Addition of β-Cyclodextrin or Gelatin Ιmproves Organoleptic and Physicochemical Attributes of Aronia Juice

Abstract

Aronia juice is well-known for its high nutritional and biological value, due to its polyphenol content, which has a powerful antioxidant effect. However, the high polyphenol content of aronia juice is associated with an astringent flavor, which diminishes consumer acceptance. To improve the flavor of aronia juice, β-cyclodextrin (0–2% w/v) or gelatin (0–0.4 mg/L) were added before pasteurization. The juice samples were first examined organoleptically, and monitored for total phenolic compounds, antioxidant capacity, total flavonoids, total monomeric anthocyanins, polymeric color, pH, total soluble solids, and color. The organoleptic test demonstrated that both β-cyclodextrin and gelatin juice aroma reduced astringency and increased sweetness, whereas β-cyclodextrin also reduced juice aroma. β-cyclodextrin significantly increased polymeric color and total soluble solids (p < 0.05), whereas antioxidant activity, total flavonoids, and monomeric anthocyanins remained unchanged compared to the unpasteurized control. In contrast, the addition of gelatin dramatically reduced total phenolic compounds, antioxidant capacity, and total flavonoids, while enhancing polymeric color and maintaining monomeric anthocyanins with minor decreases relative to pre-pasteurization levels (p < 0.05). A consumer study was conducted with control juice and juices with 2% w/v β-cyclodextrin or 0.4 mg/L gelatin added. The results confirmed the change in flavor profile by masking or removing astringency and astringent aftertaste, as well as increasing sweetness, which significantly improved overall acceptability (p < 0.05).

1. Introduction

Aronia juice is the most commonly processed product derived from aronia berries (Aronia melanocarpa). It has been proven to provide potential health advantages for consumers due to its antioxidant, anticancer, cardioprotective, antidiabetic, hypertension control, and anti-inflammatory properties [1,2,3,4,5,6,7]. Its health-promoting characteristics have recently attracted the interest of both consumers and researchers [8,9,10,11].

Among other constituents, the juice of A. melanocarpa berries include high levels of anthocyanins, procyanidins, flavonoids, and phenolic compounds, as well as vitamins and minerals [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. Despite its high antioxidant content, aronia juice has powerful astringent tastes, limiting its consumer preference [19,20,21]. It has been reported that such impact on sensory properties is often compound-specific [22]. Also, it is known that the astringent taste of aronia fruit and juice is caused by the high content of polyphenols, particularly procyanidins, which exhibit a high degree of polymerization. Procyanidin oligomers bind to proteins, inducing denaturation and contributing to the astringent, choking, and dry mouth sensations [23]. Astringency is a complex sensation induced by the shrinking, drawing, or puckering of the oral epithelium [24]. Aronia originates its strong astringent flavor from procyanidins, which exist in both oligomeric and polymeric forms (degree of polymerization > 10) [25] and at concentrations that are among the highest in foods [26]. Proanthocyanidins with a high degree of polymerization were identified as the primary astringent chemicals in aronia juice due to their better capacity to bind and precipitate salivary proteins when compared to other phenolic compounds [27].

Aronia juice is typically subjected to traditional pasteurization, a thermal treatment that has long been used in processed juices [28,29]. Nevertheless, many naturally occurring phytochemicals that offer health benefits, like phenolic compounds and ascorbic acid, are heat sensitive and readily destroyed [30]. The same is true for color, flavor, and taste characteristics, which are easily detectable by the consumer. Those effects have an immediate impact on juice quality, reducing health-conscious consumers’ willingness to accept the product. To address this, non-thermal approaches for preserving the bioactive characteristics of aronia juices have been proposed, including high pressure [31], pulsed electric fields [32], and high-pressure carbon dioxide [33].

Various methods of minimizing the unpleasant tongue sensations of nutrient-dense berry juices have been found to increase consumer acceptance. The most popular technique is to employ additives that mask unpleasant flavors, such as additional sweetness. However, adding too much sugar lowers its health benefits because it has been related to an increased risk of weight gain and type 2 diabetes [34]. Additional sugar might render the juice less attractive to health-conscious consumers [35]. Duffy et al. [20] reported that increased sweetness with added sugar improved aronia juice preference, but astringency was not reduced to a sufficient degree to increase consumer attractiveness [20]. As a result, different sensory modifiers are required to increase consumer sensorial attractiveness. Increasing sweetness with sugar and sweet olfactory flavoring has been shown to be insufficient to mitigate the disagreeable astringency of aronia juice [20].

To mitigate polyphenol-induced astringency in pasteurized juice, standard fining agents have been employed. These include animal proteins such as gelatin, egg albumin, and whey protein, as well as vegetable proteins such as wheat, pea, and potato proteins, all of which have a high affinity for proanthocyanidins [36,37].

Gelatin, as a fining agent, has a positive charge in low-pH fruit juices, allowing it to react with negatively charged phenolics. It is used to remove liquids of high-molar-mass polyphenols by forming complexes with tannins, proanthocyanidins, and other phenolic compounds, but it can also bind to anthocyanins, causing color degradation [38,39]. Although gelatin has been successfully used to eliminate undesirable flavors from pomegranate juice in multiple studies [38,40,41,42,43,44], its use in aronia juice has been proven to be ineffective in reducing astringency, and egg white powders have been proposed as an effective agent [21].

β-cyclodextrin can replace water molecules with fewer polar compounds in its hydrophobic cavity, resulting in their inclusion and stability [44,45]. β-cyclodextrin treatment has been shown to prevent anthocyanin losses of an anthocyanin-rich extract obtained from Hibiscus sabdariffa [46], acting as improver or modifier of color. Similarly, addition of 3% β-cyclodextrin in aronia juice [47] or 10g/L β-cyclodextrin in lingonberry juice [22] protected anthocyanins. The β-cyclodextrin effect on juice aroma is determined by its level of addition; low levels do not appear to alter or generate a significant drop in the aroma of pear juice [48]. The use of cyclodextrins (β-cyclodextrin) in mandarin juice mixed with pomegranate extract and goji berries juice have been shown to effectively encapsulate bi-active/antioxidant compounds [49]. When bitter compounds, like flavonols or hydroxycinnamic acids, form an inclusion complex with β-cyclodextrin, their bitterness is likely muted due to diminished interaction with bitterness receptors [50]. Adding 10 g/L of β-cyclodextrin to lingonberry juice [22] reduced its perceived astringency.

Although β-cyclodextrin has been used in aronia juice [47], its impact on organoleptic quality and physicochemical properties has not been fully investigated. Gelatin, on the other hand, has been utilized as a side treatment at extremely low doses with unclear outcomes [51]. This study examined the effectiveness of β-cyclodextrin or gelatin in lowering the astringent flavors of aronia juice. To reduce astringency in juice, β-cyclodextrin (0–2%) or gelatin (0–0.4 mg/L) were added and examined for sensory quality, total phenolic content, antioxidant activity, and other physicochemical characteristics. Pasteurization was studied to protect juice antioxidants after β-cyclodextrin and gelatin processing, as these juices are consumed as processed. Effective β-cyclodextrin or gelatin treatments on sensory quality were evaluated for consumer acceptance.

2. Materials and Methods

2.1. Chemicals and Reagents

Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid), DPPH (2,2-diphenyl picrylhydrazyl), gallic acid, NaNO₂, NaOH, KCl and β-cyclodextrin (MW = 1134.99, purity > 98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocalteu phenol reagent, sodium carbonate, sodium acetate, potassium persulfate, al chloride, and glacial acetic acid, as well as HPLC grade methanol, were purchased from Chem-Lab (Zedelgen, Belgium). Gelatin (Aquacol Fish Gelatin) was obtained from Martin Vialatte (Magenta, France).

2.2. Plant Material

The Agricultural Cooperative of Aronia Producers in the Serres area, Central Macedonia, Greece, supplied frozen aronia juice for the production of the required samples.

2.3. β-Cyclodextrin and Gelatin Treatments

Frozen aronia juice was thawed and filtered (using filter paper) before being utilized to create experimental design treatments in two separate sets.

Table 1. β-CD and gelatin addition treatments of aronia juice including pasteurization and organoleptic or physicochemical analyses.

			First Set of Samples		Second Set of Samples
UPA/PAS	β-CD (%)	Gelatin (mL/L)	Organoleptic Evaluation (5-Member Trained Panel)	Physico-Chemical Analysis	Consumer Preference (28-Member Untrained Panel)
UPA	0 (control)	0 control	-	√	-
PAS	0 (control)	0 control	√	√	√
PAS	0.5	0.1	√	√	-
PAS	1.0	0.2	√	√	-
PAS	1.5	0.3	√	√	-
PAS	2.0	0.4	√	√	√

UPA: unpasteurized, PAS: pasteurized.

shows the treatments as well as analysis of samples.

The first set of samples was formed by adding 0 (control), 0.5, 1, 1.5, and 2% w/v β-cyclodextrin (β-CD) to raw juice after filtering. The juice was then continuously stirred in a Agimatic-S magnetic stirrer (P-SELECTA, Barcelona, Spain) at 700 rpm for 60 min at room temperature (25 °C). Also, following filtration, the raw juice was treated with 0 (control), 0.1, 0.2, and 0.4 g/L gelatin. The samples were vortexed for 2 min and refrigerated at 4 °C for 24 h. The samples were then re-filtered to eliminate the generated sediment (Figure 1). Gelatin and β-cyclodextrin levels were chosen based on earlier research. Both β-CD- and gelatin-treated samples were used for organoleptic evaluation by trained members as well as for physicochemical analysis.

The second set of samples was prepared after the addition of β-CD and gelatin at maximum concentrations (2% w/v and 0.4 g/L, respectively) following exactly the steps for each case as mentioned above. These samples were used for the organoleptic consumer preference test.

Both set of samples (first and second) after treatment with β-CD or gelatin were preheated to 90 °C using microwaves (9 min, 700 watts), followed by immediate bottling in 100 mL glass containers, and pasteurized for 10 min using a water bath at 90 °C. After their pasteurization, the samples were first cooled with cold water to 50 °C and then remained in a dry and dark place at room temperature until used. Unpasteurized control juice was kept for comparison with untreated juice (control) following pasteurization with regard to physicochemical characteristics (Table 1).

2.4. Organoleptic Evaluation and Consumer Preference Test

A panel of trained members evaluated the organoleptic properties of the aronia juice prepared in the first sample set, while untrained members analyzed the juice prepared in the second set for consumer preferences (Table 1). Both tests were carried out in accordance with the Ethics and Research Integrity Committee’s Regulation of Principles and Operations at Aristotle University of Thessaloniki. Juice samples of both sets were processed (thawed, filtered, treated, and pasteurized) on the day prior to the organoleptic preference test.

The organoleptic test of aronia juice, prepared in the first sample set, was conducted by a group of five trained members (2 male and 3 female) who had previously been trained and participated in descriptive sensory analysis of aronia juice or fruit smoothies. The panel evaluation of the juices included a total of four sessions: The first session’s goal was to familiarize the panel with the aronia juice used in this experiment, and opinions on early impressions of juice consumption were asked. The second session’s purpose was to train the panel on the basic flavors (sweet, bitter, sour, and astringent) of aronia juice, as well as the astringent aftertaste, the intensity of the red color, and the juice’s distinctive fruit aroma. The testers were requested to differentiate the intensity of the standard solutions before characterizing the aronia juice given to them for each basic flavor, as well as rating the intensity of the astringent aftertaste, aroma, and red color. The instructions for evaluating the typical astringent aftertaste of aronia juice were to do so 10–15 s after swallowing the sample. Juice characterization was done on a scale of 1 to 9 (1 = little/not at all, 5 = moderate/neutral, 9 = excessively much). In the third session, the treated aronia juice (β-CD, gelatin) was evaluated, and in the fourth session, the treated aronia juice was evaluated again; in both sessions, the intensity of the red color and aroma, the taste (sweet, astringent), and the aftertaste of the aronia juice were assessed. Grading was done on a scale of 1 to 9 (1 = little/not at all, 5 = moderate/neutral, and 9 = extremely much). In sessions three and four, the samples were given in random order with three-digit codes, and the sample coding varied between tests for each treatment. Scores from sessions three and four were used to calculate the average.

The organoleptic consumer preference test of aronia juice, prepared in the second sample set, was held by twenty-eight (28) non-trained participants (students, both male and female). The students were chosen at random, and those who volunteered were given a consent form to sign before performing the test. The juice samples were examined after being treated with β-CD and gelatin at maximum levels (2% w/v and 0.4 g/L, respectively), as well as a control juice, prior to pasteurization. The participants (consumers) were instructed to score the red color, aroma, taste (sweet, astringent, sour), aftertaste (astringent), and overall acceptability of aronia juice on a nine-point hedonic scale, as indicated above. The testers were given water to drink and green apple slices to eat between tests on the samples to be examined.

2.5. Physicochemical Analyses

2.5.1. Determination of Total Phenolic Compounds and Antioxidant Activity

In this study, total phenolic compounds were determined using the method described by Singleton and Rossi [52]: 0.2 mL of properly diluted sample was placed in a volumetric cylinder which was then filled with distilled water to a final volume of 10 mL. Then, 0.5 mL of Folin–Ciocalteu reagent was added, and the mixture was lightly agitated for 5 min. Subsequently, 5 mL of Na₂CO₃ (5%) was added and then was made up to 25 mL by distilled water; gentle shaking was performed before the samples were maintained in dark conditions and at room temperature for 90 min. Distilled water was utilized to prepare the control sample. The absorbance at 760 nm was then measured using a Genesys 80 UV-VIS (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer. The total phenolic compounds were calculated using the linear regression equation of a standard curve (y = 0.0008x + 0.0875, R² = 0.9971), and the results were expressed as mg of gallic acid equivalent (GAE) per L of juice.

The antioxidant activity of the samples was measured using the DPPH technique, as described by Brand-Williams et al. [53] and Kim et al. [54]: In 2 mL Eppendorf tubes were placed 50 µL of diluted sample and 1950 µL of methanolic DPPH solution (1.6 M). The mixture was gently stirred and kept for 30 min at room temperature in darkness. The absorbance of the solution was monitored at 515 nm using a Genesys 80 UV–Vis (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer. Methanol was used as a control. Juice sample antioxidant activity values (%RSA) were determined by using the formula %RSA = [Abs515(t = 0) − Abs515(t)] × 100/Abs515(t = 0) after correction with appropriate blanks. Trolox equivalents were obtained using the linear regression equation of a calibration curve (y = 56.649x − 0.011, R² = 0.9971), and results were expressed in mmol Trolox Equivalents per mL of juice.

2.5.2. Determination of Total Flavonoids

Total flavonoids were determined according to Zhishen et al. [55] as follows: In a 25 mL container, 1 mL of adequately diluted sample was introduced, followed by 4 mL of distilled water. Then, 0.3 mL of NaNO₂ solution (5%) was added and the mixture was gently agitated. After 5 min, 0.3 mL of 10% AlCl₃ solution was added, followed by gentle stirring for 6 min at room temperature. Finally, 2 mL of 1 M NaOH was added, followed by 10 mL of distilled water. The product was gently mixed again before being stored in the dark at room temperature for 10 min. The control was produced in the same way, but with distilled water instead of the juice sample. The absorbance of all samples was measured at 510 nm with a Genesys 80 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The total flavonoids were quantified as mg of catechin equivalents (CE) per L of juice sample using a standard curve linear regression equation (y = 0.0021x − 0.0459, R² = 0.9974).

2.5.3. Determination of Total Anthocyanin Monomers

Total anthocyanin monomers were determined according to Lee et al. [56].

For each sample, two 50 mL containers were employed, each holding 0.8 mL of adequately diluted material. Then, in the first one, 7.2 mL of KCl solution (0.025 M) at pH 1.0 was added, followed by 7.2 mL of CH₃COONa solution (0.4 M) at pH 4.5. Following gentle stirring, the generated sample solutions were stored for 15 min in dark conditions at room temperature before being measured at 510 nm and 700 nm with a Genesys 80 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Distilled water was utilized as a control. Total monomeric anthocyanins were estimated using the following equation:

$$TMA \left(\mathrm{m}\mathrm{g}/\mathrm{L}\right)=(A\times MW\times DF\times 1000)/\varepsilon \times d$$

(1)

where:

$$A={(A_{510\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}})}_{pH=1}-{(A_{510\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}})}_{pH=4.5}$$

A—Absorbance

MW—Molecular weight of cyanidine 3-O-glucoside) = 449.2 g/mol

$\varepsilon$—Molar extinction coefficient of cyanidine 3-O-glucoside = 26,900 L/mol cm

1000—conversion factor from g to mg

DF—Dilution factor (Final volume/Sample volume)

d—Cell length (1 cm).

2.5.4. Determination of Color Due to Polymeric Pigments

Color due to polymeric pigments (CPP) was determined according to Giusti & Wrolstad [57] as follows: to 2.8 mL of appropriately diluted material was added 0.2 mL of K₂S₂O₅ solution (0.9 M). This was followed by gently stirring the produced solutions for 15 min in dark conditions at room temperature before measuring of the absorption spectrophotometrically at 420 nm, 520 nm, and 700 nm with a Genesys 80 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A solution with distilled water added was used as a control. The following formula was used to obtain the color density (using the control sample), CPP (using the bisulfite-bleached sample), and the associated percentage of CPP:

$$Color Density=\left[\left(A_{420\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}}\right)+\left(A_{512\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}}\right)\right]\times DF$$

(2)

$$Color due to polymeric pigments=\left[\left(A_{420\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}}\right)+\left(A_{512\mathrm{n}\mathrm{m}}-A_{700\mathrm{n}\mathrm{m}}\right)\right]\times DF$$

(3)

$$\% Color due to polymeric pigments=\left(Color due to polymeric pigments)/color density\right)\times 100$$

(4)

where:

DF—Dilution factor = iFinal volume/Sample volume.

2.5.5. Color, pH, and Total Soluble Solids

The color of juice samples was measured using the CIELab coordinate color space system. The brightness was determined by the parameter L* (lightness), with values that range from 0 (black) to 100 (white), and the two color components were determined by the parameters a* (redness) and b* (yellowness), which range from −120 to 120. The a* parameter’s positive values represent colors of red, whereas negative values represent shades of green. Positive values of the parameter b* correspond to yellow tones and negative to blue shades. In this study, the color CIELab characteristics of aronia juice were assessed using a chromameter (Minolta CR-400, Minolta, Osaka, Japan) with an 8 mm measuring head and a D65 illuminant. The instrument was calibrated with a white reference tile (L* = 97.52, a* = −5.06, b* = 3.57) prior to measurements.

Juice pH was measured using a Hanna HI 221 Calibration Check Microprocessor pH Meter (HANNA Instruments, Ann Arbor, MI, USA), and total soluble solids (°Brix) using a digital refractometer (ATAGO CO., Ltd., Itabashi, Tokyo, Japan).

2.6. Statistical Analysis

The data from the organoleptic evaluation and physicochemical analyses were evaluated using analysis of variance (ANOVA) and the General Linear Model. Using Minitab 16.1.1 (Minitab, Inc., State College, PA, USA), the averages were compared with Tukey’s test with a 5% confidence interval (p ≤ 0.05).

3. Results and Discussion

3.1. Organoleptic Evaluation

Trained tasters found significant changes in aronia juice samples following β-CD or gelatin treatment before pasteurization compared to the control. These changes were related to juice aroma, taste (astringency/sweetness), and astringent aftertaste, but not to color, as shown in Figure 2A,B.

Panelists rated the hue of all samples as dark red, choosing values that were equal to the control (9 on a 1–9 scale). β-CD and gelatin treatment resulted in subtle color changes that were undetectable to the naked eye. Deshaware et al. [29] demonstrated that β-cyclodextrin treatment had no effect on coloration of bitter melon juice. Similar findings were reported for mandarin juice fortified with pomegranate and goji berry (after pasteurization) [48] and orange juices [58]. However, Hayoglu et al. [59] and Vardin and Fenercioglu [40] reported that the organoleptic color evaluation of sour green grape juice was altered, whereas the color of pomegranate juice was improved following treatment. Organoleptic changes in juice color were related to the composition of phenolics and flavonoids, as well as to the heat treatment method applied [60].

According to the trained panelists’ scores, the strength of the aroma decreased in proportion to the amount of β-CD added to aronia juice (Figure 2A). When 0.5% w/v β-CD was added to juice, the intensity of the aroma was reduced (p < 0.05); at 2% w/v β-CD, the reduction was as high as 73.3% (p < 0.05) (Figure 2A). This could be due to the encapsulation of amygdalin by β-CD, a cyanogenic glycoside that gives aronia products their characteristic aroma [61,62]. Langorieux and Crouzet [63] showed that adding β-CD to limonene solution, a crucial aromatic component, decreases flavor. According to Navarro et al. [49], the flavor and aroma of mandarin juice were altered by the addition of β-CD at 1% w/v. Additionally, based on customer preference, Deshaware et al. [29] found that adding more β-CD (0.25–2% w/v) to bitter melon juice decreased the unpleasant odor. On the other hand, there was only a slight variation in the intensity of the distinct aroma of aronia juice by gelatin treatment when compared to the control (Figure 2B). According to Hayoglu et al. [59], adding gelatin to sour green grape juice, pasteurizing it at 85 °C for 15 min, and then chilling it decreased both aroma and flavor. Furthermore, Rinaldi et al. [64] showed that the distinctive botanical aroma of “Sangiovese” red wine is reduced when the wine is clarified with gelatin.

Aronia juice treated with β-CD or gelatin exhibited significant changes in astringency, sweetness, and aftertaste astringency (Figure 2). Adding gelatin or β-CD to juice before pasteurization reduced the astringent taste/aftertaste by as high as 64.6% (p < 0.05). The reduction was proportional to the degree of addition (0.5–2.0% for β-CD and 0.1–0.4 g/L for GEL). β-CD or gelatin treatment resulted in an increase in sweetness; adding gelatin concentrations of 0.3–0.4 g/L or 1.5–2% β-CD to aronia juice increased sweetness (p < 0.05) by 41.1–32.2% and 35–25%, respectively, compared to the control (Figure 2). Several studies have been conducted to investigate the reduction in unpleasant flavor of food and beverages by addition of different agents [65]. β-CD has been reported to reduce the bitterness of citrus juices by encapsulating juice naringin and limonene [66], as well as of grapefruit and mandarin juices [67], while the bitter taste and aftertaste of produced bitter melon juices was improved by 44.5 and 37.8%, respectively [29]. Kelanne et al. [22] found that adding 1% w/v β-CD was not sufficient to significantly alter the taste or aftertaste of lingonberry juice; however, when combined with gelatin (1 g/L), there was a significant difference in flavor and aftertaste strength compared to the control, with most panel members rating this sample as pleasant [22]. Pomegranate juice treated with gelatin (1 g/L) reduced the astringency following its processing to wine [40]. Furthermore, Curko et al. [68] and Rinaldi et al. [64] revealed that using gelatin as a clarifying agent reduced the unwanted strong astringent organoleptic flavor and subsequent astringent aftertaste in red wines.

It is clear that using 1.5–2% or β-CD as a blocking agent and 0.3–0.4 g/L gelatin as a removing agent for the compounds responsible for the astringency taste of aronia juice may result in not only a significant improvement in this attribute but also the uncovering of other taste attributes such as sweetness.

3.2. Effect of Pasteurization

Physicochemical parameters of aronia juice, shown in

Table 2. Physicochemical traits of unpasteurized (UPA) and pasteurized (PAS) aronia juices (control—no β-CD or gelatin added).

Juice	Total Phenolic Content (mgGAE/L)	DPPH Scavenging Activity (mgTE/L)	Total Flavonoids (mg CE/L)	Total Monomeric Anthocyanins (mg cyn-3-glu/L)	Total Color Due Topolymeric Pigments (%)
UPA	1613.1 ± 37.6 A	2255.6 ± 64.8 A	1699.4 ± 23.8 A	340.7 ± 7.96 A	61.6 ± 0.6 A
PAS	1591.9 ± 36.2 A	2003.1 ± 127.1 B	1716.9 ± 13.3 A	281.4 ± 4.42 B	64.0 ± 0.3 B
	Lightness (L*)	Redness (a*)	Yellowness (b*)	Soluble Solids Content (%)	pH
UPA	17.08 ± 0.23 A	2.65 ± 0.11 A	1.76 ± 0.03 A	20.2 ± 0.20 A	4.00 ± 0.01 A
PAS	16.70 ± 0.26 A	2.69 ± 0.19 A	1.67 ± 0.10 A	20.5 ± 0.12 A	4.00 ± 0.01 A

Different letters within each column represent statistically significant difference according to Tukey’s test.

, are compared between the unpasteurized (UPA) and pasteurized (PAS) controls (no β-CD or gelatin treatment).

Pasteurization did not appear to have had a statistically significant effect on total phenolic compounds or total flavonoids. Mayer-Miebach et al. [12] reported that pasteurization did not induce any change in phenolic compounds and total procyanidin contents. Further, color attributes (L*, a*, b*), soluble solids content, or pH also did not show changes due to pasteurization [69,70].

However, pasteurization did have a significant effect on DPPH scavenging activity, total monomeric anthocyanins, and color due to polymeric pigments (CPP). The UPA contained 2300 mgTE/L juice, whereas the PAS had only 1950 mgTE/L juice for DPPH radical scavenging activity (p < 0.05). Pasteurization of aronia juice also resulted in a 16.2% decrease in total monomeric anthocyanins and an increase in CPP (Table 2). Kulcan et al. [71] reported significant reduction in the antioxidant capacity of the juice produced of 11.2% (along with a decrease in total phenolic compounds) as well as an increase in the CPP from 2.05% to 2.15% due to pasteurization (85 °C/15 min) in pomegranate juice. A decrease in phenolic compounds has been reported by Mayer-Miebach et al. [12], and Wilkes et al. [13] reported an increase in CPP by 16% and a simultaneous decrease in flavonols by 9% in aronia juice post pasteurization.

Further, the PAS juice had a 26.9% decrease in total anthocyanin monomers and a simultaneous 8.6% increase in CPP (Table 2). Losses in total monomeric anthocyanins in aronia juice after applying high pressures reached 8% [72]. Szalóki-Dorkó et al. [73] and Dubrovi et al. [74] reported decreases in anthocyanin monomers in fruit juices after pasteurization by 38% and 0.7–4.4%, respectively. Monomeric anthocyanins are considered as unstable, water-soluble phenolic compounds that are particularly affected by high temperatures, such as those of pasteurization [75].

Furthermore, the formation of polymeric tannins is slow at room temperature because of the large reaction initiation energy required, but accelerates at higher temperatures, such as those used in pasteurization [76]. As a result, enhanced CPP values seen following pasteurization reflect larger amounts of polymeric anthocyanins, which are responsible for some monomer loss [77]. The above findings are corroborated by the application of pasteurization to pomegranate [41] and to aronia juice [13] to an 18% and 16% increase in CPP, respectively, and a 55.7% decrease in juice redness (color attribute a*) for both. Such a loss may be due to the loss of phenolic compounds responsible for the bright red-purple color, such as anthocyanins [43].

3.3. Effect of β-CD and Gelatin Treatment

The total phenolic compound concentration in pasteurized control juice was 1591.9 mgGAE/L (Figure 3A). In general, the concentration of phenolic components in juice varies depending on how the plant is grown [78], as well as post-harvest procedures for the fruit, such as the pressure used to extract the juice and the temperature of clarity [40].

Phenolic compounds of aronia juice were maintained at control juice levels after β-CD treatment (Figure 3A). β-CD treatment has been found to maintain initial total phenolic compounds following pasteurizing pomegranate juice [71], tangerine [49], or orange juice [58], but this was not obvious throughout the 3-month storage life. β-CD possesses a cavity volume in which the phenolic content of aronia juice is encapsulated and preserved after pasteurization [44,46].

Adding gelatin to juice before pasteurization significantly reduced total phenolic compounds (p < 0.05) (Figure 3A). The reduction corresponded to the amount of gelatin added to the juice. This was also correlated with the relative level of sentiment observed after treatment (Figure 1). At the highest gelatin content (0.3–0.4 g/L juice), juice total phenolic compounds were reduced to as low as 900–1000 mg GAE/L (Figure 3A). On the other hand, the addition of clarifying agents such as gelatin and bentonite (300 mg/L) or 1–2 g/L gelatin resulted in a reduction in total phenolic compounds in pasteurized pomegranate juice by 16% [41] or by 18.22–26.6% [40]. In low-pH fruit juices (pH = 4.22), gelatin’s positive charge combines with negatively charged particles like phenolics, forming hydrogen bonds and settling clumps. The precipitate is removed during the filtration process, which reduces the total phenolic content [45]. The purpose of aronia juice clarifying is to reduce turbidity and bad taste while preserving strong antioxidant activity, even though it may result in a drop in total phenolic content [40].

Figure 3B compares the DPPH radical scavenging activity of pasteurized aronia juices previously treated with β-CD or gelatin. Adding 0.5% and 1% β-CD, or 0.1–0.3 g/L gelatin, to aronia juice had no effect on its DPPH radical scavenging activity as compared to the control. However, adding 1.5% or 2% β-CD did not affect the unpasteurized control’s DPPH radical scavenging activity (Table 1). In contrast, juice treated with 0.4 g/L gelatin decreased DPPH radical scavenging activity to as low as 1700 mgTE/L. Treating bitter melon juice with 2% β-cyclodextrin improved its antioxidant activity [29]. Aree and Jongrungruangchok’s [79] study found that green tea flavanols, such as (+)-catechin and (−)-epicatechin, have higher antioxidant activity when complexed with β-CD. In other studies, employing β-CD in pomegranate juice did not increase the antioxidant capacity of fruit juices [49,71]. However, gelatin treatment of pomegranate juice at a concentration of 0.1 g/L reduced the antioxidant content by 16–18% [44,45]. The above findings are associated with changes in total phenolic compounds.

The increases in total flavonoids followed a similar pattern as those in total phenolic compounds (Figure 3C). There was no change between the control juice and the juice with 0.5% β-CD or 0.1–0.2 g/L gelatin, which were kept at 1650–1750 mg CE/L. In contrast, adding 1–2% β-CD to aronia juice resulted in small increases in total flavonoids, reaching 1800 mg CE/L juice, whereas 0.3–0.4 g/L gelatin drastically decreased to as low as 1000 mg CE/L juice. Kelanne et al. [22] reported that adding β-CD to lingonberry juice resulted in a small rise in total flavonoids, specifically flavonols, from 462.3 mg/L to 475.0 mg/L.

The total monomeric anthocyanins in control aronia juice after pasteurization were as low as 280 mg cyn-3-glu/L (Figure 3D). β-CD or gelatin treatment of juice maintained monomeric anthocyanins according to the quantity added. The greatest percentages of added β-CD (2%) maintained the original unpasteurized juice levels (Table 1), as did gelatin (0.4 g/L), but at slightly lower levels (Figure 3D, p < 0.05); The total monomeric anthocyanin losses in aronia juices treated with 0.4 g/L gelatin compared to the unpasteurized control were as low as 3.9% (Table 2). Anthocyanins are water-soluble and heat-sensitive; therefore, β-CD added at low levels was ineffective for preserving them after pasteurization [80]. Mourtzinos et al. [46], reported that β-CD prevented anthocyanin losses of an anthocyanin-rich extract obtained from Hibiscus sabdariffa. The findings of this study are also compatible with those of Kelanne et al. [22], who showed that adding β-CD to lingonberry juice maintained the monomeric anthocyanin content. In contrast, Bagci [44] and Vardin and Fenercioglu [40], using gelatin, observed losses of total anthocyanin monomers in pomegranate juice of 6.8% and 6.18%, respectively. Adding gelatin to pomegranate juice has also been shown to reduce anthocyanin monomers by 10.5% [45], 21% [43], and even 32% [41].

The values of color due to polymeric pigments (CPP) indicate the proportion of anthocyanin-procyanidin polymers that are resistant to bleaching in the presence of potassium bisulfite, as well as the degree of color loss induced by the polymerization process. Pasteurization resulted in a 3.07% increase in CPP (p < 0.05). Adding 0.5–2% β-CD boosted the color to 65% (Figure 3E). Kulcan et al. [71] observed that β-CD did not minimize anthocyanin losses during polymerization in pasteurized pomegranate juice, and that 2% β-CD resulted in a slight increase in polymer color. Howard et al. [47] discovered that adding 3% w/v β-CD to aronia juice promoted CPP formation by 6% compared to the control; also, juices with 3% β-CD exhibited greater percent polymer color values than all other juices while preserving high amounts of anthocyanins. To explain, they hypothesized that the high concentration of β-CD inhibited bleaching of monomeric anthocyanins in the CPP assay by either encapsulating the pyran ring, preventing procyanidin attachment, or functioning as a strong copigment. The addition of 0.1–0.2 g/L gelatin to aronia juice increased the polymer color to comparable values. Adding gelatin (0.3–0.4 g/L) increased the polymer color by up to 78% (p < 0.05) (Figure 3E).

To identify color changes or losses, organoleptic evaluation of food is frequently insufficient. Therefore, in order to identify any alterations or undesirable changes that are not immediately apparent to the human perception, it must also be measured using the proper tools. The variations in colorimeter parameters L*, a*, and b* of pasteurized and treated juice are shown in Figure 4A, 4B, and 4C, respectively. The brightness levels for all treatments and the control ranged from 16.5 to 17.5, with no discernible differences. For both the β-CD treatment and the controls, the redness (a*) and yellowness (b*) levels were kept at 2.5 and 1.7, respectively. Nonetheless, there were minor but noteworthy (p > 0.05) increases in redness (to 6—Figure 4B) and yellowness (to 2.4—Figure 4C) recorded in juice treated with 0.4 mg/L gelatin.

Similar outcomes were obtained with citrus juices containing β-CD at 1% and 1.5% w/v strengths [49,58]. Andreu-Sevilla et al. [81] found that α-, β-, and γ-CD significantly affect pear juice; cyclodextrins enabled to reduce the juice’s enzymatic degradation and limit the change in L*, a*, and b* characteristics. Turfan et al. [43] discovered that the fast polymerization rate and the statistically significant loss of total anthocyanin monomers caused the parameters a* and b* to rise.

The effects of β-CD and gelatin on the pH and total soluble solids of the produced juices are shown in Figure 4D,E. As anticipated, total soluble solids increased significantly in proportion to the amount of β-CD added. Juices treated with gelatin, however, showed no changes in total soluble solids (Figure 4E). Juice pH was not considerably impacted by β-CD or gelatin treatment in comparison to control (Figure 4D). In pomegranate juice added with gelatin, Vardin and Fenercioglu [40] and Erkan et al. [45] saw similar results, whereas a study by Jafari et al. [82] using Indian gooseberry juice corroborated the findings above.

3.4. Second Organoleptic (Preference) Test

The initial organoleptic investigation provided numerous key discoveries. The main results showed that treating juice with β-CD or gelatin at the highest levels (2% w/v or 0.4 mg/L, respectively) reduced astringency and aroma while increasing sweetness. As a result, these amounts were employed to prepare the juices for the subsequent organoleptic (consumer) research. It also offered early sensory data on some aspects of the juices, and panelists learned to identify and verbalize tastes and flavors, particularly those employed in the consumer study.

This subsequent consumer study evaluated the aronia juice, untreated and treated with β-CD or gelatin, by utilizing an organoleptic preference test performed by twenty-eight untrained testers.

Figure 5 shows statistically significant differences in the samples’ organoleptic properties for aroma, astringency, sweetness, sourness, and overall acceptability as a result of the application of β-CD and gelatin compared to the control. Color and sourness, however, did not differ substantially (p > 0.05) amongst the juices.

The consumer-preference test found that aroma-treated juices had a significantly lower intensity compared to the control (p < 0.05). β-CD had a greater impact on the shift in aroma in aronia juice, receiving the lowest scores from panelists compared to the control or gelatin. Aronia juice’s aroma was reduced by 31.1% with β-CD and 16.7% with gelatin treatment.

This organoleptic test confirmed that β-CD and gelatin improve taste balance and astringent aftertaste. More specifically, treating juice with either agent (β-CD or gelatin) resulted in a 25.6% decrease in astringent flavor and a 30% decrease in astringent aftertaste. Panelists found that juices treated with β-CD or gelatin had a considerably higher sweet taste (p < 0.05), with increases of 21.1% and 23.3%, respectively, with no variation between them.

The panelists of the preference test discovered an interrelationship between flavor in treated and untreated aronia juice; this was demonstrated by the fact that customers who evaluated aronia juice with enhanced acceptability rated high on the same attributes developed by the initial organoleptic panel. Again, the use of blocking (in the case of β-CD) or removing (in the case of gelatin) agents led in juice modifications and, as a result, altered the sensory attributes by masking or removing astringency taste/aftertaste, respectively, while also disclosing the underlying sweetness. There was no sourness detected in this particular juice combination or level of additional agents (Figure S1). The introduction of such juice processing significantly enhanced consumer acceptance.

4. Conclusions

This study aimed to assess the impact of gelatin and β-CD on the organoleptic and physicochemical qualitative attributes of pasteurized aronia juice. The study conducted on aronia juices showed that according to preliminary organoleptic analysis, both gelatin and β-CD can improve sweetness while lessening the distinctive astringent aftertaste that aronia juice leaves on the tongue.

Overall, aronia juice with added β-CD kept higher antioxidant potential, total flavonoids, and total monomeric anthocyanins as compared to gelatin after pasteurization. However, the addition of both agents maintained monomeric anthocyanins with minimal losses as compared to pre-pasteurization levels, while also producing greater juice color because of polymeric pigments since polymerization occurs at a rapid rate and anthocyanin monomers are lost. Although this study did not address storage and shelf-life difficulties, this could be an ongoing research topic.

Aronia juice has the potential to appeal to people looking for fruity flavors and nutritious foods. To match such consumer preferences, consistent quality acquired by proper processing optimization is needed. Both β-CD and gelatin have the ability to improve the astringent flavor of aronia juice, making it more appealing to consumers. For consumers who adhere to modern requirements for the consumption of food with high antioxidant activity and nutritional content, processed aronia juice may be a preferable option because the polyphenolic load and antioxidant activity are maintained at significantly high levels. Although gelatin is an animal product, its use is restricted because of the vegan concept. In the United States, it must be approved by the FDA as a food additive before being used in commercial juice production, and EU regulations, specifically those pertaining to the use of animal-derived materials, also require careful consideration of sourcing, manufacturing, and labeling. However, β-CD is a food additive that is allowed in the EU (E 459) [83] and has special rules for using it in different food categories. Because it contains extra carbohydrates, its usage in food is somewhat restricted. Costwise, β-CD is found to be three-fold more expensive than gelatin.