乳酸菌特色資源及乳酸菌發酵劑和代謝工程技術研究課題2013年度執行情況報告

孟和畢力格 孔健 劉文俊 王計成

摘 要：按照計劃任務，課題針對2013年度計劃任務書內容進行了研究，現已完成各項任務已完成，具體完成情況如下：從俄羅斯和國內新疆和西藏地區采集樣品290余份，鑒定乳酸菌1 534株，使內蒙古農業大學乳酸菌菌種資源庫保藏菌種資源增至4 924株，包括8個屬、65個種和亞種，成為中國最大的乳酸菌菌種資源庫。通過比較基因組學分析，選取dnaA、pyrG和pyrB等12個持家基因，完成Lactobacillus helveticus和Lactobacillus fermentium等8個種1 726株乳酸菌的多位點序列分型研究。優化了乳酸菌多位點序列分型技術，完善了新的乳酸菌分型方法，構建了8種乳酸菌菌株MLST數據庫。通過比較基因組分析，確定了嗜熱鏈球菌產生風味物質代謝途徑上與乙醛生成相關的7個關鍵酶基因和與雙乙酰生成相關的5個關鍵酶基因。對333株嗜熱鏈球菌中篩選出30株產香型菌株經q-PCR定量分析發酵和后熟不同時間段，各關鍵功能基因的表達量與乳酸、甲酸、雙乙酰和乙醛生成量的相關性研究。對333株嗜熱鏈球菌和194株保加利亞乳桿菌，經發酵時間、黏度、蛋白水解能力、噬菌體抗性和后酸化等特性以及其發酵乳感官評價，篩選出用于發酵乳生產的L. bulgaricus19株、S. thermophilus15株。其中L. bulgaricus ND02和S. thermophilus ND03各項指標表現最為優良。以L. bulgaricus ND02和S. thermophilus ND03作為發酵劑菌株，進行了高密度培養、冷凍干燥以及直投式發酵劑的研發，其發酵劑活菌數達1.83×1 011 CFU/g，凍干存活率提高至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等菌株開發出活性益生菌豆乳飲料和益生菌發酵乳產品及復合菌劑等4種。完成乳球菌klds4基因組測序及比較基因組學分析；完成植物乳桿菌和短乳桿菌厭氧和有氧條件下的代謝調控，證實了短小乳桿菌有氧代謝由乳酸→丙酮酸→乙酸轉化的新途徑，而丙酮酸脫氫酶具有關鍵作用；對于植物乳桿菌在有氧條件下，自身過氧化氫酶的激活對細胞的存活具有強的保護作用。

關鍵詞：乳酸菌 生物多樣性 多位點序列分型技術 發酵劑 代謝工程 新產品

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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