纯培养微生物荧光原位杂交技术检测的影响因素探究

为了研究纯培养微生物荧光原位杂交技术（FISH）检测的影响因素,该实验以大肠杆菌（Escherichia coli）及植物乳杆菌（Lactobacillus planetarium）为研究对象,研究不同种属、不同生长期菌株、菌体预处理方式及杂交条件对基于探针EUB338的FISH检测结果的影响。结果表明,菌体种类及菌体生长阶段均较明显影响FISH计数的准确性;确定超声分散菌体时间60 s（100 W、每30 s间歇）、溶菌酶处理60 min可较大幅度提高FISH对菌体的检出率;杂交时间和洗脱液中NaCl浓度对FISH检测结果影响小,杂交温度、杂交液中甲酰胺浓度对大肠杆菌影响小而对植物乳杆菌有一定影响。     

鼠李糖乳杆菌Probio-M9的特异性荧光原位杂交探针设计及应用

宿主口服益生菌的定性和局部检测具有重要意义。本研究通过设计株水平特异性荧光原位杂交（FISH）探针来研究鼠李糖乳杆菌在宿主肠道中的定殖。根据鼠李糖乳杆菌Probio-M9和其它相关物种的基因组序列,分别设计了鼠李糖乳杆菌种水平特异性探针（Probe-16S）和鼠李糖杆菌Probio-M9菌株水平特异性探针（Probe-SP）。在使用15株鼠李糖乳杆菌菌株和15株其它益生菌验证这些特异性探针后,通过荧光原位杂交（FISH）验证鼠李糖乳杆菌Probio-M9菌株的特异性探针在体外和体内的适用性。结合荧光D-氨基酸（FDAA）探针,在大鼠的肠道中检测到鼠李糖乳杆菌Probio-M9的活细胞。本研究设计的FISH探针可从肠道菌群中特异性鉴定鼠李糖乳杆菌Probio-M9,该方法可与FDAA探针结合使用,以进一步探索该益生菌菌株在宿主肠道中的定位。     

速冻水饺细菌总数FISH检测技术的优化

探索荧光原位杂交(FISH)技术检测速冻水饺中细菌总数的可行性及特异性,优化样品的快速、准确和便捷的检测体系。采用TAMRA标记的EUB338探针与大肠杆菌进行FISH实验条件的优化,并利用优化的FISH技术对4种速冻水饺中的8个样品进行检测和计数。应用FISH技术检测速冻水饺中细菌总数,理想的样品预处理实验条件为:热固定2 h,4%多聚甲醛固定20 min,系列乙醇脱水5 min,杂交条件为杂交温度为46℃,杂交液中去离子甲酰胺浓度为20%,杂交时间3h,洗脱液中NaCl浓度为225 mmol/L;检测的8个样品中,有2例细菌总数超标。FISH可以特异性的检出速冻水饺中的细菌总数,简便,检测周期短,灵敏度高,为保障食品安全提供更多的理论支持。     

地衣芽孢杆菌MYS68的鉴定及发酵培养基优化

对筛选出的MYS68菌株进行了16SrDNA序列分析鉴定,确定该菌为地衣芽孢杆菌。为了得到地衣芽孢杆菌MYS68发酵培养基的最佳配方组分,试验采用单因素试验和正交试验优化其摇瓶发酵培养基。结果表明,碳源和氮源对地衣芽孢杆菌发酵影响的显著性次序为:玉米粉豆饼粉蛋白胨酵母膏。发酵培养基的最佳组分和配比为:玉米粉0.5%,蛋白胨1.0%,豆饼粉0.3%,酵母膏0.5%,硫酸锰0.001%,磷酸二氢钾0.1%,硫酸镁0.02%,起始pH值为7.5。此条件下发酵液的菌体浓度可达2.11×109 CFU/ml,经检测芽孢率为96%。说明地衣芽胞杆菌发酵培养基的最佳配比可在一定程度上提高地衣芽孢杆菌发酵液的菌体浓度和芽孢率。     

基于荧光原位杂交法剖析窖泥微生物的预处理条件探究及应用

该研究首先对浓香型白酒窖泥样品的料液比、离心转速、超声波分散时间、Al2（SO4）3添加量等预处理条件进行优化;然后将纯培养菌体添加到窖泥样品,采用荧光原位杂交（FISH）法检测菌体回收率;最后按最优预处理条件处理不同窖龄窖池窖泥,并采用FISH法研究窖泥样品中的微生物。结果表明,窖泥样品的最优预处理条件为料液比1∶16（g∶mL）、离心转速800 r/min、超声分散时间60 s、Al2（SO4）3添加量为4.17×10-5 mol/g窖泥。在此条件下,FISH法检测大肠杆菌（Escherichia coli）、植物乳杆菌（Lactobacillus plantarum）的菌体回收率分别为62.50%、55.44%,表明优化后的预处理条件对于窖泥微生物的FISH检测是可行的。FISH法检测窖泥微生物,结果显示随着窖池窖龄的增加,窖泥样品中细菌、古菌及菌体总数差异明显,总体呈现出先增加后降低的趋势,在100年窖龄的窖泥样品中,微生物总量最高。     

火腿肠细菌总数FISH检测技术的优化

为了建立快速的火腿肠中细菌检测和计数方法,本文采用细菌通用探针EUB338作为标记,优化了荧光原位杂交(FISH)技术检测火腿肠中细菌的方法。比较了三种细胞固定方法,结果表明最佳的样品预处理方法为:4℃4%多聚甲醛固定15 min,46℃热固定2 h;最佳的杂交条件:杂交温度为46℃,杂交时间为3 h,杂交洗脱液浓度为225 mmol/L;优化的FISH方法应用于火腿肠样品的总菌数的检测,并且将FISH方法对火腿肠样品的细菌计数与传统平板计数方法进行比较,结果显示FISH方法相较于平板计数方法检出数高,而且所需时间大大缩短,操作起来更加方便、简捷。本实验充分体现了FISH快速定量的优势,FISH可以作为食品微生物快速检测的一种有效工具。     

地衣芽孢杆菌BL-5芽孢生成条件优化

地衣芽孢杆菌（Bacillus licheniformis）作为微生态制剂在食品或饲料工业中具有广泛的应用前景。通过温度对地衣芽孢杆菌BL-5致死率的影响研究得出无芽孢菌体的致死温度为75 ℃。以芽孢率和芽孢数为评价指标,得到地衣芽孢杆菌BL-5芽孢生成的最佳培养基配方为：蔗糖29.85 g/L、硫酸铵10.00 g/L、酵母膏3.00 g/L、磷酸氢二钾3.42 g/L、硫酸镁1.5 g/L、pH 8.0。最佳培养条件为：接种量5%、发酵时间48 h,发酵温度30 ℃、转速150 r/min。     

脉冲强光对枯草芽孢杆菌NG-2灭活机理

采用脉冲强光对枯草芽孢杆菌NG-2及芽孢进行灭活并研究其机理。在脉冲强光辐照枯草芽孢杆菌NG-2菌体及其芽孢后,利用扫描电子显微镜和透射电子显微镜观察形态特征变化,利用紫外分光光度法测定细胞内物质含量,利用荧光光谱法检测细胞内Ca~(2+)浓度和非特异性酯酶活力。结果表明,辐照距离为5 cm,闪照频率为5次/s,辐照15 s时,脉冲强光对NG-2菌体及其芽孢灭活率超过50%;辐照40 s时,脉冲强光对NG-2菌体及其芽孢灭活率超过99.98%。辐照枯草芽孢杆菌及芽孢后菌体结构损伤,芽孢壁破裂,细胞内核酸、蛋白质、Ca~(2+)和2,6-吡啶二羧酸含量显著减少,非特异性酯酶活力降低,研究结果为脉冲强光对枯草芽孢杆菌及芽孢灭活机理提供理论依据。     

荧光原位杂交(FISH)技术研究窖泥微生物群落

为了探索荧光原位杂交(FISH)技术,研究窖泥微生物群落结构特征多样性,进行了纯培养微生物及窖泥微生物的FISH检测。以Escherichia coli、Bacillus subtilis、Lactobacillus planetarium、Acetobacter rancens、Clostridiumacetobytylicum 5株不同种属的细菌为对象,研究了影响荧光原位杂交(FISH)技术对其定量表征的因素,并将该技术应用于窖泥微生物群落的研究。实验结果表明,E.coli及L.planetarium的定量表征受到生长期的影响,在对数和稳定期的菌体检出率>82%,衰退期的L.planetarium则明显减少;死菌体比活菌体的检出信号显著减弱;经硫酸铝处理,除去腐植酸的窖泥样品明显地提高了可辨力,加入窖泥中的E.coli,FISH检出量为光学显微镜计数量的46.89%。采用FISH技术可视化定量表征了窖泥中细菌和古菌的特征,有益于从细胞水平研究其微生物群落的关系。     

介质阻挡放电等离子体对不同pH值生长的酸土脂环酸芽孢杆菌的灭活作用

该研究探讨了介质阻挡放电等离子体对不同pH值（3.2、3.6、4.0和4.4）下生长的酸土脂环酸芽孢杆菌的杀灭作用。研究发现等离子体对最适生长pH值4.0下的酸土脂环酸芽孢杆菌杀灭效率最低,pH值升高（4.4）或降低（3.2、3.6）均引起致死率增加（P<0.05）。例如30 kV处理2 min条件下,等离子体对pH值为4.0生长的酸土脂环酸芽孢杆菌致死率为4.8个对数,对pH值为3.2、3.6和4.4生长的菌体致死率分别为5.9、5.4和5.1个对数。等离子体对不同pH值培养的酸土脂环酸芽孢杆菌致死率的差异可能与菌体形态和膜脂质成分有关。扫描电镜观察发现,pH值3.2和3.6培养的菌体出现异常伸长形态,且酸土脂环酸芽孢杆菌在pH值3.2条件下更为明显,更易被等离子损伤而导致死亡。气相-质谱联用结果表明相比于最适pH值4.0,pH值4.4生长条件下环己烷十一酸相对含量显著提高（67.86%）,而环戊烷十三酸降低（22.21%）（P<0.05）,使得等离子体产生的氧自由基等成分在细胞膜中的堆积,而导致菌体更易被杀灭。该研究表明菌体耐受性增加与酸土脂环酸芽孢杆菌细胞膜脂肪酸成分和形态变化存在一定的相关性。     

Improved detection of Salmonella spp. in foods by fluorescent in situ hybridization with 23S rRNA probes: a comparison with conventional culture methods

This report describes a new technique for the detection and identification of Salmonella species in food with the use of fluorescent in situ hybridization (FISH) with 23S rRNA-targeted oligonucleotide probes. Two species-specific 23S rRNA-targeted oligonucleotide probes (Sal-1 and Sal-3) were selected, and one (Sal-544) was newly designed. The relative specificities of these probes were compared with those of bacterial 23S rRNA sequences from the GenBank database and tested by in situ hybridization with bacterial cell smears of pure cultures. Fifty-one tested reference strains of Salmonella serovars belonging to subspecies I (enterica) hybridized with these probes. No cross-reactions with 46 other strains of the family Enterobacteriaceae or with another 14 bacterial strains from other families were observed. Storage of a Salmonella Panama test strain under various environmental conditions (2, 5, and 15% NaC1; -20 degrees C, 4 degrees C, and room temperature; pHs of 3.3 to 7.4) did not adversely affect the FISH method. No matrix effects were observed with 18 different kinds of foods. FISH was able to detect Salmonella spp. in 52 (probe Sal-1), 56 (probe Sal-3), and 35 (probe Sal-544) of 225 naturally contaminated food samples after 16 h of incubation in a preenrichment broth. When conventional culture and detection methods were used, Salmonella could be isolated from only 30 of these 225 samples. In contrast, FISH failed to identify Salmonella in only two of the culture-positive samples when Sal-1 and Sal-3 were used and in only three of the culture-positive samples when Sal-544 was used.     

肽核酸荧光原位杂交技术快速检测食品中的李斯特菌属及单增李斯特菌

目的建立利用肽核酸荧光原位杂交技术(PNA-FISH)快速检测食品中李斯特菌属及单增李斯特菌的方法。方法针对李斯特菌属、单增李斯特菌分别设计合成2份PNA探针lis-16S-1、lm-16S-2,并建立荧光原位杂交技术,优化杂交条件,对选取的13株李斯特菌和其他9株非李斯特菌进行检测,验证探针的特异性和灵敏度,并对118份食品样品用LB肉汤2次增菌培养后进行PNA-FISH检测。结果探针灵敏度和特异性均为100%,从118份食品中检出14株李斯特菌和8株单增李斯特菌,结果与API方法和VITEK方法鉴定结果一致。结论 PNA-FISH方法可靠易行,对从食品中检测致病性单增李斯特菌有较高的实用性。     

Development and application of oligonucleotide probes for in situ detection of thermotolerant Campylobacter in chicken faecal and liver samples

Based on Campylobacter 16S- and 23S-rRNA sequence data oligonucleotide probes specific for thermotolerant campylobacters and for members of the genus Campylobacter have been developed. The 16S-rRNA-targeted probe CAMP 653, recommended for a comprehensive detection of members of the genus Campylobacter, specifically detected all Campylobacter strains used in this study. Detection of thermotolerant species has been achieved by the 23S-rRNA-targeted probe CAJECO1427. Optimal hybridisation conditions have been derived for both probes from melting profiles of fluorescence-labelled probe-target hybrids recorded in fluorescence in situ hybridisation experiments (FISH). The FISH assay was evaluated both by spiking poultry faecal samples with Campylobacter jejuni and by detecting Campylobacter in naturally colonized chickens. C. jejuni was reliably detected at levels of 10(6) cfu/g faeces after a 3- h enrichment step in Blood Preston Selective broth. Low level contaminations (相似文献   

Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora

In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures.     

Optimization of conditions for profiling bacterial populations in food by culture-independent methods

In this study we used culture-independent methods to profile bacterial populations in food products. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were employed in order to identify bacterial species without the need of isolation and biochemical identification. The protocols used to extract the DNA, subsequently subjected to PCR amplification for DGGE, as well as the hybridization procedure for FISH, were optimised. Moreover, an extensive study on the primers and probes to be used for the direct detection and identification of microorganisms commonly found in food, was carried out. Meat and cheese samples, fresh or processed, were subjected to DGGE and FISH analysis and the results obtained highlighted how the processing in food industry is decreasing the bacterial biodiversity. Not only processed cheese or meat but also fermented products were dominated by only one or few species. Lactobacillus sakei, Lactobacillus curvatus and Brochothrix thermosphacta were the main species found in meat products, while in cheese(s) Lactococcus lactis, Streptococcus thermophilus and Leuconostoc spp. were repeatedly detected. The results obtained by the two culture-independent methods used always correlated well.     

地衣芽孢杆菌分批培养降解氯氰菊酯动力学研究

分析10 L发酵罐中地衣芽孢杆菌降解氯氰菊酯的分批培养动力学。以装料系数0.65、体积分数6.25%的接种量、初始pH 7.5、培养温度37 ℃、转速300 r/min为发酵条件,探讨地衣芽孢杆菌B-1分批培养过程中菌体生长、氯氰菊酯降解酯酶合成、底物消耗及氯氰菊酯降解的规律。在Logistic方程、Luedeking-Piret方程、Luedeking-Piret-Like方程及动力学一级模型的基础上进行非线性拟合,建立了菌株B-1菌体生长、氯氰菊酯降解酯酶生成、基质--葡萄糖消耗和氯氰菊酯降解动力学模型。将实验实测值与模型理论值进行比较,结果表明模型能较好地反映菌株分批培养过程的动力学特征。