Occurrence of certain mycotoxins in corn and corn-based products and thermostability of fumonisin B1 during processing

A total of 57 samples of corn and corn-based products collected from various districts of Egypt were analyzed for Fusarium mycotoxins (T-2, diacetoxyscripenol (DAS), deoxynivalenol (DON) and fumonisin B1 (FB1) and aflatoxins. FB1 was detected in about 80%, 53.85%, 33.3%, and 28.57% of yellow corn, corn meal, white corn, and popcorn samples, respectively. The levels of FB1 ranged from 10 to 780 microg/kg. T-2 and DAS were detected in 5% and 10% of yellow corn samples, respectively, and DON was detected in white corn and popcorn samples at levels of 28.8 and 10.1 microg/kg, respectively. Starch samples were found to be free from Fusarium mycotoxins. Baking balady bread at 450 degrees C/min reduced FB1 to 72.4% while baking Franco bread at 250 degrees C/20 min reduced FB1 to 57.4%. Boiling of macaroni and corn in water completely removed FB1 from contaminated samples. On the other side, corn flakes samples were found to be contaminated with aflatoxins B1 and G1 at levels ranging from 6 to 10 ppm, whereas 2.9% of samples were contaminated with aflatoxin B1 > 35 ppm and G1 > 16 ppm.     

Screening of fermentative bacteria for their ability to bind and biotransform deoxynivalenol, zearalenone and fumonisins in an in vitro simulated corn silage model.

Fermentative bacteria can potentially be utilized to detoxify corn silage contaminated by Fusarium toxins. The objective of the present study was to test a large number of these bacteria for their ability to bind and/or biotransform deoxynivalenol (DON), zearalenone (ZEN) and fumonisins B(1) and B(2) (FB(1), FB(2)) in conditions simulating corn silage. A total of 202 strains were screened in contaminated, pH 4, corn infusion inoculated with 5 x 10(8) CFU ml(-1). Eight Lactobacilli and three Leuconostoc biotransformed ZEN into alpha-zearalenol, but no biotransformation was detected for DON and fumonisins. In contrast, most strains were capable of binding Fusarium toxins. The most effective genera were Streptococcus and Enterococcus, capable of binding up to 33, 49, 24 and 62% of DON, ZEN, FB(1) and FB(2), respectively. The ability to bind Fusarium toxins seems to be a common property of fermentative bacteria and could help to decrease their toxicity in animals.     

Preliminary evaluation of fumonisins by the Nordic countries and occurrence of fumonisins (FB1 and FB2) in corn-based foods on the Danish market

Experts from the Nordic countries (Denmark, Norway, Sweden, Finland and Iceland) have carried out an evaluation of fumonisins. The working group members concluded that, at that time point, it was not possible to carry out a complete risk assessment. However, it was recommended that the human daily' intake of fumonisins should be less than 1 microg/kg bw/day. Subsequently, the presence of the Fusarium mycotoxins fumonisin B1 and B2 (FB1 and FB2) in corn-based food on the Danish retail market has been determined. A total of 70 samples were analysed and 37% contained FB1 and 21% contained FB2. No fumonisins were found in sweet corn (canned or frozen), corn-on-the-cob, corn starch or gruel powder for babies. FB1 was found in about half of the corn flakes, corn snack and popcorn (not popped) samples, whereas FB2 was seen to a lesser extent. Both FB1 and FB2 were found in 75% or more of the corn flour, tacos and polenta samples. In general, the content of FB1 was in the range of 1-1000 micro/kg and the content of FB2 was in the range of 4-250 microg/kg. Corn-based foods are consumed in rather low amounts and irregularly among the Danish population and therefore it is not meaningful to calculate an average daily funonisin intake. An estimate for an 'eater' shows that the intake of fumonisins will not exceed 0.4 microg/kg bw/day.     

Recent research on fumonisins: a review

Fumonisins are well known mycotoxins produced by Fusarium verticillioides, F. proliferatum and other Fusarium species. Many new fumonisins and fumonisin-like compounds have been detected by mass spectrometry in cultures of F. verticillioides. Recently, fumonisins B(2) and B(4) were produced by Aspergillus niger isolated from coffee and fumonisin B(2) in A. niger from grapes. Fumonisin B(2) was itself detected in coffee beans, wine and beer, adding to the list of foodstuffs and feedstuffs other than corn (maize) and sorghum in which fumonisins have been found in recent years. Fumonisin B(1) (FB(1)) can bind to proteins (PB FB(1)) and to other matrix components during food processing involving heat. The occurrence of bound fumonisins in processed corn foods is common. Another type of binding (or association) relates to observed instability of fumonisins in rice flour, corn starch and corn meal at room temperature; this can affect the immunoaffinity column clean-up procedure in analysis of naturally contaminated starch-containing corn foods for fumonisins. The occurrence of N-fatty acylated fumonisin derivatives in retail fried corn foods has also been demonstrated. Bioaccessibility of free FB(1) and total bound FB(1) (TB FB(1)) present in corn flakes has been estimated by in vitro digestion experiments. Intentional binding of fumonisins to cholestyramine has been demonstrated in vivo and is a potential means of detoxification of animal feed.     

Co-occurrence of Fusarium mycotoxins in mouldy and healthy corn from Korea

A total of 71 samples consisting of 36 mouldy and 35 visibly healthy corn were collected from Kangwon province of Korea and analysed for 8-ketotrichothecenes, zearalenone (ZEA), and fumonisins, including fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3). Five 8-ketotrichothecenes, namely deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), nivalenol (NIV), and 4-acetylnivalenol (4-ANIV), ZEA, FB1, FB2, and FB3 were detected in corn samples. DON, 15-ADON, 3-ADON, NIV, 4-ANIV, ZEA, FB1, FB2, and FB3 were detected in mouldy corn with mean values of 4.0, 0.9, 0.2, 1.7, 0.4, 0.6, 23.2, 7.5, and 6.3mug/g, respectively. Visibly healthy corn samples were contaminated with lower levels of 8-ketotrichothecenes, ZEA, and fumonisins than mouldy corn samples. However, 5 of 35 healthy corn samples analysed were contaminated with fumonisins at high levels up to 12.5mug/g for FB1, 5.4mug/g for FB2, and 0.5mug/g for FB3. This is the first report on the simultaneous occurrence of trichothecenes, ZEA, and fumonisins in corn from Korea.     

Natural occurrence of Fusarium species, fumonisin production by toxigenic strains, and concentrations of fumonisins B1, and B2 in conventional and organic maize grown in Spain

Sixty samples of corn from both conventional and organic farms were tested for internal fungal contamination. Molds were identified to genus, and those belonging to the genus Fusarium were identified to species. Twenty isolates of Fusarium verticillioides were tested with a high-performance liquid chromatography-naphthalene dicarboxaldehyde-fluorescence method for their ability to produce fumonisins B1 and B2. The internal fungal infection in organic maize (63.20%) was significantly higher than that in conventional maize (40.27%) (P < 0.05). However, the distribution of fungal genera indicated a significantly higher prevalence of Fusarium in conventional (34.93%) than in organic (18.15%) maize, making Fusarium the predominant fungus in conventional maize. This difference in mold distribution between organic and conventional maize was attributed to the difference in cultivation system. The dominant Fusarium species in both conventional and organic samples was F. verticillioides. There were no significant differences in the ability of 20 selected isolates of F. verticillioides to produce fumonisins on conventional or organic corn. Up to 13.3% of the conventional corn samples contained fumonisins B1 and B2 at mean concentrations of 43 and 22 ng/g, respectively. Organic corn samples had somewhat lower levels of contamination: 35 ng/g fumonisin B1 and 19 ng/g fumonisin B2 (P > 0.05). The organic farming system, with well-balanced crop rotation, tillage, and compost fertilization, produced corn that was less likely to be contaminated with Fusarium species, although no significant difference in fumonisin concentrations was found between the two types of contaminated corn.     

Occurrence of fumonisins and moniliformin in corn and corn-based food products of U.S. origin

Food-grade corn and corn-based food products intended for human consumption were analyzed for the incidence and levels of fumonisin B1 (FB1), fumonisin B2 (FB2), moniliformin, and Fusarium molds. A total of 100 food-grade commercial corn samples were obtained from two corn processing companies at five different locations in the United States. Seventy-one percent of the samples contained FB1 with concentrations ranging from 43 to 1,642 microg/kg. None of the samples contained FB2. Fifty percent of the samples contained moniliformin with concentrations ranging from 26 to 774 microg/kg. All samples were infected by Fusarium molds, and the infection rates ranged from 8 to 88%. Thirty-four samples of corn-based food products were purchased from supermarkets in Arizona, California, Nebraska, and Ohio. Sixty-five percent of the samples contained FB1, ranging in concentrations from 28 to 2,679 microg/kg. FB2 was detected in 29% of the samples with concentrations ranging from 30 to 797 microg/kg. Sixty-eight percent of the samples contained moniliformin with concentrations ranging from 31 to 858 microg/kg. Sixty-two percent of the samples contained viable Fusarium mold propagules ranging from 9.5 x 10(1) to 5.5 x 10(5)/g. The simultaneous occurrence of FB1 and moniliformin was observed in 34% of corn samples and 53% of corn-based food products. This study has shown co-occurrence of fumonisins and moniliformin in food-grade corn and corn-based foods that indicates a risk of simultaneous exposure of consumers to both toxins.     

伏马毒素生物合成和降解的研究进展

伏马毒素(Fumonisins,FBs)一种由串珠镰刀菌(Fusarium moniliforme)、轮状镰孢菌(Fusarium verticillioides)、多誉镰刀菌(Fusarium proliferatum)和其他一些镰孢菌种产生的水溶性次级代谢产物.广泛存在于玉米等农产品中,在较低的浓度范围内,可干扰植物正常生理代谢功能,不仅会造成农业经济损失,其严重的致癌性对人畜危害极大,因此开始受到广泛关注.目前国际上对伏马菌素的研究已经深入到分子水平,通过对产伏马菌素菌株的遗传学研究,鉴定出与伏马菌素生物合成相关基因及合成协同调节基因簇.本文介绍了伏马毒素的理化性质,以及伏马毒素作用机理,生物合成途径.阐述了伏马毒素生物合成的基因序列群以及基因簇的表达方式.论述了伏马毒素的降解方法,并为今后伏马毒素生物工程降解研究提出了建议.     

Effects of muffin processing on fumonisins from 14C-labeled toxins produced in cultured corn kernels

The persistence of fumonisins during cooking is known to be affected by several factors, including thermal degradation and the presence of various ingredients in corn-based food recipes that can react with the toxin. A method for the production of corn kernels containing 14C-fumonisins was developed. The corn kernels were colonized by Fusarium verticillioides MRC 826 and supplemented with 1,2-14C-sodium acetate. The specific activity of 14C-FB1 produced made the study of its fate in cornmeal muffins possible. The double-extraction acetonitrile-water-methanol/immunoaffinity column/o-phthaldialdehyde high-performance liquid chromatography (HPLC) method was used to determine FB1 levels in cornmeal muffins. Reductions in FB1 levels in muffins spiked with 14C-labeled and unlabeled FB1 (43 and 48%, respectively) were similar, indicating that the extraction method was efficient and consistent with previous reports. However, with the labeled corn kernel material, recovery levels based on the 14C counts for the eluate from an immunoaffinity column were much higher (90%). This finding indicates that some fumonisin-related compounds other than FB1 that were present in the cornmeal were recognized by the antibodies but not by the HPLC method.     

Natural occurrence of fumonisins b1 and b2 in corn from three main production provinces in China

A total of 255 corn samples collected in 2010 from three main corn production provinces of China (Liaoning, Shandong, and Henan) were analyzed for contamination with fumonisins (FB(1) and FB(2)). The incidence of contamination was significantly higher in samples from Liaoning than in samples from the other two provinces. Approximately 80.0% of the samples from Liaoning were contaminated with fumonisins, with a mean total fumonisin concentration of 3,990 ng/g. In contrast, the mean total fumonisin concentrations were 845 and 665 ng/g in samples from Shandong and Henan, respectively. The probable daily intake of fumonisins (0.3 μg/kg of body weight) is within the provisional maximum tolerable daily intake of 2.0 μg/kg of body weight set by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives.     

Fumonisin production on irradiated corn kernels: effect of inoculum size.

Production of fumonisins B1, B2, and B3 by Fusarium moniliforme was evaluated on irradiated corn kernels inoculated with different spore concentrations (10, 10(2), 10(3), 10(5), and 10(6)), a water activity of 0.97, and a temperature of 25 degrees C. There was a direct relationship between the level of toxin produced and inoculum size. The highest levels of total fumonisin produced after 35 days of incubation were 5,028 and 9,063 ng/g at 10(5) and 10(6) spores per ml, respectively. The pattern of fumonisin production (FB1 > FB2 > FB3) in cultures growing from different inocula was not affected during the 35 days of incubation. The ratio between FB2 and FB1 varied from 0.15 to 0.42, whereas the ratio between FB3 and FB1 varied from 0.34 to 0.87.     

Distribution of fumonisins in dry-milled corn fractions in Argentina.

Corn samples and different dry-milled fractions collected from an industrial mill in Argentina were analysed. Average contaminations were FB(1) 1540 microg kg(-1), FB(2) 716 microg kg(-1) and FB(3) 152 microg kg(-1) in whole corn; FB(1) 135 microg kg(-1), FB(2) 39.1 microg kg(-1) and FB(3) 10.2 microg kg(-1) in corn grits; FB(1) 358 microg kg(-1), FB(2) 122 microg kg(-1) and FB(3) 45.9 microg kg(-1) in 'C' flour; FB(1) 148 microg kg(-1), FB(2) 52.5 microg kg(-1) and FB(3) 28.3 microg kg(-1) in corn meal; and FB(1) 4210 microg kg(-1), FB(2) 2010 microg kg(-1) and FB(3) 447 microg kg(-1) in germ and bran together. The fumonisin contamination level was approximately three times higher in germ and bran than in whole corn, 13 times higher than in 'C' flour and 29 times higher than in corn meal and corn grits. Taking into account the distribution of fumonisins in commercial dry-milled corn fractions and corn meal consumption in Argentina, a theoretical whole corn level of 6640 microg kg(-1) maximum of total fumonisins could be processed to obtain products considered safe for human health.     

Fumonisins B1 and B2 in black tea and medicinal plants

Fumonisins are mycotoxins produced by Fusarium moniliforme that are prevalent in cereals and other agricultural products. These mycotoxins have been pointed to as a natural cause of equine leukoencephalomalacia, porcine pulmonary edema, and human esophageal cancer. A total of 87 samples, 18 black tea samples and 69 samples of four different medicinal plants (chamomile, leaves of the orange tree, leaves and flowers of the linden tree, and corn silk), for infusions preparations were acquired from supermarkets in Lisbon, Portugal. The samples were analyzed for the incidence and levels of fumonisin B1 (FB1) and fumonisin B2 (FB2) by high-performance liquid chromatography. The detection limit was 20 microg/kg for both FB1 and FB2. FB1 was detected in 55 (65.5%) of the 87 samples. The highest number of positive samples was found in black tea (88.8%). with levels ranging from 80 to 280 microg/kg. Relative to the medicinal plants, the leaves of the orange tree had higher concentrations of FB1 (range, 350 to 700 microg/kg) followed by leaves and flowers of the linden tree (range, 20 to 200 microg/kg). The samples of corn silk and chamomile had less contamination of FB1, with concentrations ranging from 50 to 150 microg/kg and 20 to 70 microg/kg, respectively. None of the samples tested had contamination of FB2. This is the first report of the natural occurrence of fumonisins in black tea and medicinal plants in Portugal. We reinforce the necessity to implement risk management measures for safety control of this kind of product.     

Fumonsin B1, B2, and B3 content of commercial unprocessed maize imported into South Africa from Argentina and the USA during 1992.

The widespread occurrence of F. moniliforme and the toxic effects of its secondary metabolites, the fumonisins B1(FB1), B2(FB2) and B3(FB3), make it imperative that fumonisin contamination of maize, a major constituent of animal feed as well as the staple diet of many populations, be closely monitored to reduce the risk of fumonisin exposure. Equine leukoencephalomalacia and porcine pulmonary oedema have been associated with the intake of feed heavily contaminated with fumonisins. In addition, high levels of fumonisins in the maize-based staple diets of certain populations have been linked to a high incidence of oesophageal cancer in the Transkei region of South Africa and in Linxian and Cixian Counties, China. Bulk shipments of maize imported into South Africa from the USA and Argentina during 1992 were sampled at the port of entry to determine fumonisin levels. Of the 79 samples from two US shipments, all were positive for fumonisins, with FB1 constituting approximately 71% of the total fumonisins with an overall mean of 2.35 micrograms/gFB1. The maximum FB1 level observed was 3.9 micrograms/g. These levels contrast with those obtained from two Argentinian bulk shipments, which also were all positive for fumonisins, but had a mean FB1 level of 0.31 microgram/g and a maximum observed level of 0.7 microgram/g FB1 measured over 47 composite samples.     

Occurrence of fumonisins in foods

Fumonisins (FB) are synthesized mainly by different species of the genus Fusarium and FB1 is the predominating fumonisin isolated in foods. FB1 shows different toxicological effects in humans and animals and has been associated with esophageal cancer in different countries. This review presents an overview of the worldwide occurrence of fumonisins in crops and human foods being corn and corn meal the most contaminated foods, and reviews variations of the presence of these mycotoxins in foods and in agricultural crops. In foods, these variations are due to the alkaline solutions, water and temperature. For agricultural crops, the role of climatic conditions and latitude, genotypes, spoilage and other fungal diseases is reviewed.     

Fumonisins in Brazilian corn-based foods for infant consumption.

A survey of 196 samples of corn-based infant foods from 13 cities of Sao Paulo State, Brazil, was carried out to investigate the fumonisin contamination in the products. Based on their ingredients, the products were divided into seven groups: infant cereal designated as types A-D, corn meal, corn starch and instant cereal baby food. Although certain infant food samples were free of fumonisin contamination (<20 microg kg(-1); corn starch and infant cereals of type A, B and D), contamination levels in the other products (corn meal, instant corn-based baby food and cereal type C) were of concern, particularly those in corn meal. All samples in these categories contained fumonisins. The mean level for total fumonisins (FB1 + FB2 + FB3) in corn meal was 2242 microg kg(-1) (maximum 8039 microg kg(-1)), in instant corn-based baby food was 437 (maximum 1096) microg kg(-1) and in infant cereal type C was 664 (maximum 1753) microg kg(-1).     

Comparison of methods and optimisation of the analysis of fumonisins B₁ and B₂ in masa flour, an alkaline cooked corn product

A comparison study of different extraction and clean-up procedures for the liquid chromatographic analysis of fumonisins B(1) (FB(1)) and B(2) (FB(2)) in corn masa flour was performed. The procedures included extraction (heat or room temperature) with acidic conditions or EDTA-containing solvents, and clean-up by immunoaffinity or C18 solid-phase extraction columns. Thereafter an analytical method was optimised using extraction with an acidic mixture of methanol-acetonitrile-citrate/phosphate buffer, clean-up through the immunoaffinity column and determination of fumonisins by liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde reagent. Recovery experiments performed on yellow, white and blue masa flours at spiking levels of 400, 800 and 1200 μg kg(-1) FB(1) and of 100, 200 and 300 μg kg(-1) FB(2) gave overall mean recoveries of 99% (±6%) for FB(1) and 88% (±6%) for FB(2). Good recoveries (higher than 90% for both FB(1) and FB(2)) were also obtained with corn tortilla chips. The limits of quantification of the method (signal-to-noise ratio of 10) were 25 μg kg(-1) for FB(1) and 17 μg kg(-1) for FB(2). The method was tested on different commercial corn masa flours as well as on white and yellow corn tortilla chips, showing fumonisin contamination levels (FB(1) + FB(2)) up to 1800 μg kg(-1) (FB(1) + FB(2)) in masa flour and 960 μg kg(-1) in tortilla chips. Over 30% of masa flours originating from Mexico exceeded the European Union maximum permitted level.     

Liquid chromatographic method for fumonisin B1 in sorghum syrup and corn-based breakfast cereals.

The fungus Fusarium verticillioides has been found on corn and sorghum, so it is possible that one or more of these toxins may be found in corn products such as breakfast cereals and syrup prepared from sorghum. Published methods when applied to syrups spiked with fumonisins gave low recoveries, less than 50%. A method was therefore developed which would be applicable to the syrup and breakfast cereals as well. Test samples were extracted with methanol-0.1 M sodium phosphate buffer (pH3) (1 + 1). The extract was diluted with water and applied to a 1 g C18 column. The column was washed with acetonitrile-water (2 + 8). Fumonisin B1 (FB1) was eluted with acetonitrile-trifluoroacetic acid (1000 + 1). The purified extract was evaporated and the toxin was derivatized with ophthaldialdehyde mercaptoethanol. The reaction mixture was resolved on a C18 liquid chromatographic column using acetonitrile-water-acetic acid (500 + 550 + 10.5) as the mobile phase at 37 degrees C, and FB1 measured with a fluorescence detector (excitation 335 nm, emission 440 nm). Recoveries of FB1 added to samples of sorghum syrup at levels ranging from 0.1 to 1.0 microgram/g were 94-132%. Recoveries of FB1 added to samples of breakfast cereal (corn flakes) at levels ranging from 0.2 to 1.6 micrograms/g were 96-100%. The method was applied to the analysis of 35 samples of sorghum syrup collected from 15 states in the US. One sample was found to contain FB1 at 0.12 microgram/g. A total of 32 samples of breakfast cereals collected by the Food and Drug Administration inspectors from grocery stores around the Kansas City area were analysed; no FB1 was found in the breakfast cereals (< 0.01 microgram/g). Results of this study indicated that FB1 possibly is not a problem in sorghum syrup and corn-based breakfast cereals in the US.     

Improving extraction of fumonisin mycotoxins from Brazilian corn-based infant foods

The current AOAC International methods for the determination of fumonisins have been validated for corn and cornflakes but have produced low recoveries and high variability when applied to processed corn products for infants. Hence, an investigation was undertaken to improve the extraction efficiency for fumonisins by investigating the use of different extraction solvents. Corn-based infant foods containing cornmeal, corn starch, and corn flour were purchased in the city of Campinas, state of Sao Paulo, Brazil, and were analyzed for fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) following extraction with a range of solvents. Comparison of the results from each of the samples indicated that acidified 70% aqueous methanol at pH 4.0 provided the best overall performance, whereas a methanol/boric acid (pH 9.2) mixture displayed poor extraction efficiency. Extraction with acidified 70% aqueous methanol showed seven of eight test samples to be positive for FB1 (range, 30 to 6,127 microg/kg; relative SD, 4.2 to 51.7%), two of eight samples to be positive for FB2 (range, 53 to 1,738 microg/kg; relative SD, 4.5 to 5.3%), and one of eight samples to be positive for FB3 (575 microg/kg). For samples in which extraction with phosphate-buffered mixtures (pH 3) proved superior, the method suffered from poor chromatography due to interfering compounds. The findings indicate that matrix interferences play a significant role in the extractability, cleanup, and chromatography of the fumonisins.     

A survey of fumonisins, deoxynivalenol, zearalenone and aflatoxins contamination in corn-based food products in Argentina.

The presence of mycotoxins in corn-based foods available in Argentina was determined in order to make a preliminary exposure assessment. Thirty-eight samples [corn meal ('polenta') and corn flakes] of different local brands were analysed for zearalenone, deoxynivalenol and aflatoxins by TLC and fumonisins (FB1, FB2 and FB3) by HPLC. None of the 38 samples contained any detectable amount of aflatoxins (< 2 micrograms/kg), zearalenone (< 50 micrograms/kg) and deoxynivalenol (< 50 micrograms/kg). By contrast fumonisin contamination was found in 95% of the samples. The highest fumonisin levels were found in corn meal: FB1 (range positives: 60-2860 micrograms/kg; mean positive value: 556 micrograms/kg), FB2 (61-1090 micrograms/kg; 232 micrograms/kg) and FB3 (18-1015 micrograms/kg; 150 micrograms/kg). Low levels of fumonisin B1 were detected in 16/17 corn flakes samples (2-38 micrograms/kg). Total fumonisin levels in corn meal were more than 1000 micrograms/kg in 24% (5/21) of the samples. Although it is not the staple food in Argentina, maize consumption is very important, especially among children. A daily fumonisin intake of 11.3 micrograms/kg of body weight was estimated for child consumers (1-5 years old) based on an average consumption of 200 g of corn meal/day. Calculated at an average rate for all children (consumers or not) the intake estimate was 0.9 microgram/kg of body weight.