植物乳杆菌CMCC-P0002代谢产物中抑菌物质的研究

目的通过对一株植物乳杆菌代谢产物的抑菌作用以及该产物的某些物理特性的研究,为进一步发现可替代现有抗生素的抗菌物质奠定基础。方法首先利用低温离心和超滤法对植物乳杆菌培养后的上清液进行初步提取,用打孔法行抑菌试验,明确其对铜绿假单胞菌、大肠埃希菌、肺炎克雷伯菌和金黄色葡萄球菌等临床分离菌株的抑菌活性;并进一步经加热、调整pH及过氧化氢酶等处理上清液后,再验证其活性变化。结果植物乳杆菌的培养上清液对铜绿假单胞菌等多株临床分离菌株均具有一定的抑菌活性,且以pH在6以内时该物质抑菌活性较好,过氧化氢酶处理后或加热至100℃、30min,上清液的抑菌活性依然存在。结论在植物乳杆菌的培养上清液中存在着具有抑菌活性的物质,对从临床标本所分离的部分革兰阴性菌及革兰阳性菌有抑菌作用,该物质对热耐受,其活性受pH变化的影响。     

南极适冷菌Pseudomonassp．C抑菌活性物质的分离纯化与结构鉴定北大核心CSCD

本文对南极适冷菌Pseudomonassp．c发酵液上清中的抗植物病原菌活性物质进行了研究,结果表明在温度40-70℃和pH5—9的范围内发酵液上清的抑菌活性稳定。根据活性追踪,通过硅胶柱层析和高效液相色谱对发酵液上清中的抑菌活性物质进行了分离纯化,得到了3个对常见植物病原真菌尖孢镰刀菌具有抑菌活性的化合物,并对其中的两个化合物进行了结构鉴定。化合物1为环二肽类（环（苯丙氨酸-脯氨酸））,化合物2为壬基酚聚氧乙烯醚类（14-壬基苯氧基．3,6,9,12．四氧十四烷-1-醇）,两者对枯草芽孢杆菌也具有一定的抑菌活性。     

产抑菌物质SDLH菌株的鉴定及发酵产物稳定性研究

目的：对1株产抑菌物质的中华稻蝗内生菌SDLH进行鉴定,并对其发酵产物稳定性进行研究。方法：通过观察生长情况及菌落特征形态学、氨基酸利用、糖发酵、脱羧酶反应生理等生化检测以及16S rDNA序列测定对菌株SDLH进行分类鉴定,并在不同条件（温度、pH、光照等）下测定其发酵产物的抑菌稳定性。结果：菌株SDLH符合肠杆菌科细菌的一般特征;生理生化特征均与Serratia marcescens的特征基本一致;菌株SDLH的16S rDNA序列长度为1 457bp。Gene bank序列登录号为EU525929。其序列在1 457bp范围内与已知的模式菌株粘质沙雷氏菌（AB244291.1）16S rDNA序列部分有100%的相似性。其发酵产物在温度（40℃~60℃）下抑菌活性稳定,在高温（≥80℃）下失去活性;在酸性条件（3≤pH〈7）下,抑菌活性无明显变化,在碱性条件（pH≥10）下,抑菌活性下降;光照2~6h后抑菌活性下降。结论：菌株SDLH属于沙雷氏菌属（Serratia）粘质沙雷氏菌（Serratia marces-cens）,其发酵产物具有较强稳定性,可置避光、pH中性、常温环境中短期保存,经提高活性、纯化等处理后可能作为新型天然防腐剂应用于食品领域。     

一株抗MRSA内生细菌的鉴定及其活性物质

根据生理生化特征和16S rDNA序列,对一株从新疆苦豆子Sophora alopecuroides中分离的、对耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)具有较强拮抗作用的内生细菌菌株KDNB6进行鉴定;并通过硅胶柱、凝胶柱层析从该菌发酵产物中分离纯化了抗MRSA的活性物质。结果表明菌株KDNB6各项特征与粘质沙雷氏菌Serratia marcescens最为接近,鉴定为S.marcescens。从菌株KDNB6的代谢产物中分离到一个抗MRSA的活性成分。     

虎奶菇发酵液抑菌成分的理化性质及其分离鉴定

为探明虎奶菇发酵液中的抑菌成分,本文以金黄色葡萄球菌(Staphylococcus aureus)为指示菌,研究了温度、pH、紫外线、蛋白酶K对虎奶菇发酵液抑菌活性的影响;并采用硅胶柱层析及反相中压液相层析追踪分离抑菌活性物质,通过核磁共振并结合相关资料对分离到的单体化合物进行了结构鉴定。结果显示,发酵液抑菌成分对蛋白酶较为稳定,对紫外照射敏感,可在90℃以下、pH1~9的范围内较好的保持其抑菌活性;活性追踪分离得到1个抑菌物质PtrA,经鉴定为2-呋喃甲酸(2-furoic acid)。     

芽孢杆菌dhs-330产脂肽-糖脂混合型表面活性剂的抑菌性能

目的:芽孢杆菌dhs-330分离自海水,能够产生脂肽-糖脂混合型表面活性剂。考察该菌株产物的抑菌性能。方法:采用抑菌圈法考察dhs-330发酵液对不同指示菌的抑菌活性,对提取的dhs-330产物进行最小抑菌浓度(MIC)测定,并考察该产物对温度、盐度和pH值的稳定性。结果:菌株dhs-330产物对嗜水气单胞菌、枯草芽孢杆菌、短小芽孢杆菌和金黄色葡萄球菌有抑菌作用,MIC分别为0.005、0.01、0.01和0.02 g/L;经100℃处理20 min、pH2~10处理30 min,或盐度为0~50的条件下,产物仍具有抑菌活性或略有下降。结论:海洋来源的芽孢杆菌dhs-330胞外脂肽-糖脂混合型产物具有抑菌活性,且在高温、低pH值和高盐度条件下具有稳定性。     

海洋短小枯草芽胞杆菌的鉴定及抑菌活性分析

对从连云港东西连岛海泥样品中分离得到的菌株Bacillus pumilus HX2-2的分类地位、生长条件和抑菌活性进行了研究。经过形态特征、生理生化性质及16S r DNA序列分析鉴定,该菌属于短小芽胞杆菌。不同温度、盐度、pH培养条件下测定菌液吸光度OD600值,表明该菌是一株轻度嗜盐菌,最适温度、盐度、pH分别为30℃、3%、7.0~8.0。在不同病原真菌的平板抑菌活性试验中,该菌对草莓尖胞镰刀菌、马铃薯炭疽病菌和水稻立枯丝核菌表现出显著的抑菌作用。菌株B.pumilus HX2-2是一株短小芽胞杆菌,具有广谱抑菌活性,具有进一步研究的价值。     

一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性

【目的】为发掘和利用蜂粮中拮抗菌资源,对分离获得的拮抗细菌菌株PC2进行分类鉴定,并测定其发酵液抑菌物质基本特性。【方法】采用改良牛津杯双层平板法测定菌株发酵液抑菌谱及温度、p H、紫外线和蛋白酶对其抑菌活性稳定性的影响,菌株鉴定结合形态学、生理生化特征和16S r RNA基因序列分析,硫酸铵沉淀法和盐酸沉淀有机溶剂提取法进行抑菌活性物质的初步分离。【结果】从3种蜂粮中分离筛选得到17株拮抗菌株,其中1株细菌PC2以马铃薯葡萄糖液体培养基发酵制备的无菌发酵液对7种供试菌株具有较强抑制作用,经形态、生理生化特征及16S r RNA基因序列分析,将其初步鉴定为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。菌株发酵液抑菌活性对温度、酸和紫外线具有较强的稳定性,对蛋白酶K、胃蛋白酶、碱性蛋白酶处理敏感。菌株发酵液存在抑菌蛋白和脂肽类物质。【结论】菌株PC2在食品保鲜和农业生防中具有潜在的开发应用价值。     

内生拮抗放线菌FRo2的鉴定及抑菌活性物质的分离

【目的】鉴定从东乡野生稻根部分离得到的对多种农作物病原真菌具有拮抗活性的内生放线菌株FRo2,并对其抑菌活性物质进行分离。【方法】根据FRo2形态特征观察、生理生化特性、细胞壁组分和16S rRNA基因序列对其进行鉴定。采用管碟法和菌丝生长速率法测定了该菌株的抗菌活性,活性追踪法结合正相硅胶柱层析及凝胶(Sephadex LH-20)柱层析等技术对抑菌组分进行分离,并通过NMR对抑菌活性物质进行解析。【结果】菌株FRo2属于链霉菌属,与娄彻氏链霉菌(Streptomyces rochei)极为相似。该菌株发酵液对小麦赤霉菌、立枯丝核菌等7种主要农作物致病菌具有较好的抑制活性;从菌株FRo2发酵液中分离得到抑菌活性化合物AW2,结构鉴定为邻苯二甲酸二丁酯。【结论】研究阐明了内生放线菌FRo2抑菌活性物质,也为该菌今后的农业生防应用提供物质基础。     

海洋链霉菌GB-2发酵产物的抗细菌活性及性质研究

从连云港海域潮间带采集的样品中筛选得到一株产高活性抗细菌物质的链霉菌GB-2。该菌的发酵产物对蜡样芽孢杆菌AS1.1846、金黄色葡萄球菌ATCC25923及6株耐药性金黄色葡萄球菌等11 株革兰氏阳性菌,大肠杆菌AS1.487、荧光假单胞菌AS1.1802等4 株革兰氏阴性菌有显著拮抗作用。纸层析对抗细菌物质分析结果表明,菌株GB-2所产抗细菌物质是中性的水溶性物质,其产生与海水的存在有显著相关性。发酵液稳定性研究表明,该物质在121℃,pH1 和pH12条件下抑菌活性均不变;紫外线照射也不影响其抑细菌活性。显示菌株GB-2产物在生防、食品及医药方面潜在的应用价值。     

副干酪乳杆菌HD1.7产生抗菌物质的初步研究

对从乳酸菌酸菜发酵液中分离到的能产生抑菌物质的菌株进行了鉴定,并对该菌的发酵产物进行了提取和性质研究。经形态学、生理生化与16SrDNA序列分析鉴定该菌株为副干酪乳杆菌(Lactobacillus paracasei)。抑菌谱试验表明:除酵母菌外,该抑菌物质对革兰氏阳性、阴性菌和多种致病菌均有较强的抑制作用。经有机溶剂萃取法获得了抑菌物质的粗提物,胰蛋白酶水解证明抑菌物质具有蛋白质性质,尿素-SDS-PAGE不连续凝胶电泳法测得抑菌物质的分子量为14000左右。     

疮痂链霉菌拮抗菌株BU396的分离鉴定与抗菌性质分析

【背景】随着马铃薯种植面积的扩大,疮痂病的发生也日益严重,且化学防治存在环境污染等诸多弊端,因此利用安全和高效的生物防治手段对该病害进行防治成为研究热点。【目的】对疮痂链霉菌(Streptomyces scabies)拮抗菌株进行分离、鉴定和功能基因分析,对抗菌物进行分离及抗菌特性的研究。【方法】从马铃薯疮痂病的病害土壤中分离、筛选并鉴定得到拮抗菌株,采用PCR法对菌株进行抗菌物合成基因检测。通过硫酸铵沉淀法分离得到抗菌物,进行抗菌谱和稳定性检测,并通过盆栽试验进一步验证该菌株的生防效果。【结果】通过抗菌试验筛选得到拮抗细菌BU396,根据形态学、生理生化性质和分子生物学鉴定结果,确定其为贝莱斯芽孢杆菌(Bacillus velezensis)。功能基因分析表明BU396中含有表面活性素等4种抗菌物的合成相关基因。采用硫酸铵沉淀法对其培养液上清进行分离,当硫酸铵的饱和度为75%时,抗菌物在沉淀中析出。抗菌谱试验结果显示该抗菌物对多种动植物病原菌均具有抗菌效果。稳定性试验表明该抗菌物耐热,不易被酶解,对多种金属离子不敏感,具有宽泛的pH活性范围,盆栽试验结果显示菌株BU396处理能够明显降低马铃薯疮痂病的发病率。【结论】分离并鉴定了一株对疮痂链霉菌具有显著拮抗作用的贝莱斯芽孢杆菌BU396。该菌株含有多种抗菌物合成的相关基因,其抗菌物具有广谱的抗菌活性和良好的稳定性,对马铃薯疮痂病的生防效果显著。本研究为马铃薯疮痂病的生物防治及后续抗菌物质的深入研究奠定了基础。     

Characterization of a broad range antimicrobial substance from Bacillus cereus

AIMS: The aim of this research was to isolate and characterize an antimicrobial substance from the Bacillus cereus type strain ATCC 14579. METHODS AND RESULTS: A substance with antimicrobial activity was isolated from B. cereus ATCC 14579. The substance was produced during late exponential growth and well into the stationary phase with a maximum 9 h after inoculation. The inhibitory substance was purified by reverse-phase HPLC and shown to be highly active against closely related Bacillus spp. Clinically relevant species such as Staphylococcus aureus and Micrococcus luteus were also inhibited. The substance was characterized as a bacteriocin-like inhibitory substance (BLIS) with a molecular mass of ca 3.4 kDa. The BLIS was very heat stable, and sensitive only to pronase E and proteinase K. Antimicrobial activity was stable and high in the pH range of 2.0-9.0, and relatively unaffected by organic chemicals. CONCLUSIONS: An antimicrobial substance produced by the B. cereus type strain ATCC 14579 was characterized, with a wide spectrum of activity and the potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a substance with antimicrobial activity from the B. cereus type strain.     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。     

Direct antimicrobial properties of substance P

The structural similarity between substance P (SP, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH(2)) and Arg/Pro rich bactericidal peptides suggests a possible direct effect of SP on invasive microbes. We now present evidence that substance P possesses direct antimicrobial activity, highest against S. aureus. A substance P antagonist also possesses such activity but while less potent than substance P agonist S. aureus, is more potent than substance P against C. albicans. Our data also show that the endogenous peptides bradykinin and neurotensin, that also play role in modulation of the host-defense system in situ, have antimicrobial properties but are less potent than substance P.     

Antimicrobial activity of Paenibacillus kribbensis POC 115 against the dermatophyte Trichophyton rubrum

In a search for an antifungal substance with activity against the dermatophyte fungus Trichophyton rubrum, strain POC 115 was chosen among different Paenibacillus strains for its phenotypic and genetic characterization and for preliminary characterization of its antimicrobial substance. Strain POC 115 was identified as belonging to Paenibacillus kribbensis. Physico-chemical characterization of the antimicrobial substance showed that it was not stable during heat and organic solvents treatments, but its activity was preserved at a wide range of pH and after treatment with pronase E, trypsin and DNase I. The crude concentrated supernatant of POC 115 culture was partially purified and the fraction presenting antimicrobial activity was further analyzed by UPLC/Mass Spectrometry. Two peaks were observed at 2.02 (mass 1,207 D) and 2.71 (mass 1,014 D) min in the mass chromatogram. The antimicrobial substance produced by POC 115 was correlated to iturin family compounds based on a set of primers designed for the amplification of PKS operon in the POC 115 genome. As happens with the mode of action of the antibiotics of the iturin group, the AMS produced by POC 115 caused the disruption of cytoplasmic membrane of T. rubrum and the subsequent withdraw of the intracellular material. This is the first report on the production of antimicrobial substances in P. kribbensis, and it may be of great relevance as an alternative or supplementary substance to antifungal drugs currently used against dermatophytes.     

Characterization of a bacteriocin-like substance produced by Bacillus amyloliquefaciens isolated from the Brazilian Atlantic forest.

A Bacillus strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. Phylogenetic analysis indicated that the strain has high sequence similarity with Bacillus amyloliquefaciens. The antimicrobial substance was inhibitory to pathogenic and food-spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus, Serratia marcescens, and Pasteurella haemolytica. It was stable over a wide temperature range, but lost activity when the temperature reached 121 degrees C/15 min. Maximum activity was observed at acidic and neutral pH values, but not at alkaline pH. The antimicrobial substance was sensitive to the proteolytic action of trypsin, papain, proteinase K, and pronase E. Except for iturins, other antimicrobial peptides have not been described for B. amyloliquefaciens. The identification of a bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection.     

Purification and characterization of an antimicrobial peptide produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.     

福寿螺不同生长发育阶段的抗菌活性物质及化学成分分析

福寿螺(Pomacea canaliculata)不同生长发育阶段(螺苗、仔螺、中螺、成螺)的软体匀浆物用乙酸乙酯进行提取.提取物分别用硅胶柱进行柱层析,并分别用不同极性的有机溶剂进行洗脱,分离出不同极性组分:非极性组分(石油醚洗脱组分)、弱极性组分(苯洗脱组分)、强极性组分(乙醇洗脱组分).然后用11种细菌对不同极性组分进行抗菌活性检测.结果表明,不同生长发育阶段组分抗菌作用的共同点是:强极性组分的抗菌活性最强,弱极性组分次之,非极性组分无抑菌作用.将不同生长发育阶段抗菌活性最强的乙醇洗脱组分分别进行薄层层析(TLC)分析并进行抗菌实验.薄层层析所用的展层剂不同,分离出条带数不同,各条带抗菌活性也存在差异.将抗菌活性最强的条带用气相色谱.质谱法进行化学成分鉴定,结果表明,各不同生长发育阶段抗菌物质的化学成分大部分是酸类物质,相同的化学成分在不同生长发育阶段的含量及相似度都不一样.     

Isolation and preliminary characterization of a nonbacteriocin antimicrobial compound from Weissella paramesenteroides DFR-8 isolated from cucumber (Cucumis sativus)

A non-proteinaceous antimicrobial substance (nonbacteriocin) produced by Weissella paramesenteroides DFR-8, an isolate from cucumber (Cucumis sativus), was purified using cell adsorption–desorption and gel permeation chromatography methods. A single peak observed in the purity check by analytical RP-HPLC strongly indicates the homogeneity of the nonbacteriocin preparation. The active substance is insensitive to proteolytic enzymes, lipase, amylase and catalase and shows a broad spectrum of activity towards food-borne/spoilage pathogens including Gram-negative organisms. The non-proteinaceous antimicrobial molecule has a molecular mass ～2.5 kDa and is thermostable up to 121 °C at pH ～4.0. A central composite rotatable design (CCRD) was employed to study the interactive effect of temperature and pH on antimicrobial activity and an equation was developed to deduce the residual activity of inhibitory substance under any conditions of temperature and pH within the experimental domain. The broad inhibitory spectrum and heat stability of the antimicrobial substance advocates its application as food-biopreservative.