乳酸菌特色资源及乳酸菌发酵剂和代谢工程技术研究课题度执行情况报告

摘 要：按照计划任务，课题针对2013年度计划任务书内容进行了研究，现已完成各项任务已完成，具体完成情况如下：从俄罗斯和国内新疆和地区采集样品290余份，鉴定乳酸菌1 534株，使内蒙古农业大学乳酸菌菌种资源库保藏菌种资源增至4 924株，包括8个属、65个种和亚种，成为中国最大的乳酸菌菌种资源库。通过比较基因组学分析，选取dnaA、pyrG和pyrB等12个持家基因，完成Lactobacillus helveticus和Lactobacillus fermentium等8个种1 726株乳酸菌的多位点序列分型研究。优化了乳酸菌多位点序列分型技术，完善了新的乳酸菌分型方法，构建了8种乳酸菌菌株MLST数据库。通过比较基因组分析，确定了嗜热链球菌产生风味物质代谢途径上与乙醛生成相关的7个关键酶基因和与双乙酰生成相关的5个关键酶基因。对333株嗜热链球菌中筛选出30株产香型菌株经q-PCR定量分析发酵和后熟不同时间段，各关键功能基因的表达量与乳酸、甲酸、双乙酰和乙醛生成量的相关性研究。对333株嗜热链球菌和194株保加利亚乳杆菌，经发酵时间、黏度、蛋白水解能力、噬菌体抗性和后酸化等特性以及其发酵乳感官评价，筛选出用于发酵乳生产的L. bulgaricus19株、S. thermophilus15株。其中L. bulgaricus ND02和S. thermophilus ND03各项指标表现最为优良。以L. bulgaricus ND02和S. thermophilus ND03作为发酵剂菌株，进行了高密度培养、冷冻干燥以及直投式发酵剂的研发，其发酵剂活菌数达1.83×1 011 CFU/g，冻干存活率提高至84.3%。利用L.bulgaricus ND02和S. thermophilus ND03及益生菌L.casei zhang、L. plantarum P-8和B. lactis V9等菌株开发出活性益生菌豆乳饮料和益生菌发酵乳产品及复合菌剂等4种。完成乳球菌klds4基因组测序及比较基因组学分析；完成植物乳杆菌和短乳杆菌厌氧和有氧条件下的代谢调控，证实了短小乳杆菌有氧代谢由乳酸丙酮酸乙酸转化的新途径，而丙酮酸脱氢酶具有关键作用；对于植物乳杆菌在有氧条件下，自身过氧化氢酶的激活对细胞的存活具有强的保护作用。

关键词：乳酸菌 生物多样性 多位点序列分型技术 发酵剂 代谢工程 新产品

2013 Annual Performance Report on the Study of Characteristic Resources of Lactic Acid Bacteria and Its Technology of Starter Culture and Metabolic Engineering

Menghe Bilige1 Kong Jian2 Liu Wenjun1 Wang Jicheng1

（1.Inner Mongolia Agricultural University； 2.Shandong University）

Abstract： The research was carried out in accordance with the scheduled task. Many aspect of scheduled task have been completed. And the detailed information was respectively described as follows： 290 samples were collected from different regions of Russia， Xinjiang and Tibet of China. 1 534 strains of LAB were isolated and identified from these samples. The number of LAB increased to 4 924 in the LABCC， These stains included eight genera and 65 species and subspecies. Multi-locus sequence typing （MLST） studies were completed concerning 1 726 strains including Lactobacillus （Lb.） helveticus and L. fermentium etc. The study was carried out based on 12 housekeeping genes of LAB. An MLST technique of Lactobacillus was optimized； new typing method was improved. MLST database of 8 species of LAB were constructed. Identification of 7 key functional genes of flavor compounds formation of S. thermophilus， and 5 key enzyme genes of diacetyl production. q-PCR of functional genes of flavor production of 30 S. thermophilus was conducted. The correlation analysis was performed for key functional gene expression and the content of lactic acid， formic acid， diacetyl， and acetaldehyde during the fermentation and ripening time periods. 19 L. bulgaricus and 15 S. thermophilus were screened out used for fermented milk production. Among which ND02 and ND03 were the most excellent stains with good indicators. High-density culture， freeze-drying technology and direct-to-vat yoghurt culture was developed based on the starter culture stains of ND02 and ND03， in which ferment viable count of starter culture reached 1.83×1 011 CFU/g， and the freeze-dried rate was improved to 84.3%. 4 kinds of products such as active probiotic soybean beverage， fermented milk and compound strains preparation was developed based on the strains of ND02， ND03 and probiotics strains L. casei zhang， L. plantarum P-8， and B. lactis V9. Complete genome sequencing of Lactococcus klds4 and comparative genomics analysis was developed. Metabolic regulation of a L. plantarum and L. brevis was finished under anaerobic and aerobic conditions. A new ways of metabolism and transformation was confirmed by the aerobic metabolism of L. brevis. It was shown pyruvate dehydrogenase plays a key role for self-activation of catalases for L. plantarum on cell survival under aerobic conditions.

Key Words： Lactic acid bacteria； Biodiversity； MLST； Starter culture； Metabolic engineering； New products

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