Internatio~ml Journal of Food Microbwlo~, 13 (1991) 75-80 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
75
FOOD 00397
Short communication
Natural occurrence of zearalenone and toxicogenic fungi in amaranth grain G i s e l a Bresler, G r a c i e l a V a a m o n d e a n d Silvia BriT~,Jo Laborcuorio de Microbiologla de Alimentoa, Departamento de ~hdmica Orgdnica, Area Bromatologla, Facu/tad de Ciencias Exaclas y Natur~es, Universi__~_dde Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina
(Received II June 1990; accepted 4 January 1991)
Zearalenone was detected as the natural contaminant in two samples of Amaram/ms crucmus ilrains (1980 t~g/kg and 420 /~g/Iql. respoetively). Fungi i~iated from these samples were aereened for myeotoxin pmduetio~ Two of eight iaolat~ of Fmarmm (F. ~ and F mon///j'orme) produced z~u-aknoae. One of four isolates of 4sper~'I/~ J/av~ and all four iwlates of A. parm/ticm produced aflatoxinL Other ~ poumfuflly toxa:ogcaic such as Aspergiflua re.color, Penicillwan viridicamm, P. puberulunt P. cruatosunt P. citrinunt P. expanmm and Fusarium solani were also found. Key words: Amaranth grain: ZearaJenone: Toxicogeaic fungi
lntroduet~ Amaranth is one of the oldest food crops known. Probably originating in Central and South America, it was a major grain crop in the pre-conquest Aztec empire. Amaranth leaves are eaten as a vegetable, while the seeds are used as cereal. The potential of amaranth as a valuable food resource has recently been redi.w.overed (Teutbnico and Knott, 1985; NAS, 1984). The Leed__scontain high quality protein. It could be a nutritional complement to conventional cereals, which are defficient in lysine, because of its high content in this amino acid. This plant resists drought, heat and pests, and adapts readily to new enviroments, even those adverse to other crops. Argentina has wide areas where amaranth can be cultivated. The promising perspectives of this crop encouraged extensive research at the Universidad Nacional de La Pampa about its genetic improvement. Both the chemical compo~tion of American species of amaranth grown in Argentina and their nutritional features have also been investigated (Bertoni et al., 19Ma, 19Mb; Seldes et al., 1987; C_nSmez Correspoadmce addr~: G. Bresle~, Laboratorio de Microbiologia de Aiimento~ Departamento de Quimica ~ Area Bromatoiogia, Facultad de Ciencias Exactas y Naturales, Univer~ia8 de Buenos Aires, Ciudad umv~r~mia, 1428 Buenos Aires, Argentina.
76
et al., 1986). Data on microbial aspects are very scarce in the literature (Ugarte de Mollepaza and Vargas Espinosa. 1986), and no reports on the natural occurrence of mycotoxins in this cereal-like grain have been found. Two samples of Amaranthus cruentus were received last year which were harvested on the Estaci6n Experimental Agropecuaria INTA, Anguil, La Pampa. These samples, which had been stored moist, had become moldy, and their temperature had started to increase within the sacks. Therefore, it was considered an interesting material in which the mycoflora and the possible presence of mycotoxins could be studied. Materials and Methods
Analysis of rnycotoxins Samples (50 g) were analysed as described by Waltking et al. (1968), whereby aflatoxins Ba, B2, G l and G 2, zearalenone, sterigmatocystin and ochratoxin A can be detected simultaneously (Scott et al., 1972). Extraction was performed with 250 ml of a methanol/water (55:45) mixture and 100 ml of bexane in a mechanical shaker during 30 rain. Sodium chloride (2.5 g) was added to avoid emulsion formation. The extract was filtered through Whatman No 4 filter paper. 25 ml of the aqueous phase were collected, transferred to a separatory funnel and extracted three times with 25 ml chloroform. The chloroform extracts were filtered through anhydrous Na 2SO4 and evaporated to dryness. Mycotoxin detection was performed by thin-layer chromatography on Silicagel G-60 plates (0.25 mm Merck No 5721) at room temperature in a nonsaturated chamber. Chloroform/acetone/water (90:9:1) was used as developing solvent. Toxins were estimated by visual comparison with known amounts of standards (Sigma Chemical Co.., St. Louis, MO, U.S.A.) spotted on the same plate.
Isolation and identification of fungi The dilution plating method (Pitt and Hocking, 1985) was used both for counting and isolation of fungi. 20 g of seeds were comminuted and blended (2 rain) in a high-speed blender with 180 ml of sterile 0.1% peptone. Serial dilutions were carried out in the same diluent and 0.1 ml of each dilution was spread (duplicate) on Petri dishes with appropriate culture media. Dichloran-chlorampherticol peptone agar (DCPA), proposed for isolation of Fusarium species from cereals (Andrews and Pitt, 1986) and dichloran-18% glycerol agar (DG18), for ennumeration of xerophilic fungi (Hocking and Pitt, 1980) were used. An additional portion of 20 g of seeds from each sample were surface disinfected for 1 min in 5% NaOCI, rinsed three times with sterile distilled water and dried aseptically in a laminar flow cabinet for 2 h before blending and plating as described above. Plates stored upright were incubated at 25 ° C, and mold counts were carried out after 5 days. Fungi were isolated from ennumeration plates showing well separated colonies. Penicilliurn and Aspergillus species were identified according to Pitt and Hocking (1985). Booth's key (1971) was used to identify Fusarium species. Isolates of other genera were rarely identified.
77
Mycotoxin production by fungal isolates Since zearalenone was detected in the samples, isolates of Fmarium species were screened qualitatively for zearalenone production according to Vaamonde et at. (1987). Aflatoxin production by A. flavus and A. parasiticus strains was also tested by the method of Shotwell et al. (1966) using sterile rice as substrate.
Results and discussion
Afiatoxins, ochratoxin A and sterigmatocystin were not found in the two samples investigated, but they contained zearalenone at levels of 1980 p g / k g and 420 pg/kg, respectively. Similar levels of this toxin were found in corn and animal feed involved in outbreaks of hyperestrogenism in farm animals (Mirocha et at., 1976). Tables I and II list mold genera and species found in the two samples, both before and after surface disinfection using two different culture media. Mold counts were always reduced by surface disinfection of the grains. In non surface-disinfected (NSD) grains plated on DCPA medium, a Scopulariosis sp. was among the predominant fungi. Fusar/um species were most often detected in surface-disinfected grains. In DG18 medium the mycoflora was dominated by Asper$illus and Penicillium, while Fusarmm occurred less frequently.
TABLE I Mold ~ and Ipecics isolated from Amaranthus cruentus grains m Dichloran Chloramphenic~! Peptone Agat (DCPA)
Sample (No. and u ~ u n ~ t )
Mold count (mioni~/I)
C.,¢~ra
Speci,.~
I(SD) "
2.5 x 10 2
Aspergilim Poucillium Mucor Ckaetomiun Fmanum
fla~m c. venicolor c
Fmanum Sco~anopsis Asp,.r~llus Rhizopus
solani c monili/ormt c
I(NSD) b
6 x 103
2(SD)
4 x 102
2(NSD)
4 x 103
• SD, surfsce disinfected. b NSD, no~surfsce di~nfe~:d.
~s ~
~
oo~ ~ ~
.
c ~ ~ solani c
flatms ", parasiticus c
Penicd/mm
parasitic~ c ~.rsicolor c crusto.mm c ~r/d/canon c corytophilum, chrysogenum
Peni¢illium Fuaarmm Scoptdanop$is
solani c
Asperloilus
md~,n~u~, g,mo/utm~
78 TABLE I1 Mold genera and speoes isolated from Amaranthus cruentus grains in dichloran-18% glycerol agar (DGI8) Sample (No and treatment)
Mold count (colonies/g)
Genera
Speoes
I(SD) "
1.6x 102
Aspergillus Penicillium
candldu$ ch~.,sogenum, citrtnum c brewcompactum equtseti c
Fusanum
I(NSD) b
3 × 10 3
Aspersillas P enicillium Cladosporium
flavus ¢ chrysogenum
2(SD)
3 x 101
Penicillium Aspergillus Fusanum
crastosum c, ch~. sogenum parasiticus c
2(NSD)
3 X 10 3
Aspergillux
tiaras ¢, versicolor c parasiticus c expansum c
Panwiliium Mucor
• SD, surface disinfected. b NSD, non-surface disinfected. c Species that could be toxicogenic.
Fusarium species were screened for zearalenone production (Table III). One F. moniliforme strain was a weak producer. The F. equiseti isolate produced a large quantity of toxin on sterile rice used as substrate. Toxicogenic capability of the Aspergillus flavus group species was also investigated (Table III). Only one of the four isolates of A. flavus produced aflatoxins (BI, G 1 and traces of G 2). All four isolates of A. parasiticus produced variable amounts of aflatoxins B 1, Bz, G 1 and G 2. Some of them were potent toxicogenic isolates, but
TABLE II1 Toxicogenic strains of Fusarium and Aspergillasflavus group isolated from Amaranthus cruentus grains Species
No. of toxin producers/ No of isolates examined
Mycotoxins
A spergillus flamm
1/4
A. parasitic'us
4/4
Aflatoxins B1, G~ and G 2 Aflatoxins
Fusarium solani F. moniliforme F. equiseti Fusarium sp.
0/3 1/2 1/1 0/2
B~, B2, GI, G2 Zearalenone Zearalenone -
79
they appeared to be unable to synthesize the toxin in the amaranth grains analyzed. The climatic conditions during pre- and post-harvest were probably not appropriate for A. flavus growth and aflatoxin formation. Temperature, one of the most important factors influencing mycotoxin formation, might have been too low. The temperature limit for aflatoxin production lies between 12 and 15°C (Diener and Davis, 1967; Boiler and Schroeder, 1974; Northolt et al., 1977). Only a few cultures grow at 10°C without toxin production. On the other hand, Fusar/um growth and biosynthesis of some Fmar/um toxins such as zearalenone are favored by relatively low temperatures. Normally, zearalenone has been detected in cereal grains infected by Fusar/um species during periods of wet weather and temperatures between 12 and 14°C (Smith and Moss, 1985). In this paper we report for the first time the natural occurrence of zearalenone in grains of an American species of cultivated Amaranthus. Among fungi listed in Tables I and II, several species that could be toxicogenic can be seen. Therefore, the possibility of contamination with other mycotoxins (e.g., ochratoxin, sterigmatocystin, patulin, citrinln~ and some trichotecenes) exists. Further studies to determine the mycotoxin-producing potential o[ the mycoflora of different American species of Amaranthus ( A. cruentus, A. caudatus and A. mantegazzianus ) are bC-mg performed at present.
Ackao~d~.~ts The authors are grateful to Ing. Agr. G. Covas for having sent samples of Amaranthus cruentm grmfins. This work was carried out with the financial support of Consejo Nacional de Investipciones Cientificas y T~nicas (CONICET) and Universidad de Buenos Aires. Thanks are also extended to Merck Qulmica Argentina for donating chemicals and culture media.
References Andrews, S. and Pitt, J.l. (1986) Selective med/um for isolation of Fmamon species and demafiaceous Hyphomyceles. Appl. Environ. ~ o L 51, 1235-1238. Benon/, M.H., ~ P,.G., Cattaneo, P. and Covas, G. (1984a) Estudios sobre sem/llas de espec/es americanas de An~rantb~ l--AceJ~s de A. cm~ana, A. ~
y A. mann,sazzums~. Anal Asoc,
Quire. Arg. 72. 391-397. Bertom, M.H., Gbnmz, P.O., Cattaneo, P. and Covas, G. (1984b) Estuclie6 sobre semilias de especies americanas de Amaranthm li--Harinas de extracci6n de A. ~ t m , A. ¢audmm y A. mamegazzianus. Anal. Asoc. ~ ~ 92, 599-605. Boiler. P..A. and Sehroeder, H.W. (19'74) lnflu,mce of temperature on production of al"latox/n in rice by
AspergiOmparasiticus. Phylopalhoio~ 64, 283-286. Booth, C. (1971) The l~US Fmmfum. Commonwealth M ~ Imtitute, Kew, England. Dianer, U.L and Davis, N.D. (1967) Limiting tempe~mre and relative humidity for growth and production of aflatoxins and free fatty acids by Aspergillm flmms m sterile peanuts. J. An~ Oil. Chem. Soc. 44, 259-263. F.uganio, C.P., ~ , C.M. and Mirocha, J.C. (1970) Factors affectin$ production of the mycotoxin F-2 by Ftz.,z~am roseum. PhytopatholoiD, 60, 1055-1057.
80 G6mez, R.G., Bertoni, M.H. and Covas, G. (1986) Composicibn quimica general y c,ontenidos de oxalatos y mtratos totales y reraanentes despuc~s de coc~6n del follaje en espec~es amencanas de amaramos (Amaranthus spp.), Anal. Asoc. Quire. Arg. 74, 333-338. Hocking, A.D. and Pitt, J.I. (1980) Dichloran-glycerol medium for ennumeration of xerophilic fungi from low moisture foods. Appl. Environ. Microbiol. 32, 488-492. Mirocha, J.C., Pathre, S.V., Schauerhamer, B. and Christensen, C.M. (1976) Natural occurrence of Fusanum toxins in feedstuff. Appl. Environ. Microbiol. 32, 553-556. N.A.S. (1984) Amaranth: modem prospects for an ancient crop. National Academy of Sciences.
Washington, DC. Northoh, M.D., van Egmond, H.P. and Paulsch, W.E. (1977) Differences between Aspergillusflavus strains in growth and aflatordn B I production in relation to water activity and temperature. J. Food Plot. 40, 778-781. Pitt, J.I.and Hocking, A.D. (1985) Fungi and Food Spoilage. Acadewac Press, Australia. Scott, P.M., van Walbeek, W., Kennedy, B. and Anyetti, D. (1972) Mycotoxms (Ochratoxin A, Citrinin and Sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J. Agr. Food Chem. 20, 1103-1109. Seldes. A.M., Bertoni, M.H. and Cattaneo, P. (1987) Studies on seeds of american species of Amaranthus llI-Sterol composition of extracted (hexane) seed oils from A. caudatus, A. cruentus and A. mantegazzianus. Anal. Asoc. Quire. Arg. 75, 91-94. Shotwell, O.L., Hesseltine, C.W., Stubblefield, ILD. and Sorenson, W.G. (1966) Production of aflatoxin on rice.Appl. Microbiol. 14, 425-428. Smith, J.E. and Moss, M.O. (1985) Mycotoxins. Formation, Analysis and Significance. John Wiley and Sons Ltd., Chichester, U.K. Teut6mco, R. and Knorr, D. (1985) Amaranth: composition, properues and applications of a rediscovered food. Food Technol. 39, 49-61. Ugarte de Mollepaza, N. and Vargas Espinosa, F.M. (1986) F.studios de la calidad microbiolbgica de los granos de kiwicha (Amaranthus caudarus L) en almacenamiento. Programa Nacional de la Kiwicha. Universidad Nacional del Cuzco, Peril, Repone 86-4. Vaamonde, G., Scarmato, O. and Bonera, N. (1987) Zearalenone production by Fusarium species isolated from soybeans. Int. J. Food M/crobiol. 4, 129-133. Waltking, A.E., Bleffert,G. and Kiernan, M. (1968) An improved rapid physicochemical assay method for aflatoxin in peanuts and peanut products. J. Am. Oil. Chem. SOc. 45, 880-884.
75
FOOD 00397
Short communication
Natural occurrence of zearalenone and toxicogenic fungi in amaranth grain G i s e l a Bresler, G r a c i e l a V a a m o n d e a n d Silvia BriT~,Jo Laborcuorio de Microbiologla de Alimentoa, Departamento de ~hdmica Orgdnica, Area Bromatologla, Facu/tad de Ciencias Exaclas y Natur~es, Universi__~_dde Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina
(Received II June 1990; accepted 4 January 1991)
Zearalenone was detected as the natural contaminant in two samples of Amaram/ms crucmus ilrains (1980 t~g/kg and 420 /~g/Iql. respoetively). Fungi i~iated from these samples were aereened for myeotoxin pmduetio~ Two of eight iaolat~ of Fmarmm (F. ~ and F mon///j'orme) produced z~u-aknoae. One of four isolates of 4sper~'I/~ J/av~ and all four iwlates of A. parm/ticm produced aflatoxinL Other ~ poumfuflly toxa:ogcaic such as Aspergiflua re.color, Penicillwan viridicamm, P. puberulunt P. cruatosunt P. citrinunt P. expanmm and Fusarium solani were also found. Key words: Amaranth grain: ZearaJenone: Toxicogeaic fungi
lntroduet~ Amaranth is one of the oldest food crops known. Probably originating in Central and South America, it was a major grain crop in the pre-conquest Aztec empire. Amaranth leaves are eaten as a vegetable, while the seeds are used as cereal. The potential of amaranth as a valuable food resource has recently been redi.w.overed (Teutbnico and Knott, 1985; NAS, 1984). The Leed__scontain high quality protein. It could be a nutritional complement to conventional cereals, which are defficient in lysine, because of its high content in this amino acid. This plant resists drought, heat and pests, and adapts readily to new enviroments, even those adverse to other crops. Argentina has wide areas where amaranth can be cultivated. The promising perspectives of this crop encouraged extensive research at the Universidad Nacional de La Pampa about its genetic improvement. Both the chemical compo~tion of American species of amaranth grown in Argentina and their nutritional features have also been investigated (Bertoni et al., 19Ma, 19Mb; Seldes et al., 1987; C_nSmez Correspoadmce addr~: G. Bresle~, Laboratorio de Microbiologia de Aiimento~ Departamento de Quimica ~ Area Bromatoiogia, Facultad de Ciencias Exactas y Naturales, Univer~ia8 de Buenos Aires, Ciudad umv~r~mia, 1428 Buenos Aires, Argentina.
76
et al., 1986). Data on microbial aspects are very scarce in the literature (Ugarte de Mollepaza and Vargas Espinosa. 1986), and no reports on the natural occurrence of mycotoxins in this cereal-like grain have been found. Two samples of Amaranthus cruentus were received last year which were harvested on the Estaci6n Experimental Agropecuaria INTA, Anguil, La Pampa. These samples, which had been stored moist, had become moldy, and their temperature had started to increase within the sacks. Therefore, it was considered an interesting material in which the mycoflora and the possible presence of mycotoxins could be studied. Materials and Methods
Analysis of rnycotoxins Samples (50 g) were analysed as described by Waltking et al. (1968), whereby aflatoxins Ba, B2, G l and G 2, zearalenone, sterigmatocystin and ochratoxin A can be detected simultaneously (Scott et al., 1972). Extraction was performed with 250 ml of a methanol/water (55:45) mixture and 100 ml of bexane in a mechanical shaker during 30 rain. Sodium chloride (2.5 g) was added to avoid emulsion formation. The extract was filtered through Whatman No 4 filter paper. 25 ml of the aqueous phase were collected, transferred to a separatory funnel and extracted three times with 25 ml chloroform. The chloroform extracts were filtered through anhydrous Na 2SO4 and evaporated to dryness. Mycotoxin detection was performed by thin-layer chromatography on Silicagel G-60 plates (0.25 mm Merck No 5721) at room temperature in a nonsaturated chamber. Chloroform/acetone/water (90:9:1) was used as developing solvent. Toxins were estimated by visual comparison with known amounts of standards (Sigma Chemical Co.., St. Louis, MO, U.S.A.) spotted on the same plate.
Isolation and identification of fungi The dilution plating method (Pitt and Hocking, 1985) was used both for counting and isolation of fungi. 20 g of seeds were comminuted and blended (2 rain) in a high-speed blender with 180 ml of sterile 0.1% peptone. Serial dilutions were carried out in the same diluent and 0.1 ml of each dilution was spread (duplicate) on Petri dishes with appropriate culture media. Dichloran-chlorampherticol peptone agar (DCPA), proposed for isolation of Fusarium species from cereals (Andrews and Pitt, 1986) and dichloran-18% glycerol agar (DG18), for ennumeration of xerophilic fungi (Hocking and Pitt, 1980) were used. An additional portion of 20 g of seeds from each sample were surface disinfected for 1 min in 5% NaOCI, rinsed three times with sterile distilled water and dried aseptically in a laminar flow cabinet for 2 h before blending and plating as described above. Plates stored upright were incubated at 25 ° C, and mold counts were carried out after 5 days. Fungi were isolated from ennumeration plates showing well separated colonies. Penicilliurn and Aspergillus species were identified according to Pitt and Hocking (1985). Booth's key (1971) was used to identify Fusarium species. Isolates of other genera were rarely identified.
77
Mycotoxin production by fungal isolates Since zearalenone was detected in the samples, isolates of Fmarium species were screened qualitatively for zearalenone production according to Vaamonde et at. (1987). Aflatoxin production by A. flavus and A. parasiticus strains was also tested by the method of Shotwell et al. (1966) using sterile rice as substrate.
Results and discussion
Afiatoxins, ochratoxin A and sterigmatocystin were not found in the two samples investigated, but they contained zearalenone at levels of 1980 p g / k g and 420 pg/kg, respectively. Similar levels of this toxin were found in corn and animal feed involved in outbreaks of hyperestrogenism in farm animals (Mirocha et at., 1976). Tables I and II list mold genera and species found in the two samples, both before and after surface disinfection using two different culture media. Mold counts were always reduced by surface disinfection of the grains. In non surface-disinfected (NSD) grains plated on DCPA medium, a Scopulariosis sp. was among the predominant fungi. Fusar/um species were most often detected in surface-disinfected grains. In DG18 medium the mycoflora was dominated by Asper$illus and Penicillium, while Fusarmm occurred less frequently.
TABLE I Mold ~ and Ipecics isolated from Amaranthus cruentus grains m Dichloran Chloramphenic~! Peptone Agat (DCPA)
Sample (No. and u ~ u n ~ t )
Mold count (mioni~/I)
C.,¢~ra
Speci,.~
I(SD) "
2.5 x 10 2
Aspergilim Poucillium Mucor Ckaetomiun Fmanum
fla~m c. venicolor c
Fmanum Sco~anopsis Asp,.r~llus Rhizopus
solani c monili/ormt c
I(NSD) b
6 x 103
2(SD)
4 x 102
2(NSD)
4 x 103
• SD, surfsce disinfected. b NSD, no~surfsce di~nfe~:d.
~s ~
~
oo~ ~ ~
.
c ~ ~ solani c
flatms ", parasiticus c
Penicd/mm
parasitic~ c ~.rsicolor c crusto.mm c ~r/d/canon c corytophilum, chrysogenum
Peni¢illium Fuaarmm Scoptdanop$is
solani c
Asperloilus
md~,n~u~, g,mo/utm~
78 TABLE I1 Mold genera and speoes isolated from Amaranthus cruentus grains in dichloran-18% glycerol agar (DGI8) Sample (No and treatment)
Mold count (colonies/g)
Genera
Speoes
I(SD) "
1.6x 102
Aspergillus Penicillium
candldu$ ch~.,sogenum, citrtnum c brewcompactum equtseti c
Fusanum
I(NSD) b
3 × 10 3
Aspersillas P enicillium Cladosporium
flavus ¢ chrysogenum
2(SD)
3 x 101
Penicillium Aspergillus Fusanum
crastosum c, ch~. sogenum parasiticus c
2(NSD)
3 X 10 3
Aspergillux
tiaras ¢, versicolor c parasiticus c expansum c
Panwiliium Mucor
• SD, surface disinfected. b NSD, non-surface disinfected. c Species that could be toxicogenic.
Fusarium species were screened for zearalenone production (Table III). One F. moniliforme strain was a weak producer. The F. equiseti isolate produced a large quantity of toxin on sterile rice used as substrate. Toxicogenic capability of the Aspergillus flavus group species was also investigated (Table III). Only one of the four isolates of A. flavus produced aflatoxins (BI, G 1 and traces of G 2). All four isolates of A. parasiticus produced variable amounts of aflatoxins B 1, Bz, G 1 and G 2. Some of them were potent toxicogenic isolates, but
TABLE II1 Toxicogenic strains of Fusarium and Aspergillasflavus group isolated from Amaranthus cruentus grains Species
No. of toxin producers/ No of isolates examined
Mycotoxins
A spergillus flamm
1/4
A. parasitic'us
4/4
Aflatoxins B1, G~ and G 2 Aflatoxins
Fusarium solani F. moniliforme F. equiseti Fusarium sp.
0/3 1/2 1/1 0/2
B~, B2, GI, G2 Zearalenone Zearalenone -
79
they appeared to be unable to synthesize the toxin in the amaranth grains analyzed. The climatic conditions during pre- and post-harvest were probably not appropriate for A. flavus growth and aflatoxin formation. Temperature, one of the most important factors influencing mycotoxin formation, might have been too low. The temperature limit for aflatoxin production lies between 12 and 15°C (Diener and Davis, 1967; Boiler and Schroeder, 1974; Northolt et al., 1977). Only a few cultures grow at 10°C without toxin production. On the other hand, Fusar/um growth and biosynthesis of some Fmar/um toxins such as zearalenone are favored by relatively low temperatures. Normally, zearalenone has been detected in cereal grains infected by Fusar/um species during periods of wet weather and temperatures between 12 and 14°C (Smith and Moss, 1985). In this paper we report for the first time the natural occurrence of zearalenone in grains of an American species of cultivated Amaranthus. Among fungi listed in Tables I and II, several species that could be toxicogenic can be seen. Therefore, the possibility of contamination with other mycotoxins (e.g., ochratoxin, sterigmatocystin, patulin, citrinln~ and some trichotecenes) exists. Further studies to determine the mycotoxin-producing potential o[ the mycoflora of different American species of Amaranthus ( A. cruentus, A. caudatus and A. mantegazzianus ) are bC-mg performed at present.
Ackao~d~.~ts The authors are grateful to Ing. Agr. G. Covas for having sent samples of Amaranthus cruentm grmfins. This work was carried out with the financial support of Consejo Nacional de Investipciones Cientificas y T~nicas (CONICET) and Universidad de Buenos Aires. Thanks are also extended to Merck Qulmica Argentina for donating chemicals and culture media.
References Andrews, S. and Pitt, J.l. (1986) Selective med/um for isolation of Fmamon species and demafiaceous Hyphomyceles. Appl. Environ. ~ o L 51, 1235-1238. Benon/, M.H., ~ P,.G., Cattaneo, P. and Covas, G. (1984a) Estudios sobre sem/llas de espec/es americanas de An~rantb~ l--AceJ~s de A. cm~ana, A. ~
y A. mann,sazzums~. Anal Asoc,
Quire. Arg. 72. 391-397. Bertom, M.H., Gbnmz, P.O., Cattaneo, P. and Covas, G. (1984b) Estuclie6 sobre semilias de especies americanas de Amaranthm li--Harinas de extracci6n de A. ~ t m , A. ¢audmm y A. mamegazzianus. Anal. Asoc. ~ ~ 92, 599-605. Boiler. P..A. and Sehroeder, H.W. (19'74) lnflu,mce of temperature on production of al"latox/n in rice by
AspergiOmparasiticus. Phylopalhoio~ 64, 283-286. Booth, C. (1971) The l~US Fmmfum. Commonwealth M ~ Imtitute, Kew, England. Dianer, U.L and Davis, N.D. (1967) Limiting tempe~mre and relative humidity for growth and production of aflatoxins and free fatty acids by Aspergillm flmms m sterile peanuts. J. An~ Oil. Chem. Soc. 44, 259-263. F.uganio, C.P., ~ , C.M. and Mirocha, J.C. (1970) Factors affectin$ production of the mycotoxin F-2 by Ftz.,z~am roseum. PhytopatholoiD, 60, 1055-1057.
80 G6mez, R.G., Bertoni, M.H. and Covas, G. (1986) Composicibn quimica general y c,ontenidos de oxalatos y mtratos totales y reraanentes despuc~s de coc~6n del follaje en espec~es amencanas de amaramos (Amaranthus spp.), Anal. Asoc. Quire. Arg. 74, 333-338. Hocking, A.D. and Pitt, J.I. (1980) Dichloran-glycerol medium for ennumeration of xerophilic fungi from low moisture foods. Appl. Environ. Microbiol. 32, 488-492. Mirocha, J.C., Pathre, S.V., Schauerhamer, B. and Christensen, C.M. (1976) Natural occurrence of Fusanum toxins in feedstuff. Appl. Environ. Microbiol. 32, 553-556. N.A.S. (1984) Amaranth: modem prospects for an ancient crop. National Academy of Sciences.
Washington, DC. Northoh, M.D., van Egmond, H.P. and Paulsch, W.E. (1977) Differences between Aspergillusflavus strains in growth and aflatordn B I production in relation to water activity and temperature. J. Food Plot. 40, 778-781. Pitt, J.I.and Hocking, A.D. (1985) Fungi and Food Spoilage. Acadewac Press, Australia. Scott, P.M., van Walbeek, W., Kennedy, B. and Anyetti, D. (1972) Mycotoxms (Ochratoxin A, Citrinin and Sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J. Agr. Food Chem. 20, 1103-1109. Seldes. A.M., Bertoni, M.H. and Cattaneo, P. (1987) Studies on seeds of american species of Amaranthus llI-Sterol composition of extracted (hexane) seed oils from A. caudatus, A. cruentus and A. mantegazzianus. Anal. Asoc. Quire. Arg. 75, 91-94. Shotwell, O.L., Hesseltine, C.W., Stubblefield, ILD. and Sorenson, W.G. (1966) Production of aflatoxin on rice.Appl. Microbiol. 14, 425-428. Smith, J.E. and Moss, M.O. (1985) Mycotoxins. Formation, Analysis and Significance. John Wiley and Sons Ltd., Chichester, U.K. Teut6mco, R. and Knorr, D. (1985) Amaranth: composition, properues and applications of a rediscovered food. Food Technol. 39, 49-61. Ugarte de Mollepaza, N. and Vargas Espinosa, F.M. (1986) F.studios de la calidad microbiolbgica de los granos de kiwicha (Amaranthus caudarus L) en almacenamiento. Programa Nacional de la Kiwicha. Universidad Nacional del Cuzco, Peril, Repone 86-4. Vaamonde, G., Scarmato, O. and Bonera, N. (1987) Zearalenone production by Fusarium species isolated from soybeans. Int. J. Food M/crobiol. 4, 129-133. Waltking, A.E., Bleffert,G. and Kiernan, M. (1968) An improved rapid physicochemical assay method for aflatoxin in peanuts and peanut products. J. Am. Oil. Chem. SOc. 45, 880-884.
