真菌多糖研究进展

真菌多糖具有多种生物活性和保健功能,近年来成为生命科学、医药科学、食品科学等领域研究的热点.本文对真菌多糖的生物活性即抗肿瘤、抗病毒、降血压、降血脂、降血糖、抗氧化、抗疲劳、抗辐射等活性作了介绍,阐述了真菌多糖的提取方法即热水浸提法、酸提法、碱提法、酶法、超声波辅助法等6种方法,以及分离纯化方法即膜分离技术和色谱技术,最后对真菌多糖的应用前景进行了探讨.     

提取方法对库拉索芦荟多糖的抗氧化活性影响研究

文章探讨了水提法和超声波辅助复合酶法对芦荟多糖抗氧化活性的影响。以多糖提取率和多糖含量为评价指标，分别得到水提法和超声波辅助复合酶法的提取产物，采用体外抗氧化活性体系评价2种提取方法对芦荟多糖抗氧化活性的影响。抗氧化活性评价结果显示，超声波辅助复合酶法对于多糖提取物的羟基自由基清除率和DPPH自由基清除率均高于水提法。     

提取防风多糖的工艺优化

采用超声波强化和微波辅助提取2种方法提取防风多糖,并与传统热水浸提法在多糖的提取率上进行比较。防风多糖超声提取的最佳工艺条件为超声功率1 000 W、提取时间25 min、液固体积质量比为25,防风多糖微波提取的最佳工艺条件为微波功率560 W、液固体积质量比为30、提取时间10 min,在最佳提取工艺下,2种方法的提取率分别为6.103%和7.639%。与传统热水浸提法相比,超声法和微波法提取防风多糖具有迅速、节能、高效、提取率高等诸多优点。     

真菌多糖水提及化学辅助提取方法研究进展

真菌多糖具有抗肿瘤、抗衰老、免疫调节等多种功效, 有较高的食、药用和商业价值。本文通过对实例的总结, 介绍热水浸提法、稀酸提取法、碱提取法、酶消化提取法等真菌多糖提取技术。通过比较阐述其优缺点、影响因素和适用范围, 为类似试验提供建议。     

一种改良的CTAB法提取产多糖真菌DNA

真菌胞外多糖由于其高吸附高粘稠特点,是困扰从胞外多糖产生菌分离高纯度DNA的难点之一。本文以生产硬葡聚糖的齐整小核菌生产菌为代表,采用改良的CTAB法获得了高质量的基因组DNA。通过分层隔离等培养方法的优化降低硬葡聚糖的产生,并在传统CTAB法的基础上,用高浓度的醋酸钾和无水乙醇共同作用初步沉淀多糖,再用CTAB/NaCl溶液再次去除多糖。相比于商业的DNA提取试剂盒和传统的CTAB法,该方法得到的基因组DNA产率大幅提高,纯度较好,可充分排除胞外多糖的干扰,为各典型产胞外多糖的真菌DNA提取提供重要的参考。提取的基因组DNA可用于基因组文库构建、PCR等分子生物学实验。     

桑黄菌丝体粗多糖的提取方法研究

采用正交试验设计,以桑黄菌丝体粗多糖含量为考察指标,用苯酚—硫酸法,分别确定了热水浸提法、微波辅助提取法和超声提取法的最佳工艺。通过极差分析得出:热水浸提法的最优工艺为浸提时间3 h、浸提3次、液料质量比50∶1、浸提温度90℃,粗多糖提取率为2.10%;微波提取法的最优工艺为微波处理15 min、液料质量比50∶1、提取3次,提取率为4.18%;超声提取法的最优工艺为超声30 min、提取2次、液料质量比60∶1、温度60℃、频率60 Hz,提取率为3.02%。微波辅助法与热水浸提法相比,时间缩短,且提取率提高近1倍;与超声提取法相比,时间缩短1/2,但提取率提高40%。因此,微波辅助提取法速度更快、提取效率更高、操作更简便,优于其他2种方法。     

灰树花多糖的提取及抗氧化活性

采用单因素和正交试验法对热水浸提、超声波辅助和微波辅助提取灰树花多糖的工艺进行研究,分别获得了3种方法的最佳条件,并发现微波辅助法最佳,其最优条件为:微波功率800 W、微波时间3 min,浸提2h,多糖得率为13.05％,比热水浸提法提高了51.22％.利用提取的灰树花多糖进行抗氧化性研究,发现灰树花多糖对O2-·和·OH都具有一定的清除作用,且多糖浓度与其对O2-·的清除作用存在量效关系,但不如·OH明显.     

树舌胞内粗多糖的提取及其抗炎活性研究

采用正交试验设计,对微波辅助法提取树舌胞内多糖的工艺进行了优化;采用二甲苯致小鼠耳肿胀模型、角叉菜胶致小鼠足肿胀模型对多糖抗炎活性进行了研究。根据极差分析确定了微波辅助法的最优工艺为微波处理10 min、液料比40∶1、提取2次。按此工艺多糖提取率为24.92%,多糖得率和含量分别为188.3 mg/g和78.47%。抗炎活性试验结果表明:树舌胞内多糖高、中、低剂量组对二甲苯致小鼠耳肿胀有明显的抑制作用,抑制率分别为52.16%、37.80%、27.97%,与空白对照组均有极显著性差异(P0.01);树舌胞内多糖高、中、低剂量可显著抑制角叉菜胶致小鼠足肿胀,具有良好的抗炎作用。     

黄芪多糖超声-微波协同提取研究

本论文采用超声-微波协同提取新工艺,通过单因素实验分别考察提取时间、微波功率、料液比等因素对黄芪多糖提取率及纯度的影响;通过正交实验得出最佳提取工艺参数;通过平行提取实验,与水提法、微波及超声波辅助提取进行比照。得出最佳提取条件为微波功率120 W,提取时间为150 s,料液比1∶25(g/mL)时,黄芪多糖的提取率最高达4.25%,并且证明了超声微波协同提取法的提取效率高于水提法、微波法及超声波法等传统的提取方法。     

姬松茸胞内多糖碱提取工艺

为了进一步提高姬松茸胞内多糖得率,采用单因素实验和响应曲面法,对姬松茸菌丝体胞内多糖碱提取工艺进行了研究。结果表明优化的碱提取工艺为提取时间4．63h,碱液浓度1．03mol／L,提取温度60．90℃,液料比85．85mL／g,在此条件下胞内多糖的提取得率为（273．49±1．59）mg／g干菌体,明显高于水提取所得到的胞内多糖得率。这为姬松茸碱提胞内多糖理化性质、生理活性等方面进一步的研究提供切实可行的高效提取方法。     

真菌生物量指示剂麦角固醇的分离及测定方法

麦角固醇作为真菌细胞膜的重要组成成分,结构稳定,可作为真菌生长的指示物质。综述了表征真菌生物量的麦角固醇的分离及测定方法,其中麦角固醇的萃取方法有传统的皂化回流法、快速物理萃取、超声波萃取、超临界流体萃取等,测定方法有高效液相色谱法、气相色谱-质谱法、液相色谱-质谱法、薄层色谱法等。并对这些方法的应用及在堆肥过程中运用麦角固醇估计真菌生物量的可行性进行了展望。     

Development of a sample preparation method for fungal proteomics

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.     

Application of molecular techniques for identification of fungal communities colonising paper material

Archives and libraries all over the world suffer from biodeterioration of writings caused by microorganisms, especially fungi. With traditionally used culture-dependent methods, only a small amount of effectively colonising organisms is detected. Restoration and maintenance of written cultural heritage is therefore problematic due to incomplete knowledge of the deterioration agents.In the present study, culture-independent molecular methods were applied to identify fungal communities colonising paper samples of different composition and age. Nucleic-acid-based strategies targeting the internally transcribed spacer (ITS) regions, which are nested in the nuclear rDNA repeats, were selected to investigate the fungal diversity on paper. The ITS regions possess a high variation among taxonomically distinct fungal species and even within the species.With this aim, several molecular biological methods were optimised for working with paper materials. Here, we introduce a DNA extraction protocol, which allowed the direct extraction of PCR-amplifiable DNA from samples derived from different kinds of paper. The DNA extracts were used to amplify either the ITS1 or ITS2 region by using different fungi-specific primer sets. The ITS-amplified regions were subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Conditions for DGGE analysis, gradient, voltage, and running time, were established to accurately discriminate different fungal species in complex communities. Pure fungal strains were used to constitute a marker for further comparative investigations of historic papers.     

海藻多糖化学及生物活性的研究进展

海藻多糖具有多种生物活性,在生物体内起着重要作用,现已成为海藻研究的热点。本文综述了海藻多糖的种类,海藻多糖的提取、分离与提纯技术,海藻多糖性质的测定方法及海藻多糖的生物活性。     

食药用真菌多糖抗氧化作用研究进展

食药用真菌多糖由于其独特的生理活性,目前正成为国内外众多学科领域研究的热点之一。综述了食药用真菌多糖抗氧化作用研究现状、作用机理及其构效与量效关系,并对其发展前景进行了展望,旨在为其深入研究和开发利用提供参考。     

Efficacy of extraction methods for lipid and fatty acid composition from fungal cultures

The efficacy of three extraction methods for determining the lipid and fatty acid composition of six fungal cultures was studied. The extraction methods were: chloroform/methanol (2:1), hexane/isopropanol (3:2) and Soxhlet extraction by using hexane. The total lipid and fatty acid composition varied in fungal cultures depending on the extraction conditions. Of the three methods, chloroform/methanol (2:1) was found to be the best for extraction of lipid and fatty acids from fungal cultures.     

具抑菌活性连翘内生真菌的分离与鉴定

【目的】从药用植物连翘内生真菌中筛选抑菌活性菌株, 并对其主要活性成分进行检测分析。【方法】采用常规组织分离法分离内生真菌, 滤纸片法测定其发酵产物对3种指示细菌的抑菌活性。TLC、HPLC和HPLC-MS检测活性菌株代谢产物中连翘苷成分。根据形态学特征和ITS序列分析鉴定目的菌株。【结果】分离得到的24株内生真菌中抑菌圈直径超过10 mm的菌株, 发酵液组有11株(45.8%), 菌丝体组有19株(79.2%), 活性菌株主要集中在果实内生真菌中, 其中G5、G7和G10表现出较强的抑菌活性。从菌株G10的胞内产物中发现活性成分连翘苷, 将其鉴定为胶孢炭疽菌Colletotrichum gloeosporioides。【结论】连翘内生真菌是天然抗菌活性物质的重要筛选来源。     

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).     

Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions

The isolation of chitosan from a fungal source offers the potential of a product with controlled physicochemical properties not obtainable by the commercial chemical conversion of crustacean chitin. A variety of culture and processing protocols using Mucor rouxii were studied for their effects on biomass yield and chitosan molecular weight. Weight-averaged molecular weight determined by gel permeation chromotography ranged from 2.0 x 10(5) to approximately 1.4 x 10(6) daltons. The chitosan yield ranged from 5% to 10% of total biomass dry weight and from 30% to 40% of the cell wall. Of the culture parameters studied, length of incubation and medium composition effected biomass production and molecular weight. Modification of the processing protocol, including the type and strength of acid, and cell wall disruption in acid prior to refluxing were used to optimize the efficiency of chitosan extraction.The degree of deacetylation of fungal and commercial chitosans was compared using infrared spectrometry, titration, and first derivative of UV absorbance spectrometry. The chitosan obtained directly from the fungal cell wall had a higher degree of deacetylation than commercial chitosan from the chemical conversion process.