Fermentation condition optimization of isopropylstilbene and its inhibition effect against Botrytis cinerea
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摘要:
isopropylstilbene (3, 5-二羟基-4-异丙基二苯乙烯) 是发光杆菌Photohabdus temperata产生的一种二苯乙烯类化合物，具有多种生物活性。为促进isopropylstilbene在农业抗生素领域应用价值的开发，采用固相萃取、柱层析等技术从发光杆菌P. temperata SN 35的发酵液中分离纯化得到1个化合物，通过核磁共振氢谱对其结构进行了确认，并采用高效液相色谱测定了其纯度；通过Plackett-Burman试验和响应面法对发光杆菌SN 35摇瓶发酵培养条件进行了优化，并初步研究了所分离化合物对灰葡萄孢Botrytis cinerea的离体抑菌活性及对灰霉病的防治效果。结果表明：所分离的化合物为isopropylstilbene，纯度为93%；对发光杆菌SN 35摇瓶发酵培养条件进行优化后，isopropylstilbene的产量是优化前的2.22倍。离体抑菌试验结果表明：isopropylstilbene对灰葡萄孢的菌丝生长具有较好的抑制作用，其EC50值为 (9.17 ± 0.17) μg/mL，但低于对照药剂百菌清[EC50值为 (0.90 ± 0.08) μg/mL]；在10 μg/mL下，isopropylstilbene对灰葡萄孢孢子萌发的抑制率为63.01%。活体盆栽试验结果表明，在200 μg/mL下，isopropylstilbene对黄瓜灰霉病的防治效果达92.19%，与百菌清的防效 (97.63%) 相当，对番茄灰霉病的防治效果为72.12%，略低于百菌清 (81.61%)。本研究通过对isopropylstilbene发酵条件的优化，为其工业化生产奠定了理论基础。该化合物对灰霉病具有很好的防治效果，可为天然杀菌剂的研究与开发提供参考。
Abstract:Isopropylstilbene (3, 5-dihydroxy-4-isopropyl stilbene) is a stilbene compound produced by Photohabdus temperata, which has various biological activities. In order to investigate the potential application of isopropylstilbene in the field of agricultural antibiotics, this compound was isolated from the fermentation broth of Photohabdus temperata SN35 by solid phase extraction and and purified by column chromatography. Its structure was confirmed by 1H NMR and the purity was determined by high performance liquid chromatography. The shake flask fermentation conditions of the Photohabdus temperata SN35 were optimized by the Plackett-Burman test and the response surface method. The in vitro and in vivo antifungal activities of the compound against Botrytis cinerea were studied. The results showed that the isolated compound was isopropylstilbene with the purity of 93%. The yield of isopropylstilbene was 2.22 times higher than that before the optimization. The in vitro test results showed that the EC50 value of isopropylstilbene against the mycelial growth of B. cinerea was (9.17 ± 0.17) μg/mL, which was lower than that of the control, chlorothalonil [EC50 value (0.90 ± 0.08) μg/mL]. And at 10 μg/mL, the inhibition rate against spore germination was 63.01%. The results of pot experiments showed that the control effect of isopropylstilbene on cucumber gray mould was 92.19% at 200 μg/mL, which was almost the same with the control effect of chlorothalonil (97.63%). The control effect on tomato gray mold was 72.12%, which slightly lower than that of chlorothalonil (81.61%). This study laid a theoretical foundation for the industrial production by optimizing the fermentation conditions of isopropylstilbene. The compound has a good control effect against gray mold which will be useful for the research and development of natural fungicides.
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图 1 isopropylstilbene标准品和样品高效液相谱图
Figure 1. HPLC chromatograms of standard of isopropylstilbene and sample
图 2 蛋白胨和氯化钠之间的响应面图 (a) 和等高线图 (b)
Figure 2. Response surface graph (a) and the contour map (b) between peptone and sal communis
图 3 未经处理 (a) 和经 50 µg/mL isopropylstilbene 处理 (b) 的番茄灰霉病菌菌丝显微镜图 (10×40)
Figure 3. Microscopic image of mycelium of Botrytis cinerea untreated (a) and treated with 50 μg/mL isopropylstilbe (b) (10×40)
表 1 在M培养基中添加不同碳源、氮源、氨基酸和氯化钠时发酵液中isopropylstilbene产量
Table 1. isopropylstilbene yield in the fermentation broth when different carbon sources, nitrogen sources, amino acid and sodium chloride were added to M medium
类别
Categorg因素
Factor添加水平
Fortified level/(g/L)isopropylstilbene 产量
isopropylstilbene yield/(mg/L)碳源 Carbon sources 蔗糖 Sugar 3.06 20.75 淀粉 Starch 6.13 18.16 葡萄糖 Glucose 6.15 38.80 氮源 Nitrogen sources 蛋白胨 Peptone 21.29 41.24 酵母浸粉 Yeast extract 25.71 40.78 牛肉膏 Extractum carnis 45.00 39.51 胰蛋白胨 Tryptone 24.69 30.48 氨基酸 Amino acid 苯丙氨酸 Phe 3.00 11.26 亮氨酸 Leu 3.00 141.44 脯氨酸 Pro 3.00 20.64 氯化钠 Sodium chloride 氯化钠 Sodium chloride 3.40 53.07 
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表 2 Plackett-Burman试验设计及结果
Table 2. Experimental design and results of Plackett-Burman design
Run X1 X2 X3 X4 X5 X6 X7 Y 1 +1 +1 –1 +1 +1 –1 +1 30.00 2 +1 –1 –1 –1 +1 +1 +1 55.00 3 +1 –1 +1 –1 –1 –1 +1 70.00 4 –1 –1 –1 –1 –1 –1 –1 45.00 5 +1 +1 –1 +1 –1 –1 –1 11.00 6 –1 +1 +1 +1 –1 +1 +1 82.00 7 +1 –1 +1 +1 –1 +1 –1 82.00 8 +1 +1 +1 –1 +1 +1 –1 53.00 9 –1 –1 +1 +1 +1 –1 +1 46.00 10 –1 +1 +1 –1 +1 –1 –1 22.00 11 –1 +1 –1 –1 –1 +1 +1 43.00 12 –1 –1 –1 +1 +1 +1 –1 47.00 注：X1 = 亮氨酸，X2 = 苯丙氨酸，X3 = 氯化钠，X4 = 空白，X5 = 葡萄糖，X6 = 蛋白胨，X7 = 温度，Y = isopropylstilbene发酵产量 (mg/L)。Note: X1 = leucine, X2 = phenylalanine, X3 = sodium chloride, X4 = control, X5 = glucose, X6 = peptone, X7 = temperature. Y is isopropylstilbene fermentation yield (mg/L).
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表 3 Plackett-Burman试验设计的效应分析
Table 3. Evaluation for the effect of Plackett-Burman design
编号 No. 水平 Level 效应 Effects t 值 t-value P 值 P-value –1 1 X1 1.0 5.0 0.002 67 0.39 0.719 X2 1.0 5.0 –0.017 33 –2.51 0.066 X3 2.4 4.0 0.020 67 2.99 0.040** X4 – – 0.001 67 0.24 0.082 X5 6.0 10.0 –0.013 33 –1.93 0.126 X6 10.0 30.0 0.023 3.33 0.029** X7 24.0 29.0 0.011 1.59 0.187 注：X1 = 亮氨酸，X2 = 苯丙氨酸，X3 = 氯化钠，X4 = 空白，X5 = 葡萄糖，X6 = 蛋白胨，X7 = 温度。**表示显著。Note: X1 = leucine, X2 = phenylalanine, X3 = sodium chloride, X4 = control, X5 = glucose, X6 = peptone, X7 = temperature. ** Indicated significance.
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表 4 响应面法试验设计因素水平
Table 4. Factors and levels of response surface design
水平 Level 蛋白胨 Peptone/(g/L) NaCl/(g/L) –1 15 1 0 25 3 1 35 5
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表 5 响应面法试验设计及结果
Table 5. Experimental design and results of response surface design
Run A/(g/L) B/(g/L) Y 1 25 1 70.21 2 35 1 68.42 3 25 5 72.63 4 25 3 80.95 5 15 1 57.82 6 35 5 68.46 7 15 5 63.67 8 25 3 80.95 9 25 3 80.91 10 15 3 75.11 11 35 3 88.37 12 25 3 80.92 13 25 3 80.94 注：A = 蛋白胨，B=氯化钠，Y = isopropylstilbene发酵产量 (mg/L)。Note: A=peptone, B=sodium chloride, Y is isopropylstilbene fermentation yield (mg/L).
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表 6 方差分析结果
Table 6. ANOVA (analysis of variance) of regression model
来源 Source 自由度 df 平方和 ss 均方 ms F 值 F-Value 显著性 Prob > F 回归模型 Model 5 839.28 167.86 23.25 0.000 3** A 1 136.33 136.33 18.89 0.003 4** B 1 11.21 11.21 1.55 0.252 8 AB 1 8.41 8.41 1.17 0.316 2 A2 1 23.12 23.12 3.20 0.116 6 B2 1 480.73 480.73 66.60 < 0.000 1** 残差 Residue 7 50.53 7.22 注：A = 蛋白胨,，B = 氯化钠，**表示极显著。Note: A = peptone, B = sodium chloride, ** Indicated significance at 0.01 level.
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表 7 在黄瓜和番茄盆栽试验中isopropylstilbene对灰霉病的防治效果
Table 7. Control effect of isopropylstilbene on gray mould in pot experiment of cucumber and tomato
处理方式
Treatment黄瓜盆栽 Pot experiment of cucumber 番茄盆栽 Pot experiment of tomato 病斑直径
Spot diameter/cm防治效果
Control effect/%发病指数
Disease index防治效果
Control effect/%空白 Control 2.495 — 11.80 — isopropylstilbenea 0.748 92.19 3.29 72.12 百菌清 chlorothalonilb 0.645 97.63 2.17 81.61 注：a isopropylstilbene为5%乳油制剂，有效成分为200 µg/mL；b 百菌清为5%乳油制剂，有效成分为200 µg/mL。Note: aIsopropylstilbene is 5% emulsifiable preparation, and the effective concentration is 200 µg/mL. b Chlorothalonil is 5% emulsifiable preparation, and the effective concentration is 200 µg/mL.
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