贝莱斯芽孢杆菌FJ17-4发酵培养基和发酵条件优化

兰成忠; 林雄; 甘林; 代玉立; 刘晓菲; 杨秀娟; 蒋军喜

doi:10.19303/j.issn.1008-0384.2022.010.013

贝莱斯芽孢杆菌FJ17-4发酵培养基和发酵条件优化

doi: 10.19303/j.issn.1008-0384.2022.010.013

兰成忠^{1, 2,},
林雄¹,
甘林¹,
代玉立¹,
刘晓菲¹,
杨秀娟¹,
蒋军喜^2, ,

1.
福建省作物有害生物绿色防控工程中心/福建省农业科学院植物保护研究所，福建　福州　350013
2.
江西农业大学农学院，江西　南昌　330045

基金项目: 福建省科技计划公益类专项（2022R1024008）；福建省农业科学院科技创新团队建设专项（CXTD2021027）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021011）

详细信息

作者简介:
兰成忠（1979−），男，博士，副研究员，研究方向：植物病害防治技术研究（E-mail：lczhong7911@126.com）

通讯作者:
蒋军喜（1964−），男，博士，教授，博士生导师，研究方向：植物病理学(E-mail：jxau2011@126.com)

中图分类号: S 435.72
计量
- 文章访问数: 745
- HTML全文浏览量: 756
- PDF下载量: 120
- 被引次数: 0
出版历程
- 收稿日期: 2022-02-10
- 修回日期: 2022-09-15
- 网络出版日期: 2022-11-29
- 刊出日期: 2022-10-30

Optimization of Bacillus velezensis FJ17-4 Fermentation

1.
Fujian Engineering Research Center for Green Pest Management, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
College of Agronomy, Jiangxi Agricultural University, Nanchang, Jiangxi　330045, China

摘要

摘要: 目的贝莱斯芽孢杆菌FJ17-4对许多病原菌具有较强的抑制作用，为提高其生防作用，开展FJ17-4发酵技术研究。方法以发酵液的OD₆₀₀值为评估指标，采用单因素和正交试验方法对发酵培养基和发酵条件进行筛选和优化，获得最佳发酵培养基和发酵条件后，进一步对优化后发酵液的菌体数、病原菌抑制率和室内盆栽防治效果进行测定和分析。结果菌株FJ17-4的最佳培养基配方为黄豆粉12.5 g·L⁻¹、玉米粉5.0 g·L⁻¹、K₂HPO₄ 12.5 g·L⁻¹，最佳发酵条件为：初始pH 7.0，培养温度30 ℃，装液量20% （50 mL/250 mL），接种量12.5%，转速180 r·min⁻¹，发酵培养时间40 h。优化后发酵液的OD₆₀₀值和菌体数量分别为1.52×10¹⁰、1.03×10¹⁰ cfu·mL⁻¹，比优化前分别提高了25.62%和21.95%。50倍优化发酵液对病原菌菌丝生长抑制率和室内盆栽防治效果分别为42.35%和 72.14%，比优化前分别提高了56.38%和13.46%。结论优化后发酵培养基和发酵条件有效提高菌株FJ17-4的发酵效果，降低了发酵成本，研究结果为贝莱斯芽孢杆菌FJ17-4的开发和工业化生产及应用提供了理论依据。
- 贝莱斯芽孢杆菌 /
- 发酵培养基 /
- 发酵条件 /
- 优化
Abstract: Objective Fermentation of Bacillus velezensis FJ17-4 known with a high inhibitory activity against several pathogens was optimized for potential application as a biocontrol agent. Methods Spectrometric measurement at OD₆₀₀ of the fermentation broth was used as the index for evaluation. Culture medium and conditions were optimized by single factor and orthogonal experiments. Bacterial quantity, pathogenic inhibition, and indoor potted control effect of the optimized fermentation product were determined. Results The optimized FJ17-4 fermentation used a medium that consisted of 12.5 g·L⁻¹ soybean meal, 5.0 g·L⁻¹ corn flour, and 12.5 g·L⁻¹ potassium dihydrogen phosphate at an initial pH of 7.0 to ferment a liquid loading (medium volume) of 50 mL with an inoculum size of 12.5% for 40 h at 30 ℃ in a 250 mL flask that rotated at a constant speed of 180 r·min⁻¹. The OD₆₀₀ of the optimized fermentation broth was 1.52 representing a cell load of 1.03×10¹⁰ cfu·mL⁻¹, which were 25.62% and 21.95%, respectively, higher than those prior to the optimization. The inhibition rate on the mycelial growth of Fusarium oxysporum f. sp. cucumerinum and the indoor potted control on cucumber fusarium wilt of the 50× optimized fermentation broth were 42.35% and 72.14%, representing 56.38% and 13.46% increases, respectively, over the original. Conclusion The FJ17-4 fermentation was significantly improved by the optimization. An operational cost reduction was also achieved.
- Bacillus velezensis /
- culture medium /
- fermentation conditions /
- optimization

HTML全文

图 1 菌株FJ17-4的生长曲线

Figure 1. Cell growth of FJ17-4

下载: 全尺寸图片幻灯片

图 2 不同C、N源和无机盐对菌株FJ17-4发酵的影响

A：碳源，B：氮源，C：无机盐。

Figure 2. Effects of C and N sources and inorganic salts on fermentation of FJ17-4

A: carbon source; B: nitrogen source; C: inorganic salt.

下载: 全尺寸图片幻灯片

图 3 不同初始pH值、温度、接种量、装液量、转速和培养时间对菌株FJ17-4发酵的影响

A：初始pH，B：温度，C：接种量，D：装液量，E：转速，F：培养时间。

Figure 3. Effects of initial pH, temperature, inoculum concentration, liquid loading, rotating speed, and culture time on FJ17-4 fermentation

A: initial pH; B: temperature; C: inoculum size; D: liquid loading; E: rotating speed; F: culture time.

下载: 全尺寸图片幻灯片

图 4 无菌滤液对黄瓜枯萎病菌FOC-1菌丝生长的抑制作用

a: 发酵体系优化后无菌滤液；b: 基础（原始）培养基无菌滤液；c: 无菌水。

Figure 4. Inhibitory effect of cell-free fermentation filtrate on mycelium growth of FOC-1

a: cell-free filtrate of optimized fermentation broth; b: cell-free filtrate of original fermentation broth; c: sterile water.

下载: 全尺寸图片幻灯片

图 5 发酵液对黄瓜枯萎病的防治效果

A: 50倍优化发酵液；B：10倍优化发酵液；C：2000倍98%噁霉灵可溶粉剂；D：无菌水；E: 50倍基础培养基发酵液。

Figure 5. Control effects of fermentation broth on cucumber fusarium wilt

A: 50× optimized fermentation broth; B: 10× optimized fermentation broth; C: 2000×98% hymexazol soluble powder; D: sterile water; F: 50× original fermentation broth.

下载: 全尺寸图片幻灯片

表 1 培养基组分的正交试验因素与水平

Table 1. Factors and levels of orthogonal test for culture medium formulation

水平 Level	各因素含量 Content of each factor /(g·L⁻¹)
水平 Level	A：玉米粉 Cornmeal	B：黄豆粉 Soybean flour	C：K₂HPO₄
1	5.0	10.0	10.0
2	7.5	12.5	12.5
3	10.0	15.0	15.0

下载: 导出CSV

表 2 培养基各组分比优化的正交试验

Table 2. Orthogonal test on all factors of medium

处理 Treatments	A：玉米粉 A: Cornmeal /(g·L⁻¹)	B：黄豆粉 B: Soybean flour /(g·L⁻¹)	C：K₂HPO₄ C: K₂HPO₄ /（g·L⁻¹）	OD₆₀₀值 OD₆₀₀ Value
1	5.0	10.0	10.0	0.98±0.01
2	5.0	12.5	12.5	1.02±0.02
3	5.0	15.0	15.0	1.12±0.00
4	7.5	10.0	12.5	1.28±0.01
5	7.5	12.5	15.0	0.86±0.01
6	7.5	15.0	10.0	0.96±0.02
7	10.0	10.0	15.0	1.03±0.01
8	10.0	12.5	10.0	0.95±0.01
9	10.0	15.0	12.5	1.06±0.02
K1	3.12	3.36	2.98
K2	3.16	3.12	3.12
K3	2.86	2.99	3.05
R	0.30	0.37	0.14
重要性顺序 Order of importance	B＞A＞C
优水平 Optimal levels	A2	B1	C2
优组合 Optimal combination	A2 B1 C2

下载: 导出CSV

表 3 FJ17-4无菌滤液对黄瓜枯萎病菌菌丝生长的抑制效果

Table 3. Inhibitory effect of FJ17-4 cell-free fermentation filtrate on mycelial growth of Fusarium oxysporum f. sp. cucumerinum

处理 Treatments	菌落直径 Colony diameter/cm	抑制效果 Inhibition effect/%	差异显著性 Difference significance (P＜0.05)
10倍优化后无菌发酵滤液 10 times optimized sterile fermentation filtrate	4.90±0.38	42.35±2.13	a
10倍基础培养基（LB）无菌滤液 10 times sterile fermentation filtrate of basic medium (LB)	6.2±0.29	27.08±1.45	b
无菌水对照 Sterile water	8.50±0.46	/	/

下载: 导出CSV

表 4 FJ17-4无菌滤液对黄瓜枯萎病菌分生孢子萌发的抑制效果

Table 4. Inhibitory effect of FJ17-4 cell-free fermentation filtrate on conidial germination of F. oxysporum f. sp. cucumerinum

处理 Treatments	孢子萌发率 Spore germination rate /%	抑制效果 Inhibition effect /%)	差异显著性 Difference significance (P＜0.05)
50倍优化后无菌发酵滤液 50 times optimized sterile fermentation filtrate	13.13±0.76	85.32±0.35	a
50倍基础培养基（LB）无菌滤液 50 times sterile fermentation filtrate of basic medium (LB)	14.82±1.03	83.43±1.44	a
100倍优化后无菌发酵滤液 100 times optimized sterile fermentation filtrate	22.14±1.41	75.26±0.24	b
100倍基础培养基（LB）无菌滤液 100 times sterile fermentation filtrate of basic medium (LB)	24.93±1.22	72.12±0.36	c
无菌水对照 Sterile water	89.42±2.43	/	/

下载: 导出CSV

表 5 FJ17-4发酵液对黄瓜枯萎病的室内盆栽防治效果

Table 5. Control effect of FJ17-4 fermentation broth on cucumber fusarium wilt in potted experiment

处理 Treatments	发病率 Incidence rate/%	防治效果 Control effect /%	差异显著性 Difference significance (P＜0.05)
10倍优化发酵液 10 times optimized fermentation broth	24.02±0.86	73.68±0.52	a
50倍优化发酵液 50 times optimized fermentation broth	24.51±1.12	72.14±0.36	b
50倍基础培养基发酵液 50 times basic medium fermentation broth	33.24±2.13	63.58±1.23	d
2000倍98%噁霉灵可溶粉剂 2000 times 98% hymexazol soluble powder	27.62±1.79	69.73±0.54	c
无菌水对照 Sterile water	91.26±3.26	/	/

下载: 导出CSV

参考文献(23)

[1]	黄曦, 许兰兰, 黄荣韶, 等. 枯草芽孢杆菌在抑制植物病原菌中的研究进展 [J]. 生物技术通报, 2010(1):24−29. doi: 10.13560/j.cnki.biotech.bull.1985.2010.01.026 HUANG X, XU L L, HUANG R S, et al. Research advance in controlling plant disease by Bacillus subtilis [J]. Biotechnology Bulletin, 2010(1): 24−29.(in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2010.01.026
[2]	KHALIL M S, HAIKAL N Z, HAFEZ E. Biological controls of cucumber wilt disease caused by Fusarium oxysporum [J]. Research Journal of Pharmaceutical Biological and Chemical Sciences, 2017, 8(6): 413−422.
[3]	RUIZ-GARCÌA C, BÉJAR V, MARTINEZ-CHECA F, et al. Bacillus velezensis sp. nov. a surfactant-producing bacterium isolated from the river Vélez in Málaga, souther Spain [J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(1): 191−195. doi: 10.1099/ijs.0.63310-0
[4]	刘洋, 刘晓昆, 陈文浩. 贝莱斯芽孢杆菌(Bacillus velezensis)物种名称的“前世今生” [J]. 生物技术通报, 2019(7):230−232. LIU Y, LIU X K, CHEN W H. “past and present” species name of Bacillus velezensis [J]. Biotechnology Bulletin, 2019(7): 230−232.(in Chinese)
[5]	沙月霞, 隋书婷, 曾庆超, 等. 贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力 [J]. 中国农业科学, 2019(11):1908−1917. doi: 10.3864/j.issn.0578-1752.2019.11.006 SHA Y X, SUI S T, ZENG Q C, et al. Biocontrol potential of Bacillus velezensis strain E69 against rice blast and other fungal diseases [J]. Scientia Agricultura Sinica, 2019(11): 1908−1917.(in Chinese) doi: 10.3864/j.issn.0578-1752.2019.11.006
[6]	LUO W J, LIU L D, QI G F, et al. Embedding Bacillus velezensis NH-1 in microcapsules for biocontrol of cucumber Fusarium wilt [J]. Applied and Environmental Microbiology, 2019, 85(9): e03128−e03118.
[7]	YE M, TANG X, YANG R, et al. Characteristics and application of a novel species of Bacillus: Bacillus velezensis [J]. ACS Chemical Biology, 2018, 13(3): 500−505. doi: 10.1021/acschembio.7b00874
[8]	刘芳, 廖先清, 周荣华, 等. 响应面法优化贝莱斯芽孢杆菌CY30发酵培养基的研究 [J]. 湖北农业科学, 2019(23):91−94. doi: 10.14088/j.cnki.issn0439-8114.2019.23.021 LIU F, LIAO X Q, ZHOU R H, et al. Study on optimization of fermentation medium of Bacillus velezensis CY30 by using response-surface method [J]. Hubei Agricultural Sciences, 2019(23): 91−94.(in Chinese) doi: 10.14088/j.cnki.issn0439-8114.2019.23.021
[9]	杨可, 司文, 林海, 等. 利用响应面分析法优化贝莱斯芽孢杆菌TCS001的发酵条件 [J]. 农药学学报, 2019, 21(4):444−452. doi: 10.16801/j.issn.1008-7303.2019.0066 YANG K, SI W, LIN H, et al. Fermentation condition optimization of Bacillus velezensis TCS001 using response surface methodology [J]. Chinese Journal of Pesticide Science, 2019, 21(4): 444−452.(in Chinese) doi: 10.16801/j.issn.1008-7303.2019.0066
[10]	黎燕珊, 崔文艳, 张陈芳, 等. 抗金银花白粉病菌贝莱斯芽孢杆菌HC-8菌株培养基及发酵条件优化 [J]. 南方农业学报, 2021, 52(8):2148−2157. doi: 10.3969/j.issn.2095-1191.2021.08.013 LI Y S, CUI W Y, ZHANG C F, et al. Optimization of culture medium and fermentation parameters of Bacillus velezensis HC-8 antagonistic to Erysiphe lonicerae [J]. Journal of Southern Agriculture, 2021, 52(8): 2148−2157.(in Chinese) doi: 10.3969/j.issn.2095-1191.2021.08.013
[11]	张晓, 张艳军, 陈雨, 等. 嘧菌酯对番茄早疫病菌的抑制作用 [J]. 农药学学报, 2008, 10(1):41−46. doi: 10.3321/j.issn:1008-7303.2008.01.007 ZHANG X, ZHANG Y J, CHEN Y, et al. Inhibitory effect of azoxystrobin on Alternaria solani [J]. Chinese Journal of Pesticide Science, 2008, 10(1): 41−46.(in Chinese) doi: 10.3321/j.issn:1008-7303.2008.01.007
[12]	韦巧婕, 郑新艳, 邓开英, 等. 黄瓜枯萎病拮抗菌的筛选鉴定及其生物防效 [J]. 南京农业大学学报, 2013, 36(1):40−46. doi: 10.7685/j.issn.1000-2030.2013.01.008 WEI Q J, ZHENG X Y, DENG K Y, et al. Screening and identification of antagonistic Bacillus vallismoritis B against cucumber Fusarium wilt and its biological effect [J]. Journal of Nanjing Agricultural University, 2013, 36(1): 40−46.(in Chinese) doi: 10.7685/j.issn.1000-2030.2013.01.008
[13]	王朝恩, 刘婉慧, 陆蓝翔, 等. 短小芽孢杆菌HR10产孢培养基及发酵条件优化 [J]. 微生物学杂志, 2021, 41(2):37−45. WANG C E, LIU W H, LU L X, et al. Optimization of sporulation medium and fermentation conditions for Bacillus pumilus HR10 [J]. Journal of Microbiology, 2021, 41(2): 37−45.(in Chinese)
[14]	赵鹏鹏, 雷淑珍, 徐晓光, 等. 培养基组成对贝莱斯芽孢杆菌产抑真菌成分的影响 [J]. 食品与发酵工业, 2020, 46(5):147−151. doi: 10.13995/j.cnki.11-1802/ts.022174 ZHAO P P, LEI S Z, XU X G, et al. Effect of medium compositions on the production of antifungalcomponents by Bacillus velezensis [J]. Food and Fermentation Industries, 2020, 46(5): 147−151.(in Chinese) doi: 10.13995/j.cnki.11-1802/ts.022174
[15]	吴志美, 兰明先, 高熹, 等. 除草活性成团泛菌ZLSY20菌株发酵条件的优化 [J]. 南方农业学报, 2019, 50(9):1990−1997. WU Z M, LAN M X, GAO X, et al. Screening of fermentation conditions for herbicidal activity of Pantoea agglomerans strain ZLSY20 [J]. Journal of Southern Agriculture, 2019, 50(9): 1990−1997.(in Chinese)
[16]	李姝江, 王淋敏, 谯天敏, 等. 利用响应面法优化贝莱斯芽孢杆菌ZJ20发酵参数 [J]. 西北农林科技大学学报(自然科学版), 2019, 47(2):88−96. doi: 10.13207/j.cnki.jnwafu.2019.02.011 LI S J, WANG L M, QIAO T M, et al. Optimization of Bacillus velezensis ZJ20 fermentation parameters by response surface methodology [J]. Journal of Northwest A & F University (Natural Science Edition), 2019, 47(2): 88−96.(in Chinese) doi: 10.13207/j.cnki.jnwafu.2019.02.011
[17]	YUAN Q P, WANG J D, ZHANG H, et al. Effect of temperature shift on production of xylanase by Aspergillus niger [J]. Process Biochemistry, 2005, 40(10): 3255−3257. doi: 10.1016/j.procbio.2005.03.020
[18]	周洋子, 邱慧珍, 董莉, 等. 马铃薯疮痂病高效生防芽孢杆菌的筛选及发酵条件优化 [J]. 干旱地区农业研究, 2020, 38(5):259−267. doi: 10.7606/j.issn.1000-7601.2020.05.36 ZHOU Y Z, QIU H Z, DONG L, et al. Screening of highly effective biocontrol Bacillus strains for potato scab and optimization of its fermentation conditions [J]. Agricultural Research in the Arid Areas, 2020, 38(5): 259−267.(in Chinese) doi: 10.7606/j.issn.1000-7601.2020.05.36
[19]	徐世荣, 陈骧, 吴云鹏. 细菌芽孢形成机制在微生态制剂生产中的应用 [J]. 食品与生物技术学报, 2007, 26(4):121−126. doi: 10.3321/j.issn:1673-1689.2007.04.025 XU S R, CHEN X, WU Y P. Application of the mechanism of sporulation in production of pharmaceutical probiotics [J]. Journal of Food Science and Biotechnology, 2007, 26(4): 121−126.(in Chinese) doi: 10.3321/j.issn:1673-1689.2007.04.025
[20]	CHEN F, WANG M, ZHENG Y, et al. Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579 [J]. World Journal of Microbiology and Biotechnology, 2010, 26(4): 675−684. doi: 10.1007/s11274-009-0222-0
[21]	CAO Y, XU Z H, LING N, et al. Isolation and identification of lipopeptides produced by B. subtilis SQR9 for suppressing fusarium wilt of cucumber [J]. Scientia Horticulturae, 2012, 135: 32−39. doi: 10.1016/j.scienta.2011.12.002
[22]	郑爱萍, 闫敏, 李平, 等. 黄瓜枯萎病新型抑制蛋白L37的研究 [J]. 园艺学报, 2005, 32(6):1102−1104. doi: 10.3321/j.issn:0513-353X.2005.06.029 ZHENG A P, YAN M, LI P, et al. Research on new antagonistic protein L37 against Fusarium oxysporum [J]. Acta Horticulturae Sinica, 2005, 32(6): 1102−1104.(in Chinese) doi: 10.3321/j.issn:0513-353X.2005.06.029
[23]	张美君, 吴庆, 尹翠, 等. 尖镰孢黄瓜专化型枯萎病菌拮抗菌的筛选、鉴定及培养条件优化 [J]. 生物技术通报, 2020, 36(9):125−136. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0578 ZHENG M J, W Q, YIN C, et al. Screening and identification of an antagonistic strain against Fusarium oxysporum f. sp. cucumerinum and optimization of culture conditions [J]. Biotechnology Bulletin, 2020, 36(9): 125−136.(in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2020-0578