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Chitosan nanocapsules containing polyunsaturated fatty acid (PUFA) concentrates from tuna oil, with EPA + DHA contents around 57% (w/w), were developed by emulsification process, using different chitosan concentration (1.0%, 1.5%, 2.0%, w/v) and stirring speed (10,000, 15,000, 20,000 rpm). The effects of these parameters on particle size and zeta potential were evaluated. The physical and oxidative stabilities were used to measure the product quality during storage. Chitosan concentration, stirring speed and its interaction significantly affected (p < 0.05) the particle size. In addition, chitosan concentration significantly affected (p < 0.05) the zeta potential of nanocapsules emulsion. Based on the results of physical and oxidative stabilities, the nanocapsules were stable for 30 days under refrigeration temperature (7 °C), and with 1.5–2% chitosan resulted in improved protection against oil oxidation. The nanocapsules produced with 2% chitosan and 10,000 rpm showed the lowest variations of polydispersity index and nanocapsules size after 30 days of storage (221.8 ± 3.0 nm). These conditions can be considered the most suitable to produce nanocapsules of PUFA concentrates from tuna oil using chitosan as wall material. These nanocapsules showed physical characteristics and oxidative stability, which could enable their application in the food industry, representing an important source of EPA and DHA fatty acids. [ABSTRACT FROM AUTHOR]