Incidence and consumer awareness of toxigenic Aspergillus section Flavi and aflatoxin B1 in peanut cake from Nigeria

Peanut cake samples were collected from major markets in five states of Nigeria and evaluated for incidence of toxigenic Aspergillus section Flavi populations, and aflatoxin B₁ (AFB₁) levels by liquid chromatography tandem mass spectrometry (LC–MS/MS). The awareness of consumers to the presence of aflatoxin in the snack and potential health risks of its regular ingestion was evaluated by questionnaire analysis. Aspergillus section Flavi populations were recovered from 83% of the peanut cake samples. Aspergillus flavus L-strain was the most predominant (>56%) across the states while Aspergillus tamarii had the least mean incidence (2.7%). The incidence of atoxigenic strains was significantly (p < 0.05) higher than that of toxigenic strains in samples from Lagos and Kaduna, while the toxigenic strains had a significantly (p < 0.05) higher incidence than the atoxigenic strains in Niger. All analyzed cake samples contained AFB₁ in concentrations exceeding the NAFDAC recommended level for AFB₁ in food and reaching up to 2824 μg/kg. There was a weak positive correlation (r = 0.32, p = 0.03) for the relationship between the incidence of toxigenic strains in the samples and AFB₁ concentration. The consumer awareness data showed that 64% of the respondents consumed peanut cake; majority of who are youth of economic and reproductive age. Eighty-five percent of the consumers lacked awareness of aflatoxin contamination in the snack and possible health risks associated with its ingestion.     

Occurrence of toxigenic fungi and ochratoxin A in Ethiopian coffee for local consumption

Information on the presence of ochratoxin A (OTA) in local Ethiopian coffee is scarce. Therefore, the objective of this study was to type the fungal and mycotoxin contamination levels in Ethiopian coffee consumed by the local community. Coffee samples were collected from six major coffee growing districts in Jimma zone of Oromiya regional state in Ethiopia. Total fungal incidence mounted up to 87% and Aspergillus (79%), Fusarium (8%) and Penicillium (5%) were the predominant toxigenic genera. OTA producing species A. westerdijkiae, Aspergillus ochraceus, and Aspergillus steynii were identified for the first time in Ethiopia and were shown to be the predominant species while aflatoxin (AF) producing Aspergillus species were scarce. Using an in vitro approach, the OTA production potential was assessed under standardized conditions. Based on this experiment, it was concluded that Aspergillus westerdijkiae isolates were clearly the most potent producers of OTA. The median OTA level in the locally sold Ethiopian coffee was 1.53 μg/kg. Notwithstanding this low median value, significant differences in fungal and toxin incidences were observed between the different coffee processing types, coffee sample types, and storage characteristics.     

Revision of ochratoxin a production capacity by the main species of Aspergillus section Circumdati. Aspergillus steynii revealed as the main risk of OTA contamination

Ochratoxin A (OTA) is a highly toxigenic mycotoxin commonly present in a number of diverse agroproducts. Aspergillus Section Circumdati includes some of the most important OTA-producing species: Aspergillus ochraceus, Aspergillus elegans and the recently described Aspergillus steynii and Aspergillus westerdijkiae. In this work, OTA production by several strains of these four species from diverse origins and food matrices was examined. Identification of all the strains was carried out by specific PCR assays. The strains were cultivated in CYA solid medium and OTA was measured by HLPC. This study demonstrated for the first time the high capacity of A. steynii strains to produce OTA at higher levels than A. westerdijkiae,A. ochraceus and A. elegans strains showed low levels or no production at all. In conclusion, the results of this study suggest that the occurrence of A. steynii and A. westerdijkiae might represent the major potential risk for OTA contamination due to their high production and the diversity of commodities that these species may contaminate.     

Isolation and identification of toxigenic fungi from infected peanuts and efficacy of silver nanoparticles against them

Peanuts are vulnerable to fungal infections during long term storage. Fungi infecting peanuts are toxigenic and cause health hazards. The goal of this study was isolation and identification of fungi in peanuts and use of silver nanoparticles (AgNPs) to inhibit them. For this purpose firstly, we have isolated fungi from infected peanuts and identified on the basis of morphological and molecular study. Out of the total 54 fungal isolates, 47 were found to be Aspergillus spp. and other belongs to Penicillium spp. and Macrophomina phaseolina. Biochemical assay was performed to identify cultures of Aspergillus flavus from other species of this genus by inoculating it on Aspergillus Differentiation Medium (ADM). Thirty-one isolates were found to be A. flavus. Toxicity of Aspergillus spp. was evaluated on Yeast Extract Sucrose agar (YES) medium with an additive methylated β-cyclodextrin and nine isolates were found toxigenic. Secondly, AgNPs were synthesized from ten different plants and their characterization was performed using various analytical techniques such as UV–Visible spectrophotometer, Fourier Transform Infrared Spectroscopy, Zetasizer and Nanoparticle Tracking Analysis, etc. Further, antifungal potential of thus synthesized AgNPs was evaluated. All the synthesized AgNPs possess ability to inhibit fungal growth. Cymbopogon citratus leaf extract mediated AgNPs were found to have prominent antifungal potential against all test fungi and its minimum inhibitory concentration (MIC) was found to be 20 μg/ml. The biogenic approach proposed in the present study is eco-friendly, safe and economical viable. AgNPs also reported to have significant antifungal activity against toxigenic isolates of peanuts, hence such AgNPs can be effectively used for the management of toxigenic pathogens.     

Natural co-occurrence of aflatoxins and ochratoxin A in spices

The present study aims to determine the co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in spices marketed in Turkey. During the period from November 2010 to August 2011, 105 samples of spices were checked for targeted mycotoxins. The mycotoxins were determined by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) after immunoaffinity column (IAC) clean-up. The method was validated for selectivity, sensitivity, accuracy and repeatability as relative standard deviation (RSD). Good recoveries (80.7–95.7%) were obtained for mycotoxins in each food matrix, with RSD values lower than 15%.Aflatoxins were detected in 79.2% of red chilli flake, 63.6% of red chilli powder, 30.4% of black pepper powder and 21.1% of cumin samples, while none of the cinnamon powder samples contained AFs at detectable levels. Four red chilli flake and three red chilli powder samples were above the EU regulatory limit of 5 μg kg⁻¹ for AFB₁. OTA was found in 75% of red chilli flake, 54.5% of red chilli powder, 17.4% of black pepper powder, with four red chilli flake and three red chilli powder samples exceeding EU limit of 30 μg kg⁻¹. No OTA was found in cinnamon powder samples, while detectable levels of OTA were found in only one cumin sample. The co-occurrence of AFB₁ and OTA was detected in 62.5% of red chilli flake, 40.9% of red chilli powder and 4.3% of black pepper powder samples. This study reports for the first time the natural co-occurrence of AFs and OTA in spices from Turkey.     

Mycological quality and mycotoxin contamination of Sri Lankan peppers (Piper nigrum L.) and subsequent exposure assessment

The aim of the study was to characterize the toxigenic moulds and to screen different mycotoxins in peppers (Piper nigrum L.) of Sri Lankan origin. Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Penicillium spp. were found to be the most dominant fungi. Characterization of the moulds was carried out in A. flavus and parasiticus agar (AFPA) and malt extract agar (MEA) in 77 black pepper (BP) and 11 white pepper (WP) samples. In total, 73% of the BP and 64% of the WP samples were contaminated with A. flavus and/or A. parasiticus (AfAp). A BP sample with water activity (a_w) 0.70 recorded the highest count of AfAp (4.3*10⁴ CFU/g). Moreover, 75% of the BP samples exceeded the safe a_w limit (0.65) set by the European Spice Association (ESA). The frequency of occurrence of A. niger in BP was 62% with counts up to 1.3*10³ CFU/g. Penicillium spp. were found in 61% and 55% of the BP and WP samples, respectively. In BP 94% of the samples had a Penicillium contamination below 10³ CFU/g. Other Aspergillus spp, found in peppers included, Aspergillus terrus, Aspergillus tamarii, Aspergillus candidus, Aspergillus penicilloides, Aspergillus sydowii and Aspergillus fumigatus. Mould counts in BP (10²–10⁴ CFU/g) were significantly higher than that of WP (<10² CFU/g). Apart from the occurrence of “classical mycotoxins” of spices, aflatoxins (<LOQ-18 μg/kg) and ochratoxin A (<LOQ-79 μg/kg), other toxins including fumonisin B1 (<LOQ-135 μg/kg), sterigmatocystin (<LOQ-49 μg/kg) and citrinin (<LOQ-112 μg/kg) were detected in peppers. In total, 63% of the BP samples were contaminated with at least one mycotoxin. Mycotoxin contamination in WP was significantly less compared to BP. The exposure to aflatoxins and ochratoxin A by consuming pepper remains harmless considering the existing pepper dietary intake data of the Sri Lankan population.     

Ochratoxin A in coffee beans (Coffea arabica L.) processed by dry and wet methods

The incidence of ochratoxin A was studied in different coffee (Coffea arabica L.) samples. A higher incidence of filamentous fungi was observed in the coffee swept from ground and floating coffee samples. The species Aspergillus ochraceus, Aspergillus sulphureus and Aspergillus sclerotiorum were ochratoxin A producing. In 128 (44%) samples ochratoxin A was not detected; however, in 89 samples (31%), ochratoxin A was detected at 0.1–5.0 μg/Kg levels. Other 25% samples presented contamination above 5.0 μg/Kg. This study showed that the fractions coffee swept from ground and floating coffee represents a serious risk of ochratoxin A contamination.     

Inhibition of non-toxigenic Aspergillus niger FS10 isolated from Chinese fermented soybean on growth and aflatoxin B1 production by Aspergillus flavus

A non-toxigenic strain FS10 was isolated from Chinese fermented soybean to control AFB₁ contamination and conformed as Aspergillus niger by 18S rDNA. To elucidate the biocontrol mechanism, the ability of culture filtrate of strain FS10 against growth and AFB₁ production by toxigenic Aspergillus flavus was evaluated in vitro. Meanwhile, the influence of filtrate on spore morphology of A. flavus was analyzed by optical microscope and atomic force microscope (AFM). Results demonstrated that the culture filtrate of FS10 significantly suppressed A. flavus growth, sporulation and AFB₁ biosynthesis. After 9 days, the inhibition of mycelia growth, sporulation and AFB₁ production reached to 37.7%, 100% and 94.5%, respectively. In addition, optical microscopy and AFM showed that the culture filtrate serious damaged spore morphology, accompanying with spore analysis and cell wall collapse, resulted in inhibition of spore germination. The degradation activity of FS10 culture filtrate on AFB₁ was also estimated. When AFB₁ was incubated with the culture filtrate, 85.8% of AFB₁ could be degraded after 5 days. It is concluded that the culture filtrate of strain FS10 has pleiotropic effects on control AFB₁ contamination. As a safe, effective and economic biological agent, the filtrate could be applied in fermented food to prevent toxigenic Aspergillus spp. and AFB₁ contamination.     

Aflatoxins contamination in chilli samples from Pakistan

The aim of this study was to prioritise disease and pest constraints in chilli by highlighting aflatoxin concentrations to assist local farmers in control. All samples contained aflatoxin B₁ and high levels were obtained from all ground samples. A direct relationship was observed between aflatoxin B₁ and aflatoxin B₂ concentrations. There was no relation between aflatoxin and Aspergillus flavus detection. Chilli production in Pakistan may be heavily constrained by aflatoxin contamination. Simply removing A. flavus may be insufficient for control. Aflatoxins from chilli may be a threat to the health of populations and a constraint on development in Pakistan.     

Investigation of regional differences of the dominant microflora of spice paprika by molecular methods

As microbial contamination of spices comes primarily from the soil, and soil microbiota are dependent on climate, geography, agriculture, etc a qualitative focus on the dominant microflora of spices instead of the quantitative determination of commonly studied spoilage and pathogenic microflora may give a better insight into the geological origin of the sample. The aim of the present study was to identify using molecular microbiological methods the dominant bacteria of spice paprika produced at different countries and to look for species characteristic of spices from the given regions. According to our data, no differentiation could be made among spice paprika samples of different geographical origin using the total bacterial count or extent of mould contamination. However, when the dominant microflora is examined, bacterial species could be identified in the spice paprika samples characteristic of a particular climate. According to our study, the presence of Bacillus mycoides and Bacillus licheniformis was characteristic of the microflora of spice paprika grown in Central Europe; Bacillus safensis could be detected in all four paprika samples examined from the tropical monsoon climate; the species common to all three samples of the tropical climate group were Bacillus amyloliquefaciens subsp. plantarum and subsp. amyloliquefaciens, while Bacillus mojavensis was detected as being characteristic of Spanish origin for paprika. No common species were found in the paprika samples originating from China.     

Natural contamination of cyclopiazonic acid in dried figs and co-occurrence of aflatoxin

Natural cyclopiazonic acid (CPA) and aflatoxin (AF) contamination were investigated in Aspergillus section Flavi positive dried fig samples collected from orchards after harvesting. Cyclopiazonic acid and AF were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), respectively. Cyclopiazonic acid was found in all samples of dried figs that contain AF. Cyclopiazonic acid and AF co-occurred in 23% of the samples. The number of dried fig samples contaminated with CPA was higher than the samples containing AF. Therefore CPA was a more common mycotoxin than AF in dried figs. To the best of our knowledge, this is the first report of CPA and co-occurrence of CPA and AF in dried figs.     

Aflatoxin-producing Aspergillus spp. and aflatoxin levels in stored cassava chips as affected by processing practices

Cassava chips (cassava balls, and cassava pellets) are derived cassava products traditionally produced by farmers in sub-Saharan Africa following fermentation, and drying of fresh roots of cassava, and are widely consumed in Cameroon. Once produced, this food commodity can be stored for more than two months and contaminated by a wide array of harmful microbes. In order to assess persistence of toxigenic fungi in cassava chips, aflatoxin-producing fungi (Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus) and aflatoxins were contrasted at regular intervals in home-stored cassava chips collected in two locations of southern Cameroon throughout a two-month monitoring period. Three hundred and forty-six isolates of aflatoxin-producing fungi were found to be associated with all samples. A. flavus contaminated more samples in both types of chips (267 isolates in 53 samples), followed by A. nomius (58 isolates in 15 samples), whereas A. parasiticus was rarest. A direct competitive Enzyme-linked immunosorbent assay (ELISA)-based method was implemented to quantify the content in aflatoxins. Eighteen of the samples contained some aflatoxins at detectable levels whereas 54 did not. The levels of aflatoxin ranged between 5.2 and 14.5 ppb. The distribution of aflatoxin in positive samples depended on 8 parameters including pH, moisture content, storage duration, types of chips, level of contamination by aflatoxin-producing fungi, processing practices and storage facilities. From analysis of variance results, only pH (p < 0.01), duration of storage (p < 0.01), population of aflatoxin-producing species (0.0001) and the chip type (p < 0.05) were significantly related to aflatoxin in positive samples. A stepwise regression analysis (forward selection procedure) indicated that aflatoxin levels were significantly (p < 0.01) correlated with processing practices, storage facilities, and storage duration of the chips.     

Fungal and aflatoxin contamination of marketed spices

Fourteen spice samples were collected from local markets in Doha, Qatar, during 2012, and were surveyed for the presence of potentially harmful mycoflora and for contamination with aflatoxins B₁, B₂, G₁, and G₂ by high-performance liquid chromatography (HPLC). Among the tested spice samples, chili powder showed the highest presence of fungal propagules, while ginger, curry and garlic samples did not present any fungal contamination. A total of 120 isolates, mostly belonging to Aspergillus and Penicillium genera, were collected and 33 representative species were identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Aspergillus flavus, Aspergillus nomius and Aspergillus niger were the most dominant. Thirty-seven Aspergillus strains were screened for their potential to produce aflatoxins using biochemical and molecular tools: only 9 A. flavus strains showed both fluorescence and amplification with all the three primers targeting aflP, aflM and aflR genes. Aflatoxins were detected in five spices (black pepper, chili, tandoori masala. turmeric and garam masala), and with the exception of garam masala, the tested samples of turmeric, black pepper, tandoori masala and chili powder exceeded B₁ and/or total aflatoxin maximum levels. Our results demonstrate the potential for mycotoxin biosynthesis by fungi contaminating imported spice products.     

Microorganisms associated with the spoilage of Pupuru

This study involved evaluation of physicochemical and microbiological changes in dried Pupuru (fermented, smoke dried cassava) balls under storage conditions simulating those currently used by the traditional processors. The aim was to understand the process of spoilage with a view to reducing the rate. pH ranged from 3 to 4, reducing significantly in cabinet-dried samples from 4.24 to 3.27 after 6 days of storage. Viable counts were in the range of 6–8 log cfu/g. Spoilage microorganisms included aerobic spore-forming and non-sporing bacteria as well as potentially toxigenic moulds like Aspergillus flavus and Penicillium species, which could constitute a health hazard to consumers.     

Natural mycotoxin contamination of maize (Zea mays L.) in the South region of Brazil

The natural co-occurrence of fungal metabolites in maize samples from the South region of Brazil was studied using an LC-MS/MS based multi-mycotoxin method. All maize samples (n = 148) were contaminated with fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). Aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) were detected in 38 and 11 samples, respectively, while zearalenone (ZEN) and deoxynivalenol (DON), which were first regulated in 2014, were found in 110 and 71 samples, respectively. Apart from regulated mycotoxins, a broad range of non-regulated metabolites, from Aspergillus, Fusarium, Alternaria, Penicillium and other microbes, were also detected in maize sample. Fusarin C, a possible carcinogenic compound to humans, produced by Fusarium species and not addressed by Brazilian legislation, was detected in 54.2% of maize samples. All analysed maize samples were found to be contaminated by at least ten different metabolites, with the largest number of metabolites found in the same sample being 51.     

Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas

This paper summarizes the results of a large study on the occurrence of ochratoxigenic fungi and Ochratoxin A from wine and table grapes in Tunisia. Our results revealed that Aspergillus section Nigri were the unique potential OTA producing fungi isolated from grapes. Isolates belonging to Aspergillus niger aggregate were the most abundant species followed by Aspergillus carbonarius isolates, then uniseriate aspergilli. A. carbonarius presented the highest percentage of OTA-positive strains (97%) whereas only 3% of A. niger aggregate isolates were OTA positive. Grapes were analysed for their OTA content and 58% of them contained detectable levels of OTA, between 0.05 and 5.85 μg/l. Only 4 samples out of 39 exceeded the OTA limit of 2 μg/l fixed by the EU for wine and grape juices. The most contaminated grapes were those from Raf-Raf region located in the North-Est and characterized by a humid climate. Grapes from the Regueb region, characterized by an arid climate, were rarely contaminated. Furthermore, A. carbonarius, which is the main OTA producer fungi on grapes, was rarely isolated in Regueb.     

Aflatoxin contamination of dried red chilies: Contrasts between the United States and Nigeria,two markets differing in regulation enforcement

Dried red chilies are among the world’s most consumed spices. From farm to fork, chilies go through cropping, harvest, drying, processing and storage. Chilies are susceptible to infection by aflatoxin producing fungi and subsequent contamination by aflatoxins at every stage. Aflatoxins are highly regulated, hepatotoxic carcinogens produced by fungi in Aspergillus section Flavi. The current study examined prevalence of aflatoxin B₁ (AFB₁) in chilies from markets across the United States (US) and Nigeria, and determined predisposition of chilies to aflatoxins post-harvest. Aflatoxin B₁ was detected in 64% chilies from US markets (n = 169), and 93% of Nigerian chilies (n = 55) with a commercial lateral flow assay (Limit of Detection = 2 μg/kg). Two percent of US samples exceeded the aflatoxin regulatory limit of 20 μg/kg, while the highest concentration detected was 94.9 μg/kg. Aspergillus spp. could be recovered only from 40% of samples from the US, and aflatoxin levels did not correlate with quantities of Aspergillus section Flavi (Colony Forming Units g⁻¹), suggesting fungi associated with chilies in US markets were killed during processing. Both average AFB₁ concentrations and fungal quantities were significantly higher (p < 0.01) in Nigerian chilies. The most contaminated sample contained 156 μg/kg AFB₁. Aflatoxin concentrations in Nigerian chilies increased as an exponential function of the quantities of Aspergillus section Flavi (r² = 0.76). Results indicate that high rates of chili consumption may be associated with unacceptable aflatoxin exposure.     

Influence of cold plasma on fungal growth and aflatoxins production on groundnuts

In this study, influence of cold plasma on the Aspergillus parasiticus and Aspergillus flavus growth and aflatoxins productions have been investigated as an alternative to chemical free and thermal treatments. Artificially inoculated fungal species onto the groundnuts were treated with air plasma at 40 W and 60 W power levels at various time intervals. After the plasma treatment, inactivation of A. parasiticus and A. flavus was examined and the concentration of aflatoxins produced was analyzed using high performance liquid chromatography. There is 97.9% and 99.3% reduction in the growth of A. parasiticus and A. flavus respectively, when treated at 60 W powers. Electron microscopy was also used to study the effect of cold plasma on the cellular membranes of the spores. Results showed complete disintegration of fungal spore membrane due to electroporation and etching caused by the reactive species of plasma. In 40 W 15 min and 60 W 12 min plasma treated samples more than 70% and 90% reduction in aflatoxin B1 content was observed. These results suggest that cold plasma may be considered as alternative methods for disinfestation of foods due to its strong potential for microbial inactivation.     

Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco

Sixty samples of cereals (20 of corn, 20 of barley, and 20 of wheat) and 55 samples of spices (14 of paprika, 12 of ginger, 14 of cumin, and 15 of pepper) purchased from popular markets of Rabat and Salé in Morocco were analyzed for mycotoxins.Cereals samples were all analyzed for ochratoxin A (OTA). The average levels of contamination were 1.08, 0.42, and 0.17 μg/kg for corn, wheat, and barley, respectively. Samples of corn were also analyzed for zearalenone (ZEA) and fumonisin B₁ (FB₁) the average contaminations were 14 and 1930 μg/kg, respectively. Co-occurrence of OTA, FB₁, and ZEA was also checked. Spices samples were analyzed only for aflatoxins (AFs) and the average contaminations found for aflatoxin B₁ (AFB₁) were 0.09, 0.63, 2.88 and 0.03 μg/kg for black pepper, ginger, red paprika and cumin, respectively. The higher level of contamination was found in red paprika (9.68 μg/kg).The present report is the first one ever drafted on the natural co-occurrence of OTA, FB₁ and ZEA in cereals and on the occurrence of AFs in spices from Morocco.     

Aflatoxigenic fungi and aflatoxins occurrence in sultanas and dried figs commercialized in Brazil

The presence of aflatoxins, Aspergillus flavus and Aspergillus parasiticus in dried fruits was investigated. A total of 62 dried fruit samples were analyzed (24 black sultanas, 19 white sultanas and 19 dried figs). A total of 10 A. flavus isolates were found, nine in one white sultana sample (corresponding to 18% infection) and one isolate in dried figs (2%), and all of them were aflatoxin B₁ and B₂ producers. A. parasiticus was not found. Aflatoxins were detected in 3 of 19 (16%) white sultana samples analyzed and, the limits were not higher than 2.0 μg/kg. In dried figs 11 of 19 (58%) samples were contaminated with aflatoxins and, with exception of one sample that was contaminated with 1500 μg/kg of B₁ aflatoxin, the others had less than 2.0 μg/kg. Neither aflatoxigenic or aflatoxins contaminated black sultanas.