International Journal of Food Microbiology 94 (2004) 211 215 www.elsevier.

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Aflatoxins in pozol, a nixtamalized, maize-based food

ndez-Albores a, G. Ara mbula-Villa a, J.A. Me z b, E. Moreno-Mart nez c,* R.E. Preciado-Ort
a

taro, C. P. 76230, Mexico CINVESTAV-IPN, Libramiento Norponiente 2000, Quere b INIFAP, Apartado Postal 112, C.P. 38010, Celaya-Guanajuato, Mexico c n-UNAM, Apartado Postal 25, Cuautitla n, C.P. 57740, Mexico FES-Cuautitla

Received 25 July 2003; received in revised form 8 January 2004; accepted 4 February 2004

Abstract To determine whether pozol, a nixtamalized maize-based food was contaminated with aflatoxins, samples of non-fermented pozol were collected during the period November 2002 to April 2003 from local markets at Comitan in Chiapas, Mexico. The samples were analyzed for the presence of aflatoxins. Nineteen out of one hundred and eleven samples were contaminated with aflatoxin B2 (AFB2) and traces of aflatoxin B1 (AFB1). The percentage of samples contaminated with AFB2 in pozol prepared with white maize was 5.4%. Pozol mixed with toasted cacao paste had a contamination rate of 41.5%. No aflatoxins were detected in pozol prepared with yellow maize. It was found that only 1 of 19 contaminated samples had aflatoxin concentrations above 20 ppb. D 2004 Elsevier B.V. All rights reserved.
Keywords: Aflatoxins; Maize; Pozol; Nixtamalized foods; Chiapas

1. Introduction Certain indigenous inhabitants of Mexico eat maize-based foods as the principle part of their diet. Typically, maize provides about 70% of calorie and 50% of protein intakes (Paredes-Lopez and Escobedo, 1983). Pozol (from the Aztec pozolli, foamy) can be prepared as either a fermented, non-alcoholic, acidic dough (masa) or as a non-fermented beverage. The maize is nixtamalized to prepare the masa, using the same procedure as in the preparation of
* Corresponding author. Tel.: +52-55-5880-93-16; fax: +52-555880-94-40. E-mail address: ernesto@servidor.unam.mx nez). (E. Moreno-Mart 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.02.009

tortillas. That is, it is cooked with lime in abundant water, washed and milled. The masa is formed into rolls that range from 10 to 12 cm in length and 5 8 cm in diameter, which weigh 70 170 g. The masa rolls are wrapped in banana leaves and are fermented. For nonfermented pozol, the fresh rolls are dispersed in water to produce a thin porridge (atole). This beverage is consumed both as a refreshment and with meals. Pozol is commonly consumed, in the states of Chiapas, Tabasco, Campeche and Yucatan, as a fermented or non-fermented drink. Daily consumption of pozol ranges from 80 to 1000 g per person (Ulloa et al., 1987). The pozol is prepared using white or yellow maize alone or the masa is mixed with toasted as-Urbina et cacao paste or ground cacao beans (Can al., 1993).

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A major health risk associated with maize-based foods is the consumption of mycotoxins, because maize grains are commonly infected by toxigenic fungi such as species of Fusarium, Penicillium or Aspergillus (Christensen and Kaufmann, 1969). Even when those fungi are destroyed by the nixtamalization process, the toxins can remain in the masa. As no sanitary measures are taken during pozol preparation, microbial contamination of the maize dough is inevitable. Lactic acid bacteria, Enterobacteriaceae, and aerobic mesophiles are introduced at the grinding step (Wacher et al., 1993), while Ulloa et al. (1987) recovered Geotrichum candidum, Trichosporon cutaneum and various species of Candida associated from pozol during the first few hours of fermentation. Other yeasts and yeast-like fungi found in pozol were Candida krusei, Hansenula fabiani, Candida guillermondii, Candida parapsilosis, Candida tropicalis and Saccharomyces cerevisiae. Filamentous fungi isolated from pozol included Alternaria tenuis, Aspergillus flavus , Cladosporium herbarum, Epicoccum sp., Fusarium sp., Mucor rouxianus, Paecilomyces fumosoroseus, Rhizopus stolonifer, Trichoderma viride, Penicillium claviforme, Penicillium cyclopium, Penicillium expansum, Penicillium italicum, and Penicillium lanoso-viridae. Aflatoxins are secondary metabolites produced principally by toxigenic strains of A. flavus Link and Aspergillus parasiticus Speare (Asao et al., 1963). Both those species invade maize in the field and in store. Aflatoxins in foods have been associated with the development of liver cancer in humans (Harrison et al., 1993). Also, there is evidence that air-borne particles of dust contaminated by aflatoxins contribute to the development of pulmonary cancer (Dvorackova, 1976). Four principal aflatoxins (AFs) are produced by A. flavus and A. parasiticus; AFB1, AFB2, AFG1, and AFG2. AFB1 is considered the most powerful hepatocarcinogenic agent known (Wogan and Newberne, 1967). Aflatoxins are found in maize-based feeds associated with animal toxicoses (Bodine and Mertens, 1983) and in maize-based human foods in underdeveloped countries. In Mexico, surveys of Aspergillus species and their potential to produce aflatoxins have revealed that A. flavus and A. parasiticus are the fungi most frequently isolated from maize (Carvajal and Arroyo, 1997). Commercially available feeds are

frequently contaminated with aflatoxins (Keyl and Booth, 1971). However, reports on mycotoxin contamination of foods for human consumption are still meager. This research was undertaken to obtain information about the incidence of aflatoxin contamination in maize-pozol destined for human consumption in the Mexican State of Chiapas.

2. Materials and methods 2.1. Source of samples A total of 111 samples of pozol purchased in local markets at Comitan, Chiapas, Mexico during November 2002 to April 2003 were dried in a vacuum oven at 40 jC for 24 h, then milled and sieved. Representative samples of pozol prepared with white maize (white-pozol) or yellow maize (yellow-pozol) and pozol mixed with toasted cacao paste (cacao-pozol) were collected and stored in paper bags at 4 jC. The color of the maize used in the pozol with cacao was not identified because it was obscured by the color of the cacao. 2.2. Aflatoxin analysis Total aflatoxins were determined according to the 991.31 AOAC method (AOAC, 1995) using monoclonal antibody columns for AFB1, AFB2, AFG1, and AFG2. Dry sub-samples of approximately 250 g were finely ground in a mill (Pulvex-200, sieve 0.8 mm; Pulvex, Mexico City, Mexico) and thoroughly mixed. Portions of the ground material (50 g) were extracted by blending with methanol water (80:20 v/v) using a laboratory blender (Mod. 51BL30; Waring, New Hartford, CT, USA). The mixture was filtered through a Whatman No. 1 filter paper and a 10 ml portion was diluted with 40 ml of distilled water. The diluted preparation was filtered through a micro-fiber filter, and 10 ml were applied to an immunoaffinity column (Aflatest-P; VICAM Science Technology, Watertown, MA, USA). Subsequently, the column was washed twice with 10 ml of distilled water and dried in a sterile air flow chamber. The toxins were then eluted with 1 ml of methanol. When the concentration of total aflatoxins was greater than 10 ppb,

 ndez-Albores et al. / International Journal of Food Microbiology 94 (2004) 211215 J.A. Me Table 1 Incidences, levels and types of aflatoxin in pozol samples from Chiapas Pozol type Incidence Range (ppb) 07 N.D. 0 21 Mean of positives (ppb)* 4.5 F 3.5 ab a 8.2 F 4.3 b Predominant AF type AFB2** AFB2**

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White-pozol Yellow-pozol Cacao-pozol

2/37 0/33 17/41

recovery by means of the HPLC method. With four replicates of six different aflatoxin concentrations (0.78, 1.56, 3.13, 6.25, 12.50 and 25 ppb), an aflatoxin recovery of 92% was attained, with a standard error of 1.2% and a coefficient of variation of 5.4%. These results indicated that the method used was applicable. 2.3. Statistical analysis The experiment was conducted as a completely randomized design with three treatments (whitepozol, yellow-pozol and cacao-pozol). Total aflatoxins and aflatoxin identity were determined in triplicate. Data were assessed by analysis of variance (ANOVA) and means were separated by the Tukey procedure using the Statistical Analysis System (SAS Institute, 1998). A significance value of (a = 0.05) was used to distinguish significant differences between treatments.

Means with the same letter in the same column are not significantly different (Tukey < 0.05). N.D.not detected (below detection limit 0.5 ppb). Means of three replicates F standard deviation. * Aflatoxin content (ppb) in dry-basis. ** Traces of AFB1.

dilutions from the extract were made for quantification of aflatoxins in a fluorometer (VICAM series 4. BBI; Source Scientific, Garden Grove, CA, USA) after reaction with 1 ml of 0.002% bromine solution (Candlish et al., 1991). The detection limit for total aflatoxins with the immunoaffinity method was approximately 0.5 ppb. Aflatoxin identity was confirmed by high-performance liquid chromatography. A high-performance liquid chromatograph (HPLC) system with two pumps (Mod 510; Waters Associates, Milford, MA, USA) and a reverse phase column (C18, 5 Am, 3.9 150 mm; Waters Associates) was used. Standards, as well as samples collected from the immunoaffinity column (20 Al) were injected and eluted isocratically with a mobile phase of 0.0125 N acetic acid/acetonitrile (55:50, v/v) at a flow rate of 1 ml/min. Aflatoxins were detected fluorometrically using a fluorescence detector (470 AC; Waters Associates). The excitation and emission wavelengths were 338 and 425 nm, respectively. Aflatoxins were identified without derivatization. The aflatoxins were identified by their retention times as compared with those for aflatoxin standard solutions under identical conditions. Aflatoxin standards were obtained from Sigma (St. Louis, MO, USA). Immunoaffinity columns (IAC) were used as a clean up step prior to fluorometry and HPLC. IAC plus fluorometry were used for quantification, and IAC plus HPLC were used for aflatoxins identification only. The performance of the 991.31 AOAC (1995) method was tested by the percentage of aflatoxin

3. Results and discussion Table 1 shows the total aflatoxin contents, in various types of pozol from Chiapas, as well as the frequencies of detection and the predominant aflatoxin types found. Of 111 samples tested, 19 from whitepozol and cacao-pozol contained aflatoxins at levels ranging from 0.5 to 21 ppb. AFB2 was the more prevalent and abundant toxin in all contaminated pozol samples. Only traces of AFB1 were detected. These results suggest that AFB2 is more resistant than AFB1 to the alkaline conditions that obtain during nixtamalization. These results are in agreement with those of Kamimura (1989).

Table 2 Distribution of total aflatoxin levels in pozol samples from Chiapas AF content (ppb) 0.5 10 11 20 >20 Total Sample Incidence (%) 16 2 1 19 (84.21) (10.53) (5.26) (100) Pozol class White-maize 2 0 0 2 Yellow-maize 0 0 0 0 Cacao 14 2 1 17

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ndez-Albores et al. / International Journal of Food Microbiology 94 (2004) 211215 J.A. Me Asao, T., Buchi, G., Abdel-Kader, M.M., Chang, S.B., Wick, E.L., Wogan, G.N., 1963. Aflatoxins B and G. Journal of the American Chemical Society 85, 1706 1707. Bodine, A.B., Mertens, D.R., 1983. Toxicology, metabolism, and physiological effects of aflatoxin in the bovine. In: Diener, U.L., Asquith, R.L., Dickens, J.W. (Eds.), Aflatoxin and Aspergillus flavus in Corn. Alabama Agriculture Experimental Station, Auburn University, Alabama, pp. 46 50. rzana-Garc a, E., Owens, J.D., Wacher as-Urbina, A.O., Ba Can Rodarte, M.C., 1993. Study of the variability in the methods of pozol production in the highlands of Chiapas. In: Wacher, genas de M.C., Lappe, P. (Eds.), Alimentos Fermentados Ind xico. Universidad Nacional Auto noma de Me xico, Mexico Me City, pp. 69 74. Candlish, A.A., Harran, G., Smith, J.E., 1991. Immunoaffinity column chromatography for detection of total aflatoxins in experimental situations. Biotechnology Techniques 5, 317 322. Carvajal, M., Arroyo, G., 1997. Management of aflatoxin contaminated maize in Tamaulipas, Mexico. Journal of Agricultural and Food Chemistry 45, 1301 1305. Christensen, C.M., Kaufmann, H., 1969. The role of storage fungi in the loss of quality. In: Christensen, C.M., Kaufmann, H. (Eds.), Grain Storage. University of Minnesota Press, Minneapolis, MN, p. 153. Dvorackova, I., 1976. Aflatoxin inhalation and alveolar cell carcinoma. British Medical Journal 1, 691. n de Pen a, D., Trudel, L., Wogan, G.N., 1995. Corn nixtaGuzma n and the fate of radiolabelled aflatoxin B1 in the malizacio tortilla making process. Bulletin of Environmental Contamination and Toxicology 55, 858 864. Harrison, J.C., Carvajal, M., Garner, R.C., 1993. Does aflatoxin exposure in the United Kingdom constitute cancer risk? Environmental Health Perspectives 99, 99 105. Kamimura, H., 1989. Removal of mycotoxins during food processing. In: Natori, S., Hashimoto, K., Ueno, Y. (Eds.), Mycotoxins and Phycotoxins. Elsevier, Amsterdam, pp. 169 176. Keyl, A.C., Booth, A.N., 1971. Aflatoxin effects in livestock. Journal of the American Oil Chemistry Society 48, 599 604. Paredes-Lopez, O., Escobedo, M.R., 1983. Influence of storage on the quality of maize meal for tortilla making. Journal of Food Technology 18, 53 60. Price, R.L., Jorgensen, K.V., 1985. Effects of processing on aflatoxin levels and on mutagenic potential of tortillas made from naturally contaminated corn. Journal of Food Science 50, 347 357. s guide. SAS Institute, 1998. Statistical Analysis System. SAS User Release 6.12 SAS Institute, Cary, NC. Sweeney, M.J., Dobson, A.D.W., 1998. Mycotoxin production by Aspergillus, Fusarium and Penicillium species. International Journal of Food Microbiology 43, 141 158. Ulloa, M., Herrera, T., 1970. Persistence of aflatoxins during pozol fermentation. Revista Latinoamericana de Microbioloa 12, 19 25. g Ulloa, M., Herrera, T., Lappe, P., 1987. Fermentaciones tradicionales genas de Me xico. Serie de Investigaciones Sociales, vol. 16. ind Instituto Nacional Indigenista, Mexico City, pp. 13 20.

Maize nixtamalization is known to destroy aflatoxins (Ulloa-Sosa and Schroeder, 1968; Ulloa and Herrera, 1970), but the inactivation of aflatoxins can be reversed by a low pH (Price and Jorgensen, 1985). It therefore appears that the traditional nixtamalization process is not as effective as has been suggested, and that more research is needed on the fates of aflatoxins and other mycotoxins (Sweeney and Dobson, 1998) during processing of maize-based foods. The aflatoxin levels in most contaminated samples were < 10 ppb (Table 2). Only one sample, from cacao-pozol, contained aflatoxins at >20 ppb. The relatively frequent presence of aflatoxins in cacaopozol might be due to cacao beans being contaminated with aflatoxins (Abdel-Hafez and El-Maghraby, 1992). The frequency and concentrations of aflatoxins that were found were considerably lower than those expected because of the inadequate rural maize storage techniques in this region of Mexico. Even though aflatoxin levels were low, it is desirable to have no aflatoxins in foods. Since nixtamalization may destroy or only hide 70 90% n de Pen a et al., 1995), some of the toxin (Guzma of the maize used to prepare the sampled pozol could have had higher levels of aflatoxins than those found in pozol. It would then be desirable to determine if aflatoxin toxicity is destroyed by nixtamalization, or if after nixtamalization the toxin has only lost the fluorescence properties by which it is detected. Acknowledgements We acknowledge the technical assistance from Y.A. Aguilar and A.R.M. Alvarez during the sampling as well as the financial support for this research from PAPIIT-UNAM grant 31689-B. References
Abdel-Hafez, A.I.I., El-Maghraby, O.M.O., 1992. Fungal flora and aflatoxin associated with cocoa, roasted coffee and tea powders in Egypt. Cryptogamie. Mycologie 13, 31 45. AOAC, 1995. In: Cunniff, P. (Ed.), 1995. Official Methods of Analysis of AOAC International, 16th edition AOAC International, Arlington, VA, pp. 20 21. Chapter 49.

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mented maize dough. World Journal of Microbiology and Biotechnology 9, 269 274. Wogan, G.E., Newberne, P.M., 1967. Dose response characteristics of aflatoxin B1 carcinogenesis in the rat. Cancer Research 27, 543 548.