【作者】 王霞；

【导师】 周惠明；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白， 2011， 博士

【摘要】 我国的小麦普遍存在强筋不强,弱筋不弱的特点,具有优秀焙烤品质的小麦品种较少,本研究从改善小麦粉的焙烤品质、保证小麦粉安全性的角度出发,制备一种安全高效的小麦粉改良剂。目前葡萄糖氧化酶（GOD）被认为是“最有前途的绿色小麦粉强筋剂”,其改良效果已得到广泛认同,但仍存在一定的缺陷:首先GOD属于快速氧化剂,催化速度很快,在面团吸水形成过程中就已经生成了大量的H₂O₂,后者使面团搅拌过程中二硫键断裂产生的大量游离巯基（-SH）再次发生氧化交联,而这种强且快速的交联反应是无选择性的,不利于后期大分子谷蛋白的聚合,影响了其改良效果;其次GOD在面团中稳定性较差,容易发生变性失活。基于此,本文采用海藻酸钠-壳聚糖为壁材,乳化-内部凝胶化法对GOD进行微胶囊化包埋,从三方面改善GOD的改良品质。首先微胶囊化使GOD与外界小麦粉环境隔离,提高了酶的稳定性;其次在面团加水后CAGC有一个吸水膨胀过程,延迟了GOD氧化作用起始时间;最后微胶囊对底物葡萄糖的扩散阻力减慢了H₂O₂的生成速度。首先对海藻酸钠-壳聚糖微胶囊化葡萄糖氧化酶（CAGC）制备工艺进行系统研究,考察了海藻酸钠浓度、CaCO₃添加量、Span 80浓度、搅拌速度和水油比对海藻酸钙凝胶珠（CA）粒度分布和球形度的影响;分析了壳聚糖分子量、浓度、pH和GOD吸附pH值对CAGC微胶囊化效率和蛋白装载量的影响。确定微胶囊化的最佳条件为:海藻酸钠浓度1.5%, CaCO3 4.0 mg/mL,水油比1:5, Span 80 2.0%,搅拌速度1000 rpm,在此条件下制得的CA凝胶珠平均粒径[D4,3]为87.332μm,球形度良好;以此CA作为微胶囊的内核,进一步确定分子量20万的壳聚糖为壁材,壳聚糖浓度为1.5%,复凝聚反应pH 4.0、反应时间60min该条件下GOD微胶囊化效率达81.7%、蛋白装载量为37.7mg/g、所制得CAGC的壁膜厚度约为13.22μm,体积平均粒径[D4,3]为59.332μm,4℃贮藏半衰期为105天。激光共聚焦显微镜观察表明,GOD通过离子键等次级键均匀吸附“捆绑”在CAGC内部,而非沉淀聚集在CAGC的表面,微胶囊的壁膜完整、厚度均匀。采用喷雾干燥技术将湿态CAGC进行干燥,以期提高CAGC的常温贮藏稳定性和实际应用便利性。通过响应面优化干燥条件,最终产品得率和酶活保留率分别达到72.19%和80.64%;建立4℃、25℃和45℃条件下干态CAGC酶活力衰减动力学方程,计算得相应的酶活力半衰期为240天、190天和74天。研究CAGC对面团中湿面筋含量（WG）、水溶性和水不溶性蛋白中-SH含量（W-SH,SDS-SH）、面团粉质特性、拉伸特性、动态流变学特性和微观结构的影响。结果表明:在面团形成过程中,CAGC处于吸水膨胀阶段尚未发挥其氧化能力,延迟了酶的作用起始时间,显著改善了面团伴随着GOD快速氧化而发生的变干变硬现象;而在面团保温阶段,随着保温时间的延长CAGC的氧化能力逐渐体现出来,表现在WG含量增大、W-SH和SDS-SH含量下降、面团的粘弹性、回复性和发酵持气能力等都显著提高。与未微胶囊化GOD相比,CAGC的氧化作用起始时间滞后,氧化速度减慢,作用时间延长,最终在保温阶段结束时对面团内巯基的氧化程度提高,有利于促进面团中大分子谷蛋白链间交联和面团面筋网络结构的架构,SDS-PAGE电泳和面团显微结构观察都对此给出了直观的证明。以GOD和KBrO₃为对比,考察了面团发酵过程中,在CAGC不同添加水平下,面团高度、持气率、CO₂泻出时间以及W-SH和SDS-SH含量等指标的变化,并通过面包焙烤和质构测试分析了CAGC对面团面筋网络结构与面包多项品质的影响。结果表明:CAGC在发酵过程中不断氧化W-SH和SDS-SH,使之形成二硫键,进而提高面团的持气能力,增大面包比容,改善面包芯品质。同时通过各项参数的分析比较,讨论GOD与KBrO₃在改良小麦粉品质方面的差异,并以KBrO₃氧化模式为基准,讨论了与GOD相比CAGC的优势所在。以不同分子量大小的聚乙二醇和蛋白分子为扩散底物,研究线性高分子和球状大分子在微胶囊内的扩散速度和截留分子量,并测定纯水体系下,干态CAGC的吸水膨胀性和葡萄糖的扩散曲线,为建立实际面团体系中CAGC催化速度模型打下基础。综合以上CAGC对面团面筋蛋白构架、流变学特性、发酵能力与焙烤面包的各项品质指标的影响,采用距离分析法建立面团各项参数与面包品质之间的相关性,通过揭示各参数之间的内在关系并结合简单小麦粉模拟体系,探讨微胶囊化提高GOD酶活稳定性、减慢氧化速度和延长氧化作用时间的内在机制。更多还原

【Abstract】 The gluten in wheat flour is neither strong nor weak ubiquitously in our country, with less wheat varieties with excellent baking quality. The study is to prepare a safe and efficient flour improver under the premise of baking quality and safety. At present, glucose oxidase （GOD）, whose improvement effect has been widely recognized, is considered to be "the most promising agent of green gluten flour", but there are still some drawbacks. Firstly, as a fast oxidizer, GOD catalyzes fast and large amount of H₂O₂ is generated during the dough formation process, which produces a large number of disulfide bonds to break free thiol （-SH） and be oxidized again, and this strong and fast cross-linking reaction is not selective, not conducive to post-polymerization of glutenin molecules and affecting the improvement effect.Secondly, GOD is less stable in dough, prone to degeneration and inactivation. Based on this, we embedded GOD using emulsion-internal gelation method with alginate -chitosan as wall material, improving the quality of GOD from three respects. Firstly, GOD was isolated from the outside environment by microencapsulation, improving the stability of the enzyme. Secondly, there is a swelling process for CAGC after adding water to the flour, delaying the oxidation onset. Lastly, diffusion resistance of substrate glucose from the microcapsules slows down the H₂O₂ generation rate.First, the preparation process of sodium alginate-chitosan micro-capsulated glucose oxidase （CAGC） was systematically studied. The effects of concentration of sodium alginate, CaCO₃ dosage, concentration of Span 80, stirring speed and water-oil ratio on the particle size distribution and sphericity of calcium alginate beads （CA）, as well as the chitosan molecular weight, concentration and pH on the CAGC microencapsulation efficiency and the amount of protein loaded were investigated. The ultimate optimum microencapsulation conditions are as follows: alginate concentration 1.5%, CaCO₃ 4.0mg/mL, water to oil ratio of 1:5, Span 80 2.0% and stirring speed 1000 rpm, under these conditions the CA gel beads presented average particle size [D4,3] as 87.332μm, with good sphericity. The CA as the core of microcapsules, the chitosan with molecular weight 200,000 as the wall material, chitosan concentration of 1.5%, complex coacervation reaction pH 4.0, 60min reaction time generated the microencapsulation efficiency of 81.7%, protein loading capacity of 37.7mg/g, the volume of the prepared CAGC average size of [D4, 3] for 59.332μm,wall thickness of about 13.22μm and 4℃storage half-life of 105 days. Confocal laser microscopy showed that GOD evenly distributed in the inside of CAGC and the microcapsule presented membrane integrity and uniform thickness.Wet CAGC was dried using spray drying technology to improve the storage stability at room temperature and practical convenience. The final product yield and enzyme activity retention rate reached 72.19% and 80.64%, through response surface optimization. Decay Kinetic equation of dry CAGC has been built under 4℃, 25℃and 45℃, by which the corresponding enzyme activity half-life as 240 days, 190 days and 74 days.The effects of CAGC on the wet gluten content （WG）, water-soluble and water insoluble protein-SH content （W-SH, DS-SH）, dough properties, tensile properties, dynamic rheological properties and micro-structure were investigated. The results showed that CAGC did not exert its oxidative capacity during the dough formation process due to swelling stage, delaying the enzyme functioning and significantly improving the dry and harden phenomenon of dough with the rapid oxidation of GOD. However, the oxidation capacity of CAGC gradually embodied, reflected in WG content increases, W-SH and SDS-SH content decreased and improved viscoelasticity of dough, holding gas recovery and fermentation capacity. Compared with un-microencapsulated GOD, CAGC has starting time lag, low oxidization and extended action time, leading to increased oxidization degree after insulation and promoting the dough gluten chains cross-linking and dough gluten network structure.The variations of the dough height, gas holdup, CO2 spilled out of time, and W-SH and SDS-SH content were studied during the dough fermentation with different CAGC dose, with GOD and KBrO₃ as control. And the effects of CAGC on the dough gluten network structure and bread quality were analyzed through bread baking and texture analysis. The results showed that CAGC continuously oxidized W-SH and SDS-SH during fermentation, leading to formation of disulfide bonds and improved gas-holding capacity of dough, increased bread specific volume and quality of bread core. Furthermore, the difference of GOD and KBrO₃ in improving the quality of wheat flour was discussed as well as the advantages of CAGC compared with GOD, with the oxidization model as a benchmark.The diffusion rate inside the microcapsules and the molecular weight cutoff of linear polymer and spherical molecules was investigated with PEG of different molecular weight as spreading substrate and the curves for dry CAGC expansion and glucose diffusion, laying the foundation for the establishment of the actual catalytic rate of CAGC in dough system.Integrating the effects of CAGC on the dough components, rheological properties, fermentation capacity and bread baking etc, we established the correlation between the parameters and bread quality through distance analysis, analyzed the principle of microencapsulation to increase the GOD stability and discussed the mechanism of CAGC slow oxidation relative to GOD.更多还原