大麻沤麻液细菌种群多样性

从分子水平分析大麻沤麻液中细菌种类及其优势菌群,为筛选出大麻微生物脱胶的生产菌种奠定基础。采用PCR-DGGE技术研究大麻沤麻液中细菌种群的多样性及动态变化,使用Gel-Pro Analyzer 4.5软件分析DGGE指纹图谱,并对优势条带进行分析。结果表明:从发酵初期过渡到主发酵期细菌种群多样性上升,群落演替迅速,从主发酵期进入到发酵末期,细菌种群多样性下降,群落演替缓慢;在整个发酵过程中,细菌种群多样性指数在发酵中期(72h)达到高峰,说明发酵中期细菌种群数量、优势菌群种类达到了最高值,发酵初期和末期以不可培养细菌为主,主发酵期以成团泛菌属为主。     

东北传统豆酱发酵过程中微生物的多样性

以东北传统发酵豆酱为研究对象,分析豆酱发酵过程中微生物群落的动态变化。分别选择发酵豆酱0、35、65、75和105 d(成品豆酱)作为研究材料,通过PCR-DGGE分析微生物多样性,检测了豆酱发酵过程中蛋白质和氨基酸态氮的变化。结果表明,豆酱发酵过程中主要优势细菌为芽孢杆菌和乳酸菌,芽孢杆菌包括枯草芽孢杆菌、短小芽孢杆菌、淀粉液化芽孢杆菌、地衣芽孢杆菌的近缘种等;主要乳酸菌为乳球菌、明串珠菌、魏斯氏菌等种属细菌的近缘种;东北豆酱发酵过程中优势真菌为米曲霉、散囊菌和谢瓦氏曲霉的近缘种,随发酵时间延长数量逐渐减少;粗蛋白相对含量先略有平稳上升后下降,最后成品酱粗蛋白含量下降为24.76%;氨基态氮含量一直在增加,成品酱中为101.2 g/kg。     

传统发酵豆酱发酵过程中养分动态及细菌多样性

以山东传统发酵豆酱作为研究对象,测定了发酵不同阶段的总酸、可溶性糖、有机碳、粗蛋白、氨基酸态氮、主要挥发性产物等指标,对样品中细菌进行PCR-DGGE分析,探讨了传统发酵豆酱中养分动态及细菌多样性.结果表明发酵过程中总酸含量先上升后下降而后又上升,最后成品酱中为6.26%;有机碳和可溶性糖含量都逐渐减少;粗蛋白相对含量先略有平稳上升后下降;氨基酸态氮含量一直在增加,成品酱中为101.2g/kg;乳酸和甘油含量随发酵进程而增加,成品酱中分别为5.65g/kg和14.72g/kg.DGGE分析表明发酵15d时细菌种类最多,随后一部分逐渐消失,种类趋于稳定,最后成品酱中出现有几种明显的优势种,主要包括:未培养细菌(Uncultured bacterium)、乳酸乳球菌(Lactococcus lactis)、地衣芽孢杆菌(Bacillus licheniformis)的近缘种.     

传统发酵豆酱发酵过程中养分动态及细菌多样性

以山东传统发酵豆酱作为研究对象, 测定了发酵不同阶段的总酸、可溶性糖、有机碳、粗蛋白、氨基酸态氮、主要挥发性产物等指标, 对样品中细菌进行PCR-DGGE分析, 探讨了传统发酵豆酱中养分动态及细菌多样性。结果表明发酵过程中总酸含量先上升后下降而后又上升, 最后成品酱中为6.26%; 有机碳和可溶性糖含量都逐渐减少; 粗蛋白相对含量先略有平稳上升后下降; 氨基酸态氮含量一直在增加, 成品酱中为101.2 g/kg; 乳酸和甘油含量随发酵进程而增加, 成品酱中分别为5.65 g/kg和14.72 g/kg。DGGE分析表明发酵15 d时细菌种类最多, 随后一部分逐渐消失, 种类趋于稳定, 最后成品酱中出现有几种明显的优势种, 主要包括：未培养细菌(Uncultured bacterium)、乳酸乳球菌(Lactococcus lactis)、地衣芽孢杆菌(Bacillus licheniformis)的近缘种。     

藏灵菇微生物种群结构的分子特性研究*

用PCR-DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78%~84%,酵母菌种群结构的相似性为80%~92%。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带(条带E)的序列与乳酸乳球菌的相似性为100%。     

应用DGGE技术分析青藏铁路沿线的土壤细菌种群多样性

选择青藏铁路沿线不同海拔高度的10个地点采集土壤样品,直接提取样品中的总DNA,以巢式PCR扩增细菌16S rDNA片段,应用变性梯度凝胶电泳(DGGE)技术分离PCR扩增的16S rDNA片段,研究土壤细菌的种群多样性.结果表明,青藏铁路沿线高海拔地区具有较为丰富的细菌种群多样性,植被类型是影响青藏铁路沿线土壤细菌种群多样性的重要因素,也是影响土壤细菌种群结构相似性的重要因素,而海拔高度等是次要的影响因素;具有相似植被类型的土壤样品,其细菌种群多样性随海拔的升高而下降.     

藏灵菇微生物种群结构的分子特性研究

用PCR—DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78％-84％,酵母菌种群结构的相似性为80％-92％。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带（条带E）的序列与乳酸乳球菌的相似性为100％。     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

从我国42个地区的自然发酵酱醅和酱醪样品中分离得到曲霉Aspergillus及其有性型真菌20种,其中优势曲霉菌种类为A.tubingensis、A.flavus、A.niger、A.ochraceus、A.candidus和A.fumigatus.调查研究发现,从酱醅和酱醪样品中分离出黄曲霉的频率较高,表明自然发酵豆酱存在一定的安全隐患.研究中分离获得的曲霉菌株有望用于纯种制曲或特殊风味豆酱的工业化生产.     

郫县豆瓣细菌多样性及群落结构动态变化北大核心

【目的】通过检测郫县豆瓣在不同发酵阶段细菌的种类、丰度及数量,探究郫县豆瓣的不同发酵产品发酵过程中细菌的动态变化情况。【方法】采用16S rRNA基因测序对郫县豆瓣4个发酵阶段中细菌种类及丰度进行分析,利用荧光定量PCR(quantitative real-time PCR,qPCR)方法检测不同发酵阶段的细菌总数。【结果】郫县豆瓣在初期的发酵过程中细菌群落处于动态稳定,在不同发酵阶段细菌群落组成相对丰富,从郫县豆瓣整个初期的发酵过程来看,细菌群落多样性呈现升高的趋势,Shannon指数从1.25升高到3.50;在郫县豆瓣初期发酵过程中细菌群落的数量以及多样性与发酵环境息息相关,不同发酵阶段细菌群落的多样性也有所不同,其中在干辣椒发酵阶段中泛菌属(Pantoea)为最优势菌属,占比为20%;在蚕豆瓣发酵阶段中葡萄球菌属(Staphylococcus)的相对丰度最高,占比为38%;混合发酵后,在红油豆瓣发酵阶段的最优势菌属是乳酸杆菌属(Lactobacillus),占比达到27%,郫县豆瓣发酵阶段的最优势菌属是乳酸杆菌属(Lactobacillus),占比为62%。【结论】推断在郫县豆瓣不同发酵阶段初期相对丰度较大的菌属对郫县豆瓣的质量以及产量可能会产生重大影响。     

DG-DGGE分析产氢发酵系统微生物群落动态及种群多样性

应用双梯度-变性梯度凝胶电泳(DG-DGGE)对生物制氢反应器微生物种群的动态变化及多样性进行监测。间隔7d从反应器取厌氧活性污泥,以细菌16SrDNA通用引物进行V2～V3区域PCR扩增,长约450bp的PCR产物经DGGE分离后,获得污泥微生物群落的16SrDNA指纹图谱。污泥接种到反应器后微生物群落中既有原始种群的消亡和增长,也有次级种群的强化和演变。反应器在运行初期群落演替迅速,15d时微生物群落结构变化最大。群落结构的相似性随着演替时间的增加而逐渐升高,种群动态变化后形成稳定的群落结构。29d时微生物多样性基本保持不变,微生物优势种属达到19个OTU。在细菌竞争和协同作用制约下,种群多样性降低后趋于稳定,形成顶级群落。有些种群在群落结构中一直存在,是群落建成的原始种群,原始种群与次级种群在代谢过程中具有协同作用,表现出群落的综合生态特征。     

Bacterial community involved in traditional fermented soybean paste dajiang made in northeast China

Dajiang is a traditional fermented food prepared from soybeans that is still popular in northeast China. Although recent studies have revealed that a variety of bacterial species contribute to the production of fermented soybean products, little is known about bacterial communities involved in the fermentation of dajiang made in northeast China. In this study, 14 samples of naturally fermented dajiang were analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the diversity of the bacteria involved in fermentation. Our results indicate that lactic acid bacteria, including Lactobacillus plantarum, uncultured Leuconostoc mesenteroides, Leuconostoc gasicomitatum, Enterococcus faecium, and Tetragenococcus halophilus, were the predominant species. This is the first report of Enterococcus spp. and Leuconostoc spp. in the Chinese fermented soybean paste dajiang using DGGE. The presence of Bacillus spp. (including Bacillus firmus), Oceanobacillus spp., and Paenibacillus glycanilyticus in the dajiang samples may be due to their salt tolerance. Potentially pathogenic Alphaproteobacteria and Staphylococcus epidermidis strains were also detected in this study. Moreover, three uncultured bacterial clones were found in some samples and require further study. The results reveal a high level of bacterial diversity in dajiang.     

Microbial diversity and dynamics of microbial communities during back-slop soaking of soybeans as determined by PCR–DGGE and molecular cloning

Tempe is a traditional fermented food in Indonesia. The manufacture process is quite complex, which comprises two stages, preparatory soaking of soybeans and fungal solid state fermentation. Daily addition of previous soak water (back-slopping) during the soybean soaking step is considered to be crucial in the manufacture of high quality tempe. The microbial diversity and dynamics of the microbial communities evolving during back-slop soaking of soybeans for tempe making was investigated by culture-independent PCR–DGGE and molecular cloning. Both DNA and total RNA were isolated and included in this study, to obtain a view on the succession of total and viable bacteria in the complex microbiota. DGGE profiles indicated that Enterobacter sp., Enterococcus sp., Pseudomonas putida, Leuconostoc fallax, Pediococcus pentosaceus, and Weissella cibaria, were the predominant bacteria. Their occurrence shifted dramatically during the back-slop soaking procedure. This study combined with previous culture-dependent studies could gain a better understanding of the complex microbiota of traditional fermented food and give useful information for its quality control.     

Analysis of Bacterial and Fungal Communities in Japanese- and Chinese-Fermented Soybean Pastes Using Nested PCR–DGGE

The microbial diversity of Japanese- and Chinese-fermented soybean pastes was investigated using nested PCR–denaturing gradient gel electrophoresis (DGGE). Five Japanese-fermented soybean paste samples and three Chinese-fermented soybean paste samples were analyzed for bacteria and fungi. Extracted DNA was used as a template for PCR to amplify 16S rRNA and 18S rRNA genes. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Tetragenococcus halophilus and Staphylococcus gallinarum were found to dominate the bacterial microbiota in Japanese samples, whereas Bacillus sp. was detected as the predominant species in Chinese samples. DGGE analysis of fungi in soybean pastes determined the presence of Aspergillus oryzae and Zygosaccharomyces rouxii in most of the Chinese and Japanese samples. Some differences were observed in the bacterial diversity of Japanese- and Chinese-fermented soybean pastes.     

Diversity and dynamics of bacterial species in a biofilm at the end of the Seoul water distribution system

To investigate changes in the bacterial species and hygienic safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyse the bacterial population in the biofilm of a semi-pilot galvanized iron pipe model. The presence of sequences from aerobic Sphingomonas sp., anaerobic Rhodobacter sp., and unculturable bacteria indicated that these organisms coexisted after 1 day of model operation, demonstrating the ease of biofilm formation on galvanized iron pipes in the end region of the water distribution system studied. Sequences similar to those of unculturable bacteria, E. coli, and anaerobic bacteria were detected during the course of succession on the biofilm. More complicated band patterns were observed after 70 days of operation. PCR-DGGE illustrated changes in the biofilm during succession as well as the possibilities of anaerobic conditions and faecal contamination of the drinking water system. PCR-DGGE and culture-dependent fatty acid methyl ester (FAME) analysis showed different patterns for the same samples (Lee & Kim 2003); however, PCR-DGGE showed less diversity than did FAME analysis. This study compared the culture-dependent FAME and culture-independent PCR-DGGE methods directly, and their use in promoting the hygienic safety of drinking water.     

传统豆瓣酱微生物群落发酵演替规律及其功能分析

【目的】解析中国传统豆瓣酱发酵过程中的微生物群落演替规律和理化代谢物质变化,探讨不同发酵阶段影响豆瓣酱风味的核心功能微生物。【方法】采用高通量测序解析豆瓣酱发酵过程中的微生物群落结构和演替,并跟踪检测发酵过程中的理化代谢物质,然后分析微生物群落和理化代谢物质变化之间的相关性,最后在体外分离核心微生物并对其功能特性进行验证。【结果】细菌和真菌群落结构在发酵前期显著变化,并在中后期逐渐趋于稳定。优势细菌主要是Staphylococcus、Bacillus和Weisiella,其中Staphylococcus在整个发酵过程中呈上升趋势,而Bacillus和Weisiella呈下降趋势。真菌群落结构较为简单且稳定,其中Aspergillus在整个发酵过程中的平均丰度占真菌总群落的97%以上,Zygosaccharomyces呈先上升后下降的趋势。相关性分析和体外功能验证表明,功能微生物(Aspergillus oryzae、Bacillus subtilis、Staphylococcus gallinarum、Weisiella confusa和Zygosaccharomyces rouxii)在不同发酵阶段发挥着不同的关键作用。【结论】在成曲和发酵前期Aspergillus oryzae、Bacillus subtilis分泌各种酶来降解大分子物质,Aspergillus oryzae、Staphylococcus gallinarum和Weisiella confusa导致了酱醅的酸化和氨基酸的生成,而耐盐的Zygosaccharomyces rouxii在发酵中后期对风味物质的形成起重要作用。     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Composition of Acridid gut bacterial communities as revealed by 16S rRNA gene analysis

The gut bacterial community from four species of feral locusts and grasshoppers was determined by denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments. The study revealed an effect of phase polymorphism on gut bacterial diversity in brown locusts from South Africa. A single bacterial phylotype, consistent with Citrobacter sp. dominated the gut microbiota of two sympatric populations of Moroccan and Italian locusts in Spain. There was evidence for Wollbachia sp. in the meadow grasshopper caught locally in the UK. Sequence analysis of DGGE products did not reveal evidence for unculturable bacteria and homologies suggested that bacterial species were principally Gammaproteobacteria from the family Enterobacteriaceae similar to those recorded previously in laboratory reared locusts.