Decontamination of Aspergillus flavus and Aspergillus parasiticus spores on hazelnuts via atmospheric pressure fluidized bed plasma reactor

International Journal of Food Microbiology 216 (2016) 50–59

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Decontamination of Aspergillus flavus and Aspergillus parasiticus spores on hazelnuts via atmospheric pressure fluidized bed plasma reactor Beyhan Gunaydin Dasan a, Mehmet Mutlu b,⁎, Ismail Hakki Boyaci a,c a b c

Department of Food Engineering, Faculty of Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey Department of Biomedical Engineering, Faculty of Engineering, TOBB University of Economics and Technology, Sogutozu, 06560 Ankara, Turkey Food Research Center, Hacettepe University, Beytepe, 06800 Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 14 May 2015 Received in revised form 6 August 2015 Accepted 10 September 2015 Available online 12 September 2015 Keywords: Aspergillus flavus Aspergillus parasiticus Atmospheric pressure plasma Fluidized bed reactor Decontamination

a b s t r a c t In this study, an atmospheric pressure fluidized bed plasma (APFBP) system was designed and its decontamination effect on aflatoxigenic fungi (Aspergillus flavus and Aspergillus parasiticus) on the surface of hazelnuts was investigated. Hazelnuts were artificially contaminated with A. flavus and A. parasiticus and then were treated with dry air plasma for up to 5 min in the APFBP system at various plasma parameters. Significant reductions of 4.50 log (cfu/g) in A. flavus and 4.19 log (cfu/g) in A. parasiticus were achieved after 5 min treatments at 100% V — 25 kHz (655 W) by using dry air as the plasma forming gas. The decontamination effect of APFBP on A. flavus and A. parasiticus spores inoculated on hazelnuts was increased with the applied reference voltage and the frequency. No change or slight reductions were observed in A. flavus and A. parasiticus load during the storage of plasma treated hazelnuts whereas on the control samples fungi continued to grow under storage conditions (30 days at 25 °C). Temperature change on hazelnut surfaces in the range between 35 and 90 °C was monitored with a thermal camera, and it was demonstrated that the temperature increase taking place during plasma treatment did not have a lethal effect on A. flavus and A. parasiticus spores. The damage caused by APFBP treatment on Aspergillus spp. spores was also observed by scanning electron microscopy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Aspergillus flavus and Aspergillus parasiticus are the predominant species responsible for fungal contamination and subsequent production of aflatoxins in nuts in the field, at harvest and during postharvest operations and storage (Arrus et al., 2005). Due to their relatively high contamination risk, decontamination methods for nuts are of great interest for economic and environmental reasons, as well as in public health (Ozilgen and Ozdemir, 2001; Richard et al., 1993). Improved post-harvest processing followed by further prevention of fungal growth is an effective way to restrict aflatoxin contamination and would have major impact on reducing health related risks and on production economics (Kendra and Dyer, 2007; Williams et al., 2004). Prevention of mycotoxin contamination in the field is the main goal of agricultural and food industries, and the most favorable way to bring this type of contamination under control is to reduce fungal infection in growing crops. There are several methods for decontamination and/or prevention of toxigenic fungal growth and detoxification of mycotoxins, including physical separation of contaminated crops (Bullerman, 1983), ⁎ Corresponding author at: Department of Biomedical Engineering, Faculty of Engineering, TOBB University of Economics and Technology, Sogutozu, 06560 Ankara, Turkey. E-mail address: [email protected] (M. Mutlu).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.09.006 0168-1605/© 2015 Elsevier B.V. All rights reserved.

extraction with solvents, adsorption (Elgazzar and Marth, 1987), heat treatment, irradiation, biological methods (Sinha, 1998) and chemical treatment (Bata and Lasztity, 1999; Kabak et al., 2006; Rusul and Marth, 1987). The use of many physical and chemical methods available for decontamination and detoxification of agriculture products contaminated with mycotoxins is restricted due to problems concerning safety issues, possible losses in the nutritional quality of treated commodities, and limited efficacy and cost implications. Limitations of traditional disinfection methods have motivated the development of alternative methods. A potential alternative for inactivation of microorganisms is plasma treatment, where a variety of energetic species (charged and exited species, reactive neutrals and UV photons) are formed. Each of these species alone can deactivate or disintegrate microorganisms, but they are more efficient in synergetic combinations (Fridman, 2008). The use of plasma is well established in areas such as surface modification for variable applications (Biederman et al., 2001; Mutlu et al., 1998), electrochemical sensors (Gunaydin et al., 2010), and preparation of functional surfaces e.g. amphoteric, bacterial anti-fouling (Akdogan and Mutlu, 2012; Cokeliler et al., 2007; Sen et al., 2012). It is also a promising technique for decontamination of fresh produce and packaged food, disinfection of surfaces and in healthcare (De Geyter and Morent, 2012; Deng et al., 2007; Fridman et al., 2007b; Joshi et al., 2011; Moisan et al., 2001; Sen and Mutlu, 2013; von Woedtke et al., 2013).

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It is well known that atmospheric pressure plasma is a source of reactive oxygen species [ROS; e.g. atomic oxygen (O), ozone (O3), hydroxyl radical (OH•)] and reactive nitrogen species (RNS; e.g. N2, NO, NO2, nitric oxide radical NO•) as well as UV-A and UV-B radiation which are considered to be the most important inactivation agents in plasma, and plasma induced oxidative stress is believed to cause cell damage and cell death (Fridman et al., 2008; Laroussi and Leipold, 2004; von Woedtke et al., 2013; Zhang et al., 2012). These active species are responsible for biological reactions ranging from intercellular DNA fracture and protein degeneration to oxidation of the outer membrane (Ma et al., 2008; Moisan et al., 2001). In general, plasma species only act on the surface of substrates, thus the reduction of microbial contamination is limited to the outer surface. In this way, bulk properties are not affected. Furthermore, the multitude of inactivation mechanisms in plasma treatment counteracts the evolution of microbial resistance, which might emerge over time if only one inactivation mechanism is applied (Shama and Kong, 2012). Over the last decade, interest and intensity of research on potential applications of plasma has increased in the field of food engineering and food processing. Development of new plasma sources, particularly those that allow generation of non-thermal plasma near ambient temperatures, are considered to be suitable for heat sensitive food surfaces. Plasma source, plasma feed gas and modes of application have a tremendous impact on the inactivation efficiency. Shama and Kong (2012) published a summary on plasma decontamination of foods, including bacon, pork, ham, apples, melons, mangos, lettuce, cheese, eggs and nuts. Furthermore, there have been recent studies on plasma treatment of corn salad (Baier et al., 2013), fresh fruits and vegetables (Schnabel et al., 2014), whole black pepper (Hertwig et al., 2015), and herbs and spices (Hertwig et al.). Escherichia coli on almonds was inactivated by applying plasma (Deng et al., 2007). Listeria monocytogenes on cheese and ham was reduced by 2 to 5 log units by using plasma (Song et al., 2009). Enterohemorrhagic E. coli O157:H7, which is responsible for increasing numbers of food poisoning cases and can survive for long periods under adverse conditions and refrigeration temperatures, was reduced by 7 log cycles in apple juice by igniting the plasma directly into sample (Montenegro et al., 2002). There are a few studies centered on toxigenic fungi in the literature, which use low-pressure plasma systems for decontamination of Aspergillus spp. on varying food samples. Low-pressure cold plasmas of sulfur hexafluoride (SF6) gas and air were used to inactivate A. parasiticus on nut surfaces (Basaran et al., 2008). However, efficacy of the treatment typically remained quite low; only about 2 log reduction in A. parasiticus was achieved after 5 min of air plasma and 5 log reduction even after 20 min treatment of SF6, which is a toxic precursor. A similar experiment was carried out for Aspergillus and Penicillium spp. on seeds, and 3-log reductions for both species were achieved within 15 min of SF6 plasma treatment (Selcuk et al., 2008), which is a relatively more favorable result compared to the study in which only 2.5 log spores/g reduction on A. flavus contaminated red pepper was obtained by applying low pressure nitrogen plasma at a very high power of 900 W (Kim et al., 2014). A 30 min treatment period resulted in a 3.45 log reduction of A. brasiliensis on cellulose acetate membranes by using pure oxygen or argon, whereas 5 min (at 150 W), 1 min (at 300 W) and 15 s (at 400 W) treatment periods resulted in a 5.4 log reduction by using a mixture of argon and oxygen in a low pressure plasma system. However, in the same system only 2-log reductions of naturally contaminated fungi were achieved for pistachios after a 1 min treatment at 300 W (Pignata et al., 2014), which demonstrated that the efficiency of sterilization with plasma treatment was greatly reduced when it was applied to the surfaces of fruits rather than nitrocellulose membranes. Bacillus amyloliquefaciens endospores artificially deposited on wheat grains were treated in a low pressure circulating fluidized bed reactor at 900 W by using a mixture of oxygen and argon

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gases and reduced by 2.59 log units within 73.5 s of effective treatment time, but the whole process took 111 min due to the time needed for maintaining and/or venting vacuum inside the reactor (Butscher et al., 2015). Besides, the surface temperature of the grains was found to be 90 °C at 900 W. Applicability of low-pressure plasma technology for sterilization and decontamination was confirmed with the studies carried out by other researchers. The advantage of homogeneous low-pressure plasmas which are generated in vacuum chambers is that the conditions of plasma ignition are much more controllable and reproducible compared to atmospheric pressure conditions. These advantages could compensate at least partially for the practical drawbacks of low-pressure plasma use, the need for vacuum equipment, long process time and the limitation of batch treatment processes, which bring additional operating costs. In addition, vacuum applied in the course of this method also had unfavorable effects on samples with high water content (i.e. fresh and perishable food). Samples containing volatile compounds (i.e. nuts, pistachios) could also be adversely affected by vacuum treatment and unfavorable changes could occur in organoleptic properties. With atmospheric pressure plasmas, a nonthermal treatment of surfaces without the need for additional vacuum equipment can be achieved, which in turn opens up the possibility for better integration of some devices such as those used for continuous production processes. However, unlike the mostly homogeneous lowpressure plasmas, such plasmas are either contracted or filamentary plasmas, which make them disadvantageous as the activity of atmospheric pressure plasmas is strictly localized. A great advantage of the strong localization of atmospheric pressure plasmas is that it is possible to bring the plasma reactivity specifically and well targeted to the place to be treated (Becker et al., 2005; Kogelschatz, 2004). In the present study, a fluidized bed plasma reactor system operated at atmospheric pressure was designed and decontamination effect on Aspergillus spp. was investigated. Hazelnut was chosen as the model food sample due to the fact that is susceptible to mold contamination during postharvest operations, transport and storage, and that fungal contamination may result in mycotoxin production (Arrus et al., 2005; Williams et al., 2004). Rather than removal of toxins after they have been produced, the goal should be to eliminate fungus before toxin production. It is, therefore, crucial to develop novel, practical, and cost effective post-harvest methods or processes to reduce, or if possible, completely eliminate fungus before aflatoxins are produced during storage. Development of new techniques for manufacturing high quality and safe hazelnuts and their byproducts are significant considering their financial benefits. The main aim of this study was to design an atmospheric pressure fluidized bed plasma (APFBP) reactor using the low cost and easy handling technology of atmospheric plasma and to prove the feasibility of a faster and more effective decontamination process for aflatoxigenic fungi, (A. flavus, A. parasiticus) as well as providing great convenience to scale-up to continuous systems. This APFBP system allows for the plasma treatment of a continuously flowing granular product and is, in principle, scalable to industrial dimensions. In addition, the multiple circulations of the product in the system make it possible to realize an adequate treatment time. Up to now, fluidized bed plasma reactors have mostly been used for the treatment of powders with mean particle size smaller than 300 μm and with the objectives of film deposition (Bretagnol et al., 2004; Chen et al., 2008; Jafari et al., 2011; Karches and von Rohr, 2001). In this study, we expand the range of application of fluidized bed plasma reactors to the treatment of particles in the mm range, which is a challenge due to higher particle gravity and lower particle drag. In the present study, the most common, cheap and practical precursor, “air”, was used in an atmospheric pressure fluidized bed plasma (APFBP) reactor. The use of plasma processing for inactivation of spores of Aspergillus spp. inoculated on hazelnut samples was the focus of the investigation.

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2. Materials and methods 2.1. Hazelnut samples Hazelnuts were purchased from a local supplier from the northeast region of Turkey. They were unroasted, retained their inner shells, and were 9–11 mm in diameter, 0.59 ± 0.07 g in weight, and 0.97 ± 0.06 g/cm3 in density. Samples were stored at 4 °C. 2.2. Microbial strains, inoculation and sample preparation A. flavus (ATCC 327) and A. parasiticus (ATCC 1041), capable of producing aflatoxin were obtained from TÜBİTAK MAM Culture Collection, Turkey, on slant agar media. These pure cultures were inoculated onto slants and plates of Yeast Extract Glucose Chloramphenicol (YGC) Agar (Merck KGaA, Darmstadt, Germany) and cultured for 5 days at 25–28 °C. As a pre-decontamination process, hazelnut samples were treated with 70% ethanol (Merck KGaA, Darmstadt, Germany) solution for 10 min to reduce the background microbiota on the surface. Following the ethanol treatment, no cultivable spores were observed on YGC agar after incubation for 7 days at 25–28 °C. The effect of plasma decontamination process was investigated only on hazelnuts that were artificially contaminated with pure cultures of aflatoxigenic fungi. A 10 g sample of hazelnuts was placed on A. flavus/A. parasiticus cultures grown on YGC agar and agitated for 30 s to allow the mold spores to transfer on to the sample and inoculate homogeneously. After mixing, the artificially contaminated sample was transferred to a sterile Petri dish and incubated at 25–28 °C for 18–24 h to enable mold spores to adhere to the sample surface and to remove the extra moisture acquired during fungal inoculation. 2.3. Design of the atmospheric pressure fluidized bed plasma (APFBP) reactor The atmospheric pressure plasma system (Plasmatreat GmbH, Steinhagen, Germany), illustrated in Fig. 1 consists of a 3x400V-16A power generator, a plasma jet, a high voltage transformer and a pressure supply control unit. In the atmospheric pressure plasma jets (APPJ), plasma is ignited inside a nozzle equipped with one or two electrodes and expands outside the nozzle via a high gas flow. The major advantages of APPJs are the small plasma dimensions and their ability to penetrate into narrow gaps with a high aspect ratio (Weltmann et al., 2008). This makes APPJs particularly promising for applications at

complex geometries with micro structured cavities or capillaries. Likewise, the small dimensions of the blown out discharge are advantageous for the precise treatment of sensitive spots. The atmospheric plasma device could be operated with air and nitrogen in the range of 100–70% reference voltage and 18–25 kHz frequencies with a precursor gas flow of 1000–5000 L/h. An atmospheric pressure fluidized bed plasma reactor (APFBP) was installed for the plasma treatment of granular materials (Fig. 1). This reactor was made from glass with a 49 mm diameter, 147 mm length and a L/D ratio of 3. A stainless steel stand was designed to hold the fluidized bed reactor on top of the plasma jet. Samples were repeatedly conveyed to the treatment zone by means of an aeration gas flow supplied from a compressor with a 900 L/min air yield, 8 bar working pressure, 200 L tank volume and 5.5 kW engine power (Tamsan, Ankara, Turkey) integrated into the system so as to ease the fluidization process. A stainless steel sieve was designed for homogeneous distribution of air released from the compressor and placed just at the bottom of the reactor. Additionally, a brass apparatus was designed to hold the fluidized bed reactor still and contribute to homogenous air distribution. Hazelnuts were placed in the reactor in direct contact with plasma afterglow. The schematic diagram of APFBP system and a detailed demonstration of atmospheric plasma jet were shown in Fig. 1 (a, b). 2.4. Experimental setup/evaluation of the plasma treated spore survivors on nut surface Hazelnut samples that were artificially contaminated with A. flavus and A. parasiticus and incubated for 18–24 h at 25–28 °C to enable mold spores to adhere to the nut surface were placed in the APFBP reactor and subjected to plasma decontamination process at varying plasma parameters [25 kHz; 80% V (525 W); 90% V (590 W); 100% V (655 W)/ 20 kHz; 80% V (460 W), 90% V (520 W), 100% V (575 W)] by using dry air at 3000 L/h flow rate at different time intervals (1–5 min). After plasma treatment, samples were dispersed into 0.85% sterile saline solution containing 0.1% Tween 80 and rinsed for 15 min (10 min rinsing homogeneously + 5 min vortex). The resulting wash fluid was serially diluted with 0.85% sterile saline solutions. Mold spore counts were determined by surface plating (100 μL) of appropriate aliquots in triplicate on YGC agar. The YGC plates were incubated at 25–28 °C for 3–7 days before mold colonies were counted. The results were expressed as log colony-forming units per gram (log cfu/g) and the detection limit of this method was 10 cfu/g. Three samples were taken from each experimental run and evaluated in duplicate. For statistical analyses, we averaged the duplicates first, and calculated mean and standard error of the

Fig. 1. The schematic diagram of APFBP system (a) and detailed demonstration of atmospheric plasma jet (b).

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mean from three sample results. Finally, plasma treated samples were compared to the mean of six controls, which are inoculated but untreated samples representing the initial spore concentration. The reduction of cfu is expressed by the decimal logarithm of the ratio of final spore concentration Nt and initial value N0, and the D-value expresses the time required for the cfu reduction by one logarithmic unit: Logarithmic spore reduction ¼ −D:t ¼ log

N0 : Nt

ð1Þ

To investigate the effect of airflow on mold spores adhering to the samples in the fluidized bed reactor system, the same procedure was applied to hazelnut samples by using just airflow and without generating plasma. In order to evaluate the effect of storage on recovery of spores, plasma treated hazelnut samples and control samples, which were untreated after artificially contamination with Aspergillus spp. spores were stored at 25 °C for 30 days, after which recovering spores were counted as described above. 2.5. Thermal imaging The temperature changes occurring on the surface of hazelnuts while passing close to the plasma jet in the course of APFBP treatment were monitored with a thermal camera PI400 using the software Optris PIconnect (Optris GmbH, Berlin, Germany). This compact thermal camera works with 80 Hz measurement velocity at 382 × 288 pixel optic resolution and at a temperature range between 0 and 250 ± 2 °C. Thermal camera was set up just above the APFBP reactor at a 50 mm distance (Fig. 1a) and the whole plasma process was recorded. The temperature– time profiles of the samples during the whole plasma process were investigated. 2.6. Thermal process on hazelnut samples Hazelnut samples were subjected to a thermal process under dry heat at 100 °C for 5 min to investigate the effect of temperature change occurring on A. flavus and A. parasiticus spores during plasma process in which the maximum temperature that occurred on hazelnut surfaces during their rapid passing too close to the plasma jet throughout the plasma process was about 90 °C. For this reason, the most unfavorable and severe conditions for the Aspergillus spp., which normally did not occur during the process, were provided. Hazelnuts were placed in a drying oven where the environmental conditions were stabilized and held for 5 min when 100 °C was achieved at the sample surface. The viable spore counts acquired before and after the thermal process applied to hazelnuts were compared. All experiments were conducted in triplicate. 2.7. Sample preparation procedure for scanning electron microscope (SEM) Scanning electron micrographs were obtained from control and plasma treated (at 25 kHz — 100% V (655 W) for 30 s) A. flavus and A. parasiticus spores. The suspensions consisting of fungal hyphae and spores were fixed in 2.5% glutaraldehyde overnight at 4 °C, rinsed with 0.1 M sodium phosphate buffer (PBS), and then fixed with 1% osmium tetroxide (OsO4) in 0.1 M PBS for 30 min. Following three rinses in 0.1 M PBS (10 min each) and two rinses with water (10 min each), fixed samples were dehydrated in a graded series of ethanol concentrations (from 30% to 100%) for 15 min each. Dehydration was completed by applying three 10-min washes in 100% ethanol. Ten microliters portions of the sample were placed on SEM stubs, which were air-dried in desiccators before sputter coating with a thin layer of gold palladium. After coating, the samples were examined using a scanning electron microscope (QUANTA 400F Field Emission SEM, METU Central Lab., Ankara Turkey) operated at the high vacuum mode with an acceleration voltage of 20 kV (Gong et al., 2014; Guven et al., 2011).

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3. Results and discussions 3.1. Inactivation of A. flavus and A. parasiticus In the first part of the study, the artificially contaminated hazelnut samples were exposed to air flow in the APFBP reactor, to check whether or not the population of the A. flavus and A. parasiticus spores were being changed by shear stress and drag force over the hazelnut samples created by air flow. For this purpose, unique airflow rate at 3000 L/h was applied to hazelnut samples that were artificially contaminated with spores of A. flavus and A. parasiticus without generating plasma at variable time intervals (1–5 min). After the air treatment, it was observed that the total mold spore counts on the hazelnut samples (~106 cfu/g) did not differ from the initial mold spore level (~106 cfu/g) on logarithmic basis. Thus, it was clearly seen that mold spores adhered to hazelnut samples so tightly that the spore concentration did not decrease with the applied gas flow during APFBP process. APFBP was applied at two different frequencies (25 kHz and 20 kHz) and varying reference voltages (100% V, 90% V, 80% V) for 1 to 5 min by using dry air as the precursor. At the end of the 5 min APFBP treatment at 25 kHz and 20 kHz with the reference voltages of 100% V, 90% V, 80% V, A. flavus on hazelnuts was inhibited by 4.50 ± 0.15, 3.64 ± 0.06, 3.42 ± 0.20 log (cfu/g), and 3.51 ± 0.09, 3.07 ± 0.11, 2.86 ± 0.03 log (cfu/g), respectively (Fig. 2). A 2 log (cfu/g) reduction had already been achieved in the first minute of plasma decontamination process at 100% V — 25 kHz (655 W). While reduction was rapid at the beginning, the process slowed down after the initial phase. This might be a consequence of the spore distribution on the surface of hazelnuts. A large portion of the spores was distributed over the outer side of the hazelnuts and hence easily accessible for the plasma species. Some spores however, may have been hidden in the porous surface of the hazelnut where they could be shaded from plasma species. Experimental work on the germicidal effects of atmosphericpressure plasmas has shown that survival curves can take different shapes. Some researchers observe straight-line survival curves (Choi et al., 2006; Laroussi et al., 2000); however, in most cases, just as in this study, two or three linear segments occur, each representing a different inactivation phase (Fridman, 2008; Laroussi, 2005; Laroussi et al., 2000; Moisan et al., 2001). The first phase provides the highest killing rate (smallest D-value) and is dominated mainly by the action of UV radiation on isolated spores or on the first layers of stacked spores. The second phase, which has the slowest kinetics, is attributed to erosion processes by UV photons (photo desorption) and by other active species such as atomic oxygen (etching). These erosion processes progressively remove matter from the microorganisms or from the material covering them. Finally, the third phase starts when the last living spores have been sufficiently eroded, allowing UV photons to hit their genetic material directly (Moisan et al., 2002). These results clearly reveal that microorganism inactivation by plasmas is a complex process in which several factors can affect the kinetics of the killing process. In general, higher plasma power resulted in greater inactivation at shorter times. For the lowest plasma power, each successively longer treatment yielded significant additional increases in inactivation of the toxigenic fungi. Treatment at 100% V demonstrated the greatest reduction of all the parameters used in the experiments, which might be caused by the increase in reference voltage. Total plasma power affecting the sample increases, while the applied reference voltage increases. Therefore, the detrimental effect of plasma on spores increases as the amount and variety of reactive species that are formed during plasma process intensifies. In addition to this, it was observed that since the applied frequency decreases, total power that affects the sample also decreases, which leads to a lesser reduction in A. flavus spores at 20 kHz than that at 25 kHz. In the case of A. parasiticus, APFBP treatment at 25 kHz and 20 kHz with the reference voltages of 100% V, 90% V, 80% V caused reductions by 4.19 ± 0.06, 3.48 ± 0.16, 3.16 ± 0.04 log (cfu/g), and 3.48 ± 0.07,

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Fig. 2. Logarithmic survival curves of A. flavus spores at 25 kHz (a) and 20 kHz (b) 80% V, 90% V, 100% V.

2.88 ± 0.06, 2.64 ± 0.11 log (cfu/g), respectively (Fig. 3). It was observed that A. flavus spores were more susceptible to plasma decontamination process than A. parasiticus spores. Detrimental effect of plasma active species on A. flavus spores were found to be higher than on A. parasiticus spores, thus a more considerable reduction on A. flavus spores was achieved when all other parameters were kept constant. However A. flavus differs from A. parasiticus in only a few features such as the morphological characteristics, toxins produced, and geographical range (Pitt and Hocking, 2009a). Those characteristics could not directly be attributed to plasma sterilization efficiency, so a molecular and structural study needs to be performed against response to plasma species during the process. There have been limited studies focused on the effect of plasma against toxigenic fungus in particular. A 3.42 log reduction in Aspergillus ochraceus was achieved in 5 min of atmospheric plasma treatment using argon as the plasma forming gas (Herceg et al., 2015). In another study, only 2.5 log spores/g reduction was obtained on A. flavus contaminated red pepper by applying low pressure nitrogen plasma at a very high power of 900 W (Kim et al., 2014). A. flavus counts were reduced by 3.03 log cfu/g on brown cereal bars by using argon plasma at atmospheric conditions for 5 min (Suhem et al., 2013). Low-pressure cold plasma of air was used to inactivate A. parasiticus on nut surfaces and

Fig. 3. Logarithmic survival curves of A. parasiticus spores 25 kHz (a) and 20 kHz (b) 80% V, 90% V, 100% V.

only about 2-log reduction in A. parasiticus was achieved after 5 min (Basaran et al., 2008). Following our experiments, we achieved a 4.50 ± 0.15 log (cfu/g) reduction in A. flavus spores on hazelnuts, which proves that the continuous flowing of sample during plasma treatment increases the decontamination efficacy of the process. Direct treatment brings charged energetic particles in contact with the treated sample/microorganism and a faster killing of microorganism is observed (Fridman et al., 2007a). And also, when air is used as the plasma forming gas, a reactive mix of atoms, excited molecules, charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS) and UV photons occur, all of which may contribute to its antibacterial properties (Mai-Prochnow et al., 2014). However, most of the antimicrobial effects of atmospheric pressure plasmas are attributed to the generation of reactive oxygen and nitrogen species (RONS). Recent evidence suggests that RONS are major factors in antimicrobial and antiparasitic drugs, cancer therapies and wound healing (Graves, 2012). The only fluidized bed plasma reactor constructed for decontamination of food samples so far was a low pressure system, and it was used to inactivate B. amyloliquefaciens endospores artificially deposited on wheat grains (Butscher et al., 2015). Only a 2.59 log units reduction by using an admixture of oxygen and argon gases was observed within

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73.5 s of effective treatment time, but the whole process took 111 min due to the time needed for maintaining and/or venting vacuum inside the reactor. It is not feasible to compare the reductions because of the different target microorganisms and the system features, but the total time needed for decontamination process was considerably higher because of the nature of the low-pressure systems. And also, the surface temperature of the grains was found to be 90 °C at 900 W. D-value is a kinetic measurement parameter used for characterizing the slope of survival curves and inactivation phases (Laroussi and Leipold, 2004; Selcuk et al., 2008). The D-values for A. flavus and A. parasiticus at the optimum plasma parameter (100% V — 25 kHz — 655 W) providing the highest killing rate (the smallest D value) means that the time required to kill the 90% of the initial spore concentration were 1.11 and 1.19 min, respectively. The effect of storage on the survival counts of aflatoxigenic fungi following plasma treatment was investigated after 30 days of storage at 25 °C. Fig. 4 shows the viable spore counts of Aspergillus spp. evaluated both immediately and after 30 days of storage at 25 °C following the plasma treatment. And also, hazelnut samples, which were artificially contaminated to Aspergillus spp. spores at a level close to the final concentration on hazelnuts after plasma treatment (≈ 2 log cfu/g) were evaluated as control. The results demonstrated that the damage caused by plasma process on Aspergillus spp. spores was not temporary, since the viable spore count did not increase but decreased after 30 days following the APFBP decontamination process, while Aspergillus spp. spores on control samples continued to grow at the same conditions. This result indicates that the Aspergillus spp. spores were injured following the plasma treatment and could not recover from the detrimental effect of plasma on their structure even after 30 days of storage, as the viable spore counts decreased.

3.2. Inhibition of natural flora on hazelnuts A plasma decontamination process of 5 min at 25 kHz — 100% V (655 W) with dry air in the APFBP reactor system was applied on control hazelnut samples (uncontaminated with aflatoxigenic fungi) without pre-decontamination process with 70% ethanol. The logarithmic survival curve of the natural background flora of molds was shown in Fig. 5. This plasma process inhibited the natural mold flora of hazelnuts by 3.45 log (cfu/g) in two minutes. No viable cells were counted after the plasma process. This result demonstrates that APFBP system could be also effective on the inactivation of mold flora, which was naturally found on the sample surface in lower concentrations.

Fig. 4. Logarithmic viable spores of untreated and treated Aspergillus spp. with plasma both instantly and after 30 days of storage at 25 °C.

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Fig. 5. Logarithmic survival curve of natural background mold flora of hazelnuts at 25 kHz — 100% V (655 W).

3.3. Thermal imaging The temperature change on the surface of hazelnuts while passing too close to the plasma jet in the course of the plasma decontamination process (5 min) was monitored with a thermal camera at all plasma parameters for the same conditions. Fig. 6. shows the temperature time profiles of hazelnut surfaces treated at variable plasma parameters. The mean temperature of the total hazelnuts having been treated with plasma was measured, and it was found that the temperature on hazelnuts varied between 35 and 90 °C on average at all parameters during the whole plasma process. The images obtained by the thermal camera indicated that the temperature on the nutshell surface exceeded 90 °C. However, this temperature could not be attributed to a single area due to the design and processing of the image. It was clearly observed from the other direction of the reactor that although an instantaneous temperature increase might be observed on hazelnut surfaces while passing by too close to the plasma jet at the bottom of the reactor, hazelnut surfaces were rapidly cooled while rising up to the top of fluidized bed reactor by means of airflow. Even if the surface temperature of hazelnuts was increased to 90 °C, the average temperature of the

Fig. 6. Temperature profiles of hazelnuts treated at varying plasma parameters.

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below 45 °C which does not affect the nutritional characteristics, quality and taste of hazelnuts. In further studies, a cooling system could be mounted on the APFBP system to achieve more moderate operating conditions. 3.4. Effect of thermal process on A. flavus and A. parasiticus

Fig. 7. Logarithmic viable spores of A. flavus and A. parasiticus treated for 5 min at 100 °C (dry heat).

whole grain was assumed to be far below those levels. Nonetheless, the temperature inside the fluidized bed reactor in which the decontamination process took place during the atmospheric plasma process was

Hazelnut samples were subjected to a thermal process under dry heat at 100 °C for 5 min to investigate the effect of temperature on A. flavus and A. parasiticus spores during APFBP process. The main aim was to prove that the reduction on mold spores achieved with plasma decontamination process was due to the detrimental effect of plasma active species, rather than the temperature change occurring during the treatment. For this reason, the most unfavorable and severe conditions for the Aspergillus spp., which normally did not occur during the process, were provided. Fig. 7 shows the viable spore counts acquired before and after the thermal process applied on hazelnuts. It was clearly observed that heat treatment at 100 °C for 5 min did not alter the initial spore concentration on logarithmic basis in both A. flavus and A. parasiticus. This proves that the inactivation of mold spores within this study was a consequence of effects caused by plasma decontamination process performed in the APFBP system other than heat generated during the process. The results should be evaluated by taking into consideration that the heat transfer by conduction at a stationary state took place during the thermal

Fig. 8. Scanning electron micrograms of control A. flavus (without plasma treatment) (a) and A. flavus after plasma treatment (b), (c), (d).

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process. However, the hazelnut samples were continuously flowing during the whole plasma process, which leads to temperature decreases on the sample surface by means of airflow. Many fungi belonging to the order Eurotiales, which includes the environmentally ubiquitous genera Aspergillus and Penicillium, form sexual ascospores that can survive high temperature (Dijksterhuis, 2007) and drought (Wyatt et al., 2015) and can exhibit other types of extreme stress resistance, and are therefore among the most resistant eukaryotic cells reported so far. For instance, ascospores of Neosartorya fischeri, a teleomorph of Aspergillus, survive 85 °C in an aqueous environment for more than 10 min (Beuchat, 1986; Houbraken et al., 2012). Moreover, in a dry state they survive a relative humidity lower than 0.5% at a temperature of 60 °C for more than 7 days (Wyatt et al., 2015). These properties enable fungi such as N. fischeri to survive stress under adverse natural conditions including high temperatures during wet or dry conditions. Consequently, these ascospores can survive mild food-preservation treatments such as pasteurization. Factors contributing to stress resistance of ascospores include a thick cell wall, low water content, high viscosity, and accumulation of protective compatible solutes such as trehalose and mannitol (Dijksterhuis, 2007; Wyatt et al., 2015). Factors such as pH, water activity and the presence of preservatives also have an influence on heat resistance (Pitt and Hocking, 2009b). Similar conclusions can be applied to our samples due to very low water activity (b 0.60; Guine et al., 2015; Ozay et al., 1993) resulting in resistance to higher temperatures.

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3.5. Scanning electron microscopy (SEM) images Scanning electron microscopy was used to examine the effects of the atmospheric pressure plasma decontamination process. Figs. 8 (a–d) and 9 (a–d) show the SEM imaging of A. flavus and A. parasiticus spores before and after plasma process (at 25 kHz — 100% V (655 W) for 30 s). The damage caused by the plasma process on A. flavus spores can be clearly seen in Fig. 8 (b) and (d). The integrity of the cellular structure was completely broken, and cell contents were dispersed. Consequently, spores were observed in clusters, whereas untreated spores were spread individually (Fig. 8a). Fig. 8c shows the destruction of the phialide structure of A. flavus after the atmospheric plasma process. As seen in the Fig. 9(b–d), the shape of the A. parasiticus spores changed after the plasma process and the integrity of the spore cells was broken down by the damage caused by active species formed during plasma process, whereas untreated spores were observed as individual spheres (Fig. 9a). SEM results show clear signs of major changes in the external cellular structure that cause cell death. Among the studies performed by other researchers on inactivation of Aspergillus spp. using plasma, some demonstrated the damage caused by plasma treatment by utilizing only a bright field microscope, which was not sufficient since the structure could be damaged even during the preparation of the slide (Basaran et al., 2008; Suhem et al., 2013). In another study, they observed in typical

Fig. 9. Scanning electron micrograms of control A. parasiticus (without plasma treatment) (a) and A. parasiticus after plasma treatment (b), (c), (d).

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SEM images of A. Niger that the spores had clearly ruptured and adhered to one another, before and after atomic oxygen exposure during plasma process (Yoshino et al., 2013). 4. Conclusion We, hereby, presented preliminary evaluation results of fungal reduction and fungal elimination on hazelnuts by using atmospheric plasma system accompanied by a fluidized bed reactor. The APFBP system effectively inhibited A. flavus and A. parasiticus on hazelnuts and is convenient for large-scale applications due to its low cost and operation feasibility at atmospheric conditions. With atmospheric pressure plasmas, treatment of surfaces is possible without the need for additional vacuum equipment, and this can open up the possibility for better integration of some devices such as those used for continuous production processes. The obligation of vacuum and batch processing in low-pressure plasma systems bring additional costs for both investment and operation. In addition to this, we have achieved a reasonable reduction of Aspergillus spp. and a safe storage period of 30 days at 25 °C for hazelnuts with a plasma treatment for 5 min using dry air. Plasma processing of materials for various purposes is quite well established in various industrial processes, and its use is also suggested as an innovative technology in the food sector. Due to its low-cost, adaptability to large-scale systems, its eco-friendly nature, convenience of applicability and demonstrated detrimental effect on microorganisms, atmospheric plasma treatment is a promising technology for decontamination of food and food products. In this study, we have shown that Atmospheric Pressure Fluidized Bed Plasma (APFBP) Reactor could be used for decontamination of aflatoxigenic fungi on hazelnuts with significant reductions. Thus, this process has the potential to be used as an alternative practical method for nut decontamination in the industry to enhance the microbiological safety during postharvest operations and storage. Parameter optimization of plasma processing of foodstuffs in atmospheric plasma conditions, performance tests against other types of spores and contaminants, and further scale up studies for pilot and industrial scales are still under investigation by our research group and many others. Acknowledgments This study was supported by the Scientific and Technological Research Council of Turkey; Project Number: 113O779. The authors thank Mr. Markus Hoffman and Mr. Syed Salman Asad for the atmospheric pressure plasma power measurements. References Akdogan, E., Mutlu, M., 2012. Generation of amphoteric surfaces via glow-discharge technique with single precursor and the behavior of bovine serum albumin at the surface. Colloids Surf. B Biointerfaces 89, 289–294. Arrus, K., Blank, G., Abramson, D., Clear, R., Holley, R.A., 2005. Aflatoxin production by Aspergillus flavus in Brazil nuts. J. Stored Prod. Res. 41, 513–527. Baier, M., Foerster, J., Schnabel, U., Knorr, D., Ehlbeck, J., Herppich, W.B., Schluter, O., 2013. Direct non-thermal plasma treatment for the sanitation of fresh corn salad leaves: evaluation of physical and physiological effects and antimicrobial efficacy. Postharvest Biol. Technol. 84, 81–87. Basaran, P., Basaran-Akgul, N., Oksuz, L., 2008. Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment. Food Microbiol. 25, 626–632. Bata, A., Lasztity, R., 1999. Detoxification of mycotoxin-contaminated food and feed by microorganisms. Trends Food Sci. Technol. 10, 223–228. Becker, K., Koutsospyros, A., Yin, S.M., Christodoulatos, C., Abramzon, N., Joaquin, J.C., Brelles-Marino, G., 2005. Environmental and biological applications of microplasmas. Plasma Phys. Control. Fusion 47, B513–B523. Beuchat, L.R., 1986. Extraordinary heat resistance of Talaromyces flavus and Neosartorya fischeri ascospores in fruit products. J. Food Sci. 51, 1506–1510. Biederman, H., Boyaci, I.H., Bilkova, P., Slavinska, D., Mutlu, S., Zemek, J., Trchova, M., Klimovic, J., Mutlu, M., 2001. Characterization of glow-discharge-treated cellulose

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