Aspergillus flavus Infection and Aflatoxin Production in Corn:

Transcription

1 APP~iu MICROBIOLOGY, Nov. 1974, p Copyright American Society for Microbiology Vol. 28, No. 5 Printed in U.SA. Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements E. B. LILLEHOJ, W. J. GARCIA AND M. LAMBROW Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois Received for publication 10 June 1974 Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 gg of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 Ag/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25,g of cadmium per g or 500 Ag of copper per g of germ. Fennell and co-workers (6) found that Aspergillus flavus was primarily associated with the germ region of infected corn kernels. Since corn germ contains lipids, proteins, carbohydrates, minerals, and trace elements generally required for microbial growth (2, 7, 8), it is not surprising that the fungus develops in this region of the kernel. However, significant variation has been observed in the incidence of A. flavus-infected kernels in contaminated lots of corn (6, 14). In addition, the presence ofa. flavus in a commodity is not always associated with aflatoxin contamination (5). In laboratory studies differences in aflatoxin production by A. flavus have been attributed to heterogeneity of the fungal substrate (5). Synthetic media have rountinely supported minimal toxin production (1 to 60 mg of B1 per kg of medium), whereas maximum yields (700 to 900 mg of B1 per kg) occur on such commodities as autoclaved wheat, rice, cottonseed, and corn (5). Schroeder (13) found that a constituent(s) present in corn steep liquor increased the production of aflatoxin by Aspergillus parasiticus in a Czapek's broth medium. Studies by Mateles and Adye (1, 9) of toxin production in a glucose-ammonia-salts synthetic medium showed that deletion of zinc (2 jg per g) reduced aflatoxin yield without restricting growth of the fungus. Therefore, Schroeder (13) proposed that the enhanced level of toxin detected in media enriched with corn steep liquor was the result of zinc in the corn extract. Davis et al. (4) examined the effect of trace elements on A. flavus synthesis of aflatoxin in a sucrose-nitrate-salts medium. They found that zinc (5 fg per g), iron (2,g per g), and magnesium (100 Aig per g) were required for optimal toxin yield but that manganese levels of 1 Ag per g interfered slightly with toxin production. In a similar synthetic medium Reddy et al. (12) corroborated the requirement for zinc to achieve maximum aflatoxin elaboration but no increase was observed with added iron; omission of manganese from the medium increased toxin accumulation. Trace elements in corn occur predominantly in the germ fraction (7, 10). However, phytate (inositol hexaphosphate) in the germ strongly binds several elements, particularly zinc; the bound elements are not readily available biologieally (10, 11). The ratio of phytate to metal ion levels in the corn germ and binding of trace elements by other constituents of the germ would be important factors in microelement availability. We compared the levels of trace elements in corn germ fractions from kernels naturally infected with A. flavus to germ from kernels free of the fungus. We also studied the variability in aflatoxin production by strains of Aspergillus spp. on autoclaved corn and by A. flavus on autoclaved, defatted corn germ amended with trace elements; by determining toxin yields we 763

2 764 LILLEHOJ, GARCIA, AND LAMBROW APPL. MICROBIOL. measured availability of trace elements to the fungus. This investigation was presented at the May annual meeting of the American Society for Microbiology in Chicago, Ill., MATERIALS AND METHODS Samples of whole corn, acquired from commercial dealers, were either heavily infected with A. flavus and contaminated with aflatoxin or the kernels were free of both fungus and toxin. A. flavus in the test corn was detected by surface-sterilization of kernels with 1% sodium hypochlorite for 1 min, washing the corn with sterile water, and placing the seed on ME agar (malt extract, 30 g, and agar, 15 g per liter). Petri plates were incubated for 3 days at 28 C, and fungi on the kernels were identified under a mocroscope. Corn fractions were obtained either from commercial sources or by hand dissection. Composition of similar commercial and hand-excised corn fractions has been reported (2, 7, 8). In this study manual excision of corn germ was carried out on kernels that had been steeped in water for 10 min before removal of the pericarp and subsequent dissection with special precaution to avoid contamination of the germ with other kernel constituents. Germ fractions for trace element analyses came from both A. flavus-infected and A. flavus-free corn samples. Kernels exhibiting bright greenish-yellow (BGY) fluorescence under ultraviolet light (14) were selected for germ excision; 99% of the BGY-positive kernels were infected with A. flavus. Elemental analyses were carried out by flame atomic absorption; whole kernel corn and hand-dissected germ were wet ashed with nitric acid and assayed by the procedure described by Garcia, Blessin, and Inglett (Cereal Chem., in press). Strains of A. parasiticus NRRL 2999, 5013, and 5862 and A. flavus NRRL 3251, 3357, and 5520 were supplied by the ARS Culture Collection maintained at the Northern Regional Research Laboratory. Cultures were grown on potato dextrose agar slants, and spores for inocula acquired from the slants were incubated for 10 days at 28 C. Aflatoxin production studies were carried out with 1-g samples of whole corn and commercial corn fractions in 50-ml Erlenmeyer flasks with addition of 1 ml of sterile water or the appropriate solution of a specific trace element. Test flasks were autoclaved for 20 min at 121 C and the sterile substrates were inoculated with 0.5 ml of spore suspension (7.0 x 107 spores per ml). Aqueous solutions of the following compounds were used as trace element additives: ZnSO,.7H20; CuSO4-5H20; CrO2; Pb(C2HO2)2. 3H20; CdCl2.2H20; FeCl1; and MnSO4.H20. Inoculated flasks were statically incubated at 25 C. Contents of individual flasks were extracted with 250 ml of chloroform in a Waring blender for 3 min. The extraction solvent was recovered by filtration with subsequent removal of water by addition of anhydrous sodium sulfate; the chloroform solution was vacuumevaporated to the desired volume. Fractions of test solutions were spotted on thin-layer (TLC) plates coated with 0.5-mm Adsorbisil-1 (Applied Science Laboratories Inc., State College, Pa.). The plates were individually developed in ether (3) and subsequently in chloroform:acetone:water (92:8:1). Quantitative determinations were made on developed TLC plates by comparison of fluorescent intensities of alfatoxin from the fermentation extracts with reference standards of pure toxin. RESULTS AND DISCUSSION Trace element levels in corn naturally infected with A. flavus. In preliminary studies we examined the relationship between trace element concentrations in corn and the natural occurrence of A. flavus. Analyses were carried out on whole kernel corn and on manually excised corn germ from samples that were either infected with A. flavus or free of the fungus (Table 1). In addition to trace elements, the levels of phosphorus in test corn were also determined to provide information on phytate concentration. About 90% of the phosphorus in corn germ occurs as phytate (10). Generally, element levels were similar or higher in whole kernels of A. flavus-infected corn than in kernels free of the fungus. However, chromium concentrations were an exception; A. flavus-free corn had a distinctly higher level of the element than kernels infected by the fungus. Since the chromium levels in the A. flavus-free whole kernel and germ fractions were approximately equal, it was concluded that the endospermhull component of corn in this sample contained significant quantities of the metal. A pattern of increased metal levels was observed in fungalinfected germ compared with a germ fraction free of the fungus; the greatest differences (40 to TABLE 1. Trace element levels in whole kernel and corn germ from A. flavus-infected and A. flavus-free samples Ag of element per g of corn (dry weight)a Element A. flavus infected A. flavus free Whole kernel Germ Whole kernel Germ Zinc 22.5 ± ± Manganese Copper Lead 0.29 ± Chromium ± ± Cadmium Phosphorusb 3.04 ± a Whole kernel values represent means of four independent 10-g samples; full-fat germ values were acquired from single determinations. A. flavus was observed in 75% of the infected kernels, and the fungus was not detected in A. flavus-free corn. " Levels in milligrams of corn (dry weight) per gram.

3 VOL. 28, 1974 A. FLAVUS INFECTION IN CORN 60%) were observed in manganese, cadmium, and chromium concentrations. Although the data in Table 1 suggest a correlation between enhanced trace element levels in corn germ and the presence of A. flavus, the increased phytate concentration in the fungal-infected germ indicates that more of the elements in this fraction would be biologically unavailable. Therefore, a clear cause-effect relationship between trace element levels and natural infection of corn by A. flavus cannot be established by these results. Aflatoxin production on corn. Six A. parasiticus and A. flavus strains were tested for toxin yields on a ground, whole corn substrate that was initially toxin free. Two of the three strains of A. parasiticus produced more aflatoxin B, after 7 days of static incubation at 25 C than did the A. flavus isolates (Table 2); A. parasiticus NRRL 2999 and A. flavus NRRL 3357 synthesized equivalent amounts of the toxin. This test showed that large quantities of aflatoxin are produced on autoclaved, whole corn by strains of both Aspergillus spp. during a 1-week incubation and that the pattern of toxin elaboration is similar to the distribution observed on other substrates; i.e., A. parasiticus produces aflatoxins B1, B2, G,, and G2 and A. flavus synthesizes only aflatoxin B1 and B,. Since A. flavus commonly occurs as a natural contaminant of corn and A. parasiticus only rarely, we chose A. flavus NRRL 3357 as the test organism for further studies of toxin elaboration in corn. Aflatoxin production by A. flavus on autoclaved, ground whole corn was compared with yields on other autoclaved corn fractions (Fig. 1). During the 5-day fermentation, toxin elaboration on whole corn and commercial, full-fat germ fractions was about equal. Corn endo- TABLE 2. Aflatoxin production by A. parasiticus and A. flavus on corn meala Aflatoxin Aflatoxins Strain B,1 (gg/g of corn) B1, B. G,, G, A. parasiticus NRRL NRRL NRRL A. flavus NRRL NRRL NRRL a Incubated at 25 C for 7 days. Values represent means for triplicate flasks assayed independently. 765 InClbatiol Period Iaysj FIG. 1. Production of aflatoxin by Aspergillus flavus on corn fractions incubated statically at 25 C. Toxin production studies were carried out on 1-g samples of substrate in 50-ml Erlenmeyer flasks. Values represent means of triplicate test flasks assayed independently. sperm (grits) supported toxin production comparable to whole corn and full-fat germ during the initial 3 days of incubation; after 5 days, aflatoxin yield on endosperm was 22% below the optimal levels observed in whole corn and full-fat germ fractions. Defatted germ was the poorest substrate for toxin production; the yield after 5 days was 75% below the maximum accumulation observed on both ground, whole corn and full-fat germ. Availability of trace elements in corn germ. Differences between trace element levels in germ fractions from either A. flavus-infected or A. flavus-free corn, detected in preliminary studies, indicated a possible relationship between the concentration of metals in the germ and natural occurrence of the fungus. The interaction between trace elements and A. flavus development was further examined by testing the influence of added trace metals on aflatoxin synthesis on an autoclaved sample of commercial, defatted corn germ. Corn germ was selected for the trace element studies because metals occur predominantly in this fraction of the kernel; defatted germ was chosen because the reduced rate of aflatoxin synthesis on this substrate provided a more sensitive medium for observing subtle changes in toxin yields. Binding of trace elements by phytate in corn germ would interfere with biological availability of trace metals (8, 10, 11). Aflatoxin yields were plotted on defatted corn germ with zinc and iron added at levels ranging from 50 to 1,000 gg per g of germ (Fig. 2). Addition of 250 ug of zinc per g of germ

4 766 LILLEHOJ, GARCIA, AND LAMBROW APPL. MICROBIOL. increased aflatoxin yield 2.5 times after 3 days of incubation at 25 C; maximum production of the toxin was observed at 250 to 500 /ig of zinc per g of germ. Since previous studies had shown that addition of 1 to 5,ug of zinc per ml of a submerged fermentation medium distinctly stimulated aflatoxin production (1, 4, 19), evidently the zinc in the corn germ (208 Mg per g) is not readily available for A. flavus biosynthesis because added quantities of the metal significantly increased toxin yields. Addition of iron to the corn germ substrate had no effect on toxin production. The effect of added manganese and copper (1 to 500 Ag per g) on aflatoxin production in defatted corn germ is plotted in Fig. 3. At 10 Mg per g, either metal increased toxin levels about 2.3 times and similar yields were observed with addition of up to 250 gg per g. However, addition of 500 Mg of copper per g of defatted germ reduced aflatoxin accumulation 67% from maximum levels. Obviously manganese and copper availability in defatted germ is also an 32 Z / Fe - I Trace Element )pg/g of Corn Germ) FIG. 2. Production of aflatoxin by A. flavus on defatted corn germ enriched with zinc or iron incubated statically for 3 days at 25 C. Toxin production studies were carried out on 1-g samples of germ in 50-ml Erlenmeyer flasks. Values represent means of triplicate test flasks assayed independently cm24 T16 C Trace Element luo/g Corn Germl FIG. 3. Production of aflatoxin by A. flavus on defatted corn germ enriched with copper or manganese incubated statically for 3 days at 25 C. Toxin production was carried out on 1-g samples of germ in 50-ml Erlenmeyer flasks. Values represent means of triplicate test flasks assayed independently. Mn important factor in A. flavus production of aflatoxin. Although previous studies have shown that yields of aflatoxin in submerged fermentation can be restricted by low levels of manganese (1 ttg per g) (4), the synthesis of toxin on defatted germ was increased by the addition of 10 Mtg of the metal per g with no significant inhibition after addition of 10 to 500 Mg of the substance per g of germ. Clearly, the response of the toxin-producing fungus to manganese differs in development on defatted corn germ versus liquid media. Aflatoxin production on defatted corn germ enriched with cadmium, chromium, or lead (levels ranged from 1 to 50 tig per g) is presented in Fig. 4. From 1 to 10 Mg of cadmium per g of germ increased toxin yield 2.1 times relative to unamended controls; 25 to 50 Mg of cadmium per g decreased aflatoxin accumulation to control levels. Low levels of chromium (1 Mg per g) also increased toxin yields about two times with only a slight reduction from maximum accumulation at 50 Mg per g. Low levels of lead (1 to 5 Ag per g) did not significantly increase toxin levels, but addition of 25 to 50,ug per g of the metal increased production. It appears that cadmium, chromium, and lead levels in defatted germ are not available at the concentrations required for optimal toxin synthesis. The inhibition of aflatoxin production by increased levels of cadmium resembled the effect observed with elevated concentrations of copper. Studies of aflatoxin production on defatted corn germ enriched with trace elements demonstrate a relationship between addition of several metals and significant differences in toxin yields. Our results provide preliminary information on the association between a factor in corn, trace elements, and the process of A. flavus APb.16 A ~~~~~Cd Trace Element log/g Corn Germ] FIG. 4. Production of aflatoxin by A. flavus on defatted corn germ enriched with cadmium, lead, or chromium incubated statically for 3 days at 25 C. Toxin production was carried out on 1-g samples of germ in 50-ml Erlenmeyer flasks. Values represent means of triplicate test flasks assayed independently.

5 VOL. 28, 1974 A. FLAVUS INFECTION IN CORN 767 infection and subsequent elaboration of aflatoxin. LITERATURE CITED 1. Adye, J., and R. I. Mateles Incorporation of labelled compounds into aflatoxins. Biochim. Biophys. Acta 86: Blessin, C. W., G. E. Inglett, W. J. Garcia, and W. L. Deatherage Defatted germ flour-food ingredient from corn. Food Prod. Develop. 6: Dantzman, J., and L. Stoloff Screening method for aflatoxin in corn and various corn products. J. Ass. Off. Anal. Chem. 55: Davis, N. D., U. L. Diener, and V. P. Agnihotri Production of aflatoxins B1 and G, in chemically defined medium. Mycopathol. Mycol. Appl. 31: Detroy, R. W., E. B. Lillehoj, and A. Ciegler Aflatoxin and related compounds, p In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Academic Press, Inc., New York. 6. Fennell, D. I., R. J. Bothast, E. B. Lillehoj, and R. E. Peterson Bright greenish-yellow fluorescence and associated fungi in white corn naturally contaminated with aflatoxin. Cereal Chem. 50: Garcia, W. J., C. W. Blessin, and G. E. Inglett Mineral constituents in corn and wheat germ by atomic absorption spectroscopy. Cereal Chem. 49: Garcia, W. J., H. W. Gardner, J. F. Cavins, A. C. Stringfellow, C. W. Blessin, and G. E. Inglett Composition of air-classified defatted corn and wheatgerm flours. Cereal Chem. 49: Mateles, R. I., and J. C. Adye Production of aflatoxins in submerged culture. Appl. Microbiol. 13: O'Dell, B. L., A. R. de Boland, and S. R. Koirtyohann Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J. Agr. Food Chem. 20: O'Dell, B. L., C. E. Burpo, and J. E. Savage Evaluation of zinc availability in foodstuffs of plant and animal origin. J. Nutr. 102: Reddy, T. V., L. Viswanathan, and R. A. Venkitasubramanian Factors affecting aflatoxin production on a synthetic medium. Biochem. J. 128:61 p. 13. Schroeder, H. W Effect of corn steep liquor on mycelial growth and aflatoxin production in Aspergil- Ius parasiticus. Appl. Microbiol. 14: Shotwell, 0. L., M. L. Goulden, and C. W. Hesseltine Aflatoxin contamination: association with foreign material and characteristic fluorescence in damaged corn kernels. Cereal Chem. 49: Downloaded from on January 22, 2019 by guest