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

Note: Mean values ± standard deviation over three replicates

Table 2. Effect of coatings and packaging methods on mois- ture content of kernels

Moisture content, %

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.

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

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

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

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).

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

                                                        1,000                                                                                         

crobial load than the others did. The main constituents

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                                                                                         

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

0

1                            60                          120

Time, days

1,000

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;