无细胞百日咳疫苗效价检定用攻击菌液氮保存的稳定性

为了考察无细胞百日咳疫苗效价检定用攻击菌在液氮中保存的稳定性,对液氮保存1~7年的4~9代百日咳攻击菌随机抽取7~11批次样品,采用LD50检测并经统计学处理,分析其样品的毒力变化。试验结果显示,4~9代的百日咳攻击菌液氮保存5年,毒力仍能达到《中华人民共和国药典》三部(2005版)的要求;保存6年和7年,4~7代的百日咳攻击菌毒力仍符合上述要求,8、9代的攻击菌毒力的合格率分别为84%和40%。试验证实,无细胞百日咳效价检定用攻击菌液氮保存5年具有良好的稳定性,可用于无细胞百日咳疫苗的效价检定。     

液氮保存疫霉属菌种存活期检测

对Phytophthora和Peronophythora所属12个种29株菌在液氮中保存5年零8个月至6年零3个月后检测证明有68.9％的菌种存活下来。但有些种未能存活,这些种有Phytophthora colocasiae(XH30),P.drechsleri(ATCC15428),P.erythroseptica(ATCC36302)与Peronophythora litchii(ATCC 34595)。即使存活的菌种也不一定每个保存的菌株与菌块都存活。说明在液氮中也有存活力下降的现象。除菌种本身耐深冻贮藏特性不同外,贮藏前菌种培养的旺盛程度明显影响存活。对一些菌的致病性测定证明长期保存后致病力维持不变。液氮保藏不失为保持菌种长期不变的良好方法,只是要严格按要求掌握,并对菌的耐冻力先行了解。     

具有ACC脱氨酶活性的杜仲内生细菌的分离鉴定及其抗菌活性

采用富集筛选法从杜仲根中分离到5株具有ACC脱氨酶活性的内生细菌, 利用纸片法测定它们的抑菌活性, 通过形态特征、生理生化试验和16S rRNA序列分析对分离菌株进行鉴定。结果显示, 5株杜仲内生细菌均具有较高的ACC脱氨酶活性, 其中4株菌对大肠杆菌CGMCC1.1103和枯草芽孢杆菌CGMCC1.769均有较好的抑菌活性, 通过生理生化试验和16S rRNA序列分析, 将菌株JDM-2、JDM-8、JDM-11、JDM-14和JDM-19分别鉴定为Pseudomonas koreensis、肺炎克雷伯氏菌(Klebsiella pneumoniae)、路德维希肠杆菌(Enterobacter ludwigii)、变栖克雷伯氏菌(Klebsiella variicola)和阿氏肠杆菌(Enterobacter asburiae)。     

青蒿内生真菌的抗肿瘤抗氧化活性

采用MTT法和DPPH法,分别测定已分离得到的68株贵州青蒿内生真菌乙酸乙酯粗提物的肿瘤细胞生长抑制率和DPPH自由基清除率。试验共筛选获得12株活性内生真菌,根据其形态特征进行鉴定,分别隶属于子囊菌亚门(Ascomycotina)的链格孢属(Alternaria)、刺盘孢属(Colletotrichum)、拟盘多毛孢属(Pestalotiopsis)和拟茎点霉属(Phomopsis);其中,有8株内生真菌至少对1种指示瘤株具有细胞毒活性,占总菌株数的11.8%;5株内生真菌具有不同程度的清除DPPH自由基活性,占总菌株数的7.4%;1株内生真菌同时具有细胞毒活性和抗氧化活性。     

酶和发酵工程

861949蛋白酶对黑曲霉C突变体合成葡糖糖化酶的影响【英〕/Fiedurek,J.…了ActaMierobiol.Pol一1985,34(1)一25一32〔译自DBA,1985,4(8),85一04112〕 用紫外光照射和用0.01%亚硝基肌处理黑曲霉C分生抱子悬液,这株菌具有葡糖糖化酶合成活性。由分生抱子长出的1230个培养物中选到     

药用植物内生放线菌的分离、筛选及活性菌株YIM 61470鉴定

从云南西双版纳热带雨林多种药用植物中分离到272株内生放线菌,活性筛选表明 146株菌的发酵产物具有抗菌活性,其中94株菌具有拮抗病原细菌活性,127株菌具有抑制病原真菌的功能.分离菌株YIM 61470具有广谱抗菌活性,通过形态特征、培养特征、生理生化特征、细胞化学分类特征和基于16S rRNA基因序列的相似性分析等研究,菌株YIM 61470被鉴定为链霉菌属(Streptomyces)氢化链霉菌(S.llydrogenans)的一个菌株.     

冷冻干燥保藏曲霉菌种的效果

本文报告了用冷冻干燥法保藏曲霉属（Aspergillus）5种8株曲霉的效果,并分别对这些曲霉菌株的糖化酶活力进行检测。这些曲霉菌株经过冷冻干燥保藏8年后全部保持生活能力,其培养及形态特征除一株生长稍差外,其余菌株均保留原有形状,测定其糖化酶活力未有明显变化。     

超低温冷冻对益智种子生理生化特性的影响

益智(Alpinia oxyphylla)是我国四大南药之一,其种子为顽拗性种子,不耐干燥和低温,无法常规保存。为解决益智种子长期稳定保存的问题,该研究以益智种子为材料,从种子含水量以及冷冻方式方面优化益智种子超低温保存方法,并对比液氮冷冻前后种子发芽率、α-淀粉酶活性、超氧化物歧化酶活性、过氧化物酶活性、脱氢酶活性、丙二醛含量等活力指标,分析液氮超低温冷冻对益智种子生理生化特性的影响。结果表明:直接液氮冷冻含水量13.92%~14.38%范围的种子是益智种子超低温保存的最佳条件。液氮冷冻后,随着超低温冷冻时间的延长,益智种子发芽率先由76.76%下降至52.5%,再上升至65%,最后下降并稳定在50%以上。其种子α-淀粉酶活性先下降后上升,然后又下降,最后上升并稳定至冷冻前的水平。丙二醛含量则是先上升后下降,最后趋于平稳状态。过氧化物酶活性是先下降,然后稳定在恒定水平。超氧化物歧化酶活性是随着冷冻时间的延长而持续上升,脱氢酶活性则是持续下降。以上结果说明,液氮冷冻时间对益智活力有一定的影响,但对种子有些性能具有一定的促进作用。无论是发芽率,还是生理生化指标,都不同程度表明液氮超低温保存益智种子是可行的。     

硇洲岛褐蓝子鱼肠道抗菌活性菌株的分离及多样性

【目的】了解广东湛江硇洲岛褐蓝子鱼(Siganus fuscescen)肠道抗菌物质产生菌的多样性。【方法】采用传统的分离方法与牛津杯扩散法对褐蓝子鱼肠道菌进行分离纯化与抗菌活性测定,运用16S rRNA基因序列的系统发育分析对抗菌活性菌株进行多样性分析。【结果】根据形态观察和部分生理生化实验去冗余,从褐蓝子鱼肠道样品中分离到68株细菌,其中抗菌活性菌株19株,占分离株的27.9%。19株不同种类的活性菌株分属于细菌域的放线杆菌门(Actinobacteria)、变形杆菌门(Proteobacteria)与厚壁菌门(Firmicutes)的11个科、12个属。多数菌株属于厚壁菌门(8株,42.1%),其次是变形杆菌门(7株,36.8%)和放线杆菌门(4株,21.1%)。大多数抗菌活性菌株与其系统发育关系最密切的已知典型菌株之间存在一定的遗传差异(16S rRNA基因序列相似性为96.2%-99.9%),其中有4株代表潜在的新种。【结论】广东湛江硇洲岛褐蓝子鱼肠道中存在较为丰富的细菌抗菌物质产生菌,并蕴藏着较多的微生物新类群。     

东南极格罗夫山土壤可培养细菌的分离鉴定及其产胞外酶和抗菌活性北大核心CSCD

【目的】获得东南极格罗夫山地区土壤中可培养细菌组成信息,分析菌株胞外水解酶产生及抗菌活性情况。【方法】稀释直接涂布平板法获得可培养细菌菌株并根据其16S rRNA基因序列对其进行系统发育分析。平板法初步鉴定菌株的胞外酶产生情况,琼脂块法分析菌株对5种供试菌的抗性。【结果】所有土壤样品共分离出39株可培养细菌,分属于厚壁菌门(Firmicutes,19株,48.7%)、变形菌门(Proteobacteria,分属于α-、β-、γ-变形菌纲,共计10株,25.6%)、放线菌门(Actinobacteria,8株,20.5%)、拟杆菌门(Bacteroidetes,1株,2.6%)和异常球菌-栖热菌门(Deinococcus-Thermus,1株,2.6%)等5门20个菌属,优势菌属为芽孢杆菌(Bacillus)。不同冻存温度的土壤样品分离菌株有所不同。33株细菌具有至少一种的胞外酶活力,其中产淀粉酶的菌株最多(25株,64.1%)。6株细菌可抑制至少一种供试菌的生长。【结论】格罗夫山土壤可培养细菌组成在大分类上与南极其他地区一致,但在属的水平上有所不同。分离出的具有胞外酶活性和抗菌活性的适冷菌株为进一步开发利用南极低温酶和抗菌活性物质提供了良好的菌种资源。     

利用黑曲霉单宁酶酶法制取没食子酸的研究

利用已有的 10株高单宁酶活性的菌株为起始菌 ,经活化分离选择 ,借助Ⅱ级发酵培养程序、生物转化、结合TLC分析进行筛选实验。最后选出具有高单宁酶活性的 1号和 5 0号菌株 ,开展了没食子酸 (GA)克量级生物转化法制备实验 ,结果表明 ,本酶法工艺是可行的 ,在发酵液中GA的浓度分别达到2 0 .6mg/ml和 2 1 3mg/ml,产品产率 (以从五倍子提取的单宁酸计 )达到 41 2 %和 42 6 % ,具有潜在的工业开发价值     

植酸钠对黑曲霉柠檬酸发酵产酸的促进效应

研究了植酸钠对黑曲霉柠檬酸发酵产酸的促进效应。在葡萄糖全合成培养基中添加１％的植酸钠,可使产酸比对照提高２．４倍;在薯粉、玉米粉等天然培养基中添加１％植酸钠,柠檬酸产量分别提高１．７倍和１．３倍。酶活性测定分析表明,植酸钠对柠檬酸代谢途径中的几种关键酶的活性有影响。     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

低功率He-Ne激光照射黑曲霉的方法与效应

目的:探索低功率He-Ne激光对柠檬酸生产菌黑曲霉诱变育种的简易方法。方法:应用带扩束镜的低功率He-Ne激光装置,在无菌条件下对柠檬酸生产菌黑曲霉的单孢子膜进行不同时间的垂直照射,无菌水洗脱,指示性平板筛选,液体培养,测定黑曲霉诱变菌株的柠檬酸产量。结果:不同时间的激光照射黑曲霉单孢子,其存活率与激光照射时间没有线性关系。激光照射6min,黑曲霉M2代产酸的正变率高达37.5%,而此时的存活率也高达40.0%。获得了黑曲霉柠檬酸产酸率提高了10%的突变菌株,为黑曲霉的遗传育种提供了材料。结论:这种方法便于在无菌条件下操作,保证了激光照射黑曲霉单孢子的均匀性,是一种简便易行的微生物诱变育种方法。     

Ellagic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in solid state fermentation of pomegranate residues

Two Aspergillus niger strains (GH1 and PSH) previously isolated from a semiarid region of Mexico were characterized for their effectiveness in converting pomegranate ellagitannins (ET) into ellagic acid (EA) in a solid state fermentation (SSF). Pomegranate seeds and husk were used as support for the SSF. Released EA was evaluated by liquid chromatography. Yields of 6.3 and 4.6 mg of EA per gram of dried pomegranate husk were obtained with A. niger GH1 and PSH, respectively. Total hydrolyzable polyphenols of pomegranate husk were degraded during the first 72 h of culture (71 and 61%, by GH1 and PSH strains, respectively). Tannin acyl hydrolase activity was not clearly associated with EA production. EA that accumulated in cultures of A. niger GH1 was remarkably pure after a simple extraction process. Pomegranate husk is a good support, and at the same time an excellent substrate in the production of high commercial interest metabolites like EA due the degradation of its ET content.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

Isolation and evaluation of tannin-degrading fungal strains from the Mexican desert

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source.     

Microbial transformation of dehydropinguisenol by Aspergillus sp

Two metabolites were obtained by microbial transformation of a furanosesquiterpene alcohol, dehydropinguisenol, using Aspergillus niger and Aspergillus cellulosae. Their structures were established as 10-oxo-lejeuneapinguisenol and lejeuneapinguisenol on the basis of their spectroscopic data. The latter compound was obtained after 4 and 9 days of incubation with A. cellulosae at 30 degrees C and 25 degrees C, respectively. Aspergillus niger produced both metabolites after 3 and 5 days incubation at 30 degrees C, respectively. A possible pathway for the formation of these compounds is discussed here together with their antimicrobial activity against A. niger and A. cellulosae.     

Fumonisin and ochratoxin production in industrial Aspergillus niger strains

Aspergillus niger is perhaps the most important fungus used in biotechnology, and is also one of the most commonly encountered fungi contaminating foods and feedstuffs, and occurring in soil and indoor environments. Many of its industrial applications have been given GRAS status (generally regarded as safe). However, A. niger has the potential to produce two groups of potentially carcinogenic mycotoxins: fumonisins and ochratoxins. In this study all available industrial and many non-industrial strains of A. niger (180 strains) as well as 228 strains from 17 related black Aspergillus species were examined for mycotoxin production. None of the related 17 species of black Aspergilli produced fumonisins. Fumonisins (B(2), B(4), and B(6)) were detected in 81% of A. niger, and ochratoxin A in 17%, while 10% of the strains produced both mycotoxins. Among the industrial strains the same ratios were 83%, 33% and 26% respectively. Some of the most frequently used strains in industry NRRL 337, 3112 and 3122 produced both toxins and several strains used for citric acid production were among the best producers of fumonisins in pure agar culture. Most strains used for other biotechnological processes also produced fumonisins. Strains optimized through random mutagenesis usually maintained their mycotoxin production capability. Toxigenic strains were also able to produce the toxins on media suggested for citric acid production with most of the toxins found in the biomass, thereby questioning the use of the remaining biomass as animal feed. In conclusion it is recommended to use strains of A. niger with inactive or inactivated gene clusters for fumonisins and ochratoxins, or to choose isolates for biotechnological uses in related non-toxigenic species such as A. tubingensis, A. brasiliensis, A vadensis or A. acidus, which neither produce fumonisins nor ochratoxins.     

Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production

AIMS: To exploit conidiospores of Aspergillus niger as a vector for glucose oxidase extraction from solid media, and their direct use as biocatalyst in the bioconversion of glucose to gluconic acid. METHODS AND RESULTS: Spores of A. niger (200 h old) were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found retained in the spores even after repeated washings. Average rate of reaction was 1.5 g l(-1) h(-1) with 102 g l(-1) of gluconic acid produced out of 100 g l(-1) glucose consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide. CONCLUSIONS: Spores of A. niger served as efficient catalyst in the model bioconversion reaction after permeabilization. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detailed study on the ability of A. niger spores to act as reservoir of enzyme synthesized during SSF without its release into solid media. Use of this material served as an innovative concept for enzyme extraction and purification from a solid medium. Moreover, this approach could compete efficiently with the conventional use of mycelial form of the fungus in gluconic acid production.