Studies On Bioactive Compounds Production By Submerged Fermentation Of Antrodia Camphorata

Antrodia camphorata is a value medicinal mushroom in China, and grows specifically inthe inner wall of the rotting trunk of Cinnamomum kanehirai native only in Taiwan.Polysaccharides and triterpenes are main bioactive compounds of Antrodia camphorata.Antrodia camphorata has many virtues of keep liver, anti-tumour, anti-oxidation, immunitymodulation, deoxifcation and anti-inflammation, and has medicinal merit, and has anexploiture potential and apply foreground medicinal fungus. Recently, it has been become ahotspot of research and exploitation in china and out of china. There are two ways to obtainthese bioactive compounds. One is to extract bioactive compounds from fruiting bodies; theother is to obtain them from the fermentation products. However, the quality of fruitingbodies is not stable which easy affected by environmental conditions and the cycle culturetime is long, that always is several months. The quality of fermentation product is stable bycontrolling the fermentation conditions and period is short.The fermentation conditions of Antrodia camphorata optimized according to the objectmycelium biomass and intracellular triterpene yield, the metabolic laws investigated, theoptimal fermentation parameters were obtained as follows: 40g/Lglucose, 6 g/L soybean, 1g/L K₂HPO₄, 0.5 g/L MgSO₄ and 100mg/L VB₁ .The optimum cultural period was 6 d at 26℃in 250mL shake flasks at 100r/min.The best aeration ratio was 1:2.5(medium volume:flask volume). The inoculation volume was 20%. The intracellular triterpene and myceliumbiomass of Antrodia camphorata obtained (about 15.25mg/100mL and 0.53g/100mL) inthe optimum culture respectively, and they are increased, and then decreased withfermentation time prolonging. Because Antrodia camphorata inoculated in the anaphase oflogarithm growth, the stagnancy is very short. The anaphase of logarithm growth ofAntrodia camphorata is 72h, and it enter into contabescence about after 168h, and fromthat, thalli begin to autolyze, contabescence, mycelium biomass declining slowly. Therefore,Antrodia camphorata had better to inoculate at 72h, the stagnation of mycelium growth canbe avoided ,fermentation period was shorten, and intracellular triterpene was collected atstabilization(72～168h), at that time, mycelium growth and the collection of secondarymetabolites are also tiptop. After fermentation, mycelium was extracted using different solvents so as to extractingintracellular tdterpene; the result showed that the law ethanol ultrasonic is best. Thenintracellular triterpene was extracted using different concentration ethanol and differentextracting time. This work combining HPLC analysis and cell model in vitro for the firsttime. Results indicated that at concentrations of 50μg/mL, extractions(A, B, C, D) stronglyinhibited the growth of L1210 tumor cells and inhibition rates exceeded 50%. Moreover, itis correspond with the same chromatogram peak, when the value ofαis 0.68, relativelyarea is different. The bigger relatively area strongly inhibited inhibition rate of tumor cells.At concentrations of 50μg/mL, extractions(D , F) strongly inhibited the growth of SW620tumor cells and inhibition rates exceeded 50%. Moreover, it is correspond with the samechromatogram peak, when the value ofαis 0.63 and 0.68, relatively area is pretty much thesame thing. It will establish good theoretic base for separation and purification anti-tumorcomposition at the value ofαis 0.63 and 0.68.Antrodia camphorata was cultivated in the excellent condition, after fermentation,filtrated mycelium, and left Culture filtrates. Culture filtrates (100 mL) extracted four timesusing three different organic solvents: diethyl ether (Extract E), a mixture (1:1, v/v) ofpentane/diethyl ether (Extract P/E), and a mixture (2:1, v/v) of pentane/dichloromethane(Extract P/D). After extraction, the upper organic phases were dried over anhydrous sodiumsulfate, concentrated at 42℃to 1mL, and stored at -20℃prior to gaschromatography-mass spectrometry (GC-MS) and GC-Olfactometry (GC-O) analyses. Allextractions performed in triplicate.The result of GC- MS indicated that clear differences were in the number of organiccompounds extracted from culture fluids using different solvent systems, and probablyreflected the different solvent polarities. The highest number of compounds (45) wasdetected in P/E extracts, while solvent E contained 40 and solvent P/D only 20. Organiccompounds identified in P/E extracts consisted of 8 esters, 16 alcohols, 5 acids, 2 aldehydes,5 heterocyclic compounds, 4 hydrocarbons and 5 ketenes. Solvent E extracts contained fiveesters, 15 alcohols, 5 acids, 3 aldehydes, 6 heterocyclic compounds, 2 hydrocarbons and 4ketenes.The result of GC- olfactometry indicated that ethyl acetate,γ-undecalactone, linalool and3-hydroxy-2-butanone were strong intensity, with sweet, juicy peach, Citric, flowery, fresh,spicy, buttery, milk noses. These components made up the special aroma character of A.camphorata fluids.