利用响应面法优化贝莱斯芽孢杆菌ZJ20发酵参数
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摘要
【目的】优化贝莱斯芽孢杆菌的培养条件,以提高其菌落数和抗菌活性,为该菌株工业化生产提供理论依据。【方法】以贝莱斯芽孢杆菌ZJ20为供试菌株,采用单因素试验,研究培养基、pH、温度、转速对ZJ20菌落数和抗菌活性的影响;在此基础上,以菌落数为考察指标,采用响应面法对pH、温度、转速3个因素进行进一步的优化,得到了贝莱斯芽孢杆菌ZJ20的最佳培养条件;最后在温室采用盆栽试验,研究最佳培养条件得到的贝莱斯芽孢杆菌ZJ20对杂交竹梢枯病的防治效果。【结果】单因素试验结果显示,供试培养基中,NB培养基(牛肉膏3g,蛋白胨10g,氯化钠5g,琼脂20g,蒸馏水1L,pH 7.0～7.2)上贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均最高,分别为4.11×108 CFU/mL和6.10mm,且显著高于其余培养基,说明NB培养基更有利于贝莱斯芽孢杆菌的生长。当pH为4～10时,随着pH的增加,贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均先增后降,其中当pH为7～8时ZJ20的菌落数和抑菌圈直径均显著高于其他处理;在此基础上的复筛结果显示,7.0～7.4为贝莱斯芽孢杆菌的最优pH。当转速为140～200r/min时,随着转速的增加,贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均先增后降,其中当转速为160～180r/min时,菌落数和抑菌圈直径均较高。温度为22～30℃时,随着温度的升高,菌落数和抑菌圈直径均先增加后下降,其中当温度为22～28℃时菌落数和抑菌圈直径均较高。响应面试验结果显示,3个因素中,转速对菌落生长和繁殖的影响最大,其次是温度,影响最小的是pH,贝莱斯芽孢杆菌ZJ20最优培养条件为:pH 7.27,转速160r/min,温度28℃,在此条件下测定的菌落数为769×107 CFU/mL。盆栽试验结果显示,优化培养条件下得到的ZJ20悬液防治效果明显提高,均超过50%。【结论】得到了贝莱斯芽孢杆菌的最佳培养条件,这不仅使其菌落数增加,而且极大地增强了该菌的抗病活性。
【Objective】This study optimized the culture conditions of Bacillus velezensis ZJ20 for enhancing the quantity and antibiotics activity to provide basis for the industrial production of this strain.【Method】The effects of culture medium,pH,temperature and speed on the quantity and activity of B.velezensis ZJ20 were studied by single factor method.On this basis,the quantity was used as the index,the three factors of pH,temperature and speed were further optimized by response surface method,and the optimum culture conditions of B.velezensis ZJ20 were obtained.Finally,the control effects of B.velezensis ZJ20 under the optimal conditions were studied by pot culture in greenhouse.【Result】The results of singlefactor method showed that the colony number and inhibition zone diameter of B.velezensis ZJ20 in NB medium(beef extract 3 g,peptone 10 g,NaCl 5 g,agar 20 g,distilled water 1 L,and pH 7.0-7.2)were the highest with the values of 4.11×108 CFU/mL and 6.10 mm,which were significantly higher than the other culture media.Thus,NB medium was to the best for the growth of B.velezensis ZJ20.With the increase of pH from 4 to 10,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the peak values at pH 7-8.The rescreening showed that 7.0-7.4 was the best pH to B.velezensis ZJ20.When the speed was 140-200 r/min,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the increase of speed.The colony number and inhibition zone diameter with the speed of 160-180 r/min were higher than other treatments.With the increase of temperature during 22-30 ℃,the colony number and inhibition zone diameter increased first and then decreased with the peak values at 22-28℃.The response surface method showed that speed had most influence on the growth and breeding of this strain,followed by temperature,while pH had the minimum influence.The optimum culture conditions of B.velezensis ZJ20 were pH 7.27,speed 160 r/min,and temperature 28℃.Under these conditions,the colony numbers were 769×107 CFU/mL.In the pot experiment,the control effect of the bacterial suspension of optimized parameters was significantly heightened and exceeded 50%.【Conclusion】Optimization culture conditions of B.velezensis were obtained,which not only increased the quantity,but also significantly enhanced the disease resistance.
【Objective】This study optimized the culture conditions of Bacillus velezensis ZJ20 for enhancing the quantity and antibiotics activity to provide basis for the industrial production of this strain.【Method】The effects of culture medium,pH,temperature and speed on the quantity and activity of B.velezensis ZJ20 were studied by single factor method.On this basis,the quantity was used as the index,the three factors of pH,temperature and speed were further optimized by response surface method,and the optimum culture conditions of B.velezensis ZJ20 were obtained.Finally,the control effects of B.velezensis ZJ20 under the optimal conditions were studied by pot culture in greenhouse.【Result】The results of singlefactor method showed that the colony number and inhibition zone diameter of B.velezensis ZJ20 in NB medium(beef extract 3 g,peptone 10 g,NaCl 5 g,agar 20 g,distilled water 1 L,and pH 7.0-7.2)were the highest with the values of 4.11×108 CFU/mL and 6.10 mm,which were significantly higher than the other culture media.Thus,NB medium was to the best for the growth of B.velezensis ZJ20.With the increase of pH from 4 to 10,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the peak values at pH 7-8.The rescreening showed that 7.0-7.4 was the best pH to B.velezensis ZJ20.When the speed was 140-200 r/min,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the increase of speed.The colony number and inhibition zone diameter with the speed of 160-180 r/min were higher than other treatments.With the increase of temperature during 22-30 ℃,the colony number and inhibition zone diameter increased first and then decreased with the peak values at 22-28℃.The response surface method showed that speed had most influence on the growth and breeding of this strain,followed by temperature,while pH had the minimum influence.The optimum culture conditions of B.velezensis ZJ20 were pH 7.27,speed 160 r/min,and temperature 28℃.Under these conditions,the colony numbers were 769×107 CFU/mL.In the pot experiment,the control effect of the bacterial suspension of optimized parameters was significantly heightened and exceeded 50%.【Conclusion】Optimization culture conditions of B.velezensis were obtained,which not only increased the quantity,but also significantly enhanced the disease resistance.
引文
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[6]Wang L T,Lee F L,Tai C J,et al.Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(3):671-675.
[7]Nam M H,Park M S.Biological control of strawberry Fusarium wilt caused by Fusarium oxysporumf.sp.fragariae using Bacillus velezensis BS87and RK1formulation[J].Microbiol Biotechnol,2009,19(5):520-524.
[8]连彩,郭晓军,朱宝成,等.兰花枯萎病拮抗细菌的筛选与鉴定[J].华北农学报,2012,27(2):222-225.Lian C,Guo X J,Zhu B C,et al.Screening and identification of antagonistic bacterium against Colletoichum wilt[J].Acta Agriculturae Boreali-Sinica,2012,27(2):222-225.
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[10]杜淑涛,李术娜,朱宝成.白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验[J].河北农业大学学报,2010,33(6):51-56.Du S T,Li S N,Zhu B C.Screening and identification of antagonistic strain DL-59of Bacillus velezensis against Alternaria brassicae and biocontrol efficiency[J].Journal of Agricultural University of Hebei,2010,33(6):51-56.
[11]朱天辉,李姝江,梁漫,等.贝莱斯芽孢杆菌ZJ20菌株及其液体制剂:ZL201210122696.6[P].2013-12-18.Zhu T H,Li S J,Liang M,et al.Bacillus velezensis ZJ20and liquid preparation:ZL201210122696.6[P].2013-12-18.
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[14]Xu T,Zhu T H,Li S J.β-1,3-1,4-glucanase gene fromBacillus velezensis ZJ20exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):1-9.
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[17]褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药抗生素分册,1999,20(2):58-60,66.Chu Y W.Opimization of media of microorganism and OPTIsoftware[J].World Notes on Antibiotics,1999,20(2):58-60,66.
[18]Ramkrishna S,Swaminmhan T.Response surface modeling and optimization to elucidate and analyze the effects of inoculums age and size on surfactin production[J].Biochemical Engineering Journal,2004,21(2):141-148.
[19]甄新武,乔均俭.BP神经网络结合正交试验法优化Bacillus velezensis Z-27菌株培养基组分[J].湖北农业科学,2013,52(13):3109-3112.Zhen X W,Qiao J J.Culture medium of strain-Bacillus velezensis Z-27based on BP artificiai neural network combined with orthogonal experiment[J].Hubei Agricultural Sciences,2013,52(13):3109-3112.
[20]杨秀荣,王雪莲,王敏,等.利用响应面分析方法优化生防细菌B579增殖培养基[J].微生物学杂志,2010,30(3):35-39.Yang X R,Wang X L,Wang M,et al.Optimization of fermentation medium for biocontrol bacterium B579 with response surface analysis method[J].Journal of Microbiology,2010,30(3):35-39.
[21]Li S J,Zhu T H.Purification of the toxin protein Pc fromArthrinium phaeospermumand its effect on the defence enzymes of Bambusa pervariabilis×Dendrocalamopsis grandis varieties[J].Forest Pathology,2014,44(2):96-106.
[22]Chen L,Yang X,Raza W,et al.Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil[J].Bioresource Technology,2011,102(4):3900-3910.
[23]肖荣凤,郑梅霞,刘波,等.利用响应面法优化非致病性尖孢镰刀菌FJAT-9290固体发酵培养基[J].中国生物防治学报,2017,33(2):258-265.Xiao R F,Zheng M X,Liu B,et al.Optimization of solid-state fermentation culture for biocontrol agent non-pathogenic Fusarium oxysporum FJAT-9290by response surface methodology[J].Chinese Journal of Biological Control,2017,33(2):258-265.
[24]曹可可,刘宁,马双新,等.大斑刚毛座腔菌高产漆酶条件的响应面优化及酶学特性[J].中国农业科学,2015,48(11):2165-2175.Cao K K,Liu N,Ma S X,et al.Optimization of fermentation condition for laccase production by Setosphaeria turcica using the response surface methodology and the enzymatic characters[J].Scientia Agricultura Sinica,2015,48(11):2165-2175.
[25]刘京兰,蔡勋超,薛雅蓉,等.生防解淀粉芽孢杆菌CC09合成iturin A条件的响应面优化[J].中国生物防治学报,2016,32(2):235-243.Liu J N,Cai X C,Xue Y R,et al.Optimization of iturin A synthesis by Bacillus amyloliquefacuens CC09using response surface technique[J].Chinese Journal of Biological Control,2016,32(2):235-243.
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[2]Shoda M.Bacterial control of plant disease[J].Journal of Bioscience and Bioengineering,2000,89(6):515-521.
[3]Swadling I R,Jeffries P.Antagonistic properties of two bacterial biocontrol agents of grey mould disease[J].Biocontrol Science and Technology,1998,8(3):439-448.
[4]章小洪,汪琨,朱廷恒,等.解淀粉芽孢杆菌BW-13培养基和培养条件优化[J].浙江工业大学学报,2013,41(1):35-39.Zhang X H,Wang K,Zhu T H,et al.Optimization of medium and fermentation conditions for the production of antifungal substance by Bacillus amyloliquefacuens BW-13[J].Journal of Zhejiang University of Technology,2013,41(1):35-39.
[5]Ruiz-García C,Béjar V,Martínez-Checa F,et al.Bacillus velezensis sp.nov.,a surfactant-producing bacterium isolated from the river Vélez in Málaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(1):191-195.
[6]Wang L T,Lee F L,Tai C J,et al.Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(3):671-675.
[7]Nam M H,Park M S.Biological control of strawberry Fusarium wilt caused by Fusarium oxysporumf.sp.fragariae using Bacillus velezensis BS87and RK1formulation[J].Microbiol Biotechnol,2009,19(5):520-524.
[8]连彩,郭晓军,朱宝成,等.兰花枯萎病拮抗细菌的筛选与鉴定[J].华北农学报,2012,27(2):222-225.Lian C,Guo X J,Zhu B C,et al.Screening and identification of antagonistic bacterium against Colletoichum wilt[J].Acta Agriculturae Boreali-Sinica,2012,27(2):222-225.
[9]Choi G J,Kim J C,Jang K S,et al.Bacillus velezensis PB3strain and method for the biological control of plant diseases using same[P].Europe PMC,2010,KR20080111656.
[10]杜淑涛,李术娜,朱宝成.白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验[J].河北农业大学学报,2010,33(6):51-56.Du S T,Li S N,Zhu B C.Screening and identification of antagonistic strain DL-59of Bacillus velezensis against Alternaria brassicae and biocontrol efficiency[J].Journal of Agricultural University of Hebei,2010,33(6):51-56.
[11]朱天辉,李姝江,梁漫,等.贝莱斯芽孢杆菌ZJ20菌株及其液体制剂:ZL201210122696.6[P].2013-12-18.Zhu T H,Li S J,Liang M,et al.Bacillus velezensis ZJ20and liquid preparation:ZL201210122696.6[P].2013-12-18.
[12]李姝江,梁漫,朱天辉,等.杂交竹梢枯病拮抗菌的筛选及抗菌蛋白分析[J].南京林业大学学报(自然科学版),2013,37(6):27-32.Li S J,Liang M,Zhu T H,et al.Selection of an antagonistic bacterium against Arthrinium phaeospermumand its antibacterial protein analysis[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2013,37(6):27-32.
[13]徐婷,朱天辉,李姝江,等.贝莱斯芽孢杆菌Bacillus velezensis YB15β-葡聚糖酶的抑菌作用与基因克隆[J].中国生物防治学报,2014,30(2):276-281.Xu T,Zhu T H,Li S J,et al.Fungus-inhibitory activity and gene cloning ofβ-glucanase from Bacillus velezensis YB15[J].Chinese Journal of Biological Control,2014,30(2):276-281.
[14]Xu T,Zhu T H,Li S J.β-1,3-1,4-glucanase gene fromBacillus velezensis ZJ20exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):1-9.
[15]王永宏,张强,张兴.响应面法优化Xenorhabdus nematophila发酵培养基的研究[J].西北农林科技大学学报(自然科学版),2009,37(7):140-146.Wang Y H,Zhang Q,Zhang X.Enhanced antibiotic activity of Xenorhabdus nematophila by fermentation medium optimization with response surface methodology[J].Journal of Northwest A&F University(Nat Sci Ed),2009,37(7):140-146.
[16]张海秀,杜春梅.响应面法优化链霉菌HD-010发酵产抗辣椒根腐病菌活性物质条件[J].食品科学,2010,31(17):340-344.Zhang H X,Du C M.Response surface optimization of medium composition and fermentation conditions for the production of anti-Fusarium soloni substances by Streptomyces HD-010[J].Food Science,2010,31(17):340-344.
[17]褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药抗生素分册,1999,20(2):58-60,66.Chu Y W.Opimization of media of microorganism and OPTIsoftware[J].World Notes on Antibiotics,1999,20(2):58-60,66.
[18]Ramkrishna S,Swaminmhan T.Response surface modeling and optimization to elucidate and analyze the effects of inoculums age and size on surfactin production[J].Biochemical Engineering Journal,2004,21(2):141-148.
[19]甄新武,乔均俭.BP神经网络结合正交试验法优化Bacillus velezensis Z-27菌株培养基组分[J].湖北农业科学,2013,52(13):3109-3112.Zhen X W,Qiao J J.Culture medium of strain-Bacillus velezensis Z-27based on BP artificiai neural network combined with orthogonal experiment[J].Hubei Agricultural Sciences,2013,52(13):3109-3112.
[20]杨秀荣,王雪莲,王敏,等.利用响应面分析方法优化生防细菌B579增殖培养基[J].微生物学杂志,2010,30(3):35-39.Yang X R,Wang X L,Wang M,et al.Optimization of fermentation medium for biocontrol bacterium B579 with response surface analysis method[J].Journal of Microbiology,2010,30(3):35-39.
[21]Li S J,Zhu T H.Purification of the toxin protein Pc fromArthrinium phaeospermumand its effect on the defence enzymes of Bambusa pervariabilis×Dendrocalamopsis grandis varieties[J].Forest Pathology,2014,44(2):96-106.
[22]Chen L,Yang X,Raza W,et al.Solid-state fermentation of agro-industrial wastes to produce bioorganic fertilizer for the biocontrol of Fusarium wilt of cucumber in continuously cropped soil[J].Bioresource Technology,2011,102(4):3900-3910.
[23]肖荣凤,郑梅霞,刘波,等.利用响应面法优化非致病性尖孢镰刀菌FJAT-9290固体发酵培养基[J].中国生物防治学报,2017,33(2):258-265.Xiao R F,Zheng M X,Liu B,et al.Optimization of solid-state fermentation culture for biocontrol agent non-pathogenic Fusarium oxysporum FJAT-9290by response surface methodology[J].Chinese Journal of Biological Control,2017,33(2):258-265.
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