真菌玫瑰红钠培养基平皿计数验证法的改进

为了避免玫瑰红钠培养基平皿法计数验证时细菌在培养基上生长而干扰真菌计数的准确性这一问题,采用在该培养基中选加了0.08g/L、0.10g/L和0.12g/L三种不同浓度的氯霉素的方法。结果证实,在玫瑰红钠培养基中加入浓度为0.10g/L的氯霉素可完全抑制供试大肠杆菌CMCC(B)44102株的生长,但又不影响供试真菌如白色念珠菌CMCC(F)98001、黑曲霉CMCC(F)98003的培养生长。因此,在微生物限度检查时的玫瑰红钠培养基中加入氯霉素最适宜的有效浓度为0.10g/L。     

一株卡他布朗汉姆菌的重新鉴定

目的对中国医学细菌保藏管理中心库藏的一株卡他布朗汉姆菌CMCC(B)29103株进行重新鉴定。方法用营养琼脂培养基培养CMCC(B)29103株,对其进行形态观察、生理生化特性、脂肪酸组分、分子生物学等多相鉴定,同时与模式株DSM25388T相对照;分析CMCC(B)29103株的特征属性、进化位置以及与Acinetobacter indicus模式株DSM25388T的同源性。结果形态学特性、生理生化以及脂肪酸组分构成均与DSM25388T株十分相似,仅存在个别差异;16 S rRNA基因序列比对显示,CMCC(B)29103株与Acinetobacter(不动杆菌属)相近,与模式株DSM25388T相似性最高,为99.85%。基于Acinetobacter属所有成员的16 S rRNA和rpo B基因的系统进化分析均显示CMCC(B)29103与DSM25388T稳定聚类成一个独立分支,且二者的DNA-DNA同源性为78.3%。结论CMCC(B)29103株属于Acinetobacter indicus种,与模式株DSM25388T为不同的菌株,可将其更名为Acinetobacter i ndicus。     

一株分离自铀矿酸性浸出液霉菌的鉴定与碳源代谢分析

旨在通过形态学观察、r DNA ITS序列分析以及Biolog微生物鉴定系统对霉菌进行鉴定,并通过计算平均吸光值(AWCD)了解霉菌的生长情况以及各类碳源的消耗量,运用数据分析霉菌的碳源利用情况。结果表明,结合r DNA ITS序列、形态学观察以及Biolog鉴定系统确定菌株为黑曲霉;比较8类碳源消耗情况,可知对氨基酸类、脂类、酸类的利用最多,根据碳源代谢情况列出15种最先利用碳源。鉴定菌株为黑曲霉,且该菌株的最适碳源为P-羟苯乙酸,可作为耐酸曲霉代谢研究的材料。     

一株产赭曲霉毒素A黑曲霉的筛选与鉴定

【目的】筛选出可产生赭曲霉毒素A (OTA)的霉菌菌株。【方法】利用CYA和YES培养基从实验室32个霉菌样品中筛选目的菌株。利用高效液相色谱-荧光检测法对OTA产生菌进行初筛,利用高效液相色谱-质谱联用对OTA初筛菌株进行复筛。通过菌落形态、菌丝及分生孢子形态、ITS DNA序列、β-Tubulin基因序列及Calmodulin基因序列分析等鉴定目的菌株。【结果】得到一株OTA产生菌株1062,该菌株能在25 °C条件下,在CYA、YES和CA培养基中很好生长。结合形态学、对培养基的要求以及上述3个基因序列的进化树分析,该菌株属于黑曲霉(Aspergillus niger)。【结论】菌株1062具有OTA产生能力,是一株黑曲霉。     

一株支气管炎鲍特杆菌国家标准菌种的再鉴定

通过生化试验、质谱鉴定、16S rRNA基因序列分析、全基因组序列测定及生物信息学分析等方法对国家标准菌种CMCC(B)40001进行再鉴定。对菌株的毒力、耐药和MLST进行分析，并对鲍特杆菌（Bordetella spp.）的群体进化进行分析。结合不同方法，CMCC(B)40001再鉴定为支气管炎鲍特杆菌（Bordetella bronchiseptica），且其作为标准菌种能够发挥国家标准描述的质控作用。同时获取该菌株的新ST型，在进化关系上，其与百日咳鲍特杆菌(Bordetella pertussis)和副百日咳鲍特杆菌(Bordetella parapertussis)遗传距离近。本研究再鉴定了国家标准菌种CMCC(B)40001为支气管炎鲍特杆菌，并分析了其遗传特征，为国家标准菌种的应用提供资源描述支持。     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

黑曲霉mnn9基因缺失株的构建及其功能分析

本研究通过分析比较黑曲霉基因组与酿酒酵母基因组序列同源性,分离鉴定了黑曲霉mnn9基因。通过同源重组,在黑曲霉GICC2773(ΔAP4:pGPT-laccase)菌株中敲除了mnn9基因。该黑曲霉mnn9基因缺失使外源蛋白漆酶的分泌表达提高了14%,内源蛋白葡萄糖淀粉酶的分泌表达则降低了4%。     

一株拮抗放线菌的筛选和初步分类鉴定

从杨树根际土壤样品中分离的一株放线菌,对供试菌株圆弧青霉、辣椒疫霉、镰刀菌、黑曲霉具有拮抗作用,测定了该菌株的形态学特征、培养特征以及生理生化的特征,根据所得结果发现该菌株所表现出的各类特征与链霉菌的所属特征相似,初步鉴定为链霉菌。     

金荞麦和苦荞麦抗菌活性内生真菌的筛选及鉴定

从药用植物金荞麦和苦荞麦的根、茎、叶、花中分离到62株内生真菌,并以金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)[CMCC(B)63501]、小麦赤霉病菌(Fusarium graminearum)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cu-cumerinum)和绵腐病菌(Pythium aphanidermatum)6种微生物为指示菌对分离到的内生真菌进行抗菌活性检测。结果发现,分离的内生真菌菌株KQH-01、KQH-02和JQY-1的发酵醇提取物具有较好的抑菌活性。形态学特征和分子鉴定确定菌株KQH-01为炭角菌属(Xylaria sp.)真菌,菌株KQH-02为球毛壳菌(Chaetomium globosum),菌株JQY-1为葡萄座腔菌(Botryosphaeria dothidea)。     

一株产天然蓝色素海洋链霉菌的筛选鉴定

从胶州湾附近海域水样中分离筛选到一株可产生天然蓝色素的链霉菌。采用通用引物16F27/16R1492扩增该菌株的16SrDNA,对测序结果进行序列分析,采用Neighbor-Joining(N-J)法构建系统发育进化树,同时结合链霉菌的传统形态学和生理生化特性对菌株进行鉴定。16SrDNA序列分析表明菌株与Streptomyces violaceolatus KCTC 9772相近,相似率为99%。在Neighbour-Joining法构建的系统发育进化树中,该菌株与Streptomyces violaceolatus聚类在同一分枝上。因此将该菌株鉴定为紫边链霉菌Streptomyces violaceolatus。     

Development of enzyme technology for Aspergillus oryzae,A. sojae,and A. luchuensis,the national microorganisms of Japan

This paper describes the modern enzymology in Japanese bioindustries. The invention of Takadiastase by Jokiti Takamine in 1894 has revolutionized the world of industrial enzyme production by fermentation. In 1949, a new γ-amylase (glucan 1,4-α-glucosidase, EC 3.2.1.3) from A. luchuensis (formerly designated as A. awamori), was found by Kitahara. RNase T₁ (guanyloribonuclease, EC 3.1.27.3) was discovered by Sato and Egami. Ando discovered Aspergillus nuclease S₁ (single-stranded nucleate endonuclease, EC 3.1.30.1). Aspergillopepsin I (EC 3.4.23.18) from A. tubingensis (formerly designated as A. saitoi) activates trypsinogen to trypsin. Shintani et al. demonstrated Asp76 of aspergillopepsin I as the binding site for the basic substrate, trypsinogen. The new oligosaccharide moieties Man₁₀GlcNAc₂ and Man₁₁GlcNAc₂ were identified with α-1,2-mannosidase (EC 3.2.1.113) from A. tubingensis. A yeast mutant compatible of producing Man₅GlcNAc₂ human compatible sugar chains on glycoproteins was constructed. The acid activation of protyrosinase from A. oryzae at pH 3.0 was resolved. The hyper-protein production system of glucoamylase was established in a submerged culture.     

The Pathogenesis of Aspergillus fumigatus,Host Defense Mechanisms,and the Development of AFMP4 Antigen as a Vaccine

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines’ current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.     

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans

AIMS: To determine the incidence and severity of infection by ochratoxin A (OA)-producing fungi in Vietnamese green coffee beans. METHODS AND RESULTS: Aspergillus carbonarius, A. niger and yellow Aspergilli (A. ochraceus and related species in section Circumdati) were isolated by direct plating of surface-disinfected Robusta (65 samples) and Arabica (11 samples) coffee beans from southern and central Vietnam. Significantly, more Robusta than Arabica beans were infected by fungi. Aspergillus niger infected 89% of Robusta beans, whereas A. carbonarius and yellow Aspergilli each infected 12-14% of beans. OA was not produced by A. niger (98 isolates) or A. ochraceus (77 isolates), but was detected in 110 of 113 isolates of A. carbonarius, 10 isolates of A. westerdijkiae and one isolate of A. steynii. The maximum OA observed in samples severely infected with toxigenic species was 1.8 microg kg(-1); however, no relationship between extent of infection and OA contamination was observed. CONCLUSIONS: Aspergillus niger is the dominant species infecting Vietnamese coffee beans, yet A. carbonarius is the likely source of OA contamination. SIGNIFICANCE AND IMPACT OF STUDY: Vietnamese green coffee beans were more severely infected with fungi than the levels reported for beans from other parts of the world, yet OA contamination appears to be infrequent.     

曲霉属黄绿组的一个产毒新种

本文报道曲霉属黄绿组(通常称黄曲霉群)的一个产毒新种肇庆曲霉(Aspergillus zhaoqingensis sp.nov.)。该菌分离广东肇庆土壤,在形态上近于米曲霉(A.oryzae),但作为该组的关键分类特征分生孢子纹饰很不相同:本种的分生孢子明显的粗疏粗糙至具不规则的脊状突起而米曲霉则为光滑或稍粗糙。本种能产生黄曲霉毒素B_1而米曲霉则不产生。本菌亦不同于组内其它菌种。文中对与黄曲霉密切相关诸种也作了简短的讨论。     

黄曲霉和米曲霉的多相鉴定方法

【背景】黄曲霉(Aspergillus flavus)和米曲霉(Aspergillus oryzae)形态特征相近,基因组高度相似,较难区分。【目的】旨在总结一套准确鉴别二者的分类方法。【方法】利用22株标准菌株对传统形态学、产毒培养基、酶联免疫毒素检测、系统发育分析、产毒基因检测等5种鉴别方法分别进行验证。【结果】各鉴定方法的结果存在异同,单一的鉴定方法容易出现假阴性或假阳性结果。【结论】利用单一方法区分黄曲霉和米曲霉具有潜在风险,多相鉴定方法可以准确鉴别二者。     

Development of VNTR markers for three Aspergillus species used in brewing

Using a bioinformatics approach, we developed 18 variable number of tandem repeat markers for Aspergillus oryzae for use in population genetic studies. Repeat sequences in the genome sequences of A. oryzae were identified by a tandem repeat finding program. Length polymorphisms at 18 loci were examined in 41 strains of A. oryzae. The total number of alleles per locus ranged from two to 20. Investigation of cross-species amplifications with A. sojae and A. tamarii showed success. The variable number of tandem repeat markers will be used to determine the population structure of these three Aspergillus species used in brewing.     

米曲霉和黑曲霉营养缺陷型的分离及原生质体的制备

米曲霉(Aspergillus oryzae)3042是目前国内酱油生产中广泛使用的菌种,而黑曲霉(Aspergillus niger)3350则是制醋业中广泛使用的菌种。前者具有较高的蛋白酶活性而后者具有较高的淀粉酶活性。在酱油生产中,为了提高原料利用率,改善酱油风味,希望获得一株既有较高的蛋白酶活性同时又具有较高淀粉酶活性的杂交菌株作为     

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL⁻¹, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).