Large-scale submerged fermentation of
Antrodia cinnamomea for anti-hepatoma
activity
Jih-Hung Pan, Yi-Song Chen, Lee-Yen Sheen and Been-Huang Chiang
∗Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC
Abstract
BACKGROUND: Submerged cultivation of Antrodia cinnamomea was carried out for manufacturing the fermentation product with anti-hepatoma activity. The fermentation process was optimized for different parameters at shake flask level to obtain products with high inhibition potency against Hep G2 hepatoma cells. Scale-up of the fermentation process was then achieved from 250 mL shake flask to 5 L, 500 L and 5-ton fermenters.
RESULTS: Glucose and malt extract were found to be the best carbon and nitrogen sources, respectively. The initial pH of 5.0 and an operating temperature of 22◦C were the best for a product with lowest IC50value. A shorter cultivation time was required when the scale of fermentation increased from 5 L to 5 tons. The reducing sugar and solids contents of the broth filtrate were correlated exponentially with the IC50of the ethanolic extract of mycelium for hepatoma cells, and the level of ergosterol in the mycelium extract follows the same profile as the increase in Hep G2 cells inhibition.
CONCLUSION: When Antrodia cinnamomea is cultured in a 5-ton fermenter, 4 weeks are required for the fermentation product to reach the highest anti-hepatoma activity. The solid and reducing sugar contents of the broth filtrate as well as the ergosterol content in the ethanol extract of mycelium can serve as the marker during fermentation for manufacturing product with anti-hepatoma activity.
2008 Society of Chemical Industry
Keywords: scale-up; submerged cultivation; Antrodia cinnamomea; anti-hepatoma activity; Hep G2 cells
INTRODUCTION
Antrodia cinnamomea, renamed from Antrodia campho-rata,1 is a fungus species native to Taiwan. Many studies have shown that A. cinnamomea possess various health-promoting activities including anti-inflammatory, antioxidant, anti-cancer and hepato-protective functions.1 – 5 It is generally believed that steroids and triterpenoids may be the major compo-nents for the bioactivities of A. cinnamomea. Recent evidence, however, shows that A. cinnamomea also contains other active compounds such as polysaccha-ride in the mycelium. Polysacchapolysaccha-ride from this fungus was found to exhibit anti-hepatitis B, anti-oxidative and immunomodulatory functions.6
Hepatocarcinoma is one of the most fatal cancers in Taiwan. This may be attributed to the fact that Taiwan is a hyperendemic area for hepatitis B virus infection, with approximately 16% of the general population infected with this virus.7In addition, chronic infection of Hepatitis B virus is a known risk factor of liver cancer. With such a high prevalence of liver cancer, people frequently use A. cinnamomea as an alternative
medical treatment. Nowadays many A. cinnamomea products are being sold commercially.
A. cinnamomea is a parasitic fungus that lives on the wall of the inner cavity of Cinnamonum kanehira. The fruiting body of A. cinnamomea is very expensive because it has a high medicinal value and also because of its slow growth rate. In view of this, submerged cultivation has been adopted to meet the consumption demands of this medicinal fungus.8Yang et al. (2003) cultured A. cinnamomea using submerged fermentation with different carbon sources (glucose, sucrose, potato starch, corn powder) and nitrogen sources (yeast extract, YM broth, malt extract).9They found that fermentation with corn powder as carbon source and YM broth as nitrogen source yielded the maximum amount of mycelium. Song et al. cultured A. cinnamomea in a 500 L fermenter at 27 – 30◦C for 7 days, and found that the filtrate of the fermentation broth had no inhibitory effect on human hepatoma cells, but the methanolic extract of mycelium had an anti-hepatomatic effect.3,10 Shu (2004) studied the effect of pH on the production and molecular weight distribution of exopolysaccharide produced by ∗Correspondence to: Been-Huang Chiang, Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC
E-mail: [email protected]
(Received 6 April 2008; revised version received 22 June 2008; accepted 22 June 2008) Published online 26 August 2008;DOI: 10.1002/jsfa.3332
A. cinnamomea during fermentation, and found that the exopolysaccharide yielded at pH 3.0 had a higher molecular weight (800 kDa) and pH 5.0 gave a higher exopolysaccharide yield (5.05 m g−1).11Huang (2002) cultivated a new strain of A. cinnamomea and evaluated the effect of the broth filtrate on different cancer cells.12 Through 2 weeks of cultivation, they claimed that the broth displayed a reddish color and exhibited inhibition of eight cell lines, including Hela, ES2, Hep 3B, MCF7, AGS, COLO 205, COLO 320HSR and Caco-2 to various extents.
Although the health-promoting and disease pre-ventive functions of this medicinal fungus are widely recognized, the biochemical process and components responsible for its bioactivity are still under investiga-tion. Particularly, the relationship between fermenta-tion process and the bioactivity of the fermentafermenta-tion products is still not clear. Thus the objectives of this study were to investigate the effect of fermen-tation conditions on the anti-hepatomatic activity of the products, and to identify the marker which can be used to monitor the process during fermentation of the A. cinnamomea on a commercial scale.
MATERIALS AND METHODS Materials
Malt extract and peptone were purchased from Difico (Sparks, MD, USA). Ethyl acetate, dimethyl sulfoxide (DMSO), glucose, galactose, fructose, sucrose, lactose, 3-(4, dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT), and other reagents used in the study were purchased from Sigma Chemical (St Louis, MO, USA). Trpysin, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal calf serum (FCS) were purchased from Hyclone (Logan, UT, USA).
Organism and inoculum
Antrodia cinnamomea (BCRC35716) was obtained from the Bioresources Collection and Research Center (BCRC) in Food Industry Research and Development Institute (Hsinchu, Taiwan). The culture was main-tained on potato dextrose agar (PDA) slopes. Slopes were inoculated and incubated at 25◦C for 4 weeks and stored at 4◦C. To prepare the inoculum, the mycelium of A. cinnamomea was transferred to a Petri dish containing PDA medium and incubated at 25◦C for 4 weeks. Mycelia agar discs (0.5 cm) were obtained using a self-designed cutter and used as inocula to cultivate the fungus in a shake flask.
Preparation of liquid cultures of A. cinnamomea Colonies of A. cinnamomea were observed under a light microscope to ensure that no contamination occurred. The agar covered with fungal mycelia was cut into small pieces and then homogenized aseptically with liquid medium (2% glucose, 2% malt extract and 0.1% peptone) for 30 s. The suspension was used as inoculum for submerged cultivation of A. cinnamomea.
Submerged shake flask culture
In order to discover the optimal cultivation conditions the partial medium replacement method was used. The initial cultivation medium, composed of 2% malt extract and 2% glucose with pH adjusted to 5, was prepared in 250 mL flasks. For studying the influence of the carbon source, glucose was replaced with different carbon sources, including glucose, lactose, galactose, fructose and sucrose. Different nitrogen sources were also checked for optimal fermentation conditions, including malt extract, soy peptone, peptone, yeast extract and yeast malt extract. To investigate the effect of initial pH on fermentation, the culture medium was adjusted to various pH values from 2 to 9 using HCl and NaOH. The optimum temperature for the fermentation process was investigated by incubating the shake flasks at 22, 25 and 28◦C. Then, 5 mL homogenized A. cinnamomea inocula were added to each flask. The submerged cultures were incubated for 4 weeks with constant agitation at 100 rpm on an orbital rotary shaker.
Scale-up of submerged fermentation
The 5 L fermenter was inoculated with 50 mL of A. cinnamomea cultivated in a 250 mL flask for 2 weeks. The 500 L fermenter was inoculated with 1 L of A. cinnamomea cultivated in a 2 L flask for 2 weeks. Finally, the 5-ton fermenter was inoculated with 400 L of inoculum cultivated in 500 L fermenter for 2 weeks. The fermentation conditions were glucose 2%, malt extract 2%, initial pH 5, and cultured at 22◦C, 50 rpm agitation, and an aeration of 0.5 vvm.
The dimensions of the 5 L, 500 L and 5-ton fermenters were 16 cm× 36 cm (inside diameter × height), 75 cm × 150 cm and 130 cm × 366 cm, respectively. During fermentation, the working volume was approximately 80% of the total fermenter volume.
Fermentation broth processing after cultivation At the end of the fermentation process, the fermenta-tion broth was passed through Whatman No. 1 filter paper (for shake flask and 5 L fermenter) or cen-trifuged using a decanter to separate the mycelium from the broth filtrate. The pellets of mycelium were then washed twice with 50 mL distilled water to remove the broth adsorbed on the pellets. Both the mycelium and broth filtrate were freeze dried and stored at 4◦C for further use.
To prepare the ethanolic extract of the mycelium, 1 g of dry mycelium and 50 mL of 95% ethanol were homogenized using a Polytron homogenizer (Kinematica, AG, Lucerne, Switzerland), and then agitated for 24 h to extract the ethanol-soluble components from the mycelium. The extract thus obtained was concentrated using a rotary evaporator and then stored in a refrigerator at −80◦C for component analyses and cancer cell viability test.
Influence of A. cinnamomea products on viability of cancer cell lines
Tumor cell lines were purchased from the BCRC, including AGS from human stomach adenocarcinoma (BCRC 60 102), HeLa from human cervical epithe-lioid carcinoma (BCRC 60 005), MCF7 from human breast adenocarcinoma (BCRC 60 436), Colo 320 HSR from human colon adenocarcinoma (BCRC 60 109), Hep 3B from human hepatocellular carci-noma (BCRC 60 434) and Hep G2 from human hepatoblastoma (BCRC 60 177). These cell lines were maintained as stocks in DMEM supplemented with 10% fetal bovine serum. The cells were maintained in a humidified atmosphere containing 5% CO2at 37◦C and passaged twice each week using trypsin – EDTA to detach the cells from cell culture flasks.
Cell viability was studied by MTT assay. Tumor cells were harvested, counted and inoculated into a 96-well microtiter plate at the designated concentrations. The start-up concentration of the cells was 10 000 cells per well. The total volume of the cell culture medium in each well was 100µL, and cells were maintained in a humidified atmosphere containing 5% CO2 at 37◦C. On the second day the fermentation products, including the dry matter of filtrate and the ethanolic extract of the mycelium, were diluted 20 – 20 000 times with serum-free DMEM and 0.1% ethanol. The diluted solutions were filtered through a 0.22µm filter; 200µL of the diluted samples was then applied to the culture wells, and the resultant cultures were incubated for 3 days under the same conditions as mentioned above. Subsequently, the supernatant was removed from each well, and 100µL MTT (2 mg mL−1 in phosphate-buffered saline) was added to each well. After an additional 2 h incubation at 37◦C, the supernatant was removed from each well and 100µL of 100% DMSO was added to solubilize the MTT – formazan product. After thorough mixing with a mechanical plate mixer, the absorbance at 570 nm was measured with an ELSA reader (Tecan Spectra, Wetzlar, Germany). The tumor cell viability was calculated by dividing the absorbance of each experimental sample by the corresponding control sample (the medium). The IC50 value (the concentration of the sample which inhibits 50% of the cancer cells to proliferate or the concentration of the sample which results in 50% of cell viability) was calculated by linear regression between the sample concentration in DMEM and cell viability.
High-performance liquid chromatographic (HPLC) analysis
HPLC analysis was performed using an ICI 1100 system, composed of LC1100 pump (ICI Australia), ICI LC 1200 UV-visible detector, SFD S5200 autosampler (set at 20µL) (Bad Honnef, Germany) and online degasser ERC 3415 (Tokyo, Japan). SISC (Scientific Information Service Corporation Inc., Taipei, Taiwan) chromatography software was used for data acquisition and integration. Separations were
carried out with a Lichrospher 100RP (Darmspadt, Frankfurt, Germany) column and detected at UV 280 nm. The mobile phase was methanol. All samples were filtered through a 0.45µm filter before analysis. To identify the ergosterol in the fermentation product, pure compound of ergosterol purchased from Sigma Co. (St Louis, MO, USA) was used.
Statistical analysis
Statistical analysis was performed using two-way ANOVA and Tukey’s multiple comparison test (SAS Institute Inc., Cary, NC, USA) to determine significant differences among means (P < 0.05).
RESULTS AND DISCUSSION
Influence of A. cinnamomea products on various cancer cell lines
Different cancer cell lines including MCF7, Hela, AGS, COLO, Hep 3B and Hep G2 maintained in DMEM were tested for anti-tumor activity of the ethanolic extract of A. cinnamomea mycelium cultured in a shake flask for 8 weeks. The ethanolic extract was found to be most inhibitory to the hepatoma cell lines Hep 3B and Hep G2, showing the lowest IC50 of 30µg mL−1for both of them (Fig. 1). The IC50for the other cell lines were 150, 250, 90 and 90µg mL−1for AGS, Hela, MCF7 and COLO 320 HSR, respectively. This result validated that the ethanolic extract of A. cinnamomea was more effective in inhibiting the liver cancer cells than other cancer cells, and inhibition of cell growth might be brought about by upregulation of Fas/FasL to activate the caspase-3 and caspase-8 cascades, thus inducing cell apoptosis.13
Effects of carbon and nitrogen sources
A. cinnamomea cultured in a shake flask was optimized for various growth conditions. Various carbon sources were used to check their influence on growth
0 50 100 150 200 250 300
AGS Hela MCF7 COLO 320 HSR Hep 3B Hep G2 IC 50 (µ g mL -1) Cell Lines b a c c d d
Figure 1. Effect of ethanolic extracts of A. cinnamomea mycelium cultured in a shake flask for 8 weeks on different cancer cells. AGS, human stomach adenocarcinoma (BCRC 60 102); HeLa, human cervical epithelioid carcinoma (BCRC 60 005); MCF7, human breast adenocarcinoma (BCRC 60 436); Colo 320 HSR, human colon adenocarcinoma (BCRC 60 109); Hep 3B, human hepatocellular carcinoma (BCRC 60 434); Hep G2, human hepatoblastoma (BCRC 60 177).
conditions of the fungus and IC50 values of the ethanolic extract of mycelium, as well as the broth filtrate obtained after submerged fermentation. It was found that glucose was the best carbon source for production of mycelium, with high inhibition potency towards Hep G2 cells (Table 1). In the case of broth filtrate, glucose, galactose and lactose were found to be the better carbon sources, giving a higher anti-Hep G2 activity (Table 2). The inhibitory activity of the broth filtrate might be attributed to secondary metabolites released from the mycelium such as triterpenoids and exopolysaccharide produced by A. cinnamomea.8,14 Considering the cost of the medium, glucose was chosen for the subsequent studies.
Fermentation was performed with various nitrogen sources including malt extract, soy peptone, peptone, yeast extract and yeast malt extract. It was observed that with malt extract the highest Hep G2 cell inhibition was achieved both in case of mycelium extract and broth filtrate. Although the yield of mycelium for malt extract was lower than that for yeast
extract and yeast malt extract, malt extract yielded the highest amount of dried product from the broth filtrate (Table 3). In view of the bioactivity as well as the yield of fermentation product, malt extract was chosen as the nitrogen source for submerged cultivation of A. cinnamomea.
Optimization of initial pH and temperature
Cultured medium adjusted to different pH was used for submerged cultivation of A. cinnamomea in a shake flask to optimize the initial pH for culture conditions. Maximum mycelium yield was obtained with an initial pH of 5.0, while the maximum broth filtrate dry weight was achieved with a pH of 2.0 (Table 4). These results were similar to those obtained by Shu et al., who found that the initial pH of 5.0 gave the highest mycelium yield, though no biological activity of the mycelium was reported in this study.11 The ethanolic extract of the mycelium grown at an initial pH value of 2.0 exhibited the maximum anti-hepatoma potency against Hep G2 cells. In the case of
Table 1. Effect of ethanolic extracts of mycelium of A. cinnamomea cultured with different carbon sources on Hep G2 cells
Cell viability (%)
Dosage (µg mL−1) Glucose Galactose Lactose Sucrose Fructose 1500 16.2± 1.8k 21.0± 3.0jk 37.1± 5.6ghijk 42.4± 6.5ghij 44.2± 5.0ghi 1200 20.7± 2.3jk 29.2± 4.1ijkh 38.7± 5.8ghij 49.6± 3.5fgh 51.7± 5.3efgh
900 21.0± 2.0jk 37.6± 5.3ghijk 71.8± 10.8cde 87.5± 8.2abc 81.5± 9.8bc 600 25.5± 2.7ijk 56.3± 7.9defg 98.4± 14.8ab 103.8± 12.5ab 99.6± 11.7ab
300 40.2± 4.1ghij 70.2± 9.8cdef 101.6± 15.2ab 106.2± 11.6a 101.3± 12.5ab
150 74.2± 9.2dc 106.9± 15.0a 105.7± 15.9a 107.4± 13.1a 106.2± 10.4a
Values followed by different letters are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3).
Table 2. Effect of broth filtrate of A. cinnamomea cultured with different carbon sources on Hep G2 cells
Cell viability (%)
Dosage (µg mL−1) Glucose Galactose Lactose Sucrose Fructose 1500 13.2± 1.7fg 11.0± 2.4g 12.0± 1.7g 12.2± 1.6g 15.0± 1.7fg 1200 17.0± 1.8fg 12.2± 3.1g 13.3± 1.9fg 11.3± 1.0g 17.3± 1.0fg 900 14.1± 2.1fg 13.7± 3.2fg 13.0± 1.8fg 22.4± 2.8f 35.6± 4.8e 600 18.9± 2.2fg 14.8± 3.8fg 17.4± 2.4fg 43.5± 5.8cde 46.4± 8.4cde 300 49.0± 3.2cd 41.3± 6.0de 42.5± 3.1cde 75.2± 5.1b 72.7± 9.3b 150 65.6± 2.8b 52.9± 11.1c 45.0± 3.4cde 109.0± 8.4a 104.0± 7.4a Values followed by different letters are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3).
Table 3. Influence of nitrogen source on bioactivity and yield of A. cinnamomea fermentation products
Nitrogen source
Cell viability of mycelium ethanolic extract (%)a
Cell viability of broth filtrate (%)a
Dry weight of mycelium (g 100 mL−1)
Dry weight of broth filtrate (g 100 mL−1) Malt extract 30.5± 2.8c 31.1± 0.5e 0.70± 0.01dc 2.60± 0.03b Soy peptone 72.5± 5.6b 62.7± 0.4d 0.85± 0.02c 0.82± 0.02c Peptone 68.5± 6.2b 72.0± 1.0c 0.65± 0.02d 2.72± 0.04a Ye 86.4± 7.3a 96.2± 1.3a 1.26± 0.10a 0.52± 0.01e YM 65.4± 6.2b 76.3± 1.3b 1.05± 0.08b 0.67± 0.04d Values followed by different letters in the same column are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3). aHep G2 cells were treated with 400µg mL−1A. cinnamomea fermentation product.
Table 4. Influence of initial pH on bioactivity and yield of A. cinnamomea fermentation products
pH
Cell viability of mycelium ethanolic extract (%)a
Cell viability of broth filtrate (%)a
Dry weight of mycelium (g 100 mL−1)
Dry weight of broth filtrate (g 100 mL−1) 2 16.3± 2.6e 43.2± 2.1c 0.08± 0.01d 3.21± 0.05a 3 46.3± 2.5c 42.9± 1.8c 0.45± 0.02c 2.87± 0.06b 4 47.8± 2.8c 39.6± 12.2cd 0.53± 0.04b 2.61± 0.08c 5 28.6± 2.8d 29.6± 2.5d 0.69± 0.02a 2.55± 0.04cd 6 32.1± 3.2d 30.6± 10.3d 0.65± 0.03a 2.42± 0.05de 7 45.6± 1.5c 36.5± 1.3cd 0.55± 0.04b 2.31± 0.08e 8 62.4± 1.9b 56.7± 2.5b 0.41± 0.01c 2.84± 0.05b 9 68.7± 2.1a 92.3± 8.2a 0.42± 0.02c 2.96± 0.07b
Values followed by different letters in the same column are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3). aHep G2 cells were treated with 400µg mL−1A. cinnamomea fermentation product.
broth filtrate the initial pH of 5.0 gave the highest activity against Hep G2 cells (Table 4). Although the ethanolic extract of mycelium cultured at pH 2 had the highest anti-tumor activity, its mycelium content was also the lowest one. This result indicates that the active compounds are produced in extreme culture conditions – the lowest pH. However, since the mycelium yield was considerably reduced at such a low pH, pH 5.0 was used in subsequent studies.
A. cinnamomea has been submerge cultured at temperatures ranging from 25 to 30◦C.9,14However, the fruiting body of A. cinnamomea usually grows on the inner cavity of Cinnamomum kanehirai Hay (Lauraceae) on mountains at 450 – 1200 m above sea level at temperatures normally between 22◦C (evening) and 28◦C (daytime) in the summer. Therefore, the fermentation temperatures investigated in this study were 22, 25, and 28◦C. It was found that fermentation at 22◦C yields a product with a high inhibition value both in the case of filtrate broth as well as ethanolic extract of mycelium. Conversely, maximum yield was obtained at 25◦C for both mycelium ethanolic extract and filtrate broth (Table 5). We decided to use 22◦C as the fermentation temperature for the subsequent study because bioactivity was the primary concern.
Scale-up of submerged fermentation of A.
cinnamomea
The fermentation process was scaled up in a stepwise manner. First the process was scaled up to 5 L fermentation with 50 mL inoculum broth, and the process was monitored by taking a small aliquot from the fermenter on a weekly basis. The pH, reducing sugar, mycelium content, solid content of the broth
filtrate, and IC50 of the broth filtrate as well as the ethanolic extract of mycelium for Hep G2 cells were analyzed. In a similar manner, the fermentation process was scaled up to 500 L inoculated with 2 L inoculum. Finally, the process was scaled up to 5 tons using 400 L inoculum. It was observed that the fermentation carried out in the 5 L fermenter took 4 weeks to reach stationary phase, as indicated by the mycelium content. The reducing sugar and solid content of the broth filtrate decreased continuously with decreases in IC50 for both broth filtrate and ethanolic extract of mycelium (Table 6). When the IC50of the ethanolic extract of mycelium is correlated with the contents of reducing sugar and total solids, a significant exponential relationship is found, with coefficients of determination (R2) of approximately 0.9 (Fig. 2). It appears that both the solid content and reducing sugar content of the broth filtrate can serve as the marker during fermentation of A. cinnamomea for manufacturing product with anti-hepatoma activity. Since the cultivation of A. cinnamomea in the 5 L fermenter reached stationary phase in 4 weeks and the IC50 of the mycelium extract also reached its lowest point at almost the same time, we decided to limit the cultivation time to 4 weeks when scaling up the process using commercial scale fermenters. However, it is worth noting that while the IC50of the mycelium extract remained the same after 4 weeks of fermentation, the IC50of the broth filtrate decreased continuously as the fermentation time was prolonged. It was suspected that the bioactive metabolites started to be released from the mycelium to the broth after the fungal culture reached stationary phase.
When the process was scaled up to 500 L and then to the 5-ton fermenter, it was found that the culture
Table 5. Influence of temperature on bioactivity and productivity of A. cinnamomea fermentation products
Temperature
Cell viability of mycelium ethanolic extract (%)a
Cell viability of broth filtrate (%)a
Dry weight of mycelium (g 100 mL−1)
Dry weight of broth filtrate (g 100 mL−1) 22 26.8± 2.7a 30.2± 2.5b 0.71± 0.01b 2.70± 0.04c 25 28.2± 3.5a 35.6± 2.3a 0.80± 0.03a 3.50± 0.03a
28 29.4± 2.8a 36.8± 3.2a 0.82± 0.02a 2.80± 0.02b Values followed by different letters in the same column are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3). aHep G2 cells were treated with 400µg mL−1A. cinnamomea fermentation product.
(a) y = 1.95e0.189X R2 = 0.89 y = 48.1x + 89.0 R2 = 0.98 0 100 200 300 400 500 600 700 800 900 1000 0 10 20 30 Reducing sugar (mg mL-1) IC 50 (µ g mL -1) (b) y = 0.2179e0.222X R2 = 0.90 y = 63.4x - 495 R2 = 0.97 0 100 200 300 400 500 600 700 800 900 1000 0 10 20 30 40 Solid content (mg mL-1) IC 50 (µ g mL -1)
Figure 2. Relationships between IC50of the ethanolic extracts of mycelium and broth filtrate versus the reducing sugar content (a) as well as the
solid content (b) of the filtrate of the fermentation broth of A. cinnamomea., IC50of broth filtrate;, IC50of ethanolic extracts of mycelium.
Table 6. Influence of cultivation time on mycelium content, pH, reducing sugar, IC50of broth and mycelium ethanolic extract in 5 L fermenter
Weeks PH Reducing sugar (mg mL−1) Mycelium content (mg 100 mL−1) Solid content of broth filtrate (g 100 mL−1) IC50of broth filtrate (µg mL−1) IC50of mycelium ethanolic extract (µg mL−1) 0 4.86a 34.1a 0 4.06a >1000 – 1 4.50b 33.6a 0.07e 3.53b >1000 750a 2 3.19c 32.1b 0.18d 2.98c >1000 280b 3 3.05cd 23.5c 0.24c 2.52d >1000 60c 4 2.95de 14.2d 0.35ab 1.96e 800a 3.8c 5 2.90de 8.7e 0.35ab 1.62f 450b 4.2c 6 2.85e 2.6f 0.36a 1.16g 250c 3.5c 7 2.85e 0.4g 0.35ab 0.95h 120d 2.8c 8 2.85e 0.1g 0.32b 0.89h 75d 2.9c
Values followed by different letters in the same column are significantly different (P < 0.05) by Tukey’s multiple comparison test (n= 3).
Table 7. Performance of fermentation of A. cinnamomea in a 500 L fermenter
Weeks pH Reducing sugar (mg mL−1) Mycelium content (mg 100 mL−1) Solid content of broth filtrate (g 100 mL−1) IC50of broth filtrate (µg mL−1) IC50of mycelium ethanolic extract (µg mL−1) 0 4.33 34.0 – 4.05 >1000 >1000 1 3.20 17.6 0.14 2.09 950 235 2 3.13 0.26 0.41 1.37 555 85.0 3 3.04 0.09 0.54 0.76 390 8.75 4 2.97 0.06 0.57 0.67 250 3.25
also reached its stationary phase in 3 – 4 weeks, but the mycelium content in the larger-scale fermenters appeared to be higher than that in the 5 L fermenter (Tables 7 and 8). In addition, although the IC50 of the ethanolic extract of the mycelium from large-scale fermenters was basically the same as that from the laboratory fermenter after 4 weeks of fermentation, the broth filtrate from the large-scale fermenter appeared to have higher anti-hepatoma activity (lower IC50) than that from the 5 L laboratory fermenter. It is very difficult to predict performance when the scale of fermentation changes. In this study, the surface:diameter ratio of the fermenters decreased dramatically during scale-up, which would reduce the surface aeration and might have an adverse effect on the growth of fungus. However, the oxygen
supply by surface aeration in the large-scale fermenter might not be as important as the contribution by sparging. It was suspected that effective sparging and gas dispersion (by impeller) might have compensated for the insufficient surface aeration in large-scale fermenters. Nonetheless, this study has demonstrated that the fermentation of A. cinnamomea can be carried out in commercial fermenters with similar or even better performance than the laboratory fermenter.
HPLC analysis
HPLC analyses were performed on mycelium extracts and broth filtrates every week from the 5-ton fer-menter, and the resulting profiles are given in Figs 3 and 4. It was observed that in the mycelium extract a peak at retention time 7.8 min – ergosterol – appeared
Table 8. Performance of fermentation of A. cinnamomea in a 5-ton fermenter Weeks pH Reducing sugar (mg mL−1) Mycelium content (mg 100 mL−1) Solid content of broth filtrate (g 100 mL−1) IC50of broth filtrate (µg mL−1) IC50of mycelium ethanolic extract (µg mL−1) 0 4.88 34.1 – 4.02 >1000 >1000 1 3.14 29.3 0.11 2.98 515 185 2 2.99 18.8 0.50 1.58 375 72.5 3 2.94 5.70 0.63 0.77 335 27.5 4 2.88 0.85 0.68 0.70 290 4.25 0 2 4 6 8 10 12 14 0 10 20 30 Week 0 0 2 4 6 8 10 12 14 -10 0 10 20 Week 1 mV 0 2 4 6 8 10 12 14 0 20 40 60 80 100 0 2 4 6 8 10 12 14 0 25 50 75 100 125 Week 2 Week 3 0 2 4 6 8 10 12 14 -25 0 25 50 75 100
Retention Time (min)
Week 4 Ergosterol Ergosterol Ergosterol Ergosterol Ergosterol
Figure 3. HPLC chromatograms of A. cinnamomea mycelium extract from 5-ton fermenter at different weeks.
after 3 weeks’ fermentation (Fig. 3), but this char-acteristic peak was not found in the broth filtrate (Fig. 4). Ergosterol is the predominant sterol found in the membrane of most fungi.15 Since the level of ergosterol in the mycelium extract follows the same profile as the increase in Hep G2 cell inhibition, it may also serve as a marker for fermentation product with high anti-hepatoma potency.
CONCLUSION
The process parameters for submerged cultivation of A. cinnamomea, including carbon source, nitrogen source, initial pH and temperature, were optimized to obtain mycelium and broth filtrate having the lowest IC50 values for anti-hepatoma activity. The fermentation process was then scaled up to the 5-ton fermenter. It was found that the fungal culture
0 10 20 30 40 50 Week 0
Retention Time (min) 0 20 40 60 80 100 Week 1 0 20 40 60 80 100 Week 2 -20 0 20 40 60 80 100 Week 3 mV 0 50 100 150 200 Week 4 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Figure 4. HPLC chromatograms of A. cinnamomea broth filtrate from 5-ton fermenter at different weeks.
reached stationary phase in 3 weeks, while the lowest IC50 value both in the case of broth filtrate and mycelium extract was achieved in 4 weeks. Both the reducing sugar and solid content of the broth filtrate decreased continuously with the decreases in IC50 for both broth filtrate as well as ethanolic extract of mycelium, and significant exponential relationships were found between the IC50of the ethanolic extract of mycelium and the contents of reducing sugar and total solids. Therefore, both the solid content and reducing sugar content of the broth filtrate can serve as the marker during fermentation of A. cinnamomea for manufacturing products with anti-hepatoma activity. In addition, HPLC analysis of the mycelium extract indicates that ergosterol can also serve as a marker for the fermentation of A. cinnamomea with high
anti-hepatoma potency. However, further studies are needed to fractionate and identify the bioactive metabolites responsible for anti-hepatoma activity of the fermentation products.
ACKNOWLEDGEMENTS
The research work was supported by the National Science council, the Republic of China, under grants NSC92-2321-B-002-015 and NSC94-2321-B-002-012. We are also grateful to Grape King Inc., Chung-Li, Taiwan, for allowing us to use their 500 L and 5-ton fermentation facilities to scale up the process.
REFERENCES
1 Chang TT and Chou WN, Antrodia cinnamomea reconsidered and A. salmonea sp. nov. on Cunninghamia konishii in Taiwan.
Bot Bull Acad Sin 45:347–352 (2004).
2 Mau JL, Huang PN, Huang SJ and Chen CC, Antioxidant properties of methanolic extracts from two kinds of Antrodia
camphorata mycelia. Food Chem 86:25–31 (2004).
3 Song TY and Yen GC, Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats. J Agric Food Chem
51:1571–1577 (2003).
4 Hseu YC, Chang WC, Hseu YT, Lee CY, Yech YJ and Chen PC, Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythro-cytes. Life Sci 71:469–482 (2002).
5 Shen YC, Chou CJ, Wang YH, Chen CF, Chou YC and Lu MK, Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species. FEMS
Microbiol Lett 231:137–143 (2004).
6 Chou CJ, Shen YC, Chang TT and Chen CF, Evaluation of the immuno-modulating activity of some active principles isolated from the fruiting bodies of Antrodia camphorate, in
12th International Congress of Oriental Medicine, Taipei, Taiwan
(2003).
7 Chien YC, Jan CF, Kuo HS and Chen CJ, Nationwide hepatitis B vaccination program in Taiwan: effectiveness in the 20 years after it was launched. Epidemiol Rev 28:126–135 (2006). 8 Shih IL, Pan K and Hsieh C, Influence of nutritional
compo-nents and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia
cin-namomea. Process Biochem 41:1129–1135 (2006).
9 Yang FC, Huang HC and Yang MJ, The influence of envi-ronmental conditions on the mycelial growth of Antrodia
cinnamomea in submerged cultures. Enzym Microb Technol
33:395–402 (2003).
10 Song TY and Yen GC, Antioxidant properties of Antrodia
camphorata in submerged culture. J Agric Food Chem
50:3322–3327 (2002).
11 Shu CH and Lung MY, Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures. Process Biochem 39:931–937 (2004).
12 Huang RC, Chen JC and Wang BC, Isolate of Antrodia
camphorata process for producing a culture of the same and
product obtained thereby. US Patent 6391615 (2002). 13 Song TY, Hsu SL, Yeh CT and Yen GC, Mycelia from Antrodia
camphorata in submerged culture induce apoptosis of human
hepatoma Hep G2 cells possibly through regulation of Fas pathway. J Agric Food Chem 53:5559–5564 (2005). 14 Lin ES and Sung SC, Cultivating conditions influence
exopolysaccharide production by the edible Basidiomycete
Antrodia cinnamomea in submerged culture. Int J Food Micro-biol 108:182–187 (2006).
15 Bermingham S, Malthy L and Cooke RC, A critical assessment of the validity of ergosterol as an indicator of fungal biomass.
