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Extracellular α-Galactosidase from Trichoderma sp. (WF-3): Optimization of Enzyme Production and Biochemical Characterization 木霉胞外α-半乳糖苷酶(WF-3):产酶优化及生化表征
Pub Date : 2015-11-02 DOI: 10.1155/2015/860343
A. Chauhan, Arunesh Kumar, N. J. Siddiqi, Bechan Sharma
Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0) for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (K m), maximum rate of reaction (V max), K cat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources.
木霉因其分泌胞外α-半乳糖苷酶的能力较早被报道。本通讯的重点是优化酶的最佳生产的培养条件和它的特性。评价了稳定性、pH、温度、底物浓度和孵育时间等不同酶分析参数对酶活性的影响。结果表明,柠檬酸磷酸盐缓冲液(50 mM, pH 6.0)酶活性最佳。该酶在较高温度下相当稳定,在60℃时活性为72%。该酶在室温下孵育2小时后,活性没有明显下降。它符合Michaelis-Menten曲线,与底物浓度的变化有直接关系。较高的底物浓度对酶活性无抑制作用。该酶的表观Michaelis-Menten常数(K m)、最大反应速率(V max)、K cat和催化效率值分别为0.5 mM、10 mM/s、1.30 U mg−1和2.33 U mg−1 mM−1。这些信息将有助于了解其他微生物来源的胞外α-半乳糖苷酶的生物物理和生化特性。
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引用次数: 8
In silico analysis of bioethanol overproduction by genetically modified microorganisms in coculture fermentation. 共培养发酵中转基因微生物生物乙醇生产过剩的计算机分析。
Pub Date : 2015-01-01 Epub Date: 2015-02-16 DOI: 10.1155/2015/238082
Lisha K Parambil, Debasis Sarkar
Lignocellulosic biomass is an attractive sustainable carbon source for fermentative production of bioethanol. In this context, use of microbial consortia consisting of substrate-selective microbes is advantageous as it eliminates the negative impacts of glucose catabolite repression. In this study, a detailed in silico analysis of bioethanol production from glucose-xylose mixtures of various compositions by coculture fermentation of xylose-selective Escherichia coli strain ZSC113 and glucose-selective wild-type Saccharomyces cerevisiae is presented. Dynamic flux balance models based on available genome-scale metabolic networks of the microorganisms have been used to analyze bioethanol production and the maximization of ethanol productivity is addressed by computing optimal aerobic-anaerobic switching times. A set of genetic engineering strategies for ethanol overproduction by E. coli strain ZSC113 have been evaluated for their efficiency in the context of batch coculture process. Finally, simulations are carried out to determine the pairs of genetically modified E. coli strain ZSC113 and S. cerevisiae that significantly enhance ethanol productivity in batch coculture fermentation.
木质纤维素生物质是发酵生产生物乙醇的一种有吸引力的可持续碳源。在这种情况下,使用由底物选择性微生物组成的微生物联合体是有利的,因为它消除了葡萄糖分解代谢抑制的负面影响。在这项研究中,详细分析了由不同组成的葡萄糖-木糖混合物通过木糖选择性大肠杆菌菌株ZSC113和葡萄糖选择性野生型酿酒酵母共培养发酵生产生物乙醇。基于微生物基因组尺度代谢网络的动态通量平衡模型已被用于分析生物乙醇的生产,并通过计算最佳的好氧-厌氧切换时间来实现乙醇产量的最大化。在批量共培养过程中,对大肠杆菌菌株ZSC113乙醇过量生产的一系列基因工程策略进行了评估。最后,进行了模拟实验,确定了在分批共培养发酵中显著提高乙醇产量的转基因大肠杆菌菌株ZSC113和酿酒酵母菌株对。
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引用次数: 11
Application of two-stage cultivation for exploring the nutritional requirements for sporulation of three biocontrol fungi. 应用两阶段培养法探索三种生物防治真菌产孢的营养需求。
Pub Date : 2015-01-01 Epub Date: 2015-01-29 DOI: 10.1155/2015/682839
Li Gao

Fungicide was an important part in mycopesticides, which play an important role in pest management, while their mass production and commercialization faced problem. We found that the nutrition for mycelia growth and sporulation differences a lot. So, we developed "two-step method" to define the nutrition for sporulation in this paper. The results indicated that the novel method led to a great increase of spore yields for Beauveria bassiana (IBC1201), Lecanicillium lecanii (CA-1-G), and Pochonia chlamydosporia (HSY-12-14), respectively, of about 100, 2, and 16 times and, also reduced the cycle of mass production to 1/3 compared with common time for culturing.

杀菌剂是真菌农药的重要组成部分,在害虫治理中发挥着重要作用,但其规模化生产和商业化面临着诸多问题。我们发现菌丝生长和产孢的营养差异很大。因此,本文提出了“两步法”来确定产孢营养。结果表明,该方法可使球孢白僵菌(IBC1201)、Lecanicillium lecanii (CA-1-G)和Pochonia chlamydosporia (HSY-12-14)的孢子产量分别提高约100倍、2倍和16倍,并将批量生产周期缩短至普通培养时间的1/3。
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引用次数: 2
Effect of Nitrite and Nitrate Concentrations on the Performance of AFB-MFC Enriched with High-Strength Synthetic Wastewater. 亚硝酸盐和硝酸盐浓度对高浓度合成废水富集AFB-MFC性能的影响
Pub Date : 2015-01-01 Epub Date: 2015-10-01 DOI: 10.1155/2015/798397
Jian-Sheng Huang, Ping Yang, Chong-Ming Li, Yong Guo, Bo Lai, Ye Wang, Li Feng, Yun Zhang

In order to study the effect of nitrite and nitrate on the performance of microbial fuel cell, a system combining an anaerobic fluidized bed (AFB) and a microbial fuel cell (MFC) was employed for high-strength nitrogen-containing synthetic wastewater treatment. Before this study, the AFB-MFC had been used to treat high-strength organic wastewater for about one year in a continuous flow mode. The results showed that when the concentrations of nitrite nitrogen and nitrate nitrogen were increased from 1700 mg/L to 4045 mg/L and 545 mg/L to 1427 mg/L, respectively, the nitrite nitrogen and nitrate nitrogen removal efficiencies were both above 99%; the COD removal efficiency went up from 60.00% to 88.95%; the voltage was about 375 ± 15 mV while the power density was at 70 ± 5 mW/m(2). However, when the concentrations of nitrite nitrogen and nitrate nitrogen were above 4045 mg/L and 1427 mg/L, respectively, the removal of nitrite nitrogen, nitrate nitrogen, COD, voltage, and power density were decreased to be 86%, 88%, 77%, 180 mV, and 17 mW/m(2) when nitrite nitrogen and nitrate nitrogen were increased to 4265 mg/L and 1661 mg/L. In addition, the composition of biogas generated in the anode chamber was analyzed by a gas chromatograph. Nitrogen gas, methane, and carbon dioxide were obtained. The results indicated that denitrification happened in anode chamber.

为了研究亚硝酸盐和硝酸盐对微生物燃料电池性能的影响,采用厌氧流化床(AFB)与微生物燃料电池(MFC)组合系统处理高强度含氮合成废水。在本研究之前,AFB-MFC已在连续流模式下处理高强度有机废水约一年。结果表明:当亚硝酸盐氮和硝态氮浓度分别从1700 mg/L增加到4045 mg/L和545 mg/L增加到1427 mg/L时,亚硝酸盐氮和硝态氮的去除率均在99%以上;COD去除率由60.00%提高到88.95%;电压约为375±15 mV,功率密度为70±5 mW/m(2)。当亚硝酸盐氮和硝酸盐氮浓度分别高于4045 mg/L和1427 mg/L时,亚硝酸盐氮和硝酸盐氮的去除率分别为86%、88%、77%、180 mV和17 mW/m(2),而亚硝酸盐氮和硝酸盐氮的去除率分别为4265 mg/L和1661 mg/L。此外,用气相色谱仪对阳极室产生的沼气成分进行了分析。得到了氮气、甲烷和二氧化碳。结果表明,反硝化发生在阳极室。
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引用次数: 9
Production and Multiplication of Native Compost Fungal Activator by Using Different Substrates and Its Influence on Growth and Development of Capsicum chinensis Jacq. "Bhut Jolokia". 使用不同基质生产和繁殖本地堆肥真菌活化剂及其对辣椒 "Bhut Jolokia "生长发育的影响。
Pub Date : 2015-01-01 Epub Date: 2015-01-06 DOI: 10.1155/2015/481363
Vipin Parkash, Ankur Jyoti Saikia

In vitro experiment was carried out to see the effect of saw dusts of Pinus kesiya, Shorea robusta, and Callicarpa arborea on Trichoderma harzianum, isolate TH-13 mass production, along with its biotization effect on Capsicum chinensis Jacq. "Bhut Jolokia." Early mycelium initiation (2 days) occurred in S. robusta followed by P. kesiya and C. arborea (3.5 days). The sporulation was observed earlier in S. robusta (100% after 6 days) than P. kesiya (33.4% after 8 days) and C. arborea (16.7% after 9 days) but no sporulation was observed in control. The complete sporulation was also earlier in S. robusta (100% after 10 days) than P. kesiya (33.4% after 15 days) and C. arborea (16.4% after 18 days). Higher conidial yield (86 × 10(6)) was also in S. robusta than P. kesiya (70 × 10(6)) and C. arborea (45 × 10(6)), respectively. The increase in height (60-70 cm), number of leaves (600-650), and yield of chili (120-150 fruits) were also more in inoculated C. chinensis seedlings than control. It is concluded that S. robusta saw dust is the best substrate for mass production of compost fungal activator and can be used in nursery practices for quality stock production of various crops/plantations.

我们进行了体外实验,以观察 Pinus kesiya、Shorea robusta 和 Callicarpa arborea 的锯屑对哈茨真菌(Trichoderma harzianum,TH-13 分离物)大量繁殖的影响,以及其对 Capsicum chinensis Jacq.S. robusta 的菌丝萌发较早(2 天),其次是 P. kesiya 和 C. arborea(3.5 天)。与 P. kesiya(8 天后为 33.4%)和 C. arborea(9 天后为 16.7%)相比,S. robusta(6 天后为 100%)更早观察到孢子的产生,但对照组没有观察到孢子的产生。与 P. kesiya(15 天后 33.4%)和 C. arborea(18 天后 16.4%)相比,S. robusta(10 天后 100%)的完全孢子化也更早。S. robusta 的分生孢子产量(86 × 10(6))也分别高于 P. kesiya(70 × 10(6))和 C. arborea(45 × 10(6))。接种 C. chinensis 的幼苗在株高(60-70 厘米)、叶片数(600-650 片)和辣椒产量(120-150 个果实)方面的增幅也高于对照。结论是,S. robusta 锯屑是大量生产堆肥真菌活化剂的最佳基质,可用于各种作物/种植园的优质种群生产的育苗实践中。
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引用次数: 0
Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin. 表达TMPRSS2和MSPL的MDCK稳定细胞系的建立及其在缺乏外源性胰蛋白酶的流感疫苗病毒繁殖中的应用
Pub Date : 2015-01-01 Epub Date: 2015-03-30 DOI: 10.1155/2015/402628
Zhiyuan Wen, Chao Wu, Weiye Chen, Xianying Zeng, Jianzhong Shi, Jinying Ge, Hualan Chen, Zhigao Bu

We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies.

建立了两株稳定表达人气道跨膜蛋白酶的Madin-Darby犬肾(MDCK)细胞株:跨膜蛋白酶丝氨酸2 (TMPRSS2)和大形式花叶丝氨酸蛋白酶(MSPL),它们支持H5高致病性禽流感病毒(HPAIV)重组疫苗(Re-5和Re-6)和H9禽流感病毒(AIV)重组疫苗(Re-9)在缺乏胰蛋白酶的情况下的多周期生长。数据显示,经过20次连续传代,细胞系稳定表达TMPRSS2和MSPL。MDCK-TMPRSS2和MDCK-MSPL均能蛋白裂解Re-5、Re-6和Re-9的HA,支持疫苗在没有外源性胰蛋白酶的情况下高滴度生长。Re-5、Re-6和Re-9在MDCK- tmprss2和MDCK- mspl细胞中有效感染和复制,病毒滴度与在MDCK细胞中使用TPCK-trypsin生长的病毒相当。因此,我们的研究结果表明这些细胞系在基于细胞的流感疫苗生产中的潜在应用,并可能作为HA蛋白水解裂解相关研究的有用工具。
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引用次数: 8
Ethanol Production from Extruded Thermoplastic Maize Meal by High Gravity Fermentation with Zymomonas mobilis. 流动单胞菌高重力发酵挤出热塑性玉米粉生产乙醇。
Pub Date : 2014-01-01 Epub Date: 2014-11-03 DOI: 10.1155/2014/654853
Mayeli Peralta-Contreras, Edna Aguilar-Zamarripa, Esther Pérez-Carrillo, Erandi Escamilla-García, Sergio Othon Serna-Saldívar

A comparative study of extruded and ground maize meals as raw materials for the production of regular (12°P) and high gravity (20°P) worts was devised. Extruded water solubility index (WSI) was higher (9.8 percentage units) and crude fat was lower (2.64 percentage units) compared to ground maize. Free-amino nitrogen compounds (FAN), pH, and glucose were evaluated in regular and high gravity worts produced from ground or extruded maize. Extrusion improved glucose content and ethanol yield. In 20°P mashes, extrusion is enhanced by 2.14% initial glucose compared with regular ground mashes. The 12°P and 20°P extruded treatments averaged 12.2% and 8.4% higher ethanol, respectively, compared to the uncooked counterpart. The 20°P worts fermented with Zymomonas mobilis produced 9.56% more ethanol than the 12°P counterpart. The results show that the combination of extrusion and fermentation of 20°P worts improved ethanol yield per kg flour until 20.93%. This pretreatment stimulates Z. mobilis fermentation efficiency.

对挤压玉米粉和磨碎玉米粉作为生产常规(12°P)和高重力(20°P)麦汁的原料进行了比较研究。挤压水溶性指数(WSI)高于磨碎玉米(9.8个百分点单位),粗脂肪含量较低(2.64个百分点单位)。研究人员对碾磨玉米和挤压玉米生产的普通麦芽汁和高重力麦芽汁进行了游离氨基氮化合物(FAN)、pH和葡萄糖的测定。挤压提高了葡萄糖含量和乙醇收率。在20°P的土豆泥中,与常规土豆泥相比,初始葡萄糖的挤压量提高了2.14%。与未煮熟的处理相比,12°P和20°P挤压处理的乙醇含量平均分别高出12.2%和8.4%。与12°P发酵相比,20°P发酵的麦汁乙醇产量增加9.56%。结果表明,20°P麦汁挤压与发酵相结合,每kg面粉乙醇得率提高至20.93%。这种预处理方法可以提高霉的发酵效率。
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引用次数: 8
Evaluation of novel design strategies for developing zinc finger nucleases tools for treating human diseases. 评估用于开发治疗人类疾病的锌指核酸酶工具的新型设计策略。
Pub Date : 2014-01-01 Epub Date: 2014-04-06 DOI: 10.1155/2014/970595
Christian Bach, William Sherman, Jani Pallis, Prabir Patra, Hassan Bajwa

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.

锌指核酸酶(ZFNs)与无数不希望出现的位置结合,从而导致细胞死亡和凋亡。这种细胞毒性与锌指结构域的结合能力有关,它能以高亲和力结合不同的 DNA 序列。一般来说,转录因子的结合偏好与显著的退化多样性和复杂性有关,这使得精确 DNA 结合结构域的设计和工程变得复杂。然而,天然锌指蛋白的成功进化证明,大自然创造了特定的进化特征和策略,如模块化和等级特异性识别,以应对结合的复杂性。我们的研究结果表明,当交换第二指的氨基酸时,转录因子 SP1 三指结合结构域的一般模块性得以保留,而等级特异性结合偏好发生了显著变化。
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引用次数: 0
Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3. 印度Xenorhabdus indica KB-3产纤溶碱性金属蛋白酶培养基成分的统计优化
Pub Date : 2014-01-01 Epub Date: 2014-04-23 DOI: 10.1155/2014/293434
Kumar Pranaw, Surender Singh, Debjani Dutta, Surabhi Chaudhuri, Sudershan Ganguly, Lata Nain

Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone-5.71, soya peptone-4.9, dextrose-1.45, NaCl-6.08, and dipotassium phosphate-0.47 in g/L). To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

从其昆虫病原线虫共生体嗜热斯坦纳菌中分离得到一种著名的蛋白酶产生菌——印度Xenorhabdus indica KB-3。由于培养基成分对蛋白酶生产至关重要,因此采用响应面法(RSM)对所选培养基(大豆酪蛋白消化液)的化学成分进行了旋转中心复合设计(RCCD)优化。研究了色氨酸、大豆蛋白胨、葡萄糖、NaCl和磷酸二钾这五种化学成分(自变量)对蛋白酶生成的影响(因变量),发现色氨酸和葡萄糖对蛋白酶生成的影响最大。在最佳培养基条件下(色氨酸-5.71,大豆肽-4.9,葡萄糖-1.45,NaCl-6.08,磷酸二钾-0.47,g/L),蛋白酶产量显著提高66.31%。就目前所知,还没有关于利用RSM优化籼菜KB-3生产蛋白酶的培养基成分及其在纤溶中的应用的报道。本研究为籼稻KB-3蛋白酶的工业化生产及在农业和医药领域的应用提供了理论依据。
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引用次数: 12
Biocontrol Activity of the Local Strain of Metschnikowia pulcherrima on Different Postharvest Pathogens. pulcherrima metschnikoia本地菌株对不同采后病原菌的生物防治活性研究。
Pub Date : 2014-01-01 Epub Date: 2014-04-17 DOI: 10.1155/2014/397167
Sezai Türkel, Mihriban Korukluoğlu, Mümine Yavuz

The strains of the yeast Metschnikowia pulcherrima have strong biocontrol activity against various microorganisms. Biocontrol activity of M. pulcherrima largely depends on its iron immobilizing pigment pulcherrimin. Biocontrol activity of pulcherrimin producing strain, M. pulcherrima UMY15, isolated from local vineyards, was tested on different molds that cause food spoilage. M. pulcherrima UMY15 was a very effective biocontrol agent against Penicillium roqueforti, P. italicum, P. expansum, and Aspergillus oryzae in in-vitro plate tests. However, the inhibitory activity of M. pulcherrima UMY15 was less effective on Fusarium sp. and A. niger species in biocontrol assays. In addition, M. pulcherrima UMY15 strain completely inhibited the germination and mycelia growth of A. oryzae, A. parasiticus, and Fusarium sp. spores on artificial wounds of apples when they coinoculated with M. pulcherrima UMY15. Moreover, when coinoculated, M. pulcherrima UMY15 strain also inhibited the growth of P. roqueforti, P. italicum, P. expansum, A. oryzae, Fusarium sp., and Rhizopus sp. in grape juice, indicating that M. pulcherrima UMY15 can be used as a very effective biocontrol yeast against various species of postharvest pathogens, including Penicillium, Aspergillus, Fusarium, and Rhizopus.

pulcherrima Metschnikowia酵母菌株对多种微生物具有较强的生物防治活性。pulcherrima的生物防治活性在很大程度上取决于其铁固定色素pulcherrimin。从当地葡萄园分离的pulcherrima M. UMY15产菌对引起食物腐败的不同霉菌进行了生物防治活性试验。在体外平板试验中,M. pulcherrima UMY15对罗氏青霉、意大利青霉、膨化青霉和米曲霉具有很好的防效。然而,在生物防治试验中,pulcherrima UMY15对镰刀菌和黑曲霉的抑制效果较差。此外,pulcherrima UMY15菌株与M. pulcherrima UMY15共接种时,完全抑制了米蚜、寄生和镰刀菌孢子在苹果人工伤口上的萌发和菌丝生长。同时接种后,M. pulcherrima UMY15菌株还能抑制roqueforti、P. italicum、P. expansum、a . oryzae、Fusarium sp.和Rhizopus sp.在葡萄汁中的生长,表明M. pulcherrima UMY15菌株可以作为一种非常有效的生物防治酵母,对抗青霉菌、曲霉、镰刀菌和Rhizopus等多种采后病原菌。
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引用次数: 47
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