土家腊肉中耐盐或嗜盐微生物多样性及其群落结构演替规律的研究

Tujia Bacon, a traditional fermented food of the Tujia, is a welcomed meat product in China and one of main economically significant agricultural products in Xiang Xi, Hunan Province. It has been well known that microorganisms are essentially associated with the nutritional values, aroma and other qualities of bacon, however, knowledge about microbial ecosystems in bacon is limited and the production techniques of bacon has not been normalized and modernized yet. In this project, we propose to investigate the diversity of halotolerant or halophilic microorganisms during the process and storage of traditional Tujia bacon by culture-dependent methods and culture-independent methods including PCR-DGGE (Denaturing Gradient Gel Electrophoresis) and RT-PCR-DGGE, and analyze specific community composition, the proportion and dynamics of microbes at the different processes of bacon production, in order to detect the connection between microbial metabolism activities and Tujia bacon fermentation and ripeness. Meanwhile, we will develop new cultivation strategies for uncultured microbes and isolate the previously uncultured halotolerant or halophilic microorganisms from Tujia bacon. These works will unravel the diversity of halotolerant or halophilic microbes involving in Tujia bacon fermentation and ripeness for the first time, and help us understand the effect of microbial community composition and dynamics on the quality of Tujia bacon, the outcomes will provide some basic knowledge of bacon production, which will further enhance the improvement of fermentation arts to produce high quality of bacon industry in the future. In addition, novel halolerant or halophilic microorganisms discovered from traditional Tujia bacon will be good microbial resources for studying the evolution mechanism of halophiles and developing meat culture starters.

腊肉是深受大众青睐的传统腌制食品，但是目前对其加工过程中微生态系统缺乏系统深入的研究。本研究拟从分析土家腊肉中耐盐或嗜盐微生物多样性入手，采用纯培养法和免培养法（PCR-DGGE和RT-PCR-DGGE）双重手段研究微生物的种类、数量和系统发育多样性，阐明土家腊肉加工过程中微生物群落结构、种群丰度和动态变化规律，探讨不同时期微生物代谢活动与土家腊肉发酵、成熟的关系；同时，研究未培养微生物的分离策略，建立其可培养方法，挖掘新的耐盐或嗜盐微生物类群。本课题的开展，将首次揭示土家腊肉这一传统发酵肉制品中耐盐或嗜盐微生物多样性，系统地理解微生物的种类、数量和优势菌群的消长与腊肉发酵、成熟的关系，为土家腊肉制品生产工艺改善、产品质量控制和优良性状发酵剂的开发提供理论依据。同时，从土家腊肉这一特殊生境中发现新的耐盐或嗜盐微生物资源，将为探讨生命适应环境进化机制和开发肉制品发酵剂储备种质资源。

土家腊肉是深受大众青睐的传统腌制食品，但目前对其加工工艺中微生物菌相和风味物质组成变化缺乏系统研究。鉴于此，本项目以3个样地4个工艺阶段传统土家腊肉为研究对象，开展了其理化指标与风味物质组分检测，采用纯培养法和免培养法分析了微生物群落结构、种群丰度和动态变化规律，以及筛查了可培养细菌重要生化特性等，其研究结果：(1) 在土家腊肉加工工艺中，pH值维持在6.0，水分含量和水分活度逐步下降，过氧化值和食盐含量逐步上升，最后又略有下降，酸价总体呈上升趋势。从样品中共鉴定出176种挥发性物质，主要有醇类、醚类、酚类、酮类、醛类、酸类、酯类、碳氢化合物、其他类等。分析可知，酚类、酯类、其他类为土家腊肉主要风味物质。(2) 通过传统纯培养方法分离获得365株细菌，经鉴定属于3个门、24个科、36个属。其中优势菌群为葡萄球菌和芽孢杆菌，其次是链霉菌。首次从腊肉样品中分离到放线菌。(3) 基于宏基因组测序，发现土家腊肉样品微生物分布于12个门、118个科和297个属。其中葡萄球菌是优势菌群，相对含量为54.50%；其次是鞘脂单胞菌，相对含量为16.60%。随着土家腊肉加工成熟葡萄球菌和短杆菌相对含量逐渐增加，鞘脂单胞菌、嗜冷杆菌、假单胞菌和水栖菌等相对含量逐渐减少。(4) 选取47株可培养细菌开展胞外蛋白酶、脂肪酶、接触酶、硝酸盐还原酶、亚硝酸盐还原酶、生物胺氧化酶等活性检测，发现BC-20（Dermacoccus nishinomiyaensis）等15株细菌胞外酶活性较强，可作为肉制品发酵剂候选菌种研发。采用平板显色法和邻苯二胺比色法，从47株代表性菌株中筛选获得7株产丁二酮量较高的细菌，其中BC-08（Bacillus licheniformis）产丁二酮含量达68.9 mg/L，可作为微生物发酵生产香料物质--丁二酮的种质资源。本项目首次系统揭示土家腊肉加工工艺中风味物质组成和微生物菌相变化的规律，比较全面地理解了微生物的种类、数量和优势菌群的消长对腊肉发酵品质的影响，为土家腊肉制品生产工艺改善、产品质量控制和科学化生产提供理论依据，为优良性状发酵剂的开发积累种质资源。