酵母拮抗菌的抑病效力、规模化培养及相关机理研究

我们实验室从果实表面分离获得的酵母拮抗菌已经证明能有效防治各种果实采后主要病害，为了加快生物拮抗菌的商业化应用，本文在完善拮抗菌抑病机理的基础上，重点研究了拮抗菌规模化培养条件，生物菌剂制品的稳定性，以及拮抗菌对环境胁迫的生理反应。主要研究内容包括：（1）分析酵母菌拮抗菌、病原菌与果实之间的互作效应及其影响因子;（2）筛选酵母拮抗菌规模化培养的最佳营养配方及培养条件;（3）优化酵母拮抗菌干粉与液体剂型的制备方式;（4）研究酵母拮抗菌在不同剂型中生活力下降的可能机理;（5）探讨酵母拮抗菌次生代谢产物的抑菌效果。研究结果如下： 1、单独接种Monilinia fructicola或同时接种M. fructicola和Cryptococcus laurentii均能诱导甜樱桃果实SOD、CAT和POD等抗氧化酶活性升高并加速脂质过氧化，同时伴有PPO同工酶新酶带出现。病原菌M. fructicola和Penicillum expansum在接种初期均显著促进拮抗菌C. laurentii在桃果实伤口处的生长。C. laurentii在接种24h内显著抑制桃果实LOX活性、O2•-产生与H2O2积累。单独接种病原菌能显著诱导桃果实LOX活性升高，促进O2•-产生，但抑制H2O2积累。病菌侵染后果实中 O2•-增加，以及H2O2的降低可能是桃果实对病原菌侵染的一种生理应答方式。 2、抗坏血酸钠能显著提高C. laurentii对甜樱桃果实褐腐病的防治效果，较低浓度的拮抗菌（ 1×107 cells mL-1）与200mM抗坏血酸配合使用可以达到较高浓度拮抗菌（1×108 cells mL-1）单独使用对M. fructicola的防治效果。抗坏血酸钠的协同抑病机理可能是在抑制病原菌生长的同时，也抑制了果实的抗氧化酶活性，从而加速了脂质过氧化过程。 3、酵母菌产业化培养条件的筛选结果表明，不同拮抗菌对培养基中营养物质的需求不一样，培养所需的温度有差异。在120L发酵罐的中试实验表明两种拮抗菌采用筛选出的最佳培养条件均得到浓度大于1× 109 CFU mL-1的菌悬液。 4、保护剂种类是影响Rhodotorula glutinis 和 C. laurentii两种酵母拮抗菌冷冻干燥效果的最主要因素，但保护剂效果的发挥依赖于其浓度与酵母菌生长阶段。无菌水和PBS（100mM, pH5.8）可以作为拮抗菌C. laurentii液体剂型的有效保护剂，而柠檬酸钠（100mM, pH5.8）则诱导拮抗菌C. laurentii的生活力快速丧失。 5、酵母菌拮抗菌冻干制品的研究表明，在胁迫环境下酵母菌生活力快速丧失与大量产生活性氧有关，这暗示活性氧的产生可能是导致酵母菌细胞死亡的主要因素。柠檬酸钠（100mM, pH5.8）对C. laurntii死亡的诱导效应受柠檬酸根浓度和介质酸度的双重影响。活性氧在柠檬酸钠诱导酵母菌生活力快速丧失中大量产生并发挥重要作用。 6、酵母拮抗菌能够产生某些对果实采后病原真菌具有抑制效果的挥发性和不挥发性物质。同一种酵母菌产生的物质对不同病原菌有不同的拮抗效果，而不同酵母菌对同一种病原菌的拮抗效果也不完全相同。但是，不同类型的培养基对拮抗菌产生的抑菌物质有明显的影响。

海藻糖提高酵母拮抗菌生活力和生防效力的作用机制

近年来，利用酵母拮抗菌进行果实采后病害的生物防治已经成为果实采后领域的研究热点。但是，在实际应用中生物拮抗菌制剂的防病效果远不如化学药剂稳定。由于生物拮抗菌是活体，生活力和生防效力易受诸多因素影响。在商品化制剂的剂型加工、销售、以及使用过程中的环境条件往往影响酵母拮抗菌的生活力和抑病能力，成为酵母拮抗菌产业化生产和商品化应用过程中的一个主要障碍。将酵母拮抗菌和其它化学物质配合使用可以提高拮抗菌的生防效力。另外，增加酵母拮抗菌对逆境条件的耐受力也是增加或稳定其防治效果的有效途径。本文研究了海藻糖与酵母拮抗菌生活力和生防效力的关系，通过生理手段提高了酵母拮抗菌内源海藻糖的含量，同时探讨了在多种逆境条件下内源海藻糖含量对酵母拮抗菌生活力和生防效力的影响及其作用机制。主要研究结果如下： 1. 以1 %海藻糖作为碳源培养酵母拮抗菌Cryptococcus laurentii可以提高其内源海藻糖含量。在in vitro试验中，提高内源海藻糖含量可以提高C. laurentii在低温（1 ºC）、气调（1 ºC，5 % O2，5 % CO2）条件下的生活力;在in vivo试验中，提高C. laurentii内源海藻糖含量可以提高其在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。内源海藻糖的积累还能提高C. laurentii冷冻干燥后的生活力，海藻糖对酵母细胞质膜的保护作用可能是一个主要原因。 2. 以1 %海藻糖作为碳源能提高酵母拮抗菌Rhodotorula glutinis的内源海藻糖含量。内源海藻糖含量的增加可以提高C. laurentii和R. glutinis在慢速冷冻处理中的生活力。同时，海藻糖作为外源保护剂可以明显提高两种酵母拮抗菌在冷冻干燥处理后的生活力。在快速冷冻、慢速冷冻和冷冻干燥处理中，提高酵母拮抗菌的内源海藻糖含量并使用海藻糖作为外源保护剂可以获得更高的生活力。同时，这种内、外源保护因子的综合作用也可以提高两种拮抗菌在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。 3. 脱脂牛奶和糖（葡萄糖，半乳糖，蔗糖，海藻糖）作为保护剂可以提高C. laurentii冷冻干燥后在常温（25 ºC）和低温（4 ºC）保存过程中的生活力。脱脂牛奶和糖保护剂的复合使用对C. laurentii的保护效果高于其单独使用的效果。通过对几种常用的碳源进行筛选，发现柠檬酸作为碳源对C. laurentii内源海藻糖的积累有明显的诱导作用。当使用相同的保护剂或保护剂组合时，高内源海藻糖含量的酵母拮抗菌生活力更强。当冷冻干燥前使用半乳糖 + 脱脂牛奶作为保护剂时，高内源海藻糖含量的C. laurentii在4 ºC保存90 天后对苹果青霉病的防治效果和与新鲜培养的酵母拮抗菌相当。 4. 酵母拮抗菌C. laurentii冷冻干燥后在常温(25 ºC)保存期间，细胞生活力下降，细胞膜的完整性降低，胞内活性氧水平增加，同时与抗氧化相关的超氧化物歧化酶（SOD）活性也增加，而过氧化氢酶（CAT）活性则下降。提高内源海藻糖含量和/或使用外源保护剂（5 %脱脂牛奶 + 10 %葡萄糖）可以减缓上述指标下降或上升的速度。内、外源因子的共同作用有利于提高对拮抗菌细胞的保护作用。 5. 利用褐藻酸钠制成的含酵母拮抗菌的胶球在干燥后能有效的保持C. laurentii的生活力。在3种不同粘度的褐藻酸钠中，0.5 % 3500 cp的褐藻酸钠表现出较好的保护效果。内源海藻糖的积累可以提高干胶球中C. laurentii的生活力，作为外源保护剂的4种糖（葡萄糖、半乳糖、蔗糖、海藻糖）中只有海藻糖在低温条件下可以提高C. laurentii的生活力，其它3种糖反而降低了C. laurentii的生活力。

外源物质对酵母拮抗菌生活力的影响

　　近年来，酵母拮抗菌在水果采后病害防治中展示了良好的应用前景。然而，在实际应用中,酵母拮抗菌在逆境条件下会因为发生凋亡或细胞损伤而引起生活力的下降，最终导致拮抗菌抑病能力降低。研究酵母拮抗菌生活力下降的规律，提高酵母拮抗菌的生产效率，减少剂型加工过程中的细胞损伤，增强其对逆境条件的耐受力是增加或稳定生防制剂防治效果的有效途径。本文主要研究酵母拮抗菌正常培养过程中生活力下降的规律，筛选剂型加工过程中对酵母拮抗菌具有保护作用的化学物质，并对酵母拮抗菌的培养条件进行了优化。主要研究结果如下： 　　1. 在正常培养过程中，酵母拮抗菌Rhodotorula glutinis和Cryptococcus laurentii中细胞染色质凝集或细胞膜破损的发生一般在6天以后。外源加入的N-乙酰半胱氨酸及硅酸钠等物质在超过一定浓度时会加速酵母菌的死亡。 　　2. 在不同的液体悬浮制剂中，对R. glutinis而言，使用磷酸缓冲液（PBS）悬浮时保护效果最好；而C. laurentii悬浮在NYDB培养基中或海藻糖、乳糖溶液中时的生活力最高。 　　3. 以10 %葡萄糖 + 5 %脱脂牛奶作保护剂，可以有效地保持酵母拮抗菌C. laurentii冻干制剂的生活力，配合使用的保护效果高于它们单独使用时的保护效果。添加1 mM N-乙酰半胱氨酸能更好地保持拮抗菌制剂在常温保存过程中的生活力，这可能与这种还原性物质缓解了细胞内活性氧的积累有关。 　　4. 不同酵母拮抗菌对不同碳、氮源的利用能力有明显差异。在9种不同的碳源和10种不同的氮源中，Pichia membranefaciens能够最有效利用的碳、氮源是葡萄糖、果糖和多价胨，而Candida guilliermondii的最佳碳源和氮源分别是果糖和肉蛋白胨。

酵母拮抗菌和外源水杨酸对核果类果实抗病相关基因的诱导表达

在果实采后贮藏过程中，病原真菌的侵染会引起果实腐烂，造成巨大的经济损失。利用生物和非生物因子诱导果实抗病性，已经成为采后病害防治领域的一个研究热点。本文主要利用RT-PCR和RACE技术克隆果实抗病相关基因，通过分子杂交和蛋白羰基化免疫检测技术，研究了外源SA和酵母拮抗菌诱导果实抗病性机理，结果表明： 1. 通过优化RNA提取方法，能从含有多糖的冬枣、葡萄、甜樱桃、桃、番茄等果实中提取到质量较好的RNA，用于RT-PCR和Northern杂交。 2. 采用RT-PCR和RACE方法，从甜樱桃果实克隆了两个抗氧化相关基因CAT2（Genbank：EF165590）和GPX（Genbank：EF165591）和两个PR基因GLU-1（Genbank：EF177487）和GLU-3（Genbank：EF177488)。其中CAT2全长cDNA序列为1479 bp，编码492个氨基酸；GPX全长cDNA序列为513 bp，编码170个氨基酸；GLU-1全长cDNA序列为1050 bp，编码349个氨基酸；GLU-3部分cDNA序列为454 bp，编码141个氨基酸。 3. 酵母拮抗菌Pichia membranaefaciens处理不同成熟度的甜樱桃果实，能显著降低果实贮藏期间青霉病（Penicillium expansum）的发生，并且对低成熟度果实的病害防治效果更为明显。酵母拮抗菌的抑病机理与减轻了甜樱桃果实蛋白羰基化程度，诱导了果实抗氧化酶基因（CAT和GPX）和PR基因（GLU-1）的表达和提高了抗氧化酶（CAT和GPX）和β-1,3-葡聚糖酶的活性有关。 4. 四种酵母拮抗菌P. membranaefaciens, Cryptococcus laurentii, Candida guilliermondii和Rhodotorula glutinis处理桃果实，可显著降低贮藏期间的褐腐病（Monilinia fructicola）。这是由于酵母拮抗菌能抑制病原菌侵染造成的氧化胁迫和蛋白羰基化。此外，酵母拮抗菌处理还能显著诱导CAT、POD、几丁质酶、β-1,3-葡聚糖酶活性及相应基因的表达。 5. 水杨酸（SA，2 mM）处理采后不同成熟度的甜樱桃果实，能显著降低青霉病的危害。其抑病机理与SA处理能减轻P. expansum侵染引起的果实蛋白羰基化程度，显著提高CAT、GPX和β-1,3-葡聚糖酶基因的表达和相关的酶的活性有关。而2 mM的SA处理对P. expansum的生长没有直接抑制作用。 6. 水杨酸（SA，2 mM）与P. membranaefaciens（1×108 CFU/ml）配合处理能显著降低低温贮藏期间桃果实的褐腐病，并能提高几丁质酶、β-1,3-葡聚糖酶和POD的活性和相关基因的表达。另外，2 mM的SA对拮抗菌P. membranaefaciens的生长没有影响，但能够抑制病原菌M. fructicola的孢子萌发和菌丝扩展。

自然界可能存在第二种固氮途径

<正> 近年来,发现了一些真核固氮生物,如红酵母(Rhodotorula sp.)、小球藻(Chlorococcus sp.)、卵囊藻(Oocystis sp.),报告者都以~(l5)N法测出其固氮能力,或在无氮培养基上培养,发现其含氮量提高。但一些实验室以乙炔还原法检测其固氮酶活,又否定了这些报道。那么,我们是否可以提出另外一种固氮途径,其固氮能力不能以乙块还原法测出呢?笔者对此设想如下: 在真核固氮生物中有一类氮的氧化酶,与电子传递链相联,可以把N_2氧化成NO甚至NO_2~-、NO_3~-。如果是N

染料脱色菌株筛选及染料脱色作用研究

染料废水成分复杂，色度高，是主要的环境污染源之一。应用微生物对染料废水脱色具有处理费用低和操作管理方便等优势，具有较好的发展前景。 本文采用筛选到一株短刺小克银汉霉(Cuuninghamella echinulata) KM3及一株红酵母(Rhodotorula sp.)YR-1分别对偶氮染料刚果红废水和酸性红废水进行了脱色研究，同时利用KM3及YR-1组成的复合菌种脱色体系对酸性红废水进行了脱色实验，为染料脱色真菌的应用奠定了基础。 KM3形成的菌丝球对多种染料具有较好脱色效果。KM3最适菌丝球形成条件：pH6-7、装液量90ml、孢子悬液浓度105个/ml (接种5ml)、摇床转速120rpm、培养温度30℃、培养时间72h；成球机理为凝聚型发育。KM3菌丝球对刚果红最佳脱色条件为：染料溶液初始pH值7.0，温度为33℃，摇床转速为120rpm。在刚果红50~200mg/l 浓度范围内， KM3菌丝球对刚果红吸附脱色动力学可用拟二级动力学方程描述。菌丝球对刚果红的吸附行为符合Langmuir和Freundlich 等温方程模型，Langmuir方程能更好的描述菌丝球对刚果红的吸附行为。丙酮对刚果红的解吸率最高，其解吸动力学过程符合二级动力学方程。将KM3应用在真菌生物滤床反应器中，在非灭菌和灭菌情况下接种孢子悬液后处理刚果红废水的过程中，均需长时间挂膜，处理效果一般。反应器中投加菌丝球后处理刚果红废水在3小时内达到92%以上的脱色效果。 YR-1对酸性红脱色机制属于降解脱色。YR-1对酸性红的最佳脱色培养条件为：初始pH 5.0、接种量5%、温度30℃；最佳碳源是葡萄糖，其次是蔗糖；最佳氮源是酵母膏或蛋白胨。在替换分批脱色实验和连续补料脱色实验中，脱色率一直保持在75%以上，显示YR-1在上述脱色体系中均具有良好适应性。 在三种复合菌种对酸性红的脱色体系中，体系Ⅲ（菌丝球与海藻酸钠固定化的酵母细胞混合培养）的脱色效果可达98%以上。对该体系而言，3种粒径固定化酵母小球（Ø1.1mm、1.8mm、3.1mm）中Ø1.8mm、3.1mm小球脱色率较为理想，而酵母细胞初始密度对脱色效果的影响不大。 KM3、YR-1分别具有处理刚果红废水和酸性红废水的应用前景，而KM3与YR-1联合应用对酸性红的脱色效果更好。

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

粘红酵母对铀的吸附研究

Characteristic of uranium biosorption in water solution by Rhodotorula glutinis was investigated in the present study and the optimal pH for uranium adsorption was found to be 6-7.At the same time,maximum adsorption capacity of 149.4 mgU/(g dry cell)was identified,and Langmuir adsorption models can be used to simulate the isothermal biosorption process with high correlation coefficient of 0.99.According to Fourier transform infrared spectra,a new peak at wave number of 904 cm-1,which can be assigned to the ...中文摘要：研究了粘红酵母对水溶液中铀的吸附行为,发现其吸附铀的最佳pH值为6～7,最大吸附量为149.4mgU.g-1,其吸附等温线和Langmuir吸附等温方程符合较好,相关系数R2达到0.99;比较吸附铀前后粘红酵母的红外光谱图发现,吸附过铀的菌体的红外光谱在904cm-1处出现了一个新的峰,此峰为UO2的伸缩振动峰,说明粘红酵母确实对铀发生了吸附作用。此外,氨基或羟基的伸缩振动峰由3309移至3287cm-1,细胞壁中碳水化合物或醇中C—O键伸缩振动发生位移,由1068移至1080cm-1,说明这些基团可能参与了吸附过程;蛋白质的特征吸收峰(1653,1540,1237cm-1)在吸附前后基本无明显变化,表明粘红酵母的主要成分及结构仍保持完整。吸附后的菌体利用0.1mol.L-1的NaHCO3处理后可解吸出其中96%的铀,可见该菌在铀矿废水处理方面具有广阔的应用前景。

重离子辐照对红酵母的诱变作用

应用重离子加速器的 50MeV/u12C6+重离子对胡萝卜素生产菌-红酵母(Rhodotorula RYStrain )进行辐照处理,经酵母发酵实验,发现 50MeV/u12C6+重离子对胡萝卜素生产菌-红酵母具有诱变作用。初步筛选到了胡萝卜素产量有变化的辐照变异菌株,并对这些辐照变异菌株进行了 RFLP(限制性片段长度多态性)和 RAPD(随机扩增 DNA 多态性)分析,这些工作为工业上利用重离子对胡萝卜素生产菌进行诱变育种展现了新的前景。

Inactive and mutagenic effects induced by carbon beams of different LET values in a red yeast strain

To evaluate biological action of microorganism exposed to charged particles during the long distance space exploration. Induction of inactivation and mutation in a red yeast strain Rhodotorula glutinis AY 91015 by carbon beams of different LET values (14.9-120 0 keV mu m(-1)) was investigated It was found that survival curves were exponential, and mutation curves were linear for all LET values The dependence of inactivation cross section on LET approached saturation near 120 0 keV mu m(-1) The imitation cross section saturated when LET was higher than 582 keV mu m(-1) Meanwhile, the highest RBEI for inactivation located at 120 0 key mu m(-1) and the highest RBEm for mutation was at 58.2 key mu m(-1) The experiments imply that the most efficient mutagenic part of the depth dose profile of carbon ion is at the plateau region with intermediate LET value in which energy deposited is high enough to Induce mutagenic lesions but too low to induce over kill effect in the yeast cells (C) 2010 Elsevier B V All rights reserved

重离子辐照粘红酵母诱变育种及其产油条件的优化

本论文以在微生物油脂生产中较具潜力的粘红酵母为材料，利用兰州近代物理研究所重离子研究装置（HIRFL）产生的80 MeV/u碳离子束对产油菌株粘红酵母进行辐照诱变, 采用含有脂肪酸合成酶抑制剂cerulenin的培养基进行高产油脂突变株的初选, 并通过磷酸香草醛反应和氯仿甲醇抽提法对初筛菌株油脂含量进行分析，复选出油脂产量和质量都有较大提高的突变菌株，并对其产油条件进行优化，以期获得一些其它诱变方法难以获得的高产油脂微生物突变株，大力开发离子束辐照技术在解决能源危机中的应用。通过本论文的实验研究，得到了以下初步结果 ： 1．液体培养基中，粘红酵母菌在接种后的40h－80h处于对数生长期，此时菌体的生长繁殖比较旺盛，活力最佳，为辐照诱变的最适时期。辐照后，不同来源的菌株对重离子辐射的敏感性有所不同，然而其存活趋势却大致相当。菌体的存活率都呈现出随辐照剂量的增加先减小后增加，再减小的马鞍型剂量－效应曲线。 2．cerulenin对酵母细胞的生长具有较好的抑制作用，浓度为8.96×10-6 mol/L时，抑制率达98%以上，可作为高产油脂粘红酵母菌的筛选浓度。通过磷酸香草醛反应法和氯仿－甲醇抽提法对初筛菌体油脂含量进行定量分析，结果表明初筛菌株的正突变率达66%以上。该方法快速方便，是一种较为理想的高产油脂酵母菌的筛选方法。通过这些方法，筛选出了2株油脂含量明显高于对照的突变株。 3．经实验发现，菌体培养物与磷酸香草醛试剂反应后在530nm的光吸收与氯仿－甲醇抽提法所测得的菌体油脂含量成正比，其标准曲线方程为y=38.2257x+0.67314，R2为0.995，从而建立起一种方便快捷的油脂定量测量方法，有望实现自动化分析。 4．通过对影响粘红酵母菌生长和油脂合成的几个主要因素(葡萄糖浓度、碳氮比、接种量、培养时间、温度、PH值)的初步探讨研究，得出了粘红酵母突变株的最佳产脂条件为: 葡萄糖浓度10％，碳氮比40:1，接种量10%，温度28℃，PH=5.0，培养时间为6天

Identificação dos principais fungos das sementes de trigo.

Procedimento para identificação dos fungos das sementes de trigo; Descrição diagnostica dos principais fungos das sementes de trigo; Sclerotium Tode; Rhizoctonia DC; Chaetomium Kunze; Pleospora Rabenh; Sporobolomyces Kluy. & Niel; Rhodotorula Harrison; Phoma Sacc.; Septoria tritici Rob; Stagonospora nodorum (Berk.) Cas. & Germ.; Stagonospora avenae (Frank) Bisset f. sp. triticae; Colletotrichum graminicola (Ces.) Wilson; Fusarium tricinctum (Corda) Sacc; Fusarium moniliforme Sheldon; Fusarium avenaceum (Fr.) Sacc; Fusarium acuminatum Ell. & Kellerm; Fusarium equiseti (Corda) Sacc.; Fusarium graminearum Schw.; Mucor Micheli; Rhizopus Ehrenb; Aspergillus Link.; Penicillium Link.; Alternaria Nees; Epicoccum Link; Cladosporium Link; Nigrospora Zimm; Curvularia Boedijn; Drechslera tritici-repentis (Died.) Drech; Bipolaris sorokiniana (Sacc. in Sorok.) Shoem; Chave sistemática dos principais fungos de sementes de trigo; Ilustrações dos principais fungos encontrados em sementes de trigo.

Identificação dos principais fungos das sementes de trigo.

Procedimento para identificação dos fungos das sementes de trigo; Descrição diagnostica dos principais fungos das sementes de trigo; Sclerotium Tode; Rhizoctonia DC; Chaetomium Kunze; Pleospora Rabenh; Sporobolomyces Kluy. & Niel; Rhodotorula Harrison; Phoma Sacc.; Septoria tritici Rob; Stagonospora nodorum (Berk.) Cas. & Germ.; Stagonospora avenae (Frank) Bisset f. sp. triticae; Colletotrichum graminicola (Ces.) Wilson; Fusarium tricinctum (Corda) Sacc; Fusarium moniliforme Sheldon; Fusarium avenaceum (Fr.) Sacc; Fusarium acuminatum Ell. & Kellerm; Fusarium equiseti (Corda) Sacc.; Fusarium graminearum Schw.; Mucor Micheli; Rhizopus Ehrenb; Aspergillus Link.; Penicillium Link.; Alternaria Nees; Epicoccum Link; Cladosporium Link; Nigrospora Zimm; Curvularia Boedijn; Drechslera tritici-repentis (Died.) Drech; Bipolaris sorokiniana (Sacc. in Sorok.) Shoem; Chave sistemática dos principais fungos de sementes de trigo; Ilustrações dos principais fungos encontrados em sementes de trigo.

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.