Aspergillus Flavus and Aspergillus Parasiticus: Aflatoxigenic Fungi of Concern in Foods and Feeds
Aspergillus Flavus and Aspergillus Parasiticus: Aflatoxigenic Fungi of Concern in Foods and Feeds
Aspergillus Flavus and Aspergillus Parasiticus: Aflatoxigenic Fungi of Concern in Foods and Feeds
Department of Food Science and Technology, University of Nebraska, Lincoln, Nebraska 68583-0919 USA
the length of the conidial chains. The color of the conidia chain of conidia). Generally phialides are nonseptate. Al-
determines the color of the conidial head; thus the color of though most A. jlavus species are biseriate (two layered),
A. jlavus is light to deep yellow green, or olive green. some species are uniseriate (single layered).
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).
extract agar and Czapek agar. Generally potato dextrose agar gentamicin and streptomycin. Other media can be used, such
(PDA) may also be used; however, appearance on PDA is not as malt extract agar and Czapek agar. None of these media are
considered to be consistent enough to be useful diagnosti- selective forA. jlavus, as other mold genera such as Penicil-
cally. Selective media such as Aspergillusjlavus-parasiticus lium and Fusarium also grow on these media. King et al. (65)
agar (AFPA) (91) can be used to identify A.jlavus species. The developed a medium using dichloran and rose bengal to make
major taxonomic references (67, 98, 112, 113) base the dichloran-rose bengal-chloramphenicol agar (DRBC), which
identification keys on the color and morphology of the fungi, restricts the spreading of fungal colonies without affecting
which in many cases is not reliable. The characteristics can spore germination.
change with media, growth conditions, and mutations. In 1974, Bothast and Fennell (23) developed a differen-
Kozakiewicz (68) suggested the use of scanning electron tial medium for A.jlavus species, called Aspergillus differen-
microscopy (SEM) to solve this problem. With the high tial medium (ADM). The differential ingredient of ADM is
resolution of SEM, diagnostic keys of Aspergillus could be ferric citrate (0.05%), which reacts with fungal metabolites
based on the differences in the ornamentation of the conidia. such as kojic acid and aspergillic acid to produce a bright
Kozakiewicz (68) demonstrated that the genetic stability of orange-yellow pigment on the reverse side ofthe colony. Pitt
the spore ornamentation is maintained under different condi- et al. (96) added dichloran and chloramphenicol to ADM to
tions of physiological and environmental stress. The conidium make a new medium called Aspergillusjlavus and parasiticus
ornamentation has been divided into nine categories that agar (AFPA). AFPA is composed of peptone, 10 g; yeast
varied from echinulate to smooth. In a more recent publication extract, 20 g; ferric ammonium citrate, 0.5 g; chloramphenicol,
by the same author (69), it was reported that based on conidial 100 mg; agar, 15 g; distilled water, 11; and dichloran, 2 mg.
ornamentation, A. jlavus species fall into two distinct catego- The final pH of this medium is ca. 6.2. Cultures on AFP A are
ries, echinulate or lobate-reticulate. The roughened lobate- incubated at 30°C for 42 to 48 h. Dichloran inhibits spreading
reticulate type of conidia are evident in Fig. 2. Using SEM, of fungi, while chloramphenicol inhibits bacteria. A. jlavus
Kozakiewicz (69) found that many of the A. parasiticus and A. parasiticus are identified on this medium by production
cultures obtained from different world collections were of typical yellow to olive green spores and a bright orange
misidentified. In addition, SEM is a useful tool to differentiate reverse. Another advantage of this medium is that A. jlavus
between mixed related isolates such as A. parasiticus, and A. parasiticus grow rapidly because the medium is incu-
A. oryzae and A. jlavus. Kurtzman et al. (71) described bated at 30°C, permitting identification within 3 days in most
Aspergillus nomius as a new af1atoxigenic species that is cases. This medium was recommended for use in enumerating
phenotypically similar to A. jlavus. The separation between A.jlavus species in nuts, com, spices and soil (95). Aspergillus
the two species was based on the presence of intermediate niger produces a yellow but not orange reverse color, and after
sclerotia and low growth temperature. 48 h of incubation A. niger starts to develop its dark'brown to
black conidia, which easily distinguish it from A. jlavus.
DETECTION OF A. FLA VUS IN FOODS AND FEEDS Aspergillus ochraceus grows slowly at 30°C and the yellow
color appears after 48 h (95). The use of AFPA shortens the
Generally, detection of A. jlavus in foods and feeds is
time required to isolate and identify A. jla vus species. Another
carried out by using traditional microbiological plating meth-
advantage is the isolation and identification of potentially
ods, either surface spread or direct plating of kernels and
af1atoxigenic fungi. In many laboratories it is easier and more
seeds. Various media are used for this purpose: PDA, acidi-
economical to first look for af1atoxigenic fungi to identify
fied PDA and PDA with antibiotics. Different antibiotics can
contaminated materials than to chemically test for af1atoxins.
be used: chlortetracycline, chloramphenicol, oxytetracycline,
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.
coP
o 0
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
1~
subsp. parasiticus tends to be more stable in producing
aflatoxins than A. flavus. All aflatoxin-producing fungi are
CCJCCCHS
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needed to distinguish A. flavus from A. oryzae. o
1~
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AFLATOXINS
CC;CCHS
(HZ ~CHZCH
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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
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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
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higher yields of aflatoxin are produced in shaken flasks rather
.
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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
of administration, composition of the diet, and time lapse pIing, sample preparation, extraction, clean-up, qualitative
before measurement ofLDso' In animal studies aflatoxin B. is detection, confirmation, and quantification (30, 55). Selection
the most toxic, followed by MI' Gj, Bz' and Gz. Aflatoxin- of representative samples is the first problem encountered in
induced tumors of organs other than the liver have also been mycotoxin analysis. The content of aflatoxin in grains or nuts
reported (121). Aflatoxin was also reported to inhibit the can vary from less than 1 ppb to more than 12 ppm. Aflatoxin
germination of mold spores and the growth of many bacterial can be highly concentrated in individual kernels. For example,
species (24). in a peanut lot one peanut kernel alone has been reported to
The scientific investigations suggest that metabolites of contain as much as several hundred micrograms (30). These
aflatoxins and not the aflatoxins themselves are responsible kernels can contaminate 1,000 other kernels with a high level
for the toxic effect The biotransformation of aflatoxins is of aflatoxin. Consequently, the importance of adequate sam-
necessary in order for toxicity to occur. Most of the other pling cannot be overemphasized. Park and Pohland (89) wrote
mycotoxins do not need any activation. Aflatoxin Bl-2,3 a good review of accepted procedures for sampling and
epoxide is the resulting derivative and is highly toxic (56). subsampling of various agricultural products.
Billing (5) et al. (20) used cytochrome P-460 containing the S- Aflatoxins should be analyzed by accepted methods (6).
9 metabolic activation system for rat liver to activate aflatoxin Extraction is usually done with polar solvents such as metha-
BJ" The bioactivation increased aflatoxin Bj toxicity by a nol, acetone, and chloroform. Impurities are removed during
factor of 10. Inhibition of DNA replication and of RNA and purification and clean-up by using liquid-liquid partitioning
protein synthesis by aflatoxin have also been reported (56, or column clean-up. The different components in the extract
116). Neal et al. (85) found that during acute toxicity there is are separated by thin-layer chromatography (TLC) or by gas
transformation in the liver of aflatoxin B] to the 2,3 dihydrodiol liquid chromatography (GLC) or high performance liquid
of aflatoxin B (aflatoxin B j-dhd). The reaction of aflatoxin B j- chromatography (HPLC). Samples are quantified by visual
dhd in the aldehyde resonance hybrid with primary amine estimation or instrumentally by using fluorodensitometry or
groups of proteins may explain the mechanism of aflatoxin B] fluorescence detection with HPLC. However, these physico-
acute toxicity. In addition, the 2,3 dihydrodiol of aflatoxin B] chemical methods are time consuming, require skilled ana-
has been shown in vitro to inhibit protein synthesis, which lysts, and use expensive material and equipment Quality
may explain the necrosis of the liver, which causes death. control laboratories need methods that detect aflatoxin in 15
Mycotoxins have been proven to be toxic to a wide range to 30 min. Three types of rapid methods are available: black
of animals. Different mycotoxicoses in different animal spe- light (UV) tests (30), minicolumns (30), and immunoassays
cies have been caused by different mycotoxins produced by a (52,93). In the black light test, the broken kernels are exposed
large number of different fungi. Some human diseases have to UV light to observe the bright greenish-yellow (BGY)
also been shown to be due to the ingestion of fungal metabo- fluorescence. Various investigations have shown the correla-
lites. There is some evidence that aflatoxins are involved to tion between BGY fluorescence and the presence of A. jlavus.
some degree in primary liver cancer in humans on a world- The BGY fluorescence is believed to be due to the kojic acid,
wide basis (111, 121). In 1987 the International Agency for produced by most aflatoxin-producing A. jlavus strains, and
Research on Cancer (IARC) (62) declared that aflatoxin B] is converted to a fluorescing substance by plant tissue peroxi-
a class I carcinogen, on the basis of animal assays. Evidence dase. The black light test is a presumptive rather than a
suggesting a link between aflatoxin and liver cancer is based confirmative test, since the correlation between BGY fluores-
on animal assays, epidemiological studies, and in vitro cence and the presence of aflatoxins is not perfect
mutagenicity tests. However, in the case of aflatoxins and The minicolumn methods are semiquantitative techniques
carcinogenicity, the short-term tests are not really reliable that use columns of silica gel alone or in combination with
tests for this purpose. Adamson et al. (1) reported that feeding florisil and alumina. After extraction of the sample with the
mixed aflatoxins to rhesus monkeys caused development of appropriate solvent(s), the extract is forced through the col-
hepatocellular carcinoma. Studies on possible cases of umn, which is then compared to columns containing different
aflatoxicoses in humans have been reported in many countries standard concentrations under long wavelength UV light
in southeast Asia and Africa (76, 81,91,92,102,118). Aflatox- Aflatoxins appear as a fluorescing band on the columns.
ins have been reported to cause hepatocellular carcinoma (39), The immunoassay methods use antibodies which are
acute hepatitis (70, 86), Reye's syndrome (103), cirrhosis in highly selective for aflatoxins and other mycotoxins. Most
malnourished children (4), and kwashiorkor (39). However, mycotoxins are low molecular weight and do not cause an
many scientific reports question the role of aflatoxin in liver antigenic response. Thus, it is important to couple mycotoxins
cancer because of the strong correlation between liver cancer to antigenic macromolecules before injection into an animal
and chronic hepatitis B virus in African and Asian populations in order to produce antibodies. Several kits have been devel-
(22,53). Other investigators believe that an interaction between oped based on immunochemistry technology. These immu-
hepatitis B virus and aflatoxin is responsible for the high noassays can be divided into two categories. The first is
incidence of liver damage in those parts of the world and that affinity column chromatography, in which a large amount of
carriers of hepatitis B virus may be more susceptible and antibodies is attached to an affinity column. The sample
predisposed to cancer initiation by aflatoxins (91, 92). extract is passed through the column; the bound mycotoxin is
then eluted and detected by fluorometry or HPLC. With this
Analysis method, the antibodies are used primarily to isolate and
There are several basic analytical steps for the analysis of remove the aflatoxins from the matrix. The second category
aflatoxins and most other mycotoxins. These include sam- consists of competitive immunoassays that involve radioim-
munoassay (RIA) or an enzyme-linked immunosorbent assay aflatoxin chemical structure, particularly when the pH of the
(ELISA). A number of commercial kits employing these medium is higher than 9.5. Decarboxylation must follow this
techniques are now available. treatment in order to avoid regeneration of the aflatoxins,
especially when the pH becomes acid. Many toxicological
Control and detoxification studies have shown no adverse effects from feeding ammoni-
A great deal of effort has been ex pended on finding better ated aflatoxin by-products to animals (14). However because
ways to remove or destroy aflatoxin in contaminated products ammonia lowers the grain quality, this process is suitable only
(49). The ideal approach is to prevent aflatoxin production. for animal feed and not for foods intended for human con-
However, aflatoxins are natural contaminants and in many sumption.
instances they are unavoidable contaminants. Techniques of
detoxification include chemical and biological detoxification Regulation of aflatoxins
and physical removal of aflatoxins. Aspergillusflavus spores, Following the discovery of aflatoxins in 1960, several
media, and sclerotium are usually present in soil and they countries developed legislation to regulate and control myc-
provide an early A. flavus inoculum (120). In the case of otoxins (mainly aflatoxin BI,) in food and feed. Van Egmond
peanuts, A. flavus-parasiticus produces spores during the (117) developed a listing of mycotoxin legislation in many
growing season and a large accumulation of spores are found countries. In the United States, the Food and Drug Adminis-
during harvest time. Drought conditions are favorable for tration (FDA) considers aflatoxins poisonous and deleterious
fungal contamination and aflatoxin production (40, 42). Cole substances and regulates them according to the Food, Drug,
et al. (35) reported that adequate mineral nutrition can mini- and Cosmetic Act, Section 402 (a)(1), which defines adulter-
mize aflatoxin contamination. Contamination of grain by ated food as food that contains "any poisonous or deleterious
A. flavus does not automatically lead to aflatoxin production, substance which may render it injurious to health" (100). The
because many conditions, mainly moisture and temperature, FDA established regulatory working guidelines on acceptable
have to be met. The risks of aflatoxin contamination can also levels of aflatoxin in foods and feed. The action level for food
be controlled during harvesting. Grain should be harvested at is set at 20 ppb total aflatoxins, with the exception of milk,
optimum maturity, be cleaned of foreign materials and broken which has an action level of 0.5 ppb of aflatoxin MI' This low
kernels, and dried to a moisture content that prevents mold level was set due to the fact that milk containing aflatoxin M),
growth (12 to 14%). presents a high risk to infants and young children. The action
Although development of grain varieties that would resist levels for cottonseed meal, corn, and mixed feed for beef cattle
fungal contamination and aflatoxin production have been is 300 ppb. The action level set for corn that is u.sed for
investigated in many laboratories (42), no such genotypes finishing swine is 200 ppb, while feeds used for breeding
have yet been developed. Controls during storage are also cattle, breeding swine, and mature poultry have an action level
critical. Control of moisture and insects and use of pennitted of 100 ppb. The action level for feed for dairy cattle is 20 ppb.
antifungal agents help prevent mold growth and mycotoxin More than 50 countries around the world are known to have
production. Other environmental conditions such as tempera- regulations. The maximum limits of total aflatoxins vary from
ture, atmospheric conditions, pH, competing microflora, and zero up to 50 ppb. In practice, a zero tolerance reflects the
CO2 should also be considered. limitations of the detection method used (117). In 1987, 14
Since total control of aflatoxin contamination is almost countries had actual or proposed tolerance levels for aflatoxin
impossible, other practical techniques are needed. Different MI in milk and dairy products (117).
methods of removing toxins, or detoxification, have been In 1990, the Codex Committee on Food Additives and
reported (5, 50, 88). Physical methods of separation such as Contaminants set a maximum tolerance of 10 ppb of total
electronic sorting, density separation, and dry and wet milling aflatoxins in all foods, excluding milk and dairy products.
(25, 35, 123), have been used. Aflatoxins are thermostable, Some countries consider the 10 ppb level to be too low while
and are therefore not totally inactivated by heat treatments others consider it too high.
(32,37,83). Shantha and Screenivasamurthy (104) reported
that exposure of contaminated peanut oil to UV light caused Economic aspects of aflatoxin occurrence
significant reduction in aflatoxin content. Each year 25% of the world's crops are contaminated by
Aflatoxins can be extracted using solvents. The removal mycotoxins (82). It is impossible to totally quantify the losses
can be complete under specific conditions with minimal effect due to mycotoxins. Generally, the losses remain undetected,
on the nutritional value of commodities (50, 99). The disad- and in cases of chronic mycotoxicoses in livestock, lead to
vantages of the solvent extraction are high cost and the decreases in production. Export of grains is severely affected
possible introduction of off flavors. Adsorbents such as acti- by high content of aflatoxins. There are additional costs for
vated carbon, bentonite, clays, and aluminosilicates have controlling aflatoxins through testing, regulatory enforce-
been reported to bind aflatoxins in liquid foods (38, 78). ment, research, and extension services. The fooct crops that are
Aflatoxins can be modified or inactivated using microorgan- most often affected are corn, peanuts, cottonseed, sorghum,
isms (33, 83). Numerous chemicals have been reported to wheat, other grain crops, and some tree nuts. Other losses are
degrade aflatoxins in naturally contaminated commodities; due to contaminated corn products, animal products, fruits
examples of these chemicals are strong acids and bases and and vegetables (109). Aflatoxins can cause economic losses to
oxidizing agents. Ammonia treatment is the most promising fanners and merchants, national losses through export reduc-
and practical approach. The degradation of aflatoxins by tion, increases in food and feed imports, the additional cost of
ammonia is due to the opening of the lactone ring in the returning shipments of rejected crops, cost of control, cost of
tabolism of aflatoxin by liver microsomes isolated from certain avian implication of reciprocity between ethylene and aflatoxin biogenesis
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