Postharvest Quality of Granny Smith Apples: Interplay of Harvest Stage, Storage Duration, and Shelf-Life

Abstract

Apples are the most widely consumed temperate fruit worldwide and are often stored for long-term to ensure year-round availability. However, maintaining fruit quality during storage and subsequent shelf-life remain a significant postharvest challenge. This study investigated the combined effects of the harvest stage, cold storage duration, and shelf-life on the physico-chemical properties of Granny Smith apples. Key quality attributes including texture, maturity indices, color, and starch degradation were evaluated using instrumental methods and Raman microscopy. Fruit quality was affected differently by individual factors and their interactions. Texture parameters showed varied sensitivity: the harvest stage affected several parameters, storage duration had the strongest overall impact, shelf-life influenced a moderate number of parameters, and some were affected by combined factor interactions. Maturity indices were significantly influenced by all factors individually and combined. Color parameters were consistently affected by harvest stage and storage, with shelf-life and interactions influencing fewer parameters. These findings emphasize the complex interplay of factors shaping apple quality after harvest. The study demonstrates the importance of timing harvest and tailoring postharvest handling to maintain apple quality. It also demonstrates the potential of combining traditional and advanced techniques for effective ripeness monitoring.

1. Introduction

Apples (Malus domestica Borkh.) are among the most widely consumed and economically significant fruits globally, valued for their sensory qualities, nutritional composition, and long storage potential. To meet consumer demand throughout the year and reduce postharvest losses, apples are routinely stored under cold or controlled atmospheric conditions for several months [1].

Granny Smith is a late-maturing apple variety originally discovered in New South Wales, Australia, in the 19th century, by Maria Ann “Granny” Smith. The original tree was fruiting by 1868. It was a chance seedling that originated from spontaneous hybridization between Malus sylvestris (European wild apple cultivar ‘French Crab’) and Malus domestica (domesticated apple). Significant plantings of ‘Granny Smith’ in Australia were established in the 1920s, but it became important worldwide from the 1950s [2]. It is easily recognizable by its bright green skin, firm texture, tart-sweet flavor, and juiciness [3]. Due to its high acidity, firmness, and low ethylene production, Granny Smith apples are particularly well-suited for long-term storage [1]. The skin is smooth, thick, and tough, offering resistance to mechanical damage and bruising during handling and storage [4]. However, this variety is highly susceptible to superficial scald, especially when harvested prematurely [5].

Superficial scald is a physiological disorder that typically develops during extended cold storage [6] and is most common in early-harvested or less mature fruits, although it may also occur in fully mature apples [7]. It manifests as darkened or browned areas on the peel, resulting from damage to hypodermal cells beneath the epidermis, while deeper parenchymal tissues remain largely unaffected [6]. To combat the postharvest presence of scald, 1-methylcyclopropene (1-MCP) showed promising results as being the most resistant to scald [7]. Premium postharvest quality is generally achieved when fruits are harvested at a medium-green ground color and once starch disappearance is complete in the core region [4]. Moreover, the incidence of superficial scald as well as core flush tends to be higher in apples grown under warmer climatic conditions.

Apple quality is influenced not only by variety but also by many pre-harvest treatments and the level of maturity during harvest. Ripening affects key characteristics, such as flavor, appearance, firmness, and nutritional value, and plays a central role in determining postharvest performance, storability, and shelf-life [8]. The optimal harvest date (OHD) corresponds to a physiological stage in which the apple has reached sufficient biochemical development without entering advanced ripening [9]. This stage is critical for achieving a balance between fruit quality and extended shelf life.

Apple quality continues to improve after harvesting until the fruit reaches full ripeness, at which point it expresses optimal sensory properties for fresh consumption. After reaching this peak, the quality of apples gradually begins to decline as a result of continued physiological and biochemical processes. However, when stored under appropriate conditions, this deterioration can be significantly slowed. Storage refers to maintaining harvested produce in an environment that preserves its quality until it reaches the market [10]. Among the available methods, cold storage is considered the most effective for minimizing quality and quantity losses [11]. Depending on the variety, apples can be successfully stored for 4 to 10 months under optimal conditions; for example, chilling-insensitive Granny Smith can be stored at 0–1 °C in controlled atmospheric conditions, often combined with 1-MCP treatment to maintain quality over extended storage periods [12].

Determining the OHD is challenging due to the gradual and overlapping nature of ripening stages. Although terms like unripe, ripe, and overripe are widely used, they often rely on sensory cues. In practice, harvest readiness is commonly assessed using parameters such as firmness, starch degradation, and soluble solids content (SSC)—often integrated into indices like the Streif Index [9]. An early harvest can result in undeveloped fruit with reduced flavor and color, while a late harvest increases the risk of postharvest disorders such as scald, internal breakdown, and bitter rot [13]. Therefore, a precisely timed harvest and maturity assessment are essential for maintaining quality and minimizing losses during storage. Monitoring starch-to-sugar conversion, along with SSC and firmness, remains fundamental for ripeness classification and consumer acceptability [14].

Starch is the primary storage carbohydrate in woody plants and plays a key role in fruit development and ripening [15]. While histochemical staining with iodine–potassium iodide (I₂KI) enables the visual detection of starch through color contrast, quantification remains challenging due to variability in sample size and tissue distribution [16]. To overcome these limitations, image analysis under microscopy has been employed to estimate starch levels based on optical density rather than stained area, with successful application in several fruit species, most recently in sweet cherry [16].

The demand for rapid, non-destructive fruit quality assessments has led to the increased use of optical techniques. While refractometry remains a standard for measuring SSC, near-infrared spectroscopy (NIRS) and Raman microscopy are emerging as effective non-destructive alternatives [17]. Raman microscopy, in particular, offers molecular specificity and has proven to be effective in monitoring starch degradation and ripening-related changes in apples [18].

Maintaining apple quality during storage and marketing is crucial for consumer satisfaction and postharvest profitability. Given these considerations, the present study aimed to evaluate the combined effects of harvest maturity, cold storage duration, and shelf-life on the physico-chemical characteristics of Granny Smith apples. Through a comprehensive analysis of fruit texture, maturity indices, color, starch content using staining with I₂KI, and tissue structure using Raman microscopy, the research sought to provide insights into optimizing harvest and postharvest management practices for extended storability and improved eating quality.

2. Materials and Methods

2.1. Apple Variety, Experimental Conditions, and Experimental Design

The Granny Smith apples used in this study were harvested in 2024 from the commercial orchard “Zlatni Jazak” (Ruma, Serbia), located on the Fruska Gora mountain. Trees were planted in 2013 on M9 rootstock, trained in a slender spindle system, and spaced at 3.2 m × 0.8 m. Standard orchard management included winter pruning and chemical thinning (target crop load: 110–120 fruits per tree), drip irrigation with a water amount of 1.6 L/h per emitter spaced at 60 cm, and fertilization with annual inputs of 90 kg N, 150 kg K, and 15 kg P. Foliar applications of calcium and micronutrients were also performed. The trees remained in good health throughout the season.

Fruits were harvested at three harvest stages based on the anticipated harvest window for long-term storage: unripe (10 days before the optimal harvest date), ripe (optimal date), and overripe (10 days after the optimal date), as determined using the Streif Index [9] (Equation (1)):

$$\mathrm{S}\mathrm{I}=\mathrm{F}\mathrm{I}\mathrm{R}\mathrm{M}/(\mathrm{S}\mathrm{S}\mathrm{C}\times \mathrm{S}\mathrm{P}\mathrm{I})$$

(1)

We considered the fruit firmness (FIRM, N), the soluble solids content (SSC, %), and starch pattern index (SPI, scale 1–10) as key parameters. For Granny Smith, the SI at the optimal harvest date was 0.199, with a corresponding firmness of 7.39 ± 0.73 N, soluble solids content of 9.27 ± 0.25%, and starch pattern index of 4.00 ± 0.00 on a scale of 1–10.

At each harvest stage, all fruits of the experimental trees (three replicates, two trees per replicate) were harvested. One third of the harvested fruit was stored at room temperature, while the remaining two thirds were kept for two and four months at a temperature of +1 °C and a 95% relative humidity in a cold room with a regular atmosphere (RA), in which the oxygen and carbon dioxide content was not regulated and remained at ambient air levels (approximately 21% O₂ and 0.03% CO₂).

After harvesting and each storage interval, samples were transported within 2 h to the Faculty of Agriculture laboratory and stored at room temperature (up to 22 °C), with a relative humidity of approximately 50% during the shelf-life experiments. Throughout the 15-day shelf-life period, the quality of the apples was evaluated using the following methods: texture analysis, maturity indices analysis, color measurement, starch grains quantification, and Raman microscopy.

The randomized experimental design included the observation of three key factors:

• Harvest stage: unripe, ripe, and overripe.
• Storage duration: 0, 2, and 4 months.
• Shelf-life period: days 0 (within 24 h of harvesting and removal from storage), 5, 10, and 15.

These three factors and their interactions were considered in all subsequent analyses, except for starch grain quantification, where only two shelf-life testing days (0 and 15) were evaluated, to assess their individual and combined effects on fruit quality attributes.

2.2. Texture Analysis

Fruit texture was analyzed using a Brookfield CT3 Texture Analyzer (Brookfield Engineering, Middleboro, MA, USA), employing two different tests: Texture Profile Analysis (TPA) and a puncture test using a cylindrical probe.

For the TPA, the following parameters were applied: a trigger force of 10.0 g, speed of 2 mm/s, distance of 10 mm, a TA7 knife-edge probe (Ø 25.4 mm), and a data acquisition rate of 50 points/s. For the puncture test, a TA41 cylindrical probe (Ø 6 mm, 35 mm length) was used under the same conditions: 10 mm trigger, 10 mm distance, and 2 mm/s test speed. All measurements were performed under ambient conditions (up to 22 °C).

Each fruit sample was cut in half, and the skin and surface flesh layer were punctured transversely. The texture was assessed in five replicates per sample, according to the described experimental design, considering all factors and their interactions. This resulted in a total of 180 samples per method.

From these two tests, 16 textural parameters were extracted (

Table 1. List of textural parameters related to the texture profiling.

Textural Parameters	General Description	Unit
Hardness cycle 1	The peak force recorded during the first compression cycle; represents the firmness of the sample.	N
Deformation at hardness	The displacement at which the maximum force (hardness) occurs during the first cycle; reflects the sample’s resistance to deformation.	mm
Total work cycle 1	The area under the force–time curve during the first cycle; represents the total energy required to deform the sample in that cycle.	mJ
Load at target	The force measured at a predefined deformation distance; indicates the resistance of the fruit tissue at a standardized penetration depth.	N
Deformation at target	The actual distance at which the target force is reached or maintained; used to quantify tissue yield characteristics.	mm
Adhesiveness	The negative force area following the first compression, representing the work required to overcome attractive forces between the sample and the probe during probe withdrawal.	mJ
Quantity of fractures	The number of discrete fracture events (peaks) observed during the test; associated with crispness or fracture behavior of the fruit.	-
Fracturability	The force at the first significant fracture event, usually the first drop after initial peak; associated with the brittleness or initial cracking of the surface.	N
1st fracture work performed	The energy (area under the curve) expended up to the first fracture event; indicates how much work is needed to initiate breakdown of tissue structure.	mJ
1st fracture deformation	The deformation at which the first fracture occurs; gives insight into the elastic properties of the tissue before breaking.	mm
Hardness work cycle 2	The area under the curve of the second compression cycle, reflecting the sample’s resistance after deformation.	mJ
Cohesiveness	The ratio of the area under the second compression cycle to the first one. Indicates how well the fruit withstands a second deformation relative to the first—internal bonding strength.	-
Springiness	The distance the sample recovers in height between the end of the first and the start of the second compression; reflects the elastic recovery of the sample.	Mm
Gumminess	A derived parameter calculated as Hardness × Cohesiveness. Reflects the energy required to disintegrate a semisolid food.	N
Chewiness index	A compound parameter calculated as Hardness × Cohesiveness × Springiness. Reflects the energy needed to chew a solid sample until it is ready for swallowing.	e
Average peak load	The mean value of all force peaks observed during the compression; gives an average measure of internal structural resistance across the test.	N

). Parameter definitions were adapted from previous studies [19,20]. These parameters provided insights into the fruit’s resistance to compression, structure breakdown, and mastication behavior.

2.3. Maturity Indices Analysis

To assess fruit maturity, six replications per sample were performed according to the experimental design, considering all factors and their interactions. In total, 72 samples were examined for the following parameters: firmness (kg/cm²), total soluble solids content (%), starch pattern index (scale 1–10), total and reducing sugars (%), pH, titratable acidity (%), and superficial scald.

Firmness was measured on three replicates per harvest stage and test day (three fruits per replicate), using a handheld penetrometer (T.R. Turoni srl, Forli, Italy) with an 11 mm plunger. Measurements were taken on two opposite, peeled sides of the fruit, and results were expressed in kg/cm².

For the analysis of total soluble solids (SSC), juice was extracted from the fruit and measured using a digital refractometer Pocket PAL-1 (Atago Co., Ltd., Tokyo, Japan). Starch pattern index (SPI) was determined by immersing half of a longitudinally cut apple in a solution containing 10 g potassium iodide and 2.5 g iodine dissolved in 1 L of water for 5 s. The resulting starch pattern was rated on a scale from 1 (full starch presence) to 10 (complete starch hydrolysis).

Total and reducing sugar contents were determined using the Luff–Schoorl method [21]. pH was measured with a pH meter 6173 (Jenco Instruments, Inc., San Diego, CA, USA), and titratable acidity (TA) was assessed by titrating 10 mL of fresh juice diluted in 90 mL of distilled water with 0.1 N NaOH, following the method described by Dzamic [22].

Superficial scald was assessed visually, and values ranging from 0 to 10 were assigned based on its presence on the fruit surface, with 0 indicating no physiological disorder and 10 signifying 100% of the fruit surface covered with scald.

2.4. Color Measurement

Color measurements were performed on three replicates per sample, according to the experimental design, which encompassed all relevant factors. In total, 108 samples were examined. The color was measured on both the sun-exposed and shaded side of each apple using a spectrophotometer AMT529 (Amtast USA Inc., Lakeland, FL, USA) and expressed in CIELab coordinates: L*, a*, and b* values. The L* value represented lightness and ranged from 0 (black) to 100 (white), while a* ranged from green (negative values) to red (positive values), and b* from blue (negative) to yellow (positive) [23]. These parameters are commonly used to quantify color differences and monitor changes during storage and shelf life.

From these primary values, additional color parameters were calculated:

• Browning Index (Equation (2)), which estimates the purity of brown color in products containing sugar [24]:

$$\mathrm{B}\mathrm{I}=\left[100(\mathrm{x}-0.31)\right]/0.17$$

(2)

where (Equation (3))

$$\mathrm{x}=(a^{*}+1.75L^{*})/(5.645L^{*}+a^{*}-3.012b^{*})$$

(3)

• Yellow Index (Equation (4)), which is associated with the Browning Index [25]:

$$\mathrm{Y}\mathrm{I}=142.86(b^{*}/L^{*})$$

(4)

Color variation during storage and shelf-life was further assessed by calculating the Total Color Difference (ΔE) between each sample and a reference (L₀*, a₀*, b₀*), which corresponds to optimally ripen fruits measured on day 0 (Equation (5)) [24]:

$$∆E=\sqrt{{(a^{*}-a_0^{*})}^2+{(b^{*}-b_0^{*})}^2{+(L^{*}-L_0^{*})}^2}$$

(5)

ΔE values indicate the perceptibility of color changes by human observers: ΔE < 1: imperceptible; 1 < ΔE < 2: only noticeable by experienced observers; 2 < ΔE < 3.5: noticeable to untrained observers; 3.5 < ΔE < 5: clearly distinguishable; ΔE > 5: considered different colors altogether [23].

2.5. Starch Grains Quantification

The presence and distribution of starch grains were analyzed in histological sections of Granny Smith apple fruits, following the experimental design but limited to shelf-life days 0 and 15. For each combination, three replicates were examined, resulting in a total of 54 samples.

Apple fruits were cut into cubes (approximately 1 cm³), including the peel, and then fixed in 50% ethanol and stored at +4 °C until further analysis. Tissue pieces (3–5 mm³) were embedded in paraffin using the protocol described by Chen et al. [26]. Paraffin blocks were sectioned at 10 µm thickness using a Leica RM 2155 rotary microtome (Leica Microsystems GmbH, Wetzlar, Germany), and the sections were mounted on Polysine^® adhesion slides. Staining was performed with Lugol’s iodine solution (I₂KI) following the method of Fadon and Rodrigo [16] to visualize starch grains.

Microscopic examination and imaging were conducted using an OLYMPUS BX61 microscope (Olympus Corporation, Tokyo, Japan) in a bright-field mode. Quantification of starch grains was performed by measuring the optical density (OD) on black-and-white images using ImageJ software (version 1.54g, National Institutes of Health, Bethesda, MD, USA, 2023).

2.6. Raman Microscopy

Raman microscopy was employed to analyze structural and compositional changes in the mesocarp tissue of apple fruits, according to the experimental design. One representative fruit was analyzed for each combination of harvest stage, storage duration, and shelf-life testing day (0 and 15), resulting in a total of 18 samples.

Raman spectra were recorded using an XploRA Raman spectrometer microscope (Horiba Scientific, Kyoto, Japan), equipped with a 785 nm laser (maximum power 20–25 mW), a 1200 gr/mm grating, and a 50× long-working-distance objective. The acquisition time was 30 s per measurement. For each fruit, five spectra were collected from different regions of the mesocarp after the skin was removed using a standard vegetable peeler. All analyses were completed within 20 min per sample to minimize tissue oxidation due to air exposure.

2.7. Statistical Analysis

Statistical analysis was performed using IBM SPSS Statistics 17.0 (IBM Corporation, Armonk, NY, USA, 2009). A three-way between-subjects ANOVA was used to evaluate the effects of harvest stage (H), storage duration (S), and shelf-life period (D) on textural parameters, CIELab colorimetric values, and maturity indices. Interaction effects among the three factors (H × S, H × D, S × D, and H × S × D) were also examined. The Tukey–Kramer HSD test was applied to determine the statistical significance of the main effects and interactions at the level of p ≤ 0.05.

A Principal Component Analysis (PCA) was conducted to explore spectral variations obtained from Raman microscopy across different postharvest storage periods, harvest stages, and shelf-life durations and to detect potential outliers or systematic artifacts.

3. Results and Discussion

3.1. Textural Parameters

The texture analysis of the fruit of Granny Smith apples is presented in

Table 2. Texture profile analysis of Granny Smith apple fruits at different harvest stages, storage durations, and shelf-life periods.

	HC1	DH	DT	AD	FR	COH	SPR	GUM	CHI
Harvest stage (H)
Unripe	3.71 ± 4.32	9.87 ± 0.19 b	9.93 ± 0.02	0.82 ± 0.79	45.70 ± 10.62	0.02 ± 0.01	3.22 ± 2.68	0.17 ± 0.23 a	0.05 ± 0.12
Ripe	3.55 ± 3.63	9.92 ± 0.14 ab	9.93 ± 0.02	0.88 ± 0.83	43.08 ± 8.95	0.02 ± 0.02	2.96 ± 2.95	0.10 ± 0.17 b	0.04 ± 0.08
Overripe	3.05 ± 4.97	9.94 ± 0.08 a	9.92 ± 0.01	0.91 ± 0.76	42.81 ± 9.04	0.02 ± 0.02	2.87 ± 2.18	0.09 ± 0.13 b	0.04 ± 0.06
Storage duration (S)
0 month	4.80 ± 4.00 a	9.90 ± 0.16	9.93 ± 0.02	0.86 ± 0.58	46.36 ± 9.65 a	0.02 ± 0.02	3.62 ± 2.78 a	0.20 ± 0.23 a	0.08 ± 0.12 a
2 months	3.24 ± 5.24 ab	9.91 ± 0.14	9.93 ± 0.02	0.87 ± 0.88	44.25 ± 7.80 ab	0.02 ± 0.02	3.00 ± 2.36 ab	0.09 ± 0.15 b	0.03 ± 0.05 b
4 months	2.27 ± 3.16 b	9.92 ± 0.13	9.93 ± 0.01	0.88 ± 0.89	40.98 ± 10.53 b	0.02 ± 0.02	2.43 ± 2.58 b	0.08 ± 0.14 b	0.03 ± 0.07 b
Shelf-life period (D)
Day 0	2.94 ± 3.46	9.88 ± 0.17	9.92 ± 0.01	0.84 ± 0.72	44.76 ± 10.31	0.02 ± 0.02	3.32 ± 3.08	0.15 ± 0.24	0.06 ± 0.15
Day 5	4.25 ± 6.29	9.90 ± 0.17	9.93 ± 0.02	0.85 ± 0.82	44.61 ± 8.87	0.02 ± 0.02	3.20 ± 2.41	0.13 ± 0.17	0.05 ± 0.07
Day 10	3.90 ± 3.87	9.93 ± 0.11	9.92 ± 0.02	0.88 ± 0.78	44.10 ± 10.52	0.02 ± 0.02	2.85 ± 2.84	0.11 ± 0.13	0.04 ± 0.07
Day 15	2.67 ± 2.80	9.94 ± 0.11	9.93 ± 0.02	0.90 ± 0.86	41.98 ± 8.63	0.02 ± 0.01	2.69 ± 2.03	0.10 ± 0.19	0.03 ± 0.05
H × S × D
Unripe × 0 month × Day 0	5.57 ± 6.96	9.75 ± 0.28	9.92 ± 0.01	0.78 ± 0.54	48.93 ± 12.00	0.03 ± 0.01	5.09 ± 3.06	0.56 ± 0.40	0.24 ± 0.35
Unripe × 0 month × Day 5	7.76 ± 7.48	9.82 ± 0.26	9.95 ± 0.05	1.10 ± 0.33	52.44 ± 9.22	0.03 ± 0.01	2.71 ± 2.18	0.35 ± 0.29	0.08 ± 0.06
Unripe × 0 month × Day 10	5.38 ± 2.56	9.95 ± 0.03	9.92 ± 0.00	1.28 ± 0.41	49.44 ± 13.07	0.03 ± 0.02	4.91 ± 3.58	0.21 ± 0.14	0.10 ± 0.13
Unripe × 0 month × Day 15	1.99 ± 1.60	9.89 ± 0.16	9.93 ± 0.03	0.50 ± 0.52	46.56 ± 8.73	0.03 ± 0.01	3.11 ± 1.95	0.05 ± 0.07	0.03 ± 0.05
Unripe × 2 months × Day 0	5.62 ± 5.45	9.91 ± 0.12	9.93 ± 0.02	1.44 ± 0.97	51.24 ± 4.99	0.02 ± 0.01	1.89 ± 1.53	0.29 ± 0.28	0.02 ± 0.02
Unripe × 2 months × Day 5	3.34 ± 1.12	9.97 ± 0.04	9.91 ± 0.01	2.12 ± 1.01	41.76 ± 11.18	0.02 ± 0.02	4.52 ± 3.33	0.07 ± 0.08	0.06 ± 0.08
Unripe × 2 months × Day 10	4.69 ± 5.59	9.96 ± 0.04	9.93 ± 0.03	0.84 ± 0.89	45.63 ± 6.12	0.03 ± 0.02	3.54 ± 2.93	0.14 ± 0.10	0.05 ± 0.07
Unripe × 2 months × Day 15	1.33 ± 0.98	9.82 ± 0.22	9.93 ± 0.01	0.22 ± 0.16	43.62 ± 8.39	0.02 ± 0.01	3.23 ± 3.00	0.03 ± 0.03	0.01 ± 0.01
Unripe × 4 months × Day 0	0.90 ± 0.26	9.78 ± 0.30	9.92 ± 0.01	0.26 ± 0.15	38.99 ± 5.72	0.01 ± 0.00	1.33 ± 0.60	0.09 ± 0.10	0.00 ± 0.00
Unripe × 4 months × Day 5	1.37 ± 0.72	9.94 ± 0.08	9.93 ± 0.01	0.70 ± 0.79	50.36 ± 11.23	0.02 ± 0.01	3.74 ± 2.08	0.03 ± 0.03	0.01 ± 0.02
Unripe × 4 months × Day 10	3.47 ± 5.72	9.77 ± 0.28	9.92 ± 0.00	0.56 ± 0.42	37.75 ± 13.63	0.02 ± 0.01	2.96 ± 3.93	0.20 ± 0.24	0.01 ± 0.02
Unripe × 4 months × Day 15	3.14 ± 1.61	9.93 ± 0.08	9.92 ± 0.01	0.08 ± 0.08	41.63 ± 15.84	0.01 ± 0.01	1.59 ± 1.81	0.05 ± 0.04	0.03 ± 0.03
Ripe × 0 month × Day 0	2.79 ± 1.96	9.91 ± 0.18	9.92 ± 0.01	1.12 ± 0.56	40.55 ± 9.27	0.02 ± 0.01	3.86 ± 4.86	0.06 ± 0.11	0.04 ± 0.07
Ripe × 0 month × Day 5	6.38 ± 4.65	9.85 ± 0.24	9.94 ± 0.03	0.34 ± 0.45	45.13 ± 9.30	0.03 ± 0.03	2.52 ± 2.46	0.22 ± 0.21	0.06 ± 0.06
Ripe × 0 month × Day 10	7.75 ± 3.95	9.97 ± 0.04	9.92 ± 0.02	1.14 ± 0.57	41.62 ± 4.14	0.02 ± 0.02	3.24 ± 3.96	0.17 ± 0.12	0.07 ± 0.09
Ripe × 0 month × Day 15	3.42 ± 1.95	9.95 ± 0.05	9.92 ± 0.01	0.84 ± 0.92	46.97 ± 7.73	0.02 ± 0.01	3.14 ± 1.01	0.12 ± 0.09	0.06 ± 0.06
Ripe × 2 months × Day 0	3.26 ± 2.63	9.93 ± 0.04	9.92 ± 0.01	1.12 ± 0.84	49.66 ± 5.17	0.01 ± 0.01	1.59 ± 0.67	0.03 ± 0.05	0.01 ± 0.01
Ripe × 2 months × Day 5	1.80 ± 1.32	9.74 ± 0.32	9.92 ± 0.00	0.24 ± 0.09	39.52 ± 5.57	0.03 ± 0.04	2.99 ± 3.91	0.09 ± 0.16	0.04 ± 0.09
Ripe × 2 months × Day 10	2.20 ± 2.25	9.95 ± 0.04	9.92 ± 0.01	0.62 ± 0.83	40.11 ± 6.11	0.01 ± 0.00	1.93 ± 1.45	0.03 ± 0.03	0.00 ± 0.01
Ripe × 2 months × Day 15	1.71 ± 0.97	9.97 ± 0.05	9.93 ± 0.01	0.26 ± 0.24	44.85 ± 9.48	0.02 ± 0.01	3.45 ± 2.14	0.15 ± 0.29	0.04 ± 0.06
Ripe × 4 months × Day 0	2.08 ± 1.76	9.96 ± 0.04	9.93 ± 0.02	1.06 ± 0.93	43.34 ± 16.88	0.03 ± 0.03	4.66 ± 4.42	0.12 ± 0.20	0.10 ± 0.21
Ripe × 4 months × Day 5	3.78 ± 4.57	9.94 ± 0.06	9.92 ± 0.02	1.28 ± 1.11	39.16 ± 7.19	0.01 ± 0.01	3.84 ± 3.25	0.06 ± 0.06	0.01 ± 0.01
Ripe × 4 months × Day 10	2.81 ± 4.49	9.91 ± 0.07	9.92 ± 0.01	0.68 ± 1.30	44.75 ± 14.92	0.01 ± 0.00	1.27 ± 0.97	0.04 ± 0.07	0.00 ± 0.01
Ripe × 4 months × Day 15	4.67 ± 6.48	9.97 ± 0.05	9.94 ± 0.03	1.86 ± 0.26	41.27 ± 6.44	0.02 ± 0.02	3.08 ± 3.84	0.17 ± 0.35	0.06 ± 0.10
Overripe × 0 month × Day 0	3.13 ± 2.07	9.90 ± 0.18	9.92 ± 0.00	0.72 ± 0.43	39.34 ± 12.39	0.03 ± 0.01	6.88 ± 2.27	0.12 ± 0.11	0.07 ± 0.06
Overripe × 0 month × Day 5	3.29 ± 2.21	9.92 ± 0.11	9.92 ± 0.01	0.76 ± 0.62	48.20 ± 4.11	0.02 ± 0.00	3.86 ± 1.02	0.22 ± 0.19	0.13 ± 0.09
Overripe × 0 month × Day 10	5.24 ± 2.96	9.95 ± 0.03	9.92 ± 0.02	1.22 ± 0.61	57.21 ± 3.76	0.01 ± 0.01	1.63 ± 0.79	0.07 ± 0.08	0.01 ± 0.02
Overripe × 0 month × Day 15	4.93 ± 3.11	9.97 ± 0.04	9.93 ± 0.01	0.52 ± 0.28	39.87 ± 7.61	0.02 ± 0.01	2.43 ± 1.44	0.23 ± 0.30	0.04 ± 0.03
Overripe × 2 months × Day 0	2.21 ± 2.25	9.85 ± 0.12	9.92 ± 0.01	0.78 ± 0.92	51.07 ± 7.88	0.02 ± 0.01	3.24 ± 1.65	0.05 ± 0.05	0.02 ± 0.02
Overripe × 2 months × Day 5	9.14 ± 15.70	9.97 ± 0.05	9.92 ± 0.01	0.40 ± 0.39	39.25 ± 7.09	0.03 ± 0.02	2.25 ± 1.48	0.08 ± 0.09	0.05 ± 0.07
Overripe × 2 months × Day 10	1.91 ± 1.20	9.91 ± 0.08	9.93 ± 0.01	0.68 ± 0.88	46.96 ± 4.36	0.03 ± 0.03	4.60 ± 3.20	0.09 ± 0.12	0.05 ± 0.07
Overripe × 2 months × Day 15	1.69 ± 1.63	9.95 ± 0.09	9.94 ± 0.03	1.74 ± 0.46	37.31 ± 2.57	0.02 ± 0.03	2.79 ± 0.47	0.07 ± 0.13	0.03 ± 0.05
Overripe × 4 months × Day 0	0.85 ± 0.42	9.95 ± 0.04	9.92 ± 0.00	0.32 ± 0.31	39.70 ± 8.09	0.02 ± 0.01	1.36 ± 1.54	0.03 ± 0.03	0.01 ± 0.01
Overripe × 4 months × Day 5	1.39 ± 0.88	9.94 ± 0.05	9.92 ± 0.01	0.74 ± 0.56	45.65 ± 6.43	0.03 ± 0.02	2.42 ± 1.43	0.05 ± 0.06	0.03 ± 0.04
Overripe × 4 months × Day 10	1.63 ± 1.39	9.97 ± 0.04	9.93 ± 0.01	0.92 ± 0.96	33.39 ± 5.26	0.02 ± 0.02	1.57 ± 2.05	0.06 ± 0.08	0.02 ± 0.04
Overripe × 4 months × Day 15	1.14 ± 0.67	9.98 ± 0.04	9.93 ± 0.01	2.12 ± 0.53	35.74 ± 3.64	0.02 ± 0.01	1.40 ± 1.03	0.03 ± 0.03	0.02 ± 0.03
Significance
H	n.s.	*	n.s.	n.s.	n.s.	n.s.	n.s.	*	n.s.
S	**	n.s.	n.s.	n.s.	**	n.s.	*	***	**
D	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
H × S	n.s.	n.s.	n.s.	***	n.s.	n.s.	n.s.	n.s.	n.s.
H × D	n.s.	n.s.	n.s.	***	n.s.	n.s.	n.s.	**	n.s.
S × D	n.s.	n.s.	*	**	**	n.s.	n.s.	n.s.	n.s.
H × S × D	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.

Legend: HC1—Hardness Cycle 1 (N); DH—Deformation at Hardness (mm); DT—Deformation at Target (mm); AD—Adhesiveness (mJ); FR—Fracturability (N); COH—Cohesiveness; SPR—Springiness (mm); GUM—Gumminess (N); CHI—Chewiness index (N). Parameters are reported as mean ± standard deviation (Mean ± StD). Different letters within columns indicate significant mean differences according to Tukey HSD test (p ≤ 0.05). n.s., *, **, and *** denote non-significant or significant effects at p < 0.05, 0.01, and 0.001, respectively.

and

Table 3. Instrumental textural properties of Granny Smith apple fruits at different harvest stages, storage durations, and shelf-life periods.

	TWC1	LT	DT	AD	FWP	APL
Harvest stage (H)
Unripe	117.19 ± 19.84 a	9.84 ± 2.14 a	9.91 ± 0.03	1.95 ± 0.56	15.09 ± 32.07	26.44 ± 4.37
Ripe	115.63 ± 17.02 a	9.56 ± 1.72 a	9.90 ± 0.06	1.90 ± 0.63	15.08 ± 37.02	26.69 ± 4.59
Overripe	108.70 ± 22.78 b	8.83 ± 2.04 b	9.91 ± 0.04	2.05 ± 0.80	18.76 ± 38.74	25.93 ± 4.60
Storage duration (S)
0 month	134.03 ± 10.82 a	11.53 ± 1.32 a	9.89 ± 0.08 b	1.74 ± 0.46 b	9.26 ± 32.26	27.51 ± 4.48 a
2 months	110.06 ± 14.74 b	8.86 ± 1.34 b	9.91 ± 0.01 a	2.02 ± 0.73 ab	17.75 ± 36.59	26.34 ± 4.29 ab
4 months	97.43 ± 14.37 c	7.85 ± 1.17 c	9.91 ± 0.01 a	2.13 ± 0.73 a	21.91 ± 38.01	25.21 ± 4.52 b
Shelf-life period (D)
Day 0	120.68 ± 18.81 a	10.06 ± 1.95 a	9.89 ± 0.07 b	1.96 ± 0.85	23.33 ± 43.73	26.83 ± 3.73 ab
Day 5	120.06 ± 16.81 a	9.97 ± 1.96 a	9.92 ± 0.01 a	2.00 ± 0.67	23.99 ± 44.76	28.00 ± 3.70 a
Day 10	108.21 ± 22.88 b	9.02 ± 2.04 b	9.90 ± 0.06 ab	2.03 ± 0.57	7.82 ± 17.71	25.08 ± 5.02 b
Day 15	106.41 ± 18.22 b	8.60 ± 1.74 b	9.91 ± 0.01 a	1.87 ± 0.55	10.09 ± 28.07	25.52 ± 4.94 b
H × S × D
Unripe × 0 month × Day 0	141.34 ± 7.57	12.82 ± 0.82	9.86 ± 0.12	1.74 ± 0.30	0.34 ± 0.11	28.62 ± 3.43
Unripe × 0 month × Day 5	146.86 ± 5.54	13.81 ± 0.88	9.91 ± 0.01	2.10 ± 0.48	0.36 ± 0.13	26.19 ± 5.29
Unripe × 0 month × Day 10	131.54 ± 8.09	10.94 ± 0.72	9.91 ± 0.00	1.80 ± 0.52	5.42 ± 11.45	26.99 ± 4.21
Unripe × 0 month × Day 15	126.66 ± 8.21	10.86 ± 0.63	9.91 ± 0.00	1.54 ± 0.40	0.32 ± 0.11	27.71 ± 6.51
Unripe × 2 months × Day 0	128.72 ± 10.67	10.46 ± 0.46	9.91 ± 0.00	1.88 ± 0.53	6.10 ± 12.97	25.53 ± 3.21
Unripe × 2 months × Day 5	112.80 ± 6.48	9.97 ± 0.46	9.92 ± 0.01	2.26 ± 1.10	4.60 ± 9.62	25.71 ± 1.76
Unripe × 2 months × Day 10	102.88 ± 23.61	8.78 ± 1.46	9.91 ± 0.00	1.74 ± 0.30	6.52 ± 13.91	22.51 ± 6.84
Unripe × 2 months × Day 15	102.56 ± 11.35	8.69 ± 1.11	9.91 ± 0.00	1.86 ± 0.38	4.20 ± 8.67	23.72 ± 5.72
Unripe × 4 months × Day 0	111.82 ± 5.83	8.92 ± 0.42	9.91 ± 0.00	1.86 ± 0.48	65.44 ± 59.62	28.12 ± 2.42
Unripe × 4 months × Day 5	107.48 ± 16.13	8.09 ± 1.09	9.92 ± 0.01	2.20 ± 0.47	67.50 ± 48.16	28.74 ± 3.69
Unripe × 4 months × Day 10	100.16 ± 8.59	7.87 ± 0.43	9.92 ± 0.01	2.46 ± 0.74	12.86 ± 17.02	26.93 ± 3.92
Unripe × 4 months × Day 15	93.48 ± 15.55	6.90 ± 1.39	9.91 ± 0.01	1.98 ± 0.43	7.40 ± 15.76	26.53 ± 2.12
Ripe × 0 month × Day 0	133.56 ± 7.18	12.19 ± 1.41	9.86 ± 0.12	1.24 ± 0.38	0.34 ± 0.13	26.80 ± 6.24
Ripe × 0 month × Day 5	129.06 ± 10.45	11.19 ± 0.93	9.91 ± 0.00	2.18 ± 0.58	23.72 ± 52.31	25.74 ± 2.25
Ripe × 0 month × Day 10	139.40 ± 10.24	11.67 ± 0.75	9.81 ± 0.14	1.56 ± 0.23	0.30 ± 0.07	28.34 ± 6.10
Ripe × 0 month × Day 15	126.64 ± 3.39	10.46 ± 0.47	9.91 ± 0.00	1.76 ± 0.25	0.34 ± 0.11	27.10 ± 4.93
Ripe × 2 months × Day 0	124.18 ± 10.89	10.43 ± 0.79	9.91 ± 0.01	1.44 ± 0.75	69.90 ± 63.41	24.72 ± 6.16
Ripe × 2 months × Day 5	119.08 ± 9.85	9.47 ± 0.80	9.92 ± 0.01	1.86 ± 0.79	22.42 ± 49.24	29.18 ± 3.40
Ripe × 2 months × Day 10	101.02 ± 10.91	8.25 ± 0.86	9.92 ± 0.01	2.14 ± 0.24	25.78 ± 42.85	27.21 ± 2.83
Ripe × 2 months × Day 15	107.88 ± 7.59	7.88 ± 0.47	9.91 ± 0.00	2.04 ± 0.17	0.26 ± 0.05	28.20 ± 2.77
Ripe × 4 months × Day 0	100.60 ± 8.84	8.17 ± 1.29	9.91 ± 0.00	2.32 ± 0.55	6.46 ± 13.66	25.98 ± 2.04
Ripe × 4 months × Day 5	111.72 ± 14.22	8.41 ± 1.28	9.92 ± 0.01	1.88 ± 1.02	30.68 ± 54.05	29.87 ± 4.60
Ripe × 4 months × Day 10	99.44 ± 7.06	8.50 ± 0.60	9.91 ± 0.00	2.18 ± 0.77	0.38 ± 0.11	25.08 ± 3.42
Ripe × 4 months × Day 15	94.96 ± 13.36	8.14 ± 1.05	9.91 ± 0.00	2.14 ± 0.72	0.34 ± 0.05	22.10 ± 6.25
Overripe × 0 month × Day 0	141.90 ± 14.12	11.08 ± 1.63	9.83 ± 0.12	1.68 ± 0.26	0.28 ± 0.04	28.12 ± 3.57
Overripe × 0 month × Day 5	137.36 ± 6.21	11.23 ± 1.25	9.91 ± 0.01	1.60 ± 0.40	54.98 ± 75.02	31.89 ± 2.50
Overripe × 0 month × Day 10	131.76 ± 14.36	11.70 ± 1.30	9.91 ± 0.00	1.88 ± 0.58	0.34 ± 0.11	26.72 ± 3.97
Overripe × 0 month × Day 15	122.32 ± 5.67	10.35 ± 0.39	9.92 ± 0.01	1.78 ± 0.55	24.34 ± 53.70	25.94 ± 4.18
Overripe × 2 months × Day 0	110.60 ± 12.94	8.45 ± 1.43	9.91 ± 0.00	2.76 ± 1.60	48.82 ± 65.05	28.45 ± 1.30
Overripe × 2 months × Day 5	115.66 ± 8.90	9.29 ± 0.73	9.91 ± 0.01	2.02 ± 0.48	5.66 ± 11.87	28.92 ± 1.36
Overripe × 2 months × Day 10	91.92 ± 7.16	7.11 ± 0.90	9.92 ± 0.01	2.26 ± 0.71	18.38 ± 11.61	23.62 ± 4.49
Overripe × 2 months × Day 15	103.44 ± 11.28	7.52 ± 0.69	9.91 ± 0.00	2.00 ± 0.35	0.38 ± 0.08	28.28 ± 3.85
Overripe × 4 months × Day 0	93.44 ± 3.97	8.04 ± 0.53	9.91 ± 0.00	2.68 ± 0.97	12.28 ± 16.44	25.11 ± 2.43
Overripe × 4 months × Day 5	100.52 ± 4.66	8.27 ± 0.73	9.91 ± 0.01	1.94 ± 0.67	6.02 ± 12.62	25.75 ± 3.06
Overripe × 4 months × Day 10	75.74 ± 3.66	6.36 ± 0.47	9.92 ± 0.01	2.22 ± 0.54	0.36 ± 0.09	18.28 ± 2.21
Overripe × 4 months × Day 15	79.78 ± 13.18	6.57 ± 1.42	9.92 ± 0.01	1.72 ± 1.22	53.22 ± 47.23	20.07 ± 0.92
Significance
H	***	***	n.s.	n.s.	n.s.	n.s.
S	***	***	***	**	n.s.	**
D	***	***	*	n.s.	n.s.	**
H × S	*	n.s.	n.s.	n.s.	**	***
H × D	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
S × D	*	n.s.	*	n.s.	*	n.s.
H × S × D	n.s.	**	n.s.	n.s.	*	n.s.

Legend: TWC1—Total Work Cycle 1 (mJ); LT—Load at Target (N); DT—Deformation at Target (mm); AD—Adhesiveness (mJ); FWP—1st Fracture Work Performed (mJ); APL—Average Peak Load (N). Parameters are reported as mean ± standard deviation (Mean ± StD). Different letters within columns indicate significant mean differences according to Tukey HSD test (p ≤ 0.05). n.s., *, **, and *** denote non-significant or significant effects at p < 0.05, 0.01, and 0.001, respectively.

, with additional data provided in Supplementary Table S1. All evaluated textural parameters, except cohesiveness (Table 2), were significantly affected by at least one of the studied factors: harvest stage, storage duration, and shelf-life period (p < 0.05). Notably, most parameters exhibited statistically significant interactions among these factors, highlighting the complexity and dynamic nature of texture changes during postharvest life. While the harvest stage influenced a specific subset of parameters, storage duration emerged as the dominant factor affecting the majority of texture parameters. Shelf-life had a moderate effect, and for certain parameters, the combined interaction of all three factors played a significant role. These findings reflect the multifactorial control of texture changes in apples and emphasize the importance of considering both pre- and post-storage conditions in assessing fruit quality.

Regarding TPA (Table 2 and Table S1), five parameters—hardness cycle one, the load at target, hardness work cycle one, springiness, and the chewiness index—were significantly influenced by the storage duration, with the highest values observed in freshly harvested apples (0 months). These results indicate a loss of firmness with prolonged storage.

Deformation at hardness was significantly affected by the harvest stage, while deformation at the target was significantly influenced by the interaction between storage duration and shelf-life period. This is consistent with findings that fruit ripening leads to cell wall degradation and the loss of turgor pressure, resulting in decreased firmness [27].

Total work cycle one showed statistically significant differences with respect to all three factors—H, S, and D—reaching its highest values in the unripen, freshly harvested fruit (0 months) and on day five of shelf-life.

Adhesiveness was significantly affected by several interaction terms: H × S, H × D, and S × D. These interactions suggest that the stickiness of the fruit surface is influenced by the combined effects of these factors, potentially due to changes in pectin composition and moisture content [28].

The quantity of fractures showed significant differences concerning storage duration, the interaction between the harvest stage and shelf-life period, and the three-way interaction among all factors. Fracturability, the first fracture work performed, and the first fracture deformation exhibited significant differences depending on the storage duration and the interaction between the storage duration and shelf-life period, while the first fracture deformation also showed differences regarding the harvest stage. These parameters are indicative of the fruit’s crispness and structural integrity, which are known to diminish during storage due to cell wall degradation [29]. In our study, fracturability values were higher compared to those reported by Topaiboul et al. [30], who measured the fracturability of waxed apples. Higher fracturability in our samples suggests better preservation of the fruit’s structural integrity during storage.

Both the gumminess and average peak load showed statistically significant differences based on the storage duration and the H × D interaction. These parameters reached maximum values in the freshly harvested, unripen fruit and on day 0 of shelf-life, and then gradually decreased with ripening and extended storage. This decline reflects reduced resistance to mechanical damage. High gumminess values indicate a firm and crisp texture characterized by tissue failure through cell rupturing, whereas low gumminess values correspond to a softer texture. Our results are consistent with the findings of Sabbaghi et al. [31], who examined dried apple slices.

As shown in Table 3, total work cycle one was significantly influenced by all three main factors—H, S, and D—as well as by the H × S and S × D interactions. The highest values were observed in the freshly harvested (0 month), unripen fruits at the beginning (day 0) of the shelf-life period. Although direct literature values for total work cycle one in apples are scarce, comparable trends have been reported in studies using texture profile analysis of dried apple slices [32]. In this study, higher work values were associated with firmer, more structurally intact tissue, whereas prolonged storage or drying led to a decrease in total work. Our findings are consistent with these observations, indicating that total work cycle one can serve as a reliable indicator of mechanical resistance and internal cohesion in apple tissue during postharvest storage.

The load at target was also significantly affected by all three factors and their three-way interaction (H × S × D), indicating a strong combined influence of ripening stage and storage progression on tissue resistance to compression.

The deformation at target was significantly influenced by storage duration, shelf-life period, and their interaction. Extended storage and longer shelf-life led to increased tissue deformation, reflecting progressive softening and reduced mechanical resistance, as observed in similar studies [27].

Adhesiveness was solely affected by storage duration, with the lowest values recorded in freshly harvested fruits (0 months). Both texture tests showed generally low adhesiveness values, indicating minimal force was needed to separate the fruit surface from a contacting material. This low adhesiveness is typical for fresh apple tissue, which generally exhibits limited surface stickiness due to the presence of intact cuticle and low free moisture on the surface [33]. As storage progresses, cell wall degradation, enzymatic softening, and loss of cell turgor can lead to partial leakage of intracellular contents, slightly increasing surface tackiness [34]. These changes can cause the fruit surface to become slightly stickier during storage, although the absolute values remained low, suggesting that Granny Smith apples used in this study do not develop significant surface tackiness even after prolonged storage.

The first fracture deformation showed significant differences for three interaction effects: H × S, S × D, and the three-way interaction (H × S × D). These results highlight the complexity of fracture behavior in apple tissue, which is influenced by both physiological maturity and external storage conditions.

Lastly, average peak load was significantly affected by storage duration, shelf-life period, and the H × S interaction. This parameter, which reflects the average maximum force applied before failure, decreased over time, particularly in fruits harvested at more advanced ripening stages.

Our results are consistent with a recent study on Braeburn and Golden Delicious apples, which similarly reported a decline in firmness related to both harvest stage and shelf-life duration, confirming the significant impact of these factors on fruit texture [35]. Moreover, recent advances in postharvest technology, such as modified atmosphere preservation (MAP), have shown a promising potential in maintaining fruit quality during storage by reducing respiration rates and slowing down texture degradation [36].

These findings highlight the importance of carefully managing harvest timing and storage conditions to preserve the desirable textural qualities of Granny Smith apples. The use of controlled atmosphere storage combined with optimized shelf-life conditions can effectively maintain firmness and other key texture parameters, thereby extending the fruit’s marketability [36].

3.2. Maturity Indices

Maturity indices were generally more influenced by individual factors and their interactions compared to the texture parameters, as shown in

Table 4. Maturity indices of Granny Smith apple fruits at different harvest stages, storage durations, and shelf-life periods.

	FIRM	SSC	SPI	TS	RS	pH	TA	Scald
Harvest stage (H)
Unripe	7.30 ± 0.79 a	10.53 ± 0.75 b	5.29 ± 1.49 c	6.74 ± 0.48 b	5.90 ± 0.54 b	3.18 ± 0.16 c	0.56 ± 0.10 a	3.40 ± 3.01 a
Ripe	7.15 ± 0.82 b	10.58 ± 0.60 b	6.17 ± 1.74 b	6.76 ± 0.43 b	5.93 ± 0.47 b	3.24 ± 0.17 b	0.53 ± 0.09 b	1.88 ± 2.46 b
Overripe	6.78 ± 0.89 c	10.92 ± 0.56 a	7.38 ± 1.31 a	7.00 ± 0.39 a	6.19 ± 0.46 a	3.28 ± 0.18 a	0.48 ± 0.07 c	0.67 ± 1.15 c
Storage duration (S)
0 month	7.71 ± 0.51 a	10.18 ± 0.51 c	6.28 ± 1.73	6.54 ± 0.36 c	5.63 ± 0.35 c	3.04 ± 0.05 c	0.61 ± 0.08 a	-
2 months	7.24 ± 0.75 a	10.54 ± 0.42 b	-	6.74 ± 0.30 b	5.87 ± 0.31 b	3.25 ± 0.08 b	0.48 ± 0.07 b	0.38 ± 0.96
4 months	6.28 ± 0.58 b	11.31 ± 0.48 a	-	7.21 ± 0.37 a	6.51 ± 0.37 a	3.41 ± 0.11 a	0.47 ± 0.06 b	3.58 ± 2.71
Shelf-life period (D)
Day 0	7.44 ± 0.73 a	10.51 ± 0.81 b	4.61 ± 1.09 d	6.71 ± 0.51 a	5.88 ± 0.57 b	3.19 ± 0.15 d	0.58 ± 0.08 a	0.63 ± 1.14 c
Day 5	7.13 ± 0.77 b	10.71 ± 0.62 ab	5.67 ± 1.46 c	6.87 ± 0.48 ab	6.05 ± 0.55 a	3.22 ± 0.17 c	0.52 ± 0.08 b	1.97 ± 2.69 b
Day 10	6.95 ± 0.92 bc	10.65 ± 0.54 ab	6.83 ± 1.10 b	6.81 ± 0.34 ab	5.96 ± 0.39 ab	3.25 ± 0.17 b	0.51 ± 0.09 b	2.58 ± 2.76 ab
Day 15	6.78 ± 0.89 c	10.83 ± 0.65 a	8.00 ± 1.08 a	6.94 ± 0.43 c	6.12 ± 0.48 a	3.29 ± 0.19 a	0.47 ± 0.08 c	2.74 ± 2.87 a
H × S × D
Unripe × 0 month × Day 0	7.88 ± 0.62	9.47 ± 0.31	3.83 ± 0.41	6.07 ± 0.23	5.13 ± 0.22	2.97 ± 0.02	0.74 ± 0.02	-
Unripe × 0 month × Day 5	7.83 ± 0.50	10.30 ± 0.10	4.33 ± 0.52	6.36 ± 0.05	5.46 ± 0.08	3.01 ± 0.02	0.66 ± 0.02	-
Unripe × 0 month × Day 10	7.78 ± 0.43	9.83 ± 0.35	5.67 ± 0.82	6.47 ± 0.29	5.60 ± 0.30	3.00 ± 0.02	0.69 ± 0.06	-
Unripe × 0 month × Day 15	7.54 ± 0.31	10.27 ± 0.12	7.33 ± 0.52	6.66 ± 0.12	5.81 ± 0.13	3.08 ± 0.05	0.54 ± 0.03	-
Unripe × 2 months × Day 0	8.35 ± 0.38	10.40 ± 0.10	-	6.75 ± 0.10	5.84 ± 0.11	3.05 ± 0.03	0.60 ± 0.03	0.00 ± 0.00
Unripe × 2 months × Day 5	7.65 ± 0.19	9.90 ± 0.10	-	6.43 ± 0.09	5.51 ± 0.08	3.15 ± 0.07	0.48 ± 0.03	0.00 ± 0.00
Unripe × 2 months × Day 10	7.56 ± 0.28	10.60 ± 0.53	-	6.79 ± 0.32	5.85 ± 0.31	3.31 ± 0.02	0.51 ± 0.01	1.33 ± 1.03
Unripe × 2 months × Day 15	7.21 ± 0.30	10.13 ± 0.15	-	6.36 ± 0.04	5.51 ± 0.04	3.33 ± 0.03	0.44 ± 0.01	2.75 ± 1.08
Unripe × 4 months × Day 0	7.00 ± 0.50	11.17 ± 0.55	-	7.09 ± 0.41	6.34 ± 0.40	3.27 ± 0.01	0.55 ± 0.04	2.58 ± 1.28
Unripe × 4 months × Day 5	6.57 ± 0.30	11.43 ± 0.15	-	7.30 ± 0.11	6.56 ± 0.10	3.26 ± 0.01	0.56 ± 0.04	6.67 ± 1.75
Unripe × 4 months × Day 10	6.11 ± 0.70	10.80 ± 0.10	-	6.84 ± 0.09	6.12 ± 0.08	3.30 ± 0.01	0.46 ± 0.03	7.00 ± 1.10
Unripe × 4 months × Day 15	6.19 ± 0.44	12.07 ± 0.29	-	7.74 ± 0.17	7.02 ± 0.18	3.46 ± 0.01	0.44 ± 0.02	6.83 ± 0.75
Ripe × 0 month × Day 0	7.39 ± 0.53	9.27 ± 0.25	4.00 ± 0.00	6.06 ± 0.07	5.19 ± 0.10	3.03 ± 0.01	0.67 ± 0.05	-
Ripe × 0 month × Day 5	7.58 ± 0.21	9.97 ± 0.15	5.67 ± 1.63	6.35 ± 0.15	5.50 ± 0.14	3.01 ± 0.04	0.64 ± 0.03	-
Ripe × 0 month × Day 10	7.81 ± 0.47	10.23 ± 0.25	7.67 ± 0.82	6.40 ± 0.18	5.46 ± 0.19	3.05 ± 0.03	0.62 ± 0.03	-
Ripe × 0 month × Day 15	8.15 ± 0.69	10.93 ± 0.35	7.33 ± 0.52	7.10 ± 0.29	6.15 ± 0.28	3.06 ± 0.04	0.60 ± 0.02	-
Ripe × 2 months × Day 0	7.89 ± 0.41	10.47 ± 0.12	-	6.63 ± 0.14	5.75 ± 0.15	3.27 ± 0.02	0.55 ± 0.04	0.00 ± 0.00
Ripe × 2 months × Day 5	7.93 ± 0.59	10.60 ± 0.17	-	6.70 ± 0.13	5.81 ± 0.10	3.31 ± 0.04	0.46 ± 0.02	0.50 ± 1.22
Ripe × 2 months × Day 10	7.20 ± 0.34	10.40 ± 0.26	-	6.66 ± 0.18	5.73 ± 0.16	3.30 ± 0.04	0.41 ± 0.02	0.00 ± 0.00
Ripe × 2 months × Day 15	6.44 ± 0.44	10.83 ± 0.32	-	6.94 ± 0.23	6.12 ± 0.22	3.27 ± 0.02	0.48 ± 0.01	0.00 ± 0.00
Ripe × 4 months × Day 0	6.47 ± 0.36	10.50 ± 0.10	-	6.63 ± 0.07	5.93 ± 0.08	3.25 ± 0.02	0.56 ± 0.01	0.92 ± 1.07
Ripe × 4 months × Day 5	6.36 ± 0.40	11.27 ± 0.31	-	7.47 ± 0.48	6.76 ± 0.47	3.41 ± 0.03	0.45 ± 0.04	4.00 ± 0.63
Ripe × 4 months × Day 10	6.31 ± 0.45	11.37 ± 0.31	-	7.24 ± 0.22	6.39 ± 0.16	3.47 ± 0.01	0.47 ± 0.01	4.08 ± 2.20
Ripe × 4 months × Day 15	6.28 ± 0.36	11.07 ± 0.15	-	6.97 ± 0.19	6.33 ± 0.15	3.51 ± 0.01	0.42 ± 0.02	5.50 ± 2.43
Overripe × 0 month × Day 0	7.87 ± 0.51	10.47 ± 0.15	6.00 ± 0.63	6.46 ± 0.16	5.53 ± 0.15	3.15 ± 0.03	0.52 ± 0.06	-
Overripe × 0 month × Day 5	7.60 ± 0.40	10.53 ± 0.21	7.00 ± 0.00	6.96 ± 0.24	5.99 ± 0.23	3.06 ± 0.05	0.52 ± 0.06	-
Overripe × 0 month × Day 10	7.72 ± 0.66	10.63 ± 0.06	7.17 ± 0.41	6.96 ± 0.09	6.04 ± 0.10	3.07 ± 0.02	0.52 ± 0.02	-
Overripe × 0 month × Day 15	7.41 ± 0.58	10.23 ± 0.35	9.33 ± 0.52	6.64 ± 0.25	5.69 ± 0.25	3.03 ± 0.03	0.57 ± 0.02	-
Overripe × 2 months × Day 0	7.23 ± 0.71	11.03 ± 0.12	-	7.11 ± 0.14	6.27 ± 0.13	3.25 ± 0.01	0.55 ± 0.01	0.00 ± 0.00
Overripe × 2 months × Day 5	6.58 ± 0.26	10.97 ± 0.67	-	7.01 ± 0.54	6.19 ± 0.52	3.23 ± 0.03	0.49 ± 0.01	0.00 ± 0.00
Overripe × 2 months × Day 10	6.51 ± 0.60	10.57 ± 0.23	-	6.67 ± 0.16	5.88 ± 0.15	3.26 ± 0.02	0.48 ± 0.01	0.00 ± 0.00
Overripe × 2 months × Day 15	6.31 ± 0.22	10.63 ± 0.60	-	6.87 ± 0.48	5.97 ± 0.45	3.30 ± 0.02	0.36 ± 0.03	0.00 ± 0.00
Overripe × 4 months × Day 0	6.96 ± 0.29	11.87 ± 0.42	-	7.58 ± 0.29	6.91 ± 0.28	3.47 ± 0.03	0.48 ± 0.01	0.25 ± 0.27
Overripe × 4 months × Day 5	6.07 ± 0.37	11.40 ± 0.44	-	7.26 ± 0.32	6.63 ± 0.31	3.50 ± 0.06	0.44 ± 0.04	0.67 ± 0.68
Overripe × 4 months × Day 10	5.57 ± 0.36	11.40 ± 0.30	-	7.25 ± 0.23	6.60 ± 0.22	3.49 ± 0.01	0.41 ± 0.01	3.08 ± 1.36
Overripe × 4 months × Day 15	5.53 ± 0.37	11.33 ± 0.15	-	7.20 ± 0.15	6.53 ± 0.17	3.53 ± 0.03	0.38 ± 0.03	1.33 ± 0.52
Significance
H	***	***	***	***	***	***	***	***
S	***	***	-	***	***	***	***	-
D	***	**	***	**	***	***	***	***
H × S	***	**	-	n.s.	n.s.	***	***	***
H × D	*	***	***	**	***	***	***	***
S × D	***	**	-	***	***	***	**	***
H × S × D	**	***	-	***	***	***	***	***

Legend: FIRM—Firmness (kg/cm²); SSC—Soluble solids content (%); SPI—Starch pattern index; TS—Total sugars; RS—Reducing sugars; TA—Titratable acidity; Scald—Superficial scald. Dash (-) indicates that measurements were not conducted under those conditions. Parameters are reported as mean ± standard deviation (Mean ± StD). Different letters within columns indicate significant mean differences according to Tukey HSD test (p = 0.05). n.s., *, **, and *** denote non-significant or significant effects at p < 0.05, 0.01, and 0.001, respectively.

. All main factors, as well as their interactions, had a statistically significant effect on the measured maturity indices. In commercial practice, maturity indices for each orchard block are typically assessed using a minimum of 10 fruits [37]. However, this study focused on the relationship between maturity indices and three factors—H, S, and D; thus, only six fruits per timepoint were analyzed.

Assessing fruit firmness is essential for determining how well fruits can withstand storage and how storage conditions influence their quality over time. Firmness was significantly influenced by all factors and their interactions, reaching its highest values in the freshly harvested (0 months), unripen fruits on day 0 of shelf-life, and gradually decreasing with progressing ripening and storage. Fruit firmness plays a key role in determining the storability and marketability of apples, as it directly influences consumer acceptance and shelf life. Different apple varieties exhibit varying rates of softening during storage, which is largely associated with physiological and biochemical changes in fruit tissue. Although cell wall composition was not directly measured in this study, the observed decrease in fruit firmness across harvest stages, storage durations, and shelf-life periods is consistent with previously reported mechanisms of fruit softening [38]. In addition, earlier studies have demonstrated that enzymes involved in cell wall metabolism (e.g., polygalacturonase, cellulase) modify the cell wall structure, increasing its porosity, disrupting intercellular adhesion, and leading to tissue softening [39]. Pectin, a major component of the middle lamella, is converted from insoluble protopectin to soluble forms during ripening, contributing significantly to the reduction in fruit hardness [40]. In our study, firmness values were slightly lower than those reported by Zivic et al. [41], which could be attributed to differences in ripening stage at harvest or postharvest handling conditions.

Starch pattern index (SPI) was affected by harvest stage, shelf-life period, and their interaction. The lowest value was observed in the unripen fruits at day 0 of shelf-life, while the index gradually increased during shelf-life, particularly in later harvests, indicating enzymatic hydrolysis of starch into reducing sugars (RS). This trend was reflected in the consistent rise of total and reducing sugar content throughout sequential harvests. SPI was not evaluated after cold storage, as starch is typically degraded to a level that makes the test unfeasible at that point. SPI offers a visual estimate of starch distribution, though it is less precise than direct analytical quantification methods. In the Granny Smith variety, starch degradation was characterized by small, indistinct patches that initially appeared in central areas and then expanded outward. As starch reserves diminished, the patches became lighter in color, while vascular bundles remained invisible, aligning with the findings of Szalay et al. [42].

Soluble solids content (SSC), total acidity (TA), and pH were also influenced by all three main factors and their interactions. SSC and pH were lowest in the unripen apples at the beginning of shelf-life and gradually increased during ripening and storage. This increase is largely attributed to the breakdown of insoluble polysaccharides into simpler, more soluble forms via enzymatic activity [43], supporting the results of Cárdenas-Pérez et al. [44] and Kassebi et al. [45]. In contrast, TA showed an opposite trend—being highest in the unripen fruits at the beginning of shelf-life and gradually decreasing as the fruits ripened and organic acids were metabolized. Titratable acidity is an important indicator of fruit maturity and a key contributor to flavor, as well as a minor nutritional component. Our values were slightly higher than those reported for several other varieties studied by Mehdi et al. [46] in Pakistan, which may be attributed to varietal differences and environmental conditions during fruit development. Total sugars and reducing sugars were significantly influenced by all examined factors and their interactions, except for the H × S interaction. Both parameters showed the lowest values in the freshly harvested (0 months) unripen apples on day 0 of shelf-life and progressively increased during ripening and storage, in line with expected sugar accumulation during maturation.

The accumulation of SSC may also result from moisture loss, which concentrates sugars, and from the hydrolysis of starch and degradation of cell wall components [47]. Since starch serves as a carbohydrate reserve, it is metabolized into simple sugars during respiration, contributing to increased sweetness and energy availability. At early stages of fruit development, lower SSC levels correspond to limited respiration and lower sugar content. As ripening progresses, starch breakdown leads to a noticeable increase in SSC and total sugars, often accompanied by weight loss and softening [48]. Our findings align with the work of Ali et al. [49], who observed a rise in SSC, starch degradation, and sugar accumulation in various apple varieties stored at ambient temperature.

Finally, scald was significantly affected by all factors and interactions except storage duration, as it was not assessed in freshly harvested fruits (0 months) due to its occurrence only after a period of storage. Skin browning associated with superficial scald is caused by the oxidation of α-farnesene, a volatile compound that accumulates in the wax layer during the first 8–12 weeks of cold storage [50]. Its concentration increases with ripening and ethylene production, and it is readily oxidized by atmospheric oxygen [51]. Marc et al. [52] reported that scald incidence is higher in fruit harvested at an unripe stage, which is consistent with our findings.

Our findings are also in line with the study by Ding et al. [27], who reported that firmness and other maturity indices declined during storage in all examined apple varieties, particularly in the unripen fruits at harvest, emphasizing the combined effects of harvest maturity and storage duration on postharvest quality.

Figure 1 illustrates the progressive reduction in starch-iodine-stained area in Granny Smith apples, depending on harvest stage and shelf-life duration for freshly harvested fruit (0 months). As expected, the unripen apples showed intense and uniform staining on day 0, indicating a high starch content. With later harvest stages and longer shelf-life, the staining area significantly decreased, reflecting enzymatic starch hydrolysis during ripening. This trend was in agreement with increasing SPI values.

Starch accumulation in apple fruit results from the translocation of sugars produced through photosynthesis in the leaves, which are then stored in the developing fruit [53]. As ripening progresses, this starch is enzymatically hydrolyzed back into sugars, a transformation that can be effectively visualized using the iodine staining method [54]. Each variety exhibits a specific starch degradation pattern, and the rate of stain disappearance varies accordingly. In our study, a characteristic flower pattern emerged during maturation, marked by five petal-like clear zones expanding as ripening advanced. Additionally, a relatively slow fading of dark coloration was observed in fruit left to ripen on the tree, indicating gradual starch degradation. This behavior is comparable to findings reported by Hanrahan and Galeni [55] for the WA 38 apple variety, which also exhibited delayed starch clearance during on-tree ripening, as well as flower pattern.

3.3. Colorimetric Parameters

Table 5. CIELab colorimetric parameters of Granny Smith apple fruits at different harvest stages, storage durations, and shelf-life periods.

	L*	a*	b*	BI	YI	ΔE
Harvest stage (H)
Unripe	61.57 ± 6.07 a	−5.56 ± 5.13 a	32.05 ± 4.01 a	62.99 ± 9.93 b	74.34 ± 5.64 ab	6.56 ± 5.49 b
Ripe	62.66 ± 5.51 a	−6.42 ± 4.03 b	33.09 ± 3.39 b	63.66 ± 11.45 ab	75.66 ± 7.28 b	4.95 ± 4.75 a
Overripe	64.16 ± 4.71 b	−5.68 ± 3.32 a	32.64 ± 2.85 ab	60.99 ± 9.23 a	72.93 ± 7.01 a	5.78 ± 3.37 ab
Storage duration (S)
0 month	62.82 ± 4.55 b	−9.48 ± 1.11 a	31.96 ± 2.35 a	55.48 ± 6.93 a	72.93 ± 6.15 a	4.07 ± 3.28 a
2 months	65.62 ± 4.14 c	−6.02 ± 2.34 b	34.15 ± 2.65 b	62.83 ± 8.32 b	74.59 ± 6.81 ab	5.71 ± 3.39 b
4 months	59.95 ± 6.22 a	−2.16 ± 4.51 c	31.66 ± 4.48 a	69.23 ± 10.37 c	75.41 ± 7.08 b	7.51 ± 6.10 c
Shelf-life period (D)
Day 0	62.22 ± 3.62 a	−7.73 ± 2.28 a	35.52 ± 2.41	60.31 ± 7.13 a	74.80 ± 5.85	2.81 ± 2.95 a
Day 5	62.01 ± 5.02 a	−6.18 ± 3.91 b	32.01 ± 3.62	61.40 ± 8.98 ab	73.80 ± 6.58	5.49 ± 4.34 b
Day 10	63.72 ± 6.60 b	−5.18 ± 4.91 c	32.92 ± 3.71	63.48 ± 11.57 bc	74.05 ± 7.05	7.53 ± 4.62 c
Day 15	63.23 ± 6.34 ab	−4.45 ± 4.63 c	32.91 ± 3.90	65.00 ± 12.13 c	74.59 ± 7.49	7.21 ± 4.93 c
H × S × D
Unripe × 0 month × Day 0	61.02 ± 3.22	−9.82 ± 0.35	31.94 ± 1.46	57.05 ± 4.86	74.89 ± 4.03	3.55 ± 1.78
Unripe × 0 month × Day 5	61.03 ± 2.30	−9.36 ± 1.11	31.70 ± 1.64	56.86 ± 4.01	74.22 ± 2.83	3.15 ± 1.83
Unripe × 0 month × Day 10	60.16 ± 2.21	−9.44 ± 0.78	31.36 ± 2.10	56.82 ± 3.83	74.42 ± 3.49	3.58 ± 2.42
Unripe × 0 month × Day 15	62.60 ± 2.33	−9.39 ± 0.45	31.08 ± 1.01	53.16 ± 3.98	71.04 ± 3.78	2.88 ± 1.49
Unripe × 2 months × Day 0	63.76 ± 3.82	−8.06 ± 0.84	32.76 ± 2.07	58.40 ± 5.55	73.53 ± 4.68	3.86 ± 2.10
Unripe × 2 months × Day 5	65.05 ± 2.53	−7.82 ± 0.84	34.93 ± 1.95	63.33 ± 6.97	76.84 ± 5.65	4.42 ± 1.66
Unripe × 2 months × Day 10	65.08 ± 3.64	−6.64 ± 1.61	35.06 ± 1.75	65.43 ± 7.42	77.23 ± 6.50	5.38 ± 2.04
Unripe × 2 months × Day 15	68.72 ± 2.38	−4.74 ± 1.38	37.43 ± 1.71	69.10 ± 5.85	77.88 ± 4.12	8.85 ± 2.12
Unripe × 4 months × Day 0	60.90 ± 5.13	−5.28 ± 3.47	30.19 ± 3.21	58.67 ± 6.40	70.85 ± 5.29	6.12 ± 4.20
Unripe × 4 months × Day 5	56.01 ± 8.72	1.44 ± 5.14	27.12 ± 4.51	66.56 ± 8.38	69.15 ± 3.30	13.07 ± 7.62
Unripe × 4 months × Day 10	57.46 ± 8.77	0.95 ± 5.50	30.10 ± 5.81	73.53 ± 13.09	74.87 ± 8.85	12.44 ± 6.61
Unripe × 4 months × Day 15	57.05 ± 8.22	1.47 ± 5.36	30.89 ± 5.65	76.97 ± 9.71	77.11 ± 6.25	11.40 ± 8.05
Ripe × 0 month × Day 0	62.59 ± 3.58	−9.59 ± 0.86	33.13 ± 2.41	59.48 ± 9.11	75.99 ± 8.41	0.00 ± 0.00
Ripe × 0 month × Day 5	63.23 ± 3.07	−9.33 ± 0.85	30.45 ± 2.40	51.11 ± 7.70	69.05 ± 7.47	4.33 ± 1.95
Ripe × 0 month × Day 10	73.87 ± 2.92	−11.53 ± 0.59	35.89 ± 2.20	50.83 ± 5.89	69.52 ± 5.36	12.02 ± 2.70
Ripe × 0 month × Day 15	60.41 ± 2.34	−9.39 ± 0.49	32.14 ± 1.70	59.37 ± 7.22	76.18 ± 6.03	3.53 ± 1.16
Ripe × 2 months × Day 0	63.09 ± 2.91	−8.29 ± 0.42	32.09 ± 2.63	57.07 ± 7.52	72.73 ± 6.07	0.00 ± 0.00
Ripe × 2 months × Day 5	63.25 ± 2.86	−7.02 ± 1.73	34.02 ± 2.03	64.15 ± 6.87	76.93 ± 4.74	4.03 ± 1.85
Ripe × 2 months × Day 10	63.07 ± 4.03	−5.61 ± 1.36	33.22 ± 2.22	64.16 ± 7.46	75.44 ± 5.86	5.25 ± 1.46
Ripe × 2 months × Day 15	65.55 ± 2.82	−4.97 ± 2.02	35.97 ± 2.31	69.48 ± 8.13	78.53 ± 6.08	6.61 ± 2.20
Ripe × 4 months × Day 0	60.80 ± 2.71	−5.79 ± 2.15	33.15 ± 2.23	66.82 ± 2.99	77.88 ± 3.92	0.00 ± 0.00
Ripe × 4 months × Day 5	59.90 ± 4.92	−4.32 ± 2.47	31.75 ± 4.54	66.44 ± 10.43	75.70 ± 9.26	5.84 ± 4.36
Ripe × 4 months × Day 10	60.21 ± 6.02	−1.51 ± 4.41	34.00 ± 4.57	76.97 ± 7.41	80.56 ± 6.34	8.05 ± 5.20
Ripe × 4 months × Day 15	55.91 ± 5.30	0.32 ± 4.82	31.22 ± 5.22	78.07 ± 12.78	79.38 ± 8.33	9.70 ± 6.82
Overripe × 0 month × Day 0	60.52 ± 2.48	−9.28 ± 1.55	31.93 ± 1.82	58.62 ± 6.77	75.49 ± 5.29	3.54 ± 2.14
Overripe × 0 month × Day 5	61.64 ± 3.27	−9.20 ± 0.57	31.59 ± 2.64	56.53 ± 8.83	73.48 ± 7.85	3.93 ± 2.22
Overripe × 0 month × Day 10	63.37 ± 3.17	−8.87 ± 1.31	31.71 ± 1.66	54.89 ± 6.46	71.67 ± 5.26	3.59 ± 1.98
Overripe × 0 month × Day 15	63.46 ± 4.13	−8.55 ± 1.07	30.60 ± 2.18	52.22 ± 6.67	69.15 ± 6.69	4.72 ± 2.77
Overripe × 2 months × Day 0	63.86 ± 3.47	−7.73 ± 0.99	34.11 ± 1.58	62.75 ± 6.08	76.49 ± 5.21	4.02 ± 1.81
Overripe × 2 months × Day 5	66.03 ± 3.29	−5.79 ± 1.18	34.19 ± 1.86	62.69 ± 7.34	74.19 ± 6.23	5.57 ± 1.67
Overripe × 2 months × Day 10	67.84 ± 4.37	−2.79 ± 2.82	32.03 ± 2.94	58.55 ± 10.43	67.62 ± 6.73	9.06 ± 1.93
Overripe × 2 months × Day 15	72.08 ± 3.30	−2.77 ± 1.05	33.96 ± 3.39	58.90 ± 10.45	67.64 ± 9.19	11.44 ± 2.41
Overripe × 4 months × Day 0	63.47 ± 3.51	−5.75 ± 1.45	33.34 ± 2.23	63.95 ± 8.16	75.31 ± 7.06	4.23 ± 2.77
Overripe × 4 months × Day 5	61.93 ± 4.16	−4.24 ± 1.38	32.36 ± 3.44	64.89 ± 7.42	74.61 ± 5.68	5.03 ± 2.84
Overripe × 4 months × Day 10	62.46 ± 5.77	−1.15 ± 3.07	32.92 ± 4.59	70.16 ± 8.59	75.16 ± 6.24	8.41 ± 3.63
Overripe × 4 months × Day 15	63.26 ± 2.97	−2.08 ± 1.86	32.93 ± 2.96	67.73 ± 7.42	74.41 ± 6.38	5.81 ± 2.43
Significance
H	***	**	*	*	***	***
S	***	***	***	***	**	***
D	**	***	n.s.	***	n.s.	***
H × S	***	***	***	n.s.	***	***
H × D	***	n.s.	*	***	***	***
S × D	***	***	***	***	**	***
H × S × D	***	**	n.s.	n.s.	n.s.	***

Legend: BI—Browning Index; YI—Yellow Index; ΔE—Total Color Difference. Parameters are reported as mean ± standard deviation (Mean ± StD). Different letters within columns indicate significant mean differences according to Tukey HSD test (p ≤ 0.05). n.s., *, **, and *** denote non-significant or significant effects at p < 0.05, 0.01, and 0.001, respectively.

summarizes the statistical significance of the effects of harvest stage, storage duration, shelf-life period, and their interactions on the colorimetric parameters of Granny Smith apples, highlighting the dynamic changes in external fruit appearance during storage and ripening. Both harvest stage and storage duration had a consistent and significant effect on all colorimetric parameters, while shelf-life and interaction influenced several of them.

Statistically significant differences were observed for L* and ΔE across all three main factors and their interactions. L* values, which reflect surface lightness, reached their maximum after two months of storage in the overripen apples at the end of shelf-life (days 10 and 15, with similar values). This increase is associated with tissue degradation and reduced pigment concentration, likely driven by polyphenol oxidase (PPO) activity [48]. In contrast, ΔE values, which indicate a Total Color Difference from the original state, peaked after four months of storage in the unripen apples, also at the end of shelf-life, confirming substantial visual color changes and reduced color uniformity [45]. The ΔE values obtained in our study were lower than those reported by Veleșcu et al. [56], who investigated the effects of convective drying conditions on color change in Golden Delicious apple slices. This difference is expected, as drying processes typically cause more pronounced color shifts compared to cold storage and shelf-life conditions examined in our study.

Parameter a* was significantly affected by all factors except for the H × D interaction. The negative a* values throughout the study are characteristic of green-skinned cultivars like Granny Smith, reflecting the dominance of green pigmentation. These values gradually approached zero with extended storage and shelf-life duration, indicating a progressive loss of chlorophyll and a shift toward neutral hues [48]. The obtained L* value in our study was similar to that reported by Zivic et al. [41], indicating comparable lightness of the fruit surface. However, the a* value in our samples was higher, suggesting a less intense green coloration. The b* value was slightly higher as well, reflecting a more pronounced yellow component, which confirms that differences in harvest stage or postharvest handling could have influenced the observed color parameters.

Parameters b* and the Yellow Index (YI) were significantly influenced by all factors except shelf-life period and the three-way interaction (H × S × D). The consistently positive b* values reflect the accumulation of yellow pigments, primarily carotenoids and xanthophylls, with the highest values observed in the ripe fruits after two months of storage on day 0 of shelf-life. These changes are linked to chlorophyll degradation and the unmasking of preformed yellow pigments during ripening [57]. Although the YI is commonly used in food colorimetry, especially for processed products such as mashed potatoes, onion slices, and yogurt [58], its application in fresh fruits like apples, to our knowledge, has not been reported. This highlights the potential relevance of our findings and suggests that further exploration of YI in fruit ripening and storage contexts could provide additional insights into colorimetric changes during postharvest handling.

The Browning Index (BI) showed statistically significant differences for all factors except for the H × S interaction and the three-way interaction. BI values increased with prolonged storage and shelf-life, and the highest values were observed in the ripe fruits. These results indicate a higher susceptibility of the ripe apples to enzymatic browning, especially in green cultivars like Granny Smith [59]. In the study by Shimizu et al. [60], BI values were reported for several apple varieties, including Fuji, Cripps Pink, Shinanogold, Kinshu, Tsugaru, and Aori27. These values were lower compared to those observed in our study. This discrepancy may be attributed to the different apple cultivars studied, as BI values can vary significantly between varieties due to differences in phenolic content and enzymatic activity.

These findings align with previous research showing that color evolution in apples is driven by a combination of chlorophyll degradation, pigment synthesis (e.g., carotenoids), and exposure of preformed pigments, processes that are tightly connected to starch breakdown, respiration, and climacteric ripening [61].

3.4. Starch Grains

Differences between starch concentrations and the rate of starch degradation within the fruit can be variety-specific [37]. A long time ago, Brookfield et al. [62] found that the amounts of starch in the outer cortex were higher compared to the core and the vascular bundle region. According to Doerflinger et al. [63], in the apple varieties ‘Gala’ and ‘Empire’, starch concentrations in the calyx-end tissue are higher compared to the equatorial and stem zones. This means that starch is not evenly distributed within the fruit during development. This may all be relevant to the development of physiological disorders such as flesh browning in ‘Empire’ and ‘Gala’ apples, which is first visible in the stem-end part of the fruit [64].

The quantification of starch grains in Granny Smith apples at different harvest stages, both at the beginning and at the end of the shelf-life period, is presented in Figure 2. On day 0, the unripen fruits exhibited the highest starch content (1.09 × 10³ OD), which subsequently declined with increasing maturity. By day 15 of shelf-life, the starch content also reached its maximum in the unripen fruits (0.79 × 10³ OD), but the values were overall lower compared to those recorded at the beginning of shelf-life. At the end of shelf life, starch grain values were significantly reduced across all maturity stages. After cold storage, the measured values dropped to nearly zero, indicating that starch was almost completely degraded during prolonged storage. These findings were consistent with the SPI data presented in Table 4, which also demonstrated progressive starch hydrolysis as shelf life advanced.

The starch granules in the fruit are degraded from the inside out, resulting in a rate of degradation that does not depend on the size of the granules. Therefore, the differences in granule size and/or the number of starch granules in different tissue zones of the fruit could lead to differences in starch distribution within the fruit, even if starch degradation is initiated simultaneously throughout the fruit [63].

Figure 3 shows representative microscopic images of stained starch grains in the parenchyma tissue of Granny Smith apples obtained after I₂KI staining, illustrating the decline in starch content with later harvest stages.

Starch, which plays a central role in maintaining cell turgor and firmness, was progressively degraded during shelf life and storage. This degradation contributed not only to the observed increase in soluble solids content but also to the loss of turgor pressure and overall softening of the fruit [65]. These findings align with previous reports that highlight starch hydrolysis as a key process in postharvest ripening, affecting both texture and sweetness [66]. In apples, starch is also recognized as a reliable indicator of harvest maturity, making its quantification and pattern crucial for postharvest quality assessment. The enzymatic conversion of starch into sugars during storage provides a respiratory substrate, supporting metabolic activities but ultimately contributing to tissue softening, as confirmed by the decreasing firmness values observed in this study [67].

3.5. Raman Spectra

Figure 4 displays the normalized Raman spectra of peeled Granny Smith apple samples. Several characteristic bands were identified, with prominent peaks around 478, 865, 940, and 1128 cm⁻¹, corresponding to starch components, as previously reported [17,68]. Variations in the intensity of these bands aligned with the starch indices obtained from the iodine staining.

In the initial phase of analysis, measurements were also conducted on unpeeled apples. However, the structural complexity of the apple peel hindered the Raman microscope’s ability to reliably differentiate chemical surface features. This limitation is likely due to the small laser spot size (on the order of a few micrometers, per manufacturer specifications), which restricts surface area coverage. Although some studies have employed Raman systems with larger laser spots to improve surface characterization—for example, Monago-Maraña et al. [18] used a 6 mm laser spot—challenges still persist. These authors noted that both peeled and unpeeled samples produced spectra dominated by carbohydrate signals, which they attributed to the laser’s ability to penetrate the thin apple skin and diminish the influence of surface pigments such as chlorophyll.

Figure 5 presents the Principal Component Analysis (PCA) of Raman spectral data related to starch content in Granny Smith apples, which was used to investigate the main sources of variation within the spectra. The first two principal components, PC1 and PC2, accounted for 88.7% and 7.4% of the total variance, respectively, and revealed three distinct clusters. The first cluster comprised the unripen fruits sampled at days 0 and 5, while the second cluster included the unripen fruits at day 10, along with the ripen fruits from various time points (excluding day 15) and the overripen fruits at days 5 and 10. The third cluster was specific to the overripen fruits at day 0. Notably, starch was not detected at day 15 for any of the harvest stages, indicating advanced starch degradation. Although there are indications that the samples tend to cluster according to sampling time, this pattern is not well-defined, likely due to substantial chemical variability within samples at each time point.

The PCA results, together with Raman spectral analysis, confirmed the progressive reduction in starch content during ripening. These findings are in agreement with the results obtained from SPI (Table 4) and starch grain quantification (Figure 4), supporting the overall trend of starch hydrolysis throughout the shelf-life period.

These findings have practical relevance for growers and suppliers aiming to ensure consistent fruit quality along the supply chain.

To further advance our understanding and enhance postharvest quality management, future studies should consider the following:

• Integration of non-destructive monitoring techniques: The use of hyperspectral imaging or handheld Raman spectroscopy could provide real-time insights into fruit ripening status without damaging the produce.
• Detailed cell wall profiling: Enzymatic activity and pectin solubilization could be monitored to better understand the biochemical basis of firmness loss.
• Varietal comparisons: Expanding the study to include additional apple cultivars with different storage behaviors would enable the development of variety-specific storage protocols.
• Consumer preference assessment: Linking objective texture and flavor parameters with sensory evaluation could help define optimal quality windows from the consumer perspective.
• Storage condition modeling: Predictive modeling based on multivariate data (e.g., PCA, machine learning) could support decision-making in postharvest handling and logistics.

Overall, these results lay a solid foundation for improving postharvest strategies and demonstrate the potential of combining classical techniques with advanced analytical tools for comprehensive fruit quality assessment.

4. Conclusions

This study highlights the critical influence of harvest stage, storage duration, and shelf-life period on the physico-chemical quality of Granny Smith apples. The comprehensive evaluation of these combined effects provides valuable insights for improving postharvest handling practices and guiding future studies aimed at maintaining apple quality and storability.

The influence of each factor on texture parameters varied in magnitude, with storage duration exerting the strongest overall effect, followed by harvest stage and shelf-life. The three-way interaction had a minimal effect, influencing only a few specific texture parameters. Regarding maturity indices, all factors individually, as well as their combination, showed significant effects.

For color parameters, harvest stage and storage duration showed a measurable effect across all parameters, while shelf-life and factor interactions contributed to variation in specific parameters. The use of starch pattern index, microscopic quantification, and Raman spectroscopy provided complementary information on starch degradation, supporting their relevance in monitoring postharvest ripening processes in apples.

Overall, the findings emphasize the relevance of harvest timing and storage conditions in preserving key quality parameters. Apple harvested at earlier maturity stages showed more favorable postharvest behavior under the tested conditions.