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Journal of Food Protection, Vol. 58, No. 12, Pages 1395-1404 Copyrighl©, International Association of Milk, Food and Environmental Sanitarians

Aspergillus flavus and 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 USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 (MS# 91-230: Received 22 December 19911Accepted 15 May 1995)

ABSTRACT isolates are still used as specific names, e.g., Aspergillus capitatus, Aspergillus niger, etc. (98). Aspergillus are com- Aspergillusflavus and the closely related subspeciesparasiticus have long been recognized as major contaminants of organic and mon saprobic molds, which grow in a wide range of natural nonorganic items. A. flavus, a common soil , can infest a wide substrates and climatic conditions. Austwick (8) reported that range of agricultural products. Some A. flavus varieties produce one conidial head may produce up to 50,000 . Aspergil- aflatoxins, which are carcinogenic toxins that induce liver cancer in lus spp. are of particular importance to humans. Although laboratory animals. A.flavus var.flavus, A.flavus subsp. parasiticus, many Aspergillus species are considered pathogens or spoil- and A. nomius share the ability to produce aflatoxins. Identification age organisms, many others are beneficial. Some species are of the A. flavus species group is mainly based on the color and used to prepare fermented foods (57). Aspergillus spp. can macroscopic and microscopic characteristics of the fungus. A. flavus also be allergenic, toxigenic, and pathogenic to humans and growth and aflatoxin biosynthesis depend on substrate, moisture, animals. Pathogenic Aspergillus spp. represent a real hazard temperature, pH, aeration, and competing microflora. The growth of to animal health: they can produce numerous diseases such as A. flavus and aflatoxin production are sometimes unavoidable. avian aspergillosis and bovine mycotic abortion (2). Mold Aflatoxins are considered natural contaminants; the ideal control approach is prevention of mold growth and aflatoxin production. The spores can cause hypersensitivity reactions in sensitized pa- detection of members of the A.flavus species group in foods and feed tients, fibrosis, and hypersensitivity pneumonitis. Aspergillus is generally carried out by using plate techniques such as surface spp. also produce , which are formed as secondary spread or direct plating. Research on alternative fungal detection metabolites and cause mycotoxicoses in animals and humans methods is still in its infancy. Few immunoassay techniques have (29). Aflatoxins which are produced by some Aspergillus been investigated in this regard. Aflatoxins are generally analyzed by jlavus species are considered potent carcinogens (12). chemical methods, although immunochemical methods which use antibodies are becoming common analytical tools for aflatoxins. MORPHOLOGICAL DESCRIPTION OF ASPERGILLUS FLA VUS Key words: Aspergillus flavus, Aspergillus parasiticus, aflatoxins The production of phialides and foot cells demonstrates the presence of Aspergillus species (95, 108). The production Species of Aspergillus have long been known to be of phialides alone is not enough to characterize aspergilli. common contaminants of human food and animal feeds. Although phialides are common to most Aspergillus spp. they Antonio Micheli in 1729 (84) was the first to name the genus are also formed by Penicillium species. Raper and Fennell Aspergillus. Micheli's monograph revolutionized mycology (98) reported that the presence of foot cells is evidence that a into a major science. Aspergillus species affect food products, mold isolate is Aspergillus; however, the absence offoot cells wood, leather, textiles, kerosine, paint, plastics, rubber, ce- does not prove that the isolate does not belong to Aspergillus ment, and pharmaceutical items (114). They possess a high group. Most morphological descriptions oftheA.jlavus group metabolic versatility and a great ability to disperse their were done on standard media such as Czapek's solution agar spores. Micheli noticed that the chains rise radially from and malt extract agar. the vesicle, which he thought resembled a holy-water sprin- kler (aspergillum). Micheli's Latin descriptions of the mold Conidial head The conidial head characteristics such as color, shape, t Published as Paper No.1 1009 Journal Series, Agricultural Re- and size are important key diagnostic criteria of the Aspergil- search Division, Lincoln, Nebraska 68583. Research was con- lus group (94, 98). The shape of conidial heads varies from ducted under Project 16-042. columnar to radiate and globose. The arrangement of phialides t Present Address: Food Science, Penn State Berks Campus, on the vesicle dictates the shape of the conidial head. The size Tulpehocken Road, P. O. Box 7009, Reading, PA 19610-6009. of the conidial head is determined by the size of the vesicle and

JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER 1995 1396 GOURAMA AND BULLERMAN 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).

Conidiophore Spores (conidia) The conidiophore, which is also known as the stalk, is a Conidia (singular: conidium), also called spores, are thick-walled branch which arises perpendicularly from the asexual reproductive structures. Conidia in Aspergillus spp. foot cell (Fig. 1)(67). Generally conidiophores are unbranched are one-celled structures that can be uni- or multinucleate. The and are composed of three parts: the foot cell, stipe, and phialide is the conidiogenous cell. At the initial stages of spore vesicle. Although conidiophores are usually nonseptate, formation, the tip ofthe phialide is ruptured and a cytoplasmic septation may occur in certain species (98). The character of mass moves to the neck of the phial ide, forming a bulbous the outer surface of the conidiophore can vary from smooth to structure (54). New wall material is formed to close the new rough. These features constitute a key element for the identi- structure which forms the first conidium. The new conidia are fication of Aspergillus spp. The conidiophores in the A. jlavus formed beneath the earlier ones, so the oldest conidium is at group are rough and hyaline (nonpigmented). the apex of the chain while the youngest is at its base. After Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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- Figure 1. Conidiophores characteristic of Aspergillus spe- tion. They are found in the teleopmorphs of A. nidulans and A. cies, showing a single layer of phialides or sterigmata (uniseriate) and double layer of cells, phialides and metulae amstelodami. They are not known to be produced in the A. (biseriate). (Adaptedfrom Klich and Pitt (67); drawn by A. D. jlavus group (98). Hocking). CLASSIFICA TION AND IDENTIFICATION

Vesicle Aspergillus is mainly based on morphological The vesicle is the swollen apex of the conidiophore. The characteristics. Antonio Micheli applied the name Aspergillus shape can be globose, hemispherical, elliptical, or clavate to the imperfect state (anamorph) of the fungus (81) .Later, the (club shaped). Vesicles in the A. jlavus group are elongated perfect state (teleomorph) was discovered in some Aspergil- when young and become globose as the culture ages. The lus species, which created confusion in the taxonomy of the shape varies with the composition of the substrate. The Aspergillus group. Raper and Fennell (98) classified the genus diameter varies from 10 to 65 ~m (98). These features of the Aspergillus in both the Eurotiacea and Moniliacea families. vesicle are important diagnostic characters for identification They divided the genus into 18 groups and 132 species with 18 of Aspergillus species (Fig. 1). varieties. Minor revisions have been made since then (81). Sterigmata Samson and Van Reenen-Hoekstra (101) introduced 42 addi- Sterigmata (singular sterigma) are defined as specialized tional taxa and produced a useful survey of species described conidiogenous cells which develop on the fertile area of the since 1945. Raper and Fennell (98) summarized the important vesicle. Sterigmata on the vesicle are uniseriate or biseriate, lines of demarcation into the following criteria: shape and depending on whether one (uniseriate) or two (biseriate) color of conidial heads, characteristics of conidiophores, layers of cells are present. The primary (first layer) sterigmata presence or absence of metulae, size of sterigmata, and the are called metulae, while the secondary (second layer) ones presence or absence of Hulle cells. are called phialides (Fig. I). In the uniseriate species, the The use of appropriate media is essential to achieve an phialides are produced directly from the vesicle, while for the accurate identification of the Aspergillus species. Raper and biseriate species, the phialides arise from the metulae. The Fennell's keys (98) have been universally used for the last 30 phialides are the conidia-bearing cells (each phial ide bears a years and described the species when it is grown on malt

JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER 1995 ASPERG1ILUS FLA VUS AND A. PARAS1TlCUS 1397 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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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.

BIOLOGY AND HABITAT OF A. FLA VUS Aspergillus jlavus species are present in soil and contami- Figure 2. Electron micrograph of the conidial head of As- nate a wide variety of agricultural products in the field, storage pergillus f1avus showing lobate-reticulate conidia. (Courtesy areas, and processing plants and during distribution. Ajlavus, of Dr. R. A. Samson, Centraalbureau voor Schimmelcultures, A.jlavus subsp. parasiticus, and A. nomius are the only molds Baarn, The Netherlands). that have so far been shown to produce af1atoxins (71). Aspergillus oryzae and Aspergillus tamarii were found to be

JOURNAL OF FOOD PROTECTlON. VOL. 58. DECEMBER 1995 1398 GOURAMA AND BULLERMAN

nontoxic. A. flavus strains range from nontoxic to those that o 0

produce aflatoxins B} and B , whereas A. flavus subsp. 2 1~ , , • parasiticus produces aflatoxins BI' B2 GI and G2 A. flavus subsp. parasiticus tends to be more stable in producing CCJCCCHSo 0 aflatoxins than A. flavus. All aflatoxin-producing fungi are coP liZ soil microorganisms, but there are some differences in the o o o o 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 , 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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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. o 1~ H AFLATOXINS (HZ ~CHZCH

CC;CCHS0 Introduction o o 0 -" OeHS Aflatoxicol cParasiticalc& 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 () meal (21). The main microbial contaminant of the peanut meal was identified Figure 3. Chemical structures of aflatoxins. as Aspergillusflavus. A chemical analysis of the peanut meal yielded a series of toxic compounds that fluoresced under UV considered secondary metabolites, which are defined as com- light. These compounds were named aflatoxins (for A.flavus pounds that are not essential for growth. The main precursors

, , toxin.) There are four main aflatoxins, BI B2 GI and G2 (Fig. of the secondary metabolites are acetate "polyketides." The 3). The B group are bifurano coumarins fused to different aflatoxins are produced as a family of chemical cyclopentanone, and the G group are bifurano coumarins compounds from acetate and malonate building blocks that fused to a lactone. The B group fluoresces blue in long- are produced during the idiophase. The functional role of wavelength ultraviolet light, while the G group fluoresces aflatoxin in the producing fungus is not known. The four main

, , ) green. The subscripts 1 and 2 designate the chromatographic aflatoxins (AFB" AFB2 AFGI and AFG2 are CI7 com- mobility (Rf values) pattern of the compounds on thin layer pounds classified as nonaketides (115). Most of the aflatoxin chromatography (TLC) plates. biosynthetic studies have used precursors that were isotopi- Dutton and Heathcote (46) characterized the hemiacetal cally labelled and precursor accumulating mutants which do

derivatives of aflatoxin B) (AFB)) and aflatoxin GI (AFG)) not form aflatoxins. Many investigators, by using isotope- , that were designated B2a and G2a (Fig. 3). Biotransformation labeled acetate, concluded that aflatoxin BI is derived from of aflatoxin in several animal species results in the production acetate (7, 17, 97). The methoxy group in the aflatoxin

of aflatoxin M1 (AFM) and aflatoxin M2 (AFM). These molecule is derived from methionine. The following com- , ) aflatoxins (AFM1 AFM2 were first isolated from the milk pounds have been determined to be intermediate compounds : and urine of animals fed aflatoxins (3, 63). Later on, two in the biosynthesis of aflatoxin BI norsolorinic acid, averantin, , hydroxy derivatives of aflatoxin Gl and G2 aflatoxins GM} averufin, versiconal hemiacetal acetate, versicolerin A and , and GM2 respectively, were isolated and characterized (56). sterigmatocystin (Fig. 4). Sterigmatocystin is naturally pro- The four major aflatoxins (AFB I' AFB} AFG}, AFG) and the duced by Aspergillus versicolor (16). Singh and Hsieh (107)

, , , minor aflatoxins (AFMI' AFM2 AFB2a AFG2a GM} and reported that the mutant strain AVR -1 converts versicolorin A GM ) are considered "naturally occurring" toxins. . 2 and sterigmatocystin into AFBj' and AFGI Many investigators have proposed that all the other Biosynthes is aflatoxins are derived from AFB( (58, 79, 80). However, the The biosynthetic pathways for aflatoxins have been ex- biosynthetic relationship between the AFB group and the tensively investigated (16,17,18,73,74,75). Aflatoxins are AFG group is controversial. Bhatnagar et al. (18) and Cleve-

JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER ]995 ASPERGILLUS FLAVUS AND A. PARASlTICUS 1399

"'lo 0\1 toxins occurs after 4 to 7 days at 24°C (64). Moisture content S r ~ o~ 0 -\10 ",I 10\1 of the substrate and relative humidity are also critical to aflatoxin production. Diener and Davis (42) reported that POLYKETIDE NORSOLORIN IC ACID 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 . "0 CII, 1I0~0A..--o and 88%. Aflatoxin yields increase with increasing RH up to o 0 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 "O 0100 ~rll" I higher yields of aflatoxin are produced in shaken flasks rather II~O.J...O..p. o r I I I I than stationary flasks (64). Landers et al. (72) showed that o ~ 1I,c0 '" 0 0 ~II,CO 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- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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 aflatoxin B Jand B , while a pH higher than 6 favored the intermediate between ST and AFB" Dutton et al. (47) re- 2 production of aflatoxins G and G Basappa et al. (13) re- ported that the AFBJ' and AFB biosynthetic pathways are 1 r . 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 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 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- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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-

JOURNAL OF FOOD PROTECTION, VOL. 58, DECEMBER ]995 ASPERGILLUS FLA VUS AND A. PARASITICUS 1401 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 expended 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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/58/12/1395/1665895/0362-028x-58_12_1395.pdf by guest on 02 October 2021 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 fooctcrops 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

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Food Prot. 45:1298-1301. ties are also capable of producing mycotoxins, which are 10. Badii, F., M. O. Moss, and K. Wilson. ]986. 1he effect of sodium bise]enite highly toxic to humans and animals. However, other A.jlavus on the growth and aflatoxin production of Aspergillus parasiticus and the species have useful roles, such as the use of growth of other Aspergillus. Lett Appl. Microbiol. 2:61--{j4. A. oryzae in the production of soy sauce. The growth of 11. Bahk, J., and E. H. Marth. 1983. Aflatoxin production is inhibited by selected herbal drugs. Mycopathologia 83:129-134. the A. jlavus group of species and the production of aflatoxins 12. Barnes, J. M., and W. H. Butler. 1964. Carcinogenic activity of depend on numerous factors: substrates, temperature, pH, aflatoxin to rats. Nature 202:1016. environment, relative humidity, and the presence of compet- 13. Basappa, S. C., V. Sreenivasmurthy, and H. A. B. Parpla. 1976. ing microflora. 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