Intensification of Palmaria palmata protein biorefinery using multifrequency ultrasonication and enzymes

The red seaweed Palmaria palmata is a promising source for alternative proteins. Development of mild and novel biorefinery approaches are needed for green processes yielding functional proteins. Process intensification using ultrasound-assisted unit operations can be an effective strategy to circumvent current seaweed biorefinery challenges, in particular limited protein extraction yields. In this research, first, effects of acoustic cavitation (AC) during ultrasound-assisted extraction (UAE) were studied using a multifactorial design of experiments for total protein and R-phycoerythrin (RPE) extraction from P. palmata. Secondly, novel enzyme-assisted high-frequency ultrasonic extraction (EAUE) was developed following the same approach and responses. Contrary to the traditional acoustic cavitation process, this intensification strategy allows a milder extraction of proteins from the recalcitrant P. palmata harnessing combined effects of acoustic irradiation and enzymes. Validation experiments showed the robustness of developed models. Maximum RPE yields were 2.6 mg/g_dw for both approaches. However, maximum total protein extraction efficiencies were 13.6% when applying acoustic cavitation and increased to 36.0% under high-frequency acoustic irradiation together with the Viscozyme® L enzyme preparation. Moreover, intensification phenomena were observed during EAUE and an increased extraction yield in comparison to traditional enzymatic processes (EAE). We hypothesize the synergistic effect observed is due to acoustic fields improving cell wall disentanglement and enhancing enzyme-substrate interactions. The present study provides insights into the use of ultrasound for protein extraction of P. palmata and introduces, to the best of our knowledge, for the first time EAUE as process intensification strategy of seaweed biorefinery processes.