硫磺菌原变种液体培养代谢物生物活性分析

硫磺菌原变种Laetiporus sulphureusvar.sulphureus在液体培养条件下对果蝇具有致死效应,研究发现在液体培养过程中分泌到细胞外的代谢产物是致死效应的主要原因,并且上清液对果蝇的生物活性受pH值的影响。离子交换树脂柱和高效液相色谱分离分析表明草酸存在于硫磺菌原变种培养液的上清液中并且是果蝇致死效应和培养体系pH下降的一个重要因素。硫磺菌原变种在气升式反应器ALR/ff培养体系中草酸的浓度、菌丝体量和pH值呈简单相关。进一步分析发现还有另外一种结构未知、在碱性条件下呈紫红色的色素也具有致死效应。     

蘑菇菌丝体代谢产物对果蝇的致死效应

以果蝇为靶标 ,对 15种蘑菇液体培养产物的毒性进行了检测 ,发现硫磺菌红色变种Laetiporussulphureusvar miniatus和硫磺菌原变种Laetiporussulphureusvar sulphureus液体培养中产生的代谢产物对果蝇具有致死活性。气升式生物反应器 (Airlift/ff)培养条件下 ,硫磺菌红色变种和原变种培养体系的 pH值均连续下降 ,最终都降至 2 2左右。研究发现致死活性产物被分泌到细胞外 ,离子交换树脂柱层析的线性洗脱分离结果表明活性代谢产物呈酸性 ,进一步分析表明草酸是活性代谢物之一 ,而树脂静止交换表明另外一种未知色素也具有致死活性     

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100 %. Within each cluster, the ITS rDNA sequence variation was below 3 %. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.     

Antioxidant components of Laetiporus sulphureus (Bull.: Fr.) Murr. fruit bodies

Antioxidant activity of fruit bodies of Laetiporus sulphureus (Bull.: Fr.) Murr. (Polyporales) obtained by the natural plantation growing method in Pribaikal'e (Irkutsk region) has been studied. It was determined that the ethyl acetate fraction of L. sulphureus, which was chromatographically separated into seven compounds identified as quercetin, kaempferol, (+)-catechin, p-coumaric, gallic, caffeic, and chlorogenic acids was characterized with more expressed antioxidant activity. All compounds were extracted from this basidiomycete species for the first time. The quantitative amount of the substances isolated from L. sulphureus was determined by HPLC. It was found that antioxidant activity of preparations obtained from L. sulphureus is conditioned by phenolic compounds.     

Comparison of phenanthrene and pyrene degradation by different wood-decaying fungi.

The degradation of phenanthrene and pyrene was investigated by using five different wood-decaying fungi. After 63 days of incubation in liquid culture, 13.8 and 4.3% of the [ring U-14C]phenantherene and 2.4 and 1.4% of the [4,5,9,10-14C]pyrene were mineralized by Trametes versicolor and Kuehneromyces mutabilis, respectively. No 14CO2 evolution was detected in either [14C]phenanthrene or [14C]pyrene liquid cultures of Flammulina velutipes, Laetiporus sulphureus, and Agrocybe aegerita. Cultivation in straw cultures demonstrated that, in addition to T. versicolor (15.5%) and K. mutabilis (5.0%), L. sulphureus (10.7%) and A. aegerita (3.7%) were also capable of mineralizing phenanthrene in a period of 63 days. Additionally, K. mutabilis (6.7%), L. sulphureus (4.3%), and A. aegerita (3.3%) mineralized [14C]pyrene in straw cultures. The highest mineralization of [14C] pyrene was detected in straw cultures of T. versicolor (34.1%), which suggested that mineralization of both compounds by fungi may be independent of the number of aromatic rings. Phenanthrene and pyrene metabolites were purified by high-performance liquid chromatography and identified by UV absorption, mass, and 1H nuclear magnetic resonance spectrometry. Fungi capable of mineralizing phenanthrene and pyrene in liquid culture produced enriched metabolites substituted in the K region (C-9,10 position of phenanthrene and C-4,5 position of pyrene), whereas all other fungi investigated produced metabolites substituted in the C-1,2, C-3,4, and C-9,10 positions of phenanthrene and the C-1 position of pyrene.     

Identification of very-long-chain polyunsaturated fatty acids from Amphidinium carterae by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very-long-chain polyunsaturated fatty acids (VLCPUFAs) from total fatty acids of Amphidinium carterae and their identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The combination of argentation TLC and LC-MS/APCI was used to identify unusual VLCPUFAs up to hexatriacontaoctaenoic acid. Two acids, 36:7n-6 and 36:8n-3, were also synthesized to unambiguously confirm their structure. The possibilities of VLCPUFAs biosynthesis are proposed.     

Odd-numbered very-long-chain polyunsaturated fatty acids from the dinoflagellate Amphidinium carterae identified by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

A method is described for the enrichment of odd very-long-chain polyunsaturated fatty acids (VLCPUFAs) by means of RP-HPLC and argentation TLC from total fatty acids of the dinoflagellate A. carterae and their identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The combination of argentation TLC and LC-MS/APCI was used to identify rare and unusual odd VLCPUFAs up to nonacosahexaenoic acid. Two acids, (allZ)-nonacosa-11,14,17,20,23-pentaenoic acid (29:5n-6) and (allZ)-nonacosa-11,14,17,20,23,26-hexaenoic acid (29:6n-3), were synthesized for the first time to unambiguously confirm their structure. Possible biosynthetic pathways for odd VLCPUFAs are also proposed.     

Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore LC-MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid. All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.     

Production of fruit-bodies of a mycorrhizal fungus,Lyophyllum shimeji,in pure culture

Cultivation of mycorrhizal fungus,Lyophyllum shimeji, was examined using selected strains capable of forming primordia in pure culture. Mycelia grew fastest on barley grains containing synthetic liquid medium. The primordia readily formed in test-tubes after lowering the incubation temperature from 23°C to 15°C. The co-existence of pine seedlings had no promotive effect on primordium formation. Fruit-bodies formed on a medium consisting of barley, beech sawdust, and liquid synthetic nutrients in 500-ml glass bottles. Mature fruit-bodies produced basidiospores. The spores thus produced could germinate on an agar medium and formed mycelial colonies. Thereby, the life cycle inL. shimeji was accomplished in pure culture without using the host plant.     

Antimicrobial activity of Laetiporus sulphureus strains grown in submerged culture]

Cultural conditions for growth and fruit body formation were elaborated to four strains of Laetiporus sulphureus isolated from nature. All strains demonstrated antimicrobial activity against a wide spectrum of gram-positive and gram-negative bacteria during agar and submerged cultivation including methicillin-resistant strain of Staphylococcus aureus (MRSA) and glycopeptide-resistant strain of Leuconostoc mesenteroides. Antifungal activity was not found. The level of antimicrobial activity during submerged cultivation reached maximum after seven days of growth on specific medium with soybean meal and corn liquid; the next four weeks its increasing was not so manifested. Antimicrobial activity correlated with orange pigment secretion and cultural liquid acidification to pH 2.0-2.8 that indicates on acid nature of synthesized products.     

Changes in growth,hormones levels and essential oil content of Ammi visnaga L. plants treated with some bioregulators

The effects of foliar application of different concentrations of amino acids (tyrosine and phenylalanine) and phenolic acids (trans-cinnamic acid, benzoic acid and salicylic acid) on growth, pigment content, hormones levels and essential oil content of Ammi visnaga L were carried out during two successive seasons. It is clear that foliar application of either amino acids or phenolics significantly promoted the growth parameters in terms of shoot height, fresh and dry biomass, number of branches and number of umbels per plant. The increment of growth parameter was associated with elevated levels of growth promoters (IAA, GA3, total cytokinins) and low level of ABA. The greatest increase in the previously mentioned parameters was measured in plants exposed to different concentrations of phenols particularly in benzoic acid-treated plants. Such effect was concentration dependent. All treatments led to significant increments in yield seeds and oil content. Moreover, gas liquid chromatographic analysis revealed that the main identified components of essential oil were 2,2-dimethyl butanoic acid, isobutyl isobutyrate, α-isophorone, thymol, fenchyl acetate and linalool. Phenolics and amino acid treatments resulted in qualitative differences in these components of essential oil.     

Cultivation characteristics ofIsaria japonica

Cultivation characteristics of fruit-body (synnema) formation ofIsaria japonica were examined using liquid and solid media in order to produce fruit-bodies on a large scale. Mycelia grew well at 18–28°C on PDA medium with an initial pH of 7.0. The formation of fruit-bodies ofI. japonica was induced by lowering temperature to below 20°C in PD liquid medium. In sawdust-rice bran basal medium mixed with pupal powder prepared from silkworms (Bombyx mori), the fresh weight of fruit-bodies increased with increasing content of pupal powder. The highest yields of fruit-bodies were obtained in carbon-rich barley grain medium supplemented with pupal powder. The fruit-bodies grown under CO₂ concentrations of 1,000 μl/L had coral-like, many-branched synnemata with numerous conidiospores, whereas those formed under high concentrations (9,000 μl/L) of CO₂ had unbranched and longer synnemata. High concentrations of CO₂ remarkably inhibited conidiospore formation on synnemata. Continuous high-intensity illumination at 2.93 W·m⁻² inhibited the elongation of synnemata, and low-intensity illumination at 0.088 W·m⁻² slightly inhibited the branching of synnemata. Fruit-bodies were produced on the pupa metamorphosed from living larvae ofAgrotis fucosa placed on the surface of a culture ofI. japonica incubated in sawdust-rice bran medium.     

Contents of amino acids in Tricholoma giganteum

Seventeen protein-constitutive and free amino acids were isolated and determined from the fruit-bodies of Tricholoma giganteum . Aspartic acid and alanine were the most abundant protein constitutive amino acids. Among the free amino acids, alanine, glutamic acid, serine, aspartic acid and glycine were present in the largest amounts, with aspartic acid and glutamic acids especially dominant.     

Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

作物节木代料香菇与纯木屑代料香菇蛋白质营养比较研究

采用国际通用的蛋白质营养评价方法,对2种作物节木代料和纯木屑代料(CK)香菇子实体蛋白质的营养价值进行了比较研究。结果表明,2种作物节木代料香菇子实体蛋白质的NI指标低于纯木屑代料香菇,而CS、AAS、EAAI、BV、SRCAA等指标均高于纯木屑代料香菇。结论:作物节木代料香菇子实体和纯木屑代料香菇子实体蛋白质均具有较高营养价值,且二者之间无显著差异。     

Spheroplast fusion as a mode of genetic recombination in mycobacteria

Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR9 and A11, which were obtained by the chemical mutagenesis of the wild type strain A+ with N-methyl-N'-nitro-N-nitrosoguanidine. The carotenoid pigments and the alpha- and beta-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on L?wenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 degrees C. The frequency of NgR9 X A11 recombination in optimal conditions was about 2.5 X 10(-3). The recombinants selected on the basis of their carotenoid pigment profile were also tested for their alpha- and beta-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions for spheroplast fusion as a mode of genetic recombination in M. aurum are described.     

Identification and production of a novel natural pigment, cordycepoid A, from Cordyceps bifusispora

A novel yellow pigment, cordycepoid A, was isolated and identified from the entomogenous fungi Cordyceps bifusispora. Cordycepoid A exhibited no significant toxicity against Chinese hamster ovary (CHO) cells and mice, and showed high stability against food addictives, metal ions and heat. A liquid/solid double-phase cultural process for the production of the pigment was optimized as follows: 3 days aged liquid seed, 7.5 % inoculums, incubation temperature at 25 °C, 10 days of solid culture, and the last 5 days exposed to 200 Lx scattered light. The liquid seed medium and the solid culture medium were also optimized. Ethanol was selected as extracting solvent for its scale-up production. The optimal extracting conditions were determined as liquid/solid ratio at 20:1, extracting temperature at 40 °C, ultrasonic power at 400 W, and extracting time of 40 min.     

Fixed-bed fermentation of rice straw and chicken manure using a mixed culture of marine mesophilic microorganisms

A mixture of rice straw (80%) and chicken manure (20%) was pretreated and fermented to carboxylic acids by using a mixed culture of marine mesophilic microorganisms. Two sets of four fermentors, built from PVC pipes, were used for both biomass pretreatment and fermentation. Four 1L fermentors (F1-F4) were arranged in series, where liquid fermentation products were transferred from one fermentor to the other, to form a train. A liquid volume of 10mL and 15mL were transferred every four days for Trains A and B, respectively. The maximum total acid concentration for F1 in Train A was 34.2g/L and the maximum acid concentration in F2-F4 was approximately 44g/L. The maximum total acid concentration in F1 in Train B was 30.5g/L and the maximum acid concentration in F2-F4 was approximately 48g/L. The conversion in each of the fermentors in Train A varied from 0.821 to 0.879g VS digested/g VS fed and the yield was in the range 0.489-0.609g total acids/g VS fed. The conversion and yield in Train B were 0.741-0.914g VS digested/g VS fed and 0.563-0.669g total acids/g VS fed, respectively. The continuum particle distribution model (CPDM) predicted acid concentrations and retention times in the fixed-bed fermentation system with R(2) of 0.67-0.84 in Trains A and B.     

Modeling of eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum cultures in tubular photobioreactors. Effects of dilution rate, tube diameter, and solar irradiance

A model for the prediction of eicosapentaenoic acid (EPA) productivity from Phaeodactylum tricornutum cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effects of the external irradiance on the culture surface, the average irradiance inside the culture, and the light regime at which the cells are exposed on pigments and EPA content are studied. The chlorophyll content decreases exponentially with the average irradiance, whereas the carotenoids content increases linearly with the external irradiance due to a higher extension of photoinhibition. A decrease in the fatty acid content of the biomass with irradiance on reactor surface is observed when photoinhibition becomes relevant. The average irradiance within the culture mainly influenced the fatty acid profile of the biomass. As the average irradiance becomes higher, percentages of saturated and monounsaturated fatty acids decrease, increasing the portion of EPA. By taking into account the different relationships among pigment and EPA content with the irradiance, the variation in EPA productivity over the year can be simulated as a function of average and external irradiance. For the two photobioreactors employed the maximum EPA productivity is attained in spring and fall (30 mg L(-1) day(-1) for tube diameter 0. 06 m and 50 mg L(-1) day(-1) for tube diameter 0.03 m). In winter, the biomass productivity is limited by low light availability although the EPA content is maximum. In summer, the biomass productivity is higher although the EPA content diminished by photoinhibition; the higher the dilution rate, the lower the minimum. Thus, the conditions that increase the biomass productivity and the polyunsaturated fatty acids content are in opposition, the optimum being reached by operating under photolimitation with high growth rates in order to produce a high proportion of polyunsaturated fatty acids.     

一株产槲皮素类色素的银杏内生真菌的研究

从银杏树根中分离出70余株内生真菌.将它们置于马铃薯葡萄糖(PD)液体培养基中培养,发现其中一株(Gh01)能产生橙黄色色素.经过化学反应及HPLC检测证明该橙黄色色素为槲皮素类糖苷.这是关于内生真菌产槲皮素类糖苷的首次报道.本文深入探讨了碳源,氮源,金属离子,初始PH及培养温度对色素产量的影响.PD液体培养基的最适培养温度和初始PH分别为28℃和7.0.正交设计结果显示:最适碳源和氮源分别为20g/L葡萄糖和5g/L蛋白胨.增加1g/L的氯化锌可提高色素产量.在最适培养条件下连续培养120h色素的产量可达到27.515g/L.