Study On Submerged Culture Of Antrodia Camphorata And Its Triterpenoids

Antrodia camphorata, a new basidiomycete in the Polyporaceae (Aphyllophorales) distributed only in Taiwan, is well known in Taiwan under the name"niu-chang-ku"or"niu-chang-chih"due to its potent biologic activities. Investigations revealed that Antrodia camphorata has extensive biological activities, such as hepatoprotective effect, anti-hepatitis B surface antigen, antioxidation and anticancer activities. In this study, the Antrodia camphorata strain preserved in the lab was identified by molecular biotechnology, and observed on its biological characteristics. The culture condition of Antrodia camphorata was optimized to increase the triterpenoid production. Meanwhile, the bioactive components of Antrodia camphorata cultured by 5 L fermentor were extracted and analyzed. Furthermore, the protective effect of components of Antrodia camphorata in submerged culture on ethanol-induced liver injury was evaluated.The Antrodia camphorata strain preserved in the lab was identified by analyzing its 18S rDNA and ITS rDNA sequences. When compared with the data in the NCBI database, it is conformed that the preserved strain was Antrodia camphorata. Meanwhile, the biological characteristics of the stain were evaluated, and the anthroconidia produced in submerged culture were observed.The spectrophotometric quantitative method was established to determine the content of total triterpenoid of Antrodia camphorata in submerged culture by using vanillin-perchloric acid as the developer. The reaction conditions were optimized. Taking oleanolic acid as the standard, 5% vanillin-acetic acid (0.3 mL) and perchloric acid (1 mL) as the developer, after being incubated at 60°C for 20 min the OD value of sample solution was determined at 550 nm. This method operated conveniently, and the result was stable and repeatable. In order to increase the triterpenoid production, the culture condition of Antrodia camphorata was optimized by method of one-variable-at-a-time. The optimized result was showed as following: inoculum with spores at 1.53×105 cell/mL, temperature of 26°C, rotation speed of 100 rpm, initinal pH of 4.5, culturing for 12 d. The optimized medium was glucose 20.0 g/L, peptone 10.0 g/L, MgSO4 1.0 g/L, KH2PO4 2.0 g/L, bran 2.0 g/L.Furthermore, the variables that significantly affected the biomass and triterpenoid production of Antrodia camphorata were optimized by RCCD, and the results were trained by BP artificial neural network (ANN). Genetic algorithm was used to obtain the optimal results: inoculum of 1.09×105 cell/mL, glucose of 32.93 g/L, peptone of 2.81 g/L, soybean flour of 2.01 g/L. The predicted triterpenoid production was 63.14 mg/L. The experimental result showed that the triterpenoid production under the optimal culture condition reached to 55.89±2.43 mg/L with difference 11.48% as comparing to the predicted data. It was increased to 221.6% as compared with the pre-optimized yield 25.22 mg/L.Effects of extracts from stem and leave of Cinnamomum camphora on the biomass, triterpenoid yield and triterpenoid production of Antrodia camphorata were evaluated. Petroleum ether and ethyl acetate extract of stem and leave of Cinnamomum camphora could promote the triterpenoid yield of Antrodia camphorata, 0.05 g/L petroleum ether extract of stem of Cinnamomum camphora (PES) showed the greatest activity. The triterpenoid yeild reached to 23.20 mg/g, increased 30.78% as compare to the control. A total of 39 compounds were identified in the PES by GC-MS, hereinto, terpenoids (28 kinds, 50.86%) including 9 monoterpenoids (30.57%) and 19 sesquiterpenoids (20.29%) were the main compounds. Furthermore, effect of different compound in PES on the biomass, triterpenoid yield and triterpenoid production of Antrodia camphorata was evaluated. The results showed thatα-terpineol could promote the yield and production of triterpenoids and volatile terpenoids of Antrodia camphorata, and influenced the biosynthesis pathway of terpenoids in Antrodia camphorata.Effects of rotate speed and inoculum on the triterpenoid production of Antrodia camphorata were evaluated in 5 L fermentor. The result showed that the optimal rotate speed was 50 rpm, and the optimal inoculum was 1.0×105 spores/mL. The biomass reached to the highest (6.51 g/L) at 7th day, while the triterpenoid production reached to highest (48.90%) at 8th day.The fermented product of Antrodia camphorata cultured by 5 L fermentor was analyzed. The results showed that Antrodia camphorata contained 8 kinds of minerals. Relative contents of saturated fatty acid in mycelia, spore and culture broth were 55.32%, 58.62% and 60.79%, respectively. And for unsaturated fatty acid were 43.15%, 40.52% and 39.18%, respectively. A total of 18 amino acids were detected in mycelia (26.49 g/100g) and spores (30.56 g/100g) among which glutamic acid was the highest. Both intracelluar polysaccharides (IPS) and extracelluar polysaccharides (EPS) in Antrodia camphorata were comprised of manose, glucose and galactose but in different proportion.A total of 49 and 55 kinds of volatile compounds were identified in the culture broth and mycelia of Antrodia camphorata in submerged culture, respectively including 22 alcohols, 8 ketones, 7 aldehydes, 23 esters, 5 terpene hydrocarbons and 3 aromatics. The overall flavor of the Antrodia camphorata in submerged culture was dominated by mushroom-like, fruity and flowery impressions. The impression'mushroom-like'was mainly attributed due to a series of C8 aliphatic components, including 3-octanol, 1-octen-3-ol, (E)-2-octen-1-ol, 3-octanone, 1-octen-3-one and 1-octen-3-yl acetate. Several lactones such asγ-octalatone andγ-undecalactone were accounted for the peach-like fruity odor of submergedly cultured Antrodia camphorata. Furthermore, several flower-like odorants were picked up in various intensities during the GC-O investigation of the culture broth, with L-linalool giving the strongest impact.The extraction of total triterpenoids in the mycelia of Antrodia camphorata was optimized by the method of one-variable-at-a-time and response surface methodology (RSM). The optimal conditions for the extraction of total triterpenoids included the following parameters: ethanol 86%, extraction temperature 75 ?C, liquid-solid ratio 37, and extraction twice. The predicted yield of triterpenoids was 3.23%. Furthermore, the Fick's second law of diffusion was used to establish the kinetics equation of extracting triterpenoids parameters from mycelia of Antrodia camphorata. The results showed that the kinetics on the extraction of triterpenoids agreed with the first-order rate equation, and the rate-determining step for extraction was the diffusion of triterpenoids through the holes in particles. The triterpenoids in the culture broth were adsobed by macroporous resin. Compared with static and dynamic absorption performance, macroporous resin XAD-16 was selected to adsorb the steroids from culture broth. Furthermore, the conditions of macroporous resin for adsorbing the fungal steroids from fermentation broth were optimized. The pH of solution of 6.0, the initial steroid compound concentration of 0.97 mg/mL, with a flow rate of 2 mL/min, desorption solvent of ethanol. Macroporous resin compounds for triterpenoid adsorption through van der Waals force, supplemented by the role of the hydrogen bond. The fitting equation proves that the macroporous resin adsorb triterpenoid by singer-layer adsorption. In the process of adsorption, the temperature will decrease the adsorption efficiency. The hepatoprotective effects of the extracts of Antrodia camphorata in submerged culture on ethanol-induced liver cell injury were evaluated. The results showed that ethanol extract and water extract of mycelia possessed the potent hepatoprotective effect. The survival rate of ethanol-treated liver cells incubated with ethanol extract (25μg/mL) and water extract (200μg/mL) for 48 h increased to 57.71% and 63.60%, respectively, which was significantly different with that of the group treated ethanol alone.