利用非培养技术研究芝麻香型白酒高温大曲的细菌群落多样性

采用构建16S rDNA克隆文库的非培养方法,对我国芝麻香型白酒高温大曲细菌的群落结构及其多样性进行研究。文库中含有24个分类操作单元(OTU),优势菌属(丰度)分别为Thermoactinomyces sp.(51.99%),Kroppenstedtia sp.(17.38%),Saccharopolyspora sp.(13.87%),Lactobacillus sp.(6.95%)和Weissellasp.(4.96%)。首次在高温大曲中发现Thermoactinomyc vulgaris和Kroppenstedtia eburnea;同时发现3个潜在细菌新种,分属于Saccharothrix sp.,Streptoalloteichus sp.和Pseudofulvimonas sp.。     

应用PCR-DGGE对巴氏消毒乳中腐败细菌的污染溯源研究

运用PCR-DGGE技术分析了巴氏杀菌乳从原料乳到杀菌、灌装这两个加工关键控制点的微生物动态变化和差异性,容易发生污染的环节是挤乳环节、杀菌后的加工管道和包装环节。原料乳中易携带的细菌是乳酸链球菌(Lactococcus lactis subsp.)和乳酸杆菌(Lactobacillus sp.);挤乳环节中易受无乳链球菌(Streptococcus agalactiae)污染;杀菌后的加工管道易受热杀环丝菌(Brochothrix thermosphacta)、钻黄肠球菌(Enterococcus casseliflavus)、瑞士乳杆菌(Lactobacillus helveticus)和2株非培养的假单胞菌属(Uncultured Pseudomonas sp.)污染;肉杆菌属(Carnobacterium sp.)和产乳酸菌素的肉杆菌(Carnobacterium maltaromaticum)则在挤乳环节、杀菌后的加工管道或包装环节都可能被污染。     

PCR-DGGE分析东北自然发酵酸菜中的微生物多样性

为了探明传统的自然发酵酸菜中微生物的多样性和优势菌群,通过聚合酶链式反应–变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术分析酸菜发酵过程中微生物群落动态变化。结果表明:东北自然发酵酸菜中的细菌种类比较丰富,真菌在酸菜中存在相对较少。东北自然发酵酸菜在发酵早期的活跃菌为明串珠菌(Leuconostoc sp.),而后是发酵产酸的嗜酸乳杆菌(L.acidophilus)、发酵乳杆菌(L.fermentum)和植物乳杆菌(L.plantarum),最后是由植物乳杆菌完成发酵过程。其中明串菌株为酸菜发酵前期优势细菌,植物乳杆菌为发酵中后期优势细菌,东北自然发酵酸菜发酵过程中主要真菌为汉逊德巴利酵母(Debaryomyces hansenii)、热带假丝酵母(Candida tropicalis)、扩展青霉(Penicillium expansum),真菌数量随发酵时间的增加而减少,且种类也随时间的变化而不断变化。     

应用PCR-DGGE技术分析发酵鸭肉中细菌多样性

对不同前处理的发酵鸭肉应用变性梯度凝胶电泳(DGGE)技术进行细菌菌群分析。以细菌通用引物扩增16S rDNA基因可变区V3区,进行DGGE上样,从而获得可以表征细菌群落结构的图谱。研究结果表明,热炒后发酵和未热炒发酵的2个样品菌群差异比较大,但它们都有乳杆菌(Lactobacillus alimentarius),在发酵过程中乳酸菌是主要优势菌,而且未热炒发酵相对于热炒后发酵的样品各菌群数量比较明显。热炒后发酵的鸭肉中存在乳酸菌属(Lactobacillus sp),赖氨酸芽孢杆菌属(Lysinibacillus sp),乳酸片球菌(Pediococcus acidilactici),葡萄球菌属(Staphylococcus stepanovicii),丛毛单胞菌属(Comamonas sp)。未热炒发酵的鸭肉中有赖氨酸芽孢杆菌属(Lysinibacillus sp),普氏厌氧球菌(Anaerococcus prevotii),戊糖片球菌(Pediococcus pentosaceus),吲哚嗜胨菌(Peptoniphilus indolicus),乳杆菌(Lactobacillus alimentarius)。在发酵鸭肉中还检测到了3种不可培养菌Uncultured Lactobacillus sp、Uncultured bacterium、Uncultured Comamonas sp,这在鸭肉发酵过程中的作用还有待进一步研究和验证。PCR-DGGE能快速地反应发酵鸭肉中细菌群落结构。     

5株发酵剂对熏马肠成熟过程中内源菌的影响

使用经实验室分离鉴定的4株葡萄球菌(Staphylococcus epidermidis;Staphylococcus simulans.001;Staphylococcus simulans.002;Staphylococcus simulans.003)及1株植物乳杆菌(Lactobacillus plantarum),采取不同的纯菌及复合添加组合(4株葡萄球菌分别与植物乳杆菌1∶1的比例)作为发酵剂,在发酵和成熟的不同时期取样及提取熏马肠中细菌的总DNA,采用PCR-DGGE技术研究发酵剂对内源微生物在熏马肠成熟全过程中动态变化的影响。试验结果表明,不同发酵剂添加组合的熏马肠菌相分布呈现不同变化,但是随着成熟时间的增加,优势菌趋于明显,Weissella sp.,Lactobacillus sp.,Enterococus faecium,Enterococus faecalis始终存在于菌相中。其中F组(Staphylococcus simulans.002)的抑菌效果明显,内源菌的种类和数量都有所减少,尤其是对Lactobacillus plantarum,Pseudomonas sp.,Enterococus faecalis有较强的抑制作用。     

水晶肘花长白斑现象的微生物分析

为了探索肘花长白斑的原因,分别从正常和长白斑的肘花产品中分离纯化得到14株菌,传统培养和16S rDNA的综合分析结果表明,对照组中的细菌主要为泛团菌属(Pantoea sp.)和包括Enterobacter cloacae、Enterobacter ludwigii 在内的肠杆菌属(Enterobacter sp.);而白斑组中除了肠杆菌属外,还分离出包括蜡样芽孢杆菌(Bacillus cereus)在内的芽孢杆菌属细菌(Bacillus sp.),由此推断芽孢杆菌的存在可能是引起产品变质的主要原因.     

1株产细菌素乳酸菌的鉴定及所产细菌素的诱导合成现象

从发酵大蒜中筛选到一株具有抑菌作用的菌株Br26,通过用不同蛋白酶处理判断该菌株是否为细菌素产生菌。随后,通过分析自身发酵上清液与其他乳酸菌对细菌素合成的影响,探讨该细菌素的诱导合成现象。结果表明：菌株Br26的发酵上清液经胃蛋白酶和木瓜蛋白酶处理后抑菌活性降低,确定该菌为细菌素产生菌,经16S rDNA鉴定为Weissella sp. Br26。Weissella sp. Br26在42 ℃、1/4 MRS培养时,细菌素抑菌活性消失,而当添加不同生长时期的发酵上清液时,抑菌活性显著增加,表明该细菌素可进行自我诱导。另一方面,Weissella sp. Br26与卷曲乳杆菌、瑞士乳杆菌、副干酪乳杆菌和肠膜明串珠菌的活菌共同培养后,发酵液pH值未有明显变化,但细菌素抑菌活性显著增加,表明细菌素的合成亦可受其他乳酸菌的诱导。综上,Weissella sp. Br26细菌素的合成是受自身发酵液及其他菌诱导调控的。     

四川泡菜可培养微生物菌群的研究

对四川泡菜可培养微生物进行分离鉴定,并做系统发育及菌群多样性分析。细菌共分离207株,采用16SrRNA序列分析进行鉴定,大部分可鉴定到种的水平,优势菌为乳杆菌属,包括植物乳杆菌(Lactobacillus plantarum)、干酪乳杆菌(Lactobacillus casei)、短小乳杆菌(Lactobacillus brevis)、棒状乳杆菌(Lactobacillus coryniformis)等共计11个种;还分离到片球菌属(Pediococcus sp.)、葡萄球菌属(Staphylococcus sp.)、链球菌属(Streptococcus sp.)、明串珠菌属(Leuconostoc sp.)、肠球菌属(Enterococcus sp.)。酵母共分离91株,采用26SrRNA D1/D2序列分析进行鉴定,均鉴定到种的水平,包括汉逊德巴利酵母(Debaryomyces hansenii)、异常毕赤酵母(Pichia anomala)、膜璞毕赤酵母(Pichia membranifaciens)、盔状毕赤酵母(Pichia galeiformis)、Saccharomyces bulderi、Saccharomyces servazzii。     

酱油发酵过程中细菌群落结构的动态变化

本文采用16S r DNA PCR-DGGE(变性梯度凝胶电泳)指纹图谱和系统发育分析方法,以高盐稀醪发酵工艺生产的广东酱油为研究对象,揭示了酱油生产发酵过程中细菌群落结构的多样性及动态变化。从样品中提取总细菌DNA,用降落PCR扩增16S r DNA V3片段序列,再通过分析DGGE图谱选择特异性条带,进行割胶回收、测序及Blast分析。DGGE图谱表明,发酵初期样品具有丰富的微生物群落,但之后只有少数种类细菌存活,整个酱油发酵过程微生物群落结构的演变规律是由复杂到简单,这也说明酱油发酵环境具有抑制微生物生长的作用。测序结果表明,代表最相似菌为魏斯菌属(Weissella cibaria)和非培养的肠杆菌属(Uncultured Enterobacter sp.),其次是嗜盐四联球菌(Tetragenococcus halophilus)、类肠膜魏斯氏菌(Weissella paramesenteroides)、弗氏柠檬酸杆菌(Citrobacter freundii)、产气肠杆菌(Enterobacter aerogenes)和肠杆菌属(Enterobacter sp.BF1-8)。     

山西老陈醋大曲细菌群落结构及多样性研究

山西老陈醋是以大曲为发酵剂,自然固态发酵的传统酿造食品。大曲中的微生物在其发酵过程中起重要作用。为了分析山西老陈醋大曲细菌群落结构及多样性,提取山西老陈醋大曲样品总DNA,应用Miseq高通量测序平台结合16S rDNA序列分析大曲细菌群落结构。大曲中的细菌种类丰富,包括:厚壁菌门、变形菌门、放线菌门和拟杆菌门,其中厚壁菌门和变形菌门为优势菌门,分别占大曲总细菌的69.67%和29.17%,放线菌门及拟杆菌门仅占大曲总细菌的0.86%和0.28%。厚壁菌门上仅检测到杆菌纲,主要包括乳杆菌属(32.82%)及芽孢杆菌属(29.45%)。在变形菌门上检测到γ-变形菌纲,主要包括泛菌属(27.36%)和不动杆菌属(0.80%)。放线菌门与拟杆菌门结构简单,分别检测到放线菌纲和黄杆菌纲,属水平上分别检测到糖多孢菌属(0.59%)和黄杆菌属(0.27%)。本研究揭示了山西老陈醋大曲细菌群落结构,为明确山西老陈醋微生物酿造机理提供基础数据。     

Succession of a microbial community during stable operation of a semi-continuous garbage-decomposing system

The microbial community in a garbage-decomposing system was analyzed using denaturing gradient gel electrophoresis (DGGE) on the basis of 16S rDNA. The system treated 1 kg of garbage everyday for two months at ambient temperature with almost constant decomposition efficiency, although a transient pH increase occurred. Succession of the banding pattern of the DGGE profile suggested that the bacterial community was not directly affected by the continuous addition of non-sterilized garbage into the open system, but changed with the fluctuation of pH. These resistance and resilience characteristics of the community structure may be effective to keep the decomposition efficiency stable. The analyses of the DNA sequences from the DGGE bands suggested the existence of uncultured or novel bacteria as well as Lactobacillus sp., Corynebacterium spp., Enterococcus spp., and Staphylococcus sp. A specific PCR detection was performed to evaluate the existence of Escherichia coli within the community. E. coli 16S rDNAs were not detected from the decomposing system.     

Changes of bacterial diversity and main flora in chilled pork during storage using PCR-DGGE

This study was designed to explore the bacterial diversity and the main flora in chilled pork by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Longissimus muscle was removed from pork carcasses at 24 h postmortem. The muscle was tray- and vacuum-packaged at 4 degrees C for 2, 4, 7 days to extract the bacteria total DNA, respectively. The results indicated that the bacterial diversity of chilled pork decreased with storage time regardless of packaging method. Nine types of bacteria were identified, including Arthrobacter sp., Enterococcus sp., Staphylococcus sp., Moraxella sp., Pseudomonas sp., Lactobacillus sp., Aeromonas sp., Acinetobacter sp., Brochothrix thermosphacta. For tray-packaged pork, Pseudomonas sp. and B. thermosphacta were the dominant micro-organisms. The differences in the species found were related with the presence of Lactobacillus sp. in vacuum-packaged meat. The results of the present study might be useful to study the changes of the contaminating bacteria and their characteristics in chilled pork.     

PCR-DGGE技术分析不同包装条件下鱼肉表面优势菌的菌群变化

采用聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-deformation gradient gel electrophoresis analysis,PCR-DGGE）技术研究3 种包装（空气、真空、气调）结合钴60辐照技术对鱼肉表面优势菌菌群的影响,分别选择6 种辐照剂量（0、2、4、6、8、10 kGy）照射4 ℃贮藏18 d后的鱼肉表面优势腐败菌群进行研究。方法：采集第18天不同包装不同剂量的草鱼肉样品,洗脱鱼肉表面菌;提取DNA,扩增16S rDNA的V3可变区,用PCRDGGE技术鉴定优势菌,割胶回收测序。结果表明：不同包装鱼肉表面优势菌群数量和种类不同,在样品中共同的优势腐败菌为假单胞菌,在空气包装中优势腐败菌是假单胞菌、乳酸菌和沙雷菌,沙雷菌在辐照剂量高于8 kGy时被抑制。在真空包装中优势腐败菌是假单胞菌、沙雷菌、热死环丝菌和耶尔森菌,沙雷菌辐照剂量高于6 kGy时被抑制,而耶尔森菌在2 kGy以上即被抑制,热死环丝菌在6、8 kGy辐照条件下才出现,其具有很高的辐照抗性。在气调包装中优势腐败菌是假单胞菌和乳酸菌,但乳酸菌仅在气调包装2 kGy辐照和气调包装8 kGy两种辐照包装条件下是优势腐败菌。结果表明,PCR-DGGE技术可以很好地鉴别鱼肉表面优势腐败菌。     

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4 °C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.     

冷藏带鱼贮藏期间主要微生物动态变化的PCR-DGGE分析

本文研究了冷藏带鱼贮藏期间的主要微生物种群演替规律,为延长水产品货架期提供参考。采用SDS化学裂解法,从冷藏带鱼中提取总细菌基因组DNA,并进行16SrRNA的V3可变区PCR扩增,再通过DGGE得到动态指纹图谱。结果表明,贮藏初期冷藏带鱼样品中的微生物群落丰富,嗜冷杆菌(Psychrobacter sp.)为前期的主要优势菌。随着贮藏时间的延长,仅有少数种类细菌存活并最终成为主导菌群。荧光假单胞菌(Pseudomonas fluorescens)在贮藏过程中比例逐渐增大,希瓦氏菌(Shewanella sp.)与弧菌(Vibrio sp.)比例也有明显增加,在贮藏后期发展成为优势腐败菌。     

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork.     

豆酱不同发酵阶段细菌群落多样性及动态变化分析

以东北传统自然发酵豆酱为研究对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reactiondenaturing gradient gel electrophoresis,PCR-DGGE)技术结合高通量测序方法分析豆酱发酵过程中细菌的多样性及动态变化。结果显示,细菌的可操作分类单元数值在发酵过程中上下波动,并在后发酵第21天时达到最大值,此时细菌数目最多;整理分析PCR-DGGE图谱鉴定结果以及细菌在属水平上的分布得出,明串珠菌属、肠球菌属、四联球菌属和乳杆菌属为豆酱样品不同发酵阶段的优势细菌菌属,其中四联球菌属波动性较大,易受内外环境的影响;屎肠球菌、明串珠菌属、绿色魏斯菌随着发酵时间的延长在减少,说明这些细菌主要参与豆酱发酵初期产酶分解物质过程,并在整个发酵过程中起到调整发酵内环境等作用。     

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork.     

PCR-DGGE指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物变化的影响

为探讨复合保鲜剂的作用机理和延长水产品货架期,应用聚合酶链式反应(PCR)-变性梯度凝胶电泳(DGGE)指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物种群多样性演替规律的影响。采用SDS(sodium dodecyl sulfate)化学裂解法,分别从对照组与复合保鲜剂处理组的冷藏带鱼中提取总细菌基因组DNA,同时进行16S rDNA的V3可变区PCR扩增,再通过DGGE得到动态变化的指纹图谱。DGGE图谱表明,产品在贮藏初期具有丰富的微生物群落,说明污染微生物的多样性,但经过一段时间后,只有少数种类细菌存活并最终成为主导菌群;通过对冷藏带鱼DGGE图谱上的主要条带进行测序分析,获得12种细菌。对照组与复合保鲜剂处理组在腐败后期的特定腐败微生物种类具有高度的相似性。嗜冷杆菌为带鱼贮藏初期的优势菌,随着贮藏时间的延长,希瓦氏菌与假单胞菌的比例逐渐增加,在贮藏过程中成为主要优势菌,荧光假单胞菌与弧菌在贮藏期间占有较大比例。同时,冷藏带鱼经复合保鲜剂处理后,其中的希瓦氏菌与假单胞菌受到明显抑制。