262 resultados para Trichoderma harzianum


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本文研究施化肥和海洋微生物制剂对桉树人工林土壤质量的影响。研究结果表明:施用不同的化肥对桉树人工林土壤质量影响具有明显差异;从海洋植物根际分离得到的微生物菌株制成的微生物制剂中的活性微生物菌株能够在桉树人工林土壤中定殖,对桉树生长具有一定促进作用。桉树凋落叶分解过程中是否释放化感物质是桉树人工林发展过程中人们普遍关注的问题之一,本论文也对该部分做了初步研究。 对施用长效尿素、芬兰复合肥、高峰复合肥三种不同化肥对桉树人工林土壤质量的的影响进行了初步研究,研究结果表明长效尿素在保障土壤氮素供应、促进土壤纤维素分离能力提高和增强土壤对磷元素吸收方面具有重要作用;芬兰复合肥在增强土壤呼吸作用和促进土壤酶活性提高方面优于长效尿素和高峰复合肥。 以两株海洋来源的枯草芽孢杆菌(Bacillus subtilis 3512, Bacillus subtilis 3728)和一株海洋木霉(Trichoderma TF4)为研究材料,在实验室条件下对其生防机理进行了研究,研究表明:两株枯草芽孢杆菌通过产生脂肽和蛋白酶对植物病原菌产生抑制作用;海洋木霉TF4则能够产生HCN,IAA类植物生长激素,同时还具有一定的解磷能力,具有很好的应用前途,采用传统分类学方法和分子系统学方法鉴定为棘孢木霉(T.asperellum)。这三株海洋菌株制成微生物菌剂,在原位条件和盆栽条件下考察了其对桉树生物量和土壤质量指标的影响,研究结果表明将三株海洋微生物混合后添加少量三叶草作辅剂,能有效改善桉树人工林土壤质量,并促进桉树树高和胸径的增加,具备进一步研究和开发成产品的价值。 用高效液相色谱(HPLC)对桉树凋落叶中毒性物质进行分离、纯化,以小麦、绿豆、大速生菜为指示植物,对分离到的物质进行毒性跟踪,分离到一个毒性较强的组分,经1H氢谱和NMR鉴定为3–β甲酰基–乌索酸。

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生物质燃料乙醇是一种高度清洁的交通液体燃料,是减少温室气体排放,缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全,有利于产业的可持续发展。在众多的非粮原料中,甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90%。同时,甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的,因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇,其技术水平低,发酵强度低,一般在0.7-2.5g/(L•h),乙醇浓度低,甘薯发酵乙醇为6-8%(v/v),能耗高,环境负荷大,污染严重。针对上述问题,本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中,大型发酵罐底部高压条件下,CO2对酵母乙醇发酵的影响,我们通过CO2 加压的方法进行模拟试验,研究结果表明,发酵时间随压强的升高而逐渐延长,高压CO2 对乙醇发酵效率影响不大,在0.3 MPa 以下时,发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下,酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下,酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下,乙醇发酵途径中多个基因表达量下调,同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾,实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本,实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1,进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵,发酵33h,最终乙醇浓度达到10.7%(w/w),发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右,显示菌株具有一定的耐酸性能力。同时观察到40 ℃下,菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化,乙醇发酵途径中关键酶基因表达下调,而海藻糖合成酶与热激蛋白基因表达量上调,这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料,本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂,研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液,采用分批补料的发酵方式,乙醇浓度最高达到了10.26% (w/w),发酵时间为42 h。同时观察到在糖蜜发酵中,乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下,采用10L 发酵罐进行分批乙醇发酵,经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%,平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时,拥有快速生产乙醇的能力,适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点,传统液化糖化处理很难在短时间内充分糖化原料;高粘度的醪液也难以进行管道输送,容易堵塞管路;同时,也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析,进而对预处理条件进行了研究,在最佳预处理条件下,糖化2h 后,醪液葡萄糖值最高可达99.3,粘度4.5×104 mPa.s,而采用传统糖化工艺,醪液DE 值仅为85.8,粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明,通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上,进一步将发酵规模扩大到500L,在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模,采用边糖化边发酵(SSF)工艺,在料液比为3∶1,发酵醪液最高粘度为6×104mPa.s 条件下,发酵37h,乙醇浓度达到了12.7%(v/v),发酵效率91%,发酵强度为2.7 g/(L•h)。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成,采用了高效液相色谱—蒸发光散射检测法,对乙醇发酵残糖进行了分析。结果表明,甘薯、木薯乙醇发酵残糖均为寡聚糖,主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长,寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖,过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株,对甘薯乙醇发酵残糖的降解利用率均达到了40%以上,而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析,确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发,以期形成乙醇转化率高,能耗低,生产效率高、季节适应性好,原料适应性广,经济性强,符合清洁生产机制的燃料乙醇高效转化技术,为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%(w/w)at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.

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采用微生物菌落计数方法和Pearson相关性分析,以辽河油田锦采污水处理厂稠油废水为对象,研究了不同季节稠油废水中优势微生物的种群组成及其变化规律。结果表明,在稠油废水处理过程中微生物种群组成及其作用均以细菌为主,真菌次之,放线菌最小;细菌菌落形态多样性指数(H′)与均匀度指数(E)均能较准确地反映废水中的细菌多样性,但不能反映水质状况,不宜作为该水质评价的生物指标;细菌数量与总石油烃(TPH)和COD均呈强正相关,统计学上关系显著,适宜作为废水水质评价的生物指标;经鉴定,废水中的优势菌株为液化金杆菌(Aureobateriumliquefaciens)和弗氏丙酸杆菌(Propionibacterium freudenreichii),主要真菌为青霉属(Penicillium)、曲霉属(Aspergillus)、木霉属(Trichoderma)和交链孢霉属(Alternaria)。

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土壤质量维持和改良是农林业生产面临的重要问题。利用海洋枯草芽孢杆菌(Bacillus subtilis)3728、3512、海洋木霉(Trichoderma sp.)TF4和少量白三叶草(Trrifolium repens)制成海洋微生物复合制剂,对当年生桉树人工林进行了生物调控试验。结果表明:海洋微生物复合制剂对土壤微生物数量和群落结构具有调节作用;曲霉、木霉、毛霉等与纤维素分解相关的真菌数量增加,镰刀菌数量减少;细菌数量比对照增加1.0倍,其中枯草芽孢杆菌增加2.02倍,蜡样芽孢杆菌增加1.18倍;放线菌数量增加0.3倍;土壤有机质含量、铵态氮含量和速效磷含量增加,土壤中蔗糖酶、蛋白酶、脲酶的活性显著提高;施用180d后测定桉树树高和胸径变化,树高净增加量比对照高12.6%,表明海洋微生物复合制剂作为生物调控剂对桉树生长具有一定促进作用。

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通过对江苏省云台山地区7种不同野生中草药植物烟台百里香(T.quinquecostatusCelak.)、掌叶半夏(P.pedatisectaSchott.)、何首乌(P.multiflorumThunb.)、海洲香薷(E.splendensNakai ex F.)、野葛[P.lobata(Willd)Ohwi]、紫金牛(A.japonicaBl.)和菝葜(S.sieboldiiMiq.)根区土壤真菌进行分离鉴定,共分离出真菌16属126种。多样性分析结果表明,7种野生中草药植物根区土壤真菌种群多样性丰富,其中青霉属(Penicillium)、曲霉属(Asper-gillus)和木霉属(Trichoderma)是中草药植物根区土壤中的优势种群,镰孢菌属(Fusarium)、交链孢属(Alternaria)、腐霉属(Pythium)、毛霉属(Mucor)真菌分布丰度也较高。结果也表明根区土壤真菌群落在一定程度上受到中草药植物的影响,大部分野生中草药植物根区土壤的真菌群落的均匀度指数低于裸地非根区土壤,而丰富度指数却高于裸地非根区土壤。不同野生中草药植物根区土壤真菌区系的结构和组成存在一定的差异性,紫金牛根区土壤中真菌种类最多,达到14属,而野葛根区土壤中真菌种类最少,只有8属。

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Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag7O and gluc78 from the biocontrol fungus Trichoderma atroviride were transformed into rice mediated by Agrobacterium tumefaciens singly and in all possible combinations. A total of more than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Our data indicated that gluc78 gene had negative effects on transformation frequency and plant growth. Some regenerated plants with gluc78 gene were stunted; spontaneously produced brown specks; could not tassel. The combination with either one of the two other genes (ech42, nag70) present in the same T-DNA region reduced the negative effect of gluc78 on plant growth. These results indicated that expression of several genes in one T-DNA region interfered with each other and expression of exogenous gene in recipient plant was a complex behavior. (c) 2007 Published by Elsevier Ireland Ltd.

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铊是一种有毒有害的重金属元素,已经引起了广泛的关注。本论文通过对黔西南铊矿区土壤和沉积物样品的菌株分离、铊高耐受性菌株的筛选、胞外吸附、富集、亚细胞水平区系分布、絮凝实验及ITS序列等实验研究分析,并结合铊的地球化学相关研究,较系统地阐述了真菌--铊的生物地球化学过程机理,得出以下结论: 1、与环境背景区相比,黔西南滥木厂铊矿区内的河流、土壤中铊的已有不同程度的积累,直接导致了当地微生物生物量在很大程度上的降低,微生物生物量与铊含量间有显著的负相关关系。研究区内的沉积物、土壤中的微生物区系结构和数量发生了明显变化,细菌、真菌及放线菌数量均出现显著降低,而且三大微生物对重金属污染的敏感性大小也不一样,即放线菌>细菌>真菌。从土壤样品中分离到的主要菌群仍为常见种属,如青霉属(Penicillium)、木霉属(Trichoderma)、拟青霉(Paecilomyces)等。 2、经过初筛菌株的铊耐受性实验,在1000 mg/L水平筛选得到九株高耐受性菌株。吸附实验表明:微生物菌株对铊的吸附效率在4.63~16.89%,且随着环境中铊浓度的上升而降低,这可能是因为铊浓度的升高加大了对微生物生长的抑制作用,所形成的菌丝体(或菌丝球)减少,表面积也相应减少,从而导致了吸附效率的下降。各种常量元素和铊的关系呈显著相关性,钙、钾和钠等常量元素也是微生物赖以维持生存的因子,可能由于微生物细胞对钙、钾的吸附方式与对铊的吸附方式类似。因此,随着铊处理浓度的上升,钙和钾的吸附量也随之减少,而钠则呈现相反的趋势。 3、富集实验表明,九株菌株对铊的富集量随着铊处理浓度上升而降低,其影响趋势与对生物量的影响趋势基本一致,最高可达到7189 mg/kg,最大富集系数为7.2。九株菌株对常量元素的富集与对铊的富集并无明显的相关性,但在考察铊处理浓度对常量元素的富集影响时发现,铊处理浓度的上升与对钙的富集量表现出较强的正相关;而对钾、钠、镁的富集影响并不明显。 4、亚细胞水平上的铊分布研究表明,铊的富集优先顺序为:细胞质>细胞壁>细胞器。亚细胞水平的区隔化作用是微生物对铊的主要耐受机制,细胞质是赋存铊的主要场所(53.83~79.45 %)。结合各亚细胞组分中常量元素与铊之间的相关性,并联系前人的研究,Tl+主要是通过细胞壁的Na+ -K+ ATPase和K+ -电位门通道进入细胞内的从而影响细胞的正常代谢的,而Ca2+的活化更有助于这一过程。 5、絮凝实验表明,培养三天后的发酵液对矿区废水中铊的去除率最高可达到70.49 %,最佳影响因子组合为:pH=8,温度为16℃,搅拌时间为4分钟。菌株的絮凝活性最高可达到57.32%,最佳影响因子组合为:pH=8,温度为14℃,搅拌时间为4分钟。 6、通过对九株铊高耐受性菌株的ITS序列分析及其在Gene Bank中的BLAST比对结果表明,五株菌株同属于木霉属(Trichoderma),两株菌株同属于青霉属(Penicillium)。这表明这两类真菌对铊的适应性较强,为以后寻找铊高耐受性菌株及其资源化利用提供了理论基础。

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Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2010

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Dissertation presented to obtain the Ph.D degree in Biology

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Trichoderma aggressivum f. aggressivum is a filamentous soil fungus. Green mold disease of commercial mushrooms caused by this species in North America has resulted in millions of dollars in lost revenue within the mushroom growing industry. Research on the molecular level of T aggressivum have jus t begun with the goal of understanding the functions of each gene and protein, and their expression control. Protein targeting has not been well studied in this species yet. Therefore, the intent of this study was to test the protein localization and production levels in T aggressivum with green fluorescent protein (GFP) with an intron and tagged with either nuclear localization signal (NLS) or an endoplasmic reticulum retention signal (KDEL). Two GFP constructs (with and without the intron) were used as controls in this study. All four constructs were successfully transferred into T aggressivum and all modified strains showed similar growth characteristics as the wild type non-transformed isolate. GFP expression was detected from all modified T aggressivum with confocal microscopy and the expression was similar in all four strains. The intron tested in this study had no or very minor effects as GFP expression was similar with or without it. The GFP signal increased over a 5 day period for all transformants, while the GFP to total protein ratio decreased over the same period for all transformants. The GFP-KDEL transformant showed similar protein expression level and localization as did the control transformant lacking the KDEL retention signal. The GFP-NLS transformant similarly failed to localize GFP into nucleus as fluorescence with this strain was virtually identical to the GFP transformant lacking the NLS. Thus, future research is required to find effective localization signals for T aggressivum.

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La cellulose et ses dérivés sont utilisés dans un vaste nombre d’applications incluant le domaine pharmaceutique pour la fabrication de médicaments en tant qu’excipient. Différents dérivés cellulosiques tels que le carboxyméthylcellulose (CMC) et l’hydroxyéthylcellulose (HEC) sont disponibles sur le commerce. Le degré de polymérisation et de modification diffèrent énormément d’un fournisseur à l’autre tout dépendamment de l’origine de la cellulose et de leur procédé de dérivation, leur conférant ainsi différentes propriétés physico-chimiques qui leurs sont propres, telles que la viscosité et la solubilité. Notre intérêt est de développer une méthode analytique permettant de distinguer la différence entre deux sources d’un produit CMC ou HEC. L’objectif spécifique de cette étude de maitrise était l’obtention d’un profil cartographique de ces biopolymères complexes et ce, par le développement d’une méthode de digestion enzymatique donnant les oligosaccharides de plus petites tailles et par la séparation de ces oligosaccharides par les méthodes chromatographiques simples. La digestion fut étudiée avec différents paramètres, tel que le milieu de l’hydrolyse, le pH, la température, le temps de digestion et le ratio substrat/enzyme. Une cellulase de Trichoderma reesei ATCC 26921 fut utilisée pour la digestion partielle de nos échantillons de cellulose. Les oligosaccharides ne possédant pas de groupements chromophores ou fluorophores, ils ne peuvent donc être détectés ni par absorbance UV-Vis, ni par fluorescence. Il a donc été question d’élaborer une méthode de marquage des oligosaccharides avec différents agents, tels que l’acide 8-aminopyrène-1,3,6-trisulfonique (APTS), le 3-acétylamino-6-aminoacridine (AA-Ac) et la phénylhydrazine (PHN). Enfin, l’utilisation de l’électrophorèse capillaire et la chromatographie liquide à haute performance a permis la séparation des produits de digestion enzymatique des dérivés de cellulose. Pour chacune de ces méthodes analytiques, plusieurs paramètres de séparation ont été étudiés.

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A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

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Controlling Armillaria infections by physical and chemical methods alone is at present inadequate, ineffective, or impractical. Effective biological control either alone or in integration with another control strategy appears necessary. Biological control agents of Armillaria function by the antagonists inhibiting or preventing its rhizomorphic and mycelial development, by limiting it to substrate already occupied, by actively pre-empting the substrate, or by eliminating the pathogen from substrate it has already occupied. Among the most thoroughly investigated antagonists of Armillaria are Trichoderma species. Depending on the particular isolate of a Trichoderma species, control may be achieved by competition, production of antibiotics, or by mycoparasitism. The level of control is also influenced by the growth and carrier substrate of the antagonist, time of application in relation to the occurrence of the disease, and several environmental conditions. Among a range of the other antagonists are several cord-forming fungi and an isolate of Dactylium dendroides. Integrating biological methods with an appropriate method of chemical could control the disease more effectively. However it is essential to determine whether the antagonist or the fungicide should be applied first, and the time interval between.

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Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

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The effect of a commercial cellulase preparation on phenol liberation and extraction from black currant pomace was studied. The enzyme used, which was from Trichoderma spp., was an effective "cellulase-hemicellulase" blend with low P-glucosidase activity and various side activities. Enzyme treatment significantly increased plant cell wall polysaccharide degradation as well as increasing the availability of phenols for subsequent methanolic extraction. The release of anthocyanins and other phenols was dependent on reaction parameters, including enzyme dosage, temperature, and time. At 50 degrees C, anthocyanin yields following extraction increased by 44% after 3 h and by 60% after 1.5 h for the lower and higher enzyme/substrate ratio (E/S), respectively. Phenolic acids were more easily released in the hydrolytic mixture (supernatant) and, although a short hydrolysis time was adequate to release hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCA) required longer times. The highest E/S value of 0.16 gave a significant increase of flavonol yields in all samples. The antioxidant capacity of extracts, assessed by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the oxygen radical absorbance capacity, and the ferric reducing antioxidant potential depended on the concentration and composition of the phenols present.