Traditionally Processed Beverages in Africa: A Review of the Mycotoxin Occurrence Patterns and Exposure Assessment.

African traditional beverages are widely consumed food-grade liquids processed from single or mixed grains (mostly cereals) by simple food processing techniques, of which fermentation tops the list. These beverages are very diverse in composition and nutritional value and are specific to different cultures and countries. The grains from which home-processed traditional beverages are made across Africa are often heavily contaminated with multiple mycotoxins due to poor agricultural, handling, and storage practices that characterize the region. In the literature, there are many reports on the spectrum and quantities of mycotoxins in crops utilized in traditional beverage processing, however, few studies have analyzed mycotoxins in the beverages themselves. The available reports on mycotoxins in African traditional beverages are mainly centered on the finished products with little information on the process chain (raw material to final product), fate of the different mycotoxins during processing, and exposure estimates for consumers. Regulations targeting these local beverages are not in place despite the heavy occurrence of mycotoxins in their raw materials and the high consumption levels of the products in many homes. This paper therefore comprehensively discusses for the 1st time the available data on the wide variety of African traditional beverages, the mycotoxins that contaminate the beverages and their raw materials, exposure estimates, and possible consequent effects. Mycotoxin control options and future directions for mycotoxin research in beverage production are also highlighted.

Aflatoxins and growth impairment: a review.

Aflatoxins, fungal toxins produced by Aspergillus flavus and Aspergillus parasiticus in a variety of food crops, are well known as potent human hepatocarcinogens. Relatively less highlighted in the literature is the association between aflatoxin and growth impairment in children. Foodborne aflatoxin exposure, especially through maize and groundnuts, is common in much of Africa and Asia--areas where childhood stunting and underweight are also common, due to a variety of possibly interacting factors such as enteric diseases, socioeconomic status, and suboptimal nutrition. The effects of aflatoxin on growth impairment in animals and human children are reviewed, including studies that assess aflatoxin exposure in utero and through breastfeeding. Childhood weaning diets in various regions of the world are briefly discussed. This review suggests that aflatoxin exposure and its association with growth impairment in children could contribute a significant public health burden in less developed countries.

Determination of aflatoxin in processed dried cassava root: validation of a new analytical method for cassava flour.

A new method that uses HPLC with a photochemical reactor for enhanced detection was developed and validated for the determination of aflatoxins in cassava flour. Samples were spiked with a mixture of four aflatoxins at 5, 10, and 20 microg/kg mixed with either 1 or 5 g NaCI and extracted with methanol-water (80 + 20, v/v) by shaking for 10 or 30 min. An immunoaffinity column was used for cleanup. HPLC with postcolumn derivatization, for enhancement of aflatoxin fluorescence, and fluorescence determination were used for quantitation of the toxin concentration. The method was validated for recovery, linearity, and precision at the three concentrations tested. Recovery ranges were 52-70, 69-85, and 80-89% for the spiking levels of 5.0, 10.0, and 20.0 microg/kg, respectively. It appears that the amount of salt (NaCl) and the shaking time are critical factors in this method; optimal performance was obtained when 1 g salt was used and the shaking time was 10 min. The good linearity and precision of the method allowed baseline separation from interferences, e.g., coumarins.

Aflatoxin analysis at the beginning of the twenty-first century.

Aflatoxin mycotoxins were first described in the early 1960s as important fungal toxins, which contaminate many different human foods and animal feeds. Accurate and sensitive determination of these carcinogenic compounds immediately became an important requirement to meet food safety concerns and new official legislated regulations. For these reasons, analytical methods for aflatoxins continued to develop over the decades, reflecting advances in analytical chemistry. Currently, a wide range of methods are available to analytical scientists, ranging from newly described multi-toxin liquid chromatography tandem mass spectrometry to rapid methods based on immunological principles. These latter methods can provide quantitative outputs or a simple rapid determination of contamination level above or below a pre-determined cutoff value. The newest official methods as validated by Association of Official Analytical Chemists International or Comité Européen de Normalisation rely on immunoaffinity column clean-up of conventional extracts, followed by high-performance liquid chromatography separation of the analogues with detection based on natural fluorescence or the fluorescence generated by various derivatisation methods. In selecting from this range of available methods, the analytical chemist must decide on the requirements of the analysis such that the method chosen is 'fit for purpose'.

Mycotoxins produced by Fusarium proliferatum and F. pseudonygamai on maize, sorghum and pearl millet grains in vitro.

Maize (Zea mays), sorghum (Sorghum bicolor) and pearl millet (Pennisetum glaucum) are basic staple foods for many rural or poorer communities. These crops are susceptible to plant diseases caused by multiple species of Fusarium, some of which also produce mycotoxins, including fumonisins and moniliformin that are detrimental to both humans and domesticated animals. Eighteen potentially toxigenic Fusarium strains were isolated from maize (n = 10), sorghum (n = 7) and pearl millet (n = 1) growing in the same field in Nigeria. The 17 strains from maize and sorghum were all F. proliferatum and the one strain from pearl millet was F. pseudonygamai. Under conducive conditions, the 17 F. proliferatum strains produced fumonisins, 11 in relatively large quantities (700-17,000 mg total fumonisins, i.e., FB1 + FB2 + FB3/kg culture material), and six at <45 mg/kg. Ten F. proliferatum strains produced >100 mg of moniliformin per kg culture material with a maximum of 8900 mg/kg culture material. All strains could use all grains for growth and toxin production, regardless of the host from which they were isolated. Isolates varied in the amount of toxin produced on each substrate, with toxin production a property of the strain and not the host from which the strain was recovered. However, the extent to which a toxin-producing phenotype could be altered by the grain on which the fungus was grown is consistent with subtle genetic × environment interactions that require a larger data set than the one presented here to rigorously identify. In conclusion, there is significant variation in the ability of strains of F. proliferatum to produce fumonisins and moniliformin on maize, sorghum and millet. If the amount of toxin produced on the various grains in this study reflects real-world settings, e.g., poor storage, then the consumers of these contaminated grains could be exposed to mycotoxin levels that greatly exceed the tolerable daily intakes.

Mycotoxin Crises: Fit-for-Purpose Analytical Responses in the Developing World.

In developed market economies, control of mycotoxin exposure in the general population is achieved by legislated regulations governing maximum permitted levels. Such regulations are widely enforced to prevent outbreaks of overt mycotoxicoses. In developing countries, particularly in Africa, the situation is reversed, and individual mycotoxin exposures can be high, especially in rural communities reliant on subsistence or small-holder farming and local markets. Besides the effects of chronic mycotoxin exposure, Africa in recent years has experienced outbreaks of acute toxicity, such as aflatoxicosis. Recognizing and handling mycotoxin-induced health crises requires a range of responses, many of which rely on the provision and availability of fit-for-purpose analytical methods. Although regional laboratories may be able to provide support, rapid responses require in-field test kits reliant on antibody technologies. The future development of aptamers into test systems may be an important component of these analytical responses, as they provide important advantages in terms of stability, shelf-life, and low production costs.

The Mycotox Charter: Increasing Awareness of, and Concerted Action for, Minimizing Mycotoxin Exposure Worldwide.

Mycotoxins are major food contaminants affecting global food security, especially in low and middle-income countries. The European Union (EU) funded project, MycoKey, focuses on “Integrated and innovative key actions for mycotoxin management in the food and feed chains” and the right to safe food through mycotoxin management strategies and regulation, which are fundamental to minimizing the unequal access to safe and sufficient food worldwide. As part of the MycoKey project, a Mycotoxin Charter (charter.mycokey.eu) was launched to share the need for global harmonization of mycotoxin legislation and policies and to minimize human and animal exposure worldwide, with particular attention to less developed countries that lack effective legislation. This document is in response to a demand that has built through previous European Framework Projects—MycoGlobe and MycoRed—in the previous decade to control and reduce mycotoxin contamination worldwide. All suppliers, participants and beneficiaries of the food supply chain, for example, farmers, consumers, stakeholders, researchers, members of civil society and government and so forth, are invited to sign this charter and to support this initiative.

Structure and natural occurrence of stereoisomers of the fumonisin B series mycotoxins.

1H and 13C NMR spectroscopy of both fumonisin B3 and B4, as well as high-performance liquid chromatography (HPLC) analysis of samples of fumonisin B3 used as standards, showed in each case the presence of two stereoisomers, which could not be separated by preparative chromatography. The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B3 and B4 was deduced from the NMR data, and their 2S,3R absolute configurations were established by application of Mosher's method using the fumonisin B3 sample. Samples of fumonisin B3 and B4 can contain between 10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB3, determined by HPLC with fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much lower than FB3 (< 20%). The identification of members of the 3-epi-fumonisin B series provides insight into the order and selectivity of steps in fumonisin biosynthesis.

Determination of patulin in apple juice: comparative evaluation of four analytical methods.

The performance of 4 purification methods for the analysis of patulin in apple juice was evaluated by high-performance liquid chromatography (HPLC). Samples were spiked with patulin at 10, 20, 50, 100, and 150 ppb (ng/mL) and extracted by one of 4 methods (3 solid-phase extraction and one liquid-liquid extraction), and then analyzed by HPLC-UV under the same isocratic conditions. The methods were validated for recovery, linearity, and precision at high and low concentrations. Recoveries were all >70% for spiking range 10-150 ppb. The relative standard deviation for repeatability was found to meet European Union Directive requirements. In addition, all the methods showed baseline separation from hydroxymethylfurfural.

Mycotoxin contamination of dietary and medicinal wild plants in the Eastern Cape Province of South Africa.

Nineteen dietary and 30 medicinal wild plants used by residents of the Eastern Cape Province of South Africa were investigated for the presence of fumonisin B1 and aflatoxin B1. The plants were extracted in water, and cleanup was undertaken on immunoaffinity cartridges; analysis was by HPLC using fluorescence detection. None of the plant extracts contained detectable levels of aflatoxin B1; however, eight plants, four dietary and four medicinal, were positive for fumonisin B1 at levels ranging from 34 to 524 microg/kg and from 8 to 1553 microg/kg, respectively. The presence of fumonisin B1 was confirmed by LC-MS/MS using positive ion electrospray ionization. Fumonisin B1 provided characteristic fragment ions at m/z 704, 686, 546, 528, 370, and 352 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone. These results indicate that exposure to fumonisin B1 is much more widespread than initially thought and is the first report of mycotoxin contamination in South African medicinal and dietary wild plants.

Incidence of Fusarium verticillioides and levels of fumonisins in corn from main production areas in Iran.

A total of 52 corn samples collected in 2000 from four main corn production provinces of Iran (Fars, Kermanshah, Khuzestan, and Mazandaran) were analyzed for contamination with Fusarium verticillioides and fumonisins (FB(1), FB(2), FB(3), and 3-epi-FB(3)). The mean incidence of F. verticillioides (percent of kernels infected) for these four areas was 26.7, 21.4, 24.9, and 59.0%, respectively. The incidence in Mazandaran was significantly (p < 0.05) above that of the other areas. All samples from Mazandaran were contaminated with fumonisins with a mean level of total fumonisins of 10674 microg/kg. In contrast, the incidence of fumonisin contamination above 10 microg/kg was 53 (8/15), 42 (5/12), and 57% (8/14) in the samples from Fars, Kermanshah, and Khuzestan, respectively, and the corresponding mean total fumonisin levels were 215, 71, and 174 microg/kg, respectively. No statistical differences (p > 0.05) were observed in the fumonisin levels of the corn samples from these three provinces, which were significantly (p < 0.05) lower than the fumonisin contamination in samples from Mazandaran.

Current Status of Mycotoxin Analysis: A Critical Review.

It is over 50 years since the discovery of aflatoxins focused the attention of food safety specialists on fungal toxins in the feed and food supply. Since then, analysis of this important group of natural contaminants has advanced in parallel with general developments in analytical science, and current MS methods are capable of simultaneously analyzing hundreds of compounds, including mycotoxins, pesticides, and drugs. This profusion of data may advance our understanding of human exposure, yet constitutes an interpretive challenge to toxicologists and food safety regulators. Despite these advances in analytical science, the basic problem of the extreme heterogeneity of mycotoxin contamination, although now well understood, cannot be circumvented. The real health challenges posed by mycotoxin exposure occur in the developing world, especially among small-scale and subsistence farmers. Addressing these problems requires innovative approaches in which analytical science must also play a role in providing suitable out-of-laboratory analytical techniques.

Fumonisin mycotoxins in traditional Xhosa maize beer in South Africa.

The production and consumption of home-brewed Xhosa maize beer is a widespread traditional practice in the former Transkei region of South Africa. HPLC determination of fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) in maize beer samples collected in two magisterial areas, Centane and Bizana, showed a wide range of levels. All samples were positive for FB(1), with a mean level of 281 +/- 262 ng/mL and a range from 38 to 1066 ng/mL. Total fumonisins (FB1 + FB2 + FB3) ranged from 43 to 1329 ng/mL, with a mean of 369 +/- 345 ng/mL. Data on the consumption of home-brewed beer are not available. On the basis of published data for the consumption of commercial beer in South Africa, the fumonisin exposure in these districts among the consumers of maize beer was found to be well above the provisional maximum tolerable daily intake of 2 mug/kg of body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives.

Do fumonisin mycotoxins occur in wheat?

The fumonisin mycotoxins are mainly produced by the fungi Fusarium verticillioides and Fusarium proliferatum, which are both field pathogens of maize. The natural occurrence of fumonisins has been verified in maize and a large range of maize-based products in many countries of the world. However, occasional reports have emerged of fumonisins being detected in wheat, despite the main producing fungi not being pathogens of this cereal. An investigation was conducted into a recent report of the natural occurrence of fumonisins in the 2003/2004 South African wheat crop at levels up to 1.7 mg/kg, as determined by immunoaffinity column cleanup and direct fluorometric measurement. An AOAC International high-performance liquid chromatographic (HPLC) method for the determination of fumonisins in maize was modified and validated for the determination of fumonisins in spiked wheat samples. HPLC analysis of the wheat samples previously found to be positive for fumonisins revealed no detectable (<5 microg/kg) fumonisins in the 30 samples analyzed. These results, which lay doubt on previous reports of fumonisins in wheat, emphasize the fact that screening methods, especially if used outside their range or matrix of applicability, can produce false positive results despite the use of immunoaffinity cleanup. Such results should be validated and confirmed with a more definitive technique.

Production of fumonisin B and C analogues by several fusarium species.

Six strains of Fusarium verticillioides, two of F. oxysporum, one strain of F. proliferatum, and a strain of an unidentified species were cultured on maize patties and rice and evaluated for their ability to simultaneously produce fumonisin B (FB) and C (FC) series analogues. Fumonisins were quantified by LC-MS-MS using positive ion electrospray ionization. FC1 provided characteristic fragment ions at m/z 690, 672, 654, 532, 514, and 338 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone, while FC3 and FC4 provided equivalent product ions 16 and 32 amu lower than the corresponding FC1 fragments, respectively. All isolates cultured on maize produced FC4. All isolates except for that of F. proliferatum also produced FC1, and three of the six strains of F. verticillioides produced FC3. All isolates except those of F. oxysporum produced detectable amounts of FB1, FB2, and FB3. Isolates that produced fumonisin B analogues produced at least 10 fold more of the B series analogues than they did of the C series analogues. The results confirm that at least some strains of F. oxysporum produce FC, but not FB, fumonisin analogues and also suggest that the genetics and physiological regulation of fumonisin production may be more complicated than previously envisaged since some strains of F. verticillioides and F. proliferatum as well as the strain of the unidentified species can simultaneously produce both FB and FC analogues.

Relative severity of fumonisin contamination of cereal crops in West Africa.

Traditional and improved varieties of maize, pearl millet and sorghum were planted by small-scale farmers under the direction of the International Institute for Tropical Agriculture in two Nigerian agro-ecological zones: the Sudan Savanna and the Northern Guinea Savanna. Samples were collected for the determination of Fusarium infection and fumonisin (B1, B2 and B3) contamination. A previous paper reported Aspergillus infection and aflatoxin contamination of these samples. Fusarium infection levels, measured by per cent kernels infected, were modest with mean levels for the above cereals of 16% ± 11% (SD), 12% ± 7% and 13% ± 16%, respectively. However, the Fusarium species recovered from maize were predominantly the fumonisin producers F. verticillioides and F. proliferatum, together making an infection rate of 15% ± 10%, whereas these species were present to a limited extent only in the other two cereals, 1% ± 1% for pearl millet and 2% ± 6% for sorghum. Fumonisin contamination was variable but reflected the diversity of Fusarium producers in these three cereals. Mean levels were 228 ± 579 µg kg(-1) (range < 5-2860 µg kg(-1)) for maize, 18 ± 7 µg kg(-1) (range = 6-29 µg kg(-1)) for pearl millet and 131 ± 270 µg kg(-1) (range < 5-1340 µg kg(-1)) for sorghum. Together with previous results on aflatoxin, this study confirmed the co-occurrence of aflatoxins and fumonisins in maize as well as in the traditional African cereals, millet and sorghum (89% co-occurrence across all three cereals). The low fumonisin levels may be ascribed to the use of good agricultural practices. Of the Fusarium species present, those in maize consisted mainly of fumonisin producers, the opposite of what was observed in pearl millet and sorghum. It is concluded that replacement of maize by pearl millet and sorghum could improve food safety with regards to aflatoxin B and fumonisin B exposure.

Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B1.

In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B(1) (FB(1)) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB(1) containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB(1) dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB(1) treatment in the control rats was significantly (P < 0.05) enhanced by the 2-AAF/PH cancer promotion treatment. The nodular and surrounding Sa levels returned to baseline following FB(1) initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB(1) treatment, the initiation effected by FB(1) was less (P < 0.01) sensitive to the accumulation of Sa in the nodular and surrounding tissues than DEN initiation and the 2-AAF/PH control treatment. In contrast, the So level of FB(1) initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P < 0.05) higher with the secondary FB(1) treatment. Although, the FB(1)-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P).

Quantitation of ochratoxin A in South African wines.

The natural occurrence of the carcinogenic mycotoxin ochratoxin A (OTA) in wines sold in local retail outlets in South Africa and Italy was investigated by HPLC analysis with fluorescence detection following cleanup by immunoaffinity column. All 24 local South African wines tested (15 white and 9 red) were found to contain detectable levels (>0.01 microg/L) of OTA, with a mean of 0.16 microg/L in the white wines and a mean of 0.24 microg/L in the red wines. Results were subsequently confirmed by LC-MS analysis using positive ion electrospray ionization with collision-induced dissociation of the protonated molecular ion [M + H](+) at m/z 404 and selected reaction monitoring of the resultant product ions [M + H - H(2)O - CO](+) at m/z 358 and [M + H - H(2)O](+) at m/z 386. Comparison with the fluorescence method gave a significant correlation (r = 0.87; p < 0.01). Although OTA contamination was present in all of the South African samples analyzed, levels were well below the suggested European Union limit of 0.5 microg/kg. The highest level found in a locally purchased wine was 0.39 microg/L in a blend of local and imported Spanish red wine. Of the eight Italian wines analyzed, only two red wines were contaminated above the suggested maximum level.

Mycoflora and fumonisin mycotoxins associated with cowpea [Vigna unguiculata (L.) Walp] seeds.

Cowpea seed samples from South Africa and Benin were analyzed for seed mycoflora. Fusariumspecies detected were F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. semitectum, and F. subglutinans. Cowpea seed from South Africa and Benin and F. proliferatum isolates from Benin, inoculated onto maize patty medium, were analyzed for fumonisin production. Samples were extracted with methanol/water and cleaned up on strong anion exchange solid phase extraction cartridges. HPLC with precolumn derivatization using o-phthaldialdehyde was used for the detection and quantification of fumonisins. Cowpea cultivars from South Africa showed the presence of fumonisin B(1) at concentrations ranging between 0.12 and 0.61 microg/g, whereas those from Benin showed no fumonisins. This is believed to be the first report of the natural occurrence of FB(1) on cowpea seed. Fumonisin B(1), B(2), and B(3) were produced by all F. proliferatum isolates. Total fumonisin concentrations were between 0.8 and 25.30 microg/g, and the highest level of FB(1) detected was 16.86 microg/g.

Fumonisin contamination and fusarium incidence in corn from Santa Catarina, Brazil.

In Brazil, the southern region has the highest incidence of esophageal cancer and also the highest production and consumption of corn (Zea mays) products. Corn samples intended for human consumption from the western, northern, and southern regions of the state of Santa Catarina, southern Brazil, had mean total fumonisin B (B(1), B(2), and B(3)) levels of 3.2, 3.4, and 1.7 mg/kg, respectively. Fusarium verticillioides, the predominant fungus in the corn samples, had mean incidences (percent of kernels infected) of 14, 11, and 18% for the three regions, respectively. Additional corn samples intended for animal feed from the southern region had a mean total fumonisin level of 1.5 mg/kg and a mean F. verticillioides incidence of 10%. The fumonisin levels in corn from the state of Santa Catarina, Brazil, were similar to the high levels determined in other high esophageal cancer incidence regions of the world.