Fate of Aflatoxins during Almond Oil Processing.

ABSTRACT: Almonds rejected as inedible are often used for production of almond oil. However, low-quality almonds are frequently contaminated with aflatoxins, and little is known regarding transfer of aflatoxins to almond oil during processing. In this study, oil was produced from reject almonds by hexane extraction. Of 19 almond samples that were naturally contaminated with aflatoxins, 17 oil samples contained measurable amounts of aflatoxins, and aflatoxin content of contaminated oil was correlated with aflatoxin content of the nuts. However, oil aflatoxin levels were not correlated with the oxidation level of the oil as measured by percent free fatty acids and peroxide value. Adsorbents used in oil refining were tested for their ability to remove aflatoxins from contaminated oil. Fuller's earth and bentonite were the most effective, removing 96 and 86% of total aflatoxins from contaminated oil samples, respectively. Treatment with diatomaceous earth, in contrast, had no effect on aflatoxin levels in oil. These results show that oil refining steps using mineral clay adsorbents may also function to remove aflatoxins from contaminated oil.

Effect of Blanching on Aflatoxin Contamination and Cross-Contamination of Almonds.

ABSTRACT: Blanching of almonds was examined for reducing the aflatoxin content of contaminated nuts. Almonds with intact pellicles were spiked with aflatoxin B1 (AFB1) and blanched at 85°C. Following blanching, almond kernels and pellicles contained 20 and 19% of the spiked AFB1, respectively. The blanching water contained an additional 41% of the spiked AFB1. In a separate study, postblanching water was spiked with AFB1 and used for subsequent blanching of uncontaminated almonds. The resulting blanched kernels acquired 3.3% of the AFB1 from the spiked water, demonstrating a low level of cross-contamination from reused contaminated blanching water. The effect of the blanching temperature on partitioning of AFB1 from almonds to blanching water was significant at a 20-ppb spiking level, but not at 100 ppb. AFB1 levels that were unaccounted for in the mass balance of blanching components were presumed to be lost due to binding to water-solubilized almond components and were independent of pH and blanching time. Blanching reduced total aflatoxins in naturally contaminated almonds by 13 to 76%, depending on almond quality, as well as blanching time and temperature. These results indicate that the association between almond components and aflatoxin generated through mold contamination is more complex than in spiking experiments.

Curcumin and Quercetin as Potential Radioprotectors and/or Radiosensitizers for X-ray-based Sterilization of Male Navel Orangeworm Larvae.

Two natural compounds (quercetin and curcumin) were tested as sensitizing or protecting agents for Navel Orangeworm (NOW) larvae under x-ray sterilization, with the aim to reduce required doses and thus facilitate the substitution of x-ray for radioisotopes. The compounds were added to NOW diet at concentrations between 0 and 1.0 mmol kg-1 and subsequent reared male larvae were subjected to x-ray irradiation (90 keV, 9 mA) to doses up to 15 Gy. Upon emergence as adults, surviving male NOW were paired with colony virgin females and placed in isolation for observation of deformity, mortality, and fertility. Treatments included rearing larvae on infused diet before irradiation, after irradiation, and both. Results were tabulated as percentage of insects that were dead/deformed, infertile, or fertile and subjected to chi-squared analysis. While insect populations subjected to quercetin treatments were not found to be significantly different from control at any x-ray dose, all curcumin treatments yielded significant differences at an absorbed dose of 10 Gy, both in terms of decreased mortality and fertility. While none of the treatments resulted in acceptable mortality/deformity rates, the observed effects strongly support the need for continued testing of natural compounds for their efficacy to reduce required dose levels for sterilization.

Degradation kinetics of aflatoxin B1 and B2 in solid medium by using pulsed light irradiation.

BACKGROUND: Pulsed light (PL) is a new potential technology to degrade aflatoxin. The objective of this study was to investigate the degradation characters of aflatoxin B1 (AFB1 ) and B2 (AFB2 ) treated under PL irradiation. A kinetic degradation study of AFB1 and AFB2 in solid medium was performed under PL irradiation at different initial concentrations of AFB1 (229.9, 30.7 and 17.8 µg kg-1 ) and AFB2 (248.2, 32.2 and 19.5 µg kg-1 ) and irradiation intensities (2.86, 1.60 and 0.93 W cm-2 ) of PL. A second-order reaction model was applied to describe degradation of AFB1 and AFB2 . RESULTS: The results showed that the degradation of AFB1 and AFB2 followed the second-order reaction kinetic model well (R2  > 0.97). The degradation rate was proportional to the intensities of PL irradiation and the initial concentrations of aflatoxins. CONCLUSION: It is concluded that the degradation of AFB1 and AFB2 with the use of PL could be accurately described using the second-order reaction kinetic model. © 2018 Society of Chemical Industry.

Silo-Stored Pistachios at Varying Humidity Levels Produce Distinct Volatile Biomarkers.

Fungal-contaminated tissues are known to produce volatile profiles that are different from uncontaminated tissues. Fungi require certain water activity levels before growth can occur. For nonxerophilic fungi, a water activity of 0.85 is typical for growth, and for extreme xerophilic fungi, the water activity can be as low as 0.64. Recent investigations with stored pistachios (kernels in shell, no hull tissue) at varying relative humidities showed differences among the collected volatile profiles at the tested humidities (ambient, 63, 75, and 84%). Water activities of the kernel and shell were also measured. Results showed significant changes in volatile profiles as a function of water activity of the corresponding pistachio tissue with measured water activity levels at or below that of what is considered extreme xerophilic activities. Because fungal growth, including mycotoxigenic fungi, is dependent upon water activity, the detected volatile profiles could be used for early detection of fungal presence. Multivariate analysis of the volatile data demonstrated significant differences among the volatile profiles at the tested relative humidity levels, and several volatiles were identified as biomarkers of increased humidity and likely fungal development.

Differentiation of Volatile Profiles from Stockpiled Almonds at Varying Relative Humidity Levels Using Benchtop and Portable GC-MS.

Contamination by aflatoxin, a toxic metabolite produced by Aspergillus fungi ubiquitous in California almond and pistachio orchards, results in millions of dollars of lost product annually. Current detection of aflatoxin relies on destructive, expensive, and time-intensive laboratory-based methods. To explore an alternative method for the detection of general fungal growth, volatile emission profiles of almonds at varying humidities were sampled using both static SPME and dynamic needle-trap SPE followed by benchtop and portable GC-MS analysis. Despite the portable SPE/GC-MS system detecting fewer volatiles than the benchtop system, both systems resolved humidity treatments and identified potential fungal biomarkers at extremely low water activity levels. This ability to resolve humidity levels suggests that volatile profiles from germinating fungal spores could be used to create an early warning, nondestructive, portable detection system of fungal growth.

Simultaneous decontamination and drying of rough rice using combined pulsed light and holding treatment.

BACKGROUND: Pulsed light (PL) technology has been proven effective in food disinfection. However, increasing the light intensity or treatment time could swiftly increase the temperature of the food product. Using the thermal effect in an appropriate way may achieve a simultaneous disinfection and drying effect. The objective of this study was to investigate the feasibility of simultaneous disinfection and drying of rough rice using PL and holding treatment. RESULTS: Freshly harvested rice samples were inoculated by Aspergillus flavus (A. flavus) and treated using PL under different intensities and durations followed by holding treatment. The PL treatment under intensity of 1.08 W cm(-2) for 21 s led to a reduction of 0.29 log cfu g(-1) on the population size of A. flavus spores. After holding treatment, a 5.2 log cfu g(-1) reduction was achieved. The corresponding total moisture removal reached 3.3% points. No adverse effect on milling quality was detected after the treatment. CONCLUSION: The obtained results revealed that the combined PL and holding treatment had good potential for successful application in the rice industry to simultaneously achieve disinfection and drying. © 2015 Society of Chemical Industry.

Semiochemicals from ex situ abiotically stressed cactus tissue: a contributing role of fungal spores?

Semiochemicals play a central role in communication between plants and insects, such as signaling the location of a suitable host. Fungi on host plants can also play an influential role in communicating certain plant vulnerabilities to an insect. The spiroketal conophthorin is an important semiochemical produced by developing fungal spores. Spiroketals are also used as signals for scolytid communication. Plants and fungi are known to emit varying volatile profiles under biotic and abiotic stress. This paper reports distinctive temporal-volatile profiles from three abiotic treatments, room temperature (control), -15 °C (cold), and -15 °C to room temperature (shock), of cactus tissue plugs. Volatiles from the three treatments included monoterpenes from control plugs, compounds of varying classes and origin at later stages for cold plugs, and known semiochemicals, including spiroketals, at later stages for shock plugs. The results highlight several important findings: a unique tissue source of the spiroketals; abiotic cold-shock stress is indicated as the cause of spiroketal production; and, given previous findings of spirogenesis, fungal spore involvement is a probable biosynthetic origin of the spiroketals. These findings suggest an important role of fungal volatiles as signaling plant vulnerability to insects.

Spread of Aspergillus flavus by Navel Orangeworm (Amyelois transitella) on Almond.

Navel orangeworm (NOW) damage to almond is correlated with increased incidence of aflatoxin contamination caused by Aspergillus flavus. However, no reports demonstrate a causative relationship between NOW feeding and A. flavus infection. To demonstrate the potential of NOW to act as a vector of A. flavus on almond, NOW eggs were dusted with A. flavus and incubated in microchambers adjacent to but not touching agar plates or almond kernels. Following egg hatch, A. flavus colonies developed on agar along trails left by NOW larvae. Almond kernels damaged with A. flavus-carrying NOW showed higher incidence of A. flavus colonization and aflatoxin contamination than control treatments. Interestingly, levels of aflatoxin in NOW-damaged, A. flavus-infected almond were significantly higher than control treatments, even in the absence of visible fungal growth. Commercial almond orchards had a relatively low level of contamination with Aspergillus section Flavi in spring and early summer and a high level during summer, corresponding with the higher level of NOW infestation of the crop. Our study demonstrates that NOW is capable of vectoring A. flavus to almond, and that monitoring and sorting of almond kernels for insect damage is warranted to limit aflatoxin contamination potential both before and after harvesting.

Generation of the volatile spiroketals conophthorin and chalcogran by fungal spores on polyunsaturated fatty acids common to almonds and pistachios.

The spiroketal (E)-conophthorin has recently been reported as a semiochemical of the navel orangeworm moth, a major insect pest of California pistachios and almonds. Conophthorin and the isomeric spiroketal chalcogran are most commonly known as semiochemicals of several scolytid beetles. Conophthorin is both an insect- and plant-produced semiochemical widely recognized as a nonhost plant volatile from the bark of several angiosperm species. Chalcogran is the principal aggregation pheromone component of the six-spined spruce bark beetle. Recent research has shown conophthorin is produced by almonds undergoing hull-split, and both spiroketals are produced by mechanically damaged almonds. To better understand the origin of these spiroketals, the volatile emissions of orchard fungal spores on fatty acids common to both pistachios and almonds were evaluated. The volatile emission for the first 13 days of spores placed on a fatty acid was monitored. The spores investigated were Aspergillus flavus (atoxigenic), A. flavus (toxigenic), Aspergillus niger, Aspergillus parasiticus, Penicillium glabrum, and Rhizopus stolonifer. The fatty acids used as growth media were palmitic, oleic, linoleic, and linolenic. Spores on linoleic acid produced both spiroketals, those on linolenic acid produced only chalcogran, and those on palmitic and oleic acid did not produce either spiroketal. This is the first report of the spiroketals conophthorin and chalcogran from a fungal source.

Volatile analysis of ground almonds contaminated with naturally occurring fungi.

Aflatoxigenic aspergilli inflict major economic damage to the tree nut industry of California, with the highest negative impact to almonds. Aspergilli and fungi in general are known to emit volatiles in varying quantity and composition dependent upon their growth media. The goal of the study was to determine the volatile emission of whole and blanched almonds that had been picked out and labeled as inedible by processors. The aflatoxin content and number of colony forming units of each sample were also determined. A total of 23 compounds were consistently detected and identified. Several volatiles from the blanched almonds demonstrated significant increases when compared to the emissions of whole almonds. Several of these volatiles are considered fatty acid decomposition products and included hexanal, heptanal, octanal, nonanal, 3-octen-2-one, tetramethylpyrazine, and decanal. The almond samples investigated were characteristic of a typical postharvest environment and illustrative of potential contamination within a stockpile or transport container. Volatiles indicative of fatty acid decomposition were predominant in the samples that underwent some form of blanching. The emission amounts of hexanal, heptanal, octanal, and hexanoic acid increased 3-fold in samples contaminated with aflatoxin; however, due to variability between samples they could not be considered as indicator volatiles for aflatoxin content. The emission profile of volatiles from almond kernels contaminated with naturally occurring aspergilli and associated fungi is heretofore unreported.

Inhibition of ochratoxin A production and growth of Aspergillus species by phenolic antioxidant compounds.

The phenolic antioxidants, gallic acid, vanillic acid, protocatechuic acid, 4-hydroxybenzoic acid, catechin, caffeic acid, and chlorogenic acid were studied for their effects on ochratoxin A (OTA) production and fungal growth of ochratoxigenic Aspergilli. Of the 12 strains tested, which included A. alliaceus, A. lanosus, A. ochraceus, A. albertensis, A. melleus, A. sulphureus, A. carbonarius, A. elegans, and A. sclerotiorum, the greatest inhibition of OTA production was seen in A. sulphureus, A. elegans, and A. lanosus. Vanillic acid and 4-hydroxybenzoic acid were the most inhibitory to both OTA production and growth of most of the strains tested. However, A. ochraceus was not inhibited by either compound, and A. carbonarius was not inhibited by vanillic acid. The effect of each compound on OTA production and growth differed among strains and generally was variable, suggesting that species-specific OTA production and response to phenolic compounds may be influenced by different ecological and developmental factors. In addition, inhibition of OTA production by antioxidant compounds may be useful in determining biosynthetic and regulatory genes involved in both OTA production and stress response in ochratoxigenic Aspergilli.

Isolation of bacterial antagonists of Aspergillus flavus from almonds.

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.

Identification of phenolics for control of Aspergillus flavus using Saccharomyces cerevisiae in a model target-gene bioassay.

The yeast Saccharomyces cerevisiae was used in a high-throughput bioassay to identify phenolic agents for control of the aflatoxigenic fungus Aspergillus flavus. Veratraldehyde, 1, cinnamic acid, 5, and the respective benzoic acid derivatives vanillin, 2, vanillic acid, 3, and vanillylacetone, 4, and cinnamic acid derivatives o-coumaric acid, 6, m-coumaric acid, 7, and p-coumaric acid, 8, showed significant antifungal activities (from highest to lowest, 2, 5 > 1 > 6, 7 > 4 > 3, 8) in the yeast system, with caffeic acid, 9, having little to no effect. Antifungal activity levels against A. flavus were similar. This similarity in antifungal activity demonstrated the usefulness of the S. cerevisiae bioassay for screening antifungal compounds. Assays using deletion mutants of yeast identified signal transduction and antioxidative stress response genes important to fungal tolerance. Targeting the antioxidative stress response system with certain compounds (e.g., 4) in combination with strobilurin fungicides had a synergistic effect against both fungi.

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus.

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

Aspergillus on tree nuts: incidence and associations.

California exports tree nuts to countries where they face stringent standards for aflatoxin contamination. Trade concerns have stimulated efforts to eliminate aflatoxins and Aspergillus flavus from almonds, pistachios and walnuts. Incidence of fungi on tree nuts and associations among fungi on tree nuts were studied. Eleven hundred pistachios, almonds, walnuts and brazil nuts without visible insect damage were plated on salt agar and observed for growth of fungi. Samples came both from California nut orchards and from supermarkets. To distinguish internal fungal colonization of nuts from superficial colonization, half the nuts were surface-sterilized before plating. The most common genera found were Aspergillus, Rhizopus and Penicillium. Each species of nut had a distinct mycoflora. Populations of most fungi were reduced by surface sterilization in all except brazil nuts, suggesting that they were present as superficial inoculum on (rather than in) the nuts. In general, strongly positive associations were observed among species of Aspergillus; nuts infected by one species were likely to be colonized by other species as well. Presence of Penicillium was negatively associated with A. niger and Rhizopus in some cases. Results suggest that harvest or postharvest handling has a major influence on nut mycoflora, and that nuts with fungi are usually colonized by several fungi rather than by single species.