Preparation and Release of Nattokinase Microcapsules and Evaluation of Absorption in Caco-2 Cells Model

To protect nattokinase from inactivation and improve its absorption in gastrointestinal tract,the optimum preparation condition of nattokinase microcapsules formed by double emulsion evaporation method was obtained by orthogonal experiment,with poly(ethylene glycol-b-(DL-lactic acid-co-glycolic acid)-b-ethylene glycol)(PEG-PLGA)as the wall material,and entrapment efficiency as the index. Then nattokinase microcapsules were respectively prepared with PEG-PLGA and folate-poly(ethylene glycol-b-(DL-lacticacid-co-glycolic acid)-b-ethylene glycol)(FA-PEG-PLGA)as the wall material,under the optimum condition. Finally,the gastrointestinal release in vitro and cytotoxicity and absorption in Caco-2 monolayer model of the two microcapsules were evaluated. Results showed that,the optimum preparation condition of PEG-PLGA nattokinase microcapsules were listed as follows:Wall material concentration of 5 mg/mL,PVA concentration of 1%,homogenization speed of 17500 r/min and homogenization time of 7.5 min for double emulsion. For PEG-PLGA and FA-PEG-PLGA nattokinase microcapsules,their average particle sizes were 271.33 and 255.75 nm,the encapsulation efficiency was 61.54%±2.36% and 58.76%±2.54%,the Zeta potential was(-20.17±1.42)and(-24.73±2.36) mV,respectively. Results of gastrointestinal release in vitro showed that more than 60% nattokinase was retained in the two microcapsules after 2 h in gastric pH,and the sustained release effect was good in the intestinal environment(pH7.0)within 22 h. Both microcapsules had almost no toxicity to Caco-2 cells at a concentration of 500 μg/mL,and could be well absorbed,the apparent permeation coefficients of the two microcapsules were as high as 2.367×10^-6 and 3.497×10^-6 cm/s,respectively. Results of laser confocal microscopy also showed that both microcapsules were well absorbed in Caco-2 cells,and FA-PEG-PLGA microcapsules were absorbed better than PEG-PLGA microcapsules.