Risk Assessment of Mycotoxins and Predictive Mycology in Sri Lankan Spices: Chilli and Pepper

This research contributes to a better understanding of the mycotoxin problem associated with two important spices in world trade;, chilli and pepper by a multidisciplinary approach including analytical chemistry, risk assessment, food chemistry and predictive mycology. More specifically, this work provides important insights in mycotoxin contamination of these spices and associated risks in Sri Lanka. Firstly, a simple extraction method based on the QuEChERS approach was developed and successfully validated for the simultaneous determination of multiple mycotoxins using an advanced chromatographic technique, LC-MS/MS. The method was applied on complex spices for quantitative screening of seventeen mycotoxins. In addition to the classical aflatoxins and ochratoxin A, the spices were also found to be contaminated with several other toxicologically significant mycotoxins. Chilli samples (87%) were more frequently contaminated with mycotoxins than peppers (65%). Subsequently, the mycotoxins screening results and the collected consumption data were integrated in a quantitative risk assessment study. The results showed that AFB1 exposure via chilli consumption is of a public health concern in Sri Lanka, pepper is of lesser extent a risk due to the lower consumption. The toxigenic mould characterization in black peppers showed that Aspergillus flavus and/or Aspergillus parasiticus were the predominant moulds (73%) found, with considerable contamination (60%) of Penicillium spp. and A. niger. Furthermore, predictive mould growth models on peppercorns were developed at three temperatures and seven water activity levels for both A. flavus and A. parasiticus isolates. Based on the research, suitable storage conditions for black peppercorns were suggested and the way forward in managing the risk towards mycotoxins posed by the consumption of these two spices in Sri Lanka.     

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.     

Mycotoxins in foodstuffs. XII. The influence of the water activity (a-w) of cakes on the growth of moulds and the formation of mycotoxins.

The influence of various a-w values (high, moderate and low, e.g. 0.92, 0.87, and 0.82) of cakes ("Rührkuchen", made of wheat flour, sugar, eggs, fat and water) on the development and the mycotoxin production of Aspergillus flavus (aflatoxins), A. versicolor (sterigmatocystin), A. ochraceus (ochratoxin A), Penicillium chrysogenum (citrinin) and P. expansum (patulin) was investigated. None of the moulds was able to germinate at an a-w of 0.82. An increase of the water activity resulted in a pronounced growth of the five fungi. Citrinin was not formed on the cakes at all. The yields of the other mycotoxins were not markedly influenced by the a-w (0.92 or 0.87). During the development of the moulds the a-w of the substrate was lowered at first (water requirement during germination and initial growth) and subsequently increased (initiation of the secondary metabolism).     

Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina

Aspergillus section flavi strains isolated from peanuts, wheat and soybean grown in Argentina were screened for aflatoxins (type B and G) and cyclopiazonic acid (CPA) production. Aspergillus flavus was the predominant species in all substrates, although there was almost the same proportion of A. flavus and Aspergillus parasiticus in peanuts. Aspergillus nomius was not found. Incidence of aflatoxigenic A. flavus strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%) while the ratio of CPA producers A. flavus isolated from all substrates was very high (94% in peanuts, 93% in wheat and 73% in soybeans). Isolates of A. flavus able to produce simultaneously aflatoxins type B and CPA were detected in all substrates, suggesting the possibility of co-occurrence of these toxins. Almost all isolates of A. parasiticus resulted aflatoxins (type B and G) producers but did not produce CPA. Five of sixty-seven strains isolated from peanuts showed an unusual pattern of mycotoxin production (aflatoxins type B and G simultaneously with CPA). These strains also produced numerous small sclerotia like S strains of A. flavus detected in cottonseed in Arizona and in soils of Thailand and West Africa. The atypical strains are not widely distributed in Argentina and were found uniquely in peanuts.     

Mycoflora and toxigenic Aspergillus flavus in Spanish dry-cured ham

Sixty-five dry-salted hams were analysed. Aspergillus and Penicillium were the dominant genera. In general, the mould flora was dominated by Aspergillus spp. and primarily A. glaucus, A. fumigatus, A. niger and A. flavus. A flavus was found in 16 hams and 9 out of 16 strains examined produced aflatoxins 'in vitro'. Surface samples of dry-salted hams showed growth of inoculated A. parasiticus NRRL-2999 strains and production of aflatoxins in low levels at 25 and 30 degrees C. It is concluded that the presence of toxigenic strains in Spanish dry-salted ham does not constitute a health risk.     

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.     

干腌火腿中霉菌毒素研究的进展

从干腌火腿中分离到一些霉菌菌株在培养基中生长时可以产生黄曲霉毒素、环匹阿尼酸、青霉酸、梗曲霉素、展青霉素、灰黄霉素、霉酚酸、赭曲霉毒素、橘青霉素和疣孢苷啶等多种霉菌毒素。生成霉菌毒素的霉菌都属于青酶和曲霉。鲜绿青霉可以在干腌火腿中生成环匹阿尼酸,且这种霉菌毒素在干腌火腿中具有较高的稳定性,从而对干腌火腿的安全性构成很大的威胁。将无毒性的霉菌菌株进行培养并接种到干腌火腿上,可防止干腌火腿中形成霉菌毒素。     

Occurrence of mycotoxigenic fungi and mycotoxins in animal feed from bihar,india

A total of 387 samples comprising animal feed, poultry feed and cattle cakes from India were examined in order to isolate mycotoxin-producing fungi as well as mycotoxins. Of 385 Aspergillus flavus group isolates 53% were capable of producing aflatoxins in liquid SMKY medium. Toxigenic strains of other mycotoxigenic fungi, viz A ochraceus, A versicolor, Penicillium citrinum and Fusarium moniliforme, were also recorded. In feed samples, beside the aflatoxins present in 139 samples zearalenone, ochratoxin A and citrinin were also recorded alone or as cocontaminants. A monsoon climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in animal food.     

Further mycotoxin effects from climate change

Climate change will affect mycotoxins in food. The 2007 Intergovernmental Panel on Climate Change report is reinterpreted herein to account for what may occur with mycotoxins. Warmer weather, heat waves, greater precipitation and drought will have various impacts, depending on which regions of the world and mycotoxin systems are considered. The humidity issues are more complex as some areas will experience drought and others greater precipitation: in vivo data on the effects of moisture on mycotoxins in crops are more ambiguous than those for temperature. In vitro data on fungal growth and mycotoxin production may not relate directly to the situation in the field or post harvest, but are useful for base-line assumptions. The effects of climate in various regions of the world, i.e. Africa, Europe, Asia, Latin America and North America are considered in terms of mycotoxin contamination. Crops introduced to exploit altered climate may be subject to fewer mycotoxin producing fungi (the “Parasites Lost” phenomenon). Increased mycotoxins and UV radiation may cause fungi to mutate on crops and produce different mycotoxins. Whereas there is relevant information on aflatoxins, deoxynivalenol, and ochratoxin A, more mycotoxins require to be considered: Data on patulin are missing. The current paper considers uniquely ergot alkaloids. Amelioration strategies are provided. There is considerable urgency in the need to address these issues.     

Post-harvest control strategies: minimizing mycotoxins in the food chain

Contamination of cereal commodities by moulds and mycotoxins results in dry matter, quality, and nutritional losses and represents a significant hazard to the food chain. Most grain is harvested, dried and then stored on farm or in silos for medium/long term storage. Cereal quality is influenced by a range of interacting abiotic and biotic factors. In the so-called stored grain ecosystem, factors include grain and contaminant mould respiration, insect pests, rodents and the key environmental factors of temperature, water availability and intergranular gas composition, and preservatives which are added to conserve moist grain for animal feed. Thus knowledge of the key critical control points during harvesting, drying and storage stages in the cereal production chain are essential in developing effective prevention strategies post-harvest. Studies show that very small amounts of dry matter loss due to mould activity can be tolerated. With <0.5% dry matter loss visible moulding, mycotoxin contamination and downgrading of lots can occur. The key mycotoxigenic moulds in partially dried grain are Penicillium verrucosum (ochratoxin) in damp cool climates of Northern Europe, and Aspergillus flavus (aflatoxins), A. ochraceus (ochratoxin) and some Fusarium species (fumonisins, trichothecenes) on temperate and tropical cereals. Studies on the ecology of these species has resulted in modelling of germination, growth and mycotoxin minima and prediction of fungal contamination levels which may lead to mycotoxin contamination above the tolerable legislative limits (e.g. for ochratoxin). The effect of modified atmospheres and fumigation with sulphur dioxide and ammonia have been attempted to try and control mould spoilage in storage. Elevated CO2 of >75% are required to ensure that growth of mycotoxigenic moulds does not occur in partially dried grain. Sometimes, preservatives based on aliphatic acids have been used to prevent spoilage and mycotoxin contamination of stored commodities, especially feed. These are predominantly fungistats and attempts have been made to use alternatives such as essential oils and anti-oxidants to prevent growth and mycotoxin accumulation in partially dried grain. Interactions between spoilage and mycotoxigenic fungi and insect pests inevitably occurs in stored grain ecosystems and this can further influence contamination with mycotoxins. Effective post-harvest management of stored commodities requires clear monitoring criteria and effective implementation in relation to abiotic and biotic factors, hygiene and monitoring to ensure that mycotoxin contamination is minimised and that stored grain can proceed through the food chain for processing.     

Toxigenic fungi isolated from dried vine fruits in Argentina

To evaluate the potential for mycotoxin production by fungi in dried vine fruits, the mycobiota was determined both before and after surface disinfection. Predominant genera were Aspergillus (50.2%), Eurotium (21.4%) and Penicillium (13.5%). Aspergillus section Nigri ("black aspergilli") were isolated with relatively high frequency. Aspergillus niger was the most common species but only 3 of 293 isolates screened were ochratoxin A (OTA) producers. Aspergillus carbonarius was less common but 96% of 48 strains screened were ochratoxigenic. OTA was not produced by A. japonicus. Other toxigenic fungi detected were A. ochraceus (3 strains produced OTA), Aspergillus flavus (5 strains produced cyclopiazonic acid but not aflatoxins), P. citrinum (19 strains were strong citrinin producers) and Alternaria alternata (15 strains were producers of tenuazonic acid, alternariol and alternariol methyl ether). In spite of the high incidence of A. carbonarius capable of producing OTA, low levels of this toxin were detected in the samples analysed.     

Fungal population and distribution of aflatoxigenic fungi in commercial almond powder products

The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under 1.0 x 10 colony-forming units (CFU)/g to 8.5 x 10(3) CFU/g as determined with the dilution plating technique. The predominant fungi in the mould-contaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2% yeast extract and 15% sucrose broth culture. Four different aflatoxigenic fungi were detected in the isolates; aflatoxins B1 and B2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B1, B2, G1, G2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters.     

黑曲霉抗产毒黄曲霉作用的初步研究

采用90 × 2.6 × 1013N+/cm2 注入黑曲霉筛选能抗黄曲霉(Aflavus)生长的突变菌株,以利发酵中控制被黄曲霉污染的原材料的再污染。进行产毒黄曲霉与被离子注入的黑曲霉混合对峙、原黑曲霉菌株与黄曲霉单独培养生长及混合对峙培养实验。结果显示经离子注入的菌株及未注入菌株均对黄曲霉产生抑制作用,但后者仅有微弱抑制,前者不仅表现出几乎不能使黄曲霉生长,且已长出的黄曲霉菌丝体较瘦小,并呈灰白色。从培养基中提取物检验结果显示,黄曲霉组表现出有较明显的荧光反应,而黑曲霉菌株对峙培养物提取物中有微弱的荧光反应,其黑曲霉突变株对峙培养物未见荧光反应检出。这表明黑曲霉原菌株虽然能对黄曲霉只有微弱抑制,但表现出黄曲霉产毒和合成色素能力下降。与对照组相比,突变株有较强抑制黄曲霉生长能力。     

Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest

The total mycobiota and the mycotoxin contamination of peanuts were analyzed in plants collected at different stages of the pod maturity sampled in Junqueirópolis, at S?o Paulo State (Brazil). The prevalent peanut mycobiota were Fusarium spp. and Aspergillus flavus, present in 26% and 17% respectively of the samples analyzed. In soil, the genus Penicillium and Fusarium were most frequently detected, and A. flavus was detected in 8% of the samples. The screening of mycotoxins indicated that aflatoxins and cyclopiazonic acid were present the highest incidence, being detected in 32% of the samples, in concentrations, respectively, from 4.20 microg/kg to 198.84 microg/kg and from 260 microg/kg to 600 microg/kg. Fumonisins were not detected by HPLC. All data were correlated with the occurrence of wind-dispersed fungi and the environmental and soil conditions. Results indicate that good management of the agricultural environment may offer a way to reduce mycotoxins and the toxigenic fungal contamination in peanuts preharvest because the pods are exposed to different environmental conditions during their formation until harvest, and the optimal conditions for mycotoxin production and fungal growth are frequently found in the crop fields.     

Effect of processing for saponin removal on fungal contamination of quinoa seeds (Chenopodium quinoa Willd.)

Incidence of fungal contamination of quinoa seeds from three locations (Salar de Uyuni, Bolivia; Salta and Tucumán provinces, Argentina) was analyzed in samples with and without treatment to remove saponins (wet method). In processed samples, the percentage of infection was reduced. Distribution of the different fungal genera was not homogeneous in the three locations (p<0.05), although Penicillium and Aspergillus were the most prevalent contaminants, regardless the geographic origin of the samples. Other genera, such as Eurotium, Fusarium, Phoma, Ulocladium, Mucor and Rhizopus were less frequently isolated. Absidia, Alternaria, Cladosporium, Dreschlera, Epicoccum and Monascus were sporadically encountered. Significant differences (p<0.05) in the distribution of fungal genera in samples with and without saponins from each location were observed. In all cases, processing caused a decrease of Aspergillus incidence, while increased the proportion of Penicillium, Eurotium, Mucor and Rhizopus indicating that these genera were part of the internal mycota. A. flavus and A. niger were the dominating species of genus Aspergillus. A similar pattern of prevalent Penicillium species was observed in samples with and without saponins, since P. aurantiogriseum, P.chrysogenum, P. citrinum and P. crustosum were always present in high number, although their relative density was variable according to the geographic origin of samples. Mycotoxin-producing ability of most representative species was also determined. Toxigenic strains of A. flavus (aflatoxins and cyclopiazonic acid), A. parasiticus (aflatoxins), P. citrinum (citrinin) and P. griseofulvum (cyclopiazonic acid) were found. None of the A. niger isolates was ochratoxin A producer. The above mentioned mycotoxins were not detected in the samples analyzed.     

Mycological survey for potential aflatoxin and ochratoxin producers and their toxicological properties in harvested Brazilian black pepper.

A mycological survey was carried out on 115 samples of whole dried black pepper seeds, from two main production regions of Brazil (Pará and Espírito Santo). A high incidence of contamination was verified in both regions when 99.1% of the samples showed filamentous fungi contamination. A total of 497 species of nine different genera were isolated (Aspergillus, Eurotium, Rhizopus, Penicillium, Curvularia, Cladosporium, Absidia, Emericella and Paecilomyces). The genus Aspergillus was the predominant (53.5%) followed by species from the Eurotium genus (24.5%). Eurotium chevalieri (16.4%) was the most predominant species followed by A. flavus (14.6%) present on 55 samples of black pepper (47.8%) analysed. Twenty-five samples (21.7%) were contaminated with aflatoxigenic strains of A. flavus and A. parasiticus. In relation to the types of aflatoxins produced by mycotoxigenic strains, it was observed that 25 strains (44.6%) of 56 isolated of A. flavus produced aflatoxins. From 12 samples, A. ochraceus species were isolated in low frequency (3.5%). Two strains of A. ochraceus from 16 isolated were producers of ochratoxin A. With respect to the aflatoxins and ochratoxin A natural contamination, none of the samples presented detectable levels of these mycotoxins using thin-layer chromatographic analysis.     

Incidence of microflora and of ochratoxin A in green coffee beans (Coffea arabica).

Coffee is produced in tropical countries around the Equator where climatic conditions are favourable for fungal development and mycotoxin production; however, mycotoxins do not only occur in the tropical countries. The aim was to evaluate the mycoflora and possible incidence of ochratoxin A (OTA) in 60 samples of green coffee beans from Brazil. The mycological evaluation was carried out using a conventional method and the OTA was determined using sequential phenyl silane and immunoaffinity column cleanup followed by HPLC. The detection limit was 0.2 microg kg(-1). Practically all samples (91.7%) were contaminated with moulds. The dominant fungal genus was Aspergillus, including A. niger (83.3%), A. ochraceus (53.3%) and A. flavus (25.0%). The occurrence and the levels of the genus Cladosporium (16.6%) and Penicillium (10.0%) were substantially lower than Aspergilli. Twenty samples (33.3%) of 60 were contaminated with the toxin at levels ranging from 0.2 to 7.3microg kg(-1). The average concentration was 2.38 microg kg(-1). All positive samples showed OTA levels below the limit suggested by the European Union (8 microg kg(-1)).     

重庆地区辣椒、花椒、八角中真菌毒素污染状况分析

目的 了解重庆地区辣椒、花椒和八角中真菌毒素污染状况。方法 采用随机抽样方法,在重庆地区盘溪、菜园坝和江津三个市场,抽取180批次香辛料样品,采用HPLC-MS/MS检测黄曲霉毒素（AFB1、AFB2、AFG1和AFG2）、赭曲霉毒素A（OTA）和伏马毒素（FB1和FB2）。结果 重庆地区辣椒、花椒和八角中的真菌毒素总检出率分别为60.0%、100%、96.7%。AFs和OTA是三种香辛料最主要的污染毒素,其中AFG2是最主要的黄曲霉代谢产物。盘溪市场的AFs污染总体情况稍好,菜园坝市场AFs污染最严重;江津市场的OTA污染最为严重。结论 香辛料中存在真菌毒素污染,有必要建立香辛料中真菌毒素的限量标准。     

Development of a method for direct visual determination of aflatoxin production by colonies of the Aspergillus flavus group

This report describes a simple, rapid and reliable method for screening the aflatoxin production by moulds of the Aspergillus flavus group. Strains were cultivated on yeast extract agar to which methylated beta-cyclodextrin derivative plus sodium desoxycholate was added. Production of aflatoxins was readily detectable by direct visualisation of a beige ring surrounding colonies after an incubation time of 3 days at 28 degrees C. When this ring was examined under UV light, it exhibited blue fluorescence. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by HPLC with fluorescence detection.     

Assessment for fungal,mycotoxin and insect spoilage in maize stored for human consumption in Zambia

BACKGROUND: Maize constitutes the main staple food and most important crop grown in Zambia. However, maize incurs considerable losses both in field and storage due to pathogens and insects. Some of the pathogens and resultant mycotoxins reduce the nutritional quality of the product. Mycotoxins are toxigenic fungal compounds that can cause cancer and suppress growth. In spite of this health hazard, there has been very little research to document their occurrence. Maize grains stored for human consumption were sampled from different agro‐ecosystems (forest, valley and plateau areas) of three agroecological zones (high, mid and low altitude). RESULTS: Several fungal genera were recovered among which Aspergillus flavus, A. niger, Fusarium verticillioides, F. solani, Rhizopus stolonifer and Penicillium spp. were prevalent. The weevil Sitophilus zeamais and the larger grain borer Prostephanus truncatus were the most damaging. Enzyme‐linked immunosorbent assay (ELISA) tests yielded fumonisins and aflatoxins ranging between 0.02 and 21.44 ppm, and 0.7 and 108.39 ppb in 96.4% and 21.4% of samples, respectively. Fumonisin was more pronounced in villages in forest areas whereas aflatoxin was highest in valley and forest areas in Zone II. CONCLUSION: Strategic interventions to curtail fungal, mycotoxin and insect contamination should be directed towards improved agronomic and post‐harvest practices of maize from fields to consumers. Copyright © 2009 Society of Chemical Industry