Determination of aflatoxins in by-products of industrial processing of cocoa beans

This study has examined the occurrence of aflatoxins in 168 samples of different fractions obtained during the processing of cocoa in manufacturing plants (shell, nibs, mass, butter, cake and powder) using an optimised methodology for cocoa by-products. The method validation was based on selectivity, linearity, limit of detection and recovery. The method was shown to be adequate for use in quantifying the contamination of cocoa by aflatoxins B₁, B₂, G₁ and G₂. Furthermore, the method was easier to use than other methods available in the literature. For aflatoxin extraction from cocoa samples, a methanol–water solution was used, and then immunoaffinity columns were employed for clean-up before the determination by high-performance liquid chromatography. A survey demonstrated a widespread occurrence of aflatoxins in cocoa by-products, although in general the levels of aflatoxins present in the fractions from industrial processing of cocoa were low. A maximum aflatoxin contamination of 13.3?ng?g^?1 was found in a nib sample. The lowest contamination levels were found in cocoa butter. Continued monitoring of aflatoxins in cocoa by-products is nevertheless necessary because these toxins have a high toxicity to humans and cocoa is widely consumed by children through cocoa-containing products, like candies.     

Occurrence of ochratoxin A in cocoa by-products and determination of its reduction during chocolate manufacture

This work reports an investigation carried out to assess the natural occurrence of ochratoxin A in 168 samples from different fractions obtained during the technological processing of cocoa (shell, nibs, liquor, butter, cake and cocoa powder) and the reduction of ochratoxin A during chocolate manufacture. Ochratoxin A analyses were performed with immunoaffinity columns and detection by high performance liquid chromatography. Concerning the natural ochratoxin A contamination in cocoa by-products, the highest levels of ochratoxin A were found in the shell, cocoa powder and cocoa cake. The cocoa butter was the least contaminated, showing that ochratoxin A seems to remain in the defatted cocoa solids. Under the technological conditions applied during the manufacture of chocolate in this study and the level of contamination present in the cocoa beans, this experiment demonstrated that 93.6% of ochratoxin A present in the beans was reduced during the chocolate producing.     

Natural co-occurrence of aflatoxin and cyclopiazonic acid in peanuts grown in Argentina

Natural occurrence of aflatoxins and cyclopiazonic acid (CPA) contamination in peanuts was investigated. Co-occurrence of CPA and aflatoxins was detected in two of 50 samples analysed. The levels of these toxins found in positive samples were 4300 and 493 μg kg -1 for CPA, 625 and 435 μg kg -1 for aflatoxin B (AFB 1 ), and 625 and 83 μg kg -1 for aflatoxin G (AFG 1 ), respectively. Levels of CPA contamination in the positive samples were similar to those registered in other substrates. This is the first report of natural co-occurrence of CPA and aflatoxins in Argentina.     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

Survey of aflatoxin contamination of dried figs grown in Turkey in 1986

A total of 284 dried fig samples, collected from fields during drying, and from warehouse and processing units in the Aegean region of Turkey in 1986, were examined for aflatoxin contamination. Aflatoxin B1, B2, and G1 were detected in 4, 2, and 2% of the samples, respectively, which were of the lower grade of figs taken from the drying stage. The average alfatoxin levels in positive samples were estimated to be 112.3 (B1), 50.6 (B2), and 61.4 ng/g (G1). The samples collected from storage (64 samples) and processing units (14 samples) contained no aflatoxins. The results of this survey show that aflatoxin contamination of Turkish dried figs in 1986 was highly correlated with the poorer grade of fig.     

Aflatoxin determination in commonly consumed foods in Tunisia

BACKGROUND: To investigate natural aflatoxin occurrence, a total of 180 samples of different foods widely consumed in Tunisia were analysed by an in‐house‐validated high‐performance liquid chromatography method including affinity column clean‐up and post‐column bromination techniques. RESULTS: The method used appeared to be rapid, selective and reproducible, and its performances were established. Detection limits were 0.05 ng g^?1 for aflatoxin B1 and 0.025 ng g^?1 for aflatoxins B2, G1 and G2. Aflatoxins were detected in all investigated commodities except rice, with an overall contamination frequency of 34.4% and concentrations ranging from 0.1 to 40.6 ng g^?1. Aflatoxin B1 was found in all contaminated samples. Sorghum, spices and nuts were most contaminated. CONCLUSION: This study has provided an effective analytical method for the reliable determination of aflatoxins in food samples. Over one‐third of the samples investigated were contaminated with aflatoxins. Sorghum, spices and nuts were most contaminated, whereas rice showed no contamination. Copyright © 2010 Society of Chemical Industry     

Occurrence of aflatoxins B 1 , B 2 , G 1 and G 2 in some Brazilian pet foods

The presence of cereals and grains in the formulation of pet foods suggests the need to control aflatoxin contamination in these foods. The objective of the study was to analyse domestic pet food to determine the occurrence of aflatoxins as well as their risk to animal health. One hundred food samples (45 for dogs, 25 for cats, 30 for birds) were collected at random from pet shops in Alfenas city, south-east Brazil. Thin-layer chromatography was used for separation, identification and quantification of the compounds after validation of the method. Aflatoxins were detected in 12.0% of the samples. Levels of aflatoxins (B 1 + B 2 + G 1 + G 2 ) above the maximum limit established in Brazil (50 µg kg -1 ) for animal food were detected in five of the 12 positive samples (41.7%). The concentration of total aflatoxins was 15-374 µg kg -1 (mean 131 µg kg -1 ). All samples containing peanuts were positive for aflatoxin B 1 . Aflatoxins are carcinogenic and their consumption might be a risk for domestic animal health. The high prevalence of aflatoxin B 1 in foods prepared for birds, species highly susceptible to aflatoxins, shows the need for the re-evaluation of the use of peanuts (present in seven of the eight samples positives for aflatoxin) and/or the addition of fungicides to the food.     

Distribution of fumonisins and aflatoxins in corn fractions during industrial cornflake processing

The aim of this study was to investigate the distribution of fumonisins (B(1) B(2,) and B(3)) and total aflatoxins (B(1), B(2), G(1), and G(2)) in various corn processed fractions. 92 batches of whole corn and derived dry-milled fractions (animal feed flour, flaking grits, corn flour and corn meal) and cooked and roasted cornflakes fractions were industrially obtained. Samples were analyzed for both groups of mycotoxins by enzyme-linked immunosorbent assay (ELISA). Dry milling of corn led to a heterogeneous distribution of the two groups of mycotoxins in the different parts of the grain, with increased levels in fractions processed from outer layers (animal feed flour and corn flour) and decreased levels in fractions processed from inner portions, such as corn meal and flaking grits. Levels of fumonisins in cornflakes were lower than 400 microg/kg, the maximum tolerable limit set by the EU. By contrast, three samples of final product were found to exceed the aflatoxin maximum tolerable limit of 4 microg/kg. Animal feed flour showed concentration factors of 317 and 288% for fumonisins and aflatoxins, respectively. Food traceability system was used by the industrial companies which processed corn into breakfast cereals. Nevertheless, even though the use of food traceability, which is defined as the ability to trace any food, feed, food-producing animal or substance that will be used for consumption through all stages of production, processing and distribution, only initial fumonisin contamination of whole corn and contamination of animal feed flour and corn flour were found to be correlated.     

The preparation, validation and certification of the aflatoxin content of two peanut butter reference materials.

The preparation of two peanut butter reference materials and the certification of their aflatoxins B1, B2, G1, G2 and total aflatoxin contents is described. The materials were prepared and certified within the BCR Programme of the Commission of the European Community as part of a broad activity to improve accuracy and agreement of measurements of importance in food and agriculture (Wagstaffe and Belliardo 1990). Reference material RM 385 was prepared from naturally contaminated peanuts, roasted and ground into a paste and then blended with uncontaminated peanut butter to achieve the desired aflatoxin concentrations. Details are given of the blending and canning procedure, and the checks to ensure homogeneity and stability of the material. Reference material RM 401 was similarly prepared but from an uncontaminated peanut butter. The certification exercise was carried out by nine laboratories using a variety of extraction and clean-up procedures, but all using high performance liquid chromatography (HPLC) as the determinative stage although operating under a variety of chromatographic conditions. RM 385 was certified as containing aflatoxins B1, B2, G1 and G2 at levels of 7.0 +/- 0.8 micrograms/kg, 1.1 +/- 0.2 micrograms/kg, 1.7 +/- 0.3 micrograms/kg and 0.3 +/- 0.2 micrograms/kg respectively (total aflatoxin content of 10.1 +/- 1.5 micrograms/kg) and RM 401 as containing aflatoxin B1, B2 and G2 at less than 0.2 micrograms/kg and aflatoxin G1 at less than 0.3 micrograms/kg (total aflatoxin content less than 0.9 micrograms/kg). The materials are intended for the verification of methods used to determine aflatoxins in nuts and nut products.     

Distribution of aflatoxins in product and by-products during glucose production from contaminated corn

Aflatoxins are known to be hepatotoxic, carcinogenic, and teratogenic. A positive correlation has been established between the consumption of aflatoxin-contaminated foods and the increased incidence of liver cancer worldwide. A survey of Egyptian corn and corn-based products and by-products shows that the majority of the samples had higher limits of aflatoxin. We have conducted experiments to determine the fate and distribution of aflatoxin during wet-milling process fractions and investigate the aflatoxin destruction during starch conversion to glucose syrup. The present results showed that about half of the aflatoxin content (48.1%) in the infected corn grain was found to be lost in steep liquor, depending upon the aflatoxin type, arranged in the order G1 > G2 > B1 > B2. After wet-milling aflatoxins were distributed into starch, gluten, fiber, and germ. Gluten, fiber, and germ were the most highly contaminated fractions. The loss of aflatoxin during process of starches reached 54.4% in steep water and water process. Although the gluten fraction represents only 9.6% of corn, the higher percentage (25.3%) of aflatoxin was found in this fraction, the fiber and germ account for nearly 29% of the milled corn and contain 11.6% of the aflatoxin. On the other hand, 8.7% of the total aflatoxins in start corn was found in starch fraction which accounts 61% of the milled corn. Aflatoxins G1 and G2 were found lost in higher concentrations compared to the aflatoxin B1 and B2. A higher percentage of AfG1 (86.35%) and AfG2 (78.36%) and a lower percentage of AfB1 (16.3%) and AfB2 (14.7%) were found in starch fraction. The conversion percent of contaminated starch was 89.5% compared with control starch. It can be concluded that aflatoxins were destroyed during starch conversion. Consequently, glucose syrup produced from contaminated starch was found aflatoxin-free.     

OCCURRENCE OF AFLATOXINS IN VARIOUS NUTS COMMERCIALIZED IN TURKEY

Nuts are important agricultural commodities in Turkey as they are exported and largely consumed domestically. Two hundred and seventeen samples of hazelnuts, pistachio nuts and peanuts were randomly collected from public markets, bazaars, supermarkets and retail stores in several regions of Turkey and analyzed for the incidence of aflatoxin (B1, B2, G1, G2) contamination by high-performance liquid chromatography. The levels of aflatoxin B1 (AFB1) and the total aflatoxins in the majority of samples analyzed (87.09%) were so low that they were not quantifiable. Thirty-one samples (14.28%) were found to have low levels of aflatoxins, below the Turkish National regulatory limits of 5 µg/kg for AFB1 and 10 µg/kg for total aflatoxins. However, four samples (1.84%) showed a level of contamination that exceeded the maximum tolerated levels set in the Turkish regulations. The highest value of AFB1 was 36.81 µg/kg in pistachio nuts. This article reports the data of the first survey on the presence of aflatoxins in nuts sampled in three distinct regions of Turkey.

PRACTICAL APPLICATIONS

The formation of aflatoxins depends not only on the genetic potential of mold strains but also on environmental factors, especially during post-harvest transportation and storage. Although further national surveys must be performed on a regular base, the results of the present study indicate a reduced level of aflatoxin contamination of nut-based products compared to earlier observations. The results conclude that implementation of stricter quality control measures, technical assistance to private sector actors and regulatory initiatives to support employment of these strategies undertaken in recent years by the National Authorities have paid back.     

Occurrence of aflatoxins and fumonisins in Incaparina from Guatemala.

The occurrence of aflatoxins and fumonisins in Incaparina was investigated. Incaparina is a mixture of corn and cottonseed flour with added vitamins, minerals and a preservative. It has been marketed as a high-protein food supplement, particularly for children on protein-deficient diets. According to estimates, 80% of Guatemalan children in their first year are given Incaparina to provide an adequate diet. Eight samples of Incaparina manufactured in Guatemala were collected. Five were from three different geographical locations in the USA and three were from Guatemala. Seven were examined for fungal contamination and analysed for aflatoxins and fumonisins. Aspergillus flavus was the predominant fungus in all samples purchased in the USA and in one sample purchased from Guatemala, whereas Fusarium verticillioides was present in only two samples (one from the USA and one from Guatemala). All samples contained aflatoxins, ranging from 3 to 214 ng g(-1) and <2 to 32ng g(-1) for aflatoxin B(1) and aflatoxin B(2), respectively; and one sample contained aflatoxin G(1) (7 ng g(-1)). Total aflatoxins present ranged from 3 to 244 ng g(1). All samples contained fumonisins, ranging from 0.2 to 1.7 microg g(-1), <0.1 to 0.6 microg g(-1), and <0.1 to 0.2 microg g(-1) for fumonisins B(1), fumonisin B(2), and fumonisin B(2), respectively. Total fumonisins present ranged from 0.2 to 2.2 microg g(-1). The identity of aflatoxin B(2) was confirmed using both the chemical derivatization method and liquid chromatographic (LC)/mass spectrometric (MS) analysis. Appropriate regulatory action was recommended for the import of Incaparina and has been in effect since 22 December 1998.     

Occurrence of aflatoxins M(1) and M(2) in milk commercialized in Ribeir?o Preto-SP, Brazil.

Aflatoxins are toxic metabolites found in foods and feeds. When ruminants eat foodstuffs containing aflatoxins B(1) and B(2), these toxins are metabolized and excreted as aflatoxin M(1) and M(2) in milk. The aim was to determine the incidence of these aflatoxins in commercial milk collected from supermarkets in Ribeir?o Preto-SP, Brazil, and consisting of 60 ultrahigh temperature (UHT) milk samples and 79 pasteurized milk samples. The milk samples were analysed according to method 986.16 of AOAC International. None of the milk samples analysed were contaminated with aflatoxin M(2), and aflatoxin M(1) was detected in 29 (20.9%) of samples in the range 50-240 ng l(-1). The results show that despite a high occurrence of aflatoxin M(1) in commercial pasteurized and UHT milk sold in Ribeir?o Preto in 1999 and 2000, the contamination level of these toxins could not be considered a serious public health problem according to MERCOSUR Technical Regulations. However, levels in 20.9% of the milk samples exceeded the concentration of 50 ng l(-1) permitted by the European Union. Although it is not necessary to continue monitoring the incidence and levels of aflatoxins M(1) and M(2) in milk samples, surveillance could be appropriate.     

Aflatoxins in Turkish dried figs intended for export to the European Union

Dried figs for export from Turkey from crop years 2003 through 2006 were tested for aflatoxin B1 and total aflatoxins. For export to the European Union, consignments of 0.5 to 10 tonnes of dried figs were sampled according to European Commission regulations, and high-pressure liquid chromatography (HPLC) was used to determine concentrations of aflatoxins Bl, B2, G1, and G2. For each consignment of dried figs, a 30-kg sample (comprising 100 subsamples) was divided into three 10-kg subsamples, which were separately blended and analyzed with HPLC. This monitoring effort was conducted for figs from 2003, 2004, 2005, and up to June 2006, for a total of 10,396 30-kg samples (28,489 analyses). The incidence of contamination with aflatoxin B1 at higher than 2 ng/g was on average 0.6, 2.0, 4.0, and 2.4% for 2003, 2004, 2005, and up to June 2006, respectively, whereas contamination with total aflatoxins at higher than 4 ng/g was 2.6, 3.0, 5.1, and 2.7%. There was significant variability in contamination between replicate 1-kg samples, indicating small numbers of individual contaminated figs were probably responsible. There were also substantial differences in the relative proportions of aflatoxins B1, B2, G1, and G2 among samples, suggesting different contributing fungal sources.     

Determination of aflatoxins in peanut butter and sesame samples using high-performance liquid chromatography method

In this study, total number of samples analysed were 20 packages of sesame and 20 cans of peanut butter, which were collected from Ankara local markets, Turkey. Extraction and determination of aflatoxins have been made by immunoaffinity column technique and high-performance liquid chromatography (HPLC) method. Mean levels (±SE) of aflatoxins B₁, B₂, G₁ were found to be 15.756±3.129 ng/g, 1.232±0.244 ng/g and 9.689±1.005 ng/g, respectively in peanut butter samples. Regarding the sesame samples, mean level of aflatoxin G₁ was found to be 0.754±0.213 ng/g. Our data revealed that while aflatoxin levels found in sesame samples were within the Turkish Food Codex (TFC) values, for peanut butter samples, they were higher than the TFC values.     

Survey of breakfast and infant cereals for aflatoxins B1, B2, G1 and G2.

Three hundred and forty-nine breakfast and infant cereal samples were collected at retail level across Canada from 2002 to 2005. They included rice-, soy-, barley-based and mixed-grain infant cereals, corn-, wheat-, rice-based and mixed-grain breakfast cereals, and were analysed for aflatoxins B1, B2, G1 and G2 using a modified AOAC International official method. An immunoaffinity column was used for the cleanup and purification of extracts. Determination of aflatoxins was by LC using post-column derivatization with pyridinium hydrobromide perbromide and fluorescence detection. Results indicated that 50% of both breakfast and infant cereals had detectable levels (limit of detection = 0.002 ng g-1) of aflatoxin B1, which is the most toxic of the four toxins. The levels found varied from 0.002 to 1.00 ng g-1 for aflatoxin B1, from 0.002 to 0.14 ng g-1 for aflatoxin B2, from 0.008 to 0.27 ng g-1 for aflatoxin G1, and from 0.008 to 0.048 ng g-1 for aflatoxin G2. Only 4% of the breakfast cereals and 1% of the infant cereals had aflatoxin B1 levels exceeding 0.1 ng g-1, which is the European Union maximum limit for aflatoxin B1 in baby foods and processed cereal-based foods for infants and young children.     

Determination of aflatoxin B1 levels in peanut butter using an immunoaffinity column clean-up procedure: inter-laboratory study

Ten United Kingdom laboratories participated in an evaluation of an immunoaffinity column sample preparation procedure used to prepare aflatoxin B1 containing extracts obtained from peanut butters contaminated by aflatoxins. Each laboratory was sent seven randomly numbered samples of roasted peanut butter which included two sets of undisclosed triplicates. These two peanut butters were naturally contaminated with aflatoxin B1 at levels of about 12 and 35 micrograms/kg. The other sample was a nominal blank peanut butter containing approximately 2 micrograms/kg of aflatoxin B1 which was also employed by participants for recovery experiments. Participating laboratories were instructed to follow a protocol regarding the use of the immunoaffinity columns for extract preparation, but were allowed a free choice of instrumental technique for quantification of aflatoxin levels. Mean recovery for spikes was 72%. Coefficients of variation for the results from the 10 participants for the two contaminated roasted peanut butters were, respectively, 45% (on a mean of 13.6 micrograms/kg) and 36% (on a mean of 37.2 micrograms/kg).     

Aflatoxin in betel nut and its control by use of food preservatives.

The occurrence of aflatoxins in market betel nut samples was studied. It was observed that several betel nut samples were infested with aflatoxin-producing fungus, Aspergillus flavus. Out of 32 samples collected from various places, 12 were positive for aflatoxin. Aflatoxin B1 was detected in all the positive samples. Other aflatoxins were also detected in some samples. Boric acid, propionic acid and potassium metabisulphite were used for the control of aflatoxin B1 on betel nuts. Propionic acid was most effective in inhibiting aflatoxin production on betel nut after intervals of 2 (62%) and 4 (85%) weeks. Controlling the occurrence of aflatoxin could safeguard the users from the health hazards of aflatoxins.     

Distribution of aflatoxins in shelling and milling fractions of naturally contaminated rice

The objective of this study was to determine the distribution of an economically important class of mycotoxins, the aflatoxins, in rice milling fractions. Rice plants grown under field production conditions are frequently infected with types of pathogenic fungi that produce toxic metabolites (mycotoxins). Paddy (seeds) rice from healthy plants in the field was collected and stored on a farm under humid, poorly ventilated conditions. Samples were milled into four fractions (hulls, brown rice, bran and white rice) and analysed for aflatoxins (B(1), B(2), G(1) and G(2)) using a validated method. Rice fractions from healthy plants, which contained low levels of aflatoxins (less than 1?μg?kg(-1)), were used to determine the efficiency of the extraction method. Seeds stored under poor conditions were found to be contaminated with aflatoxins B(1) and B(2) as were the fractions. The sums of AFB(1) and AFB(2) in stored paddy rice, hulls, brown rice, bran and white rice were 141, 39, 158, 367 and 56?μg?kg(-1), respectively. The ratio of aflatoxin B(1) and B(2) was about 10?:?1. AFG(1) and AFG(2) were less than 1?μg?kg(-1). Thus, brown rice contained 92.9% of the aflatoxins in paddy rice, whereas white rice contained only 27.9%.     

A survey of aflatoxin levels in peanut meal imported into Poland for animal feedingstuffs

Peanut meals (366 samples) imported into Poland from Brazil and India during 1984-86 for use in animal feedingstuffs were analysed for aflatoxins B1 and B2. Average levels of contamination were 71 micrograms per kg for aflatoxin B1 and 16 micrograms per kg for aflatoxin B2 with total concentrations ranging from 2 to 750 micrograms per kg.