Standardization of Vacuum Drying Conditions Based on Biochemical Composition of Pomegranate Peel for Polyphenol Extraction

Introduction

Pomegranate (Punica granatum L.) peel is an abundant agro-industrial by-product, recognized for its rich profile of bioactive compounds, including polyphenols, flavonoids, and potent antioxidants. Compared to the edible arils, the peel possesses significantly higher concentrations of these health-promoting agents (Venkataramanamma et al., 2016) [16] . Furthermore, pomegranate peel extracts exhibit significant anti-inflammatory and oxidative stress modulation potential, and their valorization is considered a key strategy for sustainable waste management. However, effective recovery of these compounds is constrained by thermal sensitivity. Postharvest processing, specifically drying, is a critical step in preserving the bioactive profile; however, inconsistent parameters can severely undermine phytochemical quality (Magangana et al., 2020) [8] . Vacuum drying has emerged as a superior alternative, enabling moisture removal at lower temperatures and reduced oxygen exposure, which minimizes oxidative and thermal damage to bioactive constituents (Tran et al., 2024) [15] . Despite the theoretical advantages of vacuum drying, there remains a need for precise optimization of processing parameters such as temperature and pressure to balance drying kinetics with the retention of bioactive quality. Furthermore, the literature reveals significant variability in outcomes due to differing, unstandardized operational parameters, which complicates the identification of optimal processing conditions for specific fruit varieties (Al-Rawahi et al., 2013) [1] . Consequently, this study aims to standardize vacuum tray drying conditions specifically for the Mridula variety of pomegranate peel. By systematically evaluating the influence of varying drying temperatures at a constant vacuum pressure of -600 mmHg, this research seeks to establish an optimal processing protocol that maximizes the retention of polyphenols and overall antioxidant capacity in pomegranate peel powder.

Materials and Methods

Materials

Pomegranate (Punica granatum L.) fruits of the Mridula variety were procured from a local market at commercial maturity. All analytical grade chemicals including sodium carbonate, sodium hydroxide, potassium acetate, copper sulphate, gallic acid, DPPH (High Media Laboratories Pvt. Ltd.), Folin & Ciocalteu phenol reagent (Research Lab Pvt. Ltd.), anhydrous aluminum chloride, methanol (Loba Chemie Pvt. Ltd.), quercetin, sulphuric acid, hydrochloric acid, boric acid, and methyl red indicator (Sisco Research Laboratories Pvt. Ltd.) were used as received. All reagents were prepared following standard laboratory protocols. For packaging, 50 mL black metallized vacuum stand-up pouches (without zipper) were sourced from Swiss Packaging Pvt. Ltd.

Vacuum Tray Drying of Pomegranate Peels

Pomegranate fruits were washed thoroughly under running tap water to remove surface contaminants. Arils were manually separated from the peels. The collected peels were washed with potable water, drained of excess surface moisture, and cut into uniform pieces (3-4 mm) to facilitate consistent heat and mass transfer. Drying was performed using a laboratory-scale vacuum tray dryer equipped with a condenser (0.37 kW) and a rotary vacuum pump (0.75 kW). The drying chamber contained five tray shelves. Prior to loading, the dryer was pre-equilibrated for 20 min to reach the target temperature. Samples were subjected to vacuum drying at -600 mmHg pressure at three temperature levels (40, 50, and 60 °C). The process continued until the moisture content was reduced to <10% (w.b.). Following drying, samples were cooled to room temperature, ground using a laboratory mill, and passed through a 40-mesh sieve to ensure uniform particle size. The resultant powder was vacuum packaged in 50 mL black metallized stand-up pouches (vacuum pressure: 60 mbar; sealing time: 2.3 s; cooling time: 0.5 s). The use of light-impermeable packaging minimized light-induced degradation and moisture absorption. Sealed pouches were stored at -18 °C until further analysis.

Proximate Analysis

The proximate composition of the pomegranate peel powder including moisture, crude protein, crude fat, total ash, and crude fiber, was determined according to the standard analytical procedures of the Association of Official Analytical Chemists (AOAC, 2019). Moisture content was determined by the oven drying method (Method 925.10) until a constant weight was achieved. Crude protein was estimated using the Kjeldahl method (Method 920.87), employing a conversion factor of 6.25 to convert total nitrogen to crude protein. Crude fat was analyzed via solvent extraction using a Soxhlet apparatus (Method 920.39). Total ash content was measured by dry ashing the sample in a muffle furnace at 550 °C (Method 923.03). Crude fiber was determined using the acid-base digestion method (Method 962.09). Total carbohydrate content was calculated by the difference method, subtracting the sum of moisture, protein, fat, ash, and fiber contents from 100%.

Extraction of Phenolic Compounds

Extraction was performed according to the method of Shiban et al. (2012) [14] with minor modifications. Briefly, 10 mg of the dried, powdered pomegranate peel sample was macerated with 10 mL of methanol to facilitate the extraction of bioactive compounds. The mixture was stored at 5 °C for 24 h to ensure complete extraction, followed by centrifugation at 3000 rpm for 15 min. The resulting supernatant was collected, filtered, and used as the crude extract for further analysis.

Determination of Total Phenolic Content (TPC)

The total phenolic content was estimated using the Folin-Ciocalteu (FC) colorimetric assay, following the protocol of Redha et al. (2018) with slight modifications. In a reaction vessel, 1 mL of the crude extract was mixed with 10 mL of distilled water and 2 mL of Folin-Ciocalteu reagent. The mixture was allowed to react for 2 min. Subsequently, 4 mL of 0.7 M Na₂CO₃ solution was added, and the solution was vortexed thoroughly. Finally, 8 mL of distilled water was added to the mixture. The reaction mixture was incubated at room temperature in a dark environment for 30 min to ensure color development. The absorbance of the resulting blue-colored complex was measured at 760 nm using a UV-VIS spectrophotometer against a blank prepared with distilled water and identical reagents. Gallic acid was utilized as the standard, and the total phenolic content was calculated from the standard calibration curve and expressed as mg GAE g⁻¹.

Total Flavonoid Content

The total flavonoid content (TFC) was determined using the aluminium chloride colorimetric assay, as described by Zhishen et al. (1999) with minor modifications. Briefly, 1 mL of the crude extract was mixed with 5 mL of 60% methanol. After an equilibration period of 5 min, 0.2 mL of 10% aluminium chloride (AlCl₃) and 0.2 mL of 1 M potassium acetate were added to the mixture. The final volume of the reaction mixture was adjusted to 10 mL using distilled water. The solution was vortexed thoroughly and incubated in the dark at room temperature for 15 min. The absorbance was measured at 510 nm using a UV-VIS spectrophotometer against a blank prepared with distilled water and identical reagents. The TFC was calculated from a quercetin standard calibration curve and expressed as mg QE g⁻¹.

Antioxidant Activity

The antioxidant potential of the pomegranate peel extracts was evaluated using the DPPH (2,2-diphenyl-1picrylhydrazyl) radical scavenging assay, following the protocol of Brand-Williams et al. (1995) [3] with minor modifications. Briefly, an aliquot of the methanolic extract (equivalent to 100 mg of sample) was transferred to a reaction vessel and mixed with 2.9 mL of 0.1 mM DPPH solution. The mixture was vortexed thoroughly and incubated in the dark at room temperature for 30 min to ensure complete reaction. Absorbance was subsequently measured at 517 nm using a UV-VIS spectrophotometer, with methanol serving as the blank. The percentage of DPPH radical scavenging activity was calculated as follows: % Scavenging Activity = [(blanksample) / blank] × 100 where blank is the absorbance of the DPPH solution without the extract, and sample is the absorbance of the reaction mixture containing the extract. [10,6,7] , reinforcing that the processing conditions employed successfully preserved the functional properties of the peel. Observed variations in composition compared to earlier studies (Fawole et al., 2012) [4] may be attributed to differences in genetic variety, fruit maturity at harvest, and the specific vacuum drying parameters applied.

Statistical Analysis

Vacuum Tray Drying of Pomegranate Peels

Lower drying temperatures for pomegranate peel extended drying time as shown in Table 2, whereas higher drying temperatures resulted in a loss of total phenolic content negatively impacting the responses. These findings align with those reported by Sarkar et al. (2024) [13] regarding the thermal degradation of bioactive compounds, where elevated drying temperatures resulted in reduced antioxidant capacity and phenolic stability. Instantaneous drying rates (g h⁻¹) were calculated as weight loss per hour. Initial (0-1 h) drying rates increased with temperature: 32.94 g h⁻¹ (40 °C), 62.06 g h⁻¹ (50 °C) and 79.18 g h⁻¹ (60 °C). Across all temperatures, drying rate declined over time, indicating a transition from a short constant-rate due to surface moisture removal period to a prolonged falling-rate period due internal diffusion controlled. By 6-8 h, rates at different temperatures converged to low values less than 2 g h⁻¹. The experimental data obtained was employed to generate a drying curve depicting the relationship between moisture content and drying time as presented in Fig. 3. The vacuum drying treatment conducted at 40 °C required the longest drying duration of 10 h to achieve the desired moisture content, whereas the shortest drying time of 6 h was observed at 60 °C. Drying at 50 °C required an intermediate duration of 8 h to attain comparable moisture reduction. At lower temperatures prolonged drying period was required to achieve moisture reduction in pomegranate peels whereas 60 °C accelerated the process. These observations confirm the influence of drying temperature on the moisture removal of pomegranate peels under vacuum conditions. The drying profile showed two distinct phases, an initial phase with a notable decrease in moisture content over time followed by a slower drying rate in the final stage due to reduced mass transfer driving force. This behaviour likely resulted from the faster evaporation of surface moisture initially while internal moisture required longer diffusion pathways to reach the surface. Similar findings were reported by Hameed et al. (2022) [5] .

Standardization of Vacuum Drying Process Parameters

The standardization of vacuum drying parameters was directed toward optimizing the recovery of bioactive compounds, with Total Phenolic Content (TPC) serving as the primary response variable. The experimental data (Table 3) illustrates a significant temperature-dependent impact on phenolic retention, with visual trends depicted in Fig. 4. The highest TPC yield was recorded at 40 °C (242.15 ± 3.56 mg GAE g⁻¹), followed by 50 °C (222.00 ± 5.21 mg GAE g⁻¹), and 60 °C (194.73 ± 3.75 mg GAE g⁻¹). These results reveal a clear inverse correlation between drying temperature and TPC retention, suggesting that elevated processing temperatures promote the thermal degradation of heat-sensitive compounds. This trend aligns with the findings of Mphahlele et al. (2016) [9] , who reported that extended exposure to higher temperatures during drying leads to the thermolabile breakdown of bioactive constituents.

Statistical analysis via one-way ANOVA (Table 4) confirmed that drying temperature exerted a significant influence on the TPC of the pomegranate peel powder (p < 0.05). The high F-value (31.52) indicates that temperature is the dominant factor driving the variance in phenolic retention. The application of Duncan's Multiple Range Test (DMRT) further validated distinct performance gaps between the 40 °C, 50 °C, and 60 °C treatments (Table 3), reinforcing the suitability of 40 °C as the optimal processing condition for maximizing the functional quality of the peel powder.

These findings align with studies on the vacuum drying of various vegetables, which demonstrate that elevating drying temperatures or extending processing durations consistently leads to a reduction in phenolic compounds. The reduction in TPC observed in pomegranate peels as temperatures rose from 40 °C to 60 °C can be attributed to the thermal degradation of heat sensitive phenolic compounds during extended thermal exposure (Mphahlele et al., 2016) [9] . The analysis of variance (ANOVA) presented in Table 4 confirms a statistically significant relationship (p < 0.05) between drying temperature and the TPC of pomegranate peel. The high F-value indicates that the differences in mean values across the tested temperatures are robust and not attributable to random chance. Furthermore, the sum of squares analysis reveals that drying temperature is the primary driver of variance in phenolic retention, accounting for the majority of the total observed variation. The low coefficient of variation underscores the precision and reproducibility of the experimental measurements, validating the suitability of the proposed vacuum drying protocol for high-quality product output. These bioactiverich extracts are highly promising for the food industry; recent research highlights their efficacy not only as functional ingredients but also as natural preservatives capable of enhancing shelf life in food systems.

Conclusion

This study successfully standardized vacuum tray drying conditions for the Mridula variety of pomegranate peel, identifying 40 °C at -600 mmHg as the optimal protocol for maximizing the retention of bioactive compounds. Proximate analysis confirmed that the resulting powder is a nutrient-dense functional ingredient, rich in dietary fiber (16.26%) and carbohydrates (73.31%), with a significant total phenolic content (264.80 mg GAE g⁻¹) and robust antioxidant activity (94.18%). Experimental results demonstrated an inverse correlation between drying temperature and phenolic retention; while elevated temperatures (50 °C and 60 °C) accelerated moisture reduction, they detrimentally impacted the thermolabile bioactive profile. Statistical validation via ANOVA and DMRT confirmed that the 40 °C treatment maintained significantly higher phytochemical integrity compared to higher temperature treatments. Consequently, vacuum drying at 40 °C represents an effective, scalable strategy for preserving the functional and phytochemical properties of pomegranate peel, facilitating its application as a highvalue, natural ingredient in the nutraceutical and food processing industries, consistent with industrial valorization trends.

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