樟芝产生三萜类化合物的液体培养条件的响应面法优化
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摘要
樟芝(Antrodia cinnamomea)是一种非褶菌目、多孔菌科的多年生蕈菌类,樟芝子实体中含有多种生理活性物质包括三萜类化合物,但野生樟芝只生长在台湾特有的牛樟树树干腐朽的心材内壁和枯死倒伏的牛樟树表面,生长极其缓慢,很难获得,价格昂贵。本研究的目的是通过液体培养樟芝的菌丝体以产生三萜类化合物,用响应面法优化了樟芝产生三萜类化合物的液体发酵条件。首先对樟芝的液体发酵的培养基成分及培养条件进行了单因素优化,研究发现樟芝液体发酵产生三萜类化合物的最佳碳源和氮源分别为葡萄糖和酵母提取物,最佳浓度分别为100 g/L和9 g/L,优化的培养基配方为(g/L):葡萄糖100.0,酵母提取物9.0,Mg SO_4·7H_2O 0.5,KH_2PO_41.0。优化的培养条件为:初始pH值6.0,温度28℃,接种量10%(v/v),摇床转速180 r/min。在此基础上,用响应面法对樟芝液体发酵产生三萜类化合物影响最大的三个因素即碳源、氮源和培养温度进行了优化,结果优化的三种因素条件为:葡萄糖104.71 g/L,酵母提取物9.93 g/L,温度28.42℃。用优化的培养条件液体培养樟芝,其三萜类化合物的产量达到391.45 mg/L,比优化前的产量223.39 mg/L提高了75.23%,为进一步研究液体培养樟芝产生三萜类化合物奠定了基础。
Antrodia cinnamomea is a kind of perennial mushroom, belonging to Polyporaceae in Aphyllophorale.The fruitbody of this mushroom contains many kinds of physiological active substances to human body including triterpenoids. However, the wild fruitbody of A. cinnamomea can grow only on the inner sides of the decaying trunk of Cinnamomum kanehirai and the wet surface of the dead laid tree. The fruitbody develops extremely slowly. It is very difficult to get the mushroom and the price of the mushroom is too expensive. In this study, the conditions for liquid cultivating the mycelium of A. cinnamomea to produce triterpenoids were optimized using surface response methodology. The cultivation conditions for producing triterpenoids in submerged culture fermentation were first optimized by the single factor experiment. As a result, the best carbon source and nitrogen source for A. cinnamomea to produce triterpenoids were glucose and yeast extract, respectively. The optimal concentration for glucose and yeast extract was 100 g/L and 9 g/L, respectively. The optimized medium composition(g/L)was: glucose 100.0, yeast extract 9.0, Mg SO_4·7 H_2 O 0.5, KH_2 PO_41.0. The optimized culture conditions were as below: initial p H6.0, temperature 28℃, amount of inoculum at 10%(v/v), shaking speed at 180 r/min. Based on the results from the single factor experiment, the effect of three most influential factors on the production of triterpenoids in submerged culture fermentation of A. cinnamomea were tested with surface response methodology.As a result, the optimal conditions for the three factors for submerged culture fermentation of A. cinnamomea were as below: 104.71 g/L of glucose, yeast extract 9.93 g/L, the temperature of 28.42℃. Under the optimized condition,the yield of triterpenoids in submerged culture fermentation of A. cinnamomea reached 391.45 mg/L, which was75.23% higher than the yield before optimization(223.39 mg/L), which provided solid basis for further industrial production of triterpenoids by A. cinnamomea.
Antrodia cinnamomea is a kind of perennial mushroom, belonging to Polyporaceae in Aphyllophorale.The fruitbody of this mushroom contains many kinds of physiological active substances to human body including triterpenoids. However, the wild fruitbody of A. cinnamomea can grow only on the inner sides of the decaying trunk of Cinnamomum kanehirai and the wet surface of the dead laid tree. The fruitbody develops extremely slowly. It is very difficult to get the mushroom and the price of the mushroom is too expensive. In this study, the conditions for liquid cultivating the mycelium of A. cinnamomea to produce triterpenoids were optimized using surface response methodology. The cultivation conditions for producing triterpenoids in submerged culture fermentation were first optimized by the single factor experiment. As a result, the best carbon source and nitrogen source for A. cinnamomea to produce triterpenoids were glucose and yeast extract, respectively. The optimal concentration for glucose and yeast extract was 100 g/L and 9 g/L, respectively. The optimized medium composition(g/L)was: glucose 100.0, yeast extract 9.0, Mg SO_4·7 H_2 O 0.5, KH_2 PO_41.0. The optimized culture conditions were as below: initial p H6.0, temperature 28℃, amount of inoculum at 10%(v/v), shaking speed at 180 r/min. Based on the results from the single factor experiment, the effect of three most influential factors on the production of triterpenoids in submerged culture fermentation of A. cinnamomea were tested with surface response methodology.As a result, the optimal conditions for the three factors for submerged culture fermentation of A. cinnamomea were as below: 104.71 g/L of glucose, yeast extract 9.93 g/L, the temperature of 28.42℃. Under the optimized condition,the yield of triterpenoids in submerged culture fermentation of A. cinnamomea reached 391.45 mg/L, which was75.23% higher than the yield before optimization(223.39 mg/L), which provided solid basis for further industrial production of triterpenoids by A. cinnamomea.
引文
Cai W.M.,Jin Q.L.,Cai Q.S.,and Chen J.S.,2015,Artificial cultivation of Antrodia camphorata and its active components and pharmacological effects,Shiyaoyong Jun(Edible and Medicinal Mushrooms),(1):17-23(蔡为明,金群力,蔡青松,陈景荣,2015,牛樟芝人工培养及活性成分与药理作用的研究进展,食药用菌,(1):17-23)
Chang C.Y.,Lee C.L.,and Pan T.M.,2006,Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures,Applied Microbiology and Biotechnology,72(4):654-661
Chang T.T and Chou W.N.,1995,Antrodia cinnamomea sp.nov on Cinnamomum kanehirai in Taiwan,Mycological Research,99(6):756-758
Chen F.,Liu X.H Yang N.Y.,Fu X.S.,Xu H.,and Zhou Y.Z.2011,An overview of the research on Antrodia camphorata Zhongyaocai(Journal of Chinese Medicinal Materials),34(11):1804-1808(陈菲,刘训红,杨念云,傅兴圣,许虎,周逸芝,2011,樟芝的研究概述,中药材,34(11):1804-1808)
Chen J.,2011,Study on Submerged fermentation and selenium-enrich technologcial conditions of Antrodia camphorata mycelium,Thesis for M.S.,Nanchang University,Supervisor:Fan Q.S.,pp.37-39(陈娟,2011,樟芝菌丝体发酵与富硒工艺条件研究,硕士学位论文,南昌大学,导师:范青生,pp.37-39)
Chiu H.H,2007,Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region,Antonie Van Leeuwenhoek,91(3):267-276
Chou M.J.,2007,Ruby in the forest-Antrodia camphorata,Shipin Yu Shenghuo(Food and Life),(7):48(仇明金,2007,森林中的红宝石-樟芝,食品与生活,(7):48)
Fazenda M.L.,Seviour R.,Mcneil B.,and Harvey L.M.,2008Submerged culture fermentation of“higher fungi”:the macrofungi,Advances in Applied Microbiology,63:33-103
Huang D.B.,Yang J.,Huang J.H.,and Weng J.H.,2001,Biological characteristics of Antrodia camphorata,Shiyongjun Xuebao(Acta Edulis Fungi),8(2):24-28(黄大斌,杨菁,黄进华,翁景华,2001,樟芝生物学特性研究,食用菌学报,8(2):24-28)
Lin E.S.and Chen Y.H.,2007,Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media,Bioresource Technology,98(13):2511-2517
Lin E.S.,Wang C.C.,and Sung S.C.,2006,Cultivating conditions influence lipase production by the edible Basidiomycete Antrodia cinnamomea in submerged culture,Enzyme and Microbial Technology,39(1):98-102
Liu H.,Zhou K.,Jia W.,Zhang J.S.,and Pan Y.J.,2007,Technology of active medicinal fungi and polysaccharide extraction from mycelia of Antrodia camphorata,Shipin Kexue(Food Science),28(6):243-245(刘华,周堃,贾薇,张劲松,潘迎捷,2007,药用真菌樟芝菌丝体多糖提取工艺及活性的研究,食品科学,28(6):243-245)
Lu M.C.,Du Y.C.,Chuu J.J.,Hwang S.L.,Hsieh P.C.,Hung C.S.,Chang F.R.,and Wu Y.C.,2009,Active extracts of wild fruiting bodies of Antrodia camphorata(EEAC)induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A,Archives of Toxicology,83(2):121-129
Lu Z.M.,2009,Study on submerged culture of Antrodia camphorata and its triterpenoids,Thesis for Ph.D.,Jiangnan University,Supervisor:Tao W.Y,pp.35-39(陆震鸣,2009,樟芝深层液态发酵及其三萜类化合物的研究,博士学位论文,江南大学,导师:陶文沂,pp.35-39)
Madamanchi G.,and YewMin T.,2010,Review of pharmacological effects of Antrodia camphorata and its bioactive compounds,Evidence-Based Complementray and Alternative Medicine,2011(1741-427X):212641
Madamanchi G.,Fang S.H.,Lai C.H.,Rao Y.K.,Hsiuman L.,and Yewmin T.,2010,Inhibitory effect of Antrodia camphorata constituents on the Helicobacter pylori-associated gastric inflammation,Food Chemistry,119(1):149-153
Peng C.C.,Lin Y.T.,Chen K.C.,Chyau C.C.,and Peng R.Y.,2015,Antrodan,aβ-glucan obtained from Antrodia cinnamomea mycelia,is beneficial to benign prostate hyperplasia,Food and Function,6(2):635
Shen Y.C.Wang Y.H.,Chou Y.C.,Chen C.F.,Lin L.C.,Chang T.T.,Tien J.H.,and Chou C.J.,2004,Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata,Planta Medica,70(4):310
Shen Y.C.,Chen C.F.,Wang Y.H.,Chang T.T.,and Chou C.J.,2003,Evaluation of the immuno-modulating activity of some active principles isolated from the fruiting bodies of Antrodia camphorata,Chinese Pharmaceutical Journal,55(5):313-318
Shih I.L.,and Hsieh P.C.,2006,Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea,Process Biochemistry,41(5):1129-1135
Sinica A.,1990,Ganoderma comphoratum,a new taxon in genus ganoderma from Taiwan China,Acta Botanica Yunnanica,12:395-396
Wang W.M.,Wu R.Y.,and Ko W.H.,2005,Variation and segre gation following nuclear transplantation in Antrodia cinnamomea,Botanical Bulletin of Academia Sinica,46(3):217-222
Wu S.H.,Ryvarden L.,and Chang T.T.,1997,Antrodia camphorata(niu-chang-chih),new combination of a medicinal fungus in Taiwan,Botanical Bulletin-Academia Sinica Taipei,38(4):109-116
Yang F.C.,Yang Y.H.,and Lu H.C.,2013,Enhanced antioxidant and antitumor activities of Antrodia cinnamomea cultured with cereal substrates in solid state fermentation,Biochemical Engineering Journal,78(37):108-113
Yu Z.J.,Bao S.M.,and Li R.T.,2006,The content of triterpenoids in Pleurotus tuber-regium(Fr.)Sing determined by colorinetry,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),17(6):911(余志坚,包水明,李荣同,2006,比色法测定菌核侧耳中三萜类成分的含量,时珍国医国药,17(6):911)
Zhao W.,Xu J.W.,and Zhong J.J.,2011,Enhanced production of ganoderic acids in static liquid culture of Ganoderma lucidum under nitrogen-limiting conditions,Bioresource Technology,102(17):8185
Chang C.Y.,Lee C.L.,and Pan T.M.,2006,Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures,Applied Microbiology and Biotechnology,72(4):654-661
Chang T.T and Chou W.N.,1995,Antrodia cinnamomea sp.nov on Cinnamomum kanehirai in Taiwan,Mycological Research,99(6):756-758
Chen F.,Liu X.H Yang N.Y.,Fu X.S.,Xu H.,and Zhou Y.Z.2011,An overview of the research on Antrodia camphorata Zhongyaocai(Journal of Chinese Medicinal Materials),34(11):1804-1808(陈菲,刘训红,杨念云,傅兴圣,许虎,周逸芝,2011,樟芝的研究概述,中药材,34(11):1804-1808)
Chen J.,2011,Study on Submerged fermentation and selenium-enrich technologcial conditions of Antrodia camphorata mycelium,Thesis for M.S.,Nanchang University,Supervisor:Fan Q.S.,pp.37-39(陈娟,2011,樟芝菌丝体发酵与富硒工艺条件研究,硕士学位论文,南昌大学,导师:范青生,pp.37-39)
Chiu H.H,2007,Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region,Antonie Van Leeuwenhoek,91(3):267-276
Chou M.J.,2007,Ruby in the forest-Antrodia camphorata,Shipin Yu Shenghuo(Food and Life),(7):48(仇明金,2007,森林中的红宝石-樟芝,食品与生活,(7):48)
Fazenda M.L.,Seviour R.,Mcneil B.,and Harvey L.M.,2008Submerged culture fermentation of“higher fungi”:the macrofungi,Advances in Applied Microbiology,63:33-103
Huang D.B.,Yang J.,Huang J.H.,and Weng J.H.,2001,Biological characteristics of Antrodia camphorata,Shiyongjun Xuebao(Acta Edulis Fungi),8(2):24-28(黄大斌,杨菁,黄进华,翁景华,2001,樟芝生物学特性研究,食用菌学报,8(2):24-28)
Lin E.S.and Chen Y.H.,2007,Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media,Bioresource Technology,98(13):2511-2517
Lin E.S.,Wang C.C.,and Sung S.C.,2006,Cultivating conditions influence lipase production by the edible Basidiomycete Antrodia cinnamomea in submerged culture,Enzyme and Microbial Technology,39(1):98-102
Liu H.,Zhou K.,Jia W.,Zhang J.S.,and Pan Y.J.,2007,Technology of active medicinal fungi and polysaccharide extraction from mycelia of Antrodia camphorata,Shipin Kexue(Food Science),28(6):243-245(刘华,周堃,贾薇,张劲松,潘迎捷,2007,药用真菌樟芝菌丝体多糖提取工艺及活性的研究,食品科学,28(6):243-245)
Lu M.C.,Du Y.C.,Chuu J.J.,Hwang S.L.,Hsieh P.C.,Hung C.S.,Chang F.R.,and Wu Y.C.,2009,Active extracts of wild fruiting bodies of Antrodia camphorata(EEAC)induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A,Archives of Toxicology,83(2):121-129
Lu Z.M.,2009,Study on submerged culture of Antrodia camphorata and its triterpenoids,Thesis for Ph.D.,Jiangnan University,Supervisor:Tao W.Y,pp.35-39(陆震鸣,2009,樟芝深层液态发酵及其三萜类化合物的研究,博士学位论文,江南大学,导师:陶文沂,pp.35-39)
Madamanchi G.,and YewMin T.,2010,Review of pharmacological effects of Antrodia camphorata and its bioactive compounds,Evidence-Based Complementray and Alternative Medicine,2011(1741-427X):212641
Madamanchi G.,Fang S.H.,Lai C.H.,Rao Y.K.,Hsiuman L.,and Yewmin T.,2010,Inhibitory effect of Antrodia camphorata constituents on the Helicobacter pylori-associated gastric inflammation,Food Chemistry,119(1):149-153
Peng C.C.,Lin Y.T.,Chen K.C.,Chyau C.C.,and Peng R.Y.,2015,Antrodan,aβ-glucan obtained from Antrodia cinnamomea mycelia,is beneficial to benign prostate hyperplasia,Food and Function,6(2):635
Shen Y.C.Wang Y.H.,Chou Y.C.,Chen C.F.,Lin L.C.,Chang T.T.,Tien J.H.,and Chou C.J.,2004,Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata,Planta Medica,70(4):310
Shen Y.C.,Chen C.F.,Wang Y.H.,Chang T.T.,and Chou C.J.,2003,Evaluation of the immuno-modulating activity of some active principles isolated from the fruiting bodies of Antrodia camphorata,Chinese Pharmaceutical Journal,55(5):313-318
Shih I.L.,and Hsieh P.C.,2006,Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea,Process Biochemistry,41(5):1129-1135
Sinica A.,1990,Ganoderma comphoratum,a new taxon in genus ganoderma from Taiwan China,Acta Botanica Yunnanica,12:395-396
Wang W.M.,Wu R.Y.,and Ko W.H.,2005,Variation and segre gation following nuclear transplantation in Antrodia cinnamomea,Botanical Bulletin of Academia Sinica,46(3):217-222
Wu S.H.,Ryvarden L.,and Chang T.T.,1997,Antrodia camphorata(niu-chang-chih),new combination of a medicinal fungus in Taiwan,Botanical Bulletin-Academia Sinica Taipei,38(4):109-116
Yang F.C.,Yang Y.H.,and Lu H.C.,2013,Enhanced antioxidant and antitumor activities of Antrodia cinnamomea cultured with cereal substrates in solid state fermentation,Biochemical Engineering Journal,78(37):108-113
Yu Z.J.,Bao S.M.,and Li R.T.,2006,The content of triterpenoids in Pleurotus tuber-regium(Fr.)Sing determined by colorinetry,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),17(6):911(余志坚,包水明,李荣同,2006,比色法测定菌核侧耳中三萜类成分的含量,时珍国医国药,17(6):911)
Zhao W.,Xu J.W.,and Zhong J.J.,2011,Enhanced production of ganoderic acids in static liquid culture of Ganoderma lucidum under nitrogen-limiting conditions,Bioresource Technology,102(17):8185