Vortex fluidic mediated encapsulation of functional fish oil featuring in situ probed small angle neutron scattering

The study addresses the challenge of improving fish oil delivery by enhancing bioavailability, masking odor, and preventing oxidation of omega-3 PUFAs. Conventional encapsulation methods often require multiple steps or costly equipment and can yield large, unstable particles. The research investigates whether a continuous-flow vortex fluidic device (VFD) can produce stable, nanoscale encapsulations of fish oil at high concentrations using food-grade Tween 20, thereby protecting omega-3s from oxidation and enabling fortification of water-based foods without sensory compromise. The work also explores simultaneous encapsulation of sparingly water-soluble bioactives (curcumin, quercetin) and, uniquely, employs in situ small-angle neutron scattering (SANS) to monitor shear-induced self-assembly in real time within the VFD.

Background highlights include: (1) Fish oil omega-3 PUFAs benefit health but are odorous and readily oxidize; capsules protect PUFAs but are large. (2) Prior nanoencapsulation approaches often involve multi-step processing, high surfactant-to-oil ratios (e.g., up to 10:1), or expensive equipment; stable emulsions via homogenization have been reported but with limitations in scalability and cost. (3) The VFD is a continuous-flow thin film platform that delivers high shear, intense micromixing, and mass transfer, with growing applications across synthesis, materials processing, biocatalysis, and prior proof-of-concept for fish oil encapsulation at low concentration. (4) Post-VFD SANS studies exist, but real-time in situ SANS during VFD processing had not been reported before this work.

- Materials: Fish oil enriched with omega-3 PUFAs; food-grade surfactant Tween 20; Milli-Q water. For SANS, D2O used to enhance contrast. - Encapsulation (VFD): Oil-in-water with Tween 20:fish oil at 1:1 (w/w), total concentration 0.2 g/mL (10 mL batch: 1 g oil, 1 g Tween 20, 8 mL water). VFD tube (quartz/borosilicate, 20 mm OD, ~17–17.5 mm ID) operated at optimized conditions: 9000 rpm, 0.3 mL/min flow, 45° tilt, ~25 °C. Parameters systematically varied (4000–9000 rpm, 0.3–0.5 mL/min, 30–60°) to select optimum; 1:1 ratio chosen for cost-effectiveness (1:2 slightly better but more surfactant). Continuous flow processing; scale-up trials from 10 mL to 100–200 mL performed. - Control (homogenization): Same formulation; T25 ULTRA-TURRAX at 13,500 rpm for 10 min at 25 °C; batch process. - Emulsion stability: Visual assessment and quantitative stability (% stable fraction), immediate and after 24 h. - Particle size and imaging: DLS at 25 °C (He–Ne 633 nm, backscatter 173°) for hydrodynamic size and PDI; epi-fluorescence microscopy (Nikon DS-Qi1Mc, 20× objective) for droplet size comparison. - Energy consumption: Calculated from device power and processing time for 10 mL and extrapolated to 420 mL for VFD (continuous) vs homogenizer (batch). - SANS (in situ and post-VFD): Conducted on Bilby (ANSTO). Samples: 10 wt% Tween 20 in D2O with/without 10 wt% fish oil. Real-time scans at 0, 4000, 7000, 9000 rpm; post-shear scans after each speed. Detector distances 1.3/12/20 m; q-range 0.00216–0.38125 Å−1; 60 min acquisition per condition. Data reduced with Mantid; modeled in Igor Pro (NIST macros). Models: Gauss Sphere with screened Coulomb interaction (Tween 20 alone, during/after); Power Law + Gauss Sphere with screened Coulomb (fish oil post-VFD); Power Law + Gauss Sphere (fish oil during VFD). Screened Coulomb structure factor used to fit correlation peak (electrostatic intermicellar repulsions). Extracted micelle radii and polydispersities. - Fatty acid profiling: Fresh fish oil and VFD-encapsulated (freeze-dried) before and after 14 days ambient storage. FAMEs analyzed by GC-FID (HP 6890, BPX-70 column), standard identification and external calibration. - Encapsulation efficiency (fish oil): Freeze-dried product oil content measured; efficiency = oil recovered relative to 1 g starting oil, assessed immediately and after 14 days. - Food fortification and sensory: Apple juice (250 mL) enriched with 0.2 g omega-3 (either 0.2 g free fish oil or 0.4 g encapsulated powder containing 0.2 g oil) to 0.8% w/v oil. Sensory tests with 20 trained panelists; hedonic scales for color, taste, aroma, overall acceptance; randomized coded samples; ANOVA/LSD (p < 0.05). - Co-encapsulation of bioactives: Curcumin or quercetin (30 mg) dissolved with 1 g fish oil + 1 g Tween 20 in 10 mL water; VFD at 9000 rpm, 0.3 mL/min, 45°. Post-process centrifugation (3000 rpm, 20 min). Encapsulation capacity determined by measuring unencapsulated precipitate dissolved in 80% ethanol (A420 nm) against standard curve. Confocal microscopy of supernatant (excitation 420 nm for curcumin, 370 nm for quercetin). Fluorescence spectroscopy with specified excitation/emission ranges. - Statistics: Triplicate measurements for stability and encapsulation capacity; 20 replicates for sensory; mean ± SD; one-way ANOVA and LSD via MINITAB v15; significance at p < 0.05.

- Emulsion stability and size: VFD produced markedly smaller droplets than homogenization (microscopy and DLS) and much higher immediate emulsion stability (VFD 96% vs homogenization 72%). VFD emulsions remained homogeneous after 24 h whereas homogenized samples phase-separated. - Throughput and energy: Continuous VFD processing scaled from 10 mL to 100–200 mL with consistent product stability. For 10 mL, energy use was 162.4 Wh (VFD) vs 136 Wh (homogenizer). For 420 mL, VFD 5711 Wh vs homogenizer 5712 Wh, indicating competitive energy consumption under continuous flow. - In situ SANS insights: Tween 20 (10 wt%) showed a weak correlation peak indicating intermicellar interactions; micelle radius ~2.59 nm and largely insensitive to shear, with a slight decrease during 9000 rpm (to ~2.57 nm) and minor polydispersity changes. With fish oil present (10 wt%), baseline micelle radius increased (to ~2.65 nm). During VFD shear (4000–9000 rpm), micelles grew and polydispersity decreased (more uniform), reflecting enhanced homogenization; correlation peak disappeared during shear and reappeared post-shear. After 7000 rpm, micelles became smaller than baseline (radius ~2.29 nm) and more polydisperse (0.31), suggesting shear-induced micelle breakup, likely from Faraday-wave eddies. Strong low-q scattering was observed for fish oil samples due to larger globules (~tens of nm) consistent with DLS. - Protection of omega-3s: Fresh fish oil contained 69.3% omega-3 fatty acids; after 14 days ambient storage, free oil dropped to 31.0%. VFD-encapsulated (freeze-dried) omega-3 content was 62.6% (fresh) and 61.9% after 14 days, indicating substantial protection against oxidation. Encapsulation efficiency was ~99.06% immediately post-VFD, decreasing to ~88.92% after 14 days, while omega-3 content remained essentially unchanged (62.6% to 61.9%). - Food application and sensory: Fortifying apple juice (0.8% w/v oil) with encapsulated fish oil did not significantly alter taste, color, aroma, or overall acceptance compared to plain juice, and both were preferred over juice with free fish oil. - Co-encapsulation of bioactives: Curcumin and quercetin were solubilized in fish oil and encapsulated. Encapsulation capacities were ~67.9% (curcumin) and ~51.2% (quercetin). DLS indicated curcumin-loaded droplets 200–500 nm; quercetin-loaded 700 nm–2 µm; confocal fluorescence confirmed homogeneous dispersion of fluorescent droplets in water. - Industrial relevance: VFD offers continuous processing, lower capital cost relative to high-pressure microjet homogenizers (approx. USD 15k per VFD vs ~USD 150k for homogenizer), and scalability by parallelization.

Findings demonstrate that VFD-generated intense shear and micromixing in a dynamic thin film yield smaller, more uniform oil-in-water droplets than conventional homogenization, translating to improved emulsion stability and reduced propensity for coagulation. Real-time SANS established that at high Tween 20 concentrations, intermicellar electrostatic repulsions (screened Coulomb) contribute to a correlation peak and that VFD shear modulates micelle size and polydispersity, with notable, potentially permanent restructuring near 7000 rpm due to Faraday-wave-induced eddies. Encapsulation effectively mitigated omega-3 oxidation during ambient storage, supporting use of freeze-dried encapsulated fish oil for food fortification without sensory penalties, as evidenced by consumer preference over free oil fortification in apple juice. The VFD also enabled one-step encapsulation of poorly water-soluble polyphenols into fish-oil droplets, creating homogeneous aqueous suspensions and broadening potential applications for delivering lipophilic bioactives in foods. Collectively, the results support the VFD as an efficient, continuous, and scalable platform for nanoencapsulation and provide mechanistic insight into shear-induced self-assembly.

A one-step, continuous-flow VFD process produced stable, nanoscale fish-oil emulsions (≈100–200 nm) with significantly enhanced protection of omega-3 fatty acids during storage and maintained sensory acceptability in fortified juice. In situ SANS provided the first real-time observation of VFD-driven structural evolution, revealing shear-dependent micellar interactions and size changes, including a notable restructuring near 7000 rpm. The approach co-encapsulated lipophilic bioactives (curcumin, quercetin) with appreciable capacities, yielding homogeneous aqueous dispersions. The method is solvent-free, time- and cost-efficient, and scalable from tens to hundreds of milliliters, suggesting direct applicability to industrial food processing. Future work could explore longer-term stability, broader surfactant systems, diverse food matrices, higher-throughput scaling, and optimization of shear regimes for targeted structuring.

- Storage stability was evaluated over 14 days at ambient conditions; longer-term stability and performance under varied environmental conditions were not reported. - A single surfactant (Tween 20) and fixed oil:surfactant ratio (1:1 w/w) were primarily studied; broader surfactant chemistries and ratios could affect generalizability. - Comparative benchmarking was limited to a laboratory homogenizer; while a high-pressure microjet system might yield similar outcomes, it was not experimentally tested here. - Sensory evaluation involved 20 trained panelists and one model beverage (apple juice); broader consumer studies across different food matrices are needed. - SANS data exhibited differences in overall scattering intensity due to solvent scattering subtraction complexities arising from the experimental setup, which may affect absolute intensity comparisons.