乳酸乳球菌和魏斯氏菌的分离及鉴定

试验以生牛奶、自制泡菜水、青贮料、市售奶酪为样品,进行乳酸乳球菌和魏斯氏菌的筛选与鉴定。通过培养基中菌落形态观察和镜检细胞形态观察,共筛得6株疑似乳酸球菌（分别命名为ST1、ST2、ST6、ST7、ST8、ST9）。经生理生化、耐盐性、耐热性试验以及16S rD-NA序列分析鉴定,这6株菌分属两个属：ST1、ST2、ST7、ST9为乳酸乳球菌（Lactococcus lac-tis）,其中ST2为乳酸乳球菌乳脂亚种（Lactococcus lactis subsp. Cremoris）,ST7为乳酸乳球菌乳酸亚种（Lactococcus lactis subsp. Lactis ）;ST6、ST8属于魏斯氏菌属（Weissella）,其中ST6为食窦魏斯氏菌（Weissella cibaria）。研究表明,生牛奶和泡菜水分别是乳酸乳球菌和魏斯氏菌的优良生活环境,传统方法与分子生物技术相结合可更准确快速地分离及鉴定菌株。     

一株蚕丝脱胶酶产生菌的筛选鉴定及产酶条件优化试验

从丝厂周边土壤中筛选到一株能高效生产脱胶酶的菌株。通过形态学观察和16S rDNA碱基序列测定及同源性分析,鉴定该菌株为革兰阳性杆状细菌,有芽孢,好氧且不具运动性,属于芽孢杆菌属(Bacillus)。对scw2-1菌株产脱胶酶发酵条件的优化结果是:培养基组分的最佳碳源为0.03 g/mL可溶性淀粉、氮源为0.03 g/mL大豆粉、金属离子为K+(0.01 g/mL KHPO),发酵最适温度为37℃,培养基初始pH值为10.0,发酵时间为72 h。     

一株桑树内生拮抗细菌的分离鉴定与抑菌活性

以大肠杆菌为指示菌,从野生型鲁桑(Morus multicaulis Perr.)桑树枝条韧皮部组织中分离得到具有抑菌活性的内生细菌ME1。形态与生理生化特性鉴定ME1菌株为有芽孢和鞭毛的革兰阳性菌,细胞呈杆状,大小为0.6～0.7μm×1.8～1.9μm;能够利用葡萄糖、蔗糖和乳糖等碳水化合物,所产淀粉酶活力可达4 182.60 U/g;耐盐性较高,可耐受100 g/L NaCl。进一步通过16S rDNA序列及其系统进化分析,初步鉴定ME1菌株为枯草芽孢杆菌(Bacillus subtilis)。抑菌试验表明ME1菌株的代谢产物具有广谱抗菌活性,对大肠杆菌(Escherichia coli)、苏云金芽孢杆菌(Bacillus thuringiensis)、金黄色葡萄球菌(Staphylococcus aureus)、蕈状芽孢杆菌(Bacillus mycoides)和白僵菌(Beauveria bassiana)的体外抑制率分别为59.92%、75.70%、51.60%、87.25%和69.81%,该菌株产生的抗菌活性物质具有潜在的医用开发价值。     

山羊瘤胃内产乳酸菌的分离鉴定及其产D-、L-乳酸特性的研究

为阐明瘤胃中产乳酸菌的种类及其产乳酸特性,本试验以饲喂精粗比例为6:4的健康山羊瘤胃液为接种物,利用厌氧分离技术,经形态学和16s rRNA序列分析法,从山羊瘤胃中分离获得6株细菌(分别命名为L2、L3、L5、L8、L10和L12).菌株主要为3类:L2、L8、L10为牛链球菌,L5为马链球菌,L3和L12为食窦魏斯氏...     

一株致病性粪肠球菌的分离与鉴定

从1羽发病雏鸡中分离到1株溶血性革兰氏阳性球菌。初步分离鉴定后经攻毒试验能够造成小白鼠和雏鸡死亡,具有致病性。药敏试验表明,该菌对多种药物均表现耐药性,仅对庆大霉素、青霉素、氧氟沙星敏感。通过BLAST软件分析该菌株的16S rRNA序列,确定该致病性细菌为粪肠球菌。     

几株家蚕蛹油降解细菌的分离鉴定与降解能力测定

利用稀释培养法从缫丝废水中分离到5株可降解家蚕蛹油的细菌,采用紫外分光光度法测定了其对家蚕蛹油的降解能力,H73、H23、H1、H5和H43菌株在72 h内对家蚕蛹油的降解率分别达到76.36%、62.04%、60.58%、53.85%和51.20%。通过形态特征、生理生化特性测定和16S rDNA序列系统发生分析对各菌株进行鉴定,其中H23、H1和H43为芽孢杆菌属(Bacillus sp.),H5为微杆菌属(Microbacterium sp.),H73为肠杆菌属(Enterobacter sp.)。     

1株利用乳酸的猪源丁酸梭菌的分离鉴定及基因组序列分析

【目的】 研究1株利用乳酸的猪源丁酸梭菌的基因组信息。【方法】 采用厌氧培养法, 通过乳酸分解培养基(LADM)初筛、梭菌增殖培养基(RCM)复筛, 从饲喂乳酸菌发酵饲料的健康仔猪肠道内容物中分离利用乳酸快、丁酸转化率高的菌株, 对所筛选的菌株进行形态鉴定、16S rDNA序列分析、乳酸转化特性试验、基因组重测序和框架图测序, 并对其编码蛋白进行功能注释。【结果】 分离得到1株分离菌LY33, 基于形态和16S rDNA序列分析鉴定为丁酸梭菌。菌株LY33对乳酸具有较好的利用能力, 在生长第6天可将66 mmol的乳酸基本转化完全, 生成17 mmol左右的丁酸。LY33菌株的重测序表明, 其编码基因整体变异较小, 与参考基因组相比, LY33菌株全基因组杂合比率为0.022‰, 单核苷酸多态性(SNP)位点变异总数21 590个(占整个基因组0.4666%), 插入缺失(InDel)突变总和594个(占0.0128%), 不存在拷贝数变异(CNV), 结构变异(SV)注释的变异总数103个(占0.0003%)。突变基因主要为与菌株生长、能量代谢调节、维生素合成(特别是生物素)有关的基因, 分布于2条染色体的多条序列上。LY33菌株框架图测序及其编码基因蛋白的功能注释表明, 其基因组序列长度4 627 127 bp, GC含量28.60%, 含有4 171个基因, 编码区总长度占比84.12%, 参与了糖代谢(carbohydrate metabolism)、膜转运(membrane transport)和氨基酸代谢(amino acid metabolism)等多种代谢途径转化过程, 基因组中抗大环内酯类药物、抗杆菌肽、VanI糖肽抗性基因个数较多。【结论】 本研究从饲喂4%乳酸菌发酵饲料的健康仔猪肠道内容物中分离到1株利用乳酸快、丁酸转化率高的LY33菌株, 经鉴定为丁酸梭菌, 其具有良好的应用前景, 可作为一种新的微生物资源用于微生态制剂产品。     

固态发酵银杏叶生产蛋白饲料的研究

试验选择地衣芽孢杆菌作为发酵菌种,以粗蛋白含量为考查指标,通过L9(34)正交试验优化固态发酵银杏叶蛋白饲料工艺.结果显示,固态发酵银杏叶生产蛋白饲料的最佳工艺参数为接种量8％、含水量50％、发酵温度31℃、发酵时间84 h.经固态发酵后,银杏叶的粗蛋白、粗灰分、粗脂肪、总氨基酸含量分别提高102.16％、49.03％...     

单菌和混菌固态发酵三七渣生产蛋白饲料的动力学比较

试验对康宁木霉纯种及康宁木霉/热带假丝酵母混菌发酵三七渣生产蛋白饲料的动力学过程进行了比较分析.结果表明,与康宁木霉纯种发酵相比,康宁木霉/热带假丝酵母混菌固态发酵三七渣生产蛋白饲料无明显优势.在发酵前期,混菌发酵体系可在一定程度上促进总糖消耗,提高还原糖生成的速率;在发酵中后期,单菌发酵体系的酶活力和真蛋白质含量更高.     

Identification of lactic acid bacteria isolated from corn stovers

One hundred and twenty‐six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram‐positive, catalase‐negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover‐associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage.     

1株犬类志贺邻单胞菌的分离鉴定及序列分析

从病死犬病料中分离到1株类志贺邻单胞菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。结果表明,本菌对多种抗生素耐药,其庆大霉素耐药机制与所携带的质粒相关;本菌对小白鼠有强致病性,其LD50为1．6×10^7.4CFU。用PCR方法扩增分离菌株16SrDNA基因并测序,并将其与GenBank上其他细菌16SrDNA核苷酸序列进行同源性分析。结果表明,分离株的16SrDNA核苷酸序列与类志贺邻单胞菌（GQ359962．1）的同源性为98％,因此将该分离菌株鉴定为致病性肠球菌,命名为YN-1株（云南-1株）。     

芽孢杆菌FJ-01-R的分离鉴定及固体发酵条件的探讨

从池塘底泥分离到11株菌,3株菌产碱性蛋白酶菌,经鉴定碱性蛋白酶活性最高的FJ-01-R为芽孢杆菌,并对该菌株固体发酵产酶条件进行了优化。优化结果为发酵终止时间48h,温度32℃,初始含水量60%,初始pH值8,细菌分泌碱性蛋白酶酶活由原来的250U/g增加到540U/g,提高1.15倍。     

Classification of lactic acid bacteria isolated from chigee and mare milk collected in Inner Mongolia

One hundred and seventeen isolates from chigee and 191 isolates from mare milk collected in Inner Mongolia were classified into six and 10 groups, respectively, based on the protein profiles obtained by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A dendrogram on the basis of the molecular sizes of the protein bands was drawn and the similarity among the groups ranged from 96% to 44%. Representative isolates from all groups were identified by 16S rDNA sequencing and a phylogenetic tree was drawn. Furthermore, the results of carbohydrate fermentation tests of all groups were compared with those obtained by 16S rDNA sequencing and SDS‐PAGE analyzes. The isolates from chigee consisted of mainly Lactobacillus sp., Lactobacillus (Lb.) plantarum, Lb. pentosus and Lactococcus (Lc.) lactis ssp. cremoris were isolated at the rates of 48, 33 and 19%, respectively. The isolates from mare milk consisted of coccus, namely, Leuconostoc (Leuc.) mesenteroides, Leuc. pseudomesenteroides, Lc. garvieae, Lc. lactis ssp. lactis, Streptococcus (Sc.) parauberis and Enterococcus (Ec.) faecium. The isolation rates were 45, 19, 7, 8, 16 and 6%, respectively. The results demonstrated that the species isolated from chigee were different from those in mare milk.     

丁鲑鱼维氏气单胞菌的鉴定和生物学特性研究

对引起丁鲑鱼发病的菌株——XN602进行鉴定和生物学特性研究,经形态特征与生理生化鉴定,结果表明:该菌为革兰氏阴性杆菌,其16SrDNA序列与GenBank中多株维氏气单胞菌相似性高达99%,系统发育树显示该菌与维氏气单胞菌聚为一簇。因此,分离菌株XN602在分类学上应属于维氏气单胞菌。     

海南天然饲草青贮饲料中乳酸菌分离鉴定及优良菌株筛选

为筛选适合热带牧草的高温发酵优良乳酸菌,改善青贮发酵品质,本试验从海南岛不同气候区采集野生草本植物斑茅(Saccharum arundinaceum)、蔓生莠竹(Microstegium vagans)和木本植物银合欢(Leucaena leucocephala)、构树(Broussonetia papyrifera),青贮60 d后分离纯化乳酸菌,以培养36 h条件下最终发酵液pH≤4且OD(Optical density,吸光度)≥2作为条件初筛乳酸菌。用温度T=40℃,45℃,50℃和pH=3.0,3.5,4.0,6.0,8.0,9.0进行复筛,选取OD值较高的菌株作为优势菌株,再对其进行16S rDNA序列分析、生长和产酸速率测定以及生理生化实验。结果表明,本试验共分离到98株天然乳酸菌,经筛选得到4株优良乳酸菌(15B,10D,13C,14B)。经鉴定,10D,13C,14B为植物乳杆菌(Lactobacillus plantarum),15B为乳酸片球菌(Pediococcus acidilactici)。菌株15B,10D,13C,14B均耐酸、耐高温、且生长及产酸性能优良,具有良好的青贮潜力并对致病菌具有良好的抑制作用,可作为热带地区制备青贮添加剂的备选菌种。     

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.