Effects of temperature and packaging atmosphere on total antioxidants and colour of walnut (Juglans regia L.) kernels during storage

Scientia Horticulturae 131 (2011) 49–57

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Effects of temperature and packaging atmosphere on total antioxidants and colour of walnut (Juglans regia L.) kernels during storage M.V. Christopoulos ∗ , E. Tsantili Laboratory of Pomology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece

a r t i c l e

i n f o

Article history: Received 10 May 2011 Received in revised form 22 September 2011 Accepted 23 September 2011 Keywords: Phenolics Antioxidant capacity Colour Walnut kernels Storage Walnut browning

a b s t r a c t The effects of cultivar and storage conditions (time, temperature and O2 availability) on total phenolics (TP), total antioxidant capacity (TAC) and colour in walnut kernels of three cultivars (Chandler, Hartley and Ioli) were investigated. Harvested walnuts were dried at 36 ◦ C for 24 h, packaged in polyethylene||polyamide pouches flushed with dry air or N2 or CO2 and stored at 1 ◦ C or 20 ◦ C for up to 12 mo. Before storage, dried kernels exhibited the highest values of L*, h◦ and whiteness index (WI) colour parameters, as well as the highest TP content and TAC assessed either with FRAP or DPPH assays. Chandler and Hartley exhibited much higher antioxidant levels than Ioli during the whole experiment, while Ioli exhibited the highest h◦ at least before storage. During storage, browning (decreases in L*, h◦ , WI) and antioxidant losses were observed by advanced time. After 12 mo, the greatest losses of TP, FRAP and DPPH in all studied cultivars were observed in kernels stored at 20 ◦ C under air, and averaged approximately 48%, 38% and 40%, respectively. Low temperature and packaging under N2 or CO2 prevented additively both antioxidant losses and browning. Pairwise correlation and Principal Component Analyses revealed strong relationships among TP, FRAP and DPPH values, as well as, between TP and colour parameters (L*, h◦ and WI). These relationships indicated that the decreases in antioxidants of stored walnut kernels are responsible not only for the nutritional loss, but also for the quality deterioration in relation to consumer visual perception. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Walnut (Juglans regia L.) tree cultivation is widely distributed worldwide and on a global basis, walnuts rank third in nut production after cashews and almonds (FAOSTAT, 2011). During the last decade, the worldwide walnut production was doubled, probably reflecting on the increase in consumers demand for this nut. Among plant foodstuffs and especially nuts, walnut kernels are an excellent source of antioxidants since they exhibit high total phenolic concentration (TP) and total antioxidant capacity (TAC) (Halvorsen et al., 2006; Kornsteiner et al., 2006). Recent studies have supported a substantial beneficial impact of walnut antioxidants on human health (Carvalho et al., 2010; Papoutsi et al., 2008).

Abbreviations: DPPH, radical scavenging capacity (2,2-diphenyl-1picrylhydrazyl); FRAP, Ferric Reducing Antioxidant Power; GAE, gallic acid equivalents; TAC, total antioxidant capacity; TAE, trolox acid (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) equivalents; TP, total phenolics; TPTZ, 2,4,6-tripyridyl-s-triazine; WI, whiteness index. ∗ Corresponding author. Tel.: +30 210 529 4612; fax: +30 210 529 4592. E-mail addresses: [email protected], [email protected] (M.V. Christopoulos). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.09.026

Dried walnuts are usually stored for prolonged time, and it is reported that they should be exposed below 10 ◦ C, with the lower temperature being more effective (Kader and Thompson, 2002). However, exposure of kernels to ambient temperature during storage or disposal to market places often occurs in practice. Temperature and O2 availability are two of the most important external factors that affect postharvest nut quality during storage. Behaviour of walnuts during storage has been evaluated at temperatures from 3 ◦ C to 37 ◦ C and in O2 concentration from ∼0% up to 50% controlled by packaging or O2 absorbers (Jensen et al., 2003; López et al., 1995; Maté et al., 1996; Mexis et al., 2009). These studies reported that increased temperature and O2 availability advanced product deterioration in terms of lipid oxidation (rancidity evolution and volatiles production) and sensory attributes. Although the effect of storage on the above quality attributes related to consumer perception is well documented, there are limited data concerning the effect of storage on walnut antioxidants. Moreover, there is a lack of data concerning the storage stability of different walnut cultivars, since the most studies were referred to walnuts of unknown cultivars and/or unknown postharvest handling until sampling from the market. During storage, antioxidants and especially phenolics are prone to oxidation resulting to nutritional or sensory deterioration

50

M.V. Christopoulos, E. Tsantili / Scientia Horticulturae 131 (2011) 49–57

(browning) of a product (Manzocco et al., 2000). During 12 mo storage, low temperature (1 ◦ C instead of 20 ◦ C) and packaging atmosphere with N2 prevented additively the losses of TP and TAC in eight pistachio cultivars tested (Tsantili et al., 2011). In peanuts stored at 20 ◦ C TP losses were restricted in comparison with those stored at 35 ◦ C (Talcott et al., 2005). Levels of TP and TAC have been studied in walnuts after harvest (Pereira et al., 2008) or from market (Halvorsen et al., 2006; Kornsteiner et al., 2006), but to the best of our knowledge, changes in antioxidants during storage under particular conditions were limited to Franquette cultivar (Christopoulos et al., 2010b). The aim of the present work was to investigate changes in TP, TAC and colour, as well as, the relationships among these antioxidants and colour attributes since a possible correlation between them could be useful to optimize processing and/or storage conditions, as suggested by Manzocco et al. (2000). In order to obtain more information in relation to storage conditions, kernels were packaged under dry air, N2 , or CO2 , and stored at 1 ◦ C or 20 ◦ C for up to 12 mo. To investigate the cultivar effect on all these attributes three cultivars grown under the same field conditions were included in the experiments. Chandler and Hartley cultivars were selected due to their worldwide cultivation, and Ioli as a relatively new Greek promising cultivar due to its agronomical traits (Christopoulos et al., 2010a).

2. Materials and methods 2.1. Plant material and postharvest handling Walnut (Juglans regia L.) fruits were harvested from trees cultivated on the Agricultural Research Station of Vardates (N.AG.RE.F., Vardates) at Central Greece (lat. 38◦ 49 37 N, long. 22◦ 26 10 E, altitude 12 m). The studied cultivars were: Chandler, Hartley and Ioli (cultivated only in Greece). Chandler (Pedro × UC 56-224) was released by Californian breeding program and is one of the most important commercial cultivars due to very high productivity, lateral bearing habit, and bright and large kernel. Hartley is a seedling selection originated from California with high productivity, intermediate bearing habit (5–10% lateral bud flowering) and large bright-yellow kernel. Ioli is a seedling selection released by breeding program of N.AG.RE.F. and is one of the most important genotypes derived from Greek germplasm repository. Ioli has very high productivity, lateral bearing habit, and kernels of excellent visual quality with bright colour and large size. More agronomical and genetic information was presented in a previous work (Christopoulos et al., 2010a). Under the present field conditions, the fruits of these cultivars mature from 25 September to 5 October. Walnuts, from four trees per cultivar, were harvested at the same maturity stage, when the husk was just beginning to split and the packing tissue between and around the kernel halves has just turned brown (Kader and Thompson, 2002). Nuts were immediately hulled with tap water and dried in a commercial bin drier with an air velocity of 1500 m3 h−1 at 36 ◦ C for 24 h. Dried walnuts were manually cracked, shelled and only healthy kernels, macroscopically free of disorders and diseases, were selected for the experiment. For each replication approximately 100 g of kernels in halves obtained from 20 nuts were placed in pouches (Rovac A, CASFIL, Portugal) of 80 ␮m thickness (60 ␮m polyethylene, 20 ␮m polyamide), 76.28 g m−2 density and 46.65 mL m−2 day−1 atm−1 O2 permeability (measured at 23 ◦ C, 0% R.H.). The pouches were flushed with dry air or N2 , or CO2 and then heat-sealed using a vacuum sealer (Henkovac 1900, Howden Food Equipment, The Netherlands) in a final volume of sealed pouch of 300 mL. To test

O2 concentration in the packaging atmosphere a silicone septum was glued as a sampling port on the surface of the pouch. The packaged kernels were stored at 1 ◦ C or 20 ◦ C for up to 12 mo so six different combinations of temperature with flushing gas were obtained: 1-AIR, 1-N2 , 1-CO2 , 20-AIR, 20-N2 and 20-CO2 . Drying, packaging and storing were all carried out randomly within 48 h from harvest. On each sampling date, pouches were removed from store, kernel colour was measured and then samples were placed randomly at −80 ◦ C for up to one month until TP and TAC measurements. Kernel moisture was measured twice, before and after drying. Colour, TP and TAC were estimated after drying and after 4, 8, and 12 mo storage. For each combination of temperature with flushing gas, on each sampling date and for each cultivar all measurements were carried out in four replicates. 2.2. Kernel moisture content and O2 concentration in packaging atmosphere Moisture was determined by drying 10 g of chopped kernels in an oven with air circulation at 100–105 ◦ C until constant weight. For O2 concentration in the packaging atmosphere, a headspace gas sample of 0.5 mL from the packages was drawn with a syringe and the gas composition was measured using a gas chromatograph (HP 5890 Series II, HP, USA). The instrument was equipped with a Molecular Sieve (40–60 M) column, a Thermal Conductivity Detector (TCD) and a Hewlett Packard 3395 laboratory computing integrator. Helium carrier gas was used at a flow rate of 50 mL min−1 , the temperatures of injector and detector were 150 ◦ C and 200 ◦ C, respectively, and an isotherm oven temperature program at 60 ◦ C was used. A standard gas mixture of 2% CO2 + 2% O2 + 1 ␮L L−1 ethylene diluted in N2 was used for calibration. 2.3. Colour Colour kernel was measured on the upper part of the outer surface on each half by a chromatometer (CR-300; Minolta, Germany) in a dark chamber. The recorded CIE-L*a*b* values were converted into hue angle (h◦ ) and chroma (C*) according to McGuire (1992). Furthermore the whiteness index (WI) was calculated according to the equation WI = 100 − [(100 − L*)2 + ˛*2 + b*2 ]1/2 (Boun and Huxsoll, 1991). 2.4. Total phenolic concentration and total antioxidant capacity The extraction for antioxidant measurements was conducted by homogenizing frozen chopped tissue (−80 ◦ C) with 80% acetone (v/v) in deionised water (5 mL g−1 tissue) using an Ultra-Turrax (T 25, Kika Labortechnik, Germany) for 2 min (1 min at 9500 rpm and 1 min at 13,500 rpm). The homogenate was placed in a supersonic bath for 30 min, incubated in the dark for 2 h and filtered in a büchner funnel (90 mm i.d.) using #1 Whatman paper. During incubation the head-space of the vials was filled with N2 while all the above procedure was conducted at 4 ◦ C. After incubation the acetone was evaporated at 38 ◦ C under N2 , the filtrate was recovered and then brought to the desired volume (10 mL). The TP concentration was measured by a modified FolinCiocalteu colorimetric method (Singleton et al., 1999). Briefly, 0.2 mL of diluted extract was added into a tube containing 2.6 mL of deionised water and 0.2 mL of Folin-Ciocalteu reagent, and the tube was stirred and allowed to stand at room temperature for 6 min. Then, 2 mL of Na2 CO3 (7%, w/v) was added to the mixture. Absorbance was measured at 750 nm using a spectrophotometer (He␭ios Gamma & Delta, Spectronic Unicam, UK) versus a blank after incubation for 90 min at room temperature. The results were

M.V. Christopoulos, E. Tsantili / Scientia Horticulturae 131 (2011) 49–57

expressed as gallic acid equivalents on a dry weight basis (mg GAE g−1 D.W.). TAC was evaluated according to both FRAP (Benzie and Strain, 1996) and DPPH (Brand-Williams et al., 1995) assays in extracts diluted with deionised water. For FRAP assay, 0.1 mL of diluted extract was added into a screw tap tube containing 3 mL of preheated at 37 ◦ C FRAP reagent [300 mM acetate buffer (C2 H3 NaO2 ·3H2 O, C2 H4 O2 ), pH 3.6; 10 mM TPTZ (2,4,6-tripyridyls-triazine) in 40 mM HCl; 20 mM FeCl3 ·6H2 O; in 10:1:1 (v/v)]. The reaction mixture was incubated at 37 ◦ C for 30 min and then the absorbance was measured at 593 nm versus a blank. For DPPD assay, 0.1 mL of diluted sample of the extract was added into a screw tap tube containing 3.9 mL DPPH solution (2,2-diphenyl1-picryhydrazyl, 60 ␮M in MeOH). The decrease in absorbance at 515 nm was recorded versus blank after 30 min incubation at room temperature. For both antioxidant capacity assays the selected incubation time (30 min) was the required time for the reaction to reach a plateau and the results were expressed as trolox acid (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents on a dry weight basis (␮mol TAE g−1 D.W.). 2.5. Statistical analyses The significance of the effects of cultivar, storage time, temperature and flushing gas was estimated by three-way factorial analyses of variance (ANOVA), in combinations of three factors out of four. For each combination of temperature with flashing gas, two-way ANOVA (cultivar × storage time) was conducted for the evaluation of changes in kernel attributes during storage. The significance of the cultivar effect before storage was estimated by one-way ANOVA. Standard error (SE) was calculated from the residual variances and mean separations were performed by Tukey-HSD test. Principal Component Analyses (PCA) and pairwise correlations were applied in order to get an overview of the main variation in the data and to interpret variable relationships. Data analyses were made using JMP 7.0.1 (SAS Institute, Cary, NC, USA). 3. Results 3.1. Moisture content of kernels and gas composition in the packaging atmosphere At harvest and before drying, the moisture content of kernels varied from 29.65% to 32.50% (w/w), but the differences among cultivars were not significant (Table 1). After the drying process the content of moisture in kernels reduced below 5% in all cultivars. Chandler had the highest moisture content (4.91%) followed by Hartley (4.57%) and Ioli (4.30%), but these slight differences were significant (P < 0.001). The initial O2 concentration in the packaging atmosphere was 20.72% (v/v) in pouches flushed with dry air and below 0.01% (v/v)

51

Table 1 Content of moisture in three walnut cultivars before and after drying. Cultivar

Moisture (%) Raw kernels

Dry kernels

32.50 29.65 31.47

4.91 4.58 4.29

HSD a

3.93

0.19

P

NS

***

Chandler Hartley Ioli

NS, not significant. a Honest significant difference (Tukey-HSD test). *** Significant at P < 0.001.

in those flushed with N2 or CO2 (Table 2). During storage the atmosphere did not change in pouches at 1-AIR and 20-AIR (P > 0.05), whereas a progressive increase in O2 concentration was observed in all other pouches by advanced storage duration (P < 0.001) and temperature (P < 0.001). Indeed, in these pouches the O2 concentration increased up to approximately 10% (v/v) at 20 ◦ C after 12 mo. However, on each sampling date the concentration of O2 in pouches flushed with N2 was similar with that flushed with CO2 (P > 0.05). 3.2. Colour Before storage the values of kernel colour parameters among cultivars ranged approximately from 61 to 62 for L*, 79 to 82 for h◦ , 29 to 32 for C* and 50 to 52 for WI (Table 3, Fig. 1 A–C). The differences among cultivars were significant for h◦ (P < 0.05) and C* (P < 0.01), but not for L* (P > 0.05) and WI (P > 0.05). In particular, Ioli had the highest h◦ and Hartley exhibited the highest C* among the cultivars. Storage resulted in decreased h◦ values at all storage conditions (P < 0.001) except for 1-N2 and 1-CO2 (P > 0.05) (Fig. 1A–C). In Hartley and Ioli decreases in h◦ were observed in all kernels stored at 20 ◦ C from the first storage interval, but at 1-AIR after 8 mo. In Chandler, the value of h◦ decreased at 20-AIR after 4 mo and at 20-N2 and 20-CO2 after 8 mo, but h◦ did not change during the whole storage period at 1-AIR. After 12 mo, the lowest values of h◦ were 70.35, 69.77 and 69.84 in Chandler, Hartley and Ioli, respectively, all recorded on kernels stored at 20-AIR. During storage, decreases in L* and WI were observed in stored kernels at 1-AIR (P < 0.001) and 20-AIR (P < 0.001). The values of L* did not changed in kernels stored under N2 (P > 0.05) or CO2 (P > 0.05) at both temperatures, whereas WI decreased at 20-N2 (P < 0.05) and 20-CO2 (P < 0.01), but not at 1-N2 (P > 0.05) and 1CO2 (P > 0.05) (Table 3). In particular, both Hartley and Ioli showed significant decreases in L* and WI values at 20-AIR after 4 mo and at 1-AIR after 12 mo, whereas L* and WI decreased in Chandler only at 20-AIR after 8 mo. After 12 mo, for all three cultivars the lowest

Table 2 Oxygen concentration in package atmosphere of walnut kernels stored under various conditions. Storage temperature (◦ C)

Flushing gas

O2 (%) Months (mo) 0

4

8

12

1

Air N2 CO2

20.72 ± 0.08a <0.01 ± 0.00 <0.01 ± 0.00

20.75 ± 0.07 2.01 ± 0.05 2.07 ± 0.04

20.68 ± 0.06 3.90 ± 0.09 3.86 ± 0.11

20.79 ± 0.07 5.60 ± 0.28 5.52 ± 0.24

20

Air N2 CO2

20.78 ± 0.11 <0.01 ± 0.00 <0.01 ± 0.00

20.70 ± 0.05 4.38 ± 0.19 4.32 ± 0.17

20.72 ± 0.07 7.62 ± 0.17 7.69 ± 0.14

20.75 ± 0.07 10.15 ± 0.22 10.21 ± 0.25

a

Numbers represent the mean of four replicates ± standard deviation.

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M.V. Christopoulos, E. Tsantili / Scientia Horticulturae 131 (2011) 49–57

Table 3 Effects of cultivar, storage temperature, flushing gas and their interactions on L*, C* and WI colour parameters in walnut kernels stored under various conditions. Cultivar

T (◦ C)

Gas

L*

C*

WI

Months (mo)

Chandler

Hartley

Ioli

1

Air N2 CO2

20

Air N2 CO2

1

Air N2 CO2

20

Air N2 CO2

1

Air N2 CO2

20

Air N2 CO2

0

4

8

12

0

4

8

12

0

4

8

12

60.69

59.43 64.26 62.90

59.38 65.04 62.41

58.31 64.94 63.71

30.09

30.49 30.43 30.53

31.47 30.57 31.26

31.62 30.97 31.54

50.48

49.25 53.06 51.93

48.61 53.56 51.11

47.68 53.22 51.92

58.35 63.63 61.90

54.39 †a 63.33 60.55

53.95 † 61.63 60.45

30.76 30.23 30.89

31.20 30.05 30.98

31.39 30.28 30.94

48.22 52.50 49.48

44.74 † 52.59 49.83

44.27 † 51.12 49.78

60.69 63.53 62.66

57.78 64.14 61.51

52.89 † 61.27 60.18

32.84 33.47 33.22

32.92 34.03 † 33.87 †

33.59 34.22 † 34.21 †

48.76 50.49 49.91

46.37 50.50 48.72

42.14 † 48.31 47.50

56.48 † 62.79 61.95

52.42 † 60.73 59.49

49.42 † 59.71 59.33

32.18 33.31 33.28

32.14 33.52 † 33.27 †

32.08 33.71 † 33.41 †

45.87 † 50.05 49.43

42.56 † 48.37 47.57

40.10 † 47.47 47.37

59.43 62.02 62.00

58.38 62.68 61.64

55.92 † 61.85 61.27

29.52 29.54 29.20

29.90 30.02 30.39

30.84 30.74 † 30.25

49.82 51.87 52.06

48.75 52.10 51.05

46.20 † 51.01 50.85

57.80 † 61.04 60.65

53.67 † 61.16 60.03

51.57 † 60.18 59.28

30.31 29.48 29.83

30.44 29.91 30.12

30.64 † 29.44 29.83

48.04 † 51.13 50.60

44.56 † 50.98 49.95

42.69 † 50.47 49.52

61.55

61.95

32.18

29.01

49.81

52.13

HSDb

4.87

4.32

4.60

2.54

1.58

1.94

1.60

1.05

3.34

3.25

3.47

2.35

Pcc Pt Pg Pc × t Pc × g Pt × g Pc × t × g

NS

NS

NS

***

**

***

***

***

NS

**

***

***

***

***

***

*

***

***

***

***

***

***

***

***

*

NS NS

***

NS NS

NS NS

***

NS NS NS NS

*

*

**

**

NS NS

NS

NS NS NS NS

NS NS

**

NS NS NS NS NS NS

**

**

NS

NS

NS

NS NS

**

NS

NS, not significant. a Means with a dagger (†) within rows indicate a significant difference from 0 derived from two-way ANOVA (cultivar × storage time) for each combination of storage temperature and flushing gas. b Honest significant difference (Tukey-HSD test). c Probabilities of the effects: cultivar, (Pc); storage temperature, (Pt); flushing gas, (Pg). * Significant at P < 0.05. ** Significant at P < 0.01. *** Significant at P < 0.001.

L* (51.6 in average) and WI (42.4 in average) values were observed in kernels stored at 20-AIR. During storage the changes of C* had a different trend than that of the other colour parameters. Minor and non-consistent increases in C* values in stored kernels were observed by advanced time (Table 3). After 12 mo in all storage conditions Hartley exhibited

the highest C* (33.5, in average), followed by Chandler (31.1, in average) and Ioli (30.3, in average). During the whole storage period, the effects of both storage temperature (P < 0.001) and flushing gas (P < 0.001) on L*, h◦ and WI were significant. This was confirmed by appropriate statistical three-way ANOVA (cultivar × temperature × gas), which

Fig. 1. Hue angle (h◦ ) parameter in walnut kernels stored under various conditions. A, Chandler; B, Hartley; C, Ioli. Vertical bars indicate HSD values at P = 0.05 from three-way ANOVA (storage time × temperature × flushing gas) conducted by cultivar and correspond to data after 4, 8 and 12 mo storage. Values of HSD(0.05) = 1.96, HSD(0.05) = 2.44 and HSD(0.05) = 1.67 correspond to all data (in A, B and C) after 4, 8 and 12 mo storage, respectively.

M.V. Christopoulos, E. Tsantili / Scientia Horticulturae 131 (2011) 49–57

53

conducted for each storage date (Table 3). In most cases, kernels stored at 1 ◦ C exhibited higher L*, h◦ and WI values than those stored at 20 ◦ C. At each storage temperature, kernels packaged under either N2 or CO2 showed higher L*, h◦ and WI values than those under air. The differences in L*, h◦ and WI between 1 ◦ C and 20 ◦ C, as well as, between packaging under air and N2 or CO2 , were greater by advanced storage time. During storage for up to 8 mo, kernels stored at 1-AIR, 20-N2 and 20-CO2 had similar values for all colour parameters. After 12 mo, kernels stored at 1-AIR showed higher h◦ in Chandler and lower L* and WI in both Hartley and Ioli than kernels at 20-N2 or 20-CO2.

3.3. Total phenolics (TP) and antioxidant capacity (TAC) Before storage, the concentration of TP was approximately 22 mg GAE g−1 D.W., 20 mg GAE g−1 D.W. and 11 mg GAE g−1 D.W. in Chandler, Hartley and Ioli, respectively, and these differences among cultivars were significant (P < 0.001) (Fig. 2 A and B) (Table 4). During storage, the levels of TP decreased progressively by advanced storage time (Fig. 2A and B). When data were analyzed by two-way ANOVA (storage time × cultivar) for each storage condition, the effects of both storage time (P < 0.001) and cultivar (P < 0.001) were significant. Indeed, the pattern of decreases in TP was quite similar among cultivars. These decreases at 20 ◦ C and/or under air atmosphere were significant from the first storage interval, while at 1-N2 and 1-CO2 the reduced TP levels were significant after 8 mo. On each sampling date, higher TP losses were observed on the higher temperature used, by comparison, and losses were also greater in packages with air than with other gases. Indeed, the positive effects of lower temperature and packaging under N2 or CO2 on prevention of TP losses were additives (Fig. 2A and B). For each cultivar and during storage for up to 8 mo, the concentrations of TP at 1-AIR were similar with those at 20-N2 and 20-CO2 , while after 12 mo kernels at 20-N2 and 20-CO2 exhibited higher TP than those stored at 1-AIR. After 12 mo, TP losses fluctuated from 14% at 1-N2 in Hartley to 59% at 20-AIR in Ioli. The present results showed that storage at 20 ◦ C resulted in averaged losses approximately 1.5fold greater than at 1 ◦ C. Also, at both temperatures, the averaged losses under air were approximately 1.7-fold greater than under N2 or CO2 , while packaging with N2 flushing had very similar effect on TP with CO2 flushing. The initial levels of TAC assessed by FRAP assay before storage were 153.4 ␮mol TAE g−1 D.W., 181.2 ␮mol TAE g−1 D.W. and 95.4 ␮mol TAE g−1 D.W. in Chandler, Hartley and Ioli, respectively, while by DPPH assay the TAC values were slightly lower than FRAP values (Fig. 1C–F). The differences in FRAP (P < 0.001) and DPPH (P < 0.001) values among cultivars were significant. During storage, decreases in both FRAP and DPPH values observed followed a pattern similar with that of TP. Cultivar, storage time, temperature and flushing gas affected FRAP and DPPH similarly to TP and significantly (P < 0.001 for all four factors) (Fig. 1C–F). However, some differences between either FRAP or DPPH and TP were observed. Hartley exhibited higher FRAP and DPPH values than Chandler during the entire storage period, although Hartley had TP concentration lower than Chandler at the same storage conditions, by comparison. Furthermore, the percentage of FRAP and DPPH losses were lower than the TP ones. In particular, after 12 mo the lowest losses of both FRAP and DPPH were observed in Chandler at 1-CO2 (13% and 11%, respectively), whereas the highest in Ioli at 20-AIR (45% and 49%, respectively). Storage at 20 ◦ C resulted in averaged losses of FRAP and DPPH approximately 1.4-fold and 1.5-fold higher than at 1 ◦ C, respectively, while during storage in air, the averaged losses of either FRAP or DPPH were 1.8-fold higher than storage in other gases.

Fig. 2. Total phenolics (TP), total antioxidant capacity (TAC) evaluated by FRAP and DPPH assays, in walnut kernels stored under various conditions. A, TP at 1 ◦ C; B, TP at 20 ◦ C; C, FRAP at 1 ◦ C; D, FRAP at 20 ◦ C; E, DPPH at 1 ◦ C; F, DPPH at 20 ◦ C. Vertical bars indicate HSD values at P = 0.05 from three-way ANOVA (cultivar × storage time × flushing gas) conducted by temperature and correspond to all data after 4, 8, 12 mo storage. Values of HSD(0.05) = 1.92, HSD(0.05) = 1.54 and HSD(0.05) = 2.65 correspond to all data (in A and B) after 4, 8 and 12 mo storage, respectively. Values of HSD(0.05) = 9.85, HSD(0.05) = 7.77 and HSD(0.05) = 11.13 correspond to all data (in C and D) after 4, 8 and 12 mo storage, respectively. Values of HSD(0.05) = 7.68, HSD(0.05) = 10.24 and HSD(0.05) = 12.06 correspond to all data (in E and F) after 4, 8 and 12 mo storage, respectively.

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Table 4 Probabilities of the effects of cultivar (Pc), storage temperature (Pt), flushing gas (Pg) and their interactions on h◦ colour parameter (Fig. 1A–C), total phenolics (TP), and total antioxidant capacity (TAC), evaluated by FRAP and DPPH assays (Fig. 2A–F), in walnut kernels stored under various conditions. Source of variation

Variable h◦

TP

FRAP

DPPH

Months (mo)

Pc Pt Pg Pc × t Pc × g Pt × g Pc × t × g

4

8

12

4

8

12

4

8

12

4

8

12

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

***

**

***

***

***

***

***

***

***

***

NS NS NS NS

NS NS

***

NS NS

NS NS

NS NS

***

***

**

***

***

*

***

NS

NS

NS

NS

NS

NS

NS

**

NS NS NS NS

**

NS

NS NS

***

NS NS NS NS

***

NS

NS NS

NS NS NS

NS, not significant. * Significant at P < 0.05. ** Significant at P < 0.01. *** Significant at P < 0.001.

3.4. Principal component analysis overview and relationships among walnut attributes The PCA showed two interpretable components, explaining together 89% (eigenvalue 2.69) of the total variation in TP, FRAP, DPPH and colour parameters of walnuts (Fig. 3). In the score plot the three cultivars were separated clearly (Fig. 3) and in consequence the relationships among variables could be interpreted performing partial multivariate analyses by cultivar. Strong relationships were found among the antioxidant attributes of walnuts. Significant, positive and strong correlations were found between TP and TAC, either assessed by FRAP (P < 0.001, r2 = 0.901) or DPPH method (P < 0.001, r2 = 0.900), while higher r2 were found in partial analyses by cultivar (data not shown). Concerning TP and colour parameters, significant and positive correlations were found, and analyses by cultivar showed correlation coefficients (r2 ) between TP and L*, and h◦ and WI from 0.684 to 0.819 (data not shown). These relationships were supported by PCA

since TP, L*, h◦ and WI were shown to be situated close together near the right axes of load plots of each cultivar (Fig. 4A–C). 4. Discussion 4.1. Moisture content of kernels Proper harvesting and post-harvest handling are the keys for achieving maximum yield of good quality nuts. Drying of walnuts is a common practice in order to reduce the moisture within the prerequisite limits (3–13%) for long storage (Kader and Thompson, 2002). The most important factor during walnut drying is the air temperature that should be in the range of 32–43 ◦ C (Hassan-Beygi et al., 2009). In the present work, drying at 36 ◦ C resulted in moisture content slightly below 5% that ensured the proper storage stability of kernels. Besides drying conditions (temperature, air velocity and time), different physical properties of in-shell walnuts could be responsible for the slight, but significant differences in kernel moisture content among cultivars (Hassan-Beygi et al., 2009). 4.2. Total phenolics and antioxidant capacity

Fig. 3. Principal Component Analysis in walnut kernels stored under various conditions according to the variables of total phenolics (TP), total antioxidant capacity (TAC) evaluated by FRAP and DPPH assays, and colour parameters (L*, h◦ , C* and WI). Left and bottom axes correspond to the score plot. Right and upper axes correspond to load plot. Circle, square and triangle indicate the position of each walnut sample in score plot. Dagger indicates the position of each variable in load plot. Numbers in parentheses correspond to the percentage of the total variance explained by each component.

Chandler exhibited the highest level of TP before storage, and in most cases higher than Hartley during storage under the same conditions. Ioli showed much lower TP levels than the other cultivars during the whole experiment. The range among cultivars presented here agreed well with most other studies (Arranz et al., 2008; Christopoulos et al., 2010b; Kornsteiner et al., 2006). During storage the TP concentration decreased progressively with a pattern quite similar among cultivars. In case of exposure at 20-AIR a great nutritional deterioration was observed, since all cultivars lost approximately 50% of their initial TP after 12 mo storage. Storage of packaged kernels at low temperature and low O2 restricted the TP losses additively. The present results are in agreement with another walnut study on Franquette cultivar (Christopoulos et al., 2010b), as well as on pistachios (Tsantili et al., 2011). The beneficial effect of low temperature on prevention of TP losses was observed in peanuts even when temperature range was much higher than the one here (Talcott et al., 2005). In this work, it is remarkable that storage at 1-N2 and 1-CO2 inhibited the reduction of TP levels up to 4 mo. The inhibition or prevention of TP loss could be ascribed to the restriction of oxidation reactions by the low temperature and/or low O2 availability, since TP are prone to the chemical or enzymatic oxidation (Manzocco et al., 2000). Unfortunately, the packaging material used here was not much

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Fig. 4. Load plots of Principal Component Analysis in walnut kernels stored under various conditions according to variables of total phenolics (TP), total antioxidant capacity (TAC) evaluated by FRAP and DPPH assays, and colour parameters (L*, h◦ , C* and WI). A, Chandler; B, Hartley; C, Ioli. Dagger indicates the position of each variable in load plot. Numbers in parentheses correspond to the percentage of the total variance explained by each component.

effective on prevention of oxygen permeation inside the package, resulting in progressive O2 increases during storage. Furthermore, at 20 ◦ C, the O2 level was approximately 2-fold higher than at 1 ◦ C, and this could be attributed to the increased material permeability with temperature elevation (Gomes et al., 2010). The similar level of O2 concentration in pouches flushed with either N2 or CO2 at each temperature and for the same time intervals during storage suggested that the O2 permeation was affected mainly by the changes in physical properties of the packaging material due to temperature. In consequence, the higher TP loss at the higher temperature could be ascribed not only to the direct effect of temperature on phenolic oxidation, but also to its indirect effect via the increase in O2 concentration in the pouches stored at the higher temperature. The presented differences in TP levels among cultivars could be rather attributed to background genetics since drying and storage conditions were similar throughout the entire experiment. In walnuts, individual phenolic compounds have been determined at various quantities each among cultivars (Colaric et al., 2005; Li et al., 2006). Also, individual phenolics may have differentiated response to storage conditions, as it has been observed in walnuts (Christopoulos et al., 2010b) and peanuts (Talcott et al., 2005). Indeed, phenolic oxidation rate depends on the number and nature of hydroxyl groups attached to phenolic molecule (Yokoyama et al., 1998). Before storage, walnut kernels exhibited very high TAC assessed by either FRAP or DPPH. The present TAC levels were in general agreement with other reports on walnuts (Arranz et al., 2008; Halvorsen et al., 2006). Chandler and Hartley exhibited much higher FRAP and DPPH than Ioli during the whole experiment. This could be important in practice since Chandler and Hartley stored under the most unfavourable conditions (20-AIR for 12 mo) showed antioxidant values similar to or higher than the highest found in Ioli before storage. Even though, Ioli exhibited high antioxidant levels in comparison with other nuts or various foodstuffs (Halvorsen et al., 2006; Kornsteiner et al., 2006). Two antioxidant methods here were selected because each one assesses different antioxidant properties (Apak et al., 2007). It is known that FRAP assesses the reducing ability, while DPPH assesses the antiradical activity. Both methods are quick, reliable and widely used for the TAC evaluation of plant foodstuff. A strong relationship between DPPH and FRAP was revealed by correlation and PCA in this study, probably because these methods are based on the same principle (Single Electron Transfer) (Apak et al., 2007). The similarity of the pattern of changes in TP, FRAP and DPPH could be explained by the fact that phenolics contributed

significantly to TAC in walnuts, as reported by others (Li et al., 2006). Moreover, phenolic oxidation is responsible for a loss of antioxidant capacity according to Manzocco et al. (2000). Indeed, in the present work significant correlations were found between TP and DPPH or FRAP that were further confirmed by PCA. Similar correlations between TP and TAC were also found in other studies on dried walnuts (Li et al., 2006; Pereira et al., 2008). Hartley exhibited higher FRAP and DPPH values than Chandler during the entire storage period, although Hartley had TP concentration lower than Chandler under the same storage conditions, by comparison. This discrepancy could be ascribed to differences in composition between the two cultivars concerning either nonphenolic compounds that contribute to TAC, as tocopherols, or to their different phenolic profile. It was also observed that the contribution of each individual phenolic compound to antioxidant capacity was different (Zhang et al., 2009). The percentage of TAC losses, assessed either by FRAP or DPPH, were slightly lower than the TP ones. This could be explained by the different quantitative changes in each phenolic during storage (Christopoulos et al., 2010b; Talcott et al., 2005). Furthermore, in some cases the partially oxidized polyphenols could exhibit higher antioxidant capacity than that of non-oxidized ones (Manzocco et al., 2000). 4.3. Colour The colour of walnut kernel is generally classified from ‘extra light’ to ‘amber’. In this work, values of h◦ were near 80, indicating a yellowish colour (McGuire, 1992). Values of L* were higher than 40 before and after storage, and this ensures the good colour quality according to walnut industry standards (Wang et al., 2007), while a sum of L*, h◦ and C* equal to or higher than 155 corresponds to kernels of ‘light’ or ‘bright’ colour of good quality (Warmund et al., 2009). The last authors reported a value of 166 for Chandler that is considered the brightest cultivar derived from the Californian breeding program. Here, Ioli exhibited the highest h◦ before storage and it could be considered as the brightest cultivar among the studied. Kernel colour of walnuts can be affected by postharvest handling and/or field conditions (Koyuncu et al., 2003; Warmund et al., 2009). In this work, the differences in colour among cultivars could be mainly ascribed to genetic factors, as in the case of antioxidants, since field conditions and postharvest handling were similar for all cultivars. During storage, L*, h◦ and WI showed decreasing trends that indicated the evolvement of browning and quality deterioration of kernels. Walnut browning was also observed during storage in

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other studies either by visual estimation (Koyuncu et al., 2003; López et al., 1995) or by objective measurement (Christopoulos et al., 2010b). The browning of a product is ascribed mainly to the enzymatic or chemical oxidation of phenolics (Manzocco et al., 2000). Indeed, in the present work the most intense browning (the lowest L*, h◦ and WI values) was observed in kernels stored at 20-AIR that also showed the greatest TP losses. The relationship between TP and browning was supported by both pairwise correlation and PCA analyses, and in consequence TP losses seemed to be responsible for the observed browning. Both low temperature and O2 restricted the kernel browning additively, as shown earlier on Franquette walnut cultivar (Christopoulos et al., 2010b) and other species (Nasar-Abbas et al., 2008). In the present work, L*, h◦ and WI parameters remained almost stable at 1-N2 and 1-CO2 during 12 mo storage. These conditions appeared to be the most suitable for the prevention of browning. The similar colour values under either N2 or CO2 suggested that the reduction of O2 was rather the main factor for browning prevention than the kind of the flushing gas.

5. Conclusion This work dealt with the effects of cultivar and storage conditions on TP concentration, TAC (assessed by FRAP and DPPH methods), colour and the probable relationships among these variables in walnut kernels. Chandler and Hartley exhibited much higher antioxidants than Ioli. In terms of kernel colour, Hartley was the darkest during the whole experiment, while Ioli exhibited the brightest colour at least before storage. Initial levels of antioxidants and colour values along with their changes during storage should be taken under consideration in cases of cultivar selection. Main results of this work were also referred to the positive and additive effects of the lower temperature tested (1 ◦ C) and reduced O2 in the packaging atmosphere during storage. Indeed, the best results for all measured variables were achieved at 1-N2 and 1-CO2 , and the beneficial effects of these conditions increased progressively by advanced storage duration. On the contrary, the quality deterioration was highest at 20-AIR. The rest conditions (1-AIR, 20-N2 , 20-CO2 ) showed intermediate results, but similar among them. Temperatures below 10 ◦ C are recommended for walnut storage, but in industries and market places walnuts are exposed to higher temperature because of the high cost of cooling infrastructure and operation. The present data showed that the expensive storage at 1-AIR could be replaced by storage at 20-N2 or 20-CO2 . During the entire experiment, relationships among the variables showed that the nutritional deterioration, in terms of antioxidant losses, was responsible for the simultaneous kernel browning that is a quality characteristic according to consumer perception. The increased oxidation rates of the higher TP levels during storage at higher temperature and oxygen concentration seemed to be responsible for the darkening susceptibility. The increasing demand for walnut consumption due to the beneficial effect on human health necessitates further investigation for improved quality of stored walnuts. A first step for quality improvement of stored walnuts could be the packaging in materials of lower O2 permeability than the presented here.

Acknowledgements The authors thank Dr. D. Rouskas for the supply of nuts from the Agricultural Research Station of Vardates (N.AG.RE.F. Vardates) and Prof. G.J.E. Nychas of the Agricultural University of Athens for the use of vacuum sealer Henkovac 1900.

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