Improvement of ganoderic acid production by fermentation of Ganoderma lucidum with cellulase as an elicitor

Two-stage cultivation of Ganoderma lucidum was performed for the enhanced production of ganoderic acid (GA). Cellulase was identified to be an effective elicitor for the improvement of GA production, and GA titer reached 1334.5 mg/l compared to the control (779.6 mg/l) using lactose as the substrate without cellulase addition. Loading of 5 mg/l cellulase on day 3 resulted in the maximal GA titer of 1608 mg/l. To our knowledge, this is the first time that cellulase was used as the elicitor to enhance GA production. Submerged fermentation in a 2.0-l bioreactor was also conducted with cellulase as the elicitor, and as a result the maximal GA titer of 1252.7 mg/l was obtained on day 12. This is so far the best GA production obtained in submerged fermentation of G. lucidum.     

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Abstract

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.     

Polysaccharides of Ganoderma lucidum: factors affecting their production

The conditions of polysaccharide production by the fungus Ganoderma lucidum were optimized. The maximal yield of endopolysaccharides and exopolysaccharides was observed at 25-30 degrees C, initial pH of culture medium 4.0-6.0, and at a C : N ratio of approximately 18 : 1 and 25 : 1, respectively. The greatest yield of mycelium was reached at a more intensive aeration, and the maximal yield of polysaccharides was observed at a less intensive aeration. The optimal ratio between fungus growth and polysaccharide production was observed at 100 rpm and an aeration of 1.0-1.5 1/1 medium min.     

变转速策略调控灵芝菌丝体发酵高产三萜

灵芝发酵过程中,采用变转速调控策略,对振荡发酵阶段进行优化,以期达到高产三萜的目的。振荡阶段最佳条件为转速由150r/min变为100r/min,该策略与液体静置培养相结合,最终菌丝体三萜产量高达678.0g/L,比优化前提高了21%。振荡发酵阶段的变转速策略有效地提高了三萜的产量。     

Significance of inoculation density control in production of polysaccharide and ganoderic acid by submerged culture of Ganoderma lucidum

Control of inoculation density was significant for cell growth, morphology, and production of polysaccharide and ganoderic acid in submerged culture of the higher fungus Ganoderma lucidum. A maximal cell concentration of 15.7 g dry cell weight (DW)/l was obtained at an inoculation density of 330 mg DW/l. For inoculation density within the range of 70–670 mg DW/l, a large inoculation density led to a small pellet size and high production of extracellular and intracellular polysaccharides, while a relatively big pellet size and high accumulation of ganoderic acid were observed at a low inoculation density. It was also shown that small pellet size resulted in high polysaccharide production, while large pellet size led to high production of ganoderic acid.     

Anti-inflammatory and anti-tumor-promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum

A series of lanostane-type triterpene acids, including eleven lucidenic acids (3, 4, 9, 10, 13-19) and six ganoderic acids (20-22, 24, 26, 27), as well as six sterols (28-33), all isolated from the fruiting bodies of the fungus Ganoderma lucidum, were examined for their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, a known primary screening test for anti-tumor promoters. All of the compounds tested, except for ganolactone (27) and three sterols (29-31), showed potent inhibitory effects on EBV-EA induction, with IC(50) values of 235-370 mol ratio/32 pmol TPA. In addition, nine lucidenic acids (1, 2, 5-8, 11, 12, 18) and four ganoderic acids (20, 23-25) were found to inhibit TPA-induced inflammation (1 microg/ear) in mice, with ID(50) values of 0.07-0.39 mg per ear. Further, 20-hydroxylucidenic acid N (18) exhibited inhibitory effects on skin-tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.     

Effects of different graphene-based nanomaterials as elicitors on growth and ganoderic acid production by Ganoderma lucidum

Graphene-based nanomaterials (GBNs) have attracted considerable interest nowadays due to their wide range of applications. However, very little attention has been paid to the application of nanomaterials as potential elicitors for production of valuable metabolites. Herein, aiming to earn insight into effects of nanomaterials on secondary metabolite biosynthesis by medicinal fungi, we evaluated the influence of GBNs on growth and production of ganoderic acid (GA) by Ganoderma lucidum in submerged culture. Graphene oxide (GO), reduced graphene oxide (rGO), and rGO/Fe₃O₄ nanocomposite were synthesized successfully and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy analysis. The prepared nanomaterials were added to the culture of G. lucidum at final concentrations of 50, 100, and 150 mg/L on Day 5. The results showed that the elicitation of G. lucidum with GO and rGO decreased the cell dry weight and GA production slightly, especially in higher concentrations. However, rGO/Fe₃O₄ nanocomposite not negatively affected cell growth and improved GA production. G. lucidum growth rate responded to elicitation experiments differently and depended on the type of nanomaterials and their concentrations, but almost all GBNs caused an increase in GA content (mg/100 mg dry weight). Also, field emission scanning electron microscopy morphological study showed that under elicitation, mycelia were more condensed and tightly stacked together. The findings from this study may suggest that GBNs in low concentrations could be applied as elicitors to secondary metabolites production from higher fungus, but further environmental, physiological, and biological studies required.     

灵芝三萜酸分批发酵的非结构动力学模型

研究了灵芝胞内和胞外三萜酸在30L发酵罐中分批发酵的动力学特征。利用Sigmoid函数构建了灵芝细胞生长、底物消耗、胞内和胞外三萜酸的非结构动力学模型,并根据Boltzmann拟合求解出各模型参数。结果表明,各模型预测值能够较好地吻合实验实测值。灵芝细胞比生长速率在第2.5天达到最大值（μmax）,为0.700d?1;葡萄糖比消耗速率在第2.4天达到最大值（qS, max）为1.060d?1;胞内三萜酸比合成速率在第4.7天达到最大值（qITA, max）为11.345mg/(g·d);胞外三萜酸比合成速     

A novel membrane-surface liquid co-culture to improve the production of laccase from Ganoderma lucidum

In this work, a laccase producer, Ganoderma lucidum, was separated and identified according to its morphological characteristics and phylogenetic data. A 4000 U/l and 8500 U/l of laccase activity was obtained in 500 ml flask by submerged culture and biomembrane-surface liquid culture (BSLC), respectively. Furthermore, the novel biomembrane-surface liquid co-culture (BSLCc) was developed by adding Saccharomyces cerevisiae to reactor in order to shorten the fermentation period and improve laccase production. Laccase activity obtained by BSLCc, 23 000 U/l, is 5.8 and 2.7 times of that obtained by submerged culture and BSLC, respectively. In addition, laccase production by BSLCc was successfully scaled-up to 100 l reactor, and 38 000 U/l of laccase activity was obtained on day 8. The mechanism of overproducing laccase by BSLCc was investigated by metabolism pathway analysis of glucose. The results show glucose limitation in fermentation broth induces the secretion of laccase. The addition of S. cerevisiae, on one hand, leads to an earlier occurrence of glucose limitation state, and thus shortens the fermentation time; on the other hand, it also results in the appearance of a series of metabolites of the yeast including organic acids, ethanol, glycerol and so forth in fermentation broth, and both polyacrylamide gel electrophoresis analysis and enzyme activity detection of laccase show that these metabolites contribute to the improvement of laccase activity.     

On immunotherapies and cancer vaccination protocols: A mathematical modelling approach

In this paper we develop a new mathematical model of immunotherapy and cancer vaccination, focusing on the role of antigen presentation and co-stimulatory signaling pathways in cancer immunology. We investigate the effect of different cancer vaccination protocols on the well-documented phenomena of cancer dormancy and recurrence, and we provide a possible explanation of why adoptive (i.e. passive) immunotherapy protocols can sometimes actually promote tumour growth instead of inhibiting it (a phenomenon called immunostimulation), as opposed to active vaccination protocols based on tumour-antigen pulsed dendritic cells. Significantly, the results of our computational simulations suggest that elevated numbers of professional antigen presenting cells correlate well with prolonged time periods of cancer dormancy.     

The intracellular pH of the thermophilic bacterium Thermoanaerobacter wiegelii during growth and production of fermentation acids

The thermophilic glycolytic anaerobe Thermoanaerobacter wiegelii grows over the pH range 5.1–7.7, and no growth is observed below pH 5.0 or above 7.7. When T. wiegelii was grown in pH-uncontrolled batch culture, glucose was fermented to ethanol, acetate, and lactate. Small amounts of lactic acid were produced once the external pH reached 6.0, and a fructose-1,6-diphosphate (FDP) activated lactate dehydrogenase (LDH) was detected in cell-free crude extracts. Maximal activation of LDH by FDP was observed at pH 6.2. As the pH of the medium declined from 6.7 to 5.1 due to the production of acetate and lactate, the total protonmotive force (Δp) remained between −110 and −130 mV, and the membrane potential (ΔΨ) decreased from −104 to −65 mV. This decrease in ΔΨ was paralleled by an increase in the chemical gradient of protons (ZΔpH) from −31 to −62 mV at pH 5.1. Based on these results, T. wiegelii maintained a small ΔpH (0.3–0.9 units, inside alkaline) as the medium pH declined and interconverted ΔΨ to ZΔpH to maintain the total Δp relatively constant. Intracellular potassium decreased from 150 mM at pH 6.70 to 50 mM at pH 5.1, and this represented a 33-mV decline in the transmembrane chemical potential of potassium. The ability to synthesize ATP remained constant as the external pH declined, and therefore metabolic energy per se was not a critical aspect of pH sensitivity. Received: March 16, 2000 / Accepted: May 8, 2000