Fungal community and toxigenic taxa in chestnut fruits in postharvest conditioning process and storage.

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.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

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.

Impacts of Gaseous Ozone (O3) on Germination, Mycelial Growth, and Aflatoxin B1 Production In Vitro and In Situ Contamination of Stored Pistachio Nuts.

Pistachio nuts can become colonized by mycotoxigenic fungi, especially Aspergillus flavus, resulting in contamination with aflatoxins (AFs). We examined the effect of gaseous O3 (50-200 ppm; 30 min; 6 L/min) on (a) in vitro germination, (b) mycelial growth, and (c) aflatoxin B1 (AFB1) production on a milled pistachio nut-based medium at different water activity (aw) levels and at 30 °C. This was complimented with in situ studies exposing raw pistachio nuts to 50-200 ppm of O3. Exposure of conidia to gaseous O3 initially resulted in lower germination percentages at different aw 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 O3 dose, with significant inhibition only at 0.98 aw. The production of AFB1 after O3 treatment and storage for 10 days was stimulated in A. flavus colonies at 0.98 aw. Raw pistachio nuts inoculated with A. flavus conidia prior to O3 exposure showed a significant decrease in population after 20 days of storage. However, AFB1 contamination was stimulated in most O3 treatments. The relationship between exposure concentration, time and prevailing aw levels on toxin control needs to be better understood for these nuts.

Heat Inactivation of Listeria monocytogenes on Pecans, Macadamia Nuts, and Sunflower Seeds.

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.

Dietary Risk Assessment and Consumer Awareness of Mycotoxins among Household Consumers of Cereals, Nuts and Legumes in North-Central Nigeria.

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 B1 was the most prevalent mycotoxin in peanut (64%) and rice (57%), while fumonisin B1 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.

Growth Behavior of Probiotic Lactobacillus casei 01 in Tiger Nut Milk Prepared with Milk By-Products.

<b>Background and Objective:</b> 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 <i>L. casei</i>-01 in tiger nut milk made with milk permeate or cheese whey as an extraction medium. <b>Materials and Methods:</b> 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 <i>L. casei</i>-01 at 2%. The Titratable Acidity (TA) and <i>L. casei</i>-01 were determined during fermentation at 37°C for 8 hrs as well as during cold storage at 4°C for 20 days. <b>Results:</b> 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 <i>L. casei</i>-01 were the highest in fermented whey-tiger nut milk after 10 days. <b>Conclusion:</b> <i>Lactobacillus casei</i>-01 can grow with high viability in permeate or whey-tiger milk.

Antimicrobial activity of ClO2 gas against Salmonella Enteritidis on almonds.

Nuts, including almonds, are occasionally contaminated with Salmonella spp. In this study, we used chlorine dioxide (ClO2) 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 ClO2 gas generated from 1.0 or 1.5 mL ClO2 solution in a sealed container at 50 or 60 °C (43% relative humidity) for up to 10 h. The concentration of ClO2 gas peaked at 354-510 and 750-786 ppm within 0.5 h upon deposition of 1.0 and 1.5 mL of aqueous ClO2, 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 ClO2 gas and decreased to below the detection limit (1.7 log cfu/sample) at all ClO2 concentrations after 8 h. At 60 °C, the microbial population fell below the detection limit within 1 h, regardless of the volume of ClO2 solution supplied. Microbial survival on almonds treated with ClO2 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 ClO2 gas treatment, but yellowness (b value) increased. Results showed that Salmonella on almonds was successfully inactivated by ClO2 gas treatment and the microbial survival did not occur during storage.

Microbiomes of commercially-available pine nuts and sesame seeds.

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.

Prevalence of lipase producer Aspergillus niger in nuts and anti-biofilm efficacy of its crude lipase against some human pathogenic bacteria.

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.

Analysis of E.U. Rapid Alert System (RASFF) Notifications for Aflatoxins in Exported U.S. Food and Feed Products for 2010-2019.

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.

Survey of Salmonella in raw tree nuts at retail in the United States.

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.

Potential probiotic and food protection role of wild yeasts isolated from pistachio fruits (Pistacia vera).

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.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillusflavus and A. parasiticus in Hazelnut.

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.

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt.

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.

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.

Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from selected groundnut growing agro-ecological zones of Uganda.

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 (χ2 = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ2 = 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 (χ2 = 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.

Inactivation of Salmonella and Listeria monocytogenes on dried fruit, pistachio nuts, cornflakes and chocolate crumb using a peracetic acid-ethanol based sanitizer or Advanced Oxidation Process.

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/cm2 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 H2O2 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.

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.

HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts.

Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.