基于宏基因组学的老面酵头微生物菌相分析及酸化途径研究

Sourdough preparing is the most important step during the processing of traditional steamed bread. However, the changes of microbial strains, metabolism and fermentation of the components, and the interaction occurred in the dough involved in the physiological and biochemical process in sourdough, have not been clearly explained yet, leading to no standardized fermentation process.This study, around microbial composition and the mechanism of acidification in sourdough, analyze biodiversity and genome-wide of microorganism in sourdough by metagenomic and bioinformatics analytical tools, analyze and regulate gene expression profiles of microbial acidification metabolic pathways by using real-time fluorescent quantitative PCR, pinpoint the relationship between strains interaction,and establish critical strains. Build liquid "dough" model system, combined with metabolic flux analysis, build up metabolic flux balance model for all the major cellular response by material balance, and calculate metabolic flux distribution of acidification metabolic pathways. Thereby optimize and analyze the metabolic network, reveal regulation and intervention mechanisms of sourdough microbial metabolism and acidification by different components and critical control, establish contacts between sensory quality characteristics of traditional sourdough steamed bread and micro-ecology acidification metabolism, and provide direct theory basis for realizing industrial production of traditional steamed bread.

传统老面馒头加工最重要的过程就是老酵的制备，但老酵的生理生化过程涉及微生物菌系变化、代谢发酵和主面团体系相互作用关系等环节，目前还未被阐释清楚，无法形成标准化的发酵工艺。本课题围绕老面酵头微生物菌相组成及酸化途径研究，采用宏基因组和生物信息学分析手段对老酵微生物进行生物多样性和全基因组解析，通过利用实时荧光定量PCR分析调控微生物酸化代谢途径基因表达图谱，明确菌系互作关系，确立关键菌株；构建液态"面团"模式体系，结合代谢流分析方法，通过物料衡算对所有主要胞内反应建立代谢流平衡模型，计算得出酸化代谢途径的代谢流分布，从而对代谢网络进行优化分析，揭示不同组分和关键控制点对老酵微生物代谢酸化的调控和干预机制，建立传统老酵馒头感官品质特性与微生态酸化代谢之间的联系，为传统馒头工业化生产提供直接理论依据。

馒头作为中国传统发酵主食，通常采用老面酵头进行加工制作，尤其在我国北部地区。然而，老面酵头的生理生化过程涉及微生物菌系变化、代谢发酵和主面团体系相互作用关系等环节，目前还未被阐释清楚，无法形成标准化的发酵工艺。本项目围绕老面酵头微生物菌相组成以及酸化代谢途径对产品品质的影响开展研究。采用宏基因组学和传统分离技术方法，阐明不同酸化水平老面酵头微生物菌群组成存在显著差异，其中，I型酸面团中主要以旧金山乳杆菌为主，而酵子则主要以食窦魏斯氏乳杆菌为主。不同酸化水平老面酵头还对终产品风味特性和物性品质有着重要的影响。基于非靶向性代谢组学分析和风味阈值计算方法，乙酸乙酯，乳酸乙酯，乙酸己酯，（E）-2壬烯醛和2-戊基呋喃为I型酸面团制得产品的关键香气组分，且酸化过程中伴随的亚油酸和棕榈油酸氧化是醛类物质生成的主要因素。通过构建γ-谷酰基连接酶缺失突变菌株，阐明酸化过程中γ-谷酰基二肽代谢形成机制，结果表明Gcl对于γ-谷酰基二肽的合成具有氨基酸底物选择差异性，Gcl1和Gcl2分别调控参与γ-谷酰基异亮氨酸与γ-谷酰基半胱氨酸的合成途径。研究通过比较分析不同酸化水平对馒头物性品质与感官品质影响发现，面团发酵终点pH与馒头硬度、咀嚼性和胶着性同样呈极显著正相关。研究证实，适度酸化与面筋蛋白降解及面筋网络形成有着直接联系，过度酸化导致复杂蛋白大聚合体的严重降解，麦谷蛋白及醇溶蛋白分子间的二硫键被破坏，从而导致产品出现质地过于松软，弹性差等问题。