The effect of chitosan coating combined with cold plasma on the quality and safety of pistachio during storage

Abstract Pistachios are one of the most important agricultural and export products of Iran. Fresh pistachio fruit has soft skin, is highly perishable, and therefore has a short life after harvesting, which has made traders and consumers have a great desire to increase the shelf life of this product. For this purpose, in this study, the effect of different concentrations of chitosan as an edible coating (0.5 and 1.5% w/v) and the duration of cold plasma treatment (60 and 120 s) were investigated during 180 days of pistachio storage. The effect of treatments on the shelf life of pistachio fruit was evaluated by determining moisture content, color components, peroxide value, total mold and yeast, hardness, aflatoxin content, and sensory evaluations. The results showed that the treatment with 1.5% chitosan coating and 120 s of cold plasma treatment preserved the hardness of the pistachio and the color indices in the best way (p < .05). Also, this treatment had the minimum number of peroxide, aflatoxin, and mold and yeast counts during the storage time. The treatments with chitosan coating and under plasma application did not cause any unpleasant odor or taste during the storage time. In conclusion, according to the results of this research, it was determined that the simultaneous use of chitosan coating and cold plasma treatment can potentially be used as a new approach for commercial applications and the export of fresh pistachios.

Decontamination of dry products such as pistachios is difficult because microorganisms, especially spore-bearing microorganisms, are more resistant in an environment with low water activity. To sterilize and reduce the microbial load of pistachios, methods such as irradiation, and use of ethylene oxide, and steam are also used, and each of the mentioned methods has disadvantages. The radiation method is very expensive and people have a negative attitude toward it. Ethylene oxide is banned as a carcinogenic substance in the European Union. The steaming method also causes clumping, color change, etc. Considering the mentioned disadvantages and existing problems, there is a need to develop new processes to reduce the microbial load and replace the existing methods.
One of the developed antimicrobial technologies for sterilizing contaminated surfaces is the use of atmospheric nonthermal plasma.
This technology, which is based on the use of ionized gases and produced at room temperature and atmospheric pressure, has been noticed since the mid-1990s, but its use as a method of microbial decontamination of food still attracts the attention of researchers (Pankaj et al., 2018). Plasma is the fourth state of matter after solid, liquid, and gas. Plasma is created by the increase in molecular energy and the interaction of ionized particles, and these particles include atoms, free radicals, electrons, photons, and positive and negative ions. In order to produce plasma, ionized gases such as oxygen, air, nitrogen, or argon can be used under the influence of high-frequency electromagnetic or electric fields and by placing in direct (or alternating) currents and waves (radio or microwave) (Bourke et al., 2018).
The most common method to produce and stabilize a cold plasma is to apply an electric field to a neutral gas. This process may take place under atmosphere, vacuum, high pressure, heat, and chemical reactions. Cold plasma by electrons and ion bombardment, as well as the thermal effect and free radical production of ultraviolet rays, causes the destruction of the bacterial cell membrane and denatures the proteins, and also causes the destruction of DNA (Pankaj & Keener, 2017;Zhu et al., 2020). Cold plasma technology is considered one of the emerging alternative techniques for preserving food commodities, extending shelf life, and retaining bioactive compounds in foods. Besides, due to its nonthermal nature, CPT is a useful technology for the sterilization process, especially for heat-sensitive foods (Siciliano et al., 2016;Wu et al., 2021). There are fewer reports on aflatoxins degradation by cold plasma. In particular, relevant research for aflatoxins degradation is still at the laboratory stage, with most experiments on vessels containing a few food samples (Wu et al., 2021).
Previous studies have used cold plasma to reduce the microbial load of different products (Guo et al., 2023;Mahnot et al., 2020;Mandal et al., 2018;Misra & Jo, 2017;Niveditha et al., 2021); however, no study has investigated the joint effect of coating with chitosan and cold plasma on pistachios. Therefore, as aflatoxins contamination is a vital challenge facing the pistachio as well as considering the importance of cold plasma technology and edible coatings as novel technologies to extend the shelf life of food products, the purpose of this study is to use the simultaneous application of plasma treatment and chitosan coating on pistachio and investigating the effect of these treatments on the physicochemical, microbial, sensory characteristics, and reducing aflatoxin of pistachio.

| Materials
Akbari variety pistachios were purchased from Rafsanjan, Iran. The hard shell of the pistachio was separated and the healthy kernels were selected and stored in polyethylene and black nylons in the refrigerator (4°C) until use. Chitosan powder with two low molecular weights (70 KD) from crab shells, each in 50-g cans was purchased from Sigma Aldrich. The powders of aflatoxins were obtained from Sigma. All other chemicals used in this study were of analytical grade and purchased from chemical suppliers.

| Preparation of chitosan solution
In order to prepare chitosan solutions with concentrations of 0.5% and 1.5% (w/v), respectively, 5 and 15 g of chitosan powder were added slowly and in several stages to 1000 mL of 1% acetic acid on a stirrer. Stirring continued for 5-6 h (until all chitosan particles were dissolved and the solution became clear). After the solution became clear, glycerol was added to the solution as a plasticizer equal to half the weight of chitosan and stirring was continued for another 15 min. Then the solution was removed from the magnetic stirrer and it reached the ambient temperature. After that, the solution was filtered with the help of Whatman no. 3 filter paper and a vacuum pump (Maghsoudlou et al., 2012).

| Pistachio coating
At first, pistachios were weighed and placed in mesh containers. By placing the mesh container containing the pistachio kernels in the container containing the chitosan solution, the pistachio kernels were immersed in the chitosan solution for 40 s and removed. For this purpose, concentrations of 0.5%, 1%, and 1.5% of each type of chitosan with low molecular weights were used. The control sample was also prepared by immersing pistachio kernels in water. After coating, in order to remove excess moisture, the pistachios were dried for 4 h in an oven at 40°C (until the moisture content was 3.5% or 4%) (Zhang et al., 2017).

| Cold plasma treatment
The main structure of plasma products in this article is the discharge of the dielectric barrier (Nik Fanavaran Plasma, Iran). This structure includes two cylindrical copper electrodes with a height of 12 mm and a diameter of 40 mm. Each of the electrodes was mounted between two circular plates. Air was used as a gas source to form a uniform plasma in the space between two electrodes by applying a voltage between two electrodes. Argon gas type, oxygen gas pressure of 0.4 millibars equivalent to 0.3 Torr and power of 89 watts which were equivalent to radiometric waves. In order to treat pistachios with plasma, about 100 g of coated pistachios was placed in the machine, observing the hygiene requirements (Zhu et al., 2020).
Then they were treated for 60 and 120 s ( Figure 1).

| Storage stability
The prepared pistachio kernels were randomly divided into 50 g units and packed in 20 × 6 cm polyethylene bags. The packed pistachio kernels were kept at room temperature (25-27°C) for 6 months.
During the storage period, every month, one package was randomly taken from each treatment and chemical and microbial tests were performed on all treatments. The treatments used in this study are shown in Table 1.

| Moisture content
Moisture content was measured according to AOAC (2000) method.
First, metal containers for measuring humidity were washed and placed in a 105°C oven for 2 h; then they were transferred to the desiccator and reached the ambient temperature. From each treatment, 5 g of pistachio nuts was crushed and after transferring to a container, their weight was recorded; then they were transferred to a 105°C oven. After 5 h, the dishes containing crushed pistachio kernels were removed from the oven and transferred to the desiccator; after reaching the ambient temperature, their weight was recorded. The moisture was obtained through Equation 1. The amount of moisture was reported based on wet weight.
where M 1 is the weight of the dish and pistachio kernels before entering the oven at 105°C, M 2 is the weight of the container and pistachio kernels after leaving the oven at 105°C and reaching the ambient temperature in the desiccator and M 0 is the initial weight of pistachio nuts.

| Peroxide value (PV)
Before performing the peroxide value test, pistachio oil was extracted using Rabadán et al. (2017). The Peroxide value was determined according to the official methods of AOCS (2011). Briefly, the oil sample (3 g) was dissolved in glacial acetic acid (30 mL) and chloroform (20 mL) (3:2 v/v). Then saturated KI solution (1 mL) was added.
The mixture was kept in the dark for 1 min, after adding distilled water (50 mL), the mixture was titrated against sodium thiosulfate (0.01 N). The PV value (mEq of oxygen/kg) was calculated using the following equation: where S is the volume of sodium thiosulfate solution (blank corrected) in mL, N is the normality of sodium thiosulfate solution, and W is the weight of the oil sample (gram).

| Total count of mold and yeast
For this purpose, 10 g of pistachio nuts was pounded with a sterile mortar next to the flame and under the microbial hood. Pistachio powder along with 90 mL of sterilized 0.85% sodium chloride solution was transferred to the Stomaker bag and mixed completely in the Stomaker. From dilutions 10 −1 , 10 −2 , and 10 −3 were also prepared. Yeast Glucose Chloramphenicol Agar (YGC) culture medium was used for the cultivation and counting of mold and yeasts in pistachio samples. After preparing the culture medium, it was sterilized.
After that, the temperature reached 45°C. Under the hood, 10 to 15 mL of the medium was added to the disposable plates, and a pe-

| Determination of aflatoxins
The measurement of aflatoxin was done by HPLC method using immunoaffinity column clean up-Test method (Balsini et al., 2021;Rezaie & Zareie, 2021). For minimizing the sub-sampling error in aflatoxins analysis, water slurry of pistachio samples were prepared.
For that matter, 1.5 L of water was added to 1 kg of pistachio. The resulting mixture was blended for 15 min with the slurry machine. Aflatoxins were eluted from the column by passing 1.5 mL of HPLC grade methanol and then 1.5 mL of HPLC grade water and using gravity to collect the eluate into a glass vial. A 50 μL aliquot of the eluate was injected into the HPLC.
(2) PV value = 1000 (S × N) × W TA B L E 1 Treatments used in this study.

| Statistical analysis
Each experiment was carried out at least in duplicate and measurements were performed at least in triplicate. Statistical analysis of data was performed using Microsoft Excel. Analysis of variance was calculated using the SPSS (Version 26.0. Armonk, NY: IBM Corp.) with a confidence level of 0.05, to find any significant difference between treatments.

| Moisture
The moisture content is one of the important factors in determining the quality of dried fruit. The moisture content of pistachios during harvesting is 35-40%, which decreases to 4-6% during the drying process; in this moisture, the product is stable and its water activity is less than 0.6. Changes in the moisture content of pistachio samples are shown in Figure 2. Due to the low initial moisture content of the product (4.82%) and the absence of a hard pistachio shell as a protector, there is a possibility of moisture absorption by the pistachio kernel during the storage period. By using edible chitosan coating and polyethylene bags as secondary packaging, the rate of moisture absorption in pistachio nuts can be reduced. During the storage period at room temperature and relative humidity (25-27°C and 35-45%), the amount of moisture absorption in the samples without chitosan coating was always higher than the samples with coating and a significant difference was observed between them, which is in agreement with the results of previous studies (Hamasalih & Rasul, 2022;Jafari & Javadi, 2020;Kaviani et al., 2015). Also, the moisture content of chitosan-coated samples remained in the range of 4% (initial product moisture) from the beginning to the end of the storage period. While in the samples without chitosan coating, due to the lack of barrier against moisture transfer, at the end of the storage period, the pistachio kernel moisture content reached about 6%.
As seen in Figure 2, changes in moisture with different plasma treatments do not follow a specific pattern. Therefore, it can be said that plasma treatment had no significant effect in preventing moisture transfer and only the presence or absence of chitosan coating was effective in moisture transfer. In general, the chitosan coating, like a barrier, reduces the rate of moisture transfer between pistachio kernels and the surrounding atmosphere. Due to the low moisture level of pistachio kernels and considering that the pistachio kernels were packed in polyethylene bags and were not in direct contact with the humidity of the environment, therefore chitosan concentrations did not show a significant effect on the moisture content of pistachios.

| Color parameters
The color of the pistachio kernel is the natural color of the third skin of a healthy pistachio kernel, which usually varies depending on the variety, the length of growth, the time of picking, and storage, and is a mixture of green, purple pink, and red. Anthocyanin, chlorophyll, and xanthophyll are the main pigments of the pistachio kernel, and their amount varies depending on the ripening of the pistachio fruit (Molamohammadi et al., 2020). Since no adverse enzymatic or nonenzymatic reactions occur during the storage of dried pistachio nuts, the main reason for checking the color of pistachio nuts during storage was to ensure that the coating on the pistachio nuts was colorless and that the nuts did not change color by covering them with chitosan. Table 2 shows the results of color parameters L*, a*, and b* of pistachios, which represent their lightness, redness, and yellowness, F I G U R E 2 Moisture changes of pistachios treated with chitosan and plasma during storage. gradually, from the third month onwards, the difference between them became significant. Also, according to the results of the plasma treatment, there was no effect on the color of any of the treatments.

| Peroxide value
Considering that about 53.5% of pistachio weight is made up of fat, and of this amount of fat, 56-70% is a monounsaturated fatty acid (oleic acid), and 18-31% is an unsaturated fatty acid (linoleic acid).
This product is very ready for oxidation. If the environmental conditions are unfavorable during the pistachio storage period, spoilage reactions such as spontaneous, optical, and enzymatic oxidation begin and free radicals are formed. The production of these radicals, as well as the production of side compounds such as free fatty acids, hydroperoxides, aldehydes, ketones, and volatile alcohols, create an unpleasant taste and smell in the pistachio kernel (Kaviani et al., 2015). Peroxide value represents primary reaction products of lipid oxidation, which can be measured by their ability to liberate iodine from potassium iodide (Nor et al., 2008). According to  Increasing the duration of plasma treatment has slightly in-

| Microbial count
During the storage period, the growth of mold and yeast in the sample showed an increasing trend. As seen in Figure 4, the sud-

| Hardness
The results of pistachio hardness after 180 days of storage are shown in Figure 5. Accordingly, no significant difference was observed between the different treatments. In some studies, an increase in hardness in coated samples was reported, which is related to the higher moisture content (Li et al., 2022;Wang et al., 2015). Whereas some other studies did not observe a significant effect between chitosan coating and hardness (Kazemian-Bazkiaee et al., 2020;Maghsoudlou et al., 2012).

| Aflatoxin content
Aflatoxins are toxins that are mainly produced by a number of different species of Aspergillus such as Flavus and Parasiticus under certain conditions and have different types, the most important of which are B1, B2, G1, and G2. In this method, aflatoxins are meant to be B1, B2, G1, and G2 types. In the aflatoxin test, since it was not possible to analyze all the treatments, the ones that showed the best results in other tests, that is, P 120 C 0 and P 0 C 1.5 along with the control sample were analyzed on days 0, 90, and 120 (Iran, 2002;Regulation, 2010).
The results revealed ( Table 3) that, the total aflatoxin levels in control treatment after 90 days of storage were above the Codex F I G U R E 4 Total mold and yeast changes in pistachios treated with chitosan and plasma during storage. Several studies have been conducted on the ability to remove aflatoxins by chitosan (Chaudhari et al., 2020;Cota-Arriola et al., 2011).
In a similar study, Pirouz et al. (2020) and the overall efficiency of cold plasma degradation for specific food contaminants (Wu et al., 2021). The aim of this study was to investigate the mechanism of degradation of food contaminants by cold plasma, and the benefits and drawbacks of this method (Tasouji et al., 2021).
Results available up to now show that the mechanisms of degrading aflatoxins by cold plasma treatment are mainly from the perspective of aflatoxins structure. Aflatoxins toxicity is related to the C8 = C9 double bond on the furan ring (toxicity site), and the degradation products are regarded as less toxic than the original due to losing the double bond (Tasouji et al., 2021;Wu et al., 2021).

| Sensory evaluation
The results of sensory analysis after 180 days of storage are presented in Figure 6 by a radar chart. The compared attributes were placed on pentagonal sides and the treatments were marked with different colors. The taste evaluation of the control sample was TA B L E 3 Color changes of pistachios treated with chitosan and plasma during storage. F I G U R E 6 Sensory evaluation results of pistachio treated with chitosan and plasma after 180 days of storage. not done due to the contamination of the sample with aflatoxin, and this sample was rejected in terms of taste. According to the obtained results, it was found that in all the investigated parameters, the control sample and the plasma treatments received the lowest and highest scores, respectively. No significant difference was observed between different concentrations of chitosan and different times of plasma treatment. Chitosan is a tasteless and colorless compound, and in the concentrations used, the panelists did not feel any special taste or smell regarding the treatments containing chitosan. Sensory analyses of texture and color also confirm the results of instrumental analysis. In general, the evaluation of overall acceptance showed that the lowest and highest scores were related to the control sample and the P 120 C 1.5 treatment, respectively.

| CON CLUS ION
The present study indicated that the cold plasma treatment and chitosan coating can be used as a potent method to increase the storage stability of pistachio. The results showed that the concentration of 1.5% chitosan and cold plasma treatment for 120 s could significantly reduce the amount of mold and yeast after 120 days of storage. It also had a significant effect on reducing the amount of aflatoxin. In addition, other physicochemical characteristics of pistachios did not change significantly. In addition, the sensory evaluation also showed that these treatments did not have an adverse effect on the sensory characteristics of pistachios. Thus, it successfully increased the shelf life of fresh pistachios. This study will provide bases for future studies in this area. It is recommended that other nuts and perishable food products be examined for microbial and aflatoxin content.

ACK N OWLED G M ENTS
The first author expresses her sincere gratitude to Zahra Safaei from iQneiform Oy for her valuable scientific collaboration in this research project.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.