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Pistachio (Pistacia vera L.) is an economically important tree nut. Due to its nutritional properties and health benefits, it is considered a healthy food and thus widely consumed worldwide. However, fungal contamination of the commodities has received considerable attention because of possible contamination by toxigenic fungi, important source of mycotoxins, resulting from secondary metabolism and hazards to health consumer. Members of the genus Aspergillus, mainly Aspergillus flavus and Aspergillus niger, are reported as occurring most frequently on pistachio nuts, because able to grow in the presence of low amounts of water and to produce mycotoxins (aflatoxins and ochratoxins), that are well known for their harmful health effects on humans. Monitoring the contaminating fungal species is particularly worthy of note also in climate change scenario, allowing to notice changes in fungal population composition through the time. This study aimed to contribute to collect data about fungal population and mycotoxins occurred in pistachio samples collected in Turkey: prevalence of 2 species, A. flavus and Aspergillus tubingensis, was assessed. The A. flavus strains consisted of a mixed population of aflatoxin producers and non-producing strains in vitro, with evidence of a new genotype in gene cluster within strains of aflatoxin non-producing chemotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Almonds are susceptible to infestation by Aspergillus flavus , an aflatoxin-producing fungus. The objective of this study was to investigate the effects of kernel type (inshell, shelled and split almonds) on the ability of A. flavus to grow and produce aflatoxins at different combinations of temperature (20, 27 and 35 °C), water activity (0.85, 0.92, 0.95 and 0.98 a w ) and incubation period (10, 20 and 30 days). There was no fungal growth at 0.85 a w on any of the kernel types. At 0.92 a w , only the split kernels supported growth and aflatoxin synthesis. The fungus was able to grow and produce aflatoxins on all three kernels at 0.95-0.98 a w and 20-35 °C. At 0.98 a w , high total aflatoxin concentrations (>300 µg/kg) were found on the shelled and split kernels at all temperatures. On the inshell nuts, the fungus produced up to 372 µg/kg of total aflatoxins at 0.98 a w and 27 °C. Regression analysis showed that significantly higher levels of aflatoxins were produced at 27 °C (as compared to at 20 and 35 °C) on shelled and split almonds. Incubation time was also a significant predictor of aflatoxin accumulation. The results of this study indicated that shipping almonds below 0.85 a w and reducing storage time would significantly decrease the risk of infestation and aflatoxin production by A. flavus .

Background: Chestnut fruit quality is affected by fungal contamination. The study of the patterns of contamination in the postharvest is crucial to individuate the critical phases and propose solutions. To understand how fungal colonization varies on fruits, the composition of mycobiota was investigated in postharvest handling and in between tissues (shell and kernel)., Results: Fungal sequences were clustered into 308 operational taxonomic units (OTUs). Biodiversity was higher in shell than kernel tissues. Results evidenced the risk of new contamination in specific phases such as the 'cold bath' and storage. Genera known as mycotoxin producers were detected in all phases. Specifically, 47 OTUs belonging to Penicillium, eight to Fusarium and two to Aspergillus genera were identified. While Fusarium spp. was sensitive to 'warm bath' phase, Penicillium spp. was largely insensitive and accumulated in storage conditions. Surprisingly, Aspergillus spp. was poorly represented. Aflatoxin, ochratoxin A, fumonisins and T-2/HT-2 detection was performed for shell and kernel, and process phases. Higher contamination was observed on shell than in kernel samples. While aflatoxins were within the European Union (EU) limits for dry fruits, Ochratoxin exceeded the EU limits. The present study represents the first report of fumonisins and T-2/HT-2 detection in chestnuts., Conclusion: Fungal contamination taxa is high in chestnut fruits following postharvest handling and storage. A parametrization of process phases such as the 'warm bath' is functional to reduce the risk for some taxa. For other spoilage and mycotoxigenic genera strict sanitation procedures of equipment and water must be individuated and implemented to reduce their impact. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hazelnut ( Corylus avellana ), a nut crop that is rapidly expanding worldwide, is endangered by a rot. Nut rot results in hazelnut defects. A survey was conducted in northwestern Italy during 2020 and 2021 to identify the causal agents of hazelnut rots. Typical symptoms of black rot, mold, and necrotic spots were observed on hazelnuts. The prevalent fungi isolated from symptomatic hazelnut kernels were Diaporthe spp. (38%), Botryosphaeria dothidea (26%), Diplodia seriata (14%), and other fungal genera with less frequent occurrences. Among 161 isolated Diaporthe spp., 40 were selected for further analysis. Based on morphological characterization and multilocus phylogenetic analysis of the ITS, tef-1α , and tub2 , seven Diaporthe species were identified as D. eres , D. foeniculina , D. novem , D . oncostoma , D. ravennica , D. rudis , and D. sojae . D. eres was the main species isolated from hazelnut rots, in particular from moldy nuts. The pathogenicity test performed on hazelnuts 'Tonda Gentile del Piemonte' using a mycelium plug showed that all the Diaporthe isolates were pathogenic on their original host. To our knowledge, this work is the first report of D. novem , D . oncostoma , and D. ravennica on hazelnuts worldwide. D. foeniculina , D. rudis , and D. sojae were reported for the first time as agents of hazelnut rot in Italy. Future studies should focus on the comprehension of epidemiology and climatic conditions favoring the development of Diaporthe spp. on hazelnut. Prevention and control measures should target D. eres , representing the main causal agents responsible for defects and nut rot of hazelnuts in Italy., Competing Interests: The author(s) declare no conflict of interest.

Pistachio nuts can become colonized by mycotoxigenic fungi, especially Aspergillus flavus , resulting in contamination with aflatoxins (AFs). We examined the effect of gaseous O 3 (50-200 ppm; 30 min; 6 L/min) on (a) in vitro germination, (b) mycelial growth, and (c) aflatoxin B1 (AFB 1 ) production on a milled pistachio nut-based medium at different water activity (a w ) levels and at 30 °C. This was complimented with in situ studies exposing raw pistachio nuts to 50-200 ppm of O 3 . Exposure of conidia to gaseous O 3 initially resulted in lower germination percentages at different a w levels. However, 12 h after treatment, conidial viability recovered with 100% germination after 24-48 h. Growth rates of mycelial colonies were slightly decreased with the increase of the O 3 dose, with significant inhibition only at 0.98 a w . The production of AFB 1 after O 3 treatment and storage for 10 days was stimulated in A. flavus colonies at 0.98 a w . Raw pistachio nuts inoculated with A. flavus conidia prior to O 3 exposure showed a significant decrease in population after 20 days of storage. However, AFB 1 contamination was stimulated in most O 3 treatments. The relationship between exposure concentration, time and prevailing a w levels on toxin control needs to be better understood for these nuts.

This project was undertaken to determine the kinetic parameters of thermal inactivation of Listeria monocytogenes on pecans, macadamia nuts, and sunflower seeds subjected to heat treatments simulating industry processes. Five strains were grown in nonselective medium, mixed, and resuspended before inoculating macadamia nuts, pecans, and sunflower seeds (6 to 9 Log CFU/g). Redried inoculated pecans and macadamia nuts were heated in an oven at a temperature range of 90 to 140°C. Unshelled sunflower seeds were heated in sunflower seed oil. The thermal inactivation was determined by measuring viable cell counts using standard microbiological methods. Average count data were fit to the log-linear model, and thermal-death kinetics were calculated. On pecans, the viable Listeria counts were reduced by 3 and 3.5 Log CFU/g after 40 min at 110°C and 8 min at 140°C, respectively. On macadamia nuts, the L. monocytogenes population was reduced by 5 Log CFU/g after 20 min at 120°C. Unshelled sunflower seeds were subjected to heat treatment via a hot-oil bath. On sunflower seeds, >7 Log CFU/g reductions were observed after 15 min at 120°C. The thermal resistance ( D value) for inactivation on pecans at 140°C was 3.1 min and on macadamia nuts at 120°C was 4.4 min. The inactivation of L. monocytogenes was influenced by the kind of nut or seed. These results suggest that L. monocytogenes has a relatively high thermal tolerance. The findings from this study will contribute to the assessment of the effectiveness of heat treatment for control of this pathogen on nuts and seeds. IMPORTANCE Listeria monocytogenes is a major concern for the food industry in ready-to-eat (RTE) foods. In recent years, large-scale recalls have occurred with contaminated sunflower seeds and macadamia nuts that triggered product withdrawals. These events stress the importance of understanding Listeria' s ability to survive heat treatments in these low-water activity foods. Nuts and seeds are subjected to a variety of thermal treatments typically referred as roasting. To date, no listeriosis outbreak has been linked to nuts and seeds, but the recent recognition that this pathogen can be detected in commercial products stresses the need for research on thermal treatments. The characterization of heat inactivation kinetics at temperatures typically used during roasting processes will be very beneficial for validation studies. This manuscript reports inactivation rates of L. monocytogenes strains inoculated onto macadamia nuts, sunflower seeds, and pecan halves subjected to temperatures between 90 and 140°C.

Nuts, including almonds, are occasionally contaminated with Salmonella spp. In this study, we used chlorine dioxide (ClO 2 ) gas to inactivate S. enterica subsp. Enterica serovar Enteritidis on almonds. Almonds inoculated with a single strain of S. Enteritidis (8.95 log cfu/mL) were exposed to ClO 2 gas generated from 1.0 or 1.5 mL ClO 2 solution in a sealed container at 50 or 60 °C (43% relative humidity) for up to 10 h. The concentration of ClO 2 gas peaked at 354-510 and 750-786 ppm within 0.5 h upon deposition of 1.0 and 1.5 mL of aqueous ClO 2 , respectively, and gradually decreased thereafter. Population of S. Enteritidis on almonds treated at 50 °C decreased to 1.70-2.32 log cfu/sample within 1 h of exposure to ClO 2 gas and decreased to below the detection limit (1.7 log cfu/sample) at all ClO 2 concentrations after 8 h. At 60 °C, the microbial population fell below the detection limit within 1 h, regardless of the volume of ClO 2 solution supplied. Microbial survival on almonds treated with ClO 2 gas and stored at 12 or 25 °C was observed for up to 8 weeks and the organism was not recovered from the almonds treated for 10 h and stored at 12 °C for 2-8 weeks. The lightness (L value) and redness (a value) of almonds treated for 10 h were not changed by ClO 2 gas treatment, but yellowness (b value) increased. Results showed that Salmonella on almonds was successfully inactivated by ClO 2 gas treatment and the microbial survival did not occur during storage., (Copyright © 2021. Published by Elsevier Ltd.)

This study characterized the health risks due to the consumption of mycotoxin-contaminated foods and assessed the consumer awareness level of mycotoxins in households in two north-central Nigerian states during the harvest and storage seasons of 2018. Twenty-six mycotoxins and 121 other microbial and plant metabolites were quantified by LC-MS/MS in 250 samples of cereals, nuts and legumes. Aflatoxins were detected in all food types (cowpea, maize, peanut and sorghum) except in millet. Aflatoxin B 1 was the most prevalent mycotoxin in peanut (64%) and rice (57%), while fumonisin B 1 occurred most in maize (93%) and beauvericin in sorghum (71%). The total aflatoxin concentration was highest in peanut (max: 8422 µg/kg; mean: 1281 µg/kg) and rice (max: 955 µg/kg; mean: 94 µg/kg), whereas the totals of the B-type fumonisins and citrinin were highest in maize (max: 68,204 µg/kg; mean: 2988 µg/kg) and sorghum (max: 1335 µg/kg; mean: 186 µg/kg), respectively. Citrinin levels also reached 51,195 µg/kg (mean: 2343 µg/kg) in maize. Aflatoxin and citrinin concentrations in maize were significantly ( p < 0.05) higher during storage than at harvest. The estimated chronic exposures to aflatoxins, citrinin and fumonisins were high, resulting in as much as 247 new liver cancer cases/year/100,000 population and risks of nephrotoxicity and esophageal cancer, respectively. Children who consumed the foods were the most vulnerable. Mycotoxin co-occurrence was evident, which could increase the health risk of the outcomes. Awareness of mycotoxin issues was generally low among the households.

Metagenomic analysis of food is becoming more routine and can provide important information pertaining to the shelf life potential and the safety of these products. However, less information is available on the microbiomes associated with low water activity foods. Pine nuts and sesame seeds, and food products which contain these ingredients, have been associated with recalls due to contamination with bacterial foodborne pathogens. The objective of this study was to identify the microbial community of pine nuts and sesame seeds using targeted 16S rRNA sequencing technology. Ten different brands of each seed type were assessed, and core microbiomes were determined. A total of 21 and 16 unique taxa with proportional abundances >1% in at least one brand were identified in the pine nuts and sesame seeds, respectively. Members of the core pine nut microbiome included the genera Alishewanella, Aminivibrio, Mycoplasma, Streptococcus, and unassigned OTUs in the families of Desulfobacteraceae and Xanthomonadaceae. For sesame seeds, the core microbiome included Aminivibrio, Chryseolina, Okibacterium, and unassigned OTUs in the family Flavobacteriaceae. The microbiomes of these seeds revealed that these products are dominated by environmental bacterial genera commonly isolated from soil, water, and plants; bacterial genera containing species known as commensal organisms were also identified. Understanding these microbiomes can aid in the risk assessment of these products by identifying food spoilage potential and community members which may co-enrich with foodborne bacterial pathogens., Competing Interests: The authors have declared that no competing interests exist.

Nuts are the natural source of healthy lipids, proteins, and omega-3. They are susceptible to fungal and mycotoxins contamination because of their high nutritional value. Twenty-five species comprising 12 genera were isolated from 80 samples of dried fruits and nuts using the dilution plate method. Peanut recorded the highest level of contamination followed by coconut; almond and raisin were the lowest. Aspergillus was the most prevalent genus and A. niger, was the most dominant species. The morphological identification of the selected A. niger isolates as they were detected in high frequency of occurrence was confirmed by using 18SrRNA sequence. Ochratoxin biosynthesis gene Aopks was detected in the tested isolates. Lipase production by the selected A. niger isolates was determined with enzyme activity index (EAI) ranging from 2.02 to 3.28. A. niger-26 was the highest lipase producer with enzyme activity of 0.6 ± 0.1 U/ml by the trimetric method. Lip2 gene was also detected in the tested isolates. Finally, the antibacterial and antibiofilm efficiency of crude lipase against some human pathogens was monitored. Results exhibited great antibacterial efficacy with minimum bactericidal concentration (MBC) of 20 to 40 µl/100 µl against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Methicillin-resistant Staphylococcus aureus (MRSA). Interestingly, significant anti-biofilm efficacy with inhibition percentages of 95.3, 74.9, 77.1 and 93.6% was observed against the tested pathogens, respectively.

Background: The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties., Results: Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of < 22 h. Additionally, some strains showed better kinetic parameters than Saccharomyces boulardii (positive control). Complementary tests were done to determine their auto-aggregation capacity, cell surface hydrophobicity, behaviour in a sequential simulated digestion, biofilm formation capability and carbon source assimilation. Finally, 67% and 13% of the studied yeasts showed biocontrol and antioxidant activities, respectively., Conclusions: Diutina rugosa 14 followed by Diutina rugosa 8 were the best wild yeast from Pistacia vera as potential probiotic and in carbon source utilization. However, Hanseniaspora guilliermondii 6 and Aureobasidium proteae 5 could be used to improve food or feed product preservation because of their notable biocontrol and antioxidant capabilities. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

The most common, toxic, and carcinogenic mycotoxins found in human food and animal feed are the aflatoxins (AFs). The United States is a leading exporter of various nuts, with a marketing value of $9.1 billion in 2019; the European Union countries are the major importers of U.S. nuts. In the past few years, border rejections and notifications for U.S. tree nuts and peanuts exported to the E.U. countries have increased due to AF contamination. In this work, we analyzed notifications from the "Rapid Alert System for Food and Feed (RASFF)" on U.S. food and feed products contaminated with mycotoxins, primarily AFs, for the 10-year period 2010-2019. Almost 95% of U.S. mycotoxin RASFF notifications were reported for foods and only 5% for feeds. We found that 98.9% of the U.S. food notifications on mycotoxins were due to the AF contamination in almond, peanut, and pistachio nuts. Over half of these notifications (57.9%) were due to total AF levels greater than the FDA action level in food of 20 ng g -1 . The Netherlands issued 27% of the AF notifications for U.S. nuts. Border rejection was reported for more than 78% of AF notifications in U.S. nuts. All U.S. feed notifications on mycotoxins occurred due to the AF contamination. Our research contributes to better understanding the main reasons behind RASFF mycotoxins notifications of U.S. food and feed products destined to E.U. countries. Furthermore, we speculate possible causes of this problem and provide a potential solution that could minimize the number of notifications for U.S. agricultural export market.

&lt;b&gt;Background and Objective:&lt;/b&gt; Different researches have been achieved on non-dairy products as an alternative to dairy products. The interest in tiger nut tubers has considerably increase in recent years due to its nutritional and health benefits. Fermented drinks of non-dairy origin play an important role in diets worldwide. The aim of this study was to investigate the growth behavior and viability of &lt;i&gt;L. casei&lt;/i&gt;-01 in tiger nut milk made with milk permeate or cheese whey as an extraction medium. &lt;b&gt;Materials and Methods:&lt;/b&gt; Tiger nut milks were prepared using tiger nut tubers at ratios 1 to 3 (w/v) of water, milk permeate or cheese whey as extract media. Tiger nut milks and Skimmed milk were inoculated with &lt;i&gt;L. casei&lt;/i&gt;-01 at 2%. The Titratable Acidity (TA) and &lt;i&gt;L. casei&lt;/i&gt;-01 were determined during fermentation at 37°C for 8 hrs as well as during cold storage at 4°C for 20 days. &lt;b&gt;Results:&lt;/b&gt; Results showed that the substitutions of water with permeate or whey led to the change of chemical composition of tiger nut milk. Fermented permeate or whey-tiger nut milk significantly had higher rate of titratable acidity development during fermentation or during cold storage as compared with fermented water-tiger nut milk or skimmed milk. The total viable counts of &lt;i&gt;L. casei&lt;/i&gt;-01 were the highest in fermented whey-tiger nut milk after 10 days. &lt;b&gt;Conclusion:&lt;/b&gt; &lt;i&gt;Lactobacillus casei&lt;/i&gt;-01 can grow with high viability in permeate or whey-tiger milk.

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan ® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus . The limit of detection of the LAMP assay was 100-999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Two decontamination methods were evaluated for inactivating a cocktail of Salmonella or Listeria monocytogenes inoculated onto model low moisture foods (LMFs; dried strawberry, dried apple, raisins, chocolate crumb, cornflakes, shell-on or deshelled pistachio nuts). One treatment was based on a peracetic acid-ethanol (PAA-ethanol) sanitizer combination with the other being an Advanced Oxidation Process (AOP) that simultaneously applied UV-C (254 nm), ozone and hydrogen peroxide. The low moisture food was spray inoculated then dried prior to treatment. With Salmonella it was found that a pre-incubation step in 1% w/v glycerol-tryptic soy broth for 1 h prior to plating, significantly increased recovery of the pathogen compared to TSB alone. However, no increased recovery of L. monocytogenes was observed using the TSB-glycerol pre-incubation step. No Salmonella was detected on cornflakes, chocolate crumb and strawberry using 1.25 parts per thousand (‰) PAA-ethanol. The inactivation of Salmonella on deshelled pistachio was significantly higher using 2.5‰ PAA-ethanol sanitizer compared to the AOP treatments tested. Only negligible reductions of Salmonella (<1 log cfu) were obtained with shell-on pistachio treated with PAA-ethanol sanitizer or AOP. Salmonella could be reduced on dried apple slices by >4 log CFU when 5.0‰ PAA-ethanol was applied. L. monocytogenes was more sensitive to PAA-ethanol compared to Salmonella and could be eliminated on all the LMFs apart from shell-on pistachio. An AOP treatment applied 10% v/v hydrogen peroxide, ozone and 54 mJ/cm 2 UV-C could significantly reduce Salmonella on dried apple slices compared to when the individual elements (hydrogen peroxide, ozone or UV-C) were applied. Salmonella was also eliminated by AOP on the other LMFs (apart from shell-on pistachio) although the same level of inactivation was achieved by spraying with 10% v/v hydrogen peroxide alone. L. monocytogenes was sensitive to hydrogen peroxide and AOP being eliminated from all the LMFs. Although this may suggest that hydrogen peroxide spray was equivalent to AOP treatment it was noted that no residual H 2 O 2 or changes in visual appearance was evident on samples treated with the latter process. The study has demonstrated that the two decontamination methods assessed can be applied to reduce Salmonella and L. monocytogenes on LMFs although efficacy is dependent on the pathogen and product type., Competing Interests: Declaration of competing interest The authors declare that the submitted work was conducted in the absence of any commercial or financial relationships that could be construed as a conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers . Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1-3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

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.

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 B 1 , B 2 , G 1 , and G 2 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.

Background: Groundnut pre- and post-harvest contamination is commonly caused by fungi from the Genus Aspergillus. Aspergillus flavus is the most important of these fungi. It belongs to section Flavi; a group consisting of aflatoxigenic (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic (A. oryzae, A. sojae and A. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites of Aspergillus species associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure of A. flavus in different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap., Results: The overall pre- and post-harvest groundnut contamination rates with A. flavus were 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates with A. flavus across AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ 2  = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ 2  = 6, p = 0.199) was observed in the pre- and post-harvest contamination of groundnut with A. flavus across the three AEZs. The LKB had the highest incidence of aflatoxin-producing Aspergillus isolates while WN had no single Aspergillus isolate with aflatoxin-producing potential. Aspergillus isolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ 2  = 2.667, p = 0.264) than those from the post-harvest groundnut samples. Overall, A. flavus isolates exhibited moderate level (92%, p = 0.02) of genetic diversity across the three AEZs and low level (8%, p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241, p = 0.045) between genetic distance and geographic distance among A. flavus populations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01, p = 0.471), LVB farming system (r = 0.0141, p = 0.412) and WN farming system (r = 0.02, p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similar A. flavus isolates., Conclusions: These findings provide evidence that genetic differentiation in A. flavus populations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producing A. flavus.

In recent years, very many incidences of contamination with aflatoxin B 1 (AFB 1 ) 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 AFB 1 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 AFB 1 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 AFB 1 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 AFB 1 contamination risk throughout the pistachio growing season.

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.

Salmonella enterica is a common contaminant of macadamia nut kernels in the subtropical state of Queensland (QLD), Australia. We hypothesized that nonhuman sources in the plantation environment contaminate macadamia nuts. We applied a modified Hald source attribution model to attribute Salmonella serovars and phage types detected on macadamia nuts from 1998 to 2017 to specific animal and environmental sources. Potential sources were represented by Salmonella types isolated from avian, companion animal, biosolids-soil-compost, equine, porcine, poultry, reptile, ruminant, and wildlife samples by the QLD Health reference laboratory. Two attribution models were applied: model 1 merged data across 1998-2017, whereas model 2 pooled data into 5-year time intervals. Model 1 attributed 47% (credible interval, CrI: 33.6-60.8) of all Salmonella detections on macadamia nuts to biosolids-soil-compost. Wildlife and companion animals were found to be the second and third most important contamination sources, respectively. Results from model 2 showed that the importance of the different sources varied between the different time periods; for example, Salmonella contamination from biosolids-soil-compost varied from 4.4% (CrI: 0.2-11.7) in 1998-2002 to 19.3% (CrI: 4.6-39.4) in 2003-2007, and the proportion attributed to poultry varied from 4.8% (CrI: 1-11) in 2008-2012 to 24% (CrI: 11.3-40.7) in 2013-2017. Findings suggest that macadamia nuts were contaminated by direct transmission from animals with access to the plantations (e.g., wildlife and companion animals) or from indirect transmission from animal reservoirs through biosolids-soil-compost. The findings from this study can be used to guide environmental and wildlife sampling and analysis to further investigate routes of Salmonella contamination of macadamia nuts and propose control options to reduce potential risk of human salmonellosis.

Nuts and seeds have been increasingly associated with recalls due to contamination with Listeria monocytogenes. Storage of these food commodities occurs at various relative humidity (RH) conditions for months or years. The objective of this study was to assess L. monocytogenes survival on four commodities representing dried legumes, seeds, and spices categories: chickpeas, sesame seeds, pine nuts, and black pepper kernels. Inoculated products at 10 log CFU/g were stored for 180 days (6 months) at 25°C and different relative humidity (RH) levels: 25% (low), 45% (ambient), and 75% (high). After 180 days at 25% RH, L. monocytogenes populations decreased to 2.67-6.59 log CFU/g; the highest survival of the pathogen was observed on pine nuts and sesame seeds with decay rates of -0.014± 0.001 log CFU/g per d. Significantly greater population reductions on all products were observed during storage at 45 and 75% RH. At 45% RH, L. monocytogenes levels decreased to 1.90-6.36 log CFU/g. On chickpeas and black pepper stored at 75% RH, the pathogen population decreased to below the limit of enumeration (1 log CFU/g) yet were still detected via enrichments. The lowest survival of L. monocytogenes occurred at 75% RH on black pepper with a decay rate of -0.058±0.003 log CFU/g per d. Overall, regardless of RH level, the ability of the products to support survival of the pathogen may be expressed in the following order: pine nuts > sesame seeds > chickpeas > black pepper. The results of this study can aid in understanding how L. monocytogenes survives on dried legumes, seeds, and spices, and the data can contribute to the risk assessment of this pathogen., Competing Interests: The authors have declared that no competing interests exist.

Background: Chestnuts are gluten-free, low-fat, cholesterol-free products. Postharvest decay reduces chestnut shelf life and can cause severe economic losses. In this study we investigated the effect of ozone (O 3 ) gaseous treatment on chestnut rot caused by Gnomoniopsis castanea and the quality parameters of chestnuts., Results: The results showed that ozone treatment (150 ppb during the day, and 300 ppb during the night) reduced the decay of chestnuts and had a fungistatic effect on isolates of G. castanea. The exposure of chestnuts to ozone did not alter weight losses, sugar content and titratable acidity. The concentration of total phenolics decreased during the storage period, both for treated and untreated nuts. However, after 150 days of treatment the polyphenol content of the chestnuts exposed to ozone was significantly higher than in control nuts., Conclusions: Our results suggested that ozone is an appropriate and economical tool to maximize the quality of chestnut shelf life, enabling it to be stored for long periods. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The savanna cashew (Anacardium humile A. St. Hil.) nut is a native shrub species distributed in the savanna biome of Central Western Brazil. This species is similar to the cashew (Anacardium occidentale L.) nut, and its edible pseudo-fruits have been used for various purposes. However, A. humile is at risk of extinction. One reason for this is the phytosanitary issues. Therefore, phytosanitary studies of the serious plant diseases caused by fungal pathogens are necessary. The aim of this study was to survey the savanna cashew nut (Anacardium humile) in Central Western Brazil from August 2013 to October 2014. Serious damage caused by anthracnose to the plant's leaves and fruits was consistently detected. The pathogen fungal species was Colletotrichum gloeosporioides. It was identified by morphological characteristics and molecular analyses. Additional experiments showed evidence that C. gloeosporioides can be transmitted by cashew seeds. We observed anthracnose symptoms on the surfaces of the floral peduncle, leaves, and peduncle of the A. humile cashew nuts. The genomic DNA sequences of the selected strains for molecular characterization have had 99% identity with the analogous sequences of C. gloeosporioides. Naturally occurring C. gloeosporioides infection of the leaves of A. humile was low (20.3%) compared with that of the nuts (79.7%). Our results can be used to design strategies to prevent the introduction and establishment of C. gloeosporioides in new areas. They can also be helpful in monitoring programs in areas with a current occurrence of C. gloeosporioides. Finally, these results can be used in future research plans of C. gloeosporioides infection management.

The Botryosphaeriaceae family is considered a fungal family that includes pathogens causing latent infection of woody plants, and a number of species were identified as causal pathogens of canker and shoot blight diseases. To better understand the process of latent infection of major canker-causing pathogens in woody tissues in different tree crops important in California, shoot and bud samples were randomly collected from four tree crops: almond, dried plum, pistachio, and walnut. The previously developed DNA primers and quantitative real-time PCR (qPCR) assay systems were applied to detect six canker-causing pathogen groups, including Botryosphaeria dothidea , and species of Cytospora , Diplodia , Lasiodiplodia , Neofusicoccum , and Phomopsis. The concepts of molecular severity (MS) and latent infection index (LII) were introduced and applied to quantify the latent infection levels for these samples. Variation in incidence of latent infection among pathogen groups was observed, whereas the incidences were relatively low among species of Phomopsis and Diplodia. High incidences of Cytospora spp. were observed in two dried plum (prune) orchards. Most orchards showed high incidences of B. dothidea and Lasiodiplodia spp. and moderate incidences of Neofusicoccum spp. Variations in MS were observed among samples of the studied orchards, ranging from 4 to 8. The overall results of LII demonstrated that species of Diplodia and Phomopsis were less important in population development of canker-causing pathogens at the latent phase. Lasiodiplodia spp. were the most aggressive and had been well developed in populations among the studied tree crops. Cytospora spp. became predominant in two of the three dried plum orchards, whereas B. dothidea and Neofusicoccum spp. showed trends of increase in incidence across various tree crops. This study also demonstrated the usefulness of this sensitive qPCR approach in providing evidence of the latent phase of major canker-causing pathogens of stone fruit and nut crops at an early stage of latent infection in woody plant tissues.

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.

In recent decades, the cultivated area and production of nuts and olives have increased, driven by an increasing consumer interest in healthier food. Diseases of almond, pistachio, olive, and walnut crops caused by species belonging to the Botryosphaeriaceae family have caused concern worldwide. Although considerable progress has been made in elucidating the etiology of these diseases, scientific knowledge of other aspects of these diseases is more limited. In this article, we present an overview of the most important diseases caused by Botryosphaeriaceae fungi affecting almond, pistachio, olive, and walnut crops by focusing on ecology and epidemiology, primarily in California and Spain.

Almonds and peanuts are a rich source of proteins, vitamins and unsaturated fatty acids. However, they can be also contaminated by mycotoxigenic fungi; a reason that has enhanced to investigate efficient strategies of management of these fungal contaminations. Some Lactic acid bacteria have been proven capable of inhibiting growth and mycotoxin production in livestock and transform it into nontoxic derivatives. In this work, four lactic acid bacteria (LAB) were tested for their abilities to inhibit the growth and mycotoxin production of Aspergillus flavus and Aspergillus carbonarius. Antifungal activity was evaluated in agar medium as well as in almonds and peanuts. Results showed that LAB significantly inhibited Aspergillus flavus and Aspergillus carbonarius in agar medium but none of the strains were able to completely inhibit fungal growth. The highest fungal growth inhibition was obtained using L. kefiri FR7 (51.67% and 45.56% growth inhibition of A. flavus and A. carbonarius, respectively). The cell-free supernatants (CFS) from LAB reduced fungal growth with average growth inhibitions ranging from 13.33% to 40.56% and 12.78% to 37.78% for A. flavus and A. carbonarius, respectively. We noted also that cell-free supernatants at pH7 (CFS-pH7) from the entire tested LAB did not inhibit fungal growth. L. kefiri FR7 was the most effective strain in mycotoxin suppression with a reduction percentage reaching 97.22%, 95.27% and 75.26% for AFB1, AFB2 and OTA respectively. Moreover, the inoculation of L. kefiri FR7 in almonds artificially contaminated with A. flavus decrease 85.27% of AFB1 and 83.94% of AFB2 content after 7 days of incubation. On the other hand, application of L. kefiri FR7 in peanuts artificially contaminated with A. carbonarius reduced OTA content to 25%. Our study revealed the potential use and application of L. kefiri FR7 in the control of fungi growth and mycotoxins production in almonds and peanuts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The feasibility of using gaseous ozone, alone or in combination with other treatments, to decontaminate in-shell almonds and pistachios, prepared under different pathogen-inoculation conditions, was explored. Nuts were inoculated with either Salmonella enterica serovar Enteritidis or a potential Salmonella surrogate, Enterococcus faecium OSY 31284. The effect of inoculation method (with or without vacuum application), and of drying inoculated nuts (up to three days) on treatment efficacy was investigated. Inoculated nuts were subjected to gaseous ozone alone (almonds, pistachios) or ozone in combination with heating in brine solution (pistachios). Ozone treatment included application of vacuum (10 in Hg), followed by vessel pressurization to 12.5 psig with ozone‑oxygen mixture (160 g ozone/m 3 gas mixture) and holding for 30 min. Heating was conducted in a brine solution (5% NaCl) at 70 °C, for 10 min. Ozone-based treatments were significantly more effective (P < 0.05) on almonds than pistachios, with maximum S. Enteritidis reduction of 2.9 vs. 0.8 log CFU/g, regardless of inoculation method or the drying time. Treatment of inoculated pistachios with heated brine and gaseous ozone reduced S. Enteritidis population by 5.0 to 7.0 log CFU/g and was not significantly more effective than treatment with heated brine alone (reduction of 4.8 to 7.1 log CFU/g). Application of vacuum during inoculation increased bacterial population on nut kernels by approximately 1.2 log CFU/g, but the increase in inoculum population had no effect on inactivation of either species of inoculated bacteria. Decontamination treatments were less effective against both bacteria by up to 2 log CFU/g when drying time of inoculated nuts increased. E. faecium was significantly more resistant to heat and ozone treatment (P < 0.05) than was S. Enteritidis on pistachios, but not on almonds. Results of this study show that laboratory methodology affects observed treatment effectiveness. Considering its high resistance to the heat-ozone combination, E. faecium may not be a suitable surrogate for S. Enteritidis during processing of pistachios by this treatment. Efficacy of ozone gas to decontaminate S. Enteritidis-inoculated nuts depends heavily on the type of nut. Although reductions of S. Enteritidis populations on in-shell pistachios are low, treatment of in-shell almonds resulted in greater reductions, indicating the promise of this technology to enhance the safety of specific nut products., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A tremorgenic syndrome occurs in dogs following ingestion of moldy walnuts, and Penicillium crustosum has been implicated as the offending fungus. This is the first report of suspected moldy walnut toxicosis in man. An adult male ingested approximately eight fungal-infected walnut kernels and after 12 h experienced tremors, generalized pain, incoordination, confusion, anxiety, and diaphoresis. Following symptomatic and supportive treatment at a local hospital, the man made an uneventful recovery. A batch of walnuts (approximately 20) was submitted for mycological culturing and identification as well as for mycotoxin analysis. Penicillium crustosum Thom was the most abundant fungus present on walnut samples, often occurring as monocultures on isolation plates. Identifications were confirmed with DNA sequences. The kernels and shells of the moldy walnuts as well as P. crustosum isolates plated on yeast extract sucrose (YES) and Czapek yeast autolysate (CYA) agars and incubated in the dark at 25 °C for 7 days were screened for tremorgenic mycotoxins and known P. crustosum metabolites using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. A relatively low penitrem A concentration of only 1.9 ng/g was detected on the walnut kernels when compared to roquefortine C concentrations of 21.7 μg/g. A similar result was obtained from P. crustosum isolates cultured on YES and CYA, with penitrem A concentrations much lower (0.6-6.4 μg per g mycelium/agar) compared to roquefortine C concentrations (172-1225 μg/g). The authors surmised that besides penitrem A, roquefortine C might also play an additive or synergistic role in intoxication of man.

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB₁ and AFB₂ were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth ( a w 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days ( a w 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production.

A collection of 124 isolates of Penicillium spp. was created by monitoring fresh chestnuts, dried chestnuts, chestnut granulates, chestnut flour and indoor chestnut mills. Sequencing of the ITS region, β-tubulin and calmodulin, macro-morphology and secondary metabolite production made it possible to determine 20 species of Penicillium. P. bialowiezense was dominant in the fresh chestnuts, while P. crustosum was more frequent in the other sources. A pathogenicity test on chestnut showed that around 70% of the isolates were virulent. P. corylophilum and P. yezoense were not pathogenic, while the other 18 species had at least one virulent isolate. P. expansum and P. crustosum were the most virulent. The isolates were characterized to establish their ability to produce 14 toxic metabolites in vivo: 59% were able to produce at least one mycotoxin. P. expansum was able to produce patulin, chaetoglobosin A and roquefortine, while P. bialowiezense produced C. Mycophenolic acid. Cyclopenins and viridicatins were produced by most of the P. crustosum, P. polonicum, P. solitum and P. discolour isolates. Some of the P. crustosum isolates were also able to produce roquefortine C or penitrem A. Information about the occurrence of Penicillium spp. and their mycotoxins will help producers to set up management procedures that can help to control the fungal growth and the mycotoxin production of chestnuts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The in situ effect of microencapsulated 2(3)-tert-butyl-4 hydroxyanisole (BHA) on stored peanuts (Arachis hipogaea) intended for human consumption was evaluated. Peanut were stored unshelled in flexible containers called "big bags" that were made of polypropylene raffia. 100 kg of peanuts were used in each big bag and stored in refrigerated cells (<18 °C) for about 5 months in two different peanut processing companies during 2015/2016 period. Fungal populations, aflatoxin accumulation, BHA residues, acidity and fatty acid profile, sensory analyses, insect damage and environmental factors variation, were evaluated. At the end of the storage period, significant (p < 0.05) fungitoxic effects of the BHA formulation were observed in the order of 30 and 15% for the first and second company, respectively. Cladosporium, yeasts, Penicillium, Fusarium, Alternaria and Aspergillus were the main fungal isolates. No aflatoxins were found for both companies and years evaluated. In addition, taste of the peanuts was not significantly affected (p < 0.05) by formulation used and insect damage was always lower than 3%. However, different levels of BHA were detected throughout the experiment in the two companies, with final levels of 2.5 for the C1 and 275 ng BHA/g peanuts in C2. Formulation did not affect acidity and organoleptic properties of peanuts. These results show that BHA formulation could be used as part of alternative strategy for control of fungal contamination storage period., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Salmonellosis is a leading cause of hospitalisation due to gastroenteritis in Australia. A previous source attribution analysis for a temperate state in Australia attributed most infections to chicken meat or eggs. Queensland is in northern Australia and includes subtropical and tropical climate zones. We analysed Queensland notifications for salmonellosis and conducted source attribution to compare reservoir sources with those in southern Australia. In contrast to temperate Australia, most infections were due to non-Typhimurium serotypes, with particularly high incidence in children under 5 years and strong seasonality, peaking in summer. We attributed 65.3% (95% credible interval (CrI) 60.6-73.2) of cases to either chicken meat or eggs and 15.5% (95% CrI 7.0-19.5) to nuts. The subtypes with the strongest associations with nuts were Salmonella Aberdeen, S. Birkenhead, S. Hvittingfoss, S. Potsdam and S. Waycross. All five subtypes had high rates of illness in children under 5 years (ranging from 4/100 000 to 23/100 000), suggesting that nuts may be serving as a proxy for environmental transmission in the model. Australia's climatic range allows us to conduct source attribution in different climate zones with similar food consumption patterns. This attribution provides evidence for environment-mediated transmission of salmonellosis in sub-tropical regions.

Venturia effusa, which causes pecan scab, has developed resistance to fungicides that were once effective. Over 2 years, laboratory-based sensitivity of fentin hydroxide (TPTH) and tebuconazole in V. effusa and their efficacy under field conditions were compared. Leaf and nut scab were assessed on pecan trees receiving 10 applications of TPTH, tebuconazole, azoxystrobin, azoxystrobin plus tebuconazole, TPTH plus tebuconazole, or no fungicide (NTC) per year. Sensitivity of V. effusa on leaflets collected from treated and nontreated trees was assessed in June and September, respectively. The mean relative germination (RGe) on TPTH at 30 µg/ml was 10.9 and 40.9% in 2016 and 4.2 and 0.6% in 2017. Mean relative growth (RGr) on tebuconazole at 1 µg/ml was 45.5 and 34.6% in 2016 and 69.3 and 56.3% in 2017. In both years, leaf and nut scab were significantly lower on trees treated with azoxystrobin, azoxystrobin + tebuconazole, or TPTH + tebuconazole when compared with NTC and tebuconazole-treated trees. Compared with the NTC, tebuconazole did not significantly reduce leaf scab in 2017 or nut scab in either year, indicating that an RGr value between 34.6 and 69.3% is likely to result in a control failure on tebuconazole-treated trees. Although better activity was expected, TPTH reduced scab with RGe values between 0.6 and 40.9%. These results are valuable for developing fungicide sensitivity thresholds to better predict fungicide performance.

The use of plant associated, indigenous beneficial microbes for sustainable agriculture is getting worldwide acceptance as they successfully colonize at different plant niche under stress conditions to enhance the crop productivity. They also generate several plant growth regulators and protect plants from adversity like presence of salts and metals. In the present study, indigenous, halotolerant, plant growth promoting (PGP) bacterial isolates were isolated from the saline rhizospheric soil of groundnut plants aiming to investigate its in-vitro metal remediation capabilities under saline stress condition. Two pigmented bacteria were selected based on their phenotypic, biochemical, physiological and PGP characters and identified as members of family Bacillaceae (Bacillus and Halobacillus) based on 16S rRNA gene sequence similarity. The pigments were extracted, tested for different antioxidant properties and identified by GC-MS and FT-IR spectra. Simultaneously, both strains exhibited a wide range of salinity (NaCl≥25%), metal resistance (Zinc≈1700mgkg -1 , Aluminium≈1800mgkg -1 , Lead≈1800mgkg -1 ), pH (6-10), PGP attributes (indole - 1.05-3.15μgml -1 , ammonia - 0.13-19.95mmolml -1 , nitrite - 0.07-0.26mmolml -1 ) and antibiotics sensitivity revealing their wide range of metabolic diversity. In-vitro inoculation of groundnut seedlings with selected isolates under salinity (1% NaCl) and metal (Zn, Al and Pb) stress had a positive impact on different plant physiological parameters (lesser lignification, intact proto xylem and cortical parenchyma) which was correlated with PGP attributes. Microwave plasma atomic emission spectroscopy analysis of seedling samples also detected less amount of metals in plants treated with bacteria indicating, an establishment of plant-microbe protocooperation to withstand salinity and metal stress. This strategy can be implemented to improve crop production in saline metal polluted agriculture fields., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 μm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 μm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters., Competing Interests: The authors have declared that no competing interests exist.

Interactions between pathogenic and nonpathogenic fungal species in the tree canopy are complex and can determine if disease will manifest in the plant and in other organisms such as honey bees. Seasonal dynamics of fungi were studied in an almond orchard in California where experimental release of the atoxigenic biopesticide Aspergillus flavus AF36 to displace toxigenic Aspergillus strains has been conducted for five years. The presence of the vegetative compatibility group (VCG) YV36, to which AF36 belongs, in the blossoms, and the honey bees that attend these blossoms, was assessed. In blossoms, A. flavus frequencies ranged from 0 to 4.5%, depending on the year of study. Frequencies of honey bees carrying A. flavus ranged from 6.5 to 10%. Only one A. flavus isolate recovered from a blossom in 2016 belonged to YV36, while members of the VCG were not detected contaminating honey bees. Exposure of pollinator honey bees to AF36 was detected to be very low. The density of several Aspergillus species was found to increase during almond hull split and throughout the final stages of maturation; this also occurred in pistachio orchards during the maturation period. Additionally, we found that AF36 effectively limited almond aflatoxin contamination in laboratory assays. This study provides knowledge and understanding of the seasonal dynamics of Aspergillus fungi and will help design aflatoxin management strategies for almond. The evidence of the low levels of VCG YV36 encountered on almond blossoms and bees during pollination and AF36's effectiveness in limiting aflatoxin contamination in almond provided additional support for the registration of AF36 with USEPA to use in almond in California., Competing Interests: Having funding support from the Almond Board of California does not alter our adherence to PLOS ONE policies on sharing data and materials.

The presence of Aspergillus section Flavi and aflatoxin (AF) contamination was investigated in 112 samples of peanuts, almonds and dried figs collected in Algeria. The occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in different commodities has been determined with a sensitive method based on high performance liquid chromatography (HPLC) coupled with fluorescence detection with post-column photochemical derivatisation. Analytical results indicated that 28 samples of peanuts, 16 samples of almonds and 26 samples of dried figs contained detectable levels of AFs. A total of 69 samples (61.6%) were contaminated with AFB1 ranging from the limit of quantification to 174 µg kg -1 . AFB2 was found in 12 samples (10.7%) and varied from 0.18 to 193 µg kg -1 . Seven samples revealed AF concentrations lower than the limit of quantification. Eleven peanut and fourteen dried fig samples exceeded the European maximum limits for AFB1.

The multistate Escherichia coli ( E. coli ) O157:H7 outbreak associated with in-shell hazelnuts highlights the pathogen's ability to involve non-traditional vehicles in foodborne infections. Furthermore, it underscores significant gaps in our knowledge of pathogen survivability and persistence on nuts. Therefore, this study investigated the ability of E. coli O157:H7 to attach and survive on in-shell hazelnuts. In-shell hazelnuts were inoculated with a four-strain mixture of E. coli O157:H7 at 7.6 log colony forming units (CFU)/nut by wet or dry inoculation, stored at ambient conditions (24 ± 1 °C; 40% ± 3% relative humidity (RH) and sampled for twelve months. For the attachment assay, in-shell hazelnuts were inoculated and the adherent population was enumerated at 30 s -1 h following inoculation. Irrespective of the inoculation method, ~5 log CFU of adherent E. coli O157:H7 was recovered from the hazelnuts as early as 30 s after inoculation. Conversely, pathogen survival was significantly reduced under dry inoculation with samples being enrichment negative after five months of storage ( p < 0.05). On the other hand, wet inoculation led to a significantly longer persistence of the pathogen with ~3 log CFU being recovered from the in-shell nuts at 12 months of storage ( p < 0.05). These results indicate that E. coli O157:H7 can survive in significant numbers on in-shell hazelnuts when stored under ambient conditions.

Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus and a few other closely related species of Aspergillus. These highly toxigenic and carcinogenic mycotoxins contaminate global food and feed supplies, posing widespread health risks to humans and domestic animals. Field application of nonaflatoxigenic strains of A. flavus to compete against aflatoxigenic strains has emerged as one of the best management practices for reducing aflatoxins contamination, yielding successful commercial products for corn, cotton seed, and peanuts. In this study, we sequenced the genome and transcriptome of atoxigenic (does not produce aflatoxin or cyclopiazonic acid) A. flavus strain WRRL 1519 isolated from a tree nut orchard to define the genetic characteristics of the strain in relation to aflatoxigenic and other nonaflatoxigenic A. flavus strains. WRRL 1519 strain was similar to other strains in size (38.0 Mb), GC content (47.2%), number of predicted secondary metabolite gene clusters (46), and number of putative proteins (12 121). About 87.4% of the predicted proteome had high shared identity with protein sequences derived from other A. flavus genomes. However, the atoxigenic A. flavus strain WRRL 1519 had deletions, or low shared identity, for many genes in the clusters required for aflatoxins and cyclopiazonic acid (CPA) synthesis. Over half of the aflatoxin synthesis gene cluster was missing, and none of the components of the CPA gene cluster were identified with high sequence similarity. Importantly, the strain appeared to maintain functional sequences of several genes thought to be required for high infectivity. Since the ability to grow on target crop is an important attribute for a successful biocontrol agent, these results indicate that the nonaflatoxigenic A. flavus strain WRRL 1519 would be a good candidate as a biocontrol agent for reducing aflatoxin and CPA accumulation in high-value nut crops.

Low-water-activity foods have been involved in recalls and foodborne disease outbreaks. Increased consumption; better detection methods and reporting systems; improved surveillance, trace-back, and ability to connect sporadic foodborne illnesses; and inadequate implementation of food safety programs are some of the likely reasons for the increase in frequency of recalls and outbreaks linked to dry foods. Nuts and grains can be contaminated with foodborne pathogens at any stage during production, processing, storage, and distribution. Focusing on preharvest contamination, the various potential sources of contamination include soil, animal intrusion, contaminated harvesting equipment, harvest and preharvest handling, storage conditions, and others. The low water activity of nuts and grains prevents the growth of most foodborne pathogens on their surfaces. The long-term survival of bacterial foodborne pathogens ( Salmonella , Escherichia coli O157:H7, and Listeria monocytogenes ) on dry foods has been documented in the literature for different nut types. Preventing contamination is the key to avoiding foodborne disease risks linked to dry foods. The implementation of good agricultural practices and other food safety systems provides a proactive approach to address concerns thoroughly. A plethora of research is available on preventing the growth of mycotoxin-producing fungi on the surface of nuts and grains. Milling is an effective mechanism to reduce the microbial load on grains. This review focuses on providing information about associated foodborne microorganisms, preharvest contamination sources, and good agricultural practice recommendations for nuts and grains.

Due to recent foodborne outbreaks, peanuts have been considered a potential risk for Salmonella transmission. For this reason, the aim of this study was to determine the prevalence and contamination load of Salmonella, Escherichia coli and Enterobacteriaceae throughout the peanut supply chain in Brazil. Samples of peanuts and peanut-containing processed products from post-harvest (n=129), secondary processing (n=185) and retail market (n=100) were analyzed. The results showed high Enterobacteriaceae counts in the post-harvest samples. At the end of the secondary processing, 16% of the samples remained contaminated by this group of microorganisms. Six peanut samples from primary production and one sample of peanut butter were tested positive for E. coli while Salmonella was detected in nine samples (2.2%): six from post-harvest, two from the initial stage of the secondary processing and one from retail. The Salmonella counts ranged between 0.004 and 0.092MPN/g and five serotypes were identified (Muenster, Miami, Javiana, Oranienburg, Glostrup). The results demonstrated a high prevalence of Enterobacteriaceae and low prevalence of E. coli throughout the peanut supply chain. Furthermore, it was verified that peanuts may become contaminated by Salmonella during different stages of the supply chain, especially at post-harvest., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Propylene oxide (PPO), a chemical fumigant, has been validated to reduce Salmonella on bulk almonds but has not been evaluated for other tree nuts. There is a need to identify nonpathogenic surrogate microorganisms whose inactivation is comparable to that of Salmonella to assure effective PPO processing parameters in different packaging configurations without introducing Salmonella into the pasteurization facility. The objective of this research was to compare the reduction of Salmonella and three potential surrogate bacterial strains, Enterococcus faecium ATCC 8459, Pediococcus acidilactici ATCC 8042, or Staphylococcus carnosus ATCC 51365, on cashews and macadamia nuts processed by using PPO. Whole cashews and macadamia nuts were coinoculated with a five-strain cocktail of Salmonella and one surrogate, dried to the original water activity of 0.44 to 0.51 before being packaged in woven polypropylene bags (2.3 kg), and shipped overnight in Styrofoam containers under ambient conditions to a commercial facility for PPO treatment. Salmonella and surrogates were recovered by vigorous shaking in phosphate buffer (1:1, m/v), serial diluted, and plated onto tryptic soy agar with an overlay of xylose lysine Tergitol 4 for Salmonella or mannitol salt agar or bile esculin azide agar for each surrogate. The mean log reductions of Salmonella and each surrogate ( n = 18), within a sample and among all trials (three independent), were compared by using a matched pairs t test. Reduction in log CFU per gram of Salmonella was significantly greater than that of E. faecium on both macadamia nuts (7.3 ± 0.19 versus 6.4 ± 0.31) and cashews (5.4 ± 0.15 versus 5.1 ± 0.25) and significantly greater than P. acidilactici on both nuts (7.8 ± 0.22 versus 6.3 ± 0.33 on macadamia nuts and 4.9 ± 0.22 versus 4.1 ± 0.25 on cashews). Reduction of S. carnosus exceeded that of Salmonella. E. faecium and P. acidilactici may be considered as surrogates for Salmonella on whole macadamia nuts and cashews processed by using PPO.

An extensive sampling of Aspergillus section Flavi considered to be the main agent responsible for aflatoxin contamination, was carried out in the field and along the processing phases of chestnut flour production in 2015. Fifty-eight isolates were characterized by means of biological, molecular and chemical assays. The highest incidence of Aspergillus section Flavi was found in the field. The identification of the isolates was based on β-tubulin and calmodulin gene sequences. A. flavus was found to be the dominant species, and this was followed by A. oryzae var effusus, A. tamarii, A. parasiticus and A. toxicarius. Nineteen percent of the strains produced aflatoxins in vitro and forty percent in vivo. The pathogenicity assay on chestnut showed 56 virulent strains out of 58. The molecular, morphological, chemical and biological analyses of A. flavus strains showed an intraspecific variability. These results confirm that a polyphasic approach is necessary to discriminate the species inside the Aspergillus section Flavi. The present research is the first monitoring and characterization of aflatoxigenic fungi from fresh chestnut and the chestnut flour process, and it highlights the risk of a potential contamination along the whole chestnut production chain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the present study, two different fractions of pine nut (Pinus koraiensis) protein hydrolysate powder (PNPHP 1-3kDa and PNPHP 3-10kDa ) were prepared and stored for fifteen days at 25°C and 50% relative humidity. Changes in moisture absorption, secondary structure and flavor compounds were measured at various time intervals over fifteen days. Results showed that the PNPHP 3-10kDa had higher moisture absorption capacity from day 6 to day 15 and that the secondary structure of two PNPHP fractions was changed. Major nine and eight flavor compounds were identified from PNPHP 1-3kDa and PNPHP 3-10kDa , respectively. PNPHP 1-3kDa mainly generated aldehydes and PNPHP 3-10kDa generated pyrazines. Finally, the formation of pyrazines was conjectured. The data suggests that Maillard reaction, microbial fermentation and lipid oxidation occurred during storage. The reactions appeared to be strongest by the sixth day., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nuts are rich in nutrients and mounting evidence shows that consumption reduces cardiovascular disease (CVD) incidence. Nuts may also be a major source of aflatoxin B₁, a potent liver carcinogen and the risk/benefit balance is unknown. Based on national statistics and data from the PREDIMED intervention trial, we estimated the potential CVD-reduction if Swedes aged 55-79 consumed 30 g nuts/day, instead of the current national average of five grams per day. We also assessed the reduction in disability-adjusted life years (DALYs) due to myocardial infarction (MI) and stroke. We estimated the aflatoxin B₁ exposure from nuts and calculated the margin of exposure. The approximation that one nanogram aflatoxin B₁/kg body weight/day results in one additional liver cancer case/10 million person-years was used to estimate the number of liver cancer cases. The increased nut consumption scenario prevented more than 7000 CVDs in 2013 (306/100,000 person-years) and contributed to about 55,000 saved DALYs for stroke and 22,000 for MI. The concomitant increase in aflatoxin B₁ exposure caused an estimated zero to three additional cases of liver cancer, corresponding to 159 DALYs spent, emphasizing the associated risks. Increased nut consumption, as part of a varied healthy diet, is warranted even when aflatoxin B₁ exposure is taken into account. However, efforts to reduce aflatoxin exposure from food are essential., Competing Interests: The authors declare no conflict of interest.