Research On Optimization Of Nattokinase Fermentation And In Vitro Simulation Of Gastrointestinal Digestion And Absorption

Nattokinase is a traditional Japanese food,an alkaline serine protease with a molecular weight of about 28kDa found in natto.It is derived from the protein in Bacillus natto fermented soybeans.Nattokinase has a good thrombolytic activity,which can be taken orally to play a good effect and is safe,non-toxic and free of side effects.At present,however,the production of nattokinase still relies on the traditional solid-state fermentation process,with low cost and high efficiency.It is not clear how nattokinase,an active protein,enters the bloodstream through gastrointestinal digestion.This study focused on the optimization of deep fermentation of nattokinase and the study of the digestion and absorption process of nattokinase by building a simulation model in vitro.In this study,the medium and optimum conditions for nattokinase liquid fermentation were determined by using nattokinase activity as an index through multi-level optimization.Firstly,the optimum concentrations of sucrose and soybean powder were determined by single factor test,and the optimum concentrations of glycerin,CaC12 and MgC12 were determined.Then the three factors with the most significant influence on nattokinase activity were selected by Plackett-Burman test,namely dipotassium hydrogen phosphate,culture temperature and culture time.The three significant factors were optimized by box-behnken test.After several optimization,the medium formula and culture conditions were bean powder 12g/L,sucrose 24/L,glycerin 4g/L,CaCl2 0.2g/L,MgCl2 0.3g/L,KH2P04 2.3g/L,K2HP04 14.109g/L,culture temperature 31.063?,culture time 37.479h.After ultrafiltration and freeze-drying in nattokinase liquid fermentation medium,the nattokinase freeze-dried powder NK was detected by lc-ms/MS,and the presence of nattokinase was confirmed by comparison with the UniProt protein database,with a molecular weight of 27.74kDa.A static in vitro simulated digestion model was constructed to simulate the digestion of NK in the stomach and small intestine.After 1.5h of simulated digestion reaction,it was concluded that nattokinase could maintain its own state well in the environment of the small intestine,but could not tolerate the gastric environment with high acidity.The absorption model of small intestinal epithelial cells was constructed by caco-2 cells,and the modeling was completed through morphological observation,transmembrane resistance value(TEER)and marker leakage detection.After NK on the AP side,nattokinase can be detected on the BL side after 2 hours,which proves that nattokinase can be absorbed by the human body through the small intestinal epithelial cells.