Two-step enzymatic acidolysis tandem preparation of OPO/OPL-enriched human milk fat substitutes via serial continuous packed-bed reactors.

Region-specific human milk fat substitutes (HMFS) are essential for mimicking native human milk fat and improving nutrient absorption in infants. Herein, a serial continuous packed-bed reactor (CPBR) system employing Candida sp. lipase (CSL) and Lipozyme TL IM was established for the controllable preparation of HMFS via tandem two-step enzymatic acidolysis. Key parameters, including substrate molar ratio, feed flow rate, and lipase loading were systematically optimized to balance target acidolysis reaction and adverse undesirable acyl migration. Benefiting from stable mass transfer, the CPBR system achieved higher product yields and better sn-2 palmitic acid retention than traditional batch reactors. Precise two-stage flow regulation enabled the directional fabrication of HMFS tailored to the lipid profiles of both Chinese and Western human milk fat. Long-term operation and industrial scale-up validation confirmed the excellent stability and scalability of the system, providing a feasible industrial strategy for the large-scale production of customized high-quality HMFS.