Walnuts play an important part in human diet since ancient times. The walnut (Juglans regia L.) is quite widespread  in  Iran.  During  storage,  kernels  undergo a series of biochemical, physiological, and structural changes, which make them unacceptable to consume- rs. Walnut is a nutrient-rich food mainly because of its high biological value proteins (low lysine/arginine ratio), high levels of oil (60 g/100 g in average mainly polyunsa- turated fatty acids, or PUFA) [1]. Although fatty acids in walnuts have nutritional value, higher amounts of PUFA (owning unsaturated bands) may cause a poorer quality resistance and a shorter shelf-life [2]. Low oxygen pre- vent lipid oxidation. The most common oxidation indica- tors in oils are peroxide value (PV) and conjugated diene   value (CDV) [3]. Walnut kernels contain bioactive com- pounds such as phenols, so polyphenols are subject to oxidation [4, 5]. The walnut kernel can darken due to oxi- dation of phenolic compounds. L* index shows bright- ness of products [6]. Moisture is one of the important factors of the quality of nuts [7]. Moisture content (MC) of nuts has a profound effect on their physical, chemical, mechanical, aerodynamic, and thermal properties [8]. Postharvest operations are expected to have a major im- pact on the microbial contamination of nuts [9]. Among various microbes, fungi are known to play a significant role in the spoilage and loss of stored plant products [10]. Food safety issue requires safe methods with  no toxic substances. In recent years, edible coatings have been one of the most innovative ways to improve the     Copyright © 2019, Talebi Habashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.       commercial shelf life of fruits. An edible coating, such as chitosan, makes a barrier against moisture, oxygen, and dissolved materials and protects foodstuff from microbial, chemical, and mechanical damages [11, 12]. Chitosan has a higher expansion and elasticity, as well as anti-viral, anti-bacterial, anti-fungi, and antioxidant effects due to different amounts of free amine groups. It can participate in the reactions by forming hydrogen and ionic bonds [13]. Chitosan reduced the growth of Asper- gillus flavus, the absorption of moisture, and the rate of oxidative reactions [14]. Essential oils (EOs) have been extensively studied as additives in bio-based emulsified coatings. One example is the study by Campos-Requena et al. based  on  carvacrol  and  thymol,  both  included in HDPE/modified  montmorillonite  nanocomposite films [15]. Thyme essential oil (TEO) contains high le- vels of phenolic compounds, such as thymol and carvac- rol. The main component of non-phenolic compounds in TEO is paracymin. Thymol, carvacrol, and paracymin are all antioxidant agents [16]. Although edible coatings create a barrier against oxygen and moisture, they are not perfect replacement of synthetic packaging [17]. A large variety of active packaging systems have been de- veloped and. Today, numerous reviews have emphasized the potential of active packaging technologies to supply safer, ‘healthier’, and higher-quality foods to consumers [18]. Active packaging is characterized by changing the inside atmosphere of the packed food [19]. Unfavorable flavors, caused by rancidity during storage of the pro- duct, did not appear when oxygen adsorbents were ap- plied [2].   ging in polypropylene bags (PP), and active packaging in polypropylene bags containing sachets (AP). At the end, packets were stored in a dark cold room (55% RH, 4°C for 120 days) and tested every 60 days. Compositional  analysis.  The   kernels   were ground using a home  grinder  (La  Moulinette; Moulinex, Lyon, France). The protein was de- termined by means of the  micro-Kjeldahl  pro- cedure, using 5.4 as a conversion factor. The fat contents were evaluated by Soxhlet extraction. The total ash was determined by weighing the dry mine- ral residue of the samples obtained  at  500–550°C. The total amount of carbohydrate was measured by sub- tracting the amount of ash, protein, and fat from the to- tal dry matter. The moisture contents of the kernels were determined by oven-drying at 103 ± 2°C [21]. Oil extraction and quality analyses. The oil was extracted from kernels using n-hexane solvent without additional heat treatment. About 50 g of ground walnut was mixed with 50 ml of n-hexane (J.T. Baker, Deventer, Holland) and stirred for 30 min. The n-hexane extract was filtered, and the solvent was removed under reduced pressure using a rotavapor (RE 111; Büchi, Flawil, Swit- zerland) [22]. Peroxide value (PV). First, the acetic acid-chloro- form solution and the saturated potassium iodide (KI) solution were added to the oil sample. Second, 30 ml of distilled water was added, then 0.01-N sodium thiosul- fate was slowly titrated while shaking the flask vigorous- ly near the end point which was indicated by a faint blue color. Third, the sodium thiosulfate (Na S O ) was added   2   2    3   This study investigates the effects of chitosan coating enriched with thyme essential oil and types of packaging on the postharvest quality of Persian walnut under cold storage.   STUDY OBJECTS AND METHODS The study was conducted with walnuts (Juglans re- gia L.) purchased from the local market. Walnuts were shelled manually. The kernels were dried at room tem- perature till moisture contents of 3.16 ± 0.03%. The chemicals were supplied by Merck and AppliChem Companies. Sachets for active packaging were prepared with ascorbic acid, sodium bicarbonate, and iron powder with the 1:1:1 ratio. The chitosan solution (1%, w/v) was prepared by dis- solving chitosan powder in glacial acetic acid (1%, v/v). The solution was heated, and then glycerol was added as a plasticizer [20]. Tween 80 was used to achieve uniform distribution of essential oil inside the coating solution. TEO (500 and 1,000 μl L–1) was added to the solution; finally the uniform solutions were exposed to UV light for 1 hour for sterilization. First, the kernels were soaked in the coating solu- tions for 60 s. Second, the samples were dried at room temperature. The treatments resulted in four samples: control, i.e.  uncoated  (C);  coated with  chitosan  (CT); coated with chitosan containing 500 μl L–1  of thyme es-   dropwise until the blue color disappeared. Finally, the peroxide value (meq/kg–1) of oil was calculated accor- ding to the following equation: V × N × 1000 PV =                                      , W where V is the volume of the applied sodium thiosulfate, (ml); N is the normality of the thiosulfate, and W is the oil weight, g [23]. Conjugated diene value (CDV). The CDV was de- termined at a wavelength of 233 nm, using isooctane as an oil solvent. 0.1–0.3 g of oil was mixed with the isooc- tane solution. The amount of solution adsorption was determined at a wavelength of 233 nm with a spectro- photometer (Pharmacia, England) [24]. L*  index.  L*  index   (black/white)   of   the   ker- nel was measured using a HunterLab colorimeter (model D65/10) [25]. Mold count. 5 g of each sample was transferred into a sterile stomacher bag under aseptic conditions and di- luted 1:10 (w/v) with sterile peptone water (0.1%, w/v, Sigma-Aldrich, Darmstadt, Germany). Then the samples were homogenized for 2 min by means of a stomacher (Seward Laboratory, London, UK). The series of dilu- tions were prepared by adding 1 ml of each concentration to 9 ml of sterile peptone water (0.1% w/v). In order to count mold, 0.1 ml of each dilution was transferred onto   sential oil (CT   ); and coated with chitosan containing   the potato dextrose agar (PDA) medium using the surface   500 1,000 μl L–1  of thyme essential oil (CT   ). Third, each   culture method and was incubated at 25°C for 5 days [21].   1,000 sample was divided into three equal parts, and then they were packed as follows: loose packaging (LP), packa-   Sensory evaluation. A panel of 10 members eva- luated the overall acceptance using the 9-point Hedo-     Table 1. Compositional analysis of walnut kernels   Ash, % Moisture, % Fat, % Protein, % Carbohydrate, % 1.79 ± 0.11 3.16 ± 0.03 59.14 ± 0.66 15.07 ± 0.65 20.39 ± 0.56 Note: Mean values ± standard deviation over three replicates     Table 2. Effect of coatings and packaging methods on mois- ture content of kernels   Moisture content, % Packaging Coatings   Storage time, days       1 60 120 LP C CT CT 500 CT 1,000 2.25Ca 5.56Ba 5.97ABa 6.86Aa 2.06Cab 4.21Bb 4.35Bb 4.81Bb 1.30Db 2.97Cc 3.53BCc 3.73Bc PP C CT CT 500 CT                                            1,000                                                                                                     2.25Ca 5.56Ba 5.97ABa 6.86Aa 2.09Ca 4.95Ba 5.89ABa 6.04Aba 2.02CDa 4.68Ba 5.46Aa 5.96Aa AP C CT CT 500 CT 1,000 2.25Ca 5.56Ba 5.97ABa 6.86Aa 2.21Ca 5.42Ba 5.73ABa 6.62Aa 2.18Ca 5.20ABa 5.85Aa 6.29Aa 500   Note: C is the control sample; CT is coated with 1% chitosan; CT Moisture absorption in the coating can be effective- ly decreased due to the hydrophobic characteristics of TEO, which were placed in empty spaces between the polymer chains [28]. In agreement with the previously reported data, the water vapor permeability of coatings was reduced by adding coriander, citronella, tarragon, and TEO [29]. Active packaging ensures a high concen- tration of carbon dioxide and a high relative humidity in- side the package atmosphere [30]. Peroxide value (PV) and conjugated diene value (CDV). The analysis of variance showed the significant interaction effects (p < 0.05) for coating treatments, packaging methods, and time of storage. Coating treat- ments and time of storage, packaging methods and time of storage, coating treatments and packaging methods influenced the peroxide value and conjugated diene value considerably. The trend of CDV was similar to trends obtained for PV. There is a positive correlation between peroxide value and conjugated diene content in walnut [31].   and CT   are coated with 1% chitosan containing 500 and 1,000 μl   1,000 L–1  TEO, respectively. LP, PP, and AP are loose packaging, packa- ging in polypropylene bags, and active packaging, respectively. Su- perscript lower letters (a–d) beside mean values in the same row and superscript upper letters (A–D) beside mean values in the same co- lumn show the difference in Duncan’s multiple range test (p < 0.05). Standard error mean = 0.35   nic scale: 1 = dislike extremely; 2 = dislike very much; 3 = dislike moderately; 4 = dislike slightly; 5 = neither like nor dislike; 6 = like slightly; 7 = like moderately; 8 = like very much; 9 = like extremely. The panelists had sensory evaluation experience and were trained in de-   The initial PV in the walnut was about 0.04 meq/kg–1. 500                      1,000   The fresh walnut kernels in different cultivars had the peroxide values between 0.015–0.29 meq/kg–1; the di- versity can be related to the variety of the walnut trees and the weather conditions they grew up in [1]. During storage, the PV and CDV increased in all samples, the maximum amounts were detected in the C and the mini- mum amounts, in the CT and CT samples (Table 3). Chitosan prevents the reactive oxygen  species  (ROS) and the lipid oxidation in food and biological systems because of its antioxidant capacity [32]. Chitosan has antioxidant activity against free radicals [33]. The low   scriptive evaluation of nuts.   PV and CDV of CT   and CT   samples can be at-   Statistical analysis. The research employed a facto-   500                        1,000 tributed to antioxida       ropertie            TEO. Baldwin et al.   nt p   s of   rial method in the form of a complete randomized design with four replications. Data were subjected to analysis of variance (ANOVA) followed by LSD test (p < 0.05) to distinguish differences among the treatments. Statistical analyses and Pearson correlation coefficients between traits were analyzed using SPSS software 20.00.   also reported similar results in reducing the PV in oil of pecans [34]. The increasing of PV in the walnut during storage has also been reported [35].   Table 3. Effects of coating treatments and time of storage on PV and CDV of kernels   RESULTS AND DISCUSSION     Test     Coating             Storage time, days           The  chemical  compositions  of  the  kernels  were   treatments   1   60           120   shown in Table 1. Peroxide value, C 0.04Ac 1.15ABb 2.82Aa Moisture. As a result of the analysis of variance, meq/kg oil CT 0.04Ac 1.45Ab 2.09Ba the interaction effect of coating treatments and pack-   CT 500 0.04Ac 1.03Bb 1.62Ba aging methods on the moisture content was significant   CT 1,000 0.04Ac 0.97Bb 1.25Ca (p < 0.05). At the end of storage, the coated samples Conjugated diene C 4.88Ac 5.94ABb 7.543Aa had the highest moisture. The minimum and maximum moisture content was observed in LP and AP, respective- value, µmol/g CT CT 500 4.88 Ac 4.88 Ac 6.231Ab 5.828Bb 6.844Ba 6.393BCa ly (Table 2). The hydrophobicity characteristic of chi-   CT 1,000 4.88 Ac 5.771Ba 6.039Ca   500   tosan is conditioned by the acetyl groups in its structure, 1,000   which has not been completely deacetylated. Besides, re- sidual acetyl groups in chitosan play a role in preventing water vapor transmission [26]. Chitosan also decreases weight loss of guava [27]. 20   Note: C is the control sample; CT is coated with 1% chitosan; CT and CT      are coated with 1% chitosan containing 500 and 1,000 μl L–1 TEO, respectively. Superscript lower letters (a–c) beside mean va- lues in the same row and superscript upper letters (A–C) beside mean values in the same column show the difference in Duncan’s multiple range test (p < 0.05). Standard error mean = 0.15       Table 4. Effects of packaging methods and time of storage on PV and CDV of kernels   Table 6. Effects of coating treatments and packaging methods on L* index of walnuts                               Test   Pack-   Storage time, days   Packaging   Coatings   Storage time, days               aging 1 60 120       1 60 120 Peroxide value, meq/kg oil LP 0.04 Ac 1.33Ab 2.92Aa   LP C 74.87Aa 70.09Aab 59.76Cb   PP 0.04 Ac 0.95Bb 1.99Ba     CT 62.73Bb 68.42Ab 70.03Ba   AP 0.04 Ac 0.58Cb 1.30Ca     CT 500 76.34Aa 74.23Aa 72.74Ba Conjugated diene value, LP 4.88 Ac 6.11Ab 7.639Aa     CT 61.61Bb 72.94Aa 75.72Ba                                              1,000                                                                                                          µmol/g   PP         4.88 Ac AP        4.88 Ac   5.752Bb 5.395Bb   6.748Ba                 PP 6.08Ba   C                  74.87Aa CT                62.73Bb   72.95Aa 69.08Ab   65.05BCb 74.46Ba   Note: LP, PP, and AP are loose packaging, polypropylene bags, and active packaging, respectively. Superscript lower letters (a–c) beside mean values in the same row and superscript upper letters (A–C) be- side mean values in the same column show the difference in Duncan’s   AP multiple range test (p < 0.05). Standard error mean = 0.13 for PV and 0.15 for CDV   Tables 4 and 5 showed that the maximum PV and     CT   CT 500   1,000 CT C CT CT 500   1,000 76.34Aa 61.61Bb 74.87Aa 62.73Bb 76.34Aa 61.61Bb 78.18Aa 74.86Aa 67.81Bb 69.80ABab 77.85Aa 65.44Cb 79.85ABa 79.43ABa 72.58Bab 78.87Ba 83.17Aba 86.25Aa   CDV values were with LP and the minimum, with AP. In fact, oxygen is an oxidation resonator; the degree of rancidity was reduced by increasing the amount of car- bon dioxide inside the package. The mixture of ascorbic acid and sodium bicarbonate is used in active packa- ging, so carbon dioxide is produced by combining them. This system is used to increase the shelf-life of fresh meat and fish [36]. L* index. As a result of the analysis of variance, the effect of coating treatments and packaging methods on the L* index was significant (p < 0.05). The maximum value was in the CT1,000 samples  with  active  packa- ging and the minimum, in the control sample with loose packaging (Table 6). The walnut kernel has bioactive compounds such as phenols. The dark color of the walnut kernel in the con- trol sample may be caused by the enzymatic oxidation of carotenoids and phenolic compounds in the kernels [37]. Enzymatic browning of phenols in peanuts correlated   Note: C is the control sample; CT is coated with 1% chitosan; CT and CT     are coated with 1% chitosan containing 500 and 1,000 μl L–1  TEO, respectively. LP, PP, and AP are loose packaging, packa- ging in polypropylene bags, and active packaging, respectively. Su- perscript lower letters (a–d) beside mean values in the same row and superscript upper letters (A–D) beside mean values in the same co- lumn show the difference in Duncan’s multiple range test (p < 0.05). 500   1,000 Standard error mean = 2.13   pounds. The highest L* index in AP resulted from the low oxygen content in the package. The acceptable value of the L* of walnut color was above 40 [39]. The L* in- dices of all samples were higher than 40, and the coated and actively packed samples had the highest L* index. Mold count. As shown in Fig. 1a, the counts of mold in all samples increased during storage, but the con- trol sample count had the highest. In all the treatments, the growth of molds increased within 60 days, but af- ter that in the coated samples,  no  significant  change was observed in the growth of fungi. To the contrary, in the control sample, the number of fungi increased to   with the decrease in the L* index during storage [38].   log10   3.67 CFU/g–1 by the end of storage.   The reason of high L* in CT500  and CT1,000  samples can be attributed to the antioxidant properties of chitosan and TEO which inhibited the oxidation of phenolic com-   Table 5. Effects of coating treatments and packaging methods on PV and CDV of walnut   Chitosan extends the product shelf life directly af- fecting the growth of fungi and other defense opera- tions, such as chitinase  accumulation,  which  reduces the inhibitory effect of fungal cell wall proteinase. Chi- tosan inhibits microorganisms such as gram-positive and gram-negative bacteria and fungi [40]. According to [41] and [42], chitosan coating in artichoke seeds reduced the activity of various fungi and microorganisms on toma- toes. Antifungal effect of chitosan coating on pears was reported by Xianghong et al. [43]. TEO in combination with chitosan coating decreased     CT   Test Coatings   Package       LP PP AP Peroxide value, meq/kg oil C CT 3.48Aa 2.95Ba 2.91Ab 2.25ABb 2.48Ac 2.01Ab   500 CT 2.52BCa 2.34Ca 2.03BCb 2.06Cb 1.16Bc 1.06Bc the fungi count: CT1,000  and CT500  samples had less mi-                                                             1,000                                                                                            crobial load than the others did. The main constituents Conjugated diene C 8.175Aa 7.629Ab 7.217Ab of TEO are thym value, µmol/g CT 7.668Ba 6.997Bb 6.767Ab bial effects [44].     ol and carvacrol, which have antimicro-           CT   500 CT   7.256Ba 7.08Ba   6.784Bb 6.815Bb   5.95Bc 5.857Bc   As  shown  in  Fig.  1b,  with  the  LP  method,  mold growth was significantly higher than with other packa-                                                           1,000                                                                                          500   1,000 Note: C is the control sample; CT is coated with 1% chitosan; CT and CT     are coated with 1% chitosan containing 500 and 1,000 μl L–1 TEO, respectively. Superscript lower letters (a–c) beside mean va- lues in the same row and superscript upper letters (A–C) beside mean values in the same column show the difference in Duncan’s multiple range test (p < 0.05). Standard error mean = 0.15 for PV and 0.17 for CDV   ging methods, but there was no significant difference be- tween PP and AP methods. As shown in Fig. 1c, with the LP method, the hi- ghest mold growth was observed in the control sample, but there was no significant difference between the oth- er  treatments.  The  maximum  and  minimum  growth     C CT CT CT 500 LP PP AP 4     Mold, log CFU/g) 3     2   1     0 1                            60                          120 Time, days     1,000   CT500   CT1000 CT 500 CT 1,000 C                 CT   (a) 4   Mold, log CFU/g) 3     2 Fig. 2. Effects of treatments and packaging methods on overall 1                                                                                                        acceptability score of walnut kernels. C is the control sample;   500   CT is coated with 1% chitosan; CT   and CT   1,000 are coated   0 1                            60                           120 Time, days LP                          PP                          AP   (b)   3   Mold, log CFU/g) 2       1     0 LP                          PP                          AP Packaging method CT500 CT1000 CT 500 CT 1,000 C                CT (c) Fig. 1. Effects of coating treatments (a) and packaging meth- ods (b) during storage and the effect of coating treatments and with 1% chitosan containing 500 and 1,000 μl L–1 TEO, respectively. LP, PP, and AP are loose packaging, packaging in polypropylene bags, and active packaging, respectively (p < 0.05).   Sensory evaluation. As shown in Fig. 2, the AP and LP control samples had the highest and lowest overall acceptability score, respectively. The high overall accep- tability score in active packaging (AP) could be attribu- ted to the low peroxide content in the samples. Although peroxides themselves do not directly play a role in off-fla- vour, but the ingredients of their decomposition produce an undesirable flavour. Hydroperoxides break down to form short-chain compounds, including aldehydes, ke- tones, alcohols, acids, esters, lactones, ethers and hydro- carbons, which contribute to odor and taste [47]. 500 There was no significant difference in CT and CT samples in all three packages. The results indicated that chitosan did not adversely affect the general acceptance score of the walnut kernel, which was similar to the pre- vious studies on walnut kernels, strawberries, and peeled lychee [35, 48, 49].   packaging methods (c) on the growth of molds in the kernels. C is the control sample; CT is coated with 1% chitosan; CT     CT   1,000 treatment resulted in the lowest sensory pro-   and CT   500 are coated with 1% chitosan containing 500 and   perties  with  all  three  kinds  of  packaging.  Therefore,   1,000   1,000 μl L–1   TEO, respectively. LP, PP, and AP are loose pa-   treatments  containing  high  levels  of  TEO  were  eva-   ckaging, packaging in polypropylene bags, and active packa- ging, respectively (p < 0.05).   luated as undesirable. This can be related to a high con- centration  of  the  essence  oil.  The  overall  acceptance score for this treatment was higher with LP than with   was  in  CT  and  CT   samples,  respectively.  Active   other packaging methods. With LP, because of volatile   1,000 packaging had a better effect on controlling the growth 2   of molds. This could be due to creating an atmosphere with  higher  CO ,  which  inhibits  growth  of  microor- characteristics of TEO, some of the TEO evaporated. It made the flavour score of the sample comparable to those with other packaging methods.   ganisms. CO   is the only gas that has a direct antimi-   2 crobial effect and increases the lag phase and growth time during the logarithmic growth stage [45]. Packa- ging with the modified atmosphere (with a low oxy- gen and high carbon dioxide content) was effective in controlling fungal rot and protecting the quality in the post-harvest period of fruits [46].   CONCLUSION The shelf-life and quality of  the  walnut  kernel can be affected by some environment factors during storage.  Coating  materials  and  packaging   methods can be useful  for  prolonging  the  postharvest  quali- ty  of  crops.  The  study  results  showed  that  chitosan       and thyme essential oil coating  combined  with  ac- tive  packaging  had  a  significant  effect   on   redu- cing oil oxidation and growth of molds. They also pre- vented the loss of moisture and a decrease in L* va- lue, improving the sensory properties of the samples during storage. An increase in the essential oil up to 500 μl L–1 also improved the functional properties of chitosan coating. Compared with loose packaging, poly- propylene packaging was also  effective  in  protecting the qualitative properties of walnut. Active packaging   offered the considerable potential and proved the most efficient.   CONFLICT OF INTEREST The authors declare no conflict of interest.   ACKNOWLEDGMENTS The authors would like to thank the department of hor- ticultural and food industry, science and research branch, Islamic Azad university, Tehran, for their help and support.