酸面团发酵过程中麦谷蛋白大聚体降解及调控机理研究

Protein degradation is one of the main factor influencing flavor and quality of sourdough. Glutenin macropolymer(GMP) is the protein with the most complex structure in wheat flour sourdough system. Wheat flour, lactobacillus plantarum and yeast are used to prepare for sourdough in the present study. The changes in growth of lactobacillus plantarum and yeast are analyzed during sourdough fermentation. GMP is separated by SE-HPLC and content is determined. Relationship between GMP degradation and microbial growth in sourdough fermentation will be analyzed. Change in composition of GMP is analyzed by SDS-PAGE, CE and RP-HPLC during sourdough fermentation. Degradation change of GMP will be clarified, and distribution of resistant peptide bond in GMP will be established. The change in secondary structure of GMP in fermentation is determined by FTIR and CD techniques. And then, effect of the structure of resistant peptide bond on GMP degradation is studied. After the contents of free -SH/S-S bond are analyzed in GMP, the relation of S-S bond to resistant peptide bonds will be studied. According to the change in characteristics and quality of sourdough in fermentation, the relationship between GMP degradation and characteristics, quality of sourdough will be revealed. GMP degradation in sourdough fermentation will be finally clarified. The research will not only lead to a much better understanding of development mechanism and quality control of sourdough, but also eventually lead to the standardization of sourdough process.

蛋白质降解是影响酸面团风味及品质的主要因素之一,麦谷蛋白大聚体(GMP)是小麦粉酸面团中结构最为复杂的蛋白质。本项目以小麦粉-植物乳酸菌-酵母菌制备酸面团,在研究酸面团发酵过程中植物乳酸菌及酵母菌生长变化规律的基础上，利用SE-HPLC分析GMP含量,分析酸面团中GMP降解与微生物生长之间的量值关系。以SDS-PAGE、CE及RP-HPLC分析其组成变化,阐明GMP降解规律并确立“阻抗性肽键”的分布;以FTIR、CD图谱表征GMP二级结构的变化,分析“阻抗性肽键”结构对GMP降解的影响,根据-SH/-S-S-的变化,分析-S-S-与“阻抗性肽键”之间的关系。本研究将为酸面团形成机理及控制提供基础理论信息,也将为酸面团标准化生产起到指导作用。

本研究以小麦粉-植物乳酸菌-酵母菌酸面团发酵体系作为研究对象，研究了酸面团发酵过程中麦谷蛋白大聚体(GMP)的降解规律。酸面团发酵过程中，乳酸菌生长代谢使体系中pH降低、总酸度增加、面团发生酸化，对酵母菌生长有明显的抑制作用，酵母菌对乳酸菌的生长速率也有一定的影响。麦谷蛋白组分包括麦谷蛋白大聚体（GMP）含量随着酸面团发酵时间延长而降低、SDS可溶性麦谷蛋白含量增加、肽含量显著降低。GMP经RP-HPLC分离形成HMW-GS和LMW-GS两部分：HMW-GS 2个峰、LMW-GS7个峰组成，酸面团发酵发酵12 h时LMW-GS一个峰消失，其它峰面积减小，高Mr的HMW-GS峰面积减小，低Mr的HMW-GS峰面积增加。SDS-PAGE图谱中，GMP只有部分HMW-GS和Mr 39.8 KDa和42.4 KDa谱带，在初始发酵4 h内颜色变浅，LMW-GS部分变化显著，GMP中只有部分HMW-GS在发酵过程中降解，部分Mr高的亚基始终没有分解，这部分属于“阻抗性肽键”形成的HMW-GS亚基，限制了GMP的分解。在CE图谱中GMP也分为LMW-GS和HMW-GS 2个峰，酸面团发酵过程中在二者之间出现1个由HMW-GS分解产生的新峰。发酵过程中，GMP中氨基氮随着发酵的进行持续下降，可溶性氮含量在发酵初期快速随着发酵的进行快速升高，达到最大值后缓慢增加，而肽氮含量则是在达到最大值，开始出现下降。随着发酵时间的进行，游离巯基含量升高，而二硫键含量下降，二级结构发生改变。酸面团中蛋白质经SE-HPLC分离后分为 4个峰：分别表示聚合体的HMW-GS（Fl），LMW-GS（F2），醇溶蛋白（F3, 单体）和低分子量的非储藏蛋白质（F4），F1和 F2组成GMP。发酵过程中，F1和 F2峰面积随着发酵时间的进行均有所下降，发酵4h后在F1和 F2之间出现了一些新峰。酸面团发酵过程中GMP平均粒度减小，形成了更小粒径的分子。酸面团发酵过程中面筋的网状结构破裂，GMP出现断裂，CLSM图中以膜状、网络结构存在的面筋蛋白，在发酵过程中逐步变薄，出现不连续相和断裂状态，网络结构变得疏松、空洞面积增大。结果表明乳酸菌对酸面团中GMP降解起主导作用，酵母菌抑制乳酸菌对其降解，HMW-GS中的“阻抗性肽键”限制了GMP的彻底分解。