青枯病植物疫苗生产菌FJAT-1458发酵工艺的优化

doi:10.16409/j.cnki.2095-039x.2019.01.017

摘要/Abstract

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%、K₂HPO₄ 0.05%和MgSO₄ 0.025%，发酵48 h后，发酵液活菌含量可达3.87×10⁹ 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4.68×10⁸ cfu/g。

关键词: 植物疫苗, 液体发酵, 固体发酵, 培养基优化, 培养条件优化

Abstract: 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%, K₂HPO₄ 0.05% and MgSO₄ 0.025%. The average bacterial yield of FJAT-1458 could reach up to 3.87×10⁹ 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 4.68×10⁸ cfu/g.

Key words: plant vaccine, liquid fermentation, solid fermentation, medium optimization, culture condition optimization

中图分类号:

S43
S476

陈燕萍, 肖荣凤, 刘波, 朱育菁, 郑雪芳, 陈德局. 青枯病植物疫苗生产菌FJAT-1458发酵工艺的优化[J]. 中国生物防治学报, 2019, 35(1): 81-88.

CHEN Yanping, XIAO Rongfeng, LIU Bo, ZHU Yujing, ZHENG Xuefang, CHEN Deiju. Culture Optimization for Fermentation of Avirulent Ralstonia Solanacearum Strain FJAT-1458 Used as Plant Vaccine against Crop Bacterial Wilt[J]. journal1, 2019, 35(1): 81-88.

参考文献

[1] Poueymiro M, Cazale A C, Francols J M, et al. A Ralstonia solanacearum type Ⅲ effector directs the production of the plant signal metabolite trehalose-6-phosphate[J]. Mbio, 2014, 5(6):e02065-14.
[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),2015, 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.