Abstract
β-sitosterol (βsit) is a plant-derived phytosterol with recognized health-promoting properties, but its low aqueous solubility and limited bioavailability constrain its use in nutraceuticals and functional foods. This study aimed to precipitate and encapsulate βsit within polycaprolactone (PCL) using the Supercritical AntiSolvent (SAS) process in order to develop a controlled delivery system for nutraceutical bioactives. First, βsit was precipitated from a 2% ethyl acetate solution at 40 °C and pressures ranging from 9 to 13 MPa to identify suitable operating conditions; 9 MPa was selected as optimal due to its lower βsit solubility in the CO2/solvent mixture. These conditions were then applied for the precipitation of βsit with PCL. The SAS mechanism relied on the anti-solvent action of supercritical CO₂, promoting rapid precipitation and the formation of βsit:PCL microparticles. The inclusion of Tween 80 (T80) enhanced incorporation efficiency, promoted uniform microparticle morphology, and increased overall process yield. Formulation variables modulated both the release kinetics and underlying mechanisms. Incorporation within PCL slowed βsit release, while T80 accelerated it, likely due to reduced matrix crystallinity. Overall, βsit was successfully micronized along with PCL using SAS-based precipitation, highlighting its potential for the combination of bioactive compounds in functional foods and nutraceutical applications.
Highlights
- β-sitosterol was successfully precipitated by the supercritical antisolvent process.
- Optimal precipitation occurred at 9 MPa due to low β-sitosterol solubility in sc-CO2
- β-sitosterol was also encapsulated in polycaprolactone microparticles
- Polycaprolactone enabled the controlled release of β-sitosterol
- Tween 80 enhanced incorporation efficiency, process yield and accelerated release
Introduction
β-sitosterol (βsit), is a plant-derived phytosterol widely recognized for its potent cholesterol-lowering effects and broad therapeutic potential. Its incorporation into functional foods and nutraceutical products has been extensively explored, particularly due to its ability to reduce low-density lipoprotein (LDL) cholesterol levels, support cardiovascular health (Pavani et al., 2022), to help manage diabetes (Babu & Jayaraman, 2020) and urinary symptoms associated with benign prostatic hyperplasia (Berges et al., 2000).
Structurally, βsit possesses a steroidal structure with multiple carbon rings and only a hydroxyl group (Fig. 1), which confers low polarity and pronounced hydrophobicity, resulting in high solubility in organic solvents (Wei et al., 2010), but poor solubility in aqueous media of only 2–4 mg/L (Malaviya & Gomes, 2008). Consequently, the effective application of βsit is restricted, limiting its bioavailability and overall physiological efficacy. In response to these limitations, recent studies have focused on food-grade encapsulation strategies for βsit to improve its stability and gastrointestinal bioaccessibility. Protein–polysaccharide nanoparticles and core–shell systems have shown particular promising, as intermolecular interactions can induce partial or complete amorphization of βsit, resulting in enhanced dissolution and in vitro release behavior (Li et al., 2025; Lu et al., 2023). In addition, food-grade colloidal systems such as Pickering emulsions stabilized by protein–polysaccharide complexes have been explored to improve the physical stability and retention of βsit in oil–water systems, offering an alternative strategy for its incorporation into functional food matrices (Pokorski et al., 2025). Lipid-based carriers, particularly liposomes, have also been investigated for βsit delivery, where the phytosterol may contribute to membrane stabilization and enhanced oxidative stability, while enabling controlled release in food and nutraceutical applications (Chang et al., 2025; Jovanović et al., 2025). Despite these advances, many βsit delivery systems still rely on multistep wet-processing routes or complex carrier architectures, and offer limited control over particle morphology and crystallinity through processing variables.
Micronization has been proposed as an effective strategy to enhance dissolution rates and reduce the required daily dose of βsit (Moreno-Calvo et al., 2014). Conventional solvent-based micronization techniques often present drawbacks such as poor control over particle morphology and size distribution, difficulties in removing residual solvents, and the potential for thermal degradation of the compound during processing (Prosapio et al., 2018).
In this context, micronization techniques using supercritical CO2 (sc-CO2) have emerged as a sustainable alternative. These processes minimize the use of toxic organic solvents, offer enhanced control over particle size and morphology, and yield solvent-free final products (Padrela et al., 2018). Sc-CO2 is particularly attractive due to its moderate critical conditions (31 °C, 7.4 MPa), and its tunable physicochemical properties, which can be modified by adjusting the operating conditions (Zahran et al., 2014). Its low viscosity and high diffusivity promote uniform particle formation while minimizing thermal degradation (Cerro et al., 2024). Moreover, CO2 is non-toxic, non-flammable and easily removed upon depressurization, ensuring the absence of harmful residues in the final product (Cerro et al., 2023).
Among micronization techniques, the Supercritical AntiSolvent (SAS) process is particularly suitable for compounds that are poorly soluble or insoluble in sc-CO2. The sc-CO2 acts as an antisolvent by being introduced into a solution of the target compound dissolved in an organic solvent. Efficient precipitation requires that the solute be fully soluble in the organic solvent but insoluble in sc-CO2. Additionally, the organic solvent must be fully miscible with sc-CO2 to enable phase expansion and reduce the solubility of the solute, thereby promoting its controlled precipitation. Particle size and morphology can be tuned by adjusting pressure, temperature, flow rates, as well as nozzle diameter. This method produces micronized particles with tailored physicochemical properties, while enhancing the compound’s physiological efficiency through increased specific surface area and improved dissolution (Moreno-Calvo et al., 2014). It also allows co-precipitation with polymeric excipients, providing protection against degradation and potential controlled release (Prosapio et al., 2018; Zahran et al., 2014).
Although SAS has not been previously applied to the micronization of βsit, related supercritical techniques provide useful insights into its phase behavior and precipitation feasibility. According to Turk et al. the solubility of βsit in CO2 at 50 °C ranges from 2.99 × 10−5 to 1.88 × 10−4 in mol fraction at 14 and 25 MPa (Türk et al., 2006). Although relatively low, this solubility was sufficient to enable the coprecipitation of βsit with L-poly(lactic acid) using the Rapid Expansion of Supercritical Solutions (RESS) method. Concerning the combined use of βsit and organic solvents in CO2, Temelli et al. have studied the phase behavior of phytosterols in carbon dioxide-expanded ethanol. CO2 acted as a co-solvent at concentrations of up to ∼0.8 mol fraction but showed an antisolvent effect at higher CO2 concentrations (Temelli et al., 2012). These data were used to guide the precipitate of phytosterols using the Depressurization of an Expanded Liquid Organic Solution (DELOS) technique developed by Moreno-Calvo et al. (Moreno-Calvo et al., 2014).
In this study, polycaprolactone (PCL) was used as the encapsulating agent to coprecipitate along with βsit. PCL is a biocompatible and biodegradable polymer approved by the Food and Drug Administration (FDA) (Prieto and Calvo, 2017a, Prieto and Calvo, 2017b). Previous studies have successfully trapped bioactive compounds such as α-lipoic acid, eugenol, resveratrol, green tea polyphenols, and β-carotene in PCL using supercritical techniques, producing microparticles with controlled dissolution and high encapsulation efficiency (De Paz et al., 2013; Palazzo et al., 2020; Sakata et al., 2021; Sosa et al., 2011). In previous studies, we have successfully produced βsit/PCL microparticles using the Supercritical Fluid Extraction of Emulsions (SFEE) process with process efficiencies higher 60% (Cerro et al., 2024). Moreover, PCL microcapsules have been successfully applied in functional foods, such as yogurt, to encapsulate polyphenols extracted from olive leaves, enhancing their stability and enabling controlled release of the bioactive compounds (El-Messery et al., 2021).
To our knowledge, the micronization of βsit using the SAS method and its co-precipitation with PCL, has not been previously reported. In this work, sc-CO₂ is employed as an antisolvent to precipitate βsit from ethyl acetate or acetone solutions using food-compatible materials. Initially, the antisolvent effect of sc-CO₂ in βsit was evaluated using a high-pressure view cell, guiding subsequent precipitation with the SAS process. The influence of pressure, PCL incorporation, and Tween 80 surfactant (T80) addition on the structural, thermal, and functional properties of the particles, is systematically investigated, with the aim of establishing structure–process–function relationships relevant to food and nutraceutical applications.
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Materials
The materials employed were CO2 (99.98 mol% pure) obtained from Carburos Metálicos, and βsit (≥70%, Product No. 85451-100G, Merck), which contains campesterol and β-sitostanol as residual plant-derived sterol impurities. Polycaprolactone (PCL) (MW 80,000) and Tween®80 (T80) were purchased from Sigma Aldrich. Ethyl acetate, acetone (≥99.5% (GC)), sulfuric acid (≥95%), dichloromethane (≥95%) acetic anhydride (≥95%) and methanol (≥99.9%) were purchased from Merck. All water used was pretreated using the Milli-Q Elix water purification system (Millipore Ibérica, Madrid, Spain). For the dissolution test Phosphate Buffer Solution (PBS) pH 7 was obtained from Merck.
Daniela Cerro, Albertina Cabañas, Alejandra Torres, Fouad Zahran, Luisa Sepúlveda, Patricia Rivera, Julio Romero, Precipitation and encapsulation of β-sitosterol using supercritical antisolvent (SAS) method for controlled nutraceutical release., Food Research International, Volume 228, 2026, 118378, ISSN 0963-9969, https://doi.org/10.1016/j.foodres.2026.118378.
