柱花草内生细菌ZW21发酵条件优化及其抑菌物质稳定性测定

doi:10.13560/j.cnki.biotech.bull.1985.2024-1043

摘要/Abstract

摘要：

目的蜡样芽胞杆菌（Bacillus cereus）拮抗菌株ZW21对柱花草炭疽病菌有较大的生防潜力，为获得菌株ZW21的最优发酵条件，提升其活性次级代谢产物的产量，增强对柱花草炭疽病（Stylo anthracnose）的防治效果。方法以菌株ZW21的生长量及其发酵滤液抑制柱花草炭疽病菌活性为指标，通过单因素试验筛选培养基组分、正交试验确定其最佳组合；采用单因素试验对发酵条件（pH值、转速、温度、接种量、装液量和发酵时间）逐项进行优化；测定优化后无菌发酵液在不同温度、紫外线照射时间、酸碱环境和蛋白酶处理下的稳定性；通过温室盆栽实验，对比优化前后发酵液对柱花草炭疽病的防治效果。结果菌株ZW21的最优培养基配方为0.5%可溶性淀粉、1%苯丙氨酸、0.5%硫酸镁，最适发酵条件为初始pH值4、转速180 r/min、温度28 ℃、装液量200 mL、接种量7%、发酵时间96 h；无菌发酵液可耐受100 ℃高温，紫外线照射8 h抑菌活性不受影响，不受胃蛋白酶、胰蛋白酶的影响，耐强酸，但不耐强碱；与优化前LB发酵液相比，优化后发酵液抑菌活性提高了约10倍，对柱花草炭疽病的防效提高了4倍。结论菌株ZW21发酵液稳定性良好，优化后发酵液的OD₆₀₀值与抑菌率分别为7.05与73.18%，对柱花草炭疽病的防效达到62.5%。

关键词: 胶孢炭疽菌, 生物防治, 芽胞杆菌, 炭疽病, 抑菌活性, 稳定性, 生防效果

Abstract:

Objective The antagonistic strain Bacillus cereus ZW21 has significant potential for biological control against the pathogen causing stylo anthracnose. To improve the yield of active secondary metabolites and the bio-control effect of strain ZW21, the optimal fermentation conditions were investigated. Method The medium composition were optimized by single factor test and orthogonal test, and the fermentation conditions (pH, rotating speed, temperature, inoculation amount, liquid volume, and fermentation time) were optimized by single factor test. The stability of fermentation filtrate under the treatment of temperature, ultraviolet irradiation, acid-base and protease was determined. The optimization effect was tested by pot biological control test. Result The optimized results showed that the optimal medium composition was 0.5% soluble starch, 1% phenylalanine, and 0.5% magnesium sulfate. The optimal fermentation conditions were as follows: initial pH 4, rotating speed 180 r/min, temperature 28 ℃, liquid volume 200 mL, inoculation amount 7%, and fermentation time 96 h. The results of stability showed that the fermentation filtrate was resistant to the high temperature of 100 ℃, ultraviolet irradiation for 8 h, pepsin and trypsin treatment and strong acid, but susceptible to strong alkali. Under the optimized culture conditions, the antagonistic activity and bio-control effect of fermentation filtrate increased by 10 times and 4 times, respectively. Conclusion The fermentation broth of strain ZW21 has fine stability. The optimized fermentation broth has an OD₆₀₀ value of 7.05 and an inhibition rate of 73.18%, with a control efficacy of 62.5% against stylo anthracnose.

Key words: Colletotrichum gloeosporioides, biological control, Bacillus, anthracnose, anti-fungal activity, stability, biological control effect

张吉昌, 许云凤, 蒋凌雁. 柱花草内生细菌ZW21发酵条件优化及其抑菌物质稳定性测定[J]. 生物技术通报, 2025, 41(5): 280-289.

ZHANG Ji-chang, XU Yun-feng, JIANG Ling-yan. Optimization of Fermentation Conditions of Endophytic Bacterium ZW21 Isolated from Stylosanthes and Stability Analysis of Antimicrobial Substances[J]. Biotechnology Bulletin, 2025, 41(5): 280-289.

图/表 8

表1 正交试验因素

Table 1 Factors of orthogonal test

编号 No.	因素 Factor
编号 No.	可溶性淀粉 Soluble starch（A）（%）	苯丙氨酸 L-phenylalanine（B）（%）	硫酸镁 MgSO₄（C）（%）
1	0.25	0.5	0.5
2	0.5	1	1
3	1	1.5	1.5

图1 菌株ZW21生长曲线

Fig. 1 Growth curve of ZW21 strain

图2 碳源、氮源和无机盐种类对菌株ZW21生长量及无菌发酵液抑菌活性的影响不同小写字母表示在0.05水平差异显著。下同

Fig. 2 Effects of carbon sources, nitrogen sources and types of inorganic salts on the growth of strain ZW21 and the antibacterial activity of its sterile fermentation brothDifferent lowercase letters indicate significant difference between treatments at 0.05 level. The same below

表2 菌株ZW21营养条件正交试验结果

Table 2 Results of orthogonal test on nutritional conditions of strain ZW21

编号 No.	因素 Factor			抑菌率 Inhibition rate （%）	OD₆₀₀
编号 No.	可溶性淀粉 Soluble starch （A）（%）	苯丙氨酸 L-Phenylalanine （B）（%）	硫酸镁 MgSO₄（C）（%）	抑菌率 Inhibition rate （%）	OD₆₀₀
1	0.25	0.5	0.5	41.53±4.65^d	5.5±0.18^b
2	0.25	1	1.5	46.82±2.14^cd	4.96±0.11^b
3	0.25	1.5	1	51.181.62^bcd	4.44±0.14^b
4	0.5	0.5	1	52.82±0.71^bcd	5.49±0.21^b
5	0.5	1	0.5	65.76±1.45^a	7.44±0.23^a
6	0.5	1.5	1.5	61.18±2.78^ab	6.25±0.14^a
7	1	0.5	1.5	44.94±4.4^d	5.8±0.07^b
8	1	1	1	58.24±1.7^abc	7.19±0.16^a
9	1	1.5	0.5	57.41±0.68^abc	7.06±0.44^a
K1	418.59	417.87	494.1
K2	539.28	512.46	486.72
K3	481.77	509.31	458.82
k1	46.51	46.43	54.9
k2	59.92	56.94	54.08
k3	53.53	56.59	50.98
R	13.41	10.51	3.92
K1′	44.7	50.37	60
K2′	57.54	58.77	51.37
K3′	60.15	53.25	51.03
k1′	4.97	5.6	6..67
k2′	6.39	6.53	5.71
k3′	6.68	5.92	5.67
R	1.72	0.93	1

图3 不同发酵条件对菌株ZW21生长量及无菌发酵液抑菌活性的影响

Fig. 3 Effects of different fermentation conditions on the growth of strain ZW21 and the inhibitory activity of sterile fermentation filtrate

图4 菌株 ZW21无菌发酵液的稳定性分析

Fig. 4 Stability analysis of sterile fermentation filtrate of strain ZW21

图5 菌株 ZW21 无菌发酵液抑菌活性对比A：空白对照；B：优化前发酵滤液；C：优化后发酵滤液

Fig. 5 Comparison of inhibition activity of strain ZW21 fermentation filtrateA: Blank control; B: fermentation filtrate before optimization; C: optimized fermentation filtrate

图6 喷施 ZW21 优化前后发酵液盆栽防治效果比较A：对照（接种炭疽菌）；B：外施优化前发酵液后接种炭疽菌；C：外施优化后发酵液后接种炭疽菌

Fig. 6 Comparison of control effects of potted plant sprayed with fermentation liquids before and after ZW21 optimizedA: Control (inoculation of C. gloeosproioides only); B: treatment of unoptimized fermentation filtrate before inoculation of C. gloeosporioides; C: treatment of optimized fermentation filtrate before inoculation of C. gloeosporioides

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