青枯病植物疫苗生产菌FJAT-1458发酵工艺的优化
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摘要
为提高防治青枯病的植物疫苗菌FJAT-1458的发酵工艺,本文采用液固双相发酵方式,以活菌体数量为指标,通过单因子和正交设计试验对菌株FJAT-1458液体发酵培养基进行优化,并利用农业副产物——微生物发酵床养猪垫料为发酵培养基主成分进行固体培养工艺优化,为研制植物疫苗菌剂提供基础。结果表明,菌株FJAT-1458液体发酵的最适培养基配方为玉米粉2.5%、鱼骨粉0.5%、蔗糖0.1%、蛋白胨0.15%、K_2HPO_4 0.05%和MgSO_4 0.025%,发酵48 h后,发酵液活菌含量可达3.87×10~9 cfu/mL。该菌固体发酵最佳培养基配方为垫料:黄豆饼粉=7:3(质量比),最佳发酵条件为温度35℃、含水量45%、接菌量15%、通气量70%时,培养40 h后,固体菌剂活菌量达4.68×10~8 cfu/g。
FJAT-1458 is an avirulent Ralstonia solanacearum strain used as plant vaccine against crop bacterial wilt disease. In order to improve the fermentation level of the strain FJAT-1458, the liquid and solid fermentation culture media and culture conditions were optimized by using single factor and orthogonal test analysis. The solid-state fermentation medium was mainly the litters from microbial fermentation bed for pig-raising, and the bacterial yield was used as the indicator in all tests. The results showed that optimal liquid fermentation medium formula for the strain was corn flour 2.5%, fishbone meal 0.5%, sucrose 0.1%, peptone 0.15%, K_2HPO_4 0.05% and MgSO_4 0.025%. The average bacterial yield of FJAT-1458 could reach up to 3.87×10~9 cfu/mL for 48 h. The optimal solid fermentation medium formula for the strain was a mass ratio of 7:3 for litters and soybean powder. The optimal fermentation conditions were temperature 35 ℃, relative moisture 45%, inoculation quantity 15%, ventilation 70% and fermentation time for 40 h. The average bacterial yield of strain FJAT-1458 could reach up to 4.68×10~8 cfu/g.
FJAT-1458 is an avirulent Ralstonia solanacearum strain used as plant vaccine against crop bacterial wilt disease. In order to improve the fermentation level of the strain FJAT-1458, the liquid and solid fermentation culture media and culture conditions were optimized by using single factor and orthogonal test analysis. The solid-state fermentation medium was mainly the litters from microbial fermentation bed for pig-raising, and the bacterial yield was used as the indicator in all tests. The results showed that optimal liquid fermentation medium formula for the strain was corn flour 2.5%, fishbone meal 0.5%, sucrose 0.1%, peptone 0.15%, K_2HPO_4 0.05% and MgSO_4 0.025%. The average bacterial yield of FJAT-1458 could reach up to 3.87×10~9 cfu/mL for 48 h. The optimal solid fermentation medium formula for the strain was a mass ratio of 7:3 for litters and soybean powder. The optimal fermentation conditions were temperature 35 ℃, relative moisture 45%, inoculation quantity 15%, ventilation 70% and fermentation time for 40 h. The average bacterial yield of strain FJAT-1458 could reach up to 4.68×10~8 cfu/g.
引文
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[10]郑雪芳,朱育菁,刘波,等.番茄青枯病植物疫苗胶悬菌剂的制备及其对病害的防治效果[J].植物保护,2017,43(2):208-211.
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[15]刘波,林营志,朱育菁,等.生防菌对青枯雷尔氏菌的致弱特性[J].农业生物技术学报,2004,12(3):322-329.
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[19]冉淦侨,田云龙,郭萍,等.响应面法优化青枯病生防菌Hrp-菌株的发酵条件[J].生物加工过程,2012,10(4):1-6.
[20]孙义,居正英,林玲,等.茄子黄萎病生防内生菌29-12发酵条件的初步研究[J].江苏农业学报,2008,24(4):425-430.
[21]李今煜,陈聪,关雄.苏云金芽孢杆菌发酵培养基的筛选[J].福建农林大学学报(自然科学版),2003,32(4):490-492.
[22]刘美,王刘庆,廖美德,等.球形芽孢杆菌BS-10高产培养基的优化[J].西北农林科技大学学报(自然科学版),2 015,43(11):214-220.
[23]李玉洋,辛寒晓,范学明,等.菌糠固态发酵法培养多粘类芽孢杆菌SH15的工艺[J].山东农业科学,2017(12):119-125
[24]Zhao G,Niu M,Lu S,et al.Cultivation of paenibacillus polymyxa by solid-state fermentation of pear residues[J].Transactions of the Chinese Society of Agricultural Engineering,2016,32(7):303-308.
[25]徐速,孙立斌,赵清霞,等.替代抗生素枯草芽孢杆菌菌剂固态发酵工艺优化[J].中国食品学报,2016,16(8):132-139.
[26]欧阳江华.利用微生物发酵床养猪垫料研制生物肥药的研究[D].福州:福建农林大学,2010.
[27]陈燕萍,肖荣凤,刘波,等.利用微生物发酵床养猪垫料制备蔬菜育苗基质的研究[J].福建农业学报,2015(8):802-809.
[28]郑永德,卢翠香,邱春锦,等.猪场废弃垫料栽培秀珍菇的适宜添加比例试验[J].食药用菌,2015(3):199-200.
[29]曾庆才,肖荣凤,刘波,等.以微生物发酵床养猪垫料为主要基质的哈茨木霉FJAT-9040固体发酵培养基优化[J].热带作物学报,2014,35(4):771-778.
[30]陈燕萍,肖荣凤,刘波,等.基于发酵床养猪垫料的地衣芽胞杆菌FJAT-4固体发酵培养条件的优化[J].中国生物防治学报,2017,33(1):128-133.
[31]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.
[32]冉淦侨,田云龙,戴佳锟,等.防治青枯病工程菌Hrp-菌株的固体剂型研究[J].中国生物防治学报.2014,30(3):385-392.
[33]胡海燕,于勇,张玉静,等.发酵床养猪废弃垫料的资源化利用评价[J].植物营养与肥料学报,2013,19(1):252-258.
[34]程本亮,车建美,刘波.青枯雷尔氏菌Tn5转座子无致病力突变株构建及其生物学特性[J].农业生物技术学报,2011,19(1):26-35.
[35]姚伟芳,弓爱君,邱丽娜,等.苏云金芽胞杆菌固态发酵条件的筛选[J].化学与生物工程,2006,23(11):42-44.
[2]Mansfield J,Gemom S,Magori S,et al.Top 10 plant pathogenic bacteria in molecular plant pathology[J].Molecular Plant Pathology,2012,13(6):614-629.
[3]Jones J D G,Dangl J L.The plant immune system[J].Nature,2006,444(16):323-329.
[4]Chang Y H,Yan H Z,Liou R F.A novel elicitor protein from phytophthora parasitica induces plant basa immunity and systemic acquired resistance[J].Molecular Plant Pathology,2015,16(2):123-136.
[5]郑雪芳,刘波,林乃铨,等.青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析[J].植物病理学报,2013,43(5):518-531.
[6]肖田,姚廷山,于庆涛.青枯无致病力菌株对烟草青枯病的诱导抗性与控病作用[J].西南农业学报,2015,28(1):207-211.
[7]马健尧,李焕秀,李立佼,等.番茄青枯雷尔氏菌强致病力菌株变异的研究[J].广西植物,2014(4):530-534.
[8]张文州.番茄青枯病植物疫苗工程菌的筛选及其免疫抗病机理研究.硕士学位论文[D].福州:福建师范大学,2012.
[9]郑雪芳,朱育菁,刘波,等.番茄青枯病植物疫苗菌株FJAT-1458的培养条件优化及发酵过程状态参数研究[J].福建农业学报,2016,31(8):858-862.
[10]郑雪芳,朱育菁,刘波,等.番茄青枯病植物疫苗胶悬菌剂的制备及其对病害的防治效果[J].植物保护,2017,43(2):208-211.
[11]Yang S T.Bioprocessing for value-added products from renewable resources[J].Chinese Journal of Chemical Engineering,2008(4):619.
[12]Rodriguez D P.Redesigning the production of the Bacillus thuringiensis Bio-Pesticide within the context of subsistence agriculture in Andhra Pradesh,India[J].Asian Biotechnology and Development Review,2007,9(3):55-81.
[13]Vimala Devi P S,Rao M L N.Tailoring production technology:Bacillus thuringiensis(Bt)for localized production[J].Tailoring Biotechnologies,2005,1(2):107-120.
[14]陈燕萍,刘波,葛慈斌,等.作物青枯病植物疫苗工程菌FJAT-T8发酵过程生长适合度研究[J].福建农业学报,2012,27(3):287-293.
[15]刘波,林营志,朱育菁,等.生防菌对青枯雷尔氏菌的致弱特性[J].农业生物技术学报,2004,12(3):322-329.
[16]Dale W,Adrian N,Gary L.Induced resisitance for plant defence[M].Oxford:Blackwell Publishing,2007,31-81.
[17]Peng X,Liu H,Luo,et al.Fermentation conditions for antagonistic endophytic bacteria against Ralstonia solanacearum[J].Tobacco Science and Technology,2007,40(6):56-58.
[18]赵晓燕,杨欢,陈建刚,等.一株烟草青枯病生防菌的筛选鉴定及其发酵优化[J].湖北农业科学,2014(8):1810-1814.
[19]冉淦侨,田云龙,郭萍,等.响应面法优化青枯病生防菌Hrp-菌株的发酵条件[J].生物加工过程,2012,10(4):1-6.
[20]孙义,居正英,林玲,等.茄子黄萎病生防内生菌29-12发酵条件的初步研究[J].江苏农业学报,2008,24(4):425-430.
[21]李今煜,陈聪,关雄.苏云金芽孢杆菌发酵培养基的筛选[J].福建农林大学学报(自然科学版),2003,32(4):490-492.
[22]刘美,王刘庆,廖美德,等.球形芽孢杆菌BS-10高产培养基的优化[J].西北农林科技大学学报(自然科学版),2 015,43(11):214-220.
[23]李玉洋,辛寒晓,范学明,等.菌糠固态发酵法培养多粘类芽孢杆菌SH15的工艺[J].山东农业科学,2017(12):119-125
[24]Zhao G,Niu M,Lu S,et al.Cultivation of paenibacillus polymyxa by solid-state fermentation of pear residues[J].Transactions of the Chinese Society of Agricultural Engineering,2016,32(7):303-308.
[25]徐速,孙立斌,赵清霞,等.替代抗生素枯草芽孢杆菌菌剂固态发酵工艺优化[J].中国食品学报,2016,16(8):132-139.
[26]欧阳江华.利用微生物发酵床养猪垫料研制生物肥药的研究[D].福州:福建农林大学,2010.
[27]陈燕萍,肖荣凤,刘波,等.利用微生物发酵床养猪垫料制备蔬菜育苗基质的研究[J].福建农业学报,2015(8):802-809.
[28]郑永德,卢翠香,邱春锦,等.猪场废弃垫料栽培秀珍菇的适宜添加比例试验[J].食药用菌,2015(3):199-200.
[29]曾庆才,肖荣凤,刘波,等.以微生物发酵床养猪垫料为主要基质的哈茨木霉FJAT-9040固体发酵培养基优化[J].热带作物学报,2014,35(4):771-778.
[30]陈燕萍,肖荣凤,刘波,等.基于发酵床养猪垫料的地衣芽胞杆菌FJAT-4固体发酵培养条件的优化[J].中国生物防治学报,2017,33(1):128-133.
[31]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.
[32]冉淦侨,田云龙,戴佳锟,等.防治青枯病工程菌Hrp-菌株的固体剂型研究[J].中国生物防治学报.2014,30(3):385-392.
[33]胡海燕,于勇,张玉静,等.发酵床养猪废弃垫料的资源化利用评价[J].植物营养与肥料学报,2013,19(1):252-258.
[34]程本亮,车建美,刘波.青枯雷尔氏菌Tn5转座子无致病力突变株构建及其生物学特性[J].农业生物技术学报,2011,19(1):26-35.
[35]姚伟芳,弓爱君,邱丽娜,等.苏云金芽胞杆菌固态发酵条件的筛选[J].化学与生物工程,2006,23(11):42-44.