• Title/Summary/Keyword: submerged cultures

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Antifibrotic Effect of Extracellular Biopolymer from Submerged Mycelial Cultures of Cordyceps militaris on Liver Fibrosis Induced by Bile Duct Ligation and Scission in Rats

  • Nan, Ji-Xing;Park, Eun-Jeon;Yang, Byung-Keun;Song, Chi-Hyun;Ko, Geonil;Sohn, Dong-Hwan
    • Archives of Pharmacal Research
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    • v.24 no.4
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    • pp.327-332
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    • 2001
  • The antifibrotic effects of hot water extract (WEC), intracellular biopolymer (IPC) and extracellular biopolymers (EPC) from mycelial liquid culture of Cordyceps militaris on liver fibrosis were studied. Liver fibrosis was induced by a bile duct ligation and scission (BDL/S) operation, duration of 4 weeks in rats. In BDL/S rats, the levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin in serum and hydroxyproline content in liver were dramatically increased. The WEC or IPC treatment (30mg/kg/day for 4 weeks, p.o.) in BDL/S rats reduced the serum AST, ALT and ALP levels significantly (p<0.01). The EPC treatment (30 mg/kg /day for 4 weeks, p.o.) reduced the serum ALT, AST and ALP levels significantly (p<0.01). Malondialdehyde contents in liver treated with WEC, IPC or EPC were significantly reduced (p <0.05). But Liver hydroxyproline content was decreased only in EPC treated BDL/S rats to 55% that of BDL/S control rats (p < 0.01). The morphological characteristics and expression of alpha smooth muscle like actin in fibrotic liver, which appeared in BDL/S control group were improved in EPC treated fibrotic liver. These results indicate that IPC (30 mg/kg /day for 4 weeks, p.o.) has an antifibrotic effect on fibrotic rats induced by BDL/S.

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Effects of temporary and periodic emersion on the growth of Fucus spiralis and Pelvetia canaliculata germlings

  • Kim, Bo-Yeon;Park, Seo-Kyoung;Norton, Trevor A.;Choi, Han-Gil
    • ALGAE
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    • v.26 no.2
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    • pp.193-200
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    • 2011
  • The stress tolerance ability of Pelvetia canaliculata (L.) Dcne. Et Thur. and Fucus spiralis L. to temporary and periodic emersion stress was examined in order to test the following hypotheses: The upper shore alga, P. canaliculata is more tolerant to desiccation than F. spiralis in the germling stage and the former outgrows the latter under desiccation stress; Germling stress tolerance of the two species is age-specific; Crowding of germlings protects them from desiccation, irrespective of the species involved. Germling growth of the two species was retarded with increasing exposure period and was age-specific, as they were air-exposed at an earlier stage. After 16 days, the length of Pelvetia germlings was similar between 2-day-old germlings (125-140 ${\mu}m$) and 7-day-old germlings (134-140 ${\mu}m$), which were air-exposed during the same period (0, 6, 12, 24, 48, and 72 h) at the two different ages. However, Fucus germlings were significantly larger at 7-day-old germlings (211-277 ${\mu}m$) than at 2-day-old germlings (184-278 ${\mu}m$), especially in the treatments of 48 and 72 h. These results indicate that Fuscus grow faster than Pelvetia and that the growth response of germlings to temporary emersion stress is more sensitive in Fucus than that in Pelvetia. Growth of germlings of both species was reduced with increasing density under favorable growth conditions (submerged control and 6 h / 12 h exposure treatments) in the periodic air-exposed experiments using tidal tanks, but was enhanced under severe emersion stress conditions. P. canaliculata showed better growth at 6 h exposure treatment than that of the control, under continuous submergence, indicating that Pelvetia germlings require a periodic exposure period. Fucus germlings always grew faster than those of Pelvetia and did best in mixed cultures, whereas Pelvetia did least well when mixed with Fucus germlings. The adverse effects of F. spiralis on P. canaliculata were greater than those of Pelvetia cohorts. The outcome of interspecific competition between F. spiralis and P. canaliculata gemlings was slightly altered by exposure period but not to such an extent as to change the outcome.

Citric Acid Production from Glucose and Pumpkin by Using Immobilized Bead of Aspergillus niger (고정화 Aspergillus niger Bead를 이용한 포도당과 호박으로부터 구연산 생성)

  • 방병호
    • The Korean Journal of Food And Nutrition
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    • v.13 no.4
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    • pp.328-333
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    • 2000
  • The spore of Aspergillus niger KCTC-6144 was immobilized on alginate gel beads. When pumpkin powder was used with glucose for a medium of citric acid fermentation by Aspergillus niger beads, the beaded Aspergilus niger grew up inside the bead and mycelia penetrated through the pore of the bead membrane. The bead size became largely from 2.0∼2.5mm to 6∼8mm after growing at 30$\^{C}$ for 4 days. Studies of optimum culture conditions on citric acid fermentation using Aspergillus niger beads on pumpkin medium (pumpkin powder 1% +glucose 7%, pH 6.0) were carried out in submerged cultures on 250m1 Erlenmeyer flask. As a result, it was found that to reinforce 12% as carbon source was good for citric acid production and that 1% pumpkin powder was good as nitrogen and mineral source in orbital shaker (150rpm) at 30$\^{C}$ for 5 days. The optimum initial pH on citric acid production was pH 6.0 and it was found that 100 beads of immobilized Aspergillus niger was adequate for citric acid production in a 250ml Erlenmeyer flask containing 50m3 of pumpkin medium solution with orbital shaker at 30$\^{C}$ for 5 days. We also found that maximal production of citric acid was 23.5g/ℓ at optimal condition (at 30$\^{C}$ for 5 days, pH 6.0, and 100 beads and medium containing 1% pumpkin powder plus 12% glucose).

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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Utilization of Opuntia ficus-indica as a Substrate for the Growth of Mushroom Mycelia and the Functional Properties of its Culture Extracts (버섯균사체 배양기질로서의 손바닥선인장의 활용과 그 배양추출물의 기능적 특성)

  • Moon Sang-Wook;Park Soo-Yeong;Choi Soo-Youn;Hwang Joon-Ho;Jang Mi-Kyoung;Jin Yeong-Jun;Chung Wan-Seok;Kim Se-Jae
    • Journal of Life Science
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    • v.16 no.3 s.76
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    • pp.477-484
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    • 2006
  • This study was performed to know the potentialities of the fruits of Opuntia ficus-indica, as a medium for mushroom mycelial culture. Five mushroom mycelial (Agrocus blazei, Grifola frondosa, Hericium erinaceum, Innonotus obliquus, Phellinus linteus) were frown on the malt extract broth (MEB) and the cactus broth medium (CB). The submerged culture mixtures were extracted using equal volume of ethyl acetate, and their extract yields, total polyphenol contents, and some physiological activities were compared with each other Each extract from mycelial culture grown on CB medium showed remarkable enhancement in physiological activities compared with each counterpart grown on MEB. Among five mycelial cultures grown on CB medium, the extract yield and polyphenyl content were highest in the extract from Grifola frondosa (extract yield, 0.4 g/L and polyphenol content, 22.7%). Also, the extracts from Grifola frondosa showed the highest physiological activities, such as DPPH radical scavenging ($IC_{50}=362.9{\mu}g/ml$), xanthine oxidase inhibition (about 80% at $500{\mu}g/ml$), and superoxide radical scavenging (about 80% at $500{\mu}g/ml$), and NO production inhibition ($IC_{50}=43.1{\mu}g/ml$) in LPS-stimulated RAW 264.7 cells. This result suggests that the fruit of Opuntia ficus-indica can be used as a culture medium for improving the functional properties of various mushroom mycelia.