Effects of High Pressure Processing and Ultrasound-Assisted Extraction on Physicochemical Properties, Antioxidant Activity and Flavor Compounds of Cold Brew Citri Reticulatae Pericarpium Beverage

Abstract

Highlights

• HPP and UAE-assisted extraction enhanced the extraction efficiency of cold brew CRP water.

• The combined HPP+UAE approach maximized the diversity and total content of VOCs of CRP water.

• Aging years induced a clear shift in the aroma profile of CRP water from a fresh type to a woody type.

• The flavor quality and antioxidant activity of CRP water were synergistically improved by the combined HPP+UAE approach.

Abstract

High pressure processing (HPP) and ultrasound-assisted extraction (UAE) can effectively shorten extraction time and increase extraction efficiency of the cold brew (CB) process. However, their application in CB citri reticulatae pericarpium (CRP) and the underlying mechanisms of flavor modulation remain poorly understood. In this study, CB-CRP beverage was prepared with HPP-assisted, UAE-assisted, and HPP+UAE-assisted extraction from 1, 3, 5, and 10 years CRP. Results revealed that the total soluble solids (TSS), total sugars, flavonoids, polyphenols, and volatile organic compounds (VOCs) and antioxidant activity of CB-CRP increased after assisted extraction. The combined application of HPP and HPP+UAE-assisted extraction exhibited the most pronounced effects. The kinds and total content of VOCs of CB beverages prepared from 10-year-aged CRP increased from 45 to 81, and from 2.44 to 5.98 μg/mL, respectively. Moreover, the combined HPP+UAE extraction promoted the enrichment of fatty and woody aroma-related compounds, which drove a shift in the flavor profile from fresh to a richer woody type. And this endowed the CB-CRP water with a more complex and multidimensional aroma profile.

1. Introduction

In recent years, cold brew (CB) beverages, such as CB coffee, CB tea, and other variants, have gradually gained in popularity due to their pure taste and flavor, and CB beverages with functional and health-oriented effects are becoming a research hotspot [1,2]. CB is a slow, static, and full-immersion extraction process, and it is typically performed with water at room temperature or even lower temperatures [3]. The volatile aromatic compounds of traditional hot brewing are easily vaporized at high temperatures. Additionally, new compounds may be generated during high-temperature reactions, altering the original aroma profile of plant-based raw materials [4]. CB tea is typically brewed at low temperatures (4–25 °C) for extended periods, which helps reduce bitterness and caffeine content, resulting in a smoother taste. It also better preserves the original aroma and bioactive compounds of the tea leaves [2,5]. Previous studies have shown that, compared with traditional hot extraction methods, CB coffee has a smoother taste and a sweeter flavor with an overall lower intensity of bitterness [6]. However, a longer extraction time and lower extraction efficiency are typically required by the CB process, which increases production costs and limits the further development of CB beverages [7]. To date, research on CB has primarily focused on raw materials such as tea and coffee, whereas its application in beverages prepared from citri reticulatae pericarpium (CRP) remains relatively limited, warranting further investigation.

CRP is a traditional medicinal and edible material made from the dried peel of citrus fruits and their cultivated varieties in the Rutaceae family. CRP has been regarded as an important ingredient in traditional medicine in China, Japan, and Korea [8]. In China, CRP has been used for centuries, and it is generally believed that the longer it ages, the higher its medicinal value in traditional Chinese medicine. Its processing technology is well-developed, and it is rich in bioactive compounds such as flavonoids, volatile oils, and alkaloids, which exhibit strong antioxidant effects. There are a number of studies that have reported the hypotensive, hypolipidemic, and anti-inflammatory effects of CRP [9,10,11]. With changing dietary practices, CRP has increasingly been incorporated into the daily diet as a spice, beverage ingredient, or functional food component and recognized as a representative material with medicine and food homology [12]. Currently, heating is frequently applied during the preparation of CRP-based beverages; heat-sensitive components and volatile organic compounds (VOCs) may therefore be partially degraded or lost. Under a CB framework, high pressure processing (HPP) and ultrasound-assisted extraction (UAE) can be introduced as non-thermal-assisted extraction techniques to improve extraction efficiency at low temperatures.

HPP is a non-thermal technology in which high pressure (typically 100–600 MPa) is applied via a liquid medium (usually water) [13]. As a green, non-thermal technology, HPP has been widely used in food processing [14]. HPP can directly disrupt cell structures and accelerates the release of active ingredients. It has been widely applied to extract bioactive compounds from animals, plants, and fungi [13,15,16,17]. Compared with traditional extraction techniques, the bioactivity of compounds can be better preserved with higher extraction efficiency under HPP because of its low processing temperature [18]. UAE is also a typical non-thermal processing method and has been widely used to assist in the extraction of thermosensitive active ingredients [19,20]. By UAE, plant cell membranes and cell walls can be disrupted through ultrasound-induced cavitation and mechanical effects, thereby increasing the contact area between the tissue and solvent and accelerating the release of bioactive compounds [21,22]. In addition, ultrasound can also promote chemical reactions such as esterification and protein hydrolysis, thereby enhancing the aromatic properties of food [23]. In summary, HPP and UAE, as typical non-thermal assisted extraction technologies, can enhance cell permeability through physical actions under low-temperature conditions, promote the release of bioactive compounds, and effectively reduce the damage to their structure and function caused by heat treatment. The application of HPP and UAE for the preparation of CRP water may hold promise for retaining bioactive components and optimizing flavor quality, while providing a theoretical basis and technical support for the CB processing of medicinal and edible beverages.

CRP samples aged for 1, 3, 5, and 10 years were used as raw materials, and CB-CRP water was prepared by HPP, UAE, and the combined treatment of HPP and UAE, respectively. The effects of different assisted extraction approaches on the functional properties and flavor quality of CB-CRP water were systematically evaluated using physicochemical indices (color, total sugar, soluble solids, and flavonoid and polyphenol contents), antioxidant capacity assays (DPPH, ABTS, and FRAP), and flavoromics analyses (GC-MS and E-nose). The results are expected to provide a theoretical basis for process optimization and the functional development of CRP-based beverages.

2. Materials and Methods

2.1. Materials

CRP was obtained from Guangdong Yixiang Chenpi Co., Ltd. (Xinhui, Guangdong, China). The total sugar assay kit (G0503W), DPPH free radical scavenging assay kit (DPPHFRS.S-W96-N, 2621), ABTS cation radical scavenging assay kit (TAOCA-W96-N, 1720), and ferric reducing antioxidant power (FRAP) assay kit (TAOCF-W96-N, 1720) were purchased from Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Methods

2.2.1. Sample Preparation

CRP samples aged for 1, 3, 5, and 10 years were used as raw materials. Samples were stored at room temperature (65% relative humidity) in the dark, protected from strong light and UV exposure, with automatic ventilation to maintain air circulation during aging. The CRP samples were all cut into pieces, immersed in liquid nitrogen, and ground to powder in a pre-cooled mortar. Then, CRP powders were screened through 60 mesh, and 8 g of CRP powder and 200 mL deionized water were added to a 250 mL bottle. The mixture was placed at room temperature for 30 min to allow sufficient hydration. Subsequently, the mixture was subjected to different assisted extraction approaches as follows: HPP-assisted extraction (500 MPa) for 10 min, UAE-assisted (500 W, 20 kHz) extraction for 10 min, or sequential HPP-assisted (500 MPa) extraction for 10 min followed by UAE-assisted (500 W, 20 kHz) extraction for 10 min. The directly soaked mixture for 10 min without any assisted processing was used as control group (CG). All extraction processes were performed at 25 °C. After extraction, all mixtures were stored at 4 °C for 6 h. Subsequently, the extraction solution was centrifuged at 10,000 rpm for 10 min. The supernatant was collected and sterilized by filtration before analyses. The processing parameters (pressure for HPP and ultrasonic power/frequency for UAE) and the processing durations for HPP, UAE, and HPP+UAE were optimized based on preliminary experiments, and the selected optimal conditions were used in this study.

2.2.2. Color

The color of CRP water was determined with a colorimeter (JMCM-828N, CANY, Shanghai, China). 10 mL of CRP water was transferred into a quartz cuvette for measurement. After background calibration was performed with a white standard plate, the cuvette containing the sample was placed under the same background conditions for measurement, and the L* value was recorded [24].

2.2.3. TSS

The total soluble solids (TSS) content was determined with an automatic refractometer (DFT-F10V55H23, DiFluid, Shenzhen, China). Before measurement, the instrument was calibrated with distilled water, and each sample was measured in triplicate.

2.2.4. Total Sugar

Total sugar content was determined using a DNS-based total sugar assay kit (G0503W; Suzhou Grace Biotechnology Co., Ltd., Suzhou, China). Briefly, 0.1 mL of liquid sample was mixed with 400 μL of Reagent I in an EP tube and incubated in a 90 °C water bath for 30 min with intermittent shaking (every 15 min). After cooling in an ice-water bath to room temperature, 400 μL of Reagent II was added, and the mixture was brought to 1.0 mL with distilled water. The solution was centrifuged at 12,000 rpm for 10 min at 25 °C, and the supernatant was collected. For color development, 100 μL of supernatant was mixed with 100 μL of Reagent III, while the blank contained 100 μL of distilled water and 100 μL of Reagent III. The mixtures were incubated at 95 °C for 10 min, immediately cooled to room temperature in an ice-water bath, and the absorbance was measured at 500 nm. A glucose standard curve was generated, and samples were diluted as needed to ensure absorbance values fell within the linear range. Total sugar content was calculated using the equation y = 7.6134x − 0.0321, where y denotes ΔA and x denotes glucose mass (mg).

2.2.5. Total Flavonoid Content

The total flavonoid content was determined according to the method described by Liu et al. [25], with minor modifications. Briefly, 1.0 mL of the supernatant was accurately transferred into a 10 mL centrifuge tube, followed by the addition of 0.30 mL of 5% sodium nitrite solution. The mixture was shaken thoroughly and allowed to stand for 6 min. Subsequently, 0.30 mL of 10% aluminum nitrate solution was added, mixed well, and allowed to react for 6 min. Then, 4.00 mL of 4% sodium hydroxide solution was added, and the mixture was diluted to volume with 70% ethanol, mixed thoroughly, and allowed to stand for 10–15 min. The absorbance was measured at 510 nm. Rutin was used as the standard to construct the calibration curve (y = 7.602x + 0.0003, R² = 0.9997).

2.2.6. Total Polyphenol Content

The polyphenol content of CRP water was determined using high-performance liquid chromatography (LC-20ADXR, Shimadzu Corporation, Kyoto, Japan). Briefly, 1.0 mL of CRP water was filtered through a 0.22 μm aqueous membrane filter and transferred into a 1.5 mL autosampler vial. The injection volume was 10 μL. The mobile phase consisted of solvent A (water–methanol–acetic acid, 88:10:2, v/v/v) and solvent B (water–methanol–acetic acid, 10:88:2, v/v/v). Gradient elution was applied as follows: 0.01–16.5 min, 20% B; 16.5–30.0 min, 80% B; and 30.0–40.0 min, 0% B. A mixed phenolic standard solution containing neochlorogenic acid, chlorogenic acid, scopoletin, and nobiletin was used for retention time calibration and peak identification. Quantification was performed by an external standard method under identical analytical conditions, using peak areas as the quantitative response. Concentrations of individual phenolics were calculated from the sample-to-standard peak area ratios and the known standard concentrations, and reported in μg/mL. Total phenolics were obtained as the sum of the four quantified compounds.

2.2.7. Antioxidant Activity

The DPPH free radical scavenging activity was determined using a commercial kit (DPPHFRS.S-W96-N, 2621; Suzhou Grace Biotechnology Co., Ltd., Suzhou, China). In a 96-well plate, 80 μL of sample solution was mixed with 120 μL of DPPH working solution, with a sample control (80 μL sample + 120 μL extraction solvent) and a blank (80 μL extraction solvent + 120 μL DPPH working solution) included to correct for background absorbance. After incubation at room temperature (25 °C) in the dark for 30 min, absorbance was measured at 515 nm. A Trolox standard curve (0–25 μg/mL) was generated (y = 0.0287x − 0.003, R² = 0.9983), and results were calculated based on liquid volume and expressed as μg Trolox/mL: DPPH scavenging capacity = {[(scavenging rate/100) + 0.003]/0.0287} × D, where D is the dilution factor.

The ABTS cation radical scavenging assay kit (TAOCA-W96-N, 1720) was purchased from Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China). ABTS radical scavenging activity was measured at 734 nm using a 96-well plate by mixing 10 μL of sample with 190 μL of ABTS working solution; a sample control (10 μL sample + 190 μL extraction solvent) and a blank (10 μL extraction solvent + 190 μL ABTS working solution) were included. After incubation at room temperature in the dark for 6 min, absorbance was recorded. A Trolox calibration curve (0–1.0 μmol/mL) was generated (y = 0.626x − 0.0022, R² = 0.9993), and total antioxidant capacity was expressed as μmol Trolox/mL: [(ΔA + 0.0022)/0.626] × D, where D is the dilution factor.

The ferric reducing antioxidant power (FRAP) assay kit (TAOCF-W96-N, 1720) was purchased from Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China). FRAP was measured at 593 nm using a 96-well plate by mixing 10 μL of sample with 190 μL of freshly prepared FRAP working solution (Reagents I–III, preheated to 37 °C). A blank (10 μL extraction solvent + 190 μL FRAP working solution) was included. After incubation at room temperature (25 °C) for 10 min, absorbance was recorded and ΔA was obtained after blank subtraction. A Trolox calibration curve was generated (y = 0.1053x + 0.0089), and total antioxidant capacity was calculated based on liquid volume and expressed as μmol Trolox/mL: [(ΔA − 0.0089)/0.1053] × 10⁻³/V₁ × D (= 0.9497 × (ΔA − 0.0089) × D), where V₁ = 0.01 mL and D is the dilution factor.

2.2.8. Amino Acid Composition

The amino acid content of CRP water was analyzed using an automatic amino acid analyzer (V388, MembraPure GmbH, Berlin, Germany). Briefly, 1.0 mL of CRP water was filtered through a 0.22 μm aqueous membrane filter and transferred into a 1.5 mL autosampler vial prior to analysis. The analytical conditions were set as follows: detector temperature, 125 °C; sampler temperature, 40 °C; and flow rate, 240 μL/min. Ninhydrin reagent and sodium buffer reagents (A, B, C, D, and F) were used for amino acid derivatization and detection. An amino acid mixed standard solution containing Asp, Thr, Ser, Glu, Gly, Ala, (Cys)₂, Val, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, and Pro was used for retention time calibration and peak identification. Quantification was carried out by external single-point calibration. The mixed standard solution and samples were injected under identical analytical conditions, and peak areas were used as the quantitative responses. Amino acid concentrations in the samples were determined by comparing sample peak areas with those of the corresponding amino acids in the standard solution and referencing the known standard concentrations. Results were reported in μg/mL.

2.2.9. E-Nose

E-nose (C-Nose, Bosin, Shanghai, China) analysis was performed to evaluate the volatile profiles of CRP water. An quantity of 10 mL of CRP water was transferred into a 20 mL headspace vial, which was then sealed and allowed to equilibrate at room temperature for 30 min prior to analysis. The measurement conditions were set as follows: detection time, 60 s; cleaning time, 300 s; and carrier gas flow rate, 0.6 L/min [26].

2.2.10. HS- SPME-GC-MS

VOCs in CRP water were identified with a Triple Quadrupole GC-MS/MS (TSQ Quantum XLS, Thermofisher, Waltham, MA, USA) equipped with a capillary column (DB-5MS 60 m × 0.25 mm × 1.0 μm, Agilent, Santa Clara, CA, USA). A quantity of 5 mL of CRP water was filtered through a 0.22 μm aqueous membrane filter and transferred into a 20 mL sample vial. Ethyl phenylacetate (15 μL, 1 mg/mL) was added as an internal standard, and the vial was immediately sealed prior to analysis. Gas chromatography conditions were set as follows: injector temperature, 290 °C; split ratio, 10:1; carrier gas flow rate, 1.5 mL/min; and injection volume, 1 μL. The oven temperature program was as follows: the initial temperature was held at 60 °C, then increased to 250 °C at a rate of 2 °C/min, and further increased to 290 °C at a rate of 5 °C/min and held for 20 min. Mass spectrometry conditions were as follows: ion source temperature: 230 °C; interface temperature: 290 °C; solvent delay time: 1.5 min; and data acquisition mode: full-scan and selected ion monitoring (SIM).

2.2.11. Sensory Evaluation

The sensory evaluation form was designed according to GB/T 23776-2018 (Table S1). The sensory panel consisted of ten members (five males and five females), aged 20–40 years, all of whom received systematic sensory training for three months. Sensory evaluation was conducted in a standardized sensory evaluation room that was clean, well lit, and equipped with individual booths, with each panelist performing the evaluation independently.

2.3. Statistical Analysis

Data were processed with Microsoft Excel to calculate means, sums, and standard deviations. Graphs were generated with Origin 2021, and statistical significance was analyzed with SPSS Statistics 25, with a significance level set at p < 0.05. All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (SD).

3. Results

3.1. Effects of Extraction Methods on Physicochemical Properties

The effects of different assisted extraction approaches, including HPP, UAE, and the combined HPP+UAE approach, on the physicochemical properties (color, L* value, TSS, total sugars, flavonoids, and polyphenols) of different years-aged CRP water were evaluated. The changes in physicochemical properties of different years-aged CRP water under different assisted extraction approaches (CG, HPP, UAE, and HPP+UAE) were illustrated (Figure 1A–F). In the untreated CRP water, the color gradually darkened and the L* value decreased with increasing aging years, while the contents of TSS, total sugars, flavonoids, and polyphenols exhibited an increasing trend. After different assisted extraction approaches, the L* values of CRP water of all aging years decreased, whereas the contents of TSS, total sugars, flavonoids, and polyphenols were significantly increased (p < 0.05). This may be attributed to the fact that HPP weakened the integrity and binding properties of cell walls by applying uniform high pressure, leading to the disruption of pressure-sensitive noncovalent interactions within cell walls or membranes. As a result, cell wall structures were damaged and cell permeability was increased, which in turn accelerates the release of intracellular constituents [18]. Under UAE, the cellular structure of CRP was disrupted by shear forces generated through ultrasonic cavitation, thereby facilitating solvent penetration into the cells and promoting the rapid release of soluble compounds [21]. In addition, the degradation of macromolecules such as starch and pectin within CRP cells may be induced by HPP and UAE extraction, thereby further accelerating their dissolution [13,27]. After the combined HPP+UAE treatment, the physicochemical properties of different years-aged CRP water exhibited the most pronounced changes. Specifically, the TSS content increased from 0.47% to 0.68% (44.68%) in the 1-year sample, from 0.47% to 0.85% (80.85%) in the 3-year sample, from 0.27% to 0.92% (240.74%) in the 5-year sample, and from 0.25% to 0.78% (212.00%) in the 10-year sample. Consistent increases were also observed in total polyphenols. The polyphenol content increased from 4.86 to 6.63 ng/mL (36.42%) in the 1-year sample, from 5.88 to 6.89 ng/mL (17.18%) in the 3-year sample, from 6.81 to 14.21 ng/mL (108.66%) in the 5-year sample, and from 7.02 to 11.41 ng/mL (62.51%) in the 10-year sample following HPP+UAE treatment. HPP and UAE exhibited a synergistic effect when applied as assisted extraction techniques during the preparation of CB-CRP water. This synergy may be attributed to the initial disruption of CRP cell wall structures by HPP, thereby rendering the cells more susceptible to subsequent physical damage during UAE and promoting the release of a greater amount of soluble constituents [28]. Overall, the quality of CRP water was significantly improved by HPP, UAE, and the combined HPP+UAE approach, among which the combined HPP+UAE approach exhibited a distinct advantage in improving color attributes, increasing the content of soluble substances, and promoting the release of antioxidant-related bioactive compounds. Similar results were also reported in a previous study [29], where HPP combined with UAE was employed to extract bioactive compounds from date palm fruits. Compared with the sole application of HPP or UAE, the combined extraction method yielded higher contents of total sugars, polyphenols, and flavonoids in date palm juice. After 30 min of treatment, the maximum contents reached 699.71 ± 0.08 g/L, 308.16 ± 0.27 mg gallic acid equivalent (GAE)/100 g fresh weight (FW), and 328.54 ± 0.12 mg quercetin equivalent (QE)/100 g FW, respectively.

Figure 1.

Changes in color (A), L* value (B), total soluble solids (TSS) (C), total sugars (D), flavonoids (E), and polyphenols (F) of different years-aged CRP water (1, 3, 5, and 10 years) under different assisted extraction approaches. The different letters indicate a significant difference (p < 0.05).

3.2. Effects of Extraction Methods on Amino Acid Composition

The variation trends in amino acid contents of different years-aged CRP water were illustrated (Figure 2A–F). In the untreated CRP water, the total amino acid content exhibited a decreasing trend with increasing aging years, this result was consistent with the previous study [30], the amino acid content in CRP reduced with the extension of aging years, which may be associated with the gradual utilization or degradation of amino acids by microorganisms during the aging process of CRP [31]. Among the detected amino acids, sweet- and umami-related amino acids are generally associated with favorable taste attributes, whereas bitter-, acidic-, and aromatic-related amino acids are often linked to less favorable or more complex taste perceptions. Therefore, the relative composition of these amino acids is an important determinant of the overall flavor characteristics of CRP water. Among the detected amino acids, sweet- and umami-related amino acids accounted for a relatively high proportion, whereas the contents of acidic-, bitter-, and aromatic-related amino acids were comparatively lower. After different assisted extraction approaches, the amino acid content of CRP water increased across all samples, with the combined HPP+UAE approach resulting in the most pronounced enhancement. Specifically, the total amino acid content increased from 66.29 to 88.91 μg/mL (34.13%) in the 1-year sample, from 56.13 to 95.15 μg/mL (69.52%) in the 3-year sample, from 21.41 to 40.30 μg/mL (88.27%) in the 5-year sample, and from 1.86 to 5.55 μg/mL (198.39%) in the 10-year sample.

Figure 2.

Amino acids content (A), sweet-related amino acids (B), umami-related amino acids (C), acidic-related amino acids (D), bitter-related amino acids (E), and aromatic-related amino acids (F) in different years-aged CRP water, and the HCA heatmap (G–J). The different letters indicate a significant difference (p < 0.05).

Hierarchical clustering analysis (HCA) was performed on the amino acid profiles of different years-aged CRP water (Figure 2G–J). In 1-year-aged CRP water, the untreated and singly assisted extraction samples were dominated by bitter-related amino acids (Phe, Leu, and Arg), whereas enrichment of sweet-related amino acids (Gly, Ala, and Pro) and the umami-related amino acid Glu was observed after the combined HPP+UAE approach. In 3-year-aged CRP water, the UAE samples were still dominated by bitter- or aromatic-related amino acids, whereas the combined HPP+UAE approach markedly increased the content of sweet-related amino acids (Gly, Thr, Ser, Ala, and Pro) and umami-related amino acids (Glu and Asp). In 5- and 10-year-aged CRP water, bitter-related amino acids still contributed to a certain extent in the singly assisted extraction samples, whereas sweet- and umami-related amino acids became the dominant components again after the combined HPP+UAE approach. Overall, the enrichment of sweet- and umami-related amino acids in CRP water across all aging years was consistently promoted by the combined HPP+UAE approach, which was beneficial for improving flavor harmony and palatability. A similar phenomenon was also observed by Cabral et al. [32], compared with HPP treatment alone, the combined treatment of UAE and HPP led to a significant increase in the content of all amino acids extracted from hempseed meal, and facilitated the extraction of lysine rather than arginine.

3.3. Effects of Extraction Methods on Antioxidant Activity

The changes in DPPH free radical scavenging activity (A), ABTS cation radical scavenging activity (B), and ferric reducing antioxidant power (FRAP) (C) of different years-aged CRP water were illustrated (Figure 3). Chemical antioxidant assays were widely used to evaluate the antioxidant activity of citrus samples, and their mechanisms were mainly based on hydrogen atom transfer and electron transfer reactions [33]. In the untreated CRP water, the antioxidant activity gradually increased with increasing aging years, which was consistent with the trends observed for flavonoid and polyphenol contents. For citrus plants, flavonoids and polyphenols in the peel were all secondary metabolites, mainly stored in the vacuoles of parenchymal cells in the peel [34]. During the aging process, on the one hand, the action of pectinase led to the gradual relaxation or partial degradation of cell wall structures in citrus peel. Citrus peel cells lost water and shrank, followed by vacuole rupture. Bound phenols and flavonoids, originally combined with cell wall polysaccharides or proteins, were more easily released and converted into free forms, thus increasing the total polyphenols and total flavonoids in CRP water [35]. On the other hand, under the condition of long-term storage with light avoidance, good ventilation and trace oxygen, some flavonoid and phenolic precursors underwent slow non-enzymatic oxidation and structural transformation, thereby generating new flavonoids and polyphenols. In addition, the metabolic activities of microorganisms partly consumed polysaccharides and other substances in CRP, which promoted the production of flavonoids and polyphenols [36,37]. Multiple factors worked synergistically to facilitate the enrichment of flavonoids and polyphenolic compounds in CRP.

Figure 3.

DPPH free radical scavenging activity (A), ABTS cation radical scavenging activity (B), and ferric reducing antioxidant power (FRAP) (C) of CRP different years-aged water. The different letters indicate a significant difference (p < 0.05).

After different assisted extraction approaches, the DPPH free radical scavenging activity, ABTS cation radical scavenging activity, and ferric reducing antioxidant power of CRP water were all significantly enhanced, with particularly pronounced increases observed in ABTS cation radical scavenging activity and ferric reducing antioxidant power (p < 0.05). After the combined HPP+UAE approach, the ABTS cation radical scavenging activity increased from 2.60 to 3.53 μmol Trolox/mL in the 1-year sample, from 2.67 to 3.43 μmol Trolox/mL in the 3-year sample, from 2.84 to 4.39 μmol Trolox/mL in the 5-year sample, and from 2.95 to 5.60 μmol Trolox/mL in the 10-year sample. These results indicate that both HPP and UAE effectively enhanced the antioxidant activity of CRP water and exhibited a synergistic effect, which was consistent with the variation trends observed for flavonoid and polyphenol contents. It might be due to both the HPP and ultrasonic treatment disrupting the cell structure of CRP. HPP exerted high pressure on CRP cells through physical action, leading to direct vacuole rupture [38]. Ultrasonic treatment achieved a similar effect through cavitation [39]. The synergistic effect of the two further enhanced this process, realizing the rapid dissolution of flavonoids and polyphenols, and ultimately improving the antioxidant capacity of CRP infusions.

3.4. Effects of Extraction Methods on VOCs

3.4.1. Flavor Compounds

There are abundant VOCs in CRP, mainly including alcohols, olefins, esters, ketones, aldehydes, phenols, and acids, which collectively determine the characteristic citrus aroma and flavor [40]. In CRP, these VOCs are typically sequestered in lipid-rich compartments (e.g., cellular membranes and oil glands/oil bodies), stored as glycosidically bound precursors in vacuoles, or associated with cell-wall polysaccharides, which limits their release into the aqueous phase under mild conditions [40]. The numbers and total contents of VOCs in different years-aged CRP water were illustrated (Figure 4A–B). In the untreated CRP water, the numbers of detected VOCs were 30, 57, 55, and 45 in the 1-, 3-, 5-, and 10-year samples, respectively. The VOCs profiles were dominated by alcohols and olefins across all aging years, consistent with previous findings [41]. After different assisted extraction approaches, the numbers of VOCs in CRP water were significantly increased (p < 0.05), with the greatest increase observed after the combined HPP+UAE approach. Specifically, the numbers of VOCs in CRP water increased to 45, 88, 80, and 81 in the 1-, 3-, 5-, and 10-year samples, respectively. Moreover, 11, 23, 16, and 16 flavor compounds were uniquely detected after the combined HPP+UAE approach in the corresponding samples (Figure 4C–F). In the untreated CRP water, the total VOC contents at different aging years were 3.10, 5.45, 4.96, and 2.44 µg/mL, respectively, showing an initial increase followed by a decrease with increasing aging years. Among the detected VOCs, methyl methylanthranilate, which was responsible for the characteristic citrus aroma, exhibited the highest content. Previous studies have reported that methyl methylanthranilate was widely distributed in citrus peels and represents a key contributor to the characteristic aroma of CRP [40]. After HPP, UAE, and the combined HPP+UAE approach, the total VOC contents in CRP water were all increased. Among them, the combined HPP+UAE approach resulted in the highest VOC content, reaching 5.06, 9.15, 7.84, and 5.98 µg/mL in the 1-, 3-, 5-, and 10-year samples, respectively, corresponding to increases of 63.23%, 67.89%, 58.06%, and 145.08%.

Figure 4.

Number (A) and total content (B) of VOCs, Venn diagrams of VOC species (C–F), and PCA plots (G–J) of different years-aged CRP water.

To further investigate the differences in VOC profiles among CRP water prepared under different assisted extraction approaches, principal component analysis (PCA) was performed on the VOC data. The results showed that the cumulative contribution rates of the first two principal components were 63.0%, 71.9%, 66.4%, and 78.2% for the 1-, 3-, 5-, and 10-year-aged CRP water samples, respectively, indicating that the first two principal components adequately captured the major information in the response signals and clearly distinguished the differences among the samples. Although both HPP and UAE could promote the release of VOCs from CRP, their mechanisms differed. In CRP, these VOCs are typically sequestered in lipid-rich compartments (e.g., cellular membranes and oil glands/oil bodies), stored as glycosidically bound precursors in vacuoles, or associated with cell-wall polysaccharides, which limits their release into the aqueous phase under mild conditions [42]. HPP could disrupt the cellular structure of CRP, leading to the formation of microcracks and hollow openings, thereby increasing cell permeability and facilitating the release of VOCs [43]. UAE enhanced solvent penetration by disrupting cell walls through cavitation effects, thereby accelerating the release of intracellular constituents [44,45,46]. In addition, ultrasound may further increase the release rate of VOCs by weakening intermolecular interactions within the cells [44]. After the combined HPP+UAE approach, the numbers and total content of VOCs increased to the greatest extent, indicating a synergistic effect between HPP and UAE during the assisted extraction process for the preparation of CRP water. Similar phenomena have been reported in previous studies [47].

3.4.2. Differential Flavor Compound Analysis

Partial least squares discriminant analysis (PLS-DA) was performed on the flavor compounds of CRP water prepared under different assisted extraction approaches, as shown in Figure 5A–D. For 1-year-aged CRP water, the explained variance of the independent variables (R²X) was 0.816, the explained variance of the dependent variables (R²Y) was 0.975, and the predictive ability of the model (Q²) was 0.919. For samples from 3-year-aged CRP, the R²X, R²Y, and Q² values were 0.817, 0.983, and 0.860, respectively. Similarly, the R²X, R²Y, and Q² values were 0.809, 0.982, and 0.860 for 5-year-aged CRP water, and 0.806, 0.988, and 0.868 for 10-year-aged CRP water. In all cases, R² and Q² values greater than 0.5 were obtained, suggesting acceptable goodness of fit and predictive performance of the PLS-DA models. Model stability was further validated by a 200-time permutation test. It was shown that the intercepts of the Q² regression lines with the y-axis were all less than zero, indicating that no overfitting occurred and that the models were valid and reliable for the analysis of flavor compounds in different years-aged CRP water.

Figure 5.

Permutation validation plots of the PLS-DA models (A–D) and HCA heatmaps of VOCs (E–H) of different years-aged CRP water.

Based on the PLS-DA results, key differential VOCs among different years-aged CRP water were screened using a VIP > 1 and p < 0.05 as the selection criteria. The results were summarized in Supplementary Table S2. The numbers of differential flavor compounds in different years-aged CRP water (1, 3, 5, 10) were 24, 29, 30, and 29, respectively. HCA of the differential flavor compounds at different aging years was shown in Figure 5E–H. For 1-years-aged CRP water, the differential flavor compounds were mainly concentrated in alcohols, esters, olefins, and some ketones, exhibiting floral, fresh, and citrus-like aroma characteristics, such as 2,6-octadien-1-ol, 3,7-dimethyl-, acetate, (Z)-dihydrocarvone, and decanal. These were typical characteristic flavor components of early-stage CRP. Among these flavor components, decanal was a characteristic aldehyde in citrus raw materials. It played a dominant role in the formation of a fresh and clean aroma [48]. For 3-years-aged CRP water, the contents of olefins and aldehydes were significantly increased (p < 0.05), with limonene and dodecanal as the representative components. Limonene is a signature terpene in CRP. Its content rose slightly due to enzymatic reactions during the early aging stage [49]. This content variation directly affected the mildness of CRP aroma. It was the core reason for the distinct retention of a citrus-like aroma in 3-years-aged CRP water. As a saturated fatty aldehyde, dodecanal presents an oily aroma [50]. In general, the aroma of 3-years-aged CRP water was still dominated by fruity and citrus-like notes, with oily aroma components emerging initially.

For 5-years-aged CRP water, alcohols, phenols, and fatty aroma-related compounds were predominant. Woody and aged aroma characteristics, such as those associated with spathulenol, 2-ethyl-1-hexanol, and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, were markedly enhanced, indicating a gradual shift in the aroma profile from a fresh type toward a more aged, woody, and mellow type. Spathulenol exhibits a strong herbal aroma and is widely distributed in the essential oils of various plants. 1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester presents an oily aroma [51]. For 10-years-aged CRP water, phenols, alcohols, and fatty aroma-related compounds (such as 2-ethyl-1-hexanol, tetradecanoic acid, and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester) were present at higher proportions, resulting in a more stable and heavy aroma profile, while the citrus-like aroma became relatively less pronounced. Overall, with increasing aging years, a gradual shift in the flavor profile of CRP water was observed, from a fresh type toward a more intense and complex type, accompanied by enhanced aroma complexity and richness. In samples subjected to HPP, fatty aroma-related compounds, alcohols, and phenols were relatively enriched in most aging years, suggesting that the extraction efficiency of hydrophobic and high-boiling-point compounds may be enhanced by HPP through alterations in the tissue structure of CRP. Such structural modification was likely associated with increased body and aroma stability of CRP water, thereby contributing to a more rounded and well-balanced flavor profile [38]. In samples subjected to UAE, highly volatile VOCs, including alcohols, esters, olefins, and some ketones, were more prominent, particularly those associated with herbal, fresh, and citrus-like aromas. It might be attributed to the cavitation effect of ultrasonic treatment, which can accelerate the release of intracellular flavor components and the mass transfer process between the solid and liquid phases, ultimately facilitating the dissolution of highly volatile components [39]. In samples prepared using the combined HPP+UAE approach, a greater diversity of flavor compounds was observed, accompanied by a more complete and well-structured aroma profile.

By integrating the effects of CRP aging years and assisted extraction approaches on key flavor compounds, the flavor profiles of CRP water were modulated in a directional manner. After HPP and UAE, enhanced floral, refreshing, and citrus aroma characteristics were observed in CRP water prepared from 1-year-aged CRP, suggesting its suitability for the development of light and refreshing CRP water products. For CRP aged for 3 years, a better balance between citrus and fatty aroma notes was achieved following the same assisted extraction approaches, indicating its potential for the development of refreshing citrus-flavored CRP water for daily consumption or functional beverage applications. After assisted extraction, typical aged and woody aroma characteristics were observed in 5-years-aged CRP water, with a more rounded and harmonious aroma profile, indicating its suitability for the development of CRP water products emphasizing aged flavor attributes. For 10-year-aged CRP subjected to HPP, increased levels of polyphenols, alcohols, and fatty aroma-related components were observed, resulting in a more stable and persistent aroma and suggesting its suitability for the development of high-end or tonic CRP water products. In contrast, the combined HPP+UAE approach could further enhance fruity or citrus aroma notes on the basis of a rich background. In summary, important theoretical support and practical guidance are provided for the flavor design and differentiated development of CRP water products through the investigation of the synergistic effects of CRP aging years and assisted extraction approaches.

3.5. E-Nose Results

The E-nose, as a biomimetic sensing technology, can effectively discriminate and analyze flavor compounds in samples [52]. The response characteristics of the electronic nose to different years-aged CRP water samples were illustrated (Figure 6A–D), among which sensors S5 (biological compounds), S7 (aliphatic hydrocarbons), S11 (VOCs), S12 (sulfides), S13 (ethylene), and S18 (sulfides) exhibited relatively strong responses. In untreated CRP water, no pronounced changes were observed in the signal intensities of major flavor-related compounds with increasing aging years. After different assisted extraction approaches, the signal intensities of major flavor compounds in CRP water were enhanced, with the most pronounced increases observed after the combined HPP+UAE approach (p < 0.05). The PCA plots of different years-aged CRP water based on E-nose data were presented (Figure 7). Based on the E-nose response signals, CRP water prepared using different assisted extraction approaches was effectively discriminated, indicating that significant effects on the flavor of CRP water were exerted by HPP, UAE, and the combined HPP+UAE approach. For the 1-, 3-, 5-, and 10-years-aged CRP water samples, the variance contributions of the first two principal components were 97.22%, 95.30%, 96.70%, and 94.00%, respectively, indicating that the first two principal components sufficiently captured the major information in the response signals and clearly distinguished the differences among samples.

Figure 6.

E-nose radar plots (A–D) and PCA plots (E–H) of different years-aged CRP water.

Figure 7.

Correlation heatmaps between key differential flavor compounds and E-nose sensor responses of different years-aged CRP water, (A) one year CRP; (B) three years CRP; (C) five years CRP; (D) ten years CRP.

3.6. Correlation Analysis Between Antioxidant Indices and Physicochemical Parameters

Correlation analysis was performed between the antioxidant capacities and physicochemical parameters of different years-aged CRP water, and the results were shown (Figure 8A–D). In 1-year-aged CRP water, the DPPH radical scavenging activity was significantly positively correlated with the L* value and total sugar content (p < 0.05). In 3-years-aged CRP water, the DPPH radical scavenging activity was significantly positively correlated with amino acid content, the L* value, and total sugar content (p < 0.05); meanwhile, the ferric reducing antioxidant power was significantly positively correlated with total sugar content (p < 0.05). In 5-years-aged CRP water, the ABTS radical cation scavenging activity was significantly positively correlated with the L* value (p < 0.05); meanwhile, the ferric reducing antioxidant power was significantly positively correlated with TSS (p < 0.05). In 10-years-aged CRP water, the DPPH radical scavenging activity, ABTS radical cation scavenging activity, and ferric reducing antioxidant power were all significantly positively correlated with the L* value, TSS, and total sugar content (p < 0.05). Overall, the CRP water extract from various vintages treated with HPP+UAE exhibited the strongest antioxidant activity. This was concomitant with the highest levels of flavonoids, polyphenols, amino acids, TSS, total sugar, and L* value. This phenomenon suggests that the HPP+UAE treatment, acting through physical mechanisms, maximally disrupts the cellular structure of CRP, thereby releasing a variety of bioactive components, including flavonoids, polyphenols, amino acids, and polysaccharides.

Figure 8.

Correlation analysis between antioxidant capacity indices and physicochemical parameters of different years-aged CRP water samples, (A) one year CRP; (B) three years CRP; (C) five years CRP; (D) ten years CRP.

The enhancement of antioxidant activity is directly attributed to the increased content of flavonoids and polyphenols, which are the primary antioxidant constituents. Furthermore, the elevated amino acid content is likely due to increased cell membrane permeability and accelerated proteolysis under HPP+UAE treatment. This mechanism is supported by studies on citrus juice, where sonication-microwave treatment significantly increased amino acid levels (e.g., alanine, arginine, proline, and glycine) by exposing internal molecular regions and accelerating peptide bond cleavage [53]. The disruption of cell walls and membranes also leads to the massive release of intracellular contents—such as soluble sugars, organic acids, and minerals—directly increasing the TSS. Finally, the variation in the L* value may be associated with the release of cellular contents (e.g., polyphenols) and potential mild Maillard reactions occurring during processing.

3.7. Correlation Analysis Between E-Nose and GC-MS

Based on the previous analyses, the E-nose response characteristics of CRP from different aging years were obtained, and six sensors with relatively high response intensities (S5, S7, S11, S12, S13, and S18) were selected for correlation analysis with the screened differential flavor compounds (VIP > 1, p < 0.05). Correlation network heatmaps illustrating the relationships between differential flavor compounds identified by GC-MS and the responses of E-nose sensors are shown in Figure 7A–D. In 1-year-aged CRP water, sensors S11, S12, and S13 were significantly positively correlated with key flavor compounds associated with fresh and herbal aromas, such as (Z)-dihydrocarvone, myrcene, perilla alcohol, and piperitone (p < 0.05), indicating that low-aged CRP water was dominated by highly volatile, low-molecular-weight flavor compounds and that the E-nose signals mainly reflected fresh aroma characteristics. In 3-years-aged CRP water, all six sensors were significantly positively correlated with multiple classes of flavor compounds associated with fatty, citrus, floral, and woody aromas, such as nonanal, decanal, α-sinensal, methyl anthranilate, epi-γ-eudesmol, and spathulenol (p < 0.05). These results indicate that, with progressive aging, the composition of VOCs in CRP water gradually shifted from a single fresh type toward a multi-aroma synergistic profile, and that the E-nose responses transitioned from being dominated by a limited number of channels to coordinated responses across multiple sensors.

In 5-years-aged CRP water, all six sensors were significantly positively correlated with flavor compounds associated with woody, fatty, and phenolic aromas, such as (E)-caryophyllene, 1-octanol, decyl alcohol, fenchol, and thymol (p < 0.05). These results indicate that aroma characteristics gradually became dominated by structurally stable and less volatile VOCs, and that the overall flavor profile of CRP water tended to be more mellow and full-bodied. In 10-years-aged CRP water, all six sensors were significantly positively correlated with flavor compounds associated with phenolic and fatty aromas, such as dibutyl phthalate, methyl 3,5-di-tert-butylsalicylate, 2,4-di-tert-butylphenol, and thymol (p < 0.05), indicating the presence of pronounced aging characteristics. Overall, the electronic nose sensors exhibited good selectivity towards different volatile compounds in CRP aqueous extracts. The signal variations effectively reflected the flavor evolution: a transition from highly volatile, fresh notes to less volatile, mellow profiles with aging.

This transformation is essentially a complex process driven by time, environment, and biochemistry, governed by three core factors. First, intrinsic compositional transformation: high-volatility monoterpenes (e.g., limonene) gradually volatilize and oxidize over time, while low-volatility sesquiterpenes, alcohols, ketones, and phenols become relatively enriched, establishing the material basis for “mellowness.” This chemical evolution involves the dynamic rearrangement of volatiles, where low-boiling-point components decrease and high-boiling-point, stable oxygenated derivatives accumulate, causing the aroma to shift from sharp to mellow [54]. Second, microbial mediation: specific fungal and bacterial communities (e.g., Aspergillus) metabolize macromolecules into secondary metabolites with herbal and woody notes, driving the “removal of harshness.” Studies show fungal diversity peaks during years 2–4 of aging, with significant increases in aliphatic aldehydes (e.g., nonanal, decanal) that correlate highly with sensory “roundness,” indicating direct microbial involvement in flavor remodeling [55]. Third, external storage environment regulation: temperature, humidity, oxygen, light, and packaging constitute the “reaction field.” Optimal conditions (e.g., 20–25 °C, 40–60% RH) and breathable containers (e.g., pottery jars) promote oxidation and microbial activity, whereas improper conditions (e.g., high heat/humidity or plastic packaging) may induce mold or phthalate migration, compromising quality [56].

3.8. Sensory Evaluation Results

The sensory evaluation results of tangerine peel water prepared with CRP at different years via HPP, UAE, and combined treatments were shown in Figure 9. The evaluation dimensions included color, aroma, taste, and overall coordination. In the untreated CRP water, the comprehensive sensory score with lower aging years samples was significantly higher than that from higher aging years samples. This difference was closely related to the changes in components during CRP aging. For high aging years CRP, long-term aging intensified browning reactions, leading to the accumulation of brown substances and a significant decrease in color score. Meanwhile, flavonoids, polyphenols, and other bitter and astringent substances gradually accumulated during aging, which directly reduced the taste score and ultimately resulted in poor overall sensory performance.

Figure 9.

Total sensory evaluation score of CRP water prepared from CRP with different aging years.

After single treatment with HPP or UAE, there was no significant difference in the comprehensive sensory score of different years-aged CRP water. It indicated that the two single treatment methods had limited regulatory effects on the sensory quality of CRP water and did not change the sensory difference pattern between samples of different years. In contrast, the comprehensive sensory score of CRP water slightly increased after HPP+UAE combined treatment. The core reason was that the combined treatment synergistically enhanced cell wall breaking, promoted the release of volatile flavor substances in CRP, improved the aroma score, and further enhanced overall sensory coordination. This result provides a theoretical reference for optimizing the processing technology of tangerine peel water and balancing the sensory quality of CRP of different years.

4. Conclusions

In this study, the effects of HPP, UAE, and the combined HPP+UAE approach on flavor compounds and bioactive compounds in different years-aged CRP water (1, 3, 5, and 10) were investigated. The results demonstrated that the diversity of flavor compounds gradually increased with increasing aging years of CRP, with more abundant and complex flavor profiles particularly observed in samples with longer aging years. Shorter years-aged CRP water (1 and 3 years) was mainly characterized by citrus and herbal notes, whereas those from higher aging years (5 and 10 years) exhibited more pronounced fatty and woody flavor attributes. The combined HPP+UAE approach significantly enhanced the release of flavor compounds, particularly in longer years-aged CRP water, with both their diversity and concentration being markedly increased. These results indicate that the combined HPP+UAE approach effectively promoted flavor enhancement in CRP water. In addition, the combined HPP+UAE approach not only enhanced the release of flavor compounds but also significantly increased the contents of TSS, total sugars, flavonoids, and polyphenols in CRP water, thereby further improving its body and antioxidant capacity. Overall, HPP, UAE, and the combined HPP+UAE approach improved both the flavor quality and antioxidant capacity of CRP water, with these effects being particularly pronounced in samples prepared from higher aging years. This study provides a novel technological approach for optimizing the quality of CRP water and offers a theoretical basis for enhancing its nutritional value and health-related functions.

Supplementary Materials

The detailed volatile compounds, the sensory evaluation criteria and the sensory evaluation scores of different CB-CRP samples can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040757/s1.

Author Contributions

Methodology, J.Z.; software, K.S.; validation, C.C.; formal analysis, C.C.; investigation, B.L. and S.J.; data curation, K.S.; writing—original draft preparation, J.Z.; writing-review & editing, B.L. and Y.L.; supervision, S.J. and Y.L.; project administration, Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that no conflict of interests.

Funding Statement

This study was funded by the Science and Technology Program of Sichuan Province (2024YFTX0066) and Jiangsu Provincial Key R&D Program (BE2023306).