DISCUSSION

This study demonstrated that CUR delivered through an NLC system produced compound-specific functional outcomes in Nile tilapia, including improved growth trajectory, feed utilization, metabolic indices such as HSI and growth allometry, and enhanced survival following S. agalactiae challenge. Although lipid-based nanocarriers and other nano-CUR systems have been explored for phytogenic delivery in aquatic species, CUR is a polyphenolic compound with distinct physicochemical limitations and biological targets. Therefore, sustained dietary delivery of CUR may influence oxidative balance, liver metabolism, and immune responsiveness in ways that cannot be directly extrapolated from other phytobiotic–nanocarrier studies.

Physicochemical characteristics and implications for CUR delivery

CUR-NLCs exhibited favorable physicochemical properties suitable for oral delivery. The increase in particle diameter from 177.0 ± 3.2 nm in blank NLCs to 215.6 ± 2.6 nm in CUR-NLCs supports successful incorporation of CUR into the lipid matrix, consistent with NLC loading behavior reported for hydrophobic bioactive compounds [56, 57]. The reduced polydispersity index of CUR-NLCs (0.145 ± 0.003) compared with blank NLCs (0.269 ± 0.034) indicates a more uniform dispersion [58, 59], which is advantageous for consistent gastrointestinal exposure and uptake. Both formulations exhibited moderately negative zeta potentials (CUR-NLCs: −17.5 ± 0.78 mV; blank NLCs: −23.12 ± 0.9 mV), suggesting adequate colloidal stability through electrostatic repulsion that limits aggregation [60–62]. The high encapsulation efficiency (98.79 ± 1.51%) further confirms effective CUR entrapment [30, 63, 64]. Transmission electron microscopy imaging supported these findings by showing predominantly spherical, uniformly distributed particles of approximately 200 nm in diameter, consistent with dynamic light scattering results and indicating the structural integrity of the nanocarrier system [40, 41, 43, 65, 66].

The in vitro release profile supports a key delivery advantage of CUR-NLCs. CUR-NLCs exhibited a controlled and sustained-release pattern, whereas free CUR showed a rapid initial burst release followed by a slower phase. For polyphenols such as CUR, for which stability and bioavailability are major constraints, controlled-release may enable prolonged physiological exposure and improved functional effects during chronic feeding [67–69]. Despite these favorable characteristics, long-term storage stability and CUR degradation under different conditions, including temperature, pH, and feed incorporation, were not evaluated in this study. These aspects are important for practical application and should be addressed in future studies.

Molecular interactions supporting the stabilization of CUR

Fourier-transform infrared spectroscopy results further supported effective incorporation of CUR into the lipid matrix. Shifts in the O–H stretching band at approximately 3508 cm⁻¹ suggest hydrogen bonding between CUR and lipid components, which can enhance stabilization and dispersion [70, 71]. Shifts in the C=O stretching region at approximately 1736 cm⁻¹ indicate interactions with ester linkages in the lipid matrix [72, 73]. Broadening and reduced intensity of C=C stretching at 1600–1625 cm⁻¹ and aromatic ring vibrations, together with changes in C–O stretching and C–H bending peaks at 1300–1400 cm⁻¹, reflect a modified microenvironment and strong compound–carrier interactions [74, 75]. These molecular interactions are consistent with improved stability and solubility, which are critical limitations for CUR use in aquafeeds [63, 67–69].

Antibacterial performance of CUR-NLCs

CUR-NLCs produced larger IZ values (14–15 mm) than free CUR (6 mm), indicating improved diffusion and localized antibacterial activity on solid media. This finding is consistent with reports that nanoencapsulation improves dispersibility and contact efficiency [76, 77]. The antibacterial activity of CUR has been associated with mechanisms such as membrane disruption and interference with virulence-related processes [78]. However, MIC and MBC values remained unchanged between free CUR and CUR-NLCs, suggesting that nanoencapsulation did not fundamentally shift bactericidal thresholds under broth conditions. This pattern is biologically plausible because sustained-release systems may enhance localized exposure and prolonged interaction without necessarily achieving the rapid high concentrations required in endpoint MIC and MBC assays [79–81]. Therefore, the antibacterial findings are best interpreted as improved delivery and dispersion effects rather than altered intrinsic potency.

Growth and feed utilization benefits

The feeding trial demonstrated that CUR-NLCs produced early improvements at 30 days and sustained improvements at 60 days in growth performance and feed utilization compared with free CUR, blank NLCs, and control diets. These effects address a key limitation of CUR, namely poor solubility and low oral bioavailability, and support the interpretation that NLC delivery improved functional exposure to CUR during chronic feeding [82]. Mechanistically, NLCs may protect CUR from degradation in the gastrointestinal tract and facilitate uptake by enhancing interactions with intestinal surfaces, thereby improving absorption efficiency [69, 83]. Sustained availability may also support the antioxidant, physiological, and health-promoting effects of CUR, reducing physiological stress and enabling more efficient nutrient partitioning toward growth [84].

The improvement in FCR in the CUR-NLC group, particularly by day 60, indicates enhanced feed efficiency, potentially linked to improved nutrient utilization and metabolic efficiency under sustained CUR exposure [34, 47, 85]. The increased PER further suggests improved protein utilization, potentially reflecting reduced oxidative catabolism and improved nitrogen retention under the antioxidant and immune-supportive effects of CUR [47, 85, 86]. Gut microbiota composition was not evaluated in the present study; therefore, potential microbiome-mediated contributions to improved feed efficiency and health performance associated with CUR-NLC supplementation remain unclear. Although free CUR improved performance relative to the control, its effects were consistently lower than those of CUR-NLCs, highlighting the importance of delivery constraints in determining in vivo efficacy. Because identical nominal CUR concentrations were used in the free CUR and CUR-NLC diets, the improved biological responses observed in the CUR-NLC group are more likely attributable to enhanced delivery efficiency and sustained bioavailability rather than differences in dietary inclusion level alone. The absence of visible feed rejection and apparent CUR leaching during feeding further supports the practical suitability of CUR-NLC-coated pellets for dietary application in Nile tilapia culture systems.

Metabolic indices and growth allometry

HSI outcomes provided additional evidence of functional metabolic effects. By 60 days, both CUR and CUR-NLCs increased HSI compared with the control, with CUR showing the highest HSI. These findings may reflect metabolic responses associated with prolonged dietary CUR exposure [87, 88], although direct evidence supporting a hepatoprotective effect was not evaluated in the present study. Histopathological evaluation of hepatic or intestinal tissues and serum biochemical markers, such as alanine aminotransferase and aspartate aminotransferase, were not assessed. Therefore, interpretations regarding hepatoprotective effects remain preliminary and require further investigation.

The positive allometric growth observed in the CUR-NLC group (b = 3.278) indicates proportionally greater WG relative to length, suggesting favorable nutrient allocation and tissue deposition under sustained CUR delivery [30, 89, 90]. In contrast, the control and NLC groups exhibited negative allometry, supporting the conclusion that CUR, rather than the carrier alone, contributed to the improved growth pattern [50]. Although free CUR produced the highest Kn value, CUR-NLCs enhanced growth dynamics the most, indicating that the effects of CUR on body condition and weight deposition may not be identical and may vary with delivery form and exposure kinetics [30, 90].

Disease resilience and survival modeling

The most biologically and commercially relevant outcome was improved disease resilience following S. agalactiae challenge. CUR-NLC-fed fish exhibited the greatest survival and lowest mortality, with an RPS of 82.4 ± 2.8%. Cox regression further indicated an approximately 86% reduction in mortality risk relative to infected controls, whereas free CUR reduced the risk by approximately 50%. The minimal effect of blank NLCs confirms that the protective outcome was primarily mediated by CUR rather than the carrier matrix.

Given that MIC and MBC values were unchanged, the improved survival response observed in the CUR-NLC group may be associated with enhanced CUR bioavailability and sustained physiological exposure, which could contribute to improved host resilience during infection rather than direct bactericidal activity alone [30, 34, 91]. In practical terms, these findings support CUR-NLCs as a phytobiotic-based approach to reducing antibiotic dependence in streptococcosis-prone production systems, particularly in regions where S. agalactiae is endemic and recurrent.

Although improved post-challenge survival may suggest enhanced physiological resilience, hematological parameters, antioxidant biomarkers, innate immune responses, tissue bacterial burden, and immune-related molecular responses were not evaluated in the present study. Therefore, the precise mechanisms underlying enhanced disease resistance, including potential effects on systemic bacterial colonization, immunomodulation, and antioxidant-related pathways, remain unclear and require further investigation. Furthermore, although no overt adverse effects were observed in fish fed the blank NLC diet, a comprehensive toxicological evaluation of the nanocarrier system was beyond the scope of this study. Future investigations should therefore include detailed immunophysiological assessments together with long-term biosafety evaluations, including histopathological analysis, nanoparticle accumulation, and environmental fate associated with prolonged dietary exposure.

Translational relevance and future application

From a translational perspective, the CUR-NLC formulation used in this study may be adaptable for aquaculture applications because it uses commonly available lipid excipients and a relatively simple preparation process. Although nanoencapsulation may increase production costs compared with free CUR, improved bioavailability, feed efficiency, and post-challenge survival could potentially offset these costs. However, the economic feasibility and scalability of CUR-NLC incorporation into industrial feed manufacturing systems require further investigation through techno-economic analyses and farm-scale validation studies.

In addition, although CUR and the lipid excipients used in the present NLC formulation are generally regarded as biodegradable and biocompatible, the current study did not directly assess tissue residue accumulation, degradation behavior, or potential release of formulation components into surrounding aquatic systems. Because lipid-based nanocarriers may influence the stability, delivery efficiency, and biological distribution of encapsulated compounds, further studies evaluating residue kinetics, biodegradation dynamics, and environmental safety under commercial aquaculture conditions are warranted to support the sustainable application of CUR-NLCs.