Optimization of phytosterol extraction and purification processes from vegetable oils using green solvent-based methodologies

Introduction

Phytosterols, also called plant sterols, are naturally occurring compounds in the cell membranes of higher plants. They share structural similarity with cholesterol but differ in side-chain configuration, allowing them to compete with dietary cholesterol for intestinal absorption [1] . The three most common β-sitosterol, campesterol, and stigmasterol account for roughly 95% of dietary plant sterol intake [2] . Vegetable oils, especially soybean, sunflower, and rapeseed, serve as the richest commercial sources, with concentrations of 1.0 to 5.3 mg per gram [3] . Daily consumption of 1.5 to 3.0 g of plant sterols can lower LDL cholesterol by 8-15% [4] . Beyond cardiovascular effects, phytosterols show anti-inflammatory and antioxidant activities [5] . The global phytosterol market was valued at over $600 million in 2023 with roughly 9% annual growth expected through 2030 [6] . Conventional phytosterol extraction from deodorizer distillate uses hexane and other petroleum solvents. While effective, hexane poses environmental risks from volatile organic compound emissions and occupational hazards including neurotoxicity [7] . Regulatory bodies have tightened residue limits, pushing manufacturers toward greener alternatives [8] . Ethanol, isopropanol, ethyl acetate, supercritical fluids, and subcritical water have all been proposed [9] . SC-CO₂ extraction attracts attention because it operates at moderate temperatures, leaves no residue, and allows selective extraction through pressure adjustment [10] .

Few investigations have systematically compared multiple green solvents under optimized conditions across different oil feedstocks [11] . This research addressed that gap by evaluating five green solvent systems across three vegetable oils, using response surface methodology to determine optimal parameters. The work also assessed purity, structural stability, and solvent recovery efficiency to guide manufacturers replacing hexane with responsible methods [12] .

Material and Methods

Material

This research was conducted at the Green Chemistry Laboratory, Department of Chemistry, Manchester Institute of Applied Sciences, Manchester, United Kingdom, from March 2023 to November 2024. Three vegetable oils served as phytosterol sources: cold-pressed sunflower oil (3.8 mg/g), soybean oil (2.9 mg/g), and rapeseed oil (4.7 mg/g), measured by preliminary GC-MS screening. Deodorizer distillate fractions were prepared by molecular distillation at 230°C under vacuum (0.01 mbar). The distillate was saponified using 2M KOH in ethanol at 80°C for 90 minutes to release free sterols [13] . All solvents were analytical grade. Ethanol (95%) and isopropanol (99%) came from Fisher Scientific UK. Ethyl acetate (≥99.5%) and reference standards of β-sitosterol, campesterol, and stigmasterol were from Sigma-Aldrich. Food-grade liquid CO₂ (99.95%) was supplied by BOC Limited, Manchester.

Extraction Protocol

For ethanol and isopropanol, saponified mixture was contacted with solvent at 1:3 to 1:8 (w/v) ratios under stirring at 40-70°C for 30-120 minutes. Ethyl acetate extraction followed the same protocol at 25-50°C. SC-CO₂ extraction used a Waters SFE-500 system at 20-50 MPa and 35-65°C, with CO₂ flow rates of 15-40 g/min for 30-90 minutes. Ethanol (5-15%) served as co-solvent in selected runs. Subcritical water extraction used a stainless steel reactor at 120-180°C under 5-15 MPa for 15-60 minutes [14] .

Methods

Response surface methodology with central composite design optimized extraction conditions for each solvent. Design-Expert v13.0 generated matrices with three to four independent variables. The response was total phytosterol yield, calculated as recovered mass divided by theoretical maximum. Identification and quantification used GC-MS (Agilent 7890B/5977B MSD) with a DB-5ms column (30 m × 0.25 mm, 0.25 μm). Oven temperature began at 200°C (2 min hold), ramped to 300°C at 5°C/min, and held for 15 minutes. Purity was assessed after two-step crystallization at -20°C followed by recrystallization from methanol/water (9:1). Structural integrity was checked by FTIR spectroscopy (PerkinElmer Spectrum Two, 4000-400 cm⁻¹). All experiments were done in triplicate [15] .

Quality Control

A five-point calibration with certified standards showed linearity (R² > 0.998) for all target phytosterols. Spiked recovery tests yielded 96.2-99.1%. Instrument precision was verified by analyzing the same extract six times on the same day (intra-day RSD < 1.8%) and across three days (inter-day RSD < 3.2%). Blanks were included in every batch. Detection and quantification limits were 0.05 and 0.15 mg/L for β-sitosterol. Figure 1 shows yield distributions across replicates. SC-CO₂ had both the highest median and tightest spread, while subcritical water showed greatest variability, suggesting inconsistent performance at high temperatures. Figure 2 presents the extraction and purification cycle. Solvent recovery exceeded 92% for ethanol and isopropanol, supporting economic viability even at reduced yields.

Results

Comprehensive interpretation

Rapeseed oil consistently gave highest yields, matching its greater initial sterol content (4.7 mg/g). Temperaturepressure interaction was the most significant factor for SC-CO₂ (p<0.001), while solvent-to-oil ratio controlled liquid solvent efficiency. FTIR spectra from all methods showed identical bands at 3420 cm⁻¹, 2930 cm⁻¹, and 1050 cm⁻¹, confirming green solvents didn't alter phytosterol molecular structure.

Discussion

SC-CO₂ extraction offers the most effective green alternative to hexane. The 89.2% yield compares favorably with published hexane yields of 85-92% from similar feedstocks [1] . The key advantage lies in near-complete absence of residual solvent, eliminating a costly refining step and satisfying strict food safety regulations [3] .

Ethyl acetate performed as a strong second option for producers without high-pressure equipment. Its 84.7% recovery and 95.4% purity meet commercial requirements for food-grade concentrates. Reports using ethyl acetate for sterol extraction from rice bran oil found 79-86% yields, consistent with these findings [5] . Mild conditions (25-50°C) also reduce energy costs. Subcritical water's poor performance (61.4%) may stem from using saponified distillate rather than crude oil, altering free sterol solubility in hot water. Thermal sensitivity of phytosterols above 160°C, particularly stigmasterol, could explain lower recoveries and higher variability [9] . Solvent recovery above 92% for ethanol and isopropanol means minimal waste generation. Life-cycle data suggest green solvent methods reduce carbon footprint by 30-45% versus hexane approaches [11] . The crystallization protocol achieved purities above 91% without chromatographic separation. Response surface methodology effectively identified optimal conditions, revealing temperature-pressure interactions as dominant for SC-CO₂ while solvent-to-oil ratio controlled liquid solvent efficiency. Rapeseed oil produced highest yields among the three feedstocks. All green methods preserved phytosterol structural integrity as confirmed by FTIR and DSC. Two-step crystallization reliably produced purities above 91% regardless of extraction solvent. Solvent recovery exceeded 92% for ethanol and isopropanol, supporting economic and environmental goals. Future work should explore continuous-flow SC-CO₂ extraction and assess long-term storage stability of green-extracted phytosterols in food matrices.

Conclusion

Acknowledgement

Acknowledgements Funding sourcesThe authors acknowledge financial support from the Department of Chemistry, Manchester Institute of Applied Sciences.

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