一株高ACE抑制活性乳杆菌的筛选鉴定、培养条件优化及其基因组分析

Screening, identification, optimization of culture condition and genome analysis of a bacterium with high ACE inhibitory activity

摘要:

【背景】乳杆菌是人体肠道益生菌，其发酵乳中可检测到血管紧张素转换酶(Angiotensin converting enzyme，ACE)抑制肽。海洋蕴藏着丰富的微生物种质资源，分布着大量的乳杆菌。【目的】从高通量测序结果中发现渤海沉积物中分布着乳杆菌资源。为了进一步开发具有ACE抑制活性的海洋乳杆菌资源，提高乳杆菌发酵乳的ACE抑制活性，筛选瑞士乳杆菌(Lactobacillus helveticus)并对其特性进行研究。【方法】采用高通量测序技术从渤海沉积物中检测乳杆菌，并对其进行富集分离，对筛选出的乳杆菌进行16S rRNA基因鉴定和全基因组测序分析，测定该菌发酵乳的ACE抑制活性，并采用正交实验优化发酵条件。【结果】渤海沉积物中含有乳杆菌并成功筛选出一株瑞士乳杆菌GY-3，其发酵乳具有较高的ACE抑制活性。该菌在发酵温度37 °C，接种量3%，且在脱脂乳培养基中添加1.0%葡萄糖，0.6%大豆蛋白胨，1.0%酵母浸粉，0.04% MnSO4·4H2O时，抑制活性最高，可达79.52%。通过对该菌基因组进行测序研究，发现其产ACE抑制肽涉及蛋白酶系统、多肽转运系统和肽酶系统。【结论】为扩大海洋源产ACE抑制肽的乳杆菌种质资源、开发高产ACE抑制活性的发酵菌株奠定了基础，进一步研究了如何提高乳杆菌产ACE抑制肽的水平，并对其基因组进行了研究，为今后生物学特性和ACE抑制活性机理的研究奠定了基础，并对降血压相关产品的开发具有重要意义。

Abstract:

[Background] Lactobacillus is intestinal probiotics with the frequent observation of high angiotensin converting enzyme (ACE) inhibitory activity in its fermented milk. The marine is an optimal source for biological flora, including abundant types of Lactobacillus. [Objective] Lactobacillus resources were distributed in sediments of the Bohai Sea through the results of high-throughput sequencing. In order to exploit the sources for producing ACE inhibitory peptide and increase the ACE inhibitory activity in fermented milk of Lactobacillus, the present study was performed by using Lactobacillus helveticus GY-3 selected from sediments of the Bohai Sea. [Methods] Lactobacillus resources were detected by using high-throughput sequencing in sediments of the Bohai Sea. After the enrichment separation, 16S rRNA gene identification and genome sequencing analysis were then done on the selected strain. We also measured the ACE inhibitory activity of the fermented milk of the selected strain, and optimized the fermentation conditions of the selected strain through orthogonal test. [Results] Lactobacillus helveticus bacterium (GY-3) was identified and high ACE inhibitory activity was detected in its fermented milk. The optimal conditions for producing ACE inhibitory peptides by strain GY-3 are as follows: 37 °C, 3% inoculation, and 1.0% glucose, 0.6% soy protein, 1.0% yeast extract and 0.04% MnSO4·4H2O in skim milk medium. The maximal ACE inhibitory activity could reach up to 79.52%. On the basis of genome sequence data of the bacterium, it was found that the ACE-inhibiting peptide were involved in the protease system, the polypeptide transport system and the peptidase system. [Conclusion] The present study provides a broader source and foundation for the development of lactic acid bacteria that are capable of producing ACE inhibitory peptide. Studying the genome lays a foundation for the study of biological characteristics and mechanism of ACE inhibition activity in the future. Additionally, this study is of great significance for the development of antihypertensive products.