Fluorescent competitive aptasensor for detection of aflatoxin B1.
Aflatoxin B1 (AFB1) is one of the most commonly found mycotoxins in food commodities, particularly cereals, oilseeds, spices and tree nuts. In the past decade, aptamers have come into limelight and emerged as a new biosensing element replacing antibodies in various detection formats. Herein we report a faster, more sensitive, high throughput method for the detection of AFB1 using AFB1 -specific aptamers. The assay format was based on a competitive reaction of the fluorescent tagged aptamer specific to AFB1 with the aflatoxin conjugate. Under optimal conditions, a linear range of detection (50 ng to 50 pg) was achieved with a limit of detection (LOD) of 10 pg/mL in the buffer system. Results of inter- and intra-assay revealed that the assay was repeatable with standard deviation in acceptable range. The assay was also validated in food samples such as dried red chilies, groundnut and whole pepper with recovery in the range of 92 to 102% at 10 ng/mL and 100 pg/mL levels. The aptasensor assay was also compared with standard analytical method of HPLC and was found to be more sensitive. This detection technique has the potential to be developed into a biosensor platform for AFB1 detection.
Investigation of Pseudomonas fluorescens strain 3JW1 on preventing and reducing aflatoxin contaminations in peanuts.
Pseudomonas fluorescens strain 3JW1, which has a broad-spectrum antimicrobial activity, was studied to investigate whether it affects the amounts of aflatoxin B1 (AFB1) produced by Aspergillus flavus. It was found that the bacterium reduced the amounts of AFB1 in potato dextrose broth (PDB) and peanut medium by 97.8% and 99.4%, respectively. It also reduced AFB1 by ~183 μg/kg (55.8%) when applied onto peanut kernels. This strain reduced AFB1 via three mechanisms. First, it significantly inhibited A. flavus growth; second, our data showed that strain 3JW1 inhibits aflatoxin biosynthesis by A. flavus; and third, P. fluorescens strain 3JW1 is capable of degrading AFB1 at a rate as high as 88.3% in 96 hours. This is the first report demonstrating that Pseudomonas fluorescens can reduce toxin contamination caused by A. flavus on peanut kernels. Our findings indicate that P. fluorescens strain 3JW1 had multiple effects including reducing A. flavus infection and aflatoxin contamination. And the results also highlight the potential applications of the strain 3JW1 for the biological control of aflatoxin contamination in peanuts and other susceptible crops.
An on-site, ultra-sensitive, quantitative sensing method for the determination of total aflatoxin in peanut and rice based on quantum dot nanobeads strip.
An on-site, ultra-sensitive, and quantitative sensing method was developed based on quantum dot nanobeads (QDNBs) and a test strip for the determination of total aflatoxins (AFTs) in rice and peanuts. The monoclonal antibody against AFT (mAbAFT) was homemade and labeled with QDNB. After the pre-coating of the AFT antigen on the test line (T line), the competitive immunoreactions were conducted between AFT and AFT antigen on the T line with QDNBs-mAbAFT. Under optimal conditions, this approach allowed a rapid response towards AFT with a considerable sensitivity of 1.4 pg/mL and 2.9 pg/mL in rice and peanut matrices, respectively. The put-in and put-out durations were within 10 min. The recoveries for AFT in rice and peanut sample matrices were recorded from 86.25% to 118.0%, with relative deviations (RSD) below 12%. The assay was further validated via the comparison between this QDNB strip and the conventional HPLC method using spiked samples. Thus, the design provided a potential alternative for on-site, ultra-sensitive, and quantitative sensing of AFT that could also be expanded to other chemical contaminants for food safety.
Rapid Characterization of Trace Aflatoxin B1 on Groundnuts, wheat and maize by Dispersive Liquid‐Liquid Microextraction Followed by Direct Electrospray Probe Tandem Mass Spectrometry.
RATIONALE: Aflatoxins are poisonous and cancer-related chemical compounds commonly existed in crops and plants. Aflatoxin B1 is the most toxic compound among aflatoxins and has been classified as Group 1 carcinogenic to human, especially on liver cancer. Herein, an ambient mass spectrometric method was developed for rapid characterization of trace aflatoxin B1 on peanuts. METHODS: Direct electrospray probe tandem mass spectrometry was used to detect aflatoxin B1 on peanuts. To avoid the matrix effect, the aflatoxin B1 in the samples was extracted and concentrated by dispersive liquid-liquid microextraction. The mass spectrometer was operated in the positive ion mode to monitor intact molecular ion (m/z 313, MH+ ) and daughter ion (m/z 241) of aflatoxin B1 using multiple reaction monitoring. RESULTS: Since no clean up procedure of the sample was required, the sampling step, and the subsequent mass spectrometric detection of the aflatoxin B1 was completed less than 5 minutes. The limit-of-detection of aflatoxin B1 is at sub-ppb level. The results obtained by direct electrospray probe tandem mass spectrometry were also validated by liquid chromatography-tandem mass spectrometry. Recovery of aflatoxin B1 in the sample was evaluated by analyzing spiked aflatoxin B1 with liquid chromatography-tandem mass spectrometry to be 85% and direct electrospray probe tandem mass spectrometry to be 84%. CONCLUSION: The direct electrospray probe tandem mass spectrometry combined with a simple dispersive liquid-liquid microextraction procedure was successfully used for the quantitative analysis of AFB1 in nut samples. Due to its highly efficient feature, it is promising in providing important toxicological information for food safety in real world.
Static Hot Air and Infrared Rays Roasting are Efficient Methods for Aflatoxin Decontamination on Hazelnuts.
Aflatoxins are a group of secondary metabolites produced by members of Aspergillus Section Flavi that are dangerous to humans and animals. Nuts can be potentially contaminated with aflatoxins, often over the legal threshold. Food processes, including roasting, may have different effects on mycotoxins, and high temperatures have proven to be very effective in the reduction of mycotoxins. In this work, two different roasting methods-traditional static hot air roasting and infra-red rays roasting-were applied and compared for the detoxification of hazelnuts from Italy and Turkey. At the temperature of 140 °C for 40 min of exposure, detoxification was effective for both roasting techniques. Residual aflatoxins after infra-red rays treatments were lower compared to static hot air roasting. On Italian hazelnuts, residual aflatoxins were lower than 5%, while for Turkish hazelnuts they were lower than 15% after 40 min of exposure to an infra-red rays roaster. After roasting, the perisperm was detached from the nuts and analyzed for aflatoxin contents. Residual aflatoxins in the perisperm ranged from 80% up to 100%. After roasting, the lipid profile and the nutritional quality of hazelnuts were not affected. Fatty acid methyl esters analyses showed a similar composition for Italian and Turkish hazelnuts.
Simultaneous determination of six mycotoxins in peanut by High-performance Liquid Chromatography with Fluorescence detector.
BACKGROUND: Mycotoxins, which may contaminate peanut and peanut products, are responsible for many diseases to human beings. AflatoxinB1(AFB1), aflatoxinG1(AFG1), aflatoxinB2(AFB2), aflatoxinG2(AFG2), ochratoxin A(OTA) and zearalenone(ZEN) are considered the most relevant groups of mycotoxins found in food. This work aimed to develop a high-performance liquid chromatography with fluorescence detector(HPLC-FLD) combined with dispersive liquid-liquid microextraction(DLLME) method for the simultaneous determination of the six mycotoxins in peanuts. The six mycotoxins were simultaneously determined under their best wavelength by means of changing wavelength. RESULTS: Under the optimum conditions, the linear ranges were 1 ~ 100 ng mL^-1 for AFB1, AFG1 and OTA, 0.3 ~ 30 ng mL^-1 for AFB2 and AFG2, 5 ~ 1000 ng mL^-1 for ZEN, with the correlation coefficient(R2) of 0.9969 ~ 0.9997. Limits of detection(LODs) were 0.10, 0.10, 0.30, 0.03, 0.03 and 1.0 µg kg^-1, respectively, and the mean recoveries were in the range of 83.1% to 99.3% with RSD<10%(n=6, independent analysis).Thirteen(46%) of these tested samples were contaminated with at least one mycotoxin. CONCLUSION: The proposed method was demonstrated to be simple, highly selective, accurate, reliable, and was successfully applied to simultaneously analyze of the six mycotoxins in real peanut samples from China.
Effects of different drying treatments on fungal population and ochratoxin A occurrence in sultana type grapes.
This study aimed to determine the changes in mould and ochratoxin A (OTA) occurrence in sultanas under three different conventional drying conditions. Five different vineyards were chosen, and the three different treatments were applied to these grapes while drying. At the end of the drying process, total mould and black aspergilli (BA) populations in the samples varied from 2.45 to 5.61 log colony-forming units (CFU) g-1 and from 0 to 4.92 log CFU g-1, respectively. Significant increases (p<0.05) occurred in mould loads depending on the extending drying period. However, independent of vineyard location, all of the samples treated with cold dipping solution showed the lowest fungal loads. These results indicate that dipping solution treatment was the most effective drying method to minimize fungal infection of grapes. The expected results could not be achieved by drying grapes artificially contaminated with ochratoxigenic Aspergillus carbonarius spores. Seventy-one of 96 isolates (73.95%) obtained during drying were Aspergillus spp., and the remaining (n=25, 26.05%) belonged to other genera, such as Penicillium, Trichoderma and Cladosporium. Grape juice-based agar medium was used to determine the realistic OTA production capacities of the isolated mould strains. The highest OTA production capacities were 809.70±9.19, 87.58±16.89 and 45.44±18.78 ng/g in 50% grape juice agar (GJ50), all 5 of which were from A. niger isolates. OTA was not present in any sample during the drying period; however, OTA was detected in two samples at 0.32±0.15 and 0.52±0.36 µg kg-1 after the end of the drying process. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the method used for detecting OTA in samples were 0.1 and 0.3 µg kg-1, respectively.
Aflatoxins in hazelnuts and dried figs: Occurrence and exposure assessment.
A total of 300 samples of hazelnuts and dried fig were analysed for the incidence of any aflatoxins (AFs). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used to quantify the amounts of AFs. The limit of quantification varied from 0.21 to 0.30μgkg(-1). No AFs were detected in shells of the hazelnuts, while six raw hazelnut kernel samples (12%) and five roasted hazelnut kernel samples (8.3%) contained AFs ranging from 0.09 to 11.3μgkg(-1) and from 0.17 to 11.2μgkg(-1), respectively. Sixteen dried fig samples (12.3%) contained AFs ranging from 0.1 to 28.2μgkg(-1) and a mean value of 3.8μgkg(-1). Three hazelnuts and six dried fig samples exceeded the European maximum limits (MLs) of 5 and 2μgkg(-1) for aflatoxin B1 (AFB1), respectively. The contribution of hazelnuts to AFs exposure is higher than that of dried figs.
Non-aflatoxigenic Aspergillus flavus as potential biocontrol agents to reduce aflatoxin contamination in peanuts harvested in Northern Argentina.
Biological control is one of the most promising strategies for preventing aflatoxin contamination in peanuts at field stage. A population of 46 native Aspergillus flavus nonaflatoxin producers were analysed based on phenotypic, physiological and genetic characteristics. Thirty-three isolates were characterized as L strain morphotype, 3 isolates as S strain morphotype, and 10 isolates did not produce sclerotia. Only 11 of 46 non-aflatoxigenic isolates did not produce cyclopiazonic acid. The vegetative compatibility group (VCG) diversity index for the population was 0.37. For field trials we selected the non-aflatoxigenic A. flavus AR27, AR100G and AFCHG2 strains. The efficacy of single and mixed inocula as potential biocontrol agents in Northern Argentina was evaluated through a 2-year study (2014-2015). During the 2014 peanut growing season, most of the treatments reduced the incidence of aflatoxigenic strains in both soil and peanut kernel samples, and no aflatoxin was detected in kernels. During the 2015 growing season, there was a reduction of aflatoxigenic strains in kernel samples from the plots treated with the potential biocontrol agents. Reductions of aflatoxin contamination between 78.36% and 89.55% were observed in treated plots in comparison with the un-inoculated control plots. This study provides the first data on aflatoxin biocontrol based on competitive exclusion in the peanut growing region of Northern Argentina, and proposes bioproducts with potential use as biocontrol agents.
Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins.
Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N₂, 0.1% O₂ and 1% O₂, 21% O₂), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB₁ of over 70% was obtained. Aflatoxins B₁ and G₁ were more sensitive to plasma treatments compared to aflatoxins B₂ and G₂, respectively. Under plasma treatment, aflatoxin B₁ was more sensitive compared to aflatoxin G₁. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.