Isolation And Preparation Of Rice Bran Protein/Esterase By Natural Eutectic System And Its Application In Deacidification Of Rice Bran Oil

The larger amount of rice bran concerned for its lower cost,wide source and higher nutritionvalues However,the most part of rice bran has not been deeply processed and utilized due to the lack of research on its utilization,resulting in the waste of rice bran.Natural deep eutectic solvents(NADES),as a new generation of "green solvents",have great potential applications in food processing.In this paper,the simultaneous extraction of rice bran protein(RBP)and rice bran esterases(RBE)using NADES was investigated,as well as the extraction mechanism and esterase characteristics were preliminarily explored.The free fatty acids(FFA)of high-acid rice bran oil(HRBO)were removed by means of enzymatic esterification using Lipozyme TL IM,Lipozyme RM IM and Novozym 435 in NADES.A facile efficient and green enzymatic-deacidification strategy was established.The results have been provided the theoretical basis for further research on rice bran processing and applications in food fields.The main research contents and results are as follows:(1)A simultaneous extraction process of RBE and RBP by NADES assisted with a water bath method was established.The results showed that this method could simultaneously extract RBE and RBP.The optimal conditions were as follows: The solvent of proline-glycerol(molar ratio 1:2,containing 5% water),70.0℃,and the ratio of rice bran/ solvent was 9:30.The relative activity of RBE and extraction rate of the RBP was 2.97 U,85.74% respectively.The results of FT-IR and SEM showed that there was no reaction between RBP and PG.The microstructure damage of rice bran increased with the extraction rate of the RBP.After purified by anion exchange resin DEAE,the higher purity of RBE was obtained,and its purification yield and the yield was 1.74,70.01%,respectively.The molecular weight of RBE was about 35 k Da.The optimal substrate of RBE was p-nitrophenyl acetate.The optimal temperature and p H was 40.0℃,8.0,respectively.The better stability of RBE at 30.0～40.0 ℃ and p H 7.0～9.0 could be maintained,as well as the similar performance forcholine chloride-glycerol,betaine-glycerol and proline-glycerol.(2)A facile,green and sustainable strategy for enzymatic-deacidification was established using NADES as co-substrate and reaction media combined with Novozym 435 as a catalyst.The results showed that could catalyze the raction of The esterification for HRBO could be efficiently catalyzed by Novozym 435 in NADES system.Two NADES(choline chloride-glycerol and betaine-glycerol)and three lipases(Novozym 435,Lipozyme TL IM and Lipozyme RM IM)were compared to the deacidification efficiency.Novozym 435 or Lipozyme RM IM in glycerol-based NADES system was better than that of Lipozyme TL IM.The molecular docking simulation showed that reaction between Novozym 435 and glycerol withhigher binding affinity and capability to was might a reason for the potential mechanism of higher deacidification efficiency.The optimal reaction conditions were as follows: 5% of enzyme loading for Novozym 435,30 g choline chloride-glycerol(molar ratio 1:3),60℃ for 3 h.Under the optimal conditions,the retention rate of γ-oryzanol was 87.69%,and the enrichment ratio of tocopherols was 1.47.The reusability of Novozym 435 and Novozym 435-NADES systems was 7 and 4 batches,respectively.