长足大竹象雄性附腺提取物抑菌作用研究

以3种细菌(大肠杆菌Escherichia coli、金黄色葡萄球菌Bacillus subtilis、枯草芽孢杆菌Staphyloccocus aureus)为供试菌,测定了长足大竹象Cyrtotrachelus buqueti雄性附腺提取物的抑菌活性。结果表明:长足大竹象雄性附腺提取物对革兰氏阳性菌如枯草芽孢杆菌、金黄色葡萄球菌均有一定的抑菌活性。不同浓度的粗提物对枯草芽孢杆菌及金黄色葡萄球菌抑菌影响差异均具有高度统计学意义(P0.01),抑菌活性随着粗提物浓度的增加而增强。粗提物对金黄色葡萄球菌和枯草芽孢杆菌的最小抑菌浓度分别为0.2 mg·m L~(-1)、0.4 mg·m L~(-1)。不同处理温度对长足大竹象雄性附腺粗提物的抑菌作用有一定影响。     

家蝇幼虫血淋巴粗提物抑菌活性初步研究

以4种细菌(大肠杆菌、金黄色葡萄球菌、巨大芽孢杆菌、枯草芽孢杆菌)为供试菌,测定了家蝇幼虫血淋巴粗提物的抑菌活性。结果显示,家蝇幼虫血淋巴各浓度粗提物对4种供试细菌均有一定的抑菌活性,其中对金黄色葡萄球菌的抑菌率最大,其他依次为大肠杆菌>枯草芽胞杆菌>巨大芽孢杆菌,在0.8mg/mL时的抑菌率分别为51.09%、48.66%、43.27%和40.62%;在各浓度下不同菌种之间的抑菌率差异不显著,抑菌活性随着血淋巴粗提物浓度的增加而增强。在0.8mg/mL时对各细菌的抑菌圈直径分别为12.00mm、12.22mm、9.11mm和10.33mm,均显著大于对照,但低于头孢霉素。     

拮抗菌株的筛选及其发酵滤液抑菌活性考察

从土壤中筛选出3株具有抑菌活性的菌株,其中1#菌株对金黄色葡萄球菌、枯草芽孢杆菌、灰霉葡萄孢菌、变异链球菌、大肠杆菌均有较强抑制作用,初步鉴定为地衣芽孢杆菌。对其性质进行考察结果表明,在30℃,pH 7.0,150 r/min的条件下,恒温振荡培养24 h,测其发酵滤液的抑菌活力约为2 000 IU/mL。该发酵滤液具有较强的热稳定性,60℃放置1 h,抑菌活性下降了29%;100℃放置30 min,抑菌活性下降了45%,放置1 h,抑菌活性下降了48%;121℃放置1 h,抑菌活性仍能保持40%。该发酵滤液抑菌作用的最适pH值为7.0,在pH 3.0时能保持71%的抑菌活性,在pH 11.0时,仍能维持67%的抑菌活性,此发酵滤液对蛋白酶K不敏感,并且蛋白酶K对该发酵滤液的抑菌活性也起一定的促进作用。     

桑叶水提浸膏的抑菌作用研究

研究桑叶水提浸膏对五种食品常见污染菌的抑菌作用。通过滤纸片扩散法测定其相对抑菌活性。结果表明,桑叶水提浸膏对金黄色葡萄球菌有较强的抑制作用,对大肠杆菌作用次之,对枯草芽孢杆菌、荧光假单孢杆菌、巨大芽孢杆菌抑菌效果较差。桑叶水提浸膏对金黄色葡萄球菌的最小抑菌浓度是5%,抑菌作用受pH和温度的影响较大,在酸性条件下抑菌效果较好,低于60℃处理样品对抑菌效果影响不大,但高于80℃处理,样品抑菌活性明显降低。     

番茄生物碱粗提物抑菌作用的研究

本文研究了番茄生物碱粗提物对食品中常见腐败菌的抑菌效力,发现它对细菌和酵母菌具有较强的抑制作用,但对霉菌的抑菌作用较弱。番茄生物碱在中性环境中抑菌作用显著,经过121℃的高温处理仍具有较强的抑菌效力,且比苯甲酸钠的抑菌作用强。     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

松材线虫拮抗细菌的杀线活性及其发酵培养特性

旨为筛选具有高效杀松材线虫活性的拮抗细菌。采用浸测法对3株芽孢杆菌进行杀线活性测定,并对高效杀线菌株发酵滤液处理线虫后的虫体进行形态观察及该菌株培养条件和杀线物质稳定性测定。结果表明3株细菌发酵滤液处理松材线虫(Bursaphelenchus xylophilus)48 h后,线虫校正死亡率均达100%,且松材线虫在蜡样芽孢杆菌(Bacillus cereus)JK-XZ3发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率分别达99.55%和88.32%,明显高于短小芽孢杆菌(B. pumilus)HR10和瓦雷兹芽孢杆菌(B. velezensis)YH-20;该JK-XZ3菌株发酵滤液处理过的线虫虫体断裂,内含物外溢,体壁消解;该菌株在30℃培养4 d的发酵滤液杀线活性最高,且松材线虫在发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率均达100%;松材线虫在40℃、60℃、80℃和100℃处理后的发酵滤液8倍稀释液中处理48 h后,其校正死亡率均在84%以上;在pH为8-10的发酵滤液8倍稀释液处理48 h后,其校正死亡率均在88%以上;说明该菌株发酵滤液具有耐高温和耐碱特性。蜡样芽孢杆菌JK-XZ3是一株具有开发和应用潜力的高毒力杀线菌株。     

猪源产细菌素芽孢杆菌的筛选及抑菌特性

【背景】抗生素作为生长促进剂在畜牧业中的滥用,出现了严重的耐药基因富集和扩散问题,发掘新兴的生长促进剂作为饲料添加剂市场潜力巨大,目前益生菌制剂的开发最具潜力。【目的】通过对散养健康育肥猪粪便中芽孢杆菌的分离筛选,获得对典型肠道病原菌具有显著抑菌活性的芽孢杆菌,确定其产生的细菌素特性,以此对芽孢杆菌作为猪养殖业生长促进剂的潜力进行评价。【方法】采用梯度稀释涂平板法分离可培养细菌,利用牛津杯法检测菌株的抑菌活性。通过微生物形态及16S r RNA基因序列分析,确认6株产细菌素菌株的分类地位,并对其抗生素耐药性、细菌素稳定性及生理生化特征进行比较分析。【结果】从116株纯培养物中筛选得到6株对指示病原细菌具有显著抑菌效应的产细菌素芽孢杆菌,其中2株为贝莱斯芽孢杆菌(Bacillus velezensis),3株为枯草芽孢杆菌(Bacillus subtilis),1株为地衣芽孢杆菌(Bacillus licheniformis)。菌株B.licheniformis DY7和B.subtilis FX4对致泻、产肠毒素、出血性Escherichia coli均有显著的抑制效果,对头孢噻肟和红霉素高度敏感,其细菌素在p H 3.0-9.0、50-100°C水浴处理后仍具有明显的抑菌活性。【结论】猪源产细菌素芽孢杆菌DY7和FX4具有高效的病原细菌抑菌能力,所产细菌素稳定性较好,具有作为动物生长促进剂的应用潜力。     

一株植物病原拮抗细菌的分离筛选及拮抗物质

从黄豆酱中分离筛选对植物病害有防治效果的拮抗菌,提取发酵液中的拮抗物质,研究其抑菌机理及基本性质。采用稀释平板法分离细菌、平板对峙法筛选拮抗菌,通过形态特征、生理生化特征及16S rRNA鉴定菌种,硫酸铵沉淀法提取发酵液中的拮抗物质,考马斯亮蓝法测得蛋白含量,扫描电镜观察拮抗物质抑菌作用,琼脂孔扩散法测定拮抗物质抑菌谱及其活性的稳定性。从黄豆酱中分离出18株细菌,通过筛选,得到一株拮抗效果最好的菌株,菌体抑菌率62.78%,发酵液抑菌率53%,命名为4-z-3,经鉴定为Bacillus siamensis。4-z-3发酵液在硫酸铵饱和度为20%~30%时提取的粗提物抗菌活性最大,为60.8%。最适饱和度下提取的粗提物的蛋白质含量为3.45%。拮抗物质对多种病原菌有良好的拮抗效果。经过4-z-3粗提物处理的病原真菌,菌丝受到破坏,细胞形状改变,细胞完整性缺失。粗提物的稳定性方面,对热不敏感,仅在90和100℃时抑菌活性下降明显;对酸碱不敏感,pH值为4时仍有81.6%的抑菌活性;对蛋白酶不敏感,抑菌活性最低时降低了19.6%;对紫外线不敏感,照射12 h之后抑菌活性仍有92.5%。本研究表明,菌株4-z-3是一株具有发展潜力的农业生防菌,分泌的胞外拮抗物质能够有效防治多种植物病害,且抑菌活性稳定。     

解淀粉芽孢杆菌抗菌机制研究进展

自然界中,细菌与细菌之间、细菌与其他生物之间存在广泛的相互作用。细菌产生的代谢物质可以提高自身在自然界中的竞争/生存能力。解淀粉芽孢杆菌(Bacillus amyloliquefaciens)能够产生种类繁多的抑菌物质,有效地抑制真菌和其他细菌的活性,是一类应用较广的生防菌。到目前为止,解淀粉芽孢杆菌的抑菌作用研究已经广泛开展,但是生物体是复杂多变的,仍然有很多东西是未知的,并且利用解淀粉芽孢杆菌进行生物防治的前景十分广阔,所以加强对抑菌物质的机制研究、提取相关抗菌物质尤为关键。本文综述了国内外有关解淀粉芽孢杆菌的抑菌机制研究,为进一步探究解淀粉芽孢杆菌的抑菌机制、抑菌基因和基因工程菌做好铺垫。     

应用DNA分子标记鉴定黄颡鱼、瓦氏黄颡鱼及其杂交种的研究

为区分黄颡鱼（Pelteobagrus fulvidraco Richardson）、瓦氏黄颡鱼（Pelteobagrus vachelli Richardson）及其杂交种，前期研究构建了一个YY超雄黄颡鱼的BAC文库并筛选到了一个包含性别连锁标记Pf62-Y的BAC克隆。研究对该BAC克隆进行测序并鉴定到了一个新基因Inad-like，而且Inad-like的外显子序列在X和Y染色体上基本一致。通过黄颡鱼Inad-like的外显子序列及序列的保守性我们设计引物在瓦氏黄颡鱼中扩增获得了其部分内含子序列。通过内含子序列比对，发现瓦氏黄颡鱼比黄颡鱼少了一个DNA片段。基于黄颡鱼和瓦氏黄颡鱼的显著遗传差异，设计了一对引物，该引物在黄颡鱼和瓦氏黄颡鱼基因组中分别扩增出1908和1178 bp DNA片段，而且在杂交黄颡鱼中同时扩增出这两个DNA片段。总之，这个新的遗传标记提供了一种稳定、高效识别黄颡鱼及其与瓦氏黄颡鱼杂交种的方法。     

The use of probiotics in shrimp aquaculture

Shrimp aquaculture, as well as other industries, constantly requires new techniques in order to increase production yield. Modern technologies and other sciences such as biotechnology and microbiology are important tools that could lead to a higher quality and greater quantity of products. Feeding and new practices in farming usually play an important role in aquaculture, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice of many fish and shrimp feed manufacturers and farmers. Probiotics, as 'bio-friendly agents' such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are nonpathogenic and nontoxic microorganisms without undesirable side-effects when administered to aquatic organisms. These strains of bacteria have many other positive effects, which are described in this article.     

Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments

Many recent papers have deepened the state of knowledge about lactic acid bacteria (LAB) in fish gut. In spite of high variability in fish microbiota, LAB are sometimes abundant in the intestine, notably in freshwater fish. Several strains of Streptococcus are pathogenic to fish. Streptococcus iniae and Lactococcus garvieae are major fish pathogens, against which commercial vaccines are available. Fortunately, most LAB are harmless, and some strains have been reported for beneficial effects on fish health. A major step forward in recent years was the converging evidence that LAB can stimulate the immune system in fish. An open question is whether viability can affect immunostimulation. The issue is crucial to commercialize live probiotics rather than inactivated preparations or extracts. There has been a regain of interest in allochthonous strains used as probiotics for terrestrial animals or humans, due to economical and regulatory constraints, but the short survival in sea water may limit application to marine fish. If viability is required, alternative treatments may include the incorporation of prebiotics in feed, and other dietary manipulations that could promote intestinal LAB. Antagonism to pathogens is the other main feature of candidate probiotics, and there are many reports concerning mainly carnobacteria and Enterococcus. Some bacteriocins were characterized which may be of interest not only for aquaculture, but also for food preservation.     

氧化鱼油对黄颡鱼黑色素合成酶及内分泌激素的影响

实验模拟自然氧化条件制备氧化鱼油, 替代普通鱼油加入饲料中, 研究其对黄颡鱼(Pelteobagrus fulvidraco)表皮黑色素、酪氨酸酶活力、血清与脑内分泌激素含量的影响。处理组分别为未替代组(C组)、50%氧化鱼油替代组(Y50组)和100%氧化鱼油替代组(Y100组), 结果表明: 随着氧化鱼油替代普通鱼油的比例提高, 黄颡鱼5级(黑色素占细胞面积80%以上)黑色素细胞比例上升; 酪氨酸酶活力上升, 但各组差异不显著(P>0.05); 皮质酮(CORT)在血清和脑组织中各组含量差异均不显著(P>0.05); 肾上腺皮质激素(ACTH)在脑组织中含量增加 (P<0.05), 而血清中Y50组含量高于C组及Y100组(P<0.05); 血清中促肾上腺皮质激素释放激素(CRH)含量降低, Y50与Y100组之间差异不显著(P>0.05), 而脑组织中各组差异不显著(P>0.05); 血清中α-黑素细胞刺激素(α-MSH)含量, Y100组高于其他组(P<0.05), 而脑组织中各组差异不显著(P>0.05)。上述结果表明, 随着氧化鱼油替代普通鱼油的比例提高, 5级黑色素细胞比例上升, 黄颡鱼血清和脑组织中能互相转换的ACTH与α-MSH总量提高, 变化趋势与表皮黑色素含量和酪氨酸酶活性相一致。     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

黄颡鱼卵致病性绵霉的分离鉴定与药敏特性

【目的】对黄颡鱼水霉病病原进行分离鉴定,并对其药敏特性进行研究。【方法】参照传统方法对患水霉病的黄颡鱼卵上的丝状真菌进行分离,通过人工感染实验证实分离菌株的致病性,然后根据形态学特征和ITS rDNA序列分析对致病菌株进行鉴定,并进一步采用倍比稀释法研究其药敏特性。【结果】从患水霉病的黄颡鱼卵上分离到8株丝状真菌,经人工感染试验证实菌株YC对黄颡鱼卵具有致病性,并进一步研究了其形态与药敏特性,开展了ITS rDNA序列分析。结果表明,菌株YC为透明管状结构,中间无横隔;游动孢子囊多呈圆筒形、棍棒形或穗状,游动孢子发育成熟后不断从孢子囊顶端释放出来,成团聚集在游动孢子囊口,并经过一个时期的静休后,成团脱落或直接分散在水中游动;次生孢子囊具有典型的侧生现象;藏卵器呈球形或梨形,大多与雄器异枝,少数与雄器同枝,含1?15个卵孢子;成熟卵孢子中生或亚中生,偏生一个大油球。其ITS rDNA序列与GenBank基因库中绵霉属菌株自然聚类,同源性高达99%,与异丝绵霉(Achlya klebsiana)菌株CBS101.49(GenBank登录号AF119579)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株YC为异丝绵霉(Achlya klebsiana)。此外,在实验选用的中草药和消毒剂中,黄连和异噻唑啉酮分别对菌株YC的抑菌效果最好,其对菌株YC的最小抑菌浓度分别为256 mg/L和2 mg/L。【结论】首次分离了黄颡鱼卵致病性异丝绵霉菌株YC,并确定了其药敏特性,可以作为该病防治用药的依据。     

A study of the antibacterial mechanism of pinocembrin against multidrug-resistant Aeromonas hydrophila

International Microbiology - Aeromonas hydrophila is a common pathogen in fish that has caused severe economic losses in aquaculture worldwide. With the emergence of bacterial resistance, it is...     

水产养殖用解淀粉芽孢杆菌微胶囊的安全性评价

目的:评价水产养殖用解淀粉芽孢杆菌微胶囊的安全性。方法:参照《GB/T21805-2008化学品藻类生长抑制试验》、《GB/T13266-91水质物质对蚤类(大型蚤)急性毒性测定方法》、《GB/T13267-91水质物质对淡水鱼(斑马鱼)急性毒性测定方法》、《渔药临床试验技术规范》等国家标准及相关法规,观察了解淀粉芽孢杆菌微胶囊对小球藻生长的抑制作用以及对大型蚤、斑马鱼和草鱼的急性毒性,分析了其对养殖水体主要理化因子的影响。结果:解淀粉芽孢杆菌微胶囊在终浓度为0.2～2 000mg/L时对小球藻生长具有促进作用,对小球藻的半数抑制浓度大于2 000mg/L,而且其对大型蚤、斑马鱼和草鱼的半数致死浓度也大于2 000mg/L(或mg/kg体重)。此外,在养殖水体中加入解淀粉芽孢杆菌微胶囊至终浓度为0.2～2 000mg/L后14天内,各浓度组的氨氮含量、硫化物和pH均缓慢下降,仅亚硝酸盐氮含量稍微升高后逐渐缓慢降低,但这些理化因子的变化与解淀粉芽孢杆菌微胶囊的加入量呈负相关关系。结论:解淀粉芽孢杆菌微胶囊实际无毒,对养殖水中氨氮、亚硝酸盐氮、硫化物和pH等理化因子的影响均控制在虾虎鱼仔鱼、黄颡鱼、白斑狗鱼、克氏原螯虾等水产养殖动物的安全浓度范围内,为其在水产养殖中的安全应用提供了重要的科学依据。     

Complete Genome Sequence of Aeromonas hydrophila Phage CC2

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.