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Article

Effect of Temperature and Storage Time on Some Biochemical Compounds from the Kernel of Some Walnut Cultivars Grown in Romania

by
Loredana Elena Vijan
1,
Simona Giura
2,
Ivona Cristina Mazilu
2,3,* and
Mihai Botu
4,*
1
Faculty of Sciences, Physical Education and Computer Science, University of Pitesti, 1 Targu din Vale Street, 110142 Pitesti, Romania
2
Doctoral School of Plant and Animal Resources Engineering, University of Craiova, 13 A.I. Cuza Street, 200585 Craiova, Romania
3
Research Institute for Fruit Growing Pitesti-Maracineni, 402 Marului Street, 117450 Maracineni, Romania
4
Department of Horticulture and Food Science, Faculty of Horticulture, University of Craiova, 13 A.I. Cuza Street, 200585 Craiova, Romania
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(5), 544; https://doi.org/10.3390/horticulturae9050544
Submission received: 20 March 2023 / Revised: 19 April 2023 / Accepted: 28 April 2023 / Published: 30 April 2023
(This article belongs to the Special Issue Bioactive Compounds in Horticultural Plants)
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Abstract

:
Walnut kernels are appreciated not only for their mineral content, vitamins, proteins, and valuable lipids, but also for the presence of polyphenolic compounds and biogenic amines. The temperature and storage time effects on some biochemical compounds from kernels collected from eight walnut genotypes grown at the Fruit Growing Research and Extension Station (SCDP) Valcea, Romania, were studied. In general, the phenolic and carotenoid compounds followed opposite trends during short-term storage and in some cases in medium-term storage. In both cases, there was a reduction in concentration during long-term storage, which was more pronounced for carotenoids. The most efficient method for preserving the walnut kernel’s phenolic content was freezing. Keeping the walnut kernel at room temperature assured the smallest carotenoid content decrease. Depending on the walnut cultivar, the preservation of the walnut kernel can be extended to five months by storage at −20 to −18 °C without loss of phenolic compounds, while a period shorter than five months (but certainly longer than three months) could be recommended for storage at 3–4 °C. Keeping it at room temperature can be an option for a short period (about three months). None of the preservation methods was adequate if the losses recorded for carotenoids were taken into account.

1. Introduction

As a natural functional food, the fruits of the common walnut (Juglans regia L.), also known as Persian, English, or Carpathian walnut, have a low glycemic index and reduce serum cholesterol concentrations [1], and they also have protective properties associated with their chemical composition. Nut consumption reduces oxidative stress, is beneficial in the treatment of cardiovascular diseases, diabetes, and for improving blood lipid profile, and reduces adiposity and low-grade systemic inflammation [2,3,4,5].
In addition to water (3–5%), walnuts contain proteins (12–25%), polyunsaturated fatty acids (omega 3 and omega 6), biogenic amines (melatonin and serotonin), vitamins (B, C, E, and P complex), minerals (magnesium, potassium, calcium, iron, zinc), polyphenols, and fiber [1,3,6,7]. Topala et al. (2020) [8] cites authors that justified the classification of the walnut as a strategic nut crop for human nutrition as well as the inclusion in the dedicated FAO list of priority plants.
The walnut is currently cultivated in more than 60 countries in both hemispheres. Its importance is not limited to the commercial value of in-shell walnuts and walnut kernels. Its wood is used for furniture, panels, veneer, sculpture, and also for the manufacture of weapon beds [9]. Dried and ground shells (walnut endocarp), recovered after breaking and separating the walnut core, are used as abrasive materials for polishing and cleaning, filtering, or even cosmetic products [10,11]. Walnut leaves and the green shell (walnut mesocarp) can also be exploited for their polyphenol content [11,12,13,14], having practical applications not only in the para-pharmaceutic industry but also in the culinary field [11,15,16,17]. Unripe green walnuts can be used to make liqueur and green walnut jam [11,15,18,19,20]. From the walnut kernel, walnut oil can be obtained (by cold or hot pressing), a product used in culinary preparations [21,22,23] with multiple medical benefits [11,16,17].
Commerce with in-shell and kernel nuts is very important worldwide. Thus, in 2021, the amount of in-shell walnuts exported was 442,238 tons, worth USD 1.21 billion, and the amount of walnut core exported amounted to 389,860 tons, worth USD 2.45 billion [24].
Internationally recognized standards are used for the trade and quality control of unprocessed nuts, coming from cultivated varieties or non-grafted nuts from semi-spontaneous or spontaneous flora (UNECE Standard DDP-1 edition 2014 for in-shell walnuts and UNECE Standard DDP-2 edition 2019 for walnut kernels) [25].
Among the top-producing countries, only the USA (second place worldwide), Chile (sixth place), and France (ninth place) mainly obtain nuts from organized plantations with grafted trees. These three countries produce 26.6% of the world nut production (2017–2021 average). Nut productions from countries such as China (first place), Iran (third place), Turkey (fourth place), Ukraine (fifth place), Romania (seventh place), and Uzbekistan (eighth place) mostly come from non-grafted walnut trees. The share of these countries in the world total is 58.5% (2017–2021 average) [24]. Consequently, the walnut productions are non-uniform and of variable quality. Given that a large part of the production comes from non-grafted walnut trees, from cultivated and spontaneous flora, their collection is difficult and the conditioning, packaging, and preservation of nuts in-shell and kernel are very different.
The most important aspects of post-harvest walnut quality refer to the minimal exposure to field heat during the harvest, forced-air drying at relatively low temperatures (<43 °C), and cold storage (<2 °C) at a relative humidity designed to maintain low nut moisture in a reduced oxygen atmosphere [26,27].
Guiné et al. (2015) [1] analyzed shelled walnut kernels from Chile, Portugal, Romania, and the United States, stored for 90 days at room temperature, 30 °C, and 50 °C, in low-density polyethylene (LDPE) plastic packaging or linear low-density polyethylene (LLDPE) and found that samples from Romania had a higher moisture content and water activity than the other analyzed samples. They found that after 90 days of storage, the storage conditions were adequate, except for samples at 50 °C, where dehydration of the product and a large color change occurred. Regarding the type of packaging, it was observed that the use of plastic bags did not improve the characteristics of the products compared to the non-packaged samples, except for color, in which case LDPE plastic is preferable.
Jensen et al. (2003) [28] analyzed walnut kernels packed in three laminated packaging materials with different oxygen permeability for 13 months at 11 °C and 21 °C and found that storing nuts at a high-oxygen concentration resulted in rancid nuts, while storage at a low-oxygen concentration results in fine-tasting nuts. They concluded that the optimum storage temperature for nuts is 11 °C or lower, possibly combined with an oxygen absorber, and that without cold storage and the use of an oxygen absorber, it is possible to obtain acceptable nut quality with a packaging material with very low oxygen permeability combined with nitrogen flushing.
Storing walnut kernels in light and at room temperature has a negative effect on the sensory quality and shelf life of walnut kernels [29].
Mexis et al. (2009) [30] highlighted that nuts keep an acceptable quality for about two months in polyethylene terephthalate (PE) bags packed with atmospheric air (PE-air), 4–5 months in polyethylene terephthalate//polyethylene with nitrogen flushing (PET//PE-N2), and at least 12 months in PET-silicon-oxide//PE-N2 at 20 °C, with samples stored in the dark retaining a slightly higher quality than those exposed to light. The effect of the investigated parameters followed the sequence: temperature > degree of O2 barrier > lighting conditions.
Lipid oxidation can be inhibited by using a packaging material with low oxygen permeability or by storing nuts in low-oxygen atmospheres [29,31].
Rizzolo et al. (1994) [32], in a study regarding the storage of shelled and in-shell almonds, concluded that almond storage for a year and over a year without quality loss could only be attained with cultivars that possessed high concentrations of natural antioxidants, such as α-tocopherol, which protect kernel quality. Zacheo et al. (2000) [33] analyzed four different varieties of almond seeds kept over two years in the dark at room temperature and found that fatty acid oxidation only becomes significant after kernel antioxidants were depleted, as the total concentration of tocopherols decreases during storage. Deterioration reactions may occur during storage, which include lipid oxidation and browning reactions [1,34,35]. Water activity influences spoilage reactions, especially in foods with water activities between 0.65 and 0.85, with lipid oxidation occurring rapidly, also being influenced by the temperature during storage [36,37].
Oxidation of lipids can lead to loss of flavor (due to rancidity) and decreased nutritional value and functionality by decreasing the content of bioactive compounds, while the accumulation of compounds harmful to health could be observed [38].
As can be seen, lipid oxidation is a process influenced by many factors, some determined by the walnut kernel composition (lipid composition, degree of unsaturation, free fatty acids, trace metals, etc.) and others determined by walnut storage conditions (oxygen concentration, temperature, light, relative humidity, etc.) [33,39,40,41,42]. Low oxygen concentrations and its absolute absence would prevent the oxidative rancidity of lipids, oxygen being essential to propagate the reaction [31,42]. Moreover, the rate of oxidation increases exponentially with temperature [42]. Light, depending on its intensity, duration of exposure, absorption by the product, temperature, and the amount of available oxygen, may induce oxidation in nuts and nut oils [39,40,43,44] but tocopherols and tocotrienols, naturally present in these products, together with other antioxidant compounds exert effective protection against oxidative stress [45,46]. Fourie and Basson (1989) [43] studied variations in tocopherol concentrations in several nuts and found that almond kernels, with a higher tocopherol concentration than other nuts, had better storage stability. These results highlight the importance of preserving the freshness of products, which could be possible by using processing techniques that minimize the loss of tocopherols and other natural antioxidants.
Unfortunately, storing nuts at 11 °C is difficult under typical commercial storage conditions for the retail market because of the costs of refrigeration, oxygen absorbers, as well as the cold room in the store, and if an oxygen absorber is used, the consumer has to get used to it. However, without cooling and the use of an oxygen absorber, according to Jensen et al. (2003) [28], it appears possible to obtain high quality using a low oxygen permeability packaging material combined with nitrogen flushing.
Through the present study, we aimed to analyze the behavior of some Romanian and foreign walnut cultivars grown in Romania, regarding the effect of the most commonly used storage temperatures and storage period on the kernel’s quality, by quantification of polyphenol, flavonoid, tannin, lycopene, and β-carotene content for kernels collected from eight walnut cultivars grown at the Fruit Growing Research and Extension Station (SCDP) Valcea, Romania.

2. Materials and Methods

2.1. Plant Material and Sampling

The cultivars sampled for this study are located in the walnut trial of the Fruit Research and Extension Station (SCDP) Valcea, belonging to the University of Craiova. The trial is situated in Bujoreni (45°08′23 N; 24°22′35 E), north of Ramnicu Valcea City. The elevation of the site is 262 m, and the soil is alluvial, medium fertile, with a pH of 6.8.
The climate of the area is of Cfb Köppen-eiger type [47] and the annual average temperature is 10.2 °C with an annual rainfall of 715 mm [48].
The trial was set up in 1997, using planting distances of 9.0 by 8.0 m (density of 139 trees ha−1). Each walnut cultivar is represented by five trees grafted on Juglans regia L. seedling rootstocks.
Four Romanian (‘Jupanesti’, ‘Sarmis’, ‘Unival’, and ‘Valcor’), three French (‘Fernor’, ‘Franquette’, and ‘Ferjean’), and one cultivar from the U.S.A. (‘Vina’) were used in this study. ‘Franquette’ is a classic cultivar, still responsible for 70% of the French walnut production, and in other countries, it has very good nut quality and taste. ‘Fernor’ and ‘Ferjean’ are newer French cultivars with lateral bearing and are more productive. ‘Fernor’ (‘Franquette’ × ‘Payne’), issued in 1995, has larger fruits, stores well, and has very good kernel quality, and it is the most planted French cultivar. ‘Ferjean’ (‘Lara’ × ‘Grosvert’), issued in 1999, has smaller fruits, designated for the kernel market. ‘Vina’ (‘Franquette’ × ‘Payne’) is a lateral bearing cultivar, obtained in 1968 at the University of California-Davis. It has medium- to large-quality fruits and is productive [48,49].
The Romanian cultivars were initially selected from local walnut populations of Arges (‘Jupanesti’), Hunedoara (‘Sarmis’), and Valcea (‘Valcor’ and ‘Unival’) Counties. These cultivars are well adapted to the local climate conditions, are productive, and have medium to large fruits of good quality. Walnut samples were collected from the orchard at ripening time in mid-September for Romanian cultivars and ‘Vina’ and late September to early October for the French cultivars.
The walnuts were kept in their shells between the harvest moment and mid-December 2021 in boxes in a ventilated room. In mid-December 2021, the walnuts were cracked and the kernels were put in plastic bags for short-term (until mid-March 2022), medium- (until mid-May 2022), and long-term storage (until mid-June 2022) in the freezer (−20 to −18 °C, very low-temperature treatment, VLTT), refrigerator (3–4 °C, low-temperature treatment, LTT), or stored at room temperature (20–22 °C, RTT).
The walnut kernel stored in the freezer was brought to room temperature and thawed before being subjected to laboratory analysis. Then, the walnut kernel samples were transformed into a homogeneous mixture with a vertical mixer and were stored at 3–4 °C to perform all analyses.

2.2. Chemicals and Reagents

All reagents used in this study were analytical grade reagents and were obtained from Merck-Sigma-Aldrich, Darmstadt, Germany.

2.3. Extraction Procedures and Biochemical Determinations

For the extraction of polyphenols and flavonoids, samples from one gram of crushed and homogenized walnut kernels in 10 mL of ethanol were used and were subjected to the following procedures: vortexing for 5 min at 3000 rpm, ultrasonication for 30 min at 40 kHz, and centrifugation at 6500 rpm for 30 min. The ethanolic extracts were filtered and the determination of the two bioactive compounds was performed immediately after extraction, according to the methodology proposed by Giosanu et al. (2018) [50] for the determination of polyphenols and the methodology proposed by Giura et al. (2019) [12] for the determination of flavonoids.
The method used for total phenolic content determination is based on the reaction between phosphotungstic acid and polyphenols in an alkaline medium resulting in a blue-colored compound. Thus, in a volumetric flask with a volume of 10 mL, a quantity of 0.5 mL of ethanolic walnut kernel extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2 mL of distilled water. After homogenizing the mixture, 2 mL of 10% sodium carbonate solution was added to create basic conditions for the redox reaction between the phenolic compounds and the Folin-Ciocalteu reagent and it was filled up to the mark with distilled water. Similarly, the blank sample was prepared using 0.5 mL of ethanol instead of the ethanolic walnut kernel extract. The samples were kept in the dark at room temperature for 2 h. After the resting period, the absorbance of the samples was measured in relation to the control sample, with the absorption maximum located at 750 nm, and the concentration of polyphenols was estimated using the calibration curve built with standard solutions of gallic acid solubilized in ethanol. Finally, the content of phenolics (TPC) was expressed as mg gallic acid equivalents (GAE) 100 g−1 walnut kernel.
The principle of the flavonoid determination method is based on the formation of a yellow-orange-colored compound following the reaction of these bioactive compounds with aluminum chloride. In a volumetric flask with a volume of 10 mL, 1 mL of ethanolic walnut kernel extract was mixed with 0.5 mL of 5% sodium nitrite solution and 2 mL of distilled water. After homogenizing the mixture, 0.5 mL of 10% aluminum chloride solution was added to the volumetric flask. After resting for a few minutes, 2 mL of 1 M sodium hydroxide solution was added and was made up to the mark with distilled water. Similarly, the blank sample was prepared using 0.5 mL of ethanol instead of the ethanolic extract of the walnut kernel. Immediately after preparation, the absorbance of the samples was measured in relation to the control sample (with ethanol instead of the ethanolic extract of the walnut kernel), with the absorption maximum located at 510 nm, and the concentration of flavonoids was estimated using the calibration curve built with solutions catechin hydrate standard. Finally, the flavonoid content (TFC) was expressed as mg catechin equivalents (CE) 100 g−1 walnut kernel.
For the determination of tannins, aqueous extracts prepared from 1 g of crushed walnut kernels and homogenized in 10 mL of distilled water were used, following the same work procedure as for the ethanolic extract. The methodology proposed by Giura et al. (2019) [12] and the same detailed working protocol for the determination of polyphenols were followed but with 0.5 mL walnut kernel aqueous extract. The samples were kept in the dark at room temperature for 60 min. After this period, the absorbance of the samples was measured in relation to the control sample, with the absorption maximum located at 750 nm. The concentration of tannins was estimated, using the calibration curve built with standard solutions of gallic acid solubilized in distilled water. Finally, the content of tannins (TTC) was expressed as mg gallic acid equivalents (GAE) 100 g−1 walnut kernel.
For the quantitative determination of carotenoids, the supernatant obtained by magnetic stirring for 30 min at 1500 rpm from 4 g of crushed walnut kernels and homogenized in 25 mL volumetric mixture of hexane: ethanol: acetone in a 2:1:1 ratio was used, followed by magnetic stirring for another 10 min at 1500 rpm of the mixture after adding 10 mL of distilled water. After the separation of the phases, carried out after 10–15 min of rest, the volume of the supernatant was measured. The absorbance spectrum was recorded in the 350–550 nm range for the supernatant, using hexane as a control. Knowing the molar absorption coefficients for the two carotenoids as 503 nm and 470 nm, presented in our previous works [12,51], the lycopene and β-carotene content was calculated, taking into account the amount of plant material taken in the analysis and the volume of the supernatant obtained after the separation of the phases.
All analyses were performed in three replicates. Practically, the extracts necessary for the determination of TPC, TTC, TFC, lycopene, and β-carotene were analyzed in triplicate at each moment of analysis.

2.4. Statistical Analysis

Statistical analysis was performed with the IBM SPSS Statistics 29.0 software package. Data were expressed as means ± standard deviation (SD). Comparisons between means between groups were performed with Duncan’s Multiple Range Test. Differences were considered significant when p < 0.05. Microsoft Excel 2021 and Daniel’s XL Toolbox version 7.3.4. were used for graphical representation. Three-Way Analysis of Variance (ANOVA) and Interaction Plot Graphs were performed with the help of Minitab 21.4 statistical software.

3. Results and Discussions

3.1. Determination of Some Biochemical Parameters

Table 1 shows the levels of TPC, TFC, TTC, lycopene, and β-carotene, determined in the walnut cultivars ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ depending on the storage environment temperature, at four analysis moments: at the start of the experiment (mid-December 2021, the nuts kept in the shell 2–3 months after harvesting); in mid-March 2022, after 3 months of storage as a walnut kernel in a plastic bag (short-term storage); in mid-May 2022, after 5 months of storage as a walnut kernel in a plastic bag (medium-term storage); and in mid-June, at the end of the 6 months of storage as a walnut kernel in a plastic bag (long-term storage). Also, Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5 graphically represent the increases and decreases, respectively, in the contents of polyphenols, flavonoids, tannin, lycopene, and β-carotene recorded for the walnut kernel of the studied cultivars during storage, depending on the storage temperature.
As an overview, a comparison between the eight walnut cultivars indicated that the group consisting of ‘Valcor’ (4454.66 mg GAE 100 g−1), ‘Vina’ (4342.16 mg GAE 100 g−1), and ‘Franquette’ (3915.31 mg GAE 100 g−1) presented the highest TPC, but also the highest TFC (1131.93 mg CE 100 g−1, ‘Vina’; 981.76 mg CE 100 g−1, ‘Valcor’; and 973.46 mg EC 100 g−1 ‘Franquette’). In addition, next to ‘Valcor’, with 2100.53 mg GAE 100 g−1, ‘Ferjean’ stood out with the highest level of tannin, 2278.12 mg GAE 100 g−1. Last but not least, the tannin fraction dominated the phenolic compound profile compared to flavonoids (the tannin/flavonoid ratio ranged from 1.25, in the ‘Vina’ cv., to 2.46, in the ‘Ferjean’ cv.).
The results obtained in this study regarding TPC are comparable to those reported in the walnut kernel by Bakkalbaşı et al. (2012) [29] for seven walnut varieties grown in Turkey, 1062.37–3180.88 mg GAE 100 g−1 (2004) and 931.35–3029.60 mg GAE 100 g−1 (2005), as well as to those from the more recent study of Jan et al. (2022) [52] in walnut kernel samples from various eco-geographical regions of Jammu and Kashmir, India, 1980–5040 mg GAE 100 g−1. Also, in fifteen walnut selections from Inner Anatolia, Turkey, higher TPC (1107–1876 mg GAE 100 g−1) was found [53] than those previously discussed. In addition, in a study by Trandafir et al. (2016) [54] for twelve walnut genotypes, six with a red pellicle and six with a light-yellow pellicle, from Romania, TPC content ranged from 1131 to 2892 mg GAE 100 g−1 in kernels (without pellicles) and from 11,525 to 33,833 mg GAE 100 g−1 in pellicles.
TFC content of our samples was close to that reported by Yang et al. (2009) [55], Trandafir et al., 2016 [54], and Jan et al., 2022 [52]. For other varieties of Romanian walnut, Trandafir et al. (2016) [54] reported a TFC of 81.03–164.78 mg quercetin equivalent (QE) 100 g−1 in kernels (without pellicles) and 18.30–68.89 mg QE 100 g−1 in pellicles. Jan et al. (2022) [52] presented TFC values ranging from 188.5 to 815 mg QE 100 g−1 in the walnut kernel from India.
Of the two determined carotenoids, lycopene predominated, with levels up to 8.1-times higher than β-carotene, except for the ‘Jupanesti’ cv. in which β-carotene showed a very low concentration. The cultivars with lycopene levels exceeding 3 mg 100 g−1 were ‘Ferjean’ and ‘Fernor’ (with 3.41 and 3.10 mg 100 g−1, respectively), and the highest β-carotene contents were dosed for the cultivars ‘Ferjan’ (0.58 mg 100 g−1) and ‘Franquette’ (0.60 mg 100 g−1). There are only a few works with results regarding the total content of carotenoids, lycopene, or β-carotene in walnut kernels [56,57]. Some authors declare that they did not detect the two carotenoids. In our study, it was possible to determine the content of lycopene and β-carotene in the walnut kernel by increasing the amount of walnut kernel (4 g) compared to the amount generally used (1–2 g) for vegetable materials rich in carotenoids, which could allow obtaining some absorbance values in the detection limits of the PerkinElmer Lambda25 UV-Vis-spectrophotometer.
In a study by Özrenk et al. (2012) [56], the total carotenoid level for 14 walnut genotypes grown in Erzincan, Eastern Turkey, was found to be between 0.08 and 0.49 mg 100 g−1.
Analyzes carried out on walnut kernels obtained from four foreign cultivars (‘Franquette’, ‘Parisienne’, ‘Hartley’, and ‘Lozeronne’) and two landrace varieties from Tunisia [57] showed that the levels of β-carotene were in general lower than those found in our research and ranged between 0.022 mg 100 g−1 and 0.062 mg 100 g−1.
Figure 1, Figure 2 and Figure 3 present the oscillations of the phenolic compounds and carotenoids analyzed in the study for the walnut kernel kept for three, five, and six months, respectively, at very low, low, and room temperatures. As can be observed, regardless of the duration of storage, on an average in the eight walnut cultivars, in the case of very low-temperature treatment, the highest levels of phenolic compounds (TPC, TFC, and TTC) were recorded compared to low-temperature treatment and especially compared to room temperature treatment. However, in the case of flavonoids and tannins, the efficiency of the storage methods at very low temperatures and low temperatures varied depending on the cultivar.
Regarding the content of carotenoids (Figure 4 and Figure 5), on an average in the eight cultivars, storage at room temperature was the method that ensured minimal losses of lycopene and β-carotene and was followed by low-temperature treatment in the case of lycopene and by very low-temperature treatment in the case of β-carotene. Nevertheless, the efficiency of low and very low-temperature treatments, had different effects, depending on the cultivar.

3.2. Analysis of TPC Variation Depending on Temperature Treatment and the Storage Period

3.2.1. Short-Term Storage

As Figure 1 indicates, in the case of short-term storage (three months), variation of the phenolic compound concentrations was observed between 10.9% (‘Vina’) and 105.3% (‘Unival’) for walnut kernels stored in the freezer, at 7.2% (‘Vina’)–73.4% (‘Unival’) for refrigerator storage, and from 3.6% (‘Ferjean’) to 56.7% (‘Unival’), for samples kept at room temperature, respectively. Under these conditions, except for ‘Vina’ cv., the highest contents of phenolic compounds were recorded for storage in the freezer, followed, with a few exceptions, by storage at low temperature (3–4 °C). The cultivar with the highest amount of phenolic compounds accumulated at this stage of the experiment was ‘Unival’.
Horticulturae 09 00544 g001a 550Horticulturae 09 00544 g001b 550
Figure 1. Variation of walnut kernel TPC (expressed as % of increase or decrease, relative to TPC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letters in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Figure 1. Variation of walnut kernel TPC (expressed as % of increase or decrease, relative to TPC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letters in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Horticulturae 09 00544 g001aHorticulturae 09 00544 g001b

3.2.2. Medium-Term Storage

Analyzes carried out on walnut kernels stored until May (five months, medium-term storage) in the case of frozen storage indicated a TPC accumulation between 1.3% (‘Vina’) and 82.1% (‘Unival’) compared to the moment of initiation of the experiment, while for the samples kept at room temperature and in the refrigerator, reductions of 21.6% (‘Unival’), up to 53.3% (‘Franquette’), and from 1.6% (‘Valcor’) to 28.4% (‘Vina’), respectively, were recorded. Therefore, in the medium term, without exception, the highest TPC levels were determined for the frozen samples. And in this case, the cultivar ‘Unival’ accumulated the highest level of phenolic compounds (at very low temperature, followed by low-temperature treatment), and at the opposite pole, with the greatest losses were ‘Franquette’ and also ‘Vina’ and ‘Ferjean’. No uniform trend of cultivars to accumulate or reduce TPC was observed under low-temperature storage conditions.

3.2.3. Long-Term Storage

The data recorded for the long-term stored samples (six months) indicated decreases in the level of phenolic compounds (compared to the December determinations) from 31.6% (‘Jupanesti’) to 74.8% (‘Vina’) in the case of frozen storage, 46.9% (‘Franquette’)–77.6% (‘Vina’) for refrigerator storage, and reached 63.6% (‘Valcor’)–82.5% (‘Sarmis’) when stored at room temperature. Although the extension of the storage period (from May to June) led to high losses of TPC, freezing was the most efficient storage method in the long term. Storage at room temperature accentuated the tendency to reduce TPC in the kernel of the analyzed walnut cultivars.

3.3. Analysis of TFC Variation Depending on Temperature Treatment and the Storage Period

The general tendency of flavonoids was to concentrate during short-term storage, regardless of the method approached (Figure 2). There were increases between 7.9% (‘Vina’) and 55.5% (‘Unival’) (on very low-temperature treatment), between 15.6% (‘Vina’) and 63.2% (‘Fernor’) (on low-temperature treatment), and from 1.9% (‘Vina’) to 51.5% (‘Valcor’) (at room temperature), respectively. In the medium term, TFC increase was observed only in the case of very low-temperature treatment (by 6.5%, at ‘Vina’ up to 63.1%, at ‘Fernor’) and in low-temperature treatment (by 2.7%, at ‘Jupanesti’, up to 42.4%, in ‘Unival’), while in the long-term storage, TFC increases only in the case of freezer storage in some cultivars (‘Jupanesti’, ‘Unival’ and ‘Sarmis’) by 2.2–4.5%.
Horticulturae 09 00544 g002 550
Figure 2. Variation of walnut kernel TFC (expressed as % of increase or decrease, relative to TFC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter, and letters in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Figure 2. Variation of walnut kernel TFC (expressed as % of increase or decrease, relative to TFC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter, and letters in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
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3.3.1. Short-Term Storage

For the short-term stored samples, the efficiency of the storage methods (temperatures) varied depending on the cultivar (p ˂ 0.001). The low-temperature storage method produced the best results in the ‘Fernor’ cv. For ‘Unival’, the highest concentrations of TFC were produced primarily by frozen storage, followed by refrigeration, and for ‘Valcor’, inexplicably, room temperature treatment was more effective compared to refrigerator and freezer.

3.3.2. Medium-Term Storage

TFC determinations in walnut kernel stored until May indicated, only in the ‘Vina’ cv., refrigerated storage as the most effective, with a TFC increase of 12.9%. In all other cases, storage at very low-temperature resulted in the retention of the highest concentrations of flavonoids and was seconded by low-temperature storage. The lowest levels of flavonoids were determined in this phase in the case of storage at room temperature, as being due to the reduction of TFC by 12.8% (‘Vina’) to about 62% (‘Fernor’). In the ‘Fernor’ cv., the most intense effects of the storage environment temperature on TFC were observed, ranging from the maximum increase at very low-temperature treatment to the maximum reduction at room temperature. As for short-term storage, the cultivar least influenced by the storage method was ‘Vina’.

3.3.3. Long-Term Storage

The flavonoid content of the samples stored until June (long-term storage, six months) was generally lower compared to the values reported at the start of the experiment (December). The exception was the samples kept at a very low-temperature (freezer) of the cultivars ‘Jupanesti’, ‘Unival’, and ‘Sarmis’, with slight increases in TFC compared to the initial moment. The least effective method of long-term storage was also in this case the room temperature treatment, with reductions in the flavonoid levels from 58.7% (‘Vina’) to 80.1% (‘Fernor’).

3.4. Analysis of TTC Variation Depending on Temperature Treatment and the Storage Period

Tannins accumulated in walnut kernels are stored for a short period, regardless of environmental temperature (Figure 3). When extending the duration of the storage period until May (medium-term storage, five months), TTC level variations depended on both the cultivar and the storage temperature. Finally, in June, except for ‘Franquette’ cv. (long-term storage, six months), all the other cultivars recorded lower TTC levels compared to their initial content.
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Figure 3. Variation of walnut kernel TTC (expressed as % of increase or decrease, relative to TTC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter, and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Figure 3. Variation of walnut kernel TTC (expressed as % of increase or decrease, relative to TTC levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter, and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
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3.4.1. Short-Term Storage

In general, the tannin concentrations of the short-term stored samples (three months) showed the greatest increases under the very low-temperature treatment. The exception was represented by the ‘Franquette’ cv., with an increase of 57.2% TTC in the refrigerator and 54.1% in the freezer. The cultivars in which short-term storage in the freezer increased the most tannin level compared to the initial moment of the study (December) were ‘Unival’ (with an increase of 182.3%) and ‘Vina’ (with 152.5%). Although room temperature treatment generally had the lowest increases in TTC, in the case of ‘Vina’ cv., it was the second most efficient storage method. At the opposite pole, with the lowest variation depending on storage temperature treatment, was ‘Jupanesti’ cv.

3.4.2. Medium-Term Storage

For medium-term storage (until May, five months), very low-temperature storage led to the highest TTC increases, from 17.6% (‘Sarmis’) to 149.7% (‘Vina’). Results close to frozen storage (102.6% increase in TTC), although higher, were obtained for refrigerated storage in the case of ‘Franquette’ cv. (117.0% increase in TTC). Last but not least, in the case of the ‘Sarmis’ cv., the TTC had the smallest variation depending on the storage method, and walnut storage at room temperature resulted in TTC reduction in ‘Jupanesti’, ‘Franquette’, and ‘Ferjean’ cvs. by 25.4% to 50.0%.

3.4.3. Long-Term Storage

In the case of long-term stored samples (six months), only for ‘Franquette’ cv., a TTC accumulation of 1.9% in the low-temperature treatment and 29.3% at the very low-temperature treatment was observed. In all other cases, regardless of the cultivar and the method of preservation of the walnut kernel, the tannin content decreased. In these circumstances, the method that favored keeping the TTC as high as possible was the very low-temperature treatment (with TTC reductions from 4.2% in ‘Unival’ to 60.3% in ‘Ferjean’). Completely different was the room temperature treatment, with TTC reductions of 46.1% in ‘Franquette’ up to 81.8% in ‘Jupanesti’.

3.5. Analysis of the Variation of Lycopene and β-Carotene Content Depending on Temperature Treatment and the Storage Period

The analysis of the variation in the walnut kernel carotenoid content (Figure 4 and Figure 5) indicated an intense reduction, directly proportional to the increase in storage time in all studied cvs. In addition, the cultivar significantly influenced the dynamics of the carotenoid level in the case of low- and very low-temperature storage. The storage at room temperature recorded the lowest losses of lycopene and β-carotene.
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Figure 4. Variation of walnut kernel lycopene content (expressed as % of increase or decrease, relative to lycopene levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Figure 4. Variation of walnut kernel lycopene content (expressed as % of increase or decrease, relative to lycopene levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
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Figure 5. Variation of walnut kernel β-carotene content (expressed as % of increase or decrease, relative to β-carotene levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
Figure 5. Variation of walnut kernel β-carotene content (expressed as % of increase or decrease, relative to β-carotene levels determined in December) depending on storage duration and temperature for ‘Vina’, ‘Jupanesti’, ‘Unival’, ‘Sarmis’, ‘Franquette’, ‘Valcor’, ‘Ferjean’, and ‘Fernor’ cultivars. Values followed by the same lower, italic, upper case letter and letter in a box are not significantly different (p < 0.05) by Duncan’s Multiple Range Test.
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3.5.1. Short-Term Storage

Exposure of walnut kernels to room temperature until March (three months) proved to be the most effective storage method, as it resulted in the lowest decrease in lycopene levels in the ‘Franquette’ cv. (by 4.1%), followed by ‘Valcor’ cv. (with 3.4%) and ‘Vina’ cv. (with 6.7%). In five of the eight analyzed cases, the low-temperature treatment favored the retention of a higher level of lycopene compared to the very low-temperature treatment. The most drastic reductions in the level of lycopene in this phase were recorded in the ‘Fernor’ cv. in the case of low-temperature treatment (by 72.4%) and in the ‘Ferjean’ cv. when stored at very low-temperature (by 71.6%).
Regarding the dynamics of the β-carotene level, in the samples stored for 3 months (March, short-term storage), the lowest losses were determined at room temperature in ‘Unival’ (11.2%), ‘Jupânești’ (11.4%), and ‘Franquette’ cvs. (11.5%). In five cases out of eight, very low-temperature treatment reduced both carotenoid levels the most, by 41.3% (‘Vina’)–71.2% (‘Unival’). However, the highest loss of β-carotene content in the short-term stored walnut kernels was recorded for ‘Ferjean’ cv. for very low-temperature treated samples (by approximately 85%).

3.5.2. Medium-Term Storage

Storage for an average period of five months (until May) accentuated the loss of lycopene. The smallest reduction in lycopene concentration compared to the initial moment (16.9%) was observed in ‘Sarmis’ cv. kept at room temperature, while the highest lycopene loss under the same room-temperature treatment reached the maximum in the ‘Jupanesti’ cv. (81.8%). Except for ‘Franquette’ cv. (with a maximum reduction of lycopene at low-temperature), in all others, the very low-temperature treatment led to lycopene losses in this phase, which varied from 71.6% (‘ Sarmis’) up to 92.5% (‘Vina’).
In May (medium-term storage), the samples kept at room temperature showed the lowest losses of β-carotene (with 81.7%, in ‘Sarmis’, up to 85.4%, in ‘Ferjean’). The effect of low- and very low-temperature treatment on β-carotene level dynamics depended on the walnut cultivar. High losses of β-carotene were observed at this moment in samples of ‘Ferjean’ and ‘Fernor’ cvs. stored at low-temperature (by 88.1% and 86.6%, respectively), as well as for ‘Jupanesti’ and ‘ Valcor’ (of 87.6% and 87.1%, respectively) under very low-temperature treatment.

3.5.3. Long-Term Storage

When the storage period was extended until June (6 months, long-term storage), a very intense reduction in the level of lycopene was observed, especially in ‘Vina’, ‘Jupanesti’, ‘Sarmis’, and ‘Unival’ cvs., under both very low-temperature and low-temperature treatments. The smallest reductions in the lycopene level were recorded under room temperature storage conditions and ranged between 58.6% (‘Unival’) and 92.8% (‘Jupanesti’).
The determinations of the level of β-carotene in walnut kernels stored for 6 months (June, long-term storage) revealed the efficiency of room temperature with lower losses of 69.5% (‘Sarmis’) to 91.6% (‘Ferjean’), compared to very low- and low-temperature treatment. An exception was the ‘Fernor’ cv., for which the best long-term preservation method was refrigeration.
Very low- and low-temperature-treatment effects on β-carotene varied depending on the cultivar. The strongest reductions of β-carotene were recorded for ‘Ferjean’ when stored frozen (by 99%) and in ‘Fernor’ when stored refrigerated (by 99.5%).

3.6. Three Factor Analysis of Walnut Kernel Data

Three-way ANOVA has been performed to check the interactions between the three factors (cultivar, storage type, and storage time). Significant three-way interaction effects have been obtained (p < 0.05) in the case of TPC, TFC, TTC, lycopene, and β-carotene content. Tables with Analysis of Variance (Tables S1–S5) and Interaction Plot Graphs (Figures S1–S5) are attached as Supplementary Materials.
As the interaction plot graphs present, the lines are not parallel and this type of interaction effect indicates that there is a relationship between cultivar, storage type, and storage time regarding TPC, TFC, TTC, lycopene, and β-carotene contents.

4. Conclusions

The analysis of the dynamics of the phenolic compounds in the walnut kernel for the eight cultivars depending on the storage temperature and duration showed that, for the total content of phenolic compounds, freezing (very low-temperature treatment) was the most favorable short-term storage method (except for ‘Vina’ cv.), but also in the medium and long term. Although initially an increase of TPC was observed, later (due to the reduction of biochemical processes as a result of lipid oxidation during storage) their level decreased. Also, at the end of the storage period, the cultivars with the lowest TPC reduction were ‘Franquette’ and ‘Valcor’ (both registered in very low-temperature treatment).
Although in contrast to TPC, an increase in flavonoid levels (compared to their December registered levels) was observed in short- and medium-term and, under certain conditions, long-term storage; the recommended storage method of walnut kernels for flavonoid preservation was similarly very low-temperature treatment.
The most favorable method for keeping a high level of tannin was very low-temperature treatment, except for ‘Franquette’ cv., where the low-temperature treatment was the most suitable. The tendency followed by tannin during storage was a short-term increase. The same tendency towards increase was registered on medium-term storage, except for ‘Ferjean’, ‘Franquette’, and ‘Jupanesti’ cvs. (whose tannin levels were still favored by very low temperatures). Extending the storage period to 6 months led to a reduction of TTC regardless of the storage method and cultivar, except for the ‘Franquette’ cv. (with higher increases in TTC under very low-temperature compared to low-temperature treatment).
Without exception, storage negatively impacted the level of carotenoids, which is accentuated by the extension of the storage time. The most effective storage method was in this case room-temperature and only one exception was observed for ‘Fernor’ cv. (for β-carotene, in long-term storage, very low-temperature kept samples).
Therefore, walnuts are not sensitive to chilling and they may be stored at or below freezing. Moreover, walnuts’ low water content when properly stored makes them relatively inert metabolically (respiration rates are very low). These observations are similar to the results presented by Beaudry et al. (1985) [58], which measured the respiratory rates of kernels of 19 pecan genotypes at harvest and after drying them to a 3% moisture level. They found that the respiration was genotype-dependent and the respiratory rates of kernels varied logarithmically with moisture content. At harvest, the respiratory rates of kernels ranged from 26.9 to 0.3 mg CO2 kg−1 h−1 but after drying to 3% moisture, values declined, ranging from 0.21 to 0.06 mg CO2 kg−1 h−1.
Depending on the walnut cultivar, the preservation of the walnut kernel can be extended to 5 months by storage at −18 °C without loss of phenolic compounds, and a period shorter than 5 months (but certainly longer than 3 months) could be recommended for storage at 3–4 °C. Keeping it at room temperature can be an option for a short period of about 3 months. None of the preservation methods are adequate if the losses recorded for carotenoids are taken into account.
The two classes of compounds determined in this study (phenolic compounds and carotenoids) are favored by storage under diametrically opposite conditions. Based on the biological activity of the phenolic compounds, present in appreciable quantity, and the low level of carotenoids, the only compromise solution that could be considered is keeping the walnut kernel at a very low temperature (to the detriment of carotenoids).
Therefore, the biological activity assessment of the walnut kernel depending on the storage temperature and duration is also necessary to provide a more complete understanding of the storage condition effect on walnut quality. Moreover, taking into account the high-value lipid content of walnuts, which is not only nutritious but that also have implications in the physiological processes of the human body, there is a need to continue studies on storage conditions, with an emphasis on the dynamics of this class of compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9050544/s1, Figure S1: Interaction Plot for TPC (mg GAE/100 g); Figure S2: Interaction Plot for TFC (mg EC/100 g); Figure S3: Interaction Plot for TTC (mg GAE/100 g); Figure S4: Interaction Plot for lycopene (mg/100 g); Figure S5: Interaction Plot for β-carotene (mg/100 g); Table S1: Cultivar, storage type, storage time, and their interactions` effect on walnut kernel TPC (Three-Way ANOVA); Table S2: Cultivar, storage type, storage time, and their interactions` effect on walnut kernel TFC (Three-Way ANOVA); Table S3: Cultivar, storage type, storage time, and their interactions’ effect on walnut kernel TTC (Three-Way ANOVA); Table S4: Cultivar, storage type, storage time, and their interactions` effect on walnut kernel lycopene content (Three-Way ANOVA); Table S5: Cultivar, storage type, storage time, and their interactions` effect on walnut kernel β-carotene content (Three-Way ANOVA).

Author Contributions

Conceptualization, L.E.V., S.G. and M.B.; methodology, L.E.V., S.G., I.C.M. and M.B.; software, L.E.V., I.C.M. and M.B.; validation, L.E.V. and M.B.; investigation, L.E.V. and S.G.; resources, L.E.V., S.G. and M.B.; writing—original draft preparation, L.E.V., S.G., I.C.M. and M.B.; writing—review and editing, L.E.V., I.C.M. and M.B.; supervision, L.E.V. and M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the principal author. The data are not publicly available due to privacy.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Variations in the cultivars’ content of phenolic compounds (TPC), flavonoids (TFC), tannin (TTC), lycopene, and β-carotene depending on temperature and storage time (TPC = total phenolic content, TFC = total flavonoid content, TTC = total tannin content; 20–22 °C, room temperature treatment; 3–4 °C, low-temperature treatment; −20 to −18 °C, very low-temperature treatment). Data are presented as mean ± SD (standard deviation).
Table 1. Variations in the cultivars’ content of phenolic compounds (TPC), flavonoids (TFC), tannin (TTC), lycopene, and β-carotene depending on temperature and storage time (TPC = total phenolic content, TFC = total flavonoid content, TTC = total tannin content; 20–22 °C, room temperature treatment; 3–4 °C, low-temperature treatment; −20 to −18 °C, very low-temperature treatment). Data are presented as mean ± SD (standard deviation).
Biochemical ParametersStorage DurationStorage TemperatureCultivar
‘Vina’‘Jupanesti’‘Unival’‘Sarmis’‘Franquette’‘Valcor’‘Ferjean’‘Fernor’
TPCBefore the storage experiment (mid-December) 4342.16 ± 0.522294.12 ± 0.542339.27 ± 0.902793.96 ± 0.573915.31 ± 0.734454.66 ± 0.783640.32 ± 0.872798.16 ± 0.19
After
three months		20–22 °C5725.86 ± 0.612698.91 ± 0.843665.91 ± 4.533392.25 ± 0.564621.87 ± 4.074815.86 ± 0.603771.59 ± 0.662992.58 ± 0.68
3–4 °C4653.41 ± 0.832713.39 ± 0.934056.37 ± 0.543540.83 ± 0.234357.99 ± 0.615593.81 ± 0.804715.42 ± 0.763985.98 ± 0.69
−20 to −18 °C4815.43 ± 0.492873.18 ± 0.534801.48 ± 0.643573.25 ± 0.645190.36 ± 0.595729.20 ± 0.195543.58 ± 0.504535.65 ± 6.33
After
five months		20–22 °C2095.74 ± 1.521647.16 ± 0.791834.20 ± 0.981789.78 ± 1.501828.97 ± 1.902551.76 ± 1.591841.40 ± 1.631585.68 ± 1.21
3–4 °C3110.05 ± 1.001787.42 ± 0.33496.21 ± 0.592484.07 ± 0.604759.50 ± 4.804383.84 ± 0.964033.09 ± 0.433118.39 ± 0.33
−20 to −18 °C4399.04 ± 0.882884.38 ± 0.464259.77 ± 0.422865.06 ± 0.185335.45 ± 0.874692.62 ± 0.505277.94 ± 0.123623.63 ± 0.47
After
six months		20–22 °C781.35 ± 0.05484.93 ± 0.06530.26 ± 0.08489.54 ± 0.041142.38 ± 0.451621.81 ± 1.96797.87 ± 0.04771.22 ± 0.14
3–4 °C970.86 ± 0.20975.29 ± 0.161095.76 ± 0.19685.64 ± 1.122078.17 ± 0.352009.56 ± 0.771282.29 ± 0.691333.54 ± 0.61
−20 to −18 °C1092.77 ± 0.241569.20 ± 6.211202.90 ± 0.41988.95 ± 0.642388.58 ± 0.572515.19 ± 1.821728.35 ± 0.691655.69 ± 0.78
TFCMid-December 1131.93 ± 6.45740.36 ± 3.83828.50 ± 4.64821.04 ± 4.17973.46 ± 4.18981.76 ± 6.20924.91 ± 5.46700.67 ± 3.83
After three months20–22 °C1153.92 ± 8.54859.61 ± 1.881046.19 ± 4.481014.61 ± 2.621352.87 ± 28.761487.33 ± 22.581172.88 ± 1.89875.00 ± 2.62
3–4 °C1309.00 ± 14.64998.89 ± 6.131263.45 ± 2.821082.47 ± 6.641243.74 ± 12.331418.62 ± 65.781270.25 ± 8.281143.27 ± 5.64
−20 to −18 °C1221.21 ± 6.831061.69 ± 5.61287.95 ± 14.15999.69 ± 4.501326.47 ± 11.451372.22 ± 11.371237.91 ± 9.021037.91 ± 9.02
After
five months		20–22 °C986.70 ± 11.81342.16 ± 9.72381.88 ± 3.51364.66 ± 1.37582.91 ± 4.67636.58 ± 3.31367.43 ± 3.77267.19 ± 3.72
3–4 °C1278.06 ± 12.89760.42 ± 5.011180.06 ± 5.52906.42 ± 5.151093.79 ± 7.301205.93 ± 48.081070.32 ± 5.28937.30 ± 4.62
−20 to −18 °C1206.03 ± 6.82858.29 ± 8.931245.61 ± 31.19962.79 ± 3.661278.62 ± 6.291231.56 ± 7.701205.02 ± 10.921142.99 ± 2.21
After
six months		20–22 °C467.14 ± 3.53188.54 ± 3.49267.87 ± 1.46183.89 ± 3.00285.55 ± 1.61283.40 ± 2.13268.43 ± 1.54139.64 ± 1.15
3–4 °C633.52 ± 0.32640.41 ± 5.19803.02 ± 1.50733.99 ± 0.63816.29 ± 1.09754.23 ± 1.88749.10 ± 0.73608.27 ± 0.44
−20 to −18 °C618.70 ± 1.39756.85 ± 3.08856.85 ± 3.08857.71 ± 11.92925.23 ± 7.50768.49 ± 9.89830.40 ± 12.97704.02 ± 15.08
TTCMid-December 1420.42 ± 1.461815.07 ± 1.61996.89 ± 0.421723.66 ± 2.161685.12 ± 1.592100.53 ± 1.122278.12 ± 0.431395.67 ± 0.34
After
three months		20–22 °C3367.82 ± 2.831904.91 ± 1.281307.13 ± 0.251943.79 ± 0.671943.79 ± 0.672251.15 ± 3.632511.56 ± 2.001894.37 ± 1.49
3–4 °C2257.23 ± 3.651912.58 ± 4.232207.02 ± 0.042017.97 ± 0.872649.48 ± 0.153076.83 ± 0.543180.70 ± 1.342510.22 ± 1.73
−20 to −18 °C3586.81 ± 4.792154.39 ± 2.782813.77 ± 2.172374.94 ± 2.192596.77 ± 2.283299.57 ± 3.443486.87 ± 12.323011.90 ± 3.04
After
five months		20–22 °C1824.54 ± 2.811354.25 ± 2.531261.57 ± 2.731684.97 ± 3.131158.22 ± 2.532188.79 ± 4.651138.75 ± 0.331380.89 ± 0.32
3–4 °C1752.91 ± 0.301786.81 ± 0.191629.85 ± 0.571642.33 ± 2.593656.50 ± 3.932420.72 ± 1.992659.09 ± 5.012196.55 ± 0.15
−20 to −18 °C3546.46 ± 2.872439.05 ± 2.591968.66 ± 1.192026.81 ± 2.233413.98 ± 3.012971.60 ± 1.233051.34 ± 5.402339.48 ± 1.49
After
six months		20–22 °C556.65 ± 2.54330.85 ± 0.33426.75 ± 0.73365.61 ± 0.30908.17 ± 2.54968.27 ± 0.70638.46 ± 2.88616.95 ± 2.27
3–4 °C823.82 ± 3.02592.19 ± 0.29933.98 ± 0.59548.47 ± 1.681716.86 ± 2.301212.31 ± 1.85762.34 ± 0.25964.49 ± 0.32
−20 to −18 °C990.55 ± 2.551266.3 ± 3.94954.81 ± 0.97845.84 ± 0.512178.27 ± 2.761791.83 ± 0.27904.06 ± 1.311118.81 ± 0.35
LycopeneMid-December 2.56 ± 0.001.81 ± 0.001.33 ± 0.002.56 ± 0.002.49 ± 0.002.84 ± 0.003.41 ± 0.013.10 ± 0.01
After
three months		20–22 °C2.39 ± 0.000.64 ± 0.001.22 ± 0.002.17 ± 0.002.45 ± 0.002.74 ± 0.001.08 ± 0.001.64 ± 0.00
3–4 °C2.01 ± 0.000.58 ± 0.000.83 ± 0.001.98 ± 0.001.41 ± 0.001.46 ± 0.001.05 ± 0.000.86 ± 0.00
−20 to −18 °C1.96 ± 0.000.61 ± 0.000.74 ± 0.011.34 ± 0.001.47 ± 0.001.43 ± 0.000.97 ± 0.001.10 ± 0.00
After
five months		20–22 °C0.69 ± 0.000.33 ± 0.000.88 ± 0.012.13 ± 0.001.46 ± 0.000.56 ± 0.000.92 ± 0.001.28 ± 0.00
3–4 °C0.39 ± 0.000.23 ± 0.000.29 ± 0.000.92 ± 0.000.78 ± 0.000.28 ± 0.001.02 ± 0.000.67 ± 0.00
−20 to −18 °C0.19 ± 0.000.23 ± 0.010.27 ± 0.000.73 ± 0.000.90 ± 0.000.24 ± 0.000.80 ± 0.000.60 ± 0.00
After
six months		20–22 °C0.41 ± 0.000.13 ± 0.000.55 ± 0.000.59 ± 0.000.72 ± 0.000.30 ± 0.000.49 ± 0.000.63 ± 0.00
3–4 °C0.04 ± 0.000.02 ± 0.000.09 ± 0.000.05 ± 0.000.44 ± 0.000.21 ± 0.000.35 ± 0.000.29 ± 0.00
−20 to −18 °C0.03 ± 0.000.02 ± 0.010.05 ± 0.000.03 ± 0.000.47 ± 0.000.18 ± 0.000.35 ± 0.000.24 ± 0.00
β-caroteneMid-December 0.43 ± 0.010.07 ± 0.000.27 ± 0.000.35 ± 0.010.60 ± 0.010.44 ± 0.020.58 ± 0.010.38 ± 0.01
After
three months		20–22 °C0.37 ± 0.010.06 ± 0.000.24 ± 0.010.26 ± 0.000.53 ± 0.030.34 ± 0.010.42 ± 0.010.30 ± 0.02
3–4 °C0.29 ± 0.000.05 ± 0.000.11 ± 0.000.19 ± 0.000.19 ± 0.000.14 ± 0.000.09 ± 0.000.13 ± 0.00
−20 to −18 °C0.25 ± 0.000.02 ± 0.000.08 ± 0.000.18 ± 0.000.19 ± 0.000.12 ± 0.010.37 ± 0.000.22 ± 0.00
After
five months		20–22 °C0.12 ± 0.000.02 ± 0.000.13 ± 0.000.17 ± 0.000.22 ± 0.000.09 ± 0.000.08 ± 0.000.15 ± 0.01
3–4 °C0.07 ± 0.000.02 ± 0.000.06 ± 0.000.06 ± 0.000.11 ± 0.010.07 ± 0.000.07 ± 0.000.05 ± 0.00
−20 to −18 °C0.07 ± 0.000.01 ± 0.000.06 ± 0.000.08 ± 0.000.10 ± 0.000.06 ± 0.000.14 ± 0.000.12 ± 0.00
After
six months		20–22 °C0.08 ± 0.000.01 ± 0.000.05 ± 0.000.11 ± 0.000.10 ± 0.000.07 ± 0.000.05 ± 0.000.02 ± 0.00
3–4 °C0.04 ± 0.010.00 ± 0.000.01 ± 0.000.03 ± 0.000.03 ± 0.010.03 ± 0.000.04 ± 0.000.00 ± 0.00
−20 to −18 °C0.04 ± 0.000.00 ± 0.000.02 ± 0.000.02 ± 0.010.03 ± 0.000.03 ± 0.000.01 ± 0.010.07 ± 0.01
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Vijan, L.E.; Giura, S.; Mazilu, I.C.; Botu, M. Effect of Temperature and Storage Time on Some Biochemical Compounds from the Kernel of Some Walnut Cultivars Grown in Romania. Horticulturae 2023, 9, 544. https://doi.org/10.3390/horticulturae9050544

AMA Style

Vijan LE, Giura S, Mazilu IC, Botu M. Effect of Temperature and Storage Time on Some Biochemical Compounds from the Kernel of Some Walnut Cultivars Grown in Romania. Horticulturae. 2023; 9(5):544. https://doi.org/10.3390/horticulturae9050544

Chicago/Turabian Style

Vijan, Loredana Elena, Simona Giura, Ivona Cristina Mazilu, and Mihai Botu. 2023. "Effect of Temperature and Storage Time on Some Biochemical Compounds from the Kernel of Some Walnut Cultivars Grown in Romania" Horticulturae 9, no. 5: 544. https://doi.org/10.3390/horticulturae9050544

APA Style

Vijan, L. E., Giura, S., Mazilu, I. C., & Botu, M. (2023). Effect of Temperature and Storage Time on Some Biochemical Compounds from the Kernel of Some Walnut Cultivars Grown in Romania. Horticulturae, 9(5), 544. https://doi.org/10.3390/horticulturae9050544

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