Abstract <p>Squid viscera, a waste product from squid processing, is a marine source of omega-3 fatty acid–rich oil suitable for nutritional supplement use. Squid visceral oil requires extraction and neutralisation of free fatty acids prior to consumption. Immobilised lipase (Lipozyme RMIM) is suitable for green neutralisation with high acylglyceride yields, high oxidative stability and retention of the antioxidant astaxanthin. Long-term utility of this method depends on lipase stability and reusability over multiple reaction cycles. To assess stability, this study monitored the performance of immobilised Lipozyme RMIM over 35 successive cycles of reuse in a custom-built one-litre reactor. This effectively reduces enzyme cost to 2.9% compared to single cycle use costs. We found a maximum of 97% free fatty acids in the crude oil were converted to acylglycerols under optimised reaction conditions in the first cycle, reducing to 86% after 35 reaction cycles. The partial loss of enzyme activity after each cycle appears to be a combination of enzyme unfolding and aggregation with a physical fracturing and breakdown of the resin, particularly after 30 cycles.</p> Key points <p>• <i>Immobilised Lipozyme RMIM neutralises squid oil effectively for 32 repeat cycles</i></p> <p>• <i>Fatty acid composition of re-esterified squid oil is consistent over repeat cycles</i></p> <p>• <i>Enzyme denaturation and carrier breakdown led to changes in lipase performance</i></p>