6株猪源芽孢杆菌的分离鉴定及其益生性研究

本研究旨在通过高温处理和常规厌氧菌分离法从健康肥猪粪便中分离芽孢杆菌,并通过培养性状、生化试验、PCR、肠道耐受、抗菌活性和接种试验动物等方法鉴定筛选性状稳定、优良的益生性芽孢杆菌。结果显示,共分离到6株芽孢杆菌,其中1株巨大芽孢杆菌、1株死谷芽孢杆菌、2株解淀粉芽孢杆菌和2株枯草芽孢杆菌;分离菌对多黏菌素和青霉素耐药性较强,对其他12种常用药物较敏感,对致病性大肠杆菌和金黄色葡萄球菌的抑菌效果较好;4种芽孢杆菌均可在酸性、高胆盐和高温条件下生存,特别是巨大芽孢杆菌和枯草芽孢杆菌耐受能力较强;分离的芽孢杆菌对小鼠均无致病性,其中巨大芽孢杆菌和死谷芽孢杆菌有较明显的促进试验动物生长的作用。结果提示,巨大芽孢杆菌和死谷芽孢杆菌可作为益生性芽孢杆菌的初步选择。     

猪源芽孢杆菌的分离鉴定及生物学特性研究

从自然生长的健康猪肠道中分离到13株芽孢杆菌,通过生理生化试验初步鉴定其中9株为枯草芽孢杆菌,2株为巨大芽孢杆菌,1株为地衣芽孢杆菌,1株为凝结芽孢杆菌;对这13株芽孢杆菌分别进行了耐酸性试验、耐胆盐试验、体外抑菌试验和动物安全性试验。结果表明,其中1株地衣芽孢杆菌B7株和1株枯草芽孢杆菌B9株均有较强的耐酸、耐胆盐能力,对致病性大肠杆菌具有较好的体外抑制能力,且均安全无毒,可作为动物用益生菌制剂的候选菌种。     

弧菌颉颃菌的筛选及其在对虾养殖中的初步应用

试验从养殖环境中分离到22株细菌,以副溶血弧菌为病原指示菌,点种法筛选到1株弧菌颉颃菌A4;通过生理生化试验和16S rRNA序列分析,鉴定该菌为枯草芽孢杆菌。抑菌性能试验结果显示,分离菌株对副溶血弧菌、溶藻弧菌及灿烂弧菌均具有较好的颉颃作用。在对虾养殖试验中发现,使用枯草芽孢杆菌制剂2 d后可显著减少水体中的弧菌数量（P＜0.05）,改善对虾生长状态。该菌株可作为益生菌开发成微生态制剂,用于对虾养殖中弧菌病的预防。     

6株芽孢杆菌的生物学特性比较研究

以实验室分离保存的6株芽孢杆菌为材料,对其主要生物学特性进行了研究,包括产酶活力,对NaCl、牛胆盐、人工胃液和人工肠液的耐受性及对抗生素的敏感性。结果显示,枯草芽孢杆菌1、凝结芽孢杆菌和蜡样芽孢杆菌均能产生较高活性的中性蛋白酶和淀粉酶;2株枯草芽孢杆菌和凝结芽孢杆菌能耐较高浓度的NaCl;枯草芽孢杆菌2、凝结、纳豆和蜡样芽孢杆菌对牛胆盐的耐受性较强;除地衣芽孢杆菌外,其他5株芽孢杆菌对胃液和肠液的耐受性较好;6株芽孢杆菌均对常用饲用抗生素敏感。结果表明,枯草1、凝结、蜡样芽孢杆菌是优良的候选益生菌菌株。     

西藏牦牛源枯草芽孢杆菌的分离培养及鉴定

从西藏健康牦牛的新鲜粪便中分离、纯化枯草芽孢杆菌,并筛选出耐受性最好的菌株,并进行形态学观察、生化试验、PCR扩增、电泳检测、16s rRNA序列测定、构建进化树并进行同源性分析,对通过鉴定的枯草芽孢杆菌进行耐肠液、耐胆盐、耐高温等耐受性试验.结果显示:分离出疑似枯草芽孢杆菌4株,革兰氏染色为阳性;芽孢染色液染色菌体为...     

甘草内生真菌分离及其抑菌活性初探

为了解药用植物甘草(Glycyrrhiza uralensis)内生真菌资源多样性及其抑菌活性特征,通过组织块法对内生真菌进行分离,并选择小麦全蚀病菌（Gaeumannomyces graminis var.tritici）、枸杞黑果病菌（Colletotrichum gloeosporioides）、番茄灰霉病菌（Botrytis cinerea）、黄瓜枯萎病菌（Fusarium oxysporium f.sp.cucumeris）、黄瓜立枯病菌（Rhizoctonia solani）5种植物病原真菌和枯草芽孢杆菌（Bacillus subtilis）、大肠杆菌（Escherichia coli）、金黄色葡萄球菌（Staphylococcus aureus）、铜绿假单胞菌（Pseudomonas aeruginosa）4种细菌作为供试指示菌,采用对峙法和改进的菌块法测定抑菌活性。试验结果显示,从甘草根、茎、叶中分离出20株内生真菌,其中根部最多,占分离菌株总数的65.0%,其次为茎部,叶部最少;经形态学初步分类鉴定归于2目2科5属,梭孢霉属为优势菌属,占分离菌株总数的70.0%;在分离的内生真菌中有15株菌对1种或1种以上供试植物病原真菌有不同程度的抑制作用,为分离内生真菌总数的75.0%,19株菌对1种或1种以上供试细菌有不同程度的抑制作用,为分离内生真菌总数的95.0%;有7株内生真菌对枯草芽孢杆菌拮抗活性差异显著（P＜0.05）,有 8株内生真菌对金黄色葡萄球菌拮抗活性差异显著,有3株内生真菌对大肠杆菌拮抗活性差异显著,有1株内生真菌对革兰氏阴性铜绿假单胞菌拮抗活性差异显著,其中菌株RLEFR015对番茄灰霉病菌、金黄色葡萄球菌拮抗活性差异显著,菌株RLEFR010对大肠杆菌、金黄色葡萄球菌拮抗活性差异显著,菌株RLEFR002对枯草芽孢杆菌、金黄色葡萄球菌供试细菌拮抗活性差异显著;菌株RLEFR015、RLEFR002、RLEFR010均为梭孢霉属,为高活性菌株。甘草内生真菌具有多样性和抑菌活性,多数菌株对供试病原真菌和病原细菌具有拮抗活性,对革兰氏阳性金黄色葡萄球菌和革兰氏阳性枯草芽孢杆菌拮抗活性较强,预示着甘草内生真菌具有重要的资源价值。     

饲用玉米根际促生菌资源筛选及其特性研究

为获得玉米根际促生菌(PGPR)菌株并明确其功能特性;以玉米根系及根际土壤为材料,利用选择性培养基分离筛选溶磷菌与固氮菌,测定所选菌株溶磷能力和固氮酶活性,并测定其产IAA的能力,从中筛选综合性能优良的菌株,在此基础上,运用生理生化鉴定和16SrDNA分子生物学鉴定相结合的方法鉴定优良菌株的种属。结果表明:玉米根际有大量PGPR菌,分布特征表现出根表(RP)根表土(RS)远根土(NRS)根内(HP)的数量分布规律,呈根际效应。最终获得262株PGPR菌,其中固氮菌48株,溶解无机磷细菌108株,溶解有机磷细菌106株,有分泌IAA能力的菌株15株。筛选出综合性能优良、有进一步开发应用潜力的菌株7株。经鉴定其中3株为Pseudomonas fluorescens;2株为Bacillus subtilis;Klebsiella oxytoca和Bacillus megaterium各1株。     

植物根际促生菌对两种真菌病害病原的抑制作用及其鉴定

利用钼蓝比色法、乙炔还原法和高效液相色谱法分别测定了分离自5种植物根际的19株细菌溶磷、固氮和分泌生长素特性;用平板对峙生长法测定了19株细菌对黄瓜枯萎病菌和小麦根腐病菌的抑制作用;结合菌株形态、生理生化反应,利用分子生物学方法鉴定了其中8株优良细菌。结果表明,菌株Bacillus subtilis LM4-3固氮酶活性较高[244.88 nmol C₂H₄/(mL·h)];菌株B. subtilis LHS11-1溶磷能力较强(205.77 mg/L);菌株PGRS-3分泌IAA能力较好(40.78 μg/mL)。5株菌株能够有效拮抗黄瓜枯萎病菌,其中B. subtilis FX2-1抑菌活性较强(活菌抑制率69.07%,发酵液抑制率51.73%)。7株菌株能够有效拮抗小麦根腐病菌,其中B. subtilis FX2-1抑菌活性较强(活菌抑制率 78.17%),B. subtilis LHS11-1发酵液抑制率高达81.52%。其他具有较好促生或拮抗上述两种病原真菌的菌株经鉴定,分别属于蜡样芽孢杆菌(B. cereus,菌株XX1)、短小芽孢杆菌(B. pumilus,菌株F1-4和LX22)、简单芽孢杆菌(B. simplex,菌株XX6)和荧光假单胞杆菌(Pseudomonas fluorescens,菌株XX5)。     

Study on the Isolation,Identification and Biological Characteristics of a Strain of Bacillus pumilus Isolated from Tibetan Sheep

In order to select probiotic strains with probiotic potential isolated from Tibetan sheep,fresh excreta of the healthy female Tibetan sheep was processed with 90 ℃ water-curing treatment for 5 min,and Bacillus with resistance were screened.The morphological,physiological and biochemical characteristics and 16S rDNA sequence of the Bacillus were comprehensively analyzed and identified.Biochemical characteristics were studied by using antimicrobial test,acid resisting tests and bile salt resisting tests,hydrolytic enzyme test and other methods.The results showed that the Bacillus SWUN-G43 isolated from fresh excreta of Tibetan sheep had a significant antibacterial effect on E.coli and Salmonella.Through analysis and identification,the Bacillus SWUN-G43 was Bacillus pumilus.Its survival rate was 50.43% under the environment of pH 2.0 and 44.35% under the environment of 0.3% bile salt.It could produce protease and cellulase and was sensitive to 12 kinds of commonly used antibiotics.By studying,the Bacillus pumilus(SWUN-G43) had a certain degree of resistance and potential probiotic ability.Besides,the strain didn't contain R-plasmid and would not cause horizontal transmission of antibiotic resistance in the intestinal microecosystem.It also had the potential to be developed as a micro ecological additive.     

一株藏绵羊源短小芽孢杆菌的分离鉴定及生物学特性研究

为获得来源于藏绵羊的益生菌菌株,对健康母藏绵羊新鲜粪便进行90 ℃水浴处理5 min以筛选具有抗逆性的芽孢杆菌,再综合形态学、生理生化特征及16S rDNA序列分析进行鉴定,通过抑菌试验、耐酸、耐胆盐试验及产水解酶试验等方法对其生物学特性进行研究。结果显示,从新鲜粪便样品中分离到的芽孢杆菌菌株SWUN-G43对大肠杆菌和沙门氏菌有明显的抑菌效果,通过分析鉴定其为短小芽孢杆菌,该菌株在pH 2.0的环境中存活率为50.43%,在0.3%胆盐环境中存活率为44.35%,可产生蛋白酶和纤维素酶,对12种常用的抗生素具有很高的敏感性。从试验结果可分析出该短小芽孢杆菌(SWUN-G43)有一定的抗逆性和潜在的益生能力,且该菌株不含耐药质粒,不会在动物肠道微生态系统中造成抗生素抗性的水平传递,具有作为微生态添加剂开发的潜力。     

益生芽孢杆菌脂肽类抑菌成分的高效液相色谱-电喷雾串联质谱分析

本文旨在探明一株具有益生潜质的枯草芽孢杆菌N2-10对肠道菌的抗菌作用及其活性成分,为深入评价该菌株的益生功能和益生机理奠定基础。以常见的肠道致病菌和有益菌为指示菌,采用抑菌圈法对该菌株发酵液成分的抑菌活性进行测定,并通过高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)对其中的活性成分进行分析。结果表明:枯草芽孢杆菌N2-10发酵液的氯仿和正丁醇提取液对供试菌无明显的抑制作用;脂肽类对大肠杆菌、痢疾杆菌和沙门氏菌等肠道致病菌表现出明显的抑菌作用,而对肠道有益菌保加利亚乳杆菌抑制作用很弱,对嗜热链球菌则无抑制作用。脂肽粗提物的HPLC-ESI-MS/MS分析结果显示,脂肽粗提物中的脂肽类抗生素为伊枯草素家族中的C14~C17抗霉枯草菌素(Mycosubtilin)的同系物。因此推断,枯草芽孢杆菌N_2-10对肠道致病菌产生抑制作用依赖于其产生的脂肽抗生素Mycosubtilin。     

Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection

The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.     

几种商用益生芽孢杆菌特性的比较研究

为比较几种商用益生芽孢的菌株特性，为芽孢杆菌的选育提供理论基础，本试验收集整理了15种药用（Y1~Y6）和饲用益生芽孢杆菌（S7~S15）产品，对其芽孢杆菌进行分离鉴定，分别从产淀粉酶、纤维素酶和蛋白酶等酶活特性，抗菌活性，耐酸和耐0.3%胆盐稳定性，抗生素敏感性，耗氧及生长速率测试等方面对芽孢杆菌特性进行了比较研究。结果显示，分离的15株芽孢杆菌均能产蛋白酶、纤维素酶、淀粉酶，但不同菌株产酶能力差异较大，S9菌株表现出较高的产蛋白酶、纤维素酶、淀粉酶活力；所有分离的芽孢杆菌均能对金黄色葡萄球菌及藤黄微球菌产生显著抑制活性，但只有7株芽孢杆菌（Y2、Y6、S7、S9、S10、S11、S12）可对大肠杆菌产生抑制作用，尤其是S9菌株综合抗菌能力最强。S9、Y2和S8的营养体细胞具有较高的耐酸能力，S9、Y2菌株在0.3%胆盐环境中存活率超过90%。15株芽孢杆菌中有3株饲用来源菌株（S10、S12、S13）表现出对红霉素的耐受，S10菌株对四环素耐受。此外，S9菌株具有较快的耗氧速率和生长速率。上述结果表明，S9菌株（解淀粉芽孢杆菌）的益生特性更为优良，本试验结果可为芽孢杆菌益生菌的选育及活性评价提供参考依据。     

Survival and retention of the probiotic properties of Bacillus sp. strains under marine stress starvation conditions and their potential use as a probiotic in Artemia culture

The probiotic properties of Bacillus strains isolated from Artemia culture and the effect of marine stress on viability and survival were investigated, as well as the changes occurring in their properties. Analyses showed that these bacteria corresponded to the genus Bacillus sp. Antagonism and adherence assays revealed that Bacillus strains have an inhibitory effect against tested pathogenic bacteria and are fairly adherent. Normal and starved cells showed different enzymatic profiles. Challenge tests performed with Artemia larvae provided evidence that the tested Bacillus strains were neither pathogenic nor toxic to the host and conferred protection for Artemia culture against pathogens. The tested strains maintained their viability and their probiotic properties during the period of study. The results suggest that the tested strains have suffered changes allowing them to survive in seawater in the absence of nutrients and outside their natural host, identifying them as potential probiotic candidates for Artemia culture.     

一株芽孢杆菌的分离鉴定及其益生潜质分析

 从健康肉鸡的粪便中分离出一株BF3芽孢杆菌,经培养特征、形态观察、生理生化试验以及16SrDNA序列分析相似度为99%,确定该菌株为枯草芽孢杆菌,对该株芽孢杆菌对肠道致病菌的拮抗作用进行研究。结果表明BF3菌株在培养过程中,可以抑制鸡白痢沙门氏菌和鸡大肠杆菌的生长。拮抗作用的强弱与芽孢杆菌和致病菌的接种时间有关。先接种芽孢杆菌后接种致病菌,拮抗作用最强;芽孢杆菌和致病菌同时接种次之;先接种致病菌后接种芽孢杆菌,拮抗作用最弱。并且BF3菌株代谢产物对鸡白痢沙门氏菌和鸡大肠杆菌具有非常明显的拮抗作用。     

刘家峡网箱鲟鱼鱼苗主要病原菌的分离鉴定

为查明夏季甘肃省永靖县刘家峡水库西伯利亚鲟鱼鱼苗爆发性疾病病原菌,以无菌方法从甘肃省永靖县刘家峡不同鲟鱼网箱养殖场共采集了30份濒死期及死亡鲟鱼的组织样品,采用常规细菌分离、分子生物学鉴定方法对病原菌进行了分离鉴定,并进行了人工感染实验和药敏实验。结果表明:从采集到的鲟鱼组织样品中分离到57株细菌,分别为铜绿假单胞菌、异常嗜糖气单胞菌、弗氏柠檬酸杆菌、河生莱略特氏菌、枯草芽胞杆菌、嗜水气单胞菌、鲁氏耶尔森菌、迟钝爱德华氏菌和海豚链球菌9种,除枯草芽胞杆菌及迟钝爱德华氏菌外,其余7种细菌对健康鲟鱼均具有不同程度的致病性;药敏实验分离到的7种病原菌均对诺氟沙星、氧氟沙星、环丙沙星、哌拉西林、庆大霉素、氯霉素等抗生素高度敏感,对青霉素、万古霉素、克林霉素、苯唑西林等不敏感,甚至完全耐药。该研究初步查明了甘肃省永靖县刘家峡西伯利亚鲟鱼鱼苗夏季爆发性疾病的病原菌种类,为该地区鲟鱼疾病的预防和合理用药提供了理论依据。     

枯草芽孢杆菌的分离筛选

本文基于从秸秆垛底层土壤中分离枯草芽孢杆菌的目的,为发酵秸秆提供菌株,从陕西杨凌农村秸秆垛中采取底层土样并进行分离。分离出11株菌,经过形态特征、生理生化特征等方法从中分离初步鉴定了3株疑似菌株。经对液态发酵条件的优化,对3株疑似菌进行液态发酵试验,以粗蛋白和粗纤维含量为标准,筛选出一株发酵效果最好的菌株b-1进行了16SrDNA序列分析,最终鉴定b-1菌株为枯草芽孢杆菌(Bacillus subtilis),其同源性≥99%。     

一株羊源性枯草芽孢杆菌的筛选鉴定及安全性评价

为了进一步鉴定、分析和评价从绵羊粪便中分离的一株芽孢杆菌,通过形态学观察、16S rDNA测序分析、耐酸性实验,以及小鼠灌胃和腹腔注射两种方式进行安全性评价。结果发现该菌株为枯草芽孢杆菌;在适宜条件下,培养8 h可达到最大生长速度;在pH值为3.0时,长势良好;在15 d饲喂期间,小鼠生理活动正常,无死亡;各组小鼠主要脏器均未发现明显的病变,脏器系数也无显著差异。由此可知,该枯草芽孢杆菌具有良好的生物学特点,具有用于促进羊群生产的应用潜力。     

Study on Probiotic Characteristics of Eleven Bacillus subtilis Strains

The purpose of this study was to investigate the property of antibacterial, production of enzymes, tolerance and in vitro digestibility of 11 Bacillus subtilis strains.9 Bacillus subtilis strains were screened out according to antibacterial property and production of enzymes. After tests of heat, pH tolerance, artificial gastrointestinal fluid and pig bile salt,6 Bacillus subtilis strains had good performance in simulation of animal digestive tract and strong heat tolerance in the process of production. Meanwhile, 5 strains BLCC1-1, BLCC1-2, BLCC1-3,BLCC1-4 and BLCC1-6 had good digestion effects on different combinations of feeds. Based on the above results,it suggested that BLCC1-1, BLCC1-2, BLCC1-4 and BLCC1-6 had excellent production of enzymes, tolerance and in vitro digestibility, which might be suitable for using as feed additives and further research.     

11株枯草芽孢杆菌益生特性研究

试验旨在研究11株枯草芽孢杆菌的抑菌、产酶、耐受性和体外消化率等益生特性。从11株枯草芽孢杆菌中初筛得到9株体外抑菌特性和产酶特性均较好的芽孢杆菌;经过热、人工胃肠液、胆盐等耐受性复筛,结果表明9株芽孢杆菌中有6株对胆盐、胃酸、肠液等人工模拟的消化道内环境和生产过程中的热耐受性较强;经模拟动物体消化道的试验结果表明,9株芽孢杆菌中有5株（BLCC1-1、BLCC1-2、BLCC1-3、BLCC1-4、BLCC1-6）对不同组合饲料的消化效果较好。综合以上试验结果,既表现出较好的产酶、抑菌和耐受性,又对组合饲料有良好的消化效果的枯草芽孢杆菌有BLCC1-1、BLCC1-2、BLCC1-4和BLCC1-6,这4株枯草芽孢杆菌具有作为饲料添加剂应用于畜禽饲料中的潜力,可进一步开展研究。