Fermentation Optimization and Antifungal Activity of Bacillus methylotrophicus Z21
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摘要:
目的 芽孢杆菌Z21发酵液对黑曲霉、康氏木霉等真菌有拮抗性，探究Z21菌抑菌活性物质稳定性及抑菌活性分析，可为开发天然防腐剂提供参考。 方法 通过牛津杯法，以康氏木霉为指示菌，优化Z21菌产抑菌活性物质的发酵条件，探讨不同温度、紫外线、pH、作用时间、酶和金属离子对Z21菌抑菌活性物质稳定性的影响；测定硫酸铵沉淀法提取的粗提物MIC及对康氏木霉孢子萌发和菌丝生长的影响。 结果 当发酵条件为500 mL锥形瓶装液体培养基150 mL、接种量3%、发酵时间60 h、发酵温度30 ℃、转速140 r·min−1时，150 μL无菌发酵液对康氏木霉菌丝生长的抑制率为72%左右，粗提物的MIC为25 mg·mL−1。在紫外光波长为260 nm和280 nm时，经25 mg·mL−1的粗取物处理后的康氏木霉菌液紫外吸光值升高，说明菌丝的核酸和蛋白质有泄露。Z21菌发酵液耐热，耐酸、碱，对紫外线、许多酶和金属离子不敏感。扫描电镜观察显示：Z21菌处理后的康氏木霉菌丝比较纤细，不分化孢子梗。 结论 Z21菌发酵液对康氏木霉的生长和繁殖均受到抑制，抑菌活性物质稳定性良好。 Abstract:Objective Fermentation process of Bacillus methylotrophicus Z21, which exhibited antifungal activities on Aspergillus niger and Trichoderma koningii among others in a preliminary study, was optimized, and the stability and antibacterial properties of the broth as a nature preservative examined. Method The fermentation conditions, including temperature, UV, pH, time, enzymes, and metal ions, were optimized based on the antifungal activity of the broth against T. koningii determined by the Oxford cup method. Effects of the ammonium sulfate-precipitated crude extract on the MIC, inhibition rate, spore germination, and hypha growth of T. koningii were also examined. Result The optimized process was conducted in a 500 mL flask to hold 150 mL liquid medium with 3% inoculum for the 60 h fermentation at 30 ℃ under constant 140 r·min−1 stirring. The inhibition rate of the 150 μL sterile fermentation broth on the hypha growth was 72%. The increased UV absorbances of the T. koningii culture liquid at OD260 and OD280 by the treatment of the extract at MIC of 25 mg·mL−1 indicated a leakage of nucleic acids and proteins into the liquid from the mycelia. The Z21 fermentation broth was resistant to heat, acid, and alkali but insensitive to UV, enzymes, and metal ions. The scanning electron microscopic images showed that the hyphae of T. koningii treated by the broth became slender without spore stems. Conclusion The growth and reproduction of T. koningii were significantly inhibited by the presence of B. methylotrophicus Z21 fermentation broth. The antifungal activity of the broth remained stable under elevated temperature and/or acidic or alkali conditions. -
Key words:
- antifungal bacterium /
- fermentation optimization /
- anti-fungal property /
- stability
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图 1 不同发酵条件对Z21菌无菌发酵液抑菌活性的影响
注：图中不同小写字母表示不同处理间差异显著（P＜0.05）。下同。
Figure 1. Effects of fermentation conditions on antifungal activity of sterile Z21 fermentation broth
Note: Data with different lowercase letters indicate significant difference between treatments (P＜0.05). Same for the following.
图 2 Z21菌发酵液抑菌活性稳定性
Figure 2. Stability of antibacterial activity of Z21 fermentation broth
图 3 受Z21菌影响的康氏木霉菌丝表面扫描电镜图
注：A：对照组，B：处理组。放大倍数从左到右依次为×800、×2 500、×5 000。
Figure 3. SEM images of T. koningii hyphae surface as affected by Z21
Note: A: Control; B: Treatment group. Magnifications are 800x, 2 500x, and 5 000x from left to right.
图 4 Z21菌发酵液粗提物对康氏木霉菌丝细胞内蛋白质泄露的影响
注：CK：对照组，CL：处理组。下同。
Figure 4. Effect of crude Z21 fermentation broth extract on protein leakage of T. koningii hyphae
Note: CK: Control; CL: Treatment group. Same for the following.
图 5 Z21菌发酵液粗提物对康氏木霉菌丝细胞内核酸泄露的影响
Figure 5. Effect of crude Z21 fermentation broth extract on nucleic acid leakage of T. koningii hyphae
表 1 正交试验的因素及水平设计
Table 1. Factors and levels of orthogonal test
水平
LevelsA装液量
Liquid volume/mLB接种量
Inoculation amount/%C发酵时间
Fermentation time/hD发酵温度
Fermentation temperature/℃E转速
Revolution speed/（r·min−1）1 100 2 36 30 120 2 150 3 48 32 140 3 200 4 60 35 160 
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表 2 Z21菌发酵条件正交试验结果
Table 2. Orthogonal test results on Z21 fermentation conditions
试验
TestA装液量
Liquid
volume/mLB接种量
Inoculation
amount/%C发酵时间
Fermentation
time/hD发酵温度
Fermentation
temperature/℃E转速
Revolution speed/
(r·min−1)F空白
Blank抑制率
Inhibition
rate/%1 100 2 36 30 120 1 52.56 2 100 3 48 32 140 2 40.41 3 100 4 60 35 160 3 23.86 4 150 2 36 32 140 3 60.85 5 150 3 48 35 160 1 39.67 6 150 4 60 30 120 2 55.73 7 200 2 48 30 160 2 13.13 8 200 3 60 32 120 3 56.54 9 200 4 36 35 140 1 34.92 10 100 2 60 35 140 2 19.19 11 100 3 36 30 160 3 13.25 12 100 4 48 32 120 1 57.8 13 150 2 48 35 120 3 25.45 14 150 3 60 30 140 1 70.61 15 150 4 36 32 160 2 15.08 16 200 2 60 32 160 1 16.67 17 200 3 36 35 120 2 22.11 18 200 4 48 30 140 3 51.75 k1 34.512 31.308 33.128 42.838 45.032 45.372 k2 44.565 40.432 38.035 41.225 46.288 27.608 k3 32.52 39.857 40.433 27.533 20.277 38.617 R 12.045 9.124 7.305 15.305 26.011 17.764 主次因素
Secondary factorsE＞D＞A＞B＞C 最优方案
Optimal alterativeA2B2C3D1E2
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表 3 正交试验结果方差分析
Table 3. ANOVA analysis on orthogonal test results
方差来源
Variance Source平方和
Squares自由度
Freedom均方
Mean
squareF值
F valueP值
P value校正模型 Calibration model 4410.504 10 441.050 1.708 0.246 A 500.237 2 250.118 0.968 0.425 B 313.280 2 156.640 0.607 0.572 C 166.381 2 83.190 0.322 0.735 D 848.615 2 424.308 1.643 0.260 E 2581.992 2 1290.996 4.999 0.045* 残差 Residuals 1807.867 7 258.267 总和 Sum 31126.004 18
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表 4 Z21菌发酵液粗提物抑制康氏木霉孢子萌发的MIC
Table 4. MIC of crude Z21 fermentation extract on T. koningii spore germination
不同质量浓度提取物的抑制效果
Antifungal effects of extracts of different concentrations/(mg·mL−1)0 1.56 3.125 6.25 12.5 25 50 100 200 400 800 1000 + + + + + − − − − − − − 注：“+”表示长菌，无抑制效果；“−”表示不长菌，有抑制效果。
Note: “+” means growth of bacteria with no inhibitory effect; “−” means inhibition on bacterial growth.
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