Nanoencapsulation and delivery of bioactive ingredients using zein nanocarriers: approaches, characterization, applications, and perspectives

Abstract

Zein has garnered widespread attention as a versatile material for nanosized delivery systems due to its unique self-assembly properties, amphiphilicity, and biocompatibility characteristics. This review provides an overview of current approaches, characterizations, applications, and perspectives of nanoencapsulation and delivery of bioactive ingredients within zein-based nanocarriers. Various nanoencapsulation strategies for bioactive ingredients using various types of zein-based nanocarrier structures, including nanoparticles, nanofibers, nanoemulsions, and nanogels, are discussed in detail. Factors affecting the stability of zein nanocarriers and characterization methods of bioactive-loaded zein nanocarrier structures are highlighted. Additionally, current applications of zein nanocarriers loaded with bioactive ingredients are summarized. This review will serve as a guide for the selection of appropriate nanoencapsulation techniques within zein nanocarriers and a comprehensive understanding of zein-based nanocarriers for specific applications in the food, pharmaceutical, cosmetic, and agricultural industries.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-023-01489-6.

Introduction

Bioactive ingredients, such as phenolics (Zhu et al., 2021), vitamins (Weissmueller et al., 2016), carotenoids (Li et al., 2020), and essential oils (Tang et al., 2022), are known for their health-promoting properties. However, these bioactive ingredients are typically hydrophobic due to their non-polar chemical structure, which limits their applicability in food processing and impedes their absorption within the human body (Yuan et al., 2022). Additionally, these compounds are highly unstable and vulnerable to environmental stresses such as heat or light, which can greatly reduce their functional properties and efficacy when consumed. To address these challenges, nanoencapsulation of bioactive compounds in nanocarriers have emerged as a valuable approach for protecting and controlling the release of these compounds (Jayan et al., 2019). Nanocarriers, serving as nanoscale transport entities for substances like bioactive ingredients, offer additional advantages attributable to their diminutive size. This size advantage fosters a strong affinity for the mucus layer enveloping intestinal epithelial cells, which consequently leads to an extended residence within the gastrointestinal (GI) tract (Chen et al., 2006; Jayan et al., 2019). Moreover, the presence of nanocarriers with larger surface areas promotes the dispersion of bioactive ingredients, thereby enhancing their bioavailability (Gomez-Estaca et al., 2012). A potential concern related to nanocarriers involves the risk of inadvertent toxicity stemming from the specific nanomaterial employed. Inorganic nanomaterials, in particular, may pose a threat to the human body if they accumulate within specific cellular organelles (Wang et al., 2012). Ongoing research efforts are dedicated to crafting more efficient and safer nanocarrier solutions. Food-grade proteins emerge as particularly attractive candidates for the encapsulation and delivery of bioactive compounds.

Zein, a generally recognized as safe (GRAS) protein, is the primary storage protein from corn and accounts for 44–79% of the endosperm proteins (Lawton, 2002). Zein can be categorized into four components based on solubility, including α- zein (19–22, 23–24 kDa), β-zein (14 kDa), γ-zein (27 and 16 kDa), and δ-zein (10 kDa) (Lawton, 2002; Wang et al., 2022). α-Zein, which accounts for 80% of the total prolamine content in corn, is soluble in 95% ethanol and widely utilized in commercial zein products (Shukla and Cheryan, 2001). β- and γ-zein constitute approximately 10–15% and 5–10%, respectively (Luo and Wang, 2014). The isoelectric point (pI) of zein is 6.2, rendering it typically unstable within the pH range of 5–7, resulting in aggregation (Yuan et al., 2022). Moreover, below its pI, zein carries a positive charge, whereas pH levels above the pI yield negatively charged zein. Zein, characterized by a high proportion of hydrophobic amino acids, exhibits water insolubility and solubility in ethanol–water binary solvent environments ranging from 60 to 90% ethanol (Dai et al., 2018). This unique amino acid composition makes zein an ideal nanocarrier material for encapsulating hydrophobic bioactive compounds. Furthermore, zein possesses self-assembly capabilities, biocompatibility, and bioadhesive characteristics (Kasaai, 2018). Its resistance to digestive enzymes enables controlled release of bioactive ingredients loaded in zein nanocarriers within the gastrointestinal fluid (Patel and Velikov, 2014).

Numerous techniques have been studied for nanoencapsulation of active ingredients in zein nanocarriers, including emulsification, coacervation, and electrospinning, each presenting its distinct advantages and limitations. Bioactive compound-loaded zein nanocarriers typically range from 50 to 500 nm in size. Depending on the techniques applied, zein-based nanocarriers can form various structures, such as nanoparticle (Lei et al., 2023; Yang et al., 2022), nanofiber (Dehcheshmeh and Fathi, 2019), nanogel (Cardoso et al., 2022; Seok et al., 2018), and nanoemulsion (Tang et al., 2022). Characterizing zein-based nanocarriers is essential for assessing the stability of the bioactive compounds entrapped within them under diverse environmental conditions. Zein nanocarriers loaded with bioactive compounds find applications in delivering bioactive compounds in Pickering emulsion (Meng et al., 2020), food packaging (Mo et al., 2021), drug delivery (Correa et al., 2016) and pest control (Hanna et al., 2022).

To date, there is limited comprehensive information on the nanoencapsulation and delivery of bioactive ingredients using zein nanocarriers. This review aims to provide up-to-date insights into approaches, characterization, applications, and perspectives of zein-based nanocarriers loaded with bioactive ingredients. Various procedures for preparing zein nanocarriers are described, along with characterization approaches for bioactive ingredient-loaded zein nanocarrier structures. Furthermore, we summarize the additional applications of zein nanocarriers loaded with bioactive compounds in the food, pharmaceutical, cosmetic, and agricultural fields. This review article serves as a comprehensive and current reference for researchers in both academic and industrial settings.

Zein-based nanocarriers for nanoencapsulation and delivery of bioactive ingredients

Nanoparticle

Zein solid nanoparticles are prepared by dissolving zein and the bioactive compound in a solvent, typically ethanol, followed by controlled solvent removal, resulting in solid nanoparticles. These zein solid nanoparticles typically range from 100 to 400 nm, with the specific size contingent on the preparation method (Luo and Wang, 2014). Under scanning electron microscopy (SEM) observation, they exhibit a round shape, a smooth surface, and a compact structure, as shown in Fig. 1. Fish oil-loaded zein nanoparticles were reported with sizes ranging from 354.1 ± 8.1 to 456.5 ± 7.3 nm (Zhong et al., 2009). Nonetheless, zein solid nanoparticles are susceptible to limited stability in high-temperature and high-salt environments. Their vulnerability to degradation by proteases in the harsh gastrointestinal (GI) tract compromises their ability to effectively shield encapsulated bioactive compounds (Yuan et al., 2022). To address these limitations, researchers have investigated core–shell structures by applying biopolymer-based coatings to the surface of zein solid nanoparticles, enhancing encapsulation efficiency and stability. Zein serves as the core material encapsulating bioactive ingredients, while the shell, primarily composed of polysaccharides, imparts repulsion forces between the nanoparticles (Chen et al., 2019a). Round shapes and smooth surfaces were observed using SEM in curcumin and piperine-loaded zein-carrageenan core/shell nanoparticles, with a particle size of 408.8 ± 3.1 nm (Chen et al., 2020b).

Fig. 1.

Schematic representation of common zein nanocarrier.

Adapted with permission from Peñalva et al. (2015). Copyright 2015, Elsevier; Adapted with permission from Wang et al. (2022). Copyright 2022, Elsevier; Adapted with permission from Fereydouni et al. (2021) (CC BY 4.0). Copyright 2021, Springer Nature

Hollow nanoparticles represent another class of nanoparticles, featuring an inner lighter hollow core and a darker shell under transmission electron microscopy (TEM) observation (Khan et al., 2021), with particle sizes ranging from 50 to 500 nm (Khan et al., 2021). They possess a solid structure with an empty internal space and are characterized by dimensions in the nanoscale range, offering distinct advantages over their solid counterparts (Wang et al., 2016a, b). These benefits encompass lower density, increased loading and encapsulation capacity, and heightened efficiency (Wang et al., 2016a, b). Therefore, researchers have studied the use of hollow zein nanoparticles for encapsulating and delivering bioactive compounds such as curcumin (Hu et al., 2016), caffeic acid (Wusigale et al., 2021), folic acid (Wusigale et al., 2021), resveratrol (Khan et al., 2021). The typical production process of bioactive compound-loaded zein hollow nanoparticles involves the initial preparation of a sacrificial template, often sodium carbonate, as the primary core. Subsequently, a bioactive compound-loaded zein solution is applied to the template, and, finally, the templates are removed, resulting in the formation of a hollow core (Hassan et al., 2022; Wang et al., 2016a, b). However, a notable drawback of hollow nanoparticles lies in the complexity of their fabrication and the necessity for toxic solvents during template removal (Hassan et al., 2022). Sodium carbonate has been the most commonly studied sacrificial template as it can nanoprecipitate in ethanol and provide uniform nuclei for directing the hollow particle self-assembly process (Wusigale et al., 2021). However, using a sodium carbonate solution can increase the pH (above 10) due to the alkali nature of the sodium carbonate, making it unsuitable for encapsulating polyphenol compounds as they can get oxidized and damaged at high pH (Friedman and Jürgens, 2000; Wusigale et al., 2021). Therefore, other safe and inexpensive substitutes may need to be considered. Wusigale et al. (2021) proposed the use of calcium ions, sourced from calcium phosphate (CaP), to replace sodium carbonate as the template in the preparation process of folic acid and caffeic acid-loaded zein/chitosan hollow nanoparticles. Results showed that the particle size of zein hollow nanoparticles was affected by the pH and CaP concentration. At pH 9, zein is negatively charged, and the presence of CaP causes a decrease in particle size due to electrostatic interactions (Wusigale et al., 2021). An increase in CaP concentration from 0.625 to 1.250 mM leads to an increase in particle size, from 169.4 ± 8.7 to 254.1 ± 10.2 nm, due to the formation of larger crystal templates at higher calcium ion contents, resulting in a larger hollow space for the encapsulation of bioactive ingredients (Wusigale et al., 2021). Moreover, under TEM, the resulting hollow nanoparticles showed a larger particle size (110 nm) compared to the solid nanoparticles (~ 70 nm) due to the presence of an extra electron-dense layer (Wusigale et al., 2021).

Nanofiber

Bioactive compounds-loaded zein nanofibers are produced via the electrospinning technique, a rapid, economical, and scalable method that leverages electrostatic forces. Zein nanofibers exhibit a bead-free morphology. Higher bioactive compounds loading leads to increased viscosity and fiber diameter, resulting in nanofibers ranging from 100 to 1000 nm in size. Diverse properties of zein nanofibers can be obtained by adjusting electrospinning and fluid parameters, such as flow rate, spinning distance, applied potential, viscosity, dielectric constant, electrical conductivity, and surface tension (Mohammadian et al., 2020). Zein nanofibers possess distinctive characteristics, including an extracellular matrix-like network, high surface-to-volume ratios, tunable mechanical and wetting properties, notable porosity, and controlled cargo release profiles. These qualities make them outstanding nanocarriers for bioactive compounds (Mohammadian et al., 2020; Paliwal & Palakurthi, 2014). Researchers have extensively used zein nanofibers to encapsulate bioactive compounds for gastrointestinal delivery. Dehcheshmeh and Fathi (2019) investigated the encapsulation of saffron extract using the coaxial electrospinning technique to produce zein/tragacanth core–shell nanofibers. The obtained nanofibers were able to protect and delay the release of saffron. Furthermore, the researchers evaluated the release characteristics of the nanofibers under various media conditions, yielding distinct release values: 21.66% in saliva, 27.75% in hot water, 43.88% in gastric media, and 16.12% in intestinal media for saffron extract. These thermostable nanofibers have potential applications in the food industry, such as in chewing gum or tea bags (Dehcheshmeh and Fathi, 2019).

Nanogel

Nanogel is the nanoscale hydrogel, a three-dimensional network of cross-linked polymer chains that can hold a large amount of water or other fluids. Nanogels can create porous structures with variable pore sizes and adjustable configurations, offering a high loading capacity (Rezaei et al., 2019). They feature enhanced functionality, targetability, and controlled release properties (Seok et al., 2018). Nanogels typically exhibit spherical morphology and fall within the size range of 200–500 nm. They are frequently engineered to be responsive to specific stimuli, such as alterations in pH or temperature, rendering them versatile tools in the field of nutraceutical delivery systems. Zein nanogels have been studied for encapsulation and delivery of various bioactive ingredients, including curcumin (Ding et al., 2023; Seok et al., 2018), shikonin (Cardoso et al., 2022), and quercetin (Freitas et al., 2020). In 2018, Seok et al. (2018) proposed a novel hyaluronic acid (HA) cross-linked zein nanogel for the encapsulation of curcumin. The nanogels were prepared using the nanoprecipitation method, where curcumin and zein were first dissolved in 70% isopropyl alcohol solution, then rapidly poured into the HA solution, followed by oven evaporation to remove isopropyl alcohol, and finally centrifuged to wash the obtained zein/HA nanogels. The nanogels had a size range of 200–250 nm and exhibited minimal or zero toxicity on healthy fibroblast cells. In vivo experiments utilizing a CT26 tumor xenograft model demonstrated that curcumin-loaded Zein/HA nanogels significantly reduced tumor volume and weight when compared to Zein/HA nanogels alone, without causing any immunotoxicity (Seok et al., 2018). Later on, shikonin was encapsulated inside the Zein/HA nanogels, achieving a high encapsulation efficiency of 90% (Cardoso et al., 2022). The shikonin-loaded Zein/HA nanogels exhibited the potential to control heightened reactions in innate immune cells and in human tissues when the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is inappropriately active due to injury or illness (Cardoso et al., 2022). Zein nanogels is still in its early stages, future studies can focus on refining zein nanogel formulations for enhanced bioactive ingredient encapsulation.

Nanoemulsion

Nanoemulsions are oil in water (o/w) emulsions at the nanoscale (Ahmed et al., 2012). The zein nanoemulsion delivery system involves the solubilization of hydrophobic bioactive ingredients in the oil phase, which is then mixed with an aqueous phase containing an emulsifier. Nanoemulsions typically have a size range of 80–250 nm, owing to their tiny droplet sizes that grant enhanced stability against particle aggregation and gravitational separation (Ahmed et al., 2012). The morphology of nanoemulsions is generally spherical, as observed under SEM. A variety of bioactive compounds have been encapsulated and delivered within the zein nanoemulsion system, including ginger essential oil (Tang et al., 2022), resveratrol (Zhu et al., 2021), alga oil (Wang et al., 2016a, b), eugenol (Wang and Zhang, 2017), and lutein (Liu et al., 2021a, b). Ginger essential oil was encapsulated inside the zein/sodium caseinate nanoemulsion with high encapsulation efficiency (86.48%) and strong stability (Tang et al., 2022). The resulting antibacterial nanoemulsion was applied to chicken breast preservation, extending its shelf life by six days (Tang et al., 2022). In a study conducted by Zhu et al. (2021), resveratrol was encapsulated in a zein/polyglycerol-stabilized soybean oil-in-water nanoemulsion. The nanoemulsion showed remarkable stability during a 34-day storage period and had an encapsulation effectiveness of over 90%. Encapsulation prolonged the release of resveratrol and enhanced its antioxidant activity in simulated gastrointestinal environments (Zhu et al., 2021).

Overall, there are four primary types of zein nanocarriers, each suited to specific applications and bioactive compounds. Nanofibers necessitate specialized equipment, such as electrospinning, which can elevate production costs. Nanoemulsion may exhibit physical and chemical instability over time, primarily due to Ostwald ripening. Hollow nanoparticles excel in encapsulation efficiency, making them an excellent choice for delivering bioactive ingredients due to their high loading capacity. Nanogels represent a relatively recent development in the field of bioactive encapsulation and delivery, warranting further investigation in the future.

Nanoencapsulation techniques for bioactive ingredients-loaded zein nanocarriers

Numerous techniques have been explored to nanoencapsulate bioactive compounds within zein matrices. These techniques include electrohydrodynamic processes (electrospinning, electrospraying), coacervation, microfluidic systems, and antisolvent precipitation (Joye and McClements, 2014). The choice of encapsulation approach should be guided by the properties of the bioactive compounds and potential applications. Table 1 summarizes the process steps, pros, and cons of various nanoencapsulation techniques that have been applied to the formation of zein nanocarriers.

Table 1.

Comparison of techniques for formation of bioactive loaded—zein nanostructure

Techniques	Process	Advantages	Disadvantages	Examples
Anti-solvent precipitation	Adding a non-solvent to zein solution, causing the solute to supersaturate and form nanostructure	Low cost, simplicity, and scalability	Difficulty in controlling the size and size distribution, and potential for agglomeration or clustering	Chen et al., 2018; Joye & McClements, 2014; Lei et al., 2023
Electrohydrodynamic	Application of an electric field to zein solution to produce charged droplets then solidify into nanostructure	High yield and control over particle size and surface properties	Limited scalability, possible solvent residues, and potential for particle aggregation or deformation	Jayan et al., 2019; Karim et al., 2021; Torkamani et al., 2018
Coacervation	Phase separation of zein solution into a dense coacervate phase that encapsulates the target molecule	High encapsulation efficiency, and low toxicity	Limited control over particle size and distribution, potential for particle aggregation, and difficulty in scaling up the process	Chen et al., 2020b, a; Wang et al., 2018
pH-driven	Deprotonation and dissolution of hydrophobic bioactive ingredients under alkaline conditions, followed by neutralization to encapsulate the bioactive ingredients	Simplicity, organic solvent-free, and low energy	Limited control over particle size and shape, potential for low encapsulation efficiency, and sensitivity to pH changes	Cheng et al., 2017; Dai et al., 2019; Liu et al., 2021a, b; Yuan et al., 2021; Zhan et al., 2020
Layer-by-layer	Sequential adsorption of oppositely charged polymers on the colloidal particle	Control over particle size, shape, and surface properties, and potential for multi-functionalization	Complexity, and time-consuming	Chen et al., 2019b, c, d, a; Correa et al., 2016
Emulsification	Emulsification of the polymer in a solvent and followed by the evaporation of the solvent under certain conditions	Simplicity and scalability	Limited control over particle size and distribution, and particle aggregation	Wei et al., 2018, 2020

Anti-solvent precipitation

Anti-solvent precipitation (ASP), also referred to as liquid–liquid dispersion or desolvation, is a generally used method for producing zein nanoparticles (Sharif et al., 2019). This process has gained significant attention for encapsulating bioactive ingredients into the zein matrix because of its low cost, simplicity, and scalability (Joye and McClements, 2014). The general process involves dissolving the bioactive compounds in a solvent, followed by the addition of an antisolvent into the system to reduce the solubility of zein. This process leads to a supersaturated solution, resulting in zein precipitation (Joye and McClements, 2014; Shishir et al., 2018). The driving force behind this process is the imbalance in the molecular interactions between zein, solvent, and antisolvent (Joye and McClements, 2014; Sharif et al., 2019). Ethanol and water are commonly used as the solvent and antisolvent, respectively, for zein. Precipitation occurs at certain combinations of water, ethanol, and zein. However, zein nanoparticles produced via the ASP approach are unstable and prone to aggregation under extreme conditions such as high ionic strength, pH near the isoelectric point of zein (pH ~ 6.2), and elevated temperatures (Dai et al., 2018). Dai et al. (2018) observed that curcumin-loaded zein/rhamnolipid nanoparticles maintained a relatively small particle size at ionic strengths between 25 and 100 mM but exhibited larger particle sizes at higher NaCl concentrations (100 mM), indicating nanoparticle aggregation. To address this instability, researchers have employed methods like adding surfactants or blending with other biopolymers. Surfactants increase steric and electrostatic repulsion while reducing surface hydrophobicity, thus mitigating aggregation issues (Dai et al., 2017). Figure S1 illustrates the potential stabilization mechanism of tween-80, a non-ionic surfactant, on zein nanoparticles. Surfactants such as rhamnolipid (Dai et al., 2018), lecithin (Rodsuwan et al., 2021), tween-20 (Wang et al., 2019a, b) and tween-80 (Hu and McClements, 2014; Sun et al., 2020) have been reported in the literature for this purpose.

Anti-solvent co-precipitation (ASCP) is a novel approach that involves mixing zein with other polymers to create zein composite nanoparticles with improved stability and higher encapsulation efficiency (Dai et al., 2018). Biopolymers such as sodium caseinate (Davidov-Pardo et al., 2015), shellac (Chen et al., 2018), ethyl cellulose (Hasankhan et al., 2020), hyaluronic acid (Chen et al., 2019d), etc., have been incorporated into zein-based composite nanoparticles. Chen et al. (2019d) synthesized a zein/hyaluronic acid binary nanocomplex, which exhibited improved thermal stability and encapsulation efficiency compared to zein nanoparticles, with the encapsulation efficiency increasing from 36.34 to 93.56%.

Electrohydrodynamic processes

The electrohydrodynamic technique is a simple, cost-effective, and no-heat-required process for obtaining nanoencapsulation structures in the dry form (Anu Bhushani and Anandharamakrishnan, 2014), with electrospinning and electrospraying being the two most common methods used for this purpose. By applying a high electric field between the polymer solution and a grounded collector, encapsulated structures can be obtained (Gómez-Mascaraque et al., 2019), and various types of structures can be produced by adjusting solution parameters (pH, molecular weight, concentration, surface tension, and conductivity), process parameters (power voltage, flow rate, and distance between the needle tip and the collector), and ambient parameters (temperature, humidity, and air velocity) (Anu Bhushani and Anandharamakrishnan, 2014). Depending on the concentration of the polymer solution, electrospraying can produce particles, while electrospinning can produce fibers. At lower polymer concentrations, the jet from taylor cone becomes destabilized because of the varicose instability, and thus particles are formed (Anu Bhushani and Anandharamakrishnan, 2014). With higher concentrations of polymer solution, the jet becomes stabilized, and the whipping instability mechanism causes elongation and hence fibers are obtained (Anu Bhushani and Anandharamakrishnan, 2014).

In recent years, there has been continuous improvement and advancement in the electrospinning technique used to nanoencapsulate bioactive compounds in zein nanofibers. Electrospinning is especially beneficial since it does not require heating, and high encapsulation efficiency (usually above 80%) can be achieved for heat-sensitive bioactive compounds, such as flavonoids, vitamins, probiotics, fatty acids, and phenolic compounds (Jacobsen et al., 2018). Curcumin-loaded zein nanofibers were successfully prepared at room temperature with 50% relative humidity by electrospinning at a voltage of 15 kV and a tip-to-collector distance of 12 cm (Wang et al., 2017a, b). The obtained zein nanofibers showed excellent encapsulation efficiency, i.e., 98.67 ± 1.5%, and good antibacterial property against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Coaxial electrospinning has been proposed for encapsulation as it can greatly enhance the shelf life of bioactive compounds (Yang et al., 2017a), encapsulation efficiency (Anu Bhushani and Anandharamakrishnan, 2014) and stability (Torkamani et al., 2018) of the bioactive compounds. Compared with single electrospinning, coaxial electrospinning can manufacture continuous double-layer nanofibers by electrospinning two materials in a single step (Yang et al., 2017a). Yang et al. (2017b) discovered that coaxial electrospinning effectively enhanced the oxidative stability and shelf life of fish oil compared to single electrospinning. However, the release of encapsulated fish oil in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) environment was altered, with less fish oil released from the coaxial electrospun zein nanofibrous mat. To maintain both the improved oxidative stability and release behavior, the authors implemented a modified coaxial electrospinning technique, incorporating ferulic acid into the polymer solution. The results showed that the addition of ferulic acid enhanced oxidative stability without impacting the release behavior of the encapsulated fish oil (Yang et al., 2017a). Notably, the modified coaxial electrospinning technique allowed for the continuous and smooth generation of fish oil-loaded zein nanofibers, with no clogging problems (Yang et al., 2017a).

Electrospraying technique has also been studied for the nanoencapsulation of bioactive compounds in zein nanoparticle matrix. Gomez-Estaca et al. (2012) demonstrated the successful production of zein nanoparticles containing curcumin via electrospraying at a flow rate of 0.15 mL/h and a voltage of 14 kV. The curcumin-loaded zein nanoparticles exhibited excellent stability (~ 3 months) at room temperature, 43% relative humidity, and in the dark. Coaxial electrospraying has also been extensively researched for the encapsulation of bioactive compounds due to the benefits of developing a multilayer structure and enhanced protection (Gómez-Mascaraque et al., 2019). For example, zein and shellac have been employed as shell biopolymers to encapsulate thymol via coaxial electrospraying (Liu et al., 2021a, b). The results indicated that the thymol-loaded zein-shellac nanocapsules showed improved long-term storage stability and controlled-release property of thymol (Liu et al., 2021a, b).

Coacervation

Coacervation is a process that involves separating a colloidal system into two phases: a polymer-rich phase and a polymer-poor phase (Wang et al., 2018). The coacervate phase, or polymer-poor phase, can be collected and applied as the shell material for nanoencapsulation processes (Wang et al., 2018). This method has been thoroughly investigated for the development of zein nanoparticles loaded with bioactive compounds. Coacervation can be divided into two methods based on the number of polymers used in the encapsulation process: simple coacervation, which is based on one polymer, and complex coacervation, which involves two or more polymers. Complex coacervation offers the advantage of enhancing core material protection, improving encapsulation efficiency, and providing controlled-release properties (Shishir et al., 2018; Wang et al., 2018). Zein-polysaccharide complex coacervation is an effective approach to encapsulate bioactive compounds, improving encapsulation efficiency (Liu et al., 2022; Ren et al., 2019), loading capacity (Liu et al., 2022), and stability (Chen et al., 2020a). During complex coacervation, a polysaccharide coating is applied onto the zein surface, increasing surface hydrophilicity, electrostatic repulsion, and steric hindrance, ultimately leading to improved stability of the complex nanoparticles (Chen et al., 2020a). Chitosan, a cationic polysaccharide derived from shrimp shells, has emerged as a highly promising polymer for bioactive compound encapsulation. Numerous studies have reported on the self-assembly of zein-chitosan complexes for bioactive ingredients encapsulation (Chen et al., 2020a; Li et al., 2018; Ma et al., 2023; Ren et al., 2019).

pH-driven

The pH-driven method, also known as pH-shifting or pH-cycle (Zhan et al., 2020), is an eco-friendly, low-energy, organic solvent-free, and straightforward approach for encapsulating bioactive compounds (Yuan et al., 2021). This technique has been employed in the preparation of zein nanoparticles. This process involves the deprotonation and dissolution of hydrophobic bioactive compounds under alkaline conditions (pH ~ 12), followed by neutralization (pH ~ 7) to encapsulate them (Cheng et al., 2017). To date, the pH-driven method has been used with zein to encapsulate various bioactive compounds, including curcumin (Dai et al., 2019; Wang et al., 2019a, b; Yuan et al., 2021; Zhan et al., 2020), pterostilbene (Liu et al., 2021a, b), and eugenol (Wang and Zhang, 2017). It's noteworthy that the pH-driven method may not be suitable for all bioactive ingredients due to potential degradation in the alkaline environment (Yuan et al., 2022). Curcumin, characterized by a low degradation rate under alkaline conditions (Pan et al., 2014; Yuan et al., 2022), is a frequently studied core material. Additionally, given zein's isoelectric point approximately 7, employing zein in the pH-driven approach can lead to precipitation. Hence, surfactants or biopolymers have been employed to enhance zein nanoparticle stability (Dai et al., 2019; Yuan et al., 2021). For example, Dai et al. (2019) added rhamnolipid, a glycolipid surfactant produced by bacteria, to the fabrication process of curcumin-loaded zein nanoparticles and found that it enhanced the stability against aggregation of the nanoparticles. In another study, Yuan et al. (2021) added tea saponin, a biosurfactant extracted from camellia seed by-products, to develop stable zein/tea saponin composite nanoparticles for encapsulating curcumin. The resulting nanoparticles were stable under various conditions, such as pH ranges of 5–8, heating at 80 ℃, and 100 mM of ionic strength, and were able to remain stable for 30 days at 25 ℃.

Layer-by-layer assembly

The layer-by-layer (LBL) assembly technique involves sequential depositing of positively and negatively charged biopolymers onto colloidal particles (Correa et al., 2016). It has been used for bioactive ingredient encapsulation within zein nanoparticles. Depending on the polarity of the bioactive ingredient, it can be encapsulated at various locations within an organized delivery system. For example, hydrophilic bioactive ingredients are typically enclosed within a hydrophilic shell, while hydrophobic bioactive ingredients are usually located in a hydrophobic core (Chen et al., 2019b). Chen et al. (2019b) proposed using the LBL approach to co-encapsulate and co-deliver two water-insoluble nutraceuticals, curcumin, and quercetagetin. They encapsulated curcumin inside the zein nanoparticle, followed by absorbing quercetagetin onto the surface of the zein nanoparticle, which was then coated with hyaluronic acid (HA), a polysaccharide. Due to quercetagetin's higher polarity, attributed to its greater number of hydroxyl groups compared to curcumin, quercetagetin was adsorbed onto the surface of the zein core, while curcumin was encapsulated within the zein core (Chen et al., 2019b). The resulting nanoparticle exhibited excellent encapsulation efficiency (69.8%, 90.3%) and loading capacity (2.5%, 3.5%) for curcumin and quercetagetin, respectively, with enhanced stability to light, thermal, and storage conditions. In another study, the same research group investigated the co-encapsulation of two nutraceuticals with opposite polarity, curcumin, and piperine (Chen et al., 2019b). They coated curcumin-encapsulated zein nanoparticles with HA, followed by coating of piperine on the HA layer, and finally coated the particles with chitosan as the outer layer. The resulting nanoparticles also demonstrated improved light, thermal, and storage stability at 4 ℃ for 2 months.

Emulsification-evaporation

The emulsification-evaporation process involves emulsifying zein and bioactive compounds in a solvent, typically ethanol, followed by solvent removal through rotary evaporation under specific conditions, including high temperature, vacuum, or stirring, to create zein nanocarriers. This method has been utilized in the development of bioactive compounds-loaded zein nanoparticles (Wei et al., 2018, 2020). For example, Wei et al. (2018) encapsulated β-carotene in zein-propylene glycol alginate nanoparticles using this process, which resulted in good encapsulation efficiency and loading capacity of 69.37 ± 0.04% and 1.73 ± 0.00%, respectively. However, it is important to note that this process can lead to structural changes in the zein. Wei et al. (2020) reported that after the emulsification-evaporation process, the α-helix content decreased while the β-sheet content increased, indicating a transition from a disordered to an ordered secondary structure state (Wei et al., 2020).

In summary, six nanoencapsulation techniques for developing bioactive ingredient-loaded zein nanocarriers have been discussed above. Electrohydrodynamic processes demand specialized equipment, which can result in increased costs. LBL encapsulation exhibits complexity and is time-consuming. Emulsification, coacervation, and anti-solvent precipitation methods may lead to particle aggregation. pH-driven encapsulation is a favorable strategy as it necessitates no organic solvents and consumes less energy.

Characterization of bioactive ingredients-loaded zein nanocarrier structure

Morphology

A range of techniques have been utilized to examine the morphology of zein nanocarriers loaded with bioactive ingredients. These include optical/light microscopy (OM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and confocal laser scanning microscopy (CLSM). A comparative summary of these characterization techniques for bioactive-loaded zein nanocarriers is presented in Table 2.

Table 2.

Comparison of characterization techniques for bioactive—loaded zein nanocarriers

Techniques	Pre-treatment methods	Measurement environment	Maximum magnification	Results	Advantages	Disadvantages	References
Optical/light microscopy	-	Air/liquid	1000 ×	Microstructure	Cost-effective	Low resolution, insufficient comprehensive insights into structures and attributes	Klang et al., 2012; Vernon-Parry, 2000
Scanning electron microscopy	Sample coated with conductive material	Vacuum	500,000 ×	Three-dimensional surface characteristics	Visualizing large specimens at high magnification	Characterize sample surface only	Eaton et al., 2017; Falsafi et al., 2020; Inkson, 2016
Transmission electron microscopy	Dilute samples, place on carbon-coated copper grids; stain required for specific samples	Vacuum	1,000,000 ×	Two-dimensional surface characteristics	Visualize sample internal structure	Relative high cost, observe thin samples only	Eaton et al., 2017; Inkson, 2016
Atomic force microscopy	Deposit sample on a mica and dry	Vacuum/air/ liquid	100,000,000 ×	Three-dimensional surface structure	Measure interphase thickness	Limited magnification and vertical range	Eaton et al., 2017; Luo, 2020; West, 2007)
Confocal laser scanning microscopy	Sample requires staining	Air/liquid	1000 ×	Structural characteristics	Measure wall thickness	Slow imaging	Falsafi et al., 2020; Mu et al., 2018

Optical microscopy (OM), also known as light microscopy, is a technique employed to visualize tiny materials using visible light and a set of lenses. In a study by Chen et al. (2014), OM was applied to capture the image of tangeretin (TGN)-loaded zein/β-lactoglobulin nanoparticles, as shown in Fig. 2. At higher concentrations of TGN, specifically at 0.8% (w/v), large crystals were observed, which can be attributed to the supersaturation of TGN in the organic and aqueous phases mixture (Chen et al., 2014; McClements, 2012). Whereas OM's reliance on visible light, characterized by a wavelength in the range of hundreds of nanometers, imposes limitations on achievable resolution while inspecting nanostructures. It becomes challenging to differentiate features smaller than the wavelength of light. Consequently, optical microscopy may not offer comprehensive insights into nanoscale attributes and structures, leading to potential inaccuracies or incomplete depiction of nanostructures. To overcome this limitation, advanced techniques like electron microscopy is frequently employed, delivering enhanced resolution and more profound insights into nanoscale structures. SEM is used to focus on the study of the surface morphology, which has been used to observe the three-dimensional morphology of the zein nanocarrier structure before and after encapsulation of bioactive compounds. For instance, Fereydouni et al. (2021) reported that encapsulation of curcumin (CUR) nanoemulsion at a volume of 15% (v/v) within zein nanofibers resulted in the formation of thinner and more uniform nanofibers (Fig. 2). Besides, the absence of aggregates on the nanofiber surface suggested that the Nano-CUR was effectively mixed with the polymer solutions (Fereydouni et al., 2021). The AFM provides more precise information about a single protein particle over other types of scanning probe microscopies (Kasaai, 2018). However, SEM and AFM are only able to observe the surface topography, and it is hard to visualize the encapsulation property inside the zein nanocarrier structure (Fig. 2).

Fig. 2.

Morphology characterization of bioactive ingredients loaded—zein nanocarrier: Optical microscopy (OM) images of the tangeretin (TGN)-loaded zein/β-lactoglobulin nanoparticles with tangeretin at different concentrations, i.e., 0.1% and 0.8% (w/v). Adapted with permission from Chen et al. (2014). Copyright 2014, Elsevier; scanning electron microscopy (SEM) of zein nanofibers (NFs) and zein/nano-emulsion of curcumin (Nano-CUR) 15% NFs (v/v). Adapted with permission from Fereydouni et al. (2021) (CC BY 4.0). Copyright 2021, Springer Nature; atomic force microscopy (AFM) images of zein/lecithin 1:1 and zein/lecithin 5:1 composite colloidal nanoparticles. Adapted with permission from Dai et al. (2016) (CC BY 4.0). Copyright 2016, Public Library of Science; transmission electron microscopy (TEM) image of vitamin E-acetate (VitE-Ac) loaded-zein/sodium casinate (CAS) nanocarriers. Adapted with permission from Weissmueller et al. (2016). Copyright 2016, American Chemical Society; confocal laser scanning microscopy (CLSM) images of the emulsions prepared with algae oil to zein (O/Z) at ratio of 5:1. Green corresponds to the oil environment, and red corresponds to the zein environment. Adapted with permission from Chen et al. (2019c). Copyright 2019, Royal Society of Chemistry

TEM can be a very useful technique to observe the structure of the zein nanocarrier for encapsulation by observing the internal composition of the material and creating a two-dimensional image by applying an electron beam passing through the sample. For instance, Weissmueller et al. (2016) characterized nanocarriers loaded with vitamin E-acetate (VitE-Ac) using zein and sodium caseinate (CAS) using TEM (Fig. 2). The particles exhibited a uniform size distribution with a dark core composed of VitE surrounded by a faint halo of zein and casein. CLSM is a commonly used technique in cell biology that relies on fluorescence excitation to visualize the structural characteristics and protein distribution in fixed tissue (Pygall et al., 2007). However, it can also be applied to observe the structure of nanocarriers for encapsulation. For example, Chen et al. (2019c) used CLSM to visualize the encapsulation inside the zein-based emulsions. In this study, algae oil was encapsulated inside the zein shell structure, and the developed emulsions were stained by a mixture of Nile red and Nile blue and observed under the excitation wavelengths of 488 and 633 nm. CLSM images of the zein particle without and with loading of algae oil are shown in Fig. 2, where the red and green areas correspond to the zein and algae oil environments, respectively. From the CLSM characterization, the authors concluded that the algae oil-zein core–shell structure was formed, which formed larger droplets compared with the zein particles without oil loading. CLSM could also be used to measure the thickness of the shell, as marked with an arrow in Fig. 2.

Factors affecting stability

Zein nanoparticles are the most prevalent type of zein nanocarrier structure. However, food and beverage products that contain various functional ingredients can interact in ways that result in instability (McClements, 2015). Despite the benefits of using zein nanoparticles to encapsulate bioactive ingredients, their stability under environmental conditions is still problematic. Zein nanoparticles are prone to forming aggregates, precipitating in formulations, failing to encapsulate and protect the bioactive ingredients (Chen and Zhong, 2014). The inter-particle hydrophobic interaction causes a thin layer of particles to form at the bottom of the container (Donsì et al., 2017). Surface-coating techniques have been explored to enhance the stability of zein nanocarrier structures. An anionic material can be coated onto the surface of zein nanoparticles by electrostatic attraction to stabilize and prevent sedimentation or aggregation. Various coating materials, such as polysaccharides, proteins, and surfactants applied to zein nanoparticles, have been extensively reviewed (Yuan et al., 2022). In this section, we will focus on the factors that have been mostly studied to impact the stability of bioactive compounds loaded in zein nanoparticles (Fig. 3).

Fig. 3.

Factors affecting the stability of bioactive compounds loaded-zein nanocarriers

pH stability

The pH levels present during food production, storage, and digestion can vary greatly, altering the characteristics of nanoparticles used in different food systems (Zhan et al., 2020). Therefore, it is necessary to study the pH stability of zein nanocomplex under different pH levels. To characterize the stability, zein nanoparticle dispersion is typically adjusted to various pH values, and the properties of dispersion, such as particle size (Li et al., 2020; Rodsuwan et al., 2021; Yao et al., 2018; Zhan et al., 2020), polydispersity index (PDI) (Li et al., 2020; Yao et al., 2018) and zeta-potential (Li et al., 2020; Rodsuwan et al., 2021; Zhan et al., 2020), are measured. For example, in a study on curcumin loaded-whey protein isolate (WPI)/zein composite nanoparticles, the particle size and zeta-potential of the dispersions were evaluated after adjusting the pH to 2–8 (Zhan et al., 2020). The nanocomposites were found to be more stable at pH 2, 3, 6, 7, and 8, as no aggregation was observed, and the particle size remained the same as the original dispersion. This result may be partially attributed to the comparatively large electrostatic repulsion between nanoparticles at pH values distant from WPI's isoelectric point (Zhan et al., 2020).

Ionic strength stability

Ionic strength is a term used to describe the concentration of ions present in a solution. In the case of gastrointestinal fluids, they are characterized by a relatively high ionic strength and comprise both monovalent and multivalent ions, including sodium, potassium, calcium, chloride, and bicarbonate (McClements, 2015). These mineral ions can have a significant impact on the properties of colloidal delivery systems. Specifically, they can either decrease the electrostatic repulsion between colloidal particles, resulting in particle aggregation and precipitation of nanoparticles, or they can alter the electrostatic interactions between different components within a colloidal particle, affecting its stability and the release of bioactive compounds (Israelachvili, 2011; McClements, 2015). Thus, it is crucial to investigate the ionic strength stability of zein nanoparticles to optimize their performance under different environments. To evaluate their stability, sodium chloride at different concentrations was typically mixed with zein nanocarriers. Gali et al. (2022) conducted a study on the ionic strength stability of the rutin-rich extract from Ruta chalepensis L.-loaded zein/gum arabic nanoparticles and assessed the stability by measuring the hydrodynamic diameter of zein-based nanoparticles under various ionic strengths (i.e., 25–100 mM at pH 4). The results indicated that zein-based nanoparticles were stable at low ionic strengths but exhibited aggregation tendencies at high ionic strengths, while zein/gum arabic nanoparticles demonstrated enhanced stability compared to zein nanoparticles. The presence of sodium chloride can give rise to attractive interactions, such as Van der Waals forces and hydrophobic effects, which can result in the particle aggregation if they overpower the repulsive forces (Dai et al., 2016; Gali et al., 2022).

Thermal stability

The thermal stability of zein nanoparticles was assessed by subjecting zein nanoparticle dispersions to varying temperatures for set periods of time, including 37 °C (Luo et al., 2013), 60 °C (Yu et al., 2020a, b), 75 °C (Wei et al., 2018), 80 °C (Yu et al., 2020a, b), 85 °C (Wei et al., 2018), and 95 °C (Wei et al., 2018). The properties of the dispersions, such as the retention rate of bioactive ingredients, were then analyzed. For instance, the effect of heating on the stability of curcumin-loaded zein/carboxylic curdlans nanoparticles was studied by subjecting the nanocomplex to 60 °C and 80 °C (Yu et al., 2020a, b). The findings showed high curcumin retention rates of 77.5% and 89.2% when heated at 60 °C for 30 min and 80 °C for 1 min, respectively. These results demonstrate that curcumin-zein/carboxylic curdlans nanoparticles exhibit improved thermostability compared to unencapsulated curcumin (Yu et al., 2020a, b).

Storage stability

The purpose of storage stability is to assess the extent to which bioactive ingredients loaded into zein nanoparticles are retained over a specified period, such as 15 (Yuan et al., 2020), 30 (Gong et al., 2021), 84 (Zhang et al., 2019) or 120 (de Oliveira et al., 2019) days. To prevent the growth of mold or bacteria during storage, a solution of sodium azide (NaN₃) was added to the dispersion. Yuan et al. (2020) examined the storage stability of curcumin loaded into zein/dextran sulfate nanoparticles for 15 d and observed that the retention rate of curcumin inside zein nanoparticles coated with dextran sulfate was significantly higher than curcumin entrapped inside zein nanoparticles or free curcumin, indicating that encapsulation inside the zein nanocomposite improved the storage stability of curcumin.

Photostability

The assessment of photostability aims to evaluate the retention of bioactive compounds loaded in zein nanoparticles when exposed to UV or natural light sources. In a study by Lin et al. (2020), natamycin-loaded zein/carboxymethyl chitosan nanoparticle dispersions were placed in a petri dish and exposed to UV light with a wavelength of 365 nm from a height of 30 cm. The retention rate of natamycin was calculated after UV treatment every 15 min for 90 min. The results indicated that the retention rate of natamycin was enhanced after being encapsulated inside the zein nanocomplex as compared to the pure natamycin. The authors suggested two possible explanations for the improved photostability. Firstly, the hydrophobic cavity of zein may entrap the unstable and hydrophobic macrocyclic region of natamycin, blocking UV light-induced degradation. Secondly, the amino acids with aromatic rings or double bonds present in the zein structure may absorb UV light (Lin et al., 2020). In addition to UV light, natural light sources have also been used to examine the photostability of zein nanoparticles. Ren et al. (2022) conducted a study on the impact of natural light on the photostability of zein–N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC) complexes loaded with curcumin. The findings revealed that the absorbance of free CUR decreased significantly after 3 h of natural light exposure and dropped to almost zero after 12 h, whereas the curcumin enclosed inside the Zein-HTCC complexes remained stable and demonstrated high absorption intensity even after 12 h of treatment.

Stability characterization

Visual properties, particle size and shape, polydispersity index, and zeta potential are tools for assessing the stability of zein nanoparticles loaded with bioactive ingredients. Two methods are employed to evaluate the visual observation of zein nanoparticles, namely photographs and microscopy. Photographs of zein nanoparticle dispersions are used to intuitively display the results. Any sedimentation or aggregation observed at the bottom of the dispersion indicates an unstable dispersion system. SEM, TEM, and CLSM are used to visualize the shape of the nanoparticles, as previously discussed. The round shape of zein nanoparticles implies good stability. The dynamic light scattering (DLS) technique is typically employed to measure particle size and PDI. Because the zein nanoparticle dispersion is usually concentrated, samples are diluted using DI water with a pH similar to the nanoparticle dispersion before measurement. Various dilution factors were used depending on the sample type, such as a dilution of 40 (Olenskyj et al., 2017) or 50–100 times (Chen and Zhong, 2014). A lack of significant change in particle size and PDI after treatment suggests good stability of the zein nanoparticle dispersion. The zeta potential, which is typically measured using a zeta-potential analyzer, is a valuable tool for characterizing the stability of colloidal dispersions. A zeta potential value higher than ± 30 mV indicates strongly charged particles generating physically stable dispersions because of the load-imposed electrostatic repulsion (da Rosa et al., 2015). Dilution is required before the measurement, similar to DLS characterization, to reduce particle contact and prevent isoelectrostatic precipitation (Feng and Lee, 2016).

Biological activity

Figure 4 presents the relationships between bioavailability, bioaccessibility, and bioactivity, which are the three primary biological activities considered when evaluating bioactive ingredients loaded in zein nanocarriers. Bioaccessibility is defined as the proportion of a nutrient or compound that is released from the food matrix and made available for intestinal absorption in the gastrointestinal lumen (Rein et al., 2013; Saura-Calixto et al., 2007). It is evaluated using in vitro digestion models that mimic gastric and small intestine digestion, and Caco-2 cell uptake is sometimes used as the final step (Carbonell-Capella et al., 2014). In a study by Yang et al. (2022), zein-Mesona chinensis polysaccharide nanoparticles with entrapped curcumin were first digested in SGF to separate indigestible substances from the sample, and then transferred to SIF. The bioaccessibility of curcumin was calculated by determining the ratio of the curcumin content in the supernatant to the total amount. The authors found that the bioaccessibility of curcumin was significantly improved in zein-Mesona chinensis polysaccharide nanoparticles compared to curcumin entrapped inside zein nanoparticles (Yang et al., 2022).

Fig. 4.

Relationship between bioavailability, bioaccessibility, and bioactivity and their prospective investigation models.

Adapted with permission from Dima et al. (2020). Copyright 2020, John Wiley and Sons

Bioactive components can be difficult to absorb in the GI system causing their low bioavailability, which refers to the rate and extent that the bioactive substances are absorbed and utilized by the body (Rein et al., 2013). However, nanoencapsulation has emerged as an effective approach to improving the bioavailability of bioactive ingredients. By encapsulating the compounds in nanocarriers, it becomes easier for the bioactive ingredients to bypass biological barriers and prevent metabolic changes that can result in poor absorption (Rein et al., 2013). Astilbin, a type of flavonoid found in plants, is known for its bioactivity, but has low oral bioavailability due to poor solubility and permeability (Zheng and Zhang, 2019). To address this limitation, nanoencapsulation techniques using zein-caseinate nanoparticles have been developed to enhance the bioavailability of astilbin in rats. The bioavailability increased from 0.32% (free astilbin) to 4.40% (Zheng and Zhang, 2019). Similarly, Peñalva et al. (2017) reported a significant increase in the oral bioavailability of quercetin in rats after it was encapsulated in zein nanocarriers, from 4% (free quercetin) to 60%.

Bioactivity refers to the specific physiological reaction (e.g., antioxidant, anti-inflammatory) that occurs upon exposure to a component, taking into account tissue absorption (Carbonell-Capella et al., 2014). This property could be assessed using in vivo, in vitro and ex vivo models (Carbonell-Capella et al., 2014). The antioxidant, antimicrobial, and anticancer properties of zein nanocomplex-encapsulated bioactive compounds have been investigated. Generally, the antioxidant capacity of the bioactive compounds after encapsulation is determined using assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS), or ferric reducing antioxidant potential (FRAP) activities. Based on the results of ABTS and FRAP experiments, the in vitro antioxidant property of resveratrol inside zein-pectin nanoparticles was greatly improved compared to free resveratrol (Huang et al., 2019). The antimicrobial activity of encapsulated bioactive compounds is evaluated by their ability to inhibit the growth of the microorganisms, such as gram-positive bacteria, i.e., staphylococcus aureus (S. aureus) (Gonçalves da Rosa et al., 2020), Listeria monocytogenes (Chen et al., 2015), gram-negative bacteria, i.e., Pseudomonas aeruginosa (P. aeruginosa) (de Souza Tavares et al., 2021), Escherichia coli (Karim et al., 2021), Salmonella enterica serovar Typhimurium (Gonçalves da Rosa et al., 2020), as well as fungi, namely, Candida spp (Araujo et al., 2021). Nanoencapsulation of anacardic acid inside zein matrix doubled its inhibitory action against S. aureus and rendered it active against Candida auris and P. aeruginosa (Araujo et al., 2021). Bioactive ingredients can interact in combination to produce different effects, such as antagonistic, synergistic, or additive effects. Combining two bioactive compounds generally results in synergy, which increases the antimicrobial activity beyond the total antimicrobial activities of the separate components (Chouhan et al., 2017). Co-encapsulation of thymol and eugenol inside zein nanoparticles exhibited a synergistic effect against E. coli O157:H7 and L. monocytogenes, outperforming non-encapsulated samples in terms of long-term efficacy against E. coli O157:H7 (Chen et al., 2015). The anticancer/antitumor activity of bioactive ingredients inside zein nanocomplexes has been reported in various studies (Dong et al., 2016; Huang et al., 2017; Kaushik et al., 2020; Shinde et al., 2020; Wang et al., 2017a, b; Yang et al., 2022). The most common method for evaluating the anticancer properties of entrapped bioactive ingredients inside zein nanocomplexes is by studying their effect on cancer cell viability. For instance, resveratrol was nanoencapsulated inside zein/pectin core–shell nanoparticles, and its anticancer activity was assessed by its anti-proliferative activity against hepatocarcinoma cells using the tetrazolium reduction (MTT) assay method (Huang et al., 2017). The results showed that nanoencapsulated resveratrol exhibited significantly greater cytotoxicity than free resveratrol, with a lower half-maximal inhibitory concentration (IC50) value of 77.2 μM compared to 112.0 μM for non-encapsulated resveratrol (Huang et al., 2017).

Applications of bioactive ingredients-loaded zein nanocarrier

Pharmaceutical

The use of drug-loaded nanocarriers has emerged as a promising strategy in pharmaceutical applications, particularly in cancer therapy. In this regard, Ghobadi-Oghaz et al. (2022) have reported on the development and evaluation of zein-chitosan nanoparticles for the simultaneous delivery of curcumin and berberine, two natural compounds with potential health benefits. The authors assessed the efficacy of the nanoparticles on A549 and MDA-MB-231 breast cancer cells and HFF normal cells in vitro. The results showed that the nanoparticles effectively inhibited cancer cell growth while exhibiting low toxicity on normal cells. The researchers suggest that the co-delivery of curcumin and berberine using zein-chitosan nanoparticles exhibits synergistic effects on multiple cancers, indicating a promising strategy for cancer therapy (Ghobadi-Oghaz et al., 2022). In another study, Wang et al. (2017a, b) developed zein nanocomplexes by employing gold nanoparticles as the core and folate-conjugated zein–polydopamine shell to realize the targeted delivery of HCPT, a potential anti-cancer drug (Fig. 5a). The nanocomplexes exhibited favorable characteristics including small dimensions, robust stability, and substantial drug loading capacity. Moreover, they demonstrated potent inhibition of cancer cell growth and displayed commendable biocompatibility. Notably, zein has also been explored as a carrier for delivering anti-COVID-19 drugs. For example, Rejinold et al. (2021) have reported on the development of Bovine Serum Albumin (BSA)-coated niclosamide-zein nanoparticles as a potential injectable medicine against COVID-19. The study found that these nanoparticles effectively inhibited the replication of the SARS-CoV-2 virus in vitro, indicating their potential as a candidate for further investigation as a treatment for COVID-19. The use of nanomaterials to encapsulate nanocrystal drugs offers a promising approach to administer drugs in a controlled and predictable manner, with potential applications in targeted drug delivery such as cancer therapy and COVID-19 treatment.

Fig. 5.

(A) In vivo biodistribution of cy5-labeled hydroxycamptothecin (HCPT)-loaded zein nanocomplexes at various time intervals. Adapted with permission from Wang et al. (2017a, b). Copyright 2017, Elsevier; (B) schematic diagram of different interfacial arrangements at various zein: sodium caseinate ratios. Adapted with permission from Feng and Lee (2016). Copyright 2016, Elsevier; (C) opened avocados from representative samples of the 5 treatment groups on day 15 and 21 of ambient storage. Adapted with permission from Garcia et al. (2022) (CC BY 4.0). Copyright 2022, Elsevier; (D) operational principle and pathway of zein-hyaluronic acid-tetrahydrocurcumin (Z-HA-THC) nanoparticles proposed to relate anti-aging efficacy with intracellular changes of skin cells. Adapted with permission from Zhu et al. (2022). Copyright 2022, Elsevier; (E) zein nanoparticles (ZNPs) entrapped with methoxyfenozide (MFZ) as effective delivery systems capable of enhancing the translocation of nonsystemic agrochemicals from the roots to the leaves of soybean plants. Adapted with permission from Hanna et al. (2022). Copyright 2022, American Chemical Society

Food

Pickering emulsion

Pickering emulsions, first introduced by Pickering in 1907, utilize solid particles instead of surfactants for stabilization (Pickering, 1907). Edible colloidal particles have gained significant attention in recent years due to their advantages over conventional emulsions, such as the ability to utilize natural particles and greater long-term stability against coalescence and Ostwald ripening (Gao et al., 2014). Zein, a protein derived from corn, has been proposed as a suitable stabilizing agent for Pickering emulsions due to its adjustable properties and potential for adsorption on droplets. The characteristics of zein particles can be modified during the manufacturing process by adjusting factors such as pH, protein concentration, solvent type, and preparation methods. However, complexation or surface modification using polysaccharides, proteins, surfactants, and polyphenols can improve zein particle properties such as wettability, zeta potential, and size, resulting in more stable emulsions. The performance of Pickering emulsions is influenced by several factors, such as the ratio of zein to the coating polymers, the particle concentration, interfacial composition, ultrasound treatment, and the property of the modification polymers. For instance, Feng and Lee (2016) demonstrated that the surface modification of zein colloidal particles with sodium caseinate improved the stability of oil-in-water Pickering emulsions. Similarly, Meng et al. (2020) investigated the potential of zein/carboxymethyl dextrin (CMD) nanoparticles (ZCPs) to stabilize Pickering emulsions and serve as delivery vehicles. The study revealed that the wettability of zein nanoparticles was influenced by the ratio of zein to CMD. The contact angle of ZCPs decreased from 95.6° to 62.0° with a change in the zein:CMD ratio from 4:1 to 2:1, indicating an improvement in the hydrophilicity of the zein nanoparticles due to the presence of CMD. Remarkably, a contact angle of around 90° was observed for ZCPs with a zein:CMD ratio of 2:1, suggesting that this ratio could be considered the optimal Pickering stabilizer. Those findings underscore the importance of controlling the ratio of zein to the coating polymers, as well as other parameters, in order to optimize the performance of Pickering emulsions.

Interestingly, zein-based colloidal particles have also been utilized in high internal phase emulsions (HIPEs), which are characterized by having a high-volume fraction of internal, or dispersed phase (with volume fraction greater than 0.74) (Cameron and Sherrington, 1996). prepared high internal phase Pickering emulsion gels stabilized by glycyrrhizic acid-zein composite nanoparticles, which exhibit excellent stability and mechanical strength and can be used in three-dimensional (3D) printing applications. Pickering emulsions have been shown to be more stable against lipid oxidation than conventional emulsions, making them well-suited for use in the food industry.

Edible film

In recent years, the development of edible food packaging films using natural materials has received considerable attention due to increasing concerns about plastic pollution and food safety (Lei et al., 2019; Wu et al., 2019). Active packaging can be created by incorporating functional ingredients, such as zein-based nanoparticles, into film-forming solutions to introduce bioactive compounds and improve the physicochemical properties of the packaging films. Mo et al. (2021) recently developed antifungal gelatin-based nanocomposite films loaded with natamycin using zein/casein nanoparticles. The nanocomposite films exhibited excellent antifungal activity against common food spoilage fungi, Aspergillus flavus and Penicillium expansum, while maintaining good mechanical properties. The incorporation of natamycin-loaded zein/casein nanoparticles in the gelatin-based films improved their antifungal activity compared to films without nanoparticles.

Numerous studies have highlighted the potential applications of zein nanoparticle-based edible films in the food industry, particularly in meat (Ali et al., 2023; Xavier et al., 2021) and produce (Garcia et al., 2022; Xiang et al., 2021; Zhang et al., 2022) preservation. For example, Garcia et al. (2022) prepared coatings made of zein nanoparticles and ε-polylysine and investigated their effectiveness as postharvest treatments on the shelf-life of avocados. The coatings were found to reduce weight loss, delay ripening, and maintain the quality of the fruit, while also exhibiting antifungal activity against fungi responsible for postharvest decay. These promising results suggest that zein nanoparticle-based coatings have the potential for widespread application in the food industry to extend the shelf life of food products.

Cosmetic

The application of nanotechnology in the cosmetic industry has been shown to enhance the efficacy of cosmetic products through various mechanisms, such as improving the dermal penetration and encapsulation efficiency of active ingredients, as well as repairing UV-induced skin damage and controlling drug release (Salvioni et al., 2021; Zhu et al., 2022). One promising approach involves using eco-friendly tetrahydrocurcumin (THC)-loaded zein-hyaluronic acid nanoparticles as cosmetic ingredients to improve skin photoaging, as reported by Zhu et al. (2022). The study demonstrated that zein nanoparticles effectively enhanced collagen production, reduced levels of inflammatory cytokines that may cause skin damage, improved skin moisture content, and enhanced the skin's barrier function. These results suggest that zein nanoparticles can be used as a natural and effective way to improve skin photoaging in cosmetics. However, further research is needed to explore the full potential of zein-based nanoparticles in the cosmetic industry.

Agriculture

Nanotechnology can also be applied in the agricultural field to provide sustainable solutions for agriculture by enhancing the efficiency of nutrient utilization, improve plant growth, increase crop yields, control pests, monitor plant health, reduce the negative impact of climate change, and minimize the harmful environmental effects of food production (Hofmann et al., 2020). In recent years, zein-based nanoparticles have gained attention for their potential use in agriculture. Hanna et al. (2022) studied the effect of zein nanoparticles on pesticide translocation in soybean plants and found that the application of zein nanoparticles before pesticide treatment resulted in higher translocation of the pesticide, leading to improved pest control. This approach could potentially reduce the amount of pesticide needed for effective pest control and contribute to sustainable agricultural practices. Similarly, Oliveira et al. (2018) explored the use of zein nanoparticles as a carrier system for botanical repellents to control pests in a sustainable and environmentally friendly manner. The study demonstrated that zein nanoparticles enhanced the stability and effectiveness of botanical repellents against pests. Additionally, Monteiro et al. (2021) discussed the use of zein-based nanoparticles to deliver botanical pesticides, i.e., limonene and carvacrol, in the presence of trypsin enzyme and its effects on Spodoptera frugiperda, one of the most significant pests worldwide. Zein nanoparticles were enzyme stimuli-responsive, meaning that they release the pesticide in response to specific enzymes present in the pest's digestive system. This approach is intended to reduce the amount of pesticide needed for effective pest control and minimize the risk of environmental contamination due to its greater targeting. The study found that the use of zein-based nanoparticles loaded with botanical pesticides improved the effectiveness of pest control and reduced the amount of pesticide needed. The article highlights the potential of this approach to develop more sustainable and environmentally friendly pest control strategies. Overall, these studies suggest that zein-based nanoparticles have significant potential to improve the efficiency and sustainability of pest control strategies in agriculture.

Future direction

Zein nanoencapsulation holds significant promise in enhancing the delivery of bioactive ingredients to improve human health. While bioactive compounds offer valuable health benefits, they are vulnerable to degradation in challenging environments, such as high temperatures, varying pH levels, salt-rich conditions, and limited solubility in water. Consequently, nanoencapsulation has emerged as a valuable technique for safeguarding these compounds and enhancing their bioavailability.

Our review has highlighted a variety of production methods and structural considerations for bioactive ingredient-loaded zein nanocarriers. The selection of a suitable method depends on specific requirements and the characteristics of the bioactive compounds. Factors to contemplate when choosing these methods include production costs, safety considerations, time efficiency, and the desired zein nanocarrier structure. The approaches presented here, with a particular focus on the pH-driven method, offer several advantages in addressing issues encountered with alternative encapsulation strategies for bioactive compound delivery. This technique eliminates the need for solvents, is timesaving, and does not require specialized equipment. Furthermore, there is ample room for exploration in utilizing zein nanogels for bioactive ingredient encapsulation and delivery, providing a rich avenue for further research.

The future of bioactive-loaded zein nanocarriers in the food, pharmaceutical, cosmetic, and agricultural sectors is promising as these versatile carriers have the potential to revolutionize each field. In the pharmaceutical realm, zein nanocarriers are poised to assume pivotal roles in advancing drug delivery systems, biologics delivery, and precision therapies. The cosmetic and personal care industry stands to benefit from the introduction of enhanced skincare and haircare products, alongside more effective sunscreen formulations. In agriculture, zein nanocarriers possess the potential to revolutionize crop protection, enhance fertilizer efficiency, and bolster soil health applications. Meanwhile, the food industry envisions tailored nutritional products, clean-label solutions, and extended shelf-life applications with great anticipation.

Particularly in the food industry, addressing safety concerns related to organic solvents in specific production methods is imperative, presenting unique challenges. Moreover, the critical role of particle size in the food sector is pronounced, given its significant impact on sensory attributes. Striking a harmonious balance between sensory characteristics and protective qualities becomes paramount when integrating nanocarriers into food products. Consequently, the successful development of nanoencapsulated bioactive compounds via zein-based nanocarrier delivery systems necessitates comprehensive evaluations encompassing factors such as production costs, safety measures, production methodologies, and consumer preferences before embarking on large-scale production. We anticipate that ongoing research and development endeavors will continually explore the latent applications of these precision delivery systems within the food sector.

In conclusion, the relentless progression of research and innovation in zein nanocarrier technology holds the promise of yielding sustainable and consumer-friendly solutions across these diverse sectors. Ultimately, these advancements stand to enrich human health, preserve the environment, and foster industry growth.

Supplementary Information

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Declarations

Conflict of interest

The authors declare no conflicts of interest.