市售冷却牛肉中主要细菌的常规分离与鉴定

利用常用纯培养的方法, 根据细菌的菌落形态、菌落颜色、革兰氏染色等常见特征, 从市售冷却牛肉中, 选取菌落形态差别比较明显的菌株共32株, 其中保鲜膜包装冷却牛肉样品共12株, 未包装冷却牛肉样品共20株; 同时选取两样品中的优势菌株各4株进行进一步的研究(8株细菌编号为：S01~S08, 其中S01~S04为未包装冷却牛肉样品; S05~S08为保鲜膜包装冷却牛肉样品), 通过ARDRA(Amplified ribosomal DNA restriction analysis) 以及16S rDNA 序列等进行分类研究, 确定该细菌的分类地位, 并结合形态、常规生理生化特性进行鉴定, 确定各细菌所属种。实验表明：S01为假单胞菌属中的恶臭假单胞菌(Pseudomonas putida), S02为希瓦氏菌属下的(Shewanella cincia sp.), 而S03和S05为希瓦氏菌属下的腐败希瓦氏菌(Shewanella putrefaciens), S04为窄食单胞菌属中的嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia), S06为嗜冷杆菌(Psychrobacter sp.), S07为葡萄球菌属中的松鼠葡萄球菌(Staphylococcu sciuri), S08为微杆菌属中的产左聚糖微杆菌(Microbacterium laevaniformans)。证明两样品的共有优势菌为希瓦氏菌属。通过对样品可培养微生物情况进行初步的调查分析, 为冷却肉加工工艺提供一定的理论基础。     

两种包装市售冷却牛肉中微生物多样性的比较分析

本文比较分析了相同销售条件下的未包装冷却牛肉与保鲜膜包装冷却牛肉中的微生物群落结构。采用16S rDNA的V3区片段进行PCR-DGGE(变性梯度凝胶电泳), 同时利用16S rDNA全长序列, 通过克隆文库技术对两个样品的微生物群落结构进行研究。研究发现: 微生物群落结构表现出较大的差异。保鲜膜包装冷却牛肉共有6个OTU (Operational taxonomic unit), 主要为Lactococcus(乳球菌属, 28%), Lactobacillus(乳杆菌属, 26%), Carnobacterium(肉品杆菌属, 18%)和Brochothrix(索丝菌属, 10%); 未包装冷却牛肉则共得到18个OTU, 主要是Lactococcus(乳球菌属, 28%), Brochothrix(索丝菌属, 18%)和Acinetobacter(不动杆菌属, 11%)。结果表明, 保鲜膜包装对于葡萄球菌属以及冷杆菌属等一些细菌都起到了一定的抑制作用。本研究为肉类加工生产中微生物的控制提供一定的理论依据。     

南京小龙山钾矿区植物根际可培养细菌的遗传多样性分析

摘要:【目的】矿区优势植物可培养细菌生物多样性研究将有助于了解植物根际细菌与矿物,植物根系相互作用及对矿物风化和土壤形成的重要影响。【方法】采用纯培养法分离南京小龙山废钾矿区野生植物野塘蒿,千金子和栽培植物甘薯根内与根周围土壤的可培养细菌, 通过16S rDNA限制性酶切多态性分析(amplified rDNA restriction analysis, ARDRA)和16S rDNA序列分析研究了可培养细菌的多样性。【结果】分离纯化到60株具不同菌落形态的可培养细菌, 在60%相似水平上可分为18个OTU. 19株代表菌株分别属于3个门, 10个科, 11个属。多数菌株属于变形菌门(α-proteobacteria, 4株, 21.1%; β-proteobacteria, 2株, 10.5%; γ-proteobacteria, 6株, 31.6%)。假单胞菌属(Pseudomonas), 泛菌属(Pantoea)和根瘤菌属(Rhizobium)为优势种群。【结论】小龙山废矿区优势植物根围具有丰富的微生物种群多样性。     

黑龙江大豆胞囊线虫胞囊可培养细菌多样性

【目的】了解黑龙江省大豆田大豆胞囊线虫胞囊可培养细菌的多样性。【方法】运用稀释平板法和16SrDNA基因序列的系统发育分析对胞囊可培养细菌多样性进行研究。【结果】用NA培养基从胞囊上分离90株具有不同菌落形态的细菌。16S rDNA序列分析结果表明:90株菌株分属于7个属22个种。46株属于变形菌门γ亚群(Gammaproteobacteria),32株属于厚壁菌门(Firmicutes),10株属于变形菌门β亚群(Betaproteobacteria),2株属于变形菌门ɑ亚群(Alphaproteobacteria)。假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为优势菌属。【结果】黑龙江省大豆胞囊线虫胞囊中存在丰富的细菌物种多样性,这些细菌对大豆胞囊线虫可能具有一定的生理生态作用。     

新疆野生胀果甘草内生细菌多样性的非培养初步分析

摘要：【目的】了解新疆野生甘草内生细菌多样性,为开发新的微生物资源奠定基础。【方法】采用改进的CTAB (十六烷基三甲基溴化铵)法提取新疆野生胀果甘草根部总DNA,利用细菌16S rDNA 基因通用引物对甘草总DNA 进行16S rDNA 基因扩增,构建甘草内生细菌16S rDNA基因文库;挑选具有不同酶切图谱的克隆进行测序、比对并构建16S rDNA 基因系统发育树。【结果】构建的甘草内生细菌16S rDNA基因文库中, 150个克隆分属于32个不同的分类单元,Blast结果表明大部分克隆与已知细菌的16S rDNA基因序列相似性较高,分别归属于变形杆菌门(Proteobacteria)的alpha、gamma亚群,厚壁菌门(Firmicutes),放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes)中的鞘脂菌属(Sphingobium),叶杆菌属(Phyllobacterium),生丝单胞菌属(Hyphomonas),土壤杆菌属(Agrobacterium)等14个属, 其中26%的克隆与已知细菌16S rDNA 基因相似性小于96%,可能代表新的分类单元.【结论】甘草内生细菌多样性丰富且存在尚未被认识的新物种。     

云南干巴菌子实体内可培养微生物

【背景】微生物在菌根真菌的孢子萌发、菌丝体生长、菌根形成以及子实体发育等过程中起到一定作用。【目的】对采自云南省昆明市嵩明县和楚雄彝族自治州禄丰县的8个干巴菌子实体内的微生物进行分离培养鉴定,为后期研究微生物与干巴菌之间的相互作用奠定基础。【方法】采用传统平板分离法从干巴菌子实体内分离获得微生物群落,t检验分析不同地区采集的干巴菌子实体内微生物菌落总数的差异,16S r RNA基因和ITS序列进行系统发育树构建和微生物多样性分析。【结果】采自嵩明县和禄丰县的8个干巴菌子实体内共分离获得282株可培养的细菌,两个地区的细菌菌落总数无显著差异(P=0.22)。所有细菌分属2门12属15种。其中80%的细菌属于变形菌门,且以γ-变形菌为优势菌群,假单胞菌属(Pseudomonas)为优势菌属。其余20%的细菌属于拟杆菌门。从干巴菌子实体中分离获得114株真菌,两个地区的真菌菌落总数无显著差异(P=0.65)。所有真菌分属2门10属10种。其中62%的真菌属于子囊菌门(Ascomycota),并以分离自禄丰县干巴菌子实体内的Lophiostoma为优势属。38%的真菌属于担子菌门(Basidiomycota),并以Asterotremella为优势属。【结论】两个不同地区采集的干巴菌子实体内细菌和真菌在菌落总数上无显著差异。所有细菌都以γ-变形菌为优势菌群,假单胞菌属为优势菌属。嵩明干巴菌子实体内真菌以担子菌门为优势菌群,Asterotremella为优势属。而禄丰干巴菌子实体内真菌则以子囊菌门为优势菌群,Lophiostoma为优势属。     

聚赖氨酸和1-MCP联合处理对草菇贮藏期间表面细菌变化的影响

【背景】草菇具有很高的营养价值和药用价值,草菇采后品质易劣变主要是由其表面腐败菌引起的。保鲜处理条件下草菇表面菌的研究目前未见相关报道。【目的】探究聚赖氨酸(ε-polylysine,ε-PL)和1-甲基环丙烯(1-methylcyclopropene,1-MCP)联合处理对草菇贮藏期间表面细菌变化的影响。【方法】采用平板培养与16S rRNA基因高通量测序技术两种方法,分离、鉴定ε-PL和1-MCP联合处理后草菇贮藏期间的表面微生物,确定草菇贮藏期间菇体表面优势腐败菌。【结果】平板培养法结果表明,草菇子实体表面的菌落总数随着贮藏时间的延长逐渐上升。贮藏6d时,处理组(PC)草菇表面菌落总数为7.16 lg (CFU/g),极显著(P<0.01)低于CK组[7.42 lg (CFU/g)];从PC组、CK组分离鉴定的细菌分别为16株和19株,以假单胞菌属、金黄杆菌属、芽孢杆菌属和寡养单胞菌属为主;16S rRNA基因高通量测序结果表明,PC组共鉴定出370个属的细菌隶属于27个门;CK组共鉴定出366个属的细菌隶属于25个门;寡养单胞菌属为相对丰度最高的优势菌。【结论】寡养单胞菌...     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

对Bt蛋白抗性和敏感的棉铃虫中肠细菌群落的比较

【目的】通过比较Cry1Ac蛋白抗性及敏感棉铃虫中肠细菌群落的结构组成,研究中肠微生物是否与棉铃虫Bt抗性产生有关。【方法】首先提取了棉铃虫中肠微生物基因组DNA,通过PCR扩增获得了16S rDNA全长片段及V3区。采用基于16S rDNA的免培养技术-16S rDNA文库建立和变性梯度凝胶电泳(DGGE)研究了国内特有的Bt抗性和敏感品系棉铃虫中肠细菌群落组成,并对其进行分析和比较。【结果】16SrDNA文库测序结果表明,抗性品系与敏感品系棉铃虫中肠细菌群落特别是优势菌群非常相似,但在部分劣势菌群上存在差异。抗性品系中主要优势菌有:不可培养微生物(Uncultured bacterium)占56.4%,鹑鸡肠球菌(Enterococcus gallinarum)占17.0%,铅黄肠球菌(Enterococcus casseliflavus)占17.0%;敏感品系中主要优势菌为不可培养微生物(Uncultured bacterium)60.2%,鹑鸡肠球菌(Enterococcus gallinarum)占19.3%,铅黄肠球菌(Enterococcus casseliflavus)占14.7%。随后进行的PCR验证表明,部分有差异的劣势菌在两种品系虫体都存在。DGGE图谱分析表明,这两个品系棉铃虫中肠菌群相似性达到92.3%。【结论】敏感品系与抗性品系棉铃虫肠道菌群组成极其相似,推测抗性的产生与肠道微生物无直接关系。     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Changes in the Spoilage-Related Microbiota of Beef during Refrigerated Storage under Different Packaging Conditions

The microbial spoilage of beef was monitored during storage at 5°C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O₂ and 40% CO₂ (MAP2), and (iii) 20% O₂ and 40% CO₂ (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

Monitoring of microbial metabolites and bacterial diversity in beef stored under different packaging conditions

Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [(1)H NMR]), and microbial diversity (by PCR-denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. (1)H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.     

毛竹根际可培养微生物种群多样性分析

[目的]为了了解天然毛竹林根际可培养微生物种群的多样性信息,[方法]采用稀释平板法,对浙江天目山和重庆缙云山天然毛竹林根际细菌和放线菌进行了分离,并对其16S rDNA序列进行了分析.[结果]分别从天目山和缙云山天然毛竹林根际分离得到51株和31株菌落形态差异的细菌和放线菌.16S rDNA序列分析表明,天目山和缙云山毛竹根际细菌主要包括厚壁菌门(Firmicutes,分别为40%和58%)、放线菌门(Actinobacteria,分别为36.7%和10.52%)、变形菌门-亚群(Alphaproteobacteria,分别为10%和5.26%)和变形菌门 --亚群(Gammaproteobacteria,分别为10%和26.32%),其中芽孢杆菌属(Bacillus sp.)为共同的优势菌属(分别为34.38%和42.11%).分离的菌株中,B188、B171和B152等6株与GenBank中已报道16S rRNA基因序列的相似性从90%到96%不等,可能代表着新属或种.[结论]这表明,天然毛竹林根际具有较为丰富的可培养微生物种群多样性,并存在一些潜在的新的微生物菌种资源.     

贡嘎蝠蛾幼虫肠道细菌多样性分析

[目的]对实验室养殖条件下的重要经济昆虫冬虫夏草寄主-贡嘎蝠蛾(Hepialus gonggaensis,Hg)幼虫肠道微生物群落的多样性进行了研究.[方法]采用常规分离培养与分子鉴定的方法和基于16S rRNA作为分子标记的变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)的方法.[结果]用常规分离与分子鉴定方法获得8个属的细菌类群,其中肠杆菌属(Enterobacter)是优势菌群,肉食杆菌属(Carnobacterium)是次优势菌群.对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对和系统进化树分析,结果表明肉食杆菌属(Carnobacterium)的丰度最高,是肠道细菌中主要的优势菌群,芽孢杆菌属(Bacillus)是次优势菌群.DGGE图谱还显示Hg幼虫不同虫龄肠道细菌菌群的结构存在差异,推测可能与其发育生理状态的差异有关系.[结论]结合常规分离法与DGGE法能够更有效的分析肠道微生物的多样性,获得更多更全面的微生物多样性信息.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

八门湾红树林土壤芽胞杆菌分离与多样性分析

【目的】了解八门湾红树林海漆林区土壤中可培养芽胞杆菌资源的多样性。【方法】采用水浴处理与直接涂布相结合的方法选择性分离土壤中的芽胞杆菌;利用16S rDNA PCR-RFLP与16S rDNA序列分析技术研究可培养芽胞杆菌资源的遗传多样性和系统发育关系。【结果】16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析表明,在100%的相似性水平上,分离的155株芽胞杆菌分属21个遗传类群,显示了较为丰富的遗传多样性;由21种遗传类型代表菌株的16S rDNA序列分析结果得知,这些芽胞杆菌主要分布在Bacillaceae和Paenibacillaceae科下的Bacillus、Halobacillus、Virgibacillus和Paenibacillus 4个属,其中Bacillus为优势属;有8株芽胞杆菌的16S rDNA序列与数据库中相应模式菌株的最大相似性在95.1%-99.0%之间。【结论】八门湾红树林土壤可培养芽胞杆菌有着较为丰富的遗传多样性,并存在新的芽胞杆菌物种资源。