Preparation And Enzymatic Modification Of Rice Protein From Rice Dreg

Rice protein, which could be extracted from rice flour, rice bran and rice dregs, is an excellentsource of vegan, hypoallergenic protein. Rice dreg was a by-product from rice starch syrupmanufacture and organic acid or antibiotics fermentation. It contained about 40% protein. Butbecause of its poor solubility, it was mainly used for animal protein feed. There was little researchabout the utilization of rice dreg in food industry so far.In this paper, the prepatation methods, structure characteristics, enzymatic hydrolysis modeland mechanism of rice dreg protein from rice syrup production were systematically investigated soas to obtain high purity protein and improve its applicable properties,which were innovative.The analytical results indicated lower contents of every kind of proteins (Albumin, Globulin,Prolamin and Glutelin) in rice dreg than in rice flour. About 48 percentage of rice dreg proteincould be resolved in alkaline solution(named RDG), Cystein and Methionine quantity in rice dregwere increased 83% and 81% respectively compared with that in rice flour glutelin(RFG), but theseamino acids quantity in RDG were decreased 23% and 37.8% respectively compared with that intotal rice dreg, which meant that -S-S-linkage was internality factor affecting solubility of rice dregprotein, and high temperature was main factor resulting in the linkage.Based on the results above, a new idea, purification approach, was put forward for get highpurity protein from rice dreg and the purification condition was systematically investigated. Theresult showed that the protein content of the product manufactured by α-amylase hydrolysis was86%(named Rice Protein Isolates, RPI), and 17 amino acid total quantity was 79.13%, the recoveryof protein was 93%. The separation mechanism between carbohydrate and protein was studied withchange of oligo-saccharide compositions and protein conformation, These results proved thatpurification technique was better than traditional method with alkali dissolving and acidprecipitation.The analysis by HPLC showed that the relative molecular mass (Mr.) of RFG was lower than93KDa, but that of RPI was mainly about 115-211KDa. Gel chromatography in Sepharose CL-4Band UV spectra showed the differences in whole molecular, but the homology in theirsub-compositions between RPI and RFG, which meant heating caused linkage of molecular in RPIby disulphide bond. The research by gel chromatography and IR spectrum proved RPI and RFG to be sugar-proteincomplex. But sugar quantity in RPI was higher than that in RFG, heating was main reason. Gaschromatography (GC) analysis indicated that RPI contained glucose and arabinose,andβ-elimination reaction revealed that the glycoprotein was not O-glycoside peptide bond. Thedecrease of Lysine quantity meant Maillard reaction had happened in RPI, which was anotherreason for the poor solubility of RPI.The subunit composition of the protein was researched with SDS-PAGE, seven subunits(mainly 13KDa and 38KDa) were found in RPI and nine in RFG. The subunits in RPI were veryeasy to re-aggregate large molecular, which proved again that -S-S-was major reason why RPI hadpoor solubility in water and even in NaOH solution. CD spectra also indicated the evidentdifferences between RPI and RFG in secondary structure.In order to dissolve the rice protein and improve its physicochemical properties, the method ofenzymatic hydrolysis was investigated, and result showed that the efficiency of hydrolysis withAlcalase and Protamex together was better than that with single Alcalase. The effect of hydrolysisfactors on the solubility, emulsification and foaming activity of hydrolysates was analyzed withMathematica statistic software. The best condition was 0.02% Protamex plus 0.08% Alcalase and2.5h reaction time. With the analysis of Mr., it was concluded that certain large molecular wasimportant for physicochemical properties of hydrolysates.The mechanism of enzymatic hydrolysis and changes of protein molecular during hydrolysiswere also investigated. The distribution of enzyme in supernatant and insoluble protein pellet wasdifferent according to the content of enzyme and the reaction process. 57.8KDa,39KDa,26KDaand 22KDa subunits in RPI would gradually disappear,otherwise the content of 13KDa increasedduring the process of hydrolysis, but that of 29KDa and 27KDa was constant, which meant the twosubunits could resist enzymatic hydrolysis. The results could explain why RPI could not bethoroughly hydrolyzed and dissolved in water.The composition of amino acids in soluble part and residue of hydrolysate was markedlydifferent. The content of Cystein and Methionine in the residue was increased 44.8 and 92.8percentage, but Lysine, Aspartic acid and Serine decreased 25, 18.8 and 6.9 percentage respectivelythan that in the soluble part. In otherwise the residue exhibited evident slice gel structure, whichmight be formed by cross -S-S-bond in protein molecular. It was the reason why Alcalase couldselectively hydrolyze some subunits in RPI。The investigation about conjunctive property of protein and sugar showed that protein insoluble hydrolysates was still jointed with sugar, and the ratio protein/sugar was about 100:7.2, butthe connective form was not O-glycoside peptide bond;The subunits of proteins in the residue wereseparated with SDS-PAGE and stained by periodic acid-SchiffS reaction(PAS staining), the resultsshowed that 13,29,39KDa subunits were also glycoproteins. The same conclusion also derivedfrom the IR spectra of the soluble portion and the residue. The specificity of 13KDa glycoproteinsubunit maybe the reason why it could resist hydrolysis of Alcalase.Some physicochemical characterization of RPI hydrolysate(HRPI) in different applicableconditions were investigated. The higher pH and concentration of HRPI were, the higheremulsifying activity (EA) and foaming activity (FA) had. EA could be improved by adding glucose,sucrose, soluble starch and Na2SO3 in mild pH solution of HRPI. The mechanism may be thesuitable membrane formed by starch-protein and disruption of –S-S-bond in HPRI respectively.The distribution of HRPI components in water and emulsified phase was researched by pH8.0and pH12.0 emulsification systems. The results showed that Mr. of protein components in water andemulsified phase was almost equal, but composition of amino acids was very different under sameemulsification system. In different system, the components of protein and amino acids in emulsifiedphase were also different, so did in water phase, which meant that a same protein molecular couldexist in different phase under different pH emulsification systems.