Bioactive Compounds and the Performance of Proteins as Wall Materials for Their Encapsulation

Abstract

The encapsulation of bioactive compounds using proteins as wall materials has emerged as an effective strategy to enhance their stability, bioavailability, and controlled release. Proteins offer unique functional properties, including amphiphilic behavior, gel-forming ability, and interactions with bioactives, making them ideal candidates for encapsulation. Animal-derived proteins, such as whey and casein, exhibit superior performance in stabilizing lipophilic compounds, whereas plant proteins, including soy and pea protein, demonstrate greater affinity for hydrophilic bioactives. Advances in protein modification and the formation of protein–polysaccharide complexes have further improved encapsulation efficiency, particularly for heat- and pH-sensitive compounds. This review explores the physicochemical characteristics of proteins used in encapsulation, the interactions between proteins and bioactives, and the main encapsulation techniques, including spray drying, complex coacervation, nanoemulsions, and electrospinning. Furthermore, the potential applications of encapsulated bioactives in functional foods, pharmaceuticals, and nutraceuticals are discussed, highlighting the role of emerging technologies in optimizing delivery systems. Understanding the synergy between proteins, bioactives, and encapsulation methods is essential for developing more stable, bioavailable, and sustainable functional products.

1. Introduction

The term “functional foods” was first proposed in Japan in the 1980s and refers to foods that, in addition to providing essential nutrients, also offer specific health benefits when consumed regularly [1]. These benefits are directly related to the presence of bioactive compounds—substances capable of interacting with components of living tissue, promoting metabolic and physiological effects that contribute to well-being and disease prevention [2]. For food, bioactives can range from small molecules like flavonoids to bacteria used as probiotics (

Table 1. Bioactive compounds, health benefits, and source.

Chemical Class	Bioactive Compounds	Metabolic and Physiological Effects	Health Benefit Claims	Commercial Product Containing the Bioactive Compound	Authors
Flavonoids	Anthocyanins	Antidiabetic; Anti-obesity; Antihypertensive; Antiproliferative; Antimicrobial; Anti-inflammatory.	Reduction in the risk of chronic diseases such as diabetes	Grape juice, açaí, wine	[3]
Carotenoids	Carotenoids	Anticarcinogenic; Immunomodulatory; Anti-inflammatory; Antibacterial; Antidiabetic; Neuroprotective.	Protection of the nervous system	Annatto seasoning, meat products	[4]
Alkaloids	Caffeine	Neuromuscular action.	Improves performance in physical activities	Dietary supplements, coffee, energy drinks	[5]
Polyphenols	Curcuminoids	Antioxidant; Antibacterial.	Management of irritable bowel syndrome and peptic ulcers	Turmeric	[6,7,8]
	Hydroxytyrosol	Antioxidant; Anti-inflammatory; LDL oxidation reduction; Anticarcinogenic activity	Reduced risk of cardiovascular, inflammatory, and degenerative diseases	Olive, olive oil	[9]
Sterols	Phytosterols	Regulate cholesterol and lipid metabolism; Anticarcinogenic.	Cholesterol control	Fruits, vegetables, vegetable oils, nuts, and seeds	[10,11]
Polysaccharides	Inulin	Anti-inflammatory.	Improves gut health	Roots and tubers such as chicory and Jerusalem artichoke	[12,13]
Fatty Acids	Omega-3 and Omega-6	Anti-inflammatory and antifungal; Reduces the risk of cardiovascular death and myocardial infarction.	Reduction in the risk of cardiovascular death and myocardial infarction. Supports brain function; lowers triglycerides	Fish oil, flaxseed, soybean, and canola oils	[14,15]
Vitamins	Vitamin D	Promotes calcium absorption and supports the immune system.	Reduces the risk of osteoporosis and autoimmune diseases	Fortified foods	[16]
	Vitamin C	Potent antioxidant and stimulates white blood cell production.	Reduces the risk of chronic diseases, especially cancer	Citrus fruits	[16,17]
	Vitamin E	Protects cells from oxidative damage.	Reduces the risk of chronic diseases, especially heart disease and cancer	Nuts and seeds	[18]
Gram-positive Bacteria	Probiotics (Lactobacillus and Bifidobacterium)	Anti-inflammatory; Antimicrobial; Antioxidant; Antihypertensive; Immunomodulatory; Improve intestinal function.	Gut regulation, digestion improvement, skin health, immune function, and potential impact on mental health through gut–brain axis modulation	Yogurt, kefir, sauerkraut, kimchi, kombucha, dietary supplements	[19]

).

For a food to be considered functional, it is not enough to merely contain bioactive compounds, they must be present in adequate concentrations to ensure health benefits. The recommended daily intake (RDI) for a bioactive compound depends on several factors, such as bioavailability, bioaccessibility, individual metabolism, supplementation purpose, the individual’s nutritional status, and the available scientific evidence [20]. The market offers a wide variety of these products, including probiotic yogurts (Activia^®—Danone with Bifidobacterium animalis lactis DN-173 010/CNCM I-2494, Yakult^® (Yakult Honsha), with Lactobacillus casei Shirota, Vigor Viv^®—Vigor with Lactobacillus acidophilus and Bifidobacterium lactis) that support gut health, antioxidant-enriched beverages such as whole grape juices (Vita Suco^®, Native^®, Welch’s^®) and green teas (Leão Fuze^®—Coca-Cola^®, Feel Good^®, Lipton^®), omega-3-rich foods like fortified margarines (Becel^®—Flora Flood Group, Qualy Vida^®—Seara, Delícia Supreme^®—Bunge, Benecol^®—Raisio Group) promote cardiovascular health, and cereal bars with prebiotic fibers (Levittá^®) that help maintain a balanced gut microbiota.

Although interest in functional foods is growing, the application of bioactive compounds in food remains limited due to the fragility of these substances under processing, transportation, and storage conditions. This often requires high doses to achieve the desired effects [21]. Additionally, bioactive compounds may have a bitter and astringent taste, and their oral bioavailability can be low, limiting their practical use in food products [22]. Furthermore, the physicochemical properties of bioactive compounds can hinder their application in certain products, as in the case of lipophilic compounds that need to be incorporated into aqueous matrices [23].

A promising solution to overcome these limitations is encapsulation in protein matrices, which provides protection against adverse conditions, minimizes sensory and nutritional losses, and masks undesirable flavors [24]. This process involves encapsulating bioactive substances within a carrier material, which can enhance the bioavailability of these compounds, thereby maximizing their health benefits [25,26] have shown that encapsulated bioactive compounds exhibit better absorption and retention in the body compared to their non-encapsulated forms, which is particularly beneficial for compounds with low chemical stability or solubility.

Proteins, in turn, exhibit ideal characteristics for this application, such as buffering capacity, the presence of both hydrophilic and hydrophobic amino acids, and the ability to form gel matrices or films that ensure controlled release [27]. Additionally, their water solubility and the possibility of being sourced from renewable materials make proteins a sustainable and effective option for enhancing the stability and bioavailability of bioactive compounds [28]. Their ability to form capsules resistant to gastric digestion, combined with the increased bioavailability of the compounds, makes them an even more promising solution for overcoming the limitations associated with the application of these compounds in functional foods [29].

This review aims to explore the physical and chemical characteristics of bioactive compounds and some food proteins, investigating their interactions in the encapsulation process while also addressing the main techniques used for their application in functional foods. Through this review, the goal is to understand how encapsulation can overcome the limitations associated with bioactive compounds, contributing to the development of functional foods.

2. Bioactive Compounds and Their Structural Characteristics

Bioactive compounds belong to various chemical classes, such as phenolic compounds (flavonoids, phenolic acids), carotenoids, organosulfur compounds, alkaloids, bioactive peptides, and microorganisms, whose structures determine their functionality, solubility, and metabolic fate [30].

The study of the chemical structure of bioactive compounds is fundamental for their effective application in food production. The molecular characteristics of these compounds determine their functionality, bioavailability, and stability, influencing their interactions with food components and encapsulation matrices. The chemical nature of the bioactive molecule also plays a key role in defining the appropriate encapsulation method and carrier material. Hydrophilic bioactives, such as polyphenols and peptides, often require hydrophilic carriers like whey proteins, while lipophilic compounds, such as carotenoids and omega-3 fatty acids, benefit from lipid-based encapsulation systems, including plant proteins as zeins and gliadins [31].

For instance, curcumin, a hydrophobic polyphenol with antioxidant and anti-inflammatory properties, exhibits poor water solubility and rapid degradation under light and heat [32]. Encapsulation in protein systems improves its stability and bioavailability, allowing its incorporation into functional foods [33]. Similarly, probiotics require encapsulation techniques, such as microencapsulation with milk proteins, to protect them from gastric acidity and ensure their viability in the intestinal tract [34].

A thorough understanding of the chemical structure and stability of bioactive compounds enables food scientists to design innovative functional foods with enhanced health benefits. By leveraging advanced encapsulation technologies and selecting appropriate formulation strategies, it is possible to improve the bioefficacy of bioactives while ensuring their stability during processing and storage.

In the following sections, we will discuss the structural and chemical characteristics of the main hydrophilic and hydrophobic bioactives used in food production.

2.1. Hydrophilic Bioactive Compounds

Water-soluble bioactives tend to possess polar functional groups, such as hydroxyl (-OH), carbonyl (C=O), carboxyl (-COOH), amino (-NH₂), and sulfate groups (-SO₃H), which can interact favorably with the polar water molecules [35]. These polar groups create dipole–dipole interactions and hydrogen bonds with water, enhancing solubility [36]. In general, these compounds also contain hydrophilic (water-attracting) functional groups, such as hydroxyl, amine, or carboxyl groups, facilitating the dissolution process [37]. These groups enable the compound to form hydrogen bonds with water, increasing its solubility.

Many bioactive compounds, particularly those that are acidic (e.g., phenolic acids) or basic (e.g., alkaloids), can ionize in water, forming charged species that are highly soluble in aqueous solutions. The presence of ionic groups such as carboxylates (-COO⁻) or ammonium groups (-NH₃⁺) increases the compound’s solubility in water [38]. In addition, compounds with more flexible molecular structures can interact more easily with water molecules. For example, some peptides or amino acids, due to their ability to adopt various conformations, can enhance solubility in aqueous environments [39].

Another factor related to water solubility is the presence of charged or polar substituents [40]. Compounds that are conjugated with charged or polar substituents, such as sulfate, phosphate, or glucoside groups, exhibit better solubility. For instance, glucosides (e.g., flavonoid glycosides) have improved water solubility compared to their aglycone counterparts due to the hydrophilic sugar moieties attached to them [38].

The primary water-soluble bioactive compounds present in functional foods include polyphenols, peptides, amino acids, vitamins, and certain carbohydrates, each of which plays a significant role in promoting health and preventing disease [41].

2.1.1. Polyphenols

The polyphenols are a large group of bioactive compounds widely found in plant-based foods, such as fruits, vegetables, tea, and whole grains. Polyphenols are a large and diverse group characterized by the presence of multiple phenolic rings, which are aromatic structures containing hydroxyl (-OH) groups attached to a benzene ring. They are highly water-soluble due to their hydroxyl groups, which enable hydrogen bonding with water molecules [42].

The chemical structure of polyphenols is generally defined by the number and arrangement of hydroxyl groups attached to aromatic rings, as well as the presence of additional functional groups, such as methoxy (-OCH₃), carbonyl (C=O), or hydroxymethyl (-CH₂OH) groups [43]. The flavonoids are a subgroup of polyphenols with a common flavonoid skeleton consisting of two aromatic rings connected by a three-carbon bridge [44]. Flavonoids are subdivided into several subgroups, including anthocyanins, flavonols, flavones, and isoflavones, each distinguished by variations in the hydroxylation and substitution patterns on the aromatic rings [45]. The non-flavonoid polyphenols include phenolic acids (e.g., caffeic acid, ferulic acid), stilbenes (e.g., resveratrol), and lignans, which also possess multiple hydroxyl groups and exhibit various degrees of aromaticity [46].

Polyphenols are particularly sensitive to oxidation due to the presence of hydroxyl groups, which are prone to donate electrons to reactive oxygen species (ROS), leading to oxidation and the formation of quinones, semiquinones, or polymerized products [47]. The stability of polyphenols can also be affected by pH, with acidic environments making them more stable, as the hydroxyl groups are less prone to ionization and oxidation [48]. However, in alkaline conditions, polyphenols can undergo degradation processes, such as hydrolysis or rearrangement of their structures, leading to the loss of their bioactivity [49]. For instance, anthocyanins are highly sensitive to pH, with their color changing from red in acidic conditions to blue or purple in alkaline environments [50].

High heat can cause the breaking of bonds in the aromatic rings or the transformation of the polyphenolic structure into less stable or inactive forms, which is particularly relevant during food processing or storage [51]. On the other hand, light exposure can generate reactive oxygen species, leading to the oxidation of polyphenols. This phenomenon is often observed in the degradation of anthocyanins in fruits and vegetables, where exposure to sunlight causes fading and loss of antioxidant activity [52].

Factors related to food processing or storage can affect the chemical structure of polyphenols, leading to a reduction in their bioavailability in the final product. Thermal treatments, exposure to oxygen, pH variations, and interactions with other food components may cause degradation, oxidation, or polymerization of polyphenols, ultimately limiting their absorption and biological activity.

2.1.2. Water-Soluble Vitamins

Water-soluble vitamins are essential micronutrients that play crucial roles in various metabolic and physiological functions in the human body. Unlike fat-soluble vitamins, they are not stored in significant amounts and must be regularly obtained from the diet [53]. Their bioactivity depends on their chemical structure, which can be influenced by various processing and storage conditions.

Water-soluble vitamins include the B-complex group (B1, B2, B3, B5, B6, B7, B9, and B12) and vitamin C. They are characterized by their polar nature, allowing them to dissolve in aqueous environments. These vitamins generally contain functional groups such as hydroxyl (-OH), carboxyl (-COOH), amine (-NH₂), and phosphate (-PO₄), which facilitate their participation in enzymatic reactions and cellular metabolism [54,55].

In terms of structure, vitamin B1 (Thiamine) consists of a thiazole and a pyrimidine ring linked by a methylene bridge, while vitamin B2 (Riboflavin) features a flavin ring system attached to a ribitol side chain. Vitamin B3 (Niacin) exists in two forms—nicotinic acid and nicotinamide—both derived from pyridine, whereas vitamin B5 (Pantothenic acid) comprises pantoic acid moiety linked to β-alanine [54].

Similarly, vitamin B6 (Pyridoxine, Pyridoxal, and Pyridoxamine) consists of pyridine derivatives with varying functional groups, and vitamin B7 (Biotin) is a bicyclic ureido compound with a valeric acid side chain. Vitamin B9 (Folate) contains a pteridine ring, p-aminobenzoic acid, and a glutamate moiety, while vitamin B12 (Cobalamin) is distinguished by a corrin ring with a central cobalt atom [54]. Finally, vitamin C (Ascorbic Acid) is a lactone ring structure with enediol groups that provide antioxidant properties [17].

Water-soluble vitamins act as coenzymes or precursors to coenzymes, playing a vital role in enzymatic reactions across various biochemical pathways. The B-complex vitamins are essential for carbohydrate, lipid, and protein metabolism, while specific vitamins serve additional critical functions [17,56]. Thiamine (B1), pyridoxine (B6), and cobalamin (B12) support neurotransmitter synthesis and nerve function, whereas folate (B9) and cobalamin (B12) are key to nucleotide biosynthesis and erythropoiesis [57]. Meanwhile, vitamin C functions as a powerful antioxidant, scavenging free radicals, promoting collagen synthesis, and strengthening immune defenses [58].

The chemical structure of water-soluble vitamins is highly susceptible to degradation under certain conditions, which can significantly compromise their bioavailability. High temperatures can break down thermolabile vitamins like thiamine (B1) and folate (B9), while vitamin C is particularly vulnerable to alkaline conditions, leading to oxidative degradation [59]. Riboflavin (B2) is photolabile and deteriorates when exposed to ultraviolet or visible light [60]. Additionally, ascorbic acid and several B vitamins are prone to oxidation, reducing their effectiveness [61].

2.1.3. Peptides

Bioactive peptides, typically composed of two to twenty amino acid residues, exhibit diverse functional properties that depend on their sequence and composition [62]. They originate from various protein sources, each contributing distinct health benefits. Caseins and whey proteins act as precursors for peptides with antihypertensive, antimicrobial, and immunomodulatory properties [63]. Collagen-derived peptides are linked to improved skin health and joint function, while fish proteins generate peptides with both antihypertensive and antioxidant effects [64,65]. Similarly, plant-based proteins from soy, rice, wheat, and legumes produce peptides with antioxidant, anti-inflammatory, and cholesterol-lowering activities [66]. Additionally, cereals and egg proteins serve as valuable sources of peptides with potential antihypertensive and antimicrobial functions [55].

The bioactivity of peptides is determined by their amino acid sequence, which influences their interaction with biological targets. For instance, some peptides inhibit angiotensin-converting enzyme (ACE), helping to regulate blood pressure, while others (tuna-derived peptides, black soybean peptides, and cumin-derived peptides) scavenge free radicals, reducing oxidative stress and protecting cells [67]. Peptides such as nisin and melittin exhibit antimicrobial activity by disrupting bacterial cell membranes, whereas others (peptides IDR-1018 and IDR-1002) modulate immune responses, potentially enhancing defense mechanisms and reducing inflammation [68]. Additionally, peptides such as casein-derived peptides, soy protein peptides, and milk-derived peptides play a role in lipid metabolism, lowering cholesterol absorption and synthesis [69].

The functional effectiveness of bioactive peptides depends on their stability during processing, storage, and digestion. Several factors influence their bioavailability, including resistance to enzymatic degradation in the digestive system, stability under varying pH and temperature conditions, and interactions with other food components, which can either enhance or inhibit absorption [70].

Bioactive peptides are increasingly being incorporated into functional food products to maximize their health benefits. Examples include dairy-based functional foods, such as peptide-enriched yogurts, cheeses, and fermented milk products; protein supplements and beverages, including fortified drinks and powders; bakery and snack products, where peptides are added to breads, biscuits, and protein bars; and nutraceuticals and dietary supplements, designed to address specific health conditions [71].

As a promising class of functional food ingredients, bioactive peptides offer a wide range of health benefits. Understanding their structure-function relationships, stability, and bioavailability is essential for optimizing their application in food products. Continued research and technological advancements will further enhance their potential in promoting human health and preventing chronic diseases.

2.1.4. Polysaccharides

Bioactive polysaccharides vary in structure, which directly influences their functionality. Their chemical composition includes factors such as a monosaccharide composition, where they can either be homopolysaccharides, composed of a single type of monosaccharide, or heteropolysaccharides, containing a variety of monosaccharide units [72]. The type of glycosidic linkage, whether α- or β-linked, is another critical factor, as it determines solubility, digestibility, and bioactivity [73]. The degree of branching also plays a role; polysaccharides like amylopectin and arabinoxylans have branched structures that influence their ability to form gels and interact with other components in the food matrix [74]. Additionally, the molecular weight of a polysaccharide affects its viscosity and gel-forming properties, with high molecular weight polysaccharides such as β-glucans exhibiting enhanced viscosity, which can impact their bioavailability and functional effects in the body [75].

The physiological effects of bioactive polysaccharides arise from their interactions with biological systems, leading to several key bioactivities. Prebiotic effects are exhibited by polysaccharides like inulin and fructooligosaccharides (FOS), which serve as substrates for beneficial gut microbiota, enhancing the production of short-chain fatty acids (SCFAs) that support intestinal health [76]. Immunomodulatory properties are seen in β-glucans and fucoidans, which stimulate immune responses by activating macrophages, dendritic cells, and natural killer (NK) cells [77]. In terms of cholesterol and lipid metabolism regulation, soluble fibers such as pectins and β-glucans form viscous gels that reduce cholesterol absorption and bile acid reabsorption [78]. Some polysaccharides, including fucoidans and arabinogalactans, exhibit antioxidant and anti-inflammatory effects by scavenging free radicals and mitigating oxidative stress [79]. Lastly, polysaccharides can help modulate glucose absorption, improving insulin sensitivity and reducing postprandial glycemic responses [80].

Bioactive polysaccharides interact with proteins, lipids, and minerals, influencing both their functionality and bioavailability. Some polysaccharides bind with proteins, enhancing emulsification properties but potentially reducing protein digestibility [81]. Additionally, polysaccharides like alginates and pectins can chelate minerals such as calcium, iron, and zinc, which may affect their bioavailability, while chitosan interacts with lipids by trapping dietary fats, aiding in weight management [82,83].

Low molecular weight polysaccharides tend to exhibit higher solubility and absorption, while some require microbial fermentation in the gut to become bioactive, making gut microbiota composition a crucial factor [84]. Storage conditions, including temperature, humidity, and light exposure, also play a role in determining polysaccharide stability throughout shelf life.

2.1.5. Saponins

Saponins are a diverse group of naturally occurring compounds found in a variety of plant species, particularly in legumes, cereals, and medicinal plants [85]. These compounds are characterized by their soap-like properties, including their ability to form stable foams in aqueous solutions due to the amphipathic nature of their molecular structure [86]. Saponins are of growing interest in functional foods due to their bioactive properties, which have been linked to various health benefits, including cholesterol-lowering effects, antioxidant activity, immunomodulation, and anticancer potential [87].

Saponins are glycosides composed of two primary components: a hydrophobic aglycone (also called the sapogenin) and one or more hydrophilic sugar moieties [85]. The aglycone part can be either a steroid or a triterpenoid structure, while the sugar portion typically consists of one or more monosaccharides, such as glucose, galactose, or rhamnose [88]. The structure of saponins can be broadly classified into steroidal and triterpenoid saponins. The steroidal saponins contain a steroidal aglycone and are typically found in species like Solanum (e.g., tomatoes, potatoes) and asparagus [88]. On the other hand, the triterpenoid saponins have a triterpenoid aglycone and are commonly found in plants like ginseng, quinoa, and soybeans [85].

The bioactivity of saponins arises from their ability to interact with biological membranes and modulate various physiological processes [89]. Saponins are known to bind cholesterol and bile acids in the gastrointestinal tract, preventing their absorption and promoting excretion [90]. This reduces serum cholesterol levels and can help in the management of hypercholesterolemia. Many saponins also possess antioxidant properties, scavenging free radicals and reducing oxidative stress. This contributes to cellular protection against damage and may help in the prevention of chronic diseases such as cardiovascular disease and cancer [91]. In addition, the saponins can modulate the immune system by enhancing macrophage activity and increasing the production of cytokines. This property makes them valuable in enhancing immune responses and potentially reducing inflammation [15]. Finally, saponins from ginseng and quinoa have been shown to inhibit cancer cell proliferation and induce apoptosis (programmed cell death) [92,93]. They may also enhance the effectiveness of conventional chemotherapy drugs.

In a food matrix, saponins can chelate essential minerals like calcium, iron, and magnesium, which may affect their bioavailability [94]. This interaction is particularly relevant in the context of plant-based diets, where mineral absorption can be influenced by saponins. They can also interact with lipids in the gastrointestinal tract, disrupting lipid absorption and altering lipid metabolism [95]. This is one of the mechanisms by which saponins exert their cholesterol-lowering effects.

High temperatures during food processing, such as boiling or frying, can degrade saponins, reducing their antioxidant and cholesterol-lowering properties [96]. However, some saponins may become more bioavailable following heat treatment due to the breakdown of complex molecules. They are also sensitive to changes in pH, and extreme pH conditions (usually below 3 or above 9) can hydrolyze the glycosidic bond, leading to the breakdown of the saponin structure [97]. This may alter both their bioactivity and potential toxicity. In addition, prolonged storage, especially at high temperatures or humidity, and exposure to light can accelerate the degradation of saponins [97].

2.2. Hydrophobic Bioactive Compounds

Hydrophobic bioactive compounds in foods are a diverse group of molecules characterized by their low solubility in water and high affinity for lipophilic environments. Their chemical structures often contain nonpolar hydrocarbon chains, aromatic rings, or other hydrophobic moieties that contribute to their lipid solubility [98]. The degree of saturation, branching, and functional groups attached to these structures influence their physicochemical properties and bioactivity [99].

Many hydrophobic bioactive compounds contain long hydrocarbon chains or fused aromatic rings, which contribute to their low water solubility. Among the main hydrophobic bioactives found in functional foods are the carotenoids (β-carotene, lycopene, lutein, astaxanthin), fatty acids (e.g., omega-3 (DHA, EPA), omega-6, conjugated linoleic acid (CLA), tocopherols (Vitamin E), and alkaloids with hydrophobic properties as the caffeine in coffee beans [100].

2.2.1. Carotenoids

Carotenoids are a class of naturally occurring tetraterpenoid pigments with a general molecular formula of C₄₀H₅₆. Their structure is composed of eight isoprene units (C₅H₈) joined in a head-to-tail arrangement, forming a linear or cyclic hydrocarbon backbone with a highly conjugated system of alternating single and double bonds [101]. This extensive conjugated π-electron system is responsible for their strong light absorption in the visible spectrum, leading to their characteristic yellow, orange, and red colors [19].

Carotenoids are classified into carotenes, such as lycopene and β-carotene, which contain only carbon (C) and hydrogen (H), and xanthophylls, which incorporate oxygen (O) atoms in the form of hydroxyl (-OH), epoxide, or keto groups [37]. Their extensive system of conjugated double bonds enables them to scavenge reactive oxygen species (ROS) and absorb light, providing strong antioxidant activity [46]. The structural variation between cyclic and acyclic ends influences both their stability and bioavailability, with cyclic carotenoids, like β-carotene, playing a key role in provitamin A activity [102]. Beyond their structural significance, carotenoids contribute to plant pigmentation and photoprotection while offering essential health benefits, particularly in vision (as precursors to retinoids), immune function, and oxidative stress reduction [103].

The high degree of unsaturation in carotenoids makes them vulnerable to oxidative degradation, particularly in the presence of oxygen (O₂) and reactive oxygen species (ROS). Oxidation leads to cleavage of the polyene chain, generating colorless or low-molecular-weight oxidation products, including apo-carotenoids and epoxycarotenoids, which lack the biological activity of the parent compounds [104]. Lipid oxidation products, such as hydroperoxides, can further accelerate carotenoid degradation, reducing their antioxidant potential [103].

Carotenoids naturally occur in the all-trans configuration, which is thermodynamically more stable and exhibits greater bioactivity [105]. However, exposure to heat during food processing (e.g., pasteurization, cooking, and drying) can induce cis-isomerization, altering their spatial configuration and potentially reducing their antioxidant capacity [106]. While some cis-isomers exhibit increased bioaccessibility due to enhanced solubility, excessive thermal treatment can lead to depolymerization and loss of functional integrity.

Carotenoids are highly sensitive to light, particularly ultraviolet (UV) and visible light, which promote photooxidation by generating singlet oxygen (O₂). This reactive species interacts with the polyene backbone, leading to the formation of epoxides and peroxides, which compromise color stability and bioactivity [107]. Light exposure is particularly detrimental in low-moisture foods, oils, and beverages, where carotenoid degradation is accelerated.

Carotenoids remain relatively stable in neutral pH conditions, but extreme acidic or alkaline environments can promote their hydrolysis and oxidative degradation [108]. Enzymes such as peroxidases and lipoxygenases, commonly present in fresh plant tissues, catalyze carotenoid breakdown, contributing to pigment loss in minimally processed fruits and vegetables [109].

2.2.2. Fatty Acids

Fatty acids are aliphatic monocarboxylic acids that serve as the building blocks of lipids. These compounds can be classified as saturated, monounsaturated, or polyunsaturated, depending on the number of double bonds in their structure. They are also distinguished by chain length, which can range from four to twenty-eight carbons. Long-chain fatty acids (LCFAs) are those with aliphatic tails of sixteen or more carbons [110]. Among bioactive fatty acids, omega-3 and omega-6 stand out as polyunsaturated fatty acids, meaning they contain two or more double bonds in their structure [111]. Their stability depends on several factors, including degree of unsaturation, temperature, oxygen exposure, light, pH, enzymatic activity, and interactions with other food components. Changes in their chemical structure, such as oxidation, hydrolysis, isomerization, and polymerization, can impact the sensory, nutritional, and functional properties of lipid-containing foods, ultimately affecting their bioavailability and health benefits.

Among the primary factors affecting fatty acid stability, oxidation leads to the formation of off-flavors, toxic compounds, and loss of nutritional value. Saturated fatty acids are relatively stable due to the absence of double bonds while monounsaturated fatty acids, such as oleic acid, are moderately susceptible to oxidation but remain more stable than polyunsaturated fatty acids [112]. Polyunsaturated fatty acids, including omega-3 (α-linolenic acid) and omega-6 (linoleic acid), are highly prone to oxidative damage due to the presence of multiple double bonds, which serve as reactive sites for oxygen attack.

The chemical stability of fatty acids is highly influenced by temperature and storage conditions, leading to oxidation, hydrolysis, isomerization, and polymerization, which compromise their nutritional and functional properties. Polyunsaturated fatty acids are particularly vulnerable due to their multiple double bonds, making them more susceptible to lipid oxidation, which generates hydroperoxides that decompose into aldehydes and ketones, causing rancidity [113]. High temperatures accelerate oxidation, promote cis-trans isomerization, leading to trans fatty acid formation, and induce polymerization, forming high molecular weight compounds that reduce lipid digestibility and quality [113].

2.2.3. Fat-Soluble Vitamins

Hydrophobic vitamins are a class of organic molecules characterized by their non-polar nature, which allows them to dissolve in lipids rather than water. These vitamins, which include vitamins A, D, E, and K, exhibit bioactive properties essential for physiological functions such as vision, calcium homeostasis, antioxidant defense, and blood coagulation [114].

These vitamins typically possess long hydrocarbon chains or fused ring systems with minimal polar functional groups, contributing to their lipid solubility [114]. They may also contain some polar moieties (e.g., hydroxyl or carbonyl groups); their structures are predominantly composed of alkyl chains, isoprenoid units, or sterane cores, which facilitate their interaction with lipids [115].

Many fat-soluble vitamins, such as vitamin A (retinoids) and vitamin E (tocopherols and tocotrienols), are derived from isoprenoid biosynthesis, making them structurally related to terpenes [116]. Vitamin D is unique among fat-soluble vitamins in that it is derived from sterols, featuring a sterane (cyclopentanoperhydrophenanthrene) core that undergoes photochemical transformation in the skin [117].

The stability of hydrophobic (fat-soluble) vitamins (A, D, E, and K) during food processing and storage is influenced by various physicochemical and environmental factors. Heat exposure, in particular, accelerates oxidation, isomerization, and hydrolysis, leading to nutrient loss. Vitamins A, D, and E are highly sensitive to high temperatures, with prolonged heating causing oxidative cleavage [117]. Meanwhile, vitamin K shows moderate heat stability but gradually breaks down with extended cooking [118].

Photodegradation, driven by the formation of free radicals, leads to the structural breakdown of fat-soluble vitamins. Vitamin A and carotenoids are highly sensitive to light, undergoing isomerization and oxidation [119]. Vitamin D degrades under UV exposure, forming biologically inactive compounds. Vitamin E is prone to photodecomposition, which diminishes its antioxidant capacity, while certain forms of vitamin K, such as phylloquinone, also degrade when exposed to light [120].

Oxygen exposure accelerates oxidative degradation through lipid peroxidation, radical reactions, and molecular cleavage. Vitamin A and carotenoids undergo auto-oxidation, leading to activity loss and the formation of non-functional derivatives [121]. Although vitamin E acts as an antioxidant, it is gradually depleted as it combats lipid oxidation. Similarly, vitamin K is susceptible to oxidative breakdown, especially in the presence of pro-oxidants such as metals [122].

Extreme pH conditions further compromise vitamin stability, accelerating hydrolysis and isomerization. In highly acidic or alkaline environments, vitamin A undergoes oxidative cleavage, while vitamins D and K, though moderately stable, degrade under extreme pH conditions [123]. Vitamin E, particularly sensitive to alkaline environments, undergoes ester hydrolysis, reducing its effectiveness. This degradation diminishes the bioavailability and bioaccessibility of these vitamins, ultimately impairing their physiological functions.

2.3. Probiotics

Probiotics are live microorganisms that provide health benefits when consumed in adequate amounts. They mainly belong to bacterial genera such as Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus, as well as yeast probiotics like Saccharomyces. Below is a detailed overview of their morphological characteristics and key properties.

In general, the bacteria are Gram-positive microorganisms classified as lactic acid bacteria, with a cellular structure that includes a bilipid plasma membrane embedded with proteins, surrounded by a robust cell wall. This cell wall consists of a thick, multilayered peptidoglycan sacculus, supplemented by teichoic and/or lipoteichoic acids, exopolysaccharides, protein filaments (pili), and other surface proteins, which contribute to the microorganism’s resistance and functionality [124].

Among probiotic yeasts, Saccharomyces boulardii stands out, with its structure studied by [125], who highlighted the following aspects: the cell wall exhibits a complex structural organization, predominantly composed of mannoproteins, β-glucans, and chitin, which play essential roles in integrity and interaction with the external environment. Mannans, present in mannoproteins, form an outer layer that protects the cell and facilitates adhesion to surfaces. β-glucans, glucose polymers with β-1,3 and β-1,6 linkages, provide mechanical resistance to the cell wall, while chitin, present in smaller amounts, contributes to additional structural stability.

High temperatures during food processing (e.g., pasteurization, spray drying) can inactivate probiotic cells by denaturing essential enzymes and structural proteins, disrupting metabolic functions and cell integrity. Heat also increases membrane permeability, causes cytoplasmic coagulation, damages DNA and ribosomes, and induces oxidative stress via reactive oxygen species (ROS) [126]. Oxygen exposure—particularly harmful to anaerobic strains like Bifidobacterium spp.—can cause lipid peroxidation while acidic conditions, common in fermented foods, along with antimicrobial compounds (e.g., organic acids, bacteriocins), can inhibit probiotic growth [127].

The interaction between probiotics and proteins improves the survival of probiotic cells under various stress conditions encountered during food processing and storage. Proteins exhibit strong buffering properties, which help to stabilize the pH around the probiotic cells. This is particularly important in acidic environments (e.g., fermented products), where proteins can mitigate the harmful effects of low pH on cell membranes and intracellular enzymes [128]. The surface charge and hydrophobicity of both proteins and probiotic cells influence their interaction. These interactions can lead to stronger adhesion and encapsulation, improving structural stability and stress tolerance.

3. Proteins as Encapsulating Material

For the encapsulation of bioactives, the most extensively studied proteins originate from dairy, eggs, meat, and plant sources which present both advantages and disadvantages in the production of food capsules (

Table 2. Wall materials for encapsulating bioactives.

Source	Advantages	Disadvantages	Authors
Dairy proteins	Good stability and compatibility with a variety of food ingredients.	Potential allergenicity, moderate gelation properties that may be affected by pH.	[129]
Egg proteins	High nutritional quality, gelation and water retention capacity, and cost-effectiveness.	Potential allergenicity, incompatibility with some ingredients, sensitivity to processing, and possible contribution to undesirable taste or odor in certain applications.	[130]
Meat proteins	Ability to form stable gels and emulsions.	Potential allergenicity for some individuals, as well as sustainability and environmental impact concerns related to meat production.	[130]
Plant proteins	Promising for release in the gastrointestinal tract due to reduced digestibility and high surface hydrophobicity, allowing transport of hydrophobic bioactive substances.	The preparation of these delivery systems generally requires high-energy equipment or the use of organic solvents, which limits their application in the food industry.	[131]

).

Proteins are macromolecules composed of amino acids linked by peptide bonds, organized into four structural levels. The primary structure is a linear amino acid sequence, while the secondary structure forms α-helices and β-sheets stabilized by hydrogen bonds. The tertiary structure is the three-dimensional folding of a single polypeptide, shaped by various molecular interactions. The quaternary structure consists of multiple polypeptide subunits held together by similar forces.

In terms of protein interactions, hydrophilic bioactives, such as polyphenols, vitamins, and peptides, form hydrogen bonds with amino acids that contain hydroxyl, carboxyl, or amide groups, like serine and glutamine [132]. Electrostatic interactions also occur with charged residues such as lysine, arginine, and glutamate [133].

Lipophilic bioactives, including carotenoids, fat-soluble vitamins, and omega-3 fatty acids, interact with nonpolar amino acids like leucine, isoleucine, and phenylalanine [134]. These interactions are primarily driven by van der Waals forces and hydrophobic interactions, facilitating the stability and functionality of lipophilic compounds in food matrices.

The interaction between proteins and microbial cell surfaces (both bacteria and yeasts) is governed by a combination of non-covalent chemical forces. These interactions play essential roles in adhesion, encapsulation, biofilm formation, stabilization in food systems, and probiotic delivery.

Bacterial and yeast cell surfaces are typically negatively charged due to the presence of teichoic acids (in Gram-positive bacteria), lipopolysaccharides (in Gram-negative bacteria), or mannoproteins and glucans (in yeast cell walls). Proteins, depending on their isoelectric point (pI) and the pH of the environment, can carry positive or negative charges. When the protein is positively charged (at a pH below its pI), it can interact electrostatically with the negatively charged microbial surface, promoting adhesion or coating [135]. Hydrophobic patches on proteins can interact with hydrophobic regions of the microbial membrane or cell wall components (e.g., lipids, hydrophobic amino acid residues, or surface proteins), and these interactions are especially important in hydrophobic environments [136].

Functional groups on proteins (-OH, -NH₂, -COOH) can form hydrogen bonds with complementary groups on the microbial surface, such as peptidoglycan, polysaccharides, or surface proteins [137]. Weak attractions, such as van der Waals forces, become significant when many such interactions occur simultaneously over large surface areas [138]. In some specialized systems (e.g., engineered delivery systems or biofilms), covalent crosslinking between proteins and microbial surface components may occur, often facilitated by enzymatic action (e.g., transglutaminases or crosslinkers), which results in strong, irreversible attachment, useful in immobilization or encapsulation strategies [139].

As encapsulating materials, proteins exhibit ideal characteristics for this application, such as buffering capacity, the presence of both hydrophilic and hydrophobic amino acids, and the ability to form gel matrices or films that ensure controlled release [27]. Additionally, their water solubility and the possibility of being sourced from renewable materials make proteins a sustainable and effective option for enhancing the stability and bioavailability of bioactive compounds [28]. Their ability to form capsules resistant to gastric digestion, combined with the increased bioavailability of the compounds, makes them an even more promising solution for overcoming the limitations associated with the application of these compounds in functional foods [40].

Several encapsulation techniques have been extensively employed for the protection and controlled delivery of hydrophilic, hydrophobic, and probiotic bioactives using proteins as encapsulating agents. Methods such as spray drying, freeze-drying, coacervation, emulsification, electrospraying, and complexation have demonstrated efficiency in stabilizing sensitive compounds and improving their bioavailability [17]. The choice of technique depends on the physicochemical properties of the bioactive compound, the desired release profile, and the functional characteristics of the protein matrix. These techniques and their fundamental mechanisms have been thoroughly documented in previous comprehensive reviews and, therefore, will not be the central focus of this article [140].

The interaction between proteins and bioactive compounds determines the encapsulation efficiency, primarily through the nature and strength of the physicochemical interactions established during the encapsulation process. The structural flexibility of proteins, their ability to undergo conformational changes, and their amphiphilic nature further enhance their encapsulation performance by promoting favorable spatial arrangements around the bioactives. These interactions not only improve the physical entrapment of bioactive compounds but also contribute to their stabilization by protecting them from environmental stressors such as oxidation, heat, and pH fluctuations.

Table 3. Main protein sources used as encapsulating materials for bioactive compounds and the encapsulation efficiency.

Bioactive Compound	Encapsulating Material (Protein)	Encapsulation Technique	Encapsulation Efficiency (%)	Reference
Vitamina A	Whey protein	Spray drying	85	[141]
Omega-3 fatty acids	Soy protein	Coacervation	92	[142]
Polyphenols	Casein	Emulsification	80	[59]

summarizes the main protein sources used as encapsulating materials for bioactive compounds, along with the encapsulation efficiency reported for each application. In the following sections, we present the chemical structures of the principal proteins employed as encapsulating agents and discuss the possible mechanisms of protein–bioactive interactions that influence encapsulation efficiency.

3.1. Dairy Proteins

Caseins, which account for approximately 80% of the total proteins in milk, form colloidal structures composed of αs1-casein, αs2-casein, β-casein, and κ-casein. These micelles are stabilized by colloidal calcium phosphate [143] and exhibit high thermal stability, making them ideal for encapsulating heat-sensitive compounds [144].

These micelles create a hydrophobic core and a hydrophilic surface, enabling them to encapsulate various bioactives. Lipophilic compounds (e.g., curcumin, carotenoids, and omega-3 fatty acids) can be trapped in the hydrophobic core of casein micelles, protecting them from oxidation and improving solubility in aqueous environments [144]. Hydrophilic bioactives (e.g., polyphenols, peptides, and vitamins) interact with the charged and polar regions on the casein surface, stabilizing the compounds in solution [145].

Caseins interact with bioactive compounds through various molecular forces, enhancing their stability and solubility. Nonpolar bioactives, such as fat-soluble vitamins (A, D, E, and K) and flavonoids, associate with casein’s hydrophobic regions. Meanwhile, positively or negatively charged bioactives bind to caseins due to their amphiphilic nature, as seen in electrostatic complexes formed with polyphenols and peptides [146]. Additionally, bioactives containing hydroxyl or carboxyl groups, such as phenolic compounds and antioxidants, establish hydrogen bonds with caseins, further improving their solubility and stability [147].

Several studies have explored the use of caseins as encapsulating agents for bioactive compounds. Ref. [148] developed casein nanocapsules to encapsulate curcumin, resulting in significantly enhanced dispersibility and antioxidant activity. Furthermore, ref. [149] encapsulated resveratrol in casein nanoparticles, reporting a 10-fold increase in oral bioavailability compared to the free compound.

Encapsulation within casein micelles shields bioactive compounds from environmental stressors such as pH fluctuations, temperature variations, oxidation, and enzymatic degradation. As casein undergoes controlled digestion in the gastrointestinal tract, it enables the gradual release of bioactives, enhancing their absorption and bioavailability.

In contrast, whey proteins include β-lactoglobulin, α-lactalbumin, immunoglobulins, and lactoferrin [150]. β-lactoglobulin stands out for its binding potential to minerals, lipophilic vitamins, and lipids, facilitating the incorporation of calcium, zinc, and antioxidants such as α-tocopherol and retinol. This property is leveraged in encapsulation processes, ensuring the efficient release of bioactive compounds [151].

α-lactoalbumina is a globular glycoprotein composed of 123 amino acid residues, with a molecular weight of approximately 14.2 kDa [152]. Its tertiary structure is stabilized by hydrophobic interactions, disulfide bridges, and its ability to bind metal ions such as calcium [153]. With moderate thermal stability, this protein becomes susceptible to denaturation at temperatures above 80 °C, especially in the absence of stabilizing cations [154]. The presence of calcium plays a crucial role in maintaining its structure and functionality, particularly in processes involving heat treatment and encapsulation of bioactive compounds [155].

Hydrophilic bioactive compounds, including water-soluble vitamins and phenolic acids, can associate with the polar regions of whey proteins primarily through hydrogen bonding and electrostatic interactions [51]. This binding confers improved stability to the bioactives by protecting them from degradation caused by oxidation, pH fluctuations, or light exposure. For instance, polyphenols such as caffeic and gallic acid have been shown to form stable complexes with β-lactoglobulin, enhancing their antioxidant capacity and retention in food matrices [156].

Ref. [157] investigated the impact of gastrointestinal digestion on the release and bioavailability of carotenoids and phenolic compounds isolated from red pepper residue, encapsulated with whey proteins using spray-drying and freeze-drying techniques. The phenolic compounds were more stable than carotenoids, exhibiting a gradual release compared to the rapid release of carotenoids under intestinal conditions within the first six hours. Encapsulation with isolated whey proteins protected the bioactive compounds from changes in intestinal pH and enzymatic activity, leading to significant improvements in bioavailability as well as nutritional and visual properties.

Conversely, hydrophobic bioactives, including fat-soluble vitamins (e.g., vitamin D), carotenoids, and curcumin, can be entrapped within the hydrophobic cores of whey protein structures [158]. The binding capacity of β-lactoglobulin is particularly notable due to the presence of a hydrophobic pocket capable of accommodating small nonpolar molecules [159]. For example, ref. [160] optimized the production of β-lactoglobulin nanostructures and applied them as carriers for bioactive molecules, achieving high encapsulation efficiencies for compounds such as quercetin, rutin, naringin, and vitamin B2. Additionally, ref. [161] developed β-lactoglobulin-based nanocomplexes composed of chitosan oligosaccharides for the delivery of astaxanthin, improving its stability and providing prolonged release in simulated gastrointestinal juices.

At pH values below their isoelectric point, whey proteins acquire a net positive charge, allowing electrostatic interaction with the negatively charged surface of probiotic cells [162]. This leads to the formation of a protective coating around the cells, which reduces susceptibility to thermal and acidic stresses. Moreover, hydrophobic interactions between whey protein molecules and microbial surfaces contribute to the formation of stable encapsulation matrices. For example, ref. [163] demonstrated that at pH values below their isoelectric point, whey proteins acquire a net positive charge, enabling electrostatic interactions with the negatively charged surface of probiotic cells. This results in the formation of a protective coating around the cells, reducing their susceptibility to thermal and acidic stresses.

Whey proteins are widely used in microencapsulation systems—such as spray-drying, freeze-drying, or complex coacervation—to enhance probiotic viability by providing a physical and biochemical barrier against adverse environmental conditions. These systems can also be tailored to achieve controlled release properties in gastrointestinal conditions. Additionally, whey proteins may serve as a nutritional substrate, promoting the growth and metabolic activity of probiotics, thereby contributing to their functional performance in symbiotic formulations [164].

Ref. [165] developed an electrospun whey protein nanofiber mat for the nanoencapsulation of omega-3, which enhanced the thermal stability of the compound by promoting strong interactions between the bioactive molecule and the wall material.

Overall, the multifunctional nature of whey proteins makes them valuable components in the design of delivery systems for bioactive compounds and probiotics, improving their stability, bioaccessibility, and therapeutic efficacy in functional food and nutraceutical applications.

3.2. Egg Proteins

Eggs are a rich source of high-quality proteins, comprising a wide variety of structural and functional proteins with diverse physicochemical properties. These proteins are distributed primarily between the egg white (albumen) and the egg yolk, each contributing distinct molecular structures and biological functionalities [166]. The primary proteins found in eggs include ovalbumin, ovotransferrin, ovomucoid, lysozyme, and avidin in the albumen, and lipovitellins, phosvitin, and livetin in the yolk [167].

Ovalbumin is the most abundant protein in egg white, accounting for approximately 54% of total albumen proteins [168]. It is a monomeric glycoprotein with a molecular weight of ~45 kDa, consisting of 385 amino acid residues, and its primary structure includes several serine, aspartic acid, and glutamic acid residues, contributing to its relatively acidic nature (pI ≈ 4.6) [169]. Ovalbumin has a high α-helical content (~30–40%) and forms a compact globular tertiary structure stabilized by hydrogen bonds and hydrophobic interactions but lacks disulfide linkages [170]. Ovotransferrin accounts for approximately 12–13% of egg white proteins and is a glycoprotein with a molecular weight of ~77 kDa that consists of two lobes, each capable of binding one ferric ion (Fe³⁺) in a synergistic complex with a bicarbonate anion [171]. The protein has extensive β-sheet regions and is structurally homologous to serum transferrin, whose iron-binding activity contributes to its antimicrobial properties [172]. On the other hand, ovomucoid represents about 11% of egg white proteins and is a highly glycosylated protein with a molecular weight of ~28 kDa, and it consists of three tandem Kazal-type serine protease inhibitor domains, which confer resistance to proteolytic enzymes like trypsin [173]. The molecule contains nine disulfide bonds and significant carbohydrate content (~25%), which contribute to its high thermal stability [82].

Egg proteins can form electrostatic and hydrogen bonds with hydrophilic molecules such as polyphenols, peptides, and vitamins (e.g., B-complex) [132]. Their polar side chains (e.g., serine, threonine, and glutamine) facilitate interactions with hydroxyl and amino groups of hydrophilic compounds, increasing the protection of the bioactive molecules [174].

Due to their amphiphilic nature, ovalbumin, ovotransferrin, and ovomucoid can encapsulate or bind lipophilic compounds such as carotenoids, essential oils, curcumin, and fatty acids [175]. Non-polar amino acid residues (e.g., leucine, isoleucine, and phenylalanine) form hydrophobic pockets that entrap lipophilic molecules. For instance, ref. [175] fabricated curcumin-loaded ovalbumin nanoparticles and characterized their structural and bioactive properties. The study demonstrated that the encapsulation process enhanced the antioxidant activity of curcumin, indicating that ovalbumin nanoparticles could serve as a viable carrier for curcumin delivery in food and other bioactive delivery systems.

3.3. Meat Proteins

Meat proteins are broadly classified into myofibrillar, sarcoplasmic, and stromal proteins [176]. The myofibrillar proteins (e.g., myosin, actin, and tropomyosin) account for 50–55% of total muscle protein, which exhibits fibrous structure, high molecular weight, and multiple domains with hydrophobic and charged residues [177]. The sarcoplasmic proteins (e.g., myoglobin, enzymes) represent 25–30% of muscle proteins, and they are generally globular, water-soluble, and rich in polar amino acids [178]. Finally, the stromal proteins (e.g., collagen, elastin) are found in connective tissue, characterized by repetitive sequences of glycine, proline, and hydroxyproline, forming triple helices [179].

These proteins display functional groups (e.g., amine, carboxyl, hydroxyl, and thiol) that participate in various chemical interactions. As an example, hydrophilic bioactives such as polyphenols, peptides, and water-soluble vitamins interact with polar residues (e.g., serine, threonine, and glutamine) in meat proteins form hydrogen bonds with hydroxyl, carboxyl, or amino groups of bioactives [180]. Furthermore, positively or negatively charged side chains (e.g., lysine, arginine, and glutamate) bind to oppositely charged molecules.

Hydrophobic compounds such as carotenoids, essential oils, and lipid-soluble vitamins interact with non-polar amino acid residues (e.g., leucine, valine, and phenylalanine) to create hydrophobic regions that accommodate lipid-soluble bioactives [181]. A recent chapter published by Springer discusses the use of bioactive compounds in meat products, including their encapsulation in forms such as nanoemulsions and microcapsules. The chapter highlights that encapsulation can enhance the stability of these compounds during processing and storage and may also aid in controlled release mechanisms. While the chapter focuses on various encapsulation techniques, it provides insights into the potential applications of meat proteins in encapsulating bioactive ingredients [45].

3.4. Vegetable Proteins

With the growing interest in sustainable and plant-based alternatives, plant-derived proteins have gained prominence as encapsulating agents due to their wide availability, environmental sustainability, and distinctive amino acid compositions and techno-functional properties [182]. Major sources include legumes (e.g., soy, peas, and lentils), cereals (e.g., wheat, rice, and corn), and pseudocereals (e.g., quinoa, amaranth) [183].

Plant proteins are primarily composed of polypeptides with a wide range of molecular weights and structural conformations. They are commonly classified based on solubility into the following four main categories: albumins (water-soluble), globulins (salt-soluble), prolamins (alcohol-soluble), and glutelins (alkali-soluble) [184].

Soy protein consists mainly of glycinin and β-conglycinin, both of which are globular proteins stabilized by disulfide bonds [185]. These proteins exhibit excellent emulsifying capacity and thermal stability, making them widely used in encapsulation processes [186]. Pea proteins, composed predominantly of legumin and vicilin, are known for their strong gel-forming ability and stable interactions with bioactive compounds [187]. In contrast, rice protein is primarily composed of prolamins—hydrophobic proteins that facilitate interactions with lipophilic compounds and demonstrate high thermal resistance [188].

Hydrophilic compounds such as polyphenols, peptides, and water-soluble vitamins (e.g., B-complex, vitamin C) interact with plant proteins through non-covalent mechanisms. Hydrogen bonding occurs via the hydroxyl, amide, and carboxyl groups of protein side chains interacting with polar functional groups of bioactives. Electrostatic interactions are established between charged amino acids (e.g., lysine, arginine, and glutamic acid) and ionized functional groups of bioactives, with the strength and nature of these interactions influenced by factors such as pH and ionic strength [189].

In a study by [190], soy proteins demonstrated a higher encapsulation efficiency for sour cherry pomace extract (94.9%) compared to whey proteins (90.1%). Although both types of microcapsules showed similar polyphenol retention over six weeks of storage at 25 °C, the particles formed with soy proteins were more effective in enhancing anthocyanin retention and color stability. Additionally, soy protein microparticles exhibited a slower rate of antioxidant degradation, highlighting their superior ability to stabilize hydrophilic compounds. Supporting these findings, ref. [191] reported that soy protein isolate effectively stabilized anthocyanin-rich extracts from jabuticaba (Plinia cauliflora) pomace, extending their shelf life during storage.

Plant proteins also show affinity for hydrophobic molecules such as carotenoids, essential oils, curcumin, and fat-soluble vitamins (A, D, E, and K). Non-polar amino acid residues (e.g., leucine, valine, and phenylalanine) located in the hydrophobic core of the protein interact with lipophilic bioactives. Certain proteins—particularly prolamins like zein—can self-assemble into colloidal structures capable of encapsulating and stabilizing hydrophobic compounds [192].

Regarding the protein source, ref. [193] evaluated soy and whey proteins as wall materials for the microencapsulation of curcumin (Table 3). The study demonstrated better gastrointestinal stability and curcumin absorption for microcapsules composed of soy protein compared to whey proteins, indicating a greater potential for the use of plant-based proteins in the encapsulation of bioactive compounds. However, the encapsulation efficiency of soy proteins was significantly lower than that of whey protein, highlighting a critical aspect to be further explored in the application of plant-based proteins.

Ref. [194] employed a pH variation modification (7-12-7) to encapsulate curcumin in reassembled soy protein nanoparticles. This process involved protein unfolding and refolding, resulting in an encapsulation efficiency of 97.43%, along with improvements in water solubility, thermal stability, and curcumin photostability (Table 3). Complementing these findings, ref. [73] demonstrated that self-assembled soy protein nanoparticles, obtained through partial enzymatic hydrolysis, protect curcumin during simulated gastrointestinal digestion and significantly enhance its gastrointestinal bioaccessibility (80%) compared to free curcumin (10%).

On the other hand, plant proteins can influence the viability and functionality of probiotics (Lactobacillus, Bifidobacterium) through interactions between protein surface residues and the microbial cell wall or membrane, potentially affecting adhesion and offering protective effects [195]. Additionally, during gelation or encapsulation processes, plant proteins form matrices that immobilize probiotic cells, shielding them from harsh conditions such as acidic or oxidative environments [196].

Ref. [197] developed nanoparticles from pea protein isolate encapsulated with chlorogenic acid using the anti-solvent method. The nanoparticles exhibited an encapsulation efficiency of 61.2% and increased the bioavailability of chlorogenic acid by 7.75% compared to free chlorogenic acid. Additionally, the antioxidant activity of chlorogenic acid was improved during simulated in vitro digestion. These results suggest that pea protein isolate nanoparticles can reduce the degradation of chlorogenic acid and enhance its biological activity.

Rice proteins are predominantly composed of prolamins and glutelins, characterized by their hydrophobic nature and high thermal resistance, attributes that favor the encapsulation of lipophilic compounds and stability under simulated gastrointestinal conditions [198]. The relatively low solubility of these proteins at neutral pH can be mitigated through physicochemical modifications such as thermal denaturation and pH adjustments, enhancing their applicability in micro- and nanoencapsulation systems [150,188]. Ref. [192] investigated enzymatic modifications of rice proteins for the microencapsulation of linseed oil and demonstrated that this strategy enhances the functional properties of the proteins, making them more effective in encapsulation systems and expanding their potential use in functional foods and nutritional supplements.

Wheat proteins, mainly composed of glutenins and gliadins, form three-dimensional protein networks with high gelation potential and mechanical resistance, attributes exploited in microencapsulation to protect sensitive bioactive compounds [199]. The glutenin fraction particularly contributes to structural stability through disulfide bonding, enhancing microcapsule robustness during thermal processing and prolonged storage [200].

Oat proteins mainly consist of avenalins and avenins, which exhibit significant solubility and notable emulsifying and film-forming properties [201]. These characteristics are fundamental for forming effective encapsulating matrices for hydrophilic compounds such as polyphenols and water-soluble vitamins. Additionally, oat proteins display inherent antioxidant activity, which can enhance the stability of encapsulated bioactives against oxidative degradation [72,202] investigated the effects of pH on the formation of complexes between oat protein isolate and high methoxyl pectin, evaluating their emulsifying properties and ability to encapsulate curcumin. The study demonstrated that pH adjustments significantly influence the interactions between these biopolymers, affecting emulsion stability and encapsulation efficiency. Results showed that, under specific pH conditions, the complexes exhibited enhanced stability and curcumin protection, highlighting the potential of combining plant proteins and polysaccharides in controlled release systems for functional food applications.

Proteins extracted from other legumes such as beans, chickpeas, and lentils exhibit diverse compositions, including albumins, globulins, and prolamins, conferring versatile functional properties applicable in microencapsulation [203]. These proteins are notable for their gel-forming ability and thermal stability, as well as their capacity to establish electrostatic and hydrophobic interactions with both hydrophilic and lipophilic bioactive compounds [204]. The study by [205] evaluated the thermal stability of bioactive compounds from annatto seed extract through ionic gelation microencapsulation using plant proteins from quinoa, lentil, soy, and sodium caseinate as wall materials. Encapsulation efficiencies ranged from 58% to 80%, depending on the protein structure and amino acid composition. The microcapsules exhibited enhanced thermal stability of polyphenols and bixin, with reductions of approximately 4% and 20% after 12 days at 65 °C, demonstrating the protective capacity of these encapsulating agents.

4. Encapsulation Techniques

The encapsulation of bioactive compounds is a widely used strategy to improve their stability, solubility, bioavailability, and controlled release. Different techniques can be employed depending on the properties of the bioactive compound and the intended application [206]. Each encapsulation technique has specific advantages and applications depending on the bioactive compound and desired functionality. The choice of technique depends on factors such as stability, release profile, scalability, and intended use in food, pharmaceutical, or cosmetic industries [207] (

Table 4. Encapsulation techniques for bioactives.

Technique	Principle	Advantages	Disadvantages	Reference
Spray Drying	Involves atomizing an emulsion containing the bioactive compound and the wall material into a hot air stream, forming dry microparticles.	Cost-effective, fast, scalable, continuous process, improves stability of bioactive, enhances solubility and dispersibility, customizable particle properties.	High thermal exposure can degrade sensitive compounds, low encapsulation efficiency for some hydrophobic compounds, high energy consumption, powder agglomeration and high hygroscopicity.	[25,208]
Complex Coacervation	Based on electrostatic interactions between positively and negatively charged polymers, forming a capsule around the bioactive compound.	High encapsulation efficiency, protection against degradation, targeted and controlled release of bioactives, biodegradability and biocompatibility, suitable for hydrophilic and hydrophobic compounds.	Highly dependent on pH and salt concentration, not always suitable for organic solvents, requires fine-tuning of polymer ratios, pH, temperature, and mixing conditions; instability during storage, potential allergenicity of some biopolymers (gelatin).	[209,210,211]
Liposomes	Vesicles composed of lipid bilayers that can encapsulate both hydrophobic and hydrophilic compounds, protecting them from degradation and enhancing their absorption.	Protection from enzymatic degradation, light, and pH variations, extending shelf life; ability to encapsulate both hydrophilic and hydrophobic compounds; generally non-toxic, biodegradable, and well-tolerated in biological systems; targeted and controlled release.	High production costs, limited stability, short circulation time in the body, risk of drug leakage when stored for long periods or exposed to temperature fluctuations, potential for oxidation and hydrolysis.	[212]
Nanoemulsions	Colloidal systems composed of nanometric oil-in-water or water-in-oil droplets stabilized by surfactants. They enhance the solubility of lipophilic compounds.	Improved bioavailability of hydrophobic bioactives, protection of bioactives from degradation by oxidation, light, and heat, increasing the shelf-life; controlled release, ormulated with plant-based oils and surfactants, considered biocompatible and non-toxic.	Stability issues, such as phase separation or coalescence; expensive production; some surfactants and oils used in the formulations could have toxicity potential; challenges in scaling up in terms of cost and stability; a lack of clear regulatory guidelines in some countries.	[142,213,214]
Cyclodextrin Complexation	Cyclodextrins form inclusion complexes with bioactive compounds, improving their solubility, stability, and bioavailability.	Improved solubility and bioavailability, protects bioactive from degradation due to environmental factors such as light, oxygen, and temperature; controlled release, low toxicity and biocompatibility.	Limited fncapsulation efficiency for compounds that do not fit well into the hydrophobic cavity of cyclodextrins; possible formation of unstable complexes; can be expensive to produce, limiting the large-scale commercialization; limited efficacy for rapid release; often require modification to improve the solubility or stability.	[215,216]
Extrusion	The bioactive compound is dispersed in a molten polymer matrix, followed by cooling to form microparticles or filaments.	Highly scalable, controlled release, protection from degradation due to light, oxygen, and moisture; wide variety of encapsulating materials (starch, lipids, proteins, and polysaccharides); homogeneity and uniformity.	High energy consumption, thermal degradation of sensitive compounds, limited control over release kinetics depending on the nature of the matrix, extrusion conditions, and the bioactive’s characteristics;possible structural changes, complicated process for highly hydrophilic compounds.	[59,217]
Interfacial Polymerization	Production of polymeric nanoparticles, where monomers react at the interface of an emulsion, forming capsules around the bioactive compound.	Controlled release, high encapsulation efficiency, protection of sensitive bioactives, versatile (can encapsulate hydrophilic, lipophilic, and amphiphilic bioactives), ability to form nanoparticles and microcapsules.	Uniformity of polymer can be challenging, potential for toxicity of residual solvents, limited scalability (effective at a laboratory scale), raw materials and equipment can be expensive, uneven polymerization.	[218,219]
Electrospinning	Uses an electric field to produce nanometric fibers containing bioactives, enabling controlled release and protection against degradation.	High surface area-to-volume ratio, manipulation of fiber diameter, morphology, and porosity, encapsulation of hydrophilic and hydrophobic bioactives; controlled release, minimal use of toxic solvents.	Requires precise control of multiple variables (polymer concentration, voltage, collector distance, and solvent evaporation), solvent residues in the final product, low yield for large-Scale production, instability of nanofibers over time; limited control over drug loading and release profile.	[46,220]
Hydrogels	Three-dimensional networks of hydrophilic polymers that can absorb large amounts of water, making them useful for controlled release of bioactive compounds.	Biocompatibility and biodegradability; high water retention capacity, controlled release and targeted delivery, non-invasive delivery of bioactive (formulated into injectable or topical forms), encapsulate wide range of bioactives (hydrophilic, hydrophobic, proteins, enzymes, or peptides).	Poor mechanical strength and be prone to deformation under stress; sensitive to environmental factors such as temperature and humidity; difficulty of scaling up production; release profile unpredictable; crosslinking agents used may be toxic or require further purification.	[221]

).

Encapsulated bioactives find diverse applications across agricultural, food, and pharmaceutical industries. These include polyphenols (such as flavonoids, phenolic acids, resveratrol, and green tea catechins), curcumin, carotenoids (including β-carotene, lycopene, and astaxanthin), probiotics (Lactobacillus spp., Bifidobacterium spp.), essential oils (like oregano, thyme, peppermint, cinnamon, and lavender), omega-3 fatty acids, vitamins (A, C, D, and E), antimicrobial peptides (e.g., nisin), as well as bioactive compounds such as quercetin, kaempferol, ibuprofen, and aloe vera gel, also benefit from encapsulation techniques [23,35].

5. Conclusions

The encapsulation of bioactive compounds using proteins has proven to be an effective solution for overcoming challenges related to the stability, bioavailability, and functionality of these compounds in functional foods. The specific interactions between proteins and bioactive compounds vary according to the chemical nature of both, with plant-based proteins, such as soy protein isolate, showing superior performance for hydrophilic compounds, while animal proteins, such as whey protein, exhibit better efficiency in encapsulating lipophilic compounds. To address these challenges, the formation of protein–polysaccharide complexes and protein modification prior to encapsulation have shown promising results in improving the encapsulation of lipophilic compounds. The application of technologies, such as self-assembly and protein modification has expanded the possibilities for encapsulation, leading to more stable and efficient systems. These studies highlight the importance of understanding the structure of the target bioactive compounds, the selection of appropriate proteins as wall materials, and the techniques and technologies available for obtaining bioactive particles. Therefore, future research should prioritize the development of more efficient encapsulation systems by exploring the synergy between proteins, bioactives, and advanced technologies, while also considering the scalability and sustainability of the proposed solutions.