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
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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

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图 1 菌株FJ17-4的生长曲线

Figure 1. Cell growth of FJ17-4

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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.

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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.

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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.

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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.

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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

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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

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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	/	/

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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	/	/

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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	/	/

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