Chitooligosaccharides (COSs) with a degree of polymerization (DP)>3 are invaluable for functional foods, biomedicine and agriculture. However, controllable techniques for regulating the enzymatic process of chitosanase in preparing high-DP COS (HdpCOS) are not available currently. To control the preparation of HdpCOS, we constructed a continuous flow enzyme membrane reactor (EMR) based on a 10 kDa tangential flow membrane module with an intermittent enzyme replenishment mode in this study. This system made the real-time separation and controllable synthesis of HdpCOS during chitosan hydrolysis feasible. Using the number average molecular weight (Mn), product distribution, volumetric yield and production rate as the indicators, the effects of enzyme loading and residence time (RT) on the degradation process of chitosan were evaluated, and the preparation processes for COSs within different DP ranges were determined. The products were further separated and purified through ethanol precipitation, yielding HdpCOS with a predominant DP range from 8 to 13. The proposed EMR system exhibited operational simplicity and robust stability, demonstrating that it has potential for being used in the controllable production of COSs.

This study developed an oil-in-water (O/W) emulsion using complexes of whey protein isolate (WPI) and docosahexaenoic acid-containing phospholipid (DHAPL) as emulsifiers for encapsulating fucoxanthin (FX), aiming to enhance its thermal stability, antioxidant capacity, bioaccessibility, and protective effects against oxidative damage in RAW264.7 macrophages. The emulsions stabilized by WPI-DHAPL complexes demonstrated excellent encapsulation efficiency for FX (> 94%), significantly surpassing the WPI-stabilized emulsion, with long-chain DHAPL (e.g., DHA-containing phosphatidyl monopalmitoyl glycerol (DHAPL-MP) and DHA-containing phosphatidyl monostearate glycerol (DHAPL-MG)) being more effective in this regard. The structure of the mixed emulsifiers endowed the emulsion with outstanding thermal stability, maintaining over 80% of FX under harsh conditions (75 ℃/0.5 h), and the FX-loaded emulsion exhibited excellent storage stability at 25 ℃. Furthermore, the DHAPL-WPI system overcame the key limitation on the bioavailability of hydrophobic bioactive substances, resulting in 70% FX bioaccessibility during in vitro simulated digestion, which was 2.8 times higher than that of free FX. The dual-interfacial antioxidant mechanism of the WPI-DHAPL complex significantly enhanced the free radical scavenging capacity of FX, increasing the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation racial and 1,1-diphenyl-2-picrylhydrazyl (DPPH) racial scavenging rates by 35%–42%. It also provided strong protection against oxidative stress in RAW264.7 macrophages, with medium-and short-chain DHAPL showing more pronounced effects. The bioactive substance delivery system developed in this study provides an effective encapsulation solution for functional food development, successfully overcoming the dual bottlenecks of thermal instability and low intestinal absorption efficiency of hydrophobic active ingredients in the food industry.