从虎皮鹦鹉粪便中分离产细菌素菌株及其细菌素特性分析

从虎皮鹦鹉(Melopsittacus undulatus)的粪便中分离得到一株产广谱细菌素的菌株,其细菌素产物对革兰氏阳性和阴性细菌均有较强抑制作用。经形态学、生理生化反应和16S rDNA分子方法鉴定,该菌株初步鉴定为干酪乳杆菌。用硫酸铵分级沉淀得到的细菌素粗提物,作温度、pH值、蛋白酶的敏感性测定,结果显示该细菌素经100℃处理10 min抑菌活性不变,当pH4.0时,对大肠杆菌和金黄色葡萄球菌的抑菌率分别达到99.06%和86.14%,并且对多种蛋白酶表现出敏感性。与Nisin标准品的抑菌效价对比,得到粗提物的效价为204.87 IU/mL。     

乳酸片球菌素（Pediocin）效价测定的条件优化

目前的乳酸片球菌素（Pediocin）效价测定方法误差大、耗时较长。为了减少测定误差并缩短测定时间,研究并优化了乳酸片球菌素（Pediocin）效价测定中的几个关键因素,得到较优的实验条件：发酵液加热时间30min、菌体吸附2h、解吸附3h、指示菌加入量为400μL（30mL固体检测培养基中含菌2．1×10^8个）、试样pH为2．0时,测定的Pediocin效价误差较小;试样处理时间由12h缩短为约5h。     

产类细菌素乳酸乳球菌V528的紫外线诱变育种研究

目的获取高产抑制鸡白痢沙门菌生长的类细菌素乳酸乳球菌的突变菌株。方法在紫外线照射致死率为82.7%的剂量下,对处于对数生长后期的乳酸乳球菌V528进行紫外线二次复合诱变处理。分别随机对第一次和第二次复合处理后生长的117株和310株菌进行效价测定分析。结果在实验所用的照射剂量下,抑菌效价提高的正突变菌株占有一定优势,负突变菌株较少,其中突变菌株Lact.174的抑菌效价较V528的效价提高了8.48倍,并具有一定的遗传稳定性。结论运用紫外诱变育种技术可有效地获得高产类细菌素的突变菌株。     

植物乳杆菌R260产细菌素发酵条件的研究

目的 获取植物乳杆菌R260产细菌素的最佳发酵条件.方法用琼脂扩散法测定发酵液对苏云金芽胞杆菌的抑菌效价.结果 产细菌素的最佳培养基是MRS培养基,最适起始Ph为6.5,最适接种量和接种种龄分别为3%和12 h,产细菌素最适发酵温度和时间分别为30℃和20 h：细菌素在对数期开始产生,稳定期产量达到最大值.结论 通过优化发酵条件提高了细菌素的产量,达1656 IU/ml.     

不同提取剂对粗细菌素提取效果的影响

摸索并建立了粗提细菌素的方法。从粗提细菌素的能力和活性保留两个方面,比较了酸沉淀法和硫酸铵沉淀法的优劣。结果发现,相对于硫酸铵沉淀法,酸沉淀法能够获得更多的粗细菌素,蛋白总量提高了28.7%。且酸沉淀法得到的粗细菌素的比活性比硫酸铵沉淀法提高了55.5%,单位效价是硫酸铵沉淀法的2倍。比较了不同有机溶剂抽提以上沉淀物,综合考虑细菌素得率及活性,发现三氯甲烷的抽提效果较好。     

血链球菌细菌素抑菌活性研究

检测分离纯化的血链球菌细菌素对牙周可疑致病菌的抑制作用。通过羟基磷灰石(HA)柱层析、SephadexG-150凝胶柱层析、中空纤维柱超滤脱盐、浓缩纯化提取血链球菌细菌素,以具核梭杆菌为指示菌,洞平板法检测细菌素的抑菌活性。经HA柱层析得到4个相互分离的组分,经洞平板法检测,第Ⅱ峰的蛋白具有抑菌活性,冻干后得纯化后的细菌素,终产率为0.082%;1mg/mL的细菌素溶液可形成17mm的抑菌环,最小抑菌浓度为62.5μg/mL。血链球菌细菌素对牙周可疑致病菌具有较强的拮抗作用。     

枯草芽胞杆菌FB123细菌素的理化性质及抑菌谱的研究

研究了细菌素产生菌枯草芽胞杆菌(Bacillus subtilis)FB123 所产细菌素的理化性质及其抑菌谱.枯草芽胞杆菌FB123经过 28 ℃、32 h的发酵得到发酵上清液,用饱和度为50%的硫酸铵溶液沉淀发酵上清液中的细菌素.以革兰阴性菌大肠杆菌和革兰阳性菌金黄色葡萄球菌为指示菌,采用牛津杯法检测细菌素抑菌活性,对细菌素粗品进行理化性质的研究.结果表明该细菌素最适作用pH为 6.0,最适作用温度 40 ℃,具有较宽的pH作用范围和较好的热稳定性.各种蛋白酶、金属离子对其活性有不同程度的影响.抑菌谱试验结果表明,该细菌素对多种革兰阳性菌和革兰阴性菌有明显的抑制作用,虽然对部分真菌有抑制作用,但抑制作用较弱.     

海绵细菌B25W最佳产抗培养基及发酵条件的研究

从辽宁海域的繁茂膜海绵(Hymeniacidonperleve)中分离到1株解淀粉类芽孢杆菌(Paenibacillusamyloliquefaciens),定名为海绵细菌B25W。它产生的抗生素用白色念珠菌(Candidaalbicans)为指示菌,通过单因子和均匀设计实验,发现菌株最佳产抗摇瓶发酵培养基:玉米面0.34%,豆饼粉1.66%,葡萄糖0.1%,ZnSO40.04%,MgSO40.02%,KH2PO40.04%;发酵条件:种龄12h,接种量10%,发酵时间20h,初始pH6.0,250mL三角瓶内含25mL培养基,往复式摇床,116次/min,振幅11cm,培养温度28℃。应用二剂量法(以制霉菌素为标样)测定B25W抗生素的相对效价为13680u/mL,比优化前效价(8444u/mL)提高了62%。     

群体感应信号分子及其抑制剂快速检测方法的建立

细菌能自发产生、释放一些特定的信号分子,并能感知其浓度变化,调节微生物的群体行为,这一调控系统称为群体感应。细菌群体感应参与包括人类、动植物病原菌致病力在内的多种生物学功能的调节,群体感应抑制剂成为抗感染药物开发的靶点。利用紫色色杆菌(Chromobacterium violaceum)和根癌农杆菌(Agrobacterium tumefaciens)作为指示菌,建立检测高丝氨酸内酯(AHLs)及其抑制剂的简便方法。结果表明,通过平板交叉划线接种,使用指示菌能够有效地检测AHLs,并且通过薄层层析(TLC)与细菌生物感应器相结合的方法可以快速、方便地鉴定AHLs的种类;通过双层平板法观察指示菌色素产生情况,能够有效地检测群体感应信号分子AHLs抑制剂,且该方法简单易行。     

应用指示菌株测定鑽井时泥浆渗入岩心的深度

1．采用一株能够在原油及泥浆中存活的Serratia marcescens作为指示菌,测定泥浆污染岩心的深度。茌钻井过程中加入带指示菌的泥浆法,此把它套入取心筒中的方法有效。 2,细菌渗入岩心的深度决定于岩心的岩性、胶结度、裂隙度以及机械破坏的程度,而与取心深度无关。 3．有裂隙、破碎和松散的岩石不适于微生物学的分析和某些地质项目的分析。 4．对于完整的岩心而言,指示菌侵入岩心的深度波动在0.2—2.5厘米的范围内,除去岩心外层2.5—3.0厘米即可得到合乎要求的岩样。 5．叙述了钻井时采用少量带指示菌的泥浆测定泥浆污染岩心深度的方法。     

嗜酸乳杆菌SR-1产类细菌素发酵条件及生物学特性的研究

本研究通过正交试验获得嗜酸乳杆菌sR-1分泌类细菌素的最适培养条件,即葡萄搪2％,大豆蛋白胨2．5％,酵母粉0．4％,血浆蛋白2．5％。并首次利用流加培养的方式,改进了类细菌素产生菌的发酵条件,抑菌直径从19mm提高到25mm,同时对类细菌素生物学特性进行了初步的探索。结果表明：嗜酸乳杆菌产生的类细菌素对多种蛋白酶不敏感,在低pH下能抑制革兰阴性、阳性的致病菌,在pH6．5下吸附菌体作用最强。     

Note: Purification and characterization of the bacteriocin PsVP-10 produced by Pseudomonas sp.

A bacteriocin (bacteriocin PsVP-10) produced by Pseudomonas sp. R-10 was purified by a simple method that included an extraction of the bacteriocin with chloroform, followed by cation exchange chromatography. The purity of the bacteriocin was verified by RP-HPLC. It is a peptide of 2·4 kDa, very stable to heat, to proteolytic enzymes and to pH. It presents a very broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria.     

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.     

Use of a bacteriocin produced by Pediococcus acidilactici to inhibit Listeria monocytogenes associated with fresh meat.

A bacteriocin produced by Pediococcus acidilactici had an inhibitory and bactericidal effect on Listeria monocytogenes associated with fresh meat. MICs were significantly lower than minimum killing concentrations. When meat was inoculated with L. monocytogenes, the bacteriocin reduced the number of attached bacteria in 2 min by 0.5 to 2.2 log cycles depending upon bacteriocin concentration. Meat treated initially with the bacteriocin resulted in attachment of 1.0 to 2.5 log cycles fewer bacteria than that attained with the control. The bacteriocin, after 28 days of refrigerated storage on meat surfaces, was stable and exhibited an inhibitory effect on L. monocytogenes.     

Luminescent method for the detection of antibacterial activities

A new rapid and sensitive method for the detection of antibacterial activities was based on luminescent indicator strains. Listeria innocua 8811 and Enterococcus faecalis 32 were transformed with plasmid carrying bacterial luciferase genes. Subsequent strains became capable to emit light during the exponential growth phase. The addition of bacteriocin containing culture supernatants to such cultures induced a drop of their light emission which was correlated to the combined antibacterial activity of acid stress and bacteriocin. The detection of antagonistic activity is independent of its mode of action, i.e. bactericidal or bacteriostatic. This method allowed to directly visualize the antagonistic activity of bacteriocin producer strains toward target strains in coculture experiments. However, a control co-culture with non-producing bacteriocin mutant was necessary in order to distinguish between nutrients competition and bacteriocin activity. Finally, five class IIa bacteriocins were purified from culture supernatants of eight strains detected in 3 days from a 120 lactic acid bacteria collection.     

Production and Characterization of Nisin-Like Peptide Produced by a Strain of Lactococcus lactis Isolated from Fermented Milk

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other Gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.     

嗜酸乳杆菌产细菌素生物学特性的研究

嗜酸乳杆菌在MRS培养基中培养得到的细菌素经30、60、90、121℃处理20min后,活性几乎不变,对以金黄色葡萄球菌为代表的革兰阳性菌、大肠埃希菌为代表的革兰阴性菌有明显的抑制作用,Tricine—SDS—PAGE和不同pH值抑菌实验测定结果证明,嗜酸乳杆菌细菌素是一组低分子量,并在pH2—4时有抑菌作用的活性物质。     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

Studies on mode of action of a bacteriocin from Clostridium septicum.

A bacteriocin was found in the supernatant fluid of Clostridium septicum strain Ovinus. Sensitivity to the bacteriocin was confined to other strains of C. septicum and to strains of C. chauvoei; the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by the bacteriocin. The bacteriocin killed sensitive cells rapidly but cell lysis did not appear to be involved. The bacteriocin inhibited protein and RNA synthesis immediately after its addition to sensitive cells; DNA synthesis was inhibited 10 min later.     

Mathematical evaluation of plantaricin formation supports an auto-induced production mechanism

The production rate of a bacteriocin, produced by Lactobacillus plantarum TMW1.25 and previously named plantaricin1.25, was studied during pH-constant batch fermentations under various growth media conditions. The growth of L. plantarum and production of bacteriocin during the retardation phase were modelled, using 11 different empirical and mechanistic approaches. The optimal pH for bacteriocin production was 4.5. Among the different nitrogen sources tested, yeast extract was the most important, on the basis of the fact that the maximum growth rate decreased 16% without yeast extract, and only 7.2% or 8.1% without meat extract or peptone respectively. However, the change of nitrogen source did not have a significant effect on bacteriocin production. The progression of plantaricin1.25 production during the retardation phase and growth of L. plantarum TMW1.25 could be described by a structured model in which the bacteriocin concentration induces its own production. Among those models not implementing bacteriocin induction, only the one with an exponential increase of bacteriocin yield per unit biomass was suitable to describe bacteriocin production. Computer-aided evaluation of experimental data appears to be helpful in elucidating the relationship between the growth of lactic acid bacteria and bacteriocin production. Received: 22 May 1998 / Received last revision: 9 November 1998 / Accepted: 14 November 1998