Aflatoxin contaminated degree detection by hyperspectral data using band index.
Aflatoxin is a highly toxic and carcinogenic substance with fluorescence characteristic. To explore the feasibility of detection the degree of aflatoxin contamination using hyperspectral imaging technology, we proposed a machine learning detection method based on support vector machine (SVM) combining band index and narrow band. First, five concentrations of aflatoxin solutions (10ug/L, 20ug/L, 50ug/L, 100ug/L and 10 mg/L) were prepared and dripped onto the surface of different peanut kernels. Next, hyperspectral images with 33 bands (400-720 nm) were acquired for each sample using a hyperspectral imaging system under 365 nm ultraviolet (UV) light. Then four fluorescence indexes including Radiation Index (RI), Difference Radiation Indexes (DRI), Ratio Radiation Index (RRI) and Normalized Difference Radiation Index (NDRI) were proposed. Finally, Fisher method was used to optimize and obtain a narrowband spectrum, and RBF-SVM model was used to recognize aflatoxin and make regression analysis on the degree of aflatoxin contamination. Experimental results showed that DRI index had the optimal performance, and the accuracy rate of 5-fold cross validation of SVM were 95.5% and the mean square error (MSE) and correlation coefficient R were respectively 0.0223 and 0.9785. Results of this paper are of positive significance for the online aflatoxin detection and grading of agricultural products.
Development of an Escherichia coli-based Electrochemical Biosensor for Mycotoxin Toxicity Detection.
Mycotoxin contamination in food and feed is a global concern because mycotoxin contamination can cause both acute and chronic health effects in humans and animals. In the present work, an Escherichia coli-based biosensor is described for the toxicity assessment of aflatoxin B1 (AFB1) and zearalenone (ZEN). In this electrochemical biosensor, E. coli is used as the signal recognition element, p-benzoquinone is used as the mediator, and a two-step reaction procedure has been developed to separate the mediator from the mycotoxins. The current value of the as-prepared microbial biosensor exhibits a linear decrease with concentrations of AFB1 and ZEN in the range of 0.01-0.3 and 0.05-0.5 μg/mL, with detection limits reaching 1 and 6 ng/mL, respectively. The IC25 values obtained by the present method are 0.25 and 0.40 μg/mL for AFB1 and ZEN, which shows that the cytotoxicity of AFB1 to E. coli is more severe than the cytotoxicity of ZEN to E. coli. The combined toxic effect of these two mycotoxins has also been explored, and synergistic biotoxicity has been observed. Moreover, the biosensor is successfully applied to the toxicity evaluation of mycotoxins in real samples, including peanut and corn oils. This work could provide new insight into mycotoxin and microorganism interactions and could establish a new approach for future mycotoxin detection.
Developing Gold Nanoparticles-Conjugated Aflatoxin B1 Antifungal Strips.
Lateral flow immunochromatographic assays are a powerful diagnostic tool for point-of-care tests, based on their simplicity, specificity, and sensitivity. In this study, a rapid and sensitive gold nanoparticle (AuNP) immunochromatographic strip is produced for detecting aflatoxin B1 (AFB1) in suspicious fungi-contaminated food samples. The 10 nm AuNPs were encompassed by bovine serum albumin (BSA) and AFB1 antibody. Thin-layer chromatography, gel electrophoresis and nuclear magnetic resonance spectroscopy were employed for analysing the chemical complexes. Various concentrations of AFB1 antigen (0-16 ng/mL) were tested with AFB1 antibody-BSA-AuNPs (conjugated AuNPs) and then analysed by scanning electron microscopy, ultraviolet-visible spectroscopy, and Zetasizer. The results showed that the AFB1 antibody was coupled to BSA by the N-hydroxysuccinimide ester method. The AuNPs application has the potential to contribute to AFB1 detection by monitoring a visible colour change from red to purple-blue, with a detection limit of 2 ng/mL in a 96-well plate. The lateral flow immunochromatographic strip tests are rapid, taking less than 10 min., and they have a detection capacity of 10 ng/g. The smartphone analysis of strips provided the results in 3 s, with a detection limit of 0.3 ng/g for AFB1 when the concentration was below 10 ng/g. Excellent agreement was found with AFB1 determination by high-performance liquid chromatography in the determination of AFB1 among 20 samples of peanuts, corn, rice, and bread.
Sensitivity programmable ratiometric electrochemical aptasensor based on signal engineering for the detection of aflatoxin B1 in peanut.
Accurately monitoring of aflatoxin B1 (AFB1), the most hazardous mycotoxin in agricultural products, is essential for the public health, but various testing demands (e.g. detection range, sensitivity) for different samples can be challenging for sensors. Here, we developed a sensitivity-programmable ratiometric electrochemical aptasensor for AFB1 analysis in peanut. Thionine functionalized reduced graphene oxide (THI-rGO) served as reference signal generator, ferrocene-labelled aptamer (Fc-apt) output the response signal. During analysis, the formation of Fc-apt-AFB1 complex led to its stripping from the electrode and faded the current intensity of Fc (IFc), while the current intensity of THI (ITHI) was enhanced. And ratiometric detection of AFB1 was achieved by using the current intensity ratio (ITHI/IFc) as quantitative signal. Compared with ratiometric strategies that highly rely on the labelled aptamers, the proposed strategy could regulate the value of ITHI/IFc by changing the modification of Fc-apt. And the detection sensitivity was found to be closely related to ITHI/IFc. Under the optimal conditions, the fabricated aptasensor with a dynamic range from 0.05-20 ng mL-1 and a detection limit of 0.016 ng mL-1 for AFB1 analysis. Besides, it exhibited excellent selectivity, reliability and reproducibility. The proposed sensitivity-programmable biosensor can be applied to detect various aptamer-recognized mycotoxins in agricultural sensing.
Exogenous β‐aminobutyric acid application attenuates Aspergillus decay, minimizes aflatoxin B1 accumulation and maintains nutritional quality in fresh in hull pistachio kernels
Background: Pistachio fruits suffer from postharvest decay, caused by Aspergillus flavus, resulted in aflatoxin B1 (AFB1) accumulation in kernels, which is hazardous for human health due to its carcinogenic activity. In this study, the mechanism recruited by exogenous β-aminobutyric acid (BABA) treatment for attenuating Aspergillus decay, minimizing aflatoxin B1 (AFB1) accumulation and maintaining nutritional quality in fresh in hull pistachio kernels, infected by A. flavus, during storage at 25 °C for 18 days was investigated. Result: Results of in vivo assay showed that the spore germination and germ tube elongation of A. flavus repressed by BABA treatment at 7.5 mM. Also, Aspergillus decay accompanied by AFB1 accumulation minimized in fresh in hull pistachio kernels treated with BABA at 7.5 mM and infected by A. flavus. Also, fresh in hull pistachio kernels, infected by A. flavus, treated with BABA at 7.5 mM exhibited higher phenols and flavonoids accumulation and DPPH scavenging capacity accompanied by higher PAL enzyme activity. Conclusion: Therefore, promoting phenylpropanoid pathway activity representing by higher PAL enzyme activity in fresh in hull pistachio kernels treated with BABA not only may be operative for attenuating kernels Aspergillus decay by cell wall fortifying but also may be favorable for maintaining kernels nutritional quality by ROS scavenging capacity. Also, since oxidative stress representing by ROS accumulation is responsible for A. flavus growth and AFB1 accumulation, higher phenols and flavonoids accumulation in fresh in hull pistachio kernels treated with BABA may be beneficial for attenuating Aspergillus decay and minimizing AFB1 accumulation.
Atoxigenic Aspergillus flavus Isolates Endemic to Almond, Fig, and Pistachio Orchards in California with Potential to Reduce Aflatoxin Contamination in these Crops.
In California, aflatoxin contamination of almond, fig, and pistachio has become a serious problem in recent years due to long periods of drought and probably other climatic changes. The atoxigenic biocontrol product Aspergillus flavus AF36 has been registered for use to limit aflatoxin contamination of pistachio since 2012 and for use in almond and fig since 2017. New biocontrol technologies employ multiple atoxigenic genotypes because those provide greater benefits than using a single genotype. Almond, fig, and pistachio industries would benefit from a multi-strain biocontrol technology for use in these three crops. Several A. flavus vegetative compatibility groups (VCGs) associated with almond, fig, and pistachio composed exclusively of atoxigenic isolates, including the VCG to which AF36 belongs to, YV36, were previously characterized in California. Here, we report additional VCGs associated with either two or all three crops. Representative isolates of 12 atoxigenic VCGs significantly (P < 0.001) reduced (>80%) aflatoxin accumulation in almond and pistachio when challenged with highly toxigenic isolates of A. flavus and A. parasiticus under laboratory conditions. Isolates of the evaluated VCGs, including AF36, constitute valuable endemic, well-adapted, and efficient germplasm to design a multi-crop, multi-strain biocontrol strategy for use in tree crops in California. Availability of such a strategy would favor long-term atoxigenic A. flavus communities across the affected areas of California, and this would result in securing domestic and export markets for the nut crop and fig farmer industries and, most importantly, health benefits to consumers.
Aflatoxins in food and feed: An overview on prevalence, detection and control strategies.
Aflatoxins produced by the Aspergillus species are highly toxic, carcinogenic, and cause severe contamination to food sources, leading to serious health consequences. Contaminations by aflatoxins have been reported in food and feed, such as groundnuts, millet, sesame seeds, maize, wheat, rice, fig, spices and cocoa due to fungal infection during pre- and post-harvest conditions. Besides these food products, commercial products like peanut butter, cooking oil and cosmetics have also been reported to be contaminated by aflatoxins. Even a low concentration of aflatoxins is hazardous for human and livestock. The identification and quantification of aflatoxins in food and feed is a major challenge to guarantee food safety. Therefore, developing feasible, sensitive and robust analytical methods is paramount for the identification and quantification of aflatoxins present in low concentrations in food and feed. There are various chromatographic and sensor-based methods used for the detection of aflatoxins. The current review provides insight into the sources of contamination, occurrence, detection techniques, and masked mycotoxin, in addition to management strategies of aflatoxins to ensure food safety and security.
On-spot surface enhanced Raman scattering detection of Aflatoxin B1 in peanut extracts using gold nanobipyramids evenly trapped into the AAO nanoholes.
Simple, rapid, convenient, and economical surface enhanced Raman scattering (SERS) substrate is developed for on-site evaluation of Aflatoxin B1 (AFB1) in food matrix using handheld Raman Spectrometer. Self-assembly of gold nanobipyramids (Au NBPs) into the nanoholes of anodic aluminum oxide (AAO) template/pattern using 'drop-dry' approach provides a reliable pathway for the rapid fabrication of highly active and uniform SERS substrate. It shows enhanced and reproducible SERS signals towards the probe molecule, 4-aminothiophenol (4-ATP) with a relative standard deviation (RSD) of less than 10% and an average enhancement factor (EF) of 1 × 108. For practical application, the proposed method is demonstrated for the detection of aflatoxin B1 (AFB1) in peanut extracts. The results show that the AFB1 in peanut extracts can be identified within 1 min, with a limit of detection of 0.5 μg/L. Compared with conventional ELISA based AFB1 analysis, our method is much more efficient (1 min versus >30 min).
Molecular Characterisation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus Section Flavi Isolated from Imported Peanuts along the Supply Chain in Malaysia.
Peanuts are widely consumed in many local dishes in Southeast Asian countries, especially in Malaysia which is one of the major peanut-importing countries in this region. Therefore, Aspergillus spp. and aflatoxin contamination in peanuts during storage are becoming major concerns due to the tropical weather in this region that favours the growth of aflatoxigenic fungi. The present study thus aimed to molecularly identify and characterise the Aspergillus section Flavi isolated from imported peanuts in Malaysia. The internal transcribed spacer (ITS) and β-tubulin sequences were used to confirm the species and determine the phylogenetic relationship among the isolates, while aflatoxin biosynthesis genes (aflR, aflP (omtA), aflD (nor-1), aflM (ver-1), and pksA) were targeted in a multiplex PCR to determine the toxigenic potential. A total of 76 and one isolates were confirmed as A. flavus and A. tamarii, respectively. The Maximum Likelihood (ML) phylogenetic tree resolved the species into two different clades in which all A. flavus (both aflatoxigenic and non-aflatoxigenic) were grouped in the same clade and A. tamarii was grouped in a different clade. The aflatoxin biosynthesis genes were detected in all aflatoxigenic A. flavus while the non-aflatoxigenic A. flavus failed to amplify at least one of the genes. The results indicated that both aflatoxigenic and non-aflatoxigenic A. flavus could survive in imported peanuts and, thus, appropriate storage conditions preferably with low temperature should be considered to avoid the re-emergence of aflatoxigenic A. flavus and the subsequent aflatoxin production in peanuts during storage.
Inhibitory Effect of Volatiles Emitted from Alcaligenes faecalis N1-4 on Aspergillus flavus and Aflatoxins in Storage.
Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 μL/L, respectively. The effective concentration for MI against A. flavus is 200 μL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.