盐渍青菜的细菌菌群结构DGGE分析及产酸细菌分离鉴定

采用变性梯度凝胶电泳技术(denatured gradient gel electrophoresis,DGGE)对不同盐渍青菜样品的细菌菌群结构进行了分析。对优势条带测序分析表明,盐渍青菜中主要的优势细菌是乳杆菌属(Lactobacillus)、魏斯氏菌属(Weissella)、糖多孢菌属(Saccharopolyspora)、短芽孢杆菌属(Brevibacillus)细菌。在腌渍初期,海源菌属(Idiomarina)与海洋杆菌属(Marinobacter)细菌较丰富,在腌渍后期明显减少。乳杆菌属细菌在腌渍14、26个月的青菜中含量丰富,在腌渍2个月的青菜中含量较少。通过分离培养技术从盐渍青菜中分离获得23株产酸细菌,通过16S rDNA序列分析初步确定戊糖乳杆菌(Pediococcus pentosaceus)8株,枯草芽孢杆菌(Bacillus subtilis)与植物乳杆菌(Lactobacillus plantarum)各5株,解淀粉芽孢杆菌(Bacillus amyloliquefaciens)2株,溶血性葡萄球菌(Staphylococcus hominis)、解淀粉芽孢杆菌植物亚种(Bacillus amyloliquefaciens subsp.plantarum)、短乳杆菌(Lactobacillus brevis)各1株。DGGE操作简便,能够较好地反映盐渍青菜的细菌菌群变化,在盐渍青菜的细菌菌群结构分析中具有独特的优势。分离获得的产酸细菌可为盐渍工艺改进提供菌种资源。     

黄酒熟麦曲中细菌多样性的评价

以黄酒熟麦曲为研究对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chan reaction-denaturing gradient gel electrophoresis,PCR-DGGE)与MiSeq高通量测序技术相结合的方法对其细菌多样性进行解析。DGGE结果表明:Bacillus(芽孢杆菌)、Weissella(魏斯氏菌)、Pediococcus(片球菌)、Staphylococcus(葡萄球菌)和Lactobacillus(乳酸杆菌)普遍存在于麦曲样品中。MiSeq高通量测序结果表明:熟麦曲中的细菌门主要为Firmicutes、Proteobacteria和Actinobacteria,平均相对含量分别为87.95%、9.00%和1.16%。熟麦曲中的细菌属主要为Bacillus (芽孢杆菌属)、Weissella(魏斯氏属)、Staphylococcus(葡萄球菌)、Klebsiella(克雷伯菌属)、Pantoea(泛菌属)和Lactobacillus(乳杆菌属),其平均相对含量分别为70.70%、8.55%、2.67%、2.29%、1.32%和1.07%。由此可见,Bacillus(芽孢杆菌)为熟麦曲中的优势细菌。     

基于纯培养和高通量测序技术解析仙桃地区鲊菜细菌多样性

为解析湖北省仙桃地区鲊菜中细菌多样性,该研究在采用纯培养技术对乳酸菌菌株进行分离鉴定的基础上,进一步使用Illumina Mi Seq高通量测序技术对其细菌多样性进行解析。纯培养结果发现,分离到的44株乳酸菌隶属于乳酸杆菌属（Lactobacillus）、明串珠菌属（Leuconostoc）、魏斯氏菌属（Weissella）和乳球菌属（Lactococcus）,其中植物乳杆菌（L. plantarum）和戊糖乳杆菌（L. pentosus）分别占总分离株含量的40.91%和34.09%。Illumina Mi Seq高通量测序结果显示,鲊菜中优势细菌属主要包括乳酸杆菌属、明串珠菌属、魏斯氏菌属、假单胞菌属（Pseudomonas）和肠杆菌属（Enterobacter）,其平均相对含量分别为85.03%、3.31%、1.68%、1.50%和1.05%。由此可见,仙桃地区鲊菜中乳酸菌具有较高的多样性,且以植物乳杆菌和戊糖乳杆菌为主。     

温度和培养基对雪莲菌菌群结构的影响

该实验分别在28℃和-80℃条件下用脱脂乳和MRS培养基培养雪莲菌粒，采用高通量测序和变性梯度凝胶电泳技术分别对上述不同处理的样品的细菌和真菌菌群结构进行分析。结果表明，经过低温处理的雪莲菌粒样品细菌菌群多样性下降，变形菌门(Proteobacteria)含量减少，厚壁菌门(Firmicutes)含量上升；优势菌属由醋酸杆菌属(Acetobacteria)变为乳杆菌属(Lactobacillus)，且脱脂乳培养基更有利于雪莲菌粒细菌菌群多样性的稳定。脱脂乳培养的雪莲菌粒真菌菌群结构在冻藏前后菌群结构相似，而在MRS培养基中雪莲菌粒冻藏后菌种丰度降低，特别是单孢酿酒酵母(Saccharomyces unisporus)。综上，低温处理会造成雪莲菌粒菌群多样性的下降，脱脂乳较MRS培养基更有助于雪莲菌菌群的稳定性。     

鄂尔多斯地区酸粥细菌多样性分析及乳酸菌的分离鉴定

以采集自内蒙古鄂尔多斯地区的8个酸粥样品为研究对象,利用Illumina MiSeq高通量测序技术,对样品进行细菌菌群结构及多样性研究,分离鉴定其优势菌株。结果表明:8个样品中的细菌主要来自厚壁菌门(Firmicutes)和变形菌门(Proteobacteria),乳酸杆菌属(Lactobacillus)和醋酸杆菌属(Acetobacter)为优势菌属。乳酸杆菌属在各样品中占比最高,为42.63%～97.49%。SZ1、SZ2样品与其他样品的菌群结构差异较大,可能与不同采集时间有关。对上述样品中的乳酸菌进行分离纯化,通过生理生化试验结合分子生物学方法,鉴定获得1株干酪乳杆菌(Lactobacillus casei),1株发酵乳杆菌(Lactobacillus fermentum),2株植物乳杆菌(Lactobacillus plantarum)和4株副干酪乳杆菌(Lactobacillus paracasei)。上述研究结果为酸粥工业化生产提供菌种资源和理论依据。     

不同天然奶酪发酵剂和非发酵剂微生物多样性的高通量测序研究

为探究天然奶酪中发酵剂和非发酵剂微生物群落结构,应用Illumina-Hi Seq高通量测序技术,对江苏省主要销售的5组进口天然奶酪表皮和内部的细菌16Sr DNA序列V4区进行测序。所有样品的有效序列均在50000条以上,可操纵分类单元(OTU)数量为118~646。多样性指数分析和主成分分析结果表明,天然奶酪表皮比内部具有更高的细菌群落丰富度和不同的群落结构。样品中发酵剂细菌属为乳球菌属(Lactococcus)、链球菌属(Streptococcus)、乳杆菌属(Lactobacillus),均位于相对丰度前五,总相对丰度在51~97%。样品中非发酵剂微生物分布于细菌厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、异常球菌-栖热菌门(Deinococcus-Thermus)和软壁菌门(Tenericutes),相对丰度在2~16%。具有产耐热乳糖酶功能的非发酵剂微生物栖热菌属(Thermus,相对丰度0.02%~3.53%)在奶酪中首次被发现,为发掘天然奶酪中的微生物资源提供了可能。本研究也为解析天然奶酪中微生物与其风味质构的关系提供了基础数据。     

基于16S rDNA高通量测序技术分析水牛原料乳中细菌的多样性

为研究不同品种水牛原乳中的细菌组成,本研究采用16SrDNA高通量测序技术分析了印摩拉水牛、尼里·拉菲水牛及三品杂水牛原乳中的细菌多样性,结果显示,3品种原料乳样本中的细菌种类覆盖430个种314个属,共540个OTU,其中包括有益菌如乳酸杆菌(Lactobacillus)和致病菌如大肠埃希菌属志贺菌(Escherichia-Shigella);各样本的菌群组成和相对丰度分析结果表明,不同品种原料乳中主要菌群组成和优势菌均呈现明显差异,其中三品杂、摩拉、尼里·拉菲水牛原料乳中的优势菌属分别为肠球菌属(Enterococcus)、籍伏氏菌属(Vogesella)、假单胞菌属(Pseudomonas),样本中还有部分未鉴定的细菌,需要进一步研究。     

无锡城乡年轻居民肠道菌群多样性研究

采用PCR和DGGE相结合的技术,研究了生活于无锡城市和乡村的青年志愿者肠道菌群的多样性。基于DGGE指纹图谱,使用聚类和PCA分析对志愿者肠道微生物相似性进行分析,使用Shannon-Weine多样性指数(H)、丰度(S)和均匀度(EH)对志愿者肠道微生物多样性进行分析,对图谱中具有代表性的共性和特异性条带进行胶回收和测序以分析志愿者肠道微生物组成;基于PCR技术在种水平上对城乡志愿者肠道内乳杆菌属(Lactobacillus)和双歧杆菌属(Bifidobacterium)多样性进行定性分析。结果显示,无锡城乡青年居民间肠道微生物群落结构存在分开趋势,相似性小于城市或乡村居民内部;多样性方面差异不显著;不动杆菌属、双歧杆菌属、葡萄球菌属、乳杆菌属、梭菌属和瘤胃球菌属为城乡青年居民肠道内的共有菌属;乳杆菌属Lactobacillus(L.)和双歧杆菌属Bifidobacterium(B.)各类菌群在城乡居民肠道内分布差异不显著,其中L.plantarum,L.casei,L.salivarius和L.acidophilus以及B.longum,B.breve,B.animalis和B.adolescentis分别为无锡青年居民肠道内的优势乳杆菌和双歧杆菌。综上分析,初步揭示了无锡城市和乡村青年居民肠道微生物多样性差异不显著。     

新疆柯尔克孜族传统发酵饮料博扎中微生物群落结构的PCR-DGGE分析

采用PCR-DGGE 技术分析新疆柯尔克孜民族古老传统发酵饮料--博扎(Bozaa)中微生物种群结构,对博扎细菌和酵母菌DGGE 图谱上主要条带的DNA 进行测序和序列分析。结果表明,博扎中细菌组成包括植物乳杆菌(Lactobacterium plantarum)、短乳杆菌(Lactobacillus brevis)、蜡状芽孢杆菌(Bacillus cereus)、巴氏醋酸杆菌(Acetobacerpasteurianus)、啤酒酵母(Saccharomyces cerevisiae),初步成功解析评估了博扎中微生物的多样性。     

发酵辣椒细菌多样性的16S rDNA测序分析

以家庭自制的剁辣椒、酸辣椒、腌辣椒和辣椒酱为对象,研究不同样品中的微生物多样性,为分离其中的乳酸菌及开发发酵辣椒制品复合发酵剂提供参考依据。采用土壤基因组DNA提取试剂盒提取样品的基因组,然后PCR扩增细菌的16S r DNA V4区,琼脂糖凝胶电泳验证后回收胶,荧光定量后上机进行MiSeq高通量测序,然后分析样品中细菌的多样性。结果显示:不同样品所含细菌的种类多样性和丰度存在较大的差异。在属的水平上,剁辣椒中魏斯氏菌、明串珠菌和乳杆菌是主要的菌属,丰度在16%左右;乳杆菌(61.5%)和片球菌(28.6%)是酸辣椒中主要的细菌;腌辣椒中乳杆菌的丰度达到79.4%;辣椒酱的物种多样性最为丰富,魏斯氏菌(40.7%)和乳杆菌(11%)是其主要的微生物。     

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem.     

Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78–84% similarity, and yeasts 80–92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.     

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.     

Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard

Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described.     

Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods

Abstract: In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture‐independent and culture‐dependent methods. In the culture‐independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture‐dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence‐based PCR (rep‐PCR) fingerprinting, and the results were validated by 16S rDNA full‐length sequencing. According to the results of culture‐dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture‐dependent and culture‐independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Practical Application: Due to their artisan‐ and region‐dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from different regions of Turkey were studied using culture‐dependent and culture‐independent molecular methods.     

Diversity of bacteria present in milk kefir grains using culture-dependent and culture-independent methods

In the present work bacteria associated with milk kefir grains from several Brazilian States, Canada and the United States of America under traditional conditions have, for the first time, been studied using a combination of pheno-and genotypic methods. Conventional culturing was performed and a total of 270 isolates were obtained from all samples. Isolates were identified using biochemical tests and partial sequence analysis of 16S rDNA. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant bacterium was Lactobacillus kefiri. PCR-DGGE revealed the presence of Gluconobacter japonicus and Lactobacillus uvarum which were no isolated. Conventional isolation revealed the presence of L. helveticus, L. kefiri and Acetobacter syzygii not identified among the sequenced DGGE bands. This study is the first to report the presence of Lactobacillus satsumensis and Acetobacter syzygii in milk kefir grains.     

Genotypic diversity of bacteria and yeasts isolated from Tibetan kefir

In this study, we have investigated the intraspecies genotypic diversity of bacteria and yeasts isolated from Tibetan kefir via the (GTG)₅‐REP‐PCR technology. According to the cluster analysis of generated fingerprints, forty‐nine bacterial isolates were grouped into nine clusters and formed thirteen genotypes, and twenty‐two yeast isolates were grouped into two clusters and formed two genotypes. It's worth noting that isolates of Lactobacillus kefiranofaciens, Streptococcus thermophilus, Lactobacillus kefiri and Lactobacillus paracasei exhibited the intraspecies genotypic diversity, whereas isolates of Saccharomyces cerevisiae and Kazachstania unispora did not exhibit. These results confirmed that bacterial isolates possessed higher level of intraspecies genotypic diversity than yeast isolates studied in this research. These findings revealed that (GTG)₅‐REP‐PCR was an efficient and sensitive molecular typing tool for revealing the intraspecies genotypic diversity among the bacterial isolates originated from Tibetan kefir.     

PCR-DGGE技术对中华开菲尔微生物菌群的分析

为了解中华开菲尔微生物菌群的结构特征,本论文运用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对开菲尔菌株发酵过程中微生物菌群的结构变化进行了实验分析,结果表明:细菌菌群DGGE图谱上出现有三种不同迁移位置的斑带,而酵母菌群DGGE图谱上只有一条斑带;经过DNA序列的对比分析可知:细菌菌群分别为肠膜明串珠菌(Leuconostoc mesenteroides)、马乳样乳杆菌(Lactobacillus kefiranofaciens)和开菲尔乳杆菌(Lactobacillus kefir),它们的序列同源性都达到100%;酵母菌群为德尔布有孢圆酵母(Torulaspora delbrueckii),其序列同源性为99%。本论文首次报道了德尔布有孢圆酵母在开菲尔菌落中的存在。     

Identification of microbial flora in kefir grains produced in Turkey using PCR

Kefir grains might have different ratios and/or content of microflora according to their origin. The purpose of this study was to determine microbial flora of kefir grains produced in three regional universities in Turkey using polymerase chain reaction (PCR) and consensus sequence primers. According to the results of PCR products with the specific primers, the following were identified as the natural inhabitants of the kefir grains: Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Bifidobacterium bifidum and Kluyveromyces marxianus kefir. The results of this study revealed that traditional kefir produced by using kefir grains as natural starter cultures contains lactic acid bacteria especially Lactobacillus spp. One of the sources also contained B. bifidum. This is the first record on the presence of B. bifidum in kefir grains.     

酱香型郎酒高温大曲、酒醅和窖泥中细菌群落结构分析

该研究利用高通量测序技术对酱香型郎酒大曲、酒醅和窖泥的细菌菌群进行多样性解析和比较分析。结果表明,郎酒窖泥中细菌菌群的丰富度和多样性最高,大曲中细菌菌群的多样性远高于酒醅。窖泥中主要优势细菌属为乳杆菌属（Lactobacillus）（26.1%）、埃希氏菌-志贺氏菌属（Escherichia-Shigella）（12%）、梭菌属（Clostridium）（7.9%）等,嗜酸乳杆菌（Lactobacillus acetotolerans）作为主体菌,同时存在多种未培养菌种;大曲中的主要优势菌群包括泛菌属（Pantoea）（21.9%）、高温放线菌属（Thermoactinomyces）（21.4%）、克罗彭施泰特氏菌属（Kroppenstedtia）（19.4%）和乳球菌属（Lactococcus）（9.4%）等;酒醅中主要优势细菌属为片球菌属（Pediococcus）（42.7%）、魏斯氏菌属（Weissella）（32.1%）、芽孢杆菌属（Bacillus）（14.2%）和乳杆菌属（Lactobacillus）（8.3%）,乳酸菌占据优势地位。