Gao-Qiang Liu

Improving the Fermentation Production of the Individual Key Triterpene Ganoderic Acid Me by the Medicinal Fungus Ganoderma lucidum in Submerged Culture

Enhanced ganoderic acid Me (GA-Me, an important anti-tumor triterpene) yield was attained with the medicinal fungus Ganoderma lucidum using response surface methodology (RSM). Interactions were studied with three variables, viz. glucose, peptone and culture time using a Central Composite Design (CCD). The CCD contains a total of 20 experiments with the first 14 experiments organized in a fractional factorial design, with the experimental trails from 15 to 20 involving the replications of the central points. A polynomial model, describing the relationships between the yield of GA-Me and the three factors in a second-order equation, was developed. The model predicted the maximum GA-Me yield of 11.9 mg·L−1 for glucose, peptone, culture time values of 44.4 g·L−1, 5.0 g·L−1, 437.1 h, respectively, and a maximum GA-Me yield of 12.4 mg·L−1 was obtained in the validation experiment, which represented a 129.6% increase in titre compared to that of the non-optimized conditions. In addition, 11.4 mg·L−1 of GA-Me was obtained in a 30-L agitated fermenter under the optimized conditions, suggesting the submerged culture conditions optimized in the present study were also suitable for GA-Me production on a large scale.

Improved Production and Antitumor Properties of Triterpene Acids from Submerged Culture of Ganoderma lingzhi

Triterpene acids (TAs) are the major bioactive constituents in the medicinal fungus Ganoderma lingzhi. However, fermentative production of TAs has not been optimized for commercial use, and whether the TAs isolated from G. lingzhi submerged culture mycelia possess antitumor activity needs to be further proven. In this study, enhanced TA yield and productivity were attained with G. lingzhi using response surface methodology. The interactions of three variables were studied using a Box-Benhnken design, namely initial pH, dissolved oxygen (DO) and fermentation temperature. The optimum conditions were an initial pH of 5.9, 20.0% DO and 28.6 °C. These conditions resulted in a TA yield of 308.1 mg/L in a 5-L stirred bioreactor. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and maximum TA production and productivity of 295.3 mg/L and 49.2 mg/L/day were achieved, which represented 80.9% and 111.5% increases, respectively, compared with the non-optimized conditions. Additionally, the triterpene acid extract (TAE) from G. lingzhi mycelia was found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro, and the TAE exhibited dose-dependent antitumor activity against the solid tumor sarcoma 180 in vivo. Chemical analysis revealed that the key active triterpene compounds, ganoderic acid T and ganoderic acid Me, predominated in the extract.

Extraction of Mycelial Polysaccharides from Submerged-Cultured Agaricus blazei

Aqueous extraction and alkaline (NaOH) hydrolysis method were comparatively studied in polysaccharides extraction from submerged-cultured Agaricus blazei mycelia. The results show that the polysaccharide content and the total extract rate from alkaline extraction method were obviously higher than those from the aqueous extraction method. Optimization results by the orthogonal matrix method on polysaccharide extraction from A. blazei show that the optimum alkaline concentration was 1.0 mol/L, hydrolysis temperature 60℃, hydrolysis time 1 hour, and liquid: solid ratio 15. With the optimized condition, the polysaccharide extraction rate reached 11.93 ±± 0.68 %. The mycelial polysaccharide obtained with alkaline hydrolysis method was fractionated by the DEAE column chromatography. Five APS fractions were isolated in the polysaccharide sample with APS-1 as the main component

Polysaccharides extraction from submerged-cultured mycelium of Grifola frondosa

Alkaline (NaOH) hydrolysis and aqueous extraction method were comparatively studied in polysaccharides extraction from mycelia of Grifola frondosa obtained with submerged-culture. The results show that the polysaccharide content and the total extract rate from alkaline extraction method were obviously higher than those from the aqueous extraction method. The conditions of alkaline extraction were further optimized by the orthogonal matrix method, and the results reveal that the optimum extraction conditions were as follows: ratio of alkaline volume to mycelium weight was 15 (mL/g), alkaline concentration was 1.0 mol/L, hydrolysis temperature was 70°C, and hydrolysis time was 1.5 hour. With the optimized condition, the polysaccharide extraction rate reached 11.18 ± 0.37 %. The mycelial polysaccharide obtained with alkaline hydrolysis method was fractionated by the DEAE column chromatography. Four G. frondosa polysaccharide (GFPS) fractions were isolated in the polysaccharide sample with GFPS-1 as the main component.

Efficient Accumulation and In Vitro Antitumor Activities of Triterpene Acids from Submerged Batch−Cultured Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes)

Triterpene acids are among the major bioactive constituents of lucidum. However, submerged fermentation techniques for isolating triterpene acids from G. lucidum have not been optimized for commercial use, and the antitumor activity of the mycelial triterpene acids needs to be further proven. The aim of this work was to optimize the conditions for G. lucidum culture with respect to triterpene acid production, scaling up the process, and examining the in vitro antitumor activity of mycelial triterpene acids. The key conditions (i.e., initial pH, fermentation temperature, and rotation speed) were optimized using response surface methodology, and the in vitro antitumor activity was evaluated using the MTT method. The optimum key fermentation conditions for triterpene acid production were pH 6.0; rotation speed, 161.9 rpm; and temperature, 30.1°C, resulting in a triterpene acid yield of 291.0 mg/L in the validation experiment in a 5-L stirred bioreactor; this yield represented a 70.8% increase in titer compared with the nonoptimized conditions. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and a triterpene productivity of 47.9 mg/L/day was achieved, which is, to our knowledge, the highest reported in the large-scale fermentation of G. lucidum. In addition, the mycelial triterpene acids were found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro. Chemical analysis showed that the key active triterpene acid compounds, ganoderic acids T and Me, predominated in the extract, at 69.2 and 41.6 mg/g, respectively. Thus, this work develops a simple and feasible batch fermentation technique for the large-scale production of antitumor triterpene acids from G. lucidum.

Medicinal Insect Catharsius molossus Enhances Production of Intracellular Polysaccharides in Ganoderma lucidum SCIM 0819

Chinese medicinal insects were added into the media to investigate their effects on the mycelial growth and polysaccharide formation by Ganoderma lucidum SCIM 0819. The experiments were carried out in submerged fermentation using shake flasks. The results show that addition of C. molossus at 5.0 g/L lead to significant increase in intracellular polysaccharides (IPS) concentration from 1.48 ±± 0.12 g/L to 1.85 ±± 0.17 g/L. However, it had no stimulatory effects on cell growth (P > 0.05). Other types of insects could not stimulate IPS formation, and all the insects showed inhibitory effect with an increase in the concentration. We further studied the effect of addition of C. molossus on IPS components of G. lucidum. The results indicate that there were no new components in IPS obtained by the addition of C. molossus, suggesting the biosynthetic pathways of IPS had not been changed

Screening of oleaginous microorganisms for microbial lipids production and optimization

To screen potential oleaginous microorganisms for production of microbial lipid, thirty-one yeast strains from forest soils collected from Hunan Province, China, were tested for their potentiality to produce microbial lipid, and fourteen potential strains were further screened. The results showed that yeast strain SCIM 2.012 displayed the most potential ability in producing microbial lipid, the lipid content of cells reached 52.4 %, and the lipid yield was 7.8±0.54 g/L. Furthermore, the components profile of the lipid was analyzed. The results suggested that the lipid was abundant in low degree unsaturated long chain fatty acid (C18: 1) and saturated long chain fatty acids (C16: 0), suggesting the lipid could be used for biodiesel production. The optimized media for lipid production from strain SCIM 2.012 were as the follows: Sucrose as carbon source with initial concentration 100 g/L, NH4NO3 as nitrogen source at 3.5 g/L, the initial pH was 6.0. With the optimized media, the lipid content of cells highly reached 56.7 %, the lipid yield was 9.7±0.67 g /L, and biomass was 17.1±0.37 g/L.

Effects of Water and Chloroform Extracts of Chinese Medicinal Insects on Polysaccharides Production by Ganoderma sinense in Submerged Fermentation

Water and chloroform extracts of various Chinese medicinal insects were added into the medium of Ganoderma sinense to investigate the effects of the extracts on production of biomass, intracellular polysaccharides (IPS), and extracellular polysaccharides (EPS). The results showed that chloroform extract of E. sinensis (CES) at 55 mg/L lead to significant increase in both biomass and IPS concentration from 7.29 plusmn 0.51 to 10.36 plusmn 0.39 g/L and 591.1 plusmn 29.1 to 622.3 plusmn 24.6 mg/L, respectively. In addition, chloroform extract of E. sinensis at 35 mg/L and water extract of C. molossus at 55 mg/L showed stimulatory effects on EPS production, and their EPS yield significantly enhanced from 365.2 plusmn 18.7 to 421.8 plusmn 16.1 mg/L and 365.2 plusmn 19.1 to 413.4 plusmn 17.1 mg/L, respectively. Both the extracts can be used as easily available stimulators for the biomass or polysaccharide production by submerged fermentation of G. sinense.

Enhanced Production of Biomass and Polysaccharides in Ganoderma lucidum SCIM 0818 by the Addition of Palmitic Acid

To screen stimulators for mycelial growth and polysaccharides production of Ganoderma lucidum , the fungus was inoculated into the media with and without supplementation of several fatty acids and ether extracts of Chinese medicinal insects. Palmitic acid at 1.0 g/L lead to significant increase in both biomass and extracellular polysaccharides (EPS) concentration from 7.73 plusmn 0.41 to 10.21 plusmn 0.43 g/L and 342.6 plusmn 10.7 to 401.3 plusmn 14.9 mg/L, respectively. In addition, ether extract of Eupolyphaga sinensis at 1.5 g/L also was of great advantage to biomass and EPS production. There were no new components in the EPS obtained by the addition of palmitic acid.

Head-Space Gas Chromatographic Analysis for the Volatile Flavor Compounds from Submerged Culture Broth of Ganoderma sinense (a Medicinal Fungus)

The volatile flavor compounds from submerged culture broth of Ganoderma sinense were investigated by head-space gas chromatography-mass spectrometry (GC-MS). More than twenty-eight different volatile flavor compounds from the broth were detected, and twenty compounds were identified, which were mainly ketones, alcohols and lactones. Among all the compounds detected in the broth, the compound with its characteristic retention peaks at 22.36 min was 2,5-dihydro-3,5- dimethyl-2-furanone, and its content was 68.19 % of the total contents of all the volatile flavor compounds. In addition, 3- penten-2-one and amyl vinyl carbinol with their characteristic retention peaks at 9.74 and 18.03 min, respectively, were the known food spice substances. The results were useful to help us to know the flavor compounds of culture broth since it can be used as a convenient medicinal product, and also to preliminarily understand the mechanism of light aroma emitted from the submerged culture broth of G. sinense .