Genetic Diversity, Ochratoxin A and Fumonisin Profiles of Strains of Aspergillus Section Nigri Isolated from Dried Vine Fruits.
We investigated ochratoxin A (OTA) contamination in raisin samples purchased from Slovak markets and determined the diversity of black-spored aspergilli as potential OTA and fumonisin (FB1 and FB2) producers. The taxonomic identification was performed using sequences of the nuclear ITS1-5.8s-ITS2 region, the calmodulin and beta-tubulin genes. We obtained 239 isolates from eight fungal genera, of which 197 belonged to Aspergillus (82%) and 42 strains (18%) to other fungal genera. OTA contamination was evidenced in 75% of the samples and its level ranged from 0.8 to 10.6 µg/kg. The combination of all three markers used enabled unambiguous identification of A. carbonarius, A. luchuensis, A. niger, A. tubingensis and A. welwitschiae. The dominant coloniser, simultaneously having the highest within-species diversity isolated from our raisin samples, was A. tubingensis. Out of all analysed strains, only A. carbonarius was found to produce OTA, but in relatively high quantity (2477-4382 µg/kg). The production of FB1 and FB2 was evidenced in A. niger strains only.
Ingenious Electrochemiluminescence Bio-aptasensor based on Synergistic Effects and Enzyme-Drive Programmable 3D DNA Nanoflowers for Ultrasensitive Detection of Aflatoxin B1.
Aflatoxin B1 (AFB1), one of the most toxic mycotoxins, has undergone a wide range of studies over the years. The development of rapid, simple, and sensitive analytical methods remains a major challenge for the accurate detection of AFB1 in foodstuffs. In this study, we designed an enhanced and stable Ingenious Electrochemiluminescence Bio-aptasensor (IEC-BA) for ultrasensitive detection of AFB1 based on universal synergistic effects and enzyme-drive programmable assembled 3D DNA Nanoflowers (EPDNs). This synergistic effect encountered comprised of the competitive impact on Auxiliary Probes (AP) and the cutting effect of the Hae III. Compared to the traditional aptamer direct-competition method, the synergistic effects ensured that the aptamer was more efficiently and adequately competed away by the target, Also, the redundant double-stranded probes were removed, which greatly facilitates simple, quick, and sensitive detection of AFB1. Besides, a large chunk of positively charged Ru (II) complexes were aggregated by the utilization of EPDNs, which resulted in tremendous improvement of the sensitivity of the designed method. Thus, even in the presence of trace amounts of AFB1, a sharply visual electroluminescent signal was generated. The proposed method can realize the quantification of AFB1 with a good linear range from 1 ppt to 10 ppb with detection limit of 0.248 ppt. In addition, it can also be successfully applied for the analysis of AFB1 in peanut and wheat, with total recoveries ranging from 93.7 % to 106.6 %. Furthermore, the IEC-BA also exhibited good selectivity, reproducibility, and stability, revealing prospective applications of food safety monitoring and environmental analysis.
Development of Anti-Idiotypic Nanobody-Phage Based Immuno-Loop-Mediated Isothermal Amplification Assay for Aflatoxins in Peanuts.
Aflatoxin contamination in agricultural products has posed serious health hazards and brought huge economic loss in the food and feed industries. Monitoring aflatoxins in various foods and feeds has become a crucial means to protect public health. This study aimed to report an immuno-loop-mediated isothermal amplification (iLAMP) assay by using an anti-idiotypic nanobody-phage for on-site and rapid detection of aflatoxin in real samples. The iLAMP method was developed on the basis of a competitive immunoassay and LAMP reaction performed in a simple water bath. This method can provide visualized test results: violet color represents positive samples while sky blue represents negative. The visual detection limits of iLAMP for aflatoxin B1, B2, G1, and G2 in peanut samples were 1.6, 1.6, 3.2, and 16 μg/kg, respectively. The developed assay was verified with high performance liquid chromatography (HPLC) for the analysis of aflatoxins in peanuts, which demonstrated that the iLAMP method can be applied to the detection of aflatoxin in real samples. The novel iLAMP assay eliminates the need for aflatoxin conjugates, the antibody labeling process, and special equipment, and offers an alternative to existing methods with advantages of time-saving, cost-effectiveness, and ease-of-use.
Biocontrol potential of native yeast strains against Aspergillus flavus and aflatoxin production in pistachio.
Aspergillus flavus is the main aflatoxin producer in food and feed and has wide ecological niches. Contamination of food products such as pistachio nuts and aflatoxin secretion directly affects food safety and international food product trades. Abilities of 13 yeast strains isolated from 200 soil and pistachio nut samples collected in Iranian orchards to reduce the growth of A. flavus as well as aflatoxin production were assessed in dual culture, volatile and non-volatile compounds tests. The growth of A. flavus was reduced by 32-60%, 13-31% and 40-61% in dual culture, volatile and non-volatile compounds, respectively, while aflatoxin B1 production was diminished by 90.6-98.3%. Based on these assays, five yeast strains were selected for co-inoculation experiments using soil, pistachio hulls and leaf. A significant reduction in colony-forming units (CFU) ranging from 23% to 110% (p < .05) was observed. Molecular, physiological and morphological identification revealed these were strains of Pichia kudriavzevii and Lachansea thermotolerans. Aflatoxin biocontrol with yeast strains possesses many advantages including the ease of commercial production and organic application which is an environmental approach. More investigation is required to understand the efficiency of selective strains to inhibit A. flavus and aflatoxin production as well as withstand predominant abiotic stress in pistachio orchards and mass production in field application.
Fate of aflatoxins during almond oil processing.
Almonds rejected as inedible are often used for production of almond oil. However, low-quality almonds are frequently contaminated with aflatoxins, and little is known regarding transfer of aflatoxins to almond oil during processing. In this study, oil was produced from reject almonds by hexane extraction. Of 19 almond samples that were naturally contaminated with aflatoxins, 17 oil samples contained measurable amounts of aflatoxins, and aflatoxin content of contaminated oil was correlated with aflatoxin content of the nuts. However, oil aflatoxin levels were not correlated with the oxidation level of the oil as measured by percent free fatty acids and peroxide value. Adsorbents used in oil refining were tested for their ability to remove aflatoxins from contaminated oil. Fuller's earth and bentonite were the most effective, removing 96% and 86% of total aflatoxins from contaminated oil samples, respectively. Treatment with diatomaceous earth, in contrast, had no effect on aflatoxin levels in oil. These results show that oil refining steps using mineral clay adsorbents may also function to remove aflatoxins from contaminated oil.
Occurrence and Exposure Assessment of Mycotoxins in Ready-to-Eat Tree Nut Products through Ultra-High Performance Liquid Chromatography Coupled with High Resolution Q-Orbitrap Mass Spectrometry.
Tree nuts have become popular snacks due to their attributed benefits in the health state. Nevertheless, their susceptibility to fungal contamination lead to the occurrence of potentially dangerous mycotoxins. Hence, the aim of this work was to evaluate the presence of mycotoxins in ready-to-eat almonds, walnuts, and pistachios from Italian markets. The most relevant mycotoxin found in almonds was α-zearalanol in 18% of samples (n = 17) ranging from 3.70 to 4.54 µg/kg. Walnut samples showed frequent contamination with alternariol, present in 53% of samples (n = 22) at levels from 0.29 to 1.65 µg/kg. Pistachios (n = 15) were the most contaminated commodity, with β-zearalenol as the most prevalent toxin present in 59% of samples ranging from 0.96 to 8.60 µg/kg. In the worst-case scenario, the exposure to zearalenone-derived forms accounted for 15.6% of the tolerable daily intake, whereas it meant 12.4% and 21.2% of the threshold of toxicological concern for alternariol and alternariol monomethyl-ether, respectively. The results highlighted the extensive presence of Alternaria toxins and zearalenone-derived forms, scarcely studied in ready-to-eat tree nut products, highlighting the necessity to include these mycotoxins in analytical methods to perform more realistic risk assessments.
Effect of blanching on aflatoxin contamination and cross-contamination of almonds.
Blanching of almonds was examined for reducing aflatoxin content of contaminated nuts. Almonds with intact pellicles were spiked with aflatoxin B 1 (AFB 1 ) and blanched at 85°C. Following blanching, almond kernels and pellicles contained 20% and 19% of spiked AFB 1 , respectively. Blanching water contained an additional 41% of spiked AFB 1 . In a separate study, post-blanching water was spiked with AFB 1- and used for subsequent blanching of uncontaminated almonds. The resulting blanched kernels acquired 3.3% of the AFB 1 from the spiked water, demonstrating a low level of cross-contamination from reused contaminated blanching water. The effect of blanching temperature on partitioning of AFB 1 from almonds to blanching water was significant at 20 ppb spiking level, but not at 100 ppb. AFB 1 levels that were unaccounted for in mass balance of blanching components were presumed to be lost due to binding to water-solubilized almond components, and were independent of pH and blanching time. Blanching reduced total aflatoxins in naturally contaminated almonds by 13-76%, depending on almond quality as well as blanching time and temperature. These results indicate that the association between almond components and aflatoxin generated through mold contamination is more complex than in spiking experiments.
Profiles of fungal metabolites including regulated mycotoxins in individual dried Turkish figs by LC-MS/MS.
Fungal metabolites including regulated mycotoxins were identified by a validated LC-MS/MS method in 180 individual Turkish dried figs from 2017 and 2018 harvests. Hand-selected dried figs were subjectively classified based on the extent of fluorescence. Forty-three fungal metabolites including eight EU-regulated mycotoxins were identified and quantified. Figs classified as being uncontaminated mostly did not contain aflatoxins above 1 μg/kg. Despite being "uncontaminated" from an aflatoxin perspective, kojic acid was present in significant quantities with a maximum level of 3750 mg/kg (0.375% w/w) and tenuazonic acid was also found (2 μg/kg to 298 mg/kg) in some figs. Notable in the screening of figs has been the presence of significant amounts of aflatoxin M1 (AFM1) in figs also containing significant levels of aflatoxin B1 (AFB1), which is the first time that AFM1 has been reported as naturally occurring in dried figs.
AFLA-PISTACHIO: Development of a Mechanistic Model to Predict the Aflatoxin Contamination of Pistachio Nuts.
In recent years, very many incidences of contamination with aflatoxin B1 (AFB1) in pistachio nuts have been reported as a major global problem for the crop. In Europe, legislation is in force and 12 μg/kg of AFB1 is the maximum limit set for pistachios to be subjected to physical treatment before human consumption. The goal of the current study was to develop a mechanistic, weather-driven model to predict Aspergillus flavus growth and the AFB1 contamination of pistachios on a daily basis from nut setting until harvest. The planned steps were to: (i) build a phenology model to predict the pistachio growth stages, (ii) develop a prototype model named AFLA-pistachio (model transfer from AFLA-maize), (iii) collect the meteorological and AFB1 contamination data from pistachio orchards, (iv) run the model and elaborate a probability function to estimate the likelihood of overcoming the legal limit, and (v) manage a preliminary validation. The internal validation of AFLA-pistachio indicated that 75% of the predictions were correct. In the external validation with an independent three-year dataset, 95.6% of the samples were correctly predicted. According to the results, AFLA-pistachio seems to be a reliable tool to follow the dynamic of AFB1 contamination risk throughout the pistachio growing season.
Integrated small RNA and mRNA expression profiles reveal miRNAs and their target genes in response to Aspergillus flavus growth in peanut seeds.
Background: MicroRNAs are important gene expression regulators in plants immune system. Aspergillus flavus is the most common causal agents of aflatoxin contamination in peanuts, but information on the function of miRNA in peanut-A. flavus interaction is lacking. In this study, the resistant cultivar (GT-C20) and susceptible cultivar (Tifrunner) were used to investigate regulatory roles of miRNAs in response to A. flavus growth. Results: A total of 30 miRNAs, 447 genes and 21 potential miRNA/mRNA pairs were differentially expressed significantly when treated with A. flavus. A total of 62 miRNAs, 451 genes and 44 potential miRNA/mRNA pairs exhibited differential expression profiles between two peanut varieties. Gene Ontology (GO) analysis showed that metabolic-process related GO terms were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses further supported the GO results, in which many enriched pathways were related with biosynthesis and metabolism, such as biosynthesis of secondary metabolites and metabolic pathways. Correlation analysis of small RNA, transcriptome and degradome indicated that miR156/SPL pairs might regulate the accumulation of flavonoids in resistant and susceptible genotypes. The miR482/2118 family might regulate NBS-LRR gene which had the higher expression level in resistant genotype. These results provided useful information for further understanding the roles of miR156/157/SPL and miR482/2118/NBS-LRR pairs. Conclusions: Integration analysis of the transcriptome, miRNAome and degradome of resistant and susceptible peanut varieties were performed in this study. The knowledge gained will help to understand the roles of miRNAs of peanut in response to A. flavus.