Advancing microencapsulation strategies for bioactive compounds: Enhancing stability, bioavailability, and controlled release in food applications

2.1.1 Influence of solvent polarity

The varying polarities of bioactive compounds within intricate food matrices may hinder their concurrent extraction. The choice of solvent affected the morphology of the microparticles and microcapsules [37]. Mehta et al. [38] found that drug incorporation, matrix porosity, and solvent residues are some of the microparticle features that are impacted by the polymers’ solubilities in organic solvents, which in turn influence the pace of polymer solidification during microparticle formation (Figure 2). The influence of solvent polarity on the structure of microcapsules has been investigated in several studies. A study reported that the polarity of solvent can significantly impact the crystallinity and physicochemical properties of microcapsules. For instance, reducing solvent polarity may enhance the crystallinity of specific components within the microcapsules, potentially influencing their digestibility and functionality in various applications [39]. Moreover, in the encapsulation of gallic acid using yeast cells revealed a notable influence of the solvent type in the encapsulation media (H₂O or EtOH:H₂O) on both the encapsulation efficiency and bioactive properties of the resulting microcapsules. At higher concentrations, the effectiveness of yeast cell encapsulation was greatly improved by the solvent choice. The gallic acid loading and bioactive performance of distilled water were better than those of the ethanol–water mixture (EtOH:H₂O) [40]. Similarly, in the preparation of bovine hemoglobin-loaded nanoparticles, an aqueous system with surfactants achieved encapsulation efficiencies exceeding 97%, demonstrating the suitability of aqueous media for hydrophilic compounds [41]. A separate investigation examined alginate microcapsules for yeast in sparkling winemaking, utilizing an aqueous system with calcium chloride as the crosslinker, resulting in a high encapsulation efficiency (∼97%) and excellent structural stability, thereby illustrating the efficacy of aqueous alginate systems for hydrophilic or biologically active agents [42]. In contrast, alcohol-based solvents exhibited improvement in solubility and encapsulation of hydrophobic compounds, such as risperidone, and allowed greater control over the particle size and porosity. Due to their greater solubility in alcohol-based solvents, the encapsulation efficiency increased from 62 to 78% or 69 to 80% [43]. A study on propolis-alginate microcapsules indicated low encapsulation efficiency when sodium alginate was dissolved in water; however, altering the solvent solution to incorporate alcohol markedly enhanced encapsulation efficiency to ∼99% [44]. This indicated that alcohol-based solvents may enhance the incorporation of hydrophobic substances such as propolis in alginate microcapsules, possibly owing to greater solubility and interaction with the active chemicals in comparison to only aqueous systems [44]. These findings suggest that solvent polarity plays a significant role in determining the structure and properties of microcapsules. Quantitative comparisons demonstrate that aqueous solvents are generally more advantageous for hydrophilic and biologically active compounds, particularly when stability and gelation through ionic crosslinking are necessary, whereas alcohol-based systems are more efficacious for hydrophobic bioactives due to enhanced solubility and diminished polarity mismatch.

Figure 2

Factors influencing encapsulation efficiency (redrawn from Yeo and Park [20]).

2.1.2 Effect of solvent pH

The pH of the solvent can significantly impact the structure and properties of microcapsules. Besides its influence on the yield of bioactive compounds during extraction, it also impacts the pH in the microencapsulation system. Guo et al. [45] reported how pH levels impact the microstructure and characteristics of self-assembled graphene microcapsules and revealed the presence of core material in the shells, with the pH values significantly affecting the deposition of the core material onto the shells, which leads to changes in the morphology, size, and shell thickness. The surface morphologies and core–shell structures of microcapsules are affected by the pH of the reaction medium. A different study indicated that the characteristics and degree of interactions among microcapsule components, affected by pH, affect the overall stability of the structure [46].

In addition, pH plays a crucial role in determining the charge characteristics of encapsulating polymers such as alginate, chitosan, and proteins, which in turn impact their interactions with bioactive compounds. The changes in pH influence the ionization states of functional groups, such as carboxyl or amino groups, which in turn affect electrostatic interactions and the formation of complexes. For example, alginate shows larger pores and diminished mechanical strength in acidic environments (such as pH 2.5), which may enhance the release rate of encapsulated bioactives. At a neutral pH of approximately 6.0, alginate microspheres exhibit enhanced structural stability and decreased porosity, leading to improved retention of bioactive compounds [47].

Chitosan, as a polycation, possesses a positive charge under acidic conditions, which promotes electrostatic interactions with negatively charged polymers such as alginate, leading to the creation of polyelectrolyte complexes [48]. The encapsulation efficiency of chitosan–alginate systems reaches its peak when chitosan is introduced initially at lower pH levels [49]. In a similar way, wall materials derived from proteins exhibit responses to changes in pH relative to their isoelectric point, influencing their solubility, surface activity, and capacity to interact with active compounds.

Additionally, pH-responsive synthetic polymers, like poly(acrylic acid), demonstrate charge-switching behavior that can be adjusted for controlled encapsulation and release. For instance, carboxyl-containing polymers that exhibit weak ionization may shift from extended to globular conformations in response to pH changes, thereby improving their interaction with membranes and encapsulation capabilities [50–52]. Although research regarding the influence of the pH solvent on the microcapsule structures and properties is still limited, existing studies clearly demonstrate that the solvent pH influences polymer ionization, molecular interactions, and ultimately the structural integrity and release behavior of encapsulated bioactives (Figure 3).

Figure 3

General factors that affect microencapsulation efficiency.

2.2.1 Encapsulation through fluidized-bed spray coating

Fluidization and spray techniques can be combined to agglomerate particles into larger granules or apply coatings to agglomerate particles or apply coatings, a process often referred to as fluid bed coating or granulation (Figure 4). This includes spray drying, cooling, and chilling, where the process air both fluidizes the particles and promotes solidification, either by congealing molten materials (e.g., waxes, hydrogenated oils) or evaporating solvents. In fluidized-bed spray coating, core particles are suspended and sprayed with an encapsulating solution or molten material. The encapsulating material may adhere to the core surface through various mechanisms, which vary based on the material used and specific encapsulation techniques, including physical adsorption such as electrostatic forces and van der Waals forces in cases where the surface is rough or irregular, chemical bonding such as covalent bonds and hydrogen bonding, coating techniques such as coacervation and in situ polymerization, mechanical interlocking, and capillary forces [11,13,53]. Since it is a coating process, the resulting encapsulation typically showcases a core–shell or mononuclear microstructure [54]. In the process of encapsulation using fluidized-bed spray coating, solidification takes place through a complex combination of fluidization, droplet spraying, adhesion, and the swift drying facilitated by circulating hot air. The interplay of these processes results in the formation of uniform microcapsules that exhibit controlled release characteristics, while effectively reducing the risks of agglomeration [55,56].

Figure 4

Illustration of the fluidized-bed spray coating process.

The physical properties of microcapsules produced via fluidized-bed spray coating are strongly influenced by process parameters and have a direct impact on the coating performance and final product stability. Particle sizes typically range from 0.1 mm to several millimeters, with specific applications reporting size distributions between 20 and 120 μm, and about 80% of the particles below 100 μm [57,58]. Sphericity is critical for uniform coating – systems like polyamide powders used in fluidized-bed sintering exhibit at least 75% spherical particles, enhancing coating adhesion and flow behavior [59,60]. Density and porosity also vary depending on particle size and coating materials. For example, smaller particles (50–75 μm) generally result in lower porosity, but may compromise the mechanical strength and hardness [61]. These characteristics must be carefully optimized to ensure functional encapsulation tailored to specific applications (Figure 3).

An encapsulant solution is characterized by two critical properties: surface tension and viscosity. The efficiency of encapsulation can be considerably impacted if the viscosity is either too low or too high, resulting in imperfect microstructures such as pores, cracks, or uneven thickness[62]. The atomization of coating solutions may be limited by high viscosity. The production of uniformly coated particles in fluidized-bed systems requires effective atomization of fine droplets. An excessively viscous solution may lead to inadequate dispersion and uneven coating on the substrate, preventing it from forming small droplets [63]. To attain more refined structures, uniform wall thickness, and improved encapsulation efficiency, various studies indicate the utilization of encapsulants characterized by reduced viscosity. In general, encapsulation efficiency for fluidized-bed spray coating ranges from 75 to 95%, depending on the coating thickness and substrate type. When applied to sensitive materials such as biocontrol bacteria (e.g., Collimonas arenae), this method achieved about 6 log[CFU/g coated solids], which is slightly lower than spray drying under similar conditions (7 log[CFU/g solids]) [64]. Compared to other encapsulation methods, spray drying often yields higher encapsulation efficiency for hydrophilic ingredients, typically in the 85–99% range. Extrusion and emulsion techniques using alginate systems show efficiencies of 80–97%, especially for probiotics like Lactiplantibacillus plantarum [65].

To achieve the desired microstructure and increased encapsulation efficiency in spray coating, careful selection of the encapsulant solution's concentration, surface tension, and viscosity, as well as the temperature during the drying process, is essential. Lowering the inlet drying air temperatures below 75°C has been observed to yield high encapsulation efficiencies, reaching up to 99%. It is recommended to use a material with lower viscosity for improved results [63]. The most frequently utilized coating agents for achieving the desired microstructure of capsules include polymer-based components, specifically acrylic polymers, cellulose esters, and copolymers. Enhanced solubility, decreased viscosity and permeability, along with the ability to generate more compact films, represent some of the recognized advantages of these coating agents [66].

In terms of release profiles, fluidized-bed spray coatings offer controlled release times ranging from less than 1 h to more than 22 h, depending on the coating thickness and material properties [67]. Thicker and uniform coatings favor slower, sustained release, whereas thinner or porous coatings may lead to faster “burst” release. In contrast, spray-dried capsules typically have thinner walls and higher porosity, resulting in faster release, often within minutes to a few hours [64]. Extrusion-based alginate beads also allow for prolonged release depending on the crosslinking density and bead size, with smaller beads releasing faster due to higher surface area, and multilayered beads mimicking the slow-release behavior of fluidized-bed capsules [64,68].

It is important to note that adjusting the flow rate of the coating solution, along with factors such as glass transition temperature, atomization pressure, core/encapsulant ratio, and fluidizing air velocity, is crucial for optimal results.

2.2.2 Emulsion technique encapsulation

An emulsion is a mixture of at least two immiscible liquids – liquids that do not dissolve into each other. One of these liquids makes up the dispersed phase, which is distributed as tiny droplets within the other liquid, referred to as the continuous phase (Figure 5). The continuous phase may consist of an organic solvent, oil, a meltable solid, or any substance that is soluble in a solvent. The polymer functioning as the encapsulant is typically dissolved in the solution or within the continuous phase of a suspension or emulsion, such as sorbitans, polysorbates, sucrose esters, sodium caseinate, sodium carboxymethyl cellulose, gelatine, or natural surfactants such as soybean lecithin and saponins [69–71]. After being homogenized, the liquid core forms small droplets in the continuous phase [72].

Figure 5

Illustration of the emulsion technique encapsulation.

Commonly, oil and water are used as the two immiscible liquids to create an emulsion. Emulsions are categorized into two primary forms: single emulsions and double emulsions. Single emulsions, particularly oil-in-water (O/W) emulsions, are widely used for oil-soluble core materials [73]. In practical food applications, O/W emulsions are employed to encapsulate flavor oils, enhancing sensory properties and masking undesirable tastes [74]. They are also used to deliver nutraceuticals – such as essential vitamins and minerals – to improve the health value of food products [75], and can contribute to novel textures through gel formation, improving the overall eating experience [76].

Particles produced through emulsion encapsulation techniques exhibit a wide range of sizes and morphologies, depending on the formulation and processing parameters. Colloidosomes, for example, vary from 5 nm to several microns and often show broad size distributions due to the use of polydisperse emulsions [77]. Chitosan-based microcapsules typically display uniform spherical shapes with micron-sized, monomodal distributions, as confirmed by laser diffraction [78]. Polymeric nanoparticles produced for pharmaceutical use are generally smaller than 500 nm, offering advantages for controlled drug release [79]. The mechanical strength, permeability, and encapsulation efficiency of these particles depend heavily on the emulsion type, stabilizers, and polymer matrix, with challenges such as size uniformity and structural integrity during processing remaining critical for optimized performance.

Double emulsions (W1/O/W2), on the other hand, may be employed for specific purposes, referring to emulsions in which oil globules (O) containing small aqueous droplets (W1) are dispersed in a continuous aqueous phase (W2) [70]. In the process of incorporation, cores that are either oil-soluble or water-soluble may first be dissolved in their respective mediums – oil or water – prior to the introduction of an encapsulant or emulsifier, followed by the homogenization of the resultant mixture. Homogenization is essential in the production of emulsions, as it adeptly minimizes droplet size, amplifies surface area, boosts stability, and ensures an even distribution of core materials throughout the solution [80]. In the process of emulsion production, small droplets are effectively coated by emulsifiers, utilizing a blend of electrostatic forces, hydrogen bonding, steric stabilization, and the intricate dance of hydrophobic and hydrophilic interactions. The interplay of these mechanisms promotes a stable emulsion, effectively inhibiting droplet coalescence and ensuring a consistent dispersion throughout the continuous phase [81].

Various types of emulsions, including conventional, layer-by-layer or multilayer, microemulsions, nanoemulsions, emulsion gel, and pickering emulsion, have been developed to enhance their performance [69,82–84]. The process of creating multilayer emulsions typically commences with the creation of a main emulsion with a charged emulsifier. This comprises the homogenization of an oil and aqueous phase, wherein the ionized hydrophilic emulsifier rapidly adsorbs to the surface of the droplets generated during homogenization, yielding small charged droplets [85]. The addition of a biopolymer, electrolyte, or another oppositely charged polymer to the mixture leads to adsorption onto the surfaces of the droplets, which facilitates the formation of a secondary emulsion. This technique can be employed repeatedly to construct multiple layers. The pH of an emulsion requires meticulous regulation, as the charged state of the biopolymer may fluctuate with changes in pH [86].

The zeta potential (ζ-potential) is a critical factor in predicting emulsion stability, as it reflects the net surface charge of dispersed droplets. Stable emulsions are characterized by zeta potential values exceeding 30 mV, irrespective of whether the charge is positive or negative. The stability of these emulsions is primarily maintained through electrostatic repulsions within the colloidal system [87]. While high ζ-potential generally promotes stability, excessively high values can lead to emulsion instability due to over-repulsion, causing droplet breakup or flocculation [88–90]. On the other hand, flocculation can occur when the δ-potential is close to zero since there is not enough repulsive force within the system. Therefore, maintaining a balanced zeta potential is key to ensuring long-term emulsion stability, especially in functional food formulations.

2.2.3 Emulsion solidification process

Various atomization techniques, including spray drying, spray cooling, spray chilling, and spray-freeze drying, are frequently employed to solidify emulsions. The most common technique of this process is spray drying, which employs hot air to facilitate the evaporation of water from atomized droplets [91]. Spray cooling and chilling, on the other hand, use temperatures below the encapsulant material's melting point to solidify atomized droplets. Spray–freeze drying is a method that utilizes the properties of spray drying, which entails the atomization of a liquid to produce smaller particles, alongside freeze-drying, which is particularly beneficial for drying thermally sensitive materials, to generate dry powders with controlled size and improved stability [92].

The physical characteristics of particles produced by solidification of emulsion encapsulation techniques – such as spray drying, spray cooling, spray chilling, and freeze drying – are highly dependent on the processing method and encapsulating materials (Figure 6). Spray-dried powders typically have particle sizes ranging from 20 to 120 μm, with approximately 80% of particles measuring less than 100 μm in diameter [57]. Spray-freeze drying tends to yield spherical particles, although some aggregation may occur depending on the properties of the encapsulation agents [93]. The morphology of spray-dried particles generally falls into three categories: crystalline structures, skin-forming shells, and loose agglomerates [94]. In particular, spray-dried flavor encapsulates are known for their low surface area to volume ratio, resulting in high bulk density and good flowability [95]. Bulk density is significantly influenced by the composition of the encapsulant; for instance, milk fat with higher lipid content tends to produce powders with lower bulk density [57]. Flowability, as indicated by the angle of repose, ranges between 37 and 46° and is closely linked to the particle size and morphology. While spray drying often provides powders with desirable flow and encapsulation characteristics, freeze-drying techniques can be more effective in preserving the solubility and stability of thermally sensitive bioactive compounds, highlighting the need to carefully balance physical attributes with functional requirements [96].

Figure 6

Illustration of the emulsion technique encapsulation. (a) Spray drying illustration and (b) freeze drying illustration.

The microstructure of matrix encapsulates can exhibit considerable variation in characteristics such as surface morphology, which refers to the external structure; it may be rough, porous, hollow, fractured, or shrinking [16]. The shape may be spherical, but it can also exhibit heterogeneity in size and form based on the encapsulants employed [97]. This can be influenced by variables such as the inlet temperature, the encapsulant's properties (such as molecular weight), and the drying medium's characteristics [54]. Encapsulates with a compact microstructure, often referred to as perfect-microstructure encapsulates, are reported to have the highest encapsulation efficiency according to several studies. Conversely, encapsulates with microstructures such as holes, dents, or shrinkage may result in reduced encapsulation efficiency. This is because these features have the ability to push the core material beyond the outer surface [98]. Additionally, hollow encapsulates are prone to breakage during handling, resulting in the loss of volatile core materials. Microcapsules with structural defects such as cracks or blow holes exhibit reduced encapsulation efficiency [99]. This is primarily because these defects create pathways that allow environmental factors, such as moisture and oxygen, to penetrate and interact with the core material. Consequently, the protective function of the microcapsule is compromised, leading to a more rapid release of the encapsulated substance and diminished stability. Consequently, these types of microstructures generally lead to lower encapsulation efficiencies.

Encapsulates with a porous structure are typically achieved through processes such as spray–freeze drying or freeze drying [100]. While spray–freeze or freeze-dried encapsulates exhibit elevated encapsulation efficiencies initially, which is related to reduced drying temperatures [101], the pronounced porosity associated with these structures becomes a drawback when considering the abilities to preserve and promote sustained release of a core material. Increased porosity also implies a larger surface area, making them more susceptible to oxidative degradation for oxygen-sensitive core materials.

Incorporating high-molecular-weight encapsulants such as maltodextrin into an emulsion prior to spray drying is advisable to enhance both the efficiency of the process and the quality of the resultant powder. Low-molecular-weight sugars in food powders can induce stickiness during spray drying, which can be reduced by employing high-molecular-weight encapsulants [102]. Employing suitable coating materials, such as carbohydrates and proteins, is crucial for food-grade applications and preserves the core materials from external influences [103]. To prevent the occurrence of cracks, blow holes, and hollow structures, it is essential to meticulously regulate the temperature of the drying process. Moreover, regulating the exit air temperature can indirectly control the moisture levels, hence affecting the final particle output [104]. The inclusion of substances with a high molecular weight can result in smaller droplet sizes in emulsions [105]. Smaller droplets generally yield reduced pore diameters in the final dried products, as they facilitate a more uniform structure during the drying process. Subsequently, it is advisable for the drying process to be conducted at a temperature lower than the glass transition temperature of the resultant emulsion.

2.2.4 Liposome encapsulation technique

Liposomes are colloidal carriers characterized by their spherical structure composed of lipid bilayers that encapsulate an aqueous core (Figure 7). This structural versatility makes them highly effective in food technology applications, particularly for improving the stability, solubility, and bioavailability of sensitive bioactive compounds [106]. The ability of liposomes to encapsulate both hydrophilic and hydrophobic drugs allows for versatile therapeutic applications, including in pharmaceuticals, cosmetics, and food industries [107]. In the food industry, liposomes are used for nutrient enrichment (e.g., vitamins and antioxidants), controlled delivery of flavors and natural colorants, and protection of volatile or oxidation-prone ingredients. By shielding encapsulated materials from environmental stressors such as heat, oxygen, and light, liposomes help preserve sensory quality and extend shelf life [108,109]. Their biocompatibility and ability to target delivery make them a valuable encapsulation method in functional and fortified food products [110].

Figure 7

Illustration of the liposome encapsulation technique.

The primary classifications of liposomes structures are small unilamellar vesicles (SUVs), characterized by a single lipid bilayer and a size typically under 100 nm; large unilamellar vesicles (LUVs), which possess a single bilayer exceeding 100 nm; multilamellar vesicles (MLVs), consisting of several lipid bilayers arranged in an onion-like configuration; and multivesicular vesicles (MVVs), which feature a multilamellar structure with concentric phospholipid spheres formed by multiple unilamellar vesicles within larger liposomes [111]. MLVs generally vary in size from 500 nm to 5 μm. MLVs possess an onion-like architecture, consisting of concentric bilayer membranes [112]. MLVs and MVVs demonstrate enhanced stability compared to unilamellar liposomes [113]. MLVs and UVs, despite being similar in size, exhibit distinct physicochemical characteristics such as permeability, stability, elasticity, and toughness, resulting in varied applications [114]. LUV, possessing an extensive hydrophilic zone, adeptly encapsulates a substantial quantity of hydrophilic substances, whereas MLV successfully entraps hydrophobic materials owing to its lipophilic region. Liposomes can concurrently encapsulate both hydrophilic and hydrophobic core substances.

The internal microstructure of liposomes is determined by their composition and can be classified according to their size and the quantity of bilayers present [111], such as phospholipids, cholesterol molecules, position, and vesicle types. Phospholipids act as the primary structural element of liposomes, characterized by a hydrophilic head and a hydrophobic tail, which facilitates the development of an amphiphilic architecture. Cholesterol plays a key role in liposome formation by positioning its hydroxyl group toward the aqueous core and its hydrophobic part in the bilayer's hydrocarbon region. It enhances bilayer rigidity, strengthening its mechanical stability [115].

Liposomes' internal microstructure is predominantly determined by their distinctive composition and organization. The internal microstructures are unique and do not include mononuclear, polynuclear, or matrix components. Liposomes typically demonstrate a significant capacity for encapsulating hydrophobic core materials effectively. The encapsulation efficiency of liposomes for hydrophilic substances improves with liposome size and decreases with the number of bilayers [111]. The application of biopolymers like chitosan, pectin, and alginate to coat liposomes has demonstrated enhancements in their stability and encapsulation efficiency. The application of these coatings serves to protect the liposomes against environmental degradation while minimizing the leakage of active components [116].

Nevertheless, previous studies have suggested that liposomes generally demonstrate a lower encapsulation efficiency for hydrophilic core materials than for hydrophobic ones. Many studies have reported that encapsulation efficiencies of hydrophilic materials, e.g., Gonzalez Gomez et al. [117] have reported that the ability of liposomes to integrate hydrophobic drugs into their lipid bilayer offers them an optimal encapsulant for such drugs. Conversely, hydrophilic drugs are typically less effectively retained due to their propensity to diffuse from the liposomal aqueous core. Substances that are challenging to encapsulate in liposomes include hydrophilic antibiotics such as vancomycin, which has an encapsulation effectiveness of 33.4%. One possible explanation is that the twisted structure of liposomes acts as a barrier for certain water-soluble core components with molecular weights below 500 Da [117]. Liposome encapsulation efficiency varies based on core material properties. While larger molecules like peptides and proteins may encapsulate more effectively, high core loading can compromise liposome stability, leading to leakage. Hydrophobic cores with lower molecular weights achieve high encapsulation efficiency, whereas hydrophilic cores below 500 Da exhibit reduced efficiency.

2.2.5 Encapsulation by coacervation and ionic gelation

Two significant techniques for microencapsulation are coacervation and ionic gelation, commonly employed across multiple domains, including pharmaceuticals, food technology, and cosmetics (Figure 8). Both techniques leverage the physicochemical properties of polymers to encapsulate bioactive compounds, enhancing their stability and bioavailability.

Figure 8

Illustration of coacervation (a) and ionic gelation (b) encapsulation.

Coacervation is a microencapsulation technique that involves the separation of a colloidal solution into two liquid phases: one rich in colloid (coacervate) and the other poor in colloid. This process can be categorized into two types: simple and complex coacervation [118]. In simple coacervation, a single polymer, such as sodium alginate, is dissolved in water, and upon the addition of a core material, droplets form. Introducing these droplets into a gel-forming medium (e.g., calcium chloride) leads to the creation of calcium alginate microcapsules [118,119]. This method is particularly effective for encapsulating lipophobic molecules but has limitations due to its sensitivity to pH and electrolyte concentrations. Complex coacervation involves two or more polymers, such as alginate and gelatine, which are solubilized in water at different pH levels [118,119].

Electrostatic interactions between the polymers promote coacervate formation around the core material. This method provides improved stability for sensitive compounds like anthocyanins but requires careful control of pH and charge conditions. When the ζ-potential between biopolymers approaches zero, charge neutralization facilitates coacervation and the formation of compact microcapsules [120,121]. However, a near-neutral ζ-potential alone does not guarantee full interaction or efficient encapsulation – maintaining electrostatic balance and suitable environmental conditions is essential for stable capsule formation [122].

Ionic gelation is another encapsulation technique that has gained attention for its simplicity and effectiveness. This method relies on the ability of ionic polymers to crosslink with counter ions to form hydrogels. The mechanism of ionic gelation entails the establishment of complexes between anionic salts and charged biopolymers [118]. Ionotropic gelation typically occurs when two molecules possessing opposing charges interact with each other. In a chemical reaction, negatively charged divalent or multivalent ions interact with positively charged polymer chains. The creation of microstructured particles featuring interconnected nanofibrillar networks results from the electrostatic response. This can be achieved through one of three techniques: internal, external, or inverse gelation (Table 2) [123].

The external method is the most prevalent method employed in ionotropic gelation. This technique is also referred to as controlled diffusion [124]. The crosslinking solution is administered incrementally, dropwise, in conjunction with the polysaccharide solution. The formation of the bead matrix occurs as crosslinking agents migrate from the external continuous phase of the polymer into its internal structure. The droplets conducting the sol–gel transition are located within the outermost layer of the produced hydrogel bead. The outer layer of the produced hydrogel bead exhibits a rapid sol–gel transition, resulting in immediate gel formation. Rapidly, counter-ions start forming in the subsequent phases, and the process of gel formation initiates immediately. The subsequent steps entail the penetration of counter-ions into the particle, leading to a heterogeneous gelation profile. This profile indicates that the interaction between the ions and polymer functional groups is maximized at the surface and diminishes to zero at the core [125].

Internal and in situ gelling are interchangeable. An alternative to this procedure is polymer particle preparation. An insoluble calcium salt (CaCO₃ or CaSO₄) is incorporated into the polymer solution and extruded into an acidic crosslinking bath. Various conditions enhance the solubility of the calcium salt, facilitating its release and the formation of a gel network with the polymer. This regulates the gelling mechanism and uniformly exposes the polymer to cations, resulting in the formation of a gel network. The primary disadvantage of this strategy is significant, which generates matrices with larger pore sizes and reduced densities compared to external gelling, enhancing permeability but diminishing entrapment efficiency and elevating release rates [125,126].

The third method is reverse gelation, which involves the gradual addition of a medium containing gelling agents to the polymer solution. This approach is commonly employed for the production of polymer-based soft-shell microcapsules containing oil-in-emulsions. This technique employs minimal quantities of biopolymer, leading to the formation of a soft molecular shell. The unique characteristics of the produced microcapsules, such as mechanical qualities and pharmaceutical active compounds release, may differ based on the emulsion type employed (water-in-oil or oil-in-water) [123,125].

The outcomes of inverse gelation, internal gelation, and external gelation consistently yield equivalent levels of encapsulation efficiency. The final product's microstructures can exhibit compact, rough, or cracked characteristics, influenced by the concentrations of biopolymers and anionic salts, the degree of cross-linkage, and the specific drying processes and conditions employed. Sufficient concentrations and effective complexation between biopolymers and anionic salts yield compact complexes. Insufficient concentrations, complex formation, or cross-linking may result in diminished, uneven, or porous structures, hence decreasing encapsulation efficiency [54]. Encapsulation efficiency improves with reduced core loads. Consequently, an optimally optimized ionic gelation technique utilizing a reduced core load, adequate charged biopolymers and anionic salts, along with enhanced cross-linking, can attain very high encapsulation efficiencies.

The physical characteristics of particles formed through coacervation and ionic gelation encapsulation techniques are significantly shaped by the composition and process parameters. In ionic gelation, particles such as chitosan-based nanoparticles generally exhibit sizes in the nanometer range, with their dimensions governed by the concentrations of chitosan and crosslinking agents like tripolyphosphate, as well as by the reaction time [134,135]. The morphology is typically spherical, as confirmed by scanning electron microscopy, and remains stable even with the addition of substances like sodium fluoride, unless used at high concentrations [136]. These nanoparticles also exhibit positive zeta potential values that increase with higher chitosan concentrations, contributing to their colloidal stability, which is essential for pharmaceutical and food delivery systems [135]. In contrast, coacervation produces larger particles, generally below 1,000 μm, with morphologies ranging from spherical to elongated ellipsoids. These particles are typically mononuclear in structure and maintain integrity without aggregation, even when encapsulating heat-sensitive compounds [137]. Structural analyses using gas chromatography–mass spectrometry and infrared spectroscopy confirm their stable architecture. While both methods allow for the encapsulation of sensitive bioactive materials, ionic gelation offers enhanced control over particle stability, whereas coacervation is particularly suited for producing larger, structurally robust particles for applications requiring thermal protection.

3.1.1 Control over release rates

One of the key factors influencing release mechanisms is the composition of the microcapsule shell. Shell materials such as alginate, chitosan, and lipids contribute to the mechanical strength and permeability of the microcapsule, thus dictating the controlled diffusion and release rates of encapsulated substances [141]. The morphology of microcapsules, including regular or irregular shapes, mononuclear, polynuclear, and matrix types, also affects the release properties. Different microstructures resulting from encapsulation methods or systems, such as spray coating, co-extrusion, emulsion-based, and ionic gelation encapsulation, influence the encapsulation efficiency and retention of core materials [145]. The size of the pores in the microcapsule shell determines the release mechanism, with smaller pores leading to slower release rates [54]. Additionally, the thickness and permeability of the microcapsule shell can be adjusted to control the release kinetics of the encapsulated compounds [146]. In one study, it was observed that the size of the pores in the Ca-alginate network has an impact on the release of encapsulated bioactive compounds. The release kinetics of encapsulated compounds within the core of Ca-alginate microcapsules were noticeably influenced by the structure and physicochemical properties of the bioactive substance [141]. Jurić et al. [147] further demonstrated that the surface morphology and structure of alginate microparticles, influenced by factors such as calcium concentration and the presence of Trichoderma viride spores, can impact the release behavior of bioactive agents.

Another study stated that spray-coated encapsulates are recognized as a slow-release system, primarily attributed to the presence of additional layer(s) covering the core particles [72]. The outer layer(s) plays a crucial role in regulating the release of the encapsulated core. In order to extend the release duration of encapsulated cores, low-solubility materials are frequently employed in conjunction with fluidized-bed coating. As an illustration, ethylcellulose has been observed to significantly prolong the release of a coated drug for up to 10 h in demineralized water at 37°C [148]. Hence, the outer layer's role is crucial in determining the duration and pace of the release process. Incorporating low-solubility materials in the formulations can be a consideration to achieve desired controlled-release characteristics for various drugs.

3.1.2 pH-responsive system

The pH of the microencapsulation matrix has a significant impact on the release of the encapsulated material. In one study, pectin matrices were used to encapsulate gallic acid (GA) via spray drying. The pH-responsive release mechanism was monitored, and optimal results were obtained at a pH value of 7, indicating that the release of GA was influenced by the pH of the matrix [149]. Similarly, Baghi et al. [150] examined the ability of pea protein isolate and soybean lecithin to encapsulate trans-cinnamaldehyde (TC) at pH 3 and 7. Better thermal stability was demonstrated by the powders generated at pH 3, indicating that the matrix's pH had an impact on the TC release properties. Furthermore, one study found that the pH of the coacervation and crosslinking processes influenced the size, morphology, and release properties of geraniol-containing microcapsules. Optimal conditions for pH resulted in longer-lasting retention of geraniol, indicating that pH affected the release of the encapsulated material [151]. Moreover, Lavelli and Sri Harsha [152] also stated that the pH of the microencapsulation matrix affects the release of the encapsulated material. At pH 1.4, only 13% of the total phenolic compounds were released, while at pH 7.4, the microbeads dissolved and released the encapsulated material. By controlling the rate at which the microcapsule shells dissolve, the pH of the microencapsulation matrix influences the release of the substance that has been encapsulated. Microcapsules that react to either basic or acidic conditions are made using various polymers with varying pH values. Once exposed to a trigger pH, the shells dissolve steadily, releasing the contents in the process. By adjusting the ratios of pH-responsive and pH-unresponsive polymers in the shell composition, the rate of release can be independently adjusted [153].

3.2.1 Hydrophilic coating agents

The application of hydrophilic encapsulants facilitates the effective encapsulation of micro- or nanosized hydrophilic compounds. The utilization of the internal aqueous pockets within the capsules enhances encapsulation efficiency by maintaining the stability of the active components and inhibiting their premature release into the surrounding environment [165]. The encapsulation effectiveness of hydrophilic substances can be improved using hydrophilic encapsulants such as chitosan and alginate. Microcapsules containing alginate-coated chitosan, produced from poly (dl-lactide-co-glycolide) (PLGA), exhibited superior encapsulation rates and reduced early burst release compared to conventional methods, facilitating a more regulated and extended-release profile [166]. The choice of coating material has a significant impact on the kinetics of chemical release. As an example, chitosan coatings allow hydrophilic drugs to be released slowly, reducing the initial burst release significantly [166]. This is crucial for reaching and maintaining therapeutic levels in the bloodstream while minimizing the risk of undesirable effects caused by rapid drug release. The ability of various hydrophilic encapsulants to respond to pH variations renders them optimal for application in biological contexts as drug delivery systems. Certain tissues or pathological conditions may lead to the degradation of biodegradable nanocarriers made from poly(thiourethane-urethane). Encapsulated hydrophilic compounds can be selectively released upon reaching certain regions of the body [167].

A different study found that eugenol, an antimicrobial compound that has been encapsulated and dried by spray-drying, demonstrated a significant reduction in the populations of both Gram-negative (Escherichia coli) and Gram-positive (Listeria innocua) bacteria, decreasing from 5 to 6 log CFU/mL to 0 log CFU/mL within a time frame of just 30 min [86]. The implementation of low-solubility encapsulants facilitates the achievement of a slower, controlled release over a duration ranging from hours to days from spray-dried encapsulates. When combined with suitable encapsulation technology, this approach leads to extended-release periods. In such cases, an initial rapid release may occur, attributed to the presence of some core droplets on the surface of the encapsulates, followed by a slower and sustained release. Additionally, manipulating the release rate of the encapsulated material can be achieved by varying the core-encapsulant ratio. Decreasing the core load, for instance, contributes to a reduced release rate due to the resulting larger shell thickness of the encapsulates.

3.2.2 Hydrophobic coating agents

Lipid carriers are essential in improving the bioavailability of microencapsulated compounds. Lipid-based carriers, including nanoemulsions, liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs), have been successfully developed to enhance the solubility, stability, and release profiles of bioactive compounds within the food industry [168,169]. Lipid-based carriers improve the bioavailability of microencapsulated compounds by offering heightened chemical and physical stability, extended release duration, and protection against external influences [169].

The development of NLCs aimed to address certain issues and constraints associated with other lipid-based carriers like SLNs. NLCs were designed to overcome the limitations of SLNs, which exhibit a lower capacity to accommodate bioactive compounds compared to NLCs. Additionally, SLNs tend to have high water content, ranging from approximately 70% to 99.9%. This particular drawback renders SLNs unsuitable for delivering certain food ingredients and limits their application as a delivery system in certain food-related contexts [170]. Shishir et al. [171] stated that the distribution of solid and liquid lipid constituents in NLCs significantly impacts the transport and release rates of bioactive compounds. Due to the great mobility and solubility of bioactives inside the liquid fraction channels, it is probable that their path transpires through these channels instead of the denser, crystalline solid lipids. Consequently, an increase in oil fractions is anticipated to elevate the rate of bioactive release. The transport and release kinetics of bioactive molecules are profoundly influenced by the features of liquid fractions, including fatty acid profile, compositional heterogeneity, saturation level, and chain length [172].

There is some inconsistency in the data collected about the rate of bioactive release from NLCs. Bioactive release rates varied among experiments; in some, they increased as oil fractions increased, while in others, they declined as fractions increased. Release rates of bioactives were shown to vary over time apart from changes in the oil fraction mass, according to certain research that failed to find a correlation between the two. In essence, lipid-based encapsulation provides a range of significant advantages and proves effective in enhancing encapsulation efficiency, ensuring improved stability, elevating the bioavailability of hydrophobic bioactive compounds, and mitigating potential toxicity concerns.

3.3.1 In vitro bioavailability evaluation

In vitro digestion evaluation is extensively employed to assess the bioaccessibility of bioactive compounds. These procedures are convenient cost-effective, rapid, and do not present the same ethical concerns as clinical trials. In vitro models replicate gastrointestinal conditions by modifying ionic strength and pH, introducing enzymes, bile salts, and even fermentation reactions to emulate colon conditions. Static, semi-dynamic, and dynamic in vitro digestion models are used to study the digestion behavior of various substances. These models simulate different aspects of the human gastrointestinal system to better understand the digestion process. In static digestion models, the digestion behavior is observed without considering the dynamic variations of the gastrointestinal conditions [174]. Semi-dynamic digestion models introduce some dynamic aspects, such as pH variations, but still have limitations in replicating physiological conditions [175]. Dynamic digestion models, on the other hand, take into account parameters like gastric juice secretion, gastric emptying rate, intestinal juice secretion, and pH variations, providing a more realistic representation of the digestion process [176]. These models have been used to study the digestion of lipids, proteins, and starch [177,178]. Although static models are widely used due to their simplicity and standardization, they lack dynamic physiological elements such as gastric emptying and enzyme flow, which limits their predictive power for real-life digestion. Semi-dynamic models offer partial improvements, but they still fall short in replicating complex gastrointestinal conditions. In contrast, dynamic models provide a more physiologically relevant environment, yet their complexity and cost limit their accessibility and scalability for routine screening. Thus, the choice of model must balance realism with practicality, depending on the research goal.

Among these, the INFOGEST protocol has emerged as the most widely accepted in vitro digestion model due to its standardized, physiologically relevant design. It simulates human digestion across three stages – oral, gastric, and intestinal – and has been widely adopted in academic and industry research [179]. Its reproducibility and comprehensive simulation of digestive conditions allow for inter-laboratory consistency and more accurate evaluation of nutrient release and food matrix interactions. However, despite their utility, in vitro models face critical limitations when extrapolating to in vivo outcomes, particularly in the context of protein–polyphenol interactions. In vitro systems often apply fixed pH and lack dynamic enzyme activities that occur naturally during digestion, leading to potentially inaccurate estimates of bioaccessibility [180,181]. Moreover, complex formation mechanisms – both covalent and non-covalent – are influenced in vivo by fluctuating ionic strength, protein conformation, and compartmentalized absorption pathways that are rarely captured in vitro [182]. These discrepancies can result in over- or underestimation of bioavailability. For example, polyphenols may exhibit high antioxidant activity in vitro, yet this can be masked in vivo due to binding with proteins, altering their nutritional functionality [183]. Therefore, while in vitro digestion models are invaluable for early-stage screening, critical interpretation is necessary when translating findings to real physiological scenarios. Improvements in model complexity and physiological mimicry, especially concerning gut microbiota metabolism and compound–protein interactions, remain essential for accurate predictions.

In vitro models have been used to assess the bioaccessibility of microencapsulated bioactive compounds. Based on the simulated digestion system, the protective effect of encapsulation against environmental conditions was assessed by Vergara et al. [184], where the bioaccessibility of anthocyanin from the microencapsulated extract with maltodextrin was found to be 20% higher than that of the non-encapsulated extract, indicating the protective effect of encapsulation against environmental conditions. The rate of bioactive compounds degradation, like anthocyanins, and how the coating agents slow it down, were also observed during in vitro digestion [185]. Both increase the thermal stability of blackberry anthocyanins and reduce the rate at which these pigments degrade in simulated gastrointestinal settings. Furthermore, an in vitro enzymatic model was able to analyze the potential of the anti-hyperglycemic effect of encapsulated compound, and the results suggest that encapsulated ferulic acid has a potential anti-hyperglycemic effect [186]. In the release study model, the duration of encapsulated bioactive compound retention was prolonged by the outer coating of the particles. Gomes et al. [187] observed that the duration of encapsulated catechin retention is prolonged by the lipid outer coating of the particles. This is because the use of these lipid/particle structures is intended for techniques involving cell absorption under physiological settings, as well as the regulated release of EGCG at the lower digestive tract. Other in vitro assessments of microcapsules are presented in Table 5.

Notably, studies using static in vitro models may overestimate encapsulation stability, as they do not simulate mechanical stresses or gradual pH shifts encountered in vivo. For instance, anthocyanin retention [184] and ferulic acid functionality [186] were demonstrated under simplified conditions, which may not fully capture degradation patterns seen in dynamic digestion. Moreover, the use of lipid-based encapsulants [187] shows promise for targeted intestinal release, but further validation in dynamic systems is needed to confirm controlled release kinetics. Comparing across studies, it becomes clear that while encapsulation enhances bioaccessibility and functionality, outcomes can vary significantly depending on the model employed. This underlines the importance of selecting appropriate in vitro systems based on the physicochemical characteristics of the encapsulated compound and intended application.

3.3.2 In vivo bioavailability assessment

Currently, it remains unfeasible to completely substitute in vivo studies with in vitro models, regardless of their advancements. The inherent complexity of the human body has led to a growing recognition of animal models as a viable alternative to in vitro research. Given fundamental differences in metabolic processes between animals and humans, the findings derived from animal studies may be subject to debate. In order to gain a deeper insight into the health benefits of bioactive compounds (CBAs), in vitro studies and animal experiments remain valuable methodologies for assessing human in vivo processes such as digestion, absorption, metabolism, and tissue distribution, among others [195]. Consequently, findings from both in vitro and in vivo research involving animals or other non-human models can provide supportive evidence. It is essential to note that human clinical trials remain irreplaceable, as emphasized in the EFSA scientific and technical guidelines for health claim authorization [196]. Human intervention studies should ideally complement nutritional research after in vitro and in vivo (animal experimental) investigations have yielded strong findings supporting the proposed theory. This is considered the most effective way to evaluate the genuine significance of foods or bioactive components concerning their health benefits [195]. Human intervention studies provide insights into how these elements interact with the human body and contribute to overall health, ensuring a more comprehensive understanding of their impact.

In vivo animal models are able to examine the delivery of bioactive compounds both in digestive and non-digestive tissues, such as in Tong et al. [197], where anthocyanins encapsulated with amylopectin nanoparticles showed improved stability and delivery to the stomach and lungs, respectively. The level of microencapsulated active compound in plasma was able to be detected [193]. In that study, animals treated with quercetin-loaded casein nanoparticles displayed higher plasma levels of quercetin compared to animals receiving the solution of the flavonoid (control). Moreover, the effect of microencapsulated bioactive compounds on the blood profile can be detected [186,198]. Panwar et al. [186] reported that encapsulated ferulic acid was found to attenuate the diabetes-associated symptoms in the rats. It showed an enhancement in body weight but a decrease in blood glucose levels, and also had a regulatory effect on the blood lipid profile of the diabetic rats. Moreover, Cian et al. [199] stated that the pro-oxidant and prothrombotic effects of a diet high in sucrose on rats were prevented by microencapsulated peptides. Other in vivo assessments of microcapsules are presented in Table 6.