Role of Postharvest Oxalic Acid Treatment on Quality Properties, Phenolic Compounds, and Organic Acid Contents of Nectarine Fruits during Cold Storage

Abstract

Due to the soft texture of the nectarine fruit, there are difficulties in long-term storage of this fruit. Therefore, it is of great importance to extend the postharvest storage period of this fruit species. In this study, the effect of postharvest OA (Oxalic acid) applications (0, 0.5, 1 and 1.5 mM) on the quality and biochemical contents of nectarine fruits was investigated. On the 40th day of storage, 1.5 mM OA doses reduced weight loss (1.96%) and fruit flesh firmness (36 N) by approximately 44% and 20%, respectively, compared to the control group. The most effective dose in reducing the respiratory rate was again 1.5 mM OA. The 1 mM OA dose was determined to prevent the decay rate approximately by 16% compared to the control group. Organic acid contents showed a continuous decreasing trend during storage and malic acid was determined to be the dominant acid in nectarine fruits. At the end of storage, it was determined that a 1.5 mM OA dose prevented the decrease in malic acid content approximately 35% more than the control group. Chlorogenic acid and rutin were detected at a higher rate than other phenolics. It was observed that 1.5 mM OA dose prevented the breakdown of chlorogenic acid and rutin compounds more so than other doses during storage. In this study, it was determined that a 1.5 mM OA dose especially protected the quality properties and biochemical contents of nectarine fruits in the cold storage more than the control group.

1. Introduction

A significant part of the food produced in the world decays and turns into waste before reaching the consumer due to the inadequacy of preservation techniques and possibilities. In order to reduce postharvest product losses, it is important to make new applications depending on the development of storage technology. Due to the soft texture of the nectarine fruit, which is in the group of stone fruits, a short postharvest storage period occurs, and this makes the storage of the product important. In studies on the preservation of nectarine fruit, the lowest oxygen value and optimum carbon dioxide level are needed to slow down aging and reduce respiration rate during storage in order to prevent fruit quality losses [1,2,3]. Pre-harvest cultural practices, temperature, humidity, oxygen, and carbon dioxide ratio of the storage environment affect the postharvest storage period of nectarine fruit significantly [4,5,6,7]. Different applications such as modified atmosphere packaging [8,9] and organic and inorganic compounds are used for long-term postharvest storage of fruits. These applications include ethylene, polyamines and edible food supplements [10,11,12,13,14].

The main goals of storing fruits in cold environments include reducing ethylene production, lowering respiratory rate, minimizing biochemical changes of fruits and maintaining quality criteria [15,16]. Oxalic acid (OA), a compound among organic acids, controls the browning reaction by inhibiting ethylene biosynthesis, delays the ripening process of fruits and prolongs the storage period [17,18,19,20]. This compound also reacts against environmental stress factors and has an effect on the biochemical and quality criteria of fruits [21]. As a matter of fact, in recent studies, it has been reported that OA application delays ripening in peach [22], banana and mango fruits [17,23,24]. In this research, oxalic acid applications were made during the postharvest cold storage of nectarine fruits, and the quality characteristics and biochemical contents of the fruits were examined.

Nectarine fruits are an important food in human nutrition because they are rich in biochemical substances such as phenolic compounds [25]. Phenolic compounds and organic acids are compounds that affect many quality criteria such as color, taste and aroma in fruits [7,26,27,28]. These compounds are substances that form the defense system of plants against biotic and abiotic stress factors and are effective in many physiological events [29,30]. Since phenolic compounds have anticarcinogenic properties, the demand for fruits rich in these compounds is constantly increasing [31]. Shukitt et al. [32] also reported that organic acids form complexes with heavy metal ions and prevent oxidation catalytics. In the research literature, it was seen that the studies on oxalic acid applications in the preservation of nectarine fruits are limited and there are no studies on the effects of oxalic acid on the phenolic compound and organic acid contents of nectarine fruits. Therefore, this research reveals a new approach to the preservation of nectarine fruits.

2. Materials and Methods

2.1. Experimental Design and Treatments

In this study carried out in the year 2022, the fruits of the Fantasia Nectarine variety grown in the Bursa province İnegöl district were used as material. The fruits of the Fantasia nectarine variety were collected from a garden established at 4 × 5 m distances between rows. Nectarine fruits were collected by hand at the stage of commercial maturity [approximately 11–12% SSC (soluble solid content) value], placed in perforated plastic boxes (with three replications and 15 fruits per replication) and brought to the laboratory in a refrigerated vehicle on the same day. A selection was made to obtain fruits that are uniform in size, color, shape and absence of defects. Oxalic acid (Sigma-Aldrich (Darmstadt, Germany), with 99% purity) was administered in 0.50 mM, 1 mM and 1.5 mM doses. Oxalic acid application was made by immersing the fruits in Tween-20 (2%) added solutions for 10 min. The control group was kept in distilled water for 10 min. After oxalic acid treatments were applied to the fruits, they were stored in cold conditions (0 ± 0.5 °C and 90 ± 5% RH) for 20 and 40 days. Quality criteria, phenolic compounds and organic acid contents were examined at 20 day intervals during the 40-day storage period.

2.2. Weight Loss

In the study, the initial weights (Wi) and final weights (Wf) of the fruits (on the 20th and 40th days) were determined with a 0.01 g precision digital balance (Radwag, Radom, Poland). Weight loss of fruit during storage was determined as a percentage with the following equation (Equation (1)), based on the weight at the beginning of each measurement period [33].

$$\mathrm{WL}=\frac{\mathrm{Wi}-\mathrm{Wf}}{\mathrm{Wi}}\times 100$$

(1)

2.3. Fruit Firmness

The firmness of the nectarine fruits was measured as 10 fruits per replication. The fruit peel was cut at two different points (on the cheeks) along the equatorial portion of the fruit and hardness was determined as Newton (N) with a 7.9 mm penetrating effegi penetrometer (FT-327; McCormick, WA, USA) [33].

2.4. Decay Rate, Soluble Solids Content, Titratable Acidity, pH and Respiration Rate

At the beginning of the study, the total number of fruits (TF) was determined by counting the fruits in each replication. Then, rotting fruit (DF) was determined at each replicate in each measurement period. If mycelium has developed on the peel, the fruit is considered rotten [33]. Finally, the degradation rate (DR, %) was determined by the following equation (Equation (2)).

$$\mathrm{DR}=\frac{\mathrm{TF}-\mathrm{DF}}{\mathrm{TF}}\times 100$$

(2)

Fruit samples were homogenized with a mixer (Promix HR2653, Philips, Istanbul, Turkey) and the homogenate was filtered through cheesecloth to obtain juice filtrate. The soluble solids content (SSC) was determined with a digital refractometer (Atago PAL-1, Washington, DC, USA) and recorded as a percentage (%). The pH was determined with a pH meter. For titratable acidity (TA) measurement, 10 mL of distilled water was added to 10 mL of fruit juice. Then, 0.1 N sodium hydroxide (NaOH) was added until the pH of the solution reached 8.2. Titratable acidity was determined according to the amount of NaOH consumed in the titration and g malic acid was expressed as kg⁻¹ [33]. In determining the respiration rates of the nectarine fruits, a rubber septum was inserted into the 2 L airtight compartments. The fruits were sealed in each compartment at 20 ± 1 °C and 80% relative humidity for 1 h. The chambers were then connected to a gas sensor (Vernier, Washington, DC, USA) and the amount of CO₂ produced by the fruit was considered the respiratory rate (mg CO₂ kg⁻¹ h⁻¹).

2.5. Analysis of Phenolic Compounds

For the phenolic compound (caffeic acid, catechin, chlorogenic acid, ferulic acid, gallic acid, o-coumaric acid, p-coumaric acid, protocatechuic, quercetin, rutin, syringic acid and vanillic acid) analysis in the research, 300 g of nectarine fruit was homogenized from the homogenizer and mixed with distilled water at a ratio of 1:1. The mixture was centrifuged at 15,000 rpm for 15 min. Supernatants were filtered through coarse filter paper and twice through a 0.45 µm membrane filter (Millipore Millex-HV Hydrofilic PVDF, Millipore, Burlington, MA, USA) and injected into an HPLC (Agilent, Santa Clara, CA, USA). Chromatographic separation was performed with a 250 × 4.6 mm, 4 μm ODS column (HiChrom, USA). Solvent A methanol: acetic acid: water (10:2:28) and solvent B methanol:acetic acid:water (90:2:8) were used as mobile phase. Spectral measurements were made at 270 nm (vanillic), 280 nm (catechin, chlorogenic acid, ferulic acid, gallic acid, o-coumaric acid, p-coumaric acid, protocatechuic, quercetin, rutin and syringic acid) and 330 nm (caffeic acid) wavelengths. The flow rate and injection volume were set to 1 mL min⁻¹ and 20 µL, respectively. All phenolic compound standards were obtained from Sigma-Aldrich (Germany), with at least 99% purity in the HPLC grade [34].

2.6. Analysis of Organic Acids

In the analysis of specific organic acids (citric, malic, fumaric, succinic and tartaric acid) in this study, approximately 300 g of each sample was fractionated and 50 g of each sample was transferred to a centrifuge tube, then diluted 1:3 with distilled water. After adding 25 mL of 0.009 N H₂SO₄ to the samples, the samples were homogenized with a crusher (Heidolph Silent Crusher M, Berlin, Germany) and mixed with a shaker (Heidolph Unimax 1010, Berlin, Germany) for one hour. After centrifugation at 15,000× g for 15 min, the supernatant was passed twice through coarse filter paper on a 0.45 µm membrane filter (Millex-HV Hydrophilic PVDF, Millipore, Taufkirchen, Germany) and finally in the SEP-PAK C18 cartridge. The concentration of organic acids was determined by HPLC using an Aminex column (HPX-87H, 300 mm × 7.8 mm, Bio-Rad) attached to an 1100 series HPLC (Agilent Technologies, Waldbronn, Germany). All organic acids were detected at wavelengths of 210 nm. The mobile phase (0.009 N H₂SO₄) was passed through 0.45 µm filters in the analysis processes. The standards of organic acids used in the research were obtained from Sigma-Aldrich (Germany) with at least 99% purity in HPLC grade [35].

2.7. Statistical Analysis

This study analyzed data with two-way ANOVA using SAS Version 9.1 (SAS Institute Inc., Cary, NC, USA) software. When the F test was significant, the means were compared with Tukey’s posthoc test. Correlations between studied features were determined by Pearson’s binary correlations using the “corrplot” package of the R software (version 0.92). The interrelationships of applications and features were determined by principal component analysis (PCA) with the “ggplot2” package of R software. Heatmap analysis was performed with the R package “bioconductor” [36].

3. Results and Discussion

3.1. Weight Loss, Fruit Firmness, Decay Rate, SSC, TA, pH and Respiration Rate

Due to the sensitive skin and flesh structures of nectarine fruits, the postharvest storage life is limited. The important quality criteria, which are among the external appearance characteristics of fruits in cold storage, are weight loss, fruit firmness and decay rates. In this study, weight loss increased during storage (20 and 40 days) and the greatest weight loss was detected on the 40th day. When the postharvest OA applications were compared with the control group, the effective dose of OA was found to be 1.5 mM (F: 1147.59; p ≤ 0.001). Compared to the control group, a 1.5 mM OA dose proportionally provided a 44% retention of weight loss at day 40 (Figure 1, Table S1). As a result of the statistical analysis, it was observed that all OA doses decreased the weight loss rate of the nectarine fruits during storage. Razavi et al. [37] reported that the rate of weight loss increased with the storage period in the peach fruit, which is in the same group in the botanical classification as the nectarine fruit, and that the minimum weight loss occurred in the fruit treated with OA. It has also been demonstrated by many researchers that OA applications prevent weight loss in different fruits during storage [38,39,40].

Fruit firmness values decreased during storage and the greatest decrease was observed in the control group (30.07 N on the 40th day). In the study, it was determined that 1 mM and 1.5 mM doses were the most effective among OA applications. As a result of statistical analysis, it was determined that OA applications had significant effects on fruit firmness (F_{Storage time}: 18.65; F_oxalic: 6.84; F_{Storage time×Oxalic}: 379.93) and it was observed that fruit firmness was significantly preserved during storage compared to the control group (Figure 1, Table S1).

Decay in fruits is an increasing parameter during storage and causes postharvest product losses. In this study, while the decay rate was 4.98% in the control group on the 20th day, it was determined as 4.02% in 1 mM OA application. It was determined that 1 mM OA (6.74%) dose among applications on the 40th day of the storage period was more effective than other applications and significantly reduced the decay rate. There was no statistically significant difference between the control group and 1.5 mM OA dose in terms of decay rate on the 40th day of storage. Oxalic acid is an organic acid commonly found in plants, and many studies have demonstrated that it prevents chilling damage and decay rate in fruits and preserves biochemical changes during storage [20,22,41,42]. The soluble solid ratios of the nectarine fruits showed a tendency to increase during storage. It was determined that OA application prevents the increase in soluble solid ratio as it reduces respiration rate, decay rate and weight loss of the nectarine fruits. As a matter of fact, while SSC was 13.1% in the control group at the end of the storage period (40 d), it was found to be 12.31% in 1.5 mM OA application. This situation reveals that OA applications are effective on the postharvest biochemical changes of fruits and preserve the fruits during storage [43].

Acidity and pH are important quality parameters that affect the taste and aroma of fruits [43]. In this study, the total acidity content of the nectarine fruits showed a decreasing trend during storage. Statistically, the effect of OA applications on this decrease was found to be insignificant. However, there were differences in the statistical evaluation between the control group and the storage periods during the storage period. Considering the pH values, it was determined that the effect of OA applications on the storage time was statistically insignificant. In the research conducted by Örnek and Kaynaş [44], it was reported that the SSC rate of nectarine fruits was 12.20% and the acidity rate was 63% after 25 days of storage. It has been reported that the decrease in acidity during storage can be attributed to the use of organic acids as a respiration substrate during storage [45]. Similar results in nectarines were also found by other researchers [46,47]. The respiration rate of the nectarine fruits showed an increasing trend during storage. This situation was found to be 46.52 mg CO₂ kg⁻¹ h⁻¹ and 53.91 mg CO₂ kg⁻¹ h⁻¹ on the 20th and 40th days, respectively, with a more significant increase in the control group. Considering the effect of OA applications on the respiratory rate, it was observed that the 1.5 mM dose was the most effective dose on the respiration rate on the 20th (41.62 mg CO₂ kg⁻¹ h⁻¹) and 40th (47.32 mg CO₂ kg⁻¹ h⁻¹) days and that it decreased the respiration rate of the nectarine fruits during storage (Figure 2, Table S1). In previous studies, it has been reported that postharvest OA applications reduce the respiration rate in peach [22] and apricot [48] fruits, which are among the stone fruit species. In this study, it was determined that OA application of nectarine fruit, which is among the stone fruit species, during cold storage, decreased the respiration rate and similar results were obtained with our findings.

3.2. Organic Acid Content

In this study, the effect of OA application at different doses after harvest on malic acid, citric acid, succinic acid, fumaric acid and tartaric acid contents of the nectarine fruits was found to be statistically significant (p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001). Malic acid was determined as the major organic acid in the nectarine fruits. This was followed by citric, succinic, fumaric and tartaric acids, respectively (

Table 1. Influence of different doses of oxalic acid applications on organic acid contents of nectarine fruits during storage (mg g⁻¹).

Storage Time		Citric	Malic	Fumaric	Succinic	Tartaric
Harvest		2.39 ± 0.11 a	9.04 ± 0.12 a	1.24 ± 0.07 a	1.74 ± 0.06 a	0.62 ± 0.07 a
20		1.62 ± 0.15 b	6.28 ± 0.74 b	0.93 ± 0.11 b	1.40 ± 0.14 b	0.48 ± 0.06 b
40		1.58 ± 0.17 b	5.52 ± 0.64 c	0.84 ± 0.18 b	1.23 ± 0.11 c	0.41 ± 0.10 b
Treatments
Control		1.41 ± 0.12 c	4.97 ± 0.50 b	0.72 ± 0.12 c	1.18 ± 0.11 b	0.37 ± 0.09 c
OA 0.5 mM		1.56 ± 0.06 bc	5.93 ± 0.38 a	0.85 ± 0.08 bc	1.26 ± 0.12 ab	0.40 ± 0.07 bc
OA 1 mM		1.68 ± 0.07 a	6.58 ± 0.87 a	0.91 ± 0.08 b	1.40 ± 0.11 a	0.48 ± 0.05 ab
OA 1.5 mM		1.75 ± 0.10 a	6.12 ± 0.09 a	1.06 ± 0.10 a	1.43 ± 0.14 a	0.52 ± 0.06 a
Storage time × Oxalic acid interaction
Harvest		2.39 ± 0.11 a	9.04 ± 0.12 a	1.24 ± 0.07 a	1.74 ± 0.06 a	0.62 ± 0.07 a
Day 20	Control	1.49 ± 0.12 de	5.40 ± 0.07 e	0.81 ± 0.10 de	1.24 ± 0.12 def	0.42 ± 0.06 bcd
	OA 0.5 mM	1.55 ± 0.10 cde	6.25 ± 0.12 c	0.91 ± 0.07 bcd	1.35 ± 0.07 bcd	0.46 ± 0.04 bc
	OA 1 mM	1.69 ± 0.09 bcd	7.33 ± 0.14 b	0.95 ± 0.06 bcd	1.48 ± 0.07 bc	0.50 ± 0.06 ab
	OA 1.5 mM	1.78 ± 0.13 b	6.13 ± 0.02 c	1.06 ± 0.06 abc	1.53 ± 0.11 b	0.54 ± 0.04 ab
Day 40	Control	1.34 ± 0.10 e	4.54 ± 0.07 f	0.64 ± 0.07 e	1.11 ± 0.06 f	0.31 ± 0.08 d
	OA 0.5 mM	1.56 ± 0.04 b–e	5.61 ± 0.06 de	0.80 ± 0.06 de	1.16 ± 0.05 ef	0.35 ± 0.06 cd
	OA 1 mM	1.68 ± 0.08 bcd	5.83 ± 0.14 d	0.87 ± 0.10 cd	1.31 ± 0.05 cde	0.46 ± 0.03 bc
	OA 1.5 mM	1.72 ± 0.11 bc	6.11 ± 0.15 c	1.07 ± 0.15 ab	1.33 ± 0.08 cde	0.50 ± 0.08 ab
ANOVA
FStorage time		22.43 ***	22.05 ***	5.777 *	14.11 ***	6.11 *
FOxalic		10.20 **	8.62 **	6.29 **	3.97 *	4.26 *
FStorage time×Oxalic acid		17.31 ***	279.99 ***	8.31 **	12.37 ***	4.79 *

Different letters in the same column indicate statistical differences at p ≤ 0.05. *, **, *** indicates p ≤ 0.05, 0.01, and 0.001, respectively.

)

Different researchers have reported that malic acid is the dominant organic acid in nectarine and peach fruits, followed by citric acid [49,50,51]. A decrease was observed in general organic acids during cold storage. Due to the use of organic acids as a respiratory substrate, they can cause a decrease in organic acids content in fruits during storage [45]. While the malic acid content was 9.04 mg g⁻¹ in the harvest period, it was determined as 4.54 mg g⁻¹ in the control group on the 40th day of storage. It was observed that there was a decrease of approximately 100% in malic acid content during the cold storage process. It was determined that the most effective dose of OA on the 20th day of storage was 1 mM (7.33 mg g⁻¹). At the end of the cold storage period of the nectarine fruits (on the 40th day), the most effective dose of OA was found to be 1.5 mM. As a result of statistical analyzes performed in terms of malic acid content of the nectarine fruits, it was observed that OA applications had significant effects (F_{Storage time}: 22.05; F_oxalic: 8.62; F_{Storage time×Oxalic}: 279.99) on malic acid content and significantly prevented the decrease in malic acid content during storage compared to the control group. It was determined that the most effective dose of OA, which preserves the citric acid ratio during cold storage, is 1.5 mM. When the interaction between OA applications and storage time was examined, it was seen that the statistical differences were significant at the p ≤ 0.001 level. Changes in succinic acid content during storage and the effect of OA applications on these changes were found to be similar in citric acid. In this study, a decrease was observed in fumaric and tartaric acid contents during storage as in other organic acids. Generally effective OA doses were determined to be 1 mM and 1.5 mM. Organic acids are organic compounds that are effective on many quality criteria such as taste and aroma in fruits [28]. Therefore, changes in these compounds affect the storage life of fruits. Li et al. [52] reported that OA and 1-MCP (1-methylcyclopropene) applications applied to peach fruits stored in cold reduced the respiration rate. It is a scientifically expected physiological condition that the organic acids used as respiratory substrate change in parallel with the respiratory rate during the storage process [45]. Therefore, in this study, it was determined that OA acid was effective on the change of organic acids depending on the respiration rate and significantly prevented the decrease in organic acids during the storage process. In many studies, it has been observed that postharvest OA application affects the change in many biochemical contents of fruits such as organic acids and provides the preservation of these compounds [22,48,53].

3.3. Phenolic Fluctuation by Storage Period and Oxalic Acid

Phenolic compounds are secondary metabolites involved in physiological processes such as respiration and photosynthesis. These compounds affect quality criteria such as color and taste in fruits [43]. In this study, the changes in major phenolic compound contents of nectarine fruits were investigated during storage and the effect of OA applications on these compounds was investigated. A decrease was observed in the phenolic compound contents of the nectarine fruits during storage. It was determined that OA application prevented this decrease and 1 mM and 1.5 mM OA doses were especially effective (

Table 2. The effect of different doses of oxalic acid applications on phenolic compounds of nectarine fruits during storage (mg 100 g⁻¹).

Storage Time		Caffeic Acid	Catechin	Chlorogenic Acid	Ferulic Acid	Gallic Acid	o-Coumaric Acid
Harvest		2.25 ± 0.05 a	2.94 ± 0.17 a	13.77 ± 0.09 a	3.67 ± 0.09 a	3.11 ± 0.05 a	4.93 ± 0.08 a
20		1.83 ± 0.22 b	2.58 ± 0.20 b	11.33 ± 0.92 b	3.32 ± 0.17 b	2.29 ± 0.29 b	3.99 ± 0.57 b
40		1.75 ± 0.16 b	2.23 ± 0.54 c	9.21 ± 0.95 c	2.96 ± 0.28 c	1.38 ± 0.51 c	3.60 ± 0.51 c
Treatments
Control		1.82 ± 0.34 c	2.29 ± 0.61 b	10.78 ± 2.50 d	3.16 ± 0.45 c	1.89 ± 1.08 d	3.65 ± 1.00 c
OA 0.5 mM		1.87 ± 0.31 bc	2.55 ± 0.38 ab	11.05 ± 2.28 c	3.24 ± 0.41 bc	2.21 ± 0.84 c	4.16 ± 0.67 b
OA 1 mM		2.01 ± 0.22 ab	2.79 ± 0.35 a	11.65 ± 2.02 b	3.35 ± 0.31 ab	2.37 ± 0.68 b	4.38 ± 0.46 a
OA 1.5 mM		2.08 ± 0.16 a	2.70 ± 0.31 ab	12.26 ± 1.43 a	3.51 ± 0.16 a	2.56 ± 0.51 a	4.50 ± 0.38 a
Storage time × Oxalic acid interaction
Harvest		2.25 ± 0.07 a	2.94 ± 0.23 a	13.77 ± 0.12 a	3.67 ± 0.12 a	3.11 ± 0.07 a	4.93 ± 0.10 a
Day 20	Control	1.61 ± 0.11 d	2.33 ± 0.08 ab	10.32 ± 0.08 d	3.11 ± 0.07 cd	1.87 ± 0.08 de	3.12 ± 0.09 de
	OA 0.5 mM	1.69 ± 0.11 cd	2.57 ± 0.13 ab	10.68 ± 0.08 d	3.28 ± 0.06 bc	2.28 ± 0.09 bc	4.10 ± 0.11 bc
	OA 1 mM	1.97 ± 0.05 abc	2.61 ± 0.09 a	11.90 ± 0.10 c	3.38 ± 0.09 abc	2.43 ± 0.04 b	4.27 ± 0.14 bc
	OA 1.5 mM	2.07 ± 0.07 ab	2.82 ± 0.11 a	12.41 ± 0.13 b	3.50 ± 0.13 ab	2.58 ± 0.09 b	4.49 ± 0.08 b
Day 40	Control	1.59 ± 0.07 d	1.61 ± 0.06 b	8.24 ± 0.11 g	2.69 ± 0.06 e	0.69 ± 0.06 g	2.90 ± 0.14 e
	OA 0.5 mM	1.67 ± 0.10 cd	2.14 ± 0.15 ab	8.71 ± 0.08 f	2.78 ± 0.10 de	1.25 ± 0.10 f	3.45 ± 0.11 d
	OA 1 mM	1.80 ± 0.11 bcd	2.83 ± 0.67 a	9.29 ± 0.08 e	3.00 ± 0.07 cde	1.59 ± 0.06 e	3.94 ± 0.09 c
	OA 1.5 mM	1.92 ± 0.12 abcd	2.34 ± 0.13 ab	10.60 ± 0.11 d	3.36 ± 0.09 abc	1.98 ± 0.17 cd	4.10 ± 0.12 bc
ANOVA
FStorage Time		77.13 ***	16.14 ***	3734.61 ***	108.77 ***	849.17 ***	322.63 ***
FOxalic acid		11.67 ***	4.59 *	234.06 ***	14.73 ***	68.12 ***	74.55 ***
FStorage time×Oxalic acid		3.39 *	2.43 ns	63.97 ***	5 **	21.55 ***	20.08 ***

Different letters in the same column indicate statistical differences at p ≤ 0.05. ns: not significant. *, **, *** indicates p ≤ 0.05, 0.01, and 0.001, respectively.

and

Table 3. Table 2 continued (mg 100 g⁻¹).

Storage Time		p-Coumaric Acid	Protocatechuic	Quercetin	Rutin	Syringic Acid	Vanillic Acid
Harvest		4.49 ± 0.01 a	2.02 ± 0.07 a	6.47 ± 0.45 a	12.17 ± 0.09 a	2.00 ± 0.09 a	1.20 ± 0.07 a
20		4.08 ± 0.20 a	1.78 ± 0.28 b	6.22 ± 0.45 ab	11.08 ± 0.34 b	1.68 ± 0.18 b	0.91 ± 0.16 b
40		4.11 ± 1.44 a	1.25 ± 0.31 c	5.80 ± 0.45 b	8.71 ± 1.66 c	1.56 ± 0.15 b	0.81 ± 0.13 c
Treatments
Control		3.83 ± 0.61 a	1.40 ± 0.53 c	5.77 ± 0.63 b	9.96 ± 2.37 d	1.64 ± 0.29 a	0.87 ± 0.26 c
OA 0.5 mM		4.03 ± 0.41 a	1.67 ± 0.39 b	6.07 ± 0.49 ab	10.22 ± 2.20 c	1.70 ± 0.25 a	0.92 ± 0.22 bc
OA 1 mM		4.79 ± 1.39 a	1.78 ± 0.32 ab	6.32 ± 0.31 ab	10.94 ± 1.20 b	1.80 ± 0.20 a	1.01 ± 0.18 ab
OA 1.5 mM		4.28 ± 0.23 a	1.88 ± 0.22 a	6.49 ± 0.35 a	11.50 ± 0.60 a	1.85 ± 0.16 a	1.08 ± 0.11 a
Storage time × Oxalic acid interaction
Harvest		4.49 ± 0.01 a	2.02 ± 0.10 a	6.47 ± 0.59 ab	12.17 ± 0.11 a	2.00 ± 0.12 a	1.20 ± 0.09 a
Day 20	Control	3.84 ± 0.09 a	1.35 ± 0.06 cd	5.61 ± 0.10 ab	10.67 ± 0.18 c	1.51 ± 0.11 ab	0.76 ± 0.07 cd
	OA 0.5 mM	4.00 ± 0.13 a	1.81 ± 0.11 ab	6.16 ± 0.10 ab	11.02 ± 0.13 bc	1.60 ± 0.13 ab	0.80 ± 0.07 bcd
	OA 1 mM	4.17 ± 0.08 a	1.93 ± 0.06 ab	6.36 ± 0.08 ab	11.14 ± 0.17 bc	1.75 ± 0.20 ab	1.01 ± 0.11 abc
	OA 1.5 mM	4.32 ± 0.05 a	2.01 ± 0.09 a	6.75 ± 0.12 a	11.49 ± 0.11 b	1.86 ± 0.09 ab	1.08 ± 0.05 ab
Day 40	Control	3.15 ± 0.09 a	0.83 ± 0.07 e	5.24 ± 0.15 b	7.03 ± 0.17 e	1.41 ± 0.07 b	0.67 ± 0.07 d
	OA 0.5 mM	3.59 ± 0.11 a	1.19 ± 0.05 d	5.57 ± 0.10 ab	7.46 ± 0.11 e	1.50 ± 0.13 b	0.76 ± 0.05 cd
	OA 1 mM	3.69 ± 2.65 a	1.38 ± 0.09 cd	6.14 ± 0.11 ab	9.51 ± 0.09 d	1.64 ± 0.13 ab	0.82 ± 0.06 bcd
	OA 1.5 mM	4.02 ± 0.12 a	1.61 ± 0.05 bc	6.24 ± 0.10 ab	10.85 ± 0.16 c	1.69 ± 0.12 ab	0.98 ± 0.07 abc
ANOVA
FStorage Time		0.71 ns	180.09 ***	7.5 **	1385.17 ***	26.43 ***	54.88 ***
FOxalic acid		1.74 ns	37.03 ***	4.71 *	164.63 ***	3.43 ns	9.11 **
FStorage time×Oxalic acid		1.3 ns	9.86 ***	1.28 ns	101.81 ***	0.9 ns	2.61 ns

Different letters in the same column indicate statistical differences at p ≤ 0.05. ns: not significant. *, **, *** indicates p ≤ 0.05, 0.01, and 0.001, respectively.

). In this study, chlorogenic acid was determined as the most intense phenolic compound found in the nectarine fruits. Studies have reported that chlorogenic acid is the dominant phenolic compound in peach and nectarine fruits [43,54]. While the amount of chlorogenic acid was 8.24 mg 100 g⁻¹ in the control group at the end of the storage (40 d), it was determined as 10.60 mg 100 g⁻¹ in 1.5 mM OA application. The amount of chlorogenic acid during the harvest period was determined as 13.77 mg 100 g⁻¹. Therefore, it was observed that a 1.5 mM OA dose preserved the amount of chlorogenic acid by approximately 29% compared to the control group. In the statistical evaluation, the effect of OA application on chlorogenic acid was found to be significant at p ≤ 0.001 (F_{Storage time}: 3734.61; F_oxalic: 234.06; F_{Storage time×Oxalic}: 63.97). It was determined as the most concentrated phenolic compound in the nectarine fruits after routine chlorogenic acid. A decrease was observed in the amount of rutin during storage. The maximum decrease was detected in the control group (7.03 mg 100 g⁻¹) on the 40th day of storage. At the end of the storage (40 d), it was determined as 10.85 mg 100 g⁻¹ in 1.5 mM OA application (Table 3). A dose of 1.5 mM OA was found to retain approximately 54% of the amount of rutin. In this study, the effect of OA doses on p-coumaric and syringic acid was found to be statistically insignificant. Considering the interaction of storage time and OA applications, the changes in catechin, quercetin and vanillic acid contents were found to be statistically insignificant. When the effects of OA doses were examined alone, it was observed that OA doses were effective on all phenolic compounds except p-coumaric and syringic acid. In the study, it was determined that 1 mM and 1.5 mM OA doses were the most effective doses in general. In many studies, it has been reported that oxalic acid inhibits ethylene synthesis in fruits, delays the ripening process and reduces the change in biochemical contents [17,18,19,20]. Accordingly, Liang et al. [21] revealed that OA treatment in peach fruits is effective on the ripening processes of fruits and reduces the exchange of phenolic compounds. Similar results were obtained in banana and mango fruits [17,23,24]. Due to the limited research on postharvest OA applications to nectarine fruits, comparisons were made on fruits such as peach [52] and plum [53], which are similar in terms of agro-morphological characteristics, and our findings were similar to the results of the researchers.

3.4. Associations among Fruit Properties, Organic Acids, and Phenolic Compounds

In this study, a negative relationship was found between fruit flesh firmness and fruit weight, SSC, respiration rate and rot rate (p ≤ 0.001). While a negative relationship was determined between respiration rate and phenolic compounds and organic acids, it was observed that total acidity formed positive relationships with organic acids. A positive relationship was determined between organic acid contents and phenolic compound contents of the nectarine fruits during cold storage. It was seen that the positive statistical relationship between p-coumaric acid and catechin, one of the phenolic compounds, was significant (r = 0.77, p ≤ 0.001), but the statistical relationship between p-coumaric acid, other phenolics and organic acids was determined to be insignificant (Figure 3). There was a negative correlation between the decay rate and phenolic compounds and organic acids, while positive correlations were found between the respiration rate (r = 0.80, p ≤ 0.001) and pH (r = 0.66, p ≤ 0.01)

3.5. Identification of Interrelationships between Storage, Spermidine Treatments and Bioactive Compounds by PCA and Hatmap

The organic acid contents of the nectarine fruits changed during cold storage and the effect of OA applications on these changes differed depending on the doses. PCA (principal component analysis) is a statistical model that reveals the statistical significance of the applications and the findings obtained in the research and defines the results scientifically [30,55]. The variation resulting from the interaction between OA treatments and storage times was determined as 92.96% (PC1 + PC2) (Figure 4). According to the analysis, organic acids showed a positive relationship in the PCA plane. While malic and succinic acid formed a close group statistically, tartaric acid showed a different change from other acids. Considering the storage times, it was determined that the level of organic acids was high, especially during the harvest period; however, they showed a great decrease on the 40th day of storage. In the study, it showed a wider variation, including all OA doses of the control group. It was observed that OA doses showed a high level of intersection within themselves.

It was observed that the phenolic compound contents of the nectarine fruits decreased during storage and the lowest levels were detected on the 40th day. It was determined that the 1 mM OA dose was the most effective on this change and protects the phenolic compounds during storage. As a result of the PCA analysis, the interaction that occurred as a result of the PCA analysis performed to reveal the effect of storage times and OA applications was determined as 88.5% (PC1 + PC2). While p-coumaric acid and catechin from phenolic compounds showed a different variation from other phenolic compounds, a close correlation was detected between other phenolics. Considering the OA doses, it was determined that the groups of control, 0.5 mM and 1.5 mM doses overlapped to a large extent, whereas the 1 mM OA dose differed from other applications. In this study, it was observed that the correlation lines of most phenolics (except p-coumaric and catechin) intersect in the PCA plane (Figure 5).

Hatmap analysis was carried out to reveal the effects of storage times and OA doses on some quality criteria and biochemical properties of the nectarine fruits. In the Hatmap analysis, the color change towards the red color on the color scale shows that the statistical significance level has increased. According to this statistical analysis method, the changes in the respiration rate, weight loss, SSC and decay rate in the harvest and control group were less significant than the other applications (blue color). On the other hand, the change in organic acids and phenolic compounds (except p-coumaric) in this group were determined as the most statistically significant parameters (red color). Quality parameters (weight loss, respiration rate, SSC, pH and decay) showed a more significant change than organic acids and phenolic compounds in the control + 40th day group. Considering the OA doses, it was observed that the 1.5 mM dose has a significant effect on the decay rate on the 40th day of storage and reduces the decay rate by protecting the fruit. At the end of the storage (day 40), it was determined that a 1 mM OA dose preserved the catechin and p-coumaric acid content (Figure 6). The results obtained in this study were interpreted by Hatmap analysis and it was seen that the correlations obtained were similar to the results obtained by other researchers with the same method [28]. This situation reveals that Hatmap analysis can be used effectively in the scientific evaluation of data.

4. Conclusions

In this study, it was observed that postharvest OA applications preserve the quality properties and biochemical contents of nectarine fruits during storage. Especially on the 40th day of storage, it was observed that the weight loss increased significantly in the control group and the 1.5 mM OA dose greatly reduced the weight loss. Compared to the control group OA doses, it was determined that the 1.5 mM OA dose decreased the respiration rate and preserved the fruit firmness more. Regarding the decay rate, it was determined that the 1 mM OA dose was more effective than the other doses. In the study, organic acids showed a decrease in storage time and OA doses (especially 1.5 mM OA) were found to significantly reduce the decrease in organic acids. When the contents of phenolic compounds were examined, it was observed that they showed a similar change to organic acids in general. In this study, it was determined that the dominant phenolic compound in nectarine fruits is chlorogenic acid and the OA dose of this compound that was the most protective during storage was 1.5 mM. As a result, it was determined that increasing doses of OA applications preserved the quality and biochemical properties of nectarine fruits during cold storage.