Preparation And Purification Of Rice Immune Active Peptide

Chinese rice production was about 200 million tons in 2014,highest in the world.A lot of byproducts like broken rice,rice bran and rice residue were generated in machining processes.Fermentation and starch industry produced rice residue whose protein content was as high as 40%to 60%.However,most of these byproducts were just used as feedstuff directly,and not be sufficiently exploited.Rice protein has an amino acid composition ratio similar to the ideal model that WHO/FAO recommended,better than that of soy protein and casein better,and comparable to that of milk,eggs,and beef.Morever,rice protein is a low antigenic protein,does not cause allergic reactions,and is very suitable as a raw material for infant food.At the same time,aging,obesity,or a decline in the quality of sleep,smoking and drinking and other bad habits are likely to lead to the decrease of immunity and cause various diseases.Therefore,the search of suitable immunomodulator to enhance human immunity has become a hot spot of society's needs.Using rice processing byproducts like residue and broken rice as raw materials,rice immunologically active peptides that achieved by enzymatic preparation were not only provide hypoallergenic immunomodulatory agent for people with weakened immune systems and infants,but can also improve rice processing value-added byproducts,which has an important social benefit and a higher economic efficiency.This paper selected nine kinds of seed to be screening the enzyme protease cleavage sites based on rice protein digestion,considering the hydrolyzate of mouse macrophage cell proliferation index SI,yield and molecular weight distribution,the optimal enzyme of selected species was trypsin.Its hydrolyzate of mouse macrophage proliferation index SI,the proportion of molecular weight less than 1000 and the yield of component peptides were1.597?1 000 g/mL?,74.11%and 75.96%respectively.After the enzyme species identified in mouse macrophages SI value of the indexes,effects of temperature,time,pH,substrate concentration and enzyme dosage on rice protein hydrolyzate?Rice protein hydrolysates,RPHs?SI value by single factor test and response surface optimization of the optimum conditions for preparing rice tryptic peptide immunoreactivity temperature was 41?,enzyme dosage was 2940 U/g,hydrolysis time was 1.9 h,pH was 7.5,solid-liquid ratio was5%,under these conditions RPHs SI value was 1.418?125 ug/mL?.This paper further considered to separate and purify the hydrolyzate of rice protein trypsin by various chromatographic separation and membranes.First sequential ultrafiltration?UF?,nanofiltration?NF?and reverse osmosis?RO?membrane were used for molecular classification,desalination and concentration of RPHs.Effects of temperature,pressure and feed concentration on the UF,NF and Effect of RO membrane flux process to determine the conduct of RPHs UF,NF and RO process conditions better handled,where UF preferred conditions for the operating pressure was 0.35 MPa,operating temperature was 20?,RPHs concentration was 30 g/L;NF preferred conditions for the operating pressure was 0.7 MPa,operating temperature was 25?,RPHs concentration was 30 g/L;RO process conditions preferred operating pressure was 0.8 MPa,operating temperature was 25?,RPHs concentration was 30 g/L.At last,this paper considered the mouse macrophages SI value,and used the following five kinds of separation methods for purification:DA201-C macroporous resin,001×7-type strong cation exchange resin,Sephadex G-10 gel chromatography,NGC^TM chromatography and reverse phase high pressure High performance liquid chromatography?RP-HPLC?.Determine the condition DA201-C macroporous resin chromatography RPHs of the sample flow rate was 0.5 BV/h,sample concentration was 20 mg/mL,elution flow rate was 1.0 BV/h,and 20%,40%,60%,80%ethanol as elution stage,the RPHs were separated into four components,their hydrophobicity valueswere 90.0,120.5,191.9 and 235.3 respectively;compared with RPHs,the SI values of components RPHs-B and RPHs-C were significantly increase?P<0.05?,in which the SI RPHs-C is 1.594?125?g/mL?.001×7 strong cation exchange resin chromatography was continued to be used to separate RPHs-C.Eluting with pH stage,the RPHs-C were isolated and purified to obtain five components,wherein the value of the highest SI?1.211,62.5?g/mL?is RPHs-C-7 group points,compared with RPHs-C component,SI value?1.108,62.5?g/mL?has a significant increase?P<0.01?.Sephadex G-10 chromatography were further used to separate and purify RPHs-C-7,three component obtained,and RPHs-C-7-3 has the highest SI?1.252,62.5?g/mL?,and compared with that of RPHs-C-7,the SI value?1.105,62.5?g/m L,?has a significant increase?P<0.01?.High pressure chromatography using NGC^TM RPHs-C-7-3 for separation and purification components,compared with RPHs-C-7-3 components,the resulting component 25,26,27,SI value 28,31,36,37,39,40,41 of both significantly increased?P<0.01?which SI values were 1.350,1.364,1.292,1.338,1.322,1.389,1.410,1.427,1.432,1.434?31.25?g/m L?.Using RP-HPLC to component RPHs-C-7-3-28,RPHs-C-7-3-31 separation and purification,as compared with the component RPHs-C-7-3,only SI values have increased significantly and the two components are high purity,its peak area were 88.73%and 89.44%.After hydrolysis process optimization and order macroporous resin chromatography,ion exchange chromatography,gel chromatography,NGC^TM high pressure liquid chromatography and HPLC separation and purification,at SI equal,rice protein pancreatic required protease concentration can be reduced 32 times.