| Summary: | 碩士 === 明新科技大學 === 化學工程研究所 === 96 === Antrodia camphorata (Zang-Zhi) belongs to the aphyllophorales and the poly -poracease family with the effect of remedy for anticancer, antitumor and protection of the liver. Some bioactive compounds of A. camphorata including sesquiterpene lactone, steroids, triterpenoids have been isolated and characterized. Wild A. camphorata parasitizes specifically on Cinnamomum kanehirai hays in a small quantity and its quality is not stable. In recent years, A. camphorata was attempted to be produced on large scale by submerged culture. The main purpose of this research was to investigate the influence of L-Proline and aeration rate on polysaccharide fermentation by submerged culture of A. camphorata, and to increase production of exopolysaccharide (EPS) by regulating two-stage culture strategy. Moreover, the biological activity of polysaccharides from A. camphorate was examined by using TNF-α release capability of polysaccharides on a murine macrophage cell line, RAW264.7. A. camphorata BCRC 35396 was obtained from the Bioresources Collection and Research Center (BCRC) in Food Industry Research and Development Institute (Hsin chu, ROC).
The effects of the different amino acids at 3g/L of (i.e., L-Arginine, L-Glutamine, L-Isoleucine, L-Lysine, L-Proline, and L-Threonine) on production of biomass and EPS were conducted in this study. The result exhibited that L-Lysine and L-Proline stimulated biomass (2.97g L-1) and EPS (218.64mg L-1) production, respectively. In addition, the effects of various concentrations of L-Proline (i.e., 3, 6, 9, 12, 15 g/L) on biomass and EPS production of A. camphorata were conducted in shake-flask cultures. Comparing with controlled 0 g/L of L-Proline, the biomass production increased with L-Proline concentration ranging from 3 to 15 g/L. The optimal biomass concentration was reached to 3.85 g/L as L-Proline concentration was at 15 g/L, and the optimal EPS concentration was reached to 247.12 mg L-1 as L-Proline concentration was at 12 g/L.
The effects of different L-Proline concentration (i.e., 3, 6, 9, 12, 15 g/L) on biomass and EPS production of A. camphorata by submerged culture were conducted in stirred-tank fermentor. The maximal value of the specific growth rate (μ) was 0.29 d-1 at 0.3% L-Proline. At 9g/L of L-Proline, the maximum values were 6.57 g L-1, 204.32 g L-1, 308.33 g L-1, and 47 mg g-1 for biomass, EPS, endopolysaccharide (IPS), and IPS content production, respectively. In addition, the effects of the aeration rates (i.e., 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 vvm) on production of biomass and EPS of A. camphorata by submerged culture were also conducted in stirred-tank fermentor in this study. Controlled with aeration of 0.2 vvm, the maximum value was 188.65mg L-1 for EPS production ; controlled with aeration of 1.2 vvm, the maximum values were 5.89g L-1, 289.98mg L-1, and 50.00mg L-1 for biomass, IPS, and IPS content production, respectively. In order to improve the A. camphorata production, the two-stage culture strategy was used. The productions were reached to 6.98g L-1, 255.19g L-1, 381.23g L-1, and 55.65mg g-1 for biomass, EPS, IPS, and IPS content, respectively.
The crude EPS from submerged culture of A. camphorata was purified by using Silica gel 60 column chromatography, where two EPS peaks were eluted. The protein to polysaccharide ratios from A. camphorata by regulating two-stage culture strategy (fraction-1 of EPS:20.78% ; IPS:38.69%) were superior those of batch cultures in 9g/L L-Proline, aeration rates of 0.2vvm and 1.2vvm. The molecular weight of EPS by regulating two-stage culture strategy (1.24×106 Da of EPS in fraction-1) was higher than those by two the other cultures (i.e., culture in 9g/L L-Proline, aeration rates of 0.2vvm and 1.2vvm), and the similar result was observed in the molecular weight of IPS (3.30×105Da) from regulating two-stage culture strategy. The carbohydrate compositions of polysaccharide produced from A. camphrata consisted of glucose, xylose, and galactose ; the percentages of monosaccharide compositions from polysaccharides in each fraction were almost constant. The results indicate that the monosaccharide compositions were not changed with culture conditions including of L-Proline effect, oxygen transfer, and two-stage culture strategy used in this study.
The results of biological activities (cytokine assay) of polysaccharide from A. camphorata were directly proportion to molecular weight of polysaccharide from A. camphorata. The higher molecular weight of polysaccharide was, the more murine tumor necrosis factor-alpha (mTNF-α) concentration was achieved. The maximal mTNF-α concentration values for EPS in fraction-1, EPS in fraction-2, and IPS were 1779.91 ± 46.52 pg/mL, 544.93 ± 44.39 pg/mL, 567.42 ± 50.83 pg/mL, respectively. Besides, the protein to polysaccharide ratio was closely correlated with the biological activity of polysaccharide from A. camphorata. The higher protein to polysaccharide ratio was, the more mTNF-α concentration was obtained.
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