HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts
Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.
Bridging Fe3O4@ Au Nanoflowers and Au@ Ag Nanospheres with Aptamer for Ultrasensitive SERS Detection of Aflatoxin B1.
Aflatoxin B1 (AFB1) as the most toxic mycotoxin in contaminated food can greatly threaten human health, and sensitive and selective detection of AFB1 is thus highly desired. An ultrasensitive surface-enhanced Raman spectroscopy (SERS) aptasensor was developed for AFB1 detection in peanut oil samples. SH-cDNA modified Fe3O4@Au nanoflowers acted as capture probes, SH-Apt modified Au@Ag nanospheres and commercial Cy3-Apt were used as reporter probes. Strong SERS signals of reporter probes were produced due to the recognition of AFB1 aptamer and its complementary strand (SH-cDNA). With the preferred binding of AFB1 aptamer to AFB1, reporter probes were released from capture probes, causing a linear decrease in SERS intensity. Therefore an ultralow detection limit of 0.40 pg·mL-1 in a wide linear range of 0.0001-100 ng·mL-1 was obtained and the sensibility of this SERS aptasensor was higher than that of the Cy3-Apt based SERS aptasensor. In addition, an excellent selectivity in interfering toxins and satisfactory recoveries of 96.6-115% in peanut oil samples were obtained, proving this aptasensor is a promising analytical tool in AFB1 detection.
Effect of Drying Temperature and Exposure Times on Aspergillus Flavus Growth and Aflatoxin Production on Artificially Inoculated Hazelnuts.
Aspergillus flavus may colonise hazelnuts and produce aflatoxins in field and during storage. The main purpose of this study was to investigate the influence of drying temperature and exposure times on the viability and ability of A. flavus to produce aflatoxins during the drying process and storage. Hazelnuts were inoculated with A. flavus and dried at different temperatures to reach 6% moisture content and a w 0.71, a commercial requirement to avoid fungal development and aflatoxin contamination. Hazelnuts were dried at 30, 35, 40, 45, and 50 °C and subsequently stored at 25 °C for 14 days. After drying at 30, 35 and 40 °C, an increased number of A. flavus was evident, with the highest concentration at 35 °C (6.1 ± 2.4 x10 6 A. flavus CFU/g). At these temperatures, aflatoxins were detected only at 30 °C and 35 °C. Aflatoxins, however, were higher after drying at 30 °C with a concentration of 1.93 ± 0.77 μg/g for aflatoxin B1 (AFB1) and 0.11 ± 0.04 μg/g for aflatoxin B2 (AFB2). After 14 days of storage, the highest A. flavus concentration and the highest level of mycotoxins were detected in samples treated at 35 °C (8.2 ± 2.1 x10 7 A. flavus CFU/g, 9.30 ± 1.58 μg/g and 0.89 ± 0.08 μg/g for AFB1 and AFB2, respectively). In hazelnuts dried at 45 °C or 50 °C no aflatoxins were found both after drying and storage, and a reduction of A. flavus viable conidia was observed, suggesting that a shorter and warmer drying is essential to guarantee the nut safety. The lowest temperature that guarantees the lack of aflatoxins should be selected to maintain the organoleptic quality of hazelnuts. Therefore, 45°C should be the recommended drying temperature to limit A. flavus growth and aflatoxin contamination on hazelnuts.
Aspergillus, Penicillium and Cladosporium species associated with dried date fruits collected in the Perugia (Umbria, Central Italy) market.
A total of 20 dried date samples, chosen as representative among those available on the Perugia (Umbria, Central Italy) market, were analyzed for the possible occurrence of fungal species and related contamination by fungal secondary metabolites. Twenty-six isolates, representative of the total mycobiota, were obtained and morphologically identified as belonging to the genera Aspergillus, Penicillium and Cladosporium. Inside each genus, molecular characterization (by partial sequencing of ITS region and/or β-tubulin and calmodulin regions for Aspergillus and Penicillium isolates or actin region for Cladosporium isolates) and in vitro mycotoxigenic profile characterization (by LC-MS/MS analysis) showed the presence of the following species: A. flavus, A. tubingensis, P. brevicompactum, P. chrysogenum, P. crustosum, P. glabrum, P. solitum, P. venetum, C. cladosporioides, C. limoniforme and C. halotolerans, with A. tubingensis as the prevalent species and P. crustosum, P. solitum, P. venetum and C. limoniforme first reported here on dates. Date packaging and format showed an effect on the incidence of isolated fungi, with the lowest incidence recovered from whole dates and in hermetic bag packaging. These findings can be useful both for dried dates producers and consumers, guiding them towards choices of packaging and format with a lower risk of mycotoxigenic species presence. However, no fungal metabolites were detected in the dried date samples analyzed, which were therefore regarded as safe for human consumption, underlining the absence of correspondence between fungal isolation and mycotoxin contaminations.
Advances in Crop Improvement and Delivery Research for Nutritional Quality and Health Benefits of Groundnut (Arachis hypogaea L.).
Groundnut is an important global food and oil crop that underpins agriculture-dependent livelihood strategies meeting food, nutrition, and income security. Aflatoxins, pose a major challenge to increased competitiveness of groundnut limiting access to lucrative markets and affecting populations that consume it. Other drivers of low competitiveness include allergens and limited shelf life occasioned by low oleic acid profile in the oil. Thus grain off-takers such as consumers, domestic, and export markets as well as processors need solutions to increase profitability of the grain. There are some technological solutions to these challenges and this review paper highlights advances in crop improvement to enhance groundnut grain quality and nutrient profile for food, nutrition, and economic benefits. Significant advances have been made in setting the stage for marker-assisted allele pyramiding for different aflatoxin resistance mechanisms-in vitro seed colonization, pre-harvest aflatoxin contamination, and aflatoxin production-which, together with pre- and post-harvest management practices, will go a long way in mitigating the aflatoxin menace. A breakthrough in aflatoxin control is in sight with overexpression of antifungal plant defensins, and through host-induced gene silencing in the aflatoxin biosynthetic pathway. Similarly, genomic and biochemical approaches to allergen control are in good progress, with the identification of homologs of the allergen encoding genes and development of monoclonal antibody based ELISA protocol to screen for and quantify major allergens. Double mutation of the allotetraploid homeologous genes, FAD2A and FAD2B, has shown potential for achieving >75% oleic acid as demonstrated among introgression lines. Significant advances have been made in seed systems research to bridge the gap between trait discovery, deployment, and delivery through innovative partnerships and action learning.
Functional Biology and Molecular Mechanisms of Host-Pathogen Interactions for Aflatoxin Contamination in Groundnut (Arachis hypogaea L.) and Maize (Zea mays L.).
Aflatoxins are secondary metabolites produced by soilborne saprophytic fungus Aspergillus flavus and closely related species that infect several agricultural commodities including groundnut and maize. The consumption of contaminated commodities adversely affects the health of humans and livestock. Aflatoxin contamination also causes significant economic and financial losses to producers. Research efforts and significant progress have been made in the past three decades to understand the genetic behavior, molecular mechanisms, as well as the detailed biology of host-pathogen interactions. A range of omics approaches have facilitated better understanding of the resistance mechanisms and identified pathways involved during host-pathogen interactions. Most of such studies were however undertaken in groundnut and maize. Current efforts are geared toward harnessing knowledge on host-pathogen interactions and crop resistant factors that control aflatoxin contamination. This study provides a summary of the recent progress made in enhancing the understanding of the functional biology and molecular mechanisms associated with host-pathogen interactions during aflatoxin contamination in groundnut and maize.
Thermosensitive Hydrogel for Encapsulation and Controlled Release of Biocontrol Agents to Prevent Peanut Aflatoxin Contamination.
Starch, alginate, and poly(N-isopropylacrylamide) (PNIPAAm) were combined to prepare a semi-interpenetrating network (IPN) hydrogel with temperature sensitivity. Calcium chloride was used as cross-linking agent, the non-toxigenic Aspergillus flavus spores were successfully encapsulated as biocontrol agents by the method of ionic gelation. Characterization of the hydrogel was performed by Fourier-transform infrared spectroscopy (FTIR), scanning electron micrograph (SEM), and thermogravimetry analysis (TGA). Formulation characteristics, such as entrapment efficiency, beads size, swelling behavior, and rheological properties were evaluated. The optical and rheological measurements indicated that the lower critical solution temperature (LCST) of the samples was about 29-30 °C. TGA results demonstrated that the addition of kaolin could improve the thermal stability of the semi-IPN hydrogel. Morphological analysis showed a porous honeycomb structure on the surface of the beads. According to the release properties of the beads, the semi-IPN hydrogel beads containing kaolin not only have the effect of slow release before peanut flowering, but they also can rapidly release biocontrol agents after flowering begins. The early flowering stage of the peanut is the critical moment to apply biocontrol agents. Temperature-sensitive hydrogel beads containing kaolin could be considered as carriers of biocontrol agents for the control of aflatoxin in peanuts.
A Sensitive, Point-of-Care Detection of Small Molecules Based on a Portable Barometer: Aflatoxins In Agricultural Products.
Sensitive and point-of-care detection of small toxic molecules plays a key role in food safety. Aflatoxin, a typical small toxic molecule, can cause serious healthcare and economic issues, thereby promoting the development of sensitive and point-of-care detection. Although ELISA is one of the official detection methods, it cannot fill the gap between sensitivity and point-of-care application because it requires a large-scale microplate reader. To employ portable readers in food safety, Pt-catalysis has attracted increasing attention due to its portability and reliability. In this study, we developed a sensitive point-of-care aflatoxin detection (POCAD) method via a portable handheld barometer. We synthesized and characterized Au@PtNPs and Au@PtNPs conjugated with a second antibody (Au@PtNPs-IgG). A competitive immunoassay was established based on the homemade monoclonal antibody against aflatoxins. Au@PtNPs-IgG was used to catalyze the production of O2 from H2O2 in a sealed vessel. The pressure of O2 was then recorded by a handheld barometer. The aflatoxin concentration was inversely proportional to the pressure recorded via the barometer reading. After optimization, a limit of detection of 0.03 ng/mL and a linear range from 0.09 to 16.0 ng/mL were achieved. Recovery was recorded as 83.1%-112.0% along with satisfactory results regarding inner- and inter-assay precision (relative standard deviation, RSD < 6.4%). Little cross-reaction was observed. Additionally, the POCAD was validated by high-performance liquid chromatography (HPLC) by using peanut and corn samples. The portable POCAD exhibits strong potential for applications in the on-site detection of small toxic molecules to ensure food safety.
High Efficacy of the Volatile Organic Compounds of Streptomyces yanglinensis 3-10 in Suppression of Aspergillus Contamination on Peanut Kernels.
Aspergillus flavus and Aspergillus parasiticus are saprophytic fungi which can infect and contaminate preharvest and postharvest food/feed with production of aflatoxins (B1, B2, and G). They are also an opportunistic pathogen causing aspergillosis diseases of animals and humans. In this study, the volatile organic compounds (VOCs) from Streptomyces yanglinensis 3-10 were found to be able to inhibit mycelial growth, sporulation, conidial germination, and expression of aflatoxin biosynthesis genes in A. flavus and A. parasiticus in vitro. On peanut kernels, the VOCs can also reduce the disease severity and inhibit the aflatoxins production by A. flavus and A. parasiticus under the storage conditions. Scanning electron microscope (SEM) observation showed that high dosage of the VOCs can inhibit conidial germination and colonization by the two species of Aspergillus on peanut kernels. The VOCs also showed suppression of mycelial growth on 18 other plant pathogenic fungi and one Oomycetes organism. By using SPME-GC-MS, 19 major VOCs were detected, like in other Streptomyces, 2-MIB was found as the main volatile component among the detected VOCs. Three standard chemicals, including methyl 2-methylbutyrate (M2M), 2-phenylethanol (2-PE), and β-caryophyllene (β-CA), showed antifungal activity against A. flavus and A. parasiticus. Among them, M2M showed highest inhibitory effect than other two standard compounds against conidial germination of A. flavus and A. parasiticus. To date, this is the first record about the antifungal activity of M2M against A. flavus and A. parasiticus. The VOCs from S. yanglinensis 3-10 did not affect growth of peanut seedlings. In conclusion, our results indicate that S. yanglinensis 3-10 may has a potential to become a promising biofumigant in for control of A. flavus and A. parasiticus.
A Lateral Flow Strip Based on a Truncated Aptamer-Complementary Strand for Detection of Type-B Aflatoxins in Nuts and Dried Figs.
Type-B aflatoxins (AFB1 and AFB2) frequently contaminate food, especially nuts and fried figs, and seriously threaten human health; hence, it is necessary for the newly rapid and sensitive detection methods to prevent the consumption of potentially contaminated food. Here, a lateral flow aptasensor for the detection of type-B aflatoxins was developed. It is based on the use of fluorescent dye Cy5 as a label for the aptamer, and on the competition between type-B aflatoxins and the complementary DNA of the aptamer. This is the first time that the complementary strand of the aptamer has been used as the test line (T-line) to detect type-B aflatoxins. In addition, the truncated aptamer was used to improve the affinity with type-B aflatoxins in our study. Therefore, the lengths of aptamer and cDNA probe were optimized as key parameters for higher sensitivity. In addition, binding buffer and organic solvent were investigated. The results showed that the best pair for achieving improved sensitivity and accuracy in detecting AFB1 was formed by a shorter aptamer (32 bases) coupled with the probe complementary to the AFB1 binding region of the aptamer. Under the optimal experimental conditions, the test strip showed an excellent linear relationship in the range from 0.2 to 20 ng/mL with a limit of detection of 0.16 ng/mL. This aptamer-based strip was successfully applied to the determination of type-B aflatoxins in spiked and commercial peanuts, almonds, and dried figs, and the recoveries of the spiked samples were from 93.3%-112.0%. The aptamer-complementary strand-based lateral flow test strip is a potential alternative tool for the rapid and sensitive detection of type-B aflatoxins in nuts and dried figs. It is of help for monitoring aflatoxins to avoid the consumption of unsafe food.