少数民族传统发酵肉制品乳酸菌多样性高通量分析及抗氧化菌株筛选

Traditional minority fermented food have characteristic of long history, unique flavor, nutrition rich, originate from nature as well as plentiful microbial resources must be contained. To the question of lacking research and explore on function of LAB on molecular level, this research will design to analyze of the diversity of microbial community structure of traditional minority fermented meat products by 454 high throughput technology, genome annotation by comparative genomics, screening anti-oxidative LAB strains by efficient screening manner, so as to provides reference to explore, protective indigenous microbial resources. The research contents were as follows: 1. the total bacteria DNA was directly extracted from traditional fermented meat, shrimp and fish, a metagenomic library was constructed, the V3 and V6 region of the 16S rDNA were sequencing by 454 high throughput technology, the microbial community and diversity were analyzed by UCLUST and MOTHUR software, the species information was obtained by comparative analysis of samples; 2. primary screening LAB by enrichment culture of samples, primary screening of LAB based on DPPH free radical scavenging ability, optimization culture and enrichment to improve anti-oxidation of these strains obtained, selecting strains with higher anti-oxidation ability to functional evaluation, identification, genetic analysis and characteristic study, and than the excellent strains with the higher anti-oxidative were obtained.

少数民族传统发酵食品历史悠久、风味独特、营养丰富、源自绿色原生态，其中必然蕴藏丰富的微生物资源。针对少数民族传统发酵肉制品中乳酸菌缺乏多样性了解和发掘优良菌株问题，本项目应用高通量测序技术对传统发酵肉制品中菌群结构进行多样性分析，采用高效筛选法筛选具有较高抗氧化活性的乳酸菌菌株，为发掘、保护本土原生态肉制品微生物资源提供应用基础参考。研究内容：1. 从传统发酵酸肉、酸虾、酸鱼等样品提取总细菌DNA，构建宏基因组双末端片段文库，454高通量测序16S rDNA V3、V6可变区、利用UCLUST和 MOTHUR软件进行菌落结构与多样性分析，样品间比较分析，获得相关物种信息；2.对采集样品富集培养后进行乳酸菌初筛，基于清除DPPH自由基能力的菌株初筛，对初筛菌株进行抗氧化活性优化培养与富集，选择高抗氧化活性的菌株进行功能评价、菌株鉴定与遗传性分析，菌株特性研究，获得性能优良的高抗氧化活性菌株。

侗族传统发酵酸肉/鱼历史悠久、工艺独特、源于绿色原生态环境，蕴藏十分丰富的微生物资源。项目针对酸肉/鱼制品中乳酸菌缺乏多样性了解和发掘应用的问题，进行了乳酸菌多样性分析、抗氧化乳酸菌筛选及功能性评价研究。首先，我们提取样品中总细菌DNA，构建宏基因组双末端片段文库，通过454高通量对16S rDNA可变区序列进行测序，并进行菌落结构与多样性生物信息学分析，同时以DGGE分子指纹图谱进行对比，获得酸肉/鱼中细菌多样性及分布信息。研究共检测到细菌207个属，其中主要菌属11个；得出4种优势菌属葡萄球菌属(Staphylococcus)、乳杆菌属(Lactobacillus)、四联球菌属(Tetragenococcus)、魏斯氏菌属(Weissella)和11种乳酸菌，并构建了乳酸菌DNA指纹图谱库1个；其次，通过对样品富集培养后进行抗氧化乳酸菌初筛、复筛和优化，获得功能性乳酸菌11株（其中CCTCC保藏6株）。包括抗氧化乳酸菌2株，对DPPH自由基清除率达79%以上；降胆固醇乳酸菌1株，对胆固醇降解率达33.96%；高产胞外多糖并具有抗氧化活性乳酸菌8株。 最后，我们对2株抗氧化乳酸菌（Lactobacillus curvatus LAB26 ；Lactobacillus paracasei）进行了功能性、安全性评价和应用验证实验，证实它们具有应用于发酵肉制品的优质特性，前景广阔。