been reported to inhibit or affect either natural or laboratory media exert a strong effect on
aflatoxin production, including benzoic acid (31), sorbic acid aflatoxin production. Natural substrates such as cereal grains
(124), ferulic acid (124), oleuropein (51), propionic acid have proven to be good substrates for aflatoxin production in
(124), vanillin (19), cinnamon oil (9, 28), plant extracts (19, laboratory studies. Defined culti vation media, either simple or
60), caffeic acid (124), spices (9,59, 60, 77), herbal drugs (11) complex, are useful substrates for studying aflatoxin produc-
fungicides, insecticides and other compounds (112). How- tion. Generally, the preferred carbon sources for aflatoxin
ever, most of these compounds inhibittoxin production through production are glucose, sucrose or fructose. Glycine and
inhibition of fungal growth. The mechanism by which most of glutamic acid were found to be essential single amino acids for
these compounds inhibit aflatoxin production is not well aflatoxin production (64). Buchanan et al. (27) reported that
known and needs further investigation. Zaika and Buchanan aflatoxin biosynthesis occurs during the period of depressed
(124) published an excellent review of this subject. TCA cycle activities. The effect of minerals on aflatoxin
production is variable; zinc and manganese are essential for
Factors that affect aflatoxin production aflatoxin biosynthesis, and a mixture of cadmium and iron
Aflatoxin production is the consequence of a combina- were found to stimulate aflatoxin production; however, iron
tion of fungal species, substrate, and environment. The factors depressed mold growth and hence aflatoxin production. Nu-
affecting aflatoxin production can be divided into three cat- merous substances have been reported to inhibit aflatoxin
egories: physical, nutritional, and biological factors. production. Examples of these substances are dichlorvos (45,
Physical factors include temperature, pH, relative humid- 61), selenite (10), nitrate (124), ethylene (105), benzoic acid
ity and moisture, light, aeration, and level of atmospheric (31), oxygen (106), azide (106), epoxy derivatives, peroxy
gases. The optimum temperature for aflatoxin production derivatives (124), oleuropein (51), sorbic acid, butylated
depends on the substrate. In liquid media the optimal tempera- hydroxyanisole (BHA), potassium sulfite, trace metals, and
ture for A.flavus was shown to be 25°C, while for A. parasiticus caffeine (48, 124). Competing mycoflora such as A. niger,
the temperature varied between 25 and 35°C (41) and afla- Rhizopus oligosporus, and Neurospora spp. have also been
toxin production did not occur below 13°C and above 42°C. found to inhibit aflatoxin production (64).
Sorenson et al. (110) reported that the optimum temperature
for aflatoxin BJ and GJ production on rice was 28°C; aflatox- Biological effects
ins were not detected below 8°C or at 37°C and above. Thus, Effects of aflatoxins on animal health vary from species
the optimum temperature for aflatoxin production is generally to species. Animal species such as calves, chicks, ducklings,
accepted to be 25 to 28°C, although production can occur over guinea pigs, and pigs are susceptible to aflatoxin Bl' while
a range of temperatures. The incubation period for maximum goats, rats, mice, and sheep (90) are relatively resistant. The
production depends on the strain and the substrate or medium susceptibility to acute aflatoxicosis can be determined by the
used. Maximum levels of aflatoxins are also linked to the LDso (mglkg of body weight). LDsos for various animals are
exhaustion of sugars in the medium and the onset of mycelial as follows: duckling 0.3 to 0.5; rabbit, 0.3 to 0.5; rainbow
autolysis. It has been reported that the maximum yields occur trout, 0.5; dog, 1.0; pig, 0.62; monkey, 2.2; chicken, 6 to 16;
after 15 days at 20°C and 11 days at 30°C (41). Other rat, 7; and mouse, 9.0. The LDso values depend on many
investigators reported that the maximum production of afla- factors, such as age, sex, strain, condition of the animal, rate

JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER 1995

1400 GOURAMA AND BULLERMAN
of administration, composition of the diet, and time lapse
before measurement ofLDso' In animal studies aflatoxin B. is
the most toxic, followed by MI' Gj, Bz' and Gz. Aflatoxin-
induced tumors of organs other than the liver have also been
reported (121). Aflatoxin was also reported to inhibit the
germination of mold spores and the growth of many bacterial
species (24).
The scientific investigations suggest that metabolites of
aflatoxins and not the aflatoxins themselves are responsible
for the toxic effect The biotransformation of aflatoxins is
necessary in order for toxicity to occur. Most of the other
mycotoxins do not need any activation. Aflatoxin Bl-2,3
epoxide is the resulting derivative and is highly toxic (56).
Billing (5) et al. (20) used cytochrome P-460 containing the S-
9 metabolic activation system for rat liver to activate aflatoxin
BJ" The bioactivation increased aflatoxin Bj toxicity by a
factor of 10. Inhibition of DNA replication and of RNA and
protein synthesis by aflatoxin have also been reported (56,
116). Neal et al. (85) found that during acute toxicity there is
transformation in the liver of aflatoxin B] to the 2,3 dihydrodiol
of aflatoxin B (aflatoxin B j-dhd). The reaction of aflatoxin B j-
dhd in the aldehyde resonance hybrid with primary amine
groups of proteins may explain the mechanism of aflatoxin B]
acute toxicity. In addition, the 2,3 dihydrodiol of aflatoxin B]
has been shown in vitro to inhibit protein synthesis, which
may explain the necrosis of the liver, which causes death.
Mycotoxins have been proven to be toxic to a wide range
of animals. Different mycotoxicoses in different animal spe-
cies have been caused by different mycotoxins produced by a
large number of different fungi. Some human diseases have
also been shown to be due to the ingestion of fungal metabo-
lites. There is some evidence that aflatoxins are involved to
some degree in primary liver cancer in humans on a world-
wide basis (111, 121). In 1987 the International Agency for
Research on Cancer (IARC) (62) declared that aflatoxin B] is
a class I carcinogen, on the basis of animal assays. Evidence
suggesting a link between aflatoxin and liver cancer is based
on animal assays, epidemiological studies, and in vitro
mutagenicity tests. However, in the case of aflatoxins and
carcinogenicity, the short-term tests are not really reliable
tests for this purpose. Adamson et al. (1) reported that feeding
mixed aflatoxins to rhesus monkeys caused development of
hepatocellular carcinoma. Studies on possible cases of
aflatoxicoses in humans have been reported in many countries
in southeast Asia and Africa (76, 81,91,92,102,118). Aflatox-
ins have been reported to cause hepatocellular carcinoma (39),
acute hepatitis (70, 86), Reye's syndrome (103), cirrhosis in
malnourished children (4), and kwashiorkor (39). However,
many scientific reports question the role of aflatoxin in liver
cancer because of the strong correlation between liver cancer
and chronic hepatitis B virus in African and Asian populations
(22,53). Other investigators believe that an interaction between
hepatitis B virus and aflatoxin is responsible for the high
incidence of liver damage in those parts of the world and that
carriers of hepatitis B virus may be more susceptible and
predisposed to cancer initiation by aflatoxins (91, 92).
Analysis
There are several basic analytical steps for the analysis of
aflatoxins and most other mycotoxins. These include sam-
JOURNAL OF FOOD PROTECTION,
pIing, sample preparation, extraction, clean-up, qualitative
detection, confirmation, and quantification (30, 55). Selection
of representative samples is the first problem encountered in
mycotoxin analysis. The content of aflatoxin in grains or nuts
can vary from less than 1 ppb to more than 12 ppm. Aflatoxin
can be highly concentrated in individual kernels. For example,
in a peanut lot one peanut kernel alone has been reported to
contain as much as several hundred micrograms (30). These
kernels can contaminate 1,000 other kernels with a high level
of aflatoxin. Consequently, the importance of adequate sam-
pling cannot be overemphasized. Park and Pohland (89) wrote
a good review of accepted procedures for sampling and
subsampling of various agricultural products.
Aflatoxins should be analyzed by accepted methods (6).
Extraction is usually done with polar solvents such as metha-
nol, acetone, and chloroform. Impurities are removed during
purification and clean-up by using liquid-liquid partitioning
or column clean-up. The different components in the extract
are separated by thin-layer chromatography (TLC) or by gas
liquid chromatography (GLC) or high performance liquid
chromatography (HPLC). Samples are quantified by visual
estimation or instrumentally by using fluorodensitometry or
fluorescence detection with HPLC. However, these physico-
chemical methods are time consuming, require skilled ana-
lysts, and use expensive material and equipment Quality
control laboratories need methods that detect aflatoxin in 15
to 30 min. Three types of rapid methods are available: black
light (UV) tests (30), minicolumns (30), and immunoassays
(52,93). In the black light test, the broken kernels are exposed
to UV light to observe the bright greenish-yellow (BGY)
fluorescence. Various investigations have shown the correla-
tion between BGY fluorescence and the presence of A. jlavus.
The BGY fluorescence is believed to be due to the kojic acid,
produced by most aflatoxin-producing A. jlavus strains, and
converted to a fluorescing substance by plant tissue peroxi-
dase. The black light test is a presumptive rather than a
confirmative test, since the correlation between BGY fluores-
cence and the presence of aflatoxins is not perfect
The minicolumn methods are semiquantitative techniques
that use columns of silica gel alone or in combination with
florisil and alumina. After extraction of the sample with the
appropriate solvent(s), the extract is forced through the col-
umn, which is then compared to columns containing different
standard concentrations under long wavelength UV light
Aflatoxins appear as a fluorescing band on the columns.
The immunoassay methods use antibodies which are
highly selective for aflatoxins and other mycotoxins. Most
mycotoxins are low molecular weight and do not cause an
antigenic response. Thus, it is important to couple mycotoxins
to antigenic macromolecules before injection into an animal
in order to produce antibodies. Several kits have been devel-
oped based on immunochemistry technology. These immu-
noassays can be divided into two categories. The first is
affinity column chromatography, in which a large amount of
antibodies is attached to an affinity column. The sample
extract is passed through the column; the bound mycotoxin is
then eluted and detected by fluorometry or HPLC. With this
method, the antibodies are used primarily to isolate and
remove the aflatoxins from the matrix. The second category
consists of competitive immunoassays that involve radioim-
VOL. 58, DECEMBER ]995
 ASPERGILLUS FLA VUS AND A. PARASITICUS
munoassay (RIA) or an enzyme-linked immunosorbent assay
(ELISA). A number of commercial kits employing these
techniques are now available.
Control and detoxification
A great deal of effort has been ex pended on finding better
ways to remove or destroy aflatoxin in contaminated products
(49). The ideal approach is to prevent aflatoxin production.
However, aflatoxins are natural contaminants and in many
instances they are unavoidable contaminants. Techniques of
detoxification include chemical and biological detoxification
and physical removal of aflatoxins. Aspergillusflavus spores,
media, and sclerotium are usually present in soil and they
provide an early A. flavus inoculum (120). In the case of
peanuts, A. flavus-parasiticus produces spores during the
growing season and a large accumulation of spores are found
during harvest time. Drought conditions are favorable for
fungal contamination and aflatoxin production (40, 42). Cole
et al. (35) reported that adequate mineral nutrition can mini-
mize aflatoxin contamination. Contamination of grain by
A. flavus does not automatically lead to aflatoxin production,
because many conditions, mainly moisture and temperature,
have to be met. The risks of aflatoxin contamination can also
be controlled during harvesting. Grain should be harvested at
optimum maturity, be cleaned of foreign materials and broken
kernels, and dried to a moisture content that prevents mold
growth (12 to 14%).
Although development of grain varieties that would resist
fungal contamination and aflatoxin production have been
investigated in many laboratories (42), no such genotypes
have yet been developed. Controls during storage are also
critical. Control of moisture and insects and use of pennitted
antifungal agents help prevent mold growth and mycotoxin
production. Other environmental conditions such as tempera-
ture, atmospheric conditions, pH, competing microflora, and
CO2 should also be considered.
Since total control of aflatoxin contamination is almost
impossible, other practical techniques are needed. Different
methods of removing toxins, or detoxification, have been
reported (5, 50, 88). Physical methods of separation such as
electronic sorting, density separation, and dry and wet milling
(25, 35, 123), have been used. Aflatoxins are thermostable,
and are therefore not totally inactivated by heat treatments
(32,37,83). Shantha and Screenivasamurthy (104) reported
that exposure of contaminated peanut oil to UV light caused
significant reduction in aflatoxin content.
Aflatoxins can be extracted using solvents. The removal
can be complete under specific conditions with minimal effect
on the nutritional value of commodities (50, 99). The disad-
vantages of the solvent extraction are high cost and the
possible introduction of off flavors. Adsorbents such as acti-
vated carbon, bentonite, clays, and aluminosilicates have
been reported to bind aflatoxins in liquid foods (38, 78).
Aflatoxins can be modified or inactivated using microorgan-
isms (33, 83). Numerous chemicals have been reported to
degrade aflatoxins in naturally contaminated commodities;
examples of these chemicals are strong acids and bases and
oxidizing agents. Ammonia treatment is the most promising
and practical approach. The degradation of aflatoxins by
ammonia is due to the opening of the lactone ring in the
JOURNAL OF FOOD PROTECTION,
1401
aflatoxin chemical structure, particularly when the pH of the
medium is higher than 9.5. Decarboxylation must follow this
treatment in order to avoid regeneration of the aflatoxins,
especially when the pH becomes acid. Many toxicological
studies have shown no adverse effects from feeding ammoni-
ated aflatoxin by-products to animals (14). However because
ammonia lowers the grain quality, this process is suitable only
for animal feed and not for foods intended for human con-
sumption.
Regulation of aflatoxins
Following the discovery of aflatoxins in 1960, several
countries developed legislation to regulate and control myc-
otoxins (mainly aflatoxin BI,) in food and feed. Van Egmond
(117) developed a listing of mycotoxin legislation in many
countries. In the United States, the Food and Drug Adminis-
tration (FDA) considers aflatoxins poisonous and deleterious
substances and regulates them according to the Food, Drug,
and Cosmetic Act, Section 402 (a)(1), which defines adulter-
ated food as food that contains "any poisonous or deleterious
substance which may render it injurious to health" (100). The
FDA established regulatory working guidelines on acceptable
levels of aflatoxin in foods and feed. The action level for food
is set at 20 ppb total aflatoxins, with the exception of milk,
which has an action level of 0.5 ppb of aflatoxin MI' This low
level was set due to the fact that milk containing aflatoxin M),
presents a high risk to infants and young children. The action
levels for cottonseed meal, corn, and mixed feed for beef cattle
is 300 ppb. The action level set for corn that is u.sed for
finishing swine is 200 ppb, while feeds used for breeding
cattle, breeding swine, and mature poultry have an action level
of 100 ppb. The action level for feed for dairy cattle is 20 ppb.
More than 50 countries around the world are known to have
regulations. The maximum limits of total aflatoxins vary from
zero up to 50 ppb. In practice, a zero tolerance reflects the
limitations of the detection method used (117). In 1987, 14
countries had actual or proposed tolerance levels for aflatoxin
MI in milk and dairy products (117).
In 1990, the Codex Committee on Food Additives and
Contaminants set a maximum tolerance of 10 ppb of total
aflatoxins in all foods, excluding milk and dairy products.
Some countries consider the 10 ppb level to be too low while
others consider it too high.
Economic aspects of aflatoxin occurrence
Each year 25% of the world's crops are contaminated by
mycotoxins (82). It is impossible to totally quantify the losses
due to mycotoxins. Generally, the losses remain undetected,
and in cases of chronic mycotoxicoses in livestock, lead to
decreases in production. Export of grains is severely affected
by high content of aflatoxins. There are additional costs for
controlling aflatoxins through testing, regulatory enforce-
ment, research, and extension services. The fooct crops that are
most often affected are corn, peanuts, cottonseed, sorghum,
wheat, other grain crops, and some tree nuts. Other losses are
due to contaminated corn products, animal products, fruits
and vegetables (109). Aflatoxins can cause economic losses to
fanners and merchants, national losses through export reduc-
tion, increases in food and feed imports, the additional cost of
returning shipments of rejected crops, cost of control, cost of
VOL. 58, DECEMBER 1995
detection, and the health hazards caused by contamination.
The most tragic loss is the loss of human life through the
ingestion of contaminated foods.
CONCLUSIONS
Aspergillusjlavus species will continue to occupy a wide
variety of human habitats. Many of the A. jlavus species that
are frequent contaminants of food and agricultural commodi-
ties are also capable of producing mycotoxins, which are
highly toxic to humans and animals. However, other A.jlavus
species have useful roles, such as the use of
A. oryzae in the production of soy sauce. The growth of
the A. jlavus group of species and the production of aflatoxins
depend on numerous factors: substrates, temperature, pH,
environment, relative humidity, and the presence of compet-
ing microflora. Despite our vast knowledge of
A. jlavus and of aflatoxin production, many questions need to
be answered before any efficient control system can be estab-
lished. More research is needed to understand the physiology,
metabolism, and nutritional requirements of aflatoxigenic
fungi. Detection and identification of A. jlavus species is
another area that needs further development. The use of
scanning electron microscopy, the development of selective
media, and the rise of immunoassay techniques seem to have
promising potential in this regard.
The evidence of aflatoxin involvement in foodborne
human disease is getting stronger. Various studies around the
world have shown the relationship between the occurrence of
acute and chronic disease and ingestion of foods that are
contaminated with aflatoxins and other mycotoxins. Thus
efforts have to be made to prevent mold growth and myc-
otoxin production along the entire food chain, from field to
table. However, such efforts are almost impossible without
the further research needed to answer numerous questions.
First, there is need for total surveillance of foods and feeds for
the presence of mycotoxigenic fungi and mycotoxins. Sec-
ond, an efficient control system needs to be established. This
could be accomplished through the development of efficient
detoxification and decontamination procedures, worldwide
regulations, and improvement of storage facilities, especially
in developing countries. Third, there is need for better under-
standing of the toxicological effects of mycotoxins, and
fourth, sampling and analytical methods for mycotoxigenic
fungi and mycotoxins must continue to be improved.
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