Comparison of the Oil Quality of Light and Dark Walnuts under Different Storage Conditions.

The skin of the walnut kernels can get dark during the pre- and post-harvest stages of the production. Dark kernels are less palatable for most consumers but are still edible and maybe preferable, especially in the ground form, for industrial use. In this study, we investigated the differences between oil oxidation indexes, fatty acid and tocopherol compositions of the oils, total polyphenol contents and antioxidant capacities of the extracts of light and dark walnuts. In addition, we evaluated the effects of packaging under nitrogen and vacuum-packaging techniques and storage temperature on these characteristics of both light and dark walnuts during storage for 6 months. Peroxide values and free fatty acid contents of all samples were higher at the end of storage compared to initial values, being more noticeable at 20°C than at 4°C. Increases in the free fatty acid contents were quite higher in dark walnuts than the light ones (6.1 and 3.1 fold, respectively) and the highest values of conjugated diene and peroxide were determined in the samples packaged under air and stored at 20℃. Dark walnuts had lower total phenolic, α- and γ-tocopherol contents and antioxidant activities compared to the light ones. Total phenolic and tocopherol contents decreased over time. We conclude that due to the antioxidant compounds in the fruit, oxidation parameters of dark walnuts are still acceptable at the end of 6-month storage even in the packages with air and at non-refrigerated conditions.

required quality characteristics including ideal kernel color 11 .
In fact, darkening of walnut kernel is a typical example of enzymatic browning in which phenolic compounds serve as substrates. Zhang et al. 12 identified potential genes in Chinese walnuts associated with enzymatic browning. Oxidizable phenolic compounds, such as o-diphenols, are transformed to brownish o-quinones as a result of enzymatic browning reactions. These reactions are more likely to occur in fresh walnuts compared to dried ones due to relatively higher moisture content of the former 8 . Therefore, fresh walnuts have to be dried as soon as possible to a moisture content of 5-8 to minimize darkening of the kernels 9 .
As a matter of fact, dark walnuts are perhaps not preferable but are still edible, while moldy and insect-invaded walnuts are almost worthless. Dark walnuts are classified as second quality 13 and can be sold at relatively lower prices than the first quality products 14 . They are generally chopped or ground and used as an ingredient of bakery products, chocolate, etc. or can be used for walnut oil production for economic choices. In recent years, quality attributes of dark walnuts from different varieties have been compared with those of light ones and significant differences in important chemical and sensory properties have been reported 7,15,16 .
The effects of different packaging techniques and storage conditions on the oxidation values of inshell walnuts, walnut kernels, ground walnut and walnut oil have been evaluated by other authors 17 21 . It can be concluded from all these studies that reducing oxygen content in the package and refrigerated storage conditions can slow down the oxidation reactions. Mexis et al. 22 demonstrated that temperature was the most important parameter for oxidation reactions occurred in the walnuts and other determinative factors were oxygen availability and lighting conditions. Christopoulos and Tsantili 23 showed that walnuts became darker and their phenolic and antioxidant contents decreased during storage. They also determined that packaging under nitrogen N 2 and carbon dioxide CO 2 and refrigerated storage conditions were effective on limiting both antioxidant loss and darkening of the walnut. López et al. 24 reported that walnut type is also a crucial factor on darkening of the kernels during refrigerated storage of the walnut fruit.
As described above, the effect of packaging and storage conditions on ongoing natural processes including darkening of the kernels have been studied. However, there are no reports investigating the storage period of the walnut kernels that already have a dark color at the beginning of the storage. In addition, although they are sold in the market and used as an ingredient of many food products, the shelf life of these dark walnuts has not yet been reported. Furthermore; the effects of packaging techniques and storage conditions on quality parameters were well defined for first quality walnuts, but have not yet been revealed for dark ones.
It is known that phenolics on the pellicle have a protective role in oxidation reactions 25,26 . The amount of phenolics decreases as the oxidation reactions proceed 27 . Decrease in phenolics can cause a decrease in resistance against lipid oxidation with a concurrent darkening of the pellicle of the kernel. Since these reactions are quite complicated and probably trigger each other, in our opinion, they should be separately investigated in light and dark walnut samples.
The purpose of this study was to compare ground light and dark walnut kernels in terms of oil oxidation indexes, fatty acid and tocopherol compositions of their oils, total polyphenol contents and antioxidant capacities of their extracts. We also investigated the effects of packaging under nitrogen and vacuum-packaging techniques on quality parameters of these samples during storage for 6 months at 4 and 20 and determined if there are any differences between the oxidation status of light and dark walnuts.

Materials
The cultivar of the walnuts used in the study were Sebin , one of the most widely planted cultivar in Turkey. Fruits were harvested from trees in an orchard located in Acipayam region in Denizli, Turkey, in September 2018. The fruits were hulled and dried in open air for 4 days to a moisture content of about 3.5 . Dried walnuts were stored at 4 for further experiments. Chemicals used in the study were of analytical grade or higher purity. Anhydrous sodium sulfate, chloroform, potassium hydroxide, sodium hydroxide, sodium carbonate, 2,2-diphenyl-1-picrylhydrazyl DPPH , 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid trolox and Folin-Ciocalteau reagent were purchased from Sigma-Aldrich St. Louis, MO, USA . Starch and gallic acid were purchased from Merck Darmstadt, Germany . Potassium iodide, sodium thiosulfate, 2-propanol and methanol chromatography grade were purchased from Carlo Erba Milan, Italy . Isooctane, methanol reagent grade and acetic acid were purchased from Isolab Inc. Akron, OH, USA . Ethanol and n-hexane were purchased from Tekkim Chemicals Istanbul, Turkey .

Categorization, grinding, packaging and storage
The shells of the walnuts were cracked using a nutcracker and removed manually. The walnut kernels were visually inspected and defective kernels damaged, shrivelled and moldy ones were discarded. The surface color of the remaining kernels were measured three measurements for each kernel using a portable colorimeter CSM 1, PCE Instruments, Southampton, UK and L*, a* and b* values were determined. Measuring aperture of the colorimeter was 4 mm and the light source was D65. Chroma and hue angle values were calculated from a* and b* values according to the following equations: Hue angle arctan b*/a* 2 Categorization of the kernels was performed using a system developed by Brawner and Warmund 6 with a slight modification. Accordingly, LCH sums L* chroma hue angle were calculated and the kernels with a LCH sum ≥ 75 were described as light and the kernels with a LCH sum ≤ 50 were described as dark . Light and dark walnuts were separately ground using a coffee grinder King, K-444 Grindex, Istanbul, Turkey . The ground walnut samples were sieved through stainless steel sieves to obtain homogeneous particles with sizes in the range of 1-4 mm. Then, 75 g of the ground samples were weighed into a filter bag and the bag was placed into laminated plastic pouches polyethylene polyamide ethylene vinyl alcohol polyamide polyethylene, 25  2 Measurement of gas composition in the packages Gas composition in the packages was determined just after packaging and on each sampling day using a portable gas analyzer Dansensor, Checkpoint, PBI, Ringsted, Denmark on two replicates. The analyzer drew a gas sample from the package atmosphere via a syringe needle and detected the oxygen and carbondioxide concentrations. The nitrogen concentration was calculated by subtracting the sum of these concentrations from 100 .

Oil extraction from ground walnut samples
Walnut samples from each sampling day 75 g were further ground using a mortar and pestle and transferred to an erlenmeyer flask. Then, approximately 375 mL nhexane was added and the content of the flask was homogenized at 14000 rpm for 1 min using a homogenizer IKA T18 Ultra-Turrax, Staufen, Germany . The flasks were placed on an orbital shaker OS-20, Boeco, Hamburg, Germany and shaken at 140 rpm for 2 h at room temperature. After this period, the homogenate was filtered through a layer of anhydrous sodium sulfate and the filtrate was collected into a 250 mL round bottom flask. The remaining walnut sample was re-extracted with the same procedure described above. The extracts were combined and n-hexane was evaporated at 40 under vacuum using a rotary evaporator Büchi Rotavapor R-114, Flawil, Switzerland . The walnut oil and the defatted walnut sample were separately kept at 20 until use in the analysis.

Determination of free fatty acid and acid values
Free fatty acid FFA values of the oil samples were determined according to the Official Method Ca 5a-40 of the American Oil Chemical Society 28 . Accordingly, 1 g of oil sample was dissolved in 10 mL of ethanol and this solution was titrated with the standardized sodium hydroxide solution in the presence of phenolphthalein as an indicator. FFA value of the sample was calculated according to the following equation: where; V is the volume mL of the NaOH solution used in the titration, N is the normality of the NaOH solution and m is the weight g of the sample. The acid value is expressed as the number of milligrams of potassium hydroxide required to neutralize the free fatty acids present in one gram of oil sample. The acid value was determined by multiplying the value obtained for the percent free fatty acids FFA by 1.99 28 .
The acid value was calculated as: Acid value mg KOH g 1 1.99 FFA 4

Determination of peroxide value
Peroxide values of the oil samples were determined according to the AOCS Official Method Cd 8-53 29 . 1 g of walnut oil was weighed in an erlenmeyer flask and 6 mL of acetic acid-chloroform mixture 60:40, v/v was added. This mixture was reacted with a saturated potassium iodide solution 0.1 mL in the dark for 1 min. Afterwards, the resulting solution was titrated with a sodium thiosulfate solution in the presence of starch solution as an indicator. Peroxide value was expressed as milliequivalents of active oxygen per kilogram of oil meq O 2 kg 1 oil . Results were calculated according to the following equation: where; S and B are the volumes mL of the sodium thiosulfate solution used in the titration of the sample and blank, respectively, N is the normality of the sodium thiosulfate solution and m is the weight g of the sample. 2.2.6 Determination of conjugated diene and triene contents The conjugated diene and triene contents were determined by measuring the absorbance of the samples prepared with isooctane 1 , w/v at 232 and 270 nm using a spectrophotometer PG Instruments, Lutterworth, UK according to the AOCS Official Method Ch5-91 30 . Results were calculated using the following equation: where; K λ is the specific extinction at the wavelength tested 232 nm or 270 nm , A λ is the absorbance measured at the wavelength tested 232 nm or 270 nm ; c is the concentration of the solution g per 100 mL , and L is the path length of the quartz cuvette cm . 2.2.7 Determination of total phenolic content and antioxidant activity Extraction of phenolics and other antioxidant compounds from defatted walnut sample was done according to the method described by Bakkalbaşi et al. 31 with a slight modification. A portion 0.25 g of the sample was weighed in an erlenmeyer flask and 10 mL of methanol:water mixture 80:20, v/v was added. After homogenization IKA T18 Ultra-Turrax , the flask was shaken on the orbital shaker at 200 rpm for 2 h at room temperature. Then, the flask content was transferred to a polypropylene tube and centrifuged at 7031 g for 10 min Nuve, NF 800R, Nuve, Ankara, Turkey . The supernatant was transferred to an amber vial and the residue was re-extracted using the procedure explained above. The extracts were combined in a polypropylene tube and used for total phenolic content and antioxidant activity determination.
Total phenolic content in the samples was determined according to the Folin-Ciocalteu FC method as described by Singleton et al. 32 with a slight modification. FC solution 1:10, v/v and sodium carbonate solution 75 g L 1 were prepared with distilled water. Three hundred μL of the extract and 1500 μL of FC solution were put into a 15-mL polypropylene tube and the tube was vigorously mixed with a vortex mixer. Afterwards, 1200 μL of sodium carbonate solution was added to the tube and the tube was vigorously mixed again. After holding the tube in the dark for 2 h, the absorbance was measured at 760 nm by the spectrophotometer Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Finland . Standard solutions of gallic acid were prepared in distilled water and a calibration curve 5-100 mg L 1 was drawn. Total phenolic content of the sample was determined using this curve and expressed as mg gallic acid equivalent GAE per g defatted walnut.
Antioxidant activity of the samples was determined using DPPH method as described by Thaipong et al. 33 . A stock solution of DPPH was prepared in methanol and the absorbance of this solution at 515 nm was adjusted to 1.1 0.02 by diluting with methanol. One hundred and fifty μL of the extract and 2850 μL of DPPH stock solution was put into a 15-mL polypropylene tube and the tube was vigorously mixed with a vortex mixer. After holding the tube in the dark for 1 h, the absorbance was measured at 515 nm by the spectrophotometer. Trolox, a water-soluble analogue of α-tocopherol, was used as a standard for antioxidant activity. An exact amount of trolox was weighed in a 10-mL volumetric flask and dissolved in 1 mL ethanol. Then, the volume was adjusted to 10 mL with distilled water. This solution was used to draw a calibration curve covering the range of 10-50 μM trolox. Antioxidant activity of the sample was determined using this curve and expressed as mmol trolox equivalent mmol TEAC per g defatted walnut.

Determination of tocopherol contents
Tocopherols were determined according to the AOCS Official Method Ce 8-89 34 with a slight modification. Briefly, 0.25 g of oil samples was diluted with 1 mL of 2-propanol, filtered through a microfilter with a pore size of 0.45 μm Chromafil Xtra PTFE-45/25, Macherey-Nagel, Duren, Germany and injected into a high pressure liquid chromatography HPLC, Shimadzu LC-20AD, Kyoto, Japan . Analytical column was Zorbax Eclipse XDB-C18 column Agilent Technologies, 250 mm 4.6 mm i.d., 5 μm particle diameter, Santa Clara, USA , column temperature was 25 and the injection volume was 20 μL. The mobile phase was HPLC grade methanol with a flow rate of 1 mL min 1 . Detection was carried out at 289 nm for α-tocopherol and 297 nm for δ-tocopherol and γ-tocopherol using a photodiode array detector Shimadzu SPD-M20A . Standard solutions of α-, δand γ-tocopherol were separately prepared in ethanol using analytical standards of these compounds Supelco, Sigma-Aldrich, Bellefonte, CA, USA and 6-point calibration curves 0.25-25 mg L 1 for α-tocopherol, 1-50 mg L 1 for δ-tocopherol and 5-500 mg L 1 for γ-tocopherol were drawn. Quantification of the tocopherols in the samples was performed using these curves.

Determination of fatty acid composition
Fatty acid methyl esters FAMEs were prepared according to the AOCS Official Method Ce 2-66 35 . Accordingly, 0.2 g of the oil sample was dissolved in 2 mL of n-hexane and treated with 0.2 mL of methanolic potassium hydroxide solution. After mixing vigorously and waiting for phase separation 30 min , clear upper layer was taken with a microsyringe and injected into the injection port of the gas chromatography apparatus Agilent 7820A, Santa Clara, USA equipped with flame ionization detector. FAMEs were separated on a capillary column Agilent Technologies, DB-FATWAX UI, 30 m 0.25 mm i.d., 0.25 μm film thickness, Santa Clara, USA . The injection volume was 1 μL with a split ratio of 1:100 and the carrier gas was hydrogen at a flow rate of 1.4 mL min 1 . The column temperature was programmed to 50 for 2 min and increased to 174 for 14 min at 50 min 1 and then increased to 215 for 25 min at 2 min 1 . The temperatures of the injector and the detector were 250 and 280 , respectively. Peaks in the chromatogram were identified by comparison of their retention times with those of standard methyl esters Supelco 37-component FAME Mix, Bellefonte, PA, USA .

Statistical analysis
An analysis of variance ANOVA was employed to determine significant differences among means using a statistical package program MINITAB v. 13, MINITAB Inc., State College, PA, USA . Significant differences among means were identified by Tukey s Multiple Range Test p ≤ 0.05 using MSTAT-C statistical software MSTAT 1991, Michigan State University, MI, USA .

Gas compositions in the packages of ground light
and dark walnuts Gas composition percentages in the packages of ground light and dark walnuts during storage at 4 and 20 are shown in Table 1. O 2 , CO 2 and N 2 concentrations ranged between 19.3-21.2 , 0.0-0.7 and 78.9-80.1 , respectively, in packages with air during 6-month storage. The corresponding concentrations were 0.0-0.3 , 0.0-0.1 and 99.7-100.0 , respectively, in samples packaged with N 2 . Storage temperature did not have a marked impact on gas composition in the packages. The slight and negligible changes in the gas composition of the packages might be attributed to the high barrier properties of the packaging material we used. Laminated films with an ethylene vinyl alcohol layer were reported to have high barrier properties 36 and to be effective in retaining the atmosphere in walnut 37 and almond 38 packages.

Free fatty acid and acid values of ground light and
dark walnuts FFAs are released by decomposition of triacylglycerols as a result of chemical or enzymatic reactions which may be triggered by heat, light and moisture 39 . FFA value is used as an indicator of lipid hydrolysis and stability of the oil. Although there is no a clearly stated threshold for FFA value for walnuts, a value of 0.6 oleic acid is generally recognized as acceptable by the walnut industry 19,40 . FFA values of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Figs. 1a and 1b,  High FFA values were also reported in previous studies for walnuts 19  For example, at the end of storage at 20 , FFA values increased 2.9-3.1 and 4.5-6.1 fold compared to the initial value in light and dark walnuts, respectively. FFA values of light walnuts were low during storage at both temperatures. On the other hand, FFA values of dark walnuts were low and quite stable during storage at 4 , but gradually increased -to over 1 oleic acid in some cases-during storage at 20 . FFA values of the walnuts packaged under nitrogen were significantly p 0.05 lower except the light ones stored at 4 . Then, it can be concluded that there is no need for packaging under nitrogen if the conditions kernel color, storage temperature, etc. are favorable. However, if dark walnuts are stored or storage temperature is not low enough, packaging under nitrogen can be used as an effective method to minimize FFA increase. Although statistically not significant, FFA values of vacuum-packaged light and dark walnuts were higher compared to other samples at the end of storage at 20 . In our opinion, walnut particles might be compressed during vacuum application and oil could leak from the damaged cells of the particles. Oil leaked out of the cell could get involved in enzymatic and chemical reactions, and therefore the FFA value could increase 42 .
Acid value is dependent on FFA value and an increase in the FFA content is directly proportional to an increase in the acid value. The acid value characterizes the destruction of the quality of the oil stemming from the hydrolysis of triacylglycerol. The maximum acceptable level for acid value is 4 mg KOH g 1 oil according to the Codex Alimentarius Commission standard for cold-pressed and virgin oils 43 . Acid values of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Figs. 2a and 2b, respectively. The initial acid values of light and dark walnuts were 0.38 0.03 and 0.39 0.02 mg KOH g 1 , respectively. In the present study during storage, both light and dark walnut samples had low acid values 0.38-2.28 mg KOH g 1 which are within the Table 1 Gas composition percentages in the packages of ground light and dark walnuts during storage at 4 and 20 . a

Peroxide values of ground light and dark walnuts
Peroxides are the primary products of lipid oxidation. These peroxides are then transformed into various secondary products some of which cause undesired changes such as rancidity and off-flavor in the product. Although there is not a standard numerical threshold determined for the peroxide value for walnuts, different values have been suggested by the researchers. For instance, peroxide value should be lower than 1 meq O 2 kg 1 according to private walnut companies 19 , however, in the study conducted by Buransompob et al. 44 , it was stated that walnuts with peroxide values less than 3 meq O 2 kg 1 are acceptable. Furthermore, walnuts with even 10 meq O 2 kg 1 of peroxide value were identified as consumable according to the  results of sensory panels in the study of Mexis et al. 22 . Peroxide values of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Figs. 3a and 3b, respectively. The initial peroxide values of light and dark walnuts were 0.28 0.02 and 0.24 0.06 meq O 2 kg 1 , respectively. In general, all stored walnut samples had similar changing trends in terms of peroxide values. Peroxide values increased at the end of first month, then decreased to a minimum at the end of third month of storage. Then, peroxide values increased again at the end of fourth month. It can be concluded from these findings that the degradation rate was faster than the formation rate of peroxides during the third month of storage. Similar fluctuations in peroxide values of other nuts were also observed by other researchers 45 47 . It can be due to the unstable nature of the peroxides and their simultaneous formation and decomposition in vegetable oils 48 . According to Ding et al. 49 , increases and decreases observed in peroxide values could be related to unstable lipoxygenase activity. On the other hand, consistent increases in peroxide values of hazelnuts 50 and almonds 51 were observed during storage. It is obvious that this discrepancy could arise from differences among cultivars, pre-and postharvest applications, storage conditions, etc. Peroxide values in all samples were significantly higher p 0.05 at the end of 6-month storage than that at the beginning. Different packaging techniques caused some differences in peroxide values of both light and dark walnuts, but these differences were not statistically significant p 0.05 . Similar findings were reported for almonds 52 and peanuts 53 packaged under nitrogen. According to our results, storage temperature has a strong effect on peroxide values of the walnuts. We determined that peroxide values of the walnuts ranged from 0.15 to 1.08 meq O 2 kg 1 and from 0.10 to 1.15 meq O 2 kg 1 when stored at 4 and 20 , respectively. Bakkalbasi et al. 31 stored vacuum-packaged shelled walnuts at 3 different temperatures for 12 months. They determined that peroxide values of the walnuts were low and almost stable at 10 and 20 , however, increased systematically during storage at 30 . Villarreal-Lozoya et al. 54  The highest peroxide value 1.15 meq O 2 kg 1 was detected in dark walnut samples packaged under air and stored at 20 for 4 months. Crowe et al. 55 conducted a study with non-oxidized and oxidized stored at 60 for 8 d walnut pieces. They determined peroxide values of 0.06-1.58 and 5.02-12.37 meq O 2 kg 1 in non-oxidized and oxidized samples, respectively. Zajdenwerg et al. 56 reported that consumers rejected the nut samples with a peroxide value above 18.8 meq O 2 kg 1 due to their rancid flavor. According to our results, the peroxide values of both light and dark samples -even stored for 6 months-were below 1.5 meq O 2 kg 1 . Therefore, it can be concluded that all the samples in the present study are acceptable in terms of peroxide value.

Conjugated diene and triene values of ground light
and dark walnuts The conjugated diene and triene values represent the concentration of conjugated hydroperoxides in oils formed by the shifting of double bonds during oxidation of the unsaturated fatty acids 57 . When polyunsaturated fatty acids with three or more double bonds oxidize, conjugation may take place, and this can cause the formation of conjugated trienes. Peroxides that are formed at the beginning of oxidation are quite unstable and can undergo further changes  The conjugated diene values of the walnut samples both light and dark in the present study were far below these values even at the end of 6-month storage. It might be due to the fact that our samples were ground walnut samples ground kernels with their pellicles during storage in contrast to the studies conducted with walnut oil. It is wellknown that phenolics and other antioxidants are accumulated in the pellicle and these compounds may slow down the oxidation processes and limit the increase of conjugated diene values.
Conjugated triene values of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Figs. 5a and 5b,  3.5 Total phenolic contents of ground light and dark walnuts Phenolic compounds are secondary metabolites commonly found in plants that protect them against abiotic and biotic stress conditions. Phenolic substances in the walnut are mostly found in the pellicle surrounding the kernel. Total phenolic contents of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Table 2. As can be seen from the table, total phenolic content of dark walnuts was lower than that of light ones at the beginning of storage. Similarly, Fuentealba et al. 15 reported that total phenolic content was significantly higher in light walnut kernels than in amber kernels. This may be due to the decrease in the phenolic content in the pellicle covering the surface of the kernel as a result of enzymatic browning reactions. The phenolic compounds, which are the substrates of enzymatic browning, oxidize to quinones and then brown substances are formed as a result of the polymerization reactions of quinones 63 . In general, total phenolic contents of all samples decreased during storage. According to our results, refrigerated storage conditions has a protective effect on phenolics of light walnuts; but on the other hand, vacuum-packaging or packaging under nitrogen did not limit the decrease in the total phenolic content of the walnuts at 20 with only one exception vacuum-packaged light walnuts . Previous studies reported that factors such as packaging technique 64 and storage temperature 65 did not have a definite effect on total phenolic contents of the nuts. The protective role of phenolic compounds against oxidation reactions is well-known 66 . Our results showed that the total phenolic contents of dark walnuts were lower than that of light ones at the beginning and during the whole storage period. Then, it can be expected that dark walnuts are more susceptible to oxidation reactions. However, our findings related with lipid oxidation peroxide, conjugated diene and triene values showed that lipid oxidation occurred in the dark walnuts were not higher than that occurred in the light ones. As a results, it can be concluded that walnut phenolics even at relatively low amounts were effective in protecting from oxidation.

Antioxidant activities of ground light and dark walnuts
Antioxidants are the substances which have the ability to prevent/delay the onset and/or propagation of oxidation reactions. There are many phytochemicals with antioxidant activity in walnut fruit some of which located in defatted kernel while some others are dissolved in its oil. Arranz et al. 67 reported that the defatted part of the walnut kernel provides the bulk of the antioxidant capacity of the fruit and the contribution of its oil to the overall antioxidant capacity is less than 5 . Antioxidant activity of light and dark walnuts packaged with different techniques and stored for 6 months at 4 and 20 are shown in Table 3. At the beginning of the storage, the antioxidant activity of dark walnuts were about 2.5-fold lower than that of light ones. It is probably due to the fact that phenolics in the dark walnuts were already oxidized before storage. However, it should be noted that there were no significant differences between lipid oxidation parameters peroxide, conjugated diene and triene of light and dark walnuts according to our results. Then, in our opinion, the oxygen in the environment first meets the pellicle and the antioxidants in the pellicle i.e. phenolics react with the oxygen and prevent the reaction of oxygen with the oil of the fruit.
As long as the antioxidants in the pellicle are oxidized, walnut color darkens but oil is protected from oxidation. Antioxidant activity of dark walnuts did not change significantly during the storage at both temperatures. In case of light walnuts, even though some drastic decreases up to 40 were observed in the antioxidant activity values, this situation was not consistent throughout the storage periods. Similarly, fluctuations in the antioxidant activities during storage of some other nuts were reported 64,68 . The authors explained this phenomenon by many complex oxidation mechanisms and complicated biochemical reactions. Phenolic substances can easily undergo oxidation reactions and turn into brown colored polymers. However, prior to the formation of these brown polymers, phenolics are partially oxidized and converted to uncolored intermediate products. These intermediate products were reported to have higher antioxidant activity than the original phenolics and the brown polymers 69,70 . Boiling et al. 65 stored almonds at 4 and 20 for 15 months and periodically analyzed total phenolic content and antioxidant activity. Contrary to their hypothesis that storage would decrease the antioxidant activity, almonds stored at both temperatures had increased phenolic content and antioxidant activity values. This result was attributed to increased extractability and/or continuing synthesis of polyphenols during storage of almonds. The authors also stated that the storage temperature of almonds did not affect phenolic content and antioxidant activity. Likewise, storage temperature and packaging techniques tested in the present study did not have any significant effect on antioxidant activity of the walnuts.
3.7 Tocopherols contents of ground light and dark walnuts Tocopherols are important antioxidants which are abundantly found in nuts and vegetable oils. They compete with unsaturated fatty acids for lipid peroxy radicals and hence improve oxidative stability 71 . It has been reported that, of four isomers of tocopherols α-, β-, γ-, and δ-, γ-tocopherol is the most abundant form whereas β-tocopherol is rarely present in walnut oil 72,73 . Likewise, the concentrations of tocopherols ranked as follows: γ-, δand α-tocopherols, while β-tocopherol was not detected in our samples. γ-, δ-  light and dark walnuts were slight but significant p 0.05 according to the statistical analysis. It could be attributed to the presence of more antioxidant substances on the surface of light walnuts -compared to dark ones-limiting the oxidation of the tocopherols inside the kernel. Tocopherols are dissolved in the oil of the walnut and the oil is dispersed in the kernel as droplets surrounded by a single layer of phospholipids embedded with proteins termed oleoresins 74 . This structure provides a protection of the components in the oil from oxidation. However, it should be noted that, this protection effect is unique to the intact form of the kernel and disappears when the integrity is disrupted by a process like grinding just as in the present study. We determined that there were no significant differ-ences between the concentrations of any tocopherol types in light and dark walnuts at the end of 6-month storage of ground samples Figs. 6-8 . Probably, tocopherols in the oil leaked from the cells as a result of grinding process were oxidized in time. It was reported that the relative antioxidant activity of the tocopherols is in the order of α-γδ-tocopherol 75,76 . According to our calculations, decreases in α-, γand δ-tocopherol levels were 26.6-39.9 , 27.0-35.5 and 20.8-31.5 in light walnuts and 21.8-35.4 , 19.8-27.8 and 11.3-27.0 in dark walnuts, respectively, at the end of storage for 6 months. Then, it can be concluded that the more antioxidant activity the tocopherol has, the more pronounced decrease occurs.
Statistical analysis revealed that there were no signifi-   52 reported that neither storage temperature nor packaging atmosphere had a significant effect on α-tocopherol content of almonds packaged under nitrogen and air and stored at 8 and 36 . In our opinion, not only atmospheric oxygen but also other factors such as free radicals formed during oxidation chain reactions have an important role on the levels of tocopherols.
3.8 Fatty acid compositions of ground light and dark walnuts Fatty acid composition is an indicator of the ratios of saturated, mono-and poly-unsaturated fatty acids and can give an idea about the nutritional quality of the oil. Walnut oil is rich in unsaturated fatty acids and is famous for its high linoleic acid content. However, high content of unsaturated fatty acids makes the fruit quite susceptible to oxidation reactions.
In parallel with the previous studies 77, 78 , we found that the approximate ratios of saturated, mono-unsaturated and poly-unsaturated fatty acids in walnut samples were 9, 22 and 68 , respectively, and linoleic acid was the major fatty acid in both light and dark walnut samples. The percentages of linoleic, oleic, linolenic, palmitic and stearic acids in ground light walnut samples were 55.00, 21.45, 12.49, 7.21 and 2.58 , respectively, prior to storage. The corresponding values in ground dark walnuts were 55.49, 21.08, 12.63, 6.84 and 2.71 , respectively. Other fatty acids myristic, palmitoleic, vaccenic, arachidic and eicocenoic acids constituted a sum of about 1 . Table 4 gives the percentages of fatty acids in ground light and dark walnut samples at the end of storage for 6 months at 4 and 20 . The fatty acid compositions of light and dark walnuts were statistically similar at both the beginning and the end of the storage. Moreover, no significant differences were found among the values of any fatty acids during storage p 0.05 . Although measurement of changes in fatty acid composition is not widely used in assessing lipid oxidation, the changes occurred in this composition was reported to be an indicator of oxidative changes in highly unsaturated vegetable oils 79 . Therefore, the stability in fatty acid composition of our samples could be attributed to the protective effect of natural antioxidants such as tocopherols, phenolics, etc. found in walnuts.

Conclusion
In this study, we investigated the effects of packaging techniques and storage temperature on the chemical properties of ground light and dark walnuts. We observed that free fatty acid contents increased during storage and these Table 4 The percentages of fatty acids in ground light and dark walnuts packaged under air, nitrogen and vacuum-packaged, and stored for 6 months at 4 and 20 . a, b, c increases were marked for dark walnuts. Peroxide values of all samples fluctuated during storage. Both free fatty acid contents and peroxide values recorded at the end of storage were higher than the initial values. Storage at refrigerated temperature significantly reduced the increases of free fatty acid contents, peroxide and conjugated diene values. Fatty acid composition and triene values did not change significantly at any storage conditions tested in this study. Dark walnuts had lower phenolic and tocopherol contents than the light ones, but even this lower contents of antioxidant compounds were sufficient to protect the fruit against oxidation reactions. According to our results; ground dark walnuts, just as the light ones, can be stored for at least 6 months even in the packages with air. All walnut samples were acceptable at the end of 6-month storage in terms of oxidation parameters. Further research is needed to provide data on the acceptability of the sensory quality attributes of dark walnuts and to relate them with their chemical characteristics.