【作者】 王章存；

【导师】 姚惠源；

【作者基本信息】 江南大学 ， 粮食、油脂与植物蛋白工程， 2005， 博士

【摘要】 本论文以淀粉糖生产中的副产品米渣为原料,对米渣蛋白的制备方法、结构特征、酶法改性和酶水解机理等进行了系统研究,旨在探讨制备高纯度米渣蛋白技术,改善蛋白的物化功能,为开发利用米渣蛋白资源提供技术基础。分析表明,与大米相比,米渣中的清、球、醇溶和谷蛋白含量明显降低,碱溶液只能溶出米渣中48%的蛋白质(称为米渣谷蛋白,RDG)。米渣中胱氨酸、蛋氨酸含量分别比大米粉谷蛋白(RFG)中相应氨基酸提高83%和81%,RDG中胱氨酸、蛋氨酸分别比米渣降低23%和37.8%,表明-S-S-是影响米渣蛋白质溶解的内在因素,高温加热是大米蛋白严重变性的重要原因。基于米渣蛋白的特点,提出了用纯化工艺制备高纯度米渣蛋白的思路,本文系统地研究了制备高纯度米渣蛋白的纯化工艺和条件。结果表明,α-淀粉酶水解法所得到的蛋白含量高达86%(称作米渣分离蛋白,RPI),其中17种氨基酸的含量为79.13%,蛋白质回收率达93%,而且通过酶水解过程中低聚糖组分变化和蛋白质形态观察研究了纯化过程中糖与蛋白质的分离机制。研究了所制备的RPI分子大小、亚基组成、蛋白与糖的结合性质等结构特征。HPLC和Sepharose CL-4B凝胶色谱分析表明,RFG中主要是93KDa以下的蛋白质组分,而RPI中主要是115KDa-211KDa的大分子;两种蛋白的亚基组分具有相近的结构和性质,说明RPI通过二硫键的连接形成了高聚体,并进而影响了它的溶解性能。凝胶色谱和IR光谱分析表明,RPI和RFG是糖蛋白,且RPI中的糖含量较高,表明高温加热促进了RPI中蛋白质与糖的结合。GC分析表明糖的成分是葡萄糖和阿拉伯糖,二者含量之比约为4.3:1;β-消去反应证明蛋白与糖以N-糖肽键方式连接;赖氨酸分析表明RPI中发生了Maillard反应。所以糖类物质的存在是米渣蛋白不溶的又一重要原因。SDS-PAGE分析显示RPI中有7条谱带(其中13KDa和38KDa含量较高), RFG中有8条谱带, RPI亚基有很强的重新聚合成大分子的能力,再次说明,-S-S-的存在是米渣蛋白质不溶的重要原因。CD分析显示RPI中α-螺旋、β-转角和自由回转结构分别占5.6%、42%和42%,而RFG中分别为17.75、0和82.3%,表明高温变性的RPI二级结构与未变性的RFG有显著差异。研究了蛋白酶法水解对RPI溶解、乳化和发泡性质的影响。单一的碱性蛋白酶对米渣蛋白的水解不如碱性蛋白酶和复合蛋白酶共同作用时的水解效果好;通过数学软件分析了不同酶水解条件与蛋白质物化性能间的关系,当复合蛋白酶0.02%、碱性蛋白酶0.08%和反应时间2.5h,蛋白质溶解度可达56.62%,乳化活性(OD值)为0.493,发泡性为175ml。蛋白质组分的相对分子质量及其含量分析表明,适当大小的酶水解物分子有利于表现乳化和发泡性能。更多还原

【Abstract】 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.更多还原