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1、 米渣谷蛋白论文：米渣谷蛋白卡拉胶糖基化改性及其价接枝物乳化性能研究【中文摘要】米渣(淀粉糖等工业的副产品)具有丰富的蛋白资源,而米渣谷蛋白作为米渣蛋白的主要的蛋白(85%以上),氨基酸组成平衡合理且具有低过敏等特点,是一种优质的植物蛋白添加剂,但由于其高疏水性和低溶解性,限制了其在食品工业中的应用。本论文以米渣谷蛋白和卡拉胶为原料,利用干热美拉德接枝反应对谷蛋白进行糖基化改性,制备了米渣谷蛋白-卡拉胶共价接枝物,系统的探讨了接枝产物的理化性质、结构、功能性质及其在乳液中的应用,旨在深入研究糖基化改性的机理和改性产物的功能性质,为其应用于工业化生产提供一定的理论基础。以米渣为原料提取谷蛋白,并
2、采用-淀粉酶法纯化谷蛋白,研究了-淀粉酶添加量及酶解时间对谷蛋白纯度及提取率的影响,在此基础上,考虑酶成本及时间等因素,确定了最适的酶解条件：-淀粉酶添加量为0.25mg/mL,酶解时间为3h,谷蛋白的纯度达到90.53%,提取率为35.27%。采用干法美拉德反应,对提取的米渣谷蛋白进行糖基化改性,探讨了谷蛋白-卡拉胶的质量比和反应时间对接枝反应进程和接枝产物功能性质的影响,进一步确定了米渣谷蛋白-卡拉胶共价接枝的最佳反应条件：谷蛋白-卡拉胶的质量比为1：2,在相对湿度79%、温度60条件下,反应24h,产物接枝度达到28.84%。相比谷蛋白,功能性质得到显著改善,接枝产物的溶解性、乳化性和乳
3、化稳定性分别提高了2.04、4.84和0.63倍。氨基酸分析结果表明,米渣谷蛋白与卡拉胶的美拉德接枝反应的位点有赖氨酸、精氨酸和组氨酸,其中以赖氨酸和精氨酸为主。通过聚丙烯酰胺凝胶电泳分析,随着美拉德反应的进行,谷蛋白相对分子质量为20182Da的谱带逐渐消失,在分离胶顶端出现了新的高分子量(大于170KDa)的谱带。荧光光谱表明,接枝产物具有典型的美拉德产物的荧光特征,在激发波长340nm,发射波长423mn处有最强吸收,红外光谱的结果也表明谷蛋白以共价键的形式接入了卡拉胶分子。圆二色性光谱分析了糖基化改性对米渣谷蛋白二级结构的影响,接枝产物发生了不同程度的-螺旋和D-折叠含量减少,以及p-
4、转角和不规则卷曲含量增加。采用ANS荧光探针法测定米渣谷蛋白及谷蛋白-卡拉胶接枝物的表面疏水性,结果表明,糖基化改性显著降低了谷蛋白的表面疏水性。DSC分析表明,谷蛋白-卡拉胶接枝物的热变性温度高,热稳定性良好。从功能特性的研究发现,与米渣谷蛋白比较,谷蛋白-卡拉胶共价接枝物在广泛pH范围内的功能性质得到显著改善。将米渣谷蛋白-卡拉胶共价接枝物作为一种新型乳化剂应用到乳液中,采用超声乳化法制备了油相体积分数为20%的谷蛋白-卡拉胶共价接枝物乳液,乳液均一稳定、粒径较小、粒度分布均匀。研究了pH、盐离子强度、贮藏等环境因素对乳液稳定性的影响,发现以谷蛋白-卡拉胶共价接枝物乳化稳定的乳浊液对盐离子
5、有较强的抵抗作用,而对酸性pH依然有弱敏感性。【英文摘要】Rice dregs, an under-utilized milling co-product of rice syrup industry, contains good-quality extractable proteins. Gluten, as the only major protein of the dregs, comprising at least 85% of the total protein, is hypoallergenic, hypocholesterolemic and rich in essential
6、 amino acids. However, it was limited in the application of food industry as its high hydrophobicity and low solubility. To improve the functional properties of the gluten, rice dregs gluten(RDG) and carrageenan(Car) were used to prepare RDG-Car conjugates by dry-heating method. The physicochemical,
7、 conformational and functional properties of conjugates were investigated in order to study the mechanism of Maillard reaction and provide a theoretical basis for industrial processing, as a result, to be used to meet the need of industrialization.The gluten extracted from rice residue was purified
8、by a-amylase hydrolysis. The effects of the addition of a-amylase and the hydrolysis time were studied on the protein content and yield of gluten, and the optimum conditions were obtained: a-amylase addition 0.2mg/mL, and hydrolysis time 3h. The protein content of gluten was up to 90.53%, and the yi
9、eld was 35.27%.The gluten was glycosylated with carrageenan by dry-heated Maillard reaction, The conditions affecting the degree of graft reaction and the functional properties of conjugates were studied, such as the ratio of protein to carrageenan and reaction time. The optimal reaction conditions
10、were as follows:RDG reacted with carrageenan in weight ratios of 1:2, at 60,79% relative humidity for 24h, the graft degree of the conjugates was 28.84%. The functional properties of DRG-Car were improved obviously, the solubility, emulsifying property and emulsion stability were increased by 2.04,4
11、.84 and 0.63 times, respectively.Amino acids analysis suggested that the major locus during Maillard reaction contained lysine, arginine and histidine, with the first two contributed more. Analysis by SDS-polyacrylamide gel electrophoresis showed that, with the Maillard reaction continued, the band
12、about 20182D was disappeared gradually, and there was a new high molecular weight band (greater than 170KD) closer to the upper of separating gel. Fluorescence spectra resulted that the DRG-Car conjugates had the typical fluorescent characteristics of Maillard products, the conjugates had the strong
13、est florescence intensity when emission was 340nm and the excitation was 423nm. Also infrared spectra results indicated that carrageenan was grafted to gluten by covalent band. The effect of glycosylation modification on the secondary structure of gluten was studied, results showed that the products
14、 were destroyed seriously, of which a-helix and-strand content decreased accompany with-turns and random coil content increasing. The surface hydrophobicity of gluten determined by ANS probes was reduced rapidly when it was glycosylated with carrageenan. The DSC results suggested that the grafts sho
15、wed a good thermal stability, with a high thermal denaturation temperature. The study also found that, in comparison with gluten, the solubility, emulsifying property and emulsion stability of conjugates in a wide pH range were improved significantly.The RDG-Car conjugates as a novel emulsifier were
16、 applied to the emulsion. Oil-in-water emulsions were prepared by blending 20% soybean oil (v/v) and 80% protein solutions (v/v) together through ultrasonic emulsification. The influence of pH, ionic strength, and storage on emulsion stability was studied, results revealed that at the same protein c
17、oncentration, emulsions stabilized by conjugates produced smaller droplet size, compared to emulsions stabilized by gluten and mixtures, and the emulsion was stable against long-term storage and ionic strength, but they showed a weak sensitivity to acid pH.【关键词】米渣谷蛋白 卡拉胶 糖基化 美拉德反应 乳化性【英文关键词】gluten g
18、lycosylation carrageenan maillard emulsifying property【目录】米渣谷蛋白卡拉胶糖基化改性及其价接枝物乳化性能研究摘要3-5ABSTRACT5-6缩略语注释10-11第1章 引言11-211.1 米渣谷蛋白研究概述11-131.1.1 米谷蛋白的分子组成与结构11-121.1.2 米谷蛋白的营养价值12-131.1.3 米谷蛋白的提取131.2 蛋白质糖基化接枝改性13-191.2.1 蛋白质糖基化改性机理14-151.2.2 蛋白质糖基化改性方法15-171.2.3 糖基化改善蛋白质功能性质的研究进展17-191.2.4 米蛋白糖基化改性研
19、究进展191.3 课题研究背景与意义19-201.4 课题研究内容20-21第2章 米渣谷蛋白-卡拉胶糖基化改性21-332.1 材料与仪器21-222.1.1 材料21-222.1.2 主要仪器与设备222.2 试验方法22-252.2.1 米渣中各蛋白组分的测定22-232.2.2 -淀粉酶法纯化米渣谷蛋白23-242.2.3 米渣谷蛋白-卡拉胶共价复合物的制备242.2.4 共价接枝物接枝度的测定242.2.5 米渣谷蛋白及共价接枝物溶解性的测定24-252.2.6 米渣谷蛋白及共价接枝物乳化性的测定252.3 结果与讨论25-322.3.1 米渣蛋白的组成25-262.3.2 -淀粉酶
20、添加量及酶解时间对蛋白质纯度及提取率的影响26-282.3.3 谷蛋白与卡拉胶的糖基化反应28-322.4 本章小结32-33第3章 米渣谷蛋白-卡拉胶共价接枝物的理化性质和功能性质研究33-503.1 材料与仪器33-343.1.1 材料33-343.1.2 主要仪器与设备343.2 试验方法34-373.2.1 谷蛋白-卡拉胶共价接枝物的制备与纯化343.2.2 谷蛋白-卡拉胶共价接枝物功能性质的研究34-353.2.3 表面疏水性测定353.2.4 接枝物氨基酸组成分析353.2.5 十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析35-373.2.6 接枝物红外光谱分析373
21、.2.7 荧光光谱分析373.2.8 圆二色光谱(CD)分析373.2.9 差示扫描量热(DSC)分析373.3 结果与讨论37-483.3.1 氨基酸组成分析37-383.3.2 亚基分析38-403.3.3 荧光光谱分析40-413.3.4 红外光谱分析41-423.3.5 圆二色性光谱分析42-433.3.6 表面疏水性分析43-443.3.7 热稳定性分析44-453.3.8 谷蛋白-卡拉胶共价接枝物的功能性质的研究45-483.4 本章小结48-50第4章 米渣谷蛋白-卡拉胶共价接枝物的乳化性能研究50-644.1 材料与方法50-514.1.1 材料504.1.2 主要仪器与设备50-514.2 试验方法51-524.2.1 稳定20%油相乳浊液的蛋白最适浓度的确定514.2.2 乳析稳定性试验51-524.2.3 乳液平均粒径及粒度分布的测定524.2.4 乳液形态外观的观察524.3 结果与讨论52-634.3.1 稳定20%油相乳浊液的蛋白最适浓度的确定52-544.3.2 乳液的表征54-564.3.3 乳液的稳定性56-634.4 本章小结63-64第5章 结论与展望64-665.1 结论64-655.2 进一步工作的方向65-66致谢66-67参考文献67-74攻读学位期间的研究成果74