Development and stability analysis of a lipid-based astaxanthin nanoemulsion, its application and effect on the immune response and survival of the Pacific white shrimp

Camila Pereira Bruzinga^*Claudia Marlene BauerCaroline SchmitzThaise GerberEVA REGINA OLIVEIRACynthia ZiechNohra Constanza Bolivar RamirezCamilla Souza MirandaMaria Helena de Araujo MendesFlavio Campos de ArrudaJúlia Victória Rodriguez HeindricksonFelipe Ferrari RamosIsabela de Almeida GomesMateus de Souza GasparottoCarlos Costa CavalcanteScheila Anelise Pereira DutraFelipe Boechat VieiraLeticia MazzarinoMaria VeleirinhoMarcelo Maraschin

• Federal University of Santa Catarina, Florianopolis, Brazil

This study aimed to develop and characterize a nanoemulsion containing lipoencapsulated astaxanthin (AST) from Haematococcus pluvialis and evaluate its effects on the immune performance and survival of Penaeus vannamei shrimp when added to feed. The nanoemulsion was prepared by high-pressure homogenization using vegetable oils, AST oleoresin, and surfactants. It was characterized for size, polydispersity, Zeta potential, pH, morphology, and physicochemical stability for 90 days at 4°C, 25°C, 30°C, and 54°C. The fatty acid content was determined by gas chromatography, while the AST content was determined by ultra-high-performance liquid chromatography (UHPLC). The in vitro antioxidant activity was verified by the DPPH method. The profile of vegetable oils showed significant concentrations of PUFAs (77.18% and 78.74%, respectively). The AST concentration was 1.55 mg g⁻¹ of nanoemulsion. The colloidal dispersions showed an average particle size 319.6 ± 0.5 nm, a polydispersity index (PDI) of 0.290 ± 0.0 and spherical shape. The formulation showed a negative Zeta potential (-44 ± 0.40mV). The antioxidant activity occurred for 90 days, but varied over time (p<0.001). The nanoemulsion was stable during 90 days of storage at 25°C. Six diets were tested: 10, 30, 50, 70 mg AST/ kg⁻¹ of feed, a negative control diet (NC) (70 mg without AST/ kg⁻¹), and a control diet. After 45 days of cultivation, hemolymph samples were collected for analysis of immunological parameters (total hemocyte count (THC)), protein content (P), phenoloxidase activity (PO), and agglutination capacity (Agl). P. vannamei survival was evaluated after a 96 h challenge with Vibrio parahaemolyticus (4 x 108 CFU mL-1). There was no significant difference for THC and Agl (p<0.05). P levels were higher in the intermediate treatments (30-70 mg AST kg⁻¹) (p=0.0454). PO showed higher concentrations in the treatments with 70 and 10 mg AST kg⁻¹ (p=0.0005). In the V. challenge, there was no significant difference (p<0.05) however, a promising trend of protection conferred by the developed nanoemulsion was observed. The 70 mg AST kg⁻¹ nanoemulsion shows strong potential for use in animal feed, offering a sustainable option for shrimp farming by enhancing serum protein levels and phenoloxidase activity in P. vannamei.