基于阿拉伯木寡糖分子结构修饰的益生活性构效关系研究

Prebiotics have attracted considerable attention as healthy food additive due to their proven health-promoting effects in human diets. Arabinoxylan oligosaccharides (AXOS), a new type of prebiotics with promising prebiotic characteristics, have gained tremendous research interests. AXOS have complex oligosaccharide composition with varied structural features, such as un-uniform degree of polymerization and substitution. Meanwhile, β-D-xylopyranoside units in arabinoxylan-oligosaccharides structure could be either unsubstituted, mono-, and/or di-substituted with α-L-arabinofuranoside unit at varied positions. Even, less abundant substituents of uronic acids, phenolic acids like hydroxycinnamic acid and ferulic acid, and/or short oligomers of D-galactose and D-glucose could be attached to arabinoxylan-oligosaccharides chains. Complex AXOS structural features restrict the elucidation of structure-function mechanism involved in their prebiotic effects by far. In the current project, arabinoxylan (AX) from flaxseed gum and wheat bran etc. will be subjected for AXOS preparation. Xylanase, produced by our research group, will be employed for enzymatic hydrolysis. Structure of AXOS will be targeted modified through enzymatic hydrolysis using a combination of different debranching and depolymerisation enzymes. Subsequently, AXOS with varied but known structural features will be subjected for prebiotic tests employing human gut microbes from faeces. AXOS fermentation fingerprinting will be constructed based on the monitoring of structure and composition changes of AXOS during fermentation. The difference between fermentation process and pattern of AXOS with varied structure will be analyzed according to proteomics analysis. The enzymes involved in AXOS degradation and utilization will be identified, as well as expression level and enzymatic activity. Fermentation kinetics of human gut microbes with different AXOS as the only carbon source will be set up by detecting the solution pH, content and composition of organic acids during AXOS degradation. Meanwhile, differences in total count and composition of human gut microbes will also be investigated. Knowledges gained in this research project on structure-function relationship of AXOS bifidogenic effects will be of great importance in rational design of functional foods with tailor-made component of prebiotics and targeted bifidogenic specificity.

益生元作为健康食品配料具有促进人体健康的功效。阿拉伯木寡糖（AXOS）是一类新型益生元，由于其组成和结构复杂，迄今AXOS益生活性构效关系尚不清楚。本项目拟在亚麻籽胶阿拉伯木聚糖（AX）前期研究基础上，采用木聚糖酶水解不同来源AX制备AXOS。选择不同脱支酶及解聚酶对AXOS分子结构进行修饰，定向移除葡萄糖醛酸、酚酸、半乳糖及阿拉伯糖等侧链并改变糖链聚合度。采用肠道菌群体外酵解AXOS，建立AXOS酵解动力学指纹图谱，运用蛋白质组学方法鉴定AXOS分解酶，分析酶表达水平差异及酶活变化，研究不同结构特征AXOS酵解过程及模式的差异；监测肠道菌群数量及组成的动态变化，分析肠道菌群代谢产有机酸及pH变化，构建肠道菌群代谢动力学，研究AXOS对肠道菌群增殖及代谢的影响规律，实现基于分子结构修饰的AXOS益生活性构效关系全面解析，为新型高效及特异性的AXOS益生元发掘和健康食品应用提供理论依据。

项目执行期间研究内容主要包括对亚麻籽胶、小麦麸皮以及山楂籽来源的阿拉伯木聚糖的制备，采用阴离子交换色谱对亚麻籽胶来源的中性糖进行了分离纯化，结合傅里叶红外光谱、气相色谱-质谱联用以及一维及二维核磁共振波谱技术对纯化后的亚麻籽胶中的中性糖的精细结构进行了解析，推测为在O-2和/或O-3位置取代的富含木糖的杂聚糖。对该阿拉伯木聚糖的免疫调节活性机制进行了研究，发现其主要是通过与免疫细胞表面的TLR2受体结合，激活MAPKs和NF -κB信号通路。克隆表达了Clostridium thermocellum来源的阿拉伯木聚糖酶CtGH5-CBM6并对小麦麸皮来源的阿拉伯木聚糖进行酶解制备阿拉伯木寡糖。水解产物经HPAEC分析发现，其主要包含12种糖组分，聚合度约在5-14之间。进一步采用体外粪便发酵评价了小麦麸皮来源的阿拉伯木寡糖的益生活性并分析了肠道菌群代谢利用阿拉伯木寡糖的结构选择性。小麦来源阿拉伯木寡糖可提高粪便菌群中Actinobacteria放线菌门相对丰度，降低Bacteroidetes拟杆菌和Proteobacteria变形菌门相对丰度。粪便菌群代谢阿拉伯木寡糖产短链脂肪酸，以乙酸为主，其次是乳酸、丙酸和少量丁酸。经代谢指纹图谱分析发现，粪便菌群优先代谢利用聚合度较高的阿拉伯木寡糖，难以利用聚合度较低的组分。采用毕赤酵母对铜绿假单胞菌GY701来源的木聚糖酶基因PaXyn10A进行了高效表达并实现了高密度发酵。通过水解山楂籽来源的乙酰基化木聚糖发现，水解产物中的木寡糖产率可达18.5g/100g。此外项目执行过程中还探究了乳酸杆菌体外酵解利用昆布寡糖的菌株特异性和结构选择性，发现乳酸杆菌能够很好吸收利用昆布二糖及昆布三糖，对于聚合度较大的昆布寡糖利用较差；通过对决明子胶甘露寡糖的体外益生活性评价发现，肠道微生物代谢利用决明子胶甘露寡糖过程中，β-半乳糖苷酶和β-甘露糖苷酶发挥了重要作用。粪便菌群可以优先代谢利用DP 3−5的决明子胶甘露寡糖，随后再代谢利用DP=2的决明子胶甘露寡糖。项目相关研究结果为基于功能性低聚类益生元的生物转化和构效关系研究提供借鉴。