On-line version ISSN 1678-457X
Ciênc. Tecnol. Aliment. vol.20 n.1 Campinas Apr. 2000
Mycoflora and aflatoxigenic species in derivatives of milled rice1
Thirty samples of rough rice stored for 6, 12 and 24 months in government authorized warehouses of the state of Rio Grande do Sul, Brazil, were simultaneously collected. After milling of the product, 90 samples (30 of polished rice, 30 of rice bran and 30 of rice hull) were evaluated for their mycoflora, aflatoxigenic species and aflatoxin contamination. The following fungi, listed in decreasing order of frequency, were isolated on Potato-Dextrose Agar: Aspergillus spp., Nigrospora spp., Penicillium spp.; Fusarium spp.; Mucor spp.; Cladosporium spp.; Trichosporon spp. and non-sporulated fungi. The degree of fungal contamination (colony forming units per gram of product) was lowest in polished rice, increasing progressively in samples of rice bran and rice hull. Among the Aspergillus species, A. flavus and A. candidus were isolated most frequently. Of the A. flavus isolates, 52.6% strains were found to be toxigenic and produced only Group B aflatoxins. Analysis of the 90 samples did not reveal the presence of aflatoxins in the rice derivatives.
aflatoxin; Aspergillus flavus; mycoflora; milled rice; stored rice.
Microbiota fúngica e espécies aflatoxigênicas em derivados de arroz beneficiado. Foram coletadas trinta amostras de arroz em casca, armazenados por 6, 12 e 24 meses em armazéns oficiais situados no Estado do Rio Grande do Sul. Após beneficiamento do produto obteve-se 90 amostras (30 de arroz brunido, 30 de farelo e 30 de palha) nas quais verificou-se a microbiota fúngica, a presença de espécies produtoras de aflatoxinas e a contaminação por aflatoxinas. Os seguintes gêneros fúngicos foram isolados em ágar batata dextrose, em ordem decrescente de freqüência, Aspergillus spp., Nigrospora spp., Penicillium spp.; Fusarium spp.; Mucor spp.; Cladosporium spp.; Trichosporon spp. e fungos não esporulados. O grau de contaminação fúngica (unidades formadoras de colônia/grama de produto) foi baixo no arroz brunido, aumentando progresivamente em amostras de farelo e palha. Entre as espécies de Aspergillus, A. candidus foi isolado com maior frequência seguido de A. flavus. Das cepas isoladas de A. flavus 52,6% foram produtoras de aflatoxinas do grupo B. As análises micotoxicológicas das 90 amostras não revelaram a presença de aflatoxinas nos derivados do arroz.
aflatoxinas; Aspergillus flavus; microbiota; arroz armazenado; arroz beneficiado.
Rice (Oriza sativa L) is the staple diet in a significant part of the world, particularly in countries of the Orient. Brazil, though not belonging to this group of nations, has one of the greatest annual consumption of rice per capita, namely 45kg per inhabitant . The structure and chemical composition of this cereal grass grain make it an ideal substrate for the establishment and growth of fungal species, especially toxigenic fungi that secrete mycotoxins, including the aflatoxins .
Factors like moisture content, water activity, temperature, period of storage, initial levels of contamination, toxigenic potential of fungal strains and nature of the substrate influence the production of mycotoxins .
Considering the economic and nutritional importance of rice, the serious risks posed to human and animal health by contaminated rice grains and derivatives and finally the scarcity of studies on this matter, in Brazil, the present work was undertaken to study the mycoflora, presence of aflatoxigenic species and aflatoxin contamination of rice derivatives.
2 MATERIAL AND METHODS
Rough rice was obtained from authorized warehouses of the Instituto Rio Grandense do Arroz (IRGA), state of Rio Grande do Sul, Brazil. Samples were taken simultaneously from combining two long grained varieties (BR/IRGA 409, BR/IRGA 410, IRGA 416) kept in the warehouses for 6, 12 and 24 months with appropriate ventilation (10 samples of 4Kg for each storage time). Sampling was performed according to FONSECA . Rough rice were milled in a Suzuki mill. A total of ninety new samples of derivatives was obtained: 30 of polished rice, 30 of rice bran and 30 of rice hull. For each derivative, 10 samples per storage time were submitted to evaluation.
Moisture content (MC) was determined by direct heating of samples at 105° C until a constant weight. The water activity (aw) of the samples was determined by automated analysis with the equipment AQUALAB CX-2 (Decagon Devices Inc.).
Fungi were recovered as described by SWANSON et al. . Portions of 10g of the 90 samples were blended in 90mL of phosphate buffered saline (PBS) and serial dilutions were prepared up to a concentration of 10-5. One milliliter of each dilution was plated onto Potato Dextrose Agar (pH 5.6, aw 0.98) duplicates. The plates were incubated for 7 days at 25° C and observed daily. Fungal colonies were selected for subculturing and identified according to the methodology recommended for each genus [13, 14].
The strains of Aspergillus flavus were inoculated on to Coconut-Agar medium and incubated for 7 days at 25° C. Aliquot of 10g of this culture medium were transferred to a 200mL beaker containing 30mL of chloroform. The mixture was macerated, filtered through filter paper and the filtrate evaporated in a water bath. The dry extract was solubilized in chloroform and chromatographed as described by LIN & DIANESE .
Samples were evaluated for the presence of aflatoxins following the procedure of SOARES & RODRIGUEZ-AMAYA . The method has a detection limit of 2 mg/kg.
3 RESULTS AND DISCUSSION
Moisture content and water activity of the milling derivatives of rough rice with 6, 12 and 24 months of storage are displayed on Table 1. By and large, a reduction in moisture was observed over the storage periods.
The most frequent contaminants were members of the genus Aspergillus with colony forming units per gram of product (CFU/g) values of 1 to 60x103 for polished rice, 1 to 130x103 for rice bran and 1 to 106x103 for rice hull. A rise in CFU/g was observed during the storage period. The highest CFU/g values for Aspergillus spp. were recorded after 12 months of storage (Table 1).
Table 2 shows the fungi recovered from polished rice in decreasing order of frequency: Aspergillus (33.3%), Penicillium [13.4], non-sporulated fungi (10%), Fusarium (6.7%), Nigrospora (3.3%), Trichosporon (3.3%), Mucor (3.3%) and Cladosporium (3.3%). The genera isolated from rice bran were Aspergillus (80%) non-sporulated fungi (23.3%), Nigrospora (16.6%) Penicillium (10%) Fusarium (6.6%) and Mucor (3.3%), and those recovered from rice hull were Aspergillus (76.7%), non-sporulated fungi (30%), Nigrospora (26.7%), Penicillium (16.6%), Fusarium (6.6%) and Mucor (3.3%). Within the genus Aspergillus, A. flavus and A. candidus were the predominant species (Table 3).
Out of the 19 isolates of A. flavus recovered, 52.6% were aflatoxigenic, producing aflatoxins B1 and B2. The mycotoxicological analysis of polished rice (30 samples), rice bran (30 samples) and rice hull (30 samples) did not reveal the presence of aflatoxins.
The greater frequencies of isolation and levels of contamination (CFU/g) found for rice hull when compared with rice bran and polished rice are in agreement with data presented by other workers . Among the fungi recovered, species of the A. flavus group (A. tamarii and particularly A. flavus) are considered aflatoxigenic  and other species such as A. candidus , A. alutaceus (A. ochraceus) and A. versicolor  are also toxigenic. Representatives of the Glaucus group, like A. amstelodami, and A.ruber, are known to be involved in the biodeterioration of stored grains .
CHRISTENSEN & KAUFMAN  observed that the predominant species of the A. glaucus group (A. amstelodami, A. chevalierii, A. repens and A ruber) occurred in stored grain samples with 13% and 14% MC. The frequencies of A candidus and A. ochrceus were most marked in grains with 15% MC and A. flavus prevailed in grains with 17% and 18% MC. The Penicillium isolates were generally dominant in grains with MC values exceeding 16%.
In this work, the MC range for the samples studied was within the legal limits and technically expected values: a maximal 13.4% MC was recorded for polished rice after 6 months of storage. Mean MC values were below 13%, which is considered the limit to avoid aflatoxin contamination according to the FOOD AND AGRICULTURAL ORGANIZATION  and also the value permitted by Brazilian authorities for milling and storage of rice . With respect to water activity, a maximum of 0.66 was found in samples of rice hull after 6 months of storage. It should be pointed out that the majority of the recorded aw values (Table 1) were less than value of 0.60 accepted as the minimum necessary for fungal growth to occur and well below the level associated with the production of aflatoxins 0.83 [7, 12]. However, our data support the views of McLEAN & BERJAK  that fungal spores remain viable for some time even when water is availability inappropriate for reproduction. These viable spores will grow once isolated from sampled grains on appropriate culture media with water activities of about 0.90, as observed in this work.
Our results show that the levels of moisture recorded did not favour the occurrence of aflatoxins in the derivatives of milled rice studied, meaning at least good storage conditions were being kept at the ware houses sampled. However, the finding that 52.6% of the A. flavus isolates were able to produce aflatoxins under laboratory conditions, represent a potential hazard and demonstrates the importance of storage conditions for this commodity.
 BRASIL Ministério da Agricultura, Portaria 845. Diário Oficial, Brasília, 17 Nov. 1976. [ Links ]
 CHATTOPADHYAY, S.K.; NANDI, B.; GHOSH, P.; THAKUR, S. A new mycotoxin from Aspergillus candidus Link isolated from rough rice. Mycopathol., v. 98, p. 21-26, 1987. [ Links ]
 CHRISTENSEN, C.M.; KAUFMANN, H.H. Deterioration of stored grains by fungi. Annual Review. Phytopathol., v. 3, p. 69-84, 1965. [ Links ]
 DEL PRADO, F.A.; CHRISTENSEN, C.M. Grain storage studies 12: The fungus flora of stored rice seed. Cereal Chem., v. 29, p. 456-462, 1952. [ Links ]
 FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Recommended practices for the prevention of mycotoxins in food, feed and their products. In: FAO Food and Nutrition papers n. 10. Rome, 1979. [ Links ]
 Fonseca, H. Sistema de amostragem para análise de aflatoxinas em grãos. Rev. Microbiol., v. 21, p. 66-70, 1991. [ Links ]
 FRISVAD, J.C.; SAMSON, R.A. Filamentous fungi in foods and feeds: Ecology, spoilage and mycotoxins production. In: ARORA, D.K.; MUKERJI, K.G.; MARTH, E.M. Eds. Handbook of applied mycology: Foods and feeds. New York: Marcel Dekker, p. 31-68, 1991. [ Links ]
 GUIA DA SAFRA. In: Folha de São Paulo, 28 de março. São Paulo: p. 6, 1995. [ Links ]
 LACEY, J. Factors affeting mycotoxins production. In: STEYN, P.S. & VLEGGAAR, R. Eds. Mycotoxins and Phycotoxins. New York: Elsevier, p. 65-76, 1986. [ Links ]
 LIN, M.T. & DIANESE, J.C. A coconut agar medium for rapid detection of aflatoxin production by Aspergillus spp. Phytopathol., v. 66, p. 1466-1469, 1976. [ Links ]
 McLEAN, M. & BERJAK, P. Maize grains and their associated mycoflora - a microecological consideration. Seed Sci. and Technol., v. 15, p. 831-850, 1987. [ Links ]
 PITT, J.L. & HUCKING, A.D. Fungi and Food Spoilage, 2nd edn. Cambridge: University Press, 1997. [ Links ]
 RAPER, K.B. & FENNELL, D.I. The genus Aspergillus. Baltimore: WILLIANS & WILLKINS, 1965. [ Links ]
 SAMSON, R.A. & PITT, J.I. Advances in Penicillium and Aspergillus Systematics. NATO ASI Series 102. New York: Plenum Press, 1985. [ Links ]
 SHOTWELL, O.L.; HESSELTINE, C.W.; STUBBLEFIELD, R.D.; SORENSON, W.G. Production of aflatoxin on rice. Appl. Microbiol., v. 14, p. 425-428, 1966. [ Links ]
 SOARES, L.M.V. & RODRIGUES-AMAYA, D.B. Survey of aflatoxins, ochratoxin A, zearalenone and sterigmatocystin in some Brazilian foods by using multi-toxin thin-layer chromatographic method. J. Assoc. Off. Anal. Chem., v. 72 (1), p. 22-26, 1989. [ Links ]
 SWANSON, K.M.J.; BUSTA, F.F.; PETERSON, E.H.; JOHNSON, M.G. Colony count methods. In: VANDERZANT, C. & SPLITTSTOESSER, D.F. Eds. Compendium of methods for the microbiological examination of foods. Washington: American Public Health Association, p. 75-96,1992. [ Links ]
1 Recebido para publicação em 31/05/99. Aceito para publicação em 10/04/00.
* A quem a correspondência deve ser envida.