樟芝In-situ萃取发酵高产Antrodin C和Antroquinonol
|
摘要
樟芝含有丰富的活性成分,但是其液态发酵产物种类及含量较少。该文通过添加前体物诱导活性成分合成,结合In-situ萃取发酵技术有效提高Antrodin C (Ac)及Antroquinonol (Aq)类化合物产量。通过发酵工艺条件优化以及7 L发酵罐实验,结果显示植物油作为萃取发酵溶剂,能够有效促进前体物辅酶Q_0的转化,从而提高泛醌类组分的产量。植物油能够高效富集Ac以及Aq类化合物,于7 L发酵罐发酵结束后产量分别为486.01 mg/L和110.06mg/L,与诱导发酵相比,分别提高了272.1%和293.5%。同时,植物油在发酵过程中的萃取作用非常明显,活性成分释放率达到80%左右。通过连续发酵实验表明,发酵结束后,采用简单的回收工艺,即可将植物油重复利用。
Antrodia camphorate contains many active ingredients, however, products of its submerge fermentation are fewer in terms of types and levels. In this research, precursor was added during fermentation to induce active ingredients synthesis, combining with In-situ extractive fermentation technology to co-effectively increase the production of Antrodin C(Ac) and Antroquinonol(Aq). Optimizing experimental conditions in a 7 L fermenter showed that using vegetable oil as an In-situ extractant could effectively promote the transformation of coenzyme Q_0 and increase the production of ubiquinone. Vegetable oil could efficiently enrich Ac and Aq, and their yields were 486.01 mg/L and 110.06 mg/L, respectively. Comparing to induced fermentation, contents of Ac and Aq increased by 272.1% and 293.5%, respectively. Meanwhile, the extraction effect of vegetable oil was obvious, and the release rates of active ingredients could reach as high as 80%. Besides, the results of continuous fermentation showed that vegetable oil could be reused through a simple recycling process.
Antrodia camphorate contains many active ingredients, however, products of its submerge fermentation are fewer in terms of types and levels. In this research, precursor was added during fermentation to induce active ingredients synthesis, combining with In-situ extractive fermentation technology to co-effectively increase the production of Antrodin C(Ac) and Antroquinonol(Aq). Optimizing experimental conditions in a 7 L fermenter showed that using vegetable oil as an In-situ extractant could effectively promote the transformation of coenzyme Q_0 and increase the production of ubiquinone. Vegetable oil could efficiently enrich Ac and Aq, and their yields were 486.01 mg/L and 110.06 mg/L, respectively. Comparing to induced fermentation, contents of Ac and Aq increased by 272.1% and 293.5%, respectively. Meanwhile, the extraction effect of vegetable oil was obvious, and the release rates of active ingredients could reach as high as 80%. Besides, the results of continuous fermentation showed that vegetable oil could be reused through a simple recycling process.
引文
[1] AO Zonghua,XU Zhenghong,LU Zhenming,et al.Niuchangchih(Antrodia camphorate)and its potential in treating liver diseases[J].Ethnopharmacol,2009,121:194-212.
[2] GEETHANGILI M,TZENG Y M.Review of pharmacological effects of Antrodia camphorataand its bioactive compounds[J].Evidence-based Complementary and Alternative Medicine,2011(1741-427X):212 641.
[3] 路瑞秋,樟芝菌固态发酵产安卓奎诺尔及其提取纯化的研究[D].无锡:江南大学,2015.
[4] NAKAMURAN,HIRAKAWA A,GAO J J,et al.Five new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata and their cytotoxic effects on LLC tumor cell line[J].Journal of Natural Products,2004,67(1):46-48.
[5] PHUONG D T,MA C M,HATTORI M,et al.Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease[J].Phytotherapy Research,2009,23(4):582-584.
[6] HO C L,WANG Juiling,LEE C C,et al.Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells[J].Biomedicine & Pharmacotherapy,2014,68(8):1 007-1 014.
[7] HUANG Yaling,CHU Yunglin,HO C T,et al.Antcin K, an active triterpenoid from the fruiting bodies of basswood-cultivated Antrodia cinnamomea, inhibits metastasis via suppression of integrin-mediatedadhesion, migration, and invasion in human hepatoma cells[J].Agric Food Chem,2015,63:4 561-4 569.
[8] CHANG Chiayu,CHENG T J,CHANG Fangrong,et al.Macrophage mediated anti-proliferation effects of Antrodia camphorata non-polysaccharide based extracts on human hepatoma cells[J].Biosci Biotechnol Biochem,2011,75(4):624-632.
[9] HSEU Y C,CHAO Y H,LIN K Y, et al.Antrodia camphorata inhibits metastasis and epithelial-to-mesenchymal transition via the modulation of claudin-1 and Wnt/β-catenin signaling pathways in humancolon cancer cells[J].Ethnopharmacol,2017,208:72-83.
[10] TUNG Y T,TSAI T C,KUO YH,et al.Comparison of solid-state-cultured and wood-culturedAntrodia camphoratain anti-inflammatory effects using NF-κB/luciferase inducible transgenic mice[J].Phytomedicine,2014,21:1 708-1 716.
[11] YEN I C,LEE S Y,LIN K T,et al.In Vitro anticancer activity and structural characterization of ubiquinones from Antrodia cinnamomea mycelium[J].Molecules,2017,23(3):1-12.
[12] XIA Y,ZHANG B,LI W,et al.Changes in volatile compound composition of Antrodia camphorata during solid state fermentation[J].Sci Food Agric,2011,91:2 463-2 470.
[13] PHUONG D T,MA C M,HATTORI M,et al.Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease[J].Phytother Res,2009,23(4):582-584.
[14] LEE T H,LEE C K,TSOU W L.A new cytotoxic agent from solid state fermented mycelium of Antrodia camphorata[J].Planta Letter,2007,73:1-3.
[15] YU C C,CHIANG P C,LU P H,et al.Antroquinonol,a natural ubiquinone derivative,includes a cross talk between apoptosis,autophagy and senescence in human pancreatic carcinoma cells[J].Nutr Biochem,2011,21(3):11-15.
[16] KUMAR K J,CHU F H,HSIEH H W,et al.Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation[J].Ethnopharmacol,2011,136:168-177.
[17] HU Yongdan,ZHANG Bobo,XU Garong,et al.A mechanistic study on the biosynthetic regulation of bioactive metabolite antroquinonol from edible and medicinal mushroom Antrodia camphorata[J].Journal of Functional Foods,2016,25:70-79.
[18] LADYGINA N,DEDYUKHINA E G,VAINSHTEIN M B.A review on microbial synthesis of hydrocarbons[J].Process Biochemistry,2006,41(5):1 001-1 014.
[19] BUHLER B,BOLLHALDER I,HAUER B,et al.Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis[J].Biotechnol Bioeng,2003,81:683-694.
[20] STARK D,KORNMANN H,MUNCH T,et al.Novel type of in situ extraction: use of solvent containing microcapsules for the bioconversion of 2-phenylethanol froml-phenylalanine by Saccharomyces cerevisiae[J].Biotechnol Bioeng,2003,83:376-385.
[21] KEVIN C C,YANG Shang-han,WU Hsiang-lin,et al.Biosynthesis of antroquinonol and 4-acetylantroquinonol B via polyketide pathway using orsellinic acid as a ring precursor in Antrodia cinnamomea[J].Journal of Agricultural & Food Chemistry,2016,121(1):1-37.
[22] CHIANG Chienchi,HUANG Tzuning,LIN Yuwei,et al.Enhancement of 4-acetylantroquinonol B production by supplementation of its precursor during submerged fermentation of Antrodia cinnamomea[J].Journal of Agricultural & Food Chemistry,2013,61(38):9 160-9 165.
[23] BENTINGER M, TEKLE M G.Coenzyme Q--biosynthesis and functions[J].Biochemical & Biophysical Research Communications,2010,396(1):74-79.
[24] HU Yongdan,ZHANG Huan,LU Ruiqiu,et al.Enabling the biosynthesis of antroquinonol insubmerged fermentation of Antrodia camphorata[J].Biochemical,Engineering Journal,2014,91:157-162.
[25] KNAGGSA R.The biosynthesis of shikimate metabolites[J].Natural Product Reports,2003,34(17):119-136.
[26] JU L K,LEE J F,ARMIGER W B.Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions[J].Biotechnology and Bioengineering,1991,37(6):505-511.
[27] 王刚,贾志海,贺吉昌,等.微结构疏水表面的液滴浸润特性[J].上海理工大学学报,2015,37(6):557-562.
[28] 王启军.氧载体促进丝状真菌发酵过程氧传递研究[D].杭州:浙江大学,2002.
[29] HSIEH C,WANG Huiliang,CHEN Chiencheng,et al.Effect of plant oil and surfactant on the production ofmycelia biomass and polysaccharides in submerged cultureof Grifola frondosa[J].Biochemical Engineering Journal,2008,38:198-205.
[2] GEETHANGILI M,TZENG Y M.Review of pharmacological effects of Antrodia camphorataand its bioactive compounds[J].Evidence-based Complementary and Alternative Medicine,2011(1741-427X):212 641.
[3] 路瑞秋,樟芝菌固态发酵产安卓奎诺尔及其提取纯化的研究[D].无锡:江南大学,2015.
[4] NAKAMURAN,HIRAKAWA A,GAO J J,et al.Five new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata and their cytotoxic effects on LLC tumor cell line[J].Journal of Natural Products,2004,67(1):46-48.
[5] PHUONG D T,MA C M,HATTORI M,et al.Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease[J].Phytotherapy Research,2009,23(4):582-584.
[6] HO C L,WANG Juiling,LEE C C,et al.Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells[J].Biomedicine & Pharmacotherapy,2014,68(8):1 007-1 014.
[7] HUANG Yaling,CHU Yunglin,HO C T,et al.Antcin K, an active triterpenoid from the fruiting bodies of basswood-cultivated Antrodia cinnamomea, inhibits metastasis via suppression of integrin-mediatedadhesion, migration, and invasion in human hepatoma cells[J].Agric Food Chem,2015,63:4 561-4 569.
[8] CHANG Chiayu,CHENG T J,CHANG Fangrong,et al.Macrophage mediated anti-proliferation effects of Antrodia camphorata non-polysaccharide based extracts on human hepatoma cells[J].Biosci Biotechnol Biochem,2011,75(4):624-632.
[9] HSEU Y C,CHAO Y H,LIN K Y, et al.Antrodia camphorata inhibits metastasis and epithelial-to-mesenchymal transition via the modulation of claudin-1 and Wnt/β-catenin signaling pathways in humancolon cancer cells[J].Ethnopharmacol,2017,208:72-83.
[10] TUNG Y T,TSAI T C,KUO YH,et al.Comparison of solid-state-cultured and wood-culturedAntrodia camphoratain anti-inflammatory effects using NF-κB/luciferase inducible transgenic mice[J].Phytomedicine,2014,21:1 708-1 716.
[11] YEN I C,LEE S Y,LIN K T,et al.In Vitro anticancer activity and structural characterization of ubiquinones from Antrodia cinnamomea mycelium[J].Molecules,2017,23(3):1-12.
[12] XIA Y,ZHANG B,LI W,et al.Changes in volatile compound composition of Antrodia camphorata during solid state fermentation[J].Sci Food Agric,2011,91:2 463-2 470.
[13] PHUONG D T,MA C M,HATTORI M,et al.Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease[J].Phytother Res,2009,23(4):582-584.
[14] LEE T H,LEE C K,TSOU W L.A new cytotoxic agent from solid state fermented mycelium of Antrodia camphorata[J].Planta Letter,2007,73:1-3.
[15] YU C C,CHIANG P C,LU P H,et al.Antroquinonol,a natural ubiquinone derivative,includes a cross talk between apoptosis,autophagy and senescence in human pancreatic carcinoma cells[J].Nutr Biochem,2011,21(3):11-15.
[16] KUMAR K J,CHU F H,HSIEH H W,et al.Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation[J].Ethnopharmacol,2011,136:168-177.
[17] HU Yongdan,ZHANG Bobo,XU Garong,et al.A mechanistic study on the biosynthetic regulation of bioactive metabolite antroquinonol from edible and medicinal mushroom Antrodia camphorata[J].Journal of Functional Foods,2016,25:70-79.
[18] LADYGINA N,DEDYUKHINA E G,VAINSHTEIN M B.A review on microbial synthesis of hydrocarbons[J].Process Biochemistry,2006,41(5):1 001-1 014.
[19] BUHLER B,BOLLHALDER I,HAUER B,et al.Use of the two-liquid phase concept to exploit kinetically controlled multistep biocatalysis[J].Biotechnol Bioeng,2003,81:683-694.
[20] STARK D,KORNMANN H,MUNCH T,et al.Novel type of in situ extraction: use of solvent containing microcapsules for the bioconversion of 2-phenylethanol froml-phenylalanine by Saccharomyces cerevisiae[J].Biotechnol Bioeng,2003,83:376-385.
[21] KEVIN C C,YANG Shang-han,WU Hsiang-lin,et al.Biosynthesis of antroquinonol and 4-acetylantroquinonol B via polyketide pathway using orsellinic acid as a ring precursor in Antrodia cinnamomea[J].Journal of Agricultural & Food Chemistry,2016,121(1):1-37.
[22] CHIANG Chienchi,HUANG Tzuning,LIN Yuwei,et al.Enhancement of 4-acetylantroquinonol B production by supplementation of its precursor during submerged fermentation of Antrodia cinnamomea[J].Journal of Agricultural & Food Chemistry,2013,61(38):9 160-9 165.
[23] BENTINGER M, TEKLE M G.Coenzyme Q--biosynthesis and functions[J].Biochemical & Biophysical Research Communications,2010,396(1):74-79.
[24] HU Yongdan,ZHANG Huan,LU Ruiqiu,et al.Enabling the biosynthesis of antroquinonol insubmerged fermentation of Antrodia camphorata[J].Biochemical,Engineering Journal,2014,91:157-162.
[25] KNAGGSA R.The biosynthesis of shikimate metabolites[J].Natural Product Reports,2003,34(17):119-136.
[26] JU L K,LEE J F,ARMIGER W B.Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions[J].Biotechnology and Bioengineering,1991,37(6):505-511.
[27] 王刚,贾志海,贺吉昌,等.微结构疏水表面的液滴浸润特性[J].上海理工大学学报,2015,37(6):557-562.
[28] 王启军.氧载体促进丝状真菌发酵过程氧传递研究[D].杭州:浙江大学,2002.
[29] HSIEH C,WANG Huiliang,CHEN Chiencheng,et al.Effect of plant oil and surfactant on the production ofmycelia biomass and polysaccharides in submerged cultureof Grifola frondosa[J].Biochemical Engineering Journal,2008,38:198-205.