Quality and in vitro Biological Activities of Lipid Extracted from Sardine (Sardinella spp.) Processing Wastes by Enzymatic Hydrolysis

Introduction/Objective:: Fish are a rich source of lipids, particularly omega-3 polyunsaturated fatty acids (PUFAs), which are known to reduce cardiovascular disease risk and support human health. Processing of fish generates significant by-products, including heads, viscera, and tails, which contain recoverable lipids. Conventional extraction methods often involve high temperatures and toxic solvents, compromising lipid bioactivity and safety. This study aimed to optimize enzymatic hydrolysis using Alcalase® for lipid recovery from sardine (Sardinella spp.) processing wastes and to evaluate the quality and biological activities of the refined oil. Methods:: Oil extraction was optimized using a central composite design of response surface methodology (RSM) with three independent variables: hydrolysis time, enzyme concentration, and temperature. Recovered oil was refined through degumming, neutralization, bleaching, and deodorization. The refined oil was analyzed for physicochemical quality parameters, fatty acid profile, and bioactivities, including antioxidant and anti-inflammatory effects. Results:: Optimal hydrolysis conditions were 3 h, 0.80% enzyme concentration, and 57.5°C, yielding 8.74% ± 0.12 crude oil. Refining improved key quality indices, including peroxide value, thiobarbituric acid reactive substances, free fatty acids, fatty acid composition, and color, while retaining PUFA content. EPA and DHA concentrations increased to approximately 52–59%. Refined oil exhibited notable bioactivities such as DPPH scavenging activity (23.47– 55.97%), cyclooxygenase-2 inhibition (26.38–81.94%), 5-lipoxygenase inhibition (22.51– 70.41%), and protein denaturation inhibition (34.9–89.6%). Discussions:: Results of the study revealed that using RSM in optimizing enzymatic conditions resulted in maximum oil recovery. Oil yield was affected by the interactive effects of the hydrolysis time and temperature, and enzyme concentration. Increasing the extraction time and enzyme concentration enhanced oil yield up to the optimum point. Refining the extracted oil removed impurities and oxidation products without reducing PUFA levels, thereby increasing biological activities. Conclusions:: Enzymatic hydrolysis and subsequent refining of sardine processing waste efficiently recover bioactive lipids with retained PUFA content. The refined oil demonstrates significant antioxidant and anti-inflammatory activities, highlighting its potential as a natural alternative to synthetic drugs and as a sustainable source of functional ingredients for nutraceutical and functional food applications.