卵孢菌素产生菌株角毛壳菌(Chaetomium cupreum)CH21-20发酵培养条件优化
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摘要
卵孢菌素(Oosporein)是具有广谱抗菌活性的二联苯醌类化合物;角毛壳菌(Chaetomium cupreum)CH21-20为本实验室通过遗传改良获得的卵孢菌素高产菌株.利用该菌株进行发酵条件研究,获得其最适发酵条件为接种量(体积分数)3%、初始p H 7.0、装液量60 mL/250 mL、摇床转速180 r/min、培养温度24℃.培养基碳氮源和无机盐筛选结果表明,15.0 g/L蔗糖、5.0 g/L蛋白粉及0.5 g/L氯化钠能显著提高卵孢菌素产量.以卵孢菌素为响应值,对蔗糖、蛋白粉、氯化钠进行三因素三水平的Box-Behnken实验设计及响应面法优化研究,得到蔗糖、蛋白粉及氯化钠的最佳配比分别为15.63 g/L、5.22 g/L、0.53 g/L,预测在此发酵条件下卵孢菌素最高产量可达2 547μg/mL,实验验证得到卵孢菌素产量为2 478μg/mL,与预测值接近;发酵条件优化后,卵孢菌素产量提高了56.14%.本研究获得了卵孢菌素稳定高产的摇瓶发酵技术参数.
Oosporein is a dibenzoquinone pigment with broad-spectrum antimicrobial activities. The oosporein hyperproducing mutant strain Chaetomium cupreum CH21-20 was obtained via genetic modification. We studied the effects of the fermentation parameters on the production of oosporein in the fermentation process of CH21-20. The results showed that the optimal values of the fermentation parameters were: inoculum size 3%, initial pH 7.0, culture volume 60 mL per 250 mL shake flask, culture temperature 24 ℃, and rotation ratio 180 r/min. The results of the experiments for screening carbon and nitrogen sources and inorganic salts revealed that 15.0 g/L sucrose, 5.0 g/L protein powder, and 0.5 g/L sodium chloride had significant influences on the yield of oosporein. Accordingly, a Box-Behnken Design of three factors and three levels was designed for the production of oosporein as a response. The highest production of oosporein was 2547 μg/mL when the concentrations of sucrose, protein powder, and sodium chloride were 15.63 g/L, 5.22 g/L, and 0.53 g/L, respectively, after response surface prediction. The terminal production of oosporein reached 2478 μg/mL after the confirmatory experiment, enhanced by almost 56.14% compared to the production before the conditions were optimized. Thus, in the present study, stable and high-producing fermentation parameters of oosporein were determined in the shake-flask.
Oosporein is a dibenzoquinone pigment with broad-spectrum antimicrobial activities. The oosporein hyperproducing mutant strain Chaetomium cupreum CH21-20 was obtained via genetic modification. We studied the effects of the fermentation parameters on the production of oosporein in the fermentation process of CH21-20. The results showed that the optimal values of the fermentation parameters were: inoculum size 3%, initial pH 7.0, culture volume 60 mL per 250 mL shake flask, culture temperature 24 ℃, and rotation ratio 180 r/min. The results of the experiments for screening carbon and nitrogen sources and inorganic salts revealed that 15.0 g/L sucrose, 5.0 g/L protein powder, and 0.5 g/L sodium chloride had significant influences on the yield of oosporein. Accordingly, a Box-Behnken Design of three factors and three levels was designed for the production of oosporein as a response. The highest production of oosporein was 2547 μg/mL when the concentrations of sucrose, protein powder, and sodium chloride were 15.63 g/L, 5.22 g/L, and 0.53 g/L, respectively, after response surface prediction. The terminal production of oosporein reached 2478 μg/mL after the confirmatory experiment, enhanced by almost 56.14% compared to the production before the conditions were optimized. Thus, in the present study, stable and high-producing fermentation parameters of oosporein were determined in the shake-flask.
引文
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2 Kogl F,Van Wessem GC.Analysis concerning pigments of fungi XIV:concerning oosporein,the pigment of Oospora colorans van Beyma[J].Recl Trav Chim Pays Bas,1944,63:5-24
3 Abendstein D,Per nf uss B,St rasser H.Evaluation of Beauveria brongniartii and its metabolite oosporein regarding phytotoxicity on seed potatoes[J].Biocontrol Sci Technol,2000,10(6):789-796
4 Vining LC,Kelleher WJ,Schwarting AE.Oosporein production by a strain of Beauveria bassiana originally identified as Amanita muscaria[J].Can J Microbiol,1962,8(6):931-933
5 Divekar PV,Haskins RH,Vining LC.Oosporein from an Acremonium sp.[J].Can J Chem,1959,37(12):2097-2099
6 Cole RJ,Kirksey JW,Cutler HG,Davis EE.Toxic effects of oosporein from Chaetomium trilaterale[J].J Agric Food Chem,1974,22(3):517-520
7 Lloyd G,Robertson A,Sankey GB.The chemistry of fungi.part XXV.oosporein,a metabolite of Chaetomium aureum chivers[J].J Chem Soc(Resumed),1955:2163-2165
8 Mao BZ,Huang C,Yang GM.Separation and determination of the bioactivity of oosporein from Chaetomium cupreum[J].Afr J Biotechnol,2010,9(36):5955-5961
9 He G,Yan J,Wu XY,Gou XJ,Li WC.Oosporein from Tremella fuciformis[J].Acta Crystallogr Sect E:Struct Rep Online,2012,68(4):o1231-o1231
10 Feng P,Shang Y,Cen K,Wang C.Fungal biosynthesis of the bibenzoquinone oosporein to evade insect immunity[J].Proc Natl Acad Sci USA,2015,112(36):11365-11370
11 Klingen I,Meadow R,Aandal T.Mortality of Delia floralis,Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi[J].J Appl Entomol,2002,126(5):231-237
12 Labbe RM,Cloutier C,Brodeur J.Prey selection by Dicyphus hesperus of infected or parasitized greenhouse whitef ly[J].Biocontrol Sci Technol,2006,16(5):485-494
13 Favilla M,Macchia L,Gallo A,Altomare C.Toxicity assessment of metabolites of fungal biocontrol agents using two different(Artemia salina and Daphnia magna)invertebrate bioassays[J].Food Chem Toxicol,2006,44(11):1922-1931
14 Alurappa R,Bojegowda MRM,Kumar V,Mallesh NK,Chowdappa S.Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.[J].Nat Prod Res,2014,28(23):2217-2220
15 Terry BJ,Liu WC,Cianci CW,Proszynski E,Fernandes P,Bush K,Meyers E.Inhibition of herpes simplex virus type 1 DNA polymerase by the natural product oosporein[J].J Antibiot,1992,45(2):286-288
16 Strasser H,Vey A,ButtTM.Arethereanyrisksinusing entomopathogenic fungi for pest control,with particular reference to the bioactive metabolites of Metarhizium,Tolypocladium and Beauveria species?[J].Biocontrol Sci Technol,2000,10(6):717-735
17 Strasser H,Abendstein D,Stuppner H.Monitoring the distribution of secondary metabolites produced by the entomogenous fungus Beauveria brongniartii with particular reference to oosporein[J].Mycol Res,2000,104(10):1227-1233
18 Basyouni SHE,Brewer D,Vining LC.Pigments of the genus Beauveria[J].Can J Bot,1968,46(4):441-448
19 Michelitsch A,Rückert U,Rittmannsberger A,Seger C,Strasser H,Likussar W.Accurate determination of oosporein in fungal culture broth by differential pulse polarography[J].J Agric Food Chem,2004,52(6):1423-1426
20卢梦梦,潘清灵,李赛妮,王磊,郝再彬,章卫民.深红虫草高产卵孢菌素的培养基筛选及发酵条件优化[J].菌物学报,2017,36(4):503-511[Lu MM,Pan QL,Li SN,Wang L,Hao ZB,Zhang WM.Screening of culture medium and optimization of fermentation condition for oosporein production of Cordyceps cardinalis strain[J].Mygosystema,2017,36(4):503-511]
21何海清,杨莉娜,周金燕,谭红.紫外分光光度法和HPLC法测定角毛壳菌CH-1发酵液中卵孢菌素含量的比较研究[J].中国抗生素杂志,2015,40(8):593-598[He HQ,Yang LN,Zhou JY,Tan H.Comparison of UV spectrophotometry and HPLC on quantitative determination of oosporein in the broth of Chaetomium cupreum CH-1[J].Chin JAntibiot,2015,40(8):593-598]
22 El Basyouni SH,Vining LC.Biosynthesis of oosporein in Beauveria bassiana(Bals.)Vuill[J].Can J Biochem,1966,44(5):557-565
23 Timoumi A,Guillouet SE,Molina-Jouve C,Fillaudeau L,Corret N.Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica[J].Appl Microbiol Biotechnol,2018,102(9):3831-3848
24 Guan N,Li J,Shin HD,Du G,Chen J,Liu L.Microbial response to environmental stresses:from fundamental mechanisms to practical applications[J].Appl Microbiol Biotechnol,2017,101(10):3991-4008
25 MacLeod DM.Investigations on the genera Beauveria Vuill,and Tritirachium Limber[J].Can J Bot,1954,32(6):818-890
2 Kogl F,Van Wessem GC.Analysis concerning pigments of fungi XIV:concerning oosporein,the pigment of Oospora colorans van Beyma[J].Recl Trav Chim Pays Bas,1944,63:5-24
3 Abendstein D,Per nf uss B,St rasser H.Evaluation of Beauveria brongniartii and its metabolite oosporein regarding phytotoxicity on seed potatoes[J].Biocontrol Sci Technol,2000,10(6):789-796
4 Vining LC,Kelleher WJ,Schwarting AE.Oosporein production by a strain of Beauveria bassiana originally identified as Amanita muscaria[J].Can J Microbiol,1962,8(6):931-933
5 Divekar PV,Haskins RH,Vining LC.Oosporein from an Acremonium sp.[J].Can J Chem,1959,37(12):2097-2099
6 Cole RJ,Kirksey JW,Cutler HG,Davis EE.Toxic effects of oosporein from Chaetomium trilaterale[J].J Agric Food Chem,1974,22(3):517-520
7 Lloyd G,Robertson A,Sankey GB.The chemistry of fungi.part XXV.oosporein,a metabolite of Chaetomium aureum chivers[J].J Chem Soc(Resumed),1955:2163-2165
8 Mao BZ,Huang C,Yang GM.Separation and determination of the bioactivity of oosporein from Chaetomium cupreum[J].Afr J Biotechnol,2010,9(36):5955-5961
9 He G,Yan J,Wu XY,Gou XJ,Li WC.Oosporein from Tremella fuciformis[J].Acta Crystallogr Sect E:Struct Rep Online,2012,68(4):o1231-o1231
10 Feng P,Shang Y,Cen K,Wang C.Fungal biosynthesis of the bibenzoquinone oosporein to evade insect immunity[J].Proc Natl Acad Sci USA,2015,112(36):11365-11370
11 Klingen I,Meadow R,Aandal T.Mortality of Delia floralis,Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi[J].J Appl Entomol,2002,126(5):231-237
12 Labbe RM,Cloutier C,Brodeur J.Prey selection by Dicyphus hesperus of infected or parasitized greenhouse whitef ly[J].Biocontrol Sci Technol,2006,16(5):485-494
13 Favilla M,Macchia L,Gallo A,Altomare C.Toxicity assessment of metabolites of fungal biocontrol agents using two different(Artemia salina and Daphnia magna)invertebrate bioassays[J].Food Chem Toxicol,2006,44(11):1922-1931
14 Alurappa R,Bojegowda MRM,Kumar V,Mallesh NK,Chowdappa S.Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.[J].Nat Prod Res,2014,28(23):2217-2220
15 Terry BJ,Liu WC,Cianci CW,Proszynski E,Fernandes P,Bush K,Meyers E.Inhibition of herpes simplex virus type 1 DNA polymerase by the natural product oosporein[J].J Antibiot,1992,45(2):286-288
16 Strasser H,Vey A,ButtTM.Arethereanyrisksinusing entomopathogenic fungi for pest control,with particular reference to the bioactive metabolites of Metarhizium,Tolypocladium and Beauveria species?[J].Biocontrol Sci Technol,2000,10(6):717-735
17 Strasser H,Abendstein D,Stuppner H.Monitoring the distribution of secondary metabolites produced by the entomogenous fungus Beauveria brongniartii with particular reference to oosporein[J].Mycol Res,2000,104(10):1227-1233
18 Basyouni SHE,Brewer D,Vining LC.Pigments of the genus Beauveria[J].Can J Bot,1968,46(4):441-448
19 Michelitsch A,Rückert U,Rittmannsberger A,Seger C,Strasser H,Likussar W.Accurate determination of oosporein in fungal culture broth by differential pulse polarography[J].J Agric Food Chem,2004,52(6):1423-1426
20卢梦梦,潘清灵,李赛妮,王磊,郝再彬,章卫民.深红虫草高产卵孢菌素的培养基筛选及发酵条件优化[J].菌物学报,2017,36(4):503-511[Lu MM,Pan QL,Li SN,Wang L,Hao ZB,Zhang WM.Screening of culture medium and optimization of fermentation condition for oosporein production of Cordyceps cardinalis strain[J].Mygosystema,2017,36(4):503-511]
21何海清,杨莉娜,周金燕,谭红.紫外分光光度法和HPLC法测定角毛壳菌CH-1发酵液中卵孢菌素含量的比较研究[J].中国抗生素杂志,2015,40(8):593-598[He HQ,Yang LN,Zhou JY,Tan H.Comparison of UV spectrophotometry and HPLC on quantitative determination of oosporein in the broth of Chaetomium cupreum CH-1[J].Chin JAntibiot,2015,40(8):593-598]
22 El Basyouni SH,Vining LC.Biosynthesis of oosporein in Beauveria bassiana(Bals.)Vuill[J].Can J Biochem,1966,44(5):557-565
23 Timoumi A,Guillouet SE,Molina-Jouve C,Fillaudeau L,Corret N.Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica[J].Appl Microbiol Biotechnol,2018,102(9):3831-3848
24 Guan N,Li J,Shin HD,Du G,Chen J,Liu L.Microbial response to environmental stresses:from fundamental mechanisms to practical applications[J].Appl Microbiol Biotechnol,2017,101(10):3991-4008
25 MacLeod DM.Investigations on the genera Beauveria Vuill,and Tritirachium Limber[J].Can J Bot,1954,32(6):818-890