细菌型豆豉发酵菌株的鉴定

为了明确细菌型豆豉发酵菌株的身份,首先采用形态及生理生化方法进行鉴定,接着提取该菌株的基因组DNA,并根据芽孢杆菌属16S rDNA序列两端的保守性片段设计引物,然后用PCR方法扩增出16S rDNA部分序列,纯化、测序,最后经BLAST搜索进行序列比对和构建系统进化树.结果表明,菌株BBDC3为革兰氏阳性芽孢杆菌,生理生化特征与枯草芽孢杆菌相符;扩增出的16S rDNA部分序列长为1344bp,与序列号为EF488171的枯草芽孢杆菌16SrDNA序列的相似度最高,为99%;系统进化树中,菌株BBDC3与枯草芽孢杆菌AB018487在同一分支.因此,菌株BBI)C3属于枯草芽孢杆菌.     

一株降解呕吐毒素蜡样芽孢杆菌的筛选与鉴定

目的:分离筛选能够降解呕吐毒素(deoxynivalenol,DON)的芽孢杆菌,以用于该毒素的生物降解。方法:采集霉变秸秆、土壤和粪便样品,先将样品加热80℃后,取上清液接种到以DON为唯一碳源的分离培养基富集DON降解菌。以LB培养基分离纯化富集菌,然后对分离到的菌株进行DON毒素降解能力检测。对降解能力最强的菌株进行形态学观察、生理生化实验和16S r DNA序列鉴定。结果:从16株分离菌中筛选得到一株对DON降解能力最强的菌株B.JG05,降解率最高可达80.61%,且对含DON饲料的降解率为82.68%。该菌株呈短杆状,能形成芽孢;生理生化特性符合蜡样芽孢杆菌的基本特征;16S r DNA序列进化树分析表明该菌株与蜡样芽孢杆菌的亲缘关系最近。结论:筛选获得了一株高效降解DON的蜡样芽孢杆菌B.JG05,为饲料和食品中DON毒素的生物降解提供了可能。     

16S rRNA基因序列、生化鉴定、质谱3类方法鉴定常见微生物的结果分析

目的比较16SrRNA基因序列、生化鉴定、质谱鉴定3类实验方法分析沙门氏菌、金黄色葡萄球菌、蜡样芽孢杆菌的鉴定结果的异同。方法挑选沙门氏菌、金黄色葡萄球菌、蜡样芽孢杆菌各50株,分别进行16S rRNA基因序列测定、VITEK COMPACT 2生化鉴定、质谱鉴定3类实验,并比较3类实验的鉴定结果。结果 3类实验方法对大部分沙门氏菌鉴定在属水平,生化鉴定方法对少数血清型鉴定到种水平;对金黄色葡萄球菌均鉴定到种水平; 16SrRNA基因序列、生化方法对蜡样芽孢杆菌鉴定到属水平,质谱鉴定到种水平。结论 3类实验方法对大部分沙门氏菌、所有金黄色葡萄球菌鉴定水平相同,质谱鉴定蜡样芽孢杆菌的结果更准确,且质谱鉴定时效性更高。     

醋醅中分离菌株W321种属鉴定的研究

试验以从醋醅中分离纯化到的菌株W321为研究对象,为进一步开发利用这一菌株,根据菌株W321的形态特征、生理生化特征和16S rDNA序列,对其进行种属鉴定的研究.经形态与生理生化鉴定菌株W321属于芽孢杆菌属,16S rDNA序列与枯草芽孢杆菌(Bacillus subtilis)的同源性达到了99％,表明与枯草芽孢杆菌极为相似,因而将菌株W.鉴定为枯草芽孢杆菌(Bacillus subtilis).     

一株葡聚糖酶产生菌的分离选育及鉴定

利用葡聚糖平板初筛,三角摇瓶复筛,从土壤样品中中分离得到一株葡聚糖酶高产菌株P5,发酵酶活力可以达到2.5U/mL。菌株呈长杆状、革兰氏染色阳性、产芽孢;生理生化实验结果表明该菌株与枯草芽孢杆菌最为相近;16S rDNA基因序列包含1510个碱基对,同源性分析显示该菌株与枯草芽孢杆菌菌株最为相近,最高相似性为99%,基于16S rDNA基因序列的系统发育分析显示,该菌株与枯草芽孢杆菌菌株CD-6及YPM8的亲缘关系最近,根据以上多相分类结果鉴定该菌株为枯草芽孢杆菌。     

3株降解玉米赤霉烯酮芽孢杆菌的快速鉴定

为了快速确定筛选的3株降解玉米赤霉烯酮的芽孢杆菌(Bacillus sp. YW、Bacillus sp. CP、Bacillus sp. GJ)的分类地位,利用形态学、生理生化特性、保守持家基因的进化分析及特异性PCR对菌株进行鉴定。3株芽孢杆菌的形态特征及生理生化特性非常相似,均产芽孢;结合16S rDNA序列分析、gyrA基因的系统发育树及特异引物PCR结果显示,菌株YW和CP属于贝莱斯芽孢杆菌(Bacillus velezensis),菌株GJ属于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。本研究表明保守持家基因序列分析及特异性PCR,能够快速、灵敏地对芽孢杆菌近缘物种进行分子鉴定,为功能性微生物菌株的快速筛选鉴定提供参考。     

芽孢杆菌LWM1的分离鉴定及其对蜡样芽孢杆菌的抑制作用

从土壤样品中分离出10株芽孢杆菌（Bacillus spp.）,以蜡样芽孢杆菌（Bacillus cereus）为指示菌株,采用牛津杯扩散法筛选出一株具有较强抑菌活性的菌株LWM1。综合形态学、生理生化试验、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）及16S rDNA基因序列同源性分析,菌株LWM1被初步鉴定为一株解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,并研究了其在马铃薯葡萄糖水（PDW）和营养肉汤培养基（NB）中发酵上清液对蜡样芽孢杆菌的抑制效果。结果表明,PDW培养基更有利于菌株LWM1生长和抑菌物质产生。因此,可利用PDW培养基为主要底物大规模发酵制备菌株LWM1的抑菌物质。     

产蛋白酶菌株的鉴定及其对虾壳的脱蛋白研究

从虾塘沉积物中分离到一株产蛋白酶的菌株C1,采用菌落形态、生理生化特征和16S rDNA基因序列分析相结合的方法进行鉴定,进一步探究其在提取虾壳甲壳素工艺中脱蛋白的应用,并与枯草芽孢杆菌（Bacillus subtilis）对虾壳蛋白脱除效果进行比较分析。结果表明,菌株C1被鉴定为一株蜡样芽孢杆菌（Bacillus cereus）,其对虾壳的脱蛋白能力高于枯草芽孢杆菌。当发酵培养基中葡萄糖添加量为50 g/L,虾壳粉添加量为20 g/L,酵母膏添加量为1 g/L时,枯草芽孢杆菌和蜡样芽孢杆菌发酵5 d的蛋白酶活力分别为145.7 U/mL、220.8 U/mL,脱蛋白率分别达到80.4%、90.8%。     

一株源于果园Alicyclobacillus的分离、鉴定及其生物学特性

目的:为了解脂环酸芽孢杆菌在果园土壤中的分布情况,对陕西果园土壤进行研究。方法:以酸化的YSG培养基分离培养脂环酸芽孢杆菌,观察菌株形态,测定生理生化特性,应用API 50CHB及API ZYM系统测定糖酵解和酶反应,以气相色谱(GC)分析脂肪酸组成,并以16S rDNA序列分析法构建系统发育树。结果:分离菌株与标准菌株的形态基本一致,酶谱有很大的相似性但糖酵解反应差异较大;16S rDNA序列分析显示,分离菌株与标准菌株属于同一属的不同种,分离菌与Alicyclobacillus contaminans的同源性达到99%。结论:采用国际通用的脂环酸芽孢杆菌的分离方法,结合API系统、脂肪酸组成分析及16S rDNA序列分析,可以快速的将分离菌鉴定到种;脂环酸芽孢杆菌在果园土壤中确有分布,可能对果汁质量造成威胁。     

一株高产蛋白酶芽孢杆菌的鉴定

通过经典生理生化实验和16S rRNA序列分析,对新分离出的1株高产蛋白酶的芽孢杆菌进行系统鉴定.研究发现该菌卵磷脂酶试验和马尿酸盐反应均为阳性,并在60℃仍有蛋白酶活性.将该菌16S rRNA序列在Genbank中进行blast,发现其与枯草芽孢杆菌EHFS1-S02Hc的16S rRNA序列同源性高达99.63%,分离出的高产蛋白酶芽孢杆菌可能为一新亚种或生物型.     

Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes

This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production. Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from <50 to 250 cfu g⁻¹ homogenized paperboard. Of those frequently found, Bacillus cereus group, B. licheniformis, B. subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures. We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B. cereus, were unreliable for industrial isolates. Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B. cereus. Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B. cereus or with B. thuringiensis. 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B. cereus group isolates and in over 30 culture collection strains. The detection of these fatty acids is a secure method for the identification of B. cereus. Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B. cereus strains producing the emetic toxin.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201^T) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.     

不同来源的植物乳杆菌基因组和抗生素抗性

为了研究不同分离源对植物乳杆菌基因组和功能的影响,选取了来自发酵酱、泡菜、粪便3种环境的33株植物乳杆菌,通过比较基因组学手段研究菌株的基因组基本特征、直系同源基因、系统进化关系,并结合功能基因注释结果与表型结果分析菌株对抗生素的耐药性。系统发育树揭示了分离源对植物乳杆菌的遗传进化具有较为显著的影响;同源基因结果表明粪便源的菌株其特殊基因数量要高于泡菜源和发酵酱源菌株的数量。33株植物乳杆菌均含有环丙沙星、四环素、氯霉素、甲氧苄氨嘧啶和万古霉素的抗性基因,菌株对这些抗生素也表现出抗性;绝大多数菌株对庆大霉素、链霉素、卡那霉素、新霉素、氨苄西林敏感,在基因组中也没有相关抗性基因;而红霉素和克林霉素的基因和表型并不对应。抗生素实验结果说明,分离源对菌株影响较小,大部分基因型与表型可以对应,基因组学对研究植物乳杆菌的生理特性起一定的指导作用。     

解淀粉芽孢杆菌YP6对三种贝壳粉的促磷研究

研究了一株从贵州磷矿植物根际分离的溶磷菌Bacillus amyloliquefaciens YP6(YP6)的促生性能及其在贝壳粉和土壤溶磷中的作用。结果表明,菌株YP6具有较好的促生作用,并且经YP6发酵贝壳粉(牡蛎粉,蛤蜊粉和生蚝粉)、土壤和贝壳粉的混合物在第7天样品中可溶磷质量浓度分别为44.9、39.2、40.3、86.4、73.1、74.8 mg/L。说明YP6对土壤和3种贝壳粉均具有显著的溶磷作用。     

The gyrase B gene as a molecular marker to resolve interspecific relationships within the Acetobacter pasteurianus group and a novel target for species-specific PCR

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays.     

枯草芽孢杆菌P spovG启动子改造与表征

枯草芽孢杆菌是重要的工业生产菌株,广泛应用于外源蛋白质的表达。目前在枯草芽孢杆菌中常见组成型启动子中,P_spovG作为高强度组成型启动子在表达外源蛋白质时,具有表达量高、稳定性好和适用范围广等优势。作者首先对P_spovG上游序列截短,发现将启动子上游序列由原来的251个碱基缩短为26个,仍能保持启动子强度不降低,并确定了紧邻-35元件上游的3个碱基“AGC”为影响启动子强度的关键碱基。其次对启动子的上游A-T富含区以及spacer区域的G-C富含区进行随机突变,证明了上游激活序列中A-T碱基的排列对启动子强度有重要影响,并得到了强度提高的突变体。最后通过将不同表达时期启动子的关键调控序列分别插入突变体的上下游,得到了启动子强度为原始启动子135.1%的新型启动子SP_spovG-P_lytR。本研究为枯草芽孢杆菌表达系统开发新型强组成型启动子提供了重要的参考。     

枸杞多糖体外调节人体肠道菌群的功能研究

为研究枸杞多糖(LBP)对人体肠道菌群结构和代谢产物的影响,对6名健康人的粪便提取物进行单独样本与混合样本的体外模拟厌氧发酵,利用16S rRNA基因高通量测序技术对发酵后肠道菌群进行结构和功能分析,并利用超高效液相色谱(UPLC)检测发酵液中的短链脂肪酸(SCFAs)浓度。结果表明,LBP能够明显改变人体肠道菌群结构与功能,提高肠道菌群中益生菌乳酸杆菌属与双歧杆菌属的丰度,并促进了SCFAs的产生。因此,LBP能够显著影响人体肠道菌群结构与功能。     

蜂粮中Lactobacillus kunkeei的益生特性研究

蜂粮是植物花粉、蜂蜜和蜜蜂唾液的发酵混合物,是营养丰富的天然食材。作者从蜂粮中分离到61株乳酸杆菌,经16S rRNA基因测序,鉴定为Lactobacillus kunkeei。首先,用邻苯二甲醛法对61株L.kunkeei进行胆固醇去除能力检测,其中5株菌胆固醇去除能力均大于15%,菌株B35对胆固醇的去除率达到(29.07±1.30)%。随后,对这5株L.kunkeei进行酸耐受性、胆盐耐受性、抗生素耐药性及抑菌等益生特性进行研究。耐酸实验和耐胆盐实验结果表明,5株L.kunkeei均具有良好的耐酸性和胆盐耐受性。药敏试验结果表明,5株L.kunkeei对4种临床常用抗生素(红霉素、克林霉素、庆大霉素、万古霉素)均敏感。通过琼脂扩散法抑菌实验,发现5株L.kunkeei对大肠埃希氏菌、金黄色葡萄球菌均有一定的抑菌作用。B35菌株的细胞黏附性能良好,对Caco-2细胞的黏附率达到3.32%。从蜂粮中分离到5株具有良好益生特性的菌株,其中菌株B35的益生特性最佳,为益生菌的开发与利用提供了新的菌种资源。