Why are clams steamed with wine in Mediterranean cuisine?

The Mediterranean diet, noted for high intake of polyphenol-rich foods, includes the popular dish of steamed clams with wine. Wine provides resveratrol (Res), linked to antioxidant and anti-inflammatory benefits and potential risk reduction for non-communicable diseases. However, Res suffers from poor water solubility and instability, limiting bioavailability and efficacy. Nano-carriers can enhance solubility, stability, and delivery of polyphenols, and prior work shows proteins and polysaccharides can form micro/nano-structures that improve polyphenol stability and alter digestion. Food processing can generate organized micro/nano-structures capable of carrying bioactives. This study hypothesizes that proteoglycan-based nanoparticles in freshwater clam soup (CFNPs) can bind and carry wine-derived Res, improving its stability, bioaccessibility, and antioxidant retention. Objectives were to characterize CFNPs-Res formation and stability, quantify encapsulation, examine binding mechanisms, evaluate in vitro release kinetics against wine and free Res, and assess bioaccessibility and antioxidant retention under simulated gastrointestinal digestion.

- Food-derived nanoparticles form during processing (e.g., soups, teas), creating colloidal carriers that enhance stability and delivery of polyphenols and other bioactives. - Black tea fermentation yields nano-carrier skeletons stabilizing polyphenols; various soups produce stable colloids with sustained antioxidant capacity. - Freshwater clam (Corbicula fluminea) soup contains abundant proteoglycan–lipid nanoparticles (CFNPs) formed during heat treatment; similar structures are reported across >45 clam species. CFNPs can carry both water- and fat-soluble compounds and remain stable under high temperature and acidity; shellfish distillates also contain nanoparticles with proteins, polysaccharides, and functional lipids. - Protein–polysaccharide nano-delivery systems (e.g., gelatin, zein, casein, chitosan hybrids) have improved loading, stability, and bioavailability for resveratrol and other polyphenols; mechanisms often involve noncovalent interactions. - Red wines contain polyphenol-rich nanoaggregates/complexes (anthocyanins, proanthocyanidins, polysaccharides), suggesting some Res may be bound within colloidal structures. These findings motivate exploring CFNPs as naturally occurring food nanoparticles to carry and protect Res and to compare with wine matrices and free Res.

- Materials: Resveratrol (97% purity), ABTS, TPTZ for FRAP, digestive enzymes (pepsin, pancreatin/trypsin), bile salts; wines (Pinot Noir—Wine X, Merlot—Wine Y, Cabernet Sauvignon—Wine Z); freshwater clams; Milli-Q water; 100 kDa ultrafiltration devices. - Preparation of CFNPs and CFNPs-Res: Freshwater clams soaked 12 h, washed, cooked 60 min in water (1:1 w/v). Soup centrifuged (5000×g, 30 min) to remove solids. CFNPs isolated from supernatant by 100 kDa ultrafiltration. Res was added dropwise under stirring to form CFNPs-Res at various nominal Res concentrations (10–200 µg/mL). Free Res solutions and wines were used as comparators. - Colloidal characterization: Dynamic light scattering (DLS) for hydrodynamic size, PDI; zeta potential at 25 °C; TEM (stained with phosphotungstic acid, 80 kV) for morphology. - Encapsulation efficiency (EE) and loading capacity (LC): Quantified by UV–Vis at 305 nm using a calibration curve (R²=0.9993, 1–20 µg/mL). Free Res removed by centrifugation or membrane filtration to assess effects of purification on EE/LC. - Fluorescence spectroscopy: Quenching studies at 298 K and 310 K (excitation/emission slit 5 nm) with incremental Res to determine Stern–Volmer constants (Ksv), quenching constants (Kq), dissociation constants (Kd), binding constants (K), and thermodynamic parameters (ΔH°, ΔS°, ΔG°) via Van’t Hoff analysis to infer interaction forces. - Antioxidant activity stability during storage: ABTS and FRAP assays on day 0 and day 3 at room temperature (dark) for CFNPs-Res (150 µg/mL), free Res (150 µg/mL), and CFNPs control. - Bound Res in wines: Wines clarified (3000×g, 15 min), ultrafiltered (100 kDa, 4500×g, 30 min), retentates washed and redissolved; bound Res quantified by UV–Vis. - In vitro release in PBS: Dialysis (8–14 kDa) of samples into PBS (0.002 M, pH 7.4) at 37 °C, dark; aliquots collected up to 24 h; Res quantified by UV–Vis. Kinetic models (zero-order, first-order, Higuchi) fitted to cumulative release data. - Simulated gastrointestinal digestion and bioaccessibility: Two-stage digestion (SGF then SIF) with pepsin followed by bile salts and pancreatin/trypsin at 37 °C. At intervals (0.5 h) and at SIF end (2 h), supernatants collected and filtered (0.45 µm); solubilized Res quantified by UV–Vis to compute bioaccessibility (% dose solubilized). Antioxidant capacity retention post-digestion measured by ABTS. Statistical analysis via one-way ANOVA (p < 0.05).

- CFNPs from clam soup: DLS size 63.7 ± 0.2 nm; ζ-potential −6.9 ± 0.3 mV. - CFNPs-Res formation: Interaction strongly concentration-dependent with a critical Res concentration near 150 µg/mL, above which particle size and PDI increased and additional Res micelles likely formed. PDI remained <0.2 up to 150 µg/mL, indicating unimodal distributions. ζ-potential shifted modestly to −8.7 mV with Res, indicating slightly enhanced stability. - Morphology: TEM showed spherical particles; CFNPs-Res exhibited a corona consistent with Res binding; TEM sizes agreed with DLS (<100 nm). - Encapsulation: Maximum EE 70.8 ± 0.9% at 10 µg/mL Res; EE ~59.3 ± 0.9% at 150 µg/mL. LC increased with concentration, reaching 2.4 ± 0.01% at 200 µg/mL. Centrifugation removal of free Res yielded slightly higher EE/LC than membrane filtration, with larger differences above 150 µg/mL. - Fluorescence quenching: Concentration-dependent quenching at 298 K and 310 K. Kq and Kd decreased with temperature, supporting static (complex formation) quenching. At 298 K: R²=0.9937, Kq=2361.77 (×10¹⁰ m⁻¹·s⁻¹), Kd=236177 L·mol⁻¹; at 310 K: R²=0.9943, Kq=1604.55, Kd=160455. Thermodynamics: ΔH° and ΔS° negative at both temperatures, indicating hydrogen bonding (and possibly van der Waals) as dominant forces; binding constant decreased with temperature. - Storage stability: CFNPs-Res retained >90% antioxidant capacity over 3 days at room temperature (dark), outperforming free Res. - Wine matrix: Total Res contents within specified ranges; only ~7.3–9.1% of Res was bound to wine nanoaggregates across three wines. DLS suggested minimal distinct particle size distributions despite steady light scattering, consistent with polyphenol–polysaccharide complexes in wine. - In vitro PBS release: Free Res and wines showed burst release within 1 h; by 4 h, cumulative release reached ~80% (Wine X) to ~90% (Wines Y, Z). CFNPs-Res exhibited slower, sustained release: within 1 h cumulative release was 30–50%, with 150 µg/mL CFNPs-Res showing the most pronounced sustained profile. - Release kinetics: CFNPs-Res data fitted first-order and Higuchi models (R² near 0.9); for 150 µg/mL CFNPs-Res, Higuchi R² > 0.97. Free Res and wines fitted first-order only. Relative release capacity ranking: 150 µg/mL free Res > Wine Y > Wine Z > Wine X > 50 µg/mL CFNPs-Res > 100 µg/mL CFNPs-Res > 150 µg/mL CFNPs-Res. - Simulated GI digestion: CFNPs-Res remained chemically stable through gastric and intestinal phases; in SIF, cumulative release reached 26.9 ± 0.4% by 2 h, higher than in PBS over the same period. Free Res and wines showed rapid release and lower stability. - Antioxidant retention after digestion: CFNPs-Res retained ~90% antioxidant capacity after 120 min in SIF, whereas other samples decreased to ~75% or lower; free Res showed the largest decrease. Overall, CFNPs-Res markedly protected Res during digestion, enhancing bioaccessibility and preserving antioxidant activity.

The study demonstrates that naturally occurring proteoglycan-based nanoparticles in freshwater clam soup can bind and carry resveratrol, improving its colloidal stability, storage stability, controlled release, bioaccessibility, and antioxidant retention compared with free Res and wine matrices. Binding is primarily driven by hydrogen bonding, forming stable complexes that yield small (<100 nm), low-PDI particles suitable for gastrointestinal interaction and sustained release. Release kinetics indicate diffusion-controlled mechanisms (Higuchi) in CFNPs-Res, while free Res and wine forms follow first-order release with burst behavior. Wine nanoaggregates contain only a small bound fraction of Res and display less protection during digestion, supporting the hypothesis that CFNPs act as more effective carriers. These findings align with broader evidence that food-derived nanoparticles formed during cooking can function as delivery systems for micronutrients and antioxidants. The work provides a mechanistic rationale for enhanced nutritional functionality in traditional dishes such as steamed clams with wine and underscores the potential to design functional foods leveraging naturally derived nano-structures.

- CFNPs effectively encapsulate resveratrol with high efficiency (>60–70%), forming stable, sub-100 nm nanoparticles with improved antioxidant stability and sustained-release behavior. - CFNPs-Res enhances in vitro bioaccessibility and preserves antioxidant capacity during simulated gastrointestinal digestion, outperforming free Res and wine-derived forms. - Release profiles of CFNPs-Res fit first-order and Higuchi models, indicating diffusion-driven, controlled release, especially at higher Res loading without surpassing a critical threshold. - Food-derived nanoparticles, exemplified by CFNPs from clam soup, represent promising, accessible carriers for dietary bioactives, potentially informing functional food development and nutritional guidance. Future directions include elucidating molecular interactions at higher resolution, optimizing loading without destabilization above critical concentrations, evaluating in vivo pharmacokinetics and efficacy, and assessing broader applicability to other bioactives and food matrices, alongside safety and regulatory considerations.

- The study relies on in vitro models (PBS release; simulated gastric and intestinal fluids), which do not fully capture in vivo complexities such as absorption, metabolism, immune interactions, and transport across intestinal barriers. - Wine characterization of nanoaggregates and bound resveratrol relied on ultrafiltration and DLS; limited resolution may underestimate colloidal heterogeneity and interactions. - At higher Res concentrations (>150 µg/mL), additional Res self-assembly introduced heterogeneity and potential instability, complicating interpretation of scattering and encapsulation metrics. - Antioxidant outcomes were primarily ABTS/FRAP-based; broader bioactivity assays and in vivo oxidative stress markers were not assessed.