双歧杆菌氮源利用的选择性及特征分析

Bifidobacteria are very significant and widely accepted probiotics. However, their culture is very difficult. The key factor which restricts the effective proliferation of Bifidobacteria is nitrogen source. Different Bifidobacteria have their own specific nitrogen metabolism systems, which make them more difficult to culture. This project aims to systematically explore the characteristics of nitrogen highly utilized by different Bifidobacteria, and to clarify their corresponding relationship with the nitrogen metabolism genome. Thus, some guidance can be provided on the culture and production of Bifidobacteria and their fermentation application on the base of these researches. Specific research contents are as follows: Firstly, the proliferation efficiency of different Bifidobacteria with different proteins will be analyzed and the characteristics of the nitrogen source selected by respective strain will be summarized. Secondly, the relationship between the amino acid composition of efficient protein and the essential amino acids for growth of different Bifidobacteria, and the characteristics of the peptides used by different strains during the growth will be analyzed. The feature of nitrogen utilization of different Bifidobacteria will be revealed. At last, the amino acid synthesis genome and proteolytic system genome (encoding protease, peptide transport system and peptidase) in different Bifidobacteria will be analyzed by comparative genomics. And, their relationships with characteristics of nitrogen utilization will be developed. This project is intended to reveal the relationship between the key genomes of the nitrogen metabolism of Bifidobacteria and the selectivity and characteristics of nitrogen source.

双歧杆菌是非常重要且被广泛认可的益生菌，但是双歧杆菌的制备非常困难，氮源是制约其有效增殖的关键因素，且不同种双歧杆菌具有各自特异的氮代谢系统，增加了其培养难度。本项目力求系统探索不同双歧杆菌氮利用的特征，并阐明其与氮代谢基因组的对应关系，以指导双歧杆菌的培养生产及其发酵应用。具体研究内容包括：分析不同种双歧杆菌利用不同蛋白的增殖效率，总结其对氮源利用的选择性；分析不同种双歧杆菌高效利用的蛋白氨基酸组成与其生长必需氨基酸的关系，结合生长过程不同菌株利用的肽的特点，揭示不同种双歧杆菌氮利用的特征；比较基因组学分析不同种双歧杆菌氨基酸合成基因组和蛋白水解系统基因组（蛋白酶、肽转运系统和肽酶编码基因），解析其与氮利用特征的关系。拟通过本项目揭示不同种双歧杆菌氮代谢关键基因组与其氮源利用的选择性和特征的对应关系。

氮源是制约双歧杆菌有效增殖的关键因素之一，本项目力求系统探索不同双歧杆菌氮利用的特征，并阐明其与氮代谢基因组的对应关系，以指导双歧杆菌的培养生产及其发酵应用。主要研究结果如下：.（1）两歧双歧杆菌对胰蛋白胨利用效率最高，但几乎不利用酵母浸粉类氮源；长双歧杆菌可有效利用除胰酪蛋白胨和牛肉浸粉外的其他氮源；短双歧杆菌对所有氮源均表现较好的利用效率；所有双歧杆菌对肽的利用效率优于氨基酸。.（2）两歧双歧杆菌F35、长双歧杆菌CCFM 760和短双歧杆菌AH-WH-9M5氨基酸合成基因无差异，且均无编码细胞壁蛋白酶的基因；编码的肽酶种类及数量在菌株间差异性也相对较小；但长双歧杆菌和短双歧杆菌的氨基酸与肽的转运系统种类和数量较两歧双歧杆菌更为丰富，对底物的摄取更为广泛。.（3）三株双歧杆菌的氨基酸营养缺陷均表现为半胱氨酸营养缺陷型，但疏水性氨基酸对于两歧双歧杆菌F35以氨基酸为氮源的增殖具有抑制作用（酵母浸粉类氮源中游离的疏水性氨基酸含量为其他氮源2倍），在其它双歧杆菌中不存在该种现象。.（4）可被不同双歧杆菌转运的肽段具有高度的菌株特异性，双歧杆菌对具有亲水性、含促生长作用的氨基酸的肽表现更高的亲和性；两歧双歧杆菌F35最多可转运14肽，且对于2、3及9肽有着更高的偏好性；长双歧杆菌CCFM 760最多可转运20肽，未检测到对6、7肽的转运；短双歧杆菌AH-WH-9M5可转运的肽段主要为2~4肽以及8~20肽，未检测到对5、6、7肽的摄取。.（5）两歧双歧杆菌的耐高渗突变株有6个与谷氨酸代谢有关的SNPs，在生长至高渗环境时谷氨酸、脯氨酸合成增多。脯氨酸的胞内积累是两歧双歧杆菌应对生长过程受到高渗抑制的关键，但该菌不能转运脯氨酸，含脯氨酸的可被两歧双歧杆菌转运的肽是该菌关键的氮源。. 根据双歧杆菌的氮利用特征，设计更适宜的氮源和培养基，对促进双歧杆菌的发酵生产效率具有重要的价值。

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