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Article

Encapsulation of Chokeberry Polyphenols by Ionic Gelation: Impact of Pullulan and Disaccharides Addition to Alginate Beads

by
Mirela Kopjar
1,*,
Ina Ćorković
1,
Josip Šimunović
2 and
Anita Pichler
1
1
Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, F. Kuhača 18, 31000 Osijek, Croatia
2
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(11), 6320; https://doi.org/10.3390/app15116320
Submission received: 24 April 2025 / Revised: 2 June 2025 / Accepted: 3 June 2025 / Published: 4 June 2025
(This article belongs to the Section Food Science and Technology)
Versions Notes

Abstract

Alginate is one of the most utilized biopolymers for the encapsulation of polyphenols throughout ionic gelation. For improvement in the encapsulation of polyphenols, other biopolymers and/or fillers can be employed. The purpose of this study was to include pullulan and/or disaccharides in an alginate encapsulation mixture to monitor whether we would achieve higher encapsulation of chokeberry juice polyphenols. Alginate hydrogel beads were used as controls, and through the results for total polyphenol and proanthocyanidin contents, concentrations of individual polyphenols, and antioxidant activities, it can be observed that pullulan and/or disaccharides had an impact on the encapsulation of these bioactives. Alginate/pullulan hydrogel beads had the highest contents of total polyphenols and proanthocyanidins (8.60 g/kg and 2.37 g/kg, respectively), whereas alginate/trehalose hydrogel beads had the lowest (5.50 g/kg and 1.16 g/kg, respectively). All hydrogel beads, except alginate/pullulan/sucrose, had higher anthocyanin (cyanidin-3-galactoside and cyanidin-3-arabinoside) contents than alginate beads (404.37 mg/kg and 89.97 mg/kg, respectively), but the most efficient combination for encapsulation of chokeberry anthocyanins was alginate/pullulan (477.32 mg/kg and 109.60 mg/kg, respectively). The highest concentration of neochlorogenic acid was determined in controls (260.14 mg/kg), while the highest concentration of chlorogenic acid in alginate/pullulan/sucrose beads (229.51 mg/kg). Quercetin-3-glucoside was evaluated as having the highest concentration in alginate/pullulan hydrogel beads (35.45 mg/kg). The data obtained through this study highlight the importance of the composition of an encapsulation mixture in order to achieve high encapsulation of chokeberry juice polyphenols. High encapsulation efficiency was obtained for anthocyanins, especially when pullulan was used in combination with alginate.

1. Introduction

Recently published manuscripts [1,2,3] on chokeberries have summarized their praiseworthy nutritional profile, highlighting their polyphenol content as one of the major contributors to their potential health benefits. Generally, edible plant polyphenols have been utilized as functional ingredients or for the formulation of functional additives for the enrichment of foods and beverages, since they can prevent or decelerate the advancement of a broad variety of diseases. Chokeberries (Aronia melanocarpa) are emphasized as a highly valuable source for the formulation of functional foods [1,2]. The most representative polyphenols in chokeberry juice are red pigments, i.e., anthocyanins (especially cyanidin-3-galactoside and cyanidin-3-arabinoside), flavonols (quercetin-3-rutinoside), and phenolic acids (chlorogenic and neochlorogenic acids) [4,5]. Over the years, many studies (in vitro, pre-clinical, clinical, and epidemiological) have been conducted with the aim of exploring the interconnection between phenolic compounds and potential health benefits such as balancing the lipid profile, possible regulation of diabetes and hyperglycemia, protection from cardiovascular disorders, and anticancer, anti-inflammation and antioxidant potential [6,7,8,9,10,11,12]. Since chokeberries are not present in fresh form all year round, they are frequently processed into different products like jams, spreads, wines, juice, fruit teas, sauces, and dietary supplements [5,13].
The formulation of different functional food additives is also one of the ways to preserve valuable bioactive compounds. Nowadays, enormous attention in the food and pharmaceutical industries is directed towards finding an adequate way to preserve phenolic compounds, in general, since they are quite unstable under various environmental conditions. Implementation of the “delivery by design concept” [14] can be used to resolve this rising question of phenolic compound stability. Progressive pursuit of new formulations through combinations of different polymers and other compounds can be perceived. Ionic gelation is one of many encapsulation techniques that is used for the formation of delivery systems of valuable plant-derived bioactives, since it is characterized by simplicity and rapidity. For these purposes, alginate is the most utilized biopolymer. Alginate is a naturally present anionic polymer that is usually extracted from brown seaweed. It is quite an interesting biopolymer and has wide applications in the food industry, agriculture, and the pharmaceutical industry, as well as in medicine, due to its attributes such as low cost, low toxicity, biocompatibility, biodegradability, thermal and chemical stability, and ability to mask undesirable flavors [15,16,17]. Application of other biopolymers and fillers alongside alginate in the encapsulation mixture can boost the encapsulation of polyphenols through alternation of the highly porous structure of the alginate gel network in a way that facilitates the encapsulation of lower-molecular-weight compounds. Usually for these purposes, chitosan, pectin, cellulose derivates, and proteins are used [17,18,19,20]. Pullulan is a water-soluble, non-toxic, and non-allergenic biopolymer, without odor or taste. Like alginate, it is characterized by its biocompatibility and biodegradability [21,22,23,24,25]. In food product formulation, pullulan has gained an important place due to its versatile properties. It can be utilized as a texturizer, for stabilization purposes, as a thickening and gelling agent, and also to improve organoleptic characteristics, prolong the shelf life of foods, and ease processing [26]. In low-calorie foods and drinks, it has been applied as a filler, but significant application has been achieved in the field of functional composite films and edible coatings in food packaging [21,26]. Interestingly, past studies have implied that dietary pullulan may be used as a prebiotic for the protection of bifidobacteria and to enhance its growth [26,27]. Over the years, pullulan applications expanded; thus, we used it in combination with alginate to investigate possible enhancement in the encapsulation of chokeberry polyphenols.
Next to biopolymers, we included two disaccharides in the encapsulation mixture, sucrose or trehalose. Sucrose is a disaccharide that is ordinarily used not just in households but also in the food industry, while, over the years, trehalose has gained popularity due to its favorable features, especially its bioprotective characteristics and non-toxicity [28]. Even though these two disaccharides are chemical isomers, trehalose is characterized by lower sweetness than sucrose. Trehalose has been known for its considerably diminished cariogenic ability in comparison to sucrose, which is beneficial for children while at the same time not possessing the laxative impact of other low-cariogenic sweeteners [29]. Another important feature of trehalose is its low glycemic index, since trehalose is slowly digested, causing a lower release of insulin than sucrose [30,31,32,33].
The purpose of this research was tailoring different hydrogel beads by ionic gelation. Alginate is the most used biopolymer for this encapsulation technique, but, due to the high porosity of the alginate gel network, we employed another biopolymer, i.e., pullulan, disaccharides (sucrose or trehalose), and blends of pullulan and individual disaccharides alongside alginate in encapsulation mixture. For the estimation of their influence on the encapsulation of chokeberry polyphenols, obtained hydrogel beads were evaluated for their total polyphenol and proanthocyanidin contents, concentrations of individual polyphenols, and antioxidant activities.

2. Materials and Methods

2.1. Ingredients for Encapsulation Mixtures and Chemicals for Analysis

Chokeberry fruits were cultivated in an area positioned at 46.176799° N 16.333695° E. Chokeberry fruits were pressed, and the yielded mass was filtered (filter paper Whatman, grade 1 (Sigma-Aldrich, Darmstadt, Germany)) and thermally treated at 90 °C for 3 min to inactivate enzymes and microorganisms, producing chokeberry juice (one batch) used for the formulation of hydrogel beads. For the preparation of the encapsulation mixture, next to two biopolymers, alginic acid and pullulan, two disaccharides, sucrose (99.8%) and trehalose (99.5%), were utilized. The sodium salt of alginic acid (very low viscosity) was acquired from Alfa Aesar (Kandel, Germany). Trehalose and pullulan were obtained as a donation from Hayashibara Co. (Nagase group, Japan). Sucrose was purchased from Gram-mol (Zagreb, Croatia). From the previously mentioned supplier, ethanol, calcium chloride, sodium acetate, potassium chloride, and ammonium acetate were obtained. Acetic acid (>99.5%) was purchased from Alkaloid (Skopje, North Macedonia), while methanol and hydrochloric acid (37%) were purchased from Carlo Erba Reagents (Sabadell, Spain). Sodium carbonate was acquired from T.T.T. (Sveta Nedelja, Croatia). Folin–Ciocalteu reagent and potassium persulfate were purchased from Kemika (Zagreb, Croatia). 2,4,6-tri(2-pyridyl)-s-triazine, neocuproine, and cupric chloride were acquired from Acros Organic (Geel, Belgium). 2,2-diphenyl-1-picrylhydrazil, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 4-dimethyl-amino-cinnamaldehyd, and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mobile phases for HPLC analysis, orthophosphoric acid and methanol (both HPLC grade), were acquired from Fisher Scientific (Loughborough, UK) and J.T. Baker (Deventer, The Netherlands), respectively. Standards for polyphenol quantification by HPLC analysis, namely chlorogenic acid and quercetin-3-rutinoside, were acquired from Sigma-Aldrich (St. Louis, MO, USA), while cyanidin-3-galactoside and neochlorogenic acid were obtained from Extrasynthese (Genay, France).

2.2. Preparation of Hydrogel Beads by Ionic Gelation

For control hydrogel beads (ALG), the encapsulation mixture was prepared by dissolving 3% (m/v) alginate in the juice by stirring with a stick blender. Alginate/disaccharide beads were prepared by dissolving 3% alginate and 10% disaccharides (sucrose or trehalose) in chokeberry juice to obtain an encapsulation mixture. Consequently, alginate/sucrose hydrogel beads (ALG_S) and alginate/trehalose hydrogel beads (ALG_T) were formulated. Alginate/pullulan hydrogel beads (ALG_P) were formulated by dissolving 3% (m/v) alginate and 1% (m/v) pullulan in the juice, while for alginate/pullulan/disaccharide hydrogel beads, an additional 10% (m/v) disaccharides (sucrose or trehalose) was added into encapsulation mixture; thus, alginate/pullulan/sucrose hydrogel beads (ALG_P_S) and alginate/pullulan/trehalose hydrogel beads (ALG_P_T) were created. Following dissolution of all ingredients in the encapsulation mixture as above defined, they were complexed for 30 min prior to conducting the microencapsulation process. As a hardening solution, 5% (m/v) CaCl2 in chokeberry juice was used. Hydrogel beads were created through the application of Encapsulator B-390 (BÜCHI Labortechnik AG, Flawil, Switzerland) at room temperature under defined parameters: a vibration frequency of 200 Hz, a 1000 V electrode, a pressure of 200 mbar, and a 1000 µm vibrating nozzle. Upon the creation of hydrogel beads, they remained in the hardening solution for 30 min. Afterwards, they were filtered through strainer and utilized for selected analysis.

2.3. Extraction of Polyphenols from Hydrogel Beads

In 15 mL plastic tubes with caps, approximately 1.5 g of hydrogel beads and 10 mL of extraction solvent, i.e., acidified methanol (99:1 (v:v)—methanol:hydrochloric acid), were added. These mixtures were inserted into an ultrasonication bath (Bandelin sonorex digitec, Berlin, Germany) and sonicated for 10 min. Afterwards, samples were inserted into the vortex (MSV-3500, Biosan, Riga, Latvia) at a rotational speed of 800 rpm for 2 h. The obtained mixtures were filtered through filter paper (Whatman, grade 1), and the gained extracts were utilized for spectrophotometric evaluation of total polyphenols, proanthocyanidins, and antioxidant potential, as well as the determination of individual polyphenols by HPLC.

2.4. Determination of Total Polyphenols

Total polyphenols were evaluated by utilization of the protocol specified by Singleton and Rossi [34] with some alternations. In a glass test tube, 0.1 mL of extract and 0.9 mL of deionized water were inserted and agitated, followed by agitation with 5 mL of Folin–Ciocalteu reagent (1:10) and 4 mL of sodium carbonate (7.5%). The obtained mixtures were placed in the dark for 120 min, followed by measurement of the absorbance of samples at 765 nm using a spectrophotometer (Cary 60, UV-VIS, Agilent Technologies, Santa Clara, CA, USA). All samples were evaluated in triplicates, and the results are presented as g of gallic acid equivalents per kg of hydrogel beads (g GAE/kg).

2.5. Determination of Proanthocyanidins

The concentration of proanthocyanidins was evaluated by the utilization of the 4-(dimethylamino)cinnamaldehyde (DMAC) method [35]. DMAC reagent was prepared by dissolving 0.05 g DMAC in acidified ethanol (75% of ethanol + 12.5% of water + 12.5% of hydrochloric acid). In the glass test tube, 0.1 mL of extract, 0.4 mL of deionized water, and 1 mL of DMAC reagent were inserted and agitated. The obtained mixtures were placed in a dark place at room temperature for 30 min prior the measurement of absorbance at 640 nm. All samples were evaluated in triplicates, and the results are presented as g of procyanidin B2 equivalent per kg of hydrogel beads (g B2E/kg).

2.6. Determination of Individual Polyphenols by High-Performance Liquid Chromatography (HPLC)

Alongside spectrophotometric analysis of total polyphenols, HPLC analysis was performed to evaluate individual ones. For this purpose, the HPLC system 1260 Infinity II (Agilent Technology, Santa Clara, CA, USA) was utilized. The system is composed of a quaternary pump, a vial sampler, and a diode array detector (DAD) and equipped with a Poroshell 120 EC C-18 column (4.6 × 100 mm, 2.7 µm). Initially, extracts were filtered over PTFE filters (0.20 µm pores) and then injected into the system at a volume of 10 µL, and the flow rate was adjusted to 1.0 mL/min. The DAD was used for monitoring UV-Vis spectra in the wavelength range from 190 to 600 nm. Two mobile phases were used, orthophosphoric acid (0.1% water solution) as mobile phase A and methanol (100%) as mobile phase B. The gradient used for separation was arranged under the following conditions: 0 min 5% B, 3 min 30% B, 15 min 35% B, 22 min 37% B, 30 min 41% B, 32 min 45% B, 40 min 49% B, 45 min 80% B, 48 min 80% B, 50 min 5% B, and 53 min 5% B. Identification of polyphenols was conducted by comparing the extract and standards through retention times and UV-Vis spectra. Anthocyanins were detected at 520 nm, phenolic acids at 320 nm, and quercetin-3-rutinoside at 360 nm. A calibration curve for cyanidin-3-galactoside was generated in the range from 1 to 500 mg/L (r2 = 0.9998; LOD = 0.0006 mg/L; LOQ = 0.0019 mg/L), for cyanidin-3-arabinoside from 1 to 150 mg/L (r2 = 0.9996; LOD = 0.29 mg/L; LOQ = 0.89 mg/L), for neochlorogenic acid from 5 to 150 mg/L (r2 = 0.9992; LOD = 0.71 mg/L; LOQ = 2.15 mg/L), for chlorogenic acid from 5 to 500 mg/L (r2 = 0.9997; LOD = 0.0002 mg/L; LOQ = 0.0006 mg/L), and for quercetin-3-rutinoside from 5 to 150 mg/L (r2 = 0.998; LOD = 0.18 mg/L; LOQ = 0.55 mg/L). For each extract, two injections were conducted, and the results are presented as mg of polyphenol per kg of hydrogel beads (mg/kg).

2.7. Determination of Antioxidant Potential by Selected Methods

CUPRAC, FRAP, ABTS, and DPPH assays were selected for the determination of antioxidant potential of hydrogel beads. For all selected methods for the estimation of antioxidant potential, all samples were analyzed in triplicates, and for the expression of results, a Trolox calibration curve was generated. Results were presented as µmol of Trolox equivalents per 100 g of hydrogel beads (µmol TE/100 g). For the evaluation of ferric-reducing ability, 0.2 mL of extract and 3 mL of FRAP reagent were inserted and mixed in glass tubes. FRAP reagent was prepared by mixing 25 mL of acetate buffer (300 mM), 2.5 mL of 2,4,6-tripyridyl-s-triazine (10 mM), and 2.5 mL of FeCl3 × 6H2O (20 mM). Prior to the addition of FRAP reagent to the extract, the reagent was heated at 37 °C. The obtained mixtures were placed in the dark at room temperature for 30 min, and absorbance was measured at 593 nm [36]. Copper (II)-reducing antioxidant ability or the CUPRAC assay was performed according to the protocol specified by Apak et al. [37]. For evaluation, in a glass tube, 1 mL of copper chloride (0.01 M), 1 mL of necuproine (7.5 mM), and 1 mL of ammonium acetate buffer (at pH 7) were inserted and agitated in the given order. To the obtained mixture, 0.2 mL of extract and 0.9 mL of distilled water were added and mixed. Absorbance was measured at 450 nm after the mixture was incubated for 30 min at room temperature. The DPPH method was performed according to the protocol of Brand-Williams et al. [38]. In total, 0.2 mL of extract and 3 mL of DPPH solution (0.5 mM) were inserted and mixed in glass tubes. The obtained mixtures were placed in the dark at room temperature for 15 min, and absorbance was measured at 517 nm. The ABTS method of Arnao et al. [39] was also used according to the following procedure: 0.2 mL of extract and 3.2 mL of ABTS reagent were inserted and mixed in glass tubes. After 95 min at room temperature, absorbance was measured at 734 nm. For the development of ABTS radicals, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt solution (7.4 mM) was mixed with potassium persulfate (2.6 mM), and after 24 h, the mixture was diluted to obtain an absorbance of 1.136.

2.8. Statistical Analysis of Results

The results of the applied analyses are displayed as mean values ± standard deviations. In order to evaluate the statistical significance of our results, statistical analysis was executed through the utilization of STATISTICA 13.1 software (StatSoft Inc., Tulsa, OK, USA). The obtained data were analyzed through analysis of variance (ANOVA) and Fisher’s least significant difference (LSD), with significance defined as p < 0.05.

3. Results

3.1. Total Polyphenols and Proanthocyanidins in Hydrogel Beads

The main goal of this study was to estimate the impact of pullulan and/or disaccharide (sucrose and trehalose) addition on the encapsulation of chokeberry polyphenols in alginate hydrogel beads. Alginate is a polymer commonly used as wall material in the encapsulation of polyphenols when encapsulation is performed by ionic gelation. The results of the evaluation of total polyphenols and proanthocyanidins are displayed in Table 1. Chokeberry juice contained 19.98 g/kg of total polyphenols and 3.15 g/kg of proanthocyanidins. Alginate hydrogel beads were used as a control sample, and through the results, it can be perceived that pullulan and/or disaccharides had an impact on the encapsulation of phenolic compounds. The control sample contained 5.54 g/kg of total polyphenols. The addition of pullulan to the encapsulation mixture generally had the most positive effect, so the total polyphenol content of these beads was 8.60 g/kg. A combination of alginate and disaccharides revealed that the type of disaccharide had a significant impact on the encapsulation of phenolic compounds. Sucrose was more effective in encapsulation than trehalose. Consequently, alginate/sucrose beads had 8.17 g/kg of total polyphenols, while alginate/trehalose beads had 4.50 g/kg. The addition of both pullulan and disaccharide to the alginate mixture also affected the encapsulation of phenolic compounds. The combination of alginate/pullulan/trehalose in the encapsulation mixture was more effective than alginate/pullulan/sucrose; thus, the opposite effect of disaccharide type was observed in comparison to hydrogel beads without pullulan. Total polyphenols in alginate/pullulan/trehalose beads were 7.83 g/kg, in contrast to 4.78 g/kg in alginate/pullulan/sucrose beads. So actually, two encapsulation mixtures, alginate/trehalose and alginate/pullulan/sucrose, were less effective than the alginate encapsulation mixture. The same trend was observed for encapsulation of proanthocyanidins. Alginate hydrogel beads contained 1.47 g/kg of proanthocyanidins while alginate/trehalose and alginate/pullulan/sucrose beads had lower amounts, 1.16 g/kg and 1.23 g/kg, respectively. The remaining beads had a higher amount of proanthocyanidins than the control sample. Alginate/pullulan, alginate/sucrose, and alginate/pullulan/trehalose beads contained 2.37 g/kg, 2.27 g/kg, and 2.15 g/kg of proanthocyanidins, respectively.

3.2. Concentration of Individual Polyphenols in Hydrogel Beads

Individual polyphenols in chokeberry juice and hydrogel beads are displayed in Table 2. Comparing their concentrations with total polyphenols and proanthocyanidins, it can be perceived that a different trend was obtained depending on the structure of individual polyphenols, as well as on the composition of the encapsulation mixture.
Cyanidin-3-galactoside and cyanidin-3-arabinoside were two anthocyanins detected in chokeberry juice and all hydrogel beads, with cyanidin-3-galactoside as the dominant one. Chokeberry juice contained 598.80 mg/kg and 119.90 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside. For both anthocyanins, the same encapsulation trend was observed. Alginate hydrogel beads contained 404.37 mg/kg and 89.97 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside, respectively. Only alginate/pullulan/sucrose beads had lower concentrations of these anthocyanins (330.21 mg/kg and 73.39 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside, respectively) than control beads. All other encapsulation mixtures caused improvement in anthocyanin encapsulation. Both anthocyanins were detected in the highest concentration in alginate/pullulan beads, and those beads contained 477.52 mg/kg and 109.60 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside, respectively. Both remaining hydrogel beads with trehalose, i.e., alginate/pullulan/trehalose and alginate/trehalose, contained approximately 434 mg/kg and 98.8 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside, respectively. Hydrogel beads formulated with sucrose (alginate/sucrose) contained 410.69 mg/kg and 93.40 mg/kg of cyanidin-3-galactoside and cyanidin-3-arabinoside, respectively. Two phenolic acids were detected in chokeberry juice and hydrogel beads, neochlorogenic and chlorogenic acids, which did not follow the trend obtained for anthocyanins. In juice, much higher concentrations of neochlorogenic acid (967.65 mg/kg) and chlorogenic acid (778.15 mg/kg) were detected than in hydrogel beads. Neochlorogenic acid was detected at the highest concentration in the control sample, 260.14 mg/kg. In the remaining samples, the concentration of neochlorogenic acid decreased in following order: alginate/sucrose > alginate/pullulan > alginate/trehalose > alginate/pullulan/trehalose > alginate/pullulan/sucrose (252.38 mg/kg > 226.07 mg/kg > 210.57 mg/kg > 157.77 mg/kg > 125.59 mg/kg). The combination of both pullulan and disaccharide with alginate (alginate/pullulan/trehalose and alginate/pullulan/sucrose beads) highly decreased the encapsulation of this phenolic acid. On the other hand, those beads had the highest concentration of chlorogenic acid, 213.03 and 229.51 mg/kg for alginate/pullulan/trehalose and alginate/pullulan/sucrose beads, respectively. Next to those two beads, alginate/trehalose and alginate/pullulan beads (approximately 180.5 mg/kg) also had higher concentration of chlorogenic acid than the control sample (162.33 mg/kg). Only alginate/sucrose beads had a lower concentration (132.95 mg/kg) of this phenolic acid than the control sample. The encapsulation of quercetin-3-rutinoside in hydrogel beads was also affected by the composition of the encapsulation mixture. In juice, the concentration of this compound was 82.15 mg/kg. The control sample and alginate/trehalose beads contained 32 mg/kg of this flavonoid, while alginate/sucrose beads had a slightly lower concentration (31.30 mg/kg) and alginate/pullulan/disaccharide beads had significantly lower concentration (26.5 mg/kg). Only alginate/pullulan beads had a higher concentration (35.45 mg/kg) of quercetin-3-rutinoside than the control one.

3.3. Antioxidant Potential of Hydrogel Beads

The formulated hydrogel beads were also tested for their antioxidant potential, which was evaluated by the most commonly used assays (CUPRAC, FRAP, ABTS, and DPPH assays). The results are presented in Table 3. As was determined for total polyphenols and proanthocyanidins, as well as for individual polyphenols, the composition of the encapsulation mixture played a major part in the antioxidant potential of hydrogel beads, which can primarily be linked to the concentration of phenolic compounds and their ability to act as antioxidants. Additionally, each method is characterized by its mechanism of action. The DPPH assay revealed that the antioxidant potential of chokeberry juice was 140.85 μmol TE/100 g, while for alginate beads, it was 21.93 μmol TE/100 g. Alginate/pullulan/sucrose and alginate/trehalose beads had slightly lower antioxidant potential than the control sample (21.2 μmol TE/100 g), while other samples had higher antioxidant potential. For those beads, antioxidant potential decreased in the following order: alginate/sucrose > alginate/pullulan > alginate/pullulan/trehalose (38.54 μmol TE/100 g > 30.19 μmol TE/100 g > 26.87 μmol TE/100 g). A slightly different trend was observed for ABTS results. The antioxidant potential of chokeberry juice was 145.85 μmol TE/100 g. Alginate/trehalose and alginate/pullulan/sucrose beads had lower antioxidant potentials (17.39 μmol TE/100 g and 21.5 μmol TE/100 g, respectively) than the control sample (42.56 μmol TE/100 g). Alginate/pullulan beads (86.99 μmol TE/100 g) had considerably higher antioxidant potential than the control sample, while the antioxidant potentials of alginate/sucrose and alginate/pullulan/trehalose were 68.66 μmol TE/100 g and 66.0 μmol TE/100 g, respectively. Considering FRAP results, it was observed that alginate/trehalose and alginate/pullulan/sucrose beads had the lowest antioxidant potential (3.1 μmol TE/100 g). Other samples had higher antioxidant potential than the control sample (3.83 μmol TE/100 g). Alginate/pullulan and alginate/sucrose beads had the highest antioxidant potential (6.3 μmol TE/100 g), while the antioxidant potential of alginate/pullulan/trehalose beads was 5.78 μmol TE/100 g. Chokeberry juice had the highest antioxidant activity, at 15.39 μmol TE/100 g. CUPRAC results also revealed that alginate/trehalose and alginate/pullulan/sucrose beads had lower antioxidant potentials (184.67 μmol TE/100 g and 203.98 μmol TE/100 g, respectively) than the control sample (261.44 μmol TE/100 g). For the other hydrogel beads, antioxidant potential decreased in the following order: alginate/pullulan > alginate/sucrose > alginate/pullulan/trehalose (438.0 μmol TE/100 g > 428.34 μmol TE/100 g > 391.99 μmol TE/100 g). For this method, as well as for the others, the highest antioxidant activity was determined for chokeberry juice, at 1052.01 μmol TE/100 g.
The correlation between the investigated parameters was also assessed and is presented in Table 4. The correlation between total polyphenols and proanthocyanidins was very high (0.9996). Comparing the results of total polyphenol and proanthocyanidin content with antioxidant activity, it is evident that the results obtained through application of the FRAP and CUPRAC methods followed the trend that was observed for total polyphenol contents and proanthocyanidin contents, and the correlation coefficients were >0.995. The correlation between total polyphenol contents and proanthocyanidin contents was slightly lower, with values of antioxidant activity obtained by the ABTS method (0.9622 and 0.9586 for total polyphenol and proanthocyanidin contents, respectively). The lowest correlation was determined for antioxidant activity obtained by the DPPH method, at 0.7878 and 0.8079 for total polyphenol contents and proanthocyanidin contents, respectively. An evaluation of the correlation coefficient between antioxidant activity and specific groups of individual polyphenols determined by HPLC revealed a low correlation between anthocyanins and antioxidant activity (from 0.3525 to 0.4133). A low and negative correlation was also observed between antioxidant activities and phenolic acids (from −0.4171 to −0.3260), while quercetin-3-rutinoside showed little effect.

4. Discussion

The goal of this study was to establish possible improvements in the encapsulation of chokeberry polyphenols by ionic gelation through modification of the encapsulation mixture composition. Alginate is the most commonly used polymer for encapsulation in this process, so we modified the encapsulation mixture with the addition of pullulan or/and two disaccharides, namely sucrose or trehalose.
The encapsulation mixture contains mostly water, so its presence and interactions with added encapsulation polymers and disaccharides, as well as with phenolic compounds, played an important part in the encapsulation of phenolic compounds. The fact that there is more available water ensures conditions that are the driving force for the conduction of diffusion-limited reactions. Additionally, oxidation and molecular mobility are also enhanced due to the higher content of water, and all of these reactions result in the degradation of sensitive components such are phenolic compounds. Anthocyanins are especially sensitive to water. The glycosidic bond in the structure of anthocyanins can be hydrolyzed, leading to the generation of unstable anthocyanidins. Usually, afterward, the opening of the pyrilium ring occurs, which causes the generation of chalcones and brown end compounds [40]. Additionally, if oxygen is present, water causes an increase in the oxidation rate of anthocyanins. Combining different polysaccharides in the mixture can lead to potential synergistic effects that are favorable for specific applications [41]. Alginate and pullulan differ in their structure and their behavior in water. While alginate dissolved in water has a more open, rigid chain structure, pullulan is characterized by a high-flexibility chain structure due to the α-(1,6) linkage [41,42,43]. As was already mentioned in the Introduction, the alginate hydrogel network is characterized by a highly porous structure, so other polymers and/or fillers are often used to reduce diffusion through the alginate network, where these compounds usually serve as a barrier in order to block the transport of components from beads to surroundings [17,18,19,20]. In previous studies, it was observed that a higher concentration of total polysaccharides in an encapsulation mixture could result in the higher preservation of anthocyanins [19,20], which was also observed in this study, since alginate/pullulan hydrogel beads had a higher concentration of anthocyanins. Diffusion of anthocyanins within the gel network can be correlated to diffusion in porous media, so this phenomenon is linked with the porosity of hydrogel beads. The addition of extra polysaccharides in the mixture enhances the gelation rate prior to the setting of hydrogel, causing a decrease in the diffusion rate of anthocyanins in surrounding media through a reduction in the porosity after hydrogel is formed [19]. Through microencapsulation, physicochemical interactions occurred. On one hand, electrostatic interactions among the dissociated carboxylic groups of alginate and the flavylium cations of the anthocyanin can have a stabilizing effect on anthocyanins [44], but also, covalent bonds among alginate, pullulan, and anthocyanins and some phenolics are formed, as was already observed between pectin–alginate and anthocyanins [19], enhancing the stability of anthocyanins and some phenolics during the formation of hydrogel beads. For better comprehension of the interactions among anthocyanins and polysaccharides, one needs to keep in mind that, firstly, these compounds are interconnected, and the following binding involves the creation of a so-called stacking impact, which leads to the attachment of additional anthocyanin molecules to those already bonded with polysaccharides [45]. This effect was observed for both anthocyanins in the encapsulation mixture of alginate and pullulan, alginate and disaccharides, and alginate/pullulan/trehalose. However, the opposite effect was observed for other phenolics; the addition of disaccharides in combination with both polysaccharides highly affected those interactions, especially negatively for neochlorogenic acid and quercetion-3-glucoside. The utilization of mixtures consisting of large polysaccharides and small disaccharides ensures tight molecular packaging, i.e., disaccharides fill gaps that are formed by polysaccharide [46]. While this effect can aid in the entrapment of some polyphenol molecules, at the same time, it can decrease the number of biding sites for others. Possible interactions among compounds that provided the chemical entrapment of polyphenols in the matrix of beads are one clarification; however, disaccharides can also be involved in changing water dynamics and through this course influencing the entrapment of phenolic compounds. We used two disaccharides in the encapsulation mixture, sucrose and trehalose, which are chemical isomers. However, their impact on the encapsulation of not only anthocyanins but also other phenolic compounds was different regardless of if they were used alongside alginate or in combination with alginate and pullulan. Both anthocyanins were estimated to have higher concentrations in hydrogel beads when trehalose was added to the encapsulation mixture alongside alginate or alginate and pullulan. There was no difference in the effect of disaccharide type on the encapsulation of quercetin-3-rutinoside, whereas an impact was detected on phenolic acids. Even though chlorogenic and neochlorogenic acids are isomers, the impact of disaccharide was diverse. When disaccharides were added to the encapsulation mixture alongside alginate, neochlorogenic acid was determined in higher concentrations in hydrogel beads with sucrose and chlorogenic acid in hydrogel beads with trehalose. Interestingly, a reverse effect was observed when disaccharides were added to the encapsulation mixture alongside alginate and pullulan. Although the investigated disaccharides are chemical isomers, based on some parameters such as the formation of clusters, the radius of gyration, the hydration number, and the glycosidic dihedral angles, it was deduced that trehalose water solutions were more homogenous in comparison sucrose ones [47]. Trehalose has a larger impact on water structure because it binds with more molecules of water than sucrose, thus having a larger destructuring impact on water [48]. Disaccharides cause the development of steric hindrance in water, which can cause protection from or slowing down of the nucleophilic attacks of water on anthocyanins. Additionally, trehalose has higher stability towards hydrolysis than sucrose [49]. These properties were probably more expressed in encapsulation mixtures with the addition of trehalose, causing higher encapsulation of anthocyanins. Also, it can be expected that interactions between some phenolics and trehalose can occur. It was proven that the creation of stable intramolecular complexes among trehalose and specific unsaturated compounds can occur. These compounds possess an olefinic double bond of the cis type or are structurally similar to them [50,51,52]. Unlike other disaccharides, trehalose is characteristic for these complexations due to its specific structure, which enables the aromatic rings of compounds to approach the dehydrated, hydrophobic pocket of trehalose, causing the formation of a complex [52].
Alginate/pullulan hydrogels had the highest concentration of individual anthocyanins, followed by alginate/trehalose and alginate/pullulan/trehalose hydrogel beads; however, antioxidant activities evaluated by CUPRAC and FRAP methods were the highest for alginate/pullulan, lower for alginate/pullulan/trehalose, and significantly lower for alginate/trehalose hydrogel beads. In comparison to alginate/pullulan hydrogel beads, alginate/trehalose beads had lower concentrations of neochlorogenic acid and quercetin-3-rutinoside. In alginate/pullulan/trehalose hydrogels, in comparison to the other two above-mentioned samples, chlorogenic acid was present at a higher concentration than neochlorogenic acid, so it probably compensated for the loss of antioxidant activity caused by lower anthocyanin and quercetin-3-rutinoside concentrations. Interestingly, alginate/pullulan/sucrose hydrogel beads also had a higher concentration of chlorogenic acid than neochlorogenic acid, but, due to the fact that this sample had the lowest concentration of anthocyanins, it can be deduced that this was the reason for the lowest antioxidant activities evaluated by the CUPRAC and FRAP methods. Interestingly, alginate/sucrose had the highest antioxidant activity determined by DPPH, followed by alginate/pullulan hydrogel beads, regardless of the fact that alginate/sucrose hydrogel beads had lower concentrations of all phenolics, except neochlorogenic acid, than alginate/pullulan hydrogel beads. One reason could be that neochlorogenic acid had higher antioxidant potential than chlorogenic acid [53]; thus, it contributed to the overall potential. Another reason could be the synergistic effect between phenolic compounds. Synergism or antagonism was also observed in alginate/pullulan/sucrose and alginate/trehalose hydrogel beads, with both having the same antioxidant activity determined by the DPPH assay. This trend was noted regardless of the fact that alginate/trehalose beads contained higher concentrations of anthocyanins than alginate/pullulan/sucrose hydrogel beads, but at the same time, this sample had a higher concentration of neochlorogenic acid than chlorogenic acid, whereas in alginate/pullulan/sucrose beads, the reverse trend was observed. When applying the ABTS assay, the highest antioxidant activity was determined for alginate/pullulan hydrogel beads, followed by alginate/trehalose and alginate/pullulan/trehalose hydrogel beads, i.e., results followed concentrations of individual anthocyanins. The lowest values were determined for alginate/trehalose and alginate/pullulan/sucrose hydrogel beads, again emphasizing the importance of synergistic or antagonistic effects between compounds. The results for antioxidant activity differ among samples, and different trends were achieved for each sample with different methods. One of the most important characteristics of phenolics is their antioxidant activity. It highly depends on their chemical structures, but other factors are also important, such as the ratio between them, the presence of other compounds, and the interactions between these compounds and phenolics. Methods differ from each other based on mechanisms of action. The mechanisms of action of the DPPH and ABTS methods involve reactions between H atom donors and the corresponding free radicals, DPPH˙ and ABTS˙+. Even though these mechanisms are similar, the obtained results are different, as, in many other studies, the ABTS method usually generates higher values [54]. Our results for antioxidant activity determined by the ABTS assay were higher than for DPPH results, with the exception of alginate/trehalose and alginate/pullulan/sucrose hydrogel beads. ABTS˙+ free radicals are less selective than DPPH˙; thus, they can be involved in reactions with any hydroxylated aromatic compounds regardless of their real antioxidant ability, leading to reactions with OH groups that do not contribute to antioxidation [55]. Contrary, DPPH˙ free radicals are characterized by greater selectivity in their reactions with H donors than ABTS˙+. DPPH free radicals do interact with flavonoids that are lacking OH groups in the structure of their B-ring. Additionally, they do not react with aromatic acids with only one OH group [55]. The mechanisms of action of the FRAP and CUPRAC methods involve the reduction of metal ions by antioxidants. While the mechanism of action of the FRAP method includes the reduction of the ferric ion (Fe3+)–ligand complex to ferrous (Fe2+) complex [56], the mechanism of action of the CUPRAC method includes the reduction of the cupric ion (Cu2+) to the cuprous ion (Cu+) [57]. The total antioxidant ability of systems that contain different phenolic compounds can be primarily additive, antagonistic, or synergistic on account of the diversity in their structures. Structures of phenolic compounds, mainly the number and position of OH groups and OCH3 groups on the phenolic rings, are responsible for their antioxidant activity. However, for a complete understanding of the antioxidant ability of complex systems, other factors need to additionally be considered, such as the concentration of phenolic compounds, their dissociation and ionization, intramolecular and/or intermolecular interactions, and matrix interference [6,58,59].

5. Conclusions

The main goal of this study was to estimate the impact of the addition of pullulan and/or disaccharides (namely sucrose and trehalose) on the encapsulation of chokeberry polyphenols in alginate hydrogel beads. Results showed that the composition of encapsulation mixture and the structure of phenolics played important roles in the entrapment of phenolics into hydrogel beads. The addition of pullulan alongside alginate in the encapsulation mixture had the most positive impact on the entrapment of anthocyanins and quercetin-3-rutinoside. The addition of pullulan and/or disaccharides negatively affected the encapsulation of neochlorogenic acids, i.e., the highest entrapment of this phenolic acid was in the alginate encapsulation mixture. The positive effect of all compounds in the encapsulation mixture was revealed in the encapsulation of chlorogenic acid. Especially high encapsulation efficiency was achieved for anthocyanins, proving that pullulan can be used in combination with alginate to encapsulate these valuable pigments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15116320/s1, Figure S1. Chromatograms of alginate hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S2. Chromatograms of alginate/pullulan hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S3. Chromatograms of alginate/sucrose hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S4. Chromatograms of alginate/trehalose hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S5. Chromatograms of alginate/pullulan/sucrose hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S6. Chromatograms of alginate/pullulan/trehalose hydrogel beads (C-3-G: cyanidin-3-galactoside, C-3-A: cyanidin-3-arabinoside, NcA: neochlorogenic acid, CA: chlorogenic acid, Q-3-R: quercetin-3-rutinoside); Figure S7. Chromatograms of cyanidin-3-glucoside solutions; Figure S8. Chromatograms of cyanidin-3-arabinoside solutions; Figure S9. Chromatograms of chlorogenic acid solutions; Figure S10. Chromatograms of neochlorogenic acid solutions; Figure S11. Chromatograms of quercetin-3-rutinoside solutions; Figure S12. Representative photo of formulated hydrogel beads.

Author Contributions

Conceptualization, M.K. and J.Š.; methodology, M.K. and A.P.; formal analysis, I.Ć. and A.P.; investigation, I.Ć., M.K. and A.P.; data curation, I.Ć. and A.P.; writing—original draft preparation, M.K.; writing—review and editing, J.Š.; supervision, M.K. and A.P.; project administration, M.K.; funding acquisition, M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Croatian Science Foundation under the project (IP-2019 04-5749) “Design, fabrication and testing of biopolymer gels as delivery systems for bioactive and volatile compounds in innovative functional foods (bioACTIVEgels)”, and Young Researchers’ Career Development Project—Training New Doctoral Students (DOK-2020-01-4205).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed at the corresponding author.

Acknowledgments

The authors would like to express their gratitude to Hayashibara Co. (Nagase Group, Japan) for their generous donation of pullulan and trehalose samples and their support over the years.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Sidor, A.; Drożdżyńska, A.; Gramza-Michałowska, A. Black chokeberry (Aronia melanocarpa) and its products as potential health-promoting factors—An overview. Trends Food Sci. Technol. 2019, 89, 45–60. [Google Scholar] [CrossRef]
  2. Zhang, Y.; Zhao, A.; Liu, X.; Chen, X.; Ding, C.; Dong, L.; Zhang, J.; Sun, S.; Ding, Q.; Khatoom, S.; et al. Chokeberry (Aronia melanocarpa) as a new functional food relationship with health: An overview. J. Future Foods 2021, 1, 168–178. [Google Scholar] [CrossRef]
  3. Jurendić, T.; Ščetar, M. Aronia melanocarpa products and by-products for health and nutrition: A review. Antioxidants 2021, 10, 1052. [Google Scholar] [CrossRef] [PubMed]
  4. Jakobek, L.; Matić, P.; Ištuk, J.; Barron, A.R. Study of interactions between individual phenolics of aronia with barley beta-glucan. Pol. J. Food Nutr. Sci. 2021, 71, 187–196. [Google Scholar] [CrossRef]
  5. Ćorković, I.; Rajchl, A.; Škorpilová, T.; Pichler, A.; Šimunović, J.; Kopjar, M. Evaluation of Chokeberry/carboxymethylcellulose hydrogels with the addition of disaccharides: DART-TOF/MS and HPLC-DAD analysis. Int. J. Mol. Sci. 2023, 24, 448. [Google Scholar] [CrossRef]
  6. Haizhou, W.; Oliveira, G.; Lila, M.A. Protein-binding approaches for improving bioaccessibility and bioavailability of anthocyanins. Compr. Rev. Food Sci. Food Saf. 2023, 22, 333–354. [Google Scholar]
  7. de Mejia, E.G.; Zhang, Q.; Penta, K.; Eroglu, A.; Lila, M.A. The colors of health: Chemistry, bioactivity, and market demand for colorful foods and natural food sources of colorants. Annu. Rev. Food Sci. Technol. 2020, 11, 145–182. [Google Scholar] [CrossRef]
  8. Shen, Y.; Zhang, N.; Tian, J.; Xin, G.; Liu, L.; Sun, X.; Li, B. Advanced approaches for improving bioavailability and controlled release of anthocyanins. J. Control. Release 2022, 341, 285–299. [Google Scholar] [CrossRef]
  9. Fallah, A.A.; Sarmast, E.; Fatehi, P.; Jafari, T. Impact of dietary anthocyanins on systemic and vascular inflammation: Systematic review and meta-analysis on randomised clinical trials. Food Chem. Toxicol. 2020, 135, 110922. [Google Scholar] [CrossRef]
  10. Kimble, R.; Keane, K.M.; Lodge, J.K.; Howatson, G. Dietary intake of anthocyanins and risk of cardiovascular disease: A systematic review and meta-analysis of prospective cohort studies. Crit. Rev. Food Sci. Nutr. 2019, 59, 3032–3043. [Google Scholar] [CrossRef]
  11. Krga, I.; Milenkovic, D. Anthocyanins: From sources and bioavailability to cardiovascular-health benefits and molecular mechanisms of action. J. Agric. Food Chem. 2019, 67, 1771–1783. [Google Scholar] [CrossRef] [PubMed]
  12. Lila, M.A.; Burton-Freeman, B.; Grace, M.; Kalt, W. Unraveling anthocyanin bioavailability for human health. Annu. Rev. Food Sci. Technol. 2016, 7, 375–393. [Google Scholar] [CrossRef] [PubMed]
  13. Kapci, B.; Neradová, E.; Čížková, H.; Voldřich, M.; Rajchl, A.; Capanoglu, E. Investigating the antioxidant potential of chokeberry (Aronia melanocarpa) products. J. Food Nutr. Res. 2013, 52, 219–229. [Google Scholar]
  14. McClements, D.J. Delivery by Design (DbD): A standardized approach to the development of efficacious nanoparticle- and microparticle-based delivery systems. Compr. Rev. Food Sci. Food Saf. 2018, 17, 200–219. [Google Scholar] [CrossRef]
  15. Lee, K.Y.; Mooney, D.J. Alginate: Properties and biomedical applications. Prog. Polym. Sci. 2012, 37, 106–126. [Google Scholar] [CrossRef] [PubMed]
  16. Rathod, G.; Kairam, N. Preparation of omega 3 rich oral supplement using dairy and non dairy based ingredients. J. Food Sci. Technol. 2018, 55, 760–766. [Google Scholar] [CrossRef]
  17. Bušić, A.; Belščak-Cvitanović, A.; Vojvodić Cebin, A.; Karlović, S.; Kovač, V.; Špoljarič, I.; Mršič, G.; Komes, D. Structuring new alginate network aimed for delivery of dandelion (Taraxacum officinale L.) polyphenols using ionic gelation and new filler materials. Food Res. Int. 2017, 111, 244–255. [Google Scholar] [CrossRef]
  18. Belščak-Cvitanović, A.; Komes, D.; Karlović, S.; Djaković, S.; Špoljarić, I.; Mršić, G.; Ježek, D. Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium. Food Chem. 2015, 167, 378–386. [Google Scholar] [CrossRef]
  19. Guo, J.; Giusti, M.M.; Kaletunç, G. Encapsulation of purple corn and blueberry extracts in alginate-pectin hydrogel particles: Impact of processing and storage parameters on encapsulation efficiency. Food Res. Int. 2017, 107, 414–422. [Google Scholar] [CrossRef]
  20. Ćorković, I.; Pichler, A.; Ivić, I.; Šimunović, J.; Kopjar, M. Microencapsulation of chokeberry polyphenols and volatiles: Application of alginate and pectin as wall materials. Gels 2021, 7, 231. [Google Scholar] [CrossRef]
  21. Ghosh, T.; Priyadarshi, R.; Krebs de Souza, C.; Angioletti, B.L.; Rhim, J.-W. Advances in pullulan utilization for sustainable applications in food packaging and preservation: A mini-review. Trends Food Sci. Technol. 2022, 125, 43–53. [Google Scholar] [CrossRef]
  22. Constantin, M.; Bucatariu, S.; Sacarescu, L.; Daraba, O.M.; Anghelache, M.; Fundueanu, G. Pullulan derivative with cationic and hydrophobic moieties as an appropriate macromolecule in the synthesis of nanoparticles for drug delivery. Int. J. Biol. Macromol. 2020, 164, 4487–4498. [Google Scholar] [CrossRef] [PubMed]
  23. Feng, X.; Wang, W.; Chu, Y.; Gao, C.; Liu, Q.; Tang, X. Effect of cinnamon essential oil nanoemulsion emulsified by osa modified starch on the structure and properties of pullulan based films. LWT 2020, 134, 110123. [Google Scholar] [CrossRef]
  24. Agrawal, S.; Budhwani, D.; Gurjar, P.; Telange, D.; Lambole, V. Pullulan based derivatives: Synthesis, enhanced physicochemical properties, and applications. Drug Deliv. 2022, 29, 3328–3339. [Google Scholar] [CrossRef]
  25. Thomas, N.; Puluhulawa, L.E.; Mo’o, F.R.C.; Rusdin, A.; Gazzali, A.M.; Budiman, A. Potential of pullulan-based polymeric nanoparticles for improving drug physicochemical properties and effectiveness. Polymers 2024, 16, 2151. [Google Scholar] [CrossRef]
  26. Rashid, A.; Qayum, A.; Liang, Q.; Kang, L.; Ekumah, J.-N.; Han, X.; Ren, X.; Ma, H. Exploring the potential of pullulan-based films and coatings for effective food preservation: A comprehensive analysis of properties, activation strategies and applications. Int. J. Biol. Macromol. 2024, 260, 129479. [Google Scholar] [CrossRef]
  27. Kycia, K.; Chlebowska-Śmigiel, A.; Szydłowska, A.; Sokół, E.; Ziarno, M.; Gniewosz, M. Pullulan as a potential enhancer of Lactobacillus and Bifidobacterium viability in synbiotic low fat yoghurt and its sensory quality. LWT 2020, 128, 109414. [Google Scholar] [CrossRef]
  28. Chen, A.; Gibney, P.A. Dietary Trehalose as a Bioactive Nutrient. Nutrients 2023, 15, 1393. [Google Scholar] [CrossRef] [PubMed]
  29. Neta, T.; Takada, K.; Hirasawa, M. Low-cariogenicity of trehalose as a substrate. J. Dent. 2000, 28, 571–576. [Google Scholar] [CrossRef]
  30. Van Can, J.G.P.; Van Loon, L.J.C.; Brouns, F.; Blaak, E.E. Reduced glycaemic and insulinaemic responses following trehalose and isomaltulose ingestion: Implications for postprandial substrate use in impaired glucose-tolerant subjects. Br. J. Nutr. 2012, 108, 1210–1217. [Google Scholar] [CrossRef]
  31. Yaribeygi, H.; Yaribeygi, A.; Sathyapalan, T.; Sahebkar, A. Molecular mechanisms of trehalose in modulating glucose homeostasis in diabetes. Diabetes Metab. Syndr. Clin. Res. Rev. 2019, 13, 2214–2218. [Google Scholar] [CrossRef]
  32. Yoshizane, C.; Mizote, A.; Yamada, M.; Arai, N.; Arai, S.; Maruta, K.; Mitsuzumi, H.; Ariyasu, T.; Ushio, S.; Fukuda, S. Glycemic, insulinemic and incretin responses after oral trehalose ingestion in healthy subjects. Nutr. J. 2017, 16, 9. [Google Scholar] [CrossRef] [PubMed]
  33. Oku, T.; Nakamura, S. Estimation of intestinal trehalase activity from a laxative threshold of trehalose and lactulose on healthy female subjects. Eur. J. Clin. Nutr. 2000, 54, 783–788. [Google Scholar] [CrossRef] [PubMed]
  34. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotonutric acid reagents. Am. J. Enol. Vitic. 1965, 16, 144–158. [Google Scholar] [CrossRef]
  35. Prior, R.L.; Fan, E.; Ji, H.; Howell, A.; Nio, C.; Payne, M.J.; Reed, J. Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders. J. Sci. Food Agric. 2010, 90, 1473–1478. [Google Scholar] [CrossRef] [PubMed]
  36. Benzie, I.F.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “Antioxidant Power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [CrossRef]
  37. Apak, R.; Güçlü, K.; Ozyürek, M.; Karademir, S.E. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Sci. Food Agric. 2004, 52, 7970–7981. [Google Scholar] [CrossRef]
  38. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT 1995, 28, 25–30. [Google Scholar] [CrossRef]
  39. Arnao, M.B.; Cano, A.; Acosta, M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 2001, 73, 239–244. [Google Scholar] [CrossRef]
  40. Syamaladevi, R.M.; Sablani, S.S.; Tang, J.; Powers, J.; Swanson, B.G. Stability of anthocyanins in frozen and freeze-dried raspberries during long-term storage: In relation to glass transition. J. Food Sci. 2011, 76, 414–421. [Google Scholar] [CrossRef]
  41. Xiao, Q.; Tong, Q.; Lim, L.-T. Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions. Carbohydr. Polym. 2012, 87, 1689–1695. [Google Scholar] [CrossRef]
  42. Kato, T.; Okamoto, T.; Tokuya, T.; Takahashi, A. Solution properties and chain flexibility of pullulan in aqueous solution. Biopolym 1982, 21, 1623–1633. [Google Scholar] [CrossRef]
  43. Mancini, M.; Moresi, M.; Sappino, F. Rheological behaviour of aqueous dispersions of algal sodium alginates. J. Food Eng. 1996, 28, 283–295. [Google Scholar] [CrossRef]
  44. Hubbermann, E.M.; Heins, A.; Stöckmann, H.; Schwarz, K. Influence of acids, salt, sugars and hydrocolloids on the colour stability of anthocyanin rich black currant and elderberry concentrates. Eur. Food Res. Technol. 2006, 223, 83–90. [Google Scholar] [CrossRef]
  45. Padayachee, A.; Netzel, G.; Netzel, M.; Day, L.; Zabaras, D.; Mikkelsen, D.; Gidley, M.J. Binding of polyphenols to plant cell wall analogues—Part 1: Anthocyanins. Food Chem. 2012, 134, 155–161. [Google Scholar] [CrossRef]
  46. Tonnis, W.F.; Mensink, M.A.; de Jager, A.; van der Voort Maarschalk, K.; Frijlink, H.W.; Hinrichs, W.L.J. Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins. Mol. Pharm. 2015, 12, 684–694. [Google Scholar] [CrossRef]
  47. Lerbret, A.; Bordat, P.; Affouard, F.; Descamps, M.; Migliardo, F. How homogeneous are the trehalose, maltose, and sucrose water solutions? An insight from molecular dynamics simulations. J. Phys. Chem. B 2005, 109, 11046–11057. [Google Scholar] [CrossRef]
  48. Olsson, C.; Swenson, J. Structural comparison between sucrose and trehalose in aqueous solution. J. Phys. Chem. B 2020, 124, 3074–3082. [Google Scholar] [CrossRef]
  49. Schebor, C.; Burin, L.; del Pilar Bueras, M.; Chirife, J. Stability to hydrolysis and browning of trehalose, sucrose and raffinose in low-moisture systems in relation to their use as protectants of dry biomaterials. LWT 1999, 32, 481–485. [Google Scholar] [CrossRef]
  50. Oku, K.; Watanabe, H.; Kubota, M.; Fukuda, S.; Kurimoto, M.; Tujisaka, Y.; Komori, M.; Inoue, Y.; Sakurai, M. NMR and quantum chemical study on the OH...pi and CH...O interactions between trehalose and unsaturated fatty acids: Implication for the mechanism of antioxidant function of trehalose. J. Am. Chem. Soc. 2003, 125, 12739–12748. [Google Scholar] [CrossRef]
  51. Oku, K.; Kurose, M.; Kubota, M.; Fukuda, S.; Kurimoto, M.; Tujisaka, Y.; Okabe, A.; Sakurai, M. Combined NMR and quantum chemical studies on the interaction between trehalose and dienes relevant to the antioxidant function of trehalose. J. Phys. Chem. B 2005, 109, 3032–3040. [Google Scholar] [CrossRef] [PubMed]
  52. Sakakura, K.; Okabe, A.; Oku, K.; Sakurai, M. Experimental and theoretical study on the intermolecular complex formation between trehalose and benzene compounds in aqueous solution. J. Phys. Chem. B 2011, 115, 9823–9830. [Google Scholar] [CrossRef] [PubMed]
  53. Navarro-Orcajada, S.; Matencio, A.; Vicente-Herrero, C.; García-Carmona, F.; López-Nicolás, J.M. Study of the fluorescence and interaction between cyclodextrins and neochlorogenic acid, in comparison with chlorogenic acid. Sci. Rep. 2021, 11, 3275. [Google Scholar] [CrossRef] [PubMed]
  54. Ali, H.M.; Almagribi, W.; Al-Rashidi, M.N. Antiradical and reductant activities of anthocyanidins and anthocyanins, structure–activity relationship and synthesis. Food Chem. 2016, 194, 1275–1282. [Google Scholar] [CrossRef]
  55. Abirami, A.; Sinsinwar, S.; Rajalakshmi, P.; Brindha, P.; Rajesh, Y.B.R.D.; Vadivel, V. Antioxidant and cytoprotective properties of loganic acid isolated from seeds of Strychnos potatorum L. against heavy metal induced toxicity in PBMC model. Drug Chem. Toxicol. 2022, 45, 239–249. [Google Scholar] [CrossRef]
  56. Prior, R.L.; Wu, X.; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005, 53, 4290–4302. [Google Scholar] [CrossRef]
  57. Pinelo, M.; Manzocco, L.; Nunez, M.J.; Nicoli, M.C. Interaction among phenols in food fortification: Negative synergism on antioxidant capacity. J. Agric. Food Chem. 2004, 52, 1177–1180. [Google Scholar] [CrossRef]
  58. Hang, D.T.N.; Hoa, N.T.; Bich, H.N.; Mechler, A.; Vo, Q.V. The hydroperoxyl radical scavenging activity of natural hydroxybenzoic acids in oil and aqueous environments: Insights into the mechanism and kinetics. Phytochemistry 2022, 201, 113281. [Google Scholar] [CrossRef]
  59. Biela, M.; Kleinová, A.; Klein, E. Phenolic acids and their carboxylate anions: Thermodynamics of primary antioxidant action. Phytochemistry 2022, 200, 113254. [Google Scholar] [CrossRef]
Table 1. Contents of total polyphenols and proanthocyanidins in chokeberry juice and hydrogel beads.
Table 1. Contents of total polyphenols and proanthocyanidins in chokeberry juice and hydrogel beads.
SamplesTotal Polyphenols
(g/kg)		Proanthocyanidins
(g/kg)
CJ19.98 ± 0.01 e3.15 ± 0.01 g
ALG_CJ5.54 ± 0.17 b1.47 ± 0.02 c
ALG_P_CJ8.60 ± 0.19 d2.37 ± 0.05 f
ALG_S_CJ8.17 ± 0.58 c,d2.27 ± 0.03 e
ALG_T_CJ4.50 ± 0.34 a1.16 ± 0.01 a
ALG_P_S_CJ4.78 ± 0.17 a1.23 ± 0.02 b
ALG_P_T_CJ7.83 ± 0.21 c2.15 ± 0.03 d
CJ: chokeberry juice; ALG: alginate; P: pullulan; T: trehalose; S: sucrose. Significantly different (at p ≤ 0.05; ANOVA, Fisher’s LSD) mean values within the column are denoted by different letters (a–g).
Table 2. Concentrations (mg/kg) of individual polyphenols in chokeberry juice and hydrogel beads.
Table 2. Concentrations (mg/kg) of individual polyphenols in chokeberry juice and hydrogel beads.
SamplesC-3-GC-3-ANcACAQ-3-R
CJ598.80 ± 0.07 f119.90 ± 0.02 f967.65 ± 3.25 g778.15 ± 2.16 f82.15 ± 0.87 e
ALG_CJ404.37 ± 2.48 b89.97 ± 0.53 b260.14 ± 1.28 f162.33 ± 0.30 b32.61 ± 0.40 c
ALG_P_CJ477.52 ± 0.59 e109.60 ± 0.22 e226.07 ± 0.67 d180.39 ± 3.89 c35.45 ± 0.02 d
ALG_S_CJ410.69 ± 5.36 c93.40 ± 1.16 c252.38 ± 0.73 e132.95 ± 0.70 a31.30 ± 0.56 b
ALG_T_CJ434.28 ± 0.01 d98.96 ± 0.09 d210.57 ± 0.68 c181.03 ± 3.00 c32.71 ± 0.24 c
ALG_P_S_CJ330.21 ± 1.57 a73.39 ± 0.80 a125.59 ± 0.04 a229.51 ± 0.56 e25.95 ± 0.16 a
ALG_P_T_CJ433.30 ± 0.01 d98.74 ± 0.09 d157.44 ± 0.39 b213.03 ± 1.28 d26.91 ± 0.66 a
CJ: chokeberry juice; ALG: alginate; P: pullulan; S: sucrose; T: trehalose. C-3-G: cyanidin-3-galactoside; C-3-A: cyanidin-3-arabinoside; NcA: neochlorogenic acid; CA: chlorogenic acid; Q-3-R: quercetin-3-rutinoside. Significantly different (at p ≤ 0.05; ANOVA, Fisher’s LSD) mean values within the column are denoted by different letters (a–g).
Table 3. Antioxidant activity of chokeberry juice and hydrogel beads.
Table 3. Antioxidant activity of chokeberry juice and hydrogel beads.
SamplesFRAPCUPRACDPPHABTS
CJ15.39 ± 0.03 e1052.01 ± 1.47 g140.85 ± 0.78 f145.85 ± 0.85 g
ALG_CJ3.83 ± 0.05 b261.44 ± 1.23 c21.93 ± 0.11 b42.56 ± 0.21 c
ALG_P_CJ6.48 ± 0.08 d438.00 ± 1.93 f30.19 ± 0.56 d86.99 ± 0.07 f
ALG_S_CJ6.21 ± 0.08 d428.34 ± 1.68 e38.54 ± 0.64 e68.66 ± 0.77 e
ALG_T_CJ3.00 ± 0.08 a184.67 ± 1.29 a20.17 ± 0.90 a17.39 ± 0.48 a
ALG_P_S_CJ3.19 ± 0.10 a203.98 ± 1.49 b20.31 ± 0.95 a21.50 ± 0.72 b
ALG_P_T_CJ5.78 ± 0.25 c391.99 ± 1.48 d26.87 ± 0.51 c66.00 ± 0.23 d
CJ: chokeberry juice; ALG: alginate; P: pullulan; T: trehalose; S: sucrose. Results expressed as μmol TE/100 g. Significantly different (at p ≤ 0.05; ANOVA, Fisher’s LSD) mean values within the column are denoted by different letters (a–g).
Table 4. Correlation between investigated parameters.
Table 4. Correlation between investigated parameters.
TPPACFRAPCUPRACDPPHABTSANTPAQ-3-R
TP1
PAC0.99961
FRAP0.99570.99511
CUPRAC0.99610.99760.99111
DPPH0.78780.80790.76350.81311
ABTS0.96220.95860.96410.95670.64261
ANT0.41300.41110.41330.37110.35250.36651
PA−0.4104−0.4088−0.4171−0.3698−0.3260−0.3719−0.99321
Q-3-R−0.0531−0.0519−0.0304−0.0437−0.2028−0.0210−0.13060.08041
TP: total polyphenols content; PAC: proanthocyanidins content; ANT: sum of anthocyanins concentrations; PA: sum of phenolic acids concentrations; Q-3-R: quercetin-3-rutinoside.
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MDPI and ACS Style

Kopjar, M.; Ćorković, I.; Šimunović, J.; Pichler, A. Encapsulation of Chokeberry Polyphenols by Ionic Gelation: Impact of Pullulan and Disaccharides Addition to Alginate Beads. Appl. Sci. 2025, 15, 6320. https://doi.org/10.3390/app15116320

AMA Style

Kopjar M, Ćorković I, Šimunović J, Pichler A. Encapsulation of Chokeberry Polyphenols by Ionic Gelation: Impact of Pullulan and Disaccharides Addition to Alginate Beads. Applied Sciences. 2025; 15(11):6320. https://doi.org/10.3390/app15116320

Chicago/Turabian Style

Kopjar, Mirela, Ina Ćorković, Josip Šimunović, and Anita Pichler. 2025. "Encapsulation of Chokeberry Polyphenols by Ionic Gelation: Impact of Pullulan and Disaccharides Addition to Alginate Beads" Applied Sciences 15, no. 11: 6320. https://doi.org/10.3390/app15116320

APA Style

Kopjar, M., Ćorković, I., Šimunović, J., & Pichler, A. (2025). Encapsulation of Chokeberry Polyphenols by Ionic Gelation: Impact of Pullulan and Disaccharides Addition to Alginate Beads. Applied Sciences, 15(11), 6320. https://doi.org/10.3390/app15116320

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