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.


Introduction
Ozone (O 3 ) is commonly generated in the summer because of the reactions between photochemicals in the atmosphere in the presence of sunlight. O 3 gas has been commonly used in the food industry for sanitizing packaging materials, raw materials, and storage facilities [1]. It has been suggested that the advantage of O 3 over other remedial chemicals used in foodstuffs is that it is residue-free. It decomposes to diatomic oxygen rapidly because of its short half-life, which is about 20-50 min in the atmosphere and 1-10 min in water [2]. However, it is a very corrosive gas, and for its use, it is critical that appropriate tubing (PTFE), steel, or glass systems are used. Manning and Teidemann [3] showed that small increases in ozone (O 3 ) concentrations (40-60 ppb) can influence the mycobiota of plant surfaces and perhaps the biosynthesis of toxins. Slight increases in exposure to gaseous O 3 as a pollutant was previously shown to change the phyllosphere mycobiota on conifer needles [4].
At the present time, the EU legislation established a maximum level of 12 µg/kg aflatoxin B 1 (AFB 1 ) in nuts, including pistachios used for processing, and 8 µg/kg AFB 1 for direct human consumption [5]. The AF contamination of pistachio nuts occurs either pre-harvest, because of early splitting of the shells and insect damage, or during subsequent drying and processing [6]. For example, in Iran, an analysis of 10,000+ samples of pistachio nuts showed AFB 1 in 36%, with almost 12% exceeding the maximum level in Iran (5 µg/kg) [7]. In Algeria, between 1998 and 2002, the highest AFB 1 in 523 samples was 113 µg/kg, with a mean range of 1 to 3.8 µg/kg [8]. In Tunisia, 76% of pistachio nuts were found to be contaminated with AFs during storage [9]. Delayed harvesting and processing and storage has been shown to significantly influence and often increase the AFB 1 contamination of pistachio nuts [6,10]. Thus, there has been interest in examining treatments such as gaseous O 3 for the mitigation of AFs in nuts, including pistachios [11][12][13].
Some previous studies have examined the efficiency of electrochemically-generated O 3 on the activity of aflatoxigenic fungi and aflatoxin (AFs) in different nuts, including peanuts and brazil nuts. However, there are less studies on pistachio nuts [11][12][13]. Mylona et al. [14] showed that gaseous O 3 (100-400 ppm) was not very effective against the microconidal germination of Fusarium verticiilioides in vitro, but it was able to reduce fumonisin B 1 (FB 1 ) contamination in stored maize grain after treatment. Indeed, the initial inhibition of germination was followed by recovery, growth, and FB 1 production. However, Sultan et al. [12] found that gaseous O 3 was very effective in inhibiting the germination of conidia of A. flavus, but it had little efficacy in controlling the mycelial growth of A. flavus strains on peanut-based media, regardless of water activity (a w ). Studies have been carried out on the O 3 treatment of Brazil nuts and showed that exposure to this gas affected the growth of the mycobiota and decreased AFs [11]. They used three concentrations of O 3 (10, 14, and 31.5 mg/L) for five hours and found that this inhibited colonization by both A. flavus and A. parasiticus over a storage period of 180 days. However, at low concentrations of O 3 , the fungi were still able to grow. Care is needed with nuts because the high concentration of lipids can interact with gaseous O 3 , resulting in tainting and off-flavors [15]. The sensitivity of fungal species to O 3 exposure may vary and depends on the exposure period, concentration applied, and type of commodity. It can also be influenced by moisture content and spore morphology [16,17].
No previous studies have examined the effect of gaseous O 3 (0-200 ppm) as a control measure for germination, colonization, and AFB 1 in raw milled pistachio-based media or in stored raw pistachios. Thus, the aim of this study was to examine the use of gaseous O 3 to control (a) in vitro conidial germination, (b) in vitro mycelial growth and AFB 1 production, and (c) A. flavus populations on raw pistachio nuts when inoculated with conidia of this species and exposed to O 3 and the effects on subsequent AFB 1 contamination after 20 days of storage at different a w levels.

Effect of O 3 on Conidial Germination of A. flavus In Vitro on a Milled Pistachio-Based Medium
The mean germination of conidia of A. flavus was influenced by gaseous O 3 treatment, regardless of the a w level used ( Figure 1)

In Vitro Effect of Gaseous O 3 Treatment on Mycelial Growth
The mycelial extension of A. flavus (strain AB3) was significantly inhibited after O 3 treatment when compared with the control at 0.98 a w ( Figure 2). However, the growth rates with the different O 3 exposure concentrations were relatively similar. At 0.93 a w , growth rates were slightly higher after O 3 treatment, with little difference between the concentrations of O 3 used. Effect of gaseous ozone treatment on the mycelial colony extension rate of A. flavus (strain AB3) on a 3% milled raw pistachio nut-based medium at 0.98 and 0.93 water activity (aw) and 30 °C for 4-6 days. Colonies were exposed to gaseous ozone for 30 min at 6 L/min prior to incubation. Different letters indicate significant differences (p < 0.05). Bars indicate mean SE.

Figure 2.
Effect of gaseous ozone treatment on the mycelial colony extension rate of A. flavus (strain AB3) on a 3% milled raw pistachio nut-based medium at 0.98 and 0.93 water activity (a w ) and 30 • C for 4-6 days. Colonies were exposed to gaseous ozone for 30 min at 6 L/min prior to incubation. Different letters indicate significant differences (p < 0.05). Bars indicate mean SE.

In Vitro Effect of Gaseous O 3 Exposure of Colonies of A. flavus on AFB 1 Production
The AFB 1 productions were significantly higher after O 3 treatments at all exposure levels when compared to the untreated control in the 0.98 a w treatments ( Figure 3). However, at 0.93 a w , there was no significant effect on toxin production, with much lower AFB 1 productions on the raw pistachio-based medium.
ins 2022, 14, x FOR PEER REVIEW 3 Figure 3. Effect of ozone treatment on AFB1 production by A. flavus (strain AB3) at 0.93 and 0.9 on a raw milled pistachio-based medium after 10 days at 30 °C. Colonies were exposed to ozone 30 min at 6 L/min prior to incubation. Different letters indicate significant differences (p < 0.05). Figure 3. Effect of ozone treatment on AFB 1 production by A. flavus (strain AB3) at 0.93 and 0.98 a w on a raw milled pistachio-based medium after 10 days at 30 • C. Colonies were exposed to ozone for 30 min at 6 L/min prior to incubation. Different letters indicate significant differences (p < 0.05).

In Situ Effects of Gaseous O 3 Treatment on Populations of A. flavus in Stored Raw Pistachio Nuts
The total populations of A. flavus slightly increased in the control samples when exposed to air ( Figure 4). However, there was a significant reduction in the A. flavus CFUs from Log 10 5.3 CFUs (control after exposure to air) to Log 10 1.4 CFUs, after exposure to 50 ppm O 3 , and <Log 10

In Situ Effects of O 3 Treatment on AFB 1 Contamination of Raw Stored Pistachio Nuts
The AFB 1 contamination was found to always be higher in the treated samples compared to the controls at both a w levels and all gaseous O 3 exposure levels, except for 50 ppm ( Figure 5). At this concentration and 0.98 a w , the AFB 1 was significantly reduced.
Toxins 2022, 14, x FOR PEER REVIEW 5 of 5 Figure 5. Effect of ozone exposure on AFB1 contamination of raw pistachio nuts inoculated with A. flavus strain AB3 at 0.93 and 0.98 aw then stored for 4 weeks after treatment at 30 °C. The pistachio nuts were exposed to gaseous ozone for 30 min at 6 L/min prior to storage. Different letters indicate significant differences between treatments (p < 0.05).
1. Figure 5. Effect of ozone exposure on AFB 1 contamination of raw pistachio nuts inoculated with A. flavus strain AB3 at 0.93 and 0.98 a w then stored for 4 weeks after treatment at 30 • C. The pistachio nuts were exposed to gaseous ozone for 30 min at 6 L/min prior to storage. Different letters indicate significant differences between treatments (p < 0.05).

In Vitro Effects on Germination and Mycelial Extension
The efficacy of gaseous O 3 for the control of conidial germination and mycelial growth of A. flavus strains (AB3, AB10) was evaluated in this study on a milled raw pistachio nut-based medium at different a w levels. The a w levels chosen were based on ecological data, which showed that optimum A. flavus growth occurs at >0.97 a w and optimum AFB 1 at >0.94 a w at 25-30 • C. For the prevention of toxin contamination, pistachio nuts would need to be dried to <0.88 a w [18]. Overall, exposure of conidia to O 3 initially had lower germination percentages when compared to the controls at both the a w levels. Treatment with 200 ppm O 3 at 0.98 a w showed the complete inhibition of germinations after 12 h; however, spore viability appeared to recover, and the germination was increased after 24 h and reached 100% germination after 48 h. Sultan and Magan [12] found that there was an effective inhibition of conidial germination by O 3 treatment of A. flavus strains from peanuts with complete inhibition at 200-250 ppm O 3 (6 L/min; 30 min). However, this was on a defined yeast extract sucrose medium. Previously, Mylona et al. [14] examined the in vitro effect of O 3 treatment at 100 and 200 ppm for 30 min (6 L/min) on the spore germination of F. verticillioides. Although germinative capacity was inhibited after 24 h, over the following 72 h there was a recovery, and after 8-10 days at both 0.98 and 0.94 a w , FB 1 production occurred. They also found that doubling the exposure time (60 min) did not improve the efficacy of O 3 . Indeed, recent studies with species from the Aspergillus section Circumdati and Nigri responsible for the ochratoxin A contamination of coffee showed tolerances of up to 500 ppm O 3 [19]. It has been suggested that O 3 acts by oxidizing vital cellular components, especially unsaturated lipids in cell membranes, resulting in a leakage of cell contents and subsequent microbial lysis at high concentrations [20,21]. However, some of these studies have been performed in O 3 -treated water and not with gaseous O 3 [12].
For mycelial growth, the present study showed that the mycelial extension was inhibited by O 3 exposure at 0.98 a w . However, growth rates decreased only slightly with the increasing O 3 dose. Very few studies have examined the in vitro effect on the growth of A. flavus, and none were on nut-based media [12]. A previous study by Zotti et al. [22] found that O 3 treatment of 3-day-old A. flavus colonies for 3 h inhibited growth and spores completely. However, when the same colony reached 6 and 9 days old, the efficacy decreased. Additionally, they found that there are different sensitivity levels among species, with A. flavus being less sensitive than A. niger. However, in their study, a w modification was not considered and the O 3 concentration used was only 1 ppm. Akbar et al. [19] found that the mycelial extension of strains of A. carbonarius and A. westerdijkiae in coffee-based medium with up to 500 ppm of O 3 had little effect on the rates of colonization, regardless of a w level used or exposure period (30-60 min).
The general tolerance of aflatoxigenic and ochratoxigenic Aspergillus spp. to O 3 may in part be due to the darker pigmentation and relatively thick-walled conidia, which can provide protection against UV-light, sunlight, and toxic gases. In addition, the capacity for relatively rapid DNA repair after exposure may be quite rapid, allowing the viability of conidia to be conserved after exposure. This could be related to both pigmentation and/or repair systems that help the cells to recover viability. Indeed, Hibben and Stotzky [16] indicated that small hyaline spores are more sensitive to O 3 , while large and pigmented spores, such as the conidia of A. niger, were more resistant [23,24]. Spores of A. fumigatus have been found to be particularly resistant to O 3 [18]. In contrast, spores of Fusarium species (e.g., F. verticillioides, F. langsethiae), which are practically hyaline and have very little pigmentation, appear to be sensitive to O 3 exposure in air initially, although some recovery of viability was found [9]. A thorough comparison amongst species belonging to the same genus is important.
Reduction of fungal growth can be obtained in high moisture conditions after treatment with up to 1000-15,000 ppm O 3 for 1 h [25]. For Brazil nuts, O 3 treatment was found to affect the growth of the mycobiota and to decrease aflatoxin contamination levels [26].
However, the exposure period was 5 h, which was effective and inhibited the growth of A. flavus and A. parasiticus, although they were still able to grow during the initial few days after O 3 exposure. Thus, O 3 levels and exposure time together with the other influencing factors, including temperature and ERH, need to be examined in more detail to optimize the potential use of this gas for control of the key life cycle phases of mycotoxigenic spoilage fungi and toxin production [21].

In Vitro and In Situ Effects of O 3 on AFB 1 Contamination
In the present study, AFB 1 was analyzed after the in vitro exposure of colonies of A. flavus to O 3 for 30 min and then stored for 10 days at 30 • C. There appeared to be variable effects on AFB 1 production by exposure to O 3 treatment. The increase in toxins may be due to O 3 exposure acting as an environmental stress, resulting in the biosynthesis of more toxins as a defense reaction. In addition, it may be that the O 3 interacts with the pistachio-based medium, changing the nutritional make up, especially in relation to fatty acids. Sultan and Magan [12] examined A. flavus exposure to O 3 (100-300 ppm) on a conducive YES medium. In this case, the use of a defined medium and exposure to O 3 resulted in a significant decrease of AFB 1 production in mycelial colonies. However, they examined toxin biosynthesis after 3 days, while the present study examined it for 10 days on a pistachio-nut based medium to simulate the natural nutritional conditions as far as was possible. This could explain the differences observed. Previously, Mason et al. [2] showed that the exposure of A. flavus colonies for 5 days inhibited asexual conidial sporulation. This suggested that perhaps the effect of O 3 on the whole life cycle of A. flavus would provide useful information on which phase might be more sensitive to such treatment [27]. The present study is the first to examine in detail the in situ effect of gaseous O 3 on the colonization and toxin production by A. flavus in stored pistachio nuts. Overall, while to populations of A. flavus significantly decreased due to O 3 exposure, there was little difference between 50-200 ppm treatment levels. Indeed, a reduction in AFB 1 was only observed in the 50 ppm O 3 × 0.98 a w treatment. It may be that a reduction in overall populations of A. flavus allows more rapid subsequent colonization by the surviving inoculum of the rich nutrient source. In naturally contaminated pistachio nuts, the mycobiota is varied with a range of fungi present [28]. Thus, the niche will be occupied by a fungal community including A. flavus, and it would have to compete with these other fungi, some of which may survive O 3 treatment. A previous study exposed pistachio nuts and ground nuts artificially contaminated with aflatoxins to very low concentrations of O 3 (5-9 mg/L = 2-5 ppm gaseous O 3 ) for 140 and 420 min [10]. They found that AFB 1 and total aflatoxins were reduced by about 23 and 24%, respectively, with the highest O 3 treatment level (9 mg/L) for 420 min. The treatments were much less effective against ground pistachios [15]. However, these studies were only carried out at 70% relative humidity, and spiking with the toxins is not the same as natural occurrence in this commodity due to colonization by the mycotoxigenic species. Thus, further studies are required to better understand the relationship between commodity type, exposure concentration × time of exposure, and prevailing a w level to determine efficacy in terms of toxin control [13].
The studies by Mylona et al. [14] certainly suggest that the natural contamination of maize grain with fumonisins can be reduced by exposure to O 3 concentrations. However, it may be more difficult to reduce mycotoxin production by specific fungi without longer term exposure to O 3 . Indeed, even with 500 ppm, O 3 was found to have relatively little effect on the reduction of ochratoxin A contamination of stored green coffee contaminated with A. westerdijkiae and A. carbonarius and stored under different a w levels at 30 • C [19]. Savi et al. [27] found that the exposure of wheat to 40-60 mg/L of O 3 for up to 180 min reduced the growth of F. graminearum significantly, with deoxynivalenol in the pericarp and endosperm tissue being completely inhibited. However, the moisture content of the grain was not considered in these studies, which are important relative to colonization by Fusaria and trichothecene production. It is, however, important to consider that food ozonation may not always be a beneficial process, especially where this gaseous treatment may alter food sensory characteristics, color, cause lipid oxidation, and the degradation of phenolic compounds and vitamins [29,30].

Conclusions
This study has shown that the use of gaseous O 3 exposure of both conidia and mycelial colonies of A. flavus on a milled raw pistachio nut-based medium was not very effective. Indeed, via repair systems, the conidia recovered germinative capacity rapidly. Up to 200 ppm of gaseous O 3 for 30 min had little impact on mycelial extension and subsequent AFB 1 production. In situ studies with stored raw pistachio nuts inoculated with A. flavus conidia and exposed to O 3 reduced the isolation of the A. flavus populations but had little effect on AFB 1 contamination after 20 days of storage at different a w levels. This suggests that perhaps much longer exposure times may be required and that the efficacy may also be influenced by the moisture content and the type of commodity, in this case a lipid-rich matrix. However, this would have to be balanced against potential tainting effects and eating quality, which might be impacted if high exposure levels are used for longer time periods. In the future, for more effective and safe use in food processing, the optimum gaseous O 3 concentration, contact time, and other treatment conditions need to be defined for specific foods. Pilot scale tests would probably need to be conducted for each commodity before potential commercial application, as every food application with O 3 application may be different.
In addition, in vitro and in vivo toxicological tests need to be conducted to quantify the effects of degradation products on human and animal health.

Apparatus for Ozone Generation and Experimental System
O 3 was generated in the laboratory using a C-Lasky series O 3 generator purchased from AirTree Ozone Technology Co. (model CL010DS), Sijhih, Taiwan. This equipment generates O 3 by corona discharge between two tubs, with no metals involved for efficiency improvement, generation stability, and less energy consumption. The generated O 3 was directed into the exposure chamber using a Teflon tube, which was properly connected to the generator. For safety reasons, the experiment was carried out in a fume cupboard to prevent O 3 from spreading into the laboratory atmosphere. Two different systems were used for O 3 exposure for in vitro and in situ assays. O 3 concentration was measured using an O 3 analyzer (Model UV-100, Eco Sensor, Santa Fe, NM, USA), which was connected to the chamber to measure the exit gas accurately. It should be noted that 1 ppm of O 3 generated is equivalent to 2.14 mg/L of O 3 in the air. This allows for comparison with some other studies. Experimental set-ups were performed as follows: The exposure chamber for in situ experiments was a 100-mL volume glass tube. The tube was capable of containing about 45-50 g of pistachio nuts. These were placed inside the column, and the O 3 was forced upwards via an inlet at the bottom of the tube coming from the generator. The outlet at the top was connected to the O 3 analyzer and vented in the fume hood. This allowed accurate exposure of the pistachio nuts to the treatment O 3 concentrations for the necessary residence time. The flow rate of generated O 3 was 6 L/min for 30 min.

Fungal Strains, Media, Spore Suspension, and Water Activity
One strain of A. flavus (AB3), representative of 3-4 others isolated from pistachio nuts, was used in these studies [13,31]. This was chosen as it was representative of those studied previously [13,31]. Pistachio-based media were used for spore germination and mycelial growth studies. A 3%-milled raw pistachio nut agar (PMA) was used with 2% technical agar (Thermo Fisher Scientific Oxoid Ltd., Basingstoke, Hampshire, UK) [13].
For spore suspension, fresh cultures of the AB3 strain were prepared on PMA and incubated at 25 • C for 5-7 days. AB3 culture surfaces were gently scraped and transferred into sterile Universal vials containing sterile water + 0.1% Tween 80 solution (Tween 80 (ACROS organics). The concentration of the spore suspension was determined using a hemocytometer (Olympus BX40 microscope, Microoptical Co., Sauerlach, Germany; slide Marienfeld superior, Germany; microscope glass cover slips, No 3, 18 mm × 18 mm, Chance proper Ltd., Malvern, Worcestshire, UK) and adjusted by dilution to 10 7 spores/mL. Target a w values (0.98 and 0.93) for PNA were obtained by using glycerol/water solutions, instead of water, to modify the a w with this non-ionic solution. For these two treatments, the equivalent of 122.5 and 355 g of glycerol per L of water was used. This was mixed, and the mixture was used similar to water.

Effect of O 3 on Conidial Spore Germination of A. flavus
Four different treatments were examined, including three concentrations of O 3 : 0, 50, 100, 200 ppm of O 3 at the two different a w levels detailed previously and incubated at 30 • C. Samples exposed to the air were used as controls for each a w . The experiment was carried out in triplicate and repeated once. An amount of 100 µL of 10 6 spore suspension was spread onto PNA media treatments and replicates and allowed to dry. Lids were taken off the plates and the media were placed inside the airtight glass jar for O 3 exposure for 30 min at a flow rate of 6 L/min, as described previously. The Petri plates were separated by 2-3 cm to ensure exposure of each plate. After exposure, plates were placed into plastic boxes, which were maintained at the same a w levels with glycerol/water solutions (500 mL × 2) and stored at 30 • C. Three agar plugs were taken every 12 h from each plate using a surface-sterilized cork-borer (1 cm) and placed on a glass microscope slide (Fisherbrand, Leicestershire, UK). The agar plugs were then stained with Lactophenol Cotton Blue (ProLab Diagnostics, Birkenhead, UK) and covered with a glass coverslip. Each plug was then examined under the microscope, and germination was recorded. Spores were considered to have germinated when the length of the germ tube was longer than the diameter of the spore. A total of 3 × 50 single spores per replicate were examined (450 per treatment). The overall mean number of germinated spores (out of 50) was calculated for the different O 3 treatments.

In Vitro Effects of Gaseous O 3 on Mycelial Growth and AFB 1 Contamination
PNA media were inoculated centrally with 10 µL of spore suspension made from AB3 strain and incubated at 30 • C in replicates and allowed to grow for 2 and 5-6 days in the 0.98 and 0.93 a w treatments, respectively. Measurements of colonies were recorded, and plates were exposed to O 3 with no lids for 30 min using the system described previously. The O 3 concentrations were 50, 100, and 200, with air as a control. A w of media and ERH during incubation after O 3 exposure was adjusted to 0.93 and 0.98 a w . After exposure, Petri plates were covered with the lids and incubated at 30 • C. Colony diameters were recorded on a daily basis for each treatment and compared with the control. Agar plugs were taken after day ten from each replicate and stored at −20 • C for AFB 1 analysis.

In Situ Effect of Gaseous O 3 on Fungal Population and AFB 1 Production on Irradiated Pistachio Nuts
Irradiated raw pistachio nuts (12)(13)(14)(15) KGys; Synergy Health Sterilisation UK Ltd., Swindon, Wiltshire, UK) were weighed and place in sterilized bottles (eight bottles) for each treatment (40 g per replicate). The absence of any fungal contaminants was checked by direct-plating individual pistachio nuts on Malt Extract Agar medium (MEA; Thermo Fisher Scientific Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated for 7 days at 25 • C. This showed no contamination. The raw pistachio nuts were rewetted using a moisture adsorption curve [25] and mixed well and left overnight at 4 • C to equilibrate to the target a w levels of 0.93 and 0.98. A conidial suspension of 10 6 spores was prepared. After equilibration, 1 mL of the spore suspension was added to the pistachio nuts and mixed well. Small sub-samples were taken (1 g) and placed in 10 mL of sterile water containing tween 80 in a 25 mL Universal bottle for serial dilution to assess the populations of A. flavus present. Three separate replicates of each treatment (40 g each) were exposed to O 3 (50, 100, and 200 ppm, or air) for 30 min at a flow rate of 6 L/min. Immediately after exposure, sub-samples were taken for serial dilution of the populations. The rest of the pistachio nuts were placed in solid culture vessels with microporous lids (Magenta, Sigma Ltd., Coventry, UK). These were previously autoclaved at 121 • C for 15 min with aluminum foil covers. The glass chambers containing the treatments/replicates were then placed in plastic chambers with glycerol/water solutions to maintain the target ERH (93 and 98% ERH) and stored for 20 days at 30 • C. Samples were taken for A. flavus fungal populations after this storage period. The remaining pistachio nuts were stored at −20 • C for later AFB 1 analysis.
For serial dilution, samples were soaked for 20 min and then vigorously shaken using a vortex mixer. From each treatment/replicate serial dilutions were made. For each concentration, three replicates were made, and 100 µL was spread on MEA media using a sterile spreader and incubated at 30 • C for 7 days before colonies were counted for A. flavus populations.

Aflatoxin B 1 Quantification
Preparation of aflatoxin standards: A 200-µL stock solution of aflatoxins (B 1 , B 2 , G 1 , G 2 ) standard in methanol containing 250 ng AFB 1 was prepared and pipetted into 2-mL Eppendorf tubes for overnight evaporation until dryness in a fume hood similar to the samples.

In Vitro Aflatoxin B 1 Analyses
Colony Extraction: Initially, agar plugs were cut out across the diameter of colonies using a surface-sterilized 4-mm diameter cork-borer (approx. [4][5][6]. The agar plugs were placed in pre-weighed 2-mL Eppendorf tubes and weighed again. Five-hundred µL of HPLC-grade chloroform was added to the tubes and shaken for 30 min using a KS 501 digital orbital shaker (IKA (R) Werke GmbH & Co. KG, Esslingen, Germany). The chloroform extract was transferred to a new Eppendorf tube and dried gently under air for derivatization.
Derivatization of aflatoxin B 1 extract: Derivatization of the AFB 1 extract was performed according to the AAOC method [32]. First, 200 µL of hexane was added to the tube, followed by 50 µL of trifluoroacetic acid. The mixture was vortexed for 30 s and left for 5 min. A mixture of water:acetonitrile (9:1) was then added to the tube, vortexed for 30 s, and left for 10 min to allow for separation of the layers. Then, the aqueous layer was filtered using a syringe nylon filter (13 mm × 0.22 µm; Jaytee Biosciences Ltd., Herne Bay, UK) into amber-salinized 2-mL HPLC vials (Agilent, Santa Clara, CA, USA) before HPLC analysis. All analytical reagents used were HPLC-grade.
Quantification of aflatoxin B 1 with High Performance Liquid Chromatography HPLC: A reverse-phase HPLC with fluorescence detection was used to confirm the identity and quantify AFB 1 . An Agilent 1200 series HPLC system was used for the analysis. It consisted of an in-line degasser, auto sampler, binary pump, and a fluorescence detector (excitation and emission wavelengths of 360 and 440 nm, respectively). Separation was achieved using a C18 column (Phenomenex Gemini; 150 × 4.6, 3 µm particle size; Phenomenex, Torrance, CA, USA) with a Phenomenex Gemini C18 3 mm, 3-µm guard cartridge. Isocratic elution with methanol:water:acetonitrile (30:60:10, v/v/v) as the mobile phase was performed at a flow rate of 1.0 mL/min. The injection volume was 20 µL. A set of standards was injected (1 to 5 ng AFB 1 , AFB 2 , AFG 1 , and AFG 2 per injection), and standard curves were generated by plotting the area underneath the peaks against the amounts of AFB 1 standard injected. The run time for the each HPLC analysis was 12 min. Supplementary Figure S1 shows an example of a standard and a sample for aflatoxin B 1 quantification.

Quantification of Aflatoxin B 1 in Pistachio Nuts
The pistachio nut samples were all dried in a drying oven at 50 • C in the dark. They were subsequently ground (Waring blender, Merck Ltd., Feltham, UK) and weighed (25 g). The background aflatoxin B 1 level in the nuts used in the experiments was 0.015 ng/g. This was taken into account as a correction factor in the final quantification of the results. Acetonitrile/water 60/40 (100 mL) was used as an extraction solvent. The mixture was blended for 3 min, and the extract was filtered into a smaller sample container. PBS buffer (pH 7.4, Thermo Fisher Scientific) was used for sample dilution, then the diluted extract was passed through an Immunoaffinity Column (IAC; AflaStar™; Romer Labs, Tulln an der Donau, Austria) with a flow rate between 1-3 mL/min. The column was rinsed with 2 × 10 mL sterile distilled water. HPLC-grade methanol (1.5-3 mL) was then applied to the column, and the eluent was collected in a new amber glass vial and left to dry overnight at room temperature before the derivatization step, as detailed previously.

Statistical Analysis
Three replicates per treatment were used in all studies and repeated once. Means were obtained by taking the average of three measurements for each experiment with the standard error of the means (±SE; standard error) obtained. Analysis of variance (ANOVA) was applied to analyze the variation of means with a 95% confidence interval. Normal distribution of data was checked by the normality test Kolmogorov-Smirnov, using Minitab statistical software. Fisher's Least Significant Difference (LSD) was used to identify differences between the means, with p < 0.05 as a significant difference, using the same statistical software.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14060416/s1, Figure S1: Examples of HPLC analyses of (a) a standard and (b) a sample of pistachio nut-based agar for aflatoxin B 1 quantification.
Author Contributions: A.B. carried out the research work and analyses of the data as part of his Ph.D. thesis in the Applied Mycology Group and wrote the initial draft manuscript; A.M. was involved as co-supervisor of the project and the mycotoxin analyses and contributed to the draft manuscript; N.M. developed the original project and conceptualization of the work, was the primary supervisor of the project, and revised the manuscript for the final submitted version. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The raw data used in this study and the statistical analyses are available via the corresponding author at Cranfield University.