健康仔猪肠道乳杆菌黑龙江地方株的鉴定与种属分析

为了从健康仔猪肠道中分离筛选用于研制微生态制剂的乳酸杆菌菌株,选择黑龙江省大庆、哈尔滨及宝泉岭地区部分猪场,采集12-60日龄健康仔猪肠道粪样64份。选用MRS乳酸杆菌专用培养基分离培养乳酸杆菌,通过分离株的形态特征和培养特性筛选出革兰阳性,厌氧,无芽胞杆菌48株。再通过生化试验鉴定和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。     

应用特异PCR快速鉴定微生物肥料中4种乳酸菌

【目的】植物乳杆菌(Lactobacillus plantarum)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和德氏乳杆菌(L.delbrueckii)是微生物肥料生产中常用的乳酸菌,它们表型特征相似,若采用传统方法鉴定则费时费力,为准确、快速地鉴定这些种,建立种特异PCR方法。【方法】利用NCBI中Primer-BLAST(引物设计和特异性检验工具),以GenBank数据库中上述菌种的recA和gyrB为靶基因,设计和筛选种特异性引物从而建立相应特异PCR鉴定方法。【结果】经过乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、片球菌属(Pediococcus)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)和假单胞菌属(Pseudomonas)7个属24个种共40株标准菌株的实验验证,4个目标种分别扩增出唯一的目的产物,而其他种均无目的扩增产物。采用建立的4种特异PCR方法对产品中分离的16株乳杆菌进行鉴定,结果与16S rDNA序列分析、Biolog鉴定结果一致。【结论】建立的特异PCR鉴定方法均具有较高的种内通用性和种间特异性,可快速、准确的用于微生物制剂中植物乳杆菌、德氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌的检测和鉴定,具有较好的应用前景。     

体外拮抗幽门螺杆菌的人嗜酸乳杆菌菌株的选育

目的 探讨人嗜酸乳杆菌对幽门螺杆菌（Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ,ＨＰ）毒力株的体外拮抗作用,筛选出对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌菌株。方法 从健康人胃肠道中分离出５２株嗜酸乳杆菌可疑株,通过其培养特性,生理特性,生化反应及代谢产物测定等进行鉴定,获得２６株嗜酸乳杆菌。同时,从临床患者胃活检标本中分离出２３株ＨＰ菌株,用ＰＣＲ方法筛选出ｃａｇＡ阳性ＨＰ毒力株,然后,采用打孔法进行嗜酸乳杆菌培养上清拮抗ＨＰ毒力株的实验,以１％的乳酸作对照。结果 筛选出４株对ＨＰ毒力株有明显拮抗作用的嗜酸乳杆菌,这种拮抗作用不依赖嗜酸乳杆菌分泌的乳酸。结论 人嗜酸乳杆菌在体外对ＨＰ毒力株具有明显拮抗作用。该研究为应用微生态疗法治疗ＨＰ感染提供了理论基础。     

一株嗜酸乳杆菌的分离与鉴定

桂林市黄洛瑶寨地区人群在洗发过程中长期使用淘米水发酵液,该地区发质和头发根部头皮质量都要优于其他地区,淘米水发酵液中益生菌对头发和头皮的影响值得关注。为了研究该地区淘米水发酵液中益生菌,对淘米水发酵液进行菌株的分离与鉴定。从淘米水发酵液中分离得到3株嗜酸乳杆菌疑似菌株,通过菌株菌落形态、显微形态、革兰氏染色、46项生理生化指标和分子生物学方法进行鉴定,结果表明3号菌株菌落形态、显微形态、革兰氏染色符合嗜酸乳杆菌特征;46项生理生化指标与嗜酸乳杆菌接近,鉴定可信度达到90%;经过16S rDNA鉴定后,其与嗜酸乳杆菌的同源性高达99%。综合多项检测结果确定其为嗜酸乳杆菌,并且命名为CFC-001。     

嗜酸乳杆菌YIT2004株的抗生素敏感性 及对万古霉素耐药机制

摘要：目的 研究嗜酸乳杆菌YIT2004株的抗生素敏感性及其耐药机制。方法 最低抑菌浓度法检测嗜酸乳杆菌YIT2004株对抗生素的敏感性;提取YIT2004株基因组,用特异性引物对耐药基因进行PCR扩增。结果 嗜酸乳杆菌YIT2004株对青霉素、氨苄西林、亚胺培南、庆大霉素、红霉素和克林霉素敏感,对万古霉素耐药;YIT2004株基因组中不存在vanA、vanB耐药基因,但存在高度相似的aad、ddl万古霉素耐药基因。该耐药为固有耐药,不具备传递性。结论 嗜酸乳杆菌YIT2004株对青霉素、氨苄西林、亚胺培南、庆大霉素、红霉素和克林霉素敏感,对万古霉素固有耐药且其耐药性不可传递。嗜酸乳杆菌YIT2004株的使用具有安全性。     

猪源肠道乳酸菌的分离与生物学特性研究

目的分离猪消化道中的乳酸菌。方法无菌采集健康猪的新鲜粪便,接种MRS培养基,厌氧培养分离乳酸菌。并对分离菌形态学,生化特性,产酸性,耐酸性,耐胆汁性,高温耐受性,抑菌性等方面进行研究。结果分离到5株嗜酸乳杆菌,分离的细菌对2株致病菌明显具有抑制性。结论为研制高效专一的猪用微生态制剂奠定基础。     

复合PCR鉴定胸膜肺炎放线杆菌方法的建立及初步应用

根据胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,App)apxIVA毒素基因序列和16SrRNA序列分别设计了一对特异性引物P1P4和一对通用引物S7S10,建立了检测App全部15个血清型的复合PCR方法。对App的15个血清型国际参考株和国内的11个App菌株进行检测,都能得到363bp和692bp的两个扩增片段。而放线杆菌等13株参考菌株只能得到692bp的扩增片段。该方法能将15个血清型的App菌株鉴定到种。检测的灵敏度达9pgDNA1300CFU。用建立的方法检测临床分离的302株可疑菌株,阳性4株,与其它鉴定方法相符。结果表明复合PCR可用于App菌株的鉴定。     

双歧杆菌四联活菌片中嗜酸乳杆菌的分离及生长动力学研究

目的以双歧杆菌四联活菌片为实验材料,利用酸化的MRS培养基筛选分离得到嗜酸乳杆菌,对其进行进一步的生长动力学研究,确定嗜酸乳杆菌的生长数学模型。方法通过浓度梯度稀释法利用改良MRS培养基对双歧杆菌四联活菌片中的嗜酸乳杆菌进行分离,利用分光光度仪和平板菌落计数两种不同的方法测定嗜酸乳杆菌在发酵过程中不同发酵时间的细胞浓度的动态变化,经软件处理后拟合出嗜酸乳杆菌细胞生长的Logistic数学模型。结果Logistic方程能很好地拟合嗜酸乳杆菌细胞生长的动态变化,并得到嗜酸乳杆菌在本实验条件下的数学模型,为进一步研究、利用嗜酸乳杆菌生长能力、产酸能力和产香能力等具有重要的理论指导意义。     

嗜酸乳杆菌NCFM对Caco-2细胞基因表达谱的影响

摘要：【目的】嗜酸乳杆菌NCFM作为一株具有良好益生功能的模式菌株,采用基因芯片技术对其作用后的宿主细胞基因的变化情况进行分析,在生物、食品等领域具有较大研究价值。【方法】将嗜酸乳杆菌NCFM和Caco-2细胞共同培养2h,提取Caco-2细胞的总RNA,并将RNA反转录成cDNA,与Human Genome U133 Plus 2.0 Array基因表达谱芯片杂交,杂交后进行图像扫描和数据分析,并采用Real-time RT PCR方法对差异表达的基因进行验证。【结果】采用基因芯片方法检测了Caco-2细胞经嗜酸乳杆菌NCFM作用2h后的基因表达变化,发现差异表达基因为508个,其中有473个基因上调,35个基因下调,初步推测Caco-2细胞能诱导多个基因的表达,以发挥嗜酸乳杆菌NCFM的益生功能。并且经Real-time RT PCR验证,表达差异显著的3个免疫调节相关基因CCL2、PTX3和TNFRSF9的确在嗜酸乳杆菌NCFM作用期间高表达。【结论】以上这些结果促进了对嗜酸乳杆菌NCFM益生功能的认识,也为揭示该乳酸菌的作用机理提供了理论基础。     

突发疫情中炭疽芽孢杆菌的分离及鉴定

对疑似炭疽感染病牛牛肉标本和牛血污染土壤标本进行了病原菌分离,经菌落形态和菌体形态观察、血清学实验和生化鉴定,证明分离到的细菌为炭疽芽孢杆菌。为进一步了解其特性,分别用保护性抗原、水肿因子和荚膜基因特异性引物对2株菌进行PCR扩增。结果显示,这两株菌有两个毒力相关质粒pX01和pX02,为有毒株。序列测定表明,这两株菌基因间同源性达99%,这两株菌与GenBank中炭疽芽孢杆菌A2012株、Ames Ancestor株和A16R疫苗株同源性达99%。     

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri

Abstract Two strains of Lactobacillus acidophilus Group A1, the neotype ATCC 4356 and a human isolate NCFM-N2, widely used as a dietary adjunct in milk and cultured dairy products, were transformed with plasmid DNA by electroporation. The transformation characteristics exhibited by the two L. acidophilus strains were found to differ markedly even though they appeared similar at the genomic level based on the DNA patterns of Sma I restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the A1 DNA homology group.     

Detection of plasmid deoxyribonucleic acid in an isolate of Lactobacillus acidophilus.

Eight strains of Lactobacillus acidophilus were examined for the presence of plasmid deoxyribonucleic acid, and one, a pig intestinal isolate, showed the presence of a 13.7- and a 6.3-megadalton plasmid. This is the first reported evidence for plasmid deoxyribonucleic acid in Lactobacillus acidophilus. The functions of these plasmids are presently unknown.     

Factors involved in adherence of lactobacilli to human Caco-2 cells.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.     

嗜酸乳杆菌黏附鸡胚肠黏膜上皮细胞能力的研究

目的建立肠黏膜上皮细胞模型和测定嗜酸乳杆菌的黏附能力。方法将嗜酸乳杆菌用胃蛋白酶和HCl-H2O低pH以及反复冻融、灭活等方法处理后测定其黏附能力。结果成功地建立了鸡胚肠黏膜上皮细胞模型;经胃蛋白酶和HCl-H2O低pH以及灭活处理后,嗜酸乳杆菌的黏附能力和对照组差异有显著性。反复冻融后的嗜酸乳杆菌黏附能力与对照组差异无显著性。结论胃蛋白酶和HCl-H2O pH2.0会使嗜酸乳杆菌黏附肠黏膜上皮细胞能力下降,热灭活可使嗜酸乳杆菌的黏附能力提高,反复冻融对嗜酸乳杆菌的黏附能力无明显影响。     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

嗜酸乳杆菌GIM1.208 β-葡萄糖苷酶的异源表达、纯化及酶学性质研究

嗜酸乳杆菌(Lactobacillus acidophilus)是益生菌,前期研究发现Lactobacillus acidophilus GIM1.208所产生的β-葡萄糖苷酶(BGL)具有较高活性,为探明其结构与特性,本研究采用PCR体外扩增技术获得Lactobacillus acidophilus GIM1.208 BGL的目的基因,并在大肠杆菌中成功表达,经镍亲和层析等蛋白质纯化技术获得纯度达90%以上的蛋白质样品,进而通过圆二色光谱法(CD)和同源建模法对BGL的二级结构和三级结构进行测定分析并研究其酶学性质.结果表明,Lactobacillus acidophilus BGL的分子质量为52 ku,纯化后浓度为1.88 g/L,二级结构包括15.9%α螺旋、44.1%β折叠、18.1%β转角、27.2%无规则卷曲,其三维结构显示,该蛋白质有8个β折叠和8个α螺旋,整体呈圆锥形.Lactobacillus acidophilus BGL具有良好的葡萄糖耐受性与NaCl耐受性,最适反应温度为47℃,最适反应pH为5.6,在20℃~50℃范围内和pH2.2~6.0间有较高的稳定性,且乙酸乙酯、甲醇对酶活性抑制作用明显,Fe³⁺、Fe²⁺对酶有显著激活作用.上述研究结果为Lactobacillus acidophilus BGL后续功能发掘和应用研究提供了重要参考依据.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.