Short Cold Storage as a Sustainable Postharvest Handling Method for Natural Enrichment in Antioxidants of Fresh and Dried Walnut Kernels—Cultivar Effect

Abstract

Fresh (raw, non-dried) walnuts (kernel moisture > 17%) have unique sensory and nutritional attributes but a narrow time availability due to their rapid deterioration during storage. In the present study, the storage (1 °C, 90% RH) potential of fresh walnuts for 20 and 40 days was assessed in relation to cultivar (Chandler, Hartley, Ioli) and the form of exposure to storage (shelled or in-shell). The effect of low-temperature exposure (at 1 °C for 0, 10 and 20 days) before nut drying was also examined. Fresh walnuts from different cultivars showed diverse quality (size, color) and physiological (respiration, weight loss) traits. Using a very low storage temperature (1 °C) was feasible to store fresh walnuts marginally up to 40 days without losing the ‘fresh’ character. The form of in-shell storage compared with shelled ones helped to determine the retention of kernel moisture and had a mild protective role in the prevention of kernel browning. The storage of fresh walnuts at 1 °C resulted in increased total phenolics (TP, by 26% in average) and antioxidant capacity (by 46%, in average) of the kernels, supporting the improvement of nutritional value due to low temperatures. The dried kernels after this short cold storage showed increased TP levels by 35–40% in comparison with conventional dried ones. Therefore, the 10 d cold exposure could be proposed as a sustainable step for incorporation in the regular postharvest handling chain for the natural enrichment of fresh and dried kernels in antioxidants.

1. Introduction

Walnuts (Juglans regia L.) are considered as an excellent source of health-promoting compounds (antioxidants, fatty acids), ranking them in an imperative position in the functional food market, with widely appreciated nutritious and functional attributes [1,2,3]. Dried walnuts (kernel moisture < 5–8%) are the common consumed products but, in recent years, fresh (raw, non-dried) walnuts have gained worldwide popularity due to their unique sensory and nutritional attributes [3,4,5]. Fresh walnut kernels should have a natural moisture content of at least 17.0% (w/w) [6]—below this limit, the ‘fresh’ character is lost. Fresh walnuts can be disposed of in the form of ready-to-eat kernels after removal of the shell (shelled kernels), or in the form of in-shell kernels (nut, whole endocarp).

The high moisture content, functioning respiration and metabolic activity make fresh walnuts prone to the rapid postharvest deterioration, restricting their availability to a narrow period after harvest. The main reflection of the deterioration is the kernel browning and the loss of moisture that weakens the ‘fresh’ character. These are the main problems that restrict the shelf-life of the product. For this reason, it is important to elucidate the factors that affect the deterioration during storage to increase the shelf-life of fresh walnuts. Cold storage (<8 °C) has been proved as an essential handling method for prolonging the postharvest availability of fresh walnuts [4,7,8,9,10,11]. The existing data have shown differences in the response of fresh walnuts to cold storage depending on the cultivar, region of cultivation and form of exposure to storage (shelled or in-shell kernels). Thus, the comparative assessment of the storage behavior of different walnut cultivars cultivated under the same climatic conditions could contribute to the knowledge about fresh walnuts’ proper postharvest handling.

The high total antioxidant capacity (TAC) derived from the high total phenolic (TP) content of walnuts ranks them at the top of the scale among various fruit and foodstuffs [12]. Also, it was shown that total phenolics were higher by 20% in fresh ‘Franquette’ walnut kernels than in dried ones, while subsequent storage of fresh walnuts at 1 °C resulted in additional increases in phenolic content, reaching a content by 1.2-fold higher than before storage [4]. The ‘Qingxiang’ walnuts showed increases in TP and TAC during storage at 5 °C for 60 d [3]. When fresh walnuts were stored at 6 °C for 18 d no significant change in TP was reported in the air-stored sample but the TP was increased by 3 fold in the samples in a modified atmosphere packaging system [7]. Studies with ‘Chandler’ walnuts showed that when fresh walnuts were exposed to 4 °C, an increase in TP content was observed in the first 15 d but then TP decreased by 30 d [11]. Also, when walnuts were of low moisture (10% w/w), the TP was gradually decreased throughout the storage period [9]. The increases in the TP and TAC of fresh walnuts by exposure to low temperatures were found to occur through the induction of the phenylpropanoid pathway (PhP), as has already been reported for fresh walnuts exposed to 1 °C [1,4].

The first objective of the present study was the comparative investigation of the cold storage behavior of two widespread cultivars and one new walnut cultivar in relation to the physiological, consumer-perceived and antioxidant attributes of fresh kernels stored in the shelled and in-shell forms. The second objective was the validation of cold storage as a sustainable postharvest handling method for natural enrichment of fresh and dried kernels in phenolics exploiting the activation of the phenylpropanoid pathway (PhP) as a response of walnuts to low temperatures.

2. Materials and Methods

2.1. Plant Material and Experimental Design

Fruit from three walnut (J. regia L.) cultivars were harvested from trees in Central Greece (Lat: 39°10′04.3″ N; Long: 22°11′42.5″ E). The fruits (approximately 50 kg per cultivar) were harvested from ten trees per cultivar at the regular commercial maturity stage, when the husk was just beginning to split and the packing tissue between and around the kernel halves had just turned brown. The studied cultivars were Chandler, Hartley and the Greek local Ioli. Their selection was based on their widespread worldwide cultivation and/or good nut quality and agronomical traits [13]. Two experiments, Experiment 1 and Experiment 2, were conducted over two years, with one experiment per year. In both experiments, immediately after harvest, fruit were hulled with tap water in a commercial dehulling unit and the in-shell nuts (endocarps), without being dried, were transported to the laboratory within 6 hours.

In Experiment 1 (Chart 1), upon arrival at the laboratory, walnuts of each cultivar were randomly divided into two groups. The walnuts of the first group were carefully shelled and only non-wounded kernel halves were used in the subsequent experiment, whereas the walnuts of the second group were left as in-shell kernels (nuts). In-shell and shelled whole kernels, macroscopically free of disorders and diseases, were randomly sorted into lots (replications) of approximately 200 g kernels (shelled) or approximately 500 g nuts (in-shell), placed in open plastic pots in a cool chamber at 1 °C with 90% RH. The number of kernels or nuts per replication were 20–40 or 25–40 (depended on the cultivar), respectively. The efficiency of two different types of kernel exposure (in-shell and shelled) was assessed during 40 d storage with 20 d sampling intervals. On each sampling day, weight loss (WL), respiration and ethylene production rates were evaluated on both whole endocarps (nuts) and shelled kernels. Kernel moisture, color, total phenolics (TP) and total antioxidant capacity (TAC) were determined in stored shelled or in-shell samples. Shell color and rupture strength were measured in stored in-shell samples. All determinations were carried out on four replications of 10 walnuts each, randomly selected. Color, respiration and ethylene production rates and rupture strength were measured at 20 °C after temperature equilibration.

In Experiment 2, upon arrival at the laboratory, in-shell walnuts were randomly sorted into lots of fifty (replications), placed in open plastic pots and stored for up to 20 d in a cool chamber at 1 ± 0.2 °C with 90 ± 5% RH. On each sampling day (0, 10 and 20 d), a total of 8 lots were removed from the cool chamber and divided into 2 groups. The walnuts of the first group were carefully shelled and kernels in halves were used as non-dried kernels (fresh kernels) in the subsequent experiment. The walnuts of the second group were dried in a laboratory bench drier at 36 °C for 24 h with an air velocity of 1000 m³ h⁻¹. Then, the dried walnuts were manually cracked, shelled and kernels in halves were used as dried kernels. Moisture, color and total phenolics (TP) were determined in both fresh and dried kernels on four replications of 10 walnuts each. All group division and sampling were carried out at random.

2.2. Weight Loss, Respiration and Ethylene Production Rates (Experiment 1)

Weight loss (WL) of both whole endocarps (nuts) and shelled kernels was measured immediately after removal from storage using a digital balance with a resolution of 0.01 g and WL expressed in % (w/w). Respiration and ethylene production rates of nuts and shelled kernels were measured according to Tsantili et al. [14] following the modifications reported by Christopoulos and Tsantili [4]. Respiration, as CO₂ production, was assessed using a closed portable infrared gas analyzer (LI-6200, LI-COR, Lincoln, NE, USA) connected to a 750 mL airtight jar at a flow rate of 900 μmol s⁻¹. The CO₂ production rates were expressed in μmol kg⁻¹ h⁻¹. Ethylene production was evaluated after incubation in a 500 mL jar at 20 °C for up to 4 h. A headspace sample of 1 mL was injected into a 120 cm × 0.2 cm i.d. column of 80–100 mesh activated alumina (Restek, Bellefonte, PA, USA) in a Perkin-Elmer-Sigma 300 (Perkin-Elmer, Norwalk, CT, USA) gas chromatograph equipped with a flame ionization detector (detection limit—0.42 nmol L⁻¹).

2.3. Moisture, Color and Shell Rupture Strength (Experiment 1)

Kernel moisture was determined by drying 10 g of chopped kernels in an oven with air circulation at 100–105 °C until constant weight and moisture were expressed in % (w/w).

The color was measured by a chromatometer (CR-300, Minolta, Ahrensburg, Germany) in a dark chamber. Kernel color was recorded on the upper part of the outer kernel surface and two measurements were taken per kernel. Shell color was recorded on two points longitudinal to suture per nut. For both kernel and shell color measurements, the CIE-L*a*b* values were recorded and the color was expressed as L*, hue angle (h°), chroma (C*) and whiteness index (WI) was calculated according to the equation WI = 100 − [(100 − L*)² + a*² + b*²]^1/2 [4]. Based on the WI values, the browning rate (BR) was calculated according to the equation BR = (WI₀ − WI_d)*100/WI₀, where WI₀ is the initial value (day 0) and WI_d is the value at the day of sampling.

The shell rupture strength was measured on one point longitudinal to suture of each nut using a flat (20 mm diameter) probe mounted on a bench Chatillon DF1S 100 penetrometer (J. Chatillon and Sons Inc., New York, NY, USA). The probe direction was perpendicular to the plane bounded by the suture at 200 mm min^–1 speed and the maximum force (N) required to rupture the shell was recorded.

2.4. Total Phenolics (TP) and Total Antioxidant Capacity (TAC) (Experiments 1 and 2)

The extraction for total phenolics (TP) and total antioxidant capacity (TAC) measurement was performed according to Christopoulos and Tsantili [4]. Briefly, frozen chopped kernels (−80 °C) were homogenized with 80% acetone (v/v) in water (5 mL g⁻¹ tissue) using an Ultra-Turrax (T 25; IKA Labortechnik, Staufen, 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 through a Büchner funnel (90 mm i.d.) using #1 Whatman paper. During incubation, the headspace of the vials was filled with N₂, while the entire procedure was conducted at 4 °C. After incubation, the acetone was evaporated at 38 °C under N₂ flow and the filtrate was recovered and then diluted in 50% methanol (v/v) in water.

The TP concentration was measured by a modified Folin–Ciocalteu colorimetric method [4]. Briefly, 0.2 mL of diluted extract was incubated for 6 min in a tube containing 2.6 mL of water and 0.2 mL of Folin–Ciocalteu reagent. Then, 2 mL of Na₂CO₃ (7%, w/v) was added to the mixture and the absorbance was measured at 750 nm (Helios Gamma & Delta; Spectronic Unicam, Cambridge, UK) versus a blank after incubation for 90 min at room temperature under darkness. The results were expressed as gallic acid equivalents (GAE) on a dry weight basis (mg GAE g⁻¹ DW).

The TAC was evaluated according to both ferric reducing antioxidant power (FRAP) and radical scavenging capacity (DPPH) assays according to Christopoulos and Tsantili [4]. For the FRAP assay, 0.1 mL diluted extract was incubated at 37 °C with 3 mL of FRAP reagent [300 mM acetate buffer (C₂H₃NaO₂·3H₂O, C₂H₄O₂), pH 3.6; 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl; 20 mM FeCl₃·6H₂O; in 10:1:1 (v/v)] preheated to 37 °C, and the absorbance was measured after 30 min at 593 nm versus a blank. For the DPPH assay, 0.1 mL diluted sample was incubated at room temperature with 3.9 mL DPPH solution (2,2-diphenyl-1-picryhydrazyl, 60 μM in MeOH) and the decrease in absorbance was recorded after 30 min at 515 nm versus blank. For both antioxidant capacity assays, the results were expressed as Trolox acid (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents (TAE) on a dry weight basis (μmol TAE g⁻¹ DW).

2.5. Data Analyses

In Experiment 1, in order to assess the differences among cultivars after harvest, and the effect of storage time for each cultivar and form of storage (shelled or in-shell), the data were analyzed by separate one-way analysis of variance (ANOVA). On each sampling day (20 or 40 days), data were subjected to two-way ANOVA (cultivar × storage form). In Experiment 2, three-way ANOVA (cultivar × storage time × drying) was performed to assess the response of cultivar on cold exposure (storage) before drying, the differences between fresh and dried walnuts, and the effects of storage time. For each analysis, all means were compared by Student’s multiple range test. For all ANOVA analyses, the significance level was α = 0.05. All statistical analyses were performed using Jump 7.0.1. software (SAS, Cary Institute, Cary, NC, USA).

3. Results

3.1. Pomological Traits, Weight Loss, Moisture Content, Respiration and Ethylene Production Rates in Fresh Walnuts (Experiment 1)

The recorded nut traits in the cultivars at harvest were 38.3–41.8 mm length, 32.2–38.4 mm width and 12.7–19.2 g weight (

Table 1. Pomological traits in fresh walnuts of three cultivars at harvest (Experiment 1, day 0).

Cultivar	Length (mm)		Width (mm)		Whole Weight (g)		Kernel Weight (g)		Kernel Percentage (%)
Chandler	40.02 ± 0.87	B 1	33.36 ± 0.53	B	16.73 ± 2.14	B	9.17 ± 1.64	B	54.59 ± 2.97	B
Hartley	38.28 ± 0.32	C	32.22 ± 0.23	C	12.66 ± 0.11	C	5.39 ± 0.06	C	42.60 ± 0.87	C
Ioli	41.76 ± 0.55	A	38.44 ± 0.57	A	19.22 ± 0.72	A	11.12 ± 0.64	A	57.84 ± 1.30	A
Pc 1	***		***		***		***		***

¹ Pc, and uppercase letters correspond to the one-way ANOVA of the data within each column for the assessment of cultivar effect. *** Significant at p < 0.001.

). The corresponding kernel weight varied from 5.4 to 11.2 g, resulting in 42.6–57.8% kernel percentage. A clear separation of cultivars concerning these traits was observed, with ‘Ioli’ having the largest nut and kernel, and ‘Hartley’ the smallest ones.

The kernel moisture after harvest was 32.0–35.4% (w/w) without significant differences among the cultivars (

Table 2. Kernel moisture, weight loss and respiration of whole endocarp (nut) and kernel in fresh walnuts of three cultivars stored (1 °C, 90% RH) as shelled (kernels) or in-shell (Experiment 1).

Cultivar	Storage	Day 0		Day 20		Day 40		Pt 1
		Kernel moisture (% w/w)
Chandler	Shelled	34.48 ± 1.05	a 1 A 2	21.46 ± 1.29	bB	18.82 ± 1.23	cBCD	***
	In-shell		a	20.82 ± 0.75	bB	23.38 ± 2.77	bA	***
Hartley	Shelled	35.35 ± 0.47	aA	24.39 ± 1.86	bA	20.36 ± 0.95	cBC	***
	In-shell		a	25.41 ± 2.97	bA	21.00 ± 1.13	cAB	***
Ioli	Shelled	31.98 ± 1.26	aB	20.02 ± 0.59	bB	16.85 ± 1.90	cD	***
	In-shell		a	20.41 ± 1.83	bB	18.14 ± 1.99	bCD	***
Pc		**		***		**
Pshell		-		ns		**
Pc × shell		-		ns		ns
		Weight loss (% w/w)
Chandler	Kernel	-		13.86 ± 1.07	bC	16.66 ± 1.01	aB	**
	Nut	-		21.34 ± 0.32	bA	32.49 ± 2.65	aA	***
Hartley	Kernel	-		12.97 ± 1.11	bC	17.56 ± 0.59	aB	***
	Nut	-		19.52 ± 0.59	aB	18.66 ± 2.67	aB	ns
Ioli	Kernel	-		12.75 ± 1.08	bC	16.97 ± 1.05	aB	**
	Nut	-		21.82 ± 0.99	bA	29.82 ± 1.90	aA	***
Pc		-		*		***
Pshell		-		***		***
Pc × shell		-		*		***
		Respiration (μmol CO2 kg−1 h−1)
Chandler	Kernel	1160.3 ± 61.0	aA	177.0 ± 34.5	bD	26.3 ± 3.3	cD	***
	Nut	866.3 ± 71.7	bB	987.3 ± 36.9	aA	514.7 ± 55.8	cA	***
Hartley	Kernel	582.1 ± 44.1	aDE	45.4 ± 4.8	bE	10.7 ± 1.7	bD	***
	Nut	539.1 ± 20.0	aE	248.3 ± 37.3	bC	99.4 ± 12.3	cC	***
Ioli	Kernel	752.3 ± 41.3	aC	49.8 ± 6.3	bE	12.3 ± 2.2	bD	***
	Nut	621.1 ± 38.6	bD	690.6 ± 39.5	aB	168.6 ± 29.1	cB	***
Pc		***		***		***
Pshell		***		***		***
Pc × shell		***		***		***

¹ Pt and lowercase letters correspond to the one-way ANOVA of the data within each row for the assessment of storage time effect. ² Pc, Pshell, Pc × shell and uppercase letters correspond to the one- or two-way ANOVA of the data within each column for the assessment of cultivar and storage form effect. ns, non significant; * Significant at p < 0.05; ** Significant at p < 0.01; *** Significant at p < 0.001.

). A gradual decrease in kernel moisture and consequently an increase in WL was observed during 40-day storage. The decrease in kernel moisture was sharper in the first 20 days of storage (mean kernel moisture of 22.1%) than in the next 20 days when the final mean moisture of kernels was 19.8%. The form of kernel storage (shelled or in-shell) did not affect the kernel moisture on day 20; but on day 40, the in-shell stored kernels had significantly higher moisture than the shelled stored ones. The effect of the form of kernel storage was more intense for ‘Chandler’ in comparison with ‘Hartley’ and ‘Ioli’. On day 40, the kernel moisture of ‘Chandler’ had decreased by 40% and 32% in shelled and in-shell kernels, respectively, whereas the differences between shelled and in-shell kernels for ‘Hartley’ and ‘Ioli’ were not significant. The WL of nut (mean WL of 27% on day 40) was higher than the WL of kernel (mean WL of 17% on day 40) and ‘Hartley’ exhibited a trend of being more resistant in WL than ‘Chandler’ and ‘Ioli’.

On day 0, the respiration rates of kernels ranged from 582 to 1160 μmol CO₂ kg⁻¹ h⁻¹ and those of nuts from 539 to 866 μmol CO₂ kg⁻¹ h⁻¹ (Table 2). For all cultivars except for ‘Hartley’, the kernels exhibited higher respiration rates than nuts. During storage and independently of tissue (kernel or nut), the highest respiration rates were recorded in ‘Chandler’ and the lowest in ‘Ioli’. During storage, the kernels showed a sharp decrease in the respiration rates by a mean of 9.2 fold on day 20 and by a mean of 50.7 fold on day 40 in comparison with the initial values, this response was similar among cultivars. The decreases in respiration rates of the nuts were smaller than those in kernels, and a different behavior among cultivars was observed. ‘Chander’ and ‘Ioli’ exhibited an increase in nut respiration rates during the first 20 days and then a decrease up to day 40, whereas the respiration of ‘Hartley’ showed a gradual decrease among storage intervals. None of the samples showed any ethylene production.

3.2. Kernel and Shell Color, and Shell Rupture Strength of Fresh Wanuts (Experiment 1)

At harvest, the L*, h°, C*, WI and BR values of kernel color were 62.2–66.2, 81.5–84.4, 29.6–33.42, 49.6–55.1 and −1.8–12.3, respectively (

Table 3. Kernel color L*, h°, C*, whitening index (WI) and browning rate (BR) parameters in fresh walnuts of three cultivars stored (1 °C, 90% RH) as shelled (kernels) or in-shell (Experiment 1).

Cultivar	Storage	Day 0		Day 20		Day 40		Pt 1
		Kernel L*
Chandler	Shelled	65.56 ± 1.13	a 1A 2	65.07 ± 0.96	aA	62.95 ± 1.47	bAB	*
	In-shell		a	66.27 ± 1.13	aA	63.85 ± 1.66	aA	ns
Hartley	Shelled	62.21 ± 0.71	aB	59.81 ± 0.84	bC	58.76 ± 2.23	bC	*
	In-shell		a	60.57 ± 1.81	aBC	60.49 ± 1.74	aBC	ns
Ioli	Shelled	66.20 ± 0.58	aA	61.26 ± 1.28	bBC	58.91 ± 0.81	cC	***
	In-shell		a	62.24 ± 0.92	bB	60.09 ± 2.33	bC	***
Pc 2		***		***		***
Pshell		-		ns		ns
Pc × shell		-		ns		ns
		Kernel h°
Chandler	Shelled	83.77 ± 0.49	aA	82.95 ± 0.56	aB	80.07 ± 0.95	bB	***
	In-shell		a	84.04 ± 0.57	aA	81.77 ± 0.93	bA	**
Hartley	Shelled	81.49 ± 0.67	aB	78.52 ± 0.75	bD	77.32 ± 0.96	bD	***
	In-shell		a	78.93 ± 0.36	bD	78.42 ± 0.43	bCD	***
Ioli	Shelled	84.42 ± 0.84	aA	81.75 ± 0.48	bC	78.11 ± 0.49	cD	***
	In-shell		a	83.01 ± 0.43	aB	79.53 ± 1.20	bBC	***
Pc		***		***		***
Pshell		-		***		***
Pc × shell		-		ns		ns
		Kernel C*
Chandler	Shelled	31.63 ± 0.87	aB	32.19 ± 0.66	aB	33.05 ± 1.04	aAB	ns
	In-shell		a	30.96 ± 1.19	aC	31.24 ± 1.79	aBC	ns
Hartley	Shelled	33.42 ± 0.53	bA	34.20 ± 0.31	abA	34.87 ± 0.78	aA	*
	In-shell		b	34.23 ± 0.37	aA	34.16 ± 0.34	aA	*
Ioli	Shelled	29.55 ± 0.61	bC	30.22 ± 0.71	bC	31.31 ± 0.70	aBC	*
	In-shell		a	30.37 ± 0.53	aC	30.94 ± 2.45	aC	ns
Pc		***		***		***
Pshell		-		ns		ns
Pc × shell		-		ns		ns
		WI
Chandler	Shelled	53.24 ± 1.32	aB	52.49 ± 1.03	aB	50.33 ± 0.56	bAB	**
	In-shell		a	54.21 ± 1.45	aA	52.22 ± 2.35	aA	ns
Hartley	Shelled	49.55 ± 0.74	aC	47.23 ± 0.51	bD	45.99 ± 2.16	bC	*
	In-shell		a	47.77 ± 1.25	aD	47.76 ± 1.30	aBC	ns
Ioli	Shelled	55.10 ± 0.80	aA	50.86 ± 1.33	bC	48.33 ± 0.67	cBC	***
	In-shell		a	51.53 ± 0.56	bBC	49.50 ± 3.27	bAB	**
Pc		***		***		**
Pshell		-		*		ns
Pc × shell		-		ns		ns
		BR
Chandler	Shelled	0.00 ± 0.00	b	1.40 ± 1.94	bC	5.46 ± 1.06	aBC	***
	In-shell		a	−1.84 ± 2.71	aD	1.92 ± 4.42	aC	ns
Hartley	Shelled	0.00 ± 0.00	b	4.69 ± 1.03	aAB	7.19 ± 4.35	aABC	*
	In-shell		b	3.59 ± 2.52	aBC	3.61 ± 2.61	aC	*
Ioli	Shelled	0.00 ± 0.00	c	7.70 ± 2.40	bA	12.29 ± 1.21	aA	***
	In-shell		b	6.48 ± 1.02	aAB	10.18 ± 5.93	aAB	**
Pc		-		***		**
Pshell		-		*		ns
Pc × shell		-		ns		ns

¹ Pt and lowercase letters correspond to the one-way ANOVA of the data within each row for the assessment of storage time effect. ² Pc, Pshell, Pc × shell and uppercase letters correspond to the one- or two-way ANOVA of the data within each column for the assessment of cultivar and storage form effect. ns, non significant; * Significant at p < 0.05; ** Significant at p < 0.01; *** Significant at p < 0.001.

). ‘Ioli’ had the highest WI and the lowest C* values, and ‘Hartley’ had the lowest L*, h°, and WI and higher C* values. All kernel color parameters except for C* and BR showed a decreasing trend during storage. The significant changes for C* and BR showed an increasing trend over the storage days. The form of kernel storage (shelled or in-shell) had a significant effect only on h° (day 20 and -40), WI (day 20) and BR (day 20), resulting in higher (for h° and WI) and lower (for BR) values of in-shell kernels than of shelled ones.

The shell color parameters at harvest were 56.5–59.6, 61.9–67.88, 22.2–24.8, 46.0–52.6 and −0.8–10.4.for L*, h°, C* WI and BR, respectively (

Table 4. Shell color L*, h°, C*, whitening index (WI) and browning rate (BR) parameters, and shell rupture strength in fresh walnuts of three cultivars during storage at 1 °C and 90% RH (Experiment 1).

Cultivar	Day 0		Day 20		Day 40		Pt 1
	Shell L*
Chandler	56.54 ± 0.22	a 1 B 2	55.77 ± 0.95	aB	54.63 ± 0.27	bB	**
Hartley	59.60 ± 0.97	aA	58.89 ± 1.87	aA	59.03 ± 0.65	aA	ns
Ioli	50.73 ± 1.09	aC	52.20 ± 0.95	aC	51.61 ± 0.65	aC	ns
Pc 2	***		***		***
	Shell h°
Chandler	65.93 ± 0.25	aB	61.75 ± 1.21	aB	66.44 ± 0.70	bA	***
Hartley	67.88 ± 0.21	aA	66.40 ± 0.63	bA	67.34 ± 0.37	aA	**
Ioli	61.93 ± 0.67	aC	59.74 ± 1.50	bC	63.77 ± 1.45	aB	**
Pc	***		***		**
	Shell C*
Chandler	23.05 ± 0.49	cB	26.19 ± 0.35	aA	24.40 ± 0.48	bB	***
Hartley	24.78 ± 0.33	bA	26.95 ± 1.37	aA	25.84 ± 0.16	abA	*
Ioli	22.15 ± 0.30	cC	26.31 ± 0.56	aA	23.22 ± 0.39	bC	***
Pc	***		ns		***
	Shell WI
Chandler	50.80 ± 0.27	aB	45.54 ± 1.48	bB	48.48 ± 0.43	cB	***
Hartley	52.60 ± 0.85	aA	50.83 ± 1.86	aA	51.56 ± 0.55	aA	ns
Ioli	45.98 ± 0.97	aC	43.38 ± 0.65	bB	46.32 ± 0.52	aC	***
Pc	***		***		***
	Shell BR
Chandler	0.00 ± 0.00	cA	10.36 ± 2.91	bA	4.57 ± 0.84	aA	***
Hartley	0.00 ± 0.00	aA	3.37 ± 3.53	aB	1.98 ± 1.04	aB	ns
Ioli	0.00 ± 0.00	bA	5.65 ± 1.42	aB	−0.76 ± 1.14	bC	***
Pc	-		*		***
	Rupture strength (N)
Chandler	248.9 ± 17.0	aB	260.9 ± 11.0	aB	243.7 ± 16.7	aB	ns
Hartley	288.3 ± 14.2	aA	299.2 ± 30.3	aA	284.8 ± 11.6	aB	ns
Ioli	236.5 ± 10.5	aB	233.3 ± 7.4	aB	222.1 ± 9.3	aC	ns
Pc	***		**		***

¹ Pt and lowercase letters correspond to the one-way ANOVA of the data within each row for the assessment of storage time effect. ² Pc and uppercase letters correspond to the one-way ANOVA of the data within each column for the assessment of cultivar effect. ns, non significant; * Significant at p < 0.05; ** Significant at p < 0.01; *** Significant at p < 0.001.

). A clear separation was observed among cultivars, with ‘Hartley’ having the highest and ‘Ioli’ the lowest values for all color parameters except for BR. The effect of storage time on shell color was significant in most cases. The observed trends of the values were a decrease in L*, h° and WI in the first 20 days and then an increase up to the end of storage. The C* and BR parameter showed an increase in the first 20-day storage interval and then a significant decrease. The shell rapture strength was clearly differentiated among cultivars at harvest and during storage since the time of storage had no effect on this parameter (Table 4). ‘Hartley’ exhibited the highest values of sell rapture strength (284.8–299.2 N), followed by ‘Chandler’ (243.7–260.9 N), whereas ‘Ioli’ showed the lowest values (222.1–236.5 N) among the studied cultivars.

3.3. Total Phenolics (TP) and Total Antioxidant Capacity (TAC) of Fresh Walnuts (Experiment 1)

The values of TP, FRAP and DPPH after harvest were 12.2–26.1 mg GAE g⁻¹ DW, 104.2–194.5 μmol TAE g⁻¹ DW and 99.8–185.3 μmol TAE g⁻¹ DW, respectively, showing significant variation among the cultivars (Figure 1). The highest TP values were recorded in ‘Chandler’ and ’Hartley ‘and the lowest in ‘Ioli’. For TAC (assessed by both FRAP and DPPH methods) ‘Harley’ showed the highest values, ‘Ioli’ the lowest and ‘Chandler’ had intermediate values. An increasing trend of the TP (by 26% in average), FRAP (by 46%, in average) and DPPH (by 46%, in average) values was observed during storage of kernels, but the form of kernel storage (shelled or in-shell) did not affect these changes. The TP and TAC changes during storage were found to be varied among cultivars. The storage of ‘Chandler’ showed an almost stable TP (decrease by 6%, in average) and mean increases by 15% in the values of both FRAP and DPPH. The stored ‘Hartley’ kernels had averaged increases by 23%, 50% and 51% in the TP, FRAP and DPPH, respectively, and these increases were sharp in the first 20-day interval of storage. The storage of ‘Ioli’ caused gradual increases in TP, FRAP and DPPH levels by 59%, 74% and 72% (in average), respectively.

3.4. Drying after Cold Exposure of Fresh Walnuts (Experiment 2)

In Experiment 2, the initial moisture of fresh kernels was 32.2–37.9% (w/w), having a gradual decreasing trend during storage of the in-shell samples, and reaching the levels of 21.2–25.7% (w/w) after 20 days (Figure 2). The drying of walnuts, at harvest and just after removal from the cold room, resulted in dried kernels of 6.2–8.3% (w/w) moisture without significant differences either among cultivars or among storage intervals.

At harvest, the TP of fresh kernels was 14.5–22.3 mg GAE g⁻¹ DW, and ‘Chandler’ and ‘Hartley’ had a significantly higher TP than ‘Ioli’. The drying of walnuts just after harvest resulted in a loss of TP in dried kernels that were 11.6, 18.0 and 18.6 mg GAE g⁻¹ DW in ‘Ioli’, ‘Hartley’ and ‘Chandler’, respectively (Figure 2). The cold storage of fresh walnuts caused significant increases in the TP of both fresh and dried kernels. The mean increases in the TP of fresh kernels were by 21% and 28% after 10 and 20 days of storage, respectively. The drying process implemented after 10 days of cold storage of fresh walnuts resulted in dried kernels with TP levels by 40% (in average) higher than dried kernels just after harvest. A mean increase by 35% in the TP levels of dried kernels was observed when the drying process was performed in 20-day cold stored fresh walnuts. A different response of each cultivar in increases in TP of dried kernels was also observed. More particularly, the dried kernels of ‘Chandler’ had the lowest (by 15% in average) and kernels of ‘Ioli’ the highest (by 60% in average) increases in TP levels.

4. Discussion

4.1. Pomological Traits at Harvest, Weight Loss, Moisture Content, Respiration and Ethylene Production Rates of Fresh Walnuts during Storage

The minimum size for classification of a walnut in the supreme quality class (‘Extra’) is the threshold of 26 mm for the minimum diameter (width) of the nut [15]. In the present study, all three cultivars had a minimum diameter of nut higher than the value of 32 mm (Table 1). For the other parameters related to the size quality of the nuts, there is no legislative standard; however, in the market, large nuts with a large kernel and a high kernel percentage are preferable for processing and consumption. Based on all assessed pomological parameters (Table 1), the studied cultivars were classified in descending order for size quality as ‘Ioli’ > ‘Chandler’ > ‘Hartley’. The present data for pomological traits for ‘Chandler’ and ‘Hartley’ are in good agreement with the reported data for the same cultivars and/or other walnut genotypes in other studies [16,17,18,19].

Fresh walnut kernels should have a natural moisture content of at least 17.0% (w/w) [6] and the lowest acceptable moisture content must be at least 20.0% (w/w) for the fresh inshell walnuts [15]. In the present study, all three cultivars met this criterion at harvest and during storage (Table 2). The in-shell form had a significant effect on the retention of kernel moisture only at day 40, suggesting that shell acts as a mild barrier for kernel moisture loss. Additionally, the WL of whole nut was higher than the WL of kernel, which confirms porosity, and this character was found to be cultivar dependent based on the significant WL differences between ‘Chandler’ and ‘Hartley’ (Table 2). For ‘Chandler’ fresh kernels (initial moisture of 24%) stored without packaging at 6 °C, the WL was approximately 36% (w/w) [7], and the kernel moisture of fresh ‘Xifu No.1’ walnuts stored in-shell in glass bottles at 0 °C decreased from 22.32% on day 0 to 21.96% on day 60 [6]. The present and previous studies support the need for use of both a very low storage temperature and postharvest technologies (i.e., packaging systems, control atmosphere, etc.) to preserve the freshness character dependent on kernel moisture for the extension of shelf-life.

The data on respiration of fresh walnut kernels and nuts are very limited. The initial respiration rates measured at 20 °C in ‘Chandler’, ‘Hartley’ and ‘Ioli’ of fresh kernels (582–1160 μmol CO₂ kg⁻¹ h⁻¹) and nuts (539–866 μmol CO₂ kg⁻¹ h⁻¹) (Table 2) were close to the range previously reported for ‘Franquette’ (950 and 770 μmol CO₂ kg⁻¹ h⁻¹ for kernels and nuts, respectively) [4]. In an unknown cultivar, respiration rates of 575 μmol CO₂ kg⁻¹ h⁻¹ and 425 at 30 °C for fresh kernels and nuts, respectively, have been reported [20]. The present results confirm that fresh walnuts could be classified in fruits with low to medium respiration rates [4,21]. During storage, the sharp and immediate decrease in kernel respiration (decrease by 9.2 fold on day 20) in contrast to the gentler and/or later decreases in respiration of the nuts (Table 2) could be attributed to a protective role of walnut shell on the retention of tissue viability. A similar pattern of the respiration’s changes during storage at 1 °C of fresh kernel and nut has been reported for ‘Franquette’ walnuts [4]. The initial respiration rates and their changes during storage for both kernels and nuts, and the different values for each cultivar should be considered for the selection of proper postharvest management of fresh walnuts (i.e., energy requirements for refrigeration and selection of modified atmosphere). In the present work, no ethylene release was found in any sample and this is in agreement with previous works [4].

4.2. Kernel and Shell Color, and Shell Rupture Strength of Fresh Wanuts during Storage

Walnut kernel color may affect consumer acceptance since it is one of the most important sensory traits and it is generally classified from ‘extra light’ to ‘amber’. There are no objectively assessed color indicators for quality classification of fresh walnuts, and for dried kernels, values of L* higher than 40 ensure good color quality according to walnut industry standards [22]. Here, the recorded kernel color parameters for all three cultivars (Table 3) correspond to the very bright color of kernels. For ‘Chandler’, that is considered the brightest cultivar derived from the Californian breeding program [23]—values of L* = 69–74 and h° = 88 have been reported for fresh kernels [7,11] being slightly higher than the present ones (L* = 66 and h° = 84), probably related to differences in climatic and agronomic conditions. Based on all recorded color parameters, ‘Ioli’ had the brightest (highest WI) and ‘Hartley’ the least bright (lowest L*, h° and WI) fresh kernels, and this classification is similar to that reported for dried kernels of the same cultivars [13]. The assessed color parameters for fresh kernels at harvest are close to the reported parameters for ‘Chandler’ [7,11], whereas the L* parameter was recorded much higher than in local walnut genotypes studied in other works [24,25]. During storage, L*, h° and WI showed a decreasing trend and BR increased, which indicates the browning and quality deterioration of fresh kernels and this is in accordance with the trends reported in other storage studies [1,4,7,11,26]. However, the selection of a temperature of 1 °C was found to be effective to maintain browning at acceptable levels. The observed browning has been related to the enzymatic oxidation of phenolics in walnut kernels [26]. Storage form (shelled or in-shell) affected only h° values, indicated by only a mild protective role of the shell in the prevention of browning. Indeed a probable protective effect of the shell against kernel browning has been reported and attributed to the barrier action of the shell on oxygen availability inside the nut [4]. The results from other studies when walnuts were stored under modified atmosphere packaging, a controlled atmosphere or using edible coatings suggest that O₂ availability is one of the more important factors affecting browning [7,9,26].

Walnut shell color is important when in-shell nuts are sent to the market, and bright shell is the preferred character. In the current study, based on the recorded shell color parameters, ‘Hartley’ was the brightest and ‘Ioli’ the least bright cultivar. For all three cultivars, the assessed color values for L*, h° and C* were within the range reported for other walnut genotypes [16,26]. The gradual browning on the shell color of fresh walnuts during storage was indicated by decreasing L*, h° and WI, and increasing BR, in accordance with the shell color changes reported for ‘Xifu No.1’ walnuts [26]. The shell rapture strength of walnuts is related to kernel extraction quality during cracking and it is an important trait for the development and configuration of cracking machines [27,28]. The observed values of shell rapture strength (222–299 N) are in agreement with those reported in other studies, and the differences among the three cultivars were probably related to differences in size, shape and shell thickness [27,28]. The storage had no effect on the shell rapture strength, indicating the stability of this character without the need for extra planning and setup of cracking machines of stored nuts.

4.3. The Total Phenolics (TP) and Total Antioxidant Capacity (TAC) of Fresh Walnuts during Storage

In this study, the measured values of TP, FRAP and DPPH after harvest (Figure 1) was within the range reported for fresh walnuts by other researchers [1,4,5,7,8,29,30]. TP values lower than 2 mg GAE g⁻¹ DW have been reported in a few studies [24,25,26,31] and these differences could be attributed to extraction methods and/or studied cultivars. Indeed, a significant cultivar effect on TP levels was found here, with ‘Chandler’ and ‘Hartley’ having 2.1 and 2.0 fold, respectively, higher values than ‘Ioli’ (Figure 1). Concerning the values of TAC, ‘Chandler’ and ‘Hartley’ had by 1.6- and 1.9-fold (in average) higher values than ‘Ioli’, respectively (Figure 1). The content of phenolic compounds and TAC are among the major traits that contribute to walnuts’ nutritional value and functional properties, making them popular as a food [2,32]. The storage of fresh walnuts at 1 °C resulted in increased TP and TAC levels of the kernels, and these increases were sharp during the first 20 d of storage (Figure 1). Similar increases in TP and TAC have been reported when ‘Franquette’ fresh walnuts were exposed to 1 °C, whereas exposure to 8 °C led to no increases [1,4]. ‘Qingxiang’ walnuts showed increases in TP and TAC during storage at 5 °C for 60 d [3]. When fresh walnuts were stored at 6 °C for 18 d, no significant change in TP was reported in the air-stored sample but the TP was increased by 3 fold in the samples in a modified atmosphere packaging system [7]. Studies with ‘Chandler’ walnuts showed that when fresh walnuts were exposed to 4 °C, an increase in TP content was observed in the first 15 d and then TP decreased by 30 d [11]; also, when walnuts were of low moisture (10% w/w), the TP gradually decreased throughout the storage period [9]. The increases in TP and TAC through the induction of the phenylpropanoid pathway has already been reported for fresh walnuts exposed to 1 °C [1,4]. The final content of phenolics in a tissue is the output of two contradictory processes that evolve simultaneously—the synthesis and the oxidation of these compounds. The present and the already published results suggest that in fresh walnut kernels, the low-temperature (1–4 °C) exposure triggers the synthesis of phenolics at a rate higher than the rate of their oxidation, enhancing kernel nutritional value.

4.4. Drying after Cold Exposure of Fresh Walnuts (Experiment 2)

In Experiment 2, a practical application for the enhancement of the nutritional value of the walnut kernels was further evaluated to produce both fresh and dried kernels using a walnut raw material of similar specifications (cultivars and the initial moisture and TP levels) by another cultivation year (Figure 2). The results confirmed the increases in the TP of the fresh kernels after the exposure of the nuts to low temperature (1 °C), and most of the increases were at the first 10 d of the exposure (Figure 2). Applying a standard thermal drying protocol in nuts just after the removal from the cold room reduced the moisture (Figure 2) in the standard levels for dried walnuts [33]. This suggests that a low temperature of 1 °C exposure as a step is compatible with the regular postharvest handling chain of walnuts. The drying of nuts resulted in a loss of TP in dried kernels by 14% (in average) in comparison with fresh kernels (Figure 2). It is well known that thermal drying results in losses of compounds prone to oxidation such as phenolics and this has already been reported for walnuts [4,8]. The dried kernels produced after the exposure of fresh nuts to a low temperature for a period of 10–20 d had increased TP levels by 35–40% (in average) in comparison with conventional dried kernels without the preceding step of low-temperature exposure (Figure 2). The present results suggest an enrichment of both fresh and dried kernels in antioxidants and related enhancement of the products’ nutritional value. Moreover, the increased antioxidants protect the tissue from lipid oxidation [7,10]. Based on these results, the incorporation of the sustainable cold (at 1 °C) exposure step in the regular postharvest handling chain of walnuts could be proposed (Figure 3). The sustainability key points of the proposed cold exposure step can be summarized as: (i) the use of the already existing infrastructure (cold rooms); (ii) the improvement of the added value of a product (dried walnuts), triggering a natural endogenous mechanism (PhP) without the use of materials that burden the environment; (iii) the reduction in food waste through an increase in the flexibility of the selection of different walnut products.

5. Conclusions

Fresh walnuts from different cultivars showed diverse quality traits that should be considered in combination with consumer preferences. The ‘Ioli’ cultivar had the highest quality traits related to appearance of fresh kernels (shape and brightness) but the lowest nutritional quality (in terms of antioxidant activity and total phenolics). Using a very low storage temperature (1 °C) is feasible to store fresh walnuts up to 40 days without losing the ‘fresh’ character (kernel moisture > 20%). The form of in-shell storage compared with shelled ones helps to determine the retention kernel moisture and has a mild protective role in the prevention of kernel browning. The storage of fresh walnuts at 1 °C results in increased TP and TAC levels of the kernels, supporting the improvement of nutritional quality through exposure to low temperatures. The cultivar effect was significant for the most studied traits. The dried kernels produced after the exposure of fresh nuts to 1 °C for a period of 10–20 d increased TP levels by 35–40% (in average) in comparison with conventional dried kernels without the step of low-temperature exposure. Based on this enrichment of dried kernels in TP, exposure of fresh walnuts to 1 °C for 10 days before drying could be proposed as a sustainable step in the regular postharvest handling chain.