响应面法优化棘孢木霉产厚垣孢子发酵工艺
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摘要
采用响应面法(Box-Benhnken Design)对影响棘孢木霉(Trichoderma asperellum)ZJSX5003菌株产厚垣孢子的4个主要因素(黄豆饼粉-玉米粉用量,甘油添加量,装液量,初始pH)进行了研究。通过对试验数据进行方差分析考察了各因素及其交互作用对厚垣孢子产量的影响;进行回归分析并建立基于统计学的回归方程,计算获得最佳发酵条件:大豆饼粉-玉米粉用量33.25g/L、甘油添加量8.86ml/L、装液量99.35ml/500ml、初始pH值3.26,此时预测的厚垣孢子产量为9.56×10~7个/ml。在此条件下,实际测得厚垣孢子产量为9.84×10~7个/ml,达到理论预测值的97.07%,且比优化前提高了69.07%,进一步采用10L发酵罐进行适用性验证,厚垣孢子产量达到1.75×10~8个/ml,比摇瓶发酵的产量高,为木霉菌厚垣孢子菌剂开发提供了理论依据。
The response surface methodology(Box-Benhnken Design) was applied to investigate the four major factors(the concentration of soybean cake powder and maize powder used in medium,the volume of glycerol addition added to the medium,loading volume of liquid medium,initial pH value of medium) that have importent effect on the production of chlamydospores of Trichoderma asperellum ZJSX5003.By variance analysis on the experiment results,the influences of the the four factors and their interactions on the production of chlamydospores was investigated.By performing regression analysis and establishing regression equation based on statistics,the optimal fermentation condition was obtained,that as follow:the concentration of soybean cake powder and maize powder used in medium 33.25 g /L,glycerol 8.86ml/L,each 500ml bottles contains 99.35 ml liquid medium,initial pH 3.26.Under the optimal conditions,the yield of chlamydospore is expected to be 9.56×10~7 spores/ml,theoretically.The practical yield of chlamydospores approached 9.84×10~7spores/ml,reached97.07%of the expected value,increased by 69.07%compared with pre-optimization.In order to test its applicability,used a 10L fermentation tank under the optimal condition,the yield of chlamydospore reached up to1.75×10~8 spores/ml,which is better than shake flask fermentation.Further researches of developing microbial inoculum of Trichoderma chlamydospore could be conducted base on this research.
The response surface methodology(Box-Benhnken Design) was applied to investigate the four major factors(the concentration of soybean cake powder and maize powder used in medium,the volume of glycerol addition added to the medium,loading volume of liquid medium,initial pH value of medium) that have importent effect on the production of chlamydospores of Trichoderma asperellum ZJSX5003.By variance analysis on the experiment results,the influences of the the four factors and their interactions on the production of chlamydospores was investigated.By performing regression analysis and establishing regression equation based on statistics,the optimal fermentation condition was obtained,that as follow:the concentration of soybean cake powder and maize powder used in medium 33.25 g /L,glycerol 8.86ml/L,each 500ml bottles contains 99.35 ml liquid medium,initial pH 3.26.Under the optimal conditions,the yield of chlamydospore is expected to be 9.56×10~7 spores/ml,theoretically.The practical yield of chlamydospores approached 9.84×10~7spores/ml,reached97.07%of the expected value,increased by 69.07%compared with pre-optimization.In order to test its applicability,used a 10L fermentation tank under the optimal condition,the yield of chlamydospore reached up to1.75×10~8 spores/ml,which is better than shake flask fermentation.Further researches of developing microbial inoculum of Trichoderma chlamydospore could be conducted base on this research.
引文
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[2]Weindling R.Studies on lethal principle effective in the pamsitie action of Trichoderma hamatum on Rhisoctonia solani and other soil fungi.Phytopathology,1932,22:837-845.
[3]高克祥,刘晓光,郭润芳,等.木霉菌对五种植物病原真菌的重寄生作用.山东农业大学学报(自然科学版),2002,33(1):37-42.Gao K X,Liu X G,Guo R F,et al.Mycoparasitism of Trichoderma spp.on five plant pathogenic fungi.Journal of Shandong Agricultural University(Natural Science),2002,33(1):37-42.
[4]朱昌雄,蒋细良,姬军红,等.我国生物农药的研究进展及对未来发展建议.现代化工,2003,23(7):1-4.Zhu X C,Jiang X L,Ji J H,et al.Research progress and some development suggestions on bio-pesticides in China.Modern Chemical Industry,2003,23(7):1-4.
[5]骆健美,金志华,岑沛霖.褐黄孢链霉菌纳他霉素发酵条件优化.高校化学工程学报,2006,20(1):69-73.Luo J M,Jin Z H,Cen P L.Optimizing the fermentation conditions for producing natamycin by Streptomyces gilvosporeus.Journal of Chemical Engineering of Chinese University,2006,20(1):69-73
[6]Castro P M,Hayter P M,Ison A P.Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.Appl Microbiol Biolechnol,1992,38:84-90.
[7]Hounsa C G,Aubry J M,Dubourguier H C.Application of fuclorial and Dochlert designs for optimization of pectale lyase production by a recombinant Escherichia call.Appl Microbiol Biolechnol,1996,45:764-770.
[8]Rao K J,Kim C H.Rhee S K.Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiaeusing response surface methodology.Process Biochemistry,2000,35:639-647.
[9]Vaidya R,Vyas P,Chhatpar H S.Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans.Enzyme and Microbial Technology,2003,33:92-96.
[10]张黎骅,张文,吕珍珍,等.响应面法优化酒糟微波间歇干燥工艺.农业工程学报,2011,27(3):369-373.Zhang L H,Zhang W,LvZZ,et al.Optimization of intermittent microwave drying technology for lees by response surface methodology.Transactions of the CSAE,2011.27(3):369-373.
[11]张秀霞,吴佳东,滕芝,等.响应面法优化固定化微生物降解石油污染物.石油学报,2012,28(5):876-881.Zhang X X.Wu J D.TengZ.et al.Optimization for oil degradation by immobilized microorganism through response surface.Acta Petrolei Sinica,2012,28(5):876-881.
[12]杨晓泉,李汴生,曹劲松.计算机在食品工程中的应用.广州:华南理工大学出版社,1998.146-151.Yang X Q,Li B S,Cao J S.The Computer Application in the Food Engineering.Guangzhou=South China University of Technology,1998.146-151.
[13]Muralidhar R V,Chinimamila R R,Marchant R,et al.A response surface approach for the comparison of lipase production by Candida cylinidracea using two different carbon sources.Biochemical Engineering Journal,2001,9(1):17-23.
[14]Thompson D R.Response surface experimentation.Journal of Food Processing and Preservation,1982,(6);155.
[15]孙瑞燕.我国南方地区木霉菌资源收集、鉴定与生防功能评价研究.上海:上海交通大学农业与生物学院,2013.Sun R Y.Resource collection,identification and bioconlrol evaluation of Trichoderma isolated from southern China.Shanghai;Shanghai Jiao Tong University,School of Agriculture&Biology,2013.
[16]Meiying Zheng,Guocheng Du,Wangfang Guo,et al.A temperature-shift strategy in batch microbial transglutaminase fermentation.Process Biochemistry,2001,36(6)525-530.
[17]Young-Jin Son,Kyong-Hee Park,Sang-Yong,et al.Effect of temperature shift strategies on human preproinsulin production in the fed-batch fermentation of recombinant Escherichia coll.Biotechnology and Bioprocess Engineering,2007,12:556-561.
[18]Hyun-Woo Kang.Yule Kim,Seung-Wook Kim,et al.Cellulosic ethanol production on temperature-shift simultaneous saccharifieation and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612.Bioprocess Biosysl Eng,2012,35:115-122.
[19]齐勇,王际辉,叶淑红,等.响应面法优化芽孢杆菌LJ-7发酵产酯酶条件.大连工业大学学报,2012,31(3):177-180.Qi Y,Wang J H,Ye S H,et al.Optimization of fermentation conditions for Bacillus sp IJ-7 esterase by response surface methodology.Journal of Dalian University of Technology,2012,31(3):177-180
[20]庄敬华,高增贵,陈捷,等.不同发酵条件对木霉产孢类型的影响.中国生物防治.2005,21(1):37-40.Zhuang J H,Gao Z G,Chen J,et al.Optimization of fermentation conditions for Bacillus sp.L J-7 esterase by response surface methodology.Effect of fermentation factors on spore types of Trichoderma strain 23.Chinese Journal of Biological Control,2005.21(1):37-40.
[21]邹勇,文成敬,唐贵群,等.几个因素对木霉菌株T-33产生厚垣孢子的影响.微生物学通报,2006,33(4):43-47.Zou Y,Wen C J,Tang G Q,et al.Effect of several factors on the chlamydospores production of Trichoderma aureoviride T-33.Journal of Microbiology,2006,33(4):43-47.
[22]李锋.木霉厚垣孢子的液体发酵及其可湿性粉剂研制.四川农业大学硕士学位论文,2010.Li F.Liquid fermentation and the wettable powder development of chlamydospores of Trichoderma spp.Sichuan Agricultural University Master Dissertation,2010.
[23]潘玮.绿色木霉厚垣孢子与分生孢子生物学特性及生防效果的比较研究.北京:中国农业科学院,2006.Pan W.Green Trichoderma viride chlamydospore and conidium comparative study of the biological characteristics and bio-control effect.Beijing:Chinese Academy of Agricultural Sciences,2006.
[24]秦芸,叶华智,黄云.影响木霉菌厚垣孢子产生因素初探.云南农业大学学报,2002,17(4):416-417.Qin Y,Ye Z H,Huang Y.Analysis for factors influencing the development of Triehoderma.chlamydospore.Journal of Yunnan Agricultural University,2002,17(4):416-417.
[25]顾金刚,律雪燕,胡丹丹,等.长柄木霉ACCC30150与哈茨木霉ACCC30371产厚垣孢子的液体培养条件.中国生物防治,2008,24(3)253-256.Gu J G,Lv X Y,Hu D I),et al.Production of Chlamydospores of Trichoderma longbrachiatum ACCC30150 and Trichoderma harzianum ACCC30371 by liquid fermenlation.Chinese Journal of Biological Control,2008,24(3)253-256.
[2]Weindling R.Studies on lethal principle effective in the pamsitie action of Trichoderma hamatum on Rhisoctonia solani and other soil fungi.Phytopathology,1932,22:837-845.
[3]高克祥,刘晓光,郭润芳,等.木霉菌对五种植物病原真菌的重寄生作用.山东农业大学学报(自然科学版),2002,33(1):37-42.Gao K X,Liu X G,Guo R F,et al.Mycoparasitism of Trichoderma spp.on five plant pathogenic fungi.Journal of Shandong Agricultural University(Natural Science),2002,33(1):37-42.
[4]朱昌雄,蒋细良,姬军红,等.我国生物农药的研究进展及对未来发展建议.现代化工,2003,23(7):1-4.Zhu X C,Jiang X L,Ji J H,et al.Research progress and some development suggestions on bio-pesticides in China.Modern Chemical Industry,2003,23(7):1-4.
[5]骆健美,金志华,岑沛霖.褐黄孢链霉菌纳他霉素发酵条件优化.高校化学工程学报,2006,20(1):69-73.Luo J M,Jin Z H,Cen P L.Optimizing the fermentation conditions for producing natamycin by Streptomyces gilvosporeus.Journal of Chemical Engineering of Chinese University,2006,20(1):69-73
[6]Castro P M,Hayter P M,Ison A P.Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.Appl Microbiol Biolechnol,1992,38:84-90.
[7]Hounsa C G,Aubry J M,Dubourguier H C.Application of fuclorial and Dochlert designs for optimization of pectale lyase production by a recombinant Escherichia call.Appl Microbiol Biolechnol,1996,45:764-770.
[8]Rao K J,Kim C H.Rhee S K.Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiaeusing response surface methodology.Process Biochemistry,2000,35:639-647.
[9]Vaidya R,Vyas P,Chhatpar H S.Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans.Enzyme and Microbial Technology,2003,33:92-96.
[10]张黎骅,张文,吕珍珍,等.响应面法优化酒糟微波间歇干燥工艺.农业工程学报,2011,27(3):369-373.Zhang L H,Zhang W,LvZZ,et al.Optimization of intermittent microwave drying technology for lees by response surface methodology.Transactions of the CSAE,2011.27(3):369-373.
[11]张秀霞,吴佳东,滕芝,等.响应面法优化固定化微生物降解石油污染物.石油学报,2012,28(5):876-881.Zhang X X.Wu J D.TengZ.et al.Optimization for oil degradation by immobilized microorganism through response surface.Acta Petrolei Sinica,2012,28(5):876-881.
[12]杨晓泉,李汴生,曹劲松.计算机在食品工程中的应用.广州:华南理工大学出版社,1998.146-151.Yang X Q,Li B S,Cao J S.The Computer Application in the Food Engineering.Guangzhou=South China University of Technology,1998.146-151.
[13]Muralidhar R V,Chinimamila R R,Marchant R,et al.A response surface approach for the comparison of lipase production by Candida cylinidracea using two different carbon sources.Biochemical Engineering Journal,2001,9(1):17-23.
[14]Thompson D R.Response surface experimentation.Journal of Food Processing and Preservation,1982,(6);155.
[15]孙瑞燕.我国南方地区木霉菌资源收集、鉴定与生防功能评价研究.上海:上海交通大学农业与生物学院,2013.Sun R Y.Resource collection,identification and bioconlrol evaluation of Trichoderma isolated from southern China.Shanghai;Shanghai Jiao Tong University,School of Agriculture&Biology,2013.
[16]Meiying Zheng,Guocheng Du,Wangfang Guo,et al.A temperature-shift strategy in batch microbial transglutaminase fermentation.Process Biochemistry,2001,36(6)525-530.
[17]Young-Jin Son,Kyong-Hee Park,Sang-Yong,et al.Effect of temperature shift strategies on human preproinsulin production in the fed-batch fermentation of recombinant Escherichia coll.Biotechnology and Bioprocess Engineering,2007,12:556-561.
[18]Hyun-Woo Kang.Yule Kim,Seung-Wook Kim,et al.Cellulosic ethanol production on temperature-shift simultaneous saccharifieation and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612.Bioprocess Biosysl Eng,2012,35:115-122.
[19]齐勇,王际辉,叶淑红,等.响应面法优化芽孢杆菌LJ-7发酵产酯酶条件.大连工业大学学报,2012,31(3):177-180.Qi Y,Wang J H,Ye S H,et al.Optimization of fermentation conditions for Bacillus sp IJ-7 esterase by response surface methodology.Journal of Dalian University of Technology,2012,31(3):177-180
[20]庄敬华,高增贵,陈捷,等.不同发酵条件对木霉产孢类型的影响.中国生物防治.2005,21(1):37-40.Zhuang J H,Gao Z G,Chen J,et al.Optimization of fermentation conditions for Bacillus sp.L J-7 esterase by response surface methodology.Effect of fermentation factors on spore types of Trichoderma strain 23.Chinese Journal of Biological Control,2005.21(1):37-40.
[21]邹勇,文成敬,唐贵群,等.几个因素对木霉菌株T-33产生厚垣孢子的影响.微生物学通报,2006,33(4):43-47.Zou Y,Wen C J,Tang G Q,et al.Effect of several factors on the chlamydospores production of Trichoderma aureoviride T-33.Journal of Microbiology,2006,33(4):43-47.
[22]李锋.木霉厚垣孢子的液体发酵及其可湿性粉剂研制.四川农业大学硕士学位论文,2010.Li F.Liquid fermentation and the wettable powder development of chlamydospores of Trichoderma spp.Sichuan Agricultural University Master Dissertation,2010.
[23]潘玮.绿色木霉厚垣孢子与分生孢子生物学特性及生防效果的比较研究.北京:中国农业科学院,2006.Pan W.Green Trichoderma viride chlamydospore and conidium comparative study of the biological characteristics and bio-control effect.Beijing:Chinese Academy of Agricultural Sciences,2006.
[24]秦芸,叶华智,黄云.影响木霉菌厚垣孢子产生因素初探.云南农业大学学报,2002,17(4):416-417.Qin Y,Ye Z H,Huang Y.Analysis for factors influencing the development of Triehoderma.chlamydospore.Journal of Yunnan Agricultural University,2002,17(4):416-417.
[25]顾金刚,律雪燕,胡丹丹,等.长柄木霉ACCC30150与哈茨木霉ACCC30371产厚垣孢子的液体培养条件.中国生物防治,2008,24(3)253-256.Gu J G,Lv X Y,Hu D I),et al.Production of Chlamydospores of Trichoderma longbrachiatum ACCC30150 and Trichoderma harzianum ACCC30371 by liquid fermenlation.Chinese Journal of Biological Control,2008,24(3)253-256.