Incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs
Ninety eight dried figs; 53 sultanas and 20 dried apricots destined for export from Turkey to the European Union were tested for ochratoxin A (OTA) contamination utilizing immunoaffinity column clean-up and high-performance liquid chromatography (HPLC). While only 2 (4%) of the sultanas exceeded the 10 ng g(-1) maximum limit set by the EU; 28 (53%) of 53 sultana samples contained detectable levels of OTA; in the range of 0.51-58.04 ng g(-1). Eighteen of 98 (18%) dried figs contained detectable levels of OTA; in the range of 0.87-24.37 ng g(-1). Only one of the 20 dried apricots was contaminated; with 0.97 ng g(-1) OTA. Dried figs analyzed for OTA were also tested for aflatoxin contamination to determine the co-occurrence of both toxins. Seven samples were confirmed aflatoxin positive; in the range of 0.23-4.28 ng g(-1); and only 2 samples contained both toxins; with a maximum concentration of 24.37 ng g(-1)for OTA and 1.02 ng g(-1) for aflatoxin B(1). The average recovery and relative standard deviation (RSD) obtained from dried fruits spiked with OTA ranged from 80.5% to 91.5% and 0.99-5%; respectively. The average recovery and relative standard deviation (RSD) obtained from dried figs spiked with aflatoxin ranged from 88.78% to 93.53% and 2.54-7.25%; respectively.
Growth of aflatoxigenic moulds and aflatoxin formation in Brazil nuts
The present study aimed at gaining more knowledge of the growth of aflatoxigenic moulds and aflatoxin production in Brazil nuts in relation to humidity conditions and storage time. For this purpose; the growth of aflatoxigenic moulds and the increase in aflatoxin levels in Brazil nuts was studied in the laboratory at temperature and humidity conditions that are relevant for the Amazon region. Fresh unprocessed Brazil nuts in shell were inoculated with an aflatoxin producing strain of Aspergillus nomius previously isolated from Brazil nuts. The nuts were stored at 27 °C in combination with 97; 90 or 80 % surrounding relative humidity in a respirometer for up to 3 months. The General Linear Model (GLM) was used for evaluation of the effect of water activity and time on aflatoxigenic mould levels and on aflatoxin levels; as well as the relationship between mould and aflatoxin levels. During storage at the highest relative humidity (97 %) aflatoxin formation occurred rapidly; whereas storage at 90 % relative humidity resulted in slower aflatoxin formation. At the lowest relative humidity (80%); aflatoxin formation occurred sporadically during storage. The increase in mould and aflatoxin levels along the production chain is also described; using field data collected in the state of Pará; Brazil. The growth of aflatoxigenic moulds and aflatoxin formation increased rapidly between 40-90 days following collection of the nuts; before the nuts reached the final drying stage at the processing plant. In addition; a logistic regression model predicting the probability that the European legislative limit of 4 µg/kg for aflatoxins in nuts will be exceeded in relation to colony counts of either one selected aflatoxigenic mould strain (laboratory experiments) or of a mixture of aflatoxigenic strains (field data) was developed. The probability that total aflatoxin levels will exceed the European legislative limit of 4 µg/kg increased rapidly from approx. 30% to above 80% for both experimental and field samples at mould levels between 2 and 3 log cfu/g.
Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis
Aims: The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. Methods and Results: Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis; using arithmetic means (UPGMA). Isolates grouped according to host; with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus-specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2); and an internal amplification control was developed; to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. Conclusions: RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus; with a detection limit of 10 fg. Significance and Impact of the Study: Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.
Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment
Low pressure cold plasma (LPCP) using air gases and sulfur hexafluoride (SF(6)) was developed and tested for anti-fungal efficacy against Aspergillus parasiticus on various nut samples. Artificially A. parasiticus contaminated hazelnuts; peanuts; and pistachio nuts were treated with air gases plasma and SF(6) plasma for up to 20 min duration. The sterilizing effect of LPCP on A. parasiticus was higher during the early treatment period than the later treatment period. Air gases plasma treatment for 5 min resulted in 1-log reduction of A. parasiticus and a further 5 min treatment resulted in additional 1-log reduction. SF(6) plasma application was more effective resulting in approximately a 5-log decrease in fungal population for the same duration. When effectiveness of plasma treatment against aflatoxins were tested; 20 min air gases plasma treatment resulted in a 50% reduction in total aflatoxins (AFB1; AFB2; AFG1; and AFG2); while only a 20% reduction in total aflatoxin was observed after 20 min SF(6) plasma treatment. In this study; a rapid; functional clean-up method for the elimination of aflatoxin producing fungus from shelled and unshelled nuts was investigated as a suitable fungal decontamination method.
Aspergillus nomius; an important aflatoxin producer in Brazil nuts?
The relation between aflatoxin B1 and G1 was examined in samples from 199 aflatoxin contaminated lots of in-shell Brazil nut imported to Europe. In most of the samples; the relation between B1 and G1 were approximately 50/50 indicating that the major responsible aflatoxin producing fungi cannot be Aspergillus flavus; which produces solely B aflatoxins. Fungal strains were isolated from two batches of Brazil nuts and isolates of both A. nomius and A. flavus could be identified. The A. nomius isolates were good producers of both B and G aflatoxins; while the A. flavus strains only produced B aflatoxins. In conclusion; this study suggests that A. nomius is an important producer of aflatoxins in Brazil nuts and that its occurrence; and eventually other B and G aflatoxin producers; should be further examined since this may influence strategies for prevention and control of aflatoxins in Brazil nuts.
Factors influencing fungal and aflatoxin levels in Turkish hazelnuts (Corylus avellana L.) during growth; harvest; drying and storage: A 3-year study
The levels aflatoxins in Turkish hazelnuts have been monitored over a 3-years period (2002-2004). Periodical sampling was made in 72 different orchards at different locations representative of the hazelnut-growing areas and post-harvest applications. Various parameters (aflatoxins; water activity; moulds) were analysed and environmental conditions (temperature and relative humidity) recorded during growing and at different stages of harvest and post-harvest processing; involving three different harvesting methods (collection in nets; from the ground; etc.) and four drying techniques (traditional sun-drying; mechanical drying; etc.). Fungal and aflatoxin analyses (HPLC) showed no significant difference except between samples which had been in contact with the ground and those which had not (at 95% confidence level). Aflatoxins levels from the orchard recorded a maximum of 0.77 +/- 0.08 ng g(-1) from a total of 1624 samples. Regarding harvesting and post-harvest processes; the only application where aflatoxins were detected was in samples which had been in direct contact with the ground (max. 3.18 +/- 0.03 ng g(-1)). Aflatoxin formation was low during storage (max. 0.34 +/- 0.003 ng g(-1)). As a result of mycological studies; a total of 5546 Aspergillus flavus (89%) and A. parasiticus (11%) species were isolated and identified from samples. The results indicated that harvesting hazelnuts into a canvas by shaking the trees; manual harvesting of mature hazelnuts where possible; use of jute instead of nylon sacks and mechanical drying technique would minimize aflatoxin levels in hazelnuts. These recommendations have been implemented and about 4000 people in the hazelnut industry have been trained in these practices.
Mycotoxins in botanicals and dried fruits: A review
Botanicals are used in many countries for medicinal and general health-promoting purposes. Numerous natural occurrences of mycotoxins in botanicals and dried fruits have been reported. Aflatoxins or ochratoxin A (OTA) have been found in botanicals such as ginseng; ginger; liquorice; turmeric; and kava-kava in the USA; Spain; Argentina; India; and some other countries; while fumonisins have been found in medicinal wild plants in South Africa and in herbal tea and medicinal plants in Turkey. Zearalenone was identified in ginseng root. Dried fruits can be contaminated with aflatoxins; OTA; kojic acid; and; occasionally; with patulin or zearalenone. One main area of concern is aflatoxins in dried figs; bright greenish yellow fluorescence under ultraviolet light is associated with aflatoxin contamination. OTA in dried vine fruits (raisins; sultanas; and currants) is another concern. There are also reports of aflatoxins in raisins and OTA in dried figs; apricots; dried plums (prunes); dates; and quince. Maximum permitted levels in the European Union include 4 microg kg(-1) for total aflatoxins in dried fruit intended for direct consumption and 10 microg kg(-1) for OTA in dried vine fruit. This review discusses the occurrence of mycotoxins in botanicals and dried fruits and analytical issues such as sampling; sample preparation; and methods for analysis. Fungal contamination of these products; the influence of sorting; storage; and processing; and prevention are also considered.
Sampling almonds for aflatoxin; part II: estimating risks associated with various sampling plan designs
About 100 nations have established regulatory limits for aflatoxin in food and feeds. Because these limits vary widely from one country to another; the Codex Alimentarius Commission; working through the Codex Committee on Food Additives and Contaminants; has initiated work to harmonize aflatoxin limits and sampling plans for almonds; pistachios; hazelnuts; and Brazil nuts. Studies were developed to measure the uncertainty and distribution among test results for replicate samples taken from aflatoxin-contaminated almond shipments. The uncertainty and distribution information was used to develop a model to evaluate the performance of aflatoxin sampling plans so that harmonized sampling plans can be developed for almonds that reduce the misclassifying of lots in the export trade. Twenty lots of shelled almonds were sampled according to an experimental protocol in which sixteen 10 kg samples were taken from each lot. The observed aflatoxin distribution among the 16 sample test results was compared with 3 theoretical distributions. The negative binomial distribution was selected to model aflatoxin distribution among sample test results because it gave acceptable fits across all 20 observed sample distributions. By using the variance and distribution information; operating characteristics curves were developed to predict the effect of sample size and accept/reject limits on the probability of rejecting good lots and accepting bad lots.
Selenium and Aflatoxin levels in raw Brazil nuts from the Amazon Basin.
Whereas selenium (Se) is an important antioxidant in human metabolism to prevent cancer; aflatoxins are highly carcinogenic. Brazil nuts from Eastern and Western Amazon regions were evaluated to find any relationship between Se and aflatoxins levels. A total of 80 (in-shell and shelled) nuts samples were collected directly from different forest sites and analyzed for Se by atomic emission spectrometry and aflatoxins by liquid chromatography tandem mass spectrometry. The limit of quantification (LOQ) for Se was 2.0 mg/kg; and LOQ for total aflatoxins was 0.390 microg/kg. Nut Se levels from the Eastern region were higher than the Western; in addition to the aflatoxins. The moisture content (mc) and water activity ( a w) of the raw nuts from the two regions did not present a significant difference; for either in-shell or shelled. The mc was 24.5% (minimum of 20.1% and maximum of 30.4%) and 22.1% (minimum of 14.6% and maximum of 28.9%) and a w of 0.85 for both regions. Further studies need to be carried out to discover the role of Se on fungi growth stress and aflatoxin production mechanisms.
Characteristics of in-shell Brazil nuts and their relationship to aflatoxin contamination: criteria for sorting.
External characteristics of in-shell Brazil nuts were evaluated for dimensions (length and face width); weight; chromaticity; and shell thickness. The internal characteristics evaluated were moisture content (mc); aflatoxin contamination (analyzed by LC-MS/MS); and shell/nut ratio. According to their length; Brazil nuts were classified in three groups: I; II; and III; corresponding to large; medium; and small sizes; respectively. It was possible to establish the following parameters as standards for normal/healthy nuts: length (53.2; 43.9; and 36.6 mm); weight (12.9; 8.8; and 6.3 g); and shell chromaticity components (L*; 38.3; 39.5; and 41.6; a*; 8.0; 7.9; and 7.8; and b*; 17.6; 18.0; and 18.7); for the three groups; respectively. The mean of shell thicknesses were 1.92 and 2.68 mm taken from each face and nut top. The nuts; classified as small (Group III); presented aflatoxin B1 contamination at a level of 5.62 microg/kg. The Groups shell/nut ratios were 1.2; 1.2; and 1.3 for normal whole and healthy nuts. No aflatoxin was detected in Groups I and II. The data obtained from the Brazil nut measured characteristics can help to distinguish healthy/safe and deteriorated nuts and will be useful for Brazil nut sorting and machine development.