Postharvest Quality Maintenance of Traditional Serbian Peppers: The Impact of Heat Treatment and Storage Temperature

Abstract

Traditional Serbian pepper cultivars, prized for their nutritional value and flavor, lack established postharvest practices. The impact of storage conditions and hot water dipping (HWD) at 55 °C for 1 min on the physicochemical properties of ‘Kurtovska Ajvaruša’, ‘Grkinja Babura’, and ‘Duga Bela Ljuta’ (Capsicum annuum L. var. longum) was examined. Peppers were stored at 4 °C (with and without HWD pretreatment) and at 10 °C (without HWD) for 21 days, followed by 3 days of shelf life. The main quality parameters measured included general appearance, weight loss, chilling injury, decay incidence, color, histological characteristics of pericarp, and quinic and succinic acid contents. Storage temperature, duration, and HWD significantly affected the examined parameters. ‘Grkinja Babura’ retained higher water content with lower weight loss (5.2%) at 4 °C, while ‘Duga Bela Ljuta’ showed significant loss (8.7%) at 10 °C after 21 + 3 days. In ‘Duga Bela Ljuta’, both quinic and succinic acid contents increased during cold storage, HWD treatment, and shelf life. In contrast, in ‘Grkinja Babura’ and ‘Kurtovska Ajvaruša’, this trend was observed only for quinic acid, whereas succinic acid content had a decreasing trend. HWD shows potential to stabilize weight loss, preserve general appearance in two cultivars, and reduce chilling injury (to 4.8%), as well as decay incidence (to 3.1%), indicating its potential to maintain quality and marketability of these traditional pepper cultivars. These findings suggest that HDW could have a high potential for sustainable strategies to improve the postharvest quality of traditional pepper cultivars in the region.

1. Introduction

Peppers (Capsicum annuum L.) are widely cultivated vegetables, valued for their various culinary applications and significant nutritional value. The genus Capsicum comprises a wide range of cultivars grown for their fruits, which are consumed fresh or in processed form, e.g., as dried products, pickles, sauces, and pastes. Fresh peppers are valued for their crisp texture and the vibrant flavors. Processed forms extend availability and add unique flavors to traditional cuisines [1]. In addition to their culinary importance, peppers are rich in vitamins A and C, capsaicin, and phenolic compounds, all of which contribute to their antioxidant activity and health benefits [2].

Despite their economic and cultural importance, these peppers face significant postharvest challenges because they are highly perishable and vulnerable to water loss, chilling injury, and fungal infections [3,4]. Traditional landraces present additional complexity due to their genetic diversity in fruit morphology and pericarp structure developed through centuries of cultivation [5,6]. Cultivars with thinner pericarps and less uniform cuticle thickness, commonly observed in traditional varieties, are more susceptible to mechanical damage and water loss during storage [7], compromising fruit quality and reducing commercial value. Understanding this cultivar-specific variability is crucial for developing effective storage strategies tailored to traditional landraces.

Traditional Serbian pepper cultivars are important genetic resources for national heritage and biodiversity. The cultivars ‘Kurtovska Ajvaruša’, ‘Grkinja Babura’, and ‘Duga Bela Ljuta’ are especially prized for their distinctive shapes, flavors, and culinary versatility, ranging from mild and sweet to highly pungent and serving as central ingredients in authentic Serbian dishes, including ajvar [8]. Cultivated predominantly in southern Serbia over generations, these landraces have developed rich phenolic profiles, with elevated concentrations of quinic, succinic, and ascorbic acids that enhance their antioxidant potential and contribute to their unique taste and aroma [8,9,10]. These genetic resources demonstrate greater resilience and surpass commercial hybrids in both phenolic content and flavor complexity [2]. While their nutritional and sensory value is well recognized, the effective postharvest handling of their fresh fruits remains a challenge.

Current storage practices typically involve temperatures in the range between 7 and 13 °C and may extend shelf life but often lead to chilling injury over time, affecting texture, appearance, and flavor of peppers [11]. Moreover, the delicate pericarp structures of traditional cultivars may render them even more susceptible to such injuries compared to commercial hybrids [7]. In response to these challenges, recent studies have explored the use of heat treatments, such as pre-storage hot water dipping (HWD), as a strategy to enhance fruit tolerance to low temperatures and suppress microbial decay, with the aim of maintaining overall quality, storability, and consumer acceptability. HWD has been shown to reduce chilling injury, delay senescence, and improve overall fruit quality by activating antioxidant pathways, such as the ascorbate–glutathione cycle [12,13,14,15,16]. In combination with optimized storage conditions (e.g., 7–10 °C, 95–98% relative humidity), these treatments offer promising alternatives to reduce postharvest losses [11]. Fallik et al. [17] showed that rinsing sweet peppers at 55 °C between 12 and 28 sec improved their quality by reducing decay and maintaining firmness without causing heat damage. The study by Kantakhoo and Imahori [18] concludes that HWD at 55 °C for 1 min effectively alleviates chilling injury in red sweet pepper fruit during cold storage.

Notably, while similar HWD conditions have been applied successfully to various pepper cultivars, the specific temperature and duration applied in the present study have not previously been tested on traditional Serbian cultivars: ‘Kurtovska Ajvaruša’, ‘Duga Bela Ljuta’, and ‘Grkinja Babura’. Our preliminary trials at 55 °C for 1 min also showed positive effect on postharvest quality of the traditional pepper cultivar ’Duga Bela’ [19]. Given the significant economic and cultural value of the examined cultivars in Serbia, identification of the optimal approaches that effectively preserve their overall quality, extend storability, and thereby enhance marketability is of considerable importance not only for maintaining traditional agricultural practices but also for supporting the local growers and the regional economy.

This study evaluated how pre-storage HWD at 55 °C for 1 min affects quality preservation during storage. Peppers were stored at 4 °C for 21 days, followed by 3 days of shelf life.

2. Materials and Methods

2.1. Plant Material and Cultivation

‘Kurtovska Ajvaruša’ (also known as ‘Kurtovska kapija’) is a robust, indeterminate-growing plant reaching 80–90 cm in height, with dense foliage that provides natural fruit protection (Figure 1). Each plant produces 8–12 fruits weighing 130–150 g, with dimensions of 13–16 cm length and 5–7 cm width. The fruits are flat and smooth surfaced, maturing from dark green to dark red in 135–145 days. Due to its thick pericarp and intense red color, it is primarily used for ajvar production but also consumed fresh.

‘Grkinja Babura’ grows semi-determinately to about 50 cm tall, with medium-strength habitus (Figure 2). Each plant yields around 10 fruits weighing 130–160 g, with dimensions of ~12 cm length and ~6.5 cm width. The fruits are round, with a single tip, featuring a shiny, smooth surface and mild taste, without spiciness. Maturity occurs in 115–135 days. It is used fresh, processed, or stored for winter applications.

‘Duga Bela Ljuta’ grows indeterminately to 50–60 cm height, with a strong branching structure. Each plant produces 9–13 elongated fruits (17–19 cm), which are milky white at technological maturity and red at physiological maturity. The fruits are flat, with smooth to semi-wrinkled skin and contain capsaicin-rich placentas that provide spicy flavor. Maturity occurs in 115–135 days. It is used fresh, processed, and stored for winter (Figure 3).

Traditional pepper production was carried out in the Aleksinac region (southern Serbia, 43°30′23.4″ N 21°42′28.6″ E) using seedlings without substrate. The seeds were sown in a greenhouse on 10 April 2023, and treated with Previcur Energy 840 SL (Bayer, Leverkusen, Germany) to protect against Pythium fungi and Rhizoctonia bacteria. Standard agricultural techniques were employed, including watering, insect protection, fertilization, and hardening of the seedlings one week before transplanting. The soil type was alluvium. Agrotechnical practices included autumn plowing and pre-sowing preparation with Elixir Supreme fertilizer (12:12:17, 600 kg/ha; Zorka, Šabac, Serbia). The pepper plants were planted on June 8, with 60 cm row spacing and 15–20 cm spacing within rows. A drip irrigation system was installed, with the plants arranged alternately on either side of the drip strip for optimal surface use.

Immediately after planting, a starter foliar fertilizer (FitoFert Crystal 10:40:10, 0.5% concentration; FitoFert, Inđija, Serbia) was applied. During flowering and fruit setting, the plants were treated with calcium (Foligal Calcium, 0.5% concentration; Galenika-Fitofarmacija, Beograd-Zemun, Serbia). Bacterial spotting was managed with copper preparations (Nordox) and the microbiological preparation Bacteria. To prevent blight and anthracnose, azoxystrobin and difenoconazole-based treatments were used. Insect protection involved acetamiprid and flonicamid for leaf veins, spinosad for thrips, with no treatment for bedbugs. Basil and tagetes formed the protective belt around the plots.

The traditional cultivars ‘Duga Bela Ljuta’ and ‘Grkinja Babura’ were harvested in mid-August at technological maturity. The harvesting of the ‘Kurtovska Ajvaruša’ variety started around 5 September, at the onset of physiological maturity. For this study, the fruits of all tree cultivars were harvested on 22 September.

2.2. Experimental Design

Pepper fruits were analyzed immediately after harvest to establish baseline measurements (day 0). Following this initial assessment, the fruits were randomly assigned to one of three experimental groups: (a) low-temperature storage, held at 4 °C for 21 days; (b) HWD, treated at 55 °C, for 1 min using a large-volume bath (~30 L per batch) to minimize temperature fluctuations, followed by storage at 4 °C for 21 days; (c) usual-temperature storage, held at 10 °C for 21 days. This method was designed to be easily implemented under farm conditions, without requiring high-tech equipment. Treatment at 55 °C for 1 min provides sufficient heat exposure to activate beneficial defense mechanisms while avoiding heat damage from longer or higher-temperature treatments [20,21].

For postharvest analysis, about 25 kg of pepper fruits (approx. 160–180 fruits per cultivar) were distributed into wooden crates (50 cm × 30 cm × 8 cm) and stored for 21 days under controlled conditions (4 °C or 10 °C; 90–95% relative humidity). Relative humidity was maintained using environmental storage chambers equipped with humidity control systems and monitored daily using digital hygrometers (±2% accuracy). Water sprinklers were build-in within the storage chambers to maintain the target humidity range. Subsequently, the samples were transferred to shelf life conditions at 24 ± 2 °C and ambient relative humidity (approximately 60–70%) for an additional 3 days. Weight loss was measured after 7, 14, and 21 days, a visual quality assessment was conducted after 14 and 21 days, whereas all other analyses were conducted at the beginning of the experiment (0 days), after the periods of cold storage (day 21) and after the subsequent shelf life (day 21 + 3). The experiment followed a 3 × 3 factorial design, comprising three storage treatments (10 °C, 4 °C, and 4 °C with HWD pretreatment) and three evaluation periods (0, 21, and 21 + 3 days), with measurements taken on multiple fruits per treatment.

2.3. Weight Loss

Fruit weight loss (g) was measured for each treatment after the harvest, cold storage, and shelf life every 7 days, by weighing individual crates using a technical balance (Kern 572–35, Kern & Sohn, GmbH, Balingen, Germany). The measurements were carried out in triplicate.

2.4. Visual Quality Assessment

After 14 and 21 days of storage all pepper fruits were visually evaluated for their general appearance, as well as for the presence of rotting and chilling injury. The assessment was conducted by six trained panelists (three women and three men), aged between 20 and 65 years, according to the methodology described by Melgarejo et al. [22] and Milović et al. [23]. The panelists were trained and qualified to perform both qualitative and quantitative assessments and had extensive experience in evaluating the visual quality of horticultural products, including symptoms such as chilling injury and decay. To ensure objectivity and eliminate potential bias, the panelists were blinded to the treatments. The samples were encoded using randomly assigned three-digit numerical codes, and no information regarding treatment identity was disclosed during the evaluation process.

Specific evaluation criteria were as follows: chilling injury: pitting, surface discoloration, water-soaked areas, calyx browning, or glossiness loss; decay: visible fungal growth, soft rot, bacterial lesions, or any signs of microbial deterioration; and general appearance: overall visual quality considering color uniformity, surface integrity, firmness, and absence of defects.

To assess the overall fruit quality, all fruits were evaluated collectively as a single sample. General appearance was scored on a continuous scale from 0 (lowest score) to 5 (highest score), based on the following criteria: 5—excellent; 3—good; 1—poor. A score below 3 were considered unmarketable. Chilling injury and decay incidences were calculated as the percentage of affected fruits relative to the total number of fruits evaluated in each treatment group [24].

The evaluation was performed at room temperature (20 °C) in individual booths under white lighting. Each panelist participated in two separate sessions on the same day.

To prevent secondary infections and maintain experimental integrity, all visibly damaged fruits were promptly removed after 7 and 14 days of storage, simulating standard commercial handling practices. The remaining healthy fruits were used for continued storage and further analysis.

2.5. Color

The skin color of pepper fruits was analyzed in the CIELab color space using a CR-400 Chroma Meter (Konica-Minolta, Osaka, Japan), calibrated with a KONICA MINOLTA White Calibration Plate (CR-A43), on 10 randomly selected fruits after the harvest, as well as after cold storage and shelf life, with two measurements taken on opposite sides of each fruit in the equatorial region [7].

L (lightness) represents the lightness of the color on a scale from 0 (black) to 100 (white). C (chroma) indicates the intensity or saturation of the color, with higher values representing more vivid colors.

Delta E (ΔE*) is a quantitative metric that represents the perceived difference between two colors. It is widely used in color measurement and is calculated in the CIELAB color space, which defines color using three coordinates: lightness (L), red/green (a), and yellow/blue (b). By comparing these coordinates between two samples, ΔE* quantifies how similar or different they appear. Values below 1 indicate differences that are nearly imperceptible to the human eye, while values above 3–4 reflect noticeable color changes. Lower ΔE* values signify closer color matches, making it a reliable tool for assessing color stability or variation over time.

ΔE* could not be determined on day 0, as this time point was used to establish the initial color baseline or reference for the samples. To evaluate ΔE* as an indicator of color changes resulting from any treatments, including those in postharvest, it is necessary to compare these baseline values with measurements taken after 21 days of storage and again after an additional 3 days of shelf life.

The ΔE* formula (1976) is as follows:

$${\mathsf{\Delta }\mathrm{E}}_{\left\{\mathrm{a}\mathrm{b}\right\}}^{\mathrm{*}}=\sqrt{\left\{{\left({\mathrm{L}}_2^{\mathrm{*}}-{\mathrm{L}}_1^{\mathrm{*}}\right)}^2+{\left({\mathrm{a}}_2^{\mathrm{*}}-{\mathrm{a}}_1^{\mathrm{*}}\right)}^2+{\left({\mathrm{b}}_2^{\mathrm{*}}-{\mathrm{b}}_1^{\mathrm{*}}\right)}^2\right\}}$$

where

• L* = lightness;
• a* = red/green value;
• b* = yellow/blue value;
• Number 1—the reference/initial color measurement;
• Number 2—the second color measurement.

2.6. Analysis of Fruit Anatomy

Anatomical analyses were performed on the pepper fruits after the harvest. The segments 2.5 × 2 cm were cut from the middle part of each fruit and fixed in 50% ethanol. Cross-sections (up to 20 µm thick) were made using a Reichert sliding microtome. Temporary slides were prepared according to the standard method for light microscopy. For the permanent slides, tissue sections were stained with Safranin (1%, w/v, in 50% ethanol) and Alcian blue (1%, w/v, aqueous). All permanent slides were mounted in Canada balsam after dehydration [25]. The anatomical sections of the fruits were analyzed using an Olympus BX41 light microscope (LM) with a camera Olympus SC30 (Olympus Corporation, Tokyo, Japan).

2.7. Chemical Analysis

Prior to chemical analysis, the quarters of 10 fruits (without seeds, calyx, and pedicel) were homogenized, transferred to Ziplock PVC bags, and immediately frozen on dry ice. The samples prepared in this way were further stored in the freezer until analyzed.

The water content (%) was determined using thermo-gravimetric analysis (TGA-701, LECO Co., St. Joseph, MI, USA).

The extractable color of fresh pepper from each experimental group was determined as previously described in Ilić et al. [7], with slight modifications. Initially, 20 mL of acetone solution was added to 2 g of homogenized pepper fruit. The extraction was carried out at room temperature in dark conditions for 12 h. Following centrifugation at 13,776× g for 5 min (Centrifuge 5804 R, Eppendorf, Hamburg, Germany), the supernatant (2 mL) was collected, and its absorbance was measured at 460 nm using the SHIMADZU 1800 UV–Visible Spectrophotometer (Kyoto, Japan). The measurements were performed in triplicate. ASTA values were calculated according to the following formula:

ASTA value = Absorbance of acetone extracts × 16.4 × If/Sample weight (g)

The deviation factor of the spectrophotometer (If) refers to the instrument correction factor, which is calculated by dividing the theoretical absorbance of ASTA (0.6 absorption units) by the experimental absorbance of the standard color solution (0.001 M K₂Cr₂O₇ and 0.09 M (NH₄)₂Co(SO₄)₂·6H₂O in 1.8 M H₂SO₄) at 460 nm.

To quantify the content of quinic and succinic acids, the pepper samples were homogenized (2 g) and subjected to the extraction, in triplicate, according to the method described by Milenković et al. [26]. Separation and analysis were performed using an HPLC system (Agilent 1200 series, Agilent Technologies, Santa Clara, CA, USA), with Nucleogel Sugar 810 H (Macherey-Nagel, Dueren, Germany) and equipped with a diode array detector (DAD G1315C, Agilent Technologies, USA). The mobile phase was 0.005 M H₂SO₄, the flow rate was 0.6 mL/min, and the injection volume was 10 μL. The total analysis time was 25 min, with a 1.5 min pause between analyses. The results were expressed as mg/100 g fresh weight.

2.8. Statistical Analysis

The differences between the means of fruit quality parameters were evaluated using factorial ANOVA, after the data were tested using Cochran’s C test, Hartley’s test, Bartlett’s chi-square test, and Levene’s test. The Duncan multiple range test was used to test the differences between the individual means. Principal component analysis (PCA) was performed on selected fruit parameters: water content (WC), color (CIELab and ASTA units), quinic acid, and succinic acid to identify patterns and treatment effects across cultivars and storage conditions. Data were standardized to ensure equal weighting of variables with different scales. PCA was conducted using a correlation matrix, extracting the first two principal components (PC1 and PC2), which explained the majority of variance in quality parameters. The variable loadings and cultivar projections were analyzed to assess the impact of HWD and storage temperatures. TIBCO Software Inc. (2020) Data Science Workbench, version 14, was used for both analyses.

3. Results

3.1. Weight Loss

All cultivars showed progressive weight loss over time (Figure 4). Storage at 10 °C caused the highest weight loss. HWD combined with 4 °C storage did not reduce weight loss relative to 4 °C storage alone. These results indicated that lower temperatures combined with HWD slightly mitigated weight loss, but this combination was not substantially more effective than cold storage alone.

3.2. Visual Quality Assessment

The effects of different storage temperatures and HWD on the three traditional pepper cultivars are presented in

Table 1. Impact of storage temperature and HWD on chilling injury, decay incidence, weight loss, and general appearance of fresh fruits from three traditional Serbian pepper cultivars after 14 and 21 days of storage.

Traditional Cultivar	Storage Condition	Storage Duration
		14 Days			21 Days
		Chilling Injury (%)	Decay Incidence (%)	Weight Loss (%)	Chilling Injury (%)	Decay Incidence (%)	Weight Loss (%)	General Appearance
Kurtovska Ajvaruša	10 °C	0	2	3.9	0	10.5	6.5	2.3
	4 °C	0.5	0	2.2	5.0	3.0	2.6	3.0
	4 °C + HWD	0	0	2.5	1.2	0	2.9	3.4
Grkinja Babura	10 °C	0	2.3	5.0	0	20.0	8.0	1.5
	4 °C	0.5	0	2.5	8.0	7.2	3.0	2.6
	4 °C + HWD	0	0	2.8	2.0	2	3.4	2.8
Duga Bela Ljuta	10 °C	0	1.8	4.2	0	16.7	7.5	1.7 *
	4 °C	1.0	0	2.1	7.0	5.0	2.8	3.0
	4 °C + HWD	0	0	2.4	1.5	1.1	3.0	3.1

Visual quality assessment: chilling injury and decay incidence expressed as the percentage of affected fruits from the total number of fruits. Weight loss calculated as the percentage of fruit weight lost, based on the difference between initial and subsequent weights of fruits during storage and shelf life. General appearance evaluated on 1–5 scale, where 5 = excellent, 3 = good, and 1 = poor (scores < 3 were considered unmarketable). (*) All fruits were shriveled.

. All pepper cultivars maintained low weight loss and marketable appearance during 14 days of storage. Peppers stored at 10 °C exhibited the highest weight loss, whereas those stored at 4 °C showed significantly lower weight loss. The fruits exposed to HWD and stored at 4 °C experienced slightly higher weight loss compared to those without heat treatment. The presence of chilling injury was noticeable (0.5–1%) in peppers without HWD, and no decay incidence was observed during the first 14 days of storage.

After 21 days of storage, all three cultivars were characterized by higher losses at 10 °C, with weight loss ranging from 6.5% to 8.0% and decay incidence between 10.5% and 20.0%. At 4 °C without HWD, chilling injury was evident and varied between 5% and 8%, depending on the cultivar. HWD reduced both chilling injury and decay incidence compared to untreated fruits. Prolonged storage of ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’ for 21 days was only feasible at 4 °C with HWD, maintaining marketable quality (general appearance > 3). However, the ’Grkinja Babura’ cultivar did not preserve freshness and quality for 21 days at 4 °C, regardless of HWD (general appearance ≤ 2.8).

3.3. Comparative Histological Analysis of Pericarp

Pericarp cross-sections of the three pepper cultivars were analyzed using permanent (Figure 5) and temporary (Figure 6) slides prepared for light microscopy. The structural components analyzed included the cuticle (c), epidermis (e), collenchyma (col), parenchyma (par), and chromoplasts (chr), with particular attention given to their organization, thickness, and distribution to establish a correlation with postharvest quality and water loss.

In ‘Kurtovska Ajvaruša’ (Figure 5A and Figure 6A), the pericarp exhibited a well-developed and continuous cuticle layer, underlain by a compact epidermis and several layers of thick-walled collenchyma cells. The parenchyma tissue was dense, with tightly packed cells contributing to enhanced mechanical strength and reduced transpirational water loss. The chromoplasts in this cultivar were predominantly localized to the peripheral regions of the parenchyma cells, suggesting efficient pigment sequestration near the plasma membrane that may contribute to greater color stability during storage.

‘Grkinja Babura’ (Figure 5B and Figure 6B) demonstrated a similar pericarp architecture, with a moderately thick cuticle and collenchyma layers. However, chromoplast distribution within the parenchyma exhibited a distinct pattern. Chromoplasts were dispersed throughout the cytoplasm, with localized accumulations in the central regions of the parenchyma cells. This dispersed, yet partially centralized chromoplast localization may indicate cultivar-specific differences in plastid positioning and metabolic compartmentalization, potentially influencing pigment retention and antioxidant dynamics during storage. The dense tissue structure observed in both cultivars, coupled with specific chromoplast positioning, suggests improved capacity for maintaining postharvest quality, particularly in terms of water retention and visual appearance.

In contrast to the other cultivars, ‘Duga Bela Ljuta’ (Figure 5C and Figure 6C) exhibited a markedly thinner, less differentiated, and continuous cuticle, accompanied by fewer collenchyma cell layers and a loosely arranged parenchyma. This anatomical structure of ‘Duga Bela Ljuta’ likely contributes to increased postharvest weight loss, as the reduced tissue compactness and cuticle integrity may diminish its ability to serve as an effective barrier against transpiration. Moreover, chromoplasts in ‘Duga Bela Ljuta’ were predominantly localized in the upper tissue layers, particularly near the epidermis, with noticeably lower abundance in the deeper parenchyma. This superficial accumulation may reflect localized pigment biosynthesis or stress-induced plastid positioning, but it also suggests a potential for uneven pigment distribution and reduced color stability during storage. Together, these structural and subcellular characteristics highlight the cultivar’s relative susceptibility to postharvest dehydration and pigment degradation.

The observed structural differences among the cultivars strongly correlate pericarp anatomy with postharvest behavior. A thicker cuticle and more developed collenchyma in ‘Kurtovska Ajvaruša’ and ‘Grkinja Babura’ likely contribute to better water content retention and firmness, while the anatomical limitations of ‘Duga Bela Ljuta’ may underlie its higher susceptibility to postharvest deterioration and water loss (Table 1 and Table 3, respectively). These histological differences underscore the role of pericarp structure and chromoplast distribution in maintaining postharvest quality and highlight the importance of cultivar-specific anatomical traits for breeding and postharvest handling strategies in traditional pepper cultivars.

3.4. Color Changes

The effects of different storage conditions on the color parameters (L*, C, and ΔE*) of pepper cultivars are presented in

Table 2. Changes in fruit color parameters (L*, C, and ΔE*) of three traditional Serbian pepper cultivars exposed to different postharvest conditions (10 °C, 4 °C, and 4 °C with HWD) for 21 days and after an additional 3-day shelf life.

	L*			C			ΔE*
Days	Kurtovska Ajvaruša
0	35.6 ab	32.7 a	36.6 b	34.9 abcde	34.4 abcd	30.6 a	N.D.	N.D.	N.D.
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	36.3 b	36.3 b	36.9 bc	33.8 abc	34.2 bcde	32.5 ab	4.5	1.0	3.2
21 + 3	36.2 bcd	37.1 b	36.3 ab	30.6 a	33.1 abc	32.0 ab	6.7	2.6	3.7
	Grkinja Babura
0	40.5 cdef	42.7 efg	43.7 fg	47.8 k	43.5 ij	44.5 k	N.D.	N.D.	N.D.
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	40.9 ef	44.2 fg	45.5 g	39.3 efghi	42.7 hij	42.7 hij	10.6	3.2	3.8
21 + 3	40.5 cdef	43.3 fg	44.3 fg	36.1 bcde	38.4 defgh	40.8 fghij	13.7	6.8	6.1
	DugaBela Ljuta
0	40.4 cde	38.9 bcde	40.7 cde	41.1 ghij	35.7 bcde	38.5 defgh	N.D.	N.D.	N.D.
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	43.6 ef	40.9 ef	41.4 ef	38.4 defgh	36.5 bcdef	37.3 cdefg	5.1	2.4	2.4
21 + 3	41.1 de	42.3 efg	41.5 ef	35.0 abcde	37.2 cdefg	35.3 bcde	8.2	3.9	4.1
Treatment (T)		*			NS
Day (D)		*			**
Variety (V)		**			**
T × D		NS			**
T × V		*			**
D × V		NS			NS
T × D × V		NS			NS

Values designated with different letters are significantly different (p < 0.05). Statistical significance for effects: * p < 0.05; ** p < 0.01; NS = not significant. L* (lightness): the lightness of the color on a scale from 0 (black) to 100 (white); higher L* values indicate lighter colors. C (chroma or saturation): the intensity or purity of the color. ΔE* (color difference): the total color difference between two samples, calculated from changes in L*, a*, and b* values; a higher ΔE* value means a greater color change. N.D.—not determined; ΔE* was not determined on day 0, as this time point served as the initial color reference for subsequent comparisons/calculations (on the 21st day and on 21 + 3 days).

. For all three traditional cultivars, noticeable color differences (ΔE*) were observed, especially after 21 + 3 days of shelf life, with the highest ΔE* at 10 °C. The storage at 4 °C generally contributed to maintaining the color stability (lower ΔE*), with the exception of ‘Grkinja Babura’ at the end of shelf life.

3.5. Fruit Composition

The water content of the peppers was significantly influenced by both cultivar and treatment (

Table 3. Changes in fruit water content (%) and ASTA units in three traditional Serbian pepper cultivars during 21 days of storage and 3-day shelf life under different postharvest treatments.

	Water Content (%)			ASTA Units
Days	Kurtovska Ajvaruša
0	92.3 ghi			18.4 ij
	10 °C	4 °C	4°C HWD	10 °C	4 °C	4°C HWD
21	91.8 bcd	92.3 hi	92.2 fghi	21.2 k	12.5 cd	15.7 fg
21 + 3	91.7 bc	91.9 cde	91.8 cde	28.2 l	19.6 jk	16.2 fgh
	Grkinja Babura
0	92.8 j			10.0 ab
	10 °C	4 °C	4°C HWD	10 °C	4 °C	4°C HWD
21	92.4 hi	92.4 hi	92.7 j	17.1 ghi	9.3 a	11.3 bc
21 + 3	92.3 fghi	92.2 fghi	92.8 j	18.0 hij	12.7 cd	12.9 cd
	DugaBela Ljuta
0	92.8 j			12.9 cde
	10 °C	4 °C	4°C HWD	10 °C	4 °C	4°C HWD
21	92.1 efgh	92.0 def	92.2 fghi	11.6 bc	17.9 hij	14.8 ef
21 + 3	91.1 a	91.6 b	92.0 defg	28.6 l	13.7 de	11.2 bc
Treatment (T)		**			**
Day (D)		**			**
Variety (V)		**			**
T × D		**			**
T × V		**			**
D ×V		**			**
T × D × V		**			**

Values designated with different letters are significantly different (p < 0.05). Statistical significance for effects: ** p < 0.01.

). ‘Kurtovska Ajvaruša’ had initial water content of 92.3%, which varied slightly between the different treatments and storage conditions. Fruits stored at 10 °C showed decrease, observed after 21 days of storage, without further decrease during shelf life. The fruits of ‘Kurtovska Ajvaruša’ stored at 4 °C did not show a decrease in water content. However, water content was significantly reduced after shelf life. At the end of the shelf life, there was no difference in water content between different treatments of ‘Kurtovska Ajvaruša’. At the beginning of experiment, ‘Grkinja Babura’ had higher water content (92.8%) than ‘Kurtovska Ajvaruša’. Contrary to the stored and untreated fruits, the HWD-treated fruits of ‘Grkinja Babura’ retained water content throughout the whole experiment. Initially, ‘Duga Bela Ljuta’ had the same water content as ‘Grkinja Babura’ (92.8%), but its water loss was similar to that of ‘Kurtovska Ajvaruša’.

The ASTA values for three pepper cultivars over different storage conditions are presented in Table 3. Initially, ‘Kurtovska Ajvaruša’ had an ASTA value of 18.4, which increased when stored at 10 °C for 21 + 3 days (28.2 units). ‘Grkinja Babura’ initially had the lowest ASTA value of 10.0 and showed significant increase after 21 + 3 days after all treatments, with the highest value at 10 °C (18.0 units). ‘Duga Bela Ljuta’ started at 12.9 units, displayed varying results, and after 21 + 3 days exhibited a significant increase at 10 °C (28.6 units). The results show that each cultivar responds differently to the treatments. All cultivars showed significant increase after 21 + 3 days at 10 °C. Compared to the initial value, HWD followed by 21 days of storage at 4 °C led to a decrease in ASTA in ‘Kurtovska Ajvaruša’, an increase in ‘Duga Bela Ljuta’, and no significant change in ‘Grkinja babura’. After 21 + 3 days, the ASTA values were lower in HWD fruits for ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’, while for ‘Grkinja babura’ they remained at the same level as in fruits stored at 4 °C. The interactions among treatment, time, and variety proved to be significant (p < 0.01) and highlight the complexity effects of storage conditions on pepper color quality.

At harvest, ‘Grkinja Babura’ had the lowest quinic acid content compared to the other two cultivars. After storage and, especially, after shelf life, the quinic acid content increased in all cultivars (

Table 4. Changes in quinic and succinic acid content (mg/100 g fresh weight) in three traditional Serbian pepper cultivars during 21 days of storage and subsequent 3-day shelf life under different postharvest treatments.

	Quinic (mg/100 g FW)			Succinic (mg/100 g FW)
Days	Kurtovska Ajvaruša
0	13.9 b			43.7 i
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	16.5 def	17.8 gh	16.8 ef	18.5 a	29.3 gh	25.0 cdefg
21 + 3	15.6 cd	16.1 de	20.0 k	27.8 fgh	27.6 fgh	23.2 bcdef
	Grkinja Babura
0	11.6 a			29.7 gh
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	18.6 hij	19.2 jk	19.0 ij	22.8 abcde	21.3 abcd	19.5 ab
21 + 3	17.1 fg	18.2 hij	18.8 hij	28.8 gh	28.3 gh	29.3 gh
	DugaBela Ljuta
0	14.6 b			19.5 ab
	10 °C	4 °C	4 °C HWD	10 °C	4 °C	4 °C HWD
21	21.5 l	22.4 l	18.0 ghi	23.7 bcdef	25.6 defgh	26.5 efgh
21 + 3	24.5 m	18.6 hij	15.6 cd	20.9 abc	26.0 efgh	25.4 defgh
Treatment (T)		**			*
Day (D)		**			**
Variety (V)		**			**
T × D		**			NS
T × V		**			NS
D × V		**			**
T × D × V		**			**

Values designated with different letters are significantly different (p < 0.05). Statistical significance for effects: * p < 0.05; ** p < 0.01; NS = not significant.

). However, the effect of shelf life on this acid depended on cultivar and storage time. After storage at 10 °C and subsequent three days of shelf life, the level of quinic acid increased in ‘Duga Bela Ljuta’, decreased in ‘Grkinja babura’, while in ‘Kurtovska Ajvaruša’ it showed no change. Interestingly, HWD followed by 4 °C storage led to an increase in the quinic acid content for ‘Kurtovska Ajvaruša’.

Succinic acid levels exhibited significant variation across treatments, days, and cultivars, although not all interactions were statistically significant (Table 4). ‘Kurtovska Ajvaruša’ displayed the highest initial succinic acid content compared to the other two cultivars. Despite initial fluctuations in the level of succinic acid, there was no significant difference in its content after the storage and shelf life among the treatments, with the exception of ‘Duga Bela Ljuta’ which was stored at 10 °C and had a notably lower content.

3.6. Principal Component Analysis (PCA)

For ‘Kurtovska Ajvaruša’ PC1 explains 47.33% of the variance, while PC2 adds 35.59% (Figure 7). Together, they account for 82.92% of the total variance, and thus capture the largest part of the variability in the data. PC1 is driven by water content (0.84) and succinic acid (0.87), with positive loadings, and lightness L (−0.80), with a negative loading, thus contrasting water content with the previously mentioned two factors. PC2 highlights the ASTA units (0.89) positively and quinic acid (−0.70) negatively, differentiating samples by these factors. The case coordinates in PCA space reveal distinct groupings based on storage duration. The ‘D0’ sample (beginning of storage) scores high on PC1 and PC2, indicating high water content, succinic acid, and ASTA units and low quinic acid. The ‘D21 T10’ sample (21 days of cold storage on 10 °C) scores high on PC2, suggesting increased chromaticity C and altered quinic acid. The ‘D213 T10’ sample (21 days cold storage + 3 days shelf life at 10 °C) scores high on PC2, showing higher ASTA and lower quinic acid. After cold storage and shelf life, all fruits (‘D213 T4’, ‘D213 T10’, ‘D213 HWD’) score negatively on multiple components, reflecting lower water content, lightness, ASTA, and variations in succinic and quinic acid.

The combination of PC1 (53.84%) and PC2 (29.55%) explains 83.39% of the total variance, suggesting that these two components are sufficient to describe most of the variability in the data for ‘Grkinja Babura’ (Figure 7). PC1 is heavily influenced by the lightness parameter L (−0.91), water content (−0.84), and ASTA units (0.88). This suggests that PC1 primarily reflects a contrast between lightness and water content versus ASTA units, with higher ASTA units being associated with lower lightness and water content. PC2 is dominated by a high positive loading on quinic acid (0.97) and a strong negative loading on succinic acid (−0.62). This indicates that PC2 differentiates between samples with high quinic acid content and those with high succinic acid levels. Regarding treatments, the case coordinates for ‘Grkinja Babura’ in the PCA space show distinct groupings based on storage duration. For example, the sample ’D0‘ (beginning of storage) exhibits a high negative score on PC2, indicating lower quinic acid and higher succinic acid content. The samples after 21 days of cold storage have a moderate positive score on PC2, suggesting increased quinic acid content. After cold storage and shelf life, the sample stored at 4 °C shows a strong positive score on PC1, reflecting a profile with higher ASTA units and lower water content and lightness. The HWD-treated fruits after 21 days and 21 + 3 days stand out with a positive score on PC2 and a negative score on PC1, indicating stability during shelf life for quinic acid and ASTA units, along with lightness and water content.

The first two PCs explain 73.23% of the total variance (47.75% PC1 and 25.49% PC2) for ‘Duga Bela Ljuta’ (Figure 7). Among the factors, PC1 shows strong negative loadings for ASTA units (−0.91), quinic acid (−0.89), and lightness (−0.74). This indicates that PC1 captures a significant contrast between high ASTA units and high quinic acid content versus water content. PC2 is primarily associated with a high positive loading on succinic acid (0.90) and a negative loading on chromaticity C (−0.65). PC2 differentiates between samples with high succinic acid content and those with higher chromaticity. For the treatments of ‘Duga Bela Ljuta’, the case coordinates in the PCA space reveal distinct patterns based on storage conditions. For example, at the beginning of storage, the sample shows a high positive score on PC1, indicating higher ASTA units and quinic acid content. The fruits after 21 days of cold storage have a moderate positive score on PC2, reflecting an increase in succinic acid content during storage. However, the fruits after 21 days of cold storage at 4 °C and 3 days of shelf life display a significant negative score on PC1, suggesting a reduction in ASTA units and quinic acid over time. HWD-treated fruits after shelf life also show a complex profile, with significant contributions from water content and chromaticity, as indicated by their positive score on PC1.

The cumulative variance explained by the first two components is similar in all three traditional cultivars, suggesting a comparable level of complexity in the relationships between variables. ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’ share a common influence of water content and ASTA units on PC1, though the direction of influence is different. ‘Grkinja Babura’ shows a more balanced influence of lightness, ASTA units, and chromaticity on PC1. ‘Grkinja Babura’ and ‘Duga Bela Ljuta’ both feature succinic acid prominently in PC2, while ‘ Kurtovska Ajvaruša’ does not. Chromaticity appears on PC2 for ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’, suggesting it plays a secondary role in these traditional cultivars, while it is more influential on PC1 for ‘Grkinja Babura’. ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’ show clear differentiation between initial and final storage stages, with water content and ASTA units being key factors. ‘Grkinja Babura’ exhibits more complex shifts, indicating a broader range of influences during storage.

4. Discussion

In our experiment, HWD at 55 °C for 1 min enhanced the postharvest quality of traditional pepper cultivars by reducing chilling injury and decay during cold storage. Among the tested varieties, ‘Grkinja Babura’ showed superior storage performance, maintaining low weight loss (5.2%) and high water retention while ‘Duga Bela Ljuta’ proved most susceptible to deterioration under suboptimal storage. PCA confirmed cultivar-specific responses to HWD treatment that directly support our biological conclusions about anatomical influences on storage behavior. ‘Grkinja Babura’ showed the most favorable PCA profile, with improved color stability and elevated quinic acid, supporting its storage performance, likely due to its robust pericarp structure. ‘Duga Bela Ljuta’ displayed mixed PCA responses, reflecting its variable storage behavior and highest susceptibility to deterioration, correlating with its thinner cuticle, as observed histologically. These multivariate patterns demonstrate that cultivar-specific anatomical and biochemical differences directly influence HWD treatment efficacy, providing practical guidance for selecting appropriate postharvest treatments based on cultivar characteristics. The observed cultivar differences can be explained by their structural and biochemical characteristics. The thin cuticle of ‘Duga Bela Ljuta’ (Figure 5) likely increased its susceptibility to water loss and chilling injury compared to ‘Kurtovska Ajvaruša’ and ‘Grkinja babura’, which have a thicker cuticle that aids water retention and stress resistance, as noted by Parsons et al. [26]. Bearing in mind that the main purpose of the fruit cuticle is to reduce water loss and protect against biotic and abiotic factors [27], the results obtained are in line with previous data on the close relationship between postharvest water loss of pepper and cuticle thickness [28].

Furthermore, the beneficial effects of HWD at 55 °C for 1 min involve multiple interconnected biochemical pathways that collectively enhance stress tolerance. Heat treatment at this temperature range induces heat shock proteins (HSPs), particularly HSP70 and HSP90, which act as molecular chaperones protecting cellular proteins from denaturation during subsequent cold stress [29]. At the membrane level, brief heat exposure alters membrane lipid composition, increasing the proportion of unsaturated fatty acids and maintaining membrane fluidity during cold storage [30]. This mechanism explains the reduced presence of chilling injury, as membrane integrity is crucial for maintaining cellular compartmentalization and preventing ion leakage, characteristic of chilling injury symptoms.

Fresh pepper storage requires high relative humidity, but increased humidity raises the risk of microbial decay [11]. While chemical treatments, including hydrogen peroxide [31], potassium bicarbonate [32], aqueous chlorine dioxide [33], acidified sodium chlorite [34], and lactic acid [35] control contamination, HWD offers a simpler, non-chemical alternative [20]. Dipping at 50 °C for 3 min prevented Alternaria alternata and Botrytis cinerea decay without compromising quality [20]. Commercial hot water rinsing with brushing at 55 °C for 12 sec improved appearance, removed contaminants, and sealed epidermal cracks through cuticular wax redistribution [17]. Beyond microbial control, HWD induces defense responses reducing chilling injury. González-Aguilar et al. [36] showed that treatment at 53 °C for 4 min alleviated chilling injury in peppers, with improved outcomes when combined with polyethylene packaging [36,37]. Since visual appearance impacts consumer behavior [38], HWD’s preservation benefits are valuable.

Research indicates that HWD at 50–55 °C for 1–12 sec or up to 5 min significantly reduces chilling injury and microbial decay in pepper varieties such as ‘Yecla’ and ‘Blondy’ [39]. Peppers treated at 55 °C for 12 sec maintained superior firmness and minimized tissue browning during 3–8 °C storage, improving durability by 20–30% compared to controls [39]. Our treatment conditions (55 °C for 1 min) fall within this effective range. However, excessive exposure above 55 °C for longer periods may cause cellular damage and increase weight loss in Capsicum annuum [40]. Comparative analyses reveal consistent antioxidant benefits, with HWD enhancing ascorbate and glutathione levels to mitigate oxidative stress, as demonstrated by reduced hydrogen peroxide accumulation in treated red sweet peppers during cold storage [18]. These results align with Maalekuu et al.’s [41] findings that yellow and orange cultivars are more susceptible to physiological degradation than red cultivars. Similarly, Ilić et al. [42] found higher weight loss in ‘Sweet bite orange’, resulting in soft fruits, high decay incidence, and lowest general appearance scores, below marketable limits among three mini pepper cultivars. This study confirmed that the HWD treatment decreased the incidence of chilling injury and decay in all the cultivars tested. Kantakhoo and Imahori [18] also found that HWD alleviates chilling injury during cold storage.

The findings have direct practical implications for growers working with traditional pepper cultivars. HWD at 55 °C for 1 min represents a simple, implementable treatment that can significantly improve storage outcomes for these valuable genetic resources. The treatment requires minimal equipment investment and can be easily adopted at farm level, making it particularly suitable for traditional producers seeking to enhance marketability without major infrastructure changes. Given the cultivar-specific responses observed, growers should consider matching storage strategies to individual cultivar characteristics—with ‘Grkinja Babura’ showing excellent potential for extended storage and ‘Duga Bela Ljuta’ requiring careful handling due to its anatomical limitations.

In our experiment for two traditional cultivars, ‘Kurtovska Ajvaruša’ and ‘Duga Bela Ljuta’, HWD showed no effect on the color change during storage, similarly to ‘Sweet Cayenne’ [43]. HWD preserved the red hue of ‘Kurtovska Ajvaruša’ during storage, unlike ‘7802 F1’s variations of L* and a* over two seasons [44]. Both previously reported and present results suggest that HWD-related color stability is most probably a cultivar-dependent trait.

While color stability enhances visual appeal, the sensory quality of peppers, particularly flavor, is equally influenced by storage conditions and treatments such as HWD. The taste of sweet peppers is determined by the sugar and organic acid contents [45]. Pepper flavor is a complex trait, influenced by environmental factors during growth and postharvest processing [9]. In that light, the levels of quinic and succinic acids in the examined traditional Serbian peppers were examined (Table 4). These acids contribute subtle tartness and flavor depth, comprising less than 10% of the total acid content [46,47]. The observed variations in organic acid content among cultivars align with the findings of García-Vásquez [10], who reported significant differences in phenolic and ascorbic acid profiles among traditional Capsicum landraces, highlighting the genetic basis for this variability. HWD increased quinic acid levels in ‘Grkinja Babura’, potentially improving flavor stability, while ‘Kurtovska Ajvaruša’ showed reduced ASTA values (the content and intensity of carotenoid pigments).

Subcellular changes also significantly influence the overall quality of fresh pepper fruit. The reduction in chilling injury following the HWD treatment observed in our study supports previous reports indicating that heat treatments can enhance antioxidant activity and membrane stability during cold storage [40]. HWD (45 °C, 15 min) reduced chilling injury in ‘Miogi’ sweet peppers stored at 6 °C for 21 days, likely via enhanced ascorbate-glutathione cycle activity [16]. Similarly, our HWD treatment (55 °C, 1 min) reduced chilling injury in traditional cultivars at 4 °C. Kantakhoo and Imahori [18] showed that HWD at 55 °C for 1 min reduced chilling injury and weight loss in ‘Habataki’ red sweet peppers stored at 10 °C for 4 weeks, potentially by boosting antioxidant defenses. Namely, this treatment resulted in the production of hydrogen peroxide and malondialdehyde, indicating reduced oxidative stress and lipid peroxidation, suggesting that more underlying biochemical changes occur after HWD [18].

This study has several limitations that should be acknowledged. The research focused on only three traditional Serbian pepper cultivars, representing a limited sample of traditional pepper diversity. The evaluation was mainly performed as it would be conducted on the market for traditional cultivars, based on general appearance. If other methods for evaluation were used, different results could be obtained. Additionally, this study was limited to a single growing season, one HWD protocol (55 °C for 1 min), and a 21-day storage period, without assessment of economic feasibility or longer-term commercial storage requirements.

The results of this study suggest several directions for future research aimed at improving the postharvest management of traditional pepper cultivars. Further optimization of HWD is needed, such as testing different temperatures (45–60 °C range) and exposure times (30 sec to 5 min) and, from a practical point of view, investigating the combination of HWD with low-tech solutions directly at producers’ farms, along with an assessment of economic feasibility. Additionally, extending the analysis of biochemical and nutritional profiles, including volatile compounds, capsaicinoids, and antioxidant enzymes, can provide deeper insights into how HWD and storage conditions affect flavor, aroma, and health-related qualities. Multi-season evaluations and extension to additional traditional Serbian and regional pepper cultivars would strengthen the broader applicability of these findings. Linking anatomical features, such as cuticle thickness and pericarp structure, to postharvest performance could also inform breeding programs which target improved storability through commercial-scale validation studies.

5. Conclusions

This study showed that storage temperature, duration, and HWD significantly influence the postharvest quality of traditional Serbian pepper cultivars. Among the tested varieties, ‘Grkinja Babura’ showed the best postharvest performance, maintaining low weight loss and high water retention, while ‘Duga Bela Ljuta’ proved most susceptible to deterioration under suboptimal storage. HWD at 55 °C for 1 min improved general appearance, reduced chilling injury and decay incidence, and, in some cases, contributed to better water content stabilization, particularly during cold storage, though effects varied by cultivar. Changes in organic acid profiles, especially the cultivar-specific trends in quinic and succinic acid contents, further highlight the differential metabolic responses to storage and treatment. Overall, the results highlight the potential of HWD as a simple, pre-storage treatment whose application could enhance the shelf life and marketability of traditional pepper cultivars, offering valuable insights for sustainable postharvest handling and preservation strategies.