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Article

Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents

by
Astrid Natalia González Morales
,
Luis Javier López-Giraldo
*,
Erika Sogamoso González
and
Yaiza Moscote Chinchilla
School of Chemical Engineering, Universidad Industrial de Santander, Bucaramanga 680002, Colombia
*
Author to whom correspondence should be addressed.
Processes 2025, 13(10), 3094; https://doi.org/10.3390/pr13103094
Submission received: 18 August 2025 / Revised: 17 September 2025 / Accepted: 24 September 2025 / Published: 27 September 2025
(This article belongs to the Special Issue Microencapsulation of Food Antioxidants)

Abstract

Cocoa pod husk (CPH) has the potential to be utilized for polyphenol extraction to be used in functional food formulations and pharmaceutical formulations due to its health benefits. However, polyphenols are sensitive to environmental factors that reduce their stability and functionality. Therefore, encapsulation is necessary to protect their antioxidant capacity, mask undesirable flavours and smells, and, at the same time, allow the release of polyphenols in the gastrointestinal phases. This study encapsulated polyphenols using complex coacervation (CC) and spray drying (SD) with gum arabic (GA), sodium alginate (SA), chitosan (C), and gelatine (G), and evaluated yield (EY), encapsulation efficiency (EE), loading efficiency (LE), and bioaccessibility through in vitro digestion. The results showed that in the encapsulation using CC, the highest LE of 36.95 ± 7.63% was obtained using SA-G. In SD, significant differences in LE were observed among the tested encapsulant ratios, with the highest LE of 34.77 ± 1.2% achieved using GA (1:3). Bioaccessibility varied significantly depending on the encapsulation technique and encapsulating agent (EA) used. Using GA and spray drying (SD), the highest polyphenol release was achieved at 76.55 ± 5.10%, in contrast to only 6.41 ± 1.61% for the non-encapsulated extract. In conclusion, both techniques for encapsulating polyphenols extracted from CPH are efficient. However, SD allows for greater polyphenol bioaccessibility.

1. Introduction

The human body is an almost perfect machine that requires the support of countless nutrients for its proper functioning, among which antioxidants play a pivotal role. Without these, human health deteriorates irreparably. Antioxidants protect cells from the damaging effects of free radicals [1], which are responsible for altering biological systems, causing diseases, or accelerating ageing [2,3].
Consequently, both the food and pharmaceutical industries are increasingly incorporating antioxidants into their formulations, either to enhance the nutritional value of food products or to develop therapeutic agents aimed at preventing or mitigating oxidative stress-related disorders [4,5].
One of the main antioxidants in the human diet is polyphenols, whose intake is 10 times higher than that of vitamin C and 100 times higher than that of vitamin E or carotenoids, which are conventionally used as potent antioxidants [2]. Various studies have demonstrated that polyphenols provide several health benefits, including antioxidant, antitumoral, anti-inflammatory, and anti-atherosclerosis properties, among others [3,6]. When incorporated into food matrices, they confer functional properties that translate into numerous practical applications, benefiting both consumer health and matrix preservation [5]. Polyphenols are plant-derived compounds found in various matrices, such as fruits and vegetables, with cocoa being the fourth richest source of polyphenols among 100 sources [7].
The most abundant polyphenol groups in cocoa are flavonoid-type metabolites, particularly three basic groups: catechins (37%), anthocyanins (4%), and proanthocyanidins (58%) [8,9]. Polyphenols are present in cocoa beans, husks, and pod shells. It is worth noting that CPH represents a significant waste product in the cocoa industry (about 75% of the harvest) that can be utilized for polyphenol extraction, which has potential applications in the formulation of functional foods [10]. According to Carreño Toledo et al. [11], polyphenol extracts from CPH contain a total polyphenol content (TPC) ranging from 60 to 98 in milligrams of gallic acid equivalents per gram (mg GAE/g) for five evaluated cocoa varieties.
The stability and shelf life of polyphenols are crucial for the functionality of foods and are directly associated with changes in their chemical structure, making them sensitive to factors such as pH changes, temperature, and oxygen presence, which affect the stability of their carbonyl groups and unsaturation [12]. Additionally, polyphenols can be affected by interactions with enzymes such as polyphenol oxidases, peroxidases, glycosidases, and esterases, which catalyze reactions that transform polyphenols into [13], for example, ascorbic acid [14]. This lack of stability necessitates the development of systems to protect their antioxidant capacity, as they may be exposed to environmental conditions or processes that could reduce their stability or functionality [9]. Among the techniques used for protecting bioactive compounds and enhancing their stability is encapsulation, which can be performed using various methods such as CC or SD. CC is a physicochemical phase separation process based on electrostatic interactions between oppositely charged polyelectrolytes, allowing the formation of an encapsulating layer around the active core [15]. In contrast, SD is a physical technique that involves the atomisation of an emulsion or suspension into a stream of hot air, leading to rapid solvent evaporation and the production of dry microcapsules [16], resulting in lower degradation of the compounds of interest, a reduction in hygroscopicity compared to the extract without encapsulation, and a masking of undesirable flavours [17].
Recent studies have evaluated the encapsulation of polyphenols extracted from various sources. For instance, fig polyphenols have been encapsulated using GA–G and propolis with SA–G via CC [18,19], whereas orange and marjoram polyphenols have been encapsulated with SA–β-cyclodextrin and C [20,21], respectively, through SD. However, no research has been found that specifically examines the encapsulation of polyphenols extracted from CPH and their subsequent incorporation into food or pharmaceutical matrices. This incorporation largely depends on the LE of these polyphenols in the EA and the subsequent release of the active compound during digestive processes, ensuring their bioaccessibility. This bioaccessibility will depend on the technique and type of EA used [22,23]. Therefore, the aim of this study was to evaluate the EE and bioaccessibility of lyophilised polyphenol extract (LPE) through an in vitro gastrointestinal simulation by determining the TPC at each phase of the model (oral, gastric, and intestinal). Both non-encapsulated extracts from CPH and those encapsulated using CC and SD with GA, SA, C, and G as encapsulating agents were assessed. To the best of our knowledge, this is the first study to systematically evaluate the encapsulation of LPE using two methodologies, four encapsulating agents, and different proportions, as well as their capacity to release polyphenols across digestive phases. This comprehensive approach allowed us to identify the most suitable conditions for effective encapsulation.

2. Materials and Methods

2.1. Extraction and Characterization of Polyphenols

The methodology described by Pico et al. and Toro [7,10] was followed with some modifications. CPH (CCN-51 variety) from San Vicente de Chucurí and Rionegro, Santander (Colombia), were manually washed, cut into 1 cm2 pieces, and thermally treated to inhibit polyphenol oxidase. The samples were then cooled in ice water, rinsed thoroughly, dried, and ground in a ball mill to obtain an approximate particle size of 2 mm. Extraction was performed using 1 g of dried husk in 30 mL of 75% ethanol/water (v/v), subjected to ultrasonic treatment (20 kHz, 15 min), and then stirred at 60 °C for 30 min. The extract was centrifuged (4000× g, 4 °C, 15 min) and vacuum filtered using Whatman No. 1 paper. Ethanol was removed from the extract by vacuum evaporation (Rotavapor R210, Büchi, Flawil, Switzerlandy), and the crude extract was then lyophilised and stored under refrigeration, protected from light. The process was carried out 11 times to obtain a total of 200 g of LPE.
The extract characterization included the quantification of TPC using the Folin–Ciocalteu method, antioxidant capacity assessment via the Oxygen Radical Absorbance Capacity (ORAC) method, and the separation and identification of catechins and epicatechins groups using HPLC/UV-vis, following the approach described by Pico et al. [7], and using Ultra-high-performance liquid chromatography coupled with Orbitrap high-resolution mass spectrometry (UHPLC-ESI + -Orbitrap-HRMS). The detailed methodology is available in Supplementary Materials of this article.

2.2. Encapsulation by Complex Coacervation (CC)

A coacervation process was conducted using GA-G and SA-G as EA. For the GA–G system, Ochoa et al. [24] previously established a pH of 4.0 ± 0.01 and a fixed relationship between EA and the material to be encapsulated. Briefly, 1 g of LPE was incorporated into 50 mL of a 2.5% w/v G solution, homogenized (Ultra-Turrax, IKA-Werke, Staufen, Germany, 15,000× g, 5 min), and subsequently mixed with 50 mL of a 2.5% w/v GA solution. Cold water (200 mL) was added, and the pH was carefully adjusted to 4.0 ± 0.01 with 1 M HCl. The process was maintained at 50 ± 3 °C, after which the mixture was cooled to 10 °C under constant agitation using an ice bath. It was then stored at 3 °C for 24 h to allow complete microcapsule precipitation in covered beakers. Excess water was carefully removed by decantation, and the coacervate was frozen at −30 °C for 24 h in glass jars covered with filter paper. The material was subsequently dried by lyophilisation. The final samples were ground and stored in amber glass vials [24].
For SA-G, the same methodology was used, adjusting the concentrations and pH as suggested by Shinde et al. [25]. Briefly, 1 g of LPE was mixed with 50 mL of a G solution (5% w/v) and 50 mL of an SA solution (1.25% w/v), with the pH adjusted to 3.5 ± 0.01. All assays were performed in duplicate.

2.3. Encapsulation by Spray Drying (SD)

For SD encapsulations, each EA was tested individually using different LPE:EA ratios to optimize EE and yield. The ratios were selected based on the literature reports and the viscosity of the encapsulants. In the case of GA, its low viscosity allowed the use of higher polymer-to-core ratios (1:3, 1:7, and 1:11, with a 3% w/v solution in distilled water) [26]. For SA and C, where higher polymer concentrations increase viscosity and reduce spray-drying performance [20,27], lower ratios (1:1, 1:2, and 1:3) were employed, using 1% w/v SA in distilled water and 1% v/v C in acetic acid solution (pre-dissolved for 12 h). In all cases, the EA solution was prepared at 50 °C under stirring (1000 rpm, 20 min), after which 1 g of LPE was incorporated and stirred for 10 min before homogenisation (Ultra-Turrax, IKA-Werke, Staufen, Germany, 8000 rpm, 5 min). Spray drying was performed in a Mini Spray Dryer S-300 (Büchi, Flawil, Switzerland) at 150 °C inlet temperature, 30 m3/h drying gas flow, and 500 L/h spray gas flow. The feed rate was adjusted according to viscosity: 8 mL/min for GA, 6 mL/min for SA, and 5 mL/min for C, while monitoring outlet temperature [20,28,29]. All assays were performed in duplicate.

2.4. Quantification of Polyphenols in the Microcapsules and Surface Polyphenol Content

To quantify the TPC in the microcapsules, they were first disrupted by adding 0.1 g of microcapsules to a 1 mL solution of 10% w/v sodium citrate, vortex mixing for 15 s, and centrifuging (4000× g, 20 min). The supernatant was then filtered (0.45 µm Millipore filter, Labfil, Hangzhou, China) and analyzed using the Folin–Ciocalteu method, with results expressed as milligrams of gallic acid equivalent per gram of encapsulated polyphenol extract (mg GAE/g of EPE) [18]. Similarly, to determine the Surface Polyphenol Content (SPC), 0.1 g of microcapsules was treated with 1 mL of an ethanol–methanol (1:1) mixture, vortexed at room temperature for 1 min, and filtered. The polyphenol content in the supernatant filtered (0.45 µm Millipore filter, Labfil, Hangzhou, China) was also quantified using the Folin–Ciocalteu method [18,30]. All assays were performed in duplicate.

2.5. Encapsulation Evaluation

2.5.1. Encapsulation Yield

The encapsulation yield was determined using the following equation [30]:
E Y % = W C W E A + W L P E 100
where WC is the weight of the microcapsules, WEA is the weight of the encapsulant agent, and WLPE is the weight of the LPE.

2.5.2. Encapsulation Efficiency

It was calculated using Equation (2), based on the assumption that an efficient encapsulation method requires high retention of the active compound in the core and minimal presence on the surface of the powder particles [31]:
E E % = T P C S P C T P C 100
where TPC is the polyphenol content within the microcapsules and SPC is the polyphenol content of the surface of the microcapsules.

2.5.3. Loading Efficiency

The LE represents the percentage of polyphenols retained in the core, accounting for losses during the process. It was determined using the following equation:
L E % = T P C f W C T P C i W L P E 100
where WC is the weight of the obtained microcapsules, WLPE is the weight of the LPE to be encapsulated, TPCf is the final polyphenol content within the microcapsules, and TPCi is the initial TCP of the LPE.

2.6. Scanning Electron Microscopy (SEM)

Particle morphology was determined using SEM (JSM-6490 LV, JEOL, Tokyo, Japan) with a thermionic emission source and EDS microanalysis system (INCA PentaFETx3, Oxford Instruments, Abingdon, UK). Detectors included SEI, BES, and LVD. Images were taken at 1000× and 4000× magnifications for SD samples, and at 100× and 500× for CC samples, following the internal protocol of the Advanced Microscopy Center at the University of Antioquia.

2.7. Bioaccessibility of Microcapsules Through In Vitro Digestion

In vitro digestion tests were conducted to assess the bioaccessibility and release of the active compound in the oral, gastric, and intestinal phases. The evaluation was carried out for the non-encapsulated LPE and for the microcapsules obtained by CC. In the case of SD, it was only the encapsulated samples prepared at the ratios that achieved the highest loading efficiencies (GA 1:3, SA 1:3, and C 1:1). All assays were performed in duplicate.
The in vitro gastrointestinal digestion assays were developed following the procedure described by Minekus et al. [32] (consult Supplementary Materials). At the end of each digestion stage, the TPC was determined using the Folin–Ciocalteu method described above, and the bioaccessibility of polyphenols was determined using the following equation:
%   B i o a c c e s i b i l i t y = T P C f d   T P C i d × 100
where TPCid corresponds to the total amount of polyphenols in the sample before digestion (mg GAE/g of EPE) and TPCfd is the total amount of polyphenols in the sample at the end of digestion in mg GAE/g of EPE.

2.8. Statistical Analysis

For each result, an analysis of variance (ANOVA) was performed using the software Statistica (Version number 14.0.0.15) to identify significant differences among the evaluated variables. Additionally, a post hoc test (Tukey HSD) was conducted to determine specific differences between the means (Refer to Supplementary Materials).

3. Results and Discussion

3.1. Extraction and Characterization of Polyphenols

As shown in Table 1, approximately 244 g of lyophilised polyphenolic extract was obtained from the extraction process, with an average mass yield of 17% ± 1%. The preliminary morphological and sensory properties were visually evaluated by an untrained panel. Under the drying conditions used, a brown powder with a sweet smell was obtained. However, it was highly hygroscopic (Figure 1), meaning it readily absorbed moisture, which made it difficult to handle.

3.2. Total Polyphenol Content, Antioxidant Capacity, and Identification of Catechins and Epicatechins

The LPE obtained was used to quantify the TPC, as well as to measure the antioxidant capacity and the quantification of catechin and epicatechin, 11 times, and the averages shown in Table 2 were obtained. The detailed results of the characterization can be found in the Supplementary Materials.
The results indicate that the obtained extracts have a high average TPC (90.48 ± 12.87 mgGAE/g of LPE); this deviation corresponds to a coefficient of variation of 14.23%, which may be attributed to the use of CPH sourced from two different municipalities and harvest seasons. Our results exceed values reported by other authors; indeed, Sotelo C et al. [8] reported 16.4–23.0 mgGAE/g for CPH of the TSH-565 variety from Córdoba, while Quiceno et al. [33] found 26.64 ± 3.85 mgGAE/g for CCN-51 husks from Cundinamarca. Carreño Toledo et al. [11] obtained 60.01 ± 1.80 mgGAE/g for lyophilised polyphenols from CCN-51 from San Vicente de Chucurí (Santander, Colombia), using a solid/solvent ratio of 1/60, 50% ethanol/water, and an extraction temperature of 60 °C. Similarly, the analyzed extracts exhibit a high ORAC antioxidant capacity, surpassing values reported by Sotelo et al. [8] (251.5–342.92 µmolTE/g) and Carreño Toledo et al. [11] (1528.2 µmolTE/g for dry extracts from CCN-51 husks). It is important to note that both the antioxidant capacity and the polyphenolic content are directly related to the TPC present in the extracts [7,11], which varies due to environmental factors such as husk origin, cocoa variety, maturity, and climate [34]. It also varies depending on the extraction method used, solute/solvent ratio, temperature, and extraction time [10]. For instance, in comparison with the findings of Carreño Toledo et al., it is noteworthy that changes in the solute/solvent ratio and ethanol concentration resulted in a 33.85% increase in TPC and a 31.29% enhancement in antioxidant capacity.
HPLC UV-Vis analysis revealed low concentrations of catechin and epicatechin in the extracts compared to those reported by Quiceno et al. [33] for CCN-51 husks (2.89 and 6.07 mg/g, respectively). Catechin levels were also lower than those reported by Carreño Toledo et al. [11] (2.3 mg/g), while epicatechin levels were higher, as it was not detected in their study. Some authors have explained this difference in concentration profiles by considering an epimerization process between catechin and epicatechin, where the chemical structure of catechin slightly changes to become epicatechin, and vice versa. Since these compounds are epimers, their relative concentrations can change depending on environmental conditions such as pH, temperature, and other chemical and physical factors [35,36]. A high epicatechin concentration is desirable for products seeking to leverage polyphenols’ antioxidant potential, as its structural differences grant it a greater capacity to neutralize free radicals than its stereoisomer, catechin [29,37,38]. Additional polyphenolic compound profiles were identified using (UHPLC-ESI + -Orbitrap-HRMS), as detailed in the Supplementary Materials. The analysis revealed the presence of phenolic acids, such as vanillic acid, as well as methylxanthines like caffeine and theobromine, which are characteristic components of cocoa husk, as reported by Sotelo C et al. [8].
Compared to values reported by other authors, the extract’s high TPC suggests greater potential for food applications, as lower amounts would be needed in formulations to achieve the same functional effect as extracts with lower TPC.

3.3. Encapsulation by Complex Coacervation (CC)

LPEs were encapsulated using GA-G and SA-G as wall materials. As shown in Figure 2, both formulations produced a loose beige powder, resulting from the blend of white encapsulation materials with brown CPH polyphenols. The powders were easily manageable, non-adherent, and when visually comparing the lyophilised non-encapsulated extracts with those encapsulated by CC, it is observed that the former undergo hydration (swelling) upon contact with the environment, whereas the latter retain the characteristics of a dry powder.
Table 3 presents the total polyphenol content, yield, EE, and LE for each encapsulant; the detailed results can be consulted in Supplementary Materials. Both encapsulants achieved similar yields with no significant differences (p > 0.05), though GA-G resulted in a slightly higher yield. The GA-G microcapsules outperformed those reported by Manzanarez et al. [19], who encapsulated fig (Ficus carica) polyphenol extract using CC with GA-G, obtaining yields of 63.83 ± 0.1%–67.12 ± 0.1%.
Regarding SA-G microcapsules, the reported yield was higher than that found by Shinde U et al. [25] (30.75 ± 2.4% to 38.20 ± 1.48%), who encapsulated eugenol using CC and SA-G as wall material. Similarly, the yields obtained here were also superior to those found by Sukri et al. [39] (41.80% and 44.32%), who encapsulated polyphenols present in propolis using CC and SA-G.
LE is a key parameter in the encapsulation of polyphenols for food formulations, as it reflects the amount of polyphenols effectively incorporated per unit mass of microcapsules. Higher LE values indicate a greater concentration of bioactive compounds within the encapsulated product, enhancing the functional potential of the final formulation. No significant differences were observed between encapsulants (p > 0.05); however, SA-G exhibited higher LE, encapsulating more polyphenols than GA-G. SA-G microcapsules contained approximately 0.710 mg GAE more polyphenols per gram than GA-G, yet TCP did not differ significantly (p > 0.05). The greater retention of polyphenols in the SA–G system is attributed to the pH. At pH 3.5, type B gelatine carries a stronger positive charge due to increased protonation of its amino groups [40], which enhances its electrostatic affinity with the highly anionic sodium alginate [41]. In contrast, at pH 4.0 (in the GA–G system), the gelatine has a weaker positive charge, resulting in less intense interactions [40]. This suggests that a lower pH favours stronger polymer–polymer binding, leading to the formation of a denser coacervate matrix and, consequently, improved polyphenol retention. Neither encapsulant exceeded 50% LE, but both outperformed previous studies. Muchiutti et al. [42] encapsulated oregano essential oil polyphenols via ionic gelation, achieving efficiencies of 6–25% and a low TPC of 0.326 ± 0.03 to 0.403 ± 0.01 mgGAE/mL in water. Sukri et al. [39] reported polyphenol concentrations of 0.43–0.80 mgGAE/g in propolis encapsulated using CC and SA-G, while Manzanarez et al. [19] obtained 3.12 ± 0.11 to 5.54 ± 0.08 mgGAE/g in fig (Ficus carica) polyphenol extract microcapsules using GA-G by CC. These differences may be attributed to the high epicatechin content in CPH extracts, where multiple hydroxyl groups enhance hydrogen bonding and electrostatic interactions with coacervation polymers, resulting in more efficient and stable encapsulation than with fig, eugenol, or propolis extracts [10].
These results confirm that GA and SA, combined with G, effectively encapsulate polyphenols from CPH via CC. This method successfully retained nearly one-third of the initial polyphenols, achieving high yields and encapsulation efficiencies, with a TPC of approximately 13 mgGAE/g of EPE.

3.4. Encapsulation by Spray Drying (SD)

SD produced microcapsules for each encapsulant at three selected concentrations. Figure 3 presents the microcapsules obtained at the concentrations yielding the highest loading efficiencies. All three encapsulants generated a finer, looser beige powder compared to CC. Similarly, the resulting powder was easily manageable, non-adherent, and resistant to rapid moisture absorption, facilitating its potential inclusion in food and pharmaceutical formulations.
As shown in Table 4, the yield was lower than that obtained via CC, as expected due to losses on the equipment walls during SD. The highest yield (62.2 ± 1.8%–63.7 ± 1.6%) was achieved using GA at all tested ratios, followed by C (54.2 ± 5.6%–45.8 ± 3.3%) and SA (50.4 ± 9.4%–45.3 ± 1.9%). These results align with Ralaivao et al. [43], who reported yields of 66.3% with GA, 49.2% with SA, and 37.7% with modified C in the SD encapsulation of epigallocatechin gallate (EGCG). Across all encapsulants, a higher proportion of encapsulant resulted in a higher encapsulation yield, though differences at each ratio were not statistically significant (p > 0.05).
EE was high for all three encapsulants across the tested ratios, with no significant differences (p > 0.05). The highest EE for GA (79.22 ± 1.6%) and SA (86.81 ± 0.1%) was achieved at a 1:3 ratio, while C at a 1:1 ratio yielded the best efficiency (89.38 ± 0.8%), indicating lower polyphenol retention on the microcapsule surface. These values were lower than those reported by Ralaivao et al. [43], who obtained 99.3 ± 1% efficiency using modified C at the same ratio. The differences may arise from the complexity of encapsulating polyphenol mixtures, as opposed to a single molecule like EGCG. These mixtures can hinder the formation of uniform microcapsules, as the compounds may compete for binding sites, exhibit different solubilities, and have varying chemical stabilities. These factors lead to uneven encapsulation and reduce overall efficiency, as not all polyphenols interact equally with the encapsulating matrix [44,45]. Additionally, these results exceeded those of Siles et al. [20], who reported encapsulation efficiencies of 75.8 ± 5.3%, 73.2 ± 6.1%, and 54.8 ± 0.9% for marjoram polyphenols encapsulated with C at 1:4, 1:6, and 1:8 (E:EA) ratios.
Significant differences (p < 0.05) in LE were observed among the encapsulating agents and the ratios evaluated. Moreover, a significant interaction was found between the ratio and the type of encapsulant, indicating that the optimal LPE:EA ratio for achieving maximum LE depends on the encapsulating agent used. GA yielded the highest LE, while C had the lowest. For GA, the highest efficiency was achieved at the lowest encapsulant ratio (1:3), with a value of 34.77 ± 1.2%, which was similar to that obtained with CC (32.59 ± 0.7%), and a mean TPC of 12.65 ± 0.05 mg GAE/g EPE. This value is lower than that reported by Jafari et al. [46], who obtained a value close to 15 mg GAE/g EPE in the encapsulation of polyphenols from CPH using the same technique and a GA ratio of 1:2. This result was expected, as our findings show that lower ratios lead to higher TPC values within the microcapsules. SA showed its highest LE at the highest encapsulant ratio (25.15 ± 1.9%), which was lower than that obtained via CC (36.95 ± 7.6%), indicating superior retention with the latter technique. Although C exhibited the best EE, its LE was the lowest, ranging from 9.5 ± 1.85% to 4.14 ± 1.14%.
The higher polyphenol retention observed with GA and SA (1:3 ratio) can be attributed to the presence of hydroxyl and carboxyl functional groups that promote favourable hydrogen bond interactions with phenolic groups [47]. However, GA achieves superior retention because it is a complex matrix of polysaccharides, glycoproteins, and other compounds with a highly branched and polar structure. This allows for greater retention of both hydrophilic and hydrophobic polyphenolic compounds [26,47]. In comparison, SA lacks a protein fraction, which can result in an encapsulation network that is less dense for efficiently retaining hydrophilic polyphenols [42]. Meanwhile, C (1:1 ratio) demonstrated the lowest efficiency. This is possibly due to its poor compatibility with polyphenols under the acidic conditions in which the encapsulating solution was prepared. Under these conditions, polyphenols are typically in neutral forms or lack significant negative charge [48], while chitosan exhibits a positive charge on its amino groups, leading to weak attractive electrostatic interactions with the extract’s functional groups [20].
Considering the obtained results, both GA and SA demonstrated efficacy as wall materials for the encapsulation of polyphenols through SD, achieving high yields and encapsulation efficiencies, obtaining microcapsules with concentrations exceeding 12 mg GAE/gEPE using a 1:3 ratio (E:EA).

3.5. Scanning Electron Microscopy (SEM)

SEM images were taken at different magnifications due to the considerable variation in sample size. CC samples were visualized at lower magnifications (100×–500×), whereas the smaller SD samples required higher magnifications (1000×–4000×) to be clearly observed (Figure 4).
Micrographs revealed marked differences in capsule morphology between techniques. SD generated spherical or semi-spherical particles, some with surface-adhered structures likely representing non-encapsulated polyphenols. GA-based capsules were irregular and polydisperse (~5–10 µm), with smoother surfaces on larger particles, whereas SA produced more uniform and spherical particles (~10 µm) with fewer surface residues, consistent with SPC data (View Table 4). C-based capsules showed heterogeneous morphologies, with smaller particles (~5 µm), agglomerates, and rougher, more porous surfaces. These findings align with those reported by Ralaivao et al. [22] for EGCG encapsulation using SD with GA, SA, and modified C.
CC produced larger capsules (>100 µm) with distinct network morphologies. GA–G capsules exhibited laminated, irregular matrices with multiple cavities, whereas SA–G capsules displayed more homogeneous and porous structures, with a less-dense polymeric network and evenly distributed pores. No unencapsulated polyphenols were detected on the surface. Despite offering greater structural complexity, typical of the drying method used after CC (lyophilisation) [6], these structures may represent a disadvantage, as their larger exposed surface area provides lower protection during storage and release compared with spray-dried products [23].

3.6. Bioaccessibility of the Microcapsules Through In Vitro Digestion

For polyphenols to be bioaccessible, they must be released from the encapsulant during digestion, mainly in the intestine; therefore, it is of the utmost importance to evaluate the behaviour of the microcapsules in simulated digestive processes [49]. As shown in Table 5, it was not possible to collect samples in all simulated digestive phases. This limitation was primarily due to saturation of the digestive media, caused either by the volume occupied by the microcapsules (as in the GA and C systems) or by their swelling upon contact with the solution (as in the SA system); the detailed results can be found in Supplementary Materials. Although digestion was made, including all the phases (oral, gastric, and intestinal), the absence of data from these early stages limits a full understanding of the release mechanism of polyphenols, particularly since the oral phase represents the first contact of the microcapsules with the digestive environment and may influence the subsequent disintegration and release profile, while the gastric phase plays a key role in structural breakdown and may influence subsequent intestinal availability [50,51,52]. Consequently, the conclusions mainly focus on intestinal bioaccessibility, without providing a precise description of the overall dynamics of release and transformation across all digestive phases [53].
Table 5 illustrates how encapsulation improves the bioaccessibility of polyphenols, which, when unencapsulated, had a bioaccessibility of only 6.4 ± 1.6% in the intestinal phase. Additionally, there is a decreasing trend in polyphenol bioaccessibility, being higher in the buccal phase and lower in the intestinal phase, with a degradation or denaturation of polyphenols of over 94%. This result is similar to that obtained by Altin et al. [54], who found that by the end of the digestive process, 97% of the polyphenols from the unencapsulated freeze-dried extract of CPH had degraded. This behaviour is expected, as free polyphenols degrade more easily than when present in a food matrix or encapsulated, either due to the environmental conditions of each of the digestive stages, such as pH and temperature, or due to their interaction with simulated solutions, leading to denaturation. Cases have been reported, such as green tea polyphenols, in which a large part of these are prone to oxidation, disappearing in the intestinal phase [55]. This is why polyphenol encapsulation is important, as it is a tool that provides protection and can provide control over the release of the active compound in a matrix or a specific site, such as the gastrointestinal tract, depending on the EA and technique used for encapsulation, as well as its subsequent inclusion.
This is corroborated in this study, where a significant effect on the bioaccessibility of polyphenols loaded in microcapsules obtained through different techniques and encapsulant types was observed (p < 0.05). Microcapsules produced via SD exhibited a progressive release throughout the digestive phases and greater polyphenol bioaccessibility in the intestinal phase compared to those obtained using the CC technique. This may be attributed to the fact that, in CC, a more intricate process occurs, wherein an electrostatic barrier forms around the active compound due to the interaction of ions from two biopolymers with opposite charges, potentially delaying its release. This characteristic could be particularly advantageous in applications requiring controlled and sustained release of the active compound [19].
The highest polyphenol release was achieved with GA microcapsules produced via SD, reaching a polyphenol release of 76.55 ± 5.1%. This was superior to the findings of Sassi et al. [56], who reported a bioaccessibility of 69.59 ± 7.9% in the intestinal phase for polyphenols extracted from date seeds encapsulated with GA using SD, exhibiting a similar pattern where the highest release occurred in the intestinal phase. This value also exceeded that obtained using CC, which yielded a bioaccessibility of 41.76 ± 4.6% with gum GA-G, a result within the range of 10.36% to 46.87% reported by Peanparkdee et al. [57] in in vitro digestion of encapsulated rice-bran extracts using CC with the same encapsulants. The second-highest bioaccessibility was observed in microcapsules produced with C via SD, achieving a bioaccessibility of 64.39 ± 2.5% in the intestinal phase, comparable to the 66.7 ± 2.2% reported by Siles et al. [20] in the in vitro digestion of marjoram polyphenol microcapsules encapsulated using the same material and technique. Microcapsules produced with SA exhibited the lowest polyphenol release across both methodologies, with a bioaccessibility of 58.85 ± 8.5% using SD and 27.45 ± 1.3% with CC, lower than the 61.76 ± 8.0% found by Machado et al. [49] for the encapsulation of polyphenols with alginate via ionic gelation.
Finally, the complete release of the active compound was not achieved in any of the evaluated samples in the intestinal phase; however, the results suggest that GA is the best wall material for encapsulating polyphenols extracted from CPH by SD, as it improves the bioavailability of polyphenols, which could have significant implications for the development of nutraceutical products, protecting the active compound in gastric phases and achieving a bioaccessibility greater than 70% in the intestine during in vitro digestive processes.

4. Conclusions

Both CC and SD techniques proved to be efficient in encapsulating polyphenols extracted from CPH. The best loading efficiencies were achieved using GA at a 1:3 (E:EA) ratio through SD, reaching a value of 34.77%, and SA-G through complex CC, with a LE of 36.95%. However, SD was the most effective method for polyphenol encapsulation, providing higher bioaccessibility compared to CC. In particular, microcapsules made with GA and SD achieved a notable bioaccessibility percentage of 76.55%, standing out as the most efficient option for the release of polyphenolic compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr13103094/s1, Table S1. Final gallic acid concentration data for the calibration curve; Figure S1. Calibration Curves of Gallic acid; Table S2. Concentration data for the preparation of catechin and epicatechin calibration curves; Figure S2. Calibration Curves of (a) Epicatechin, (b) Catechin; Table S3. Composition of Simulated Biological fluids; Table S4. Total Polyphenol Content in the Extracts; Table S5. Antioxidant Capacity of the lyophilised polyphenol extract; Figure S3. Chromatogram for Identification of Functional Groups of Polyphenols, Batch 1 to 6; Figure S4. Chromatogram for Identification of Functional Groups of Polyphenols, Batch 7 to 11; Table S6. Individual quantification of Polyphenols in the Obtained Freeze-Dried Extract; Table S7. Quantification of the Main Polyphenol Groups by ultra-high-performance liquid chromatography coupled with Orbitrap high-resolution mass spectrometry (UHPLC-ESI+-Orbitrap-HRMS); Table S8. Yield, Encapsulation efficiency, and Loading efficiency using Gum Arabic-Gelatine and Sodium Alginate- Gelatine; Table S9. Encapsulation Yield using Spray Drying; Table S10. Yield, Encapsulation efficiency, and Loading efficiency using Spray Drying; Table S11. Total Polyphenols content in the sample before digestion (mgGAE/gEPE); Table S12. Percentage of Bioaccessibility through In Vitro digestion; Table S13. Analysis of variance (ANOVA) on the effect of encapsulant type on the Encapsulation Yield via complex coacervation; Table S14. Post Hoc Tukey HSD test on the effect of encapsulant type on the Encapsulation Yield via complex coacervation; Table S15. Analysis of variance (ANOVA) on the effect of encapsulant type on the Encapsulation Efficiency via complex coacervation; Table S16. Post Hoc Tukey HSD test on the effect of encapsulant type on the Encapsulation Efficiency via complex coacervation; Table S17. Analysis of variance (ANOVA) on the effect of encapsulant type on the Loading Efficiency via complex coacervation; Table S18. Post Hoc Tukey HSD test on the effect of encapsulant type on the Loading Efficiency via complex coacervation; Table S19. Analysis of variance (ANOVA) on the effect of encapsulant type and the encapsulant:active compound ratio on the Encapsulation Yield via spray drying; Table S20. Post Hoc Tukey HSD test on the effect of encapsulant type and the encapsulant:active compound ratio on the Encapsulation Yield via spray drying; Table S21. Analysis of variance (ANOVA) on the effect of encapsulant type and the encapsulant:active compound ratio on the Encapsulation Efficiency via spray drying; Table S22. Post Hoc Tukey HSD test on the effect of encapsulant type and the encapsulant:active compound ratio on the Encapsulation Efficiency via spray drying; Table S23. Analysis of variance (ANOVA) on the effect of encapsulant type and the encapsulant:active compound ratio on the Loading Efficiency via spray drying; Table S24. Post Hoc Tukey HSD test on the effect of encapsulant type and the encapsulant:active compound ratio on the Loading Efficiency via spray drying; Table S25. Analysis of variance (ANOVA) on the effect of encapsulant type and encapsulation technique on the bioaccessibility percentage of polyphenol capsules following in vitro digestion in the gastric phase; Table S26. Post Hoc Tukey HSD test on the effect of encapsulant type and encapsulation technique on the bioaccessibility percentage of polyphenol capsules following in vitro digestion in the gastric phase; Table S27. Analysis of variance (ANOVA) on the effect of encapsulant type and encapsulation technique on the bioaccessibility percentage of polyphenol capsules following in vitro digestion in the intestinal phase; Table S28. Post Hoc Tukey HSD test on the effect of encapsulant type and encapsulation technique on the bioaccessibility percentage of polyphenol capsules following in vitro digestion in the intestinal phase.

Author Contributions

A.N.G.M.: Investigation, data analysis, writing, and preparation of the original draft. L.J.L.-G.: Investigation, writing, supervision, review, and editing. E.S.G. and Y.M.C.: Investigation. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded from the General Royalties System under the project “Increase of competitiveness in the cocoa sector through the transformation of agro-industrial waste for the innovation and development of nutraceuticals and bioproducts that generate added value to cocoa beans in the Amazon Department“ with BPIN code 2021000100226.

Data Availability Statement

All data are incorporated into the article and its online Supplementary Materials.

Acknowledgments

The authors express their gratitude to the Sistema General de Regalías, the Universidad Industrial de Santander, the School of Chemical Engineering, and the CICTA Research Center for their support and resources provided during the development of this research, as well as to the Universidad Tecnológica de Pereira, the Asociación de Cacaoteros y Artesanos Chocolateros de Colombia (ARCHOCOL), the Escuela de Empresas de Quebec (EEQ), and COSMOS Scientific Corporation.

Conflicts of Interest

The authors declare that there are no conflicts of interest. The funding source had no involvement in the design of the study; the collection, analysis, or interpretation of data; the writing of the report; nor in the decision to submit the manuscript for publication. No restrictions were im-posed by the funder regarding the dissemination of the results.

Abbreviations

The following abbreviations are used in this manuscript:
CPHCocoa Pod Husk
CCComplex Coacervation
SDSpray Drying
GAGum Arabic
SASodium Alginate
CChitosan
GGelatine
EYEncapsulation Yield
EEEncapsulation Efficiency
LELoading Efficiency
EAEncapsulating Agent
TPCTotal Polyphenol Content
GAEGallic Acid Equivalents
LPELyophilised Polyphenol Extract
ORACOxygen Radical Absorbance Capacity
SPCSurface Polyphenol Content
ETTrolox Equivalents
EPEEncapsulated Polyphenol Extract
EGCGEpigallocatechin Gallate

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Figure 1. Polyphenol extract, lyophilised and macerated, obtained from 11 successive extractions of cocoa pod husk.
Figure 1. Polyphenol extract, lyophilised and macerated, obtained from 11 successive extractions of cocoa pod husk.
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Figure 2. Microcapsules of cocoa pod husk polyphenols obtained by complex coacervation. (A). Gum Arabic-Gelatine; (B). Sodium Alginate-Gelatine.
Figure 2. Microcapsules of cocoa pod husk polyphenols obtained by complex coacervation. (A). Gum Arabic-Gelatine; (B). Sodium Alginate-Gelatine.
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Figure 3. Microcapsules of cocoa pod husk polyphenols obtained by Spray dryer (A). Gum Arabic 1:3 ratio, (B). Sodium Alginate 1:3 ratio, and (C). Chitosan 1:1 ratio.
Figure 3. Microcapsules of cocoa pod husk polyphenols obtained by Spray dryer (A). Gum Arabic 1:3 ratio, (B). Sodium Alginate 1:3 ratio, and (C). Chitosan 1:1 ratio.
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Figure 4. Scanning Electron Micrographs of polyphenol microcapsules obtained by complex coacervation: (A). gum arabic–gelatin and (B). sodium alginate–gelatin; and by spray-drying: (C). gum arabic, (D). sodium alginate, and (E). chitosan.
Figure 4. Scanning Electron Micrographs of polyphenol microcapsules obtained by complex coacervation: (A). gum arabic–gelatin and (B). sodium alginate–gelatin; and by spray-drying: (C). gum arabic, (D). sodium alginate, and (E). chitosan.
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Table 1. Amount of lyophilised polyphenolic extract obtained after mixing 11 batches of cocoa pod husk extractions.
Table 1. Amount of lyophilised polyphenolic extract obtained after mixing 11 batches of cocoa pod husk extractions.
Extraction ConditionsTreated Pod Husk [g]Lyophilised Polyphenol Extract [g]Yield [g LPE/g]
1/30 S/S, 75% E/W, T 60 °C1440244.83617% ± 1%
Table 2. Total polyphenol content, antioxidant capacity, and catechin and epicatechin content of the lyophilised polyphenolic extract obtained from cocoa pod husk.
Table 2. Total polyphenol content, antioxidant capacity, and catechin and epicatechin content of the lyophilised polyphenolic extract obtained from cocoa pod husk.
AnalysisMean *Coefficient of Variation
Total Polyphenol Content [mgGAE/g LPE] *90.48 ± 12.8714.23%
Antioxidant Capacity (ORAC) [µmolTE/g LPE] *2224.06 ± 491.9122.12%
HPLC UV-Vis Catechin [mg/g LPE]0.56 ± 0.2543.87%
HPLC UV-Vis Epicatechin [mg/g LPE]3.78 ± 1.4337.77%
* Mean ± Standard Deviation, mgGAE/g LPE: Milligrams of Gallic Acid Equivalents per Gram of lyophilised polyphenol extract, µmol TE/g LPE: Micromole of Trolox Equivalents per Gram of lyophilised polyphenol extract.
Table 3. Yield, encapsulation efficiency, and loading efficiency of microcapsules of cocoa pod husk polyphenols using Gum Arabic–Gelatine and Sodium Alginate–Gelatine.
Table 3. Yield, encapsulation efficiency, and loading efficiency of microcapsules of cocoa pod husk polyphenols using Gum Arabic–Gelatine and Sodium Alginate–Gelatine.
TPC *
[mgGAE/g EPE]
SPC * [mgGAE/g EPE]EY (%) 1EE (%) 1LE (%) 1
GA-G13.4720.94075.54 a93.02 a33.28 a
13.0380.87978.00 a93.26 a32.20 a
13.0750.99260.09 a92.41 a32.29 a
13.195 ± 0.240.937 ± 0.0671.210 ± 9.7192.900 ± 0.4432.59 ± 0.66
SA-G10.8103.15667.83 a70.80 a28.68 a
16.4852.61668.60 a84.13 a43.74 a
14.4822.88668.22 a80.07 a38.42 a
13.926 ± 2.882.886 ± 0.2768.220 ± 0.3978.334 ± 6.8336.946 ± 7.63
* Mean ± Standard Deviation, TPC: Total Polyphenol Content [mgGAE/g EPE], SPC: Surface Polyphenol Content [mgGAE/g EPE], EY: Encapsulation Yield, EE: Encapsulation Efficiency, and LE: Loading Efficiency. 1 Groups with the same letter indicate that there are no significant differences between them, according to Tukey’s test (p = 0.05).
Table 4. Yield, encapsulation efficiency, and loading efficiency of microcapsules of cocoa pod husk polyphenols using gum arabic, sodium alginate, and chitosan from a spray dryer.
Table 4. Yield, encapsulation efficiency, and loading efficiency of microcapsules of cocoa pod husk polyphenols using gum arabic, sodium alginate, and chitosan from a spray dryer.
Relation (E:EA) TPC *SPC *EY (%) 1EE (%) 1LE (%) 1
Gum Arabic
R1 1:312.6912.77862.15 ± 1.80 ab79.22 ± 1.56 a34.77 ± 1.15 a
12.6182.483
R2 1:73.3000.70163.63 ± 1.56 a78.59 ± 0.22 a18.99 ± 1.06 d
3.4480.743
R3 1:113.1330.86963.67 ± 1.60 a77.94 ± 8.02 a25.75 ± 1.66 b
2.9630.486
Sodium Alginate
R1 1:116.7722.64145.33 ± 1.87 c81.16 ± 4.37 a17.17 ± 1.22 d
17.4873.834
R2 1:212.8963.02347.88 ± 0.21 bc75.54 ± 1.44 a20.11 ± 0.60 cd
12.4423.170
R3 1:312.3321.61950.40 ± 9.44 abc86.81 ± 0.08 a25.15 ± 1.85 cb
10.4851.389
Chitosan
R1 1:18.5310.85645.75 ± 3.25 c89.38 ± 0.84 a9.50 ± 1.85 e
10.1821.142
R2 1:24.9651.90754.15 ± 5.16 abc73.87 ± 17.37 a9.06 ± 1.06 ef
5.1170.708
R3 1:31.5110.30652.38 ± 3.61 abc81.68 ± 2.70 a4.14 ± 1.14 f
2.0360.334
* TPC: Total Polyphenol Content [mgGAE/g EPE], SPC: Surface Polyphenol Content [mgGAE/g EPE], EY: Encapsulation Yield, EE: Encapsulation Efficiency, and LE: Loading Efficiency. 1 Mean ± Standard Deviation, Groups with the same letter indicate that there are no significant differences between them, according to Tukey’s test (p = 0.05).
Table 5. Percentage of Bioaccessibility through In Vitro digestion of microcapsules of cocoa pod husk polyphenols.
Table 5. Percentage of Bioaccessibility through In Vitro digestion of microcapsules of cocoa pod husk polyphenols.
SamplePhasePolyphenols Released [mgGAE/g EPE] *Bioaccessibility (%) 1
Freeze-dried Extract
(Control)
Buccal9.1819 ± 1.0010.15 ± 1.10
Gastric8.6270 ± 0.389.53 ± 0.42
Intestinal5.8006 ± 1.466.41 ± 1.61
GA-G CC Gastric8.5865 ± 0.1465.07 ± 1.02 a
Intestinal5.5097 ± 0.6141.76 ± 4.63 bc
SA-G CC Gastric2.0867 ± 0.3415.01 ± 2.47 c
Intestinal3.8164 ± 1.4727.45 ± 1.25 c
GA SD Gastric5.9513 ± 0.7147.03 ± 5.60 ab
Intestinal9.6873 ± 0.6576.55 ± 5.10 a
SA SD Intestinal6.7141 ± 2.6258.85 ± 8.54 ab
C SDGastric3.1138 ± 0.8033.28 ± 8.54 cb
Intestinal6.0245 ± 0.2464.39 ± 2.53 a
CC: Complex Coacervation, SD: Spray Drying, GA: Gum Arabic, SA: Sodium Alginate, C: Chitosan, G: Gelatine. * Mean ± Standard Deviation, Polyphenols Released [mgGAE/g EPE]: milligrams gallic acid equivalent per gram encapsulated polyphenol extract. 1 Mean ± Standard Deviation, Groups with the same letter indicate that there are no significant differences between them, according to Tukey’s test (p = 0.05), letters in regular font indicate significant differences in the gastric phase, while letters in italic indicate significant differences in the intestinal phase.
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MDPI and ACS Style

González Morales, A.N.; López-Giraldo, L.J.; Sogamoso González, E.; Moscote Chinchilla, Y. Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents. Processes 2025, 13, 3094. https://doi.org/10.3390/pr13103094

AMA Style

González Morales AN, López-Giraldo LJ, Sogamoso González E, Moscote Chinchilla Y. Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents. Processes. 2025; 13(10):3094. https://doi.org/10.3390/pr13103094

Chicago/Turabian Style

González Morales, Astrid Natalia, Luis Javier López-Giraldo, Erika Sogamoso González, and Yaiza Moscote Chinchilla. 2025. "Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents" Processes 13, no. 10: 3094. https://doi.org/10.3390/pr13103094

APA Style

González Morales, A. N., López-Giraldo, L. J., Sogamoso González, E., & Moscote Chinchilla, Y. (2025). Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents. Processes, 13(10), 3094. https://doi.org/10.3390/pr13103094

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