Recovery of eicosapentaenoic acid from fungal mycelia by solvent extraction

Utilization of lipids containing eicosapentaenoic acid (EPA) produced by microorganisms requires processes for their efficient recovery from microbial cells. Recovery of EPA from mycelia of the fungusPythium irregulare by solvent extraction with hexane-isopropanol (HIP) in a pilot-plant colloid mill was investigated. Extraction efficiencies of 96% for lipid and EPA were achieved with a 3∶2 (vol/vol) HIP mixture by milling wet, filtered mycelia for 5 min at a solvent/dry solids ratio of 100 L/kg. The process yielded a crude extract that contained up to 96% lipid and an EPA content as high as 24% (with no selectivity for EPA).     

Chemical response of Picea glehnii seed-epiphytic Penicillium species to Pythium vexans under in vitro competitive conditions for mycelial growth

The potential protection of Picea glehnii seedlings from damping-off by seed-epiphytic Penicillium species was investigated. We studied the chemical response of seed-epiphytic Penicillium species (Pen. cyaneum, Pen. damascenum, and Pen. implicatum) to Pythium vexans, a damping-off fungus, in vitro. Penicillium species were cultured singly or cocultured with Pyt. vexans for 14 or 18 d, and mycelial growth, pH of culture filtrate, antifungal activity of the culture filtrate against Pyt. vexans, and the amount of antifungal compound produced by each Penicillium species, were examined. The filtrate of both the single culture of Penicillium and the coculture of Penicillium and Pyt. vexans showed antifungal activity against Pyt. vexans. In a coculture with Pyt. vexans, Pen. cyaneum produced an antifungal compound (patulin) as in the single culture. Pen. damascenum cocultured with Pyt. vexans produced an antifungal compound (citrinin), as it did in the single culture and in larger amounts on day 10. Pen. implicatum produced two antifungal compounds, frequentin and palitantin, and the ratio of frequentin (with higher antifungal activity than palitantin) to palitantin was higher in the coculture with Pyt. vexans than in the single culture. Our results indicate that these Penicillium species have the ability to produce antifungal compounds and to keep antifungal activity under competitive condition with Pyt. vexans. The chemical response of these Penicillium species to Pyt. vexans may contribute to protect P. glehnii seedlings from damage by Pyt. vexans.     

Use of Cottonseed Meal for Producing Eicosapentaenoic Acid by Pythium irregulare

Cottonseed meal (CSM), a common agricultural by‐product, was used as a nutrient source for the production of eicosapentaenoic acid (EPA) by Pythium irregulare. CSM can support good cell growth performance, as can yeast extract (YE). In terms of the maximum EPA content and EPA yield, CSM is superior to YE. Low concentrations of CSM are beneficial to lipid synthesis, and high concentrations favor the EPA content. Utilizing response surface methodology (RSM) analysis, the optimum contents of glucose and CSM in the fermentation medium were determined to be 40.2 and 16.1 g/l, respectively. After 6 days of fermentation at 25 °C and optimal conditions, the EPA yield and productivity were 245.3 and 40.9 mg/l day, respectively. Particle size of CSM was found to affect the EPA production, and a finely ground CSM (100 mesh) was determined to be best for EPA production. The variation in the fatty acid content of total fatty acid (TFA) indicates that EPA was synthesized through the n‐6 route in P. irregulare and Δ12 desaturase was the key enzyme for EPA biosynthesis. Sodium carbonate was determined to be notably good at removing free gossypol attached to biomass. After fungal biomass from each flask had been harvested from Na₂CO₃‐supplemented medium, 1 % (w/v) Na₂CO₃ solution was used to wash the mycelia three times; free gossypol (FG) was not detected (detection limit 0.0018 %). This work provides a new approach using cottonseed meal to produce EPA through fungal fermentation.     

寡雄腐霉与氟噻唑吡乙酮协同防治烟草黑胫病研究

为了探究寡雄腐霉(Pythium oligandrum,PO)与氟噻唑吡乙酮(Oxathiapiprolin,OX)联合防控烟草黑胫病的可行性,采用菌丝生长速率法和平板对峙法分别测定了PO和OX的生物相容性,及OX、PO和OX+PO对黑胫病菌的毒力,并通过盆栽试验分别测定了OX、PO和OX+PO对烟草黑胫病菌的防效,以及对烟草幼苗的促生效应。生物相容性试验结果表明,不同浓度OX处理下,PO的生长速率与空白对照无显著差异。室内毒力结果显示,8.0×10^-3 mg/L OX对烟草黑胫病菌的抑制率为91%,EC₅₀为2.19×10^-3 mg/L,而OX(1.0×10^-3 mg/L)与PO联用的抑制率达96.31%。盆栽试验结果表明,OX+PO处理对烟草黑胫病的防效最高(84.57%),比OX(77.76%)或PO(76.54%)单独处理防效分别提高8.76%和10.49%。OX和PO联用对烟草促生效果显著,其株高、茎粗、叶长、叶宽分别比对照处理提高了57.10%、20.21%、33.62%、35.08%。...     

Supercritical carbon dioxide extraction of lipids from Pythium irregulare

Lipids that contain polyunsaturated fatty acids (PUFA) have therapeutic value. PUFA, however, degrade in high-temperature, oxygen-rich conditions typical of conventional hot solvent-extraction and distillation methods. Supercritical CO₂ extraction was chosen as an alternative method to recover these valuable compounds from the lower fungus, Pythium irregulare. Freeze-dried biomass was subjected to an aqueous phase and placed into a flow-through extraction apparatus. Extraction of oil from this biomass showed some success for moisture contents as high as 30% (wet basis). The addition of a novel CO₂-philic surfactant to the wet biomass with moisture contents as high as 95% (wet basis) increased the extraction rate of fungal oil by more than an order of magnitude. For tests with extraction times of 5 to 6 h, data for the diffusion-controlled region were modeled with an analytical solution to Fick’s second law. Equilibrium data were also obtained for the fungal oil at two isotherms (40 and 60°C) over a pressure range of 13.7 to 27.5 MPa.     

原始森林高产纤维素酶刺器腐霉的筛选鉴定

采用羧甲基纤维素钠(carboxyl methyl cellulose, CMC-Na)为唯一碳源,从广西罗城县原始森林腐木下的土壤中初步筛选分离出具有降解纤维素能力的菌株,结合刚果红染色,确定透明圆的直径大小,进一步判断纤维素的分解能力;并对其进行形态描述和显微观察。最后筛选分离得到4株产酶菌株,将其接种于液体发酵培养基,通过测定葡聚糖内切酶(endo-glucanase, CMCase)酶活力,经刚果红染色法证明H-2菌株在CMC-Na 筛选平板上形成的透明圈和菌落直径最大,产羧甲基纤维素酶的能力强。液体发酵粗酶活测定发现,H-2菌株羧甲基纤维素酶活力达130 U/mL 。H-2菌株形态特征和ITS序列建系统进化树鉴定为刺器腐霉(Pythium acanthophoron)。     

响应面法优化终极腐霉生产EPA的发酵工艺

为进一步提高终极腐霉EPA产量,在初步优化发酵条件的基础上,通过Plackett-Burman和Box-Behnken实验设计优化终极腐霉生产EPA发酵工艺。获得的最优发酵工艺条件为:蔗糖8%,硝酸钾0.4%,酵母粉0.65%,磷酸氢二钠0.175%,硫酸镁0.065%,大豆油1.0%,起始p H6.0,装液量50 m L/250 m L。在最优发酵工艺条件下,EPA产量达到541.61 mg/L,比初步优化产量(456.39 mg/L)提高了85.22 mg/L。     

Influence of surfactants on an extracellular lipase fromPythium ultimum

The activity of an extracellular lipase from the fungusPythium ultimum was studied as a function of emulsion properties, and the amount and type of surfactants used in preparing the emulsions. The highest emulsified globule surface area as a function of surfactant concentration and emulsion stability were in the order taurocholic acid >Triton X-100>3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Lipase activity was dependent on both the type and the concentration of surfactant, with taurocholic acid and CHAPS giving tenfold stimulation over the control at 0.1 and 0.8 mM, respectively. The evidence suggests that the influence of surfactant is related more to interaction with the enzyme at the oil-water interface than to providing greater interfacial surface area.     

Damage-Induced Accumulation of Phytoecdysteroids in Spinach: A Rapid Root Response Involving the Octadecanoic Acid Pathway

Some plant defenses are known to be rapidly induced following attack by phytophagous insects. Plant-produced insect molting hormones, termed phytoecdysteroids, are believed to aid plant resistance; however, their dynamics are poorly understood. Using spinach (Spinacia oleracea) as a model system, we examined the inducibility of phytoecdysteroids, primarily 20-hydroxyecdysone (20E), in an effort to characterize potential interactions with herbivorous insects. Rapid phytochemical induction was investigated using damage treatments and applications of defense-related plant-signal analogs, specifically methyl jasmonate (MJ) and methyl salicylate (MSA). Within two days, mechanically damaged roots exhibited two to three fold increases in phytoecdysteroid concentrations. Four days after root damage, small increases in shoot levels were also detectable. Unlike roots, foliar 20E concentrations were unaltered over a range of shoot treatments including insect herbivory (Spodoptera exigua), mechanical damage, and MJ applications. Additions of MJ (12.5–50 g/liter) to the root systems of hydroponically grown plants stimulated accumulations of root phytoecdysteroids in a dose-dependent manner, similar in magnitude to the response induced by root damage. Under identical conditions, MSA did not affect the accumulation of 20E when added to the hydroponic solutions of undamaged plants. Moreover, MSA inhibited the induction of 20E in wounded roots, but did not interfere with the action of applied MJ. In contrast to mechanical damage, roots did not induce 20E levels when challenged with two different fungal pathogens (Pythium aphanidermatum and Phytophthora capsici).We propose that wound-induced accumulations of 20E are generated in the roots, signaled via endogenous jasmonates, and may confer enhanced resistance against subterranean herbivorous insects.     

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.)

BACKGROUND: Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing manu ailments, including cancer have been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics. RESULTS: Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high‐performance liquid chromatography and sodium dodecyl sulfate–PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 °C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N‐Cbz‐Ile‐Pro and N‐Cbz‐Phe‐Leu in a time‐dependent manner. Peptide mass fingerprint using matrix‐assisted laser desorption/ionization–time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 µg mL^?1 towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. CONCLUSION: Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics. Copyright © 2009 Society of Chemical Industry